Indian Patents
Title of Invention

"CHROMAN COMPOUNDS AND COMPOSITION THEREOF"
Abstract Novel chroman derivatives and intermediate compounds, compositions containing same, methods for their preparation and uses thereof as therapeutic agents particularly as anti-cancer and chemotherapeutic selective agents are described.
Full Text CHROMAN DERIVATIVES, MEDICAMENTS AND USE IN THERAPY
Field of the Invention
The present invention relates to certain novel chroman derivatives, compositions
containing same, methods for their preparation and uses thereof as therapeutic agents
particularly as anti-cancer and chemotherapeutic selective agents.
Background of the Invention
Over 700 different naturally occurring isoflavones are known some of which have
biological properties with potential therapeutic benefit.
US 5,726,202 generically discloses certain isoflavan compounds, particularly 3,4- •
diarylchroman and centchroman for the treatment of benign prostatic hypertrophy.
WO 01/17986 also discloses certain isoflavan compounds.
Summary of the Invention
Surprisingly, the present inventors have found a novel group of compounds of the general
formula (I) which exhibit important therapeutic activities including strong anti-cancer
activity, chemotherapeutic selectivity and radiosensitisation of cancers.
Thus according to an aspect of the present invention mere is provided a compound of the
general formula (I):
(Figure Removed)
wherein
SUBSTITUTE SHEET (RULE 26) RO/AU
(Figure Removed)
R1 is hydrogen, alkyl, cycloalkyl or C(O)R7
R2 and R3 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, halo or
OC(O)R7, with the exception that R2 and R3 are not both hydrogen,
R4, R5 and R6are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl,
amino, C1-4-alkylamino or di(C1-4-alkyl)amino, OC(O)R7 or OR8,
R7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino, and
R8 is-aryl such as phenyl or arylalkyl such as-benzyl, and
R9 is hydrogen hydroxy, alkyl, alkoxy, cycloalkyl or halo,
or a pharmaceutically acceptable salt or derivative thereof.
In a preferred embodiment of the present invention R9 is hydrogen. Accordingly, in
another aspect of the invention there is provided a compound of the formula (I-a):
wherein
RI is hydrogen, alkyl, cycloalkyl or C(O)R7,
R2 and R3 are independently hydrogen, hydroxy, alkoxy, halo or OC(O)R7, with the
exception that R2 and R3 are not both hydrogen,
R4, R5 and R6are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl,
OC(O)R7, amino, and
R7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino.
According to another aspect of the present invention there is provided a process for the
preparation of a compound of formula (I) comprising the step of reacting the keto group of
a compound of the formula (II):
or the analogue thereof including a-substituent which corresponds to R9 in compounds of
formula (I)
wherein
R1 is alkyl or a protecting group such as Si(R1o)3,
Ra and R3are independently hydrogen, alkoxy or OSi(Rjo)3, with the exception that R
and R3are not both hydrogen, and
R10 is independently alkyl or aryl,
with an arylating agent WM"1",
wherein
W" is an optionally substituted aryl radical, and
M+ is one or more counter ions, preferably [MgBr]+,
to form the intermediate tertiary alcohol of formula (III):
or protected derivative thereof or a salt thereof (or an analogue thereof including a
substituent which corresponds to R9 in compounds of formula (I)) and which is dehydrated
to form a compound of formula (IV):
(Figure Removed) (or an analogue thereof including a substituent which corresponds to Rg in compounds of
formula (I)) the double bond of which is subsequently reduced, for example, by
hydrogenation and optionally deprotected to form a compound of formula (I)-.
According to another aspect of the present invention there is provided a compound of the
general formula (III), compositions containing same and uses thereof.
In another aspect, there is provided a compound of the general formula (IV), compositions
containing same and uses thereof.
Thus, according to another aspect of the present invention there is provided the use of a
compound of formula (I) in therapy, particularly chemotherapy and/or as a radiosensitising
or chemosensitising agent.
According to another aspect of the present invention there is provided a method for the
treatment, prevention or amelioration of a disease or disorder, which comprises
administering to a subject one or more compounds of the formula (I) or a pharmaceutically
acceptable salt or derivative thereof optionally in association with a carrier and/or
excipient.
According to another aspect of the present invention there is provided the use of one or
more compounds of formula (I) or a pharmaceutically acceptable salt or derivative thereof
in the manufacture of a medicament for the treatment of a disease or disorder.
According to another aspect of the present invention there is provided an agent for the
treatment, prophylaxis or amelioration of a disease or disorder which agent comprises one
or more compounds of formula (I) or a pharmaceutically acceptable salt or derivative
thereof.
According to another aspect of the present invention there is provided a pharmaceutical
composition which comprises one or more compounds of formula (I) or a pharmaceutically
acceptable salt or derivative thereof in association with one or more pharmaceutical
carriers, excipients, auxiliaries and/or diluents.
According to another aspect.of the present invention there is provided a drink or food-stuff,
which contains one or more compounds of formula (I) or a pharmaceutically acceptable
salt or derivative thereof.
These and other aspects of the invention will become evident from the description and
claims which follow, together with the accompanying drawings.
Brief Description of the Figures
Fig. 1 represents a comparison of dehydroequol (DHE graph A), 3-(4-hydroxyphenyl)-4-
(4-methoxyphenyl)chroman-7-ol (HMC compound 1 according to the invention graph B)
and cisplatin (graph C) toxicity in neonatal foreskin fibroblasts.
Fig. 2 represents HMC efficacy in melanoma cells in comparison with cisplatin.
Fig. 3 represents a pharmacokinetic profile of free and total forms of HMC (A) and DHE
(B) after p.o (peri oral) administration to BALB/c mice (50 mg/kg).
Fig. 4 represents a comparison of the pharmacokinetic profile the HMC concentration in
serum after i.v (intravenously) and i.p (intraperitoneally) administration of HMC
formulated in 20% hydroxypropyl-beta-cyclodextrin at a dose of 50 mg/kg.
Fig. 5 represents comparative mean tumour volume data taken from nude mice bearing
HP AC pancreatic cancer rumours treated with either i.p dosed 20% HPBCD (vehicle
control, qdxlS) or HMC (100 mg/kg, qdxl5). Data represented as mean ± SEM*,
student's T-test,p Fig. 6 represents comparative mean terminal tumour mass data taken from nude mice
bearing HPAC pancreatic cancer tumours treated with either i.p dosed 20% HPBCD
(vehicle control, qdxl5) or HMC (100 mg/kg, qdxl5). Data represented as mean ± SEM*,
student's T-test, p Fig. 7 represents comparative mean terminal tumour mass data taken from nude mice
bearing-HPAC pancreatic-cancer tumours treated with either.i.p dosed.20% HPBCD
(vehicle control, qdxlS) or HMC (100 mg/kg, qdxlS). Data represented as mean ± SEM*,
student's T-test, p Fig. 8 represents a summary of apoptosis incidence in DHE and HMC treated melanoma
cells over a 24 and 48 hour period.
Fig. 9 represents selective initiation of programmed cell death in HMC and DHE treated
malignant melanoma cells (Mel-RM and Me4405). The same concentration of DHE and
HMC and exposure times do not induce apoptosis in normal fibroblasts (MRC-5).
Fig. 10 represents a 3D analysis of HMC-cisplatin synergy cytotoxicity data in the MM200
melanoma cell line. HMC-cisplatin combinations were assessed using a 5-day
combination protocol (Fig 10 A), or a 24 hr HMC-> anti-cancer sequence (Fig 10 B). For
each combination experiment HMC was assessed at 10,5,2 and 1 pM. See Table 8 for raw
data.
Fig. 11 represents the percentage inhibition of TNFa in murine macrophages by
compounds 6 and 7 of the invention.
Fig. 12 represents the H n.m.r. spectrum of 3-(4-hydroxyphenyl)-4-(4-
hydroxyphenyl)chroman-7-ol.
Detailed Description of the Invention
The present inventors have found that a class of isoflavan derivatives of the general
formula (I) show surprising and unexpected biological and pharmaceutical properties.
The compounds of formula (I) of the invention are believed to have favourable toxicity
profiles with normal cells and good bioavailability. Surprisingly the compounds of the
invention-exhibit anti-cancer activity, significantly.better than or at least comparable to
known cancer treatments.
The compounds of formula (I) are cytostatic and cytotoxic against a broad range of cancer
cells of human and animal origin. By cancer cells, it is meant cells that display malignant
characteristics and which are distinguished from non-cancer cells by unregulated growth
and behaviour which usually ultimately is life-threatening unless successfully treated.
The cancer cells that have been found to be responsive to compounds of formula (I) are of
epithelial origin (for example, prostate, ovarian, cervical, breast, gall-bladder, pancreatic,
colorectal, renal, and non-small lung cancer cells), of mesenchymal origin (for example,
melanoma, mesothelioma and sarcoma cancer cells), and of neural origin (for example
glioma cancer cells). It is highly unusual and surprising to find a related group of
compounds that display such potent cytotoxicity against cancer cells, but with low toxicity
against non-cancer cells such as keratinocytes derived from human foreskin. Such cancer
cell selectivity is highly unusual and unexpected.
Advantageously the compounds of formula (I) show cytotoxicity against cancer cells that
are well recognised for being poorly sensitive to standard anti-cancer drugs. It is highly
unusual and unexpected to find such potent activity against cancers, for example,
cholangiocarcinoma, pancreatic adenocarcinoma and melanoma.
Advantageously the compounds of formula (I) also unexpectedly display an ability to
radio-sensitise cancer cells, by which it is meant that these compounds either lower the
amount of gamma-irradiation that is required to kill the cells, or they convert cancer cells
from a state of radio-resistance to a radio-sensitive state.
Additionally the compounds of formula (I) are thought to possess chemo-sensitising
activity, that is they increase the cytotoxicity of chemotherapeutic agents, especially to
cancer cells, and/or convert cancerous cells from a state of chemo-resistance to a chemosensitive
state.
Compounds of the invention may also provide chemo and/or radio-protective properties for
non-cancerous cells. This has significant therapeutic implications because the traumatic
side-effects of chemotherapy and radiotherapy are caused by the toxicity of the traditional
treatments to non-cancerous cells.
The properties described above offer significant clinical advantages.
The radio and/or chemo-protective properties of the compounds of the invention may be
employed to protect healthly individuals from the effects of radiation and/or chemical
toxins, or lessen the effects of the same.
Thus, the invention also provides the use of compounds of formula (I) to treat patients with
cancer by either reducing the rate of growth of such rumours or by reducing the size of
such tumours through therapy with said compounds alone, and/or in combination with each
other, and/or in combination with other anti-cancer agents, and/or in combination with
radiotherapy.
The use of compounds of the present invention either alone or in combination therapy as
described above may reduce the adverse side-effects often experienced by patients when
treated with standard anti-cancer treatments. The use of compounds of the invention may
mean that lower doses can be employed in such therapy which represents an important
advance for cancer sufferers.
Preferably Rj in compounds of formula (I) is in the 3-position.
In another aspect of the invention R9 is C1-4-alkyl, such as methyl.
Preferably in compounds of formula (I-a):
RI is hydrogen, C1-4-alky! or C(O)R7,
R2 and R3 are independently hydrogen, hydroxy, C1-4-alkoxy, halo or OC(O)R7,
provided that R2 and R3 are not both hydrogen,
R4, R5 and R6 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl,
OC(O)R7, and
R; is C1-4-alkyl, phenyl or benzyl,
or a pharmaceutically acceptable salt or derivative thereof.
More preferably in compounds of formula (I-a):
RI is hydrogen, methyl, ethyl, propyl, isopropyl or acetyl,
R2 and R3 are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy,
isopropoxy, bromo, chloro, fluoro or acetyloxy, with the exception that RZ and RS
are not both hydrogen,
R4 is hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy or acetyloxy, and
RS and Re are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy,
isopropoxy, acetyl, or acetyloxy,
or a pharmaceutically acceptable salt or derivative thereof.
Particular preferred compounds of formula (I-a) have the following substituents where:
RI is hydrogen, methyl or acetyl,
R2 and R3 are independently hydrogen, hydroxy, methoxy, bromo or acetyloxy, with
the exception that R2 and R3 are not both hydrogen,
R4 and R6 are independently hydrogen, hydroxy, methoxy or acetyloxy, and
RS is hydrogen,
or a pharmaceutically acceptable salt or derivative thereof.
The invention also extends to compounds of formula (I-b):
(Figure Removed)wherein:
Ri represents hydrogen or C1-6-alkyl, more preferably hydrogen or methyl, especially
hydrogen.
R2 represents hydrogen, hydroxy or C1-6-alkoxy such as methoxy, ethoxy, propoxy,
more preferably hydroxy or methoxy, especially hydroxy.
RS represents hydrogen, hydroxy, C1-6-alkoxy such as methoxy, ethoxy, propoxy, more
preferably hydrogen or methoxy, especially hydrogen,
with the proviso that R2 and R3 do not both represent hydrogen,
R4 represents hydrogen, hydroxy, C1-6-alkoxy such as methoxy, ethoxy, propoxy,
alkyl such as methyl, ethyl, propyl, isopropyl, especially hydrogen, hydroxy,
methoxy or methyl particularly methoxy or hydroxy,
RS represents hydrogen, d-g-alkoxy, Cj_6-alkyl, especially hydrogen, methoxy,
hydroxy, particularly hydrogen,
or a pharmaceutically acceptable salt or derivative thereof.
Preferred compounds of the invention include those of the general formula (I-c):
20 wherein:
PCT/AU2005/001436
RI is hydrogen or C1-C6 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
secbutyl, tertiary butyl,
R.2 is hydroxy or C1-6-alkoxy such as methoxy, ethoxy, propoxy, isopropoxy, butoxy,
isobutoxy, secbutoxy, tertiary butoxy, and
R4 is hydroxy or C1-6-alkoxy such as methoxy, ethoxy, propoxy, isopropoxy, butoxy,
isobutoxy, secbutoxy, tertiary butoxy
or a.pharmaceutically. acceptable, salt or a derivative thereof.
More preferably in compounds of formula (I-c) RI is hydrogen or methyl, especially
hydrogen.
More preferably in compounds of formula (I-c) Ra is hydroxy or methoxy, especially
hydroxy.
More preferably in compounds of formula (I-c) R4 is hydroxy or methoxy, especially
methoxy.
In an alternative aspect the invention provides compounds of formula (I-d):
(I-d)
wherein:
RI is hydrogen, alkyl, cycloalkyl or C(O)R7) and
RS is hydroxy, alkoxy, alkyl, cycloalkyl, halo or OC(O)R7, with the exception that
and R3 are not both hydrogen,
R« is hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino, C1-4-alkylamino or
di(C1-4-alkyl)amino or OC(O)R7, and
R7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino.
In a further alternative aspect the invention provides compounds of formula (I-e):
wherein
R1 is hydrogen, alkyl, cycloalkyl or C(O)R7, and
R2 and R3 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, halo or
OC(O)R7, with the exception that R2 and R3 are not both hydrogen,
Preferably in compounds of formula (I-e) R1 represents hydrogen or methyl, especially
hydrogen.
Preferably in compounds of formula (I-e) R2 represents hydroxy or C1-C6 alkoxy such a
methoxy.
In compounds of formula (I-e) preferably R3 represents hydrogen, hydroxy or methoxy,
especially hydrogen.
20 In a further alternative aspect the invention provides compounds of formula (I-f):
(Figure Removed)wherein
R1 is hydrogen, alkyl, cycloalkyl or C(O)R7, and
R3 is hydroxy, alkoxy, alkyl, cycloalkyl, halo or OC(O)R7, with the exception that R2
and RS are not both hydrogen,
R4 is hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino, C1-4-alkylamino or
di(C1-4-nlkyl)nminn., OC(O)R7 or OR8, and
R7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino, and
Rg is aryl such as phenyl or arylalkyl such as benzyl.
Preferably in compounds of formula (I-f) R1 represents hydrogen or methyl, especially
hydrogen.
Preferably in compounds of formula (I-f) R2 represents hydroxy or C1-6-alkoxy such as
methoxy, especially hydroxy.
Preferably in compounds of formula (1-f) R.I represents hydrogen or Ci ^-alkoxy such as
methoxy, especially hydrogen.
Preferably in compounds of formula (I-f) RS is in the 3-position.
Preferably in compounds of formula (I-f) R4a represents amino, CM-alkylamino or
alkyl)amino, especially amino.
Especially preferred compounds of formula (I) include:
3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (HMC; Cpd. 1);
3-(4-hydroxyphenyl)-4-phenylchroman-7-ol (Cpd. 2);
3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 3);
3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (Cpd. 4);
3-(4-hydroxyphenyl)-4-(4-methylphenyl)chroman-7-ol (Cpd. 5);
3-(4-methoxyphenyl)-4-(4-methoxyphenyl)-7-methoxychroman (Cpd. 6);
3-(4-hydroxyphenyl)-4-(2,6-dimethoxy-4-hydroxyphenyl)chroman-7-ol (Cpd. 7);
3-(4-hydroxyphenyl)-4-(2-hydroxyphenyl)chroman-7-ol (Cpd. 8);
3-(4-hydroxyphenyl)-4-(3-acyl-2-hydrpxy-4-methoxyphenyl)chroman-7-ol (Cpd. 9);
3-(3-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 10);
3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroraan-7-ol (HHC; Cpd. 11);
3-(4-bromophenyl)-4-(4-methoxyphenyl)chroman-7-ol (Cpd. 12);
3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 13);
3-(4-hydroxyphenyl)-4-(3-aminophenyl)chroman-7-ol (Cpd. 14);
3-(4-hydroxyphenyl)-4-(4-phenoxyphenyl)chroman-7-ol (Cpd 15);
3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-8-methylchroman-7-ol(Cpd 16).
(Figure Removed)
or a pharmaceutically acceptable salt thereof.
The compounds of formula (I) according to the invention include two chiral centres. The
present invention includes all the enantiomers and diastereoisomers as well as mixtures
thereof in any proportions. The invention also extends to isolated enantiomers or pairs of
enantiomers. Methods of separating enantiomers and diastereoisomers are well known to
person skilled in the art.
It will be clear to persons skilled in the art that the in compounds of formula (I) the aryl
substituents on the heterocyclic ring can be cis or trans relative to each other. Preferably
in the compounds of formula (I) these substituents will be cis.
A particularly preferred compound of the present invention is the cis-isomer of compound
or a pharmaceutically acceptable salt thereof.
Likewise, particularly preferred compounds are compound Nos. (2) to (16) in the cisconformation.
The compounds of formulae (III) and (IV) are intermediates as set out herein. Each
corresponding isoflavan-4-ol and isoflavan-3-ene intermediate of compound Nos. (1) to
(16) are also preferred compounds of the present invention.
W in compounds of formula (III) and (IV) may, for example, represent the following
radicals: ...
or a protected derivative thereof wherein R4, R5 and R6 are as defined above for
compounds of formula (I),
The term "isoflavone" as used herein is to be taken broadly to include as isoflavones,
isoflavenes, isoflavans, isoflavanones, isoflavanols and the like.
The term "alky!" is taken to include straight chain and branched chain saturated alkyl
groups of 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
secbutyl, tertiary butyl, pentyl and the like. The alkyl group more preferably contains
preferably from 1 to 4 carbon atoms, especially methyl, ethyl, propyl or isopropyl.
Cycloalkyl includes C3-6 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl and
cyclohexyl.
The alkyl group or cycloalkyl group may optionally be substituted by one or more of
fluorine, chlorine, bromine, iodine, carboxyl, C1-C4-alkoxycarbonyl, C1-C4-alkylaminocarbonyl,
di-(C1-C4-alkyl)-amino-carbonyl, hydroxyl, C1-C4-alkoxy, formyloxy, C1-C4-
alkyl-carbonyloxy, C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl.
Preferably the alkyl group does not bear any substituents.
The term "aryl" is taken to include phenyl, benzyl, biphenyl and naphthyl and may be
optionally substituted by one or more C1-C4-alkyl, hydroxy, C1-C4-alkoxys carbonyl, Cj-
C4-alkoxycarbonyl, C1-C4-alkylcarbonyloxy, nitro or halo.
The term "halo" is taken to include fluoro, chloro, bromo and iodo, preferably fluoro and
chloro, more preferably fluoro. Reference to for example "haloalkyl" will include
monohalogenated, dihalogenated and up to perhalogenated alkyl groups. Preferred
haloalkyl groups are trifluoromethyl and pentafluoroethyl.
The compounds of the invention include all salts, such as acid addition salts, anionic salts
and zwitterionic salts, and in particular include pharmaceutically acceptable salts as would
be known to those skilled in the art. The term "pharmaceutically acceptable salt" refers to
an organic or inorganic moiety that carries a charge and that can be administered in
association with a pharmaceutical agent, for example, as a counter-cation or counter-anion
in a salt. Pharmaceutically acceptable cations are known to those of skilled in the art, and
include but are not limited to sodium, potassium, calcium, zinc and quaternary amine.
Pharmaceutically acceptable anions are known to those of skill in the art, and include but
are not limited to chloride, acetate, tosylate, citrate, bicarbonate and carbonate.
Pharmaceutically acceptable salts include those formed from: acetic, ascorbic, aspartic,
benzoic, benzenesulphonic, citric, cinnamic, ethanesulphonic, fumaric, glutamic, glutaric,
gluconic, hydrochloric, hydrobromic, lactic, maleic, malic, methanesulphonic, naphthoic,
hydroxynaphthoic, naphthalenesulphonic, naphthalenedisulphonic, naphthaleneacrylic,
oleic, oxalic, oxaloacetic, phosphoric, pyruvic, P-toluenesulphonic, tartaric, trifluoroacetic,
triphenylacetic, tricarballylic, salicylic, sulphuric, sulphamic, sulphanilic and succinic
acid.
The term "pharmaceutically acceptable derivative" or "prodrug" refers to a derivative of
the active compound that upon administration to the recipient is capable of providing
directly or indirectly, the parent compound or metabolite, or that exhibits activity itself
and includes for- example phosphate derivatives and sulphonate derivatives. Thus,
derivatives include solvates, pharmaceutically active esters, prodrugs or the like. This also
includes derivatives with physiologically cleavable leaving groups that can be cleaved in
vivo to provide the compounds of the invention or their active moiety. The leaving groups
may include acyl, phosphate, sulfate, sulfonate, and preferably are mono-, di- and per-acyl
oxy-substituted compounds, where one or more of the pendant hydroxy groups are
protected by an acyl group, preferably an acetyl group. Typically acyloxy substituted
compounds of the invention are readily cleavable to the corresponding hydroxy substituted
compounds.
Chemical functional group protection, deprotection, synthons and other techniques known
to those skilled in the art may be used where appropriate to aid in the synthesis of the
compounds of the present invention, and their starting materials.
The protection of functional groups on the compounds and derivatives of the present
invention can be carried out by well established methods in the art, for example as
described in T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,
New York, 1981.
Hydroxyl protecting groups include but are not limited to carboxylic acid esters, eg acetate
esters, aryl esters such as benzoate, acetals/ketals such as acetonide and benzylidene, ethers
such as o-benzyl and/?-methoxy ben2yl ether, tetrahydropyranyl ether and silyl ethers such
as 7-butyldimethyl silyl ether.
Protecting groups can be removed by, for example, acid or base catalysed hydrolysis or
reduction, for example, hydrogenation. Silyl ethers may require hydrogen fluoride or
tetrabutylammonium fluoride to be cleaved.
It will be clear to persons skilled in the art of medicinal chemistry that compounds of
formula (I) may be converted into other compounds of formula (I), for example, where a
compound of formula (I) bears one or more hydroxyl substituents then one or more of
these substituents can be converted in to a halo substituent such as bromo, chloro or iodo
by treating the alcohol with a halogenating agent. Halogenating agents include compounds
like NBS, hydrobromic acid, chlorine gas etc. It may be necessary during processes such
as halogenation to use protecting groups to protect other functionality in the molecule.
Phenolic type hydroxyls may not be readily convertible to the corresponding halogen
compound by treatment with a halogenating agent. However, the desired halogen
compound may be prepared by, for example, treating an appropriate aryl amine starting
material with NaNO2 in the presence of HCI under reduced temperature conditions such as
15 0°C, to form the corresponding azide salt. Subsequent treatment with CuCl, CuBr, KI or
HBF4 may be used to convert the azide into the required halo-compound.
A general process for preparing compounds of formula (I) comprises the step of treating a
compound of formula (IV):
wherein R1 R2, R3 and W are as defined above in relation to compounds of formula
with a reducing agent to provide a compounds of formula (I) or a protected derivative
thereof.
Reducing agents are well known to persons skilled in the art and can include hydride
sources like borohydrides and alkali metal borohydrides, but would include hydrogen in
catalytic hydrogenation where a suitable catalyst such as palladium on carbon may be used.
Other suitable hydride sources include sodium triacetoxyborohydride tetrabutyl ammonium
triacetoxyborohydride and sodium cyanoborohydride.
Preferably the double bond in compounds of formula (IV) is reduced by hydrogenation.
Compounds of formula (IV) are prepared by dehydrating a compound of formula (III):
wherein R1, R2 R3 and W are as defined above, in relation to compounds of formula (II) or
a protected derivative thereof.
Dehydration can, for example, be catalysed by acid, by base or facilitated by conversion of
the tertiary alcohol into a better leaving group as would be known to those skilled in the
art.
Preferably compounds of formula (III) are dehydrated, for example, by treatment with
para-toluene sulphonic acid.
Compounds of formula (III) may be prepared by treating compounds of formula (II):
(II)
O
wherein R1, R2, R3 are as defined above for compounds of formula (II) or a protected
derivative thereof with an arylating agent, for example, a compound of formula WM*
wherein W is an optionally substituted aryl radical and M* is one ore more counter ions,
preferably [MgBr]"1".
The arylating agent W"M+ may be prepared by Grignard chemistry where the haloaryl
compound (V):
or a protected derivative thereof wherein
R4, R5 and R6 are independently hydrogen, alkoxy, alkyl, acyl, OC(0)R7, a protected
hydroxy such as OSi(R1o)3 or protected amino such as trimethylsilylamino phenyl
halide, and
RIO is independently alkyl or aryl, and
X is halo, preferably bromo,
is reacted with a metal such as magnesium to form the arylating agent.
Preferably the haloaryl compound (V) is selected from:
(VA)
wherein R4, R5, R6 and X are as defined above for compounds of formula (V).
Reaction of the arylating agent with the ketone of formula (II) provides access to the
corresponding isoflavan-4-ols (III), isoflav-3-enes (IV) and isoflavans (I) of the present
invention.
Alternatively compounds of formula (III) may be prepared by reacting compounds of
formula (II) with a compound analogous to compounds of formula (V) wherein X
represents any appropriate leaving group L which is lost in the formation of the product by
nucleophilic addition of the aryl moiety to a ketone by reactions well known by those
skilled in the art.
Preferably any free alcohols, esters or other such reactive groups in the keto compounds of
formula (II) will be protected, for example, as f-butyldimethylsilyl ethers during the
nucleophilic addition reaction.
Compounds of formula (II) can be prepared by reducing the eneone double bond in
compounds of formula (VI):
(Figure Removed)or a protected derivative thereof, wherein R1, R2 and R3 are as defined above, for
compounds of formula (II).
Suitable reducing agents are described above. Preferably reduction of the carbon-carbon
double bond can be effected, for example, by hydrogenation.
Access to compounds of general formula (VI) is available by general synthetic methods as
set out in Scheme 1 below and as described in published International application No.
WO01/17986, the disclosure of which is incorporated herein by reference. A typical
synthesis is depicted in Scheme 1.
(Figure Removed)Scheme 1
Access to the variatious 3-phenyl substituted chromans is available by varying the
substitution pattern on the phenylacetic acid derived group.
Access to the 4-phenyl substituted chromans is available by varying the substitution pattern
of the arylating agent (V).
Analogues of compounds employed in the processes may be used which include a
substituent which corresponds to R9 as defined for compounds of formula (I).
As used herein, the terms "treatment", "prophylaxis" or "prevention", "amelioration" and
the like are to be considered in their broadest context. In particular, the term "treatment"
does not necessarily imply that an animal is treated until total recovery. Accordingly,
"treatment" includes amelioration of the symptoms or severity of a particular condition or
preventing or otherwise reducing the risk of developing a particular condition.
The amount of one or more compounds of formula (I) which is required in a therapeutic
treatment according to the invention will depend upon a number of factors, which include
the specific application, the nature of the particular compound used, the condition being
treated, the mode of administration and the condition of the patient.
Compounds of formula (I) may be administered in a manner and amount as is
conventionally practised. See, for example, Goodman and Oilman, "The pharmacological
basis of therapeutics", 7th Edition, (1985). The specific dosage utilised will depend upon
the condition being treated, the state of the subject, the route of administration and other
well known factors as indicated above. In general, a daily dose per patient may be in the
range of 0.1 mg to 5 g; typically from 0.5 mg to 1 g; preferably from 50 mg to 200 mg.
The length-of dosing may- range from a single dose given once every day or two, to twice
or thrice daily doses given over the course of from a week to many months to many years
as required, depending on the severity of the condition to be treated or alleviated.
It will be further understood that for any particular subject, specific dosage regimens
should be adjust over time according to the individual need and the professional judgment
of the person administering or supervising the administration of the compositions.
15 Relatively short-term treatments with the active compounds can be used to cause
stabilisation or shrinkage or remission of cancers. Longer-term treatments can be
employed to prevent the development of cancers in high-risk patients.
The production of pharmaceutical compositions for the treatment of the therapeutic
indications herein described are typically prepared by admixture of the compounds of the
invention (for convenience hereafter referred to as the "active compounds") with one or
more pharmaceutically or veterinary acceptable carriers and/or excipients as are well
known in the art.
The carrier must, of course, be acceptable in the sense of being compatible with any other
ingredients in the formulation and must not be deleterious to the subject. The carrier or
excipient may be a solid or a liquid, or both, and is preferably formulated with the
compound as a unit-dose, for example, a tablet, which may contain up to 100% by weight
of the active compound, preferably from 0.5% to 59% by weight of the active compound.
One or more active compounds may be incorporated in the formulations of the invention,
which may be prepared by any of the well known techniques of pharmacy consisting
essentially of admixing the components, optionally including one or more accessory
ingredients. The preferred concentration of active compound in the drug composition will
depend on absorption, distribution, inactivation, and excretion rates of the drug as well as
other factors known to those of skill in the art.
The formulations of the invention include those suitable for oral, rectal, ocular, buccal (for
example, sublingual), parenteral (for example, subcutaneous, intramuscular, intradermal, or
intravenous), transdermal administration including mucosal administration via the nose,
mouth, vagina or rectum, and as inhalants, although the most suitable route in any given
case will depend on the nature and severity of the condition being treated and on the nature
of the particular active compound which is being used.
Formulation suitable for oral administration may be presented in discrete units, such as
capsules, sachets, lozenges, or tablets, each containing a predetermined amount of the
active compound; as a powder or granules; as a solution or a suspension in an aqueous or
non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may
be prepared by any suitable method of pharmacy which includes the step of bringing into
association the active compound and a suitable carrier (which may contain one or more
accessory ingredients as noted above).
In general, the formulations of the invention are prepared by uniformly and intimately
admixing the active compound with a liquid or finely divided solid carrier, or both, and
then, if necessary, shaping the resulting mixture such as to form a unit dosage. For
example, a tablet may be prepared by compressing or moulding a powder or granules
containing the active compound, optionally with one or more other ingredients.
Compressed tablets may be prepared by compressing, in a suitable machine, the compound
of the free-flowing, such as a powder or granules optionally mixed with a binder, lubricant,
inert diluent, and/or surface active/dispersing agent(s). Moulded tablets may be made by
moulding, in a suitable machine, the powdered compound moistened with an inert liquid
binder.
Formulations suitable for buccal (sublingual) administration include lozenges comprising
the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and
pastilles comprising the compound in an inert base such as gelatine and glycerin or sucrose
and acacia.
Formulations suitable for ocular administration include liquids, gels and creams
comprising the active compound in an ocularly acceptable carrier or diluent.
Compositions of the present invention suitable for parenteral administration conveniently
comprise sterile aqueous preparations of the active compounds, which preparations are
preferably isotonic with the blood of the intended recipient. These preparations are
preferably administered intravenously, although administration may also be effected by
means of subcutaneous, intramuscular, or intradermal injection. Such preparations may
conveniently be prepared by admixing the compound with water or a glycine buffer and
rendering the resulting solution sterile and isotonic with the blood. Injectable formulations
according to the invention generally contain from 0.1% to 60% w/v of active compound
and can be administered at a rate of 0.1 ml/minute/kg.
Formulations for infusion, for example, may be prepared employing saline as the carrier
and a solubilising agent such as a cyclodextrin or derivative thereof. Suitable
cyclodextrins include ct-cyclodextrin, p-cyclodextrin, y-cyclodextrin, dimethyl-pcyclodextrin,
2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-cyclodextrin, 3-
hydroxypropyl-β-cyclodextrin and tri-methyl-β-cyclodextrin. More preferably the
cyclodextrin is hydroxypropyl-β-cyclodextrin. Suitable derivatives of cyclodextrins
include Captisol® a sulfobutyl ether derivative of cyclodextrin and analogues thereof as
described in US 5,134,127.
Formulations suitable for rectal administration are preferably presented as unit dose
suppositories. Formulations suitable for vaginal administration are preferably presented as
unit dose pessaries. These may be prepared by admixing the active compound with one or
more conventional solid carriers, for example, cocoa butter, and then shaping the resulting
mixture.
Formulations or compositions suitable for topical administration to the skin preferably take
the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which
may be used include Vaseline, lanoline, polyethylene glycols, alcohols, and combination
of two or more thereof. The active compound is generally present at a concentration of
from 0.1% to 5% w/w, more particularly from 0.5% to 2% w/w. Examples of such
compositions include cosmetic skin creams.
Formulations suitable for transdermal administration may be presented as discrete patches
adapted to remain in intimate contact with the epidermis of the recipient for a prolonged
period of time. Such patches suitably contain the active compound as an optionally
buffered aqueous solution of, for example, 0.1 M to 0,2 M concentration with respect to
the said active compound. See for example Brown, L., et al, (1998).
Formulations suitable for transdermal administration may also be delivered by
15 iontophoresis (see, for example, Panchagnula R, et al., 2000) and typically take the form of
an optionally buffered aqueous solution of the active compound. Suitable formulations
comprise citrate or Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2
M active ingredient.
Formulations suitable for inhalation may be delivered as a spray composition in the form
of a solution, suspension or emulsion. The inhalation spray composition may further
comprise a pharmaceutically acceptable propellant such as carbon dioxide or nitrous oxide
or a hydrogen containing fluorocarbon such as 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-
heptafluoro-n-propane or mixtures thereof.
The active compounds may be provided in the form of food stuffs, such as being added to,
admixed into, coated, combined or otherwise added to a food stuff. The term food stuff is
used in its widest possible sense and includes liquid formulations such as drinks including
dairy products and other foods, such as health bars, desserts, etc. Food formulations
containing compounds of the invention can be readily prepared according to standard
practices.
Therapeutic methods, uses and compositions may be for administration to humans or other
animals, including mammals such as companion and domestic animals (such as dogs and
cats) and livestock animals (such as cattle, sheep, pigs and goats), birds (such as chickens,
turkeys, ducks), marine animals including those in the aquaculture setting (such as fish,
crustaceans and shell fish) and the like.
-The active compound or pharmaceutically acceptable derivatives prpdrugs or salts thereof
can also be co-administered with other active materials that do not impair the desired
action, or with materials that supplement the desired action, such as antibiotics,
antifungals, antiinflammatories, or antiviral compounds. The active agent can comprise
two or more isoflavones or derivatives thereof in combination or synergistic mixture. The
active compounds can also be administered with lipid lowering agents such as probucol
and nicotinic acid; platelet aggregation inhibitors such as aspirin; antithrombotic agents
such as coumadin; calcium channel blockers such as verapamil, diltiazem, and nifedipine;
angiotensin converting enzyme (ACE) inhibitors such as captopril and enalapril, and Pblockers
such as propanolol, terbutalol, and labetalol. The compounds can also be
administered in combination with nonsteriodal antiinflammatories such as ibuprofen,
indomethacin, aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac. The
compounds can also be administered with corticosteroids or an anti-emetic such as
zofran®.
Compounds of formula (I) seem to be particularly suitable for co-administration with one
or more anti-cancer drugs such as cisplatin, dehydroequol (DHE), taxol (paclitaxel),
gemcitabine, doxorubicin, topotecan and/or camptothecin, especially cisplatin,
dehydroequol (DHE), taxol. This may result in improved effects in the treatment, for
example in the form of synergistic effects, in comparison to when only one of the
medicaments is employed. Particularly the compounds of the presently claimed invention,
especially HMC (ie compound 1) seem to be chemosensitisers and increase the
cytotoxicity of the one or more anticancer drug co-administered therewith. This seems to
be the case even though said anticancer drugs work through a variety of different
mechanisms, for example cisplatin is thought to work by interacting with nuclear DNA,
taxol is thought to work by blocking cells in the G2/M phase of the cell cycle and prevent
hem forming normal mitotic apparatus, gemcitabine is thought to work by incorporating
itself into the DNA of the cell, ultimately preventing mitosis, doxorubicin is though to be a
topoisomerase II inhibitor thereby preventing DNA replication and transcription and
topotecan is thought to be a topoisomerase I inhibitor.
Interestingly, in some situations this increased cytotoxicity to cancerous cells is not
associated with a corresponding increase in toxicity to non-cancerous cells.
Whilst this observation has important implications for the treatment of many cancers, it is
especially important to the treatment of cancers such as melanoma, which are extremely
difficult to treat.
The co-administration may be simultaneous or sequential. Simultaneous administration
may be effected by the compounds being in the same unit dose, or in individual and
discrete unit doses administered at the same or similar time. Sequential administration
may be in any order as required and typically will require an ongoing physiological effect
of the first or initial active agent to be current when the second or later active agent is
administered, especially where a cumulative or synergistic effect is desired.
The invention also extends to a pack comprising the combination therapy.
Compounds for use in the preferred synthetic methods of the present invention may be
derived from any number of sources readily identifiable to a person skilled in the art. For
example, daidzein is readily available or can be synthesised by standard methods known in
the art. Suitable methods may be found in, for example, published international patent
applications WO 98/08503 and WO 00/49009, and references cited therein, which are
incorporated herein in their entirety by reference.
Compounds of the general formulae (II), (III) and (IV) described above are intermediates
in the production of the active isoflavan compounds of formula (I). These intermediates
also represent further aspects of the present invention.
Whilst not wishing to be bound by theory the compounds of the present invention are
thought to regulate a wide variety of signal transduction processes within animal cells and
that these signal transduction processes are involved in a wide range of functions that are
vital to the survival and function of all animal cells. Therefore, these compounds have
broad-ranging and important health benefits in animals including humans, and in particular
have the potential to prevent and treat important and common human diseases, disorders
and functions, which represents .a substantial unexpected benefit.
Thus it seems that the compounds of the present invention have activity as TNFoc
inhibitors. It Is hypothesised that TNFa is part of a tightly regulated cytokine network,
activating multiple signal transduction pathways and inducing or suppressing a wide
variety of genes. TNFa can provide a survival signal for cancer cells and hence it has been
referred to as a tumour promoting factor. As a central mediator of inflammation, TNFa
provides a molecular link between chronic inflammatory stimuli and the subsequent
15 development of malignant disease. Consequently its inhibition by the compounds of the
invention may provide one mechanism by which they exert anti-cancer and/or antiinflammatory
activity. Alternatively, these compounds may be used as chemopreventative
agents.
The particular benefits of this invention lie in (a) the large range of signal transduction
processes targeted by the compounds, (b) the fact that regulation of these various processes
includes both up-regulation of some processes and down-regulation of others, and (c) that
such a broad and varied effect on signal transduction processes also is accompanied by an
independent effect on a range of important enzymes that are fundamental to metabolism
and steroidogenesis.
The isoflavan compounds of the present invention exhibit good in vitro toxicity profiles
against normal cells. The isoflavans have broad activity, markedly better than or at least
comparable with dehydroequol. The isoflavans are highly active against cancer cells
representative of leukaemia, glioma, prostate, ovarian, breast and lung cancer. The
isoflavan compounds show potent activity against melanoma and cholangiocarcinoma
(gall bladder cancer) cell lines. Good activity was observed against colorectal cancer
PCT/AU2005/001436
cells.
Radio-sensitisation in vivo may be tested for example employing human epidermoid vulval
carcinoma A431 tumours established on the upper leg and subjected to several doses of
local radiation (to the tumour bearing leg only). A radiation treatment regimen of
2.5Gy/day for 4 days will delay tumour growth, and the effect of the radiation dose in
combination with the test compound.could be assessed by monitoring tumour growth
delay. Tumour growth delay of ~6 days can be expected using radiation alone. Tumour
growth delay using orally dosed test compound can be determined separately. Evidence of
:est compound mediated radio-sensitisation of A431 tumours is then determined by
measuring tumour growth delay using a regimen of orally dosed test compound pre-treated
animals followed by the standard radiation therapy regimen described above. A mean
growth delay of up to 30 days using the combination treatment compared to up to 10 days
using either radiation or test compound monotherapy regimens is evidence of the radio-
15 sensitisation properties of the compounds of the invention.
Radio-sensitisation in vitro may be tested, for example, employing clonogenic assays using
human the human epidermoid vulva carcinoma A431 cell line to measure response to
radiation alone or in combination with test compounds. A drug dose causing 10% toxicity
to the cells may be used in combination with graded doses of radiation. The appropriate
dose of compound would be determined by clonogenic assay. Evidence of test compound
mediated radio-sensitisation is shown by, for example, a >20% toxicity to cells using
chemoradiation therapy compared to 10% toxicity using the corresponding monotherapy
regimens.
The compounds of the invention are useful in the treatment, prevention or amelioration of
diseases associated with aberrant cell survival, aberrant cell proliferation, abnormal cellular
migration, abnormal angiogenesis, abnormal estrogen/androgen balance, dysfunctional or
abnormal steroid genesis, degeneration including degenerative changes within blood vessel
walls, inflammation, and immunological imbalance.
The invention is further illustrated by the following non-limiting Examples and
accompanying drawings.
Examples
In the Examples and accompanying drawings which follow the abbreviation "DHE" is used
for dehydroequol, "HMC" is used for compound No. 1, being 3-(4-hydroxyphenyl)-4-(4-
methoxyphenyl)-chroman-7-ol and "HHC" is used for compound No. 11, being 3-(4-
hydroxyphenyl)-4-(4-hydroxyphenyl)-chroman-7-ol.
1.0. Synthesis
Example 1: 4',7-Diacetoxydaidzein
A
A mixture of daidzein (2.0 g), acetic anhydride (10 ml) and pyridine (2 ml) was heated at
105-110 C for Ih. After cooling the mixture to room temperature, it was stirred for a
further 30 min during which time the diacetate crystallised from solution. The product was
filtered, washed thoroughly with water and recrystallised from methanol to yield 4',7-
diacetoxydaidzein as colourless prisms (2.4 g, 90%).
Example 2: 7-Acetoxy-3-(4-acetoxyphenyl)chroman-4-one
Palladium-on-charcoal (5%, 0.02g) was added to a solution of 4',7-diacetoxydaidzein
(O.50g, 1.5 mmol) in ethyl acetate (80 ml) and the mixture was stirred at room temperature
under a hydrogen atmosphere for 72h. The catalyst was removed by filtration through
Celite and the resulting filtrate was evaporated in vacua. The residue was recrystallised
WO 2006/032086 PCT/AU2005/001436
from ethanol to yield 7-acetoxy-3-(4-acetoxyphenyl)chroman-4-one (0.40g, 80%) as
colourless plates.
Example 3: 7-Hydroxy-3-(4-hydroxyphenyl)chroman-4-one
Imidazole (0.63g) was added to a suspension of 4',7-diacetoxydihydrodaidzein (0.26g, 0.08
mmol) in absolute ethanol (5.0 ml) and the mixture was refluxed for 45 min under argon.
The solution was concentrated under reduced pressure and distilled water (10 ml) was
added to the residue. The mixture was left overnight under refrigeration and the resulting
precipitate was filtered. The crude product was recrystallised from ethyl
acetate/dichloromethane to yield 7-hydroxy-3-(4-hydroxyphenyl)chroman-4-one (0.14g,
71%) as a white powder.
Example 4: 7-(tert-ButyIdimethlysilyloxy)-3-(4-(tert-butyIdimethlysilyloxy)
phenyl)chroman-4-one
O
(Figure Removed)7-Hydroxy-3-(4-hydroxyphenyl)chroman-4-one42g, imidazole 130g, tertbutyldimethylsilyl
chloride 127g, and N,N-dimethylformamide (500ml) were combined in
a 2L round bottom flask and stirred under nitrogen at room temperature for 16 hours. The
reaction was quenched with the addition of chilled H2O (200ml) with the reaction mix
cooled in an ice bath. The resultant white solid was filtered and rinsed with water.
Recrystallisation from ethanol afforded the product as white fluffy crystals (35.7g).
ExampleS: 7-(/ert-ButyldimethylsiIyloxy)-3-(4-(tert-butyIdimethyIsiIyloxy)phenyI)-4-
(4-methoxyphenyl)chroman-4-ol
7-(re/Y-Dutyldimethylsilyloxy)-3-(4-(rerr-butyldimethylsilyloxy)phenyl)-4-(4-
methoxypenyl)chroman-4-ol 25g was weighed in a 2-neck round bottom flask, and flushed
under nitrogen. Anhydrous THF 80ml was added to the reaction vessel to give a clear
slightly yellow solution. A condenser was attached and the reaction vessel placed in an ice
bath. Commercial 4-methoxyphenylmagnesium bromide (0.5M solution in THF) 225ml
was added to the reaction mix dropwise over 10 minutes. The reaction was quenched by
the dropwise addition of wet ether (50:50 H2O: di ethyl ether) while still under nitrogen,
with a white precipitate forming as increasing amounts of H2O was added. A further
amount of H2O was added to the reaction mix before extraction with diethyl ether.
The organic layers were combined and washed with water, brine, dried over anhydrous
MgSO4 and solvent removed on rotovap to give clear yellow oil which solidified overnight
to give an off-white solid. The crude product was used in the next step without
purification.
Example 6: 3-(4-HydroxyphenyI)-4-(4-methoxyphenyI)-2/H-chromen-7-ol
(Figure Removed)7-(tert/-Butyldimethylsilyloxy)-3-(4-(tet-buryldimethylsilyloxy)phenyl)-4-(4-
methoxyphenyl)chroman-4-ol (42g), pTsOH (435g), boiling chips and 2.5L of ethanol
were combined in i\ 2-ncck 5T, round bottom flask with condenser attached. The reaction
was heated at reflux for 3 hours. The solvent was concentrated in vacua to ~ 100ml before
being poured into chilled, stirred water (700ml). The mixture was then extracted with
ethyl acetate, the combined organic layers washed with water (3 x 2L), brine (1 x 500ml),
dried over anhydrous magnesium sulphate and filtered and solvent removed in vacua to
give red/ brown oil. The oil was dissolved in methanol (~100ml) and put in freezer
overnight.
A white precipitate had formed overnight, which was filtered off and rinsed with methanol.
The filtrate was concentrated in vacuo to give a brown oil.
Example 7: 3-(4-HydroxyphenyI)-4-(4rmethoxyphenyI)-chroman-7-oI
H2/Pd
ethanol
OMe OMe
3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-2H-chromen-7-ol 25,5g (70mmoles), 10%
Pd/Al2O3 3.95g and 200ml of ethanol were combined in a 2-neck 500ml round bottom
flask. The reaction was hydrogenated at low pressure using standard conditions for 3
hours. The reaction was filtered through Celite to remove the catalyst, rinsed through with
ethanol (300ml). The filtrate was concentrated to ~50ml before being poured into chilled,
stirred water (1.4L). A pale orange precipitate formed which then formed a brown oil.
The mixture was then extracted with diethyl ether, the combined organic layers washed
with water (3 x 1L), brine (1 x 500ml), dried over anhydrous magnesium sulphate and
filtered: The solvent was removed in vacua to give.red/ brown, oil. The product was
recrystallised from diethyl ether (~100ml), to give brown solid which was rinsed with
chilled diethyl ether to give off-white crystals 11.3g. The 1H NMR spectrum and
numbering scheme being shown below.
(Figure Removed)In the above general methods, the structures may be optionally substituted or protected
with appropriate substituents, or synthons or derivatives thereof. The compounds may be
present as, for example, their salts, acetates, benzyl or silyloxy derivatives as can be
determined by a skilled synthetic chemist. Hydroxy groups can be readily alkylated
(Mel/base), acylated (Ac2O/Py) or silylated (Cl-SiR3/base) and likewise deprotected by
standard methods known in the art.
/"
Example 8: 3-(4-Hydroxyphenyl)-4-(4-hydroxyphenyI)-chroman-7-ol
OH AcOH
OMe
3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (3.17g) was transferred to a round
bottom flask and the flask was purged with nitrogen. 33 wt.% Hydrogen bromide in acetic
10 acid (13ml) was added drop-wise to the flask and the contents were heated to reflux at
130°C for 7 hours. The reaction mixture was placed in an ice bath and adjusted to pH 6
using sodium hydroxide solution (40%w/v). The mixture was extracted with ethyl acetate
and the ethyl acetate layer was further washed with water and brine prior to drying over
magnesium sulphate, The mixture was filtered and the solvent was removed in vacuo to
yield a brown solid (2.89g). The solid was dissolved in minimal ethyl acetate and purified
by column chromatography (Silica 60H, 200-400mesh using ethyl acetatexhloroform
(40:60) eluant). 3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol was obtained in
-80% purity and was further purified by semi-preparative high performance liquid
chromatography (HPLC). The !H n.m.r. is shown in Fig. 12.
2.0. Materials and Methods
2.1. Tissue culture
The human pancreatic cancer cell line, HP AC (CRL-2119) was routinely cultured in 1:1
mixture DMEM (Dulbecco's Modified Eagle Medium Sigma) plus Ham's F12 (Sigma)
medium containing HEPES (15 mM), insulin (0.002 mg/mlX transferrin (0.005 mg/ml),
hydrocortisone, (40 ng/ml), epidermal growth factor (10 ng/ml). The ovarian cancer cell
lines; CP70 was obtained as a gift from Dr. Gil Mor (Yale University) and cultured in a 1:1
mixture DMEM plus Ham's F12 medium, and SKOV-3 (ovarian cancer cell line) was
purchased from ATCC and cultured in McCoys 5a medium. The breast cancer cell line
MDA-MB-468 cultured in Leibovitz's L-15 medium. The melanoma cell line MM200
was obtained as a gift from Peter Hersey (University of Newcastle) and A2058 was
obtained as a gift from Dr Peter Parsons (QIMR), Both were cultured in DMEM medium.
All cultures were supplemented with 10% PCS (fetal calf serum CSL, Australia), penicillin
(lOOU/ml), streptomycin (lOOmg/ml), L-glutamine (2mM) and sodium bicarbonate (1.2
g/L), and cultured at 37°C in a humidified atmosphere of 5% COj. All cell lines were
purchased from ATCC (Maryland, USA) except where noted.
The normal cell line NFF (neonatal foreskin fibroblasls) was a gift from Dr. Peter Parsons
(Queensland Institute of Medical Research). RK (rabbit kidney) cells were obtained from
Miller Whalley (Macquarie University). Both cell lines were cultured in RPMI
supplemented with 10% PCS (CSL, Australia), penicillin (lOOU/ml), streptomycin
(lOOmg/ml), L-glutamine (2mM) and sodium bicarbonate (1.2 g/L), and cultured at 37°C
in a humidified atmosphere of 5% CO2.
2.2. Proliferation assays
IC50 values were determined for each cell line. Cells were seeded in 96-well plates at an
appropriate cell density as determined from growth kinetics analysis and cultured for 5
days in the absence and presence of the test compounds. Cell proliferation was assessed
after the addition of 20 μl of 3-4,5 dimethylthiazol-2,5-diphenyl tetrazolium bromide
(MTT, 2.5mg/ml in PBS, Sigma) for 3-4hrs at 37°C according to manufacturer's
instructions. IC50 values were calculated from semi-log plots of % of control proliferation
on the y-axis against log dose on the x-axis.
2.3. DHE and HMC Pharmacokinetics - Oral
HMC and DHE were prepared as homogenous suspensions in 1% CMC (m:v, water). Both
formulations were delivered orally by gavage to female BALB/c mice at a dosage of 50
mg/kg. Three animals were allocated to each timepoint (15 min, 30 min, Ihr, 4 hr and 24
hr). At each respective timepoint, animals were euthanased by cervical dislocation and
blood collected. Free HMC was analysed by mass spectroscopy.
2.4. HMC Pharmacokinetics - i.v. and i.p.
HMC was prepared as a solution in 20% hydroxypropyl-beta-cyclodextrin (m:v, water).
The formulation was delivered either orally by gavage or by i.p. injection to female
BALB/c mice at a dosage of 50 mg/kg. Three animals were allocated to each timepoint (15
min, 30 min, Ihr, 4 hr and 24 hr). At each respective timepoint, animals were euthanased
by cervical .dislocation and blood collected. Urine was also collected and analysed for
HMC. Free HMC was analysed by mass spectroscopy.
2.5 Pilot in vivo efficacy study — HP AC tumour bearing mice
Subconfluent (80%) flasks of HP AC cells were trypsinised, washed in Hanks balanced salt
solution (Sigma), resuspended in dubellco's minimal essential medium (Sigma) and an
equal volume of matrigel™ (Becton Dickson) at a density of 3.7 x 107cells per ml.
Athymic nu/nu BALB/c mice were s.c. inoculated with 3.7xl06 HPAC cells bi-laterally
midway along the dorsal surface. For the HMC (n=3 per dosage regimen) and control
groups (n=2), treatment commenced five days post inoculation to allow tumour formation.
HMC was formulated 20% HPBCD and delivered i.p. daily for 15 days. The control group
received equivalent (weight:weight) i.p. doses of 20% HPBCD. Tumour measurements
commenced on day 5 post inoculation (10x10 mm2) and were measured in 2 dimensions,
length (a) and width (b), using calipers. Tumor weight (W) was calculated by the formula
W=ab2 /2, where a, is the longer of the 2 measurements (Odwyer et al., 1994), Tumour
proliferation curves were analyzed with respect to maximal tumour inhibition
(treated/control, TIC). On sacrifice, liver, kidney, femur, stomach and colon tissue were
fixed in buffered formalin, embedded in paraffin, sections cut and stained with H&E.
Stained sections were then submitted to Rothwell consulting for histopathology analysis.
Serum biochemistry was conducted on bloods taken from control, vehicle control and
HMC treatment groups. Serum analysis was conducted by Veterinary Clinical pathology
(U. Syd).
2.6 Three-D model analysis of synergy
3-D model analysis of the cytotoxic interaction between drug A and drug B enables the
representation of predicted inhibitory effect of two drugs in combination in 3 dimensions
to reveal actual regions of synergy or antagonism. The 3D synergy plots are based on a
theory of "Theretical Additivity" (TA) as outlined by Kanzawa et al (Int. J. Cancer 71,
311-319 (1997)). Theoretical Additivity was calculated from the cytotoxicities of drug A
and drug B as monotherapies using the following formula which assumes the drugs are
mutually exclusive inhibitors:
(fa)ATA(
D =
Where: (fa)A
= fraction of cells affected by drug A
(fa)s - fraction of cells affected by drug B
The TA is calculated for each combination of drug concentrations and subtracted from the
observed experimental effect for each combination to give a measurement of synergistic
action. A positive difference indicates that more cells are affected by the drug combination
than would be expected in theory if the two drugs were administered together - hence
synergism. A negative difference indicates that less cells were affected than theoretically
expected - hence antagonism.
3.0. Results
3.1. Normal cell toxicity
Dehydroequol (DHE) was less toxic to both NFF and rabbit kidney cells with IC50 values
above 150 uM when compared to HMC (86 and 61 uM respectively) (Table 1 and Figure
1). In a separate study, HHC was found to be non-toxic to both NFF and RK cells (see
again Table 1). When compared to cisplatin, a benchmark chemotherapeutic agent, the
degree of toxicity exhibited by HMC and HHC is mild.
Table 1. Relative toxicity of DHE, HMC, HHC and cisplatin against Neonatal foreskin
fibroblasts (NFF) and rabbit kidney cells.
(Table Removed)
3.2. In vitro efficacy against cancer cells
When compared to DHE IC50 values, HMC demonstrated markedly superior activity (~5-
10 fold greater) against the multi-drug resistant, p53 mt ovarian cancer cell line (SKOV-3),
the AR negative, p53 Mt prostate cancer cell line (PC3), both ER positive (p53 wt) and
negative (p53 mt) breast cancer cell lines (MCF-7 and MDA-MB-468 respectively), p53
Mt Glioma (HTB-138), p53 Mt pancreatic cancer (HPAC) and p53 Mt large cell lung
cancer (Table 2). HMC exhibited anti-cancer activity comparable to that of DHE against
all other cell lines tested (Table 1). Particular efficacy of HMC was noted against
melanoma cells. (Table 2.1 and Fig 2). This represents a substantial advantage over the
prior art.
HMC was differentially active against 2 separate colorectal cell lines, with marked activity
observed against HT-29 cells and somewhat less activity against HCT-15. It is noted that
HT-29 and HCT-15 are COX-2 positive and deficient respectively. When examined
microscopically and compared to cells treated with vehicle only, HMC treated SKOV-3
cells exhibited morphological changes consistent with cells undergoing apoptosis (cell
enlargement, granular appearance in cytosol and blebbling of plasma membrane). In
contrast SKOV-3 cells exposed to 100 uM Dehydroequol after 18 hr retained a relatively
normal morphology, comparable with that of vehicle only treated cells.
Table 2.1. Comparison of Dehydroequol and HMC cytoxicity against cell lines
representative of different malignancies.
(Table Remobed)
In further studies, the cytotoxicity of various compounds described herein against various
cell lines was determined. Compound 14-ene is the 3-ene chromene precursor to .the
correspondingly reduced chroman, compound 14. It was observed that compounds 1,2
and 11 show the best efficacy against most all cancer cell lines. Compound 14 shows
slightly better efficacy in general compared to its corresponding 14-ene and to compound 6
(Table 2.2).
-44-
Table 2.2. Chroman compounds 1,2,6, 11 and 14 and chrom-3-ene compound 14-ene
cytoxicity against cell lines representative of different malignancies.
(Table Removed)
3.3.1. HMC Pharmacokinetics - oral
When compared with the pharmacokinetic profile of orally dosed DHE, HMC
administered via the same route and dosage (50 mg/kg), HMC exhibited a Cmax of
141 μM (achieved after 1 hr) compared to 511 μM for DHE (achieved after 15 min) (Table
3 and Figure 3). Like DHE, HMC is also subject to conjugation with low plasma
concentrations of the free form of the molecule observed (1.3 uM after 30 min) (Table 3
and Figure 3). This is less than half the maximum concentration of free dehydroequol
achieved using the same dosage regimen (3.3 uM after 15 min) (Figure 3). The ratio of
free:total is greater for HMC when compared to DHE (0.92 vs 0.64 respectively).
Table 3.1. Comparison of free and total plasma concentrations achieved in mice dosed
with 50 mg/kg of either HMC or DHE p.o.
(Table Removed)
3.3.2. HMC and HHC Pharmacokinetics - oral
Human patients were orally dosed with 200 mg of either HMC or HHC. For each
challenged patient, blood was taken over a 6 hour period and the results averaged to
characterise the plasma pharrnacokinetics. The initial results give an oral half life of 3.99
hours for HMC and 3.26 hours for HHC (Table 3.2).
Table 3.2. Comparison of plasma half life concentrations achieved in humans dosed with
200 mg of either HMC or HHC
(Table Removed)
3.4. HMC Pharmacokinetics — i.v. and i.p.
When formulated in HPBCD and delivered i.v., extremely high levels of HMC were
observed in the blood equating to 1 mM of drug, 15 min post administration (Figure 4).
Elimination kinetics of i.v. delivered HMC were biphasic with HMC being rapidly
excreted from blood at a rate of ~1000 uM/hr in the first hr post administration. Assuming
linear excretion, this rate slowed to 0.97 uM/hr in hours 1 -4 hr post administration. When
the same formulation was administered i.p., approximately 1 log less HMC was observed
in plasma (131 μ,M by i.p. administration vs 1069 μM by i.v. administration) up to 1 hour
post administration (Fig 4). Elimination kinetics by i.p. administration however, was much
slower during this period (112 uM/hr) thus resulting in a serum concentration some 4.5
fold higher at 1 hr post administration (18.7 by i.p. vs 3.98 by i.v.). Conversely, in hours 1-
4 post administration, elimination kinetics was faster after i.p. administration when
compared to i.v. (4.6 μM/hr by i.p. vs 0.97 f^M/hr by i.v.). These data confirm that HMC
is highly bioavailable in its free state when administered by i.v. or i.p. routes. In
conjunction with oral PK data, these data also suggest that HMC is susceptible to rapid
removal by GI detoxification enzymes. Large concentrations of free HMC were observed
in urine over 0.5, 1 and 4hr where collected (3.3 mM, 3.9 mM and 0.093 mM).
Table 4. Comparison of the pharmacokinetic profile of HMC in serum after i.v and i.p
administration of HMC formulated in 20% hydroxypropyl beta cyclodextrin at a dose of 50
mg/kg). Inset shows HMC concentrations in serum.
(Table Removed)
3.5. Pilot in vivo efficacy study - HP AC tumour bearing mice
HMC when dosed daily, i.p. at 100 mg/kg significantly retarded the proliferation of HP AC
tumours over the treatment period when compared to vehicle control (Figures). When the
mean terminal tumour mass was assessed a significant reduction in final tumour burden
(%T/C = 62) was also noted (Figure 6). Importantly, no signs of toxicity were noted in
10 animals dosed with HMC at 100 mg/kg daily for 15 days as determined by weight loss.
Indeed animals treated with HMC appeared to thrive when compared to control (Figure 7).
Organs (liver, kidney, spleen, femur, stomach and colon) were collected and submitted for
histopathological assessment by Rothwell consulting. A limited serum biochemistry
analysis was also conducted. These data confirm that HMC demonstrates antitumorigenie
activity against HPAC tumours in vivo.
3,5.1, Histpathological examination of HMC treated groups
Histopathological examination of haematoxylin and eosin-stained sections cut from
formalin-fixed tissues from two series of experimental mice was made. The liver, kidney,
stomach and colon were examined for evidence of toxic damage, the spleen and bone
marrow for evidence of myelosuppression and the tumour for degree of necrosis. A score
of 0-5 was allocated to each rumour specimen for the degree of necrosis present, a 0 score
representing, no necrosis and a score of 5, total necrosis. The sections were scored 'blind'
on two separate occasions and the final score given in the results is the mean of these two
scores.
-47-
(Table Removed)
3.5.1.1, Overview of results
No evidence of toxicity or myelosuppression was detected in sections cut from the tissues
of the drug-treated mice. However, in all the drug-treated mice there were patchy
mild/moderately severe chronic inflammatory changes affecting the serosa and attached
mesentery, as well as reactive changes of the mesothelial cells, in some of the tissues
examined. These changes are consistent with the intra-peritoneal injection of a mildly
irritant material.
Significant necrosis of tumour tissue was not detected in control specimens 1/8 and 1/11.
However, there was considerable necrosis in the rumour sections from the drug-treated
mice.
3.5.1.2. Serum biochemistry of HMC treated mice in comparison to control
Alakalines phosphatase (ALP), alanine transferase (ALT) and creatine (Cre) were assessed
in HMC treated vs control animals. ALP and Cre levels were similar to control and fell
within normal ranges (for rat) however, ALT levels in vehicle control and HMC treated
groups were much lower than no treatment control levels.
(Table Removed)
3.6. HMC induced apoptosis in melanoma cells and normal fibroblasts
3.6.1. Melanoma
HMC induced apoptosis in all TRAIL-sensitive and -resistant melanoma cells at
concentrations down to 2uM (~7-10% apoptosis) over 24 aind 48 hrs of exposure (Table 7
and Figure 8A). At the clinically significant drug concentration of 4 μM, the incidence of
apoptotic cells after 24 hr exposure to HMC rose to 25% and 39% in TRAIL sensitive
(MEL-RM) and TRAIL negative (IGR3) cell lines respectively (Table 7 and Figure 8A).
The incidence of HMC induced apoptosis at 4 uM after 24 hr exposure in the other cell
lines was ~9%. In comparison, the incidence apoptosis in DHE- treated cells at a
concentratoin of 4 μM after 24 hr exposure was 0-1 %. Over 48 hr at the same
concentration of HMC (4μM), the incidence of apoptosis rose to 21-42 % in all cell lines
examined (Table 7 and Figure 8B). DHE was the only other agent to induce moderate
levels of apoptosis after 48 hr exposure at a concentration of 4μM, but only in ME4405
(14%) and Mel-AT (15%) cell lines (Table 7 and Figure 8B).
(Table Removed)
3.6.2. Normal fibroblasts
Studies were conducted on normal fibroblasts (MRC-5) and TRAIL-sensitive melanoma
cells (ME4405 and MEL-RM) using 8μM of either DHE or HMC over 24 and 48 hr of
exposure (Figure 9). These data demonstrate that HMC, and to a lesser extent DHE,
induced marked levels of apoptosis in both melanoma cell lines over 24 and 48hr.
Importantly, while promoting programmed cell death in malignant cells, normal fibroblasts
were shown to both HMC and DHE induced apoptosis at 8 μ,M drug over 24 and 48 hr of
exposure. These data confirm that HMC is selectively cytotoxic to cancer cells.
The isoflavan compounds of the invention exhibit a superior efficacy profile against all
cancers tested when compared to DHE. While HMC is marginally more toxic than DHE in
NFF and RBC cells, HMC is markedly less toxic than cisplatin. HMC delivered orally in
mice is less bioavailable when compared to DHE but the ratio of free:total is greater.
HPBCD-formulated HMC was markedly bioavailable in its free form when delivered i.v
and i.p. Significant serum concentrations of free HMC post delivery i.p. were some 18 fold
above that of orally delivered HMC. It has been demonstrated that HMC, formulated in
20% HPBCD and delivered i.p., exerts a moderate antitumorigenic activity against HP AC
tumours in vivo. HMC when delivered at 100 mg/kg to mice is not toxic to major organs as
determined by histopathology however, in all the drug-treated mice there were patchy
mild/moderately severe chronic inflammatory changes affecting the serosa and attached
mesentery, as well as reactive changes of the mesothelial cells which are consistent with
the intra-peritoneal injection of a mildly irritant material.
HMC induced moderate-strong levels of apoptosis in TRAIL-resistant and TRAILsensitive
melanoma cells after both 24 and 48 hrs of exposure. Normal fibroblast cells
were resistant to apoptosis after 48 hrs exposure DHE induces mild-moderate levels of
apoptosis in TRAIL-resistant and TRAIL-sensitive melanoma cells after 48 hrs of
exposure. Normal fibroblast cells were resistant to apoptosis after 48 hr exposure. The
ability of both HMC and DHE to induce apoptosis in caspase negative cells suggest that an
operational extrinsic programmed cell death pathway is not essential for HMC and DHE
mediated apoptosis.
3.7 In vitro HMC synergistic toxicity in cancer cells when combined with cisplatin,
paclitaxel and gemcitabine, camptothecin, topotecan and doxorubicin
3.7.1. HMC combination with cisplatin against the MM200 melanoma cell line.
HMC synergy with cisplatin was assessed either in combination over 5 days exposure or in
sequence (HMC -> cisplatin) against the MM200 melanoma cell line. It was difficult to
assess for synergisitic toxicity using a change in IC50 as a measure of synergy due to HMC
toxicity as monotherapy (Table 8). 3D analysis of the data reveals that only additive
toxicity was apparent vising the 5-day combination protocol (Fig. 11). Evidence of synergy
using the HMC-cisplatin combination was further assessed using the HMC—> cisplatin
sequence (24hr exposure to each compound in sequence) against the melanoma cell line
MM200. Using change in IC50 to assess for synergy it was noted that HMC at
concentrations of 2 μM markedly chemosensitised the MM200 cells to cisplatin by >1000
fold (Table 8). HMC induced chemosensitisation of MM200 cells to cisplatin was
confirmed using 3D analysis of the data (Fig. 1 IB). These data demonstrate that HMC is
able to chemosensitise cancer cells, in this case melanoma, to cisplatin.
Table 8. Comparative assessment of synergy between HMC and the cisplatin against the
Mel-RM melanoma cell line. Average IC50 data for each agent when assessed as a
monotherapy or in combination are shown
(Table Removed)
3.7.2 HMC combination with gemcitabine against the Mel-RM melanoma cells.
HMC synergy with gemcitabine was assessed either in combination over 5 days exposure
or in sequence (HMC -» gemcitabine) against the Mel-RM melanoma cell line. It was
difficult to assess for synergisitic toxicity using a change in IC50 as a measure of synergy
due to HMC toxicity as monotherapy (Table 9). 3D analysis of the data reveals that 5-day
combination protocol did not elicit synergisitic toxicity against the Mel-RM cell line.
Evidence of synergy using the HMC-gemcitabine combination was further assessed using
the HMC-* gemcitabine sequence (24hr exposure to each compound in sequence) against
the melanoma cell line Mel-RM. Using change in ICSO to assess for synergy it was noted
that HMC at concentrations of 2 and luM markedly chemosensitised the Mel-RM cells to
gemcitabine by >1000 fold. HMC-induced chemosensitisation of Mel-RM cells to
gemcitabine was confirmed using 3D analysis of the data. These data demonstrate that
HMC is able to chemosesnsitise cancer cells to gemcitabine.
Table 9. Comparative assessment of synergy between HMC and gemcitabine against the
Mel-RM melanoma cell line. Average IC50 data for each agent when assessed as a
monotherapy or in combination are shown.
(Table Removed)
3.7.3. HMC combination with paclitaxel against the 4405 melanoma cell line.
HMC synergy with paclitaxel was assessed either in combination over 5 days exposure or
in sequence (HMC -» paclitaxel) against the 4405 melanoma cell line. It was difficult to
assess for synergisitic toxicity using a change in IC50 as a measure of synergy due to HMC
toxicity as monotherapy (Table 10). A 30 fold reduction in IC50 was noted in the
combination experiment when compared to the paclitaxel monotherapy. However, 3D
analysis of the data revealed that the 5-day combination protocol did not elicit synergisitic
toxicity against the 4405 cell line. Evidence of synergy using the HMC-paclitaxel
combination was further assessed using the HMC-> paclitaxel sequence (24hr exposure to
each compound in sequence) against the melanoma cell line 4405. Using change in IC50
to assess for synergy it was noted that HMC at concentrations of 2 jaM markedly
chemosensitised the 4405 cells to paclitaxel by >1000 fold (Table 10). HMC-induced
chemosensitisation of MM200 cells to paclitaxel was confirmed using 3D analysis of the
data. These data demonstrate that HMC is able to chemosesnsitise cancer cells to
pacltaxel.
Table 10. Comparative assessment of synergy between HMC and paclitaxel against the
4405 melanoma cell line. Average IC50 data for each agent when assessed as a
mono therapy or in combination are shown.
(Table Removed)
3.7.4. HMC combination with topotecan against the MM200 melanoma cell line.
HMC synergy with topotecan was assessed either in combination over 5 days exposure or
in sequence (HMC -»topotecan) against the MM200 melanoma cell line. It was difficult
to assess for synergisitic toxicity using a change in IC50 as a measure of synergy due to
HMC toxicity as monotherapy (Table 8). 3D analysis of the data confirmed that the 5-day
combination protocol did not elicit synergisitic toxicity against the MM200 cell line.
Evidence of synergy using the HMC-gemcitabine combination was further assessed using
the HMC-» topotecan sequence (24hr exposure to each compound in sequence) against the
melanoma cell line MM200. Using change in IC50 to assess for synergy it was noted that
HMC at a concentration of 2 u.M markedly chemosensitised the MM200 cells to topotecan
by > 1000 fold (Table 11). HMC-induced chemosensitisation of MM200 cells to topotecan
was confirmed using 3D analysis of the data. These data demonstrate that HMC is able to
chemosesnsitise cancer cells to topotecan. From the 3D analysis the optimum combination
of HMC and topotecan against the MM200 melanoma cell line would appear to be 2 u.M
HMC and between 1 and 0.1 uM topotecan.
-54-
Table 11. Comparative assessment of synergy between HMC and topotecan against the
MM200 melanoma cell line. Average IC50 data for for each agent when assessed as a
monotherapy or in combination are shown.
(Table Removed)
3.7.5. HMC combination with camptothecin against the Mel-RM melanoma ceil line.
HMC synergy with doxorubicin was assessed either in combination over 5 days exposure
or in sequence (HMC -» doxorubicin) against the Mel-RM melanoma cell line. It was
difficult to assess for synergisitic toxicity using a change in IC50 as a measure of synergy
due to HMC toxicity as monotherapy (Table 12). 3D analysis of the data confirmed that
the 5-day combination protocol did not elicit synergisitic toxicity against the Mel-RM cell
line, indeed evidence of antagonism was noted. Evidence of synergy using the HMCdoxorubicin
combination was further assessed using the HMC-» doxorubicin sequence
protocol (24hr exposure to each compound in sequence) against the melanoma cell line
Mel-RM. Using change in IC50 to assess for synergy it was noted that HMC at a
concentration of 2 uM chemosensitised the Mel-RM cells to camptothecin by -12 fold
(Table 12). 3D analysis of the data, however, reveal a marked degree of synergy between
HMC and doxorubicin against Mel-RM cells. These data demonstrate that HMC is able to
chemosesnsitise cancer cells to pacltaxel. From the 3D analysis the optimum combination
of HMC and camptothecin against the MM200 melanoma cell line would appear to be 2
uM HMC and between 1 and 0.1 (μ.M doxorubicin.
Table 12. Comparative assessment of synergy between HMC and doxorubicin against the
Mel-RM melanoma cell line. Average IC50 data for each agent when assessed as a
monotherapy or in combination are shown.
(Table Removed)
3.8 Inhibition of TNFa in murine macrophages (RAW 264.7) by compounds 4,6 & 7
The mouse macrophage cell line RAW 264.7 was cultured in DMEM supplemented with
PCS, 2mM glutarnine and 50U/ml penicillin/streptomycin. Subconfluent cells were
detached from the flask by gentle scraping and 24-well plates seeded at 5 x 10s cells per
well and allowed to adhere for Ihr. Cells were treated with either test agent (in 0.025°/o
DMSO) or vehicle alone, Ihr prior to the addition of 50ng/ml LPS. After incubation for 16
hrs, culture media was collected and stored at -80°C for TNFa measurement using an
enzyme immunometric assay (Becton Dickinson),
Compound 6 and compound 7 of the present invention were tested and the results are
shown in Figure 12, which indicated that the compounds tested inhibit TNFa in murine
macrophages in a dose dependent manner over the concentration ranges tested.
The raw data for compounds 6, 7 and additionally compound 4 is shown in Table 13
below.
Table 13. Inhibition of TNFa in murine macrophages by compounds 4, 6 and 7
(Table Removed)
3,9 Assessment of HHC as a chemosensitiser
HHC was screened as chemosensitiser against a panel cell lines representative of a range
cancer indications using a panel of cytotoxics commonly used in the treatment of cancer. It
has emerged that HHC has an ability to strongly chemosensitise cancer cell lines from
different pathologies to gemcitabine (ovarian, prostate, breast and pancreatic cancers, and
glioma) (Table 14). Strong synergy has been noted using HHC:cisplatin against ovarian
and prostate cancer, mild synergy against colorectal cancer cell lines and synergy was not
observed in pancreatic cancer and glioma. Moderate synergy has been noted using the
HHCrpaclitaxel combination against breast and colorectal cancer, and melanoma cell lines.
Equivocal synergy data using the HHC:paclitaxel combination has been noted against
ovarian cancer and glioma cell lines and there was no evidence of synergy against prostate
and pancreatic cancer cell lines. Data has revealed that HHC is able to strongly
chemosensitise the MM96L melanoma cell line to cisplatin, carboplatin and decarbazine
(Table 7).
Table 14. Assessment of HHC as a chemosensitiser using a panel of cancer cell lines and
standard cytotoxics
(Table Removed)
3.10. Efficacy of DHE, HMC and HHC against selected melanoma cell lines.
In comparison with DHE, both HMC and HHC showed excellent anti-cancer activity
against a range of melanoma cell lines. HHC was the most efficacious agent against all
melanoma cell lines tested to date having sub 1 uM IC50 values (Table 15).
(Table Removed)
4.0 Effect on murine macrophages (RAW 264.7) stimulated with LPS
The mouse macrophage cell line RAW 264.7 was cultured in DMEM supplemented with
foetal calf serum (PCS), 2mM glutamine and 50U/ml penicillin/streptomycin.
Subconfluent cells were detached from the flask by gentle scraping and 24-well plates
seeded at 5 x 10s cells per well and allowed to adhere for Ihr. Cells were then treated
either test compound at a concentration of lOμM (in 0.025% DMSO) or vehicle alone, and
incubated for Ihr. LPS 50ng/ml (LPS -Sigma-Aldrich) was then added. After incubation
for 16hrs, culture media was collected and stored at -80°C for ecosanoid measurements
using enzyme immunometric assays (PGE2 and TXB2 - Cayman Chemical).
Table 16: Percentage change in eicosanoid synthesis after incubating test compound at
lOμM compared with incubation with vehicle alone. Positive values indicate enhanced
synthesis; negative values indicate inhibition of synthesis and consequently suggest antiinflammatory
activity.
(Table Removed)
The invention has been described herein, with reference to certain preferred embodiments,
in order to enable the reader to practice the invention without undue experimentation.
However, a person having ordinary skill in the art will readily recognise that many of the
components and parameters may be varied or modified to a certain extent without
departing from the scope of the invention. Furthermore, titles, headings, or the like are
provided to enhance the reader's comprehension of this document, and should not be read
as limiting the scope of the present invention.
The entire disclosures of all applications, patents and publications, cited herein, if any, are
hereby incorporated by reference.
Throughout this specification and the claims which follow, unless the context requires
otherwise, the word "comprise", and variations such as "comprises" or "comprising", will
be understood to imply the inclusion of a stated integer or step or group of integers or steps
but not the exclusion of any other integer or step or group of integers or steps.
15 Those skilled in the art will appreciate that the invention described herein is susceptible to
variations and modifications other than those specifically described. It is to be understood
that the invention includes all such variations and modifications. The invention also
includes all of the steps, features, compositions and compounds referred to or indicated in
this specification individually or collectively, and any and all combinations of any two or
more of said steps or features.
The reference to any prior art in this specification is not, and should no,t be taken as, an
acknowledgment or any form of suggestion that that prior art forms part of the common
general knowledge in the field of endeavour.
Selected Reference Articles
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inhibits dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in female
Sprague-Dawley rats. Eur J Cancer. 39, 1012-8.
Constantinou AI, Husband A. 2002 Phenoxodiol (2H-l-benzopyran-7-0,l,3-(4-
hydroxyphenyl)), a novel isoflavone derivative, inhibits DNA topoisomerase II by
stabilizing the cleavable complex. Anlieaueer Res. 22,2581-5.
Gamble, JR., Xia, P., Hahn, C., Drew, J., Drogemuller, C., Carter, C., Walker, C., Brown,
DM., Vadas, MA. 2003 Phenoxodiol, a derivative of plant flavanoids, shows potent antirumour
and anti-angiogenic properties. Nature Medicine. Submitted.
Hersey, P and Zhang, X. D. 2001 How melanoma cells evade Trail-induced apoptosis.
15 Nature reviews, Cancer, 1, 142-150.
Kamsteeg, M., Rutherford, T., Sapi, E., Hanczaruk, B., Shahabi, S., Flick, M., Brown, D.M
and Mor, G. 2003 Phenoxodiol-an isoflvone analogue- induces apoptosis in chemoresistant
ovarian cancer cells. Oncogene, ;22, 2611-20.
O'Dwyer PJ, Moyer JD, Suffness M, Harrison SD Jr, Cysyk R, Hamilton TC, Plowman J.
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alone and in combination with cisplatin in vivo. Cancer Res. 54, 724-9
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cachexia induced by a human melanoma (G361). Br J Cancer. 80,1734-7.
Bellisarii, F. L., S. Gallina, et al. (2001). "Tumor necrosis factor-alpha and cardiovascular
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2(6): 408-17.
Szlosarek, P. W. and F. R. Balkwill (2003). "Tumour necrosis factor alpha: a potential
target for the therapy of solid tumours." Lancet Oncol 4(9): 565-73.
Nakata E, Hunter N, Mason K, Fan Z, Ang KK, Milas L. 2004 C225 antiepidermal growth
factor receptor antibody enhances the efficacy of docetaxel chemoradiotherapy. Int J
Radiat Oncol Biol Phys. 59(4): 1163-73.






Claims
1 . A compound of the general formula (I)

(Figure Removed)
RI is hydrogen, alkyl, cycloalkyl or C(O)R?,
R4,
R?
R2 and RS are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalky], halo or OC(0)Ry, with the exception that Ra and RS are not both hydrogen, R5and R6 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino, C1-4-alkylamino or di(C1-4-alkyl)amino, OC(O)R7 or ORg, is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino, and
Rg is aryl such as phenyl or arylalkyl such as benzyl, and
R$ is hydrogen hydroxy, alkyl, alkoxy, cycloalkyl or halo,
or a pharmaceutically acceptable salt or derivative thereof.
2. A compound of the formula (I-a):

(Figure Removed)
wherein
RI is hydrogen, alkyl, cycloalkyl or C(0)R?;
Ra and R3 are independently hydrogen, hydroxy, alkoxy, halo or OC(O)R7, with the
exception that R2 and R3 are not both hydrogen, R4, R5 and R6 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl,
OC(O)R7, amino, and R? is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino.
3. A compound of claim 2, wherein
RI . is .hydrogen, C1-4-alkyl or C(O)R7, .
R2 and RS are independently hydrogen, hydroxy, C1-4-alkoxy, halo or OC(O)R7,
provided that R2 and R3 are not both hydrogen, R4, R5and R6 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl,
OC(O)R7, and
R7 is C1-4-alkyl, phenyl or benzyl, or a pharmaceutically acceptable salt or derivative thereof.
4. A compound of claim 3, wherein
RI is hydrogen, methyl, ethyl, propyl, isopropyl or acetyl,
R2 and R3 are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy,
isopropoxy, bromo, chloro, fluoro or acetyloxy, with the exception that R2 and R3
are not both hydrogen,
R4 is hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy or acetyloxy, and R5 and R6 are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy,
isopropoxy, acetyl, or acetyloxy, or a pharmaceutically acceptable salt or derivative thereof.
5. A compound of claim 4, wherein
RI is hydrogen, methyl or acetyl,
R2 and R3 are independently hydrogen, hydroxy, methoxy, bromo or acetyloxy, with
the exception that R2 and R3 are not both hydrogen,
R4 and R6 are independently hydrogen, hydroxy, methoxy or acetyloxy, and RS is hydrogen, or a pharmaceutically acceptable salt or derivative thereof.
6. A compound of claim 1 selected from:
3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol;
3-(4-hydroxyphenyl)-4-phenylchroman-7-ol;
3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol;
3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol;
3-(4-hydroxyphenyl)-4-(4-methylphenyl)chroman-7-ol;
3-(4-methoxyphenyl)-4-(4-methoxyphenyl)-7-methoxychroman;
3-(4-hydroxyphenyl)-4-(2,6-dimethoxy-4-hydroxyphenyl)chroman-7-ol;
3-(4-hydroxyphenyl)-4-(2-hydroxyphenyl)chroman-7-ol;
3-(4-hydroxyphenyl)-4-(3-acyl-2-hydroxy-4-methoxyphenyl)chroman-7-ol;
3-(3-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol;
3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol;
3-(4-bromophenyl)-4-(4-methoxyphenyl)chroman-7-ol;
3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol;
3-(4-hydroxyphenyl)-4-(3-aminophenyl)chroman-7-ol; and
3-(4-hydroxyphenyl)-4-(4-phenoxyphenyl)chroman-7-ol.
7. A compound according to claim 6 selected from:
3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol; and
3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol.
8. A process for the preparation of a compound of formula (I-a) according to claim 2
comprising the step of reacting the 4-keto group of a compound of the formula (II):
(Figure Removed) R2 and R3 are independently hydrogen, alkoxy, halo or OSi(R10)3, with the exception
that R2 and R3 are not both hydrogen, and RIO is independently alkyl or aryl, with an arylating agent W'M4', wherein
W" is optionally substituted aryl, and M* is one or more counter ions, preferably [MgBr]+, to form the intermediate tertiary alcohol (III):
(Figure Removed)




or a salt thereof and which is dehydrated to form a compound of formula (IV):
which is subsequently hydrogenated and optionally deprotected to form a compound of formula (I).
R2 and R3 are independently hydrogen, alkoxy, halo or OSi(R10)3, with the exception
that R2 and R3 are not both hydrogen, and RIO is independently alkyl or aryl, with an arylating agent W'M4', wherein
W" is optionally substituted aryl, and M* is one or more counter ions, preferably [MgBr]+, to form the intermediate tertiary alcohol (III):
(Figure Removed)




or a salt thereof and which is dehydrated to form a compound of formula (IV):
which is subsequently hydrogenated and optionally deprotected to form a compound of formula (I).
R2 and R3 are independently hydrogen, alkoxy, halo or OSi(R10)3, with the exception
that R2 and R3 are not both hydrogen, and RIO is independently alkyl or aryl, with an arylating agent W'M4', wherein
W" is optionally substituted aryl, and M* is one or more counter ions, preferably [MgBr]+, to form the intermediate tertiary alcohol (III):
(Figure Removed)




or a salt thereof and which is dehydrated to form a compound of formula (IV):
which is subsequently hydrogenated and optionally deprotected to form a compound of formula (I).
9. Use of one or more compounds of formula (I) or (I-a) in chemotherapy or as a
radiosensitising or chemosensitising agent.
10. A method for the treatment, prevention or amelioration of disease, which comprises
administering to a subject one or more compounds of the formula (I) or (I-a) or a
pharmaceutically acceptable salt or derivative thereof optionally in association with a
carrier and/or excipient.
11. A method of claim 10, wherein the disease is cancer or a tumour mass.

12. A method of claim 11, wherein the cancer or rumour mass is of epithelial origin
(including prostate, ovarian, cervical, breast, gall-bladder, pancreatic, colorectal, renal, and
non-small lung cancer cells), of mesenchymal origin (including melanoma, mesothelioma
and sarcoma cancer cells) or of neural origin (including glioma cancer cells).
13. Use of one or more compounds of formula (I) or (I-a) or a pharmaceutically
acceptable salt or derivative thereof in the manufacture of a medicament for the treatment
of a disease or disorder.
14. An agent for the treatment, prophylaxis or amelioration of a disease or disorder,
which agent comprises one or more compounds of formula (I) or (I-a) or a
pharmaceutically acceptable salt or derivative thereof.
15. A pharmaceutical composition which comprises one or more compounds of
formula (I) or (I-a) or a pharmaceutically acceptable salt or derivative thereof in
association with one or more pharmaceutical carriers, excipients, auxiliaries and/or
diluents.
16. A pharmaceutical composition according to claim 15 which further comprises an
additional chemotherapeutic agent.
17. A drink or food-stuff, which contains one or more compounds of formula (I) or (I-
a) or a pharmaceutically acceptable salt or derivative thereof.
18. A compound of formula (I) or (I-a) or a pharmaceutically acceptable salt thereof as
herein described with reference to the Examples and/or accompanying drawings.
19. A compound of formulae (II), (III) or (IV), pharmaceutically acceptable salts
thereof and/or uses thereof as herein described.

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Patent Number 258848
Indian Patent Application Number 2155/DELNP/2007
PG Journal Number 07/2014
Publication Date 14-Feb-2014
Grant Date 11-Feb-2014
Date of Filing 20-Mar-2007
Name of Patentee MARSHALL EDWARDS , INC.
Applicant Address A DELAWARW COMPANY OF 11975 EI CAMINO REAL, SUITE 101, SANDIEGO,CALIFORNIA 92130, UNITED STATES OF AMERICA
Inventors:
# Inventor's Name Inventor's Address
1 HEATON, ANDREW 2/46-48 ABBOTSFORD PARADE, ABBOTSFORD, SYDNEY, NEW SOUTH WALES 2046, AUSTRALIA
2 HUSBAND, ALAN, JAMES 2/18 WEST RESCENT STREET, MCMAHON'S POINT, SYDNEY, NEW SOUTH WALES 2060, AUSTRALIA
PCT International Classification Number C07D 311/58
PCT International Application Number PCT/AU2005/001436
PCT International Filing date 2005-09-21
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2004906363 2004-11-05 U.S.A.
2 60/676,934 2005-05-03 U.S.A.
3 2004-315009 2004-10-29 U.S.A.
4 60/611,299 2004-09-21 U.S.A.
5 PCT/AU2004/001619 2004-11-19 U.S.A.