|Title of Invention||
METHOXY PHOSPHONATE NUCLEOTIDE PRODRUG
|Abstract||A novel method is provided for screening prodrugs of methoxyphosphonate nucleotide analogues to identify prodrugs selectively targeting desired tissues with antiviral or antitumor activity. This method has led to the identification of novel mixed ester-amidates of PMPA for retroviral or hepadnaviral therapy, including compounds of structure (5a) having substituent groups as defined herein. Compositions of these novel compounds in pharmaceutically acceptable excipients and their use in therapy and prophylaxis are provided. Also provided is an improved method for the use of magnesium alkoxide for the preparation of starting materials and compounds for use herein.|
|Full Text||FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
The Patent Rules, 2003
(See section 10 and rule 13)
TITLE OF THE INVENTION
"PRODRUGS OF PHOSPHONATE NUCLEOTIDE
ANALOGUES AND METHODS FOR SELECTING AND
We, GILEAD SCIENCES, INC., of 333 Lakeside Drive, Foster City, CA 94404, United States of America;
The following specification particularly describes the nature of the invention and the manner in which it is to he performed:
10 Prodrugs of Phosphonate Nucleotide Analogues and
Methods for Selecting and Making Same
This application relates to prodrugs of methoxyphosphonate nucleotide analogues. In particular it relates to improved methods for making and identifying 15 sucn prodrugs.
Many methoxyphospnonate nucleotide analogues are known. In general,
such compounds have the structure A-OCH2P(0)(OR)2 where A is the residue of a
nucleoside analogue and R independently is hydrogen or various protecting or prodrug functionalities. See U.S. Patent Nos. 5,663,159,5,977,061 and 5,798,340,
20 Oliyai et al, "Pharmaceutical Research" 16(11):1687~1693 (1999), Stella et al., "J. Med. Chem." 23(12):1275-1282 (1980), Aarons, L., Boddy, A. and Petrak, K. (1989) Novel Drug Delivery and Its Therapeutic Application (Prescott, L. F. and Nimmo, W. S., ed.), pp. 121-126; Bundgaard, H. (1985) Design of Prodrugs (Bundgaard, H., ed.) pp. 70-74 and 79-92; Banerjee, P. K. and Amidon, G. L. (1985) Design of Prodrugs
25 (Bundgaard, H., ed.) pp. 118-121; Notari, R. E. (1985) Design of Prodrugs
(Bundgaard, H., ed.) pp. 135-156; Stella, V. J. and Himmelstein, K. J. (1985) Design of Prodrugs (Bundgaard, H., ed.) pp. 177-198; Jones, G. (1985) Design of Prodrugs (Bundgaard, H., ed.) pp. 199-241; Connors, T. A. (1985) Design of Prodrugs (Bundgaard, H., ed.) pp. 291-316. All literature and patent citations herein are
30 expressly incorporated by reference.
i-V^ A/ UOOX/ZOJLU4-
5 Summary of the Invention
Prodrugs of methoxyphosphonate nudeotide analogues intended for antiviral or antitumor therapy, while known, traditionally have been selected for their systemic effect. For example, such prodrugs have been selected for enhanced bioavailability, i.ev ability to be absorbed from the gastrointestinal tract and
10 converted rapidly to parent drug to ensure that the parent drug is available to all tissues. However, applicants now have found that it is possible to select prodrugs that become enriched at therapeutic sites, as illustrated by the studies described herein where the analogues are enriched at localized focal sites of HtV infection. The objective of this invention is, among other advantages, to produce less toxicity
15 to bystander tissues and greater potency of the parental drug in tissues which are the targets of therapy with the parent methoxyphosphonate nucleotide analogue.
Accordingly,jpursuant to titiese observations, a screening method is provided for identifying a methoxyphosphonate nucleotide analogue prodrug conferring enhanced activity in a target tissue comprising:
20 (a) providing at least one of said prodrugs;
(b) selecting at least one therapeutic target tissue and at least one non-target
(c) administering the prodrug to the target tissue and to said at least one non-
target tissue; and
25 (d) deterrruxang'the relative antiviral activity conferred by the prodrug in the tissues in step (c).
In preferred embodiments, the target tissue are sites where HIV is actively replicated and/or which serve as an HIV reservoir, and the non-target tissue is an intact animal. Unexpectedly, we found that selecting lymphoid tissue as the target 30 tissue for the practice of this method for HIV led to identification of prodrugs that enhance the delivery of active drug to such tissues.
A preferred compound of this invention, which has been identified by this method has the structure (1),
10 where Ra is H or methyl,
and chirally enriched compositions thereof, salts, their free base and solvates thereof.
A preferred compound of this invention has the structure (2)
and its enriched diasteromers, salts, free base and solvates.
25 In addition, we unexpectedly found that the chirality of substituents on the
phosphorous atom and/or the amidate substituent are influential in the enrichment observed in the practice of this invention. Thus, in another embodiment of this
5 ' invention, we provide diastereomerically enriched compounds of this invention having the structure (3)
which are substantially free of the diastereomer (4)
0 B—E—f-^R1 I2
R is an oxyester which is hydrolyzable in vivo, or hydroxy!; B is a heterocyclic base;
R is hydroxy!, or the residue of an amino acid bonded to the P atom through' an amino group of the amino acid and having each carboxy substituent of the amino acid optionally esterified, but not both of R andR are hydroxyl; • E is-(CH2)2-,-CH(CH3)CH2-,-CH(CH2F)CH2-,-CH(CH20H)CH2-, ^CH(CH=CH2)CH2-,-CH(CsCH)CH2-,-Ck(CH2N3)CH2-,
CH2 R7 R7
-CHOl^OCHCR6).-, -CH(R9)CH20- or -CH(R8)0-, wherein the right hand bond is
Biuced to the heterocyclic base;
the broken line represents an optional double bond;
R and R are independently hydrogen, hydroxy, halo, amino or a
substituent having 1-5 carbon atoms selected from acyloxy, alkyoxy, alkylthio, allcylamino and dialkylarrrino;
5 invention, we provide diastereomericaUy enriched compounds of this invention
having the structure (3)
which are substantially free of the diastereomer (4)
R is an oxyester which is hydrolyzable in vivo, or hydroxyl; B is a heterocyclic base;
R is hydroxyl, or the residue of an amino acid bonded to the P atom through an amino group of the amino acid and having each carboxy substituent of
the amino acid optionally esterified/ but not both of R andR arehydroxyl; -"^"E is ^CH2)2-/ -CH(CH3)CH2-, -CH(CH2F)CH2-, -CH(CH20H)CH2-, -CH(CH=CH2)CH2-, -CH(CsCH)CH2-, -CH(CH2N3)CH2-,
CH(R6)OCH(R6'K -CH(R9)CH20- or -CH(R8)0-, wherein the right hand bond is
linked to the heterocyclic base;
the broken line represents an optional double bond;
R and R are independently hydrogen, hydroxy, halo, amino or a
substituent having 1-5 carbon atoms selected from acyloxy, alkyoxy, alkylthio, alkykrnino and dialkylamino;
R6 and R6' are independently H, C^-Cg alkyl, C1-C8 hydroxyalkyl, or C2-C7
R is independently H, Ca-Cg alkyl, or are taken together to form -O or
R8 is H, C1-C8 alkyl, C1-C6 hydroxyalkyl or C1-C6 haloalkyb and
10 R is H, hydroxymethyl or acyloxymethyl;
and their salts, free base, and solvates.
The diastereomers of structure (3) are designated the (S) isomers at the phosphorus chiral center.
Preferred embodiments of this invention are the diastereomericalLy enriched 15 compounds having the structure (5a)
25 which is substantially free of diastereomer (5b)
f N I ^
R5 is methyl or hydrogen;
R independently is H, alkyl, alkenyl, alkynyl, aryl or arylalkyl, or R
independently is alkyl, alkenyl, alkynyl, aryl or arylalkyl which is substituted with from 1 to 3 substituents selected from altylarnino, alkylaminoalkyl, 10 dialkylaminoalkyl, dialkylarnino, hydroxyl, oxo, halo, anuno, alkylthio, alkoxy, alkoxyalkyl, aryloxy, aryloxyalkyl, arylalkoxy, arylalkoxyalkyl, haloalkyl, nitro, nitroalkyl, azido, azidoalkyl, aJkylacyl, alkylacylalkyl, carboxyl, or alkykcylarnino;
R7 is the side chain of any naturally-occurring or pharmaceutically
acceptable amino acid and which, if the side chain comprises carboxyl, the carboxyl
15 group is optionally esterified with an alkyl or aryl group;
R11 is amino, alkylamino, oxo, or dialkylarnino; and
R12 is amino or H;and its salts, tautomers, free base and solvates.
20 A preferred embodiment of this invention is the compound of structure (6), '
(isopropoxycarbonyl)ethyl]amino]phenoxyphosphmyl]methoxy]propyl]adenine, also designated herein GS-734Q
35 Another preferred embodiment of this invention is the furnarate salt of
structure (5) (structure (7)), 9-[(R)-2-[[(S)-[[(S)-l-
fumarate (1:1), also designated herein GS-7340-2
^ H3cA -
15 The compounds of structures (l)-(7) optionally are formulated into
compositions containing pharmaceutically acceptable excipients. Such compositions are used in effective doses in the therapy or prophylaxis of viral (particularly HIV or hepadnaviral) infections.
In a further embodiment, a method is provided for the facile manufacture of
20 9-[2-(phosphonomethoxy)propyl]adenine (hereinafter "PMPA" or 9-[2-
(phosphonomethoxy)ethyl] adenine (hereinafter "PMEA") using magnesium alkoxide, which comprises combining 9-(2-hyo^oxypropyl)adenine or 9-(2-hydroxyemyl)adenine, protected p-toluenesulfonyloxymethylphosphonate and magnesium alkoxide, and recovering PMPA or PMEA, respectively.
Detailed Description of the Invention The methoxyphosphonate nucleotide analogue parent drugs for use in this
screening method are compounds having the structure A-OCH2P(0)(OH)2 wherein
30 A is the residue of a nucleoside analogue. These compounds are known per se and are not part of this invention. More particularly, the parent compounds comprise a heterocyclic base B and an aglycon E, in general having the structure
(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]propyl]adenine fumarate (1:1), also designated herein GS-7340-2
The compounds of structures (l)-(7) optionally are formulated into compositions containing pharmaceutically acceptable excipients. Such compositions are used in effective doses in the therapy or prophylaxis of viral (particularly HTV or hepadnaviral) infections.
In a further embodiment, a method is provided for the facile manufacture of 9-l2-(phosphonomethoxy)propyl]adenine (hereinafter "PMPA" or 9-[2-(phosphonomethoxy)ethyl] adenine (hereinafter "PMEA") using magnesium alkoxide, which comprises combining 9-(2-hydrDxypropyl)aderdne or 9-(2-hydroxyethyl)adenme, protected p-toluenesulfonyloxymethylphosphonate and magnesium alkoxide, and recovering PMPA or PMEA, respectively.
Detailed Description of the Invention
The methoxyphosphonate nucleotide analogue parent drugs for use in this
screening method are compounds having the structure A-OH2P(0)(OH)2 wherein
A is the residue of a nucleoside analogue. These compounds are known per se and are not part of this invention. More particularly, the parent compounds comprise a heterocyclic base B and an aglycon E, in general having the structure
WO 02/08241 PCT/US01/23104
5 wherein the group B is defined below and group H is defined above. Examples are described in U.S. Patent Nos. 4,659,825,4,808,716,4,724,233,5,142,051,5,130,427, 5,650,510,5,663,159,5,302^585,5,476,938,5,696,263,5,744,600,5,688,778,5,386,030, 5,733,896,5,352,786, and 5,798,340, and EP 821,690 and 654,037,
The prodrugs for use in the screening method of this invention are
10 covalently modified analogues of the parent methoxyphosphonate nucleotide
analogues described in the preceding paragraph. In general, the phosphorus atom of the parent drug is the preferred site for prodrug modification, but other sites are found on the heterocyclic base B or the aglycon E. Many such prodrugs are already known. Primarily, they are esters or amidates of the phosphorus atom,but also
15 include substitutions on the base and aglycon. None of these modifications per se is part of this invention and none are to be considered limiting on the scope of the invention herein.
The phosphorus atom of the methoxyphosphonate nucleotide analogues contains two valences for covalent modification such as amidation or esterification
20 (unless one phosphoryl hydroxyl is esterified to an aglycon E hydroxyl substituent, whereupon only one phosphorus valence is free for substitution). The esters typically are aryloxy. The amidates ordinarily are naturally occurring monoamino acids having free carboxyl group(s) esterified with an alkyl or aryl group, usually phenyl, cycloalkyl, or t-, n- or s- alkyl groups. Suitable prodrugs for use in the
25 screening method of this invention are disclosed for example in U.S. Patent No. 5,798340. However, any prodrug which is potentially believed to be capable of being converted in vivo within target tissue cells to the free methoxyphosphonate nucleotide analogue parent drug, e.g., whether by hydrolysis, oxidation, or other covalent transformation resulting from exposure to biological tissues, is suitable for
30 use in the method of this invention. Such prodrugs may not be known at this time but are identified in the future and thus become suitable candidates available for testing in the method of this invention. Since the prodrugs are simply candidates for screening in the methods their structures are not relevant to practicing or enabling the screening method, although of course their structures ultimately are
35 dispositive of whether or not a prodrug will be shown to be selective in the assay,
5 The pro-moieties bound to the parent drug may be the same or different
However, each prodrug to be used in the screening assay will differ structurally from the other prodrugs to be tested. Distinct, i.e> structurally different, prodrugs generally are selected onthe basis of either their stereochemistry or their covalent structure, or these features are varied in combination. Each prodrug tested,.
10 however, desirably is structurally and stereochemically substantially pure, else the . output of the screening assay will be less useful. It is of course within the scope of this, invention to test only a single prodrug in an individual embodiment of the method of this invention, although typically then one would compare the results with prior studies with other prodrugs.
15 We have found that me stereochemistry of the prodrugs is capable of
influencing the enrichment in target tissues. Chiral sites are at the phosphorus atom and are also found in its substituents. For example, amino acid used in preparing amidates may be D or L forms, and the phosphonate esters or the amino acid esters can contain chiral centers as welL Chiral sites also are found on the
20 nucleoside analogue portion of the molecules, but these typically are already
dictated by the stereochemistry of the parent drug and will not be varied as part of the screen. For example the R isomer of PMPA is preferred as it is more active than the corresponding S isomer. Typically these diasteromers or enantiomers will be chirally enriched if not pure at each site so that the results of the screen will be
25 more meaningful. As noted, distinctiveness of stereoisomers is conferred by
enriching or purifying the stereoisomer (typically this will be a diastereomer rather than an enantiomer in the case of most methoxyphosphonate nucleotide analogues) free of other stereoisomers at the chiral center in question, so that each test compound is substantially homogeneous. By substantially homogeneous or
30 dhirally enriched, we mean that the desired stereoisomer constitutes greater than about 60% by weight of the compound, ordinarily greater than about 80% and preferably greater than about 95%.
Novel Screening Method Once at least one candidate prodrug has been selected, the remaining steps of the screening method of this invention are used to identify a prodrug possessing the required selectivity for the target tissue. Most conveniently the prodrugs are
10 labeled with a detectable group, e.g. radiolabeled, in order to facilitate detection later in tissues or cells. However, a label is not required since other suitable assays for the prodrug or its metabolites (including the parent drug) can also be employed. These assays could include mass spectrometry, HPLC, bioassays or immunoassays for instance. The assay may detect the prodrug and any one or
15 more of its metabolites, but preferably the assay is conducted to detect only the generation of the parent drug. This is based on the assumption (which may not be warranted in all cases) that the degree and rate of conversion of prodrug to antivirally active parent diphosphate is the same across all tissues tested. Otherwise, one can test for the diphosphate.
20 The target tissue preferably will be lymphoid tissue when screening for
prodrugs useful in the treatment of HTV infection. Lymphoid tissue will be known to the artisan and includes CD4 cells, lymphocytes, lymph nodes, macrophages and macrophage-like cells including monocytes such as peripheral blood monocytic cells (PBMCs) and glial cells. Lymphoid tissue also includes non-lymphoid tissues
25 that are enriched in lymphoid tissues or cells, e.g. lung, skin and spleen. Other targets for other antiviral drugs of course will be the primary sites of replication or latency for the particular virus concerned, e.g., liver for hepatitis and peripheral nerves for HSV. Similarly, target tissues for tumors will in fact be the tumors themselves. These tissues are all well-known to the artisan and would not require
30 undue experimentation to select. When screening for antiviral compounds, target tissue can be infected by the virus.
Non-target tissues or cells also are screened as part of the method herein. Any number or identity of such tissues or cells can be employed in this regard. In general, tissues for which the parent drug is expected to be toxic will be used as
35 non-target tissues. The selection of a non-target tissue is entirely dependent upon
WO 02/08241 PCT/US01/23104
5 the nature of the prodrug and the activity of the parent For example, non-hepatic tissues would be selected for prodrugs against hepatitis, and untransfbrmed cells of the same tissue as the tumor will suffice for the antitumor-selective prodrug screen.
It should be noted that the method of this invention is distinct from studies typically undertaken to determine oral bioavailability of prodrugs.' In oral
10 bioavailability studies, the objective is to identify a prodrug which passes into the systemic circulation substantially converted to parent drug. In the present invention, the objective is to find prodrugs that are not metabolized in the gastrointestinal tract or circulation. Thus, target tissues to be evaluated in the method o£ this invention generally do not include the small intestines or, if the
15 intestines are included, then the tissues also include additional tissues other than the small intestines.
The target and non-target tissues used in the screening method of this invention typically will be in an intact living animal. Prodrugs containing esters are more desirably tested in dogs, monkeys or other animals than rodents; mice
20 and rat plasma contains, high circulating levels of esterases that may produce a misleading result if the desired therapeutic subject is a human or higher mammal
It is not necessary to practice this method with intact animals. It also is within the scope of this invention to employ perfused organs, in vitro culture of organs (e.g. skin grafts) or cell lines maintained in various forms of cell culture, e.g.
25 roller bottles or zero gravity suspension systems. For example, MT-2 cells can be used as a target tissue for selecting HIV prodrugs. Thus, the term "tissue" shall not be construed to require organized cellular structures, or the structures of tissues as they may be found in nature, although such would be preferred. Rather, the term "tissue" shall be construed to be synonymous with cells of a particular source,
30 origin or differentiation stage.
The target and non-target tissue may in fact be the same tissue, but the tissues will be in different biological status. For example, the method herein could be used to select for prodrugs that confer activity in virally-infected tissue (target tissue) but which remain substantially inactive in viraUy-uninfected cells
35 (corresponding non-target tissue). The same strategy would be employed to select
WO 02/08241 PCT/US01/23104
5 prophylactic prodrugs, i.e., prodrugs metabolized to antivirally active forms incidental to viral infection but which remain substantially unmetabolized in uninfected cells. Surularly, prodrugs could be screened in transformed'cells and the untransformed counterpart tissue. This would be particularly useful in comparative testing to select prodrugs for the treatment of hematological
10 malignancies, e.g. leukernias.
Without being limited by any particular theory of operation, tissue selective prodrugs are thought to be selectively taken up by target cells and/or selectively metabolized within the cell, as compared to other tissues or cells. The unique advantage of the methoxyphosphonate prodrugs herein is that their metabolism to
15 the dianion at physiological pH ensures that they will be unable to diffuse back out of the cell They therefore remain effective for lengthy periods of time and are maintained at elevated intracellular concentrations, thereby exhibiting increased potency. The mechanisms for enhanced activity in the target tissue are believed to include enhanced uptake by the target cells, enhanced intracellular retention, or
20 both mechanisms working together. However, the manner in which selectivity or enhanced delivery occurs in the target tissue is not important. It also is not important that all of the metabolic conversion of the prodrug to the parent compound occurs within the target tissue. Only the final drug activity-conferring conversion need occur in the target tissue; metabolism in other tissues may provide
25 intermediates finally converted to antiviral forms in the target tissue.
The degree of selectivity or enhanced delivery that is desired will vary with the parent compound and the manner in which it is measured (% dose distribution or parent drug concentration). In general, if the parent drug already possess a generous therapeutic window, a low degree of selectivity may be sufficient for the
30 desired prodrug. On the other hand, toxic compounds may require more extensive screening to identify selective prodrugs. The relative expense of the method of this invention can be reduced by screening only in the target tissue and tissues against which the parent compound is known to be relatively toxic, e.g. for PMEA, which is nephrotoxic at higher doses, the primary focus will be on kidney and lymphoid
WO02/08241 FC'17 USUI/23104
5 The step of determining the relative antiviral activity of a prodrug in the
selected tissues ordinarily is accomplished by assaying target and non-target tissues for the relative presence or activity of a metabolite of the prodrug, which metabolite is known to have, or is converted to, a metabolite having antiviral or antitumor activity. Thus, typically one would determine the relative amount of the
10 parent drug in the tissues over substantially the same time course in order to identify prodrugs that are preferentially metabolized in the target tissue to an antivirally or antitumor active metabolite or precursor thereof which in the target tissue ultimately produces the active metabolite. In the case of antiviral compounds, the active metabolite is the diphosphate of the phosphonate parent
15 compounds. It is this metabolite that is incorporated into the viral nucleic acid, thereby truncating the elongating nucleic acid strand and halting viral replication. Metabolites of the prodrug can be anabolic metabolites, catabolic metabolites], or the product of anabolism and catabolism together. The manner in which the metabolite is produced is not important in the practice of the method of this
The method of this invention is not limited to assaying a metabolite which per se possesses antiviral or antitumor activity. Instead, one can assay inactive precursors of the active metabolites. Precursors of the antivirally active diphosphate metabolite include the monophosphate of the parent drug,
25 monophosphates of other metabolites of the parent drug (e.g., an intermediate modification of a substituent on the heterocyclic base), the parent itself and metabolites generated by the cell in converting the prodrug to the parent prior to phosphorylation. The precursor structures may vary considerably as they are the result of cellular metabolism. However, this information is already known or could
30 be readily determined by one skilled in the art
If the prodrug being assayed does not exhibit antitumor or antiviral activity per se then adjustments to the raw assay results may be required. For example, if the intracellular processing of the inactive metabolite to an active metabolite occurs at different rates among the tissues being tested, the raw assay results with the
35 inactive metabolite would need to be adjusted to take account of the differences
. 5 among the cell types because the relevant parameter is the generation of activity in the target tissue, not accumulation of inactive metabolites. However, determining tiie proper adjustments would be within the ordinary skill. Thus, when step (d) of the method herein calls for determining the activity, activity can be either measured directly or extrapolated. It does not mean that the method herein is
10 limited to only assaying intermediates that are active per se. For instance, the
absence or decline of the prodrug in the test tissues also could be assayed. Step (d) only requires assessment of the activity conferred by the prodrug as it interacts with the tissue concerned/and this may be based on extrapolation or other indirect measurement.
15 Step (d) of the method of this invention calls for determining the "relative"
activity of the prodrug. It will be understood that this does not require that each and every assay or series of assays necessarily must also contain runs with the selected non-target tissue. On the contrary, it is within the scope of this invention to employ historical controls of the non-target tissue or tissues, or algorithms
20 representing results to be expected from such non-target tissues, in order to provide the benchmark non-target activity.
The results obtained in step (d) are then used optimally to select or identify a prodrug which produces greater antiviral activity in the target tissue than in the non-target tissue. It is this prodrug that is selected for further development
25 It will be appreciated that some preassessment of prodrug candidates can be
undertaken before the practice of the method of this invention. For example, the prodrug will need to be capable of passing largely unmetabolized through the gastrointestinal tract, it will need to be substantially stable in blood, and it should be able to permeate cells at least to some degree. In most cases it also will need to
30 complete a first pass of ihe hepatic circulation without substantial metabolism. Such prestudies are optional, and are well-known to those skilled in the art.
The same reasoning as is described above for antiviral activity is applicable to antitumor prodrugs of methoxyphosphonate nucleotide analogues as well. These include, for example, prodrugs of PMEG, the guanyl analogue of PMEA. In
r\s LI IIOUI/ZJIUI
5 this case, cytotoxic phosphonates such as PMEG are worthwhile candidates to pursue as their cytotoxicity in fact confers their antitumor activity.
A compound identified by this novel screening method then can be entered into a traditional preclinical or clinical program to confirm that the desired objectives have been met. Typically, a prodrug is considered to be selective if the
10 activity or concentration of parent, drug in the target tissue (% dose distribution) is greater than 2x, and preferably 5x, that of the parent compound in non-target tissue. Alternatively, a prodrug candidate can be compared against a benchmark prodrug. In this case, selectivity is relative rather than absolute. Selective prodrugs will be those resulting in greater than about lOx concentration or activity in the
15 target tissue as compared with the prototype, although the degree of selectivity is a matter of discretion.
Novel Method for Preparation of Starting Materials or Intermediates Also included herein is an improved method for manufacture of preferred
20 starting materials (parent drugs) of this invention, PMEA and (R)-PMPA.
Typically, this method comprises reacting 9-(2-hydroxypropyl)adenine (HPA) or 9-(2-hydroxyethyl)adenine (HEA) with a magnesium alkoxide, thereafter adding the protected aglycon synthon p-toluene-sulfonyloxymethylphosphonate (tosylate) to the reaction mixture, and recovering PMPA or PMEA, respectively.
25 Preferably, HPA is the enriched or isolated R enantiomer. If a chiral HPA
mixture is used, R-PMPA can be isolated from the chiral PMPA mixture after the synthesis is completed.
Typically the tosylate is protected by lower alkyl groups, but other suitable groups will be apparent to the artisan. It may be convenient to employ the tosylate
30 presubstituted with the prodrug phosphonate substituents which are capable of acting as protecting groups in the tosylation reaction, thereby allowing one to bypass the deprotection step and directly recover prodrug or an intermediate therefore.
The alkyl group of the magnesium alkoxide is not critical and can be any Ca-
35 C6 branched or normal alkyl, but is preferably t-butyl (for PMPA) or isopropyl (for
5 PMEA). The reaction conditions also are not critical, but preferably comprise
heating the reaction mixture at about 70-75°C with stirrine: or other moderate
If there is no interest in retaining the phosphonate substituents, the product
is deprotected (usuaJlywith bromotrimethylsilane where the tosylate protecting 10 group is alkyl), and the product then recovered by crystallization or other
conventional method as will be apparent to the artisan.
In the compounds of this invention depicted in structures (3) and (4), the 15 heterocyclic base B is selected from the structures
I '. ' or '
R15 is H, OH, F, CI, Br, I, OR16, SH, SR16, NH2, or NHR17;
25 R16 is Ci-Ce alkyl or C2-C6 alkenyl including CH3, CH2CH3, CH2CCH,
CH2CHCH2 and C3H7;
R17 is C1-C6 alkyl or C2-C6 alkenyl including CH3, CH2CH3, CH2CCH, CH2CHCH2, and C3H7;
R18 is N, CF, CQ, CBr, CI, CR19 CSR19, or COR19;
30 R19 is H, C1-C9 alkyl, C2-C9 alkenyl, C2 - C9 alkynyl, C1-C9 alkyl-Ci-C9
alkoxy, or C7-C9 aryl-alkyl unsubstituted or substituted by OH, F, CI, Br or I, R
therefore including-CH3, -CH2CH3, -CHCH2, -CHCHBr, -CH2CH2CI, -CH2CH2F, -CH2CCH, -CH2CHCH2, -C3H7, -CH2OH, -CH20CH3,-CH20C2H5, -CH2OCCH, -CH2OCH2CHCH2, -CH2C3H7, -CH2CH2OH, -CH2CH2OCH3,
5 -CH2CH2OC2H5, -CH2CH20CCH, -CH2CH2OCH2CHCH2, and -CH2CH2OC3H7;
R21 is N, CH, CCN, CCF3, CC^CH or CC(Q)NH2;
R22 is H, OH, NH2, SH, SCH3, SCH2CH3, SCH2CCH, SCH2CHCH2, SCsHz,
10 NH(CH3), N(CH3)2, NH(CH2CH3), N(CH2CH3)2, NH(CH2CCH),
NH(CH2CHCH2), NH(C3H7), halogen (F, a, Br or I) or X wherein X is
-(CH2)m(O)n(CH2)mN(R10)2 wherein each m is independently 0-2, n is 0-1, and
R10 independently is
15 C1-C15 alkyl, C2-Q5 alkenyl, C6-C15 arylalkenyl, C6-C1S
arylalkynyl, C2-C15 alkynyl, Ci-C6-alkykmino-Ci-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroaralkyl, C5-C6 aryl, C2-C6 heterocycloalkyl,
C2-G15 alkyl, C3-C15 alkenyl, C6-Q5 arylalkenyl, C3-C15 alkynyl, C7-C15 arylalkynyl, Ci-C6-altylarnino-Ci-C6 alkyl, G5-C15 aralkyl, C6-C15 20 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in the alkyl moiety not
adjacent to N has been replaced by -O-,
optionally both R10 are joined together with N to form a saturated or
unsaturated C2-C5 heterocycle containing one or two N heteroatoms and
optionally an additional O or S heteroatom,
25 or one of the foregoing R10 groups which is substituted with 1 to 3
halo, CN or N3; but optionally at least one R10 group is not H; "
R23 is H, OH, F, Q, Br, I, SCH3, SCH2CH3, SCH2CCH, SCH2CHCH2,
SC3H7, OR16, NH2, NHR17 or R22; and
R24 is 0,S or Se.
30 B also includes both protected and unprotected heterocyclic bases,
particularly purine and pyrimidine bases. Protecting groups for exocyclic amines and other labile groups are known (Greene et aL "Protective Groups in Organic ' Synthesis") and include N-benzoyl, isobutyryl, 4,4'-dimemoxytrityl (DMT) and the
WO 02/08241 PCT/US01/23104
5 like. The selection of protecting group will be apparent to the ordinary artisan and will depend upon the nature of the labile group and the chemistry which the protecting group is expected to encounter, e.g. acidic, basic, oxidative, reductive or other conditions. Exemplary protected species are N^-benzoylcytosine, N^-
benzoyladenine, N2-isobutyrylguanine and the like.
10 Protected bases have the formulas Xa.l,XIa.l, Xlb.l, XUa.l orXIHa.l
15 Or N ' R23A^R2Cr^N R39'
(Xa.l) (XIa.1) (Xlb.l) (XHa.1) (XHIa.l)
20 wherein R18, R20, R21, R24 have the meanings previously defined; R22* is R39 or R22 provided that R22 is not NH2; R23* is R^or R^provided that R23 is not NH2; R39 is NHR40, NHC(0)R86 or CR4,N(RM)2 wherein R3S is C1-C19 alkyl, Cl-Cl9 alkenyl, C3-CIQ aryl, -adamantoyl, alkylanyl, or C3-C10 aryl substituted with 1 or 2 atoms or groups selected from halogen, methyl, ethyl, methoxy, ethoxy, hydroxy and cyano;
25 R3S is C1-C10 alkyl, or both R38 together are lrmorpholino, 1-piperidine or 1-
pyrrolidine; R40 is Q-C,. alkyl, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, octyl and decanyl; and R41 is hydrogen or CH3/
39 22A 23A
For bases of structures XIa.l and Xlb.l, if R is present at R orR ,both
R groups on the same base will generally be the same. Exemplary R are phenyl,
30 phenyl substituted with one of the foregoing R aryl substituents, -C10H15 (Where
C10H15 is 2-adamantoyl), -CH2-C6H5, -C6H5, -CH(CH3)2, -CH2CH3, methyl, butyl, t-butyl, hepfanyl, nonanyl, undecanyl, or undecenyL
Specific bases include hypoxanthine, guanine, adenine, cytosine, inosine, thymine, uracil, xanthine, 8-aza derivatives of 2-arninopurine, 2,6-diaminopurine, 35 2-airuno-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza-8-aza
5 derivatives of adenine, guanine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 1-deaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6- 10 anunopurine, 2>6-diarrrinopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 6-azacytosine; 5-fluorocytosine; 5-chloroqrtosine; 5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-bromovinyiuracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5-bromouracil; 5^tri£uoromethyluracil; 5-methoxymethyluracil; 5-ethynyluracil and 5-propynyluraciL
15 Preferably, B is a 9-purinyl residue selected from guanyl, 3-deazaguanyl, 1-
deazaguanyl, 8-azaguahyl, 7-deazaguanyl, adenyl, 3-deazaadenyl, 1-dezazadenyl, 8-azaadenyl, 7-deazaadenyl, 2,6-diaminopurinyl, 2-arninopurinyl, 6-chloro-2-aminopurinyl and 6-tMo-2-arninopurinyl, or a B' is a 1-pyrixnidinyl residue selected from cytosinyl, 5-halocytosinyl, and 5-(Ci-C3-aIkyl)cytosinyL
20 Preferred B groups have the formula
R.22 independently is halo, oxygen, NH2/ X or H, uut opuonany at least one R22isX;
X is -(CH2)m(O)n(CH2)mN(R10)2 wherein m is 0-2, n is 0-1, and
30 RlO independently is
C1-Q5 alkyl, C2-C15 alkenyl, C6-C15 arylalkenyL C6-C15 arylalkynyl, C2-C15 alkynyl, Ci-C6-alkylairunc>-Ci-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroaralkyl, C5-C6 aryl, C2-C6 heterocycloalkyl,
5 ..C2-C15 alkyl, C3-Q5 alkenyl, C6-C15- arylalkenyl, C3-C15 alkynyl, ^jy ^
C7-C15 arylalkynyl, Ci-C6-aIkylaxrdiio-Ci-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroalkyl or C3-Q5 heterocycloalkyl wherein methylene in the alkyl moiety not adjacent to N^ has been replaced by -O,
optionally both RlO are joined together with N to form a saturated or 10 unsaturated C2-C5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom,
or one of the foregoing R*0 groups is substituted with 1 to 3 halo, CN or N3; but optionally at least one RlO group is not H; and
■Z is N or QHT, provided that the heterocydic nucleus varies from purine by 15. no more than one Z.
E groups represent the aglycons employed in the methoxyphosphonate nucleotide analogues. Preferably, the E group is -CH20(CH3)CH20-or-CH20CH20-.Also,
it is preferred that the side groups at chiral centers in the aglycon be substantially solely in the (R) configuration (except for hydroxymethyl, which is the enriched (S)
R1 is an in vivo hydrolyzable oxyester having the structure -OR35 or -OR6 wherein R35 is defined in column 64, line 49 of U.S. Patent No, 5,798,340, herein incorporated by reference, and R6 is defined above. Preferably R1 is aryloxy, ordrnarily unsubstituted or para-substituted (as defined in R6) pherioxy.
25 R2 is an amino acid residue, optionally provided that any carboxy group
linked by less than about 5 atoms to the amidate N is esterified. R2 typically has the structure
5 n is 1 or 2;
R" is R6 or H; preferably R° = Q-C, alkyl; Cs-C9 alkyl substituted independently with OH, halogen, O or N; C3-C6 aryl; C3-C6 aryl which is independently substituted with OH, halogen, O or N; or C3-C6 arylalkyl which is independently substituted with OH, halogen, O or N;
10 R" independently is H or Q-Q alkyl which is unsubstituted or substituted
by substituents independently selected from the group consisting of OH, O, N, COOR" and halogen; Cg-C^ary! which is unsubstituted or substituted by • substituents independently selected from the group consisting of OH, O, N, . COOR11 and halogen; or Q-Q aryl-alkyl which is unsubstituted or substituted by
15 substituents independently selected from the group consisting of OH, O, N, COOR" and halogen;
R13 independently is C(0)-OR"; amino; amide; guanidinyl; imidazolyl; indolyl; sulfoxide; phosphoryl; Q-C3 alkylarnino; Cj-Q, alkyld^amino; Cj-C6 alkenylamino; hydroxy; thiol; C,-C3 alkoxy; Q-Q alkthiol; (CH^COOR"; Q-Q
20 alkyl which is unsubstituted or substituted with OH, halogen, SH, NHj, phenyl, hydroxyphenyl or Q-C,,, alkoxyphenyl; C2-C6 alkenyl which is unsubstituted or substituted with OH, halogen, SH, NH,, phenyl, hydroxyphenyl or Q-Qo alkoxyphenyl; and C6-Cu aryl which is unsubstituted or substituted with OH, halogen, SH, NHj, phenyl, hydroxyphenyl or Q-Q,, alkoxyphenyl; and
25 R14 is H or C,-Q, alkyl or CrC9 alkyl independently substituted with OH,
halogen, COOR", O or N; Cg-Cg aryl; Q-Cg aryl which is independently substituted with OH, halogen, COOR", O or N; or C3-C6 arylalkyl which is independently substituted with OH, halogen, COOR", O or N.
Rreferably, R" is Q-Q alkyl, most preferably isopropyl, R13 is the side chain
30 of a naturally occurring amino add, n = 1, Ru is H and RM is H. In the compound of structure (2), the invention indudes metabolites in which the phenoxy and isopropyl esters have been hydrolyzed to -OH. Similarly, the de-esterified enriched phosphonoamidate metabolites of compounds (5a), 5(b) and (6) are induded within the scope of this invention.
35 Aryl and "O" or "N" substitution are defined in column 16, lines 42-58, of
WO 02/08241 PCT/US01/23104
5 United States Patent No. 5,798,340.
Typically, the amino acids are in the natural or I amino adds. Suitable specific examples are set forth in U. S. Patent No. 5,798,340, for instance Table 4 and col. 8-10 therein.
Alkyl as used herein, unless stated to the contrary, is a normal, secondary,
10 tertiary or cyclic hydrocarbon. Unless stated to the contrary alkyl is Q-Qj.
Examples are -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH2CH2CH3), -CH2CH(CH3)2, -CH(CH3)CH2CH3, -C(CH3>3, -CH2CH2CH2CH2CH3, -CH(CH3)CH2CH2CH3/ -CH(CH2CH3)2,
20 contain at least one double or triple bond, respectively.
Where enol or keto groups are disclosed, the corresponding tautomers are to be construed as taught as well.
The prodrug compounds of this invention are provided in the form of free base or the various salts enumerated in U. S. Patent No. 5,798,340, and are
25 formulated with pharmaceutically acceptable excipients or solvating diluents for use as pharmaceutical products also as set forth in U. S. Patent No. 5,798,340. These prodrugs have the antiviral and utilities already established for the parent drugs (see U.'S. Patent 5,798,340 and other citations relating to the methoxyphosphonate nucleotide analogues). It will be understood that the diastereomer of structure (4)
30 at least is useful as an intermediate in the chemical production of the parent drug by hydrolysis in vitro, regardless of its relatively unselective character as revealed in the studies herein.
The invention will be more fully understood by reference to the following examples:
WO 02/08241 PCT/US01/23104
5 Example la
Adenine to PMEA using Magnesium Isopropoxide. To a suspension of adenine (16.8g, 0.124 mol) in DMF (41.9 ml) was added ethylene carbonate (12.1g, 0.137 10 mol) and sodium hydroxide (.100g, 0.0025 mol). The mixture was heated at 130°C overnight. The reaction was cooled to below 50°C and toluene (62.1 ml) was added. The slurry was further cooled to 5°C for 2 hours, filtered, and rinsed with toluene (2x). The wet solid was dried in vacuo at 65°C to yield 20.0g (90%) of 9-(2-hydroxyethyl)adenine as an off-white solid. Mp: 238-240°C.
N 'i' ana
9-(2-Hydroxyethyl)adenine (HEA) (20.0g, 0.112 mol) was suspended in DMF (125
20 ml) and heated to 80°C. Magnesium isopropoxide (11.2g, 0.0784 mol), or
alternatively magnesium t-butoxide, was added to the mixture followed by diethyl
p-toluenesulfonyloxymethylphosphonate (66.0g, 0.162 mol) over one hour. The
mixture was stirred at 80°C for 7 hours. 30 ml of volatiles were removed via
vacuum distillation and the reaction was recharged with 30 ml of fresh DMF. After
25 cooling to room temperature, bromotrimethylsilane (69.6g, 0.450 mol) was added
and the mixture heated to 80°C for 6 hours. The reaction was concentrated to yield
a thick gum. The gum was dissolved into 360 ml water, extracted with 120 ml
dichloromethane, adjusted to pH 3.2 with sodium hydroxide, and the resulting
slurry stirred at room temperature overnight. The slurry, was cooled to 4°C for one
30 hour. The solids were isolated by filtration, washed with water (2x), and dried in
rv, x/ uau jy^Jiu*
oaeuo at 56°C to yield 2Ug (65.4%) of 9~[2-(phosphonomemoxy)emyI3aderune (PMEA) as a white solid. Mp: > 200°C dec. aH NMR (Dp) • 3.49 (t, 2H); 3.94 (t, 2H); 4.39(t, 2H); 8.13 (s, 1H); 8.22 (s, 1H).
Adenine to PMPA using Magnesium t-Butoxide. To a suspension of adenine (40g, 0.296 mol) in DMF (41.9 ml) was added (^-propylene carbonate (34.5g, 0.338 mo]) and sodium hydroxide (.480g, 0.012 mol). The mixture was heated at 130°C overnight. The reaction was cooled to 100°C and toluene (138 ml) was added followed by methanesulfonic acid (4.7g, 0.049 mol) while mamtaining the reaction temperature between 100~110°C. Additional toluene (114 ml) was added to create a homogeneous solution. The solution was cooled to 3°C over 7 hours and then held at 3°C for one hour. The resulting solid was isolated by filtration and rinsed with acetone (2x). The wet solid was dried in vacuo at 80°C to yield 42.6g (75%) of (R)-9-[2-(hydroxy)propyl]adenine (HPA) as an off-white solid. Mp: 188-190°C.
> + TwJcOEfti-ttSB*.
Cil)-9-[2-(hydroxy)propyl]adenme (HPA) (20.0g, 0.104 mol) was suspended in DMF (44.5 ml) and heated to 65°C. Magnesium t-butoxide (14.2g, 0.083 mol), or alternatively magnesium isopropoxide, was added to me mixture over one hour followed by diethyl p-toluenesulfonyloxymethylphosphonate (66.0g, 0.205 mol)
WO 02/08241 PCT/US01/23104
5 over two hours while the temperature was kept at78°C. The mixture was stirred at 75°Cfor 4 hours. After cooling to below50oC,bromotrimemylsilane (73.9g> 0.478 mol) was added and the mixture heated to 77°C for 3 hours. When complete^ the reaction, was heated to 80°C and volatiles were removed via atmospheric distillation. The residue was dissolved into water (120 ml) at 50°C and then
10 extracted with ethyl acetate (101 ml). The pH of the aqueous phase was adjusted to pH 1.1 with sodium hydroxide, seeded with authentic ORJ-PMPA, and the pH of the aqueous layer was readjusted to pH 2.1 with sodium hydroxide. The resulting slurry was stirred at room temperature overnight. The slurry was cooled to 4°C for three hours. The solid was isolated by filtration, washed with water (60 ml), and
15 dried in vacuo at 50°C to yield 18.9g (63.5%) of crude(IO-9-[2-
(phosphonomethoxy)propyl]adenine (PMPA) as an off-white solid.
The crude(R)-9-[2-(phosphonomethoxy)propyl]adenine was heated at reflux in water (255 ml) until aU solids dissolved. The solution was cooled to room 20 temperature over 4 hours. The resulting slurry was cooled at 4°C for three hours. The solid was isolated by filtration, washed with water (56 ml) and acetone (56 ml), and dried in vacuo at 50°C to yield 15.0g (50.4%) of CR>9-[2-(phosphonomethoxy)propyl]adenine (PMPA) as a white solid. Mp: 278-280°C
Example 2 Preparation of GS-7171 (ED
JH, OH K,Ha
W OH Et3N NMP L/ OH ^-^
W OH w „^ • W AH.
u: > ^J JO HO AhH ^w ° Q o
«rv\ n O °
CSjCN = ^'V°V
V O x
JC V- A/ «J»3U JL/X»Jil/*#
5 A glassrlined reactor was charged with anhydrous PMPA, (I) (14.6 kg, 50.8 mol), phenol (9.6 kg, 102 mo]), and l-methyl-2-pyrroHdinone (39 kg). The mixture was heated to 85°Cand_triethylarnine(6.3kg,.623 mol) added. A solution of1,3-dicydohexylcarbodiimide (17.1 kg, 82.9 mol) in lr-methyl-2-pyrrolidinone (1.6 kg) was then added over 6 hours at 100°G Heating was continued for 16 hours. The
10 reaction was cooled to 45°C, water (29 kg) added, and cooled to 25°C. Solids were removed from the reaction by filtration and rinsed with water (15.3 kg). The combined filtrate and rinse was concentrated to a tan slurry under reduced pressure, water (24.6 kg) added, and adjusted to pH = 11 with NaOH (25% in water). Fines were removed by filtration through diatomaceous earth (2 kg) •
15 followed by a water (4.4 kg) rinse. The combined filtrate and rinse was extracted with ethyl acetate (28 kg). The aqueous solution was adjusted to pH = 3.1 with HC1 (37% in water) (4 kg). Crude II was isolated by filtration and washed with methanol (12.7 kg). The crude II wet cake was slurried in methanol (58 kg). Solids were isolated by filtration, washed with methanol (8.5 kg), and dried under
20 reduced pressure to yield 9.33 kg II as a white powder: 'H NMR (D20) 51.2 (d, 3H), 3.45 (q, 2H), 3.7 (q, 2H), 4 (m, 2H), 4.2 (q, 2H), 4.35 (dd, 2H), 6.6 (d, 2H), 7 (t, 1H), 7.15 (t, 2H), 8.15 (s, 1H), 8.2 (s, 1H); 31P NMR (D20) 515.0 (decoupled).
GS-7171 (HI). (Scheme 1) A glass-lined reactor was charged with monophenyl 25 PMPA, (II), (9.12 kg, 25.1 mol) and acetonitrile (30.7 kg). Thionyl chloride (6.57 kg, 56.7 mol) was added below 50°C The mixture was heated at 75?C until solids dissolved. Reaction temperature was increased to 80°C and volatiles (11.4 kg) collected by atmospheric distillation under nitrogen. The pot residue was cooled to 25°C, dichloromethane (41 kg) added, and cooled to -29°C. A solution of (L)- . 30 alanine isopropyl ester (7.1 kg, 54.4 mol) in dichloromethane (36 kg) was added over 60 minutes at -18°C followed by triemylamine (7.66 kg, 75.7 moD over 30 minutes at -18 to ~11°C. The reaction mixture was warmed to room temperature and washed five times with sodium dihydrogenphosphate solution (10% in water, 15.7 kg each wash). The organic solution Was dried with anhydrous sodium sulfate 35 (182 kg), filtered, rinsed with dichloromethane (28 kg), and concentrated to an oil
5 under reduced pressure. Acetone (20 kg) was charged to the oil and the mixture concentrated under reduced pressure. Acetone (18.8 kg) was charged to the resulting oiL Half the product solution was purified by chromatography over a 38 x 38 cm bed of 22 kg. silica gel 60,230 to 400 mesh. The column was eluted with 480 kg acetone. The purification.was repeated on the second half of the oil using fresh
10 silica gel and acetone. Clean product bearing fractions were concentrated under reduced pressure to an oiL Acetonitrile (19.6 kg) was charged to the oil and the mixture concentrated under reduced pressure. Acetonitrile (66.4 kg) was charged and the solution chilled to 0 to -5°C for 16 hours. Solids were removed by filtration and the filtrate concentrated under reduced pressure to 5.6 kg III as a dark oil: 1H
15 NMR (CDClg) 81.1 (m 12H), 3.7 (m, IH), 4.0 (m, 5H), 4.2 (m, IH), 5.0 (m, IH), 6.2 (s, 2H), 7.05 (m, 5H), 8.0 (s, IH), 8.25 (d, IH); 31P NMR (CDCI3) 8 21.0,22.5 (decoupled).
Alternate Method for GS-7171(HI)
soci2 KJ TlTX Q ^
(anhydrous) O O
HOv /=/ OH
W OH ^ O CH2C12. >
0'% _/V GS"71
5 Monophenyl TMTA (II). A round-bottom flask with reflux condenser and
nitrogen inlet was placed in a 70°C oil bath. The flask was charged with anhydrous PMPA (I) (19.2 g, 67 mmol), M,N-dimemylforrriamide (0.29 g, 3.3 mmol), and tetramethylene sulfone (40 mL). Thionyl chloride (14.2 g, 119 mmol) was added over 4 hours. Heating was increased to 100°C over the same time. A homogeneous
10 solution resulted. Phenoxytrimethylsilane (11.7 g, 70 mmol) was added to the solution over 5 minutes. Heating in the 100°C oil bath continued for two hours more. The reaction was poured into rapidly stirring acetone (400 mL) with cooling at 0°C. Solids were isolated by filtration, dried under reduced pressure, and dissolved in methanol (75 mL). The solution pH was adjusted to 3.0 with
15 potassium hydroxide solution (45% aq.) with cooling in ice/water. The resulting solids were isolated by filtration, rinsed with methanol, and dried under reduced pressure to 20.4 g II (Scheme 2) as a white powder.
GS-7171 (III). Monophenyl PMPA (II) (3 g, 8.3 mmol), tetramethylene sulfone (5 20 mL), and N,N-dimethylformamide (1 drop) were combined in a round bottom flask in a 40°C oil bath. Thionyl chloride (1.96 g, 16.5 mmol) was added. After 20 minutes the clear solution was removed from heat, diluted with dichloromethane (10 ml)> and added to a solution of (L)-alanine isopropyl ester (5g, 33 mmol) and diisopropylemylamine (5.33 g, 41 mmol) in dichloromethane (20 mL) at-10°C. The 25 reaction mixture was warmed to room temperature and washed three times with sodium dihydrogenphosphate solution (10% aq., 10 mL each wash). The organic solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to a oiL The oil was combined with fumaric acid (0.77g, 6.6 mmol) and acetonitrile (40 mL) and heated to reflux to give a homogeneous 30 solution. The solution was cooled in an ice bath and solids isolated by filtration. The solid GS-7171 fumarate salt was dried under reduced pressure to 3.7 g. The salt (3.16 g, 5.3 mmol) was suspended in dichloromethane (30 mL) and stirred with potassium carbonate solution (5 mL, 2.5 M in water) until the solid dissolved. The organic layer Was isolated, then washed with water (5 mL), dried over anhydrous
5 sodium sulfate, and concentrated under reduced pressure to afford 2.4 g III as a tan foam.
Example 3 A. Diastereomer Separation by Batch Elution Chromatography
. The diastereomers of GS-7171 (III) were resolved by batch elution chromatography using a commercially available Chiralpak AS, 20 um, 21 x 250 mm semi-preparative HPLC column with a Chiralpak AS, 20 um, 21 x 50 mm guard column. Chiralpak® AS is a proprietary packing material manufactured by Diacel
15 and sold in North America by Chiral Technologies, Inc. (U. S. Patent Nos. 5,202,433, RE 35,919,5434,298,5,434,299 and 5,498,752). Chiralpak AS is a chiral stationary phase (CSP) comprised of amylosetris[(S)-o&-methylbenzyl carbamate] coated onto a silica gel support.
The GS-7171 diastereomeric mixture was dissolved in mobile phase, and
20 approximately 1 g aliquots of GS-7171 were pumped onto the chromatographic system. The undesired diastereomer, designated GS-7339, was the first major broad (approx. 15 min. duration) peak to elute from the column. When the GS-7339 peak had finished eluting, the mobile phase was immediately switched to 100% methyl alcohol, which caused the desired diastereomer, designated GS-7340 (IV),
25 to elute as a sharp peak from the column with the methyl alcohol solvent front The methyl alcohol was used to reduce the over-all cycle time. After the first couple of injections, both diastereomers were collected as a single large fractions containing one of the purified diastereomers (>99.0% single diastereomer). The mobile phase solvents were removed in vacuo to yield the purified diastereomer as a friable foam.
30 About 95% of the starting GS-7171 mass was recovered in the two
diastereomer fractions. The GS-7340 fraction comprised about 50% of the total recovered mass.
5 The chromatographic conditions were as follows:
_ Mobile Phase(Initial)
10 Run Time
Detection Temperature Elution Profile
GS-7171 - Acetonitrile: Isopropyl Alcohol (90:10)
100% Methyl Alcohol
About 45 minute
UV at 275 run
GS-7339 (diastereomer B)
GS-7340 (diastereomer A; (XV))
B. Diastereomer Separation of GS-7171 by SMB Chromatography
For a general description of simulated moving bed (SMB) chromatography, see Strube et al, "Organic Process Research and Development" 2:305-319 (1998).
GS-7340 (IV). GS-7171 (HI), 2.8 kg, was purified by simulated moving bed chromatography over 10 cm by 5 cm beds of packing (Chiral Technologies Inc., 20 micron Chiralpak AS coated on silica gel) (1.2 kg). The columns were eluted with 30% methanol in acetonitrile. Product bearing fractions were concentrated to a
25 solution of IV in acetonitrile (2.48 kg). The solution solidified to a crystalline mass wet with acetonitrile on standing. The crystalline mass was dried under reduced pressure to a tan crystalline powder, 1.301 kg IV, 98.7% diastereomeric purity: mp 117 - 120°C; 'H NMR (CDCl,) 81.15 (m 12H), 3;7 (t, IH), 4.0 (m, 5H), 4.2 (dd, IH), 5.0 (m, IH), 6.05 (s, 2H), 7.1 (m, 5H), 8.0 (s, IH); 8.2 (s, 1H),: MP NMR (CDCI3) 5 21.0
C. Diastereomer Separation by C18RP-HPLC
GS-7171 (in) was chromatographed by reverse phase HPLC to separate the diastereomers using the following summary protocol.
Chromatgraphic column : Phenomenex Lina ™ C18(2),5 um, 100 A pore
Size, (Phenomenex, Torrance, CA), or equivalent
Guard column : Pellicular C18 (Alltech, Deerfield, IL), or
A — 0.02% (85%) H3PO4 in water-: acetonitrile
B — 0.02% (85%) H3PO4 in water: acetonitrile
Mobile Phase Gradient
Time % Mobile Phase A % Mobile Phase B
0 100. 0
5 100 0
7 70 30
32 70 30
40 0 100
50 0 100
Run Time: Equilibration Delay: Flow Rate: Temperature: Detection: Sample Solution: Retention Times:
10 min at 100% mobile phase A
20 mM sodium phosphate buffer, pH 6
GS-7339, about 25 minutes
GS-7340, about 27 minutes
D. Diastereomer Separation by Crystallization
30 GS-7340 (IV). A solution of GS-7171 (HI) in acetonitrile was concentrated to an amber foam (14.9g) under reduced pressure. The foam was. dissolved in acetonitrile (20 mL) and seeded with a crystal of IV. The mixture was stirred overnight, cooled to 5°C, and solids isolated by filtration. The solids were dried to 2.3 g IV as white crystals, 98% diastereomeiic purity (31P NMR): 'H NMR (CDCy 8
35 1.15 (m 12H), 3.7 (t, IH), 3.95 (m, 2H), 4.05 (m, 2H), 4.2 (m, 2H), 5.0 (m, IH), 6.4 (s, 2H), 7.1 (m, 5H), 8.0 (s, IH), 8.2 (s, IH); MP NMR (CDCl,) 519.5 (decoupled). X-ray crystal analysis of a single crystal selected from this product yielded the following data:
Crystal Color/ Habit colorless, column
Crystal Diimnsions 0.25 X 0.12X0.08 mm
Crystal System orthorhombic
Lattice Type Primitive
Lattice Parameters a = 8.352(1) A
b = 15.574(2) A
c= 18.253(2) A
V = 2374.2(5) A3
Space Group PZ^ (#19)
Z value 4
EU 1.333 g/cm3
Preparation of Fumarate Salt of GS-7340
GS-7340-02 (V). (Scheme 1) A glass-lined reactor was charged with GS-7340 (TV), 25 (1.294 kg, 2.71 mol), fumaric acid (284 g, 2.44 mol), and acetonitrile (24.6 kg). The mixture Was heated to reflux to dissolve the solids, filtered while hot arid cooled to 5°C for 16 hours. The product was isolated by filtration, rinsed with acetonitrile (9.2 kg), and dried to 1329 g (V) as a white powder: mp 119.7 - 121.1°C; M™ -41.7° (c 1.0, acetic acid).
W . NH
A 5 L round bottom flask was charged with monophenyl PMPA, (II), (200 g, 0.55 mol) and acetonitrile (0.629 kg). Thionyl chloride (0.144 kg, 1.21 mol) was added
20 below 27°C. The mixture was heated at 70°C until solids dissolved. Volatiles (0.45 L) were removed by atmospheric distillation under nitrogen. The pot residue was cooled to 25°C, dichloromethane (1.6 kg) was added and the mixture was cooled to -20°C. A solution of (L)-a aminobutyric acid ethyl ester (0.144 kg, 1.1 mol) in dichloromethane (1.33 kg) was added over 18 minutes at -20 to -10°C followed by
25 triethylamine (0.17 kg, 1.65 mol) over 15 minutes at -8 to -15°C The reaction mixture was warmed to room temperature and washed four times with sodium dihydrogenphosphate solution (10% aq., 0.3 L each wash). The organic solution was dried with anhydrous Bodium sulfate (0.5 kg) and filtered. The solids were rinsed with dichloromethane (0.6 kg) and the combined filtrate and rinse was
30 concentrated to an oil under reduced pressure. The oil was purified by
chromatography over a 15 x 13 cm bed of 1.2 kg silica gel 60,230 to 400 mesh. The column was eluted with a gradient of dichloromethane and methanol. Product bearing fractions were concentrated under reduced pressure to afford 211 g VI (Scheme 3) as a tan foam.
WO 02/0824I PCT/US01/23104
5 Example 5a
Diastereomer Separation of GS-7120 by Batch Elutibn Chromatography
The diastereomeric mixture was purified using the conditions described for GS-7171 in Example 3A except for the following:
Mobile Phase (Initial) : GS-7120 - Acetonitrile: Isopropyl Alcohol (98:2)
(Final) : 100% Methyl Alcohol
Elution Profile : GS-7341 (diastereomer B)
: GS-7342 (diastereomer A)
Diastereomer Separation of GS-7120 by Crystallization
A1L round bottom flask was charged with monophenyl PMPA, (II), (50 g, 0.137 mol) and acetonitrile (0.2 L). Thionyl chloride (0.036 kg, 0.303 mol) was added with a 10°C exotherm. The inixture was heated to reflux until solids dissolved. Volatiles (0.1 L) were removed by atmospheric distillation under nitrogen. The pot residue
25 was cooled to 25°C, dichloromethane (0.2 kg) was added, and the mixture was cooled to-20°C. A solution of (L)~a aminobutyric acid ethyl ester (0.036 kg, 0.275 mol) in dichloromethane (0.67 kg) was added over 30 minutes at -20 to -8°C followed by triethylamine (0.042 kg, 0.41 mol) over 10 minutes at up to -6°C. The reaction mixture was warmed to room temperature and washed four times with
30 sodium dihydrogenphosphate solution (10% aq., 0.075 L each wash). The organic solution was dried with anhydrous sodium sulfate (0.1 kg) and filtered. The solids were rinsed with ethyl acetate (0.25 L, and the. combined nitrate and rinse was concentrated to an oil under reduced pressure. The oil was diluted with ethyl acetate (0.25 L), seeded, stirred overnight, and chilled to - 15°C. The solids were
35 isolated by filtration and dried under reduced pressure to afford 17.7 g of GS-7342 (Table 5) as a tan powder: JH NMR (CDCI3) 8 0.95 (t, 3H), 1.3 (m, 6H), 1.7, (m, 2H),
WO 02/08241 PCT/US01/23104
5 3J^2H),L%(^Wr4iAid^-tH);:5S (s,2H),7.1 (m,5H),8.0 (s, lH)/8.4 (s, lHY; 3iP NMR (CDCL) 8 21 (decoupled).
Example 7 Diastereomer Separation of GS-7Q97 10
The diastereomeric mixture was purified using the conditions described for GS-7171 (Example 3A) except for the following:
Mobile Phase (Initial) : GS-7120 - Acetonitrile: Isopropyl Alcohol (95:5)
15 (Final) : 100% Methyl Alcohol
Elution Profile : GS-7115 (diastereomer B)
: GS-7114 (diastereomer A)
20 Example 8
Alternative Procedure for Preparation of GS-7097
GS-7097: Phenyl PMPA, Ethyl L-Alanyl Amidate. Phenyl PMPA (15.0 g, 41.3 • mmol), L-alanine ethyl ester hydrochloride (12.6 g, 83 mmol) and triemylarnine
25 (11.5 mL, 83 mmol) were slurried together in 500 mL pyridine under dry N2. This
suspension was combined with a solution of triphenylphosphine (37.9 g, 145 mmol), Aldrithiol 2 (2,2,-dipyridyl disulfide) (31.8 g, 145 mmol), and 120 mL pyridine. The mixture was heated at an internal temperature of 57°C for 15 hours. The complete reaction was concentrated under vacuum to a yellow paste, 100 g. 30 The paste was purified by column chromatography over a 25 x 11 cm bed of 1.1 kg silica gel 60,230 to 400 mesh. The column was eluted with 8 liters of 2% methanol in dichloromethane followed by a linear gradient over a course of 26 liters eluent up to a final composition of 13% methanol. Clean product bearing fractions were concentrated to yield 12.4 g crude (5), 65% theory. This material was contaminated
35 with about 15% (weight) trtemylamine hydrochloride by :H NMR. The
(juxirautrdnation was removed by dissolving the product in 350 mL ethyl acetate, extracting with 20 mL water, drying the organic solution over anhydrous sodium
5 sulfate, and concentrating to yield 11.1 grpure GS-7097 as a white solid, 58% yield. The process also is employedto synthesize the diastereomeric rnixture of GS-7003a and GS-7003b (the phenylalanyl amidate) and the rnixture GS-7119 and GS-7335 (the glycyl amidate). These diastereomers are separated using a batch elution procedure such as shown in Example 3A, 6 and 7. 10
In Vitro Studies of Prodrug Diastereomers
The in vitro anti-HIV-1 activity and cytotoxicity in MT-2 cells and stability in 15 human plasma and MT-2 cell extracts of GS-7340 (freebase) and tenofovir
disoproxil fumarate (TDF), are shown in Table 1. GS-7340 shows a 10-fold increase in antiviral activity relative to TDF and a 200-fold increase in plasma stability. This greater plasma stability is expected to result in higher circulating levels of GS-7340 than TDF after oral administration. 20
Table 1. In Vitro Activity and Stability
HIV-1 Activity Cyto toxicity Stability T1/2 (mi m).
ICSOJIM CCsopM Human Plasma MT-2 Cell Extract (P/MT-2)
GS 7340 0.005 >40 90.0 28.3 3.2
TDF 0.05 70 0.41 70.7 0.006
Tenofovir 5 6000 — ~ —
25 In order to estimate the relative intracellular PMPA resulting from the
intracellular metabolism of TDF as compared to that from GS-7340, both prodrugs and PMPA were radiolabeled and spiked into intact human whole blood at equimolar concentrations. After 1 hour, plasma, red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) were isolated and analyzed by HPLC
30 with radiometric detection. The results are shown in Table 2.
5 After 1hour,GS-7340 results inlOx and30x thetotal intracellular
concentration of PMPA, species in PBMCs as compared to TDF and PMPA, respectively. In plasma after 1 hour, 84% of the radioactiyiiy is due to intact GS-7340,,whereas no TDF is detected at 1 hour. Smce no mtart TDF is detected in plasma, the lOx difference at 1 hour between TDF and GS-7340 is the minirnum
10 difference expected in vivo. The PIPLC chromatogram for all three compounds in PBMCs is shown in Figure 1.
Table 2. PMPA Metabolites in Plasma, PBMCs and RBCs After 1 h Incubation of PMPA Prodrugs or PMPA in Human Blood.
Compound Matrix Total C-14Recovered,UE-eq Metabolites (% of Total Peak Area)
PMPA% PMPAp,% PMPApp,% MetX,% MetY,% GS 7340,%
GS-7340 (60 Mg-e
GS-4331(TDF)(60 ng-eq) Plasma/FPPBMC RBC/EP 48.10.133 10^ 1150 93 25 7.0 18 89 7 -
PMPA PMPAp PMPApp
PMPA (60 |ig-cq) Plasma/FP PBMC RBC/FP 55.7 0.033 3.72 100 8674 14' 10 16
Figure 1. HPLC/O-14 Traces of PBMC Extracts from Hainan Blood Incubated for 1 h at 37°C with TDE^GS^7340 or EMPAv
aocri TDF/PBMC wo-
MetX PMPAP PMPApp
Met X and Met Y (metabolites X and Y) are shown in Table 5. Lower case "p" designates phosphorylation. These results were obtained after 1 hour in human 15 blood. With increasing time, the in vitro differences are expected to increase, since 84% of GS-7340 is still intact in plasma after one hour. Because intact GS-7340 is present in plasma after oral administration, the relative clinical efficacy should be related to the IC^ values seen in vitro.
In Table 3 below, IQQ values of tenofovir, TDF, GS-7340, several nucleosides 20 and the-protease inhibitor nelfinivir are listed. As shown, nelfinavir and GS-7340 are 2-3 orders of magnitude more potent than all other nucleotides or nucleosides.
Table 3. In Vitro Anti-HIV-1 Activities-of antiretrorinal compounds
Compound r ;";. iCsoOiM)
Adefovir(PMEA) 13.4 ±4.21
Tenofovir (PMPA) 6.3±3.31'
AZT 0.17 ±0.08*
d4T 8 ±2.5i
Nelfinavir 0.006 iO.0021
GS 7340 0.005
1. A. S. Mulato and J. M. Chenington, Antiviral Research 36,91 (1997)
10 Additional studies of the in vitro cell culture anti-HIV-1 activity and CCS0 of
separated diastereomers of this invention were conducted and the results tabulated below.
5 Table 4. Effect of Diastereomer
Compound Diastereomer IC10 (uM) . Fold change A/B activity CQCuM)
PMPA - ■: 5 '-:- lx P* 6000
Ala-mefhylester . Mixture 1:1 0.025 200x 20x 80
GS-6957a A 0.0075 670x
GS-6957b 0.15 33x
Phe-methylester Mixture 1:1 0.03 170x . lOx 60
GS-7003a A . 0.01 500x
GS-7003b B 0.1 50x
Gly-ethylester Mixture 1:1 0.5. lOx 20x
GS-7119 A 0.05 lOOx >100
GS-7335 B 1.0 5x
Ala-isopropyl Mixture 1:1 0.01 500x 12x
GS-7340 A 0.005 • l,000x 40
GS-7339 B 0.06 83x >100
ABA-ethyl Mixture 1:1 0.008 625x 7.5x >100
GS-7342 A 0.004 % l,250x
GS-7341 B 0.03 170x
Ala-ethyl Mixture 1:1 0.02 250x lOx 60
GS-7114 A 0.005 100x
GS-7115 B 0.05 ' l00x
Assay reference: Arimilli, MN, etaL, (1997) Synthesis, in vitro biological evaluation and oral bioavailability of 9-[2-(phosphonomethoxy)propyl]adenrne 10 (PMPA) prodrugs. Antiviral Chemistry and Chemotherapy 8(6)557-564.
"Phe-metliylester" is the mefnylphenylalaninyl monoamidate, phenyl
monoester of tenofovir; ugly-methylester" is the methylglycyl monoamidate, phenyl
monoester of tenofovir.
15 In each instance above, isomer A is believed to have the same absolute
stereochemistry as GS-7340 (S), and isomer B is believed to have the same absolute stereochemistry that of GS-7339.
The in vitro metabolism arid stability of separated diastereomers were determined in PLCE, MT-2 extract and human plasma. A biologicalsample listed below, 80 JJL, was transferred into a screw-capped centrifuge tube and incubated at 37°C for 5 min. A solution containing 02 mg/mL of the test compound in a suitable buffer, 20 JJL, was added to the biological sample and mixed. The reaction mixture, 20 uL, was immediately sampled and mixed with 60 uL of methanol containing 0.015 mg/mL of 2-hydroxymethylnaphthalene as an internal standard for HPLC analysis. The sample was taken as the time-zero sample. Then, at specific time points, the reaction mixture, 20 uL, was sampled and mixed with 60 pL of methanol containing the internal standard. The mixture thus obtained was centrifuged at 15,000 G for 5 min and the supernatant was analyzed with HPLC under the conditions described below.
The biological samples evaluated are as follows.
(1) PLCE (porcine liver carboxyesterase from Sigma, 160 u/mg protein, 21 mg protein/mL) diluted 20 fold with PBS (phosphated-buffered saline).
(2) MT-2 cell extract was prepared from MT-2 cells according to the published procedure [A. Pompon, I. Lefebvre, J.-L. Imbach, S. Kahn, and D. Farquhar, "Antiviral Chemistry & Chemotherapy", 5:91-98 (1994)] except for using HEPES buffer described below as the medium.
(3) Human plasma (pooled normal human plasma from George King Biomedical Systems, Inc.)
The buffer systems used in the studies are as follows.
In the study for PLCE, the test compound was dissolved in PBS. PBS (phosphate-buffered saline, Sigma) contains 0.01 M phosphate, 0.0027 M potassium chloride, and 0.137 M sodium chloride. pH 7.4 at 37°C.
In the study for MT-2 cell extracts, the test compound was dissolved in HEPES buffer. HEPES buffer contains 0.010 M HEPES, 0.05 M potassium chloride, 0.005 M magnesium chloride, and 0.005 M rfl-dithiothreitoL pH 7.4 at 37°C.
5 In the study for humanplasrna, the-test compound was dissolved mTBS. TBS (tris-bufferedsaline, Sigma) contains 6.05 MTris, 0.0027 M potassium chloride 0.138 M sodium chloride.; pH7.5: at 37°C.
The HPLC analysis was carried out under the following conditions.
Detection: Flow Rate: Run Time: Injection Volume:
Zorbax Rx-Qj, 4.6 x 250 mm, 5 \L
(MAC-MOD Analytical, Inc. Chadds Ford, PA)
UV at 260 run
Column Temperature: Ambient temperature
Mobile Phase A: Mobile Phase B:
50 mM potassium phosphate (pH 6.0)/CH3CN = 95/5 (v/v) 50 mM Potassium phosphate (pH 6.0)/CH3CN = 50/50 (v/v)
100% Mobile Phase A 100% Mobile Phase B 100% Mobile Phase B
The results are shown below in Table 5 (also including selected ICS0 data from Table 4).
Table 5. In Vitro Metabolism of Isomers A and B of PMPA monoamidate at 37°C '
No PMPA monoamidate structure V HIVICW (MM) PLCE: hydrolysis rate and product. MT-2 extract hydrolysis rate and product -:: Human ' Plasma Stability (HP):
1 A f 9H3k^O^P.-NH-CHCOOEt1 OPh Isomer A GS7114 0.005 t1/2 = 2.9min Met. X& PMPA tw = 2.9mln Met. X& PMPA V= 148 mln Met.Y
2 A 9 ?H3 . k^OvP,-NH-CHCOOEt1 OPh Isomer B GS7115 0.05 tw = 8.0mln Met. X& PMPA : t,fla l5Q.6min Met. X& PMPA t1/2 3 A. ? CH3k^Ov^R-NH-CHCOOiPr%. OPh Isomer A GS7340 , 0.005 t1/2 = 3.3mln ■ Met. X & PMPA \yn- 28.3 mln Met. X& PMPAj t1fi = 90.0 mln Met.Y
4 A 9 CH3V°vp\NH-CHC00iPr 1 OPh Isomer B GS7339 0.06 tw =10.1 mln Met. X& PMPA tw> 1000 mln t1/2 = 231 mln Met.Y
5 A 9 .CH2CH3 k^OvRrNH-CHCOOai OPh Isomer A GS7342 0.004 t1/a B 3.9 min Met. X tw ■ 49.2 min Met. X& PMPA V= 103 min Met.Y
6 A 9 CH2CH3 VOvR-NH-CHCOOEt1 OPh Isomer B GS7341 0.03 tw= 11.3 min Met.X tw> 1000 min tw - 257 mln Met.Y
7 A 9 9S^CX PrOCH2OCOiPr i \ 9 GS4331 0CH20G0IPr 0.05 tI/2
A ° ?13
A ? T
WO 02/08241 PCT7US01/23104
5 Example 10
Plasma and PBMC Exposures FoEowmg Oral Adiruiustraiion Of Prodrug Diastereomers to Beagle Dogs
Thepharmacokineti.es of GS 7340 were studied in dogs after, oral 10 adrninistration of a 10 mg-eq/kg dose.
Formulations. The prodrugs were formulated as solutions in 50 mM citric acid within 0.5 hour prior to dose. All compounds used in the studies were synthesized by Gilead Sciences. The following lots were used:
GSI Amidate Amino acid AA Ester Diastereoisomer Lot Number
GS-7340-2 Alanine i-Propyl Isomer A 1504-187-19
GS-7339 Alanine i-Propyl Isomer B 1509-185-31
GS7114 Alanine' Ethyl Isomer A 1509-181-26
GS7115 Alanine Ethyl Isomer B 1509-181-22
GS7119 Glycine Ethyl Isomer A 1428-163-28
GS7342 oc-Aminobutyric Acid Ethyl Isomer A 1509-191-12
GS7341 o^Armnobutyric Acid Ethyl Isomer B 1509-191-7
Dose Administration and Sample Collection. The inrlife phase of this study was conducted inaccordance with the recommendations of the "Guide for the Careand Use of Laboratory Animals" (National Institutes of Healthpublication 86-23) and
20 was approved by an Institutional Animal Care and Use Committee. Fasted male beagle dogs (10 ± 2 kg) were used for the studies. Each drug was administered as a single dose by oral gavage (1,5-2 ml/kg). The dose was 10 mg-equivalent of PMPA/kg. For PBMCs, blood samples were collected at 0 (pre-dose), 2,8, and 24 h. post-dose. For plasma, blood samples were collected at 0 (pre-dose), 5,15, and 30
25 min, and 1,2,3,4,6,8,12 and 24h post-dose. Blood (1.0 ml) was processed
immediatelyfor plasma by centrifugation at 2,000 rpm for 10 min. Plasma samples were frozen and maintained at 70°C until analyzed.
Peripheral Blood Mononuclear Cell (PBMC) preparation. Whole blood (8 ml) 30 drawn at specified time points was mixed in equal proportion with phosphate buffered saline (PBS), layered onto 15 ml of Ficoll-Paque solution (Pharmacia Biotech,) and centrifuged at 400 x g for 40 min. PBMC layer was removed and
5 washed once with PBS. Formed PMBC pellet was reconstituted in 0.5 ml of PBS, cells were resuspended, counted using hemocytometer and nmiritained at 70°C until analyzed. The number of cells multiplied by the mean single-cell volume was used in calculation of intracellular concentrations. A reported value of 200 femtoliters/cell was used as the resting PBMC volume (B, L. Robins, K.V. Srinivas,
10 C. Kim, N. Bischofberger, and A. Fridland, Antimicrob. Agents Chemother. 42,612 (1998).
Determination of PMPA and Prodrugs in plasma and PBMCs. The concentration of PMPA in dog plasma samples was determined by derivatizing PMPA with
15 chloroacetaldehyde to yield a highly fluorescent N1, N6-ethenoadenine derivative (L. Naesens, J. Balzarini, and E. De Clercq, Clin. Chem. 38,480 (1992). Briefly, plasma (100 ul) was mixed with200 ul acetonitrile to precipitateprotein. Samples were then evaporated to dryness under reducedpressure at room temperature. Dried samples were reconstitutedin 200 ul derivatization cocktail (0.34% chloroacetaldehyde inlOO
20 mM sodium acetate, pH 4.5), vortexed, and centrifuged. Supernatantwas then
transferred to a clean screw-cap tube and incubated at95°C for 40 min. Derivatized samples were then evaporated to dryness and reconstituted in 100 ul of water for HPLC analysis.
25 Before intracelluiar PMPA could be determined by HPLC, the large amounts of
adenine related ribonucleotides present in the PBMC extracts had to be removed by selective oxidation. We used a modified procedure of Tanaka et al (K. Tanaka, A. Yoshioka, S. Tanaka, and Y. Wataya, AnaL Biochem., 139,35 (1984). Briefly, PBMC samples were mixed 1:2 with methanol and evaporated to dryness under reduced
30 pressure. The dried samples were derivatized as described in the plasma assay. The derivatized samples were mixed with 20 uL of 1M rharnnose and 30 uL of 0.1M sodium periodate and incubated at 37°C for 5 min. Following incubation, 40 uL of 4M memylamine and 20 uL of 0.5M inosine were added. After incubation at 37°C for 30 min, samples were evaporated to dryness under reduced pressure and
35 reconstituted in water for HPLC analysis.
5. No mtact prodrug was detected in any PBMC samples. For plasma samples
potentially containing intact prodrugs, experiments were performed to verify that no furtherconversion to PMPA occurred during derivatization. Prodrug standards were added to drugrfreeplasma and derivatizedas described. There were no detectablelevels of PMPA present in any of the plasma samples, and the projected
10 % of conversion was less than 1 %.
The HPLC system was comprised of a P4000 solvent delivery system with AS3000 auto injector and F2000 fluorescence detector (Thermo Separation, San Jose, CA). The column was an Inertsil ODS-2 column (4.6 x 150 mm). The mobile phases used
15 were: A, 5% acetonitrile in 25 mM potassium phosphate buffer with 5 mM tetrabutyi ammonium bromide (TBABr), pH 6.0; B, 60% acetonitrile in 25 mM potassium phosphatebufferwith5mMTBABr/pH6.0. The flow rate was 2 ml/min and the column temperature was maintained at 35°C by a column oven. The gradient profile was 90% A/10% B for 10 min for PMPA and 65%A/35%B for 10 min for the
20 prodrug. Detection was by fluorescence with excitation at 236 nm and emission at 420 nm, and the injection volume was 10 uL Data was acquired and stored by a laboratory data acquisition system (PeakPro, Beckman, Allendale, NJ).
Pharmacokinetic Calculations. PMPA and prodrug exposures were expressed as 25 areas under concentration curves in plasma or PBMC from zero to 24 hours (AUC). The AUC values were calculated using the trapezoidal rule.
Plasma and PBMC Concentrations. The results of this study is shown in Figures 2 and 3. Figure 2 shows the time course of GS 7340-2 metabolism summary of plasma 30 and PBMC exposures following oral administration of pure diastereoisomers of the PMPA prodrugs.
Figure 2. PMPA and Prodrug Concentration m Plasma and PBMGs Following Oral Administration ofGS 7340-2 to Dogs at-10 mg-eq/kg.
■PMPA In Plasma ■ 7340-2 In Plasma •PMPA In PBMC
0.01 * h
Time Posldose (h)
The bar graph in Figure 2 shows the AUG (0-24h) for tenofovir in dog PBMCs and plasma after administration of PMPA s.c, TDF and amidate ester prodrugs. All of the amidate prodrugs exhibited increases in PBMC exposure. For example, GS 7340 results in a ~21-fold increase in PBMC exposure as compared to PMPA s.c and TDF; and a 6.25-fold and 1.29-fold decrease in plasma exposure, respectively.
Figure 3. Depicts Tenofovir Exposure in PBMCs and Plasma Ut>on Administration of 10 mg-eq/kg in dogs
AUG(0-24h) for PMPA In PBMC and Plasma
Following an Oral Dose of 10 mg-eq/kg
PMPA Prodrugs to Dogs.
^ 1400 H
g 1000 -
800-600 400 200 H 0
These data establish in vivo that GS 7340 can be delivered orally, minimizes systemic exposure to PMPA and greatly enhances the intracellular concentration of PMPA in the cells primarily responsible for HIV replication.
PMPA Exposure In PBMC and Plasma from Oral Prodrugs of PMPA In Dogs
GS# Moiety PMPA AUC In Plasma PMPA AUC in PBMC Prodrug In Plasma PBMC/PlasmaExposureRatio
Mean StDev N Mean StDev N
GS-7114 GS-7115 Mono-Ala-Et-A Mono-Ala-Et-B 5.8 6.6 0.9 1.5 2 2 706284 331 94 55 YES YES 122 43
GS-7340-2 GS-7339 Mono-Ala-lPr-A Mono-AIa-iPr-A 5.0 6.4 1.1 1.3 5 2 805 200 222 57 5 5 YES YES 161 31
GS-7119 Mcma-Gly-Et-A 6.11 1.86 2 530 304 5 YES 87
GS-7342 GS7341 Mono-ABA-Et-A Mono-ABA-Et-B 4.6 5.8 1.2 1.4 2■•• 2 1060 199 511 86 5 5 YES YES 230 34
20 Biodisbabution of GS-7340
As part of the preclinical characterization of GS-7340, its biodistribution in dogs was determined. The tissue distribution of GS-7340 (isopropyl aJtamnyl monoamidate, phenyl monoester of tenofovir) was examined following oral
25 administration to beagle dogs. Two male animals were dosed orally with MC=GS-7340 (8.85 mg-equiv. of PMPA/kg, 33.2 fiCi/kg; the 8-carbon of adenine is labeled) in an aqueous solution (50 mM citric acid, pH 2.2). Plasma and peripheral blood mononuclear cells (PBMCs) were obtained over the 24-hr period. Urine and feces were cage collected over 24 hr. At 24 h after the dose, the animals were sacrificed
30 and tissues removed for analysis. Total radioactivity in tissues was determined by oxidation and liquid scintillation counting.
The biodistribution oi PMPA after 24 hours after a single oral dose of radiolabelled GS 7340 is shown in Table 4 along with the data from a previous study with TDF (GS-4331). In titie case of TDF, the prodrug concentration in the
35 plasma is below the level of assay detection, and the main species observed in
plasma is the parent drug. Levels of PMPA in the lymphatic tissues, bone marrow, and skeletal muscle are increased 10-fold after administration of GS-7340.
Accumulation in lymphatic tissues is consistent with the data observed from the PBMC analyses; since these tissues are composed primarily of lymphocytes. Likewise, accumulation in boiie marrow is probably due to the high percentage of lymphocytes (70%) in thistissue.
Table 7. Excretion and Tissue Distribution of Radiolabelled GS-7340 in Dogs (Mean, N=2) Following an Oral Dose at 10 mg-eq. PMPA/kg.
Tissue/Fluid GS-4331 GS-7340 Tissue Cone. Ratio of GS 7340
% Dose Cone. %Dose Cone
(ug-eq/g) (ug-eq/g) to GS-4331
Liver 12.40 38.30 16.45 52.94 1.4
Kidney 4.58 87.90 3.78 80.21 0.9
Lungs 0.03 0.53 0.34 4.33 8.2
Iliac Lymph Nodes 0.00 0.51 0.01 5.42 10.6
Axillary Lymph Nodes 0.00 0.37 0.01 5.54 14.8
Inguinal Lymph Nodes a.oo 0.28 0.00 4.12 15.0
Mesenteric Lymph Nodes 0.00 1.20 0.04 6.88 5.7
Thyroid Gland 0.00 0.30 0.00 4.78 15.8
Pituitary Gland 0.00 0.23 0.00 1.80, 7.8
Salivary Gland (L+R) 0.00 0.45 0.03 554 12.3
Adrenal Gland 0.00 1.90 0;00 3.47 1.8
Spleen 0.00 0.63 0.17 8.13 12.8
Pancreas 0.00 0.57 0.01 351 6.2
Prostate 0.00 0.23 0.00 2.14 9.1
Testes (L+R) 0.02 1.95 0.02 2.01 1.0
Skeletal Muscle 0.00 0,11 0.01 1.12 10.1
Heart 0.03 0.46 0.15 1.97 4.3
Femoral Bone 0.00 0.08 0.00 0.28 3.5
Bone Marrow i 0.00 0.20 0.00 2.05 10.2
Skin 0.00 0.13 0.00 0.95 7.2
Abdominal fat 0.00 0.16 0.00 0.90 5.8
Eye(L+R) 0.00 0.06 0.00 0.23 3.7
Jejunum 1.34 3.01 0.79 4.16 1.4
Duodenum 0.49 4.96 0.44 8.77 1.8
Ileum 0.01 0.50 0.16 4.61 9.2
Large Intestine 1.63 5.97 2.65 47.20 7.9
Gallbladder 0.00 3.58 0.04 25.02 7.0
BUe 0.00 9.63 0.22 40.48 4.2
Feces 40.96 ltd. 0.19 n.d. n.a.
Total GI Tract Contents 5.61 n.d. 21.64 n.d. n.a.
Urine 23.72 n.d. 14.73 n.d. n.a.
Plasma at 24 h 0.00 0.20 0.00 0.20 1.0
Plasma at 0.25 h n.a. 3.68 n.a. 3.48 0.9
PBMC* 0.00 . n.d. 0.00 63.20 n.d.
Whole Blopd 0.00 0.85 0.16 1 68.96 0.20 0.2
Total Recovery 81.10
* Calculated using typical recot reryof 15x 10* cells tofc d, and mean PBMCvoli ume of 02
n.s.« no sample, n.a. = not applicable, n,
1 A diaetereomerically enriched compound having the structure (3)
which is aubslaniially free of the dltetereomer (4)
O B—E—?-*R f2
R1 is an oxyester which It hydrolytable in vivo, or hydroxyl;
8 is a heterocyclic bate.
R2 is hydroxyl, or the residue of an amino acid bonded to the P atom through an amino group of the amino acid and having each carboxy substituent of the amino acid optionally eaterified. but not both of R1 and R2 are hydroxyl; E is -(CH20)y. -CH(CH,)CH?0-. -CH(CH?F)CH^O-, -CH(CH,0H)CHr. -CH(CH=CH20)CH20-, -CH(C-CH)CH^O-. -CH(CHZN3)CH20-,
-CH(R6)OCH(R6')-, -CH(R9)CH2)- or -CH(R8)0-, wherein the right hand bond is
linked to the heterocyclic base;
the broken line represents an optional double bond;
R4 and R5 are independently hydrogen, hydroxy, halo, amino or a substituent
having 1-5 carbon atoms selected from acyloxy, alkyoxy, alkylthio, alkylamino and
fS6 and R6' are independently h, C1-C6 alkyl, CrC6 hydroxyalkyl, or C2-C7 alkanoyl;H
R7 is independently H,d-C6 alkyl, or are taken together to form -O- or-CH2-; R8 is H, d-Cealkyl, CrC6 hydroxyalkyl or Ci-C6 haloalkyl; and R9 is H, hydroxymethyl or acyloxymethyl; and their salts, free base, and solvates. 2. A diastereomerically enriched compound as claimed in
which is substantially free of diastereomer (5b)
R5 is methyl or hydrogen;
R6 independently is H, alkyl, alkenyl, alkynyl, aryl or arylakyl, or R6 independently is alkyl, alkenyl, alkylnyl, aryl or arylalkyi which is substituted with from 1 to 3 substituents selected from alkylamino, alkylaminoalkyl, dialkylaminoalkyl, dialkylamino, hydroxyl, oxo, halo, amino, alkylthoi, alkoxy,
alkoxyalkyl, aryloxy, aryloxyalkyi, arylalkoxy, arylalkoxyalkyl, haloalkyi, nitro, nitroalkyi, azido, azidoalkyi, alkylacyl, alkylacylalkyl, carboxyl, or alkylacylamino;
R7 is the side chain of any naturally-occurring or pharmaceutically acceptable amino acid and which, if the side chain comprises carboxyl, the carboxyl group is optionally esterified with an alkyl or aryl group;
R11 is amino, alkylamino, oxo, or dialkylamino; and
R12 is amino or H;
And its salts, tautomers, free base and solvents. 3. A compound as claimed in claim 1 having the structure (6)
and its salts and solvates
4. A compound as claimed in claim 1 having the structure (7)
'••«IO wn r^^X
(7) 5. A diastereomerically enriched compound of structure (6a)
6 ' (6a)
which is substantially free of diastereomer (6b)
I « ■
and its salts and solvates.
6. A diastereomericaliy enriched compound of structure (7a)
O I (7a)
Which is substantially free of diastereomer (7b)
KT ^N-^ o
7. A pharmaceutical composition whenever comprising a compound as claimed in any preceding claim.
ramaniam BATARAJ & ASSOCIATES
Attoprfeys for the Applicants
Dated this 1st day of May 2006
A novel method is provided for screening prodrugs of methoxyphosphonate nucleotide analogues to identify prodrugs selectively targeting desired tissues with antiviral or antitumor activity. This method has led to the identification of novel mixed ester-amidates of PMPA for retroviral or hepadnaviral therapy, including compounds of structure (5a) having substituent groups as defined herein. Compositions of these novel compounds in pharmaceutically acceptable excipients and their use in therapy and prophylaxis are provided. Also provided is an improved method for the use of magnesium alkoxide for the preparation of starting materials and compounds for use herein.
|Indian Patent Application Number||529/MUMNP/2006|
|PG Journal Number||29/2010|
|Date of Filing||09-May-2006|
|Name of Patentee||GILEAD SCIENCES, INC.|
|Applicant Address||333 Lakeside Drive, Foster City, CA 94404,|
|PCT International Classification Number||C07F9/6561|
|PCT International Application Number||PCT/US01/23104|
|PCT International Filing date||2001-07-20|