"STERIX LIMITED"

In one preferred aspect R3 is =0.

StJBSTITUENTS The compound of the present invention may have substituents other than those of the ring systems show herein. Furthermore the ring systems herein are given as general formulae and should be interpreted as such. The absence of any specifically shown substituents on a given ring member indicates that the ring member may substituted with any moiety of which H is only one example. The ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of the ring system is aromatic. The ring system may be carbocyclic or may contain one or more hetero atoms.
The compound of the invention, in particular the ring system compound of the invention of the present invention may contain substituents other than those show herein. By way of example, these other substituents may be one or more of: one or more sulphate group (s), one or more phosphonate group (s), one or more thiophosphonate group (s), one or more sulphonate group (s), one or more sulphonamide group (s), one or more halo groups, one or more 0 groups, one or more hydroxy groups, one or more amino groups, one or more sulphur containing group (s), one or more hydrocarbyl group (s) -such as an oxyhydrocarbyl group.
In general terms the ring system A'B'C'D'of the present compounds may contain a variety of non-interfering substituents. In particular, the ring system A'B'C'D'may contain one or more hydroxy, alkyl especially lower (Cl-C6) alkyl, e. g. methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C,-C6) alkoxy, e. g. methoxy, ethoxy, propoxy etc., alkinyl, e. g. ethinyl, or halogen, e. g. fluoro substituents.
For some compounds of the present invention, it is preferred that the ring system is substituted with a oxyhydrocarbyl group. More preferably the A'ring of the ring system is substituted with a oxyhydrocarbyl group.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the A'ring of the ring system is substituted with a oxyhydrocarbyl group.
The term"oxyhydrocarbyl"group as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an atky) group, a cydic group etc.
In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
In one embodiment of the present invention, the oxyhydrocarbyl group is a oxyhydrocarbon group.
Here the term"oxyhydrocarbon"means any one of an alkoxy group, ah oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent (s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Typically, the oxyhydrocarbyl group is of the formula Cl-60 (such as a C1 30).
For some compounds of the present invention, it is highly preferred that the A'ring of the ring system is substituted with an alkoxy group.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the A'ring of the ring system is substituted with an alkoxy group.
Preferably the alkoxy group is methoxy.
For some compounds of the present invention, it is preferred that the ring system is substituted with a hydrocarbylsulphanyl group. More preferably the A'ring of the ring system is substituted with a hydrocarbylsulphanyl group. The term "hydrocarbylsulphanyl"means a group that comprises at least hydrocarbyl group (as herein defined) and sulphur, preferably-S-hydrocarbyl, more preferably-S- hydrocarbon. That sulphur group may be optionally oxidised.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the A'ring of the ring system is substituted with a hydrocarbylsulphanyl group.
Preferably the hydrocarbylsulphanyl group is-S-C1 10 alkyl, more preferably-S-C1-5 alkyl, more preferably-S-C1 3 alkyl, more preferably-S-CH2CH2CH3,-S-CH2CH3 or-SCH3 For some compounds of the present invention, it is highly preferred that at least the A' ring of the ring system is substituted with an hydrocarbyl group.
For some compounds of the present invention, it is highly preferred that at least the 2 position of the'ring of the ring system is substituted with an alkyl group.
Preferably the alkyl group is ethyl.
For some compounds of the present invention, it is highly preferred that the compound comprises at least two or more of sulphate group, a phosphonate group, a thiophosphonate group, a sulphonate group or a sulphonamide group.
For some compounds of the present invention, it is highly preferred that the compound comprises at least two sulphate groups.
For some compounds of the present invention, it is highly preferred that the compound comprises at least two sulphate groups, wherein said sulphate groups are not on the same ring.
For some compounds of the present invention, it is highly preferred that the A'ring of the ring system comprises at least one sulphate group and wherein the D'ring of the ring system comprises at least one sulphate group.
In some aspects of the present invention, preferably the A'ring contain one or more of an alkoxy substituent and an alkyl substituent. Thus according to further aspects of the present invention, the compound has one of the following formulae Formula VI Formula Vil Formula VIII Formula IX wherein R"is an alkoxy group or an alkyl group Preferably R"is an alkyl group. Preferably R"is a Cl-Clo alkyl group, preferably a Cl- C6 alkyl group, preferably a C1-C4 alkyl group. Preferably R11 is-CH3 or-CH2CH3.
In another preferred aspect, R11 is an alkoxy group. Preferably R11 is methoxy.
FURTHER ASPECTS According to a further aspect of the present invention there is provided a method comprising (a) performing a steroid dehydrogenase assay with one or more candidate compounds having the formula as defined herein; (b) determining whether one or more of said candidate compounds is/are capable of modulating steroid dehydrogenase activity ; and (c) selecting one or more of said candidate compounds that is/are capable of modulating steroid dehydrogenase activity.
According to a further aspect of the present invention there is provided a method comprising (a) performing a steroid dehydrogenase assay with one or more candidate compounds having the formula as defined herein; (b) determining whether one or more of said candidate compounds is/are capable of inhibiting steroid dehydrogenase activity ; and (c) selecting one or more of said candidate compounds that is/are capable of inhibiting steroid dehydrogenase activity.
In any one of the methods of the present invention, one or more additional steps may be present. For example, the method may also include the step of modifying the identified candidate compound (such as by chemical and/or enzymatic techniques) and the optional additional step of testing that modified compound for steroid dehydrogenase inhibition effects (which may be to see if the effect is greater or different). By way of further example, the method may also include the step of determining the structure (such as by use of crystallographic techniques) of the identified candidate compound and then performing computer modelling studies-such as to further increase its steroid dehydrogenase inhibitory action. Thus, the present invention also encompasses a computer having a dataset (such as the crystallographic co-ordinates) for said identified candidate compound. The present invention also encompasses that identified candidate compound when presented on a computer. screen for the analysis thereof-such as protein binding studies.
According to one aspect of the present invention, there is provided a compound identified by the method of the present invention.
According to one aspect of the present invention, there is provided a compound according to the present invention for use in medicine.
According to one aspect of the present invention, there is provided a pharmaceutical composition comprising the compound according to the present invention optionally admixed with a pharmaceutical acceptable carrier, diluent, excipient or adjuvant.
According to one aspect of the present invention, there is provided the use of a compound according to the present invention in the manufacture of a medicament for use in the therapy of a condition or disease associated with steroid dehydrogenase.
According to one aspect of the present invention, there is provided the use of a compound according to the present invention in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse steroid dehydrogenase levels.
For some applications, preferably the compounds have no, or a minimal, oestrogenic effect.
For some applications, preferably the compounds have an oestrogenic effect.
For some applications, preferably the compounds have a reversible action.
For some applications, preferably the compounds have an irreversible action.
In one embodiment, the compounds of the present invention are useful for the treatment of breast cancer.
The compounds of the present invention may be in the form of a salt.
The present invention also covers novel intermediates that are useful to prepare the compounds of the present invention. For example, the present invention covers novel alcohol precursors for the compounds. By way of further example, the present invention covers bis protected precursors for the compounds. Examples of each of these precursors are presented herein. The present invention also encompasses a process comprising each or both of those precursors for the synthesis of the compounds of the present invention.
We have also identified that in some aspects of the present invention the present compounds may also inhibit the activity of steroid dehydrogenase (HSD).
By steroid dehydrogenase or HSD it is meant 17p hydroxy steroid dehydrogenase. In one aspect the 1 7ß hydroxy steroid dehydrogenase is EC 1.1. 1.62 Preferably the HSD is of Type 1,3, 5 and/or 7. Preferably the HSD converts oestrone (ketone) to oestradiol (hydroxy).
Preferably the HSD is of Type 2 and/or 8. Preferably the HSD converts oestradiol (hydroxy) to oestrone (ketone).
In some aspects of the present invention, it is preferred that the steroid dehydrogenase is steroid dehydrogenase Type I.
In some aspects of the present invention, it is preferred that the steroid dehydrogenase is steroid dehydrogenase Type il Preferably the HSD is of Type 1, 3,5 and/or 7. Preferably the HSD converts oestrone (ketone) to oestradiol (hydroxy).
Preferably the HSD is of Type 2 and/or 8. Preferably the HSD converts oestradiol (hydroxy) to oestrone (ketone).
STEROID DEHYDROGENASE Steroid dehydrogenase or"DH"for short may be classified as consisting of two types- Type I and Type II. The two types enzyme, such as oestradiol 17 (3-hydroxysteroid dehydrogenases (E2HSD), have pivotal roles in regulating the availability of ligands to interact with the oestrogen receptor. Type I reduces oestrone (E1) to the biologically active oestrogen, oestradiol (E2) while E2HSD Type in inactivates E2 by catalysing its oxidation to E1.
DH INHIBITION It is believed that some disease conditions associated with DH activity are due to conversion of a nonactive, oestrone to an active, oestradiol. In disease conditions associated with DH activity, it would be desirable to inhibit DH activity.
Here, the term"inhibit"includes reduce and/or eliminate and/or mask and/or prevent the detrimental action of DH.
DH INHIBITOR In accordance with the present invention, the compound of the present invention is capable of acting as an DH inhibitor.
Here, the term"inhibitor"as used herein with respect to the compound of the present invention means a compound that can inhibit DH activity-such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of DH. The DH inhibitor may act as an antagonist.
The ability of compounds to inhibit steroid dehydrogenase activity can be assessed using either T47D breast cancer cells in which E2HSD Type I activity is abundant or MDA-MB-231 cells for Type in inhibitor studies. In both cell lines formation of products is linear with respect to time and cell numbers. Details on a suitable Assay Protocol are presented in the Examples section.
It is to be noted that the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit DH activity.
STEROID SULPHATASE In some aspects the compounds defined herein may also inhibit steroid sulphatase.
Steroid sulphatase-which is sometimes referred to as steroid sulphate or steryl sulphate or"STS"for short-hydrolyses several sulphate steroids, such as oestrone sulphate, dehydroepiandrosterone sulphate and cholesterol sulphate. STS has been allocated the enzyme number EC 3.1. 6.2.
STS has been cloned and expressed. For example see Stein et al (J. Biol. Chem.
264: 13865-13872 (1989) ) and Yen et al (Cell 49: 443-454 (1987)).
STS is an enzyme that has been implicated in a number of disease conditions.
By way of example, workers have found that a total deficiency in STS produces ichthyosis. According to some workers, STS deficiency is fairly prevalent in Japan. The same workers (Sakura et al, J Inherit Metab Dis 1997 Nov; 20 (6): 807-10) have also reported that allergic diseases-such as bronchial asthma, allergic rhinitis, or atopic dermatitis-may be associated with a steroid sulphate deficiency.
In addition to disease states being brought on through a total lack of STS activity, an increased level of STS activity may also bring about disease conditions. By way of example, and as indicated above, there is strong evidence to support a role of STS in breast cancer growth and metastasis.
STS has also been implicated in other disease conditions. By way of example, Le Roy et al (Behav Genet 1999 Mar ; 29 (2) : 131-6) have determined that there may be a genetic correlation between steroid sulphatase concentration and initiation of attack behaviour in


mice. The authors cohclude that sulphatation of steroids may be the prime mover of a complex network, including genes shown to be implicated in aggression by mutagenesis.
STS INHIBITION It is believed that some disease conditions associated with STS activity are due to conversion of a nonactive, sulphate oestrone to an active, nonsulphated oestrone. In disease conditions'associated with STS activity, it would be desirable to inhibit STS activity.
Here, the term"inhibit"includes reduce and/or eliminate and/or mask and/or prevent the detrimental action of STS.
STS INHIBITOR In accordance with the present invention, the compound of the present invention is capable of acting as an STS inhibitor.
Here, the term"inhibitor"as used herein with respect to the compound of the present invention means a compound that can inhibit STS activity-such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of STS. The STS inhibitor may act as an antagonist.
The ability of compounds to inhibit oestrone sulphate activity can be assessed using either intact MCF-7 breast cancer cells or placenta microsomes. In addition, an animal model may be used. Details on suitable Assay Protocols are presented in following sections. It is to be noted that other assays could be used to determine STS activity and thus STS inhibition. For example, reference may also be made to the teachings of WO- A-99/50453.
Preferably, for some applications, the compound is further characterised by the feature that if the sulphate group were to be substituted by a sulphate group to form a sulphate derivative, then the sulphate derivative would be hydrolysable by an enzyme having steroid sulphatase (E. C. 3. 1. 6. 2) activity-i. e. when incubated with steroid sulphatase EC 3. 1. 6. 2 at pH 7. 4 and 37°C.
In one preferred embodiment, if the sulphate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sutphatase (E. C.
3. 1. 6.2) activity and would yield a Km value of less than 200 mmolar, preferably less than 150 mmolar, preferably less than 100 mmolar, preferably less than 75 mmolar, preferably less than 50 mmolar, when incubated with steroid sulphate EC 3.1. 6.2 at pH 7.4 and 37°C.
In one preferred embodiment, if the sulphate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E. C.
3. 1.6. 2) activity and would yield a Km value of less than 200 molar, preferably less than 150 umolar, preferably less than 100, umolar, preferably less than 75 µmolar, preferably less than 50 molar, when incubated with steroid sulphate EC 3.1. 6.2 at pH 7.4 and 37°C.
In a preferred embodiment, the compound of the present invention is not hydrolysable by an enzyme having steroid sulphate (E. C. 3.1. 6.2) activity.
For some applications, preferably the compound of the present invention has at least about a 100 fold selectivity to a desired target (e. g. STS), preferably at least about a 150 fold selectivity to the desired target, preferably at least about a 200 fold selectivity to the desired target, preferably at least about a 250 fold selectivity to the desired target, preferably at least about a 300 fold selectivity to the desired target, preferably at least about a 350 fold selectivity to the desired target.
It is to be noted that the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit HSD activity.
SULPHAMATE GROUP





In one embodiment, the ring X has a sulphamate group as a substituent. The term "sulphamate" as used herein includes an ester of sulphamic acid, or an ester of an N- substituted derivative of sulphamic acid, or a salt thereof.
If 1R1 is a sulphate group then the compound of the present invention is referred to as a sulphate compound.
Typically, the sulphate group has the formula : (R9)(R10)N-S(O)(O)-O- wherein preferably R9 and RIO are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkylen, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R9 and/or RIO is alkyl, the preferred values are those where R9 and R10 are each independently selected from lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. RI and R5 may both be methyl. When R9 and/or RIO is aryl, typical values are phenyl and tolyl (PhCH3; o). Where R9 and RIO represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R9 and R10 typically represent an alkylen group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e. g. to provide a 5 membered heterocycle, e. g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
In some embodiments, the sulphate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one sulphamate group. By way of example, there may be two sulphamates (i. e. bis-sulphamate compounds). If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) sulphamate group is located at position 17 of the steroidal nucleus.
These groups need not be the same.
In some preferred embodiments, at least one of R9 and R10 is H.
In some further preferred embodiments, each of R9 and R10 is H.
PHOSPHONATE GROUP If R'is a phosphonate group then the compound of the present invention is referred to as a phosphonate compound.
Typically, the phosphonate group has the formula : (R12)-P (0) (OH)-0- wherein preferably R12 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R12 is alkyl, R12 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R12 may be methyl. When R6 is aryl, typical values are phenyl and tolyl (PhCH3; o). Where R6 represents cycloalkyl, typical values are cycfopropyl, cyclopentyl, cyclohexyl etc. R12 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e. g. to provide a 5 membered heterocycle, e. g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
In some embodiments, the phosphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one phosphonate group. By way of example, there may be two phosphonates (i. e. bis-phosphonate compounds). If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) phosphonate group is located at position 17 of the steroidal nucleus.
These groups need not be the same.
THIOPHOSPHONATE GROUP If R2 is a thiophosphonate group then the compound of the present invention is referred to as a thiophosphonate compound.
Typically, the thiophosphonate group has the formula: (RP (S) (OH)-0- wherein preferably R13 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R'is alkyl, R'may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R may be methyl. When I'3 is aryl, typical values are phenyl and tolyl (PhCH3; o). Where 13 represents cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. 13 may even comprise an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e. g. to provide a 5 membered heterocycle, e. g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemptary non-interfering substituents include hydroxy, amino, halo, alkoxy, aldeyl and aryl.
In some embodiments, the thiophosphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one thiophosphonate group. By way of example, there may be two thiophosphonates (i. e. bis-thiophosphonate compounds). If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) thiophosphonate group is located at position 17 of the steroidal nucleus. These groups need not be the same.
SULPHONATE GROUP If Ruz ils a sulphonate group then the compound of the present invention is referred to as a sulphonate compound.
Typically, the sulphonate group has the formula: (R'4)-S (O) (O)-O- wherein preferably R14 is H, alkyl, cycloalkyl, alkenyl or aryl, or combinations thereof, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R14 iS alkyl, R14 may be a lower alkyl groups containing from 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. By way of example, R14 may be methyl. When R14 is aryl, typical values are phenyl and tolyl (PhCH3 ; o). Where R14 represents cycloalkyl, typical values are cyclopropyl, cyclop@ntyl, cyclohexyl etc. R14 may even comprise an alkylen group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e. g. to provide a 5 membered heterocycle, e. g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groups are included containing as substituents therein one or more groups which do not interfere with the HSD inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
In some embodiments, the sulphonate group may form a ring structure by being fused to (or associated with) one or more atoms in or on group X.
In some embodiments, there may be more than one sulphonate group. By way of example, there may be two sulphonates (i. e. bis-sulphonate compounds). If these compounds are based on a steroidal nucleus, preferably the second (or at least one of the additional) sulphonate group is located at position 17 of the steroids nucleus. These groups need not be the same.
COMBINATION OF SULPHONATE/PHOSPHONATE/THIOPHOSPHONATE/SULPHAMATE For some compounds of the present invention there may be present one of a sulphonate as herein defined or a phosphonate as herein defined or a thiophosphonate as herein defined or a sulphate as herein defined; and another of a sulphonate as herein defined or a phosphonate as herein defined or a thiophosphonate as herein defined or a sulphate as herein defined. By way of example, the compound of the present invention may comprise one sulphate group and one phosphonate group.
If these compounds of the present invention are based on a steroidal nucleus, preferably the other of said groups is located at position 17 of the steroidal nucleus.
ASSAY FOR DETERMINING STS ACTIVITY USING CANCER CELLS (PROTOCQL 1) Inhibition of Steroid Sulphatase Activity in MCF-7 cells Steroid sulphate activity is measured in vitro using intact MCF-7 human breast cancer cells. This hormone dependent cell line is widely used to study the control of human breast cancer cell growth. It possesses significant steroid sulphate activity (Maclndoe et al. Endocrinology, 123, 1281-1287 (1988) ; Purohit & Reed, Int. J. Cancer, 50,901- 905 (1992)) and is available in the U. S.A. form the American Type Culture Collection (ATCC) and in the U.K. (e.g. from The Imperial Cancer Research Fund).
Cells are maintained in Minimal Essential Medium (MEM) (Flow Laboratories, Irvine, Scotland) containing 20 mM HEPES, 5% foetal bovine serum, 2 mM glutamin, non- essential amino acids and 0. 075% sodium bicarbonate. Up to 30 replicate 25 cm2 tissue culture flasks are seeded with approximately 1 x 105 cells/flask using the above medium. Cells are grown to 80% confluency and the medium is changed every third day.
Intact monolayers'of MCF-7 cells in triplicate 25 cm2 tissue culture flasks are washed with Earle's Balanced Salt Solution (EBSS from ICN Flow, High Wycombe, U. K.) and incubated for 3-4 hours at 37°C with 5 pmol (7 x 105 dpm) [6,7-3H] oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., U. S. A. ) in serum-free MEM (2.5 mi) together with oestrone-3-sulphamate (11 concentrations: 0; 1fM ; 0. 01pM ; 0. 1pM ; 1pM ; 0. 01nM ; 0. 1nM ; 1nM ; 0. 01mM ; 0. 1mM ; 1mM). After incubation each flask is cooled and the medium (1 ml) is pipette into separate tubes containing [14C] oestrone (7 x 103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U. K. ). The mixture is shaken thoroughly for 30 seconds with toluene (5 ml). Experiments have shown that >90% [14C] oestrone and Results for steroid sulphate activity are expressed as the mean 1 S. D. of the total product (oestrone + oestradiol) formed during the incubation period (20 hours) calculated for 106 cells and, for values showing statistical significance, as a percentage reduction (inhibition) over incubations containing no oestrone-3-sulphamate. Unaired Student's t-test was used to test the statistica) significance of resutts.
ASSAY FOR DETERMINING STS ACTIVITY USiNG PLACENTA MICROSOMES (PROTOCOL2) Inhibition of Steroid Sulphatase Activity in Placenta Microsomes Sulphatase-positive human placenta from normal term pregnancies are thoroughly minced with scissors and washed once with cold phosphate buffer (pH 7.4, 50 mM) then re-suspended in cold phosphate buffer (5 ml/g tissue). Homogenisation is accomplished with an Ultra-Turrax homogeniser, using three 10 second bursts separated by 2 minute cooling periods in ice. Nuclei and cell debris are removed by centrifuging (4°C) at 2000g for 30 minutes and portions (2 ml) of the supernatant are stored at 20°C. The protein concentration of the supernatants is determined by the method of Bradford (Anal.
Biochem. , 72,248-254 (1976) ).
Incubations (1 ml) are carried out using a protein concentration of 100 mg/ml, substrate concentration of 20 mM [6,7-3H] oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass. , U. S. A. ) and an incubation time of 20 minutes at 37°C. If necessary eight concentrations of compounds are employed: 0 (i. e. control); 0.05mM ; 0. 1 mM ; 0.2mM ; 0.4mM ; 0.6mM ; 0. 8mM ; 1. 0mM. After incubation each sample is cooled and the medium (1 mi) was pipetted into separate tubes containing [14C] oestrone (7 x 103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U. K.). The mixture is shaken thoroughly for 30 seconds with toluene (5 ml). Experiments have shown that >90% [14C] oestrone and A portion (2 ml) of the organic phase was removed, evaporated and the 3H and 14C content of the residue determined by scintillation spectrometry. The mass of oestrone-3- sulphate hydrolyse is calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C] oestrone added) and the specific activity of the substrate.
ANIMAL ASSAY MODEL FOR DETERMINING STS ACTIVITY (PROTOCOL 3,) inhibition of oestrones sulphatase activity in vivo The compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats. in this model compounds which are oestrogenic stimulate uterine growth.
The compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). A further group received the compound EMATE subcutaneously in an amount of 10ug/day for five days. At the end of the study samples of liver tissue were obtained and oestrone sulphate activity assayed using 3H oestrone sulphate as the substrate as previously described (see



PCT/GB95/02638).





l ANIMAL ASSAY MODEL FOR DETERMINING OESTROGENIC ACTIVITY (PROTOCOL 4) Lack of in vivo oestrogenicity The compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats. In this model, compounds which are oestrogenic stimulate uterine growth.
The compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). A further group received the estrogenic compound EMATE subcutaneously in an amount of 10ug/day for five days.
At the end of the study uteri were obtained and weighed with the results being expressed as uterine weight/whoie body weight x 100.
Compounds having no significant effect on uterine growth are not oestrogenic.
BIOTECHNOLOGICAL ASSAYS FOR DETERMINING STS ACTIVITY (PROTOCOL 5 The ability of compounds to inhibit oestrone sulphatase activity can also be assessed using amino acid sequences or nucleotide sequences encoding STS, or active fragments, derivatives, homologues or variants thereof in, for example, high-through put screens.
Any one or more of appropriate targets-such as an amino acid sequence and/or nucleotide sequence-may be used for identifying an agent capable of modulating STS in any of a variety of drug screening techniques. The target employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of target activity or the formation of binding complexes between the target and the agent being tested may be measured.
The assay of the present invention may be a screen, whereby a number of agents are tested. In one aspect, the assay method of the present invention is a high through put screen.
Techniques for drug screening may be based on the method described in Geysen, European Patent Application 84/03564, published on September 13, 1984. In summary, large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected-such as by appropriately adapting methods well known in the art. A purified target can also be coated directly onto plates for use in a drug screening techniques.
Alternatively, non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
Another technique for screening provides for high throughput screening (HTS) of agents having suitable binding affinity to the substances and is based upon the method described in detail in WO 84/03564.
It is expected that the assay methods of the present invention will be suitable for both small and Iarge-scale screening of test compounds as wet) as in quantitative assays.
In one preferred aspect, the present invention relates to a method of identifying agents that selectively modulate STS, which compounds have the formula (la).
REPORTERS A wide variety of reporters may be used in the assay methods (as well as screens) of the present invention with preferred reporters providing conveniently detectable signals (e. g. by spectroscopy). By way of example, a reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
Other protocols include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non- interfering epitopes may even be used. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8: 121 1).
Examples of reporter molecules include but are not limited to (p-ga) actosidase, invertase, green fluorescent protein, iuciferase, chloramphenicol, acetyltransferase, (- glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabelled or fluorescent tag-labelled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes.
By way of further examples, a number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wl), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for assay procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837 ; US-A-3850752 ; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241.
HOST CELLS The term"host cell"-in relation to the present invention includes any cell that could comprise the target for the agent of the present invention.
Thus, a further embodiment of the present invention provides host calls transformed or transfected with a oolynucleotide that is or expresses the target of the present invention.
Preferably said polynucleotide is carried in a vector for the replication and expression of polynucleotides that are to be the target or are to express the target. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
The gram negative bacterium E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult.
In contrast to E. coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
Depending on the nature of the polynucleotide encoding the polypeptide of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e. g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
Examples of suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A- 0284603) and Trichoderma species ; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species ; and yeasts such as I The use of suitable host cells-such as yeast, fungal and plant host cells-may provide for post-translatioinal modification (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
ORGANISM The term"organism"in relation to the present invention includes any organism that could comprise the target according to the present invention and/or products obtained therefrom. Examples of organisms may include a fungus, yeast or a plant.
The term"transgenic organism"in relation to the present invention includes any organism that comprises the target according to the present invention and/or products obtained.
TRANSFORMATION OF HOST CELLS/HOST ORGANISMS As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism.
Examples of suitable prokaryotic hosts include E coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning : A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et a/., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation-such as by removal of introns.
In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et a/ (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence into a construct designed for expression in yeast.
Several types of constructs used for heterologous expression have been developed.
The constructs contain a promoter active in yeast fused to the nucleotide sequence, usually a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.
For the transformation of yeast several transformation protocols have been developed.
For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104) ; and Ito, H et al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e. g. G418.
Another host organism is a plant. The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42 : 205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.
Thus, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target. Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression of the encoded protein. The protein produced by a recombinant cell may be displayed on the surface of the cell. If desired, and as will be understood by those of skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: 441-53).
VARIANTS/HOMOLOGUES/DERIVATIVES In addition to the specific amino acid sequences and nucleotide sequences mentioned herein, the present invention also encompasses the use of variants, homologue and derivatives thereof. Here, the term"homology"can be equated with"identity".
In the present context, an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical. Although homology can also be considered in terms of similarity (i. e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be'calculated over contiguous sequences, i. e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an"ungapped"alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed.
Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting"gaps"in the sequence alignment to try to maximise local homology.
However, these more complex methods assign"gap penalties"to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible-reflecting higher relatedness between the two compared sequences-will achieve a higher score than one with many gaps. "Affine gap costs"are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default ap penalty for amino acid sequences is-12 for a gap and-4 for each extension.
Calculation of maximum % horology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Besffit package (University of Wisconsin, U. S. A.; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid-Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for off) and online searching (s@@ Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
A further useful reference is that found in FEMS Microbiol Lett 1999 May 15 ; 174 (2) : 247- 50 (and a published erratum appears in FEMS Microbio) Lett 1999 Aug 1 ; 177 (1) : 187-8).
I Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix-the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid ; positively charged amino acids include lysine and arginin ; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamin, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below.
Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar G A P ILS Polar-uncharged C S T M N Q Polar-charged D E 19 Ft AROMATIC H F W Y l EXPRESSION VECTORS The nucleotide sequence for use as the target or for expressing the target can be incorporated into a recombinant repliable vector. The vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used.
Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
The protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
FUSION PROTEINS The target amino acid sequence may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (-galaetosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.
The fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention. In this embodiment, the fusion protein may be d non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric'substance expressing a heterologous epitope that is recognised by a commercially available antibody.
THERAPY The compounds of the present invention may be used as therapeutic agents-i. e. in therapy applications.
The term"therapy"includes curative effects, alleviation effects, and prophylactic effects.
The therapy may be on humans or animals, preferably female animals.
PHARMACEUTICAL COMPOSITIONS In one aspect, the present invention provides a pharmaceutical composition, which comprises a compound according to the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptabfe carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.
1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and'standard pharmaceutical practice.
The pharmaceutical compositions may comprise as-or in addition to-the carrier,


excipient or diluent any suitable binder (s), labricant (s), suspending agent (s), coating agent (s), solubilising agent (s).
Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
COMBINATION PHARMACEUTICAL The compound of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutical active agents.
By way of example, the compounds of the present invention may be used in combination with other STS inhibitors and/or other inhibitors such as an aromatase inhibitor (such as for example, 4hydr6xyandrostenedione (4-OHA)) and/or steroids-such as the naturally occurring sterneurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and/or other structurally similar organic compounds. Exaples of other STS inhibitors may be found in the above references. By way of example, STS inhibitors for use in the present'invention include EMATE, and either or both of the 2-ethyl and 2- methoxy 17-deoxy compounds that are analogous to compound 5 presented herein.
In addition, or in the alternative, the compound of the present invention may be used in combination with a biological response modifier.
The term biological response modifier ("BRM") includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc. For some applications, preferably, the biological response modifier is a cytokine. Examples of cytokines include : interleukins (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-19 ; Tumour Necrosis Factor (TNF) -such as TNF-a ; Interferon alpha, beta and gamma; TGF-ß. For some applications, preferably the cytokine is tumour necrosis factor (TNF). For some applications, the TNF may be any type of TNF- such as TNF-a, TNF- (3, including derivatives or mixtures thereof. More preferably the cytokine is TNF-o. Teachings on TNF may be found in the art-such as WO-A-98/08870 and WO-A-98/13348.
ADMINISTRATION Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
The compositions of the present invention may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous,- subcutaneous, intraocular or transdermal administration. Depending upon the need, the agent may be administered at a dose of from 0. 01 to 30 mg/kg body weight, such as from 0. 1 to 10 m/kg, more preterablyfrom 0. 1 to 1 mg/kg body weight.
By way of further example, the agents of the present invention may be administered in accordance with a regimen of 1 to 4 times per day, preferably once or twice per day.
The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing, therapy.
Aside from the typical modes of delivery-indicated above-the term"administered" also includes delivery by techniques such as lipid mediated transfection, liposoms, immuholiposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
The term"administered"includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
Thus, for pharmaceutical administration, the STS inhibitors of the present invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates may be in the range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose. Alternatively and preferably the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
CELL CYCLING The compounds of the present invention may be usedful in the method of treatment of a cell cycling disorder.
As discussed in"Molecular Cell Biology"3rd, Ed. Lodish et al. pages 177-181 different eukaryotic cells can grow and divide at quite different rates. Yeast cells, for example, can divide every 120 min. , and the first divisions of fertilised eggs in the embryonic cells of sea urchins and insects take only 1530 min. because one large pre-existing cell is subdivided. However, most growing plant and animal cells take 10-20 hours to double in number, and some duplicate at a much slower rate. Many cells in adults, such as nerve cells and striated muscle cells, do not divide at all ; others, like the fibroblasts that assist in healing wounds, grow on demand but are otherwise quiescent.
Still, every eukaryotic cell that divides must be ready to donate equal genetic material to two daughter cells. DNA synthesis in eukaryotes does not occur throughout the cell division cycle but is restricted to a part of it before cell division.
The relationship between eukaryotic DNA synthesis and cell division has been thoroughly analysed in cultures of mammalian cells that were all capable of growth and division. In contrast to bacteria, it was found, eukaryotic cells spend only a part of their time in DNA synthesis, and it is completed hours before cell division (mitosis). Thus a gap of time occurs after DNA synthesis and before cell division ; another gap was found to occur after division and before the next round of DNA synthesis. This analysis led to the conclusion that the eukaryotic cell cycle consists of an M (mitotic) phase, a G, phase (the first gap), the S (DNA synthesis) phase, a G2 phase (the second gap), and back to M. The phases between mitoses (G1, S, and G2) are known collectively as the interphase.
Many nondividing cells in tissues (for example, all quiescent fibroblasts) suspend the cycle after mitosis and just prior to DNA synthesis ; such"resting"cells are said to have exited from the cell cycle and to be in the Go state.
It is possible to identify cells when they are in one of the three interphase stages of the cell cycle, by using a fluorescence-activated cell sorter (FACS) to measure their relative DNA content: a cell that is in G1 (before DNA synthesis) has a defined amount x of DNA ; during S (DNA replication), it has between x and 2x; and when in G2 (or M), it has 2x of DNA.
The stages of mitosis and cytokinesis in an animal cell are as follows (a) Interphase. The G2 stage of interphase immediately precedes the beginning of mitosis. Chromosomal DNA has been replicated and bound to protein during the S phase, but chromosomes are not yet seen as distinct structures. The nucleolus is the only nuclear substructure that is visible under light microscope. In a diploid cell before DNA replication there are two morphologic chromosomes of each type, and the cell is said to be 2n. In G2, after DNA replication, the cell is 4n. There are four copies of each chromosomal DNA. Since the sister chromosomes have not yet separated from each other, they are called sister chromatids. b) Early prophase. Centrioles, each with a newly formed daughter centriole, begin moving toward opposite poles of the cell ; the chromosomes can be seen as long threads. The nuclear membrane begins to disaggregate into small vesicles.
(c) Middle and late prophase. Chromosome condensation is completed ; each visible chromosome structure is composed of two chromatids held together at their centromeres. Each chromatid contains one of the two newly replicated daughter DNA molecules. The microtubular spindle begins to radiate from the regions just adjacent to the centrioles, which are moving closer to their poles. Some spindle fibres reach from pole to pole ; most go to chromatids and attach at kinetochores.
(d) Metaphase. The chromosomes move toward the equator of the cell, where they become aligned in the equatorial plane. The sister chromatids have not yet separated.

(e) Anaphase. The two sister chromatids separate into independent chromosomes.
Each contains a centromere that is tinked by a spindfe fibre to one po) e, to which it moves. Thus one copy of each chromosome is donated to each daughter cell.
Simultaneously, the cell elongates, as do the pole-to-pole spindles. Cytokinesis begins as the cleavage furrow starts to form.
(f) Telophase. New membranes form around the daughter nuclei ; the chromosomes uncoil and become less distinct, the nucleolus becomes visible again, and the nuclear membrane forms around each daughter nucleus. Cytokinesis is nearly complete, and the Spindle disappears as the microtubules and other fibres depolymerise.
Throughout mitosis the"daughter"centriole at each pole grows until it is full-length. At telophase. the duplication of each of the original centrioles is completed, and new daughter centrioles will be generated during the next interphase.
(g) Interphase. Upon the completion of cytokinesis, the cell enters the G, phase of the cell cycle and proceeds again around the cycle.
It will be appreciated that cell cycling is an extremely important cell process. Deviations from normal cell cycling can result in a number of medical disorders. Increased and/or unrestricted cell cycling may result in cancer. Reduced cell cycling may result in degenerative conditions. Use of the compound of the present invention may provide a means to treat such disorders and conditions.
Thus, the compound of the present invention may be suitable for use in the treatment of cell cycling disorders such as cancers, including hormone dependent and hormone independent cancers.
In addition, the compound of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
For some applications, cell cycling is inhibited and/or prevented and/or arrested, preferably wherein cell cycling is prevented and/or arrested. In one aspect cell cycling may be inhibited and/or prevented and/or arrested in the G2/M phase. In one aspect cell cycling may be irreversibly prevented and/or inhibited and/or arrested, preferably wherein cell cycling is irreversibly prevented and/or arrested.
By the term"irreversibly prevented and/or inhibited and/or arrested"it is meant after application of a compound of the present invention, on removal of the compound the effects of the compound, namely prevention and/or inhibition and/or arrest of cell cycling, are still observable. e. More particu) ar) y by the term"irreversibfy prevented and/or inhibited and/or arrested"it is meant that when assayed in accordance with the cell cycling assay protocol presented herein, cells treated with a compound of interest show less growth after Stage 2 of the protocol I than control cells. Details on this protocol are presented below.
Thus, the present'invention provides compounds which: cause inhibition of growth of oestrogen receptor positive (ER+) and ER negative (ER-) breast cancer cells in vitro by preventing and/or inhibiting and/or arresting cell cycling ; and/or cause regression of nitroso-methyl urea (NMU) -induced mammary tumours in intact animals (i. e. not ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in cancer cells ; and/or act in vivo by preventing and/or inhibiting and/or arresting cell cycling and/or act as a cell cycling agonist.
CELL CYCLING ASSAY (PROTOCOL 6) Procedure Stage 1 MCF-7 breast cancer cells are seeded into mufti-well culture plates at a density of 105 cells/well. Cells were allowed to attach and grown until about 30% confluent when they are treated as follows : Control-no treatment Compound of Interest (COI) 20FM Cells are grown for 6 days in growth medium containing the COI with changes of medium/COI every 3 days. At the end of this period cell numbers were counted using a Coulter cell counter.
Stage 2 After treatment of cells for a 6-day period with the COI cells are re-seeded at a density of 104 cells/well. No further treatments are added. Cells are allowed to continue to grow for a further 6 days in the presence of growth medium. At the end of this period cell numbers are again counted.
ASSAY FOR DETERMINING DH ACTIVITY USING CANCER CELLS (PROTOCOL 7) Conversion of oestrone to oestradiol (E1 o E2, E2DH Type I) and oestradiol to oestrone (E2 E1, E2DH Type II) was measured in intact cell monolayers of T47D and MDA- MB-231 breast cancer cells respectively. Cells were cultured in flasks until they were 80-90% confluent. 3H-E1 or 3H-E2 (6pmol, 90 Ci/mmol) were added to each flask in the absence (control) or presence of various test compounds (10FM) in 2. 5ml of medium. Substrate was also added to flasks without cells and incubated in parallel (blanks).
After incubation with T47D cells for 30min or MDA cells for 3h at 37°C, 2ml of the medium was added to test tubes containing 14C-E2 or 14C-E1 (#5000 cpm) and 50ug E2 or E1 respectively. Steroids were extracted from the aqueous medium with diethyl ether (4ml). The ether phase was decanted into separate tubes after freezing the aqueous phase in solid carbon dioxide-methanol mixture. The ether was evaporated to dryness under a stream of air at 40°C. The residue was dissolved in a small volume of diethyl ether and applied to TLC plates containing a fluorescent indicator. E1 and E2 were separated by TLC using DCM-Ethyl acetate (4: 1 v/v). The position of the product from each incubation flask was marked on the TLC plate after visualisation under UV light.
The marked regions were cut out and placed in scintillation vials containing methanol (0. 5ml) to elute the product. The amount of 3H-product formed and"C-El or"C-E2 recovered were calculated after scintillation spectrometry. The amount of product formed was corrected for procedural losses and for the number of cells in each flask.
CANCER As indicated, the compounds of the present invention may be useful in the treatment of a cell cycling disorder. A particular cell cycling disorder is cancer.
Cancer remains a major cause of mortality in most Western countries. Cancer therapies developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumours. However, more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours.
Hence, the development of a pharmaceutical for anti-cancer treatment of hormone dependent and/or hormone independent tumours, yet lacking some or all of the side- effects associated with chemotherapy, would represent a major therapeutic advance.
It is known that oestrogens undergo a number of hydroxylation and conjugation reactions after their synthesis. Until recently it was thought that such reactions were part of a metabolic process that ultimately rendered oestrogens water soluble and enhanced their elimination from the body. It is now evident that some hydroxy metabolites (e. g. 2- hydroxy and 16alpha-hydroxy) and conjugates (e. g. oestrone sulphate, E1S) are important in determining some of the complex actions that oestrogens have in the body.
Workers have investigated the formation of 2-and 16-hydroxylated oestrogens in relation to conditions that alter the risk of breast cancer. There is now evidence that factors which increase 2-hydroxylase activity are associated with a reduced cancer risk, while those increasing 16alpha-hydroxylation may enhance the risk of breast cancer.
Further interest in the biological role of oestrogen metabolites has been stimulated by the growing body of evidence that 2-methoxyoestradiol is an endogenous metabolite with anti-mitotic properties. 2-MeOE2 is formed from 2-hydroxy oestradiol (2-OHE2) by catechol oestrogen methyl transferase, an enzyme that is widely distributed throughout the body.
Workers have shown that in vivo 2-MeOE2 inhibits the growth of tumours arising from the subcutaneous injection of Meth A sarcoma, B16 melanoma or MDA-MB-435 oestrogen receptor negative (ER-) breast cancer cells. It also inhibits endothelial cell proliferation and migration, and in vitro angiogenesis. It was suggested that the ability of 2-MeOE2 to inhibit tumour growth in vivo may be due to its ability to inhibit tumour- induced angiogenesis rather than direct inhibition of the proliferation of tumour cells.
The mechanism by which 2-MeOE2 exerts its potent anti-mitogenic and anti-angiogenic effects is still being elucidated. There is evidence that at high concentrations it can inhibit microtubule polymerisation and act as a weak inhibitor of colchicine binding to tubulin. Recently, however, at concentrations that block mitosis, tubulin filaments in cells were not found to be depolymerised but to have an identical morphology to that seen after taxon treatment. It is possible, therefore, that liies ta, a drug that is used for breast and ovarian breast cancer therapy, 2-MeOE2 acts by stabilising microtubule dynamics.
While the identification of 2-MeOE2 as a new therapy for cancer represents an important advance, the bioavailability of orally administered oestrogens is poor. Furthermore, they can undergo extensive metabolism during their first pass through the liver. As part of a research programme to develop a steroid sulphate inhibitor for breast cancer therapy, oestrone-3-O-sulphamate (EMATE) was identified as a potent active site-directed inhibitor. Unexpectedly, EMATE proved to possess potent oestrogenic properties with its oral uterotrophic activity in rats being a 100-times higher than that of oestradiol. Its enhanced oestrogenicity is thought to result from its absorption by red blood cells (rbcs) which protects it from inactivation during its passage through the liver and which act as a reservoir for its slow release for a prolonged period of time. A number of A-ring modified analogues were synthesised and tested, including 2-methoxyoestrone-3-0-sulphamate.
While this compound was equipotent with EMATE as a steroid sulphate inhibitor, it was devoid of oestrogenicity.
We believe that the compound of the present invention provides a means for the treatment of cancers and, especially, breast cancer.
In addition or in the alternative the compound of the present invention may be useful in the blocking the growth of cancers including leukaemias and solid tumours such as breast, endometrium, prostate, ovary and pancreatic tumours.
THERAPY CONCERNING OESTROGEN We believe that some of the compounds of the present invention may be useful in the control of oestrogen levels in the body-in particular in females. Thus, some of the compounds may be useful as providing a means of fertility control-such as an oral contraceptive tablet, pill, solution or lozenge. Alternatively, the compound could be in the form of an implant or as a patch.
Thus, the compounds of the present invention may be useful in treating hormonal conditions associated with oestrogen.
In addition or in the alternative the compound of the present invention may be useful in treating hormonal conditions in addition to those associated with oestrogen. Hence, the compound of the present invention may also be capable of affecting hormonal activity and may also be capable of affecting an immune response.
NEURODEGENERATIVE DISEASES We believe that some of the compounds of the present invention may be useful in the treatment of neurodenerative diseases, and similar conditions.
By way of example, it is believed that STS inhibitors may be useful in the enhancing the memory function of patients suffering from illnesses such as amnesia, head injuries, Alzheimer's disease, epileptic dementia, presenile dementia, post traumatic dementia, senile dementia, vascular dementia and post-stroke dementia or individuals otherwise seeking memory enhancement.
TH1 We believe that some of the compounds of the present invention may be useful in TH1 implications.
By way of example, it is believed that the presence of STS inhibitors within the macrophage or other


antigen presenting cells may lead to a decreased ability of sensitised T cells to mount a TH1 (high IL-2, IFNy low IL-4) response. The normal regulatory influence of other steroids such as glucocorticoids would therefore predominate.
INFLAMATORY CONDITIONS We believe that some of the compounds of the present invention may be useful in treating inflammatory conditions-such as conditions associated with any one or more of: autoimmunity, including for example, rheumatoid arthritis, type I and 11 diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis, ulcerative colitis and Crohn's disease, skin disorders e. g. psoriasis and contact dermatitis ; graft versus host disease ; eczema ; asthma and organ rejection following transplantation.
By way of exampie, it is believed that STS inhibitors may prevent the normai physiological effect of DHEA or related steroids on immune and/or inflammatory responses.
The compounds of the present invention may be useful in the manufacture of a medicament for revealing an endogenous glucocorticoid-like effect.
OTHER THERAPIE It is also to be understood that the compound/composition of the present invention may have other important medical implications.
For example, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890-viz : In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635. For ease of reference, part of that list is now provided: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis ; periodontitis, gingivitis ; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa ; corneal ulceration, retinopathy and surgical wound healing ; rhinitis, allergic conjunctivitis, eczema, anaphylaxis ; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.
In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859. For ease of reference, part of that list is now provided: cytokine and cell proliferation/differentiation activity ; immunosuppressant or immunostimulant activity (e. g. for treating immune deficiency, including infection with human immune deficiency virus ; regulation of lymphocyte growth ; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity) ; regulation of haematopoiesis, e. g. treatment of myeloid or Iymphoid diseases ; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e. g. for heating wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration ; inhibition or activation of follicle-stimulating hormone (modulation of fertility) ; chemotactic/chemokinetic activity (e. g. for mobilising specific cell types to sites of injury or infection) ; haemostatic and thrombolytic activity (e. g. for treating haemophilia and stroke); antiinflammatory activity (for treating e. g. septic shock or Crohn's disease); as antimicrobials ; modulators of e. g. metabolism or behaviour; as analgesic ; treating specific deficiency disorders; in treatment of e. g. psoriasis, in human or veterinary medicine.
In addition, or in the alternative, the composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985. For ease of reference, part of that list is now provided: macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity; anti-immune activity, i. e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells ; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placenta insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e. g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scisritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scaring, e. g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e. g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e. g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
SULPHAMATE COMPOUND PREPARATION The sulphate compounds of the present invention may be prepared by reacting an appropriate alcohol with a suitable chloride. By way of example, the sulphate compounds of the present invention may be prepared by reacting an appropriate alcohol with a suitable sulfamoyl chloride, of the formula R9R'°NS02CI.
Typical conditions for carrying out the reaction are as follows.
Sodium hydride and a'sulfamoyl chloride are added to a stirred solution of the alcohol in anhydrous dimethyl formamide at 0°C. Subsequently, the reaction is allowed to warm to room temperature whereupon stirring is continued for a further 24 hours. The reaction mixture is poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase is extracted with dichloromethane. The combined organic extracts are dried over anhydrous MgS04. Filtration followed by solvent evaporation in vacuo and co-evaporated with toluene affords a crude residue which is further purified by flash chromatography.' Preferably, the alcohol is derivatised, as appropriate, prior to reaction with the sulfamoyl chloride. Where necessary, functional groups in the alcohol may be protected in known manner and the protecting group or groups removed at the end of the reaction.
Preferably, the sulphate compounds are prepared according to the teachings of Page et a/ (1990 Tetrahedron 46; 2059-2068).
The phosphonate compounds may be prepared by suitably combining the teachings of Page et a/ (1990 Tetrahedron 46; 2059-2068) and PCT/GB92/01586.
The sulphonate compounds may be prepared by suitably adapting the teachings of Page et a/ (1990 Tetrahedron 46; 2059-2068) and PCT/GB92/01586.
The thiophosphonate compounds may be prepared by suitably adapting the teachings of Page et a/ (1990 Tetrahedron 46; 2059-2068) and PCT/GB91/00270.
Preferred preparations are also presented in the following text.
SUMMARY In summation, the present invention provides compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same.
EXAMPLES The present invention will now be described only by way of example.
Example SECTION 1 16-(4-Dimethylamino-benzylidene)-3-hydroxy-13-methyl-6,7,8,9,11,12,13,14,15, 16-decahydro-cyclopenta [a] phenanthren-17-one STX477 (CAB02143, GMA01044) To a stirred suspension of estrone (4.05g, 15mmol) and 4-dimethylaminobenzaldehyde (2.24g, 15mmol) in ethanol (120cm3) was added sodium hydroxide (3. 0g, 75mmol) and the resulting yellow solution was stirred at room temperature for 72h. To this solution was added glacial acetic acid (20cm3) and the resulting yellow precipitate was collected by filtration and washed with water (100cm3), ethanol (50cm3), diethyl ether (50cm3) and hexane (100cm3) before being dried under reduced pressure. Yield 66%. 1H NMR (400MHz, DMSO-d6) 8 0.86 (s, 3H), 1.34-1. 61 (m, 6H), 1.82-1. 86 (m, 1H), 1.98-2. 02 (m, 1H), 2.15-2. 19 (m, 1H), 2.32-2. 34 (m, 1H), 2.73-2. 86 (m, 3H), 2.97 (s, 6H), 6.45 (d, J=2.7Hz, 1H), 6.51 (appdd, J=8.4, 2.5Hz, 1H), 6.74 (d, J=8.6Hz, 2H), 7.04 (d, J=8.6Hz, 1H), 7.20 (s, 1H), 7. 48 (d, J=8.6Hz, 2H), 9.02 (s, 1H) ; MS (FAB+) calcd. 401.2, obsd.
402.2 (M+H+) 16- (4-Dime hylamino-benzylidene)-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-3,178-diol STX511 (CAB02158, GMA01006) To a'cooied (ice bath) solution of 16-(4-dimethylaminobenzylidene estrone (0. 402g, 1mmol) in THF (20cm3) and ethanol (20cm3) was added NaBH4 (0. 100g, 2.6mmol) and the reaction was allowed to warm to room temperature and stirred overnight. The mixture was concentrated in vacuo until precipitate began to appear before water (50cm3) was added to precipitate the product as a white powder. This powder was collected by filtration and washed with water (50cm3), methanol (20cm3) and diethyl ether (50cm3) before being dried under reduced pressure. Yield 86%.'H NMR (400MHz, DMSO-d6) 8 0. 59 (s, 3H), 1.24-1. 43 (m, 6H), 1.84-1. 95 (m, 1H), 2.08-2. 20 (m, 2H), 2.26- 2.34 (m, 1H), 2.55-2. 65 (m, 1H), 2.70-2. 80 (m, 2H), 2. 89 (s, 6H), 3.95 (s, 1H), 4.99 (d, J=6.2Hz, 1H), 6.27 (d, J=2. 0Hz, 1H), 6.45 (d, J=2. 7Hz, 1H), 6.51 (appdd, J=8. 4,2. 5Hz, 1H), 6.70 (d, J=9. OHz, 2H), 7.05 (d, J=8.6Hz, 1H), 7.22 (d, J=9. OHz, 2H), 9.01 (s, 1H) ; HPLC (98%, Rt=3. 52,10 : 90 H20 : MeOH) ; MS (FAB+) calcd. 403.3, obsd. 403.2 (M+) General procedure for hydrogenation reactions: The starting 16-methylene estradiol was dissolved in a volume of THF and the same volume of ethanol added to the solution. The solution was then degassed by bubbling nitrogen through for 40min before Pd/C (5% wt., catalytic) was added and hydrogen gas (balloon) was passed over the reaction. The reaction was stirred under hydrogen at room temperature overnight before being filtered through celite.
16- (4-Dimethylamino-benzyl)-13-methyl-7, 8,9, 11, 12,13, 14,15, 16,17-decahydro- 6H-cyclopentalalphenanthrene-3, 17-diol STX594 (GMA01008) Purification by column chromatography using hexane followed by 20% ethyl acetate in hexane as eluent gave the product, Rf0. 33 (20% EtoAc in hexane), in 45% yield.'H NMR (400MHz, DMSO-d6) # 0.75 (S, 3H), 0.94-1.08 (M, 2H), 1.10-1.36 (M, 4H), 1.49- 1. 55 (m, 1H), 1.66-1.71 (m, 1H), 1.87 (d, J=12.1Hz, 1H), 2.13 (d, J=13.3Hz, 1H), 2.19- 2. 29 (m, 2H), 2. 58-2. 73 (m, 2H), 2. 83 (s, 6H), 2. 88-2. 93 (m, 1H), 3.63-3. 66 (m, 1H), 4. 60 (d, J=4. 7Hz, 1H), 6. 41 (d, J=2. 3Hz, 1H), 6. 49 (dd, J=8. 2, 2. 7Hz), 6. 64 (d, J=9. 0Hz, 2H), 6. 99 (d, J=8. 6Hz, 2H), 7. 03 (d, J=8. 2Hz, 1H), 8. 98 (s, 1H, OH) ; HPLC (92%, Rt=3. 28, 10 : 90 H20 : MeOH) ; MS (FAB+) calcd 405. 2, obsd 405. 2 (M'), Ace. Mass (FAB+) calcd 405. 2668, obsd 405. 2668 (M+) 13-Methyl-16-pyridin-2-ylmethyl-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [olphenanthrene-3-17-diol STX 567 (GMA01018) Purification by column chromatography using 100% hexane to 100% ethyl acetate as eluent gave the product, Rf 0.1 (25% EtOAc 75% hexane), in 57% yield.'H NMR (400MHz, DMSO-d6) 8 00. 76 (s, 3H), 1.04-1. 07 (m, 2H), 1.15-1. 36 (m, 6H), 1.55-1. 60 (m, 1H), 1.64-1. 71 (m, 1H), 1.87 (d, J=2. 1Hz, 1H), 2.20-2. 25 (m, 1H), 2.60-2. 72 (m, 3H), 3.12 (d, J=8. 6Hz, 1H), 3.68-3. 71 (m, 1H), 4.97 (d, J=4.7Hz, 1H), 6.41 (d, J=2.7Hz, 1H), 6.49 (dd, J=8.4, 2.5Hz, 1H), 7.04 (d, J=8. 2Hz, 1H), 7.15-7. 19 (m, 1H), 7.25 (d, J=7. 8Hz, 1H), 7. 67 (dt, J=7.6, 2. 0Hz, 1H), 8. 44-8. 465 (m, 1H), 8.99 (s, 1H) ; HPLC (98%, Rt=2.62, 10: 90 H20: MeOH) ; MS (FAB+) calcd 363.2, obsd 364.1 (M+H+), Acc. Mass (FAB+) calcd 364.2276, obsd 364. 2297 (M+H+) 13-Methyl-16-pyridin-3-ylmethyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a] phenanthrene-3, 17-diol STX 542 (GMA01022) Purification by column chromatography using 100% hexane to 100% ethyl acetate as eluent gave the product, (Rf 0. 1 (25% EtOac in hexane) in 88% yield. 1H NMR (270MHz, DMSO-d6) 8 00. 76 (s, 3H), 1.00-1.04 (m, 2H), 1.20-1.35 (m, 5H), 1.54-1.56 (m, 1H), 1. 73-1. 90 (m, 2H), 2. 13-2. 32 (m, 3H), 2. 64 (m, 2H), 3. 01 (d, J=9. 4Hz, 1H), 4. 77 (d, J=4. 5Hz, 1H), 6. 41 (d, J=2. 5Hz, 1H), 6.49 (dd, J=8. 4, 2. 5Hz, 1H), 7. 03 (d, J=8. 4Hz, 1H), 7. 25-7. 31 (m, 1H), 7.60-7. 64 (m, 1H), 8.34-8. 39 (m, 1H), 8. 41 (d, J=1.7 Hz, 1H), 9. 02 (s, 1H) ; HPLC (97%, Rt=2.50, 10: 90 H20 : MeOH); MS (FAB+) calcd 363.2, obsd 364. 1 (M+H+), Acc. Mass (FAB+) calcd 364.2276, obsd 364.2287 (M+H+) 13-Methyl-16-pyridin-4-ylmethyl-7,, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H cyclopenta [a] phenanthrene-3, 17-diol STX 543 (GMA01020) Purification by column chromatography using 100% hexane to 100% ethyl acetate as eluent. Gave the product, (Rf 0.1 (25% EtOAc 75% hexane) in 96% yield ;'H NMR (400MHz, DMSO-d6) 8110. 76 (s, 3H), 0.94-1. 10 (m, 2H), 1.12-1. 36 (m, 5H), 1.54-1. 60 (m, 1H), 1.65-1. 71 (m, 1H), 1.85-1. 89 (m, 1H), 2.20-2. 42 (m, 3H), 2.64-2. 68 (m, 2H), 2.98-3. 02 (m, 1H), 3.66-3. 70 (m, 1H), 4.75 (d, J=4.7Hz, 1H), 6.40 (d, J=2.3Hz, 1 H), 6.49 (dd, J=8. 4,2. 5Hz, 1H), 7.03 (d, J=8. 2Hz, 1H), 7.23 (d, J=6. 2Hz, 2H), 8. 41 (d, J=5.9Hz, 2H), 8. 99 (s, 1H) ; HPLC (92%, Rt=2. 44,10 : 90 H20:MeOH) ; MS (FAB+) calcd 363.2, obsd 364. 1 (M+H+), Acc. Mass (FAB+) calcd 364. 2276, obsd 364. 2294 (M+H+) Acetic ic acid 8-acetyl-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7,8- diaza-pentaleno [2, 1-a] phenanthene-2-yl ester (GMA01110) To a solution of the starting pyrazofe (0. 598g, 2mmol) in dry pyridine ($cm3) was added acetic anhydride (2cm3) and the mixture was reffuxed under nitrogen for 16h. The mixture was then cooled and poured onto ice and the resulting cream coloured precipitate was collected by filtration and purified by column chromatography using ethyl acetate/hexane (1/1), Rf 0.48. Yield : 40%.'H NMR (270MHz, CDCl3) 8 Ell. 07 (s, 3H), 1.40-1. 60 (m, H), 1.62-2. 10 (m, 5H), 2. 28-(s, 3H, OAc), 2.28-2. 48 (m, 4H), 2.64 (s, 3H, OAc), 2.64-2. 72 (m, 1H), 2. 87-2. 96 (m, 2H), 6.82 (d, J=2. 2Hz, 1H), 6.86 (appdd, J=8. 4, 2.5Hz, 1 H), 7.30 (d=8.4Hz, 1 H), 7. 86 (s, 1 H) ; LCMS (ES+) 380.23 (M+2H+) Acetic acid 8-acetyl-6a-methyl-12-oxo-4b, 5,6, 6a, 8,'10, 10a, 10b, 11,12- decahydro-7, 8-diaza-pentaleno- [2, 1-a] phenanthren-2-yl ester (GMA01120) To a solution of the bis-acetate protected pyrazol starting material (0.121g, 0. 32mmol) in acetonitrile (2. 5cm3) was added t-butylhydroperoxide (0. 14cm3 of a 70% wt. solution in water, lmmol) and chromium hexacarbonyl (0.021g, 0. 09mol). The solution was heated to reflux for 24h before being cooled to room temperature. Water (10cm3) was added and the products extracted with diethyl ether (3x10cm3). The ther extracts were combined, washed with water, aqueous saturated bicarbonate and brine before being dried (MgS04) and concentrated in vacuo. The desired product was isolated by flash column chromatography using ethyl acetate/hexane (1/1) as eluent (Rf 0.4). Yield 23%.
'H NMR (400MHz, CDCl3) 8 01. 09 (s, 3H), 1.8-2. 0 (m, 2H), 2. 2-2. 5 (m, 4H), 2. 33 (s, 3H), 2.5-2. 9 (m, 5H), 2.65 (s, 3H), 7.34 (dd, J=2.5, 8.4Hz, 1H), 7.65 (d, J=8.6Hz, 1H), 7.79 (d, J=2. 3Hz, 1 H), 7. 88 (s, 1 H) 2-Hydroxy-6a-methyl-5, 6, 6a, 8, 10, 10a, 10b, 11-octahydro-4bH-7, 8-diaza- pentaleno[2,1-a]phenanthren-12-one STX 737 (GMA01138) The bis-acetate protected starting material (0.0137g) was dissolved in ethanol (1cm3).
To this was added a solution of KOH (0.004g) in ethanol/water (2cm3 of 1/1) and the mixture was shaken then left at room temperature for 30min. TLC at this stage showed no starting material remaining therefore the mixture was acidified with glacial acetic acid, concentrated in vacuo and water added. The solution was left standing overnight during which time a pale orange precipitate formed. This powder was collected by filtration.
Yield 46%.'H NMR (400MHz, DMSO-d6) 8 110-91 (s, 3H), 1.60-1. 82 (m, 2H), 2.1-2. 35 (m, 4H), 2.4-2. 7 (m, 5H), 7.02 (dd, J=8.6, 2.7Hz, 1H), 7.29 (d, J=2.7Hz, 1H), 7.34 (d, J=8.6Hz, 1H), 9.65 (s, 1H), 12.04 (bs, 1H) ; LCMS: (ES+) 309. 99 (M+2H+) ; Ace. Mass (FAB+) calcd 309.16030, obsd. 309.16132 (M+H+) 6a-Methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno [2,1- a] phenanthrene-2, 9-diol STX 820 (GMA02024) To a suspension of the starting ester (0. 657g, 2mmol) in toluene (10cm3) was added hydrazine monohydrate (0. 11cm3, 2. 2mmol) and the mixture was heated at 150°C in a sealed tube for 4h. During this time the starting material dissolved then another white precipitate formed. The reaction was then cooled to room temperature, acidified with glacial acetic acid and water was added to ensure that all product precipitated. The white powder was collected by filtration and washed with water, a small amount of ethanol, diethyl ether and hexane. LCMS showed two compounds with masses corresponding to the desired product (312) and the hydrazone derivative of estrone (285). Flash chromatography using 10% methanol in DCM to 100% methanol failed to separate these. Recrystallisation was attempted by suspending the solid in ethanol then slowly adding aqueous NaOH until the solid dissolved, followed by acidification to pH3 with glacial acetic acid. No precipitate formed initially therefore a small amount of water was added until a fine white powder precipitated. This was shown by LCMS and HPLC to be the desired product. Yield of pure product, 18% ; total product recovered, 50%. 1H NMR (400MHz, DMSO-d6) 5 #0.89 (s, 3H), 1.30-1. 70 (m, 4H), 1.80-1. 87 (m, 1H), 1.93-2. 11 (m, 3H), 2. 17-2. 40 (m, 3H), 2.71-2. 78 (m, 2H), 6.43 (d, J=2.2Hz, 1H), 6.50 (dd, J=8.1, 2.6Hz, 1H), 7.03 (d, J=8.4Hz, 1H), 9.01 (s, 1H), 10.3 (bs, 1H) ; HPLC (100%, Rt= 2.33, 20: 80 H20: MeOH); LCMS (APCI) 311.29 (M+H+), Acc. Mass (FAB+) calcd. 311.1759, obsd. 311.1761 (M+H+) General procedure for amide coupling using a Radleys GreenHouse Synthesiser.
To a solution of the acid starting material (0.107g, 0. 25mmol) in dry DCM (2cm3) and triethylamine (0. 02cm3, 0. 18mmol) was added a solution of EDC (0. 058g, 0. 3mmol) triethylamine (0. 017cm3, 0. 16mmol) and DMAP (catalytic) in dry DCM (1cm3) under nitrogen and the reaction was stirred at room temperature for 30min. To this solution was then added amine (1. 1eq) and stirring under nitrogen was continued for 12h, followed by standing under nitrogen for 14h. The solution was then washed with saturated aqueous NaHCO3 before separation and concentration of the organic layer and purification by flash chromatography.
2-Benzyloxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid isopropylamide (GMA02038-6) Yield 16%. Rf 0. 72 (EtOAc) ;'H NMR (270MHz, CDOg) S 0. 97 (s, 3H), 1. 18 (s, 3H), 1. 20 (s, 3H), 1. 37-1. 92 (m, 5H), 2. 08-2. 46 (m, 5H), 2. 71 (dd, J=13. 9, 6. 2Hz, 1H), 2. 83-2. 90 (m, 2H), 4. 13-4. 26 (m, 1H), 4. 98 (S, 2H), 5. 83 (bs, 1H), 6. 68 (d, J=2. 9Hz, 1H), 6. 73 (dd, J=8. 4, 2. 9Hz, 1H), 7. 15 (d, J=8. 4Hz, 1H), 7. 22-7. 38 (m, 5H) ; LCMS (ES+) 470. 37 (M+H') 2-Hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8-diaza- pentalenol2, 1-a] phenanthrene-9-carboxylic acid isopropyl amide STX857 (GMA02046) A solution of the benzyl protected starting material (0.020g, 0. 04mmol) in THF 45cm3) and ethanol (5cm3) was degassed by bubbling nitrogen through for 30min before Pd/C (5% wt., catalytic) was added and hydrogen gas (balloon) was passed over the reaction. Stirring at room temperature under a hydrogen blanket was continued overnight before the mixture was filtered through celite and the celite washed with ethyl acetate and methanol. The filtrate was concentrated in vacuo to give a white solid, Rf 0. 60 (EtOAc).
Yield 0.096g, 63%.'H NMR (270MHz, CDCI3) ô 0.97 (s, 3H), 1.25 (s, 3H), 1.27 (s, 3H), 1.47-1. 95 (m, 4H), 2.13-2. 53 (m, 6H), 2.77 (dd, J=13.9, 6. 2Hz, 1H), 2.87-2. 94 (m, 2H), 4. 19-4. 30 (m, 1H), 5. 88 (d, J=8. 1Hz, 1H), 6.60 (d, J=2. 93Hz, 1H), 6.65 (dd, J=8. 3, 2. 93Hz, 1H), 7.16 (d, J=8. 4Hz, 1H) ; HPLC (95%, Rt=1. 72, 4:96 H20:MeOH ; LCMS (AP-) 378. 35 (M-H+) 2-Hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, lOb, 11, 12-decahydro-7, 8-diaza- pentaleno [2,1-a] phenanthrene-9-carboxylic acid (pyridin-3-ylmethyl)-amide STX 860 (GMA02056/3) To a suspension of acid starting material (0. 170g, 0. 5mmol) in dry DCM (10cm3) under nitrogen was added DMAP (catalytic), EDC (0. 116g, 0. 6mmol) and triethylamine (0. 06cm3, 0. 55mol) and the solution was stirred at room temperature for 20min. To this mixture was added 3- (aminomethyl) pyridine (0. 06cm3, 0. 55mmol), followed by dry DMF (10cm3) to ensure complete dissolution of starting materials, and the reaction was stirred at room temperature under nitrogen for 24h. The solution was washed with saturated aqueous bicarbonate and the organic layer separated and concentrated in vacuo. To the resulting concentrated solution (in DMF) was added hexane followed by a small amount of DCM. The resulting cream-coloured precipitate was collected by filtration and purified by flash chromatography using DCM followed by 10% MeOH in DCM as eluent, giving a white crystalline solid. Rf 0. 40 (10% MeOH in DCM), Yield 0.034g, 16%.'H NMR (400MHz, DMSO-d6) 8 00. 92 (s, 3H), 1.37-2. 36 (m, 10H), 2.63-2. 89 (m, 3H), 4.41 (appd, J=22.3Hz, 2H), 6.44 (d, J=2. 3Hz, 1H), 6.51 (dd, J=8.2, 2.3Hz, 1H), 7.04 (d, J=8.2Hz, 1H), 7.33 (apps, 1H), 7.69 (apps, 1H). 7.69-8. 63 (m, 3H), 9.02 (s, 1H), 12.65 & 12.92 (s, 0.5H, tautomers NH on pyrazol ring); HPLC (98%, Rt=2.04, 10 : 90 H20 : MeOH) ; LCMS (ES+) 429.19 (M+H+) Route to STY 820 Route to STX 737 Routes to STX 857 and STY 860 GMA03034 [3- methyl-1 7-oxo- 6,7, 8, 9,11,12,13,14,15,17-decahydro-cyclopenta[a]phenanthren-16-ylidene]- hydroxy-acetic acid ethyl ester To a, stirred solution of TBDMS protected estrone (1.275 g, 3.1 mmol) in toluene (30 cm3) was added diethyl oxalate (0.84 cm3, 6.2 mmol) and potassium t-butoxide (0.45 g, 4 mmol) and the reaction was stirred at rt overnight. The reaction was acidified with glacial acetic acid and concentrated in vacuo before ethyl acetate (30 cm3) was added.
The solution was washed with water (30 cm3) and brine (30 cm3) before being concentrated in vacuo to give a white powder. This powder was washed with ethanol/water and collected by filtration. Yield quantitative.
'H NMR 8 (270 MHz, CDCI3) 0.21 (s, 6H, (CH3) 2Si), 0.98 (s, 3H, 18-CH3), 0. 99 (s, 9H, (CH3) 3C), 1.15 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.38 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.54- 1.69 (m, 6H), 2.00-2. 05 (m, 2H), 2. 27-2.47 (m, 3H), 2.55 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.80-2. 86 (m, 2H), 3.03-3. 11 (m, 1H), 4.35 (q, J= 7.2 Hz, 2H, OCH2CH3), 6.48 (s, 1H, 4- H), 7. 03 (s, 1H, 1-H); FAB-LRMS (MH+) 513.3 m/z ; FAB-HRMS calcd for C3oH44O2Si 512. 2958 found (M+) 512. 2965 m/z GMA03035 2- (tert-Butyl-dimethyl-silanyloxy)-3-ethyl-6a-methyl- 4b, 5,6, 6a, 8, 10,10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno [2, 1-a] phenanthrene-9- carboxylic acid ethyl ester To a stirred suspension of sm (1.53 g, 3 mmol) in ethanol (25 cm3) was added hydrazine monohydrate (0. 17 cm3, 3. 4 mmol) at which time the sm dissolved. Stirring at room temperature was continued overnight during which time the solution became cloudy. To this was added p-tofuene sutphonic acid (#0. 1 g) and the mixture was heated for 5 min to aromatise the pyrazole ring. The solution was concentrated in vacuo until precipitate began to form before water was added and the white powder was collected by filtration, washed with water and air-dried. Yield #quantitative. 1H NMR # (260 MHz, CDCl3I) 0. 22 (s, 6H, (CH3)2Si), 0.98 (s, 3H, 18-CH3), 0.99 (s, 9H, (CH3)3C), 1.16 (t, J = 7. 5 Hz, 3H, ArCH2CH3), 1. 37 (t, J = 7. 1 Hz, 3H, OCH2CH3), 1. 40-2. 18 (6H), 2. 30-2. 50 (4H), 2. 56 (q, J = 7. 5 Hz, 2H, ArCH2CH3), 2. 81-2. 96 (m, 3H), 4. 35 (q, J = 7. 1 Hz, 2H, OCH2CH3), 6. 49 (s, 1 H, 4-H), 7.06 (s, 1 H, 1-H); HPLC > 96% (Rt = 6.69, 100% MeCN) ; FAB-LRMS (MH+) 509. 2 m/z ; FAB-HRMS calcd for C34H41N2O3 509. 3168 found (MH+) 509.3178 m/z STX 1066, GMA02187 3-Ethyl-2-hydroxy-6a-methyl-4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno [2, 1-a]phenanthrene-9-carboxylic acid To a stirred suspension of sm (1.27 g, 2.64 mmol) in MeOH (30 cm3) was added aqueous NaOH (0.211 g in 16 cm3) and the mixture was refluxed overnight (the sm dissolved on heating). The reaction was cooled, acidified with glacial acetic acid and concentrated in vacuo until precipitate began to form (# half volume). Water (20 cm3) was added and the resulting precipitate was collected by filtration. This was dissolved in methanol, concentrated and dried in vacuo. Yield 57%.
(400 MHz, CD3OD) 1. 02 (s, 3H, 18-CH3), 1. 16 (t, J = 7. 4 Hz, 3H, ArCH), 1.36-1. 47 (m, 1H), 1.56-1. 83 (3H), 1. 93-1.99 (m, 1H), 2.02-2.15 (m, 1H), 2. 22-2. 28 (m, 2H), 2.36- 2.43 (m, 2H), 2. 55 (q, J = 7. 4 Hz, 2H, ArCH2CH3), 2. 72-2. 90 (m, 3H), 6. 44 (s, 1H, 4-H), 6.95 (s, 1H, 1-H) ; HPLC > 92% (Rt = 1.49, 70% MeCN in H2O) ; ES-ve-MS (M-H+) 365.16 mlz ; FAB-HRMS calcd for C22H27N2O3 367 2022 found (MH+) 367. 2010 m/z General procedure for amide coupling : To a stirred suspension of acid sm (0.045 g, 0. 123 mmoi) in dry DCM (8 cm3) under 2 wa added DMAP (catalytic), EDC (0. 071 g, 0. 37 mmol) and NE (0. 02 cm3) and the solution was stirred at rt for 30 min. To this was then added amine (0. 02 cm3) and stirring was continued for 4 d. The solution was washed with saturated bicarbonate, the organic layer separated and the aqueous layer washed with DCM. The organic layers were combined and concentrated in vacuo and the product purified by flash chromatography (Flashmaster II) using an elution gradient of DCM to 10%'MeOH in DCM.
STX 1013, GMA02188 3-Ethyl-2-hydroxy-6a-methyl-4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid (pyridin-3-ylmethyl)-amide Yield 39% ;'H NMR 8 (270 MHz, CD30D) 1.03 (s, 3H, 18-CH3), 1.15 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.39-1. 52 (m, 1H), 1. 59-1.87 (m, 3H), 1.94-2. 00 (m, 1H), 2.14-2. 50 (5H), 2.55 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.78-2. 90 (3H), 4.58 (s, 2H), 6.45 (s, 1H, 4-H), 6.97 (s, 1H, 1-H), 7.38-7. 43 (m, 1H), 7.85 (d, J = 7.7 Hz, 1H), 8.43 (d, J = 3.7 Hz, 1H), 8.56 (s, 1H) ; HPLC > 97% (Rt 2.11, 70% MeCN in H2O) ; ES+ve-MS (MH+) 457. 38 m/z ; FAB- HRMS calcd for C28H33N4O2 457. 2603 found (MH+) 457.2589 m/z STX1144, GMA03074- 3-Ethyl-2-hydroxy-6a-methyl-4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza- pentaleno [2, 1-a]phenanthrene-9-carboxylic acid (pyridin-2-ylmethyl)-amide Yield 41%; 1H NMR # (270 MHz, CD3OD) 1.01 (s, 3H, 18-CH3), 1.14 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.31-1.47 (m, 1H), 1.57-1.83 (m, 3H), 1.93-2.00 (m, 1H), 2.14-2.50 (5H), 2. 54 (q, J = 7. 4 Hz, 2H, ArCH2CH3), 2. 71-2. 86 (3H), 4. 64 (s, 2H), 6. 44 4 (s, 1H, 4-H), 6.95 (s, 1H, 1-H), 7. 27-7. 31 (m, 1H), 7. 45 (d, J= 8. 0 Hz, 1H), 7. 78 (dt, J= 7. 7, 1. 6 Hz, 1H), 8. 48 (d, J = 4.7 Hz, 1H) ; HPLC > 92% (Rt 2.15, 80% MeCN in H20) ; ES-ve-MS (M-H+) 455.36 m/z ; FAB-HRMS calcd for C28H33N402 457.2603 found (MH+) 457. 2607 mlz STX 1084, GMA03056-2 3-Ethyl-2-hydroxy-6a-methyl-4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7,8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid (2-pyridin-2-yl-ethyl)-amide Yield 46% ; 1H NMR 8 (270 MHz, CD30D) 1.00 (s, 3H, 18-CH3), 1. 14 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.33-1. 49 (m, 1H), 1. 54-1.83 (3H), 1.93-2. 02 (m, 1H), 2.04-2. 43 (5H), 2.54 (q, J = 7.5 Hz, 2H, CH2CH3), 2.71-2. 90 (3H), 3.07 (t, J = 7.1 Hz, 2H, -CH2CH2-), 3.70 (dt, J = 6. 9, 1.7 Hz, 2H,-CH2CH2), 6. 45 (s, 1H, 4-H), 6.96 (s, 1H, 1-H), 7.26-7. 31 (m, 1H), 7.36 (d, J = 7.7 Hz, 1 H), 7.77 (dt, J = 7.7, 1.7 Hz, 1 H), 8.47-8. 50 (m, 1 H) ; HPLC > 97% (Rt 3.41, 70% MeCN in H2O) ; ES-ve-MS (M-H+) 469.55 m/z ; FAB-HRMS calcd for C29H35N4O2 471. 2760 found (MH+) 471. 2747 m/z STX 1085, GMA03056-1 3-Ethyl-2-hydroxy-6a-methyl-4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7,8-diaza- pentaleno [2,1-a] phenanthrene-9-carboxylic acid (2-pyridin-3-yl-ethyl)-amide Yield 51%; 1H NMR # (270 MHz, CD3OD) 1.02 (s, 3H, 18-CH3), 1. 15 (t, J = 7. 4 Hz, 3H, Arch2CH3), 1. 30-1. 49 (m, 1H), 1. 59-1. 86 (m, 3H), 1. 93-2. 01 (m, 1H), 2.10-2.45 (5H), 2. 55 (q, J = 7. 4 Hz, 2H,-CH2CH3), 2. 72-2. 85 (3H), 2. 95 (t, J=7. 1 Hz, 2H,-CH2CH2-), 3. 61 (t, J = 7.3Hz, 2H,-CH2CH2-), 6. 46 (s, 1H, 4-H), 6. 97 (s, 1H, 1-H), 7.37-7.42 (m, 1H), 7.79 (d, J = 7.7 Hz, 1H), 8. 40 (d, J = 4. 2 Hz, 1H), 8.45 (s, 1H) ; HPLC > 98%; (Rt 3.09, 70% MeCN in H2O) ; ES-ve-MS (M-H+) 469.49 m/z ; FAB-HRMS calcd for C29H35N4O2 471. 2760 found (MH+) 471. 2756 m/z STX 1145, GMA03074-2 3-Ethyl-2-hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8-diaza- pentalenot2, 1-a] phenanthrene-9-carboxylic acid (2-methoxy-ethyl)-amide Yield 36% ;'H NMR 8 (270 MHz, CD30D) 1.02 (s, 3H, 18-CH3), 1.15 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.34-1. 50 (m, 1H), 1.53-1. 85 (m, 3H), 1.93-2. 00 (m, 1H), 2.17-2. 47 (5H), 2.54 (q, J = 7. 4 Hz, 2H,-CH2CH3), 2.73-2. 87 (3H), 3.3-3. 4 (4H, -CH2CH2-), 3.53 (s, 3H, O-CH3), 6.45 (s, 1H, 4-H), 6.96 (s, 1H, 1-H), HPLC > 96% ; (R, 2.11, 80% MeCN in H2O) ; ES-ve-MS (M-H+) 422.35 m/z ; FAB-HRMS calcd for C25H34N3O3 424.2600 found (MH+) 424. 2606 m/z STS 1146, GMA03074-4 3-Ethyl-2-hydroxy-6a-methyl-4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza- pentaleno [2,1-a] phenanthrene-9-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)- amide Yield 42%; 1H NMR # (270 MHz, CD3OD) 1.01 (s, 3H, 18-CH3), 1.14 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.31-1.47 (m, 1H), 1.52-1.83 (m, 3H), 1.92-2.00 (m, 1H), 2.15-2.58 (7H), 2. 52 (s, 3H, ArCH3), 2. 69-2. 86 (3H), 4. 64 (s, 2H), 6. 44 (s, 1H, 4-H), 6.95 (s, 1H, 1-H), 7. 27-7. 31 (m, 1H), 8. 46 (d, J = 1.1 Hz, 1H), 8. 49 (s, 1H) ; HPLC > 94% (Rt 2. 08, 80% MeCN in H2O) ; ES-ve-MS (M-H+) 470.35 m/z ; FAB-HRMS calcd for C28H34N5O2 472.2713 found (MH+) 472.2718 m/z.
STX 1166, GMA03092 3-Ethyl-2-hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a,10b,11,12-decahydro-7, 8-diaza- pentaleno [2,1-a]phenanhrene-9-carboxylic acid (benzo [1, 3] dioxol-5-ylmethyl)- amide Yield 56% ;'H NMR 8 (400 MHz, CD30D) 1.08 (s, 3H, 18-CH3), 1. 19 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.48-2. 52 (10H), 2.59 (q, J = 7.8 Hz, 2H), 2.80-2. 95 (m, 3H), 4.48 (s, 2H), 5.95 (s, 2H), 6.49 (s, 1H), 6.79-6. 89 (m, 3H), 7.01 (s, 1H) ; HPLC > 98% (R, 7.29, 90% MeCN in H2O) ; APCI (M-H+) 498. 32 m/z ; FAB-HRMS calcd for C28H34N502 found (MH+) m/z.
GMA03028 (3-Benzyloxy-2-ethyl-13-methyl-17-oxo-6,7,8,9,11,12,13,14,15,17-decahydro- cyclopenta[a]phenanthren-16-ylidene)-hydroxy-acetic acid ethyl ester To a stirred solution of benzyl protected 2-ethyl estrone (2. 00 g, 5. 15 mmol) in toluene (20 cm3) was added diethyl oxalate (1. 40 cm3, 10. 20 mmol) followed by potassium t- butoxid (0. 693 g, 6. 18 mmol) and the reaction was stirred at rt for 16 h. The solution was acidified with glacial acetic acid before being concentrated in vacuo. To the residue was added ethyl acetate (30 cm3) and the solution washed with water (30 cm3) and brine (30 cm3). The organic layer was concentrated in vacuo to give a white powder, which was washed with ethanol/water and collected by filtration. Yield 97% ;'H NMR s (270 MHz, CDCi3) 0.99 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1. 38 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.41-1. 69 (6H), 2.00-2. 05 (m, 2H), 2.30-2. 50 (m, 3H), 2.66 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.88-2. 91 (m, 2H), 3.04-3. 10 (m, 1H), 4.35 (q, J = 7.2 Hz, 2H, OCH2CH3), 5.04 (s, 2H, ArCH2O), 6.64 (s, 1H, 4-H), 7.09 (s, 1H, 1-H), 7.26-7. 45 (m, 5H) ; HPLC > 97% (Rt = 2.86, 98% MeCN in H2O) ; ES-ve-MS (M-H+) 487 m/z ; FAB-HRMS calcd for C31H36O5 488. 2563 found (M+) 488.2569 m/z.
GMA03029 2-Benzyloxy-3-ethyl-6a-methyl-4b, 5,6, 6a, 8,10, 1 Oa, 1 Ob, 11, 12-decahydro-7, 8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid ethyl ester To a stirred suspension of sm (2. 44 g, 5 mmol) in ethanol (50 cm3) was added hydrazine monohydrate (0. 3 cm3, 6 mmol) at which time the sm dissolved. Stirring at room temperature was continued overnight before the white precipitate was collected by filtration and washed with water. The filtrate was acidified with glacial acetic acid and more precipitate formed and was collected. The precipitates were combined, and suspended in ethanol before p-toluene sulphonic acid (-0. 1g) was added. The mixture was heated for 5 min to aromatise the pyrazol ring then stirred at rt for 1 h. The pale yellow solution was concentrated in vacuo until precipitate began to form before water was added and the white powder was collected by filtration, washed with water and air- dried. Yield 96%; 1H NMR # (270 MHz, CDCl3) 1. 03 (s, 3H, 18-CH3), 1.21 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1. 37 (t, J = 2 Hz, 3H, OCH2CH3), 1.45-2.20 (6H), 2.32-2.50 (m, 4H), 2. 67 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.82-2.96 (m, 3H), 4. 35 (q, J = 7. 2 Hz, 2H, OCH2CH3), 5. 05 (s, 2H, ArCH2O), 6.65 (s, 1H, 4-H), 7. 12 (s, 1H, 1-H), 7. 28-7. 46 (m, 5H) ; HPLC > 92% (Rt = 5.17, 94% MeCN in H2O) ; ES-ve-MS (M-H+) 483 m/z ; FAB-HRMS calcd for C31H36N2O3 484.2726 found (M+) 484.2730 m/z.
STX 1057, GMA03032/2, GMA03062 3-Ethyl-2-hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 1 Oa, 1 Ob, 11,12-decahydro-7, 8-diaza- pentalenol2, 1-a] phenanthrene-9-carboxylic acid ethyl ester A suspension of sm (3.40 g, 7 mmol) in MeOH (70 cm3) was degassed by bubbling nitrogen through for 30 min. To this was added Pd/C (5 % wt., catalytic) and degassing was continued for a further 10 min before hydrogen gas was passed over the reaction and stirring under hydrogen was continued for a further 3 d. The mixture was filtered through celite, the celite washed with ethyl acetate and methanol and the filtrate concentrated in vacuo. The product was purified by flash chromatography (Flashmaster II) using a gradient elution of hexane to 1: 1 ethyl acetate: hexane. Rf (1/1 ethyl acetate/hexane) 0.24. Yield 89% ;.'H NMR 5 (270 MHz, CD3OD) 1.03 (s, 3H, 18-CH3), 1. 15 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.36 (t, J = 7.2 Hz, 3H, OCH2CH3), 1. 39-2.20 (6H), 2. 26-2. 44 (m, 4H), 2.53 (q, J = 7.3 Hz, 2H, ArCH2CH3), 2.79-2. 86 (m, 3H), 4.34 (q, J = 7. 1 Hz, 2H, OCH2CH3), 6.46 (s, 1H, 4-H), 6.97 (s, 1H, 1-H) ; FAB-HRMS calcd for for Cz4H3oN203 395. 2335 found (lirt) 395. 2312 m/z.
STX 1024, GMA03008, GMA03044/3, GMA03063 3-Ethyt-6a-methyt-2-sulfamoyloxy-4b, 5, 6, 6a, 8, 10, 10a, 1 Ob, 11,12-decahydro-7, 8- diaza-pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester A solution of sulfamoyl chloride (30 cm3 of a 0. 68 M solution in toluene, 20. 4 mmol) was concentrated in vacuo without heating then cooled on ice under nitrogen. To this was added DMA (15 cm3) and this mixture added to a cooled (ice/acetone bath) solution of sm (2.42 g, 6.13 mmol) in DMA (10 cm3). The stirred solution was allowed to warm to rt overnight before being diluted with water (60 cm3) and the product extracted with ethyl acetate (100 cm3), giving an oily solution in DMA. This was redissolved in ethyl acetate and washed again with water before concentration to give a cream coloured crystalline solid. Recrystallisation from DCM/hexane gave 2g, 69% yield of product.'H NMR 6 (270 MHz, CD30D) 1.04 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.36 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.39-2. 20 (6H), 2.29-2. 50 (m, 4H), 2.71 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2.81-2. 92 (m, 3H), 4.33 (q, J = 7.1 Hz, 2H, OCH2CH3), 7.09 (s, 1H, 4-H), 7.22 (s, 1 H, 1-H); HPLC > 97% (Rt = 2. 40,80% MeCN in H2O) ; FAB-LRMS (MH+) 474 mlz ; FAB-HRMS calcd for for C24H32N305S 474.2063 found (MH+) 474.2067 m/z.
GMA03064 To a suspension of 2-chlorotrityl chloride resin (2. 27g, 1. 1 mmol/g theoretical loading) swollen in anhydrous DCM (12 cm3) and DIPEA (5.34 cm3, 29.5 mmol) under nitrogen was added sm (2.00 g, 4.22 mmol) as a solid in portions followed by a further volume of DCM (5 cm3). The reaction was shaken at rt for 96 h before the resin was collected by filtration and washed three times with DCM, methanol and again with DCM. OMA0306S To the loaded resin (3.74 g) swollen in THF (25 cm3) was added slowly aqueous sodium hydroxide (30 cm§ of 1 N) and the mixture was shaken at rt for 24 h. The resin was collected by filtration, washed with THF/water (1/1) and washed with MeOH and DCM before being dried in vacuo.
STX 1080, GMA03052-1 3-Ethyl-6a-methyl-2-sulfamoyloxy-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8- diaza-pentaleno [2, 1-a] phenanthrene-9-carboxylic acid The loaded resin (0.03 g) was placed in a fritted syringe and washed with TFA/DCM (20 cm3 of 1/1, added portion-wise with 5 min between washings) followed by DCM. The washings were combined and concentrated in vacuo. The resin loading from the weight of the crude was calculated to be 0. 8 mmol/g.
Purification by flash chromatography (Flashmaster II) using a gradient elution of DCM to 10% MeOH in DCM gave the product Rf 0. 6 (10% MeOH in DCM).
'H NMR 6 (270 MHz, CD3OD) 1. 04 (s, 3H, 18-CH3), 1. 20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1. 46-1. 90 (5H), 1. 99-2. 07 (m, 1H), 2. 12-2. 23 (m, 1H), 2. 31-2. 51 (4H), 2. 71 (q, J = 7. 5 Hz, 2H, ArCH2CH3), 2.82-2. 92 (3H), 7.09 (s, 1H, 4-H), 7. 22 (s, 1H, 1-H); HPLC > 93% (Rt 1. 45,70% MeCN in H2O) ; ES+ve-MS (MH+) 468 m/z ; FAB-HRMS calcd for for C22H2gN305S 446. 1750 found (MH+) 446.1713 m/z.
General procedure for amide coupling on resin-bound starting material : To the loaded resin (0.200 g) swollen in dry DCM (3 cm3) under N2 was added bromo- tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP, 0.260 g, 0. 5 mmol) and N- hydroxybenzotriazole (HOBt, 0. 070 g, 0. 5 mmol) followed by DIPEA (0. 18 cm3, 1. 0 mmol). The mixture was shaken for 10 min before addition of amine (0.06 cm3) and shaking under N2 at d was continued for 72 h. The resin was collected in a fritted syringe and washed with DMF, DCM and MeOH (x3) and again with DCM (x3) before cleavage of the product using TFA/DCM (1/1). Purification by flash chromatography (Flashmaster II) using a gradient elution of DCM to 10% MeOH in DCM yielded the sulfamate in yields of 1-53% and HPLC purities of 88-98%.
STX 1083, GMA03052-2/3 Sulfamic acid 3-ethyl-6a-methyl-9- [ (pyridin-2-ylmethyl)-carbamoyl]- 4b, 5,6, 6a, 8,10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno [2, 1-a] phenanthren-2-yl ester Yield 53 % ;'H NMR § (270 MHz, CD30D) 1.04 (s, 3H, 18-CH3), 1.19 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.39-1. 55 (m, 1H), 1. 59-1. 88 (m, 3H), 1.98-2. 04 (m, 1H), 2.14-2. 58 (5H), 2.70 (q, J = 7.5 Hz, 2H, ArCH2CH3), 2. 78-2. 98 (3H), 4.64 (s, 2H), 7.08 (s, 1 H, 4-H), 7.20 (s, 1H, 1-H), 7. 28-7. 33 (m, 1H), 7. 42 (d, J = 7.9 Hz, 1H), 7.80 (dt, J = 7. 8, 1.7 Hz, 1H), 8. 49 (d, J = 4.5 Hz, 1H) ; HPLC > 97% (Rt 2. 11, 70% MeCN in H20) ; ES-ve-MS (M-H+) 534 m/z ; FAB-HRMS calcd for for C28H34N5O4S 536. 2332 found (MH+) 536. 2330 m/z.
STX 1082 GMA03052-3/3 Sulfamic acid 3-ethyl-6a-methyl-9-(2-pyridin-3-yl-ethylcarbamoyl)- 4b,5,6,6a,8,10,10a,10b,11,11-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester Yield 7 % ; H NMR ã (270 MHz, CD30D) 1. 02 (s, 3H, 18-CH3), 1. 20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.46-1. 89 (4H), 1.98-2. 04 (m, 1H), 2.15-2. 52 (5H), 2.65-2. 98 (7H), 3.60 (t, J = 6. 7 Hz, 2H, -CH2CH2), 7. 04 (s, 1H, 4-H), 7.21 (s, 1H, 1-H), 7.36-7. 41 (m, 1H), 7.78 (dt, J = 6.2, 1.7 Hz, 1 H), 8 : 39 (dd, J = 4.8, 1.7 Hz, 1 H), 8.44 (d, J = 1.7 Hz, 1 H) ; LC purity > 95%; ES-ve-MS (M-H+) 548 m/z ; FAB-HRMS calcd for for C29H36N5O4S 550.2488 found (MH') 550.2499 m/z.
STX 1081, GMA03052-4/3 Sulfamic acid 3-ethyl-6a-mehyl-9-(2-pyridin-2-yl-ethylcarbamoyl)- 4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno [2, 1-a] phenanthren-2-yl ester Yield 25 % ;'H NMR 5 (270 MHz, CD30D) 1.02 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1. 46-1. 88 (4H), 1. 98-2. 05 (m, 1H), 2.12-2. 52 (5H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2. 77-2. 92 (3H), 3. 07 (t, J = 6.9 Hz, 2H, -CH2CH2), 3. 71 (dt, J = 7. 2, 1. 8 Hz, 2H, -CH2CH2), 7. 09 (s, 1H, 4-H), 7. 20 (s, 1H, 1-H), 7.26-7.29 (m, 1H), 7. 36 (d, J = 7. 9 Hz, 1H), 7. 77 (dt, J = 7.7, 1.9 Hz, 1H), 8.47-8. 50 (m, 1 H) ; HPLC > 98% (ihr 1.96, 90% MeCN in H2O) ; ES-ve-MS (M-H+) 548 rn/z ; FAB-HRMS calcd for for C29H36N5O4S 550.2488 found (MH+) 550.2483 m/z.
STX 1123 GMA03072-1/2 Sulfamic acid 3-ethyl-6a-methyl-9-[(pyridin-3-ylmethyl)-carbamoyl]- 4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaz-pentaleno[2,1-a]phenanthren-2-yl ester Yield 8 % ;'H NMR 8 (400 MHz, CD30D) 1.06 (s, 3H, 18-CH3), 1.21 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1. 47-1'. 57 (m, 1H), 1.65-1. 90 (3H), 2.04-2. 07 (m, 1H), 2.14-2. 33 (2H), 2.39- 2.53 (3H), 2.72 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.90-2. 94 (3H), 4.58 (s, 2H), 7.09 (s, 1H, 4-H), 7.21 (s, 1 H, 1-H), 7.40-7. 43 (m, 1 H), 7.85 (d, J = 7.8 Hz, 1 H), 8.42 (d, J = 4.7 Hz, 1H), 8. 54 (d, J = 2. 0 Hz, 1H) ; HPLC > 99% (Rt 1. 91,80% MeCN in H2O) ; ES+ve-MS (MH+) 536.31 m/z'FAB-HRMS calcd forfor C28H34N504S 536.2332 found (MH+) 536.2330 m/z.
STX 1124, GMA03072-2/2 Sulfamic acid 3-ethyl-6a-methyl-9- [ (5-methyl-pyrazin-2-ylmethyl)-carbamoyll- 4b, 5, 6,6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno [2, 1-a]phenanthren-2-yl ester Yield 8 % ; 1H NMR 8 (400 MHz, CD30D) 1. 06 (s, 3H, 18-CH3), 1. 22 (t, J = 7. 4 Hz, 3H, ArCH2CH3), 1.47-1.57 (m, 1H), 1. 65-1. 90 (3H), 2. 03-2. 07 (m, 1H), 2. 14-2. 55 (5H), 2. 72 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.90-2. 94 (3H), 4.66 (s, 2H), 7.09 (s, 1 H, 4-H), 7. 21 (s, 1H, 1-H), 8.48 (s, 1H), 8.49 (s, 1H) ; HPLC > 99% (R, 1. 93, 80% MeCN in H20) ; ES+ve- MS (MH+) 551.18 m/z ; FAB-HRMS calcd for for C28H3sN604S 551.2441 found (MH+) 551.2447 m/z STX 1125, GMA03072-4a Sulfamic acid 3-ethyl-9-(2-methoxy-ethylcarbamoyl)-6a-methyl-

4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester Yield 10 % ;'H NMR 5 (400 MHz, CD30D) 1.06 (s, 3H, 18-CH3), 1.22 (t, J = 7. 4 Hz, 3H, ArCH2CH3), 1. 47-1.57 (m, 1H), 1.65-1. 90 (3H), 2.03-2. 09 (m, 1H), 2. 18-2. 55 (5H), 2.72 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.84-2. 95 (3H), 3.3-3. 55 (7H), 7.09 (s, 1H, 4-H), 7.22 (s, 1H, 1-H) ; HPLC > 88% (Rt 3.28, 5 to 95% MeCN in Ha0 over 10 min); ES+ve-MS (MH+) 503.24 m/z ; FAB-HRMS ca) cd for for C25H35Na05S 503.2328 found (MH+) 503.2325 mlz.
STX 1126, GMA03072-5/2 Sulfamic acid 3-ethyl-9-(ethyl-methyl-carbamoyl)-6a-methyl- 4b, 5,6, 6a, 8,10, 1 Oa, 1 Ob, 11, 12-decahydro-7, 8-diaza-pentaleno[2, 1-a] phenanthren-2-yl ester Yield 5 % ; 1H NMR 8 (400 MHz, CD30D) 1.08 (s, 3H, 18-CH3), 1.20-1. 25 (6H), 1.30-1. 6 (1H), 1.65-1. 90 (3H), 2.01-2. 09 (m, 1H), 2. 16-2. 50 (5H), 2. 72 (q, J = 7. 4 Hz, 2H, ArCH2CH3), 2. 84-2. 93 (3H), 3.06-3. 26 (3H), 3. 46-3. 64 (m, 2H), 7. 09 (s, 1H, 4-H), 7. 22 (s, 1H, 1-H) ; HPLC > 98% (Rt 2. 43, 5 to 95% MeCN in H2O over 10 min); ES+ve-MS (MH+) 487. 24 m/z ; FAB-HRMS calcd for for C25H35N4O4S 487. 2379 found (MH+) 487. 2396 mAz.
STX 1152, GMA03076-1 Sulfamic acid 3-ethyl-9-[(furan-2-ylmethyl)-carbamoyl]-6a-methyl- 4b,5,6,6a,8,10,10a,10b,11,12-decahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester Yield 6 % ;'H NMR S (270 MHz, CD30D) 1.02 (s, 3H, 18-CH3), 1. 20 (t, J = 7. 5 Hz, 3H, ArCH2CH3), 1. 46-1. 89 (4H), 1.95-2. 06 (m, 1H), 2. 12-2. 52 (5H), 2.71 (q, J = 7. 5 Hz, 2H, CH2CH3), 2.81-2. 91 (3H), 4.52 (s, 2H), 6.28-6. 36 (m, 2H), 7.09 (s, 1H, 4-H), 7.21 (s, 1H, 1-H), 7.43 (s, 1H) ; HPLC > 91 % (Rt 2. 17,70 % MeCN in H2O) ; ES-ve-MS (M-H+) 523.27 m/z; FAB-HRMS calcd for for C27H33N405S 525.2172 found (MH+) 525.2176 m/z STX 1153, GMA03076-3 Sulfamic acid 9- [ (benzo [1, 3] dioxol-5-ylmethyl)-carbamoyl]-3-ethyl-6a-methyl- 4b, 5,6, 6a, 8,10, 10a, 10b, 11, 12-decahydro-7,8-diaz-pentaleno[2,1-a]phenanthren-2-yl ester Yield 1 % ;'H NMR 5 (270 MHz, CD30D) 1.06 (s, 3H, 18-CH3), 1. 20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.46-2. 54 (10H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.81-3 (3H), 4.45 (s, 2H), 5.91 (s, 2H), 6.74-6. 85 (2H), 7.09 (s, 1H, 4-H), 7.21 (s, 1H, 1-H); HPLC > 93 % (Rt 5. 39, 5 to 95 % MeCN in H20 over 10 min); ES-ve-MS (M-H+) 577. 26 m/z ; FAB-HRMS calcd for for C30H35N4O6S 579.2277 found (MH+) 579. 2257 m/z STX 1154, GMA03076-4 Julia acid 3-ethyl-9-[2-(1H-indol-3-yl)-ethylcarbamoyl]-6a-methyl- 4b, 5,6, 6a, 8, 10, 10a, 1 Ob, 11, 12-decahydro-7, 8-diaza-pentaleno [2, 1-a] phenanthren-2-yl ester Yield 3 %; 1H NMR # (270 MHz, CD3OD) 1. 00 (s, 3H, 18-CH3), 1. 20 (t, J = 7. 5 Hz, 3H, ArCH2CH3), 1.46-2.52 (11H), 2.71 (q, J = 7. 5 Hz, 2H, CH2CH3), 2. 90-2. 92 (2H), 3. 03- 3. OS (2H, -CH2CH2), 3. 63-3. 67 (2H, -CH2CH2), 6.95-7.12 (4H), 7.21 (s, 1H, 1-H), 7. 34 (d, J = 8. 0 Hz, 1 H), 7.56-7. 59 (1H) ; HPLC > 98 % (Rt 2. 22,70% MeCN in H2O) ; ES-ve-MS (M-H+) 586.33 m/z ; FAB-HRMS calcd for for C32H38N5O4S 588.2645 found (MH+) 588.2659 m/z STX 1155, GMA03076-6 Sulfamic acid 3-ethyl-6a-methyl-9-phenethylcarbamoyl- 4b, 5, 6,6a, 8,10, 10a,10b,11,12-dicahydro-7,8-diaza-pentaleno[2,1-a]phenanthren-2-yl ester Yield 9 % ;'H NMR 8 (270 MHz, CD30D) 1.03 (s, 3H, 18-CH3), 1.20 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1. 46-1. 90 (4H), 1.98-2. 05 (m, 1H), 2.12-2. 52 (5H), 2.71 (q, J = 7.5 Hz, 2H, CH2CH3), 2.77-2. 99 (5H), 3.55-3. 61 (2H, -CH2CH2), 7.10 (s, 1H, 4-H), 7.20-7. 30 (6H); HPLC > 93 % (Rt 2.83, 70% MeCN in H2O) ; ES-ve-MS (M-H+) 547.27 m/z ; FAB-HRMS calcd for for C3oH37N404S 549.2536 found (MH+) 549.2532 m/z.
GMA03036 2-Benzyloxy-7-(2-cyano-ethyl)-3-ethyl-4b,5,6,6,7,10,10a,10b,11,12-decahydro-7,8- diazapentaleno [2, 1-a] phenanthrene-9-carboxylic acid ethyl ester To a stirred suspension of sm (0.17 g, 0. 35 mmol) in ethanol (10 cm3) was added 2- cyanoethylhydrazine (0.05 cm3) and the dark orange solution was stirred at rt overnight.
The resulting dark brown solution was acidified with glacial acetic acid and water (10 cm3) was added. The solution was concentrated in vacuo to ~ 1 cm3'before more water was added until the product precipitated as a yellowish powder. This was collected. by filtration and washed with water.'H NMR at this stage showed that the pyrazol ring was still hydrated therefore the product was redissolved in ethanol, p-toluene sulphonic acid monohydrate was added and the solution heated for a few min. The solution was then concentrated in vacuo until precipitate began to form, water (20 cm3) was added and the resulting orange powder was collected by filtration. The product was purified by flash chromatography using a gradient elution of hexane to 1: 1 ethyl acetate: hexane. Yield 0.032g, 17% 'H NMR 8 (270 MHz, CDCI3) 1.08 (s, 3H, 18-CH3), 1.21 (t, J = 7.5 Hz, 3H, ArCH2CH3), 1.38 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.35-2. 08 (6H), 2.30-2. 58 (4H), 2.67 (q, J = 7.6 Hz, 2H, ArCH2CH3), 2.80-2. 91 (3H), 3.05-3. 08 (m, 2H), 4.27-4. 41 (4H), 5.04 (s, 2H, ArCH2O), 6.65 (s, 1H, 4-H), 7.08 (s, 1H, 1-H), 7.27-7. 45 (m, 5H); ES+ve-MS (MH+) 560 m/z.
GMA03043, STX 1079 7- (2-cyano-sthyl)-3-ethyl-4b, 5, 6, 6a, 7, 10, 10a, 1 Ob, 11, 12-decahydro-7, 8- diazapentaleno[2,1-a]phenanhrene-9-carboxylic acid ethyl ester A stirred solution of sm (0.03 g, 0.06 mmol) in THF (2 cm3) and ethanol (3 cm3) was degassed by bubbling N2 through for 20 min before Pd/C (5 % wt., catalytic) was added.
Degassing was continued for a further 10 min before H2 gas was passed over the reaction and the reaction was stirred under a hydrogen blanket overnight. The mixture was filtered through celite and the product purified by flash chromatography using a gradient elution of DCM to 10% MeOH in DCM. Yield 0.025 g, 93% ;'H NMR s (270 MHz, CDCI3) 1.07 (s, 3H, 18-CH3), 1.21 (t, J = 7.4 Hz, 3H, ArCH2CH3), 1.37 (t, J = 7.0 Hz, 3H, OCH2CH3), 1. 31-1. 52 (m, 1H), 1.57-2. 08 (5H), 2.23-2. 47 (4H), 2.60 (q, J = 7.4 Hz, 2H, ArCH2CH3), 2.78-2. 87 (3H), 2. 97-3.14 (m, 2H), 4.23-4. 41 (4H), 6.51 (s, 1H, 4-H), 7.00 (s, 1 H, 1-H) ; HPLC > 94 % (Rt 2.67, 80% MeCN in H20); ES+ve-MS (MH+) 448 m/z GMA02180 To a solution of benzyl protected estrone (1.08 g, 3.0 mmol) in dry THF (20 cm3) under ~2, cooled to-78 °C (dry ice/acetone bath) was added slowly drop-wise LDA (6.0 cm3 of a 1. 8 M soin, 3.3 mmol) and the reaction was stirred at-78 °C for 30 min. To this was then added a solution of succinic anhydride (0.30 g, 3.0 mmol) in dry THF (15 cm3) and the reaction was allowed to warm slowly to rt overnight. The mixture was poured into 5 % HCI (120 cm3) and the products extracted with diethyl ether (2 x 60 cm3). The ether extracts were combined and concentrated in vacuo. Flash chromatography using a gradient elution of DCM to 10 % MeOH in DCM resulted in separation of starting material (2. 7 mmol) from a mixture which was used without further purification for the fiollowing reaction.
GMA02181 To a solution of crude sm (0.29g, assume 0.3 mmol) in EtOH (5 cm3) was added hydrazine monohydrate (0.07 cm3, 1.4 mmol) and the reaction was stirred at rt for 3h then heated to reflux for 30 min to'ensure complete reaction and aromatisation of the pyrazol ring. The reaction was cooled, acidified with glacial acetic acid and water was added until the product precipitated as a pale orange powder. Yield 0.098 g ;'H NMR 8 (270 MHz, DMSO-d6) 0.89 (s, 3H, 18-CH3), 1.24-1. 69 (5H), 2.09-2. 57 (5H), 2.73-2. 84 (m, 4H), 5.06 (s, 2H), 6.73-6. 79 (2H), 7.19 (d, J = 8.9 Hz, 1H), 7.32-7. 45 (5H); HPLC > 80% (Rt = 1. 89, 80% MeCN in H2O) ; FAB-LRMS (MH+) 457 m/z ; FAB-HRMS calcd for for CZgHg3N2Og 457. 2491 found (MH+) 457.2479 m/z.
GMA02190 To a stirred solution of sm (0.090 g, assume 0. 2 mmol) in dry DCM (5 cm3) was added DMAP (catalytic), EDC (0. 046 g, 0.24 mmol) and NEts (0.02 cm3) and the solution was stirred for 15 min before addition of tetrahydrofurfuryl amine (0.023 cm3, 0. 22 mmol).
The reaction was stirred for 40 h before being diluted with DCM (15 cm3) and the solution washed with sat. aq. bicarb. Flash chromatography using a gradient elution of DCM to 10% MeOH in DCM yielded 0.035 g, 0.064 mmol of product.
NMR 8 (270 MHz, CD30D) 0. 96 (s, 3H, 18-CH3), 1.33-2. 66 (16H), 2. 82-2. 91 (4H), 3. 16-3. 29 (3H), 3. 56-3. 93 (3H), 98 (s, 2H), 6.67 (d, J = 2.5 Hz, 1H), 6.73 (dd, J = 8.5, 2.5 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.27-7.41 (5H); FAB-LRMS (MH+) 540 m/z GMA03014, STX 1035 A stirred solution of sm (0.034 g, 0.06 mmol) in THF (2 cm3) and ethanol (3 cm3) was degassed by bubbling N2 through for 20 min before Pd/C (5 % wt., catalytic) was added.
Degassing was continued for a further 10 min before H2 gas was passed over the reaction and the reaction was stirred under a hydrogen blanket overnight. Tlc showed the presence of sm therefore stirring under H2 was continued for a further 72h. The mixture was filtered through celite and the product purified by flash chromatography using a gradient elution of DCM to 10% MeOH in DCM. Yield 0. 012 g, 44% ; 1H NMR 8 (270 MHz, CD30D) 0.99 (s, 3H, 18-CH3), 1.28-2. 12 (9H), 2.21-2. 46 (4H), 2.53 (t, J = 7. 4 Hz, 2H) 2.62 (dd, J = 13.4, 6.2 Hz, 1H), 2. 82-2. 84 (m, 2H), 2.90 (t, J = 7.4 Hz, 2H), 3.22-3. 3 (m, 3H), 3.67-3. 96 (3H), 6.50 (d, J = 2.5 Hz, 1H), 6.55 (dd, J = 8.4, 2.7 Hz, 1H), 7.09 (s, 1H) ; HPLC > 97 % (Rt 2. 03,70% MeCN in H2O) ; ES+ve-MS (MH+) 450 m/z ; FAB-HRMS calcd for for C27H36N303 450.2757 found (MH+) 450.2745 m/z.
SECTION 2 3-Benzyloxy-13-methyl-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta [a] phenanthren-17-one (JAC01002).
A solution of 15 g (55 mmol) of estrone in DMF (110 mL) was cooled to 0°C and 2. 44 g (61 mmol) of NaH (60% in oil) were added slowly. The mixture was stirred at 0°C under N2 for 1 h before addition of 13. 2 mL (111 mmol of benzyl bromine dropwise at 0°C. The reaction was then heated to 80°C for 4 h. After cooling, the mixture was poured into water. The suspension was extracted by diethyl ether (5 times). The mixed organic layer were washed with 2N HCI (3 times) and brine, dried over Na2SO4 and concentrated in vacuo. Recrystallisation of the solid residue in ethanol gave 12.33 g (62%) of JAC01002 as beige solid.'H NMR (400 MHz, CDCl3) 8 0.92 (s, 3H, CH3) ; 1.49-1. 63 (m, 6H); 1.99- 2.05 (m, 3H); 2.17 (dAB, J = 18.9, 8.9 Hz, 1H) ; 2.23 (m, 1H) ; 2.40 (m, 1H) ; 2.51 (dAB, J = 18. 9,8. 9, 1H) ; 2. 85 (m, 2H, CH26) ; 5.05 (s, 2H, CH2Ph) ; 6.74 (d, J = 2.8 Hz, 1H, H4); 6.80 (dd, J = 8. 5,2. 8 Hz, 1H, H2) ; 7.21 (d, J = 8.5 Hz, 1H, H1) ; 7.30-7. 45 (m, 5H); Acq.
Mass (FAB+) calcd 360. 2089, obsd 360.21675 (M+).
3-Benzyloxy-13-methyl-17-oxo-7, 8, 9,11, 12,13, 14,15, 16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid methyl ester (JAC01011). [J. Org.
Chem. 1991,65, 6199-6205].
3.48 g (87 mmol) of NaH (60% in oil) were covered with anhydrous THF (50 mL), treated with 6. 11 mL (72 mmol) of dimethylcarbonate, and heated to reflux white being magnetically stirred under N2. A solution of benzylestrone (10.45 g, 29 mmol) in THF (50 mL) was added dropwise, and the reaction mixture was refluxed for 8 h, cooled to 0°C, and treated with 3M acetic acid (50 mL) in advance of being poured into brine. The product was extracted into CHCI3 (3 times), dried over Na2SO4 and evaporated. Recrystallisation of the solid residue in methanol gave 9.8 g (81%) of JAC01011 as beige solid.'H NMR (400 MHz, CDCI3) 6 #0. 97 (s, 1.05H, CH3) ; 0. 99 (s, 1. 95H, CH3) ; 1. 34-1. 74 (m, 6H) ; 1. 90-2. 50 (m, 6H); 2. 92 (m, 2H, CH) ; 3. 22 (dd, J = 10. 1, 8. 6 Hz, 0. 65H, C CHCO) ; 3. 58 (dd, 0.35H, CHCO); 3.77 (s, 3H, OCH3); 5.03 (s, 2H, CH2Ph); 6.73 (d, J = 2.5 Hz, 1H, H4); 6.80 (dd, J = 8.6, 2.5 Hz, 1H, H2); 7.20 (d, 1H, J= 8. 6 Hz, 1H, H1) ; 7. 30-7. 50 (m, 5H) ; MS (FAB+) calcd 418.21, obsd 418.20 (M+).

3-Benzyloxy-17α,ß-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro- 6H-cyclopenta[a]phenanthrene-16-carboxylic acid methyl ester (JAC01013, STX 569) and (JAC01013B, STX 570).
Solid NaBH4 (456 mg, 12 mmol) was added to a cold (0°C), magnetically stirred solution of JAC01011 (5g, 12 mmol) in THF/MeOH (90: 10), and the reaction mixture was stirred for 4h at 0°C, treated with 2N HCI until acidic pH and extracted with ether (twice). The combine organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. The solid was purified by column chromatography using CHCf3/EtOAc (90: 10) as eluant to give 2. 48 g of STX 569 (48%) and 0.91 g of STX 570 (18%) as white solids.
3-Benzyloxy-17ß-hydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid methyl ester (JAC01013, STX 569) 1 H NMR (400 MHz, CDCI3) : 8 0.85 (s, 3H, CH3) ; 1.19 (ddd, J = 12.9, 10.9, 6.2 Hz, 1H) ; 1.29-1. 43 (m, 2H); 1.46-1. 56 (m, 2H); 1.76 (dt, J = 12.9, 8.6 Hz, 1H) ; 1.90 (m, 1H) ; 2.00- 2. 13 (m, 2H); 2.23 (m, 1H) ; 2.33 (m, 1H) ; 2. 86 (m, 2H, CH26) ; 3. 15 (q, J = 9. 8 Hz, 1H, CHCOO) ; 3. 34 (d, J = 8. 2 Hz, 1H, OH) ; 3. 72 (s, 3H, OCH3) ; 3. 89 (dd, J = 9. 8, 8. 2 Hz, 1H, CHO) ; 5. 04 (s, 2H, CH2Ph) ; 6. 72 (d, J = 2. 7 Hz, 1H, H4) ; 6. 79 (dd, J = 8. 4, 2. 7 Hz, 1H, H2) ; 7. 21 (d, J = 8. 6 Hz, 1H, H1) ; 7.30-7. 46 (m, 5H, Ph); Acq. Mass (FAB+) calcd 420. 2301, obsd 420. 2298 ; Anal. Calcd for C27H3204 : C, 77. 11% ; H, 7. 67% ; obsd C, 76.70% ; H, 7.62% ; purity: 99%.
3-Benzyloxy-17α-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14,15, 16, 17-decahydro- 6H-cyclopenta[a]phenanthrene-16-carboxylic acid methyl ester (JAC01013B, STX


570).

1H NMR (400 MHz, CDCl3): # 0.84 (s, 3H, CH3); 1.20-1.60 (m, 5H); 1.72 (app. q, J = 12.4 Hz, 1H), 1. 85-1. 88 (m, 1H) ; 1. 97 (dt, J = 12. 1, 3.3 Hz, 1H) ; 2. 02-2. 10 (m, 2H) ; 2. 22 (m, 1H) ; 2. 32 (m, 1H) ; 2. 77 (ddd, J = 12.1, 8. 4, 4. 0 Hz, 1H, CHCOO) ; 2. 86 (m, 2H, CH) ; 3. 75 (s, 3H, OCH3) ; 3. 89 (dd, J = 8. 1,4. 4 Hz, 1H, CHO) ; 5. 04 (s, 2H, CH2Ph) ; 6. 73 (d, J = 2. 9 Hz, 1H, H4) ; 6. 79 (dd, J = 8. 4, 2. 6 Hz, 1H, H2) ; 7. 21 (d, J = 8.1 Hz, 1H, H1) ; 7. 30- 7.46 (m, 5H, Ph); Acq. Mass (FAB+) calcd 420.2301, obsd 420.2301 ; Anal. Gated for C27H32O4 : C, 77. 11% ; H, 7.67% ; obsd C, 76.60% ; H, 7. 71% ; purity: 99%.
3-Benzyloxy-16-hydroxymethyl-13-methyl-7, 8, 9, 11,12, 13, 14, 15,16, 17- decahydro-6H-cyclopenta [a] phenanthren-17-ol (JAC01023C).
Solid NaBH4 (452 mg, 12 mmol) was added to a cold (0°C), magnetically stirred solution of JAC01011 (5g, 12 mmol) in THF/MeOH (90: 10), and the reaction mixture was stirred for 18h at 0-20°C, treated with 2N HCI until acidic pH and extracted with ether (twice).
The combine organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. The solid was purified by column chromatography using CHC13/EtOAc (90: 10) as eluant to give 1.95 g of STX 569 (39%), 0.55 g of STX 570 (18%) and 0.63g of JAC01023C (13%) as white solids.'H NMR (270 MHz, DMSO-d6) : 8 0.68 (s, 3H, CH3) ; 0.95-1. 45 (m, 4H) ; 1. 70-1.90 (m, 4H); 2.00-2. 35 (m, 4H); 2. 75 (m, 2H, CH26) ; 3.29 (m, 8H) ; 3. 67 (m, 2H); 4. 13 (dd, J = 5.9, 4.4 Hz, 1H, CHO) ; 4. 60 (d, J = 4. 4 Hz, 1H, OH) ; 5.03 (s, 2H, CH2Ph) ; 6. 69 (d, J = 2. 7 Hz, 1H, H4) ; 6. 74 (dd, J = 8. 4, 2. 7 Hz, 1H, H2) ; 7. 14 (d, J = 8.6 Hz, 1H, H1) ; 7. 25-7. 46 (m, 5H, Ph); Acq. Mass (FAB+) calcd 392. 2351, obsd 392. 2342.
3-Benzyloxy-16- (1, 3-dioxo-1, 3-dihydro-isoindol-2-ylmethyl)-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta [a] phenanthrene-16 carboxylic ac methyl d (JAC01159).
57 mg NaH (60% in oil, 1.43 mmol) was suspended in 2 mL dry THF. The suspension was cooled to 0°C in an ice bath and 500 mg of JAC01011 (1.19 mmol) solubilized in 5 mL dry THF were added dropwise. The mixture was stirred for 30 min and the N- (Bromomethyl) phtalimide (344 mg, 1. 43 mmol) in 3 mL dry THF was added dropwise : The reaction was stirred at 0°C for 1 h and allowed to react at room temperature for a further 12h. 1N HCl was added to the mixture and the acidic layer was extracted with diethylether (3 times). The combined organic layers were washed with sat. NaHCOg (3 times), brine, dried over Na2SO4 and evaporated in vacuo to give JAC01159 as a white solid. This compound was used without further purification. 1H NMR (270 MHz, CDCI3) : 8 0.85 (s, 3H, CH3) ; 1.30-1. 70 (m, 5H); 1.85-1. 95 (m, 2H); 2. 10-2.40 (m, 4H); 2. 79 (m, 2H, CH26) ; 3.75 (s, 3H, OCH3) ; 3.96 (d, J = 14.1 Hz, 1 H, NCH2) ; 4.17 (d, J = 14.1 Hz, 1H, NCH2) ; 4.96 (s, 2H, CH2Ph) ; 6.66 (d, J = 2.6 Hz, 1 H, H4) ; 6.73 (dd, J = 8.4, 2.6 Hz, 1 H, H2) ; 7.12 (d, J = 8.4 Hz, 1 H, Hl) ; 7.24-7. 37 (m, 5H); 7.67 (dd, J = 5.1, 2.7 Hz, 2H); 7.77 (dd, J=5. 1,2. 7 Hz, 2H).
Representative procedure for debenzylation : 3-Hydroxy-13-methyl-17-oxo-7, 8, 9,11, 12,13, 14,15, 16,17-decahydro-6H- cyclopenta[a] phenanthrene-16-carboxylic acid methyl ester (JAC01012, STX 568).
2.5 g (5.97 mmol) of JAC01011 were solubilized in THF (30 mL). A catalytic amount of 10% Pd/C was added and the reaction mixture was stirred at room temperature under H2 for 8 h. Pd/C was filtered over celite and the filtrat was evaporated in vacuo.
Recrystaiiisation of the solid residue in methanol gave 1.82 g (93%) of STX 568 as a white solid. 1H NMR (400 MHz, DMSO-d6): # 0. 87 (s, 1. 95H, CH3) ; 0.91 (s, 1.05H, CH3) ; 1. 20-2. 40 (m, 11H) ; 2. 75 (m, 2H, CH26); 3.39 (dd, J= 9. 8, 8. 6 Hz, 0. 65H, CHCOO) ; 3. 65 (s, 1. 05H, OCH3) ; 3. 65 (s, 1. 95H, OCH3) ; 3. 80 (dd, J = 9. 8, 8. 6 Hz, 0. 35H, CHCOO) ; 6. 45 (d, J = 2. 3 Hz, 1H, H4) ; 6.05 (dd, J = 8.6, 2.7, 1H, H2) ; 7. 04 (d, J= 8. 2 Hz, 1H, H1) ; 9.04 (s, 1H, OH); mp : 215-217°C ; Acq. Mass (FAB+) calcd 328. 1675, obsd 328. 1684 ; Anal. Calcd for C2oH3204 : C, 73. 15%; H, 7. 37% ; obsd C, 72. 9%; H, 7. 34% ; purity: 97%.
This same procedure afforded the following phenols in the indicated yields : 3, 17-Dihydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid methyl ester (JAC01027, STX 572).
Recrystallisation of the solid residue in methanol/water. Yield = 92%, white solid.'H NMR (400 MHz, DMSO-d6) : 8 0. 75 (s, 3H, CH3) ; 1.04-1. 46 (m, 5H); 1.64-1. 86 (m, 4H); 2.08 (td, J= 10.5, 3.3 Hz, 1H) ; 2.22 (m, 1H) ; 2.70 (m, 2H, CH26) ; 3.05 (q, J= 9.0 Hz, 1H, CHCO); 3.56 (s, 3H, OCH3) ; 3.83 (dd, J = 10.5, 5.5 Hz, 1H, CHO); 5.02 (d, J = 5.0 Hz, 1 H, OH); 6.42 (d, J = 2.7 Hz, 1 H, H4) ; 6.48 (dd, J = 8.2, 2.7 Hz, 1 H, H2) ; 7.03 (d, J = 8.6 Hz, 1H, H1) ; 8.99 (s, 1H, phenol) ; Acq. Mass (FAB+) calcd 330.1831, obsd 330.1831 ; mp: 222-224°C ; Anal. Calcd for C2oH2604 : C, 72.70% ; H, 7.93% ; obsd C, 72.90% ; H, 7.90% ; purity: 95%.
16-Hydroxymethyl-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-3,17-diol (JAC01049, STX 612).
Yield = 78%, white solid. 1H NMR (400 MHz, DMSO-d6) : 5 0. 72 (s, 3H, CH3) ; 1. 06 (m, 2H) ; 1. 16-1. 42 (m, 4H) ; 1. 84 (m, 3H) ; 2. 10 (td, J = 10. 0,3. 3 Hz, 1H) ; 2. 24 (m, 2H) ; 2. 72 (m, 2H, CH26) ; 3. 34 (m, 1H) ; 3. 70 (m, 2H) ; 4.18 (dd, J = 6. 2, 4. 8 Hz, 1H, CHO) ; 4. 64 (d, J = 4. 8 Hz, OH) ; 6. 45 (d, J = 2. 6 Hz, 1H, H4) ; 6. 52 (dd, J = 8. 4, 2. 6 Hz, 1H, H2) ; 7. 06 (d, J=8.4 Hz, 1H, H1) ; 9. 02 (s, 1H, phenol) ; mp: decomposition ; MS (FAB+) calcd 302. 19, obsd 302.1 (M+) ; Anal. Gated for C19H26O3: C, 75,46% ; H, 8. 67% ; obsd C, 75. 2% ; H, 8. 7% ; purity: 100%.
16- (1, 3-Dioxo-1, 3-dihydro-isoindol-2-ylmethyl)-3-hydroxy-13-methyl-17-oxo-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H-cyclopenta [a] phenanthrene-16-carboxylic acid methyl ester (JAC01171-4, STX 780). Purification by column chromatography using flash column ISOLUTE Sl 1 g and an elution gradient Hexane/Ethyl Acetate. The white solid was precipitated in Ethyl acetate /Hexane. Yield = 6.9 %, white solid. 1H NMR (270 MHz, MeOH-d3) : 5 0.89 (s, 3H, CH3) ; 1.20-1. 75 (m, 5H); 1.85-2. 05 (m, 2H); 2.10-2. 50 (m, 4H); 2.80 (m, 2H, CH26) ; 3.72 (s, 3H, OCH3) ; 4.08 (d, J = 5.5 Hz, 1 H, NCH2) ; 6.66 (d, J = 2.6 Hz, 1 H, H4); 6.73 (dd, J = 8.4, 2.6 Hz, 1H, H2) ; 7. 12 (d, J = 8. 4 Hz, 1H, H1) ; 7.75-7. 90 (m, 4H); MS (APCI+) calcd 487.20, obsd 489.36 (M+2); purity: 88%.
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyctopenta [a]phenanthrene-16-carboxylic acid (JAC01083).
2 g (4. 75 mmol) of STX 569 were solubilized in 20 mL THF and 285 mg (7. 13 mmol) NaOH solubilized in 5 mL water were added to the solution which was stirred at room temperature for 24 h. The so) vents were evaporated and 6N HO was added to the residue. The acidic layer was extracted by diethyl ether (5 times). The combined organic layers were extracted with 10% K2CO3 (5 times). This basic solution was made acidic adding 6N HCI and extracted by ethyl acetate (5 times). The combine organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. The white solid was suspended in hexane and filtered to give 720 mg (37%) of JAC01083.'H NMR (270 MHz, DMSO-d6) : 8 0. 68 (s, 1. 05H, CH3) ; 0. 72 (s, 1.95H, CH3) ; 1.00-1. 50 (m, 6H); 1.60- 1. 90 (m, 3H); 2.11 (m, 1H) ; 2.26 (m, 1H) ; 2.74 (m, 2H, CH26) ; 2.92 (q, J=8. 9 Hz, 0.65H, CHCO); 3. 68 (dd, J = 7.2, 5.3 Hz, 0.35H, CHCO); 3.80 (d, J = 10.5 Hz, 0. 65H, CHO); 4. 91 (d, J = 5.3, 0.35H, CHO) ; 5.03 (s, 2H, CH2Ph) ; 6.68 (s, 1 H, H4) ; 6. 74 (dd, J = 8. 6, 2.6 Hz, 1H, H2) ; 7. 14 (d, J = 8. 6 Hz, 1H, H1) ; 7.25-7. 50 (m, 5H, Ph).
3-Hydroxy-13-methyt-17-oxo-7, 8, 9,11, 12, 13, 14,15, 16,17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid (JAC01043, STX 610).
2. 14 g (6.51 mmol) of STX 568 were suspended in 46 mL of 5N NaOH and stirred at room temperature for 72h until complete solubilization. After cooling to 0°C, 6N HCI was added dropwise to the solution until pH 1. The acidic layer was extracted by diethyl ether (3 times). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuum at room temperature. The solid was suspended in hexane, filtered, washed with hexane and dried in vacuo to give 1.56 g (76%) of STX 610 as beige solid, which was used without further purification. 1H NMR (270 MHz, DMSO-d6) : 5 0.84 (s, 1.95H, CH3) ; 0.88 (s, 1.05H, CH3) ; 1.20-1. 55 (m, 4H); 1. 70-2. 00 (m, 3H); 2.10- 2.35 (m, 4H) ; 2.74 (m, , 2H, CH26) ; 3.22 (t, J = 8. 6 Hz, 0.65H, CHCOO); 3.60 (t, J = 8. 6 Hz, 0. 35H, CHCOO) ; 6. 43 (d, J = 2. 4 Hz, 1 H, H4) ; 6.50 (dd, J=8.4, 2. 4 Hz, 1H, H2); 7. 03 (d, J=8.4 Hz, 1H, H1) ; 9. 02 (s, 1H, phenol) ; Acq. Mass (FAB+) calcd 314.1518, obsd 314. 1524 ; purity : 95%.
NB : decomposed with time to give estrone at room temperature.
3, 17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a] phenanthrene-16-carboxylic acid (JAC01041, STX 611).
1.1 g (3.20 mmol) of STX 572 were suspended in 10 mL MeOH and 0.58 g (14 mmol) NaOH solubilized in 3 mL water were added to the suspension. The solution was stirred at room temperature for 5 h. The solvents were evaporated and 6N HCI'was added to the residue. The acidic layer was extracted by ethyl acetate (5 times) and the combined organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo.
Recrystallisation of the solid residue in acetonitrile gave 470 mg (46%) of STX 611 as a white solid.'H NMR (400 MHz, DMSO-d6) : 8 0.69 (s, 1.86H, CH3,) ; 0.73 (s, 1.14H, CH3) ; 1.20-1. 40 (m, 5H); 1. 45-1.85 (m, 4H); 2. 10 (m, 1H) ; 2.20 (m, 1H) ; 2.45 (m, 0.62H, CHCOO); 275 (m, 2H, CH26) ; 2.95 (q, J = 9.0 Hz, 0.38H, CHCOO); 3.69 (d, J = 8. 00 Hz, 0.62H, CHO); 3.60 (d, J = 10. 8 Hz, 0.38H, CHO); 4.92 (bs, 1 H, OH); 6.42 (d, J = 2.7 Hz, 1 H, H4) ; 6.48 (dd, J = 8.2, 2.7 Hz, 1 H, H2) ; 7.03 (d, J = 8.2 Hz, 1H, H1) ; 8.99 (s, 1H, phenol) ; mp: decomp.; Acq. Mass (FAB+) calcd 316.1675, obsd 316. 1677; purity: 95%.
Solid phase chemistry t//CI acid Estone, DIER /C dry DCM / wu y Y Loading = 1. 1 mmol/g resin Loading = 0. 82 mmof/g resin Loading of estrone on 2-chloro trityl resin.
500 mg of 2-chloro-trityl resin (loading of 1. 1 mmol/g resin) were swollen in 7 mL dry DCM and 3 mL DIEA for 15 min. before addition of estrone. The mixture was shacked for 72h at room temperature under 2. The resin was filtered, washed with DCM, DMF, MeOH (3 cycles) and MeOH (5 times) and dried in vacuo. R (cm-1) 1736 (C=O, ketone).
Determination of estrone's loading. 51. 4 mg of loaded resin were swollen in 1 ml DCM in a filter funnel, 10 mL DCM/MeOH/TFA (7 : 1 : 2) were added (5 fractions of 2 mL) and the filtrat was evaporated in vacuo to give 11. 5 mg of a white solid corresponding to estrone when structure checked by'H NMR. The loading was then determined : 0. 82 mmol/g resin.
NB: When addition DCM/MeOH/TFA, the resin became purple.
Combinatorial library construction : Method A : Step 1 : Loading of JAC01041 on oxime resin. ON , OH N OH 0 I RCOOH, DIC, HOBT-N dry DMF O i i NO ho Loading = 1. 06 mmol/g resin Maximum loading = 1. 06 mmol/g resin 500 mg of oxime resin (loading of 1.06 mmol/g resin) were swollen in 7 mL dry DMF for 15 min after which 251 mg of JAC01041 were added followed by the addition of 41 µL DIC and 358 mg HOBT. The mixture was shaken for 48h at room temperature under inert atmosphere and the resin was filtered, washed with DCM, DMF, MeOH (3 cycles) and MeOH (5 times) and dried in vacuo. IR (cm-1) 1750 (C=O, ester) ; 1600 (C=N, oxime).
Step2 : Amidification.
200 mg of oxime resin loaded with JAC01041 (maximum loading of 1. 06 mmol/g resin) were swollen in 4 mL dry DCM and 47, uL (0.53 mmol) furfurylamine were added to the suspension. The mixture was shacked at 40°C under nitrogen atmosphere for 24h. After filtration, the resin was washed with DCM (5 times) and the filtrated was evaporated in vacuo.
Method B : Step 1 : Active ester formation.
3-Benzyloxy-17hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopsnta [a]phenathrene-16-carboxylic acid 4-nitro-phenyl ester (JAC01177).
[Helv. Chim. Act. 1963,795-804]. 400 mg of JAC01083 were solubilized in 4 mL dry DMF and 0. 14 mL triethylamine (0.98 mmol) and 299 mg bis- (4-nitrophenyl) carbonate (0.98 mmol) were successively added. The solution was stirred at room temperature for 2h under N2 atmosphere. The solution was cooled to 0°C and 2N HCI was added. The mixture was extracted with DCM (3 times). The combined organic layers were washed with sat. NaHCO3 (5 times) and brine, dried over Na2SO4 and evaporated in vacuo. The residue was purified by column chromatography ISOLUTE Sl 20g using an elution gradient Hexane/Ethyl Acetate to give 270 mg of JAC01177 as a yellow solid (52%). 1H NMR (270 MHz CDCl3,) : 8 0.87 (s, 3H, CH3) ; 1.27-1. 50 (m, 4H); 1. 80-2. 05 (m, 4H); 2.10- 2.40 (m, 3H); 2.84 (m, 2H, CH26) ; 3.05 (ddd, J = 11.7, 7.7, 3.7 Hz, 1 H, CHCO); 4.06 (dd, J = 8. 1,5. 1 Hz, 1 H, CHO); 5.01 (s, 2H, CH2Ph) ; 6.70 (d, J = 2.6 Hz, 1H, H4) ; 6.76 (dd, J = 8. 8,2. 6 Hz, 1H, H2); 7.19 (d, J = 8.8 Hz, 1 H, Hl) ; 7.25-7. 45 (m, 7H); 8.27 (d, J = 12.1 Hz, 2H); MS (FAB+) calcd 527.23, obsd 527.4 (M+) ; 389. 4 (M-pNO2PhO) ; 91.1 (PhCH2+).
Step 2 : General procedure for Amidification.
The active ester JAC01177 (0. 17 mmol) was solubilized in 2mL dry acetonitrile and the amine (2 eq) was added to the mixture. The solution was stirred overnight at room temperature and the solvents were evaporated in vacuo. The residue was purified by column chromatography using flash column ISOLUTE Sl 5g and an elution gradient Hexane/Ethyl Acetate.
# Step 3: general procedure for debenzylation: The benzylated amide was solubilized in THF (3 mL). A catalytic amount of 5% Pd/C was added and the reaction mixture was stirred at room temperature under H2 for 24 h.
Pd/C was filtered over paper and the filtrat was evaporated in vacuo. The final product was precipitated in a mixture Ethyl Acetate/Hexane. Reference Method STX 679 A STX 779 B STX 798 B STX 799 B STX 800 B STX 801 B STX 802 B STX 803 B STX 844 B STX 845 B 3-Benzyloxy-17-hydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid (tetrahydro-furan-2-ylmethyl)- amide (JAC01163-3).
Method B, step2. Yield = 96%.'H NMR (270 MHz, CDCl3) : 5 0. 74 (s, 3H, CH3) ; 1.10- 1.60 (m, 7H); 1.70-2. 00 (m, 5H); 2.05-2. 30 (m, 3H); 2.50 (m, 1H) ; 2.74 (m, 2H, CH26) ; 3.09 (m, 1H) ; 3.50 (m, 1H) ; 3.60-3. 85 (m, 3H); 3.90 (m, 1H), 4.94 (s, 2H, CH2Ph) ; 6.42 (t, J = 5.5 Hz, 1H, NH) ; 6.63 (s, 1H, H4) ; 6. 68 (dd, J = 8. 4, 2.2 Hz, 1 H, H2) ; 7.10 (d, J = 8. 4 Hz, 1H, H1) ; 7. 20-7. 40 (m, 5H).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid (pyridin-2-ylmethyl)-amide (JAC01179-1, STX 789).
Method B, B, step2. Yield = 94%. 1H NMR (270 MHz, CDCl3): # 0.78 (s, 3H, CH3); 1.20- 1. 60 (m, 8H) ; 1. 70-1. 90 (m, 2H); 2.05-2.30 (m, 3H); 2.63 9ddd, J=11.0, 8.0, 3.7 Hz, 1H, CHCO) ; 2. 75 (m, 2H, CH) ; 3.73 (d, J=8. 0 Hz, 1H, CHO) ; 4. 30-4.43 (dAB, J=16. 5, 4. 8 Hz, 1H, NCH2) ; 4. 60-4. 75 (dAB, J = 16. 5,6. 6 Hz, 1H, NCH2) ; 4. 95 (s, 2H, CH2Ph) ; 6. 64 (d, J. = 2.9 Hz, 1H, H4) ; 6.70 (dd, J = 8. 4,2. 9 Hz, 1H, H2) ; 6.90 (t, J = 5.1 Hz, 1H, NH); 7.10 (d, J = 8.4 Hz, 1H, H1) ; 7.13-7. 40 (m, 6H); 7.59 (td, J = 7.7, 1.8 Hz, 1H, H5') ; 8.43 (d, J=5. 1 Hz, 1H, H3') ; MS (FAB+) calcd 496.27, obsd 497.3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H- cyclopenta lalphenanthrene-16-carboxylic acid (pyridin-3-ylmethyl)-amide (JAC01179-2, STX 790). Method B, step2. Yield = 82%.'H NMR (270 MHz, CDCI3) : # 0.81 (s, 3H, CH3) ; 1.10- 1.60 (m, 8H); 1.79 (m, 2H); 2.05-2. 30 (m, 3H); 2.57 (ddd, J = 11.3, 8. 4,3. 3 Hz, 1H, CHCO); 2.75 (m, 2H, CH26) ; 3.72 (d, J = 8.4 Hz, 1H, CHO); 4.25-4. 55 (dAB, J = 15.0, 6.2, 5.5 Hz, 2H, NCH2) ; 4.95 (s, 2H, CH2Ph) ; 6.63 (d, J = 2.6 Hz, 1H, H4) ; 6.70 (dd, J = 8. 4,2. 6 Hz, 1 H, H2) ; 7.10 (d, J = 8. 4 Hz, 1H, H1) ; 7.14-7. 40 (m, 6H); 7. 58 (d, J = 7.7 Hz, 1H, H4') ; 8. 40 (dd, J = 4. 8, 1.4 Hz, 1 H, H6,) ; 8. 42 (d, J = 1. 8 Hz, 1H, H2') ; MS (FAB+) calcd 496. 3, obsd 497. 3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-yl)- (4-methyl-piperazin-1-yl)-methanone (JAC01179-3, STX 791).
Method B, step2. Yield = 86%. 1H NMR (270 MHz, CDCl3): # 0.83 (s, 3H, CH3); 1.30- 1.60 (m, 7H) ; 1. 70-1. 85 (m, 2H) ; 2. 15-2. 45 (m, 10H) ; 2. 75-2. 95 (m, 3H) ; 3. 40-3. 80 (m, 4H) ; 4. 20 (d, J = 7. 0 Hz, 1H) ; 5. 00 (s, 2H, CH2Ph) ; 6. 68 (d, J = 2. 6 Hz, 1H, H4) ; 6. 75 (dd, J = 8. 8,2. 6 Hz, 1 H, H2) ; 7. 15 (d, J = 8.8 Hz, 1H, H1) ; 7.29-7. 42 (m, 5H); MS (FAB+) calcd 488.3, obsd 489. 3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid ethyl-methyl-amide (JAC01179-4, STX 794). Method B, step 2. Yield = 35%. 1H NMR (400 MHz, CDCI3) : 8 0. 88 (s, 3H, CH3) ; 1. 13 (t, J = 7. 0 Hz, 1.5H, CH2CH3) ; 1. 21 (t, J =7. 0 Hz, 1.5H, CH2CH-1. 60 (m, 8H); 1.68- 1.88 (m, 4H); 1.98 (m, 1H) ; 2.24 (m, 1H) ; 2.33 (m, 1H, CHCO); 2.70 (m, 1H) ; 2.84 (m, 2H, CH26) ; 2.96 (s, 1. 5H, NCH3) ; 3.03 (s, 1.5H, NCH3) ; 3.30-3. 48 (m, 2H, CHzCH3) ; 4.24 (app. t, J = 7.4 Hz, 1H, CHO); 5.04 (s, 2H, CH2Ph) ; 6.72 (d, J = 2.4 Hz, 1 H, H4) ; 6.79 (dd, J = 8. 6, 2. 4 Hz, 1 H, H2) ; 7.22 (d, J = 8. 6 Hz, 1 H, Hl) ; 7.36-7. 48 (m, 5H); MS (FAB+) caicd 447.3, obsd 448. 3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid isopropylamide (JAC01179-5, STX 795).
Method B, step2. Yield = 92%. 1H NMR (270 MHz, CDCl3): # 0.80 (s, 3H, CH3); 1.14 (d, J = 7. Hz, 6H, 2 x CH3); 1. 28-1. 60 (m, 6H) ; 1. 80-1. 92 (m, 3H) ; 2. 10-2. 36 (m, 3H) ; 2. 48 (ddd, J = 11. 7, 8. 4, 4. 0 Hz, 1H, CHCO) ; 2. 81 (m, 2H, CH26) ; 3.76 (dd, J = 8. 0,5. 1 Hz, 1H, CHO) ; 4. 09 (septuplet, J = 7. 0 Hz, 1H, CH (CH3) 2) ; 5. 01 (s, 2H, CH2Ph) ; 5. 72 (d, J = 8.1 Hz, 1H, NH); 6.70 (d, J = 2.7 Hz, 1H, H4) ; 6.75 (dd, J = 8. 4,2. 7 Hz, 1 H, H2) ; 7.17 (d, J=8. 4 Hz, 1H, H1); 7.29-7. 43 (m, 5H); MS (FAB+) calcd 447.3, obsd 448.3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15, 16,17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid (1-methyl-1H-pyrrol-2-ylmethyl)- amide (JAC01179-6, STX 793). Method B, step2. Yield = 86%.'H NMR (270 MHz, CDCI3) : # 0.79 (s, 3H, CH3) ; 1.27- 1.65 (m, 6H); 1.80-1. 92 (m, 3H); 2.10-2. 36 (m, 3H); 2.54 (ddd, J = 11.7, 8. 4,4. 0 Hz, 1H, CHCO); 2.82 (m, 2H, CH26) ; 4.44 (t, J = 5.1 Hz, 1 H, CH2N) ; 5. 01 (s, 2H, CH2Ph) ; 5.98 (t, J = 5.1 Hz, 1H, NH); 6.04 (m, 2H, H3', H5') ; 6.59 (t, J = 2.2 Hz, H4') ; 6.70 (d, J = 2.6 Hz, 1 H, H4) ; 6. 76 (dd, J = 8. 8, 2.6 Hz, 1H, H2) ; 7.17 (d, J = 8.8 Hz, 1H, H1) ; 7.29-7. 43 (m, 5H); MS (FAB+) calcd 498.3, obsd 499.3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cycloopenta[a]phenanthrene-16-carboxylic acid (5-methyl-pyrazin-2-ylmehtyl)- amid@ (JAC01179-7, STX 792).
Method B, step2. Yield = 85%. 1H NMR (270 MHz, CDCl3) : 8 0. 83 (s, 3H, CH3) ; 1. 20- 1. 60 (m, 9H); 1. 87 (m, 1H) ; 2. 10-2. 35 (m, 1 H) ; 2. 50 (d, J=5.1 Hz, 1H, OH) ; 2. 54 (s, 3H, HarCH3) ; 2.65 (m, 1H, CHCO) ; 2. 81 (m, 2H, CH26) ; 3. 75 (dd, J=8.4, 5. 1 Hz, CHO) ; 4. 40- 4. 80 (dAB, J = 16. 0,6. 6,4. 8 Hz, NCH2) ; 5. 01 (s, 2H, CH2Ph) ; 6. 70 (m, 2H, H4 + NH) ; 6. 74 (dd, J = 8.4, 2.6 Hz, 1H, H2) ; 7.17 (d, J = 8.4 Hz, 1H, H1) ; 7.28-7. 43 (m, 5H); 8. 36 (s, 1 H) ; 8.47 (s, 1 H) ; MS (FAB+) calcd 511.3, obsd 512.3 (M+1).
3-Benzloxy-17-hdyroxy-13-methyl-7, 8,9, 11, 12, 13, 14, 15,16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid (pyridin-3-ylethyl)-amide (JAC01185-1, STX 814).
Method B, step2. Yield = 58%.'H NMR (270 MHz, CDC13) : 8 0.73 (s, 3H, CH3) ; 1. 10- 1.60 (m, 7H); 1.81 (m, 2H); 2.05-2. 30 (m, 3H); 2.45 (ddd, J = 11.7, 8.4, 3.7 Hz, 1H, CHCO); 2.77 (m, 2H, CH) ; 3.30-3. 65 (m, 3H); 4.94 (s, 2H, CH2Ph) ; 6.19 (t, J = 5. 5 Hz, 1 H, NH); 6.64 (d, J = 2.6 Hz, 1H, H4) ; 6.68 (dd, J = 8.4, 2.6 Hz, 1H, H2); 7.10 (d, J =8. 4 Hz, 1 H, Hl) ; 7.15-7. 38 (m, 6H); 7.49 (m, 1H) ; 8. 36 (m, 2H); MS (FAB+) calcd 510.3, obsd 511.3 (M+1).
3-benny) 3-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid (1-phenyl-ethyl)-amide (JAC01185- 4A, STX 815).
Method B, step2. Yield = 46%. 1H NMR (270 MHz, CDCl3): # 0.80 (s, 3H, CH3); 1.20- 1. 60 (m, 10H); 1.84 (m, 2H); 2.10-2.35 (m, 3H); 2.57 (ddd, J = 11.7, 8.4, 3.7 Hz, 1H, CHCO) ; 2. 81 (m, 2H, CH) ; 3. 71 (dd, J = 8.8, 5.5 Hz, 0. 71H, CHO) ; 3. 80 (m, 0. 28H, CHO) ; 5.00 (s, 2H, CH2Ph) ; 5. 12 (m, 1H, CHPh) ; 6. 22 (m, 1H, NH) ; 6.69 (d, J= 2. 9 Hz, 1 H, H4) ; 6.76 (dd, J = 8. 5,2. 9 Hz, 1 H, H2) ; 7.12 (d, J = 8.5 Hz, 1H, H1) ; 7. 15-7. 45 (m, 10H) ; MS (FAB+) calcd 509.3, obsd 510.3 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid (1-phenyl-ethyl)-amide (JAC01185- 4B, STX 816). Method B, step2. Yield = 24%.'H NMR (270 MHz, CDCI3) : 8 0.79 (s, 3H, CH3) ; 1.20- 1.60 (m, 10H) ; 1 : 87 (m, 3H); 2.10-2. 38 (m, 3H); 2.53 (ddd, J = 11. 0, 8. 1,3. 3 Hz, 1H, CHCO); 2.80 (m, 2H, CH26) ; 3.81 (dd, J = 8.1, 5.1 Hz, 1H, CHO); 5.00 (s, 2H, CH2Ph) ; 5.12 (m, 1H, CHPh) ; 6.20 (m, 1H, NH); 6.69 (d, J = 2.6 Hz, 1 H, H4) ; 6.75 (dd, J = 8. 6, 2.6 Hz, 1H, H2) ; 7. 12 (d, J = 8.6 Hz, 1 H, Hl) ; 7.20-7. 50 (m, 10H) ; MS (FAB+) calcd 509.3, obsd 510.3 (M+1).
This second isomer has been isolated from the same reaction as STX 815.
3-Benzyloxy-17-hydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-
cyclopenta[a]phenanthrene-16-carboxylic acid (2-piperazin-1-yl-ethyl)-amide (JAC01185-2, STX 817). Method B, step2. Yield = 73%. 1H NMR (270 MHz, CDCl3) : 6 0. 81 (s, 3H, CH3); 1.20- 1. 60 (m, 6H) ; ; 1.80-2.05 (m, 2H); 2.10-2.60 (m, 11H); 2.75-2.95 (m, 6H); 3.10-3.40 (m, 1H) ; 3.55-3. 70 (m, 2H) ; 5.00 (s, 2H, CH2Ph) ; 6. 01 (b s, 1H, NH) ; 6.69 (d, J=2. 6 Hz, 1H, H4) ; 6. 75 (dd, J = 8. 4, 2. 6 Hz, 1H, H20 ; 7. 17 (d, J = 8. 4 Hz, 1H, H1) ; 7. 30-7. 35 (m, 10H) ; MS (FAB+) calcd 517.3, obsd 518. 4 (M+1).
3-Benzyloxy-17-hydroxy-13-methyl-7, 8,9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (pyridin-4-ylmethyl)-amide (JAC01191-1, STX 818).
Method B, step2. Yield = 92%.'H NMR (270 MHz, CDCl3)d : ô 0. 83 (s, 3H, CH3) ; 1.20- 1.65 (m, 7H); 1.80-1. 95 (m, 2H); 2.15-2. 40 (m, 3H); 2.64 (ddd, J = 12.4, 8.4, 3.6 Hz, 1H, CHCO); 2. 82 (m, 2H, CH26) ; 3.79 (d, J = 8.4 Hz, 1H, CHO); 4. 35-4.60 (dAB, J = 16.1, 8.4, 6.2 Hz, 2H, NCH2) ; 5.01 (s, 2H, CH2Ph) ; 6.44 (m, 1H, NH); 6.70 (d, J=2. 2 Hz, 1H, H4) ; 6.75 (dd, J = 8.4, 2.2 Hz, 1H, H2) ; 7.15-7. 45 (m, 8H); 8.51 (d, J = 5.8 Hz, 2H, H2', H5') ; MS (FAB+) calcd 496.3, obsd 497.3 (M+1).
3, 17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15,16, 17-decahydro-6H- cyclopenta[a] pheanthrene-16-carboxylic acid (furan-2-ylmethyl)-amide (JAC01081B, STX 679).
Method A, step2. The brown oil was purified by column chromatography using EtOAc/Hexane (50:50) as eluant gave 3 mg (3. 6% over 2 steps) of STX 679 as a yellow solid. 1H NMR (400 MHz, CDCl3) : 8 0. 82 (s, 2. 49H, CH3) ; 0. 84 (s, 0. 51H, CH3) ; 1. 08-1. 60 (m, 6H); 1. 80 (m, 2H); 2.20 (m, 2H); 2.30 (m, 1H) ; 2. 58 (m, 1H, CHCO) ; 2. 80 (m, 2H, CH26) ; 3.74 (d, J = 8.4 Hz, 0.83H, CHO) ; 3.80 (d, J = 8.9 Hz, 0. 17H, CHO); 4.41 (d, J = 5.7 Hz, 2H, CH2N) ; 6.15 (dd, J = 3.2, 1.7 Hz, 1H, furane H3) ; 6.25 (m, 1H, furane H4); 6.50 (d, J = 2.7 Hz, 1H, H4) ; 6.55 (dd, J = 8.4, 2.7 Hz, 1H, H2) ; 7.04 (d, J = 8.1 Hz, 1H, H1) ; 7.28 (dd, J = 2.0 ; 1.0 Hz, 1H, furane H5) ; MS (FAB+) ca) cd 395.21, obsd 396. 18 (M+1) ; purity: 98%.
3, 17-Dihydroxy-13-methyl-7, 8,9, 11, 12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [alhenanthrene-16-carboxylic acid (tetrahydro-furan-2-ylmethyl)-amide (JAC01171-3, STX 779).
Method B, step3. Yield = 12 %, white solid.'H NMR (MeOH-d3, 270 MHz): # 0.81 (s, 3H, CH3) ; 1.26-1. 45 (m, 6H); 1.50-1. 70 (m, 3H); 1.80-2. 00 (m, 6H); 2.15 (m, 1H) ; 2.30 (m, 1H) ; 2.60 (ddd, J= 11.4, 8. 1, 3.7 Hz, 1H, CHCO); 2. 78 (m, 2H, CH26) ; 3.70-3. 95 (m, 3H); 3. 98 (m, 1H) ; 6. 45 (d, J = 2.6 Hz, 1 H, H4) ; 6. 52 (dd, J = 8. 4, 2. 6 Hz, 1 H, H2) ; 7. 06 (d, J = 8. 4 Hz, 1H, H1) ; MS (APCF) calcd 399. 53, obsd 401. 34 (M+2) ; Purity: 99% 3, 17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid (pyridin-3-ylmehtyl)-amide (JAC01181-2, STX 798).
Method B, step3. Yield = 36%. 1H NMR (270 MHz, DMSO-d6): # 0.69 (s, 3H, CH3); 1.10- 1. 40 (m, 7H) ; 1. 45-1. 60 (m, 2H) ; 1. 60-1. 85 (m, 4H) ; 2. 20 (m, 2H) ; 2. 67 (m, 2H, CH2-I ; 2. 86 (m, 2H) ; 3.65 (m, 1H) ; 4.29 (d, J = 5. 5 Hz, 1H) ; 4.80-5.05 (m, 1H) ; 6. 41 1 (s, 1H, H4) ; 6. 48 (dd, J = 8. 4, 2. 2 Hz, 1H, H2) ; 7. 02 (d, J = 8. 4 Hz, 1H, H1) ; 7. 33 (m, 0. 5H) ; 7. 62 (m, 1H) ; 8. 26 (m, 0.5H) ; 8.42 (dd, J=4.8, 1.8 Hz, 1H) ; 8. 47 (m, 1H) ; 9.00 (bs, 1H, phenol).
MS (APCI+) calcd 406.5, obsd 408.3 (M+2); Purity: 100% I 3, 17-Dihydroxy-13-methyl-7, 8,. 9, 11, 12, 13, 14,15, 16,17-decahydro-6H- cyclopenta [a]phenanthrene-16-yl)-(4-methyl-piperazin-1-yl)-methanone (JAC01181-3, STX799).
Method B, step3. Yield = 58%.'H NMR (270 MHz, CDCI3) : 8 0.83 (s, 3H, CH3) ; 1.26- 1.55 (m, 6H); 1.65-1. 85 (m, 3H); 1. 89 (m, 1H) ; 2.25 (m, 1H) ; 2.30 (s, 3H, NCH3) ; 2.40 (m, 4H); 2.78 (m, 2H, CH26) ; 2.88 (m, 1 H, CHCO); 3.40-3. 80 (m, 4H); 4.17 (d, J = 7.7 Hz, CHO); 6.53 (d, J = 2.7 Hz, 1H, H4) ; 6.61 (dd, J = 8.4, 2.7 Hz, 1H, H2) ; 7.12 (d, J = 8.4 Hz, 1H, H1). MS (APCI+) calcd 398.5, obsd 400.3 (M+2); Purity: 100%.
3, 17-Dihydroxy-13-methyl-7, 8, 9, 11,12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid ethyl-methyl-amide (JAC01181-4, STY 800). Method B, step3. Yield = 75%. 1H NMR (400 MHZ, DMSO-d6) : # 0. 70 (s, 3H, CH3) ; 0. 98 (t, J = 6. 8 Hz, 1. 5H, CH2CH3); 1.07 (t, J = 7. 0 Hz, 1. 5H, CH2CH3); 1.10-1. 30 (m, 6H); 1. 45-1. 85 (m, 4H) ; 2.05 (m, 1H) ; 2. 20 (m, 1H) ; 2'. 67 (m, 2H, CH26) ; 2. 79 (s, 1. 5H, NCH3) ; 2. 88 (m, 1, CHCO0 ; 2. 96 (s, 1.5H, NCH3) ; 3.30-3. 48 (m, 2H, CH2CH3) ; 3. 78 (dd, J = 12. 8, 7. 0 Hz, 1H) ; 4. 76 (t, J=5. 3 Hz, NH) ; 6. 41 (d, J = 2.6 Hz, 1H, H4) ; 6. 47 (dd, J= 8. 2, 2.6 Hz, 1H, H2) ; 7.02 (d, J = 8. 2 Hz, 1H, H1) ; 8.99 (s, 1H, phenol). MS (APCI+) calcd 357.5, obsd 359.3 (M+2); Purity: 100%.
3-, 17-Dihydroxy-13-methyl-7, 8,9, 11, 12, 13,14, 15,16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid isopropylamide (JAC01181-5, STX 801).
Method B, step3. Yield = 50%. 1H NMR (270 MHz, CDCI3) : 5 0.79 (s, 3H, CH3) ; 1.14 (d, J = 6.6 Hz, 6H, 2 x CH3) ; 1.20-1. 60 (m, 6H); 1. 84 (m, 2H); 2.09-2. 20 (m, 2H); 2.22-2. 34 (m, 1H) ; 2.48 (ddd, J = 12.1, 8.4, 4. 0 Hz, 1H, CHCO); 2.77 (m, 2H, CH26) ; 3.76 (d, J = 8.4 Hz, 1H) ; 4.09 (m, 1H, CH(CH3)2) ; 5.73 (app. d, J = 7.3 Hz, 1H, NH); 6.54 (d, J = 2.7 Hz, 1 H, H4) ; 6.61 (dd, J = 8. 4,2. 7 Hz, 1 H, H2) ; 7.11 (d, J = 8.4 Hz, 1H,H1). MS (APCI+) calcd 357.5, obsd 359.2 (M+2); Purity: 100%.
3, 17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta [a]phenanthrene-16-carboxylic acid (1-methyl-1H-pyrrol-2-ylmethyl) - amide (JAC01181-6, STY 802).
Method B, step3. Yield = 45%. 1H NMR (270 MHz, CDCl3) : 8 0. 78 (s, 3H, Chug) ; 1. 20- 1. 65 (m, 8H) ; 1. 80-1. 92 (m, 3H); 2. 10-2. 20 (m, 2H) ; 2. 25 (m, 1H) ; 2. 50 (ddd, J = 11. 7, 8. 4, 4. 0 Hz, 1H, CHCO) ; 2. 84 (m, 2H, CH26) ; 3. 57 (s, 3H); 3. 78 (d, J=8.1 Hz, 1H, CHO) ; 4. 44 (t, J = 5. 8 Hz, 1 H, CH2N) ; 5. 99 (t, J = 5.1 Hz, 1H, NH) ; 6.05 (m, 2H) ; 6. 54 (d, J = 2.9 Hz, 1H, H4); 6. 57-6. 64 (m, 2H); 7.10 (d, J=8.4 Hz, 1H, H1). MS (APCl+) calcd 408. 5, obsd 408. 3 (M+), 410.3 (M+2); Purity: 100%.
3, 17-Dihydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15, 16, 17-decahydro-6H- cyclopenta [a] phenanthrene-16-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)- amide (JAC01181-7, STX 803). Method B, step3. Yield = 26%.'H NMR (270 MHz, CDCI3) : 6 0.82 (s, 3H, CH3) ; 1.20- 1.65 (m, 8H); 1.75-1. 95 (m, 2H); 2.05-2. 40 (m, 3H); 2.54 (s, 3H, HarCH3) ; 2.64 (m, 1H, CHCO); 2.81 (m, 2H, CH26) ; 3.77 (d, J = 8.4 Hz, CHO); 4. 40-4. 80 (ddAB, J = 16.5, 7.0, 5.1 Hz, NCH2) ; 6.54 (app.. s, 1H, H4) ; 6.60 (dd, J = 8.4, 2.6 Hz, 1H, H2) ; 6.72 (t, J = 5.1 Hz, 1H, NH); 7.10 (d, J = 8.4 Hz, 1H, H1) ; 8.36 (s, 1H) ; 8.48 (s, 1H). MS (APCI+) calcd 421.54, obsd 423.3 (M+2); Purity: 100%.
3, 17-Dihydroxy-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H- cyclopenta[a]phenanthrene-16-carboxylic acid (1-phenyl-ethyl)-amide (JAC01193- 3, STX 844).
[Starting material was STX 815]. Method B, tep3. Yield = 7.3%. 1H NMR (270 MHz, DMSO-d6): # 0.70 (s, 3H, CH3); 1.10-1.38 (m, 9H); 1.65-1.85 (m, 3H); 2.05 (m, 1H); 2.20 (m, 1H) ; 2. 68 (m, 2'H, CH26) ; 3.60 (m, 1H, CHCO) ; 4. 80-5. 00 (m, 2H) ; 6. 41 (app. s, 1H, H4) ; 6. 48 (dd, J = 8. 1, 2. 9 Hz, 1H, H2) ; 7. 02 (d, J = 8. 1 Hz, 1H, Hi) ; 7. 20 (m, 1H) ; 7. 25- 7.40 (m, 5H); 8. 03 (d, J = 8. 4 Hz, 1 H, NH); 8.97 (s, 1 H, OH); MS (APCI+) calcd 419. 25, obsd 420.42, Purity: 98%.
3,17-Dihydroxy-13-methyl-7, 8, 9,11, 12,13, 14,15, 16, 17-decahydro-6H- cyclopentala] phenanthrene-1 6-carboxylic acid (pyridin-4-ylmethyl)-amide (JAC001193-5, STX 845).
Method B, step3. Yield = 17 %. ¹H NMR (270 MHz, DMSO-d6): # 0.60 (s, 0.86H, CH3); 0.63 (s, 2.14H, CH3) ; 1.10-1. 25 (m, 5H); 1.35-1. 80 (m, 4H); 2.02 (m, 1H) ; 2.13 (m, 1H) ; 2.60 (m, 2H, CH26) ; 2.81 (m, 1H) ; 3.60 (dd, J = 8.1, 4.4 Hz, 1H, CHO); 4.10-4. 30 (dAB, J = 16. 1,6. 2 Hz, 2H, NCH2) ; 4.76 (d, J = 5.9 Hz, 0.29H, CHO); 4.92 (d, J = 4.4 Hz, 0. 71 H, CHO); 6. 33 (app. s, 1H, H4); 6.40 (app. d, J = 8. 4 Hz, 1H, H2) ; 6.94 (d, J = 8. 4 Hz, 1H, HI) ; 7.16 (app. d, J = 5.5 Hz, 2H); 8. 22 (t, J = 5.8 Hz, 1 H, NH); 8.39 (dd, J = 4.7, 1.4 Hz, 2H) ; 8.92 (s, 1H, OH) ; MS (APCI+) calcd 406.2, obsd 407.3 (M+1) ; Purity: 96% SECTION 3 Scheme 1: Synthesis of common intermediate acids (5 and 7) trom Estrone Experimental General Methods, Unless otherwise noted, all reactions were conducted in oven-dried glassware using HPLC grade solvents. Purification of compounds was carried out using Argonaut pre-packed columns (Flash Column Chromatography) using flash vacuum system. Thin-layer chromatography (TLC) was performed on plates Kieselgel 60 F254 (Merck). 1H NMR was measured with a Jeof 270 MHz NMR spectrometer or 400MHz Bruker NMR spectrometer in deuterated-solvents as the internal reference. Low resolution mass spectral data were obtained either on a Micromass platform LCZ (ES +, APCI +) or FAB. High resolution mass was obtained using FAB technique Waters Alliance-HT-2790 system with a column Symmetry R C18 was used to determine the purity using HPLC spectra with 10% water in Methanol as eluent, RT values are quoted for these conditions.
2 estrone 2 (HDS01-082) : To a solution of 3-hydroxy estrone (10.00 g, 0.036 mol) in THF (180 ml) under nitrogen, was added sodium hydride (1. 056 g, 0.044 mol) portion wise and stirred at room temperature for 30 minutes. Benzyl bromide (7. 52 g, 0.044 mol) was added at r. t. and the reaction mixture was refluxed overnight. The completion of the reaction was monitored by tic (ethyl acetate: hexane, 3: 7). The crude mixture was cooled to r. t., quenched by addition of water (drop wise). The organic phase was extracted with DCM (2 x 100 mi), followed by ethyl acetate (2 x 100 mi) and the combined organic extracts were dried and concentrated in vacuo to obtain a colourless solid. The crude solid was dissolved in EtOH containing 5% EtOAc, left in a refrigerator to obtain the required product 2 (10. 711 g, 82%) as a crystalline colourless solid. Analytical data see JAC01- 002 3-Benzyloxy-16-methylene estrone carboxylic acid ethyle ster 3 (HDS01-086 and HDS-126) : To a solution of Lithium Diisopropyiamide (5.66 mi, 1.8M solution in heptanes, THF, and ethyl benzene) in THF (20 mi) was added a solution of estrone benzyl ether 2 (3.90 g, 10.8 mmol) in THF (30 ml) at 0 °C. The reaction mixture was stirred at 0 °C for approximately 15 minutes and cooled to-45 °C. A solution of ethyl bromoacetate (5.344 g, 32 mmol) in THF (10.0 ml) was added drop wise over 10 minutes and stirred at-45 °C for a period of 8 h and room temperature overnight. The reaction was monitored by tlc. The crude reaction mixture was quenched with saturated NH4CI, extracted the product into DCM (2 x 100 ml), dried and concentrated in vacuo to obtain a yellow oily product. The crude product was purified by Flash Chromatography (SiO2, ethyl acetate hexane gradient elution) to obtain the required product 3 (4.91 g, 70%, a mixture of diastereoisomers) as a pale yellow solid :'H NMR (CDCI3) 6 0. 928 and 1.002 (2 x s, 3H each, Me), 1.295 (t, 3H, CH3), 1.41-1. 61 (m, 6H), 1.77-1. 82 (m, 1H), 1.93-2. 03 (m, 3H), 2.26-2. 30 (broad m, 1H), 2. 38-2. 41 (dd, J = 8.2, 5.4 Hz, 1H), 2. 42-2. 55 (m, 1H), 2. 76- 2.81 (dAB, J = 16.0, 3.90 Hz, 1H), 2.88-2. 92 (broad m, 2H), 2.98-3. 53 (m, 1H), 4.17-4.19 (q, 2H, CH2), 5.04 (s, 1H, benzylic), 6.74-6. 75 (app d, 1H, aromatic), 6.79-6. 82 (2 x d, J = 2.7 Hz each, 1H, aromatic) 7.332-7. 456 (m, 6H); LCMS [(M+2) + = 448], (M+H2O) = 465; HPLC (99%, Rif= 6. 11) 3-Hydroxy-16-methylene estrone carboxylic acid ethyl ester 4 (STX 573, HDS01- 090) : A solution of 3-Benzyloxy-16-methylene carboxylic acid ethyl ester 3 (1.50 g, 3.36 mmol) was dissolved in EtOH (15 ml) and the mixture was degassed approximately 20 minutes and debenzylation was carried out under a hydrogen balloon at r. t. for 48 hours. The completion of the reaction was monitored using tic (EtOAc: hexane, 3: 7). The crude reaction mixture was filtered through a pad of celite, the filtrate was concentrated to obtain a sticky white solid which was then purified by column chromatography (5-20% ethyl acetate in hexane) to obtain the debenzylated product 4 (0. 828 g, 70%, mixture of 2 diastereomers 3: 1) as a white solid: 1H NMR (CDCl3) [chemical shifts of major to minor isomer ratio approximately 3: 1 was determined using Me signals at 13-position], # 0. 916 and 0. 988 (major isomer) [2 x s, 3H, 2 x Me], 1. 27-1. 29 (t, integrating to more than 3H, J = 7. 0 Hz, CH3), 1. 40-1. 60 (m, 6H), 1. 75-1. 80 (m, 1H), 1. 92-2. 23 (m, 6H), 2. 28-2. 35 (dAB, J = 16. 4 and 10. 1 Hz, 1H), 2. 36-2. 39 (m, 1H), 2. 47-2. 54 (dAB, J = 18.7, 8. 5 Hz, 1H, minor isomer), 2.74-2.79 (dd, J=3. 9,4. 2 Hz, 1 H), 2. 85-2. 88 (m, 2H), 2. 99-3. 02 (m, 1H), 4. 15-4. 17 (q, 2H, CH2), 4. 63 (1H, OH), 6. 57-6. 58 (appd, 1H, aromatic),'6. 62-6. 65 (dd, J = 8. 5, 2. 7 Hz, 1 H), 7.13-7. 16 (dd, J = 8. 5,3. 9 Hz, 1 H) ; Mass (FAB) (M+1)+= 357; Acc. Mass (FAB+) calcd 357.2065, obsd 357. 2054 ; HPLC (99%, R, = 2.01).
3-Hydroxy-17-hydroxy-16-methylene estrone carboxylic acid ethyl ester 6 (S7X 574, HDS01-094) : A solution of 3-Hydroxy-16-methylene carboxylic acid ethyl ester 4 (1.20 g, 3. 3 mmol) in MeOH (15 ml) was cooled to 0 °C and added NaBH4 (0.191 g, 5. 0 mmol) over 15 minutes. The reaction mixture was warmed to r. t. , stirred overnight. The reaction was monitored by tic (EtOAC: Hexane, 1: 1). The crude mixture was quenched with water and extracted the aqueous layer with Ethyl Acetate and DCM. The combined organics were washed with saturated NaCI, dried and concentrated to obtain a white solid and then recrystalised with DCM and Hexane to obtain pure compound 6 (0.791 g, 71%) as a white solid :'H NMR (CDCI3) [chemical shifts of major to minor isomer ratio approximately 3: 1 was determined using Me signals at 13-position] 8 0.784 and 0.852 (major isomer) [2 x s, 3H each, 2 x Me], 1.280 (t, J = 7.4 Hz, integrating to more than 3H, CH3), 1.30-1. 48 (m, 6H), 1.71-1. 88 (m, 3H), 1.96-2. 05 (1H, m), 2.24-2. 56 (m, 4H) 2.54-2. 56 (dd, J = 7.4, 2.3 Hz, approximately 1.5H), 2. 79-2.83 (m, 3H), 3.01-3. 02 (d, J = 3.9 Hz, 1H), 3.37-3. 40 (dd, J = 7.4, 3.5 Hz, 1H), 4.13-4. 18 (q, J = 7.0 Hz, 2H), 4.65-4. 70 (broad 2 x s, 1H), 6.55-6. 56 (appd, 1H, aromatic), 6.61-6. 64 (dd, J = 8. 5,2. 7Hz, 1H, aromatic) 7. 14-7. 16 (d, J = 8. 2 Hz, 1 H, aromatic) ; (FAB) (M+1)+= 359; Acc. Mass (FAB calcd 359. 2222, obsd 357. 2220 ; HPLC (99%, Rt= 1. 89).
3-Hydroxy-17-hydroxy-16-methylene estrone carboxylic acid 7 (STX 603, HDS01- 096): The Ethyl Ester 6 (0.780 g, 2.1 mmol) was dissolved in a mixture of THF: MeOH: H20 (1: 1: 0.5, 10 mi) in a seal tube and added NaOH (043 g, 10.5 mmol). The mixture was heated to 80 °C overnight. The reaction was monitored by tic (EtOAc : Hexane, 1 : 1). The crude mixture was acidified to pH = 3-4, extracted with EtOAc and DCM. The combined organics were dried (MgSO4) and concentrated to obtain a white solid. The solid was recrystalised with MeOH and hexane to obtain pure acid 7 (0. 700g, 101%) as a white solid : 1H NMR (CD3)2-SO) [chemical shifts of major to minor isomer ratio approximately 3: 1 was determined using Me signals at 13-position] S 0. 649 and 0. 688 (2 x s, 3H, 2 x CH3) 1. 148-1. 308 (m, 8H), 1. 47-1. 56 (q, J= 12. 4, 1H), 1. 90-2. 19 (m, 6H), 2. 65-2. 68 (m, approximately 3H), 3. 15-3. 16 (broad m, 1 H), 4673-4. 682 (broad d, 1 H) 6. 40-6. 41 (appd, 1 H, aromatic), 6.47-6. 49 (dd, J = 8. 2,2. 3 Hz, 1H, aromatic), 7.01-7. 03 (d, J = 8. 5 Hz, aromatic), 8.98 (s, 1H), 11.9 (broad s, 1H) ; (FAB) (M)+ = 330; Ace. Mass (FAB+) calcd 330. 1831, obsd 330.1832 ; HPLC (> 95%, Rt= 2.21).
3-Hydroxy-17-oxo-16-methylene estrone carboxylic acid 5 (S7X 604, HDS01-098) : The reaction was carried out using the procedure outlined for compound 7 with 3- Hydroxy-16-methylene carboxylic acid ethyl ester 4 (1. 00g, 2.8 mmol), NaOH (0.56 g, 1.4 mmol) and THF: MeOH: H20 (1: 1: 0.5, 10 ml). The product 5 (0.940 g, 102%) was isolated as a white solid after recrystalisation : 1H NMR (CD3) 2-SO) [chemical shifts of major to minor isomer ratio approximately 3: 1 was determined using Me signals at 13- position] 8 0.786 [major isomer] and 0.896 (2 x s, 3H each, 2 x Me), 1.31-1. 52 (broad m, 8H), 1.74-1. 77 (m, 1H), 1.88-1. 91 (m, 1.5H), 2.16-2. 22 (m, 3H), 2.27-2. 33 (m, 3H), 2. 48- 2.49 (2 x d, J = 195 Hz, 1H), 2.72-2. 74 (m, 2.5H), 6.43 6.44 (appd, 1H, aromatic), 6.48- 6.51 (d, J = 8.5, 2.4 Hz, aromatic), 7.02-7. 04 (d, J = 8.5 Hz, aromatic), 9.0 (broad s, 1 H) ; (FAB) (M+1) + = 329; Ace. Mass (FAB+) calcd 329.1752, obsd 330.1751 ; HPLC (> 95%, Rt= 2.60).
3-Hydroxy-17-oxo-16-methylene estrone carboxylic acid 8 (S7W 666, HDS01-109): The reaction was carried out using the procedure outlined for compound 7 with 3- benzyloxy-16-methylene carboxylic acid ethyl ester 3 (1. 714 g, 3. 8 mmol), NaOH (1. 537 g, 38. 4 mmol) and THF: MeOH : H2O (1 : 1: 0. 5,10 ml). The product 8 (1.114 g, 90%) was isolated as a white solid after recrystalisation : Diagnostic signals 1H NMR (CD3)2-SO) # 0.81, 0.84 (2 x s, CH3), 1.36-1. 91 (m, 6H), 2.19-2. 25 (m, 2H), 2.33-2. 37 (dd, J = 9.3 Hz, 2H), 2.59-2. 63 (2 x d, J =. 2 Hz each, 0.5 H), 2.81-2. 83 (m, 2H), 5. 06 (s, 2H, benzylic), 6.72 (appd, 1H, aromatic), 6.73-6.77 (dd, J = 8.5 and 5.8 Hz, 1H, aromatic), 7.17-7.19 (d, J = 8.5 Hz, 1H, aromatic), 7.31-7.44 (5H, aromatic); LCMS [(M)+ = 417]; (FAB) (M+1)+ = 418; Acc. Mass (FAB+) calcd 418.2099, obsd 418.2148; HPLC (99%, Rt = 1.89). Scheme 2 : Syiitliesis amides on a solid support Hellied-. 4 0 DIC, DMF, HOBt j roh2 OH'OH HO ROH2 0, (D 0 où N H HO 10.
Scheme 3: Parallel synthesis of amides in solution phase Method-B Synthesis of resin bound intermediate 9 (HDS01-104) : The Oxime resin (0.500 g, loading 1. 06mmol/g) was swollen in a minimum amount of DMF (7 ml), added the estrone carboxylic acid 5 (STX 604) [0.521 g, 5.3 mmol) followed by Di-isopropyl carbodiimide- [DIC] (0.667 g, 5.3 mmol) and Hydroxy benztriazole [HOBt] (0.715 g, 5.3 mmol). The reaction mixture was shaken using a flask shaker for approximately 72 hours under N2. The yellow reaction mixture obtained after 72 hours was filtered, and washed with DCM, DMF, MeOH (3 cycles with each solvent), finally MeOH (5 times) and dried in vacuo to obtain resin bound intermediate 8 as yellow beads: IR (cm-1) 1662 (C=O), 1739 (CO-O-N=), 3502 (OH).
Parallel synthesis of estrone-amides; Method A : The resin bound estrone intermediate 9 (0. 100 g, 0. 106 mmol) was suspended in anhydrous DCM (3.0 ml) under N2 in a glass tube in RadleysGreenHouse Synthesiser, added the corresponding amines (5 molar equivalents) and heated at 40 OC'under N2 for 72 h. The reaction mixtures were filtered and the filtrates were evaporated using Genevac to obtain a paie yellow solid. The crude compounds were purified using the Flash vacuum system and pre-packed columns (5 or 10 g) with DCM: MeOH (gradient elution, starting with 2% MeOH in DCM) to obtain pure compounds (average recovery 14 mg/100mg of resin bound intermediate).
Parallel synthesis of 16-methylene estrone-amides ; Method B : The carboxylic acid 5 (0.050 g, 0.15 mmol) or 7 (0.50 g, 0.15 mmol) was suspended in DCM (1.0 mi) in a glass tube in Radleys GreenHouse Parallel Synthesiser, added a solution of EDC (0.043 g, 0.227 mmol), DMAP (catalytic amount, 5 mg, and Et3N (0.100 mi) in DCM (1.0 ml). The reaction mixture was stirred approximately for 30 minutes and the corresponding amines (5 molar equivalents) in DCM (1.0 ml) were added and stirred overnight. [The total volume of DCM used in each reaction was 3.0 ml. The completion of the reactions were monitored by tic (EtOAc: Hexane, 2: 7). The crude mixtures were evaporated in the Genevac and purified using the Flash vacuum system and pre-packed columns (5 or 10 g) with DCM: MeOH (gradient elution, starting with 2% MeOH in DCM) to obtain pure compounds.
Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-furfuryl amide (S7X 672, HDSOi-ii2-3).
Method B, Yield = 33%. 1H NMR (CDCl3) # 0.908, 0.964, 1. 021 (3 x s, 3 x Me), 1.39- 1. 59 (broad m, 6H), 1. 94-1. 97 (m, 2H), 2. 23-2. 37 (m, 2H), 2. 82-2. 87 (m, 1. 5H) 4. 28-4. 44 (d, J = 5.4 Hz, CH2), 6.23-6.24 (m, 2H, aromatic), 6.57-6.65 (m, 2H, aromatic), 7.10-7.12 (m, 1H, aromatic), 6.34-6.35 (appd. 1H, aromatic), LCMS [(M+1)+ = 407] ; Ace. Mass (FAB+,M+1) calcd 408.3172, obsd 408. 2174 ; HPLC (90%, Rt = 1.47).
Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-tetrahydro-2-furyl amide (STX 671, HDS01-112-6) Method B, Yield = 40%. 1H NMR (CDCI3) 8 0.69, 0.73, 0. 83, 0. 87 and 0.87 (4 x s, approximately 3H each, Methyl signals), 1. 2-1. 55 (broad m, 7H), 1.74-1. 94 (broad m, 4H), 2.08-2. 29 (broad m, 2H), 2.29-2. 78 (m, 3H), 3.01-3. 07 (m, 1H), 3. 50-3. 92 (m, 2.5H), 6. 15-6. 57 (m, 3. 5H, aromatic), 6.79-7. 08 (m, 1.5H, aromatic); LCMS [(M+1) + = 411]; Acc.
Mass (FAB+ M+1) calcd 412. 2487, obsd 408.2491 ; HPLC (90%, Rt= 1. 49).
Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-3-pyridylamide (S77 (670, HDS01-112-2 and HDS01-168).
Method B, Yield = 50%, 1H NMR # (CD3)2-SO), 0. 778-0. 883 (4 x s, 3H), 1. 26-1. 55 (m, 6. 5H), 1. 61-1. 84 (m, 6H), 2. 04-2. 41 (m, 6H), 2. 29-2. 89 (m, 6H), 4. 13-4. 17 (d, J = 15. 6 Hz, 1H), 4. 27-4. 29 (d, J = 5. 8 Hz, 2H), 4. 35-4.37 (d, J = 7. 0 Hz, 0.5H), 4. 48-4. 52 (d, J = 15. 2, 1H), 5. 76 (s, 1H), 6. 39 (s, 1H), 6.04-6. 50 (m, 3H, aromatic), 6.9 (2 x d, J = 8. 2 Hz, 1 H), 7.0 (d, J = 8. 5 Hz, 0.5H), 7.28-7. 35 (m, 2H), 7.63-7. 65 and 7.73-7. 75 (2 x d, J = 8.2 Hz each), 8.35-8. 54 (m, 2H), 8.9 9.0 (2 x s, 2H); LCMS [(M+1) + = 419, (Rt = 8'58)] ; HPLC (100%, Rt= 2. 08).
A single diastereomer of STX 670 shows the following analytical data.
MP = 210-211 °C, 1H NMR # (CD3OD), 0.86-0.88 (s, 3H, Me), 1.28 - 1.57 (broad m, 5H), 1. 88-2. 15 (broad m, 2H), 2. 60-2. 82 (broad m, 4H), 4. 42 (s, 1H), 6.49 - 6.51 (appd, 1H, aromatic), 6. 54-6. 55 (d, J = 2. 47 Hz, aromatic),, 7. 05-7. 08 (d, J = 8.16 Hz, 1H, aromatic), 7. 39-7. 44 (dd, J = 7. 91 and 5. 19 Hz, 1H, aromatic), 7. 80-7. 83 (d, J = 7. 91 Hz, 1H, aromatic), 8.43 - 8.45 (appd, 1H, aromatic), 8.51 (s, 1H, aromatic) LCMS [(M+1)+ = 419, Rt = 6. 70] ; HPLC (100%, Rt= 2. 26).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-3-pyridylamide (S7X 669, HDS01-112-1).
Method B, Yield = 40%,'H NMR 6 (CDCI3), 0.778-0. 883 (4 x s, 3H), 1.26-1. 55 (m, 6.5H), 1.61-1. 84 (m, 6H), 2.04-2. 41 (m, 6H), 2.29-2. 89 (m, 6H), 4.13-4. 17 (d, J = 15.6 Hz, 1H), 4.27-4. 29 (d, J = 5.8 Hz, 2H), 4.35-4. 37 (d, J = 7.0 Hz, 0.5H), 4.48-4. 52 (d, J = 15.2, 1H), 5.76 (s, 1H), 6.39 (s, 1H), 6.04-6. 50 (m, 3H, aromatic), 6.9 (2 x d, J = 8.2 Hz, 1 H), 7.0 (d, J = 8. 5 Hz, 0.5H), 7. 28-7. 35 (m, 2H), 7.63-7. 65 and 7.73-7. 75 (2 x d, J = 8. 2 Hz each), 8. 35-8.54 (m, 2H), 8. 9 9. 0. (2 x s, 2H); LCMS [(M) + = 418]; Acc. Mass (FAB+, M+1) calcd 419.2334, obsd 419.2349 ; HPLC (100%, Rt = 1.83).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-N, N- dimethylpropyl amide (STX 688, HDS01-116-1).
Method A.'H NMR (CDCl3), 8 0.768 (Major isomer) and 0.90 (2 x s, 6H, 2 x Me), 1.2- 1. 61 (broad m,, 8H), 1.71-1. 99 (broad m, 5H), 2. 25 and 2. 93 (2 x s, 6H each, 2 x Me), 2. 30-2. 36 (m, 2H), 2. 39-2. 56 (m, 2H), 2. 56-2. 78 (broad m, 2H), 2. 89-2. 90 (d, J = 3.1 Hz, 1H) 2.93-2.94 (d, J = 3. 1 Hz, 1H), 3. 40-3. 67 (m, 4H), 6. 52 (appd, 1 aromatic), 6. 54- 6.56 (dd, J = 8.5, 2.7 Hz), 7. 04-7. 06 (d, J = 8. 2 Hz, 1H, aromatic) ; LCMS [(M)+ = 411 and (M + 1) + = 412] ; HPLC (98%, Rt= 1. 51). S of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-ethyl- methoxyamide (STX 689, HDS01-116-3).
Method A.'H NMR (CDCI3), 8 0.766, 0. 852, 0. 898 and 1. 182 (Me signals, approximately), 1. 2, 10- 1. 475 (broad m, 8H), 1.57-2. 30 (m, 13H), 2.50-2. 82 (m, 5H), 3.27-3. 39 (4 x s, OMe), 4. 85-4. 93 (broad s, 1 H), 5.93-6. 08 (2 x s, 2H), 6. 50-6. 55 (m, 1 H, aromatic), 6.55-6. 57 (m, 1H, aromatic), 7. 05-7.07 (m, 1H, aromatic); LCMS [ (M + 1)+ = 386]; HPLC (99%, Rt= 1. 47).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-ethyl- methoxyamide (HDS01-100-2).
Method B. H NMR (CDCis), 8 0. 768 (Major isomer) and 0.90 (2 x s, 6H, 2 x Me), 1. 2- 1. 61 (broad m, 8H), 1. 71-1. 99 (broad m, 5H), 2. 25 and 2. 93 (2 x s, 6H), 2. 30-2. 36 (m, 2H), 2.39-2. 56 (m, 2H), 2.56-2. 78 (broad m, 2H), 2. 89-2. 90 (d, J = 3. 1 Hz, 2. 93-2. 94 (d, J = 3. 1 Hz, 1H), 3.40-3. 67 (m, 4H), 6. 5 (appd, 1H, aromatic), 6. 54-6. 56 (dd, J = 8. 5, 2. 7 Hz), 7.04-7. 06 (d, J = 8.2 Hz, 1H, aromatic); FAB [ (M + 1)+ = 399; Acc. Mass (FAB+, M+1) calcd 399.2647, obsd 399.2627 ; HPLC (99%, Rt= 1.51). Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-#-methyl benzyl amide (STX 690, HDS01-116-4). Method A. This compound exists as a mixture of 4 diastereomers. Diagnostic signals of H NMR (CDCtg), 8 0.69, 0.73, 0. 88, 0.89 (4 x s, Methyl signals), 1.41-1. 44 tm, benzylic methyl), 2.76-2. 78 (m, 2H), 5.01-5. 06 (m, 1H, Benzyic), 6.50 (appd, 1H, aromatic), 6.55- 6.57 (broad d, 2H, aromatic), 7.03-7. 06 (m, 1H, aromatic), 7. 17-7.29 (m, aromatic); LCMS [ (M + 1) + = 432]; HPLC (99%, Rt = 1.51).
Synthesis of 3-Hydroxy-17-oxo-16-methylene estrone carboxy-2-ethyl pyridylamide (STX 691, HDS01-116-5).
Method A. Diagnostic signals of'H NMR (CDCl3), ô 0.75, 0.80 and 0.92 and 1.0 (3 x Me signals), 1.20-2. 84 (broad m, approximately 14 H), 3.0-3. 03 (m, 2H), 3.36-3. 62 (m, 0.5H), 3.64-3. 69 (m, 2H), 6. 57-6.59 (m, 1H, aromatic), 6.63-6. 64 and 6.65-6. 66 (d, J = 2.7 Hz each, 1H, aromatic), 6. 72-6.75 (m, 0.5H), 7.09-7. 30 (m, 4.5H, aromatic), 7. 64- 7. 68 (m, 2H, aromatic), 8. 40-8. 42 (m, 0.5H, aromatic), 8. 52-8. 53 (m, 1H, aromatic); LCMS [(M + 1)+ = 433 and (M - OH)+ = 415]; FAB [(M + 1)+ = 433; HPLC (99%, Rt = 1.51).
Synthesis of 3-Hydroxy-17-hydroxy-16-methylene estrone carboxy-tetrahydro-2- furyl amide (S77f 668, HDS01u110-6) Method 13, Yield = 33%. 1H NMR (CDCl3) # 0. 78 (2 x s, 3H, 2 x Me), 0. 86-0. 89 (m, 1H), 1. 19-1. 55 (broad m, 7H), 1. 69-1. 96 (m, 2H), 2. 10-2. 32 (broad m, 3H), 2. 74-2. 84 (m, 2H), 3. 73-3. 74 (m, 1H), 3.73-3. 74 (d, J = 5. 8 Hz, 1H), 4. 46-4. 48 (broad m, 1H), 6. 57 (appd, 1 aromatic), 6. 62-6. 65 (m, 1H, aromatic), 7.13-7.17 (m, 1H) LCMS [ (M- C5H9NO)+ = 313] ; HPLC (95%, Rt = 1.57).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-furfuryl amide (S7X667, HDS01-110-3).
Method B, Yield = 37%. IH NMR (CDCl3) 5 0.076, 0.77, 0.79, (3 x s, approximately 3H each, Methyl signals), 1.25-1. 58 (broad m, 14H), 1.86-1. 95 (m, 1H), 2.02-2. 42 (m, 3.5H), 2.47-2. 49 (m, 0.5H), 2. 78-2. 87 (3H, m), 3.41-3. 43 (dd, J = 2.3 and 1.9Hz, 1H), 4.44-4. 45 (d, J = 5.4 Hz, 1 H), 6. 01 (broad s, 1 H), 6.24-6. 25 (d, J = 2.3 Hz, 1 H, aromatic), 6.33-6. 64 (2 sets of multiplets, 4H, aromatic), 7.14-7. 16 (d, J = 8.5 Hz, 1.5H, aromatic), 7.28-7. 35 (m, 1H, aromatic), 7.36-7. 37 (m, 1H, aromatic) ; LCMS [(M-1) + = 408 (Rt = 8.58)] ; HPLC (100%, Rt= 2.08) ; Acc. Mass (FAB+ M+1) calcd 410. 2331, obsd 410.2317 ; HPLC (90%, Rt= 1.82 and 1.90). Scheme 4 : Parallel synthesis of amides on soild supportl#ethod support Afethod-C 0 0 r DIC, DMF, HOBt ,, N02 A 41 11 R Hn/13 Synthesis of resin bound intermediate 12 (HDS01-120) : The Oxime resin (2.0 g, loading 1. 06mmol/g) was swollen in a minimum amount of DMF (7 ml), added the estrone carboxylic'acid 8 (1.428 g, 3.18 mmol) followed by Di-isopropyl carbodiimide [DIC] (1.33 g, 10.6 mmol) and Hydroxy benztriazole [HOBt] (1.431 g, 10.6 mmol). The reaction mixture was shaken using a flask shaker for approximately 72 hours under N2. The yellow reaction mixture obtained after 72 hours was filtered, and washed with DCM, DMF, MeOH (3 cycles with each solvent), finally MeOH (5 times) and dried in vacuo to obtain resin bound intermediate 8 (2.391 g) as yellow beads : IR (cm-') 1662 (C=O), 1739 (CO-O-N=), 3502 (OH).
Parallel synthesis of estrone-amides ; Method C : The resin bound estrone intermediate 12 (Scheme 4) (0.100 g, 0.106 mmol) was suspended in anhydrous DCM (3.0 ml) under N2 in a glass tube in Radleys GreenHouse Synthesiser, added the corresponding amines (5 molar equivalents) and heated at 40 °C under N2 for 72 h. The reaction mixtures were filtered and the filtrates were evaporated using Genevac to obtain a pale yellow solid. The crude compounds were purified using the Flash vacuum system and pre-packed columns (5 or 10 g) with DCM : MeOH (gradient elution, starting with 2% MeOH in DCM) to obtain pure benzylated intermediates (average recovery 15 mg/100 mg of resin bound intermediate, analysed by'H nmr). These intermediates were then debenzylated under a balloon of H2 in the GreenHouse Synthesiser to obtain final compounds in average 90% purity. Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-methyl-N- ethyl amide (STX 741, HDS01-128-1).
Method C, Yield = 40%. 1H NMR (CDCl3) # 0. 81 and 0.9 0 (2 x s, 3H, 2 x CH3, 3: 1), 1. 01-1. 24 (m, 5H), 1. 66- 2. 60 (m, 6H), 2. 75-80 (m, 2. 5H), 2. 86-2. 90 (2 x s, 4.5H, 2 x CH3), 3.25-3. 28 (m, 1 H), 4.79 (broad s, 1 H), 6.50 (app d, 1H, aromatic), 6.51-6. 56 (2 x d, J = 2.9 and 2.5 Hz, 1H, aromatic), 7.05-7. 08 (d, J = 8.4 Hz, 1H, aromatic); LCMS [(M+2)+ = 371, (R, = 6.08)] ; HPLC (85%, Rt = 2.29).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N, N-diethyl amide (STX 742, HDS01-128-2). Method C, Yield = 40%.'H NMR (CDCI3) 8 0.78 (major isomer) and 0. 89 (2 x s, 2 x CH3), 1.03-1. 13 (m, approximately, 6H), 1.39-1. 59 (m, 5H), 1. 89-1. 94 (m, 1.5H) 2.13-2. 91 (m, 5H), 3.20-3. 36 (m, 4H), 5.05-5. 27 (broad s, 1H), 6.51-6. 52 (appd, 1H, aromatic), 6.55-6. 59 (dd, J = 2.9 and 2.5 Hz, 1H, aromatic), 7.05-7. 08 (d, J = 8. 8 Hz, 1 H, aromatic); LCMS [(M+2)+ = 385, (Rt = 6.55)] ; HPLC (86%, Rt = 2.37).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-isopropyl amide (STX 743, HDS01-128-4). Method C, Yield = 40%. 1H NMR (CDCl3) # 0.83 [Major isomers and 0.95 (2 x s, CH3 signals, 3: 1), 1. 12 (d, J = 1. 4Hz, CH3), 1. 14-1. 15 (d, J = 2. 20 Hz, CH3), 1. 90-2. 64 (m, 2H), 2. 80-2. 85 (m, 2H), 4. 91 (broad s, 1H), 6. 56 (app d, 1H, aromatic), 6. 59-6. 60 and 6. 62-6. 63 (dd, J = 2. 9 and 2. 5 Hz, 1H, aromatic), 7.10-7.13 (d, J = 8. 0 Hz, 1H, aromatic) ; LCMS [(M+2)+ = 371, (Rt = 5. 83) 1 ; HPLC (93%, Rt = 2.27).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-ethyl amide (STX 744, HDS01-128-5).
Method C, Yield = 40%.'H NMR (CDCl3) # 0.78 [Major isomer] and 0.81 (2 x s, CH3 signals, 3: 1), 1.05-1. 10 (t, J = 6.9 Hz, CH3), 1.35-. 45 (m, approximately 4H), 1.89-2. 64 (m, 11H), 2.79-2. 80 (m, 2H), 3. 18-3. 24 (m, 2H, CH2), 4.83 (broad s, 1H), 6.51 (appd, 1H, aromatic), 6.54-6. 55 and 6.57-6. 59 (dd, J = 2.5 and 2.9 Hz, 1H, aromatic), 7. 05-7. 08 (d, J = 8. 4Hz, 1H, aromatic); LCMS [(M+2)+ = 357, [ (M+Na)+ = 379]; HPLC (87%, Rt= 2.17).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-methyl amide (STX 745, HDS01-128-6).
Method C, Yield = 40%.'H NMR (CDOs) 8 0.77 [major isomer], 0. 85, 0.90 (3 x s, CH3 signals), 1. 43-1. 52 (m, 5H), 1. 89-1. 93 (m, 2H), 2. 15-2. 31 (m, 3H), 2. 49-2. 68 (m, 2H), 2. 72 and 2.74 (2 x s, 3H, Me), 2. 75-2. 80 (m, 2H), 4. 94-4.95 (broad s, 1H), 5. 91 (broad s, 1H) 6. 52 (app d, 1H, aromatic), 6.55-6.56 and 6. 58-6. 59 (dd, J = 2. 9 Hz each, 1H, aromatic), 7.04-7. 08 (d, J = 8.4 Hz, 1H, aromatic), LCMS [(M-30)+ = 311]; HPLC (97%, Rt = 2. 12). Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-methyl amide (STX 746, HDS01-128-9).
Method C, Yield = 40%. 1H NMR (CDCl3) # 0. 88 (Major isomer) and 0. 93 (s, CH3), 1. 4- 1.66 (m, 6H), 1.97-2. 00 (m, 2H), 2.39-3. 15 (m, 6H), 4.94 (broad s, 1 H), 6.51-6. 53 (d, J = 3.6 Hz, 1 H, aromatic) 6.70 (app d, 1 H, aromatic), 6.71


-6. 74 and 6.77-6. 78 (2 x d, J = 2.5 Hz each, 1H, aromatic), 7.04-7. 08 (d, J = 8.4 Hz, 1H, aromatic), LCMS [(M)+ = 412] ; HPLC (77%, Rt = 2.16).
Synthesis of 3-hydroxy-1 7-hydroxy-1 6-methylene estrone carboxy-N-methyl-N- . ethyl amide (S7X 771, HDS01-138-1) Method C, Yield = 40%. 1H NMR (CDCl3) # 0.78 (Major isomer) 0. 85 (s, 2 x CH3, 2: 1), 1.08-1. 19 (m, 5H), 1.79-2. 66 (m, 9.5H), 2.77-2. 80 (m, 4H), 2.90 and 2.96 (2 x s, 4H approximately, CH3), 3.27-3. 41 (m, 4H), 3.81-3. 94 (m, 1.5H), 4.46 (s, 1.5H), 6.54 (appd, 1H, aromatic), 6. 58-6. 62 (dd, J = 8.4 and 2.5Hz, 7.12-7. 15 (d, J = 8.4 Hz, aromatic); LCMS [(M - 29)+ = 341]; HPLC (92%, Rt= 2. 32).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-N-diethyl amide (STX 772, HDS01-138-2).
Method C, Yield = 40%. 1H NMR (CDCl3) # 0.74 and 0. 80 (2 x s, 2 x CH3, 2 : 1), 1. 02- 1.15 (m, 6H), 1. 70-2. 65 (m, approximately 16H), 3. 22-3. 32 (m, 4H), 4. 05 (appd, 1H), 4. 69 and 4. 99 (2 x s, 1H), 6. 48 8 (appd, 1H, aromatic), 6.49-6. 56 (dd, J = 8. 4 and 5. 8 Hz, 1H, aromatic), 7.07-7. 10 (d, J = 8. 0 Hz, 1H, aromatic) ; LCMS [ (M + 2)+ = 387] ; HPLC (99%, Rt= 2. 48).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-N-diethyl amide (STX 773, HDS01-138-4).
Method C, Yield = 40%.'H NMR (CDCl3) # 0.77 and 0.83 (2 x s, 2 x CH3, 2: 1), 1. 16-1. 15 (2 x d, J = 2.5 Hz each, isopropyl), 1.82-2. 80 (m, approximately 14 H), 3.82-3. 85 (2 x appd, 2H), 4.02-4. 14 (m, 4H), 4.27-4. 28 (appd, 1H), 4.59 (s, 2H), 5.3-5. 4 (broad s, 2H), 6.53 (appd, 1H, aromatic), 6.54-6. (62 (dd, J = 11.0 and 2.9Hz, 1H, aromatic), 7.12-7. 15 (d, J = 8.4 Hz, 1 H, aromatic); LCMS [ (M + 2) + = 373]; HPLC (87%, Rt= 2.31).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-N-diethyl amide (S7X774, HDS01-138-5). Method C, Yield = 40%.'H NMR (CDCl3) 6 0.73 and 0.78 (2 x s, 2 x CH3, 2: 1), 1.05-1. 10 (t, J = 14.0, 6.9 Hz, CH3), 1. 78-2. 77 (approximately 16 H), 3.17-3. 27 (m, 4H), 3.77-3. 81 (d, J = 9.5 Hz 1 H), 5.55 (broad s, 1 H), 6.48 (appd, 1 H, aromatic), 6.49-6. 56 (dd, J = 8.4 and 5.5 Hz, 1H, aromatic), 7.10-7. 07 (d, J = 8. 8 Hz, 1H, aromatic) ; LCMS [ (M + 2) = 359] ; HPLC (90%, Rt= 2.10).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-N-diethyl amide (STX 775, HDS01-138-6) Method C, Yield = 40%. 'H NMR (CDC) s) 8 0.73 (s, 3H, CH3), 1.78-2. 77 (approximately 16 H), 2.75-2. 77 (2 x s, 2 x CH3), 3.77-3. 82 (d, J = 9.5 Hz, 1 H), 4.04-4. 06 (broad s, 1), 5.56 (broad s, 1H), 6.48 (appd, 1H, aromatic), 6.49-6. 56 (dd, J = 8. 0 and 5. 5 Hz, 1H, aromatic), 7.07-7. 10 (d, J = 8.4 Hz, 1H, aromatic), LCMS [(M - CH3)+ = 327]; HPLC (90%, Rt = 2. 10).
Synthesis of 3-hydroxy-17-hydroxy-16-methylene estrone carboxy-N-propylamide (S7X776, HDS01-138-8). Method C, Yield = 40%. 1H NMR (CDCI3) 8 0.91 (s, 3H, CH3), 0.84-0. 96 (m, 3H), 1.23- 1.26 (t, 2H), 1. 48-1. 51 (m, 8H), 2.13-2. 33 (m, 4H), 2.85-2. 88 (m, 2H), 3.16-3. 23 (m, 2H), 4.98 (s, CH2), 5. 27-5.43 (broad s, 1H), 5.97 (broad s, 1H), 6.71 (appd, 1H, aromatic), 6.77-6. 78 (dd, J = 8.0 and 5.5 Hz, 1 H, aromatic), 7.18-7. 21 (d, J = 8. 4 Hz, 1H, aromatic) 7.29-7. 41 (m, 5H, aromatic) ; HPLC (90%, Rt = 2.10).
Scheme 1 : Synthenc route to sultamate and phenol libraries using a solid support 0 'i i HO BnO'C,, 1 t d "ho, HO, f ; N fX vNSlO AX f g i. v CI. O zizi p p'OH lOB i-o 5, 1 9 6 11 0 .. OJJJ O." p 5. o, o OH k//.'"R H2N 0 H 11-7 O là 10A 0 0 il i YTj' TIC ho H2N-s 0 HO 9 11 8 Iw iw 0 HO/9 O Sulfamate Library Phenol Library Reagents are: a) Benzyl bromide, K2CO3, DMF, r. t. b) LDA, THF, Ethyl bromoacetate,-78 °C-r. t. c) H2, Pd/C, MeOH : THF (1: 1) d) Sulfamoyl chloride, DMA, 0 °C to r. t. e) Trityl-Cl resin, DIPEA, DCM, 72h. f) NaOH, THF : H20 (1: 1), r.t. g) EDCI, HOBt, PyBop, DIPEA, DCM, R-NH2, r. t., O/N. h) TFA: DCM (50: 50). i) Piperazine, THF. j) 50% TFA in DCM 20 min. k) Piperazine, THF, 70 °C, 72 h.
Synthesis of 2-ethyl benzyl estrone (HDS02-054) To a stirred solution of 2-ethyl estrone 1 (10.00 g, 33.5 mmol) in anhydrous DMF (100 ml) under inert atmosphere was added potassium carbonate (14. 0 g, 100 mmol) followed by Benzyl bromide (4. 78 ml, 40 0 mmol). The reaction was left to stir at r. t. over 2 days. The solid obtained was filtered using a Buchner funnel, dissolved in DCM, filtered to remove any inorganic and concentrated to obtain a white solid. The white solid obtained was re-crystallised with DCM : MeOH (90: 10) to obtain the pure product the benzyl ether estrone derivative 2 (12.00 g, 92%) also as a white solid :'H NMR (270 MHz, CDCI3) 5 0.90 (s, 3H, 18-CH3), 1.17-1. 23 (t, J = 7.4 Hz, CH2-CH3), 1.51-2. 29 (m, 14H), 2. 45-2. 55 (m, 4H, 16-CH2), 2. 62-2. 70 (q, J = 7.6 Hz, 2H, CH3-CH2), 2. 84-2. 90 (m, 2H, 6-CH2), 5. 04 (s, 2H, CH2-phenyl) 6. 64 (s, 4-H), 7. 10 (s, 1-CH), 7. 28-7. 45 (m, 5H) ; FAB-HRMS calcd for C27H3202 388. 2357 found (M+) 388. 23$9.
Synthesis of benzyl ethyl ester (HDS02-098, HDS02-108) 2 To a stirred solution of 2-Ethylbenzyl estrone (5. 00 g, 12. 8 mmol) in anhydrous THF under inert conditions was added a solution of LDA (4. 07 ml, 1. 8 M solution in heptane, THF, and Ethylbenzene) at-10 °C over a period of 20 min. The reaction mixture was cooled to-60 °C, stirred 15 min, added ethyl bromoacetate drop wise over 10 min. and allowed to warm to rt overnight. Diluted the mixture with DCM, added saturated NH4CI and extracted with DCM. The organic phases were combined, dried, and concentrated to obtain a yellow solid which was then purified using Flash Master-11 (50 g column, DCM: MeOH, gradient elution) to obtain the pure product (6. 101g, 99% yield) as a pale yellow solid :'H NMR (270 MHz, CDCI3) 6 0.869 and 0.978 (2 x s, 3H, 18-CH3), 1.17- 1. 23 (t, J = 7.6 Hz, 3H, CH2-CH3), 1.24-1. 29 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.32-2. 59 (broad m, 12H), 2. 62-2. 70 (q, J = 7.4 Hz, 2H, 2-CH3-CH2), 2.78-3. 03 (broad m, 2H, 6- CH2), 4.11-4. 19 (q, J = 7.17 Hz, 2H, CH3-CH2-O), 5.0 (s, 2H, CH2-phenyl), 6.63 (s, 4- CH), 7.09 (s, 1-CH), 7.30-7. 45 (m, 5H); FAB-MS (M+) 474 m/z.
Synthesis of 3-Hydroxy estrone derivative (HDS02-100, HDS02-110) 3 A solution of 2 (2. 80g, 5.9 mmol) in THF: MeOH (1: 1,40 ml) was degassed for 20 min. bubbling nitrogen gas. Added 5% Pd on C (10% Wt, 300 mg) and debenzylation was carried out using a balloon of H2 for 24 h. The reaction was monitored using TLC (EtOAC: Hexane, 90: 10). The crude mixture was filtered using a pad of celite, concentrated to obtain the debenzylated product 3 (2.210 g, 97%) as a colourless solid : 'H NMR (270 MHz, CDCl3) 6 0.85 and 0.96 (2 x s, 3H, 18-CH3), 1.17-1. 28 (2 x t overlapped, 6H, 2 x CH2-CH3), 1. 49-2. 49 (m, 12H), 2.54-2. 62 (q, J = 7.4 Hz, 2H, 2-CH3- CH2), 2. 72-3. 02 (m, 4H), 4. 11-4. 16 (q, J = 7. 1 Hz, 2H, CH3-CH2-O), 4. 54 (s, 1H, CH2- Phenyl), 6. 50 (s, 1H, 4-CH), 7. 03 (s, 1H, 1-CH) ; HPLC > 97% (Rt = 6. 53, 70% MeCN/water) ; ES-ve-MS (M-H+) 383 m/z; (FAB-HRMS calcd for C24H32O4 384.2255 found (Md) 384. 2288 m/z.
Synthesis of sulfamate estrone derivative (HDS02-102) 4 A solution of sulfamoyl chloride (57 ml, 0.68M solution in toluène) was evaporated bestow 30 °C using a rotary evaporator. The brown oil obtained was cooled to 0 °C under inert atmosphere, dissolved in anhydrous DMA (30 mi) and treated dropwise with 3 in anhydrous DMA (30 ml) using a cannula. The reaction mixture was left to stir at 0 °C, warmed to r. t. overnight. TLC indicated the completion of the reaction (DCM : MeOH, 98 : 2). The crude mixture was diluted with EtOAG, washed with waSr (5 X) and brine.
The organic phase was dried and concentrated to obtain a pale yellow solid. The crude product was purified using Flash Master-11 (50 g column, DCM : MeOH, gradient elution) to obtain pure product 4 (5.901 g, 98%) as a colourless foamy solid :'H NMR (270 MHz, DMSO-d6) 6 0.81 and 0.92 (2 x s, 3H each, 18-CH3), 1. 09-1.20 (2 x t, J = 4.9 and 7.4 Hz, 6H, 2 x CH2-CH3), 1.33-2. 47 (m, 12H), 2.58-2. 66 (q, J = 7. 6 Hz, 2H, 2-CH2-CH3), 2.89- 2.97 (m, 3H), 4.04-4. 09 (q, J = 6.9 Hz, 2H, 2-CH3-CH2) 7.02 (s, 1H, 4-CH), 7.21 (s, 1 H, 1-CH), 7.95 (s, 2H, SO2NH2) ; HPLC > 90% (Rt = 6.53, 70% MeCN/water); ES-ve-MS (M- H+) 462 m/z.
Synthesis of resin bound estrone sulfamate derivative (HDS02-114) 5 The trityl chloride resin (2.60 g, Novabiochem, loading 1.1 mmol/g) was swollen in anhydrous DCM (25 ml) under inert atmosphere. Added estrone sulfamate (2.00 g, 4. 31 mmol)'followed by DIPEA (7.50 ml, 43.1 mmol). The mixture was shaken for approximately 75 h. The acidic minicleavage (50% TFA/DCM, 20 min. ) of a sample from the reaction confirmed the completion of the reaction. The crude mixture was filtered, washed with DMF, DCM, MeOH (5 cycles each) and finally with MeOH (3 cycles). After drying under vacuum this resin bound intermediate 4 (3.78 g, loading 0.86 mmol/g, pale yellow) was stored in a refrigerator until taken to the next stage. The'H NMR and other analysis of the cleaved compound was same as that is reported for 4.
Synthesis of resin bound library precursor estrone sulfamate (HDS02-112) 6 The resin 5 (6.00 g, loading 0. 86 mmol/g) was swollen in THF (60 ml), and aqueous solution of NaOH (4M, 7 ml) was added slowely. The resin was shaken at r. t. for approximately 30 h, then filtered and washed with THF/H20 (1: 1, 3 x 50 ml), THF, DCM and MeOH (5 cycles each). Dried under vacuum to a constant weight to obtain resin bound acid 6 (5. 25 g, 0.86 mmol/g loading) as pale yellow beads. Acidic minicleavage (50% TFA/DCM, 20 min. ) of a sample of this resin confirmed the complete hydrolysis of the ester to the acid. This compound exist as a mixture of diastereomers 1H NMR of this compound is same as that reported for compound 10A.
Synthesis of acid acid 10 A (KRB01-137) Resin 6 (200 mg, 0. 154 mmol) was rinsed with a 50 : 50 solution of TFSCH2CI2 (10 mL, over a period of 20 min.) and the filtrate was evaporated under reduced pressure. The residue was recrystallized from hot methanol to yield 0A (50 mg, 74%) as a white solid: Mixture of Diastereomers 1H NMR (270MHz, DMSO-d6) 6 0.81 (3H, s, 18-CH3), 1.13-1. 15 (t, J = 7.5 Hz, 2-CH2CH3), 1.45-2. 50 (m, 13H), 2.57-2. 64- (2H, q, J = 7.5 Hz, 2- CH2CH3), 2.66-2. 83 (m, 2H), 7.22 (s, 1H), 7.03 (s, 1H), 7.96 (s, 2H, NH2) ; HPLC > 94% (Rt = 1.89, 10% water/acetonitrile) ; ES-ve-MS (M-H+) 434 m/z.
Synthesis of Acid (KRB01-139) Nucleophilic Cleavage to form 10B : To resin 6 (300 mg, 0.231 mmol) was added piperazine (199 mg, 2.31 mmol) and anhydrous THF (5 mL) in a round bottom flask. The mixture was heated under N2 to 65 °C for 48 h (without stirring), after which time the mixture was filtered and the resin was rinsed with MeOH and CH2CI2 (5x). The combined washings were evaporated under reduced pressure and purified by flash chromatography on silica gel (eluted with 5%#15% methanol in CH2Cl2, piperazine sticks to baseline) to afford of 10B (6, 0 mg, 73%) as a white solid :'H NMR signals (270MHz, CDCl3) 6 0.98 and 0. 87 (s, 18-CH3, two diastereomers in approx. 1: 3 ratio), 1.21 (3H, t, J = 7.5 Hz, 2-CH2CH3), 1.20-1. 38 (t, J = 7.5 Hz, 3H, 2-CH2CH3), 1.45-2. 50 (m, 13H), 2.54-2. 64 (q, J = 7.5 Hz, 2H, 2-CH2CH3), 2.79-2. 82 (m, 2H), 2.96-2. 98 (1 H, dd, J= 16.3, 4.2 Hz, 16-H), 6.51 (s, 1H), 7.03 (s, 1H) ; HPLC >99% (10% water/acetonitrile) ; ES-ve-MS (M-H+) 356 m/z.
General procedure for synthesis of suifamate amide library A stock solution of PyBOP (Benzotriazole-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate) was prepared in 14 ml of DCM and 2. 0 ml used for each reaction.
A stock solution of HOBt ((N-hydroxy benzotriazole, 0.185 g/reaction) and DIPEA (0.297 ml/reaction) prepared in 9.0 ml of DCM and 1. 0 mol used for each reaction. The resin bound acid 6 (0.400 g, loading 0.86 mmol/g) was suspended in DCM (4.0 mi) under inert atmosphere in a 25 ml round bottom flask, added the activating agent PyBOP (0. 715 g, 1. 37 mmol,), followed by HOBt and DIPEA mixture (HOBt 0. 185 g, 1.37 mmol, DIPEA 0.297 ml), and the amines (4 molar equivalents). The mixture was shaken for approximately 72 h using a flask shaker. The crude resins were then filtered, washed with DMF (5 x), DCM (5 x), MeOH (5 x) using Argonaut Flash Vacuum Manifold and fritted syringes. The washed resins were dried under vacuum, one half of each resin was then transferred to Radleys Green House Synthesiser tubes to generate the phenol library. The remaining half was treated with 50% TFA in DCM over 20 min., TFA was evaporated using the Genevac and the products were purified using Flash Master-11 (DCM/MeOH, gradient elution, 5 or 10 g pre-packed columns) to generate the sulfamate library. The compounds generated exist as a mixture of diastereomers.
General procedure for synthesis of phenol library.
The resin bound sulfamate amides (0.200 g, 0. 86 mmol/g) were suspended in anhydrous THF (4 ml) under inert atmosphere (Radleys Green House Parallel Synthesiser was used to carry out these reactions), treated with piperazine (0.75 g, 8.7 mmol) and refluxed at 70 °C for approximately 70 h. The resins were filtered, the filtrates were collected in to tubes, concentrated using the Genevac. These compounds were pre-absorbed onto silica gel, purified using the Flash Master-11 (Ethyl acetate/Hexane gradient elution) to give pure products as colourless solids. These compounds exist as a mixture of diastereomers. Compound R1 R2 HPLC Purity (%) / 12 S02NH2 j > 91 12 SO2NH2 s 91 Compound R'R2 HPLC Purity (%) 13 S02NH2 aNm > 99 oil IS S02MH2 > 91 ia S02MH2 17 SO2NH2 tY 90 Lj 17 SOzNHz"") 18 S02NH2 H > 89 19 SOzNHs-N >95 20 S02NH2 21 S02NH2 > 97 22 (STX 1040) OH HX > 99 23 OH > 88 24 OH > 95 25 OH > 99 26 OH > 99 _. _. I 28 OH"-"N--. >99 29 OH H > 99 Compound R1 R2 HPLC Purity (%) 30 OH-,-N-,, :, 90 30 OH-N >90 - 31 OH-vv HX- Compound 11 (STX 1039) HDS02-088 (TFA Salt): 1H NMR (270 MHz, DMSO-d6 and 0. 56 (2 x s, 3H each, 18-CH3), 0. 75-0. 79 (t, J = 7. 3 Hz, 3H, CH2-CH3), 1. 33-2. 47 (m, 12H), 2.26-2. 29 (q, J = 7. 3 Hz, 2H, CH2-CH3), 2. 42-2. 59 (m, 3H), 3. 98-4. 00 (m, 2H, 2- CH3-CH2), 6.65 (s, 1H, 4-CH), 6. 84 (s, 1H, 1-CH), 7.20 (broad s, 1H) 7.52 (broad m, 1H), 7.59 (s, 2H, NH2, disappeared on D20 shake), 8.21-8. 17 (m, 3H, aromatic) ; HPLC > 95 (Rt = 12.13, 5% MeCN/water to 95% MeCN/water); ES+ve-MS (M+H+) 526 m/z ; (FAB-HRMS calcd for C28H35N3O5S 526.2375 found (M+H+) 526.2379.
Compound 12 HDS02-128-8 (TFA Salt): 1H NMR (270 MHz, DMSO-d6) 0.81 (s, 3H, 18- CH3), 1. 09-1.15 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.33-2. 47 (m, 12H), 2.61-2. 64 (q, J = 7. 3 Hz, 2H, CH2-CH3), 2.82-2. 90 (m, 3H), 4.36-4. 38 (m, 2H, 2-CH3-CH2), 7.02 (s, 1 H, 4-CH), 7.21 (s, 1H, 1-CH), 7. 28-7. 30 (m, 2H, aromatic) 7.52 (broad m, 1H), 7.96 (s, 2H, NH2), 8. 48-8.51 (m, 2H, aromatic); HPLC > 91 % (Rt = 2.12, 70% MeCN/water); ES+ve-MS (M+H+) 526 m/z; (FAB-HRMS calcd for C28H35N305S 526.2375 found (M+H+) 526.2374 Compound 13 HDS02124-2 (TFA Salt) :'H NMR (400 MHz, DMSO-d6) 0.805 (s, 3H, 18- CH3), 1.10-1. 13 (t, J = 7.4 Hz, 3H, CH2-CH3), 1.37-2. 45 (m, 12H), 2. 58-2. 62 (q, J = 7.8 Hz, 2H, CH2-CH3), 2.80-2. 87 (m, 3H), 4.34-4. 36 (m, 2H, CH3-CH2), 6.99 (s, 1H, 4-CH), 7. 18 (s, 1H, 1-CH), 1H), 7.92 (s, 2H, NH2), 8.43 (2 x s, 2H, aromatic) 8. 51-8. 84 (t, J = 5.85 Hz, 1H, NH); ES+ve-MS (M+H+) 541, (M+H-18+) 523 m/z ; HPLC > 99% (Rt = 2.0, 80% MeCN/water); FAB-HRMS calcd for C28H3sN405S 541.2484 found (M+H+) 541.2478 Compound 15 HDS02128-2 (TFA Salt) :'H NMR (270 MHz, DMSO-d6) 0. 80 and 0. 83 (s, 3H, 18-CH3), 1. 09-1. 14 (t, J = 7. 4 Hz, 3H, CH2-CH3), 1. 32-1. 37 (d, J = 6. 8 Hz, 3H, CH- CH3), 1. 39-2. 45 (m, 12H), 2.60-2. 63 (q, J = 7. 6 Hz, 2H, CH2-CH3), 2.80-2.81 (m, 3H), $. 95-4. 99 (m, 1H, CH3-CH), 7.01 (s, 1H, CH), 7. 20-7. 22 (m, 2H, aromatic), 7. 30-7. 32 (m, 4H, aromatic), 7.95 (broad m, 2H, aromatic), 8. 35-8. 39 (broad t, 1H, NH); ES+ve-MS (M+H+) 539 m/z; HPLC > 91% (Rt = 2.0, 70% MeCN/water) ; FAB-HRMS calcd for C30H38N2O5S 539.2579 found (M+H+) 539. 2579 Compound 16 HDS02128-3 (TFA Salt): 1H NMR (270 MHz, DMSO-d6) 0.80 and 0. 83 (2 x s, 3H, 18-CH3), 1.09-1.15 (t, J = 7. 1 Hz, 3H, CH2-CH3), 1. 32-1. 35 (d, J = 6. 8 Hz, 3H, CH-CH3), 1. 39-2.45 (m, 12H), 2.57-2.66 (q, J = 7.9 Hz, 2H, CH2-CH3), 2.80-2.84 (m, 3H), 4. 91-4. 94 (m, 1H, CH3-C, 7. 02 (s, 1H, CH), 7. 21-7. 23 (m, 2H, aromatic), 7. 30-7. 32 (m, 4H, aromatic), 7. 94 (broad m, 2H, aromatic), 8. 31-8. 35 (broad. t, J = 3. 57 Hz, 1H, NH) ; ES+ve-MS (M+H+) 539 m/z ; HPLC > 98% (Rt = 2. 69, 70% MeCN/water) ; FAB- HRMS calcd for C3oH38N2o5S 535. 2579 found (M+H+) 539. 2574 Compound 17 HDS02120-2 (TFA Salt) :'H NMR (400 MHz, DMSO-d6) 0.81 and 0.89 (2 x s, 3H, 18-CH3), 1.09-1. 13 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.23-2. 45 (m, 16H), 2.58-2. 63 (q, J = 7.4 Hz, 2H, CH2-CH3), 2.79-2. 81 (m, 3H), 3. 10-3. 12 (m, 2H), 3 : 62-3.80 (m, 3H), 6.99 (s, 1H, CH), 7.18 (s, 1H, aromatic), 7.92 (broad m, 2H, aromatic); ES+ve-MS (M+H+) 519 m/z ; HPLC > 90% (Rt = 2.42, 70% MeCN/water).
Compound 18 HDS02128-1 (TFA Salt) :'H NMR (270 MHz, DMSO-d6) 0.80 and 0.89 (2 x s, 3H, 18-CH3), 0.98-1. 03 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.09-1. 15 (t, J = 7.4 Hz, 3H, CH2-CH3), 1.23-2. 45 (m, 12H), 2.57-2. 66 (q, J = 7.4 Hz, 2H, CH2-CH3), 2.79-2. 82 (m, 3H), 3.02-3. 08 (q, J = 7.4 Hz, CH2-CH3), 7.01 (s, 1 H, CH), 7.21 (s, 1H, aromatic), 7.88 (broad t, 1H, NH), 7.95 (s, 2H, NH2) ; ES+ve-MS (M+H+) 463 m/z; HPLC > 89% (RT = 3.51, 5% MeCN/water to 95% MeCN/water); FAB-HRMS calcd for C24H34N2O5S 463.2266 found (M+H+) 463.2282 Compound 19 HDS02128-4 (TFA Salt) : 1H NMR (270 MHz, DMSO-d6) 0.82 and 0.91 (2 x s, 3H, 18-CH3), 0.96-1. 02 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.09-1. 15 (t, J = 7.4 Hz, 3H, CH2- CH3), 1.23-2. 45 (m, 12H), 2.58-2. 66 (q, J = 7.4 Hz, 2H,. CH2-CH3), 2.79-2. 82 (m, 4H), 2.92 (s, N-CH3), 7.01 (s, 1H, CH), 7.21 (s, 1H, aromatic), 7.95 (s, 2H, NH2) ; ES+ve-MS (M+H') 477 m/z ; HPLC > 95% (Rt = 2. 44, 70% MeCN/water) ; FAB-HRMS calcd for C24H34N2O5S 477.2423 found (M+H+) 477.2414 Compound 20 HDS02128-5 (TFA Salt) : Diagnostic signals NMR (270 MHz, DMSO- d6) 0. 81 (s, 3H, 18-CH3), 0. 83-0. 90 (m, 6H) 1. 09-1. 15 (t, J = 7. 1 Hz, 3H, CH2-CH3), 1. 23- 2.45 (m, 12H), 2.59-2. 66 (q, J = 7.4 Hz, 2H, CH2-CH3), 2.79-2. 82 (m, 3H), 3.00-3. 06 (m, 2H) 7.02 (s, 1H, CH), 7.21 (s, 1H, aromatic), 7.82-7. 87 (m, 1H, NH), 7.96 (s, 2H, NH2) ; ES+ve-MS (M+H+) 491 m/z; HPLC > 92% (Rt = 2.45, 70% MeCN/water) ; FAB-HRMS calcd for C26H38N2O5S 491.2579 found (M+H+) 491. 2581 Compound (tua (TFA Salt): 1H NMR (270 MHz, DMSO-d6) 0.82 and 0.91 (2 x s, 3H, 18-CH3), 0. 96-1. 03 1.03 (t, J = 6.8 Hz, 3H, CH2-CH3), 1.05-1.15 (m, 6H, 2 x CH2-CH3), 1. 23-2. 45 (m, 12H), 2. 58-2. 63 (q, J = 7. 6 Hz, 2H, CH2-CH3), 2. 79-2. 82 (m, 3H), 3. 25- 3. 10 (q, J = 7. 1 Hz, CH2-CH3) 7. 01 (s, 1H, CH), 7.21 (s, 1H, aromatic), 7. 94 (s, 2H, NH2) ; ES+ve-MS (M+H+) 491 m/z ; HPLC > 97% (Rt = 2. 65, 70% MeCN/water) ; FAB-HRMS calcd for C26H38N2O5S 491.2575 found (M+H+) 491.2568 Compound 22 (STX 1040) HDS02-080-1 :'H NMR (270 MHz, CDCl3) 0.79 (s, 3H, 18- CH3), 1.17-1. 22 (t, J = 7.6 Hz, 3H, CH2-CH3), 1.32-2. 40 (m, 13H), 2.54-2. 63 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.67-2. 82 (m, 3H), 4.43-4. 46 (m, 2H, NH-CH2), 6.12 (broad s, 1H, OH), 6.41 (broad t, 1 H, NH), 6.51 (s, 1 H, aromatic), 6.39-6. 41 (appt, J = 5.9 Hz, 1 H, NH), 6.70-6. 72 (appt t, J = 5.9 Hz, 1H, NH), 7.01 (s, 1H, CH), 7.28-7. 30 (m, 1H, aromatic) 7.64-7. 67 (appd, J = 7.6 Hz, 1H, aromatic), 8.50-8. 53 (m, 2H ; aromatic); HPLC > 99% (R, = 5.20, 5% MeCN/water to 95% MeCN/water) ; ES-ve-MS (M+H+) 445 m/z.
Compound 23 HDS02-086-1 :'H NMR (270 MHz, CDC/g) 0.46 (s, 3H, 18-CH3), 0.81-0. 88 (m, 2H), 1.14-1. 24 (m, 4H), 1.32-2. 85 (m, 15H), 4.39-4. 90 (m, 2H, NH-CH2), 6.48 (s, 1H, aromatic), 6.90-6. 95 (broad t, 1 H, NH), 6.97 (s, 1 H, aromatic), 7.01-7. 06 (s, 1 H, aromatic), 7.27-7. 39 (m, 1H, aromatic), 7.67-7. 74 (m, 1H, aromatic), 8.52-8. 54 (m, 1H, aromatic); HPLC > 99% (Rt = 2.25 and 2.76, 75% MeCN/water); ES-ve-MS (M+H+) 445 m/z; FAB-HRMS calcd for C28H34N203 447.2647 found (M+H+) 447.2655 Compound 24 HDS02-086-4 :'H NMR (270 MHz, CDCI3) 0.81 and 0.84 (2 x s, 3H, 18- CH3), 1.17-1. 22 (m, 3H, 2 x CH2-CH3), 1. 32-2.40 (m, 13H), 2.54-2. 63 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.67-2. 82 (m, 3H), 4.55-4. 57 and 4. 80 4.82 (m, 2H, NH-CH2), 6.19 (broad s, 1H, OH), 6.49 (s, 1 H, CH), 7.02 (s, 1 H, CH), 8.24, 8.37, 8.47, 8.60, 8.61 (4 x s, 2H, CH); HPLC > 95% (Rt = 2. 16 and 2. 38, 80% MeCN/water) ; ES-ve-MS (M+H+) 445 m/z ; FAB- HRMS calcd for C28H35N3O3 462.4756 found (M+H+) 462. 2744.
Compound 25 HDS02-134-2 : 1H NMR (270 MHz, CDCl3) 0. 74 and 0.94 (2 x s, 3H, 18- CH3), 1. 16-1. 22 (t, J = 7. 6 Hz, 3H, CH2-CH3), 1.46-1.49 (d, J = 6. 8 Hz, 3H, CH-CH3), 2.65-2. 72 (dd, J = 5.4 and 5.8 Hz, 1H), 1.32-2. 40 (m, 12H), 2.53-2. 61 (q, J = 7.6 Hz, 2H, CH2-CH3), 2. 67-2.82 (m, 3H), 4.9 and 5.0 (2 x s, 1H, OH), 5.08-5. 13 (m, CH-CH3),. 6.06- 6. 03 (d, J = 8.2 Hz,. 1H, NH), 6.30-6. 33 (d, J = 7.9 Hz, 1H, NH), 6.50 (s, 1H, CH), 7.02 (s, 1H, CH), 7. 32-7. 40 (m, 5H, aromatic) ; HPLC > 99% (Rt = 3.36, 80% MeCN/water) ; ES+ve-MS (M+H+) 460 m/z ; FAB-HRMS calcd for C30H37NO3 460. 2851 found (M+H+) 460. 2841 Compound 26 HDS02-134-3: 1H NMR (270 MHz, CDCl3) 0. 84 and 0.94 (2 x s, 3H, 18- CH3), 1. 17-1. 22 (t, J = 7.4 Hz, 3H, CH2-CH3), 1. 46-1. 49 (d, J = 6. 8 Hz, 3H, CH-CH3), 1. 32-2.40 (m, 12H), 2.53-2.61 (q, J = 7. 6 Hz, 2H, CH2-CH3), 2.65-2.72 (dd, J = 5. 4 and 5. 8 Hz, 1H) 2. 79-2. 82 (m, 3H), 4. 71 and 4. 75 (2 x s, 1H, OH), 5. 06-5.11 (m, CH-CH3), 5. 59-6. 03 (d, J = 8. 2 Hz, 1H, NH), 6.30-6. 33 (d, J = 7.9 Hz, 1H, NH), 6.50 (s, 1H, CH), 7.02 (s, 1H, CH), 7.31-7. 40 (m, 5H, aromatic); HPLC > 99% (Rt = 3. 36,70% MeCN/water); ES+ve-MS (M+H+) 460 m/z; FAB-HRMS calcd for C30H37NO3 460.2851 found (M+H+) 460. 2842 Compound 27 HDS02-134-1:'H NMR (270 MHz, CDCl3) 0.81 and 0.95 (2 x s, 3H, 18- CH3), 1. 10-1. 16 (t, J = 7.4 Hz, 3H, CH-CH3), 1.16-1. 12 (t, J = 7.4 Hz, 3H, CH2-CH3), 1.42-2. 43 (m, 13H), 2.55-2. 62 (q, J = 7. 9 Hz, 2H, CH2-CH3), 2.65-2. 82 (m, 3H), 3.22- 3.32 (m, 2H, N-CH2-CH3), 4.94 and 5.03 (2 x s, 1 H, OH), 5.72 and 5.93 (2 x broad t, 1 H, NH), 6.51 (s, 1H, CH), 7. 01 (s, 1H, CH); HPLC > 95% (Rt = 5.20, 70% MeCN/water) ; ES+ve-MS (M+H+) 384 m/z; FAB-HRMS calcd for C24H33NO3 385. 2538 found (M+H+) 384.2532 Compound 28 HDS02-134-4 :'H NMR (270 MHz, CDCI3) 0.82 and 0.94 (2 x s, 3H, 18- CH3), 1.06-1. 12 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.16-1. 22 (t, J = 7.6 Hz, 3H, CH2-CH3), 1.92-2. 43 (m, 13H), 2.53-2. 61 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.78-2. 82 (m, 3H), 2.91- 2.95 (2 x s, 3H, N-CH3), 3.31-3. 38 (m, 1H), 4.78-4. 82 (broad s,. 1H, OH), 6.51 (s, 1H, CH), 7.02 (s, 1H, CH); HPLC > 99% (Rt = 5.20, 70% MeCN/water); ES+ve-MS (M+H+) 398 m/z; FAB-HRMS calcd for C2sH35NO3 398. 2695 found (M+H+) 398.2695 Compound 29 HDS02-134-5 : Diagnostic signals NMR (270 MHz, CDCI3) 0. 82 (s, 3H, 18-CH3), 0. 91-0. 94 (m, 3H), 1. 17-1. 22 (t, J = 7. 6 Hz, 3H, CH2-CH3), 1. 29-2. 43 (m, 15H), 2. 53-2. 62 (q, J = 7. 9 Hz, 2H, CH2-CH3), 2. 78-2. 82 (m, 3H), 3. 20-3. 25 (m, 1H), 4. 82 and 4. 89 (2 x broad s, 1H, OH), 5. 72 and 5.96 (2 x broad t, 1H, NH), 6. 51 (s, 1H, CH), 7. 02 (s, 1H, CH); HPLC > 99% (Pt = 2. 96, 70% MeCN/water) ; ES+ve-MS (M+H+) 412 m/z ; FAB-HRMS calcd for C26H37NO3 412.2851 found (M+H+) 412.2857 Compound 30 HDS02-134-6 :'H NMR (270 MHz, CDCl3) 0.82 and 0.94 (2 x s, 3H, 18- CH3), 1. 08-1. 22 (2 x t, J = 7.1 and 7. 4 Hz, 3H each, 2 x CH2-CH3), CH2-CH3), 1.92-2. 43 (m, 13H), 2. 53-2. 62 (q, J = 7. 6 Hz, 2H, CH2-CH3) 2. 78-2. 81 (m, 3H), 3. 25-3. 42 (m, 4H, N-CH2-CH3), 4. 77 and 4. 81 (broad s, 1H, OH), 6. 51 (s, 1H, CH), 7. 02 (s, 1H, CH) ; HPLC > 96% (Rt = 13. 41, 5% MeCN/water to 95% MeCN/water) ; ES+ve-MS (M+H) 412 m/z ; FAB-HRMS calcd for C26H37NO3 412.2851 found (M+H+) 412.2854 Compound 31 HDS02-134-7 Mixture of diastereomers 1H NMR (270 MHz, CDCl3) 0.79 and 0. 93 (2 x s, 3H, 18-CH3), 1.12-1.16 (2 x d, J = 6.3 Hz each, 6H, 2 x Chug), 1. 19-1. 21 (t, J = 7. 1 Hz, 3H, CH2-CH3), 1. 92-2. 43 (m, 13H), 2. 53-2. 62 (m, 2H, CH2-CH3), 2. 78-2. 85 (m, 3H), 4.04-4. 08 (m, 4H, NH-CH- (CH3) 2), 5. 28 and 5. 42 (2 x s, 1H, 2 x OH), 5.57-5. 59 and 5.83-5. 86 (2 x d, J = 7.6 Hz each, 1H, NH), 6.52 (s, 1H, CH), 7.01 (s, 1H, CH); HPLC > 84% (R, = 3. 90, 5% MeCN/water to 95% MeCN/water) ; ES+ve-MS (M+H+) 398 m/z; FAB-HRMS calcd for C25H35NO3 398.2695 found (M+H+) 398. 2692 Synthesis of 2-Methoxy estrone amide.
Synthesis of benzyl ether (HDS02-170) 32 The 2-Methoxy estrone (0. 521 g, 0. 00173 mol) was dissolved in anhydrous DMF under nitrogen atmosphere, added K2CO3 (0. 718 g, 0. 00225 mol) and stirred at r. t. for approximately 40 h. The crude mixture was diluted with EtOAc, washed with water and brine. The organics were dried (MgS04) and concentrated. The crude product was pre- absorbed onto silica gel, purified using Flash Master-2 system to give the benzylated compound 32 (0.651 g, 96%) as a chalky white solid : TLC (Rt, 0. 4, EtOAc: Hexane, 3: 7); 1H NMR (270 MHz, CDCl3) 6 0. 90 (s, 3H, 18-CH3 diastereomeric methyl signals), 1. 24- 1. 27 (t, J = 3. 4 Hz, 3H, CH2-CH3), 1. 24-1. 29 (t, J = 7. 1 Hz, 3H, CH2-CH3), 1. 39-2. 44 (broad m, 12H), 2. 44-2. 54 (dd, J = 18. 8 and 8. 6 Hz, 1H), 2. 77-2. 83 (m, 2H), 3. 84-3. 87 (s, 3H, OMe), 5.0 (s, 2H, PhCH2), 6. 6 (s, 1H, aromatic), 6.77 (s, 1H, aromatic), 3. 87-3. 84 (s, 3H, OMe), 4. 10-4. 16 (q, J = 7. 0 Hz, 2H, CH3-CH2-O), 5.09 (s, 2H, CH2-phenyl), 6. 61 (s, 1H, 4-CH), 6. 82 (s, 1H, aromatic 1-CH), 7. 41-7. 43 (m, 5H).
Synthesis of the ethyl ester (HDS02-174) 33 To a stirred solution of 2-Methoxy benzyl estrone 32 (150 mg, 0.38 mmol) in anhydrous THF under inert conditions was added a solution of LDA (0.234 mi, 1.8 M solution in heptane, THF, and Ethylbenzene) at-10 °C over a period of 20 min. The reaction mixture was cooled to-60 °C, stirred 15 min, added ethyl bromoacetate (0.052 ml, 0.46 mmol) drop wise over 10 min. and allowed to warm to rt overnight. Diluted the mixture with DCM, added saturated NH4CI and extracted with DCM. The organic phases were combined, dried, and concentrated to obtain a yellow solid which was then purified using Flash Master-2 (10 g column, DCM: MeOH, gradient elution) to obtain the pure product (0.120g, 65% yield) as a colourless glassy solid :'H NMR (270 MHz, CDCI3) 5 0.99 and 0.97 (2 x s, 3H, 18-CH3 diastereomeric methyl signals), 1. 24-1. 27 (t, J = 3. 4 Hz, 3H, CH2- CH3), 1.24-1. 29 (t, J = 7.1 Hz, 3H, CH2-CH3), 1.47-3. 19 (broad m, 15H), 3.87-3. 84 (s, 3H, OMe), 4.10-4. 16 (q, J = 7.0 Hz, 2H, CH3-CH2-O), 5.09 (s, 2H, CH2-phenyl), 6.61 (s, 1H, 4-CH), 6.82 (s, 1H, aromatic 1-CH), 7.41-7. 43 (m, 5H).
Synthesis of the acid (HDS02-182) 34 The estrone ester 33 (0.120 g, 0.252 mmol) was dissolved in a mixture of MeOH : THF (1: 1,6 ml), added NaOH (4M, 3.0 ml) and stirred at room temperature for 30 h. The completion of the reaction was determined by t. l. c (EtOAc: Hexane 3: 7, product-base line spot). The crude mixture was acidified to pH = 2 to 3, extracted with DCM, washed with water and brine. The organic phase was dried (MgSO4) and concentrated to obtain the product as a pale yellow solid. This product 33 (0.092 g, 0.205 mmol) was dissolved in a mixture of MeOH : THF (1 : 1, 7 ml), degassed the mixture using N2, added 5% Pd on C (10 mg) then hydrogenated under a of balloon of Hydrogen for approximately for 15 h.
The crude mixture was filtered using a pad of celite, purified (SiO2, EtOAc: hexane gradient elution using Flash Master-2) to obtain the product 34 (0.081 g, 100 %) as a pale yellow solid : The diagnostic signals of the 1H NMR (400 MHz, CDC13) 6 0. 93 (s, 3H, 18-CH3), 1w47~2 65 (broad m, 13H), 2. 83-2. 85 (m, 2H, CH2), 2. 96-3. 01 (dd, J = 16. 3 and 4. 6 Hz, CH-diastereotopic) 3.89 (s, 3H, OMe), 5.33 (broad s, 1H, OH), 6.69 (s, 1H, aromatic 4-CH), 6. 81 (s, 1H, aromatic 1-CH) ; HPLC > 99% (Rt = 1. 59, 70% MACH in water) ; ES+ve-MS (M+H+) 357 m/z. synthesis of the amide (HDS02-190) 35 (STX 1191).
The starting material acid 34 (0. 038 g, 0. 106 mmol) was suspended in DCM (5. 0 ml), added PyBop (0.062 g, 0.12 mmol), HOBt (0. 016 g, 0.12 mmol) stirred for 20 minutes and added DIPEA (0.076 mi) followed by 3-amino methyl pyridine (0.026 g, 0.24 mmol).
The reaction was left to stir 20 h, quenched with sat. Na2CO3, washed with brine. The organics were separated, dried concentrated to obtain a pale yellow oil. This crude oil was purified using the Flash Master-2 system to obtain a white solid. The product obtained at this point was re-crystalised with hexane/EtOAc to give the required product amide 35 also as a white solid : The diagnostic signals of the mixture of diastereomers of 35'H NMR (400 MHz, CDCl3) 5 0. 84 and 0.97 (2 x s, 3H, 18-CH3), 1.47-2. 81 (broad m, 13H), 3.11-3. 17 (m, 2H, CH2), 3. 84 (s, 3H, OMe), 4.44-4. 46 (m, 2H, NH-CH2-), 5.46 (broad s, 1 H, OH), 6.61 (s, 1H, aromatic 4-CH), 6.75 (s, 1H, aromatic CH), 7.63 (m, 1H, CH aromatic), 8.53 (m, 2H, 2H); HPLC > 90% (R, = 1.59, 90% MeCN in water); ES+ve- MS (M+H+) 448 m/z.
SECTION 4 3-0-Acetyl-estrone (DSF 02124) Acetic acid (60 mL, 1. 05 mmol) was added dropwise over 45 minutes to a stirred solution of estrone (3 g, 11. 1 mmol) in anhydrous pyridine (200 mL) at 0°C under an atmosphere of N2. The resulting yellow mixture was heated to reflux for 1 hour. After cooling down, this was poured into H20 and ice (300 mL) and acidified with HCI 5M. The organics were extracted with EtOAc (300 mL), washed with H20 (200 mL), aq. NaHCO3 (200 mL), then brine (200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crystalline orange crude (3. 88 g) was recrystallized from IPA to give light yellow needles (3. 25 g, 94%): TLC (chloroform/EtOAc, 4 : 1) Rf 0. 82 cf. Rf 0. 68 (E1) ; 8H CDCl3, 400 MHz) 0.91 (3H, s, C-18-H3), 1.45-2.54 (13H, m), 2.29 (3H, s. OAc), 2.89- 2. 92 (2H, m, C-6-H2), 6. 81 (1H, d, JC-2-H, C-4-H = 2.6 Hz, C-4-H), 6.85 (1H, dd, JC-1-H,C-2-H = 8.3 Hz and JC-4-H,C-2-H = 2. 6 Hz, C-2-H) and 7.28 (1H, d, JC-2-H,C-1-H = 8. 3 Hz, C-1-H).
6-Oxo-3-O-acetyl-estrone (DSF 02128) A solution of CrO3 (673 mg, 6.73 mmol) in acetic acid 10% (4 mL) was added dropwise over 2 hours to a stirred solution of 3-O-acetyl-estrone (500 mg, 1.60 mmol) in acetic acid at 10-15°C in an ice/water bath. The resulting dark brown solution was stirred overnight at room temperature. The solvent was then removed under reduced pressure and H2O (100 mL) added. The organics were extracted with EtOAc (100 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a green foam. This was recrystallized from IPA to give grey crystals (103 mg, 20%): TLC (chloroform/EtOAc, 4: 1) Rf 0. 53 cf. Rf 0. 86; sH (CDCI3, 400 MHz) 0.92 (3H, s, C-18-H3), 1.20-2. 63 (12H, m), 2.32 (3H, s, OAc), 2. 88 (1H, dd, JAB= 16. 8 Hz, JC-8-H,C-7-HB = 3. 1 Hz, C-7-HB), 7.28 (1H, dd, JC-1-H, C-2-H = 8. 6 Hz and JC-4-H, C-2-H = 2.6 Hz, C-2-H), 7.46 (1H, d, JC-2-H, C-1-H = 8.6 Hz, C-1-H) and 7.77 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H).
6-Oxo-estrone (DSF 02130, STX 416) A solution of KOH (247 mg, 4.40 mmol) in MeOH (1.5 mL) was added dropwise to a stirred solution of 6-oxo-3-0-acetyl-estrone (80 mg, 0.24 mmol) in MeOH (1.5 mL). The resulting brown solution was stirred for 2 hours at room temperature. The solvent was then concentrated under reduced pressure and H2O (20 mL) added, followed by HCl 5M.
The organics were extracted with EtOAc (50 mL), washed with H20 (20 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product (74 mg) was purified by flash chromatography with chloroform/EtOAc (7 : 3) as eluent and gave the product as a light pink powder (41 mg, 58%) : mp 241-243°C ; TLC (chloroform/EtOAc, 4 : 1) Rf 0.39 cf. R, 0. 64 ; IR (KBr) 3345 (br, OH), 2940-2880 (aliph


CH), , 1720 (C=O), 1680 (C=O), 1610-1495 (arom C=C) cm-1; #H(CDCl3, 400 MHz) 0.93 (3H, s, C-18-H3), 1.24-2. 59 (12H, m), 2. 87 (1H, dd, JC-8-H, C-7-HB = 3.3 Hz, JAB= 17. 0 Hz, C- 7-HB), 5.74 (1H, s, exchanged with D2O, OH), 7.10 (1H, dd, JC-1-H,C-2-H = 8.5 Hz and JC-4- H, C-2-H = 2. 8 Hz, C-2-H), 7.33 (1 H, d, JC-2-H, C-1-H= 8. 5 Hz, C-1-H) and 7.60 (1H, d, JC-2-H,C-4-H = 2. 8 Hz, C-4-H) ; MS m/z (FAB+) 369.2 [50], 285.1 [95, (M+H) +], 113. 1 [68], 84.0 [94]; Acc MS m/z (FAB+) 285.14917, C,, H2, 03 requires 285.14907. HPLC (methanol/water, 90: 10, #max = 223.9 nm) Rt = 2.34 min, 100%.
6, 17-Bis-oxime-estrone (DSF 02169, STX 454) NaOAc (293 mg, 3.58 mmol) followed by hydroxylamine hydrochloride (274 mg, 3.94 mmol) were added to a solution of 6-oxo-estrone (100 mg, 0.35 mmol) in a mixture of MeOH/H20 (5: 1,18 mL). The resulting solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and H20 added (50 mL). The organics were extracted with EtOAc (50 mL + 20 mL), washed with H20 (2x20 mL), then brine (2x20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a light brown crude (120 mg). This was purified by flash chromatography using a gradient of chloroform/EtOAc (7: 3 to 1: 1) then chloroform/EtOAc/acetone (2 : 2 : 1 to 1 : 1 : 2) as eluent and gave the product as a creamy powder (46 mg, 41%) : mp 341-344°C ; TLC (chloroform/EtOAc, 7: 3) Rf 0. 11 cf. Rf 0. 44; IR (KBr) 3410,3265-3050 (br, NOH, OH), 2930-2850 (aliph CH), 1705 (C=N), 1580- 1495 (arom C=C) cm-' ; 8 H (DMSO-d6, 400 MHz) 0.83 (3H, s, C-18-H3), 1.14-2. 42 (12H, m), 3.06 (1H, m, C-7-HB), 6.73 (1H, dd, JC-1-H,C-2-H = 8.3 Hz and JC-4-H,C-2-H = 2.5 Hz, C-2- H), 7.14 (1H, d, JC-2-H,C-1-H = 8. 3 Hz, C-1-H), 7.29 (1H, d, JC-2-H, C-4-H = 2.8 Hz C-4-H) and 9. 28, 10.15, 11.08 (each 1H, s, 2xNOH, OH); MS m/z (FAB+) 315.1 [82, (M+H)+], 73 [100] ; Ace MS m/z (FAB+) 315.17149, C18H23N2O3 requires 315.17087.
6,17-Bis-O-methyl-oxime-estrone (DSF 02183, STX 515) NaOAc (146 mg, 1.79 mmol) followed by 0-methyl-hydroxylamine hydrochloride (164 mg, 1.97 mmol) were added to a solution of 6-oxo-estrone (50 mg, 0. 18 mmol) in a mixture of MeOH/H20 (5: 1,9 mL). The, resulting solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and H20 added (30 mL). The organics were extracted with EtOAc (20 mL + 10 mL), washed with H20 (2x10 mL) then brine (2x10 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The white crystalline crude was recrystallized from EtOAc/hexane to give the product as white crystals (55 mg, 92%) : mp 206-208°C ; TLC (chloroform/EtOAc, 8: 2) Rf 0.70 cf. Rf 0.28 ; IR (KBr) 3134 (br, OH), 2995 (arom CH), 2935-2890 (aliph CH), 1570-1490 (arom C=C) cm-1 ; 8H (CDCI3, 400 MHz) 0.92 (3H, s, C- 18-H3), 1.24-2. 58 (12H, m), 3. 12 (1H, dd, JC-8-H,C-7-HB = 4.5 Hz, JAB= 18.1 Hz, C-7-HB), 3.84 (3H, s, OMe), 3.99 (3H, s, OMe), 4.85 (1H, br s, exchanged with D20, OH), 6. 84 (1H, dd, JC-1-H, C-2-H = 8. 6 Hz and JC-4-H, C-2-H = 2. 7 Hz, C-2-H), 7. 20 (1H, d, JC-2-H, C-1-H = 8. 6 Hz, C-1-H) and 7.29 (1H, d, JC-2-H, C-4-H = 2. 7 Hz, C-4-H); MS m/z (FAB+) 343.2 [100, (M+H)+] ; Acc MS m/z (FAB+) 343.20329, C20H27N2O3 requires 343.20217. Found: C, 70.20 ; H, 7. 57 ; N, 8. 14. C2oH26N203 requires : C, 70. 15; H, 7.65 ; N, 8. 18.
16-Oximino-estrone (DSF 02036/STX 327) Estrone (200 mg, 740 mmol) was added to a stirred solution of potassium tert-butoxide, freshly prepared by dissolving potassium metal (80 mg, 2. 05 mmol) in tert-butanol (2 mL), under an atmosphere of N2. The reaction mixture was stirred for 1 hour at room temperature before addition of isoamyl nitrite (180 µL, 1.34 mmol) in a dropwise manner.
The deep red mixture obtained was stirred overnight, then poured into H20 (20 mL). The resulting solution was extracted with ether (2x20 mL) and the aqueous layer was acidified with glacial acetic acid (10 mL) to give a light yellow precipitate. After standing for 2h, the solid was isolated by filtration to give a yellow powder (140 mg, 63%). For analysis, a sample was recrystallized from acetone to give white crystals: mp 223-225°C [lit.1 (aq. MeOH) 214-215°C] ; TLC (chloroform/acetone, 9: 1) Rf 0. 27 cf. Rf 0. 69 (E1) ; IR (KBr) 3385 (NOH), 2920-2860 (aliph CH), 1735 (C=O), 1605-1500 (arom C=C) cm-' ; 5 H (DMSO-d6, 400 MHz) 0.89 (3H, s, C-18-H3), 1.30-2. 85 (11H, m), 2. 70-2. 81 (2H, m, C-6- H2), 6.46 (1H, d, JC-2-H, C-4-H = 2. 3 Hz, C-4-H), 6.52 (1H, dd, JC-1-H, C-2-H = 8. 3 Hz and Jc-4-H, c-2- H = 2.3 Hz, C-2-H), 7.05 (1H, d, JC-2-H, C-1-H = 8. 3 Hz, C-1-H), 9.05 (1H, s, exchanged with D20, OH) and 12.39 (1H, s, exchanged with D20, NOH); 8c (DMSO-d6, 100.4 MHz) 14.09 (q, C-18), 25.09 (t), 25.46 (t), 26. 18 (t), 29.02 (t), 30.92 (t), 37.20 (d), 43.20 (d), 44.59 (d), 48. 50 (s, C-13), 112.70 (d), 114.83 (d), 125.82 (d), 129.59 (s), 136. 88. (s), 154. 84' (s, C-3 or C-16), 155.23 (s, C-3 or C-16) and 204.64 (s, C=O) ; MS m/z (FAB+) 453.2 [30, (M+H+NBA) +], 300.1 [100, (M+H) +] ; MS m/z (FAB-) 451.3 [38, (M-H+NBA)-], 298.2 [100, (M-H)-] ; Acc MS m/z (FAB+) 300. 15963, C, 8H22NO3 requires 300. 15997.
Found: C, 71.30 ; H, 7.08 ; N, 4.35. C18H21NO3.(AcOH)1/6 requires: C, 71.17 ; H, 7.06 ; N, 4.53.
1 Huffman, M. N.; Darby, H. H. J. Am. Chem. Soc. 1944,66, 150 16, 17-Bis-oximino-estrone (DSF 02110, STX 338) NaOAc (556 mg, 6. 79 mmol) followed by hydroxylamine hydrochloride (520 mg, 7. 48 mmol) were added to a solution of 16-oximino-estrone (200 mg, 0. 67 mmol) in a mixture of MeOH/H20 (5 : 1, 36 mL). The resulting pale yellow solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and brine added (100 mL). The organics were extracted with EtOAc (100 mL), washed with brine (2x50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product (236 mg). This was recrystallized from acetone to give creamy crystals (109 mg, 52%) : mp 245-247°C ; TLC (chloroform/acetone, 8: 2) Rf 0.13 cf. Rf 0.50 ; IR (KBr) 3420-3200 (br, NOH, OH), 3020 (arom CH), 2935-2870 (aliph CH), 1705 (C=N), 1620 (C=N or arom C=C), 1585-1500 (arom C=C) cm- ; #H (DMSO-d6, 400 MHz) 0.97 (3H, s, C-18-H3), 1.43-2. 83 (13H, m), 6.44 (1H, m, C-4-H), 6.51 (1H, dd, JC-1-H,C-2-H = 8.2 Hz and JC-4-H, C2-H = 2. 3 Hz, C-2-H), 7.04 (1 1H, d, JC-2-H, C-1-H = 8.2 Hz, C-1-H), 9.02 (1H, s, exchanged with D20, OH), 10. 81 (1H, s, exchanged with D2O, NOH) and 11.19 (1H, s, exchanged with D20, NOH); MS m/z (FAB+) 315.2 [100, (M+H) +], 133.1 [21] ; MS mlz (FAB-) 466.2 [66i (M-H-NBA)-], 313.2 [100, (M-H) -], 276.1 [80]; Acc MS m/z (FAB+) 315. 17108, C18H23N2O3 requires 315. 17087. HPLC (methanol/water, 70: 30, amas ~ 252.1 nm) Rt = 2.71 min, 99.2%. Found: C, 67.30 ; H, 7.41 ; N, 7.59. C18H23N2O3. (CH3) 20 requires: C, 67.72 ; H, 7.58 ; N, 7.52.
17-Oxime-estrone (DSF 03007).
NaOAc (1. 5 g, 18. 80 mmol) followed by hydroxylamine hydrochloride (1. 4 g, 20. 72 mmol) were added to a suspension of E1 (500 mg, 1. 85 mmol) in a mixture of MeOH/H20 (5 : 1, 90 mL). The resulting suspension was stirred at room temperature overnight. The solvent was then removed under reduced pressure and H20 added (100 mL). The organics were extracted with EtOAc (100 mL + 50 mL), washed with H20 (50 mL), then brine (50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a white crystalline crude product (627 mg). This was recrystallized from MeOH to give white crystals (426 mg, 81%) : TLC (chloroform/EtOAc, 4 : 1) Rf 0. 13 cf. Rf 0.55 (E1); IR (KBr) 3415 (NOH), 3270 (OH), 2930 (aliph CH), 1620 (C=N or arom C=C), 1585-1460 (arom C=C) cm-1; # H (DMSO-d6, 400 MHz) 0.85 (3H, s, C-18-H3), 1.32-2.41 (13H, m), 2. 65-2. 80 (2H, m, C-6-H2), 6. 44 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6. 50 (1H, dd, JC-1-H, C-2-H = 8. 5 Hz and JC-4-H,C-2-H = 2. 6 Hz, C-2-H), 7.05 (1H, d, JC-2-H, C-1-H = 8.5 Hz, C- 1-H), 9. 01 (1H, s, exchanged with DwO, OH) and 10.10 (1H, s, exchanged with D2O, NOH) ; MS ml-7 (FAB+) 286. 1 [100, (M+H) +], 268. 1 [23, (M+H-H2O) +] ; Ace MS mlz (FAB+) 286.18093, C18H24NO2 requires 286.18070.
3-0-Benzyl-Marrianolic acid (DSF 01042) A solution of iodine (7.6 g, 29.9 mmol) in 95 mL of MeOH and a solution of KOH (13.7 g) in 27 mL of H20 and 61 mL of MeOH were added dropwise and alternatively to a stirred solution of 3-0-benzyl-estrone (3.8 g, 10.5 mmol) in MeOH (1 L) so that the colour of the mix remains orange/brown. The addition was carried out over 45 minutes and the resulting light yellow solution was stirred overnight at room temperature under an atmosphere of N2. The resulting clearlight yellow solution was then concentrated under reduced pressure and poured into H20 (800 mL). After acidification with 5M HCI, the organics were extracted with ether (600 mL), washed with aq. Nazis203 (4x100 mL), H20 (4x100 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The resulting yellow foam (4.54 g) was then dissolved in a solution of KOH (7. 6 g) in MeOH/H20 (1: 2, 228 mL) and heated to reflux for 4 hours. The resulting orange solution was poured into H20 (800 mL) and after acidification with 5M HCI the organics were extracted with EtOAc (300 mL). After washing with brine (4x200 mL), the organic layer was dried (MgSO4), filtered and evaporated under reduced pressure to give a yellow residue (4. 32 g). This was recrystallized from CHCI3/Hexane 5: 3 to give a creamy powder (3.25 g, 75%) : mp 212-215°C [lit.2 (aq. MeOH) 226-227°C] ; TLC (chloroform/methanol, 5: 1) Rf 0. 37 cf. Rf 0. 88 ; IR (KBr) 3050-2650 (CO2H), 1700 (C=O), 1600-1500 (arom C=C) cm-' ; 8H (DMSO-d6, 400 MHz) 1.02 (3H, s,. C-18-H3), 1. 20-2. 78 (11H, m), 2.72-2. 76 (2H, m, C-6-H2), 5.05 (2H, s, OCH2Ar), 6.68 (1H, H, d, JC-2-H,C-4-H = 2. 5 Hz, C-4-H), 6. 75 (1H, dd, JC-1-H,C-2-H = 8.7 Hz and JC-4-H,C-2-H = 2.5 Hz, C-2-H), 7.18 (1H, d, JC-2H,C-1-H = 8.7 Hz, C-1-H), 7.30-7.42 (5H, m, C6H5) and 12.14 (2H, s, exchanged with D2O, CO2H); #c (DMSO-d6, 100.4 MHz) 15.37 (q, C-18), 25.84 (t), 26.53 (t), 29.73 (t), 35.77 (t), 36.10 (t), 40.73 (d), 41.84 (d), 42.55 (d), 46.21 (s, C-13), 68.93 (t, OCH2Ar), 112. 35 (d), 114.02 (d)', 126.32 (d), 127. 29 (2xd), 127. 49 (d), 128. 19 (2xd), 131. 64 (s), 137. 18 (2xs), 155. 96 (s, C-3), 173. 93 (s, CO2H) and 178. 60 (s, CO2H) ; MS m/z (FAB+) 408. 2 [41, M+], 91.1 [100, (CH2Ar)+] ; Ace MS mlz (FAB+) 408. 194 04, C25H28O5 requires 408. 08.19367.
2 Heer, J.; Miescher, K. Helv. Chim. Acta 1945, 28, 156.
Marrianolic acid (bSF 02136, STX 417) A suspension of Pd-C (10%, 90 mg) in THF (2 mL) was added to a stirred solution of 3- benzyl-O-marrianolic acid (200 mg, 0.61 mmol) in a mixture of MeOH/THF (2: 1,30 mL).
The resulting suspension was hydrogenated at room temperature for 2 hours using a hydrogen-filled balloon. After removal of the supported catalyst by filtration and evaporation of the filtrate under reduced pressure, the resulting yellow foam (142 mg) was recrystallized from EtOAc/hexane: mp 211-213°C [lit. 2 (EtOH), 223-224°C] ; TLC (chloroform/MeOH, 5: 1) Rf 0. 40 cf. Rf 0. 53 ; IR (KBr) 3600-3350 (br, CO2H), 3080-3005 (arom CH), 2940-2860 (aliph CH), 1715 (C=O), 1695 (C=O), 1610-1450 (arom C=C) cm- 1 ; # H (DMSO-d6, 400 MHz) 1.01 (3H, s, C-18-H3), 1. 18-2. 34 (11H, m), 2.65-2. 71 (2H, m, C-6-H2), 6.41 (1 H, d, JC-2-H, C-4-H = 2. 3 Hz, C-4-H), 6.51 (1H, dd, JC-1-H, C-2-H = 8. 6 Hz and Jc-4- H, C-2-H = 2. 3 HO, C-2-H), 7. 06 (1H, d, JC-2-H, C-1-H = 8. 6 Hz, C-1-H), 9.03 (1H, s, exchanged with D2O, OH) and 12. 12 (2H, br s, exchanged with D20, 2xCO2H) ; MS m/z (FAB+) 318. 1 [100, (M+H)+], 301.1 [80, (M=OH)+] ; Ace MS m/z (FAB+) 318. 14635, C18H22O5 requires 318. 14672.
2 Heer, J.; Miescher, K. Helv. Chim. Acta 1945,28, 156.
3-tert-Butyl-dimethylsilyl-estrone (DSF 03034) Imidazole (6. 3 g, 92. 4 mmol) and tert-butyl-dimethylsilyl chloride (7.8 g, 51.8 mmol) were added to a stirred solution of E1 (10 g, 37. 0 mmol) in anhydrous DMF (300 mL) at room temperature, under an atmosphere of N2. The resulting solution was stirred overnight to give a white suspension. This was poured into H20 (500 mL) and the organics were extracted with EtOAc (500 mL + 300 mL), washed with H20 (2x200 mL), then brine (2x200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude was recrystallized from EtOH to give white needles (#14g, 100%) : TLC (chloroform/EtOAc, 9: 1) Rf 0.91 cf. Rf 0.61 (E1) ; 8H (CDCl3, 400 MHz) 0.19 (6H, s, Si (CH3) 2), 0. 91 (3H, s, C-18-H3), 0. 98 (9H, s, C (CH3) 3), 1.37-2. 55 (13H, m), 2.81-2. 89 (2H, m, C-6-H2), 6.57 (1H, d, JC-2-H,C-4-H = 2. 6 Hz, C-4-H), 6.62 (1H, dd, JC-1-H,C-2-H = 8.3 Hz and JC-4-H, C-2-H = 2.6 Hz, C-2-H) and 7.12 (1H, d, JC-2-H, C-1-H = 8.3 Hz, C-1-H).
3-0-tert-Butyl-dimethylsilyl-16-formyl-estrone (DSF 03066) NaOMe (3. 8 g, 70.2 mmol) was added portionwise to a stirred solution of 3-tert-butyl- dimethylsilyl-estrone (9 g, 23.4 mmol) in dry toluene (250 mL) at room temperature, under an atmosphere of N2. Ethyl formate (13.2 mL, 164 mmol) was then added and the resulting bright yellow solution stirred overnight. The final thick yellow mixture was poured into H20 (300 mL), acidified with HO 5M. The organics were extracted with EtOAc (2x500 mL), washed with water (3x200 mL), then brine (2x200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a creamy crude (9.49 g, 98%). For analysis a sample was recrystallized from EtOH to give white crystals : TLC (chloroform/EtOAc, 9: 1) Rf 0. 61 cf. Rf 0. 84; aH (DMSO-d6, 400 MHz) 0.17 (6H, s, Si (CH3) 2), 0.83 (3H, s, C-18-H3), 0.96 (9H, s, C (CH3) 3), 1.32-2. 63 (12H, m), 2.77-2. 85 (2H, m, C-6-H2), 6. 55 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.61 (1H, dd, JC-1-H, C-2-H = 8. 3 Hz and JC-4-H, C-2-H = 2.6 Hz, C-2-H), 7.14 (1H, d, JC-2-H, C-1-H = 8. 3 Hz, C-1-H), 7. 40 (1H, s, =CH) and 10.69 (1H, s, exchanged with D2O, = COH) ; iS m/z (FAB+) 413. 2 [68, M+H)+], 355. 1 [33, (M-C(CH3)3)+], 72.9 [100]; Acc MS m/z (FAB+) 413.24921, C2gH3y03Si requires 413. 25120.
3-Hydroxy-16-formyl-estrone (DSF 03093, STX 486) A 1.0 M solution of tetrabutyl ammonium fluoride in dry THF (21.3 mL, 21.3 mmol) was added to a stirred solution of 3-0-tert-butyl-dimethylsilyl-16-hydroxymethylene-estrone (4.4 g, 10.7 mmol) in dry THF (100 mL) at room temperature, under an atmosphere of N2. The mixture was stirred overnight to give a brown solution. After removal of the solvent under reduced pressure, H20 was added (300 mL) and the organics were extracted with EtOAc (2x200 mL), washed with H20 (200 mL), then brine (200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The light yellow crude was precipitated from boiling EtOAc, yielding a pale yellow powder (2.21 g, 69%): TLC (chloroform/EtOAc, 8: 2) Rf 0. 32 cf. Rf 0. 68; aH (DMSO-d6, 400 MHz) 0.80 (3H, s, C-18- H3), 1.28-2. 61 (11 H, m), 2.69-2. 78 (2H, m, C-6-H2), 6. 44 (1 H, d, JC-2-H, C-4-H = 2.6 Hz, C-4- H), 6.51 (1H, dd, JC-1-H, C-2-H = 8. 5 Hz and JC-4-H,C-2-H = 2. 6 Hz, C-2-H), 7.04 (1H, d, JC-2-H,C-1-H = 8. 5 Hz, C-1-H), 7.38 (1H, s, =CH), 9.02 (1H, s, exchanged with D20, C-3-OH) and 10.69 (1H, br s, exchanged with D20, =COH); MS m/z (FAB+) 299.2 [36, (M+H)'], 242.3 [100]; Ace MS m/z(FAB+) 299. 16505, C19H23O3 requires 299.16472.
3-Hydroxy-estra-1, 3,5 (10)-trieno-(17, 16-c)-pyrazole (CAB 02156/DSF 03060, STX 509) Hydrazine hydrate (48 µL, 1. 00 mmoi) was added to a solution of 16-hydroxymethylene- estrone (200 mg, 0. 67 mmol) in absolute EtOH (10 mL) at room temperature, under an atmosphere of N2. The resulting intense yellow solution was heated to reflux for 30 minutes. The solvent was then removed under reduced pressure, H2O (50 mL) added and the mixture acidified with HCI 5M. The organics were extracted with EtOAc (100 mL + 50 mL), washed with H20 (2x50 mL), then brine (2x50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a paie yellow crude (212 mg). This was recrystallized from absolute EtOH to give pale yellow flaky crystals (125 mg, 64%): mp 326-328°C [lit.3 320-328°C] ; TLC (chloroform/EtOAc, 7: 3) Rf 0.11 cf. Rf 0.28 ; 5 H (DMSO-d6, 400 MHz) 0.90 (3H, s, C-18-H3), 1.32-2. 58 (11H, m), 2.69-2. 84 (2H, m, C-6- H2), 6.45 (1H, d, JC-2-H,C-4-H) = 2.6 Hz, C-4-H), 6.52 (1 H, dd, JC-1-H, C-2-H = 8.4 Hz and JC-4-H,C-2- H = 2.6 Hz, C-2-H), 7.06 (1H, d, JC-2-H, C-1-H =, 8.4 Hz, C-1-H), 7.26 (1H, s, C-5'-H), 9.02 (1H, s, exchanged with D2O, C-3-OH) and 12.00 (1H, d, J = 3. 5 Hz, exchanged with D20, NH); MS m/z (FAB+) 295.0 [100, (M+H) +] ; Acc MS m/z (FAB+) 295.18078, C19H23N2O requires 295.18104.
3 Sweet, F; Boyd, J.; Medina, 0. ; Konderski, L.; Murdock, G. Biochem. Biophys. Res.
Comm. 1991, 180, 1057-1063 16-Cyano-estradiol (DSF 03019/STX 561) Sodium borohydride (50 mg, 1. 32 mmol) was added portionwise to a stirred solution of 16-nitrile-estrone (150 mg, 0. 51 mmol) in a mixture of THF/EtOH (3: 2, 5 mL) at room temperature. The resulting pale yellow solution was stirred for 30 minutes. The solvent was then removed under reduced pressure, and H20 added (20 mL). The organics were extracted with EtOAc (20 mL + 10 mL), washed with H20. (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a light yellow crude (159 mg). This was recrystallized from IPA/H2O to give white crystals (16 mg) and a further crop of the product (40 mg) was obtained from the residue of the mother liquor upon recrystallization from absolute EtOH (overall yield 37%) : mp 252-254°C; IR (KBr) 3430-3235 (OH), 2925-2850 (aliph CH), 2255 (CN), 1610-1500 (arom C=C) cm-1 ; sH (DMSO-d6, 400 MHz) 0. 79 (3H, s, C-18-H3), 1. 10-2. 27 (11H, m), 2. 66-2. 76 (2H, m, C-6- H2), # 3. 33 (-1H, m, C-16-H, under solvent peaks), 3.68 (1H, dd, JC-16-H, C-17-H = 9.4 Hz, JC- 17-OH, C-17-H = 4.7 Hz, C-17-H), 5.50 (1H, d, JC.-17-H, C-17-OH = 4. 7 Hz, C-17-OH), 6. 43 (1H, d, JC- 2-H, C-4-H = 2.5 Hz, C-4-H), 6.50 (1H, dd, JC-1-H,C-2-H = 8. 5 Hz and JC-4. H, C-2-H = 2. 5 Hz, C-2-H), 7.03 (1H, d, JC-2-H, C-1-H = 8.5 Hz, C-1-H) and 9.01 (1H, s, exchanged with D20,'C-3-OH) ; MS m/z (FAB+) 297.1 [100, M+], 242.1 [50]; Acc MS m/z (FAB+) 297. 17239, C19H23NO2 requires 297. 17288.
16-Isobutylidene-estrone (DSF 03029, STX 571) A solution of estrone (420 mg, 1.55 mmol) in dry THF (5 mL) was added dropwise to a stirred solution of LDA (2.47 mL of a 1.8 M solution in heptane/THF/ethyl benzene, 4.44 mmol) in dry THF (2 mL) at-78°C, under an atmosphere of N2. After 2 hours of stirring at -78°C, isobutyraldehyde (185 µL, 2.03 mmol, freshly distilled from Na2SO4) was added.
The resulting mixture was stirred overnight during which it was allowed to warm to room temperature. The solvent was then removed under reduced pressure and H20 added (50 mL). The organics were extracted with EtOAc (50 mL + 20 mL), washed with H20 (20 mL) then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a crude white foam (602 mg). This was purified by flash chromatography with chloroform/EtOAc (9 : 1) as eluent and gave the product as a white foam (401 mg, 79%). This was recrystallized from EtOAc/hexane to give white crystals (325 mg, 64%): mp 188-190°C ; TLC (chloroform/EtOAc, 8 : 2) Rf 0. 65 cf. Rf 0.74 (E1); IR (KBr) 3370 (OH), 2930-2890 (aliph CH), 1710 (C=O), 1645-1445 (arom C=C and exocyclic C=C) cm-1 ; aH (CDOg, 400 MHz) 0. 92 (3H, s, C-18-H3), 1.06 (3H, d, JC-2'-H,C-3'-H = 6. 6 Hz, C-3'-H3), 1.04 (3H, d, JC-2'-H,C-4'-H = 6.6 Hz, C-4'-H3), 1.40-2. 70 (12H, m), 2.84- 2.90 (2H, m, C-6-H2), 4.67 (1H, s, exchanged with D20, OH), 6.46 (1H, ddd, JC-2'-H,C-1'-H = 9.8 Hz, JC-15-H,C-1'-H = 1-9 Hz, C-1'-H), 6.59 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.64 (1H, dd, JC-1H,C-2-H = 8.3 Hz and JC-4-H,C-2-H = 2.7 Hz, C-2-H) and 7.16 (1H, d, JC-2-H,C-1-H = 8. 3 Hz, C-1-H);##c (CDCl3, 100. 4 MHz) 14. 99 (q, C-18), 22.34 (2xq), 26.42 (t), 26. 44 (t), 27. 18 (t), 29.69 (d), 29.94 (t), 31.98 (t), 38.32 (d), 44. 38 (d), 48. 34 (d), 48. 79 (s, C-13), 113. 12 (d), 115. 54 (d), 126. 66 (d), 132. 20 (s), 134. 73 (s), 138. 12 (s), 144. 25 (d, C-1'), 1 78 (s, C-3), and 210. 04 (s, CHO ; MS m/z (FAB+) 325. 1 [100, (M+H) +] ; Ace MS mlzl (FAB+) 325. 21663, C22H290, requires 325. 21675. Found : C, 81-30 ; H, 8. 71 ; N, 0. C22H28O2 requires : C, 81. 44 ; H, 8. 70 ; N, 0.
16-Isobutylidene-estradiol (DSF 03046, STX 614) A solution of sodium borohydride (57 mg, 1. 51 mmol) in H20 (3 mL) was added dropwise to a stirred solution of 16-isobutylidene-estrone (100 mg, 0.31 mmol) in a mixture of MeOH/THF (3: 1, 9 mL) at 0°C. The resulting solution was stirred at 0°C for 20 minutes.
5 Drops of AcOH glacial were then added, followed by aq. NaCI (10% solution, 20 mL) and the white precipitate that formed was filtered and dried (102 mg). This was recrystallized from acetone/hexane to give white crystals (60 mg, 60%): TLC (chloroform/EtOAc, 8: 2) Rf 0.72 cf. Rf 0.82 ; IR (KBr) 3435-3325 (OH), 2955-2865 (aliph CH), 1695-1500 (arom C=C and exocyclic C=C) cm-1 ; 8H (CDCl3,. 400 MHz) 0.67 (3H, s, C-18-H3), 0.96 (3H, d, JC-2'-H, C-3'-H = 6.6 Hz, C-3'-H3), 0.99 (3H, d, JC-2'-H, C-4'-H = 6. 6 Hz, C-4'- H3), 1.22-2. 02 (14H, m), 2.81-2. 87 (2H, m, C-6-H2), 3.91-3. 98 (1H, m, C-17-H), 4.66 (1H, s, exchanged with D2O,'OH), 5.32 (1H, ddd, JC-2'-H, C-1'-H = 94. Hz, JC-15-H,C-1'-H = 2. 3 Hz, C- 1'-H), 6.56 (1H, d, JC-2-H, C-4-H = 2.7 Hz, C-4-H), 6.63 (1 H, dd, JC-1-H, C-2-H = 8.6 Hz and JC-4-H, C- 2-H = 2.7 Hz, C-2-H) and 7.16 (1H, d, JC-2-H, C-1-H = 8. 6 Hz, C-1-H); MS m/z (FAB+) 326.2 [95, M+], 309. 2 [100, (M-OH)+], 283. 1 [50, (M-C3H7) +]; Acc MS m/z (FAB+) 326. 22520, C22H30O2 requires 326. 22458.
16-(2', 2'-Dimethyl)-propylidene-estrone (DSF 03069A/STX 748) A solution of estrone (500 mg, 1. 85 mmol) in dry THF (5 mL) was added dropwise to a stirred solution of LDA (2. 47 mL of a 1. 8 M solution in heptane/THF/ethyl benzene, 4. 44 mmol) in dry THF (2 mL) at-78°C, under an atmosphere of N2. After 2 hours of stirring at -78°C, 2, 2-dimethyl-propionaldehyde (261 µL, 2.40 mmol, freshly distilled from Na2S04) was added. The resulting mixture was stirred for 50 hours during which it was allowed to warm to room temperature. The solvent was then removed under reduced pressure and H20 added (50 mL). The organics were extracted with EtOAc (50 mL + 20 mL), washed with H20 (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The light yellow crude was purified by flash chromatography with a gradient of chloroform/EtOAc (9: 1 to 8: 2) as eluent and gave the product as a white crystalline solid (264 mg, 42%). This, was recrystallized from EtOAc to give white crystals (113 mg, 21 %): TLC (chloroform/EtOAc, 8: 2) Rf 0.71 cf. Rf 0. 61 (E1) ; 5H (CDCI3, 400 MHz) 0.90 (3H, s, C-18-H3), 1.17 (9H, s, C (CH3) 3), 1.41-2. 45 (11H, m), 2.83-2. 91 (2H, m, C-6-H2), 4.72 (1H, s, exchanged with D20, OH), 6.59 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.63-6. 65 (2H, m, C-2-H and C-1'-H) and 7.16 (1H, d, JC-2-H, C-1-H = 8. 0 Hz, C-1-H) ; MS m/z (FAB+) 677.5 [54, (2M+H)+], 339.2 [100, (M+H) +], 492.2 [24, (M+H+NBA) +] ; Acc MS m/z (FAB+) 339.23256, C23H31O2 requires 339.23241. Found: C, 81.50 ; H, 8. 96; N, 0.
C23H3oO2 requires: C, 81. 61; H, 8.93 ; N, 0.
16-(1'-Hydroxy-propyl)-estrone (DSF 03044B, STX 665) A solution of estrone (500 mg, 1. 85 mmol) in dry THF (30 mL) was added dropwise to a stirred solution of LDA (2. 47 mL of a 1. 8 M solution in heptane/THF/ethyl benzene, 4. 44 mmol) in dry THF (20 mL) at-78°C, under an atmosphere of N2. After 2 hours of stirring at-78°C, propionaldehyde (160'pL, 2.40 mmol, dried on CaCl2 and freshly distilled from Na2SO4) was added. The resulting mixture was stirred overnight during which it was allowed to warm to room temperature. The solvent was then removed under reduced pressure and H20 added (100 mL). The organics were extracted with EtOAc (2x100 mL), washed with H20 (100 mL), then brine (100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a creamy crude oil that crystallizes (971mg). This was purified by flash chromatography with chloroform/EtOAc (8 : 2) as eluent and gave the product as a white solid (363 mg, 60%). This was recrystallized from EtOAc to give white crystals (49 mg) and a further crop of the product (170 mg) was obtained from the residue of the mother liquor upon recrystallization from EtOAc/hexane (overall yield 36%) : TLC (chloroform/EtOAc, 8: 2) Rf 0. 38 cf. Rf 0. 61 (E1) ; sH (CDCl3, 400 MHz) 0. 94 (3H, s, C-18-H3), 1.01 (3H, t, JC-2'-H, C-3'-H = 7. 5 Hz, C-3'-H3) 1. 32-2. 44 (14H, m), 2. 83-2.91 (2H, m, C-6-H2), 3.65-3. 72 (1H, m, C-1'-H), 3.99 (1H, s, OH), 4. 69 (1H, s, OH), 6.59 (1H, d, JC-2-H, C-4-H = 2.7 Hz, C-4-H), 6.65 (1H, dd, JC-1-H,C-2-H = 8.4 Hz and JC-4-H,C- 2-H= 2.7 Hz, C-2-H) and 7.15 (1H, d, JC-2-H,C-1-H = 8. 4 Hz, C-1-H); MS m/z (FAB+) 329.2 [100, (M+H) +], 311.2 [30, (M-OH) +] ; Acc MS m/z (FAB+) 329.21188, C2, H2903 requires 329. 21167.
3-O-Acetyl-16-methylene-estrone (DSF 03023) Paraformaldehyde (480 mg, 16.0 mmol for one unit) and dimethylamine hydrochloride (1.6 g, 19.6 mmol) were added to a stirred solution of 3-0-acetyl-estrone in anhydrous isoamyl alcohol (8 mL). The resulting mixture was heated to reflux for 24 hours. The resulting light yellow solution was poured into H20 (30 mL) and acidified with HCI 5M.
The organics were extracted with EtOAc (2x50 mL), washed with NaHCO3 sat. (20 mL), H20 (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a light yellow oil. This was distilled in a kugelrohr until no more isoamyl alcohol was distilled. The oily residue crystallized overnight and was purified by flash chromatography with chloroform/EtOAc (95 : 5) as eluent to give the product as a white solid (527 mg, 51%). For analysis some crystals were obtained by adding hexane to the distilled oily crude and leaving standing for 1 h: 5H (CDCI3, 400 MHz) 0.93 (3H, s, C-18-H3), 1.41-2. 72 (11H, m), 2.29 (3H, s, OAc), 2. 90-2. 95 (2H, m, C-6-H2), 5.41-5. 43 (1H, m, C-1'-Htrans), 6.09-6. 12 (1H, m, C-1'-Hcis), 6.81 (1H, d, JC-2-H, C-4-H = 2.5 Hz, C-4- H), 6.86 (1H, dd, JC-1-H, C-2-H = 8. 3 Hz and JC-4H,C-2-H = 2.5 Hz, C-2-H) and 7.29 (1H, d, Jc-2- H, C-1-H = 8. 3 Hz, C-1-H) ; MS mlz (FAB+) 325. 2 [100, (M+H) +], 282. 2 [78, (M+H-CH3CO)+] ; Ace MS mlz (FAB+) 325. 18224, C21H25O3 requires 325.18037.
16-Ethoxymethyl-estrone (DSF 03036B, STX 664) A solution of KOH (41 mg, 0.74 mmol) in H20 (2 mL) was added dropwise to a stirred solution of 16-methylene-estrone (20 mg, 0.62) in absolute EtOH (20 mL) at 0°C. The resulting light yellow solution was stirred for 30 minutes at 0°C. The solvent was then removed under reduced pressure, H20 added (40 mL) followed by a few drops of HCI 5M. The white precipitate that formed was filtered and dried (131 mg). This was purified by flash chromatography with chloroform/EtOAc (95: 5) as eluent and gave the product as a light yellow solid (26 mg, 13%). This was recrystallized from EtOH/H20 to give light yellow crystals (13 mg, 6%): mp 208-210°C ; TLC (chloroform/EtOAc, 8 : 2) Rf 0.40 cf. R, 0.86 ; 8H (CDCI3, 400 MHz) 0. 89 (3H, s, C-18-H3), 1.16 (3H, t, JC-2'-H,C-3'-H = 7.0 Hz, C-3'- H3), 1.36-2. 42 (12H, m), 2.81-2. 85 (2H, m, C-6-H2), 3.42-3. 50 (2H, m, C-2'-H2), 3.59 (2H, m, C-1'-H2), 4.66 (1H, s, OH), 6.58 (1H, d, JC-2-H, C-4-H = 2. 8 Hz, C-4-H), 6.64 (1H, dd, JC-1- H, C-2-H = 8. 1 HO and JC-4-H,C-2-H = 2.8 Hz, C-2-H) and 7.15 (1H, d, JC-2-H,C-1-H = 8. 1 Hz, C-1-H); MS m/z (FAB+) 329.1 [100, (M+H) +] ; Acc MS m/z (FAB+) 329.21242, C21H29O3 requires 329.21167. Found: C, 76.60 ; H, 8.62 ; N, 0. C21H28O3 requireS : C, 76.79 ; H, 8.59 ; N, 0.
17-O-Allyl-oxime-estrone (DSF 03048) NaOAc (3 g, 10. 17 mmol) followed by O-allyl-Hydroxylamine hydrochloride (4. 5 g, 41. 4 mmol) were added to a solution of estrone (1 g, 3. 70 mmol) in a mixture of MeOH/H2O (5: 1,180 mL). The resulting solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure and water added (200 mL). The organics were extracted with EtOAc (200 mL + 100 mL), washed with water (2x100 mL) then brine (2x100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a white crude (1. 37 g). This was recrystallized from MeOH/H2O and gave the product as white crystals (1. 13 g, 94%) : TLC (chloroform/EtOAc, 8 : 2) Rf 0. 66 cf. Rf 0.56; # H (CDCl3, 400 MHz) 0. 94 (3H, s, C-18-H3), 1.35-2.60 (13H, m), 2.80-2.87 (2H, m, C-6-H2), 4. 55 (2H, ddd, JAB = 5.5 Hz, JC-2'-H,C-1'-H = 2.7 Hz and JC-3'-H,C-1'-H = 1.4 Hz, C-1'-H2), 4.74 (1H, s, echanged with D2O, OH), 5. 19 (1H, ddd, Jcis = 10.3 Hz, JC-1'-H,C-3'-H = 1. 4 Hz, C-3'-Ha), 5. 28 (1H, d, Jtrans = 17.2 Hz, JC-1'-H,C-3'-H = 1.6 Hz, C-3'-Hb), 5.59-6.01 (1H, m, C-2'-H), 6. 56 (1H, d, Jc-2-H, C-4-H = 2.7 Hz, C-4-H), 6.63 (1H, dd, JC-1-H, C-2-H = 8.2 Hz and JC-4-H, C-2-H = 2. 7 HO, C-2-H) and 7. 15 (1H, d, JC-2-H, C-1-H = 8. 2 Hz, C-1-Hj ; MS m/z (FAB. +) 326. 1 [100, (M+H) +], 268. 1 [30, OCH2CH=CH2+] ; Acc MS m/z (FAB+) 326. 21157, C21H28NO2 requires 326.21200. Found: C, 76.40 ; H, 8.24 ; N, 4. 36. C21H27NO2 requires: C, 76.23 ; H, 8.50 ; N, 4.17.
3-Hydroxy-estra-1, 3,5 (10)-triene- (17, 16)- [1] pyrindine (DSF 03073, STX 663) 17-0-allyl-oxime (2.45 g, 7.53 mmol) was stirred and heated to 230°C (using a sand bath) for 46 hours. The resulting dark brown solid was cooled and EtOH was added until most of the solid was dissolved. The remaining undissolved material was crushed to a fine powder, silica was added and the solvent removed under reduced pressure. The light brown powder was then transferred onto a flash chromatography column (wet packed) and the column eluted with chloroform/EtOAc (9 : 1). A dark orange solid was recovered (200 mg, 9%) which was purified by performing another flash chromatography with chloroform/EtOAc (95: 5) as eluent and gave a light orange powder (146 mg, 6%): TLC (chloroform/EtOAc, 8: 2) Rf 0. 26 cf. Rf 0. 70; 8n (CDCI3, 400 MHz) 1.01 (3H, s, C-18- H3), 1. 43-2.68 (11H, m), 2. 80-2. 96 (2H, m, C-6-H2), 6. 08 (1H, s, OH), 6.61 (1H, d, JC-2-H,C- 4-H = 2.6 Hz, C-4-H), 6. 67 (1H, dd, JC-1-H,C-2-H = 8. 4 HO and JC-4-H, C-2-H = 2.5 Hz, C-2-H), 7.04 (1H, dd, JC-4'-H, C-5'-H = 7. 4 Hz and JC-6'-H, C-5'-H = 4. 8 Hz, C-5'-H), 7.18 (1H, d, JC-2-H, C-1-H = 8-4 Hz, C-1-H), 7. 54 (1H, d, JC-5'-H,C-4'-H = 7. 4 Hz, C-4'-H) and 8.33 (1H, d, JC-5'-H,C-6'-H = 4.8 Hz, C-2'-H) ; MS m/z (FAB+) 306. 1 [85, (M+H) +], 207. 0 [95], 114. 9 [100] ; Acc MS m/z (FAB+) 306.18645, C21H24NO requires 306.18579.
3-0-Acetyl-16-acetoxymethylene-estrone (DSF 03054) Acetic anhydride (8. 34 mL, 88. 4 mmol) was added dropwise over 10 minutes to a stirred solution of 16-formyl-estrone (230 mg, 0. 77 mmol) in dry pyridine (15 mL) at 0°C, under an atmosphere of N2. The resulting yellow mixture was then heated to reflux for 1 hour.
The final brown solution was cooled, then poured into H20 (50 mL) and ice and acidified with HCI 5M. The organics were extracted with EtOAc (2x50 mL), washed with H20 (30 mL), Na2CO3 10% (30 mL), then brine (30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give an orange foam (250 mg). This was purified by flash chromatography with chloroform/EtOAc (95: 5) as eluent and gave the product as a pale yellow oil that crystalizes (140 mg, 47%): TLC (chloroform/EtOAc, 9: 1) Rf 0.70 cf. Rf 0. 30 ; aH (CDCI3, 400 MHz) 0.93 (3H, s, C-18-, H3), 1.42-2. 84 (11H, m), 2.24 (3H, s, OAc), 2.29 (3H, s, OAc), 2. 89-2.96 (2H, m, C-6-H2), 6.81 (1H, d, Jc-2-H, C-4-H = 2. 6 Hz, C-4-H), 6.86 (1H, dd, JC-1-H,C-2-H = 8.3 Hz andjC-4-H, C-2-H'= 2. 6 Hz, C-2-H), 7.29 (1H, d, JC-2-H, C-1-H = 8.3 Hz, C-1-H) and 8.15 (1H, dd, JC-15-Ha, C-1'-H = 2.9 Hz, JC-15-Hb,C-1'-H = 1.7 Hz, =CH); MS m/z (FAB+) 383.0 [100, (M+H) +], 341.0 [86, (M+H-CH2C=O) +] ; Ace MS m/z (FAB+) 383.18523, C23H27O5 requires 383.18585.
6-Oxo-3-0-acetyl-16-acetoxymethylene-estrone (DSF 03062) A solution of CrO3 (132 mg, 1. 32 mmol) in acetic acid 10% (750 µL) was added dropwise over 30 minutes to a stirred solution of 3-0-acetyl-16-acetoxymethylene-estrone (120 mg, 0. 31 mmol) in acetic acid at 10-15°C in an ice/water bath. The resulting dark brown solution was stirred for 40 hours at room temperature. The solvent was then removed under reduced pressure and H20 (50 mL) and ice added. The organics were extracted with EtOAc (2x50 mL), washed with H20 (2x30 mL), then brine. (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a brown crude. This was purified by flash chromatography with chloroform/EtOAc (8 : 2) as eluent an gave the product as a light yellow solid (13 mg, 10%) :-TLC (chloroform/EtOAc, 4 : 1) Rf 0. 40 cf., 0. 63 ; aH (CDCl3, 400 MHz) 0. 95 (3H, s, C-18-H3), 1.24-2.92 (11H, m), 2. 26 (3H, s, OAc), 2. 32 (3H, s, OAc), 7. 29 (1H, dd, JC-1-H, C-2-H = 8. 4 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 7.46 (1H, d, JC-2-H,C-1-H = 8.4 Hz, C-1-H), 7.77 (1H, d, JC-2-H,C-4-H = 2. 6 Hz, C-4-H) and 8. 17 (1H, dd, JC-15-Ha,C-1'-H = 2. 9 JC-15-Hb,C-1'-H = 1. 7 Hz, =CH) ; MS m/z (FAB+) 663. 5 [48], 397. 2 [38, (M+H)+], 355.2 [30], 73.0 [100] ; Ace MS m/z (FAB+) 397. 16700, C23H25O6 requires 397. 16511.
6-Oxo-16-ethoxymethylene-estrone (DSF 03083A, STX 749) A solution of KOH (27 mg, 0.48 mmol) in H20 (0.8 mL) was added dropwise to a stirred solution of 6-oxo-3-0-acetyl-16-acetoxymethylene-estrone (80 mg, 0.20 mmol) in absolute EtOH at 0°C. The resulting bright yellow mixture was stirred at 0°C for 30 minutes. The mixture was then acidified with 3 drops of 5M HCI and the solvent removed under reduced pressure. H20 was added (50 mL) and the organics were extracted with EtOAc (2x50 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a pale yellow crude (52 mg). This was purified by flash chromatography with DCM/EtOAc (75: 25) as eluent and gave the product as a white solid (12 mg, 18%) : mp 314-316°C (dec. ); TLC (chloroform/EtOAc, 8 : 2) Rf 0. 11 cf. Rf 0. 82; 8H (CDCI3, 270 MHz) 0.94 (3H, s, C-18-H3), 1.10-2.72 (10H, m), 1.35 (3H, t, JC-2'-H,C-3'-H = 7.0 Hz, C-3'-H3), 2.85 (1H, dd, JC-S-H, C-7-HB = 3.1 Hz, JAB= 16. 7Hz, C-7-HB), 4. 05-4. 13 (2H, m, C-2'-H2), 7.09 (1H, dd, JC-1-H, C-2-H = 8. 6 Hz and I/C-4-H, C-2-H = 2. 7 Hz, C-2-H), 7. 31 (1H, s, C-1'-H), 7.34 (1H, m, C-1-H) and 7. 57 (1H, d, JC-2-H,C-4-H = 8.4 Hz, C-4-H) ; MS m/z (FAB+) 341.2 [100, (M+H)+] ; Acc MS jazz (FAB+) 341. 17466, C21H25O4 requires 341.17528.
6-Oxo-16-formyl-estrone (DSF 03105, STX 784) A solution of K2GO3 (63 mg, 0. 46 mmol) in H20 (1 mL) was added dropwise to a stirred solution of 6-oxo-3-0-acetyl-16-acetoxymethylene-estrone (30 mg, 0. 08 mmol) in MeOH (10 mL). The resulting pale yellow solution was stirred for 45 minutes. The final pale brown mixture was then acidified with 3 drops of HCI 5M and the solvent removed under reduced pressure. H20 was added (30 mL) and the organics were extracted with EtOAc (2x50 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The beige crude was recystallized from EtOAc/hexane to give a white powder (13 mg, 54%): mp 224-227°C ; aH (DMSO-d6, 400 MHz) 0.80 (3H, s, C-18-H3), 1.41-2. 65 (11H, m), 7.01 (1H, dd, JC-1-H, C-2-H = 8. 4 Hz and JC- 4-H, C-2-H = 2.7 Hz, C-2-H), 7.28 (1H, d, JC-2-H,C-4-H = 2. 7 Hz, C-4-H), 7.31 (1H, d, JC-2-H,C-1-H = 8.4 Hz, C-1-H), 7.41 (1H, s, C-1'-H), 9.64 (1H, s, exchanged with D2O, C-3-OH) and 10.77 (1 H, br s, exchanged with D20, C-1'-OH) ; MS m/z (FAB+) 313.2 [6, (M+H)+], 149.1 [100]; MS m/z (FAB-) 311.2 [100, (M-H)-] ; Acc MS m/z (FAB+) 313.14503, C19H21O4 requires 313.14398.
3-0-Benzyl-16-formyl-estrone (DSF 03091).
Potassium tert-butoxide (9.4 g, 84. 1 mmol) was added portionwise to a stirred solution of 3-0-benzyl-estrone (10 g, 27. 7 mmol) in dry toluene (250 mL) at room temperature, under an atmosphere of N2. After stirring for 20 minutes, ethyl formate (14. 8 mL, 194 mmol) was added and the resulting suspension stirred for 2.5 hours. The final thick creamy suspension was poured into H2O (300 mL) and ice, and the organics extracted with EtOAc (2x200 mL), washed with H20 (3x100 mL), then brine (2x100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude was precipitated from boiling EtOAc to give a creamy solid (9.25 g, 86%): TLC (hexane/EtOAc, 1: 1) R, 0. 57 cf. Rf 0.78 ; 8H (DMSO-d6, 400 MHz) 0. 81 (3H, s, C-18-H3), 1. 31-2. 62 (11H, m), 2. 79-2. 85 (2H, m, C-6-H2), 5.05 (2H, s, OCH2Ar), 6. 71 (1H, d, JC-2H,C-4-H = 2. 7 Hz, C-4-H), 6. 75 (1H, dd, JC-1-H, C-2-H = 8.4 Hz and JC-4H,C2-H = 2.7 Hz, C-2-H), 7.16 (1H, d, JC-2-H, C-;-H = 8. 4 Hz, C-1-H), 7.28-7. 33 (1H, m, =CH), 7. 35-7. 43 (5H, m, C6H5) and 10.6-10.9 (1H, br s, exchanged with D20, =COH) ; MS m/z (FAB+) 389. 3 [31, (M+H) +], 91. 1 [100, (CH2Ar) +] ; Ace MS mAz (FAB+) 389. 20994, C2sFH2gO3 requires 389. 21167.
3-O-Benzylo-estra-1, 3, 5 (10)-trieno- (17, 16-c)--pyrazole (DSF 03095) Hydrazine hydrate (751 pL, 15.4 mmol) was added to a suspension. of 3-O-benzyl-16- formyl-estrone (4 g, 10.3 mmol) in absolute EtOH (200 mL) at room temperature, under an atmosphere of N2. The resulting yellow solution was heated to reflux for 45 minutes.
After acidification with HCI 5M, the solvent was removed under reduced pressure until precipitation of the product. H20 was then added (20 mL) and the creamy precipitate filtered and dried (3.73 g, 94%): TLC (hexane/EtOAc, 1: 1) Rf 0. 20 cf. Rf 0. 66; 8H (DMSO- d6, 400 MHz) 0.91 (3H, s, C-18-H3), 1.36-2. 58 (11H, m), 2.78-2. 87 (2H, m, C-6-H2), 5.05 (2H, s,'OCH2Ar), 6.72 (1H, d, JC-2-H, C-4-H = 2. 7 Hz, C-4-H), 6.76 (1H, dd, JC-1-H, C-2-H = 8. 6 Hz and JC-4-H, C-2-H = 2.7 Hz, C-2-H), 7.18 (1H, d, JC-2-H, C-1-H = 8. 6 Hz, C-1-H), 7.27 (1H, s, C-5'- H), 7.29-7. 44 (5H, m, C6H5) and 12. 00 (1H, s, exchanged with D20, NH); MS m/z (FAB+) 385.3 [75, (M+H) +], 91.1 [100, (CH2Ar) +] ; Acc MS m/z (FAB+) 385.22796, C26H29N2O requires 385.22799.
General procedure for alkylation of D-ring fused pyrazoles.
NaH (60% dispersion, 1. 5 eq.) was added to a stirred solution of 3-O-benzyl-estra-1, 3,5 (10)-trieno-(17, 16-c)-pyrazole in DMF at 0°C, under an atmosphere of N2. After stirring for 20 minutes at 0°C, the alkylating agent was added (2 eq. ) and the mixture was stirred at room temperature until completion of the reaction (followed by TLC). The mixture was then poured into H20 (50 mL) and the organics extracted with EtOAc (2x50 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography using DCM/EtOAc as eluent.
3-O-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)--N-methyl-pyrazole Following the general procedure, 3-0-benzyl-estra-1, 3, 5 (10)-trieno- (17, 16-c)-- pyrazol (150 mg, 0.39 mmol) was treated with NaH (19 mg, 0. 47 mmol) and the subsequent reaction with methyl iodide (57 µL, 0.78 mmol) was complete within 50 minutes. Purification of the crude by flash chromatography with a gradient of DCM/EtOAC (98 : 2 to 95: 5) as eluent gave : 1'-Methyl- 3-O-Benzyl-estra-1, 3, 5 (10)- trieno-(17, 16-c)-pyrazole, DSF 03098A, as a light yellow oil that crystallized o n standing (52 mg, 33%): TLC (hexane/EtOAc, 1: 1) Rf 0.52 cf. Rf 0. 38 ; 8H (CDCI3, 400 MHz) 1.01 (3H, s, C-18-H3), 1. 42-2. 67 (11H, m), 2.81-2. 99 (2H, m, C-6-H2), 3.84 (3H, s, N-Me), 5.03 (2H, s, OCH2Ar), 6.73 (1H, d, JC-2-H, C-4-H = 2. 5 Hz, C-4-H), 6. 79 (1H, dd, Jc-,- H, C-2-H = 8.5 Hz and JC-4-H,C-2-H = 2. 5 Hz, C-2-H), 6.97 (1 H, s, C-5'-H), 7.22 (1 H, d, JC-2-H, CS1-H = 8.5 Hz, C-1-H) and 7.29-7. 45 (5H, m, C6H5) ; MS m/z (FAB+) 399.3 [74, (M+H) +], 91.1 [100, (CH2Ar) +], 73.. 0 [24]; Acc MS m/z (FAB+) 399.24467, C27H31N2O requires 399. 24364.
2'-Methyl-3-0-Benzyl-estra-1, 3,5 (10)-trieno-(17, 16-c)-pyrazole, DSF 03098B, was obtained as a creamy solid (55 mg, 35%): TLC (hexane/EtOAc, 1: 1) Rf 0. 46 cf. Rf 0. 38; 8H (CDCta, 400 MHz) 1.01 (3H, s, C-18-H3), 1. 44-2. 64 (11H, m), 2.85-2. 98 (2H, m, C-6- H2), 3. 82 (3H, s, N-Me), 5. 04 (2H, s, OCH2Ar), 6.74 (1H, d, JC-2-H, C-4-H = 2.8 Hz, C-4-H), 6. 80 (1H, dd, JC-1-H,C-2-H = 8. 6 Hz and JC-4-H,C-2-H = 2. 8 Hz, C-2-H), 7.15 (1H, s, C-3'-H), 7.20 (1H, d, JC-2-H, C-1-H = 8.6 Hz, C-1-H) and 7.30-7. 45 (5H, m, C6H5) ; MS m/z (FAB+) 399.3 [55, (M+H) +], 91.1 [100, (CH2Ar) +] ; Acc MS m/z (FAB+) 399.24341, C27H31N2O requires 399.24364.
1'-Isobutyl- 3-O-Benzyl-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole and 2'-Isobutyl- 3-0-Benzyl-estra-1, 3,5 (10)-trieno- (17,. 16-c)-pyrazole Following the general procedure, 3-0-benzyl-estra-1, 3,5 (10)-trieno- (17, 16-c) -- pyrazol (250 mg, 0.65 mmol) was treated with NaH (31 mg, 0. 78 mmol) and the subsequent reaction with 1-bromo-2-methyl-propane (124 µL, 1.30 mmol) was complete within 2 hours. Purification of the crude by flash chromatography with DCM/EtOAC (95 : 5) as eluent gave: 1'-Isobutyl- 3-O-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)- pyrazol, DSF 031liA, as a pale yellow oil (121 mg, 42%) : TLC (DCM/EtOAc, 9: 1) Rf 0.93 cf. Rf 0. 26; aH (CDCI3, 400 MHz) 0.87-0. 92 (6H, 2#d, JC-2"-H, C-3"-H = 6 Hz and JC-2"-H, C- 4"-H = 6.6 Hz, C-3"-H3, C-4"-H3), 1.01 (3H, s, C-18-H3), 1.42-2. 98 (14H, m), 3.81 (1H, dd, JBA = 13. 8 Hz andJC-2"-H, C-1"-H= 7.5 Hz, N-CHAHB), 3. 86 (1 H, dd, JAB = 13. 8 Hz and JC-2"-H C- 1"-H = 7.5 Hz, N-CHAHB), 5. 04 (2H, s, OCH2Ar), 6. 71-6. 75 (1H, m, C-4-H), 6. 79 (1H, dd, JC-1-H, C-2-H = 8.6 Hz and JC-4-H,C-2-H = 2.7 Hz, C-2-H), 6.94-6.99 (1H, m, C-5'-H), 7.22 (2H, d, JC-2-H,C-1-H = 8. 6 Hz, C-1-H) and 7. 31-7. 46 (5H, m, C6H5).
2'-Isobutyl- 3-O-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole, DSF 03111B, was obtained as a white crystalline solid (56 mg, 19%) : mp 126-128°C ; TLC (DCM/EtOAc, 9: 1) Rf 0.64 cf. Rf 0.26 ; #H(CDCl3, 400 MHz) 0. 92 (3H, d, JC-2"-H,C-3"-H = 6.6 Hz, C-3"-H3), 0.95 (3H, d, JC-2"-H,C-4"-H = 6.6 Hz, C-4"-H3), 1. 02 (3H, s, C-18-H3), 1. 46-2. 60 (12H, m), 2. 89-2. 94 (2H, m, C-6-H2), 3. 74 (1H, dd, JBA = 13. 3 Hz and JC-2"-H,C-1"-H = 7.8 Hz, N-CHAHB), 3. 85 (1H, dd, JAB = 13.3 Hz and JC-2"-H,C-1"-H = 7.4 Hz, N-CHAHB), 5.04 (2H, s, OCH2Ar), 6.74 (1 H,, d, JC-2-H, C-4-H= 2. 6 Hz, C-4-H), 6.79 (1H, dd, JC-1-H,C-2-H = 8.6 Hz and Jc- 4-H, C-2-H = 2. 6 Hz, C-2-H), 7.17-7. 21 (2H, m, C-1-H and C-3'-H) and 7.31-7. 45 (5H, m, C6H5).
1'-Methylacetate-3-O-Benzyl-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole and 2'- Methylacetate-3-O-Benzyl-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole Following the general procedure, 3-O-benzyl-estra-1, 3,5 (10)-trieno- (17, 16-c)-- pyrazol (300 mg, 0.78 mmol) was treated with NaH (47 mg, 1.17 mmol) and the subsequent reaction with methyl-chloro-acetate (136 pL, 1.56 mmol) was complete within 2.5 hours. Purification of the crude by flash chromatography with a slow gradient of DCM to DCM/EtOAC (8: 2) as eluent gave: 1'-Methylacetate-3-O-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole, DSF 03117-2A as a white crystalline solid (173 mg, 48%): TLC (DCM/EtOAc, 9: 1) Rf 0.53 cf. Rf 0. 09; 8, (CDCI3, 400 MHz) 1.03 (3H, s, C-18- H3), 1. 44-2.69 (11H, m), 2.85-2. 99 (2H, m, C-6-H2), 3.76 (3H, s, OMe), 4.86 (2H, s, N- CH2), 5.04 (2H, s, OCH2Ar), 6.74 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.79 (1H, dd, JC-1-H,C- 2-H= 8.6 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 7.07 (1H, s, C-5'-H), 7. 22 (1 H, d, JC-2-H, C-1-H = 8.6 Hz, C-1-H) and 7.29-7. 44 (5H, m, C6H5) ; MS m/z (FAB+) 457.3 [55, (M+H)+], 91.1 [100, (CH2Ar)+], 73.0 [72] ; Acc MS mlz (FAB+) 457.24954, C29H33N2O3 requires 457. 24912.
2'-Methylacetate-3-O-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole, DSF 03117-2B, was also obtained as a pale yellow oil (70 mg, 20%): TLC (DCM/EtOAc, 9: 1) Rio. 28 cf. Rf0. 09 ; # #H (CDCl3, 400 MHz) 1.02 (3H, s, C-18H3), 1.42-2.64 (11H, m), 2.86-2. 98 (2H, m, C-6-H2), 3.78 (3H, s, OMe), 4.69-4. 88 (2H, m, N-CH2), 5.03 (2H, s, OCH2Ar), 6.74 (1H, d, JC-2-H, C-4-H = 2.6 Hz, C-4-H), 6.78 (1H, dd, JC-1-H,C-2-H = 8.6 Hz and JC- 4-H,C-2-H = 2.6 Hz, C-2-H), 7.18 (1H, d, JC-2-HC-1-H = 8.6 Hz, C-1-H), 7.25 (1H, s, C-3'-H), and 7.31-7. 44 (5H, m, C6H5) ; MS mAw (FAB+) 457.3 [64, (M+H)+], 91.1 [100, (CH2Ar)+], 73. 0 [271 ; Ace MS mA (FAB+) 457. 25071, C29H33N2O3 requires 457.2461912.
1'-(2-Methoxyethyl)-3-O-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole, DSF 03117-3A and 2'-(2-Methoxyethyl)-3-O-Benzyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)- pyrazole, DSF 03117-3B.
Following the general procedure, 3-O-benzyl-estra-1, 3,5 (10)-trieno- (17, 16-c)-- pyrazol (300 mg, 0. 78 mmol) was treated with NaH (47 mg, 1.17 mmol) and the subsequent reaction with 1-chloro-2-methoxy-ethane (142 pL, 1.56 mmol) was complete within 4 hours. Purification of the crude by flash chromatography with DCM/EtOAC (9: 1) as eluent gave: 1'-(2-Methoxyethyl)-3-O-Benzyl-estra-1, 3, 5 (10)-trieno- (17, 16-c)- pyrazole, DSF 03117-3A as acreamy solid (92 mg, 27%): TLC (DCM/EtOAc, 9: 1) Rf 0. 38 cf. Rf 0. 12; 8H (CDCI3, 400 MHz) 1.01 (3H, s, C-18-H3), 1.46-2. 66 (11H, m), 2. 84- 2.99 (2H, m, C-6-H2), 3.33 (#3H, s, OMe), 3.72 (2H, dt, J= 5.3 Hz, J = 1. 3 Hz, C-1"-H2 or C-2"-H2), 4.23 (2H, t, J = 5.2 Hz, C-1"-H2 or C-2"-H2), 5.04 (2H, s, OCH2Ar), 6.74 (1H, d, JC-2-H, C-4-H = 2. 8 HO, C-4-H), 6.80 (1H, dd, JC-1-H, C-2-H = 8. 3 Hz and JC-4-H, C-2-H = 2. 8 Hz, C- 2-H), 7.09 (1H, s, C-5'-H), 7.23 (1H, d, JC-2-H, C-1-H = 8. 3 Hz, C-1-H) and 7. 31-7. 45 (5H, m, C6H5) ; MS m/z (FAB+) 443.3 [100, (M+H) +], 91.1 [80, (CH2Ar) +] ; Acc MS'm/z (FAB+) 443. 27119, C29H35N2O2 requires 443.26985.
2'-(2-Methoxyethyl)-3-O-Benzyl-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole DSF 03117-3B, was obtained as a creamy solid (80 mg, 23%): TLC (DCM/EtOAc, 9: 1) Rf 0.29 cf. Rf 0. 12; #H (CDCl3, 400 MHz) 1.04 (3H, s, C-18-H3), 1.42-2. 66 (11H, m), 2.84- 2.99 (2H, m, C-6-H2), 3.32 (#3H, s, OMe), 3.78 (2H, m, C-1"-H2 or C-2"-H2), 4.19 (2H, dt, J= 5.7 Hz, J = 2.9 Hz, C-1"-H2 or C-2"-H2), 5.04 (2H, s, OCH2Ar), 6.74 (1H, d, JC-2-H, C- 4-H = 2.7 Hz, C-4-H), 6.79 (1H, dd, JC-1-H, C-2-H = 8. 6 Hz and JC-4-H, C-2-H = 2. 7 HO, C-2-H), 7.19-7. 21 (2H, m, C-1-H and C-3'-H) and 7.30-7. 44 (5H, m, CHO) ; MS m/z (FAB+) 443.3 [100, (M+H)'], 91. 1 [95, (CH2Ar) +], 73.1 [44] ; Ace MS mlz (FAB+) 443. 26964, C29H35N2O2 requires 443. 26985.
General procedure for hydrogenation of benzylated derivatives.
A suspension of Pd-C (10%) in THF (2 mL) was added to a stirred solution of the benzylated precursor in MeOH/THF (2: 1,30 mL) and the resulting suspension hydrogenated at room temperature using a hydrogen-filled balloon. After removal of the supported catalyst by filtration and evaporation of the filtrate in vacuo, the product obtained was purified by recrystallization to give the parent compound.
1'-Methyl-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03100, STX 785) Following the hydrogenation conditions, a suspension of 3-0-benzyl-estra-1, 3,5 (10) - trieno- (17, 16-c)--N-methyl-pyrazole (50 mg, 0.12 mmol) and Pd-C (20 mg) was hydrogenated overnight to give the product as a pale yellow solid (31 mg). This was recrystallized from EtOH/H20 to give creamy crystals (26 mg, 68%): TLC (chloroform/EtOAc, 9 : 1) Rf 0. 16 cf. Rf 0. 50; 8H (DMSO-d6, 400 MHz) 0.89 (3H, s, C-18- H3), 1.32-2. 57 (11H, m), 2.69-2. 83 (2H, m, C-6-H2), 3.73 (3H, s, N-Me), 6.45 (1H, d, JC-2- H, C-4-H = 2.4 Hz, C-4-H), 6.52 (1H, dd, JC-1-H,C-2-H = 8. 5 Hz and JC-4-H,C-2-H = 2. 4 Hz, C-2-H), 7.06 (1H, d, JC-2-H, C-1-H = 8.5 Hz, C-1-H), 7.24 (1H, s, C-5'-H) and 9.02 (1H, s, OH); MS m/z (FAB+) 309.2 [100, (M+H) +], 219.2 [52] ; Ace MS mlz (FAB+) 309.19747, C20H25N2O requires 309.19669.
2'-Methyl-3-Hydroxy-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole (DSF 03102,. STX 786) Following the hydrogenation conditions, a suspension of 3-0-benzyl-estra-1, 3,5 (10) - trieno- (17, 16-c)--N-methyl-pyrazole (50 mg, 0.12 mmol) and Pd-C (20 mg) was hydrogenated overnight to give the product as a white solid (40 mg). This was recrystallized from EtOH to give white crystals (20 mg, 53%): TLC (chloroform/EtOAc, 9: 1) R, 0. 13 cf. Rf 0. 32; #H(DMSO-d6, 400 MHz) 0. 95 (3H, s, C-18-H3), 1. 32-2. 52 (11H, m), 2.69-2. 83 (2H, m, C-6-H2), 3.73 (3H, s, N-Me), 6.45 (1H, d, JC-2-H,C-4-H = 2. 6 Hz, C-4- H), 6.52 (1H, dd, JC-1-H, C-2-H = 8. 4 Hz and Jc-4-H, C-2-H = 2.6 Hz, C-2-H), 7.04 (1H, s, C-3'-H), 7.06 (1H, d, JC-2-H,C-1-H = 8.4 Hz, C-1-H) and 9.03 (1H, s, OH); MS m/z (FAB+) 663.5 [24], 391.3 [28], 309.2 [100, (M+H)+]; Acc MS m/z (FAB+) 309.19737, C20H25N2O requires 309.19669.
2'-Isobutyl-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03113, STX 813) Following the hydrogenation conditions, a suspension of 3-0-benzyl-estra-1, 3,5 (10) - trieno- (17, 16-c)--N-isobutyl-pyrazole (45 mg, 0.10 mmol) and Pd-C (20 mg) was hydrogenated overnight to give the product as a light yellow solid (103 mg). This was , recrystallized from EtOAc/hexane to give creamy crystals (33 mg, 92%): TLC (chioroform/EtOAc, 9 : 1) Rf 0. 30 cf. Rf 0. 57; #H(CDCl3 400 MHz) 0. 92 (3H, d, JC-2"-H, C4"-H = 6. 6 Hz, C-3"-H3), 0.95 (3H, d, JC-2"-H,C-4"-H = 6.6 Hz, C-4"-H3), 1.03 (3H, s, C-18-H3), 1.41-2. 63 (12H, m), 2.84-2. 91 (2H, m, C-6-H2), 3.77 (1H, dd, JBA = 13. 4 Hz and JC-2"-H, C-1"- H = 8.2 Hz, N-CHAHB), 3.89 (1H, dd, JAB = 13.4 Hz and JC-2"-H, C-1"-H = 7.6 Hz, N-CHAHB), -5. 50 (-1H, brs, exchanged with D20,. OH), 6.60 (1 H, d, Jc-2-H, C4-H = 2.6 Hz, C-4-H), 6.66 (1H, dd, JC-1-H, C-2-H = 8. 4 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 7. 14 (IH, d, JC-2-H, C-1-H = 8. 4 Hz, C-1-H) and 7.22 (1H, s, C-5'-H); MS m/z (FAB+) 351.3 [100, (M+H)+] ; Acc MS m/z (FAB+) 351.24437, C23H31 N2O requires 351.24364.
1'-Isobutyl-3-Hydroxy-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole (DSF 03115, STX 812). Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1, 3,5 (10)- trieno- (17, 16-c)--N-isobutyl-pyrazole (110 mg, 0.25 mmol) and Pd-C (40 mg) was hydrogenated overnight to give the product as a white solid (78 mg). This was recrystallized from MeOH to give colourless crystals (42 mg, 48%) : mp 122-123°C ; TLC (chloroform/EtOAc, 9 : 1) Rf 0. 29 cf. Rf 0. 72 ; SH (CDCl3, 400 MHz) 0. 87 (3H, d, JC-2"-H, c-3"-H = 6.4 Hz, C-3"-H3), 0.89 (3H, d, JC-2"-H,C-4"-H = 6.4 Hz, C-4"-H3), 1.01 (3H, s, C-18-H3), 1. 42-2. 65 (12H, m), 2. 81-2.95 (2H, m, C-6-H2), 3.81 (1H, dd, JBA = 13.7 Hz and JC-2"-H,C-1"- H = 7.4 Hz, N-CHAHB), 3.86 91H, dd, JAB = 13.7 Hz and JC-2"-H,C-1"-H = 7.4 Hz, N-CHAHB), 5.61 (1H, s, exchanged with D2O, OH), 6.59 (1H, d, JC-2-H,C-4-H = 2.6 Hz, C-4-H), 6.65 (1H, dd, JC-1-H,C-2-H = 8.4 Hz and JC-4-H,C-2-H = 2. 6 Hz, C-2-H), 6. 97 (1H, s, C-3'-H) and 7. 16 (1H, d, JC-2-H, C-1-H = 8. 4 Hz, C-1-H) ; MS mlz (FAB+) 351. 3 [100, (M+H)+] ; Ace MS m/z (FAB+) 351. 24485, C23H31N2O requires 351.24364. Found: C, 75. 20; H, 8. 79 ; N, 7. 42.
C23H3oN20. MeOH requires : C, 75.35 ; H, 8.96 ; N, 7.32.
1'-Methylacetate-3-Hydroxy-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole (DSF 03129, STX 806).
Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1, 3, 5. (10) - trieno- (17, 16-c)--N-methylacetate-pyrazole (90 mg, 0.20 mmol) and Pd-C (40 mg) was hydrogenated for 6 hours to give the product as a white solid (66 mg). This was recrystallized from MeOH to give colburless crystals (49 mg, 68%): TLC (DCM/EtOAc, 9: 1) Rf 0. 15 cf. RfO. 55; #H(CDCl3, 400 MHz) 1.03 (3H, s, C-18-H3), 1.41-2. 69 (11H, m), 2.81-2. 96 (2H, m, C-6-H2), 3.75 (3H, s, OMe), 4.83 (1H, d, JBA = 17.6 Hz, N-CHAHB), 4.88 (1H, d, JAB = 17.6 Hz, N-CHAHB), 5.04 (1H, s, OH), 6.58 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H) and 6.64 (1H, dd, JC-1-H, C-2-H = 8. 4 Hz and JC-4-H, C-2-H = 2.7 Hz, C-2-H), 7.07 (1H, s, C-5'-H), 7.16 (1H, d, JC-2-H, C-1-H = 8. 4 Hz, C-1-H); MS m/z (FAB+) 367. 3 [100, (M+H)+], 176.1 [23], 73.1 [292]; Acc MS m/z (FAB+) 367. 20383, C22H27N2O3 requires 367.20217.
2'-Methylacetate-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03131, STX 807). Following the hydrogenation conditions, a suspension of 3-O-benzyl-estra-1, 3, 5 (10)- trieno- (17, 16-c)--N-methylacetate-pyrazole (60 mg, 0. 13 mmol) and Pd-C (25 mg) was hydrogenated overnight to give the product as a light brown foam (34 mg). This was purified by flash chromatography with DCM/EtOAc (8: 2) as eluent and gave the prodcut as a light yellow foam (18 mg, 37%): TLC (DCM/EtOAc, 9: 1) Rf 0.18 cf. Rf 0.37 ; # 6 H (CDCI3, 400 MHz) 1.02 (3H, s, C-18-H3), 1.42-2. 64 (12H, m), 2.84-2. 91 (2H, m, C-6-H2), 3. 77 (3H, s, OMe), 4.81 (1H, d, JBA = 17.4 Hz, N-CHAHB), 4.87 (1H, d, JAB = 17.4 Hz, N- CHAHB), 6.59 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.64 (1H, dd, JC-1-H,C-2-H = 8.6 Hz and JC-4- H,C-2-H = 2.7 Hz, C-2-H), 7.12 (1H, d, JC-2-H, C-1-H = 8. 6 Hz, C-1-H) and-7. 25 (-1H, s, C-3'-H, under solvent peak).
1'- (2-Methoxyethyl)-3-Hydroxy-estra-1, 3,5 (10)-trieno- (17, 16-c)-pyrazole (DSF 03133, STX 808) Following the hydrogenation conditions, a suspension of 3-0-benzyl-estra-1, 3,5 (10) - trieno- (17, 16-c)--N- (2-methoxyethyl)-pyrazole (80 mg, 0.18 mmol) and Pd-C (35 mg) was hydrogenated for 6 hours to give the product as a white crystalline solid (58 mg).
This was recrystallized from MeOH to give white crystals (32 mg, 50%): TLC (DCM/EtOAc, 9: 1) Rf 0. 11 cf. Rf 0. 28; #H(CDCl3, 400 MHz) 1. 02 (3H, s, C-1 8-H3), 1. 43- 2. 66 (11H, m), 2. 81-2. 93 (2H, m, C-6-H2), 3.33 (3H, s, OMe), 3.71 (2H, td, J = 5.5 Hz, J - 1 Hz, CHO or C-2"-H2), 4. 23 (2H, app t, J # 5 Hz, C-1"-H2 or C-2"-H2), 5.57 (1H, br s, OH), 6.59 (1H, d, JC-2-H, C-4-H = 2.7 Hz, C-4-H), 6.64 (1H, dd, JC-1-H, C-2~H = 8.4 Hz and JC-4- H, C-2-H = 2. 7 Hz, C-2-H), 7. 09 (1H, s, C-5'-H) and 7. 16 (1H, d, JC-2-H, C-1-H = 8. 4 Hz, C-1-H) ; MS mlz (FAB+) 353.3 [100, (M+H) +]. 2'- (2-Methoxyethyl)-3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (DSF 03135, STX 809).
Following the hydrogenation conditions, a suspension of 3-0-benzyl-estra-1, 3,5 (10) - trieno- (17, 16-c)--N- (2-methoxyethyl)-pyrazole (70 mg, 0.16 mmol) and Pd-C (30 mg) was hydrogenated for overnight to give the product as a creamy foam (50 mg). This was purified by flash chromatography using DCM/EtOAc (8: 2) as eluent and gave the product as a creamy oil (25 mg, 45%): TLC (DCM/EtOAc, 9: 1) Rf 0. 15 cf. Rf 0. 27; #H (CDCl3, 400 MHz) 1.05 (3H, s, C-18-H3), 1.42-2. 61 (12H, m), 2.82-2. 95 (2H, m, C-6-H2), 3.31 (3H, s, OMe), 3.74-3. 82 (2H, m, C-1"-H2 or C-2"-H2), 4.15-4. 24 (2H, m, C-1"-H2 or C-2"-H2), 6.60 (1 H, d, JC-2-H, C-4-H = 2. 6 Hz, C-4-H), 6.66 (1 H, dd, JC-1-H, C-2-H = 8.6 Hz and JC-4-H,C-2-H = 2.6 Hz, C-2-H), 7.14 (1H, d, JC-2-H, C-1-H = 8. 6 Hz, C-1-H) and 7.23 (1H, s, C-3'-H); MS m/z (FAB+) 353.3 [100, (M+H) +] ; Acc MS m/z (FAB+) 353.22359, C22H29N2O2 requires 353.22290.
3-Benzyloxy-16-nitrile, 16-methyl-estrone (DSF03151/DSF03163) NaH (60% dispersion in mineral oil, 1. 2 eq., 0. 248 mmol, 10 mg) was added to a stirred solution of 3-benzyloxy-16-nitrile-estrone (0.207 mmol, 80 mg) in anhydrous DMF (5 mL) at 0°C under an atmosphere of N2. After 30 minutes of stirring, methyl iodide (0. 310 mmol, 19 ju. L) was added to the white suspension and the mixture was stirred for 3 h, in which time it was allowed to warm to room temperature. The resulting light yellow solution was then poured into water (50 mL) and the organics were extracted with EtOAc (3#20 mL), washed with brine (2#20 mL), dried (Na2SO4), filtered and evaporated in vacuo. The crude product was purified by flash chromatography with CH2CI2/hexane (3: 2) as eluent, to give the product as a crystalline light yellow solid (60 mg, 72%) : sH

DMSO-d6, 270 MHz) 1.02 (3H, s, C-18-H3), 1.25-2.54 (11H, m), 1.46 (#3H, s, C-16-H3), 2. 79-2. 83 (2H, m, C-6-H2), 5. 06 (2H, s, OCH2Ar), 6. 73-6. 80 (2H, m, C-4-H and C-2-H), 7.18 (1H, d, JC-1-H, C-1-H = 8.4 Hz, C-1-H), 7.29-7.46 (5H, m, C6H5) ; Found : C, 80. 80 ; H, 7. 45 N, 3.45. C27H29N2O requires: C, 81.17; H, 7.32; N, 3.51.
16-Nitrile,16-methyl-estrone (DSF03170, STX924) Pd/C (10%, 40 mg) was added to a stirred solution of 3-benzyloxy-16-nitrile, 16-methyl- estrone (90 mg, 0.225 mmol) in a mixture of MeOH/THF (1: 1,40 mL). The resulting suspension was hydrogenated at room temperature overnight using a hydrogen-filled balloon. The final mixture was then filtered, the filter cake washed several times with boiling MeOH and THF, and concentrated in vacuo to give a crude white foam. This was purified by flash chromatography with CH2CI2/EtOAc (9: 1) as eluent to give the product as a white foam (46 mg, 66%). A sample was then triturated in hexane to give a creamy powder: 8H (CDCI3, 270 MHz) 1.12 (3H, s, C-18-H3), 1.32-2. 48 (11H, m), 1.51 (#3H, s, C- 16-H3), 2.82-2. 92 (2H, m, C-6-H2), 4.55 (1H, s, C-3-OH), 6. 73-6.58 (1H, app d, C-4-H), 6. 63 (1H, dd, JC-1-H,C-2-H = 8.5 Hz, JC-4-H,C-2-H = 2.5 Hz, C-2-H), 7.13 (1H, d, JC-2-H, C-1-H = 8.5 Hz, C-1-H); MS mlz (FAB+) 309.1 [60, M+], 97.1 [50], 83.0 [65]; Acc MS m/z (FAB+) 309.12761, C2oH23NO2 requires 309.12788. HPLC (methanol/water, 80: 20, amas = 280.5 nm) Rt = 2.53 min, 98%. l'-Propionitrile-3-0-tert-Butyl-dimethylsilyt-estra-1, 3,5 (10)-trieno-(17, 16-c) - pyrazole (DSF03145A) KOC (CH3) 3 (0. 807 mmol, 91 mg) was added to a stirred solution of 3-0-tert-butyl- dimethylsilyl-estra-1, 3, 5 (10)-trieno-(17, 16-c)-pyrazole (0.734 mmol, 300 mg) in anhydrous THF (10 mL) at 0°C under an-atmosphere of N2. After 20 minutes of stirring, acrylonitrile (0. 881 mmol, 58 µL) was added and the resulting bright orange solution was stirred for 5 h, in which time at room temperature. The resulting dark orange cloudy mixture was then concentrated in vacuo and water (50 mL) was added. The organics were extracted with EtOAc (2#50 mL), washed with water (2X30 mL), then brine (2X30 mL), dried (Na2SO4), filtered and evaporated in vacuo. The crude product was purified by flash chromatography with CH2CI2/EtOAc (9: 1) as eluent to give two products, one of which was DSF03145A, as a light yellow solid (29 mg, 8%) : aH (CDCI3, 400 MHz) 0.20 (6H, s, Si (CH3) 2), 0.98 (9H, s, C (CH3) 3), 1.02 (3H, s, C-18-H3), 1.42-2. 67 (11H, m), 2.82- 2.91 (4H, m, C-6-H2 and CH2CN), 4.28-4. 40 (2H, m, NCH2), 6.57 (1H, d, JC-2-H, C-4-H = 2.6 Hz), 6.63 (1H, dd, JC-1-H, C-2-H = 8. 5 Hz, JC-4-H,C-2-H = 2. 6 Hz, C-2-H), 7.10 (1H, s, C-5'-H), 7.14 (1H, d, JC-2-H,C-1-H = 8.5 Hz, C-1-H).
3-Hydroxy-estra-1, 3, 5 (10)-trieno-(17, 16-c)-N-propionitrile-pyrazole (DSF03153, STX921).
TBAF (0.110 mmol, 0.11 mL) was added to a stirred solution of 3-TBDMS-N-propionitrile (25 mg, 0.050 mmol) in anhydrous THF (5 mL) at room temperature under an atmosphere of N2. The mixture was stirred for 3 h at room temperature. The solvent was then removed in vacuo and water added (50 mL). The organics were extracted with EtOAc (50 mL + 20 mL), washed with water (20 mL), then brine (20 mL), dried (Na2SO4), filtered and evaporated in vacuo. The crude yellow oil was purified by flash chromatography with CH2CI2/EtOAc (8 : 2) as eluent to give a light yellow oil (12 mg, 63%). This was then precipitated from EtOAc/Hexane to give a light yellow powder (8 mg) : sH (CDCI3, 400 MHz) 0. 94 (3H, s, C-18-H3), 1. 34-2. 60 (H, m), 2. 76-2. 86 (4H, m, C- 6-H2 and CH2CN), 4.19-4. 31 (2H, m, NCH2), 4.92 (1H, br s, exchanged with D20, C-3- OH), 6.50 (1H, d, JC-2-H, C-4-H= 2. 7 Hz), 6.55 (1H, dd, JC-1-H, C-2-H = 8.4 Hz, JC-4-H, C-2-H = 2. 7 Hz, C-2-H), 7.02 (1 H, s, C-5'-H), 7. 08 (1H, d, JC-2-H,C-1-H = 8.4 Hz, C-1-H) ; MS m/z (APCI) 348. 3 [32, (M+H) +], 141. 0 [20]. HPLC (methanol/water, 90: 10, #max = 229.8 nm) Rt = 2. 27 min, 96%.
16-(2',2'-Dimethyl-propylidene)-estradiol (STX948) A solution of NaBH4 (44 mg, 1.16 mmol) in H20 (2.5 mL) was added to a stirred solution of STX748 in a mixture of MeOH/THF (2: 1,15 mL) at 0°C. The colourless solution was stirred for 1 hour at 0°C, after which AcOH glacial (4 drops) was added, followed by 10% aq. NaCI solution (15 mL). The resulting white precipitate was filtered, dried (79 mg) and recrystallised from EtOAc/Hexane (1: 8) to give 68 as white crystals (63 mg, 79%): mp 219-222°C ; IR (KBr) 3550,3410 (OH), 2960-2875 (aliph CH), 1610, 1505 (aliph C=C and arom C=C) cm-1 ; #H (CDCI3, 400 MHz) 0'65 (3H, s, C-18-H3), 1.10 (9H, s, C 9CH3)3), 1. 18-2.59 (12H, m),'2. 82-2.88 (2H, m, C-6-H2), 3.85-3. 90 (1H, app d, J = 9. 4 Hz, C-17- H), 4.60 (1H, s, exchanged with D2O, OH), 5.46 (1H, dd, J = 4.9 Hz, J = 2.5 Hz, C-1'-H), 6.55 (1H, d, J = 2.8 Hz, C-4-H), 6.61 (1H, dd, J = 8.5 Hz, J = 2.8 Hz, C-2-H) and 7.14 91H, d, J = 8.5 Hz, C-1-H); 8c (CDCI3, 100.4 MHz) 11.4 (q, C-18), 26.8 (t), 27. 8 (t), 29.1 (t), 30.0 (t), 30.8 (3xq, C (CH3) 3), 33.5 (s, C (CH3) 3), 36.8 (t), 38.7 (d), 42.9 (s, C-13), 44.4 (d), 47.5 (d), 85. 0 (dj C-17), 113.0 (d), 115.5 (d), 126.7 (d), 132.7 (s), 133.7 (d, C-1'), 138.3 (s), 140.2 (s) and 153.6 (s, C-3); MS m/z (FAB+) 340.2 [49, M+], 323.2 [100, (M- OH) +], 283.2 [92, (M-C (CH3) 3) +] ; Acc MS m/z (FAB+) 340.2390, C23H32O2 requires 340.2402. HPLC (MeOH/H2O, 96: 4, amas = 280.5 nm) Rt = 2.54 min, 99.5%. Found: C, 81. 00; H, 9.54. C23H3202 requires : C, 81. 13; H, 9.47%.
Synthesis of N-propionitrile [17-16-cl-pyrazole derivatives 3-O-tert-Butyl-dimethylsilyl-estrone (1) Imidazole (6.3 g, 92.4 mmol) and tert-butyl-dimethylsilyl chloride (7.8 g, 51. 8 mmol) were added to a stirred solution of E1 (10.0 g, 37.0 mmol) in anhydrous DMF (300 mL) at room temperature, under an atmosphere of N2. After stirring overnight, the resulting white suspension was poured into H20 (500 mL). The organics were extracted with EtOAc (500 mL + 300 mL), washed with H20 (2#200 mL), then brine (2#200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was recrystallised from EtOh to give 1 as white needles (14. 0 g, 98%) : mp 171-173°C [lit. {Fevig, 1987} (EtOH) 170-172°C]; IR (KBr) 2960-2855 (aliph CH), 1730 (C=O), 1605, 1495 (arom C=C) cm-' ; Sn (CDCI3, 400 MHz) 0.19 (6H, s, Si(CH3)2), 0.91 (3H, s, C-18- H3), 0. 98 (9H, s, C (CH3) 3), 1. 37-2. 55 (13H, m), 2. 81-2. 89 (2H, m, C-6-H2), 6.57 (1H, d, J = 2. 6, Hz, C-4-H), 6.62 (1H, dd, J = 8.3 Hz, J = 2.6 Hz, C-2-H) and 7. 12 (1H, d, J = 8. 3 Hz, C-1-H); MS m/z (FAB+) 384.2 [100, M+], 327.2 [44, (M-C (CH3) 3) +], 73.0 [44] ; Acc MS m/z (FAB+) 384.2477, C24H3602Si requires 384.2485.
3-0-tert-Butyl-dimethylsilyl-16-hydroxymethylene-estrone (2) NaOMe (4.8 g, 70.2 mmol) was added portionwise to a stirred solution of 1 (9 g, 23.4 mmol) in anhydrous toluene (250 mL) at room temperature, under an atmosphere of N2.
After stirring for 20 minutes, ethyl formate (13.2 mL, 164 mmol) was added and the resulting bright yellow solution was stirred overnight. The final thick yellow suspension was poured into H20 (300 mL) and acidified with 5M HCI. The organics were extracted with EtOAc (2x500 mL), washed with H20 (3x200 mL), then brine (2x200 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give an off-white crude product (9.49 g, 98%). For analysis a sample was recrystallised from EtOH to give 2 as white crystals: mp 168-171°C; IR (KBr) 2930-2855 (aliph CH), 1710 (C=O), 1695-1500, 1495 (C=C and arom C=C) cm-1 ; #H (DMSO-d6, 400 MHz) 0.17 (6H, s, Si (CH3) 2), 0.83 (3H, s, C-18-H3), 0.96 (9H, s, C (CH3) 3), 1.32-2. 63 (11H, m), 2.77-2. 85 (2H, m, C-6-H2), 6. 55 (1H, d, J = 2. 6 Hz, C-4-H), 6.61 (1H, dd, J= 8. 3 Hz, J= 2.6 Hz, C-2-H), 7. 14 (1H, d, J = 8. 3 Hz, C-1-H), 7. 40 (1H, s, =CHOH) and 10. 69 (1H, s, exchanged with D20, =CHOH) ; 5c (DMSO-d6, 100. 4 MHz)-4. 3 (2xq, Si (CH3) 2), 14. 5 (q, C-18), 18. 0 (s, C (CH3) 3), 24.1 (t), 25.6 (t and 3xq, C (CH3) 3), 26.2 (t), 29.0 (t), 31.5 (t), 37.3 (d), 43. 6 (d), 47.7 (s, C-13), 48.4 (d), 113.0 (s, C-16), 116.9 (d), 119.4 (d), 125.9 (d), 132.5 (s), 137.3 (s), 149.9 (d, C-1'), 152. 5 (s, C-3) and 208.2 (s, C=O) ; MS m/z (FAB+) 413.2 [68, (M+H) +], 355.1 [33, (M-C (CH3) g) , 72. 9 [100] ; Ace MS m/z (FAB+) 413.2492, C25H3703Si requires 413. 2512. Found : C, 72. 77 ; H, 8. 79. C25H3603Si requires : C, 72. 40 ; H, 8. 90%.
3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,16-c]-pyrazole (3) Hydrazine hydrate (440 µl, 9. 1 mmol) was added to a suspension of 2 (2. 5 g, 6. 1 mmol) in EtOH (125 mL) at room temperature, under an atmosphere of N2. The resulting mixture was heated to reflux for 45 minutes. After cooling down, the solvent was removed under reduced pressure, H20 (200 mL) added and the mixture acidified with 5M HCI. The organics were extracted with EtOAc (2x10 mL), washed with H20 (2x50 mL), then brine (2x50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a yellow foam (2.9 g). This was recrystallised from EtOH/H20 to give 3 as yellow crystals (2.0 g, 81%): mp 122-124°C ; IR (KBr) 3190 (arom CH), 2930-2855 (aliph CH), 1605-1495 (C=N and arom C=C) cm-' ; 5H (DMSO-d6, 400 MHz) 0.18 (6H, s, Si (CH3) 2), 0.95 (3H, s, C-18-H3), 0.96 (9H, s, C (CH3) 3), 1.36-2. 61 (11H, m), 2.79-2. 87 (2H, m, C-6-H2), 6.55 (1H, d, J = 2.3 Hz, C-4-H), 6.62 (1H, dd, J = 8.5 Hz, J = 2.3 Hz, C- 2-H), 7.16 (1H, d, J = 8.5 Hz, C-1-H), 7. 28 (1H, s, C-5'-H) and 12.02 (1H, s, exchanged with D20, NH); aC (CDCI3, 100.4 MHz) -3.8 (2xq, Si (CH3) 2), 18. 6 (s, C (CH3) 3), 18. 8 (q, C- 18), 24.3 (t), 26.2 (3xq, C (CH3) 3), 26.6 (t), 27.9 (t), 30.0 (t), 34.5 (t), 38.0 (d), 41.1 (s, C- 13), 44.9 (d), 61.9 (d), 117.4 (d), 120.2 (d), 121.8 (s), 123.2 (s), 126.2 (d), 133.2 (s), 137.8 (s), 153.5 (s) and 168.6 (s); MS m/z (FAB+) 409.2 [100, (M+H) +], 73.0 [33]; Acc MS m/z (FAB+) 409. 2662, C25H37N2OSi requires 409.2675. Found: C, 71.40 ; H, 8. 93; N, 6.53. C25H36N20Si. (H20) 2/3 requires: C, 71. 38 ; H, 8.95 ; N, 6.66%.
3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,16-c]-(1'-propionitrile)- pyrazole (4) and 3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,16-c]-(2'- propionitrile)-pyrazole (5) 'BuOK (91 mg, 807 yImol) was added to a stirred solution of 3 (300 mg, 734 µmol) in anhydrous THF (10 mL) at 0°C, under an atmosphere of N2. After 20 minutes of stirring, acrylonitrile (58 µL, 881 µmol) was added and the resulting bright orange solution was stirred for 5 hours at room temperature. The resulting dark orange mixture was then concentrated under reduced pressure and H20 (50 mL) was added. The organics were extracted with EtOAc (250 mL), washed with H20 (2X30 mL), then brine (2X30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM/EtOAc, 9 : 1) to give two products : The less polar fraction gave 4 as a light yellow oil (29 mg, 8%) : IR (KBr) 2930-2860 (aliph CH), 2250 (CN), 1640-1495 (C=N and arom C=C), 1250 (C-O or Si(CH3)2) cm-1 ; aH (CDCI3, 400 MHz) 0. 20 (6H, s, Si(CH3)2), 0.98 (9H, s, C (CH3) 3), 1.02 (3H, s, C-18-H3), 1.42-2. 67 (11H, m), 2.82-2. 91 (4H, m, C-6-H2 and CH2CN), 4.28-4. 40 (2H, m, N-CH2), 6.57 (1H, d, J = 2. 6 Hz, C-4-H), 6.63 (1H, dd, J = 8.5 Hz, J = 2.6 Hz, C-2-H), 7.10 (1H, s, C-5'-H) and 7.14 (1H, d, J = 8. 5 Hz, C-1-H) ; MS m/z (FAB+) 462.2 [100, (M+H) +], 73.0 [55]; Acc MS m/z (FAB+) 462. 2914, C28H40N3OSi requires 462. 2941.
The more polar fraction gave 5 as'a light brown oil that crystallises on standing (42 mg, 12%): mp 80-82°C ; IR (KBr) 2930-2855 (aliph CH), 2255 (CN), 1705-1445 (C=N and arom C=C), 1285,1250 (C-O or Si (CH3) 2) cm-1 ; 8H (CDCI3, 400 MHz) 0.20 (6H, s, Si (CH3) 2), 0.98 (9H, s, C (CH3) 3), 1.07 (3H, s, C-18-H3), 1.41-2. 91 (13H, m), 2.99 (2H, t, J = 6.6 Hz, CH2CN), 4.21-4. 38 (2H, m, N-CH2), 6.57 (1H, d, J = 2.4 Hz, C-4-H), 6.63 (1H, dd, J = 8. 7 Hz, J = 2. 4 Hz, C-2-H), 7. 11 (1H, d, J = 8. 7 Hz, C-1-H) and 7.25 (1 H, s, C-5'- H); MS m/z (FAB+) 462. 1 [100, (M+H) +], 73. 0 [79] ; Acc MS m/z (FAB+) 462. 2922, C28H40N3OSi requires 462. 2941.
3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(1'-propionitrile)-pyrazole (STX921) A 1. 0 M solution of TBAF in anhydrous THF (110 µL, 110 µmol) was added to a stirred solution of 4 (25 mg, 50 Fmol) in anhydrous THF (5 mL) at room temperature, under an atmosphere of N2. The mixture was stirred for 3 hours at room temperature, after which the solvent was removed under reduced pressure and H20 added (50 mL). The organics were extracted with EtOAc (70 mL), washed with H20 (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude yellow oil was purified by flash chromatography (DCM/EtOAc, 8: 2) to give STX921 as a light yellow oil (12 mg, 63%). This was then triturated with EtOAc/hexane to give a light yellow powder (8 mg) : mp 92-94°C ; R (KBr) 3230 (OH), 3095 (arom CH), 2925-2860 (aliph CH), 2265 (CN), 1665-1500 (C=N and arom C=C), 1245 (C-O) cm-1 ; aH (CDCI3, 400 MHz) 0.94 (3H, s, C-18-H3), 1.34-2. 60 (11H, m), 2.76-2. 86 (4H, m, C-6-H2 and CH2CN), 4.19-4. 31 (2H, m, N-CH2), 4. 92 (1H, br s, exchanged with D20, OH), 6. 50 (1H, d, J = 2.7 Hz, C-4-H), 6.55 (1H, dd, J = 8.4 Hz, J = 2.7 Hz, C-2-H), 7.02 (1H, s, C-5'-H) and 7. 08 (1H, d, J = 8.4 Hz, C-1-H); MS m/z (FAB+) 348.1 [90, (M+H) +], 147.1 [64], 73.0 [100]; Ace MS m/z (FAB+) 348.2072, C22H26N3O requires 348.2076. HPLC (MeOH/H2O, 90: 10, AmaX = 229.8 nm) Rt = 2. 27 min, 96%.
3-Hydroxy-estra-1, 3,5 (10)-tr ! ene- [17, 16-c]- (2'-propionitrHe)-pyrazote Method A : A 1.0 M solution of TBAF in anhydrous THF (150 pL, 150 pmol) was added to a stirred solution of 5 (35 mg, 76 llmol) in anhydrous THF (5 mL) at room temperature, under an atmosphere of N2. The mixture was stirred for 3 hours at room temperature, after which the solvent was removed under reduced pressure and H20 added (50 mL). The organics were extracted with EtOAc (70 mL), washed with H20 (20 mL), then brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude brown oil was purified by flash chromatography (DCM/EtOAc, 8: 2) to give 3-Hydroxy-estra-1, 3,5 (10)-triene- [17, 16-c]- (2'-propionitrile)-pyrazole as a light brown oil (10 mg, 38%). method E : A 1. 0 M solution of TBAF in anhydrous THF (916 µL, 916 µmol) was added to a stirred solution of 6 of mg, 458 µmol) in anhydrous THF (20 mL) at room temperature, under an atmosphere of N2. The mixture was stirred overnight at room temperature after which the solvent was removed under reduced pressure and H20 added (50 mL). The organics were extracted with EtOAc (2x50 mL), washed with H2O (2x30 mL), then brine (2X30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc/CHCI3, 9: 1) to give 3-Hydroxy-estra-1,3, 5 (10)-triene- [17, 16-c]- (2'-propionitrile)- pyrazol as a pale pink foam (136 mg, 85%). This was then triturated with EtOAc/Hexane to give white crystals (99 mg, 62%) : mp 198-200°C ; IR (KBr) 3160 (br, OH), 2925-2860 (aliph CH), 2250 (CN), 1610-1500 (arom C=C) cm-1 ; aH (CDCI3, 400 MHz) 1.06 (3H, s, C-18-H3), 1.41-2. 63 (11H, m), 2.82-2. 91 (2H, m, C-6-H2), 2.99 (2H, app td, J = 6.8 Hz, J = 3.5 Hz, CH2CN), 4.22-4. 34 (2H, m, N-CH2), 5. 42 (1H, br s, exchanged with D20, OH), 6.57 (1 H, d, J = 2 : 4 Hz, C-4-H), 6.64 (1 H, dd, J = 8.3 Hz, J = 2. 4 Hz, C-2-H), 7. 12 (1H, d, J = 8.3 Hz, C-1-H) and 7.25 (1H, s, C-5'-H); 8c (CDCI3, 100.4 MHz) 18.6 (q, C-18), 19.7 (t, CH2CN), 24.5 (t), 26.5 (t), 27.6 (t), 29.8 (t), 34.8 (t), 37.7 (d), 42.4 (s, C-13), 44.4 (d), 45.9 (t, N-CH2), 62.2 (d), 113.1 (d), 115.7 (d), 117.4 (s), 124.3 (s), 126.4 (d), 132.0 (s), 135.2 (d), 138.2 (s), 154.1 (s) and 157.98 (s) ; MS m/z (FAB+) 348.1 [100, (M+H) +], 147.0 [50], 85.1 [75], 73.0 [94]; Acc MS m/z (FAB+) 348.2060, C22H26N3O requires 348. 2076. Found: C, 75.90 ; H, 7.17 ; N, 12.10. C22H25N3O requires: C, 76.05 ; H, 7.25 ; N, 12.09%.
3-O-tert-Butyl-dimethylsilyl-estra-1, 3,5 (10)-triene-[17,16-c]-(2'-propionitrile-3', 4'- dihydro)-pyrazol-3'-ol (6) Cyanoethylhydrazine (177 pL, 2. 18 mmol) was added toi a stirred solution of 2 (600 mg, 1.45 mmol) in EtOH (50 mL) under an atmosphere of N2. After 4 hours of stirring at room temperature, the resulting orange mixture was concentrated under reduced pressure and H20 (100 mL) added, followed by 5M HCI. The organics were extracted with EtOAc (2X100 mL), washed with H20 (50 mL), then brine (50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified flash chromatography (CHOg/EtOAc, 7 : 3) to give 6 as a pale yellow solid (510 mg, 73%). This was recrystallised from EtOH to give white needles (240 mg, 34%): IR (KBr) 3380 (OH),



2950-2860 (aliph CH), 2260 (CN), 1610, 1495 (C=N and arom C=C) cm-1; #H (DMSO-d6, 400 MHz) 0. 15 (6H, s, Si(CH3)2), 0. 88 (3H, s, C-18-H3), 0. 93 (9H, s, 0 (3) 3), 1. 19-3. 16 (11H, m), 2. 70-3. 16 (7H, m), 5. 97 (1H, s, exchanged with D2O, OH), 6. 48 (1H, d, J = 2.7 Hz, C-4-H), 6. 56 (1H, dd, J = 8. 2 Hz, J = 2. 7 Hz, C-2-H), 6. 74 (1H, d, J = 1. 9 Hz, C-5'-H) and 7. 10 (1H, d, J = 8. 2 Hz, C-1-H) ; 8c (CDCl3, 100. 4 MHz)-4. 4 (2xq, Si(CH3)2), 15.6 (q, C-18), 17.1 (t, CH2CN), 18. 0 (s, C (CH3) 3), 25.6 (3xq, C(CH3) 3), 25. 8 (t), 27. 0 (t), 29. 1 (t), 29.4 (t), 31.4 (t), 38.6 (d), 43.2 (d), 43. 9 (t, N-CH2), 47.1 (s, C-13), 49. 5 (d), 56.7 (d), 104.2 (s), 116.8 (d), 119.4 (d), 119. 9 (s, CN), 126.0 (d), 132.5 (s), 137.3 (s), 144.6 (s) and 152.4 (s); MS m/z (FAB+) 480.1 [100, (M+H) +], 462.1 [63, (M+H-H2O)+], 72.9 [77]; MS m/z (FAB-) 632.3 [32, (M+NBA) -] ; Acc MS m/z (FAB+) 480. 3038 C28H42N302Si requires 480. 3046.
Synthesis of 16-hydroxymethylene derivatives and corresponding 5'-alkylated [17,16-c]-pyrazoles 3-Benzyloxy-16- (1'-hydroxy-ethylidene)-estrone (99 tBuOK (438 mg, 4.14 mmol) was added to a stirred solution of 3-benzyloxy-estrone (500 mg, 1.38 mmol) in anhydrous toluene (6 mL) and anhydrous DMSO (1.5 mL). at. 0°C, under an atmosphere of N2. Ethyl acetate (1.27 mL, 17.94 mmol) was then added and the mixture was heated to reflux for 1 hour. After cooling down, the resulting brown solution was poured into H20 (200 mL) and acidified with 5M HCI. The organics were extracted with EtOAc (2x150 mL), washed with H20 (2Woo mL), then brine (2x100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM) to give 99 as a light yellow crystalline solid (447 mg, 80%). This was recrystallised from EtOH to give yellow crystals (351 mg, 63%): mp 126-128°C ; IR (KBr) 2930-2860 (aliph CH), 1660 (C=O), 1615-1455 (C=C and arom C=C) cm-1 ; aH (CDCl3, 400 MHz) 0.83, 0.95 (3H, 2xs, C-18-H3), 1.38-2. 49 (11H, m), 1.97, 2. 37 (3H, 2xs, C-2'-H3), 2. 87-2. 92 (2H, m, C-6-H2), 3. 43 (app dd, J = 9. 4 Hz, J = 8.2 Hz, C-16-H), 5.02 (2H, s, OCH2Ar), 6.71 (1H, d, J = 2. 8 Hz, C-4-H), 6. 77 (1H, dd, J = 8. 3 Hz, J = 2. 8 Hz, C-2-H), 7,17 (1H, d, J =-8. 3 Hz, C-1-H), 7. 28-. 42 (5H, m, CHO) ans 12. 91 (br s, exchanged with D2°, C-1'-OH) ; MS m/z (FAB+) 403. 1 [68, (M+HF], 91. 0 [100, (CH2Ar)+]; Acc MS m/z (FAB+) 403.2246, C27H31O3 requires 403. 2273. Found: C, 80. 20 ; H, 7. 56. C27H3003 requires : C, 80. 56 ; H, 7.51%.
3-O-tert-Butyl-dimethylsilyl-16-(1'-hydroxy-2',2',2'-trifluoro-ethylidene)-estrone (100) tBuOK (875 mg, 7.80 mmol) was added to a stirred solution of 1 (1.0 g, 2.60 mmol) in anhydrous toluene (20 mL) at 0°C, under an atmosphere of N2. After 20 minutes of stirring, ethyl trifluoroacetate (2.16 mL, 18.2 mmol) was added and the mixture heated to reflux for 2 hours. After cooling down, the resulting dark orange suspension was poured into H20 (50 mL) and acidified with 5M HCI. The organics were extracted with EtOAc (2x50 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude orange oil that crystallises on standing was used without further purification (1.4 g, quant.). An analytical sample was recrystallised from EtOH to give 100, as light yellow crystals: mp 145-146°C ; IR (KBr) 2930-2860 (aliph CH), 1690 (C=O), 1640-1495 (C=C and arom C=C) cm-1 ; aH (CDCI3, 400. MHz) 0.19 (6H, s, Si (CH3) 2), 0.98 (9H, s, C (CH3) 3), 0.99 (3H, s, C-18-H3), 1.42-2. 90 (13H, m), 6.57 (1H, d, J = 2. 6 Hz, C-4-H), 6.63 (1H, dd, J = 8.4 Hz, J = 2.6 Hz, C-2-H) and 7.11 (1H, d, J = 8. 4 Hz, C-1-H), C-1'-OH not seen; 8c (CDCI3, 100.4 MHz) -4.4 (2xq, Si (CH3) 2), 14.8 (q, C-18), 18.1 (s, C (CH3) 3), 25.2 (t), 25.6 (t), 25.7 (3xq, C (CH3) 3), 26.7 (t), 29.3 (t), 31.0 (t), 37.5 (d), 43.9 (d),. 48.5 (s, C-13), 49.5 (d), 110.9 (s, C-16), 117.4 (d),-119 (app d, J = 272 Hz, CF3), 120.0 (d), 126. 0 (d), 132. 0 (s), 137. 4 (s), 153.6 (s, C-3), # 154 (app d, J = 38 Hz, C-1') and 216. 0 (s, C=O) ; MS m/z (FAB+) 480. 1 [47, M+J, 423.0 [38, (M-F3)+], 73. 0 [100]; Ace MS m/z (FAB+) 480. 2296, C26H35F3O3Si requires 480.2308. Found: C, 64. 90; H, 7. 13. C26H35F303Si requires : C, 64. 97; H, 7. 34%.
3-0-fert-Butyl-dimethylsilyl-16- (1'-hydroxy-1"-pyridin-3"-ylmethylene)-estrone (101) 'BuOK (875 mg, 7. 80 mmol) was added to a stirred solution of 1 (1. 0 g, 2. 60 mmol) in anhydrous toluene (30 mL) at O*C, under an atmosphere of N2. After 20 minutes of stirring, ethyl nicotinate (2. 48 mL, 18. 2 mmol) was added and the mixture was heated to reflux for 1. 5 hours. After cooling down, the resulting dark red suspension was poured into H20 (200 mL) and acidified with 5M HCI. The organics were extracted with EtOAc (2x100 mL), washed with H20 (100 mL), then brine (2x100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude yellow solid was recrystallised from EtOH to give 101 as white flaky crystals (985 mg, 77%): mp 169-170°C ; IR (KBr) 2930-2855 (aliph CH), 1660 (C=O), 1605,1495 (C=C and arom C=C) cm-1 H (CDCI3, 400 MHz) 0.21 (6H, s, Si(CH3)2), 0.99 (9H, s, C (CH3) 3), 1.11 (3H, s, C-18-H3), 1.38-2. 77 (11H, m), 2. 81-2. 95 (2H, m, C-6-H2), 6.58 (1H, d, J = 2.6 Hz, C-4-H), 6. 63 (1H, dd, J = 8.5 Hz, J = 2.6 Hz, C-2-H), 7. 12 (1H, d, J = 8.5 Hz, C-1-H), 7.41 (1H, ddd, J = 8.1 Hz, J = 4. 9 Hz, J = 0.8 Hz, C-5"-H), 8.09 (1H, app dt, J = 8.1 Hz, J = 2.0 Hz ; C-4"-H), 8.69 (1H, dd, J = 4.9 Hz, J = 1.9 Hz, C-6'-H), 8.97 (1H, app dd, J = 2.1 Hz, J = 0.8 Hz, C-2'- H) and 13.63 (1H, s, exchanged with D20, C-1'-OH) ; MS m/z (FAB+) 490.1 [100, (M+H) +] ; Ace MS m/z (FAB+) 490.2775, C30H40NO3Si requires 490.2777. Found: C, 64.90 ; H, 7.13. C26H35F303Si requires: C, 64.97 ; H, 7.34%.
16-(1'-hydroxy-ethylidene)-estrone (102) Following method 2, a suspension of 99 (200 mg, 497 µmol) and Pd-C (10%, 80 mg) in MeOH/THF 2: 1 (30 mL) was hydrogenated for 2 hours to give 102 as a white solid (98 mg, 63%). This was recrystallised from acetone/hexane to give white crystals (72 mg, 46%): mp 211-214°C ; IR (KBr) 3450 (OH), 2915-2860 (aliph CH), 1705 (C=O), 1655- 1510 (C=O, C=C and arom C=C) cm-1; #H (CDCl3, 400 MHz) 0.83, 0. 96 (3H, 2xs, C-18- H3), 1.39-2. 40 (11H, m), 1.98, 2.37 (3H, 2xs, C-2'-H3), 2.83-2. 89 (2H, m, C-6-H2), 3.43 (dd, J = 9.4 Hz, J = 8.2 Hz, C-16-H), 4.70 (br s, exchanged with D20, OH), 6.55-6. 57 (1 H, m, C-4-H), 6.62 (1 H, dd, J = 8. 0 Hz, J = 2. 7 Hz, C-2-H) and 7. 11 (1 H, d, J = 8. 0 Hz, C-1-H), 1XOH not seen ; MS mlz (FAB+) 313.1 [100, (M+H)'], 73.0 [19] ; Acc MS mlz (FAB+) 313. 1799, C20H25O3 requires 313.1804. Found : C, 76. 60; H, 7. 82. G2oH2403 requires : C, 76. 89 ; H, 7. 74%. i6- (1'-hydroxy-2',2',2'-trifluoro-ethylidene)-estrone (103, STX 946) A 1. Q M solution of TBAF in anhydrous THF (4. 90 mL, 4.90 mmol) was added to a stirred solution of 100 (1.18 g, 2.45 mmol, crude product) in anhydrous THF (50 mL) under an atmosphere of N2. The resulting light brown solution was stirred at room temperature for 4 hours, after which the solvent was removed under reduced pressure and H20 added (100 mL). The organics were extracted with EtOAc (2x100 mL), washed with H20 (2x80 mL), then brine (2x80 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude brown oil was purified by flash chromatography (DCM) to give 103 as a light brown oil (535 mg, 60%). This was crystallised from cold DCM to give white crystals (249 mg, 28%): mp 91-93°C ; IR (KBr) 3430 (OH), 2930-2860 (aliph CH), 1685 (C=O), 1610,1500 (C=C and arom C=C) cm-' ; #H (CDCl3, 400 MHz) 1.02 (3H, s, C-18- H3), 1.43-2. 91 (13H, m), 6.58 (1H, d, J = 2.7 Hz, C-4-H), 6.64 (1H, dd, J = 8.4 Hz, J = 2.7 Hz, C-2-H) and 7.14 (1H, d, J = 8.4 Hz, C-1-H), C-3-OH and C-1'-OH not seen; MS m/z (FAB+) 366.1 [100, M'] ; Acc MS m/z (FAB+) 366.1476, C20H21F3O3 requires 366.1443. HPLC (MeOH/H20, 96: 4, a, max = 280.5 nm) Rt = 1.66 min, 100%. Found: C, 57.60 ; H, 5.37. C20H21F3O3.(CH2Cl2)3/4 requires : C, 57.95 ; H, 5.27%.
16- (1'-hydroxy-1'-pyridin-3'-ylmethylene)-estrone (104) A 1. 0 M solution of TBAF in anhydrous THF (1. 43 mL, 1. 43 mmol) was added to a stirred solution of 101 (350 mg, 716 µmol) in anhydrous THF (10 mL) under an atmosphere of N2. The resulting light brown solution was stirred at room temperature for 3 hours, after which the solvent was removed under reduced pressure and H20 added (100 mL), followed by a few drops of AcOH'glacial. The organics were extracted with EtOAc (100 mL), Et2O (100 mL) and CHC13 (100 mL), washed with H20 (100 mL), then brine (100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was triturated with boiling EtOH to give 104 as a pale yellow solid (125 mg, 50%) : mp 275-278°C ; IR (KBr) 2950-2855 (aliph CH), 1645 (C=O), 1610-1500 (C=C and arom C=C) cm- ; aH (DCI3, 400 MHz) 1. 09 (3H, s, C-18-H3), 1. 39-2. 76 (11H, m), 2. 85- 2. 91 (2H, m, C-6-H2), 5. 48 (1H, s, exchanged with D2O, C-3-OH), 6.59 (1H, d, J = 2. 7 Hz, C-4-H), 6. 65 (1H, dd, J = 8. 4 Hz, J = 2. 7 Hz, C-2-H), 7.15 (1H, d, J= 8. 4 Hz, C-1-H), 7. 41 (1H, ddd, J = 8. 1 dz, J = 4. 9 Hz, J = 0. 8 Hz, C-5"-H), 8. 09 (1H, dt, J = 8. 1 Hz, J = 1. 8 Hz, C-4"-H), 8.67 (1H, dd, J = 4. 9 Hz, J = 1. 8 Hz, C-6"-H), 8. 94 (1H, app d, J =. 1. 8 Hz, C-2"-H) and 13.58 (1H, s, exchanged with D2O, C-1'-OH) ; MS m/z (FAB+) 376.0 [100, (M+H) *], 242.1 [54]; Acc MS m/z (FAB+) 376.1903, C24H26NO3 requires 376.1913.
Found: C, 74.70; H, 6.80; N, 3.51. C24H24NO3.(H2O)1/2 requires: C, 74.98; H, 6.82; N, 3.64%.
3-O-tert-Butyl-dimethylsilyl-estra-1, 3,5 (10)-triene-[17, 16-c]-[5'-(1'-pyridin-3'-yl)- 3', 4'-dihydro)]-pyrazol-3'-ol (105) Hydrazine hydrate (75 µL, 1.53 mmol) was added to a refluxing solution of 101 (500 mg, 1.02 mmol) in EtOH (20 mL), under an atmosphere of N2. The resulting pale yellow solution was heated to reflux for 1 hour, after which the solvent was partially removed under reduced pressure and H20 (100 mL) added. The resulting white precipitate was filtered and dried (460 mg, 89%) and an analytical sample was recrystallised from EtOH/H20 to give 101 as white crystals: mp 144-145°C ; IR (KBr) 3480, 3330, 3190 (NH and OH), 2940-2860 (aliph CH), 1610-1495 (C=N and arom C=C) cm-1 ; aH (DMSO-d6, 400 MHz) 0. 14 (6H, s, Si (CH3) 2), 0.92 (9H, s, C (CH3) 3), 0. 94 (3H, s, C-18-H3), 1.12-2. 31 (11H, m), 2. 63-2. 71 (2H, m, C-6-H2), 3. 42 (1H, app d, J = 9.0 Hz, C-16-H), 5. 97 (1H, s, exchanged with D2O, C-17-OH), 6. 46 (1H, d, J = 2. 5 Hz, C-4-H), 6. 55 (1H, dd, J = 8. 4 Hz, J = 2.5 Hz, C-2-H), 7. 10 (1H, d, J = 8. 4 Hz, C-1-H), 7.26 (1H, s, exchanged with D2O, NH), 7.35 (1H, app ddd, J = 8. 1 Hz, J = 4.6 Hz, J = 0.8 Hz, C-5"-H), 7.91 (1H, app dt, J = 8.1 Hz, J = 1.7 Hz, C-4"-H), 8.43 (1H, dd, J= 4. 6 Hz, J = 1.7 Hz, C-6"-H) and 8 74 (1H, app d, J = 1.7 Hz, C-2"-H); MS m/z (FAB+) 504.1 [100, (M+H) +], 486.1 [27, (M+H-H2O)+] ; Acc MS m/z (FAB+) 504. 3045, C30H42N3O2Si requires 504. 3046.
3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(5'-methyl)-pyrazole (106) Hydrazine hydrate (56 µL, 1.15 mmol) was added to a refluxing solution of 102 (240 mg, 768 µmol) in EtOH (15 mL), under an atmosphere of N2. The resulting pale yellow solution was heated to reflux for 45 minutes, after which the solvent was removed under reduced pressure, H20 (50 mL) added and the mixture acidified. with 5M HCI. The organics were extracted with EtOAc (2x50 mL), washed with H20 (30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM/EtOAc, 8: 2) to give 106 as a pale brown solid (84 mg, 35%). This was triturated with acetone to give a creamy solid (32 mg, 13%): mp 234-236°C ; IR (KBr) 3295 (OH), 2930-2860 (aliph CH), 1620-1500 (C=N and arom C=C) cm-1 ; #H (DMSO-d6, 400 MHz) 1.01 (3H, s, C-18-H3), 1.34-2. 64 (11H, m), 2.71-2. 85 (2H, m, C-6-H2), 2.28 (3H, s, C-1"-H3), 6.45 (1H, d, J = 2.6 Hz, C-4-H), 6.52 (1H, dd, J = 8.6 Hz, J = 2.6 Hz, C-2-H), 7.04 (1H, d, J = 8.6 Hz, C-1-H) and 9.04 (1H, s, exchanged with D20, NH or OH), NH or OH not seen; MS m/z (FAB+) 309.2 [100, (M+H) +], 95.1 [33], 69.0 [39] ; Acc MS m/z (FAB+) 309.1978, C20H25N2O requires 309.1967. HPLC (MeOH/H2O, 96: 4, #max = 280. 5 nm) Rt = 1.98 min, 99%.
3-Hydroxy-estra-1, 3. 5(10)-triene-[17,16-c]-(5'-trifluoro-methyl)-pyrazole (107, STX949 Hydrazine hydrate (32 µL, 655 µmol) was added to a refluxing solution of 103 (160 mg, 437 ßmol) in EtOH (15 mL), under an atmosphere of N2. The resulting pale yellow solution was heated to reflux for 3 hours, after which p-toluene sulfonic acid (~ 10 mg) was added. The mixture was then heated to reflux overnight and after cooling down, the solvent was removed under reduced pressure, H20 (50 mL) added, followed by 5M HCI.
The organics were extracted with EtOAc (2x50 mL), washed with H20 (30 mL), then brine (2X30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was. purified by flash chromatography (DCiWEtOAc, 8 : 2) to give 1Q7 as a pale yellow solid (95 mg, 60%). This was precipitated from DCM/Hexane (1 : 5) to give an off-white solid (51 mg, 32%) : mp 152-155°C ; IR (KBr) 3220 (br, OH), 2930-2860 (aliph CH), 1610-1500 (C=N and arom C=C) cm-1 ; aH (CDCl3, 400 MHz) 1.01 (3H, s, C-18-H3), 1. 40-2. 74 (11H, m), 2. 78-2. 89 (2H, m, C-6-H2) , 4. 63 (1H, s, exchanged with D2O, C-3- OH), 6.53 (1H, d, J = 2. 7 Hz, C-4-H), 6.59 (1H, dd, J = 8. 2 Hz, J = 2. 7 Hz, C-2-H) and 7. 08 (1H, d, J = 8. 2 Hz, C-1-H), NH not seen ; MS m/z (FAB+) 363. 1 [100, (M+H) +] ; Ace MS m/z (FAB+) 363. 1689, C2oH22N2F30 requires 363. 1684. HPLC (MeOH/H20, 85 : 15, #max = 280.5 nm) Rt = 2.43 min, 98%.
3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5'-(1'-pyridin-3'-yl)]-pyrazole (108) A 1.0 M solution of TBAF in anhydrous THF (436 pL, 436 µmol) was added to a stirred solution of 105 (110 mg, 218 ; n. moi) in anhydrous THF (10 mL) at room temperature, under an atmosphere of N2. The resulting light brown solution was stirred overnight at room temperature, after which the solvent was removed under reduced pressure and H20 added (50 mL). The organics were extracted with EtOAc (2x50 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude foam was triturated with boiling EtOAc/CHCl3 (1: 1) to give 108 as a white powder (61 mg, 75%) : mp 298-300°C (dec.) ; IR (KBr) 2980-2840 (aliph CH), 1610-1495 (C=N and arom C=C) cm1 ; 6H (DMSO-d6, 400 MHz) 0.98 (3H, s, C-18- H3), 1.37-2. 87 (13H, m), 6.45 (1H, d, J = 2.6 Hz, C-4-H), 6.51 (1H, dd, J = 8. 5 Hz, J = 2.6 Hz, C-2-H), 7.05 (1H, d, J = 8. 5 Hz, C-1-H), 7.37-7. 48 (1H, m, C-4"-H or C-5'-H), 7. 97-8. 06 (1H, m, C-4"-H or C-5"-H), 8. 42-8. 49 (1H, m, C-2"-H or C-6"-H) and 8. 84-8. 89 (1H, m, C-2"-H or C-6'-H), 9. 01 (1H, s, exchanged with D2O, OH), 12. 65 and 12. 72 (total 1H, 2xs NH) ; MS m/z (FAB+) 372. 0 [100, (M+H)+] ; Ace MS m/z (FAB+) 372. 2080, C24H2eN30 requires 372.2076. 3-O-tert-butyl-dimethylsilyl-estra-1, 3,5 (10)-triene-[17,16-c]-(5'-trifluoromethyl-3',4'- dihydro)-pyrazol-3'-ol (109) Hydrazine hydrate (22 µL, 468 µmol) was added to a refluxing solution of 100 (150 mg, 312 µmol) in absolute EtOH (8 mL), under an atmosphere of N2. The resulting pale yellow solution was heated to reflux for 1 hour, after which the solvent was removed in vacuo and H20 (50 mL) added, followed by HCI 5M (4 drops). The organics were extracted with EtOAc (3x30 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (CH2Cl2 to CH2Cl2/EtOAc, 98: 2, gradient, flashmaster) to give a light brown crystalline solid (65 mg, 42%). This was recrystallised from EtOH/H20 to give creamy crystals (26 mg, 18%) : mp 235-238°C ; IR (KBr) 3335 (NH), 3170 (br, OH), 2930-2860 (aliph CH), 1664,1605, 1495 (C=N and arom C=C) cm- I ; 8H (DMSO-d6s 400 MHz) 0.18 (6H, s, SiMe2), 0.96 (9H, s, C (CH3) 3), 1.04 (3H, s, C-18- H3), 1.32-2. 39 (11H, m), 2.76-2. 81 (2H, m, C-6-H2), 3.53-3. 57 (1H, m, C-16-H), 6.53 (1H, d, JC-2-H,C-4-H = 2.7 Hz, C-4-H), 6.60 (1 H, dd, JC-1-H, C-2-H = 8.4 Hz, JC-4-H,C-2-H = 2.7 Hz, C-2-H), 6.63 (-2H, s, exchanged with D20, OH and NH) and 7-14 (1H, d, JC-2-H, C-1-H = 8. 4 HO, C- 1-H) ; MS m/z (FAB+) 495.0 [69, (M+H) +], 453.0 [54], 437.0 [51, (M-F3) +], 73.0 [100]; Ace MS m/z (FAB+) 495. 26311, C26H38N2F302Si requires 495.26547.
3-Hydroxy-estra-1, 3, 5 (10)-triene- (17, 16-c)-3'-trifluoromethyl-5'-hydroxy-pyrazole (110, STX947) A 1. 0 M solution of tetrabutyl ammonium fluoride in dry THF (404 µL, 404 µmol) was added to a stirred solution of 109 (100 mg, 202 µmol) in THF (20 mL) at room temperature, under an atmosphere of N2. The resulting light brown solution was stirred for 4 hours at room temperature, after which the solvent was removed in vacuo and H20 added (50 mL). The organics were extracted with EtOAc (2x50 mL), washed with H20 (2x30 mL), then brine (2x30 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The light brown crude oil was purified by flash chromatography (CH2Cl2/EtOAc, 9 : 1, flashmaster) to give a pale yellow oil (51 mg, 66%). This was then precipitated from Et2O/Hexane (1 : 5) to give a light yellow powder (7 mg): IR (KBr) 3470, 3415 (OH), 2930-2870 (aliph CH), 1640, 1615, 1500 (C=N and arom C=C) cm-1; #H (CDCl3, 400 MHz) 1. 06 (3H, s, C-18-H3), 1. 16-2. 26 (11H, m), 2. 70-2.84 (2H, m, C-6-H2), 3. 64-3. 71 (1H, app d, C-16-H), 4. 39 (1H, br s, OH or NH), 5. 88 (1H, s, OH or NH), 6. 53 ( H, d, JC-2-H, C-4-H = 2. 6 Hz, C-4-H), 6. 61-6. 65 (1H, app dd, C-2-H) and 7. 03 (1H, d, JC-2-H,C- 1-H = 8. 8 Hz, C-1-H) ; MS mlz (FAB+) 381. 1 [100, (M+H) +], 85. 1 [87] ; MS m/z (FAB-) 533.3 [100, (M+NBA]-], 379.2 [98, (M-H)-]; Acc MS m/z (FAB+) 381. 17967, C20H24N2O2F3 requires 381. 17899. HPLC (methanol/water, 96: 4, a, = 280. 5 nm) Rt = 1. 66 min, 100%.
New Pathway to STX664 3-Benzyloxy-16-ethoxymethylene-estrone (127) K2CO3 (595 mg, 4.31 mmol) was added portionwise to a stirred suspension of 3- benzyloxy-16-hydroxymethylene-estrone (250 mg, 643 mmol) in acetone (20 mL) at room temperature, under an atmosphere of N2. After 10 minutes of stirring, ethyl iodide (296 µL, 3.70 mmol) was added and the resulting mixture was stirred for 36 hours at room temperature. H20 was then added (100 mL) and the organics were extracted with DCM (3x50 mL), washed with H20 (50 mL), then brine (2x50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc/hexane, 1: 3) to give 127 as a white solid'(215 mg, 80%).
This was recrystallised from EtOAc to give white crystals (139 mg, 52%): mp 158-160°C ; IR (KBr) 2935-2850 (aliph CH), 1710 (C=O), 1640-1500 (aliph and arom C=C), 1235 (C- O) cm-' ; aH (CDCI3, 400 MHz) 0. 92 (3H, s, C-18-H3), 1.34 (3H, t, J = 7. 2 Hz, C-3'-H3), 1. 38-2. 71 (11H, m), 2. 84-2. 93 (2H, m, C-6-H2), 4.02-4. 10 (2H, m, C-2'-H2), 5. 02 (2H, s, OCHzAr), 6. 71 (1H, d, J = 2.6 Hz, C-4-H), 6. 77 (1H, dd, J = 8. 6 Hz, J = 2. 6 Hz, C-2-H), 7. 18 (1H, d, J = 8.6 Hz, C-1-H) and 7. 26-7. 43 (6H, m, C-1'-H and CHO) ; MS m/z (FAB+) 417.0 [100, (M+H) +] ; Acc MS m/z (FAB+) 417. 2425, C28H33O3 requires 417.2430.
16ß-Ethoxymethyl-estrone (STX664) A suspension of 127 (640 mgrl. 54 mmo)) and Pd-C (10%, 200 mg) in MeOH/THF (1 : 1, 60 mL) was hydrogenated (H2-filled balloon) at room temperature for 48 hours. After removal of the supporked catalyst by filtration through celite and concentration of the filtrate under reduced pressure, the product obtained was purified by flash chromatography (EtOAc/hexane, 3 : 7) to give STX664 as a white crystalline solid (232 mg, 46%). An analytical sample was recrystallised from EtOH : mp 208-210°C ; IR (KBr) 3370 (OH), 2970-2855 (aliph CH), 1730 (C=O), 1610, 1505 (arom C=C), 1225 (C-O) cm- 1 ; 6H (CDCI3, 400 MHz) 0.89 (3H, s, C-18-H3), 1.16 (3H, t, JC-2-H, C-3'-H = 7. 0 Hz, C-3'-H3), 1. 36-2.42 (12H, m), 2.81-2. 85 (2H, m, C-6-H2), 3.42-3. 50 (2H, m, C-2'-H2), 3.61 (1H, dd, JAB = 9.4 Hz, J = 3'. 9 Hz, C-1'-HAHB), 3.65 (1H, dd, JBA = 9.4 Hz, J = 5.5 Hz, C-1'-HAHB), 4.59 (1H, s, exchanged with D20, OH), 6.56 (1H, d, J= 2.8 Hz, C-4-H), 6. 62 (1H, dd, J = 8.5 Hz, J = 2.8 Hz, C-2-H) and 7.13 (1H, d, J = 8.5 Hz, C-1-H); #C (CDCI3, 100.4 MHz) 12.9 (q, C-3'), 15.0 (q, C-18), 25.8 (t), 25.9 (t), 26.6 (t), 29.4 (t), 32.0 (t), 37.7 (d), 44.0 (d), 48. 2 (s, C-13), 48. 9 (d), 50.2 (d), 66.4 (t, C-1'or C-2'), 69. 3 (t, C-1'or C-2'), 112.5 (d), 115.0 (d), 126.2 (d), 131.9 (s), 137.8 (s) and 153.0 (s, C-3), C=O not'seen ; MS m/z (FAB+) 329.1 [100, (M+H)+] ; Acc MS m/z (FAB+) 329.2124, C21H29O3 requires 329.2117.
HPLC (MeOH/H20, 90: 10, = 279.3 nm) Rt = 2.37 min, 98%. Found: C, 76.60 ; H, 8.62. C21H28O3 requires : C, 76.79 ; H, 8.59%.
Sulfamate 3-Sulfamoyloxy-16p-ethoxymethyl-estrone (20) Anhydrous DMA (1. 5 mL) was added to a freshly concentrated solution of sulfamoyl chloride (2.2 eq.) cooled to 0°C under an atmosphere of N2. STX664 (100 mg, 304 µmol) was then added and the resulting mixture was stirred at room temperature for 3 hours.
The mixture was then poured into cold brine (15 mL), and the organics were extracted with EtOAc (2x20 mL), washed with brine (5x20 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (CHCl3/EtOAc, 8: 2) to give 20 as a colourless oil (124 mg, 100%): IR (KBr) 3380, 3285 (br, NH2), 2970-2865 (aliph CH), 1730 (C=O), 1605-1495 (arom C=C), 1380, 1190 (SO2) cm-1; ##H (CDCl3, 400 MHz) 0. 89 (3H, s, C-18-H3), 1. 16 (3H, t, J = 7. 0 Hz, C-3'-H3), 1. 40-2. 43 (12H, m), 2. 89-2. 94 (2H, m, C-6-H2), 3. 43-3. 50 (2H, m, C-2'-H2), 3. 61 (1H, dd, JAB = 9.4 Hz, J = 3. 7 Hz, C-1'-HAHB), 3.66 (1 H, dd, JBA = 9. 4 Hz, J = 5. 5 Hz, C-1'-HAHB), 4.87 (2H, s, exchanged with D2O, SO2NH2), 7. 04 (1H, d, J = 2. 4 Hz, C-4-H), 7. 07 (1H, dd, J = 8. 4 Hz, J = 2. 4 Hz, C-2-H) and 7. 29 (1H, d, J = 8. 4 Hz, C-1-H) ; Oc (CDCI3, 100. 4 MHz) 12. 9 (q, C-3'), 15. 1 (q, C-18), 25. 8 (2xt), 26. 4 (t), 29. 4 (t), 32. 0 (t), 37. 4 (d), 44.3 (d), 48. 2 (s, C-13), 49. 0 (d), 50.3 (d), 66. 6 (t, C-l'or C-2'), 69. 3 (t, C-1'or C-2'), 119. 1 (d), 122. 1 (d), 126. 8 (d), 138. 8 (s), 139. 1 (s), 148. 0 (s, C-3) and 220. 8 (s, C=O) ; MS m/z (FAB+) 408.3 [39, (M+H) +], 145.1 [63]; 131.1 [61], 117. 1 [57], 85.1 [100], 68. 0 [50]; Ace MS m/z (FAB+) 408. 1841, C21H30NO5S requires 408. 1845. HPLC (MeOH/H20, 90: 10, #max = 267. 5 nm) Rt = 2.28 min, 99%. Found: C, 59.65 ; H, 6.84 ; N, 3.36. C2yH2gNO5S. (CHCI3) 1/6 requires: C, 59.48 ; H, 6.88 ; N, 3.28%.
3-Sulfamoyloxy-16-oximino-estrone (21) Following an analogous procedure to that described for the preparation of 20, reaction of 16-oximino-estrone (200 mg, 668 µmol) with sulfamoyl chloride in DMA (1.5 mL) was complete within 3 hours. The crude product was purified by flash chromatography (CHCI3/acetone, 8: 2) to give a white foam (93 mg, 37%). This was triturated with hexane to give 21 as a white powder (36 mg, 14%): mp 88-91°C; IR (KBr) 3370-3250 (NH2, NOH), 2930-2850 (aliph CH), 1740 (C=O), 1635-1495 (C=N and arom C=C), 1380, 1185 (S02) cm-' ; 8H (DMSO-d6, 400 MHz) 0.90 (3H, s, C-18-H3), 1.32-2. 48 (11H, m), 2. 78-2. 94 (2H, m, C-6-H2), 7.00 (1H, m, C-4-H), 7.03 (1H, dd, J = 8. 6 Hz, J = 2.3 Hz, C-2-H), 7.36 (1H, d, J = 8.6 Hz, C-1-H), 7.92 (2H, s, exchanged with D20, SO2NH2) and 12.41 (1H, s, exchanged with D2O, NOH) ; MS m/z (FAB+) 532. 2 [32, (M+H+NBA) +], 379. 1 [100, (M+H)+] ; MS m/z (FAB-) 531. 1 [40, (M+NBA)-], 377. 1 [100, (M-H)-] ; Acc MS m/z (FAB+) 379.1320, C18H23N2O5S requires 379. 1328. Found: C, 56. 70 ; H, 6. 02 ; N, 6.83.
C18H22N2O5S requires : C, 57. 13 ; H, 5. 86 ; N, 7. 40%.
3, 1'-Bis-sulfamoyloxy-estra-1, 3,5 (10)-triene- [17, 16-c]-pyrazole (21) and 3- Sulfamoyloxy-estra-1, 3, 5 (10)-triene-[17,16-c]-pyrazole (22) Following an analogous procedure to that described for the preparation of 20, reaction of 3-hydroxy-estra-1, 3, 5 (10)-triene- [17, 16-c]-pyrazole (170 mg, 577 mol) with sulfamoyl chloride in DMA (2 mL) was complete within 4 hours. The crude product was purified by flash chromatography (CHCl3/EtOAc, 1 : 1) to give two products : The less polar fraction gave 21 as a pale yellow solid (85 mg, 32%) : IR, (KBr) 3440,3410 (NH2), 2930-2860 (aliph CH), 1635-1495 (C=N and arom C=C), 1385,1185 (SO2) cm- 1;#H (DMSO-d6, 400 MHz) 0.99 (3H, s, C-18-H3), 1.40-2. 68 (11H, m), 2.85-2. 90 (2H, m, C-6-H2), 6.97 (1H, d, J = 2.3 Hz, C-4-H), 7.02 (1H, dd, J = 8.6 Hz, J = 2. 3 Hz, C-2-H), 7.35 (1H, d, J = 8.6 Hz, C-1-H), 7.70 (1H, s, C-5'-H), 7.88 (2H, s, exchanged with D2O, SO2NH2) and 8.30 (2H, s, exchanged with D20, SO2NH2) ; MS m/z (FAB+) 452.8 [100, M+], 373.9 [80, (M+H-SO2NH2)+], 171.0 [26], 116.0 [29] ; Acc MS m/z (FAB+) 452.1164, C19H24N4O5S2 requires 452.1188. HPLC (MeOH/H20, 80: 20, 2,, mx = 220. 4 nm) Rt = 1.95 min, 97%.
The more polar fraction gave 22 as a white solid (123 mg, 57%) : IR (KBr) 3370,3305 (NH2), 2975-2860 (aliph CH), 1590,1495 (C=N and arom C=C), 1370, 1195 (SO2) cm- 1;#H (DMSO-d6, 400 MHz) 1.17 (3H, s, C-18-H3), 1. 38-2. 59 (11H, m), 2. 85-2. 90 (2H, m, C-6-H2), 6. 97 (1H, d, J = 2. 7 Hz, C-4-H), 7. 02 (1H, dd, J = 8. 6 Hz, J = 2. 7 Hz, C-2-H), 7. 25 (1H, s, C-5'-H), 7. 35 (1H, d, J = 8. 6 Hz, C-1-H), 7. 89 (2H, s, exchanged with D20, SO2NH2) and 11. 99 (1H, br s, exchanged with D2O, NH) ; MS m/z (FAB+) 747. 3 [38, (2M+H) +], 527. 2 [80, (M+H+NBA)+], 509. 2 [30], 443.2 [28], 374.2 [100, (M+H)+] ; Acc MS m/z (FAB+) 374.1544, C19H24N3O3S requires 374.1538. HPLC (MeOH/H20, 80: 20, #max = 218.0 nm) Rt = 2.04 min, 100%.
SECTION 5 3-(tert-Butyl-dimethyl-silanyloxy)-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrene-16-carbonitrile-(3-(tert-Butyl- dimethyl)silyl-16-cyano estrone) (CAB01044) : O, N-bistrifluoroacetyl-hydroxylamine (1.35 g, 6.0 mmol) was added to a solution of 3- (tert-butyl-dimethyl) silyl-16-hydroxymethylene estrone (820 mg, 2.0 mmol, CAB01042) and pyridine (1.0 mL) in toluene (50 mL). The mixture was heated to reflux for 1 hour (120°C oilbath temperature) and then allowed to cool to room temperature (TLC-control).
Ethyl acetate (50 mL) was added and the mixture was transferred into. separation funnel, washed with water (50 mL) and brine (30 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica (eluent : ethyl acetate/hexane 1 : 2, Rf: 0.38) to give a light yellow oil, which was dissolved in a small amount of acetone (2 mL) and precipitated by addition of hexane. The light yellow solid was filtered off and dried under high vacuum. Yield: 585 mg (71%), mixture of 16a-cyano and 166-cyan diastereomers).'H-NMR (CDC13, 400 MHz) 6 : 0.19 (s, 6H), 0.98 (s, 9H,-C (CH3) 3), 0.99 (s, ca. 1H, CH3-group of minor isomer), 1.02 (s, ca. 2H, CH3- group of major isomer), 1.40-1. 71 (m, 5H), 1.94-2. 10 (m, 3H), 2.23-2. 34 (m, 1H), 2.38- 2.48 (m, 1H), 2.54-2. 64 (m, 1H), 2. 82-2. 90 (m, 2H), 3.12 (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.58 (dd, J = 10. 2,1. 6 Hz, ca. 0.35 H, H-16 of minor isomer), 6.58 (d, J = 2.3 Hz, 1H), 6.63 (dd, J = 8. 6,2. 3 Hz, 1H), 7.10 (d, J = 8. 6 Hz, 1H). LRMS (FAB+): 73. 1 (100), 352.2 (65), 409.3 (85, [M] +). LRMS (FAB-): 408. 3 (100, [M-H]-).
HRMS (FAB+) 409. 243294 C25H35NO2Si requires 409.243708 3- (tert-Butyl-dimethyl-silanyloxy)-2-methoxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, ira., 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrene-16-carbonitrile (3 Butyl-dimethyl) silyl-2-methoxy-li6-cyano estrone) (CAB0106 Sodium ethoxide (340 mg, 5.0 mmol) was added to a solution of 2-methoxy-3- (tert-butyl- dimethyl) silyl estrone (829 mg, 2.0 mmol) and ethyl formate (2.0 mL) in toluene (20 mL).
The mixture was stirred for 2 hours at room temperature (TLC-control) and acetic acid (1 mL) and ethyl acetate (60 mL) were added. The mixture was transferred into a separation funnel and washed with water (2 x 30 mL) and brine (30 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude 2- methoxy-3- (tert-butyl-dimethylsilyl)-16-hydroxymethylene estrone (785 mg, 1.773 mmol), that was obtained was dissolved in toluene (50 mL) and pyridine (1 mL) and O, N- bistrifluoroacetyl-hydroxylamine (675 mg, 3.0 mmol) were added. The mixture was heated to reflux for 1 hour and then allowed to cool to room tempereature (TLC-control).
The reaction mixture was diluted with ethyl acetate (50 mL), transferred into a separation funnel, washed with water (2 x 50 mL) and brine (30 mL), dried over Na2SO4 and concentrated under reduced pressure.. The residue was purified by column chromatography on silica (eluent : ethyl acetate/hexane 1: 2, Rf: 0.38) to give a pale yellow solid, which was dissolved in a small amount of acetone (2 mL) and precipitated by addition of hexane. The light yellow solid was filtered off and dried under'high vacuum. Yield : 620 mg. (71%, ca. 2 to 1 mixture of 16a-cyano and 160-cyan diastereomers).'H-NMR (CDCI3, 400 MHz) 6 : 0.15 (s, 6H), 0.98 (s, 9H,-C (CH3) 3), 0. 99 (s, ca. 1H, CH3-group of minor isomer), 1.08 (s, ca. 2H, CH3-group. of major isomer), 1.36-1. 70 (m, 5H), 1.90-2. 08 (m, 3H), 2. 21-2.30 (m, 1H), 2.34-2. 42 (m, 1H), 2.54-2. 62 (m, 1H), 2.76-2. 81 (m, 2H), 3.13 (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.60 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer), 3.77 (s, 3H,-OCH3), 6.57 (s, 1H), 6.73 (s, 1H). LRMS (FAB+) : 73.1 (100), 382.3 (65), 440.3 (20, [M+H] +). LRMS (FAB-): 438.4 (100, [M-H]-). HRMS (FAB+) 440.260025 C26H38NO3Si requires 440. 262098.
STX207 3-Hydroxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-


6H-cyclopenta[a]phenanthrene-16-carbonitrile (16-cyano-estrone, mixture of 16α and 16ß diastereomers) (CAB01058): Tetrabutyl ammonium fluoride (1. 0 mL, 1M in THF, 1. 0 mmol) was added to a solution of 3-(tert-Butyl-dimethyl)silyl-16-cyano estrone (350 mg, 0. 856 mmol, CAB01044) in THF (20 mL) at 0°C (icebath). The mixture was stirred for 30 minutes at this temperature, acetic acid (0. 5 m'L) and ethyl acetate (80 mL) were added and the mixture was transferred into a separation funnel. The organic phase was washed with water (2 x 25 mL) and brine (25 mL), dried over Na2S04 and concentrated under reduced pressure.
The solid residue was dissolved in ethyl acetate and precipitated by addition of hexane.
The pale yellow solid was filtered off and dried under high vacuum. Yield : 223 mg (88%, ca. 2 to 1 mixture of 16a-cyano and 16ß-cyano diastereomers).'H-NMR (CDCI3, 400 MHz) 6 : 0.97 (s, ca. 1 H, CH3-group of minor isomer), 1.07 (s, ca. 2H, CH3-group of major isomer), 1. 36-1.70 (m, 5H), 1.90-2. 16 (m, 3H), 2. 22-2.34 (m, 1H), 2.38-2. 46 (m, 1H), 2.56-2. 62 (m, 1H), 2.85-2. 92 (m, 2H), 3. 12, (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3. 60 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer), 4.76 (bs, 1H, -OH), 6.59 (d,. J = 2.3 Hz, 1H), 6.65 (dd, J = 8.6, 2.3 Hz, 1H), 7.14 (d, J = 8. 6 Hz, 1H). LRMS (FAB+): 295.2 (100, [M+H] +).
STX208 3-Hydroxy-2-methoxy-13-methyl-17-oxo-7, 8, 9, 11, 12, 13,14, 15,16, 17-


decahydro-6H-cyclopenta [a] phenanthrene-16-carbonitrile (2-methoxy-16-cyano- estrone, mixture of 16a and 16ß diastereomers) (CAB01068). Tetrabutyl ammonium fluoride (1.0 mL, 1 M in THF, 1.0 mmol) was added to a solution of 3-(tert-Butyl-dimethyl)silyl-2-methoxy-16-cyano estrone (440 mg, 1.0 mmol, CAB01066) in THF (20 mL) at 0°C (icebath). The mixture was stirred for 30 minutes at this temperature, acetic acid (0.5 mL) and ethyl acetate (80 mL) were added and the mixture was transferred into a separation funnel. The organic phase was washed with water (2 x 25 mL) and brine (25 mL), dried over Na2S04 and concentrated under reduced pressure. The solid residue was dissolved in ethyl acetate (ca. 2 mL) and precipitated by addition of hexane. The pale yellow solid was filtered off and dried under high vacuum.
Yield : 233 mg (72%, ca. 2 to 1 mixture of 16a-cyano and 16ß-cyano diastereomers) 1H- NMR (D13, 400 MHz) b : 0. 98 (s, ca. 1H, CH3-group of minor isomer), 1. 07 (s, ca. 2H, CH3-group of major isomer), 1. 38-1. 68 (m, 5H), 1. 92-2. 12 (m, 3H), 2. 24-2. 32 (m, 1H), 2. 38-2. 44 (m, 1H), 2. 54-2. 62 (m, 1H), 2. 74-2. 86 (m, 2H), 3. 13 (dd, J = 9. 8, 8. 6 Hs, ca.
0. 65 H, H-16 of major isomer), 3. 60 (dd, J = 10. 2, 1. 6 Hz, ca. 0.35 H, H-16 of minor isomer), 3. 86 (s, 3H,-OCH3), 5. 48 (s, 1H,-OH), 6. 57 (s, 1H), 6. 73 (s, 1H). LRMS (FAB+) : 325. 3 (100, [Mr). LRMS (FAB-) : 324. 3 (100, [M-H]-). HRMS (FAB+) : 325. 168983 C20H23NO3S requires 325. 167794 STX209 Sulfamic acid 16-cyano-13-methyl-17-oxo-7, 8, 9, 11, 12, 13,14, 15, 16, 17- decahydro-6H-cyclopenta [a] phenanthren-3-yl ester (3-sulphamoyl-16-cyano- estrone, mixture of 16a and 16ß diastereomers) (CAB01062) : Sulphamoyl chloride solution (1.0 mL, 0.7M, 0.7 mmol) in toluene was concentrated under reduced pressure (30°C waterbath temperature) to ca. 0.5 mL volume. The residue was cooled to 0°C (icebath) and DMA (1. 5 mL) was added to give a colourless solution. 16-cyano-estrone (89 mg, 0.30 mmol, CAB01058) was added at 0°C, the solution was allowed to warm to room temperature and was stirred overnight at this temperature. Ethyl acetate (50 mL) and water (25 mL) were added, the organic layer was separated and washed with water (2 x 25 mL) and brine (25 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in acetone (ca. 2 mL) and precipitated by addition of hexane. The white solid was filtered off and dried under high vacuum. Yield : 94 mg (84%, ca. 2 to 1 mixture of 16a-cyano and 16p-cyano diastereomers).'H-NMR (CDCI3, 400 MHz) 6 : 0.97 (s, ca. 1 H, CH3-group of minor isomer), 1.07 (s, ca. 2H, CH3-group of major isomer), 1.42-1. 70 (m, 5H), 1.94-2. 18 (m, 3H), 2. 26-2. 38 (m, 1H), 2.40-2. 48 (m, 1H), 2. 56-2. 64 (m, 1H), 2. 88-3. 00 (m, 2H), 3. 14 (dd, J = 9. 8, 8. 6 Hz, ca. 0.65 H, H-16 of major isomer), 3.60 (dd, J = 10. 2, 1. 6 Hz, ca. 0.35 H, H-16 of minor isomer), 4. 92 (bs, 2H,-NH2), 7. 07 (d, J = 2.3 Hz, 1H), 7. 11 (dd, J = 8. 6,2. 3 Hz, 1H), 7. 30 (d, J = 8. 6 Hz, 1H). LRMS (FAB+): 375.1 (100, [M]+).
STX210 Sulfamic acid 16-cyano-2-methoxy-13-methyl-17-oxo-7, 8,9, 11, 12,13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthren-3-yl ester (2-methoxy-3-

sulphamoyl-16-cyano-estrone, mixture of 16α and 16ß diastereomers) (CAB01070): Sulphamoyl chloride solution (1. 0 mL, 0. 7M, 0. 7 mmol) in toluene was concentrated under reduced pressure (30°C waterbath temperature) to ca. 0.5 mL volume. The residue was cooled to O'C (icebath) and DMA (1. 5 mL) was added to give a colourless solution. 2-methoxy-16-cyano-estrone (89 mg, 0.30 mmol, CAB01068) was added at 0°C, the solution was allowed to warm to room temperature and was stirred overnight at this temperature. Ethyl acetate (50 mL) and water (25 mL) were added, the organic layer was separated and washed with water (2 x 25 mL) and brine (25 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in acetone (ca. 2 mL) and precipitated by addition of hexane. The white solid was filtered off and dried under high vacuum. Yield : 102 mg (82%, ca. 2 to 1 mixture of 16a-cyano and 16ß-cyano diastereomers).'H-NMR (CDCI3, 400 MHz) 6 : 0.98 (s, ca. 1H, CH3-group of minor isomer), 1.07 (s, ca. 2H, CH3-group of major isomer), 1.38-1. 70 (m, 5H), 1.90-2. 16 (m, 3H), 2.22-2. 48 (m, 2H), 2. 56-2.64 (m, 1H), 2.79-2. 90 (m, 2H), 3. 15 (dd, J = 9.8, 8.6 Hz, ca. 0.65 H, H-16 of major isomer), 3.62 (dd, J = 10.2, 1.6 Hz, ca. 0.35 H, H-16 of minor isomer), 3. 88 (s, 3H, -OCH3), 5.07 (s, 2H,-NH2), 6.91 (s, 1H), 7.06 (s, 1H). LRMS (FAB+): 405. 1 (100, [M] +).
(3-Benzyloxy-13-methyl-17-oxo-6, 7,8, 9,11, 12,13, 14,15, 17-decahydro- cyclopenta [a] phenanthren-16-ylidene)-hydroxy-acetic acid ethyl ester (CAB03048): Potassium tert.-butoxide (2. 24 g, 20. 0. mmol) was added to a solution of 3-benzyl- estrone (5.77 g, 16.0 mmol) ind diethyl oxalate (5 mL) in toluene (100 mL). The resulting clear yellow solution was stirred overnight at room temperature. Acetic acid (5 mL) was added (colour changes from dark yellow to light yellow), the mixture was transferred into a separation funnel and ethyl acetate (50 mL) and water (100 mL) were added. The organic layer was separated and washed with water (100 mL) and brine (50 mL), dried over Na2SO4 and concentrated under reduced pressure. The light yellow solid residue was suspended in ethanol (50 mL) and heated to reflux for 5 minutes. The colourless crystalline solid was filtered off after cooling to room temperature and dried under high vacuum. Yield : 7. 28 g (99%). 1H-NMR (CDGI3, 400 MHz) 6 : 1. 01 (s, 3H), 1. 41 (t, J = 7.0 Hz, 3H), 1. 44-1. 72 (m, 5H), 1. 98-2. 08 (m, 2H), 2. 26-2. 35 (m, 1H), 2. 40-2. 50 (m, 2H), 2. 89-2. 98 (m, 2H), 3. 10 (dd, J = 16.0, 5.9 Hz, 1H), 4. 37 (q, J = 7. 0 Hz, 2H), 5.05 (s, 2H), 6.75 (d, J = 2. 7 Hz, 1H), 6. 80 (dd, J = 8. 6,2. 7 Hz, 1H), 7. 20 (d, J = 8. 6 Hz, 1H), 7.30- 7.46 (m, 5H), 12. 48 (S, 1H). LRMS (FAB+) : 461.2 (100, [M+H]+).
2-Benzyloxy-6a-methyl-4b, 5, 6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid ethyl ester (CAB03049).
Hydrazine hydrate (0.75, ca. 15 mmol) was added to a suspension of (3-Benzyloxy-13- methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro-cyclopenta[a]phenanthren-16- ylidene)-hydroxy-acetic acid ethyl ester (6.91 g, 15.0 mmol, CAB03048) in ethanol (100 mL) and dichloromethane (20 mL). The suspension turned into a clear, pale yellow solution after a few minutes, which was refluxed for 15 minutes and stirred overnight at room temperature. The solvents were removed under reduced pressure to give a white solid, which was dissolved in ethanol (50 mL) and p-toluene sulphonic hydrate (200 mg) was added. The mixture was heated to reflux for 5 minutes and transferred into a separation funnel after cooling to room temperature. Ethyl acetate (120 mL) and water (50 mL) were added, the organic layer was separated, washed with concentrated NaHC03-solution (30 mL) and brine (30 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was crystallised from ethyl acetate/hexane. Yield : 5.43 g (79%) fine colourless crystals, 1H-NMR(CDCl3, 400 MHz) # : 1. 05 (s, 3H,-CH3), 1. 39 (t, J = 7. 0 Hz, 3H, OCH2CH3), 1.44-1. 72 (m, 4H), 1.98-2. 06 (m, 1H), 2.10-2. 20 (m, 1H), 2.30- 2.42 (m, 4H), 2.82-3. 00 (m, 3H), 4.37 (q, J = 7.0 Hz, 2H, OCH CH3), 5.05 (s, 2H, OCH Ph), 6.73 (d, J = 2.3 Hz, 1H), 6.81 (dd, J = 8.6, 2.3 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.30-7. 46 (m, 5H) NH-proton not visible, probably very broad signal at 11 ppm.
LRMS (FAB+) : 457. 4 (100, [M+H] +).
2-Benzyloxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentateiio [2,1-a]phenanthrene-9-carboxylic acid ethyl ester (CAB03051).
Sodium hydroxide solution (7.5 mL, 2N NaOH, 15 mmol) was added to a suspension of 2-Benzyloxy-6a-methyl-4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno [2, 1.-aJphenanthrene-9-carboxylic acid ethyl ester (2.283 g, 5.0 mmol, CAB03049) in ethanol (40 mL). The mixture was heated to reflux for 30 minutes. Acetic acid (5 mL) was added after cooling to room temperature and the mixture. was stirred for 2 hours, while the product precipitated. The product was filtered off, washed with water (50 mL) and ethanol (20 mL) and dried under high vacuum for 2 days. Yield : 2.130 g (99%) white powder.'H-NMR (DMSO-d6,400 MHz) 6 : 0.95 (s, 3H,-CH3), 1.36-1. 82 (m, 5H), 1.88-1. 98 (m, 1H), 2.00-2. 10 (m, 1H), 2.12-2. 20 (m, 1H), 2.26-2. 46 (m, 4H), 2.70 (dd, J = 14.6, 6.2 Hz, 1H), 2.80-2. 94 (m, 2H), 5.06 (s, 2H, OCH2Ph), 6.73 (d, J = 2.3 Hz, 1H), 6. 78 (dd, J = 8.6, 2.3 Hz, 1H), 7.20 (d, J = 8. 6 Hz, 1H), 7.30-7. 45 (m, 5H), 12.90 (bs, 2H, NH + OH). LRMS (FAB+) : 429.2 (100, [M+H] +).
STX782 2-Hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8- diaza-pentalenol2, 1-a] phenanthrene-9-carboxylic acid ethyl ester (CAB03052): Palladium on charcoal (100 mg, 5% Pd) was added to a solution of 2-Benzyloxy-6a- methyl-4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno [2, 1- a] phenanthrene-9-carboxylic acid ethyl ester (913 mg, 2.0 mmol, CAB03049) in THF (30 mL) and ethanol (30 mL). The resulting mixture was stirred under a hydrogen- atmosphere (balloon) for 72 hours, filtered through a 3 cm layer of celite and concentrated under reduced pressure. The residue was crystallised from acetone/cyclohexane. Yield : 602 mg (82%) pate yellow crystals. 1H-NMR(DMSO-d6, 400 MHz) 6 : 0.92 (s, 3H,-CH3), 1.12-2.84 (m, 16H), 4.14-4.40 (m, 2H, OCH2CH3), 6.45 (d, J = 2.3 Hz, 1H), 6. 50 (dd, J = 8.6, 2.3 Hz, 1H), 7.05 (d, J = 8. 6 Hz, 1 H), 9. 01 (s, 1 H,- OH), 13. 07 (s, 1H, NH). LRMS (FAB+) : 367. 2 (100, [M+H]+). HRMS (FAB+) : 367. 203796 C22H27N2O3 requires 367.202168 STX783 2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8- diaza-pentaleno [2, 1-a] phenanthrene-9-carboxylic acid (CAB03053) : ): Palladium on charcoal (50 mg, 5% Pd) was added to a solution 2-Benzyloxy-6a-methyl- 4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno[2,1- a] phenanthrene-9-carboxylic acid ethyl ester (214 mg, 0.5 mmol, CAB03051) in THF (15 mL) and ethanol (15 mL). The resulting mixture was stirred under a hydrogen- atmosphere (balloon) for 24 hours, filtered through a 3 cm layer of celite and concentrated under reduced pressure. The residue was dried under high vacuum. Yield : 165 mg (98%) light grey solid.'H-NMR (DMSO-d6,400 MHz) 5 : 0.92 (s, 3H,-CH3), 1.30-2. 86 (m, 13H), 6.44 (d, J = 2.3 Hz, 1H), 6.52 (dd, J = 8. 6,2. 3 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 9.02 (s, 1H,-OH), 12.90 (bs, 2H, -COOH and NH). LRMS (FAB+) : 176.0 (100), 329.1 (45, [M+H] +). HRMS (FAB+): 339.171135 C20H23N2O3 requires 339.170868 (2-Benzyloxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8-diaza- pentalenol2, 1-alphenanthren-9-yl)-methanol (CAB03058): 2-Benzyloxy-6a-methyl-4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl ester (457 mg, 1.0 mmol, CAB03049) was added to a suspension of LiAIH4 (100 mg) in THF (20 mL). The mixture was stirred for 30 minutes at room temperature, then water (1 mL) was added (to destroy the excess of LiAlH4). Stirring was continued for 1 hour (light yellowgreen suspension), acetic acid (0. 5 mL) was added, the inorganic solids were filtered of and washed carefully with THF (ca. 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was crystallised from ethyl acetate/diethyl ether. Yield : 215 mg (52%) colorless crystals. 1H-NMR(CDCl3, 400 MHz) o : 1. 01 (s, 3H,-CH3), 1. 40-2.46 (m, 10 H), 2. 60 (dd, J = 13. 7, 6.3 Hz, 1H), 2. 82- 3.00 (m, 2H), 4.69 (s, 2H, -CH2OH), 5. 04 (s, 2H, -CH2Ph), 6. 28 (bs, 2H,-OH and NH), 6. 73 (d, J = 2. 3 Hz, 1H), 6. 80 (dd, J = 8. 6,2. 3 Hz, 1H), 7. 21 (d, J = 8. 6 Hz, 1H), 7. 30- 7. 45 (m, 5H). LRMS (FAB+): 91.1 (100), 415.3 (60, [M+H] +). HRMS (FAB+): 415.238434 C27H31N2O2 requires 415.238554 STX804 9-Hydroxymethyl-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro- 7, 8-diaza-pentaleno[2, 1-a] phenanthren-2-ol (CAB03065):) : Palladium on charcoal (50 mg, 5% Pd) was added to a solution (2-Benzyloxy-6a-methyl- 4b, 5,6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza-pentaleno [2, 1-ajphenanthren- 9-yl)-methanol (120 mg, 0. 289 mmol, CAB03058) in THF (20 mL) and ethanol (20 mL).
The resulting mixture was stirred under a hydrogen-atmosphere (balloon) for 24 hours, filtered through a 3 cm layer of celite and concentrated under reduced pressure. The residue was dried under high vacuum. Yield : 93 mg (99%) white solid.'H-NMR (DMSO- d6,270 MHz) 6 : 0. 82 (s, 3H,-CH3), 1.20-2. 80 (m, 13H), 4.31 (s, 2H, -CH2OH), 4.95 (bs, 1H, -OH), 6.37 (s, 1H), 6. 44 (d, J = 8. 5 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 8.92 (s, 1H,- OH), 11.78 (bs, 1H, NH). LRMS (FAB+) : 325. 2 (100, [M] +). HRMS (FAB+): 325.192528 C2oH25N202 requires 325.191603.
9 3-Hydroxy-13-methyl-16-pyridin-2-ylmethylene-6,7, 8,9,11,12,13,14,15,16- decahydro-cyclopenta [a] phenanthren-17-one (CAB01150) : CAB01150 Sodium hydroxide (1. 0 g, 25 mmol) was added to a suspension of estrone (1. 35 g, 5. 0 mmol) and pyridine-2-carbaldehyde (595 mg, 5.0 mmol) in ethanol (40 mL) at room temperature. The resulting dark orange solution was stirred at room temperature for 4 hours. Then glacial acetic (ca. 10 mL) acid was added with stirring. The colour changed to light yellow and a light yellow solid precipitated. The solid was filtered off and washed with water (50 mL), ethanol (20 mL), diethyl ether (50 mL) and hexane (50 mL) and dried under high vacuum. Yield : 1. 638 g (91 %) yellow powder.
'H-NMR (DMSO-d6, 400 MHz) 5 : 0.89 (s, 3H,-CH3), 1. 30-1. 65 (m, 5H), 1.84-1. 90 (m, 1H), 1.93-2. 00 (m, 1H), 2.14-2. 23 (m, 1H), 2.30-2. 38 (m, 1H), 2.52-2. 60 (m, 1H), 2.70- 2.86 (m, 1 H), 3. 24-3. 32 (m, 2H), 6.46 (d, J = 2.3 Hz, 1H), 6.52 (dd, J = 8. 6,2. 3 Hz, 1H), 7.05 (d, J = 8. 6 Hz, 1H), 7.30 (s, 1H), 7.36 (dd, J = 6.6, 4.7 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.86 (dt, J = 7.4, 1.6 Hz, 1H), 8.70 (d, J = 3.9 Hz, 1H), 9.05 (s, 1H,-OH). LRMS (FAB+): 360.2 (100, [M+H] +). LRMS (FAB-): 358.2 (100, [M-H]-). HRMS (FAB+): 360. 196640 C24H26NO2 requires 360.196354.
C24H25NO2 (359.46) calculated: C 80.19% H 7.01% N 3. 90% found: C 79. 8% H 7. 00% N 3. 92% STX321 3-Hydroxy-13-methyl-16-pyridin-4-ylmethylene-6, 7,8, 9,11, 12,13, 14,15, 16- decahydro-cyclopenta [a] phenanthren-17-one (CAB01154) : CAB01154 Sodium hydroxide (1. 0 g, 25 mmol) was added to a suspension of estrone (1. 35 g, 5.0 mmol) and pyridine-4-carbaldehyde (595 mg, 5.0 mmol) in ethanol (40 mL) at room temperature. The resulting dark orange solution was stirred at room temperature for 4 hours. Then glacial acetic (ca. 10 mL) acid was added with stirring. The colour changed to light yellow and a light yellow solid precipitated. The solid was filtered off and washed with water (50 mL), ethanol (20 mL), diethyl ether (50 mL) and hexane (50 mL) and dried under high vacuum. Yield : 1.588 g (88%) light yellow solid.'H-NMR (DMSO-d6,400 MHz) 6 : 0.92 (s, 3H,-CH3), 1.30-1. 68 (m, 5H), 1.82-1. 92 (m, 1H), 1.96-2. Q4 (m, 1H), 2.14-2. 24 (m, 1H), 2.31-2.38 (m, 1H), 2.61-2. 69 (m, 1H), 2.71-2. 84 (m, 2H), 2. 87-2. 96 (m, 1H), 6.47 (d, J = 2.3 Hz, 1H), 6.53 (dd, J = 8. 6, 2. 3 Hz, 1H), 7. 06 (d, J=8. 6HZ, 1H), 7. 28 (s, 1H), 7. 60 (d, J = 6. 2 Hz, 2H), 8. 65 (d, J = 6. 2 Hz, 2H), 9. 05 (s, 1H,-OH). LRMS (FAB+) : 360. 2 (100, [M+HD. LRMS (FAB-) : 358. 2 (100, [M-H]-). HRMS (FAB+) : 360. 196986 C24H26NO2 requires 360. 196354.
C24H25NO2 (359.46) calculated: C 80.19% H 7.01% N 3.90%. found: C 80.1% H 6.90% N 3.96% STX320 3-Hydroxy-13-methyl-16-pyridin-3-ylmethylene-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta [a] phenanthren-17-one (CAB01156) : CAB01156 Sodium hydroxide (1.0 g, 25 mmol) was added to a suspension of estrone (1. 35 g, 5.0 mmol) and pyridine-3-carbaldehyde (595 mg, 5.0 mmol) in ethanol (40 mL) at room temperature. The resulting dark orange solution was stirred at room temperature for 4 hours. Then glacial acetic (ca. 10 mL) acid was added with stirring. The colour changed to light yellow and a light yellow solid precipitated. The solid was filtered off and washed with water (50 mL), ethanol (20 mL), diethyl ether (50 mL) and hexane (50 mL) and dried under high vacuum. Yield : 1.6313 g (90%) yellow powder.'H-NMR (DMSO-d6,400 MHz) b : 0.90 (s, 3H,-CH3), 1.26-1. 66 (m, 5H), 1.82-1. 90 (m, 1H), 1.95-2. 02 (m, 1H), 2. 14-2.22 (m, 1H), 2.30-2. 38 (m, 1H), 2.57-2. 66 (m, 1H), 2.70-2. 81 (m, 2H), 2.83-2. 92 (m, 1H), 6.45 (d, J = 2. 3 Hz, 1H), 6.51 (dd, J = 8.2, 2.3 Hz, 1H), 7. 04 (d, J = 8.2 Hz, 1H), 7.34 (s, 1H), 7.47 (dd, J = 8. 2,5. 1 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 8.56 (dd, J = 4. 7, 1.2 Hz, 1H), 8. 82 (d, J = 1. 6 Hz, 1H), 9. 03 (s, 1H,-OH). LRMS (FAB+): 360.2 (100, [M+H] +). LRMS (FAB-): 276.1 (100), 358.2 (90, [M-H]-). HRMS (FAB+) : 360.196434 C24H26NO2 requires 360.196354 STX324 13-Methyl-16-pyridin-4-ylmethylene-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol (CAB01158): CAB01158 16-(pyridin-4-yl)methylene estrone (360 mg, 1.0 mmol, CAB01154) was dissolved in ethanol (20 mL) and THF (20 mL). The solution was cooled to 0°C (icebath) and sodium borohydride (100 mg) was added. The reaction mixture was allowed to warm to room temperature and was stirred overnight at this temperature. The clear and colourless solution was concentrated under reduced pressure to ca. 20 mL volume and water (50 mL) was added. The product was filtered off and washed with water (50 mL), ethanol (20 mL) and diethylether (20 mL) and dried under high vacuum. Yield : 312 mg (86%) white powder.'H-NMR (bMSO-d6, 400 MHz) 6 : 0.58 (s, 3H,-CH3), 1.30-1. 40 (m, 5H), 1. 84- 1.98 (m, 2H), 2.12-2. 34 (m, 3H), 2.64-2. 78 (m, 3H), 3.97-4. 03 (m, 1H, H-17), 5.30 (d, J = 5.9 Hz, 1H,-OH), 6.38 (s, 1H) 6.44 (s, 1H), 6.50 (dd, J = 8. 2, 2.4 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 5.5 Hz, 2H), 8.47 (d, J = 5.5 Hz, 2H), 9.00 (s, 1H,-OH). LRMS (FAB+): 362.2 (100, [M+H] +).
STX322 13-Methyl-16-pyridin-2-ylmethylene-7, 8,9, 11,12, 13, 14,. 15,16, 17- decahydro-6H-cyclopenta [a] phenanthrene-3, 1 7-diol (CAB01160) : CAB01160 16- (pyridin-2-yl) methylene estrone (360 mg, 1.0 mmol, CAB01150) was dissolved in ethanol (20 mL) and THF (20 mL). The solution was cooled to 0°C (icebath) and sodium borohydride (100 mg) was added. The reaction mixture was allowed to warm to room temperature and was stirred overnight at this temperature. The clear and colourless solution was concentrated under reduced pressure to ca. 20 mL volume and water (50 mL) was added. The product was filtered off and washed with water (50 mL), methanol (20 mL) and diethylether (50 mL) and dried under high vacuum. Yield : 328 mg (91%) white powder.'H-NMR (DMSO-d6, 400 MHz) S : 0. 58 (s, 3H,-CH3), 1. 23-1. 45 (m, 5H), 1. 81-1. 92 (m, 2H), 2. 10-2. 18 (m, 1H), 2.23-2. 37 (m, 2H), 2. 66-2. 89 (m, 3H), 3. 97-4. 03 (m, 1H, H-17), 5. 21 (d, J = 5.9 Hz, 1H,-OH), 6.41 (d, J = 1.3 Hz, 1H), 6. 48-6. 51 (m, 2H), 7.04 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 7. 0,4. 7 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7. 69 (dt, J. = 7.8, 2.0 Hz, 1H), 8.53 (d, J = 3.5 Hz, 1H), 9.00 (s, 1H,-OH). LRMS (FAB+): 362.2 (100, [M+H]+). LRMS (FAB-): 360.2 (1000, [M-H]-).
STX323 13-Methyl-16-pyridin-3-ylmethylene-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthrene-3, 17-diol (CAB01162): CAB01162 16-(pyridin-3-yl) methylene estrone (360 mg, 1.0 mmol, CAB01156) was dissolved in ethanol (20 mL) and THF (20 mL). The solution was cooled to 0°C (icebath) and sodium borohydride (100 mg) was added. The reaction mixture was allowed to warm to room temperature and was stirred overnight at this temperature. The clear and colourless solution was concentrated under reduced pressure to ca. 20 mL volume and water (50 mL) was added. The product was filtered off and washed with water (50 mL), methanol (20 mL) and diethylether (50 mL) and dried under high vacuum. Yield: 333 mg (92%) white powder. 1H-NMR (DMSO-d6,400 MHz) 6 : 0.58 (s, 3H,-CH3), 1. 15-1. 43 (m, 5H), 1.83-1. 96 (m, 2H), 2.12-2. 31 (m, 3H), 2.62-2. 80 (m, 3H), 3.99 (bs, 1H, H-17), 5.25 (bs, 1H,-OH), 6.41 (d, J = 2.0 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 6.49 (dd, J = 8. 6,2. 3 Hz, 1H), 7.04 (d, J = 8.6 Hz, 1H), 7.34 (dd, J = 8.2, 5.1 Hz, 1H), 7.80 (d, J = 8. 2 Hz, 1H), 8.36 (dd, J = 4.7, 1.2 Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H), 9.00 (bs, 1H,-OH). LRMS (FAB+): 362.2 (100, [M+H] +). LRMS (FAB-): 360.2 (1000, [M-H]-). HRMS (FAB+): 362.211723 C24H28NO2 requires 362.212004 STX452 Sulfamic acid 13-methyl-17-oxo-16-pyridin-4-ylmethylene-7, 8,9, 11,12, 13, 14,15, 16, 17-decahydro-6H-cyclopenta [a] phenanthren-3-yl ester (CAB02078): CAB02078 A solutution of sulphamoyl chloride (5 mL, 0.7M, 3.5 mmol) in toluene was concentrated under reduced pressure (waterbath temperaturen 30°C). The residue was cooled to 0°C and DMA (5 mL) was added. The resulting colourless solution was stirred for 5 minutes and 16-(pyridin-4-yl) methylene estrone (360 mg, 1.0 mmol, CAB01154) was added. The mixture was stirred overnight at room temperature and the product was precipitated by addition of diethyl ether (20 mL) and ethyl acetate (20 mL). The yellow solid was filtered off and purified by heating it with acetone (30 mL) to reflux (the product did not dissolve completely). After cooling to room temperature the soiid was tittered off and dried under high vacuum. Yield : 341 mg (78%) yellow powder. 1H-NMR(DMSO-d6, 400 MHz) 8 : 0. 94 (s, 3H,-CH3), 1. 37-1. 70 (m, 5H), 2. 01-2. 06 (m, 1H), 2. 29-2. 34 (m, 1H), 2. 41-2. 48 (m, 2H), 2. 73-3. 02 (m, 4H), 6. 98 (d, J = 1. 8 Hz, 1H), 7. 02 (dd, J = 8. 4, 1. 8 Hz, 1H), 7. 34 (d, J = 8.4 Hz, 1H), 7. 45 (s, 1H), 7. 92 (s, 2H,-NH2), 8. 18 (d, J = 6.2 Hz, 2H), 8. 89 (d, J = 6.2 Hz, 2H). LRMS (FAB+) : 439. 1 (100, [M+H]+).
STX453 Sulfamic acid 13-methyl-17-oxo-16-pyridin-3-ylmethylene-7, 8, 9, 11, 12, 13,14, 15,16, 17-decahydro-6H-cyclopenta [a] phenanthren-3-yl ester (CAB02085): CAB02085 A solution of sulphamoyl chloride (5 mL, 0.7M, 3.5 mmol) in toluene was concentrated under reduced pressure (waterbath temperaturen 30°C). The residue was cooled to 0°C and DMA (8 mL) was added. The resulting colourless solution was stirred for 5 minutes and 16- (pyridin-3-yl) methylene estrone (360 mg, 1.0 mmol, CAB01156) was added. The mixture was stirred overnight at room temperature and the product was precipitated by addition of diethyl ether (20 mL) and ethyl acetate (20 mL). The yellow solid was filtered off and purified by heating it with acetone (30 mL) to reflux (the product did not dissolve completely). After cooling to room temperature the solid was filtered off and dried under high vacuum. Yield : 333 mg (76%) yellow powder.'H-NMR (DMSO-d6,400 MHz) 6 : 0.93 (s, 3H,-CH3), 1.34-1. 74 (m, 5H), 1.86-1. 96 (m, 1H), 2.00-2. 09 (m, 1H), 2.26-2. 36 (m, 1H), 2.38-2. 46 (m, 1H), 2.66-2. 76 (m, 1H), 2.82-3. 00 (m, 3H), 6.99 (d, J = 2. 3 Hz, 1H), 7.02 (dd, J = 8.6, 2.3 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.45 (s, 1H), 7.94 (s, 2H, - NH2), 8. 01 (dd, J = 8. 2,5. 5 Hz, 1H), 8. 68 (d, J = 8.2 Hz, 1H), 8. 83 {d, J = 5.5 Hz, 1H), 9. 12 (s, 1H). LRMS (FAB+) : 439. 1 (100, [M+H] +).
STX474 16-Furan-2-ylmethylene-3-hydroxy-13-methyl-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (CAB02072) : Potassium hydroxide (2. 0 g) was added to a suspension of estrone (2. 704. g, 10 mmol) and furfuraidehyde (2 mL, ca. 20 mmol) in ethanol (40 mL). The resulting dark brown solution was stirred for 4 hours at room temperature, then acetic acid (5 mL) and water (5ml) were added. After a few minutes the product started to precipitate. The precipitation was completed by addition of more water (ca. 10 mL). The yellow solid was filtered off, washed with water, dissolved in chloroform and dried over Na2SO. The solvent was removed under reduced pressure and the residue was crystallised from ethyl acetate/hexane. Yield : 3.24 g (93%) light yellow crystals. 1H-NMR(CDCl3, 400 MHz) 5 : 0. 97. (s, 3H,-CH3), 1.42-1. 76 (m, 5H), 2.00-2. 20 (m, 2H), 2.22-2. 35 (m, 1H), 2.36-2. 51 (m, 2H), 2.82-3. 00 (m, 2H), 3.14 (ddd, J = 15.2, 6.6, 1.6 Hz, 1H), 5.08 (s, 1H,- OH), 6.52 (dd, J = 3. 1, 1.6 Hz, 1H), 6.62 (d, J = 2. 7 Hz, 1H), 6. 64-6. 70 (m, 2H), 7. 16 (d, J = 8. 2 Hz, 1H), 7.22-7. 27 (m, 1H), 7.57 (d, J, = 1. 6 Hz, 1H). LRMS (FAB+): 349.2 (100, [M+H] +).
STX475 3-Hydroxy-13-methyl-16-thiophen-2-ylmethylene-6, 7,8, 9,11, 12,13, 14, 15, 16-decahydro-cyclopenta [a] phenanthren-17-one (CAB02073) : Potassium hydroxide (2.0 g) was added to a suspension of estrone (1.352 g, 5.0 mmol) and thiophene-2-carboxaldehyde (561 mg, 5.0 mmol) in ethanol (40 mL). The resulting dark brown solution was stirred for 4 hours at room temperature, then acetic acid (5 mL) and water (5ml) were added. the product started to precipitate after a few minutes and was filtered off, washed with water (20 mL), ethanol (10 mL) and diethyl ether (10 mL) and dried under high vacuum. Yield : 1. 614 g (89%) yellow solid. 1H-NMR(CDCl3, 400 MHz) 6 : 0. 98 (s, 3H,-CH3), 1. 44-1. 78 (m, 5H), 2. 02-2. 14 (m, 2H), 2. 26-2. 46 (m, 3H), 2. 84-3. 02 (m, 3H), 4. 98 (s, 1H, -OH), 6. 62 (d, J = 2.7 Hz, 1H), 6. 67 (dd, J = 8. 6, 2. 7 Hz, 1H), 7.14 (dd, J = 5.1, 3.5 Hz, 1H), 7.17 (d, J = 8. 6 Hz, 1H), 7.35 (d, J = 3.5 Hz, 1H), 7.53 (d, J = 5.1 Hz, 1H), 7.67 (s, 1H). LRMS (FAB+): 365.2 (100, [M+H] +). STX507 13-Methyl-16-thiophen-2-ylmethylene-7, 8, 9,11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopente [a]phenanthrene-3, 17-diol (CAB02076) : Sodium borohydride (100 mg) was added to a solution of 16-(thiophenen-2- yl) methylene estrone (365 mg, 1. 0 mmol, CAB02073) in THF (20 mL) and ethanol (40 mL) at 0°C. The solution was stirred overnight at room temperature and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (40 mL), the organic layer was washed with water (40 mL) and brine (20 mL), dried over NA2S04 and concentrated under reduced pressure. The white crude product was crystallised from ethyl acetate/hexane. Yield : 208 mg (57%) colourless crystals.'H-NMR (CDCI3, 400 MHz) o : 0.71 (s, 3H,-CH3), 1.38-1. 58 (m, 6H), 1.98-2. 06 (m, 2H), 2.12-2. 22 (m, 1H), 2.26-2. 40 (m, 2H), 2.68 (dd, J = 16.9, 6.6 Hz, 1H), 2.82-2. 92 (m, 2H), 4.17 (d, J = 9.4 Hz, 1H, H-17), 4.56 (s, 1H,-OH), 6.59 (d, J = 2.3 Hz, 1H), 6.64 (dd, J = 8.6, 2.3 Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 7.00-7. 06 (m, 2H), 7.18 (d, J = 8.6 Hz, 1 H), 7. 27 (d, J = 4.7 Hz, 1 H). LRMS (FAB+): 366.2 (100, [M] +).
STX476 3-Hydroxy-13-methyl-16- (4-nitro-benzylidene)-6, 7, 8, 9,11, 12,13, 14,15, 16-decahydro-cyclopenta [a] phenanthren-17-one (CAB02142) : CAB02142 Potassium hydroxide (2.0 g) was added to a suspension of estrone (1.352 g, 5.0 mmol) and 4-nitro-benzaldehyde (755 mg, 5.0 mmol) in ethanol (80 mL). The resulting dark brown solution was stirred for 4 hours at room temperature, then acetic acid (5 mL) and water (5ml) were added (colour changes to orange). The product precipitated and was filtered off, washed with water (50 mL), ethanol (50 mL) and diethyl ether (50 mL) and dried under high vacuum. Yield : 1.682 g (83%) yellow solid. 1H-NMR (DMSO-d6,400 MHz) 5 : 0.93 (s, 3H,-CH3), 1.30-1. 68 (m, 5H), 1.86-1. 92 (m, 1H), 1.96-2. 04 (m, 1H), 2.16-2. 24 (m, 1H), 2.32-2. 39 (m, 1H), 2.62-2. 82 (m, 2H), 2.88-2. 96 (m, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6. 52 (dd, J = 8. 6, 2. 3 Hz, 1H), 7.06 (d, J = 8. 6 Hz, 1H), 7.42 (s, 1H), 7.92 (d, J = 8. 6 Hz, 2H), 8. 27 (d, J = 8. 6 Hz, 2H), 9. 04 (s, 1H,-OH). LRMS (FAB+) : 404. 1 (100, [M+HD.
STX478 4-(3-Hydroxy-13-methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro- cyclopenta[a]phenanthren-16-ylidenemethyl)-benzonitrile (CAB02144) : CAB02144 Potassium hydroxide (2.0 g) was added to a suspension of estrone (1 : 352 g, 5.0 mmol) and 4-cyano-benzaldehyde (655 mg, 5.0 mmol) in ethanol (40 mL). The resulting orange solution was stirred for 4 hours at room temperature, then acetic acid (5 mL) and water (5ml) were added (colour changes to yellow). The product precipitated and was filtered off, washed with water (50 mL), ethanol (50 mL) and diethyl ether (50,. mL) and dried under high vacuum. Yield : 1.795 g (94%) pale yellow solid.'H-NMR (DMSO-d6,400 MHz) 5 : 0.91 (s, 3H,-CH3), 1.30-1. 66 (m, 5H), 1.82-1. 90 (m, 1H), 1.94-2. 02 (m, 1H), 2.14-2. 22 (m, 1H), 2.30-2. 38 (m, 1H), 2. 58-2. 92 (m, 3H), 6.45 (d, J = 2. 3 Hz, 1H), 6.51 (dd, J = 8. 6, 2. 3 Hz, 1 H), 7.04 (d, J = 8.6 Hz, 1H), 7.36 (s, 1H), 7. 82 (d, J = 8. 6 Hz, 2H), 7.90 (d, J = 8.6 Hz, 2H), 9.03 (s, 1H,-OH). LRMS (FAB+) : 384. 1 (100, [M+H] +). LRMS (FAB-): 383. 2 (100, [M]-).
STX508 13-Methyl-16-(4-nitro-benzylidene)-7, 8,9, 11,12, 13,14, 15,16, 17- decahydro-6H-cyclopenta [a] phenanthrene-3, 17-diol (CAB02155): CAB02155 Sodium borohydride (100 mg) was added to a solution of 16- (4'-nitrophenyl) methylene estrone (404 mg, 1.0 mmol, CAB02142) in THF (20 mL) and ethanol (20 mL) at 0°C.
The solution was stirred overnight at room temperature and concentrated under reduced pressure to ca 10 mL volume. Water (50 mL) was added to the mixture, the precipitate was filtered off, washed with water (50 mL), ethanol (50 mL) and diethyl ether (50 mL) and dried under high vacuum. Yield : 401 mg (99%) pale yellow solid.'H-NMR (DMSO- d6, 400 MHz) d : 0. 60 (s, 3H,-CH3), 1. 22-1. 46 (m, 5H), 1. 84-1. 96 (m, 2H), 2. 16-2. 32 (m, 3H), 2. 62-2. 80 (m, 3H), 4. 02 (bs, 1 H, H-17), 5.36 (d, J = 5. 9 Hz, 1H,-OH), 6. 44 (d, J = 2.3 Hz, 1H), 6. 52 (dd, J = 8. 6,2. 3 Hz, 1H), 6. 56 (d, J = 2. 3 Hz, 1H), 7. 05 (d, J = 8. 6 Hz, 1H), 7. 65 (d, J = 9.0 Hz, 2H), 8. 18 (d, J = 9. 0 Hz, 1H), 9. 02 (s, 1H,-OH). LRMS (FAB+) : 405.2 (100, [M]+).
STX510 4- (3,17-Dihydroxy-13-methyl-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro- cyclopentala] phenanthren-16-ylidenemethyl)-benzonitrile (CAB02157) : CAB02157 Sodium borohydride (100 mg) was added to a solution of 16- (4'-cyano- phenyl) methylene estrone (384 mg, 1.0 mmol, CAB02144) in THF (20 mL) and ethanol (20 mL) at 0°C. The solution was stirred overnight at room temperature and concentrated under reduced pressure to ca 10 mL volume. Water (50 mL) was added to the mixture, the precipitate was filtered off, washed with water (50 mL), ethanol (30 mL) and diethyl ether (50 mL) and dried under high vacuum. Yield : 366 mg (95%) white solid.
'H-NMR (DMSO-d6,400 MHz) 6 : 0.60 (s, 3H,-CH3), 1.30-1. 42 (m, 5H), 1.84-1. 96 (m, 2H), 2.14-2. 34 (m, 3H), 2.65-2. 79 (m, 3H), 3.98-4. 02 (m, 1H, H-17), 5. 31 (d, J = 5.9 Hz, 1 H,-OH), 6.44 (d, J = 2.3 Hz, 1 H), 6.49-6. 52 (m, 2H), 7.05 (d, J = 8.6 Hz, 1 H), 7.57 (d, J = 9. 0 Hz, 2H), 7.78 (d, J = 9.0 Hz, 1H), 9.01 (s, 1H,-OH). LRMS (FAB+) : 385.2 (100,


[MD.

[3- (tert-Butyl-dimethyl-silanyloxy)-13-methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17- decahydro-cyclopenta[a]phenanthren-16-ylidene]-hydroxy-acetic acid ethyl ester (CAB01072).
CAB01072 Sodium ethoxide (340 mg, 5.0 mmol) was added to a solution of 3-O-TBDMS-estrone (700 mg,. 1. 82 mmol) and diethyl oxalate (1. 0 ml) in toluene (20 ml). The resulting yellow solution was stirred for 2 hours at room temperature (TLC-control). The solution was transferred into a separation funnel and ethyl acetate (50 ml) and 6N hydrochloric acid (20 ml) were added. intensive shaking resulted in a nearly colourless organic layer, which was separated and dried over sodium sulphate and concentrated under reduced pressure. The residue was crystallised from ethanol. Yield : 737 mg (83%) colourless crystals. 1H-NMR (CDCl3, 400 MHz) b : 0.19 (s, 6H), 0.98 (s, 9H,-C (CH3) 3), 1.00 (s, 3H), 1.40 (t, J = 7. 2 Hz, 3H), 1.43-1. 71 (m, 5H), 1.98-2. 06 (m, 2H), 2. 24-2.34 (m, 1H), 2.38- 2.46 (m, 2H), 2. 82-2. 90 (m, 2H), 3.08 (dd, J = 16. 0,6. 0 Hz, 1H), 4.36 (q, J = 7. 2 Hz, 2H), 6.57 (d, J = 2.3 Hz,, 1H), 6.63 (dd, J = 8.6, 2'. 3 Hz, 1H), 7. 11 (d, J = 8.6 Hz, 1H), 12.47 (bs, 1H). LRMS (FAB+): 73. 1 (61), 485.3 (100, [M+H] +). LRMS (FAB-): 483. 2 (100, [M- H]-). HRMS (FAB+) 484.263771 C28H4tO5Si requires 484.264503 Hydroxy- (3-hydroxy-13-methyl-17-oxo-6, 7,8, 9,11, 12,13, 14,15, 17-decahydro- cyclopenta [a] phenanthren-16-ylidene)-acetic acid ethyl ester CAB01096, STX330).
CAB01096 Tetrabutylammonium fluoride (1. 5 mi, 1.5 mmol, 1M solution in THF) was added to a solution of [3-(tert-Butyl-dimethyl-silanyloxy)-13-methyl-17-oxo-6, 7, 8, 9, 11, 12, 13, 14, 15, 17-decahydro-cyclopenta[a]phenanthren-16-ylidene]-hydroxy-acetic acid ethyl ester (485 mg, 1.0 mmol, CAB01072) in THF (25 ml) at 0°C (icebath). The resulting yellow solution was stirred for 1 h at this temperature, and then glacial acetic acid (1 ml) and ethyl acetate (75 ml) were added. The mixture was transferred into a separation funnel, washed with water (3 x 25 ml) and brine (25 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was crystallised from ethyl acetate/hexane. Yield : 303 mg (82%) colourless crystals.'H-NMR (CDCI3, 400 MHz) o : 1. 00 (s, 3H), 1. 40 (t, J = 7. 2 Hz, 3H), 1. 42-1. 70 (m, 2H), 2. 22-2. 32 (m, 1H) 2. 36-2. 48 (m, 2H), 2. 83-2. 91 (m, 2H), 3. 09 (dd, J = 16. 0, 6. 0 Hz, 4.36 (q, J = 7.2 Hz, 2H), 4.61 (bs, 1H,-OH), 6. 59 (d, J = 2. 3 Hz, 1H), 6. 64 (dd, J = 8. 2, 2. 3 Hz, 1H), 7. 14 (d, J = 8. 2 Hz, 1H), 12.47 (bs, 1H, -OH). 13C-NMR (CDCl3, 100 MHz) # : 14. 2, 15. 0, 25. 9, 26. 7, 27. 5, 29. 4, 31. 1, 37. 7, 43. 9, 48. 8, 49. 4, 62. 2, 112. 8, 115. 2, 116. 5, 126. 2, 131. 6, 134. 0, 137. 7, 152. 4, 153. 4, 162. 6, 216. 7. LRMS (FAB+) : 370. 2 (100, [M+HD.
16-Benzylidene-3-hydroxy-13-methyl-6,7, 8,9,11,12,13,14,15,16-decahydro- cyclopenta [a] phenanthren-17-one (CAB01122, STX312).
CAB01122 Potassium hydroxide (1.0 g) was added to a solution of estrone (1.350 g, 5. 0 mmol) and benzaldehyde (1.0 g, 9.4 mmol) in methanol (50 ml) at room temperature. The resulting orange solution was stirred at this temperature for 4h. Then glacial acetic acid (ca. 5 ml) was added with stirring. The colour changed to light yellow and a white solid precipitated. The solid was filtered off and washed with water (50 ml), methanol (20 ml), diethyl ether (50 mi) and hexane and dried under high vacuum. Yield 1. 578 g (88%). H-NMR (CDCl3, 400 MHz) 6 : 1.00 (s, 3H), 1.40-1. 77 (m, 5H), 2.04-2. 12 (m, 2H), 2.26-2. 34 (m, 1H), 2.38- 2.45 (m, 1H), 2.50-2. 60 (m, 1H) 2.86-3. 03 (m, 3H), 4.76 (s, 1H, -OH), 6.60 (d, J = 2.3 Hz, 1H), 6.66 (dd, J = 8.2, 2.3 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.34-7. 45 (m, 3H), 7.48 (s, 1 H), 7.54-7. 59 (m, 2H). LRMS (FAB+) : 359.2 (100, [M+H]+). LRMS (FAB-): 357. 2 (100, [M-H] -). HRMS (FAB+) 359.202332 C25H27O2 requires 359.201105 3-Hydroxy-13-methyl-16-(3,4,5-trimethoxy-benzylidene)-6, 7, 8, 9, 11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (CAB01124, STX328).
CAB01124 Potassium hydroxide (1.0 g) was added to a solution of estrone (1.350 g, 2.0 mmol) and 3, 4, 5-trimethoxybenzaldehyde (1. 00 g, 5. 10 mmol) in ethanol (50 ml) at room temperature. The resulting dark brown solution was stirred at this temperature for 4h, then glacial acetic acid (ca. 2.5 ml) was added with stirring. The colour changed to yellow and a white solid precipitated. The solid was filtered off and washed with water (50 ml), ethanol (20 ml), diethyl ether (50 ml) and hexane and recrystallised from ethanol. Yield : 2.063 g (92%) light yellow crystals. 1H-NMR (CDCl3, 400 MHz) o : 1.00 (s, 3H), 1.40-1. 78 (m, 5H), 2.00-2. 11 (m, 2H), 2.26-2. 34 (m, 1H), 2.38-2. 56 (m, 2H), 2.84- 3.04 (m, 3H), 3.90 (s, 3H), 3.91 (s, 6H), 4.93 (s, 1H,-OH), 6.60 (d, J = 2. 3 Hz, 1H), 6.66 (dd, J = 8.2, 2.3 Hz, 1H), 6.80 (s, 2H), 7.17 (d, J = 8.2 Hz, 1H), 7.40 (s, 1H). H). 13C-NMR (CDCI3, 100 MHz) 8 : 15.0, 26.4, 27.2, 29.4, 32.1, 38.4, 44.4, 48.2, 49.0, 56.6, 61.3, 108.2, 113.2, 115.6, 126.6, 131.3, 131.9, 133.9, 135.2, 137.9, 139.7, 153.4, 154.0, 209.9. LRMS (FAB+): 449.3 (100, [M+H] +). LRMS (FAB-) : 447.3 (100, [M-H] -). HRMS (FAB+): 448.223763.
C28H3205 requires 448.224974 C28H3205 (448. 6) calculated: C 74.97% H 7. 19% found: C 75.05% H 7. 18% 13-Methyl-16- (3, 4, 5-trimethoxy-benzylidene)-7, 8,9, 11,12, 13,14, 15,16, 17- decahydro-6H-cyclopenta [a] phenanthrene-3, 17-diol (CAB02001, STX329).
A solution of 3-Hydroxy-13-methyl-16- (3, 4, 5-trimethoxy-benzylidene)-6, 7,8, 9,11, 12, 13, 14, 15, 16-decahydro-cyclopenta[a]phenanthren-17-one (448 mg, 1.0 mmol, CAB01124) in THF/methanof (1 : 1, 40 ml) was cooled to 0°C (ice bath) and sodium borohydride (100 mg, 2. 64 mmol) was added. The reaction mixture was stirred for 2h at this temperature, white it was turning from slightly yellow to colourless. The solution was transferred into a separation funnel and ethyl acetate (100 ml) and water (100 ml) were added. The organic layer was separated, washed with water (50 ml) and brine (50 ml), dried over sodium sulphate and concentrated under reduced pressure. Yield 445 mg (99%) white sotid. H-NMR (CDCtg, 400 MHz) 5 : 0. 74 (s, 3H), 1. 20-1. 60 (m, 6H), 1. 90- 1. 98 (m, 1H), 2. 01-2. 08 (m, 1H), 2. 21-2. 41 (m, 3H), 2. 74-2. 90 (m, 3H), 3. 86 (s, 3H), 3. 89 (s, 6H), 4.15 (d, J = 9.4 Hz, 1H, H-17), 4.62 (s, 1H,-OH), 6.49-6. 51 (m, 1H), 6.59 (d, J = 2.3 Hz, 1H), 6.63-6. 68 (m, 3H), 7. 18 (d, J = 8.2Hz, 1H). LRMS (FAB+) : 451.3 (100, [M+H] +).
16-Benzylidene-13-methyl-7, 8, 9,11, 12,13, 14,15, 16,17-decahydro-6H- cyclopenta [a] phenanthrene-3, 17-diol (CAB01128, STX313).
CAB01128 16-Benzylidene-3-hydroxy-13-methyl-6, 7,8, 9, 11, 12,13, 14,15, 16-decahydro- cyclopenta [a] phenanthren-17-one (179 mg, 0.50 mmol, CAB01122) was dissolved in ethanol (10 ml) and THF (10 ml). The resulting light yellow solution was cooled to 0°C (ice bath) and sodium borohydride (100 mg, 2.64 mmol) was added. The reaction mixture was stirred overnight at room temperature, the solvents were removed under reduced pressure and the residue was dissolved in ethyl acetate (40 ml). The organic solution was washed with water (2 x 25 ml) and brine (25 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and precipitated by addition of hexane. The white solid was filtered off and dried under high vacuum. Yield 175 mg (97%). 1H-NMR (d6-DMSO, 270 MHz) 5 : 0. 61 (s, 3H), 1. 20-1. 50 (m, 5H), 1. 82-. 98 (m, 2H), 2. 20-2. 35 (m, 3H), 2. 58- 2.82 (m, 3H), 3.95-4. 05 (m, 1H, H-17), 5.15 (d, J = 6.2 Hz, 1H), 6.43 (s, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6.52 (dd, J = 8.2, 2.3 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 7.15-7. 20 (m, 1H), 7, 31-7. 41 (m, 4H). LRMS (FAB+) : 343.2 (80), 360.2 (100, [M] +). LRMS (FAB-): 359.3 (100, [M-H]-). HRMS (FAB+) 360. 209709 C25h28O2 requires 360.208930 Sulfamic acid 16-benzylidene-13-methyl-13-oxo-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta[a]phenanthren-3-yl ester (CAB01148, STX314).
CAB01148 5 mi of a 0.7 M sulphamoyl chloride solution in toluene (3.5 mmol) were concentrated under reduced pressure (30°C water bath temperature). The residue was cooled to 0°C (ice bath) and DMA (10 ml) and 16-Benzylidene-3-hydroxy-13-methyl-6, 7,8, 9,11, 12, 13,14, 15, 16-decahydro-cyclopenta [a] phenanthren-17-one (717 mg, 2. 0 mmol, CAB01122) were added. The resulting clear solution was stirred for 1h at 0°C, then allowed. to warm up to room temperature and stirred overnight. Ethyl acetate (50 ml) was added and the solution was washed with water (2 x 30 ml) and brine. The organic layer was dried over sodium sulphate and concentrated under reduced pressure.
The resulting off-white solid was dissolved in a small amount of ethyl acetate and precipitated by addition of hexane. Yield : 683 mg (78%) off-white solid.'H-NMR (CDCI3, 400 MHz) 5 : 0.96 (s, 3H), 1.20-2. 60 (m, 11H), 2. 80-3. 02 (m, 3H), 5. 38 (s, 2H,-NH2), 7.06 (d, J = 2.3 Hz, 1H), 7.11 (ddj J = 8.0, 2.3 Hz, 1H), 7.30 (d, J = 8. 2 Hz, 1H), 7.34- 7.47 (m, 2H), 7.52-7. 65 (m, 2H). LRMS (FAB+): 438.2 (100, [M] +).
Sulfamic acid 16-benzylidene-17-hydroxy-13-methyl-7, 8,9, 11,12, 13,14, 15,16, 17-decahydro-6H-cyclopentala] phenanthren-3-yl ester (CAB01152, STX315).
Sodium borohydride (100 mg, 2.64 mmol) was added to a solution of sulfamic acid 16- benzylidene-13-methyl-17-oxo-7, 8, 9,11, 12,13, 14,15, 16, 17-decahydro-6H- cyclopenta[a]phenanthren-3-yl ester (438 mg, 1. 0 mmol, CAB01148) in THF (20 ml) and ethanol (20 ml) at 0°B. The mixture was stirred for 1h at this temperature (TLO-control) and ethyl acetate (100 ml) and water (50 ml) were added. The organic layer was separated and washed with water (50 ml) and brine (50 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and precipitated by addition of diethyl ether. Yield : 440 mg (quant.) white powder.'H-NMR (CDCig, 400 MHz) 8 : 0. 67 (s, 3H), 1. 36-1. 62 (m, 6H), 2. 00-2. 10 (m, 2H), 2. 22-2. 41 (m, 3H), 2. 78 (dd, J = 17. 4, 7. 2 Hz, 1H), 2. 90-2. 96 (m, 2H), 4. 15 (d, J = 9.4 Hz, 1H, H-17), 4.86 (s, 2H,-NH2), 6.55 (d, J = 2.3 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 7. 10 (dd, J = 8.2, 2.3 Hz, 1H), 7.19-7. 42 (m, 6H). LRMS (FAB+): 439.2 (100, [M] +).
Sulfamic acid 13-methyl-17-oxo-16- (3,4, 5-trimethoxy-benzylidene)-7, 8,9, 11,12, 13,14, 15, 16, 17-decahydro-6H-cyclopenta [a] phenanthren-3-yl ester (CAB02069, STX692).
CAB02069 5 ml of a 0.59 M sulphamoyl chloride'solution in toluene (2.95 mmol) were concentrated under reduced pressure (30°C water bath temperature). The residue was cooled to 0°C (ice bath) and DMA (10 ml) and 3-Hydroxy-13-methyl-16- (3, 4, 5-trimethoxy-benzylidene)- 6,7, 8, 9,11, 12, 13, 14,15, 16-decahydro-cyclopenta [a] phenanthren-17-one (899 mg, 2.0 mmol, CAB01124) were added. The resulting clear solution was stirred for 1h at 0°C, then allowed to warm up to room temperature and stirred overnight. Ethyl acetate (50 ml) was added and the solution was washed with water (2 x 30 ml) and brine. The organic layer was dried over sodium sulphate and concentrated under reduced pressure.
The resulting oft-white solid was dissolved in a small amount of acetone and precipitated by addition of hexane. Yield : 913 mg (86%) light yellow solid, 1H-NMR (d6-DMSO, 400 MHz) b : 0. 91 (s, 3H), 1. 35-1. 74 (m, 4H), 1. 86-1. 92 (m, 1H), 2. 02-2. 10 (m, 1H), 2.26-2. 34 (m, 1H), 2.40-2. 46 (m, 1H) 2.60-2. 70 (m, 1H), 2.84-2. 96 (m, 3H), 3.32 (s, 9H), 6.95 (s, 2H), 6.98 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.2, 2.3 Hz, 1H), 7.29 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.90 (s, 2H,-NH2). LRMS (FAB+): 528.2 (100, [M+H] +). LRMS (FAB-): 526.2 (100, [M-H]-).
SulZmic acid 17-hydroxy-13-methyl-16-(3,4,5-trimethoxy-benzylidene)-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthren-3-yl ester(CABO2034, STX693).
Sodium borohydride (100 mg, 2.64 mmol) was added to a solution of sacid 13-methyl- 17-oxo-16- (3, 4, 5-trimethoxy-benzylidene)-7, 8,9, 11, 12, 13, 14, 15, 16, 17-decahydro- 6H-cyclopenta [a] phenanthren-3-yl ester (528 mg, 1.0 mmol, CAB02069) in THF (20 ml) and ethanol (20 ml) at 0°C. The mixture was stirred for 1h at this temperature (TLC- control) and ethyl acetate (100 ml) ahd water (50 ml) were added. The organic layer was separated and washed with water (50 ml) and brine (50 ml), dried over sodium sulphate and concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate and precipitated by addition of diethyl ether. Yield : 527 mg (99%) white powder.'H-NMR (CDCI3, 400 MHz) 6 : 0.72 (s, 3H), 1.20-1. 60 (m, 4H), 1.64-1. 72 (m, 2H), 1.88-1. 96 (m, 1H), 2.02-2. 10 (m, 1H), 2. 21-2.40(m, 3H), 2.71-2. 94 (m, 3H), 3.83 (s, 3H), 3.86 (s, 6H), 4.13 (d, J = 9.4 Hz, 1H, H-17), 5.17 (s, 2H,-NH2), 6.48 (d, J = 2.3 Hz, 1H), 6.61 (s, 2H), 7.02 (d, J = 2.3 Hz, 1H), 7.09 (dd, J = 8. 2, 2. 3 Hz, 1H), 7.30 (d, J = 8. 2 Hz, 1H). LRMS (FAB+): 529.2 (100, [M] +). LRMS (FAB-): 528.2 (100, [M-H]-).
SECTION 6 General procedure for debenzylation of protected phenol intermediates using a Radleys GreenHouse synthesiser.
Starting materials were dissolved/suspended in THF (1 cm3) and to each suspension was added ethanol (1cm3). Each suspension was degassed by bubbling nitrogen through for a few minutes before Pd/C (5% wt., catalytic) was added and each solution/suspension was degassed for a further few minutes. Hydrogen gas (balloon) was then passed over the reactions and they were stirred under a hydrogen atmosphere at room temperature for 24h.'H NMR of one of the reactions at this stage showed that the debenzylation had not gone to completion therefore all were degassed again and more Pd added, followed by stirring under hydrogen for a further 21h. Each reaction was then filtered through celite and the celite washed with ethyl acetate and methanol. The solutions were concentrated in vacuo and the products purified by flash chromatography.
2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- paniniano ntaleno[2,1-a]phenanthrene-9-carboxylic acid (tetrahydro-furan-2-ylmethyl)- amide STX 892 (GMA02058-2) Rf 0.36 (EtOAc).'H NMR (270MHz, CD30D) S : 0.92 (s, 3H), 1.25-1. 95 (multiplets, 9H), 2. 01-2. 58 (m, 5H), 2.72-2. 82 (m, 3H), 3.25-3. 45 (m, 2H), 3. 59-3. 71 (m, 1H), 3.74-3. 85 (m, 1H), 3.89-4. 00 (m, 1H), 6.40 (d, J=2.5Hz, 1H), 6.45 (dd, J=8.4, 2.7Hz, 1H), 6.99 (d, J=8. 4Hz, 1 H); HPLC 100% (Rt 1. 70, 4: 96 H20 : MeOH) ; LCMS (APCI) 422.27 (M+H+) 2-Hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid methylamide STX 893 (GMA02058-4) Yield 57%. Rf 0.40 (EtOAc) ;'H NMR (270MHz, CD30D) 8 : 1.03 (s, 3H), 1.39-2. 45 (multiplets, 10H), 2.83-2. 88 (m, 3H), 2.89 (s, 3H), 6.50 (d, J=2.5Hz, 1H), 6.54 (appdd, J=8.4Hz, 1H), 7.09 (d, J=8.2Hz, 1H); HPLC 99% (Rt= 1.66, 4 : 96 H20 : MeOH); LCMS (APCI), 350.21 (M-H+) 2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid ethyl-methyl-amide STX894 (GMA02058-5) Yield 76%. Rf 0.33 (EtOAc); 1H NMR (270MHz, CD3OD) #: 0.96 (s, 3H), 1.12 (t, J=7.2HUz, 3H), 1. 25-2. 36 (multiplets, 10H), 2.60-2.79 (m, 3H), 2. 95 and 3.09 (singlets, comb. 3H), 3. 44-3. 54 (m, 2H), 6. 40 (d, J=2. 5Hz, 1H), 6.46 (dd, J=8. 4, 2. 7Hz, 1H), 7. 00 (d, J=8. 4Hz, 1H) ; HPLC 93% (Rt=1. 69, 4 : 96 H20 : MeOH) ; Acc. Mass (FAB+) calcd 380. 233803, obsd.
380. 232979 (ii+H+) 2-Hydroxy-6a-methyl-4b, 5,6, 6a, 8, 10,10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid (1-methyl-1H-pyrrol-2-ylmethyl)- amide STX 895 (GMA02058-8) Yield 36%. Rf 0.75 (EtOAc) ;'H NMR (270MHz, CD30D) 8 : 0.94 (s, 3H), 1.05-1. 90 (multiplets, 6H), 2.15-2. 36 (m, 4H), 2.72-2. 79 (m, 3H), 3.52 (s, 3H), 4.43 (s, 2H), 5.85- 5.86 (m, 1H), 5.96-5. 98 (m, 1H), 6.40 (d, J=2.7Hz, 1H), 6.45 (dd, J=8.4, 2.7Hz, 1H), 6.51-6. 53 (m, 1H), 7.01 (d, J=8. 4Hz, 1H) ; HPLC 100% (Rt=2.11, 4: 96 H20 : MeOH); Acc.
Mass (FAB+) calcd. 431.244702, obsd. 431.244164 (M+H+) 2-Hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11,12-decahydro-7, 8-diaza- pentaleno [2, 1-a] phenanthrene-9-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)- amide STX 896 (GMA02058-9) Yield 82%. Rf 0.26 (EtOAc) ;'H NMR (400MHz, CD30D) # : 1.04 (s, 3H), 1.06-2. 50 (multiplets, 10H), 2.54 (s, 3H), 2. 78-2. 90 (m, 3H), 4.66 (s, 2H), 6.50 (d, J=2.3Hz, 1H), 6. 55 (dd, J=8.2, 2. 7Hz, 1H), 7.09 (d, J=8.6Hz, 1H), 8.48 (s, 1H), 8.49 (s, 1H) ; HPLC 89% (Rt=1. 67,4 : 96 H20: MeOH) ; LCMS (APO) 442. 30 (M-H+) (2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2,1-a]phenanthren-9-yl)-(4-methyl-piperazin-1-yl)-methanone STX 897 (GMA02058-12) Yield 96%. Rf 0. 1 (10% MeOH in DCM) ;'H NMR (270MHz, CD30D) a : 1.05 (s, 3H), 1.28- 2.02 (m, 5H), 2.09-2. 50 (m, 8H), 2.33 (s, 3H), 2.67-2. 94 (m, 3H), 3.52-3. 90 (m, 4H), 6.50 (d, J=2.5Hz, 1H), 6.55 (dd, J=8.3, 2.6Hz, 1H), 7.10 (d, J=8.7Hz, 1H) ; HPLC 94% (Rt=1.68, 4: 96 H20: MeOH); LCMS (APCI) 421.36 (M+H+) 2-Hydroxy-6a-methyl-4b, 5,6, 6a, 8,10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentalenol2, 1-a] phenanthrene-9-carboxylic acid (pyridin-2-ylmethyl)-amide
STX 915 (GMA02070-1) To a suspension of the acid starting material (0. 095g, 0. 28mmol) in dry DCM (6cm3) was added DMAP (catalytic), EDC (0.065g, 0. 3mmol) and triethylamine (0. 04cm3, 0. 37mmol). The starting material did not dissolve therefore dry DMF (2cm3) was added and the resulting solution stirred at rt for 15min. To this solution was added 2- (aminomethyl) pyridine and the reaction was stirred at rt for 4d. The solution was washed with sat. aq. NaHCO3 and the organic layer separated and concentrated in vacuo. Flash chromatography using DCM followed by 10% MeOH in DCM as eluents gave a solution of the product in DMF. Addition of hexane to this followed by a small amount of DCM gave a white powder. 1H NMR (270MHz, DMSO-d6) # : 0. 94 (s, 3H), 1.72-1. 95 (m, 5H), 2. 00-2.49 (m, 4H), 2.55-2. 97 (m, 4H), 4.45-4. 55 (m, 2H), 6.45 (d, 1 H), 6.51 (dd, J=8. 4Hz, 1H), 7.05 (d, J=8. 9Hz, 1H), 7.22-7. 33 (m, 1H), 7.74 (m, 1H), 8.28 (m, NH), 8. 52 (apps, 1H), 9. 03 (s, 1 H) ; HPLC 99. 8% (Rt=1. 67,4 : 96 H20 : MeOH) 2-Hydroxy-6a-methyl-4b, 5, 6, 6a, 8, 10, 10a, 10b, 11, 12-dicahydro-7, 8-diaza- pentaleno[2,1-a]phenanthrene-9-carboxylic acid (2-pyridin-3-yl-ethyl)-amide STX 916 (GMA02070-2) Procedure as for STX 915. Precipitation gave a cream-coloured powder.'H NMR (270MHz, DMSO-d6) 8 : 0.93 (s, 3H), 1.36-3. 5 (multiplets, 17H), 6.47 (appd, 1H), 6.52 (appdd, 1H), 7.07 (d, J=8.2Hz, 1H), 7.31-7. 36 (m, 1H), 7.67 (appd, J-. 7Hz, 1H), 8.45 (m, 2H), 9. 07 (s, 1 H) ; LCMS (APCI) 441.48 (M-H+) Alkylation Reactions 2-Benzyloxy-6a-methyl-7-phenethyl-4b, 5, 6, 6a, 7,10, 10a, 10b, 11,12-decahydro- 7, 8-diaza-pentaleno [2, 1-a] phenanthren-9-ol, GMA02076 To a stirred, cooled (ice bath) solution of the benzyl protected hydroxypyrazole starting material (0.050g, 0. 12mmol) in dry DMF (3cm3) was added sodium hydride (0. 006g of 60% dispersion, 0. 14mmol) and the reaction was stirred at 0°C for 15min. To this was added 2-bromoethyl benzene (0. 024cm3, 0. 18mmol) and the reaction was allowed to warm to room temperature and stirred overnight. Water (5cm3) was added and the product extracted with ethyl acetate. Flash chromatography using hexane followed by ethyl acetate as eluents gave an alkylated product as the second fraction, Rf 0.8 (EtOAc). 1H NMR (270MHz, CDCl3) # : 1.01 (s, 3H), 1. 29-2. 41 (multiplets, 10H), 2. 60 (dd, J= 2. 6, 9Hz, 1H), 2. 82-2. 95 (m, 2H), 3. 07 (t, J=2. 7Hz, 2H), 4. 36 (t, J=7. 2Hz, 2H), 5. 04 (s, 2H), 6. 73 (d, J=2. 7Hz, 1H), 6. 79 (appdd, J=8. 5, 2. 6Hz, QH), 7. 18-7. 45 (m, 11H) ; LCMS PCI) 505. 37 (M+H+) A : 2-Benzyloxy-9-methoxy-6a, 7-dimethyl-4b, 5, 6, 6a, 7, 10, 10a, 10b, 11, 12- decahydro-7, 8-diaza-pentaleno[2,1-a]phenanthrene B: 2-Benzyloxy-6a,7, 8-trimethyl-5, 6a, 7, 8, 10, 10a, 10b, 11, 12-decahydro-4bH- 7, 8-diaza-pentaleno[2, 1-a]phenanthren-9-one, GMA02107 To a stirred solution of the benzyl protected hydroxypyrazole starting material (0.200g, 0. 5mmol) in dry THF (2. 3cm3) was added sodium hydride (0.06g of 60% dispersion, 1. 5mmol) and the reaction was stirred at room temperature for 30min. To this was added iodomethane (0. 1cm3, 1. 5mmol) and stirring under nitrogen at room temperature was continued for 6h. Water was added and the products extracted into ethyl acetate. The combined extracts were concentrated in vacuo. Chromatography on a 20g flash column (flashmaster) using a hexane to ethyl acetate gradient followed by a methanol wash gave the title compounds as the second fraction, Rf 0.7 (EtOAc) and fifth fraction, Rf - baseiine (EtOAc) and 0.65 (10% MeOH in DCM), respectively. Yield of A: 0.046g, 21% ; 'H NMR (270MHz, CDCI3) 8 : 1.01 (s, 3H), 1. 38-1. 54 (m, 1H), 1. 56-1. 75 (m, 2H), 1. 83- 1.97 (m, 2H), 2.15-2. 44 (m, 5H), 2.50-2. 62 (m, 1H), 2. 82-2. 93 (m, 2H), 3.65 (s, 3H), 3.87 (s, 3H), 5.03 (s, 2H), 6. 74 (d, J=2. 5Hz, 1H), 6.79 (dd, J=8. 4,2. 7Hz, 1H), 7.19 (d, J=8. 4Hz, 1H), 7. 29-7. 45 (m, 5H); NOESY (40OMHz, CDCI3) showed interaction between methyl peaks at 1. 01 and 3.65ppm ; HPLC 98% (Rt=4. 70, 4 : 96 H20 : Me0H) ; LCMS (ES-) 427. 48 (M-H+) ; Acc. Mass (FAB+) calcd. 429. 254204, obsd. 429. 254517 (M+H+) Yield of B : 0. 031g. 14%; 1H NMR (270MHz, CDCl3) # : 1. 02 (s, 3H), 1. 36-1. 52 (m, 1H), 1. 55-1. 74 (m, 2H), 1.82-1. 96 (m, 2H), 2.03-2. 20 (m, 3H), 2.28-2. 43 (m, 2H), 2.47-2. 57 (m, 1H), 2. 83-2. 91 (m, 2H), 3.24 (s, 3H), 3.28 (s, 3H), 5.01 (s, 2H), 6.72 (d, J=2. 5Hz, 1H), 6.77 (dd, J=8. 5,2. 6Hz, 1H), 7.15 (d, J=8. 4Hz, 1H), 7.27-7. 69 (m, 5H) ; LCMS (ES-) 427. 42 (M-H+), 9-Methoxy-6a, 7-dimethyl-4b, 5, 6, 6a, 7, 10, 10a, 10b, 11, 12-decahydro-7, 8-diaza- pentaleno[2, 1-a]phenanthren-2-cl, GMA 02114 To a stirred solution of the benzylated starting material (0. 025g, 0.06mmol) in dry DCM (2cm3) under nitrogen, cooled to-78°C (dry ice/acetone) was added boron tribromide (0. 065cm3 of a 1M solution in DCM, 0. 065mol) and stirring at-78°C was continued for 2. 5h. Water was added, the reaction was allowed to warm to room temperature and the resulting white precipitate was collected by filtration. This was shown by'H NMR and LCMS to consist of a 3: 1 mixture of dimethylated and monomethylated products. The dichloromethane layer of the filtrate was found to contain starting material and this was redissolved in DCM and reacted further with boron tribromide at-78°C for 1.5h before quenching with water as before. The white precipitate formed in this case was solely the dimethylated product.'H NMR (270MHz, CD30D) 8 : 1. 09 (s, 3H), 1.40-1. 77 (m, 3H), 1.87-2. 01 (m, 2H), 2.23-2. 51 (m, 5H), 2.69-2. 89 (m, 3H), 3. 71 (s, 3H), 3.94 (s, 3H), 6.50 (d, J=2. 5Hz, 1H), 6.55 (appdd, J-8. 3, 2.6Hz, 1H), 7.09 (d, J=8.2Hz, 1H) ; LCMS (ES-) 337.35 (M-H+) 2-Hydroxy-6a, 7, 8-trimethyl-5, 6,6a, 7, 8, 10,10a, 10b, 11, 12-decahydro-4bH-7, 8- diaza-pentaleno [2, 1-a] phenanthren-9-one, STX942, GMA02116 To a stirred solution of the benzylated starting material (0. 031g, 0. 07moo)) in dry DCM (3cm3) under nitrogen, cooled to-78°C, was added boron tribromide (0. 15cm3 of a 1M solution in DCM (0. 15mmol) and the reaction was stirred at-78°C for 2h. The reaction was then quenched with water and the resulting white precipitate was collected by filtration and washed with water and DCM. Recrystallisation from methanol gave a paie orange powder which was collected by filtration and washed with DCM and hexane, yield


0.0125g, 53%; 1H NMR (270MHz, CD3OD) #: 1.15 (s, 3H), 1.40-1.80 (m, 3H), 1.93-2.04 (m, 2H), 2.21-2. 53 (m, 5H), 2.70 (dd, J=13.1, 5. 7Hz, 1H), 2.80-2.95 (m, 2H), 3.34 (s, 1H), 3. 71 (s, 3H), 3. 84 (s, 3H), 6. 51 (s, 1H), 6. 56 (dd, J=8. 4, 2. 7Hz, 1H), 7. 09 (d, J=8. 4Hz, 1H) ; LCMS (ES-): 337.16 (M-H+) ; HPLC 99.6% (Rt=2. 46, 10 : 90 H2O:MeOH) BIOLOGICAL DATA The following assay for determining 17B-HSD Type I and Type 11 activities was performed.
17B-HSD TYPE @ AND TYPE 11 ASSAY PURPOSE To determine 17ß-hydroxysteroid dehydrogenase activity (types I and 11) in human breast cancer cells (T-47D and MDA) in the absence or presence of regulatory agents.
SAFETY NOTES Wear gloves and a lab coat when working with human cell lines.
# 3H-E1, 3H-E2, 14C-E1, and 14C-E2 are radioactive.
'Assume Ei, Es and inhibitors to be carcinogenic and/or teratogenic.
# DMSO promotes absorption through the skin and so when used as a solvent the solute may be easily absorbed-avoid bodily contact.
THF can form potentially explosive peroxides upon long standing in air.
Diethyl ether has extremely flammable liquid and vapour. It is harmful when swallowed, inhaled or absorbed through skin. Causes irritation to skin, eyes and respiratory tract and affects the central nervous system. o Solid CQ2-contact with the product may cause frostbite or freeze burns in exposed tissues.
Methanol is highly flammable.
# Dichloromethane must be handled with caution-causes respiratory tract, skin and eye irritation and may be a carcinogen. o Ethyl acetate-flammable liquid and vapour which is harmful if swallowed or inhaled.
Causes irritation to skin, eyes and respiratory tract and affects the central nervous system. zu Ecoscint A-contact hazard - will irritate skin and eyes. o PROCEDURE Materials Supplier Cat. No. Comments a. [6,7-3H(N)]-E1 NEN NET-051 40. 40 Ci/mmol b. [6, 7-3H (N)]-E2 NEN NET-013 41. 30 Ci/mmol c. [4-14C]-E1 NEN NEC-512 51.30 mCi/mmol d. [4-14C]-E2 NEN NEC-127 52. 00 mCi/mmol e. T-47D human breast cancer cells f. MDA-MB-231 human breast cancer cells g. Oestrone Sigma E-9750 h. R-Oestradiol Sigma E-8875 i. DMSO ICN Biomedicals 196055 j. Diethyl Ether Fisher D/2450/PB15 k. Solid CO2 BOC 1. Methanol Fisher M/4000/17 m. Dichloromethane BDH Lab. Supplies 103406N 'AnalaR' n. Ethyl Acetate BDH Lab. Supplies 101086J 'AnalaR' o. Ecsocint A National Diagnostics LS-273 p. Test Tubes FisherTES-200-151 G125 x 16mm soda glass q. Test Tubes Fisher TES-200-089Y 75 x 12mm soda glass r. Scintillation Vials Starstedt 73. 662 20mi, polypropylene o Equipment o Beckman LS6000SC Scintillation Spectrometer. o SMI Multi-tube Vortexer - model 2601.
# Fume Hood. o TLC Aluminium Sheets Silica Gel 60 F254. MERCK Purification of 3H-E1/3H-E2.
Set up a TLC plate with a lane at either end and a large lane in the middle for streaking.
* Streak 100 pCi (100 ut or rather 5 x 20µl) 3H-E,/3H-E2 in the middle lane.
# Spot 20 ut cold E1/E2 in the outer lanes.
# Leave the plate in the TLC tank for 1-1 1/2 hours.
Visualise the spots of E1/E2 (in the outer lanes) under UV light and circle.
# Cut out the area where the labelled E1/E2 should be (according to the position of the cold E1/E2 on the TLC plate) and place in a glass vial.
* Elute in 5.0 ml of diethyl ether.
# Vortex mix the contents of the vial and leave for 12 hours at 4°C.
# Vortex the vial again and allow the contents to settle down.
# Add 1 ml of distilled water (to cover all the bits of plate) and freeze the aqueous phase in a solid carbon dioxide/methanol mixture.
* Decant the diethyl ether phase into another glass vial and evaporate to dryness under an airstream at 40°C.
# Dissolve the residue in 1 mi ethanol and count a 5 ul aliquot for radioactivity using a liquid scintillation counter.
Calculations Use Specific Activity of 3H-E1/3H-E2 to calculate how much 3H-E1/3H-E2 needs to be added to each flask for physiological concentration (5 pmol) per flask. Also calculate how much needs to be added to each well for physiological concentration (3 pmol) per well when carrying out the assay in 24-well plate format.
+ Assay of 17ß-hydroxysteroid dehydrogenase activity in T-47D cells (E1 # E2).
# Remove the growth medium from all flasks.
'Add 2.5 ml of substrate [ (assay medium + 3H-E1) + appropriate inhibitor] to each flask.
# Incubate the flasks at 37°C for 0.5 hours. o Take 2 ml from each flask and add directly to tubes containing 14C-oestradiol (5000 cpm). o Extract the steroids from the medium with 4 mi diethyl ether.
# Mechanically shake the tubes for 3 minutes to get a good partition. o Separate the ether phase by freezing the aqueous phase in a solid carbon dioxide/methano) mixture and decant the ether into tubes containing 50 ug (20 ui of 10 mM solution) unlabelled product to help visualise the labelled product upon TLC.
@ Evaporate the ether phase to dryness under an airstream at 40°C.
# Dissolve the residue in 6-8 drops of diethyl ether and spot onto a TLC-plate containing a fluorescent indicator.
Separate the oestrone and oestradiol by TLC using dichloromethane/ethyl acetate (4: 1 v/v) as solvents.
Visualise the spots of product steroid (3H-E2) under UV light, cut them out, and place them in scintillation vials.
Add 0.5 mi methanol to the vials to elute the pieces of TLC.
Add 0.5 ml assay medium to each vial to correct for volume.
Add 10 ml Ecoscint A to each vial.
Measure the total activity of the 3H isotope by counting 0.5 mi substrate solution containing the 3H isotope (3H-E1), 0.5 ml methanol and 10 mi Ecoscint A. Measure the total activity of the'4C isotope by counting 14C-oestradiol (5000 cpm), 0.5 ml assay medium, 0.5 ml methanol and 10 mi Ecoscint A.
# Count product and recovery radioactivity in a liquid scintillation counter using a program for dual [3H/14C] isotopes.
Calculations Overall, four corrections are applied to the raw data: Crossover Correction.
Recovery Correction.
Blank Correction.
Dilution Correction.
Use Microsoft Excel.
B & C represent columns of 3H & 14C raw data respectively: D. CxO. 14 = Crossover E. B-D = Crossover Corrected F. C/Mean Total Activity 14C = Recovery G. E/F = Recovery Corrected H. G - Blank (mean) = Blank Corrected 1. H x Constant (see below) = fmol/flask/0. 5hrs J. [((x1 + x2) x 272) / 1, 000, 000] = ceiis/fiask (millions) K. I/J = fmol/0. 5hrs/million cells L. K x 2 = fmol/hr/million cells M. Mean of each triplicate = Mean Activity N. Standard Deviation of each triplicate = Standard Deviation Activity 0. [ (100/Mean activity of control) x L] = % Activity P. 100-O = % Inhibition Q. Mean % Inhibition of each triplicate = Mean % Inhibition R. SD % Inhibition of each triplicate = Standard Deviation % Inhibition S. Label Treatments and Concentrations = Inhibitor Codes & Concentrations Graphs and statistics as required. Dilution (2. 5/2) Constant = [1. 25 x (p, mol 3H-E1/total cpm 3H-E1/flask)] Assay of 1 R-hydroxysteroid dehydrogenase activity in MDA-MB-231 cells (E2 Ei) Remove the growth medium from all flasks.
# Add 2.5 ml substrate medium [ (assay medium + 3H-E2) + appropriate inhibitor] to each flask.
* Incubate the flasks at 37°C for 3 hours.
Take 2 ml from each flask and add directly to tubes containing 14C-oestrone (5000 cpm).
Extract the steroids from the medium with 4 mi diethyl ether. o Mechanically shake the tubes for 3 minutes to get a good partition.
# Separate the ether phase by freezing the aqueous phase in a solid carbon dioxide/methanol mixture and decant the ether into tubes containing 50 ug (20 ul of 10 mM solution) unlabelled product to help visualise the labelled product upon TLC. o Evaporate the ether phase to dryness under and airstream at 40°C. o Dissolve the residue in 6-8 drops of diethyl ether and spot onto a TLC, plate containing a fluorescent indicator. o Separate the oestrone and oestradiol by TLC using dichloromethane/ethyl acetate (4 : 1 v/v) as solvents.
# Visualise the spots of product steroid (3H-E1) under UV light, cut them out, and place into scintillation vials.
* Add 0.5 mi methanol to the vials to elute the pieces of TLC.
* Add 0.5 mi assay medium to each vial to correct for volume.
* Add 10 mi Ecoscint A to each vial.
* Measure the total activity of the 3H isotope by counting 0.5 mi substrate solution containing the 3H isotope (3H-E2) 0.5 ml methanol and 10 ml Ecoscint A. Measure the total activity of the 14C isotope by counting 14C-oestrone (5000 cpm), 0.5 ml assay medium, 0.5 ml methanol and 10 ml Ecoscint A.
* Count product and recovery radioactivity in a liquid scintillation counter using a program for dual [3H/'4C] isotopes.
Calculations Overall, four corrections are applied to the raw data: Crossover Correction.
Recovery Correction.
Blank Correction.
Dilution Correction.
Use Microsoft Excel.
B & C represent columns of 3H and 14C raw data respectively.
D. C x 0. 14 = Crossover E. B-D = Crossover Corrected F. C/Mean Total Activity 14C = Recovery G. E/F = Recovery Corrected H. G - Blank (Mean) = Blank Corrected 1. H x Constant = fmol/flask/3 hrs J. [ ( (Xl + X2) x 272) /1, 000,000] = cells/flask (millions) K. I/J = fmol/3 hrs/million cells L. K /3 = fmol/hr/million cells M. Mean of each triplicate = Mean Activity N. Standard Deviation of each triplicate = Standard Deviation Activity 0. (100 x Mean Activity of Control) x L = % Activity P. 100 - O = % Inhibition Q. Mean % Inhibition of each triplicate = Mean % inhibition R. SD % Inhibition of each triplicate = SD % inhibition S. Label Treatments and Concentrations = Inhibitor Codes & Concentrations Graphs and statistics as required.
THER NOTES: Dilution (2. 5/2) Constant = [1. 25 x (pmol 3H-E1/total cpm 3H-E1/flask)] Account for all radioactive isotope usage.
'The assay of 17ß-HSD Type I activity in T-47D cells is often carried out in 24-well plate format (rather than T25 flasks). Under these circumstances only 1.5 ml substrate solution ( inhibitor) is used and the incubation time is increased to 3 hours. After incubation only 1 mi is taken from each well.
EXAMPLES OF EXPECTED MEAN % INHIBITION VALUES FOR THE POSITIVE CONTROL: E1 # E2 Typical % inhibitions obtained with 10 pM Ei are between 97% and 99% inhibition.
E2 # E1 Typical % inhibitions obtained with 10 µM E2 are between 37% and 43% inhibition.
RESULTS C_ omaoand Compound % inhibition of % inhibition of ST),. structure s 7p0X$Ds i 7n @' ! 0 @-i0 207 L-. 83, 71, 17 208 35, 31 4 0 /o w vN /O 209 o 71, 73,. 18 -N IC50 = 10 PM 2 nu2 210 95, 95, 8 o-N ICso = 2. 1 M \" r) . 313 " 26 5 HO HO 314 ° - 1 1 315 OH 4 1 Han 0 o 23 16 0 /v 0 320 44 10 HO HO 0 nô-N OU OH /\ Nô Oh 323 52 43 HO HA OH 324 81, 72, 3 IC50= 4. 1 pM HO ho O 327 96, 94, 5 IC50 = 1. 1 zM HO 3, ° 24-12 ho ho O 329 " 63, 33 3 HO ho 0 "aS ho HO 4 s 7, OH 8 47 8 337 oh 47 8 - han NOH oh Ho 0 , HO 0 416 rr\ s, 8 .. ! J./tCgo=0. 34 ! jM HO 0 0 452 28 11 --N /% HaN'O J O i 453 22 10 N-OH H : N M-OH 454 (IGSO = 1. 9 NM 27 HO HA Ho O 474 26 17 o / 0 475 67 14 Hoof S 476 15 15 HO O 477 CoX. 13 HO ho N- HA X J/ o--- HO O 486 OH 97, 15 i 0-11 PM HO ho OH 507 41 25 oh- 508 33 38 Nô HA OH 0 OH 510 57 31 vio HO oh 511 72 $1, IC50= 5. 4 pM HO ri lab zu N\ I ,. /N N S 95 48 IC50=530 nM' HO ho N 809 83 55 HOw ho 812 77 61 HO 813 70 44 820 XH 86 7 Ho NO N\ N-__ / HA ho ° o IC, (, =810 nM HO 845 ° 87 2. 5 hum HO HA Bw d 87 2-5 _. _ XN Ho N--NH N 694b'N v9 IC50=780 nM 0 HA N 87-4 0 Ha Ha --nu 893 H 62 14 N\ ho I NOH 894--NH 88 6 way ho HO 895 N 89-4 0 IC50 = 2. 3 pM Ho 897 ; 50 W07 896 I 80 61 I way ho mu 97 50-0. 7 y° ol ho N---NH H N 93 9 1050 880 nii ho NOH NH 916 N, 98. 5 3 IC5o = 300 nrul 9ns Nv Ho zon arJoD W'i . i '°orp = % f' Gd'v v HO aJ"£ ho 924 12 9. 42 32 15 Ho 946 0 OH OH 69, 87-1. 5 F F HA Hic) 947 HC) H N--N Ha 948 oh HO i ho HO / 75. 36 F/F HA HO IC50 5. 1 pM õ 0 Ft N-O O 100 ° 1040 n n =\ ICI ho 1191 0 H I, 1 96. 1. 2 N N ou A11 publications and patents and patent applications mentioned in the above specification are herein incorporated by reference.
Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.
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We Claim:
1, A substituted cyclopenta[a]phonanthrene compound of Formula Ill
(Formula Removed)
wherein
(l) R1 is a selected from
(x) amide or alkytamide wherein (a) the alkyl of the alkylamide is -CH2- or -CH2CH2, (b) the amide is di-substituted and/or (c) the amide i3 substituted with at least one of alkylheterocycle group, alkenylheterocycle group, alkylheteroaryl group, alkenylheteroaryl group, heteroaryl group, alkyfamine group, alkyloxyalkyl group, alkylaryl group, straight or branched alkyl group,
(tl) R2 is selected from
groyps capable of forming a hydrogen bond , a sulphamate group, a phospnonate group, a thiophosphonate group, a sulphonate group and a sulphonamide group; and
OH) R3 is selected from
-OH, =O, or a -C(=O)- mimetic.
2. A compound as claimed in claim 1 wherein the aryl group of the alkylaryl group is substituted by a group selected from N,N-dialkyI, alkoxy, nitro (-NO2-), nitrite, and azaalkyl group®.
3. A compound as claimed in claim 2 wherein the N,N-dialkyl is NMe2
4. A compound as claimed in claim 2 or 3 wherein the alkoxy group is methoxy.
5. A compound as claimed in any one of the preceding claims wherein the alkyiaryi group is -CH2-Ph.

6. A compound as claimed in any one of the preceding claims wherein the aryl
group of the alkylheteroaryl group is substituted by a group selected from N,N-diaIkyl,
alkoxy, nitro (-NO2), and nitrite groups.
7. A compound as claimed in claim 6 wherein the N,N-dialkyl is NMe2.
8. A compound as claimed in claim 6 or 7 wherein the alkoxy group Is methoxy.
9. A compound as claimed in any one of the preceding claims wherein the alkylheteroaryi group is selected from

(Formula Removed)


10. A compound as claimed in any one of the preceding claims wherein the amide is of the formula -C(=O)NR5R6 or -NfCO-R7R8, wherein R5, R6, R7 and R8 are independently selected from H and hydrccarbyl groups.
11. A compound as claimed in any one of the preceding claims wherein the amide or alkylamide is an alkylamide.
12. A compound as claimed in any one of the preceding claims wherein the substituents of the di-substituted amide together form a cyclic structure.
13. A compound as claimed in claim 12 wherein the substrtuents cf the di-substituted amide togetherfbrm an aryt ring.
14. A compound as claimed in claim 12 or 13 wherein the substrtuents of the di-substituted amide together form & heterocyclic ring.
15. A compound as claimed in any one of the preceding claims wherein R2 is selected from groups capable of forming a hydrogen bond and a sufphamate group,
16. A compound as claimed in claim 15 wherein the group capable of forming a hydrogen bond are selected from -OH.

17. A compound as claimed in any one of the preceding claims wherein the -G(=O)-mimetic Is selected from -CN, =N-O-alkyl group, =N-O-H group, pyridine, pyrimidine, and groups of the formula

(Formula Removed)


A compound as claimed In any one of the preceding claims wherein R3 is selected from OH, and =0.
19. A compound as claimed tn any one of the preceding claims wherein R3 is =0.
20. A compound as claimed in any one of the preceding claims wherein the compound is of Formula IV
(Formula Removed)

21. A' compound as claimed in any one of the preceding claims wherein the sulphamate group is of the formula
(Formula Removed)

wherein R9 and R10 are independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or combinations thereof, or together represent alkytene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
22. A compound as claimed in claim 21 wherein at least one of R8 and R1 is H.

23. A compound as claimed in claim 21 wherein R9 and R10 is H.
24. A compound as claimed in any one of claims 1 to 23 as and when used in a pharmaceutical composition optionally with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.