PRODRUGS OF PHOSPHONATE NUCLEOTIDE ANALOGUES AND METHODS FOR SELECTING AND MAKING SAME

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.

Description: Prodrugs of P osphonate 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 such prodrugs. Many methoxyphosphonate nucleotide analogues are known. In general, such compounds have the structure A-OCH 2 P(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, 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 Nirnmo, 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 (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 expressly incorporated by reference. Summary of the Invention Prodrugs of methoxyphosphonate nucleotide 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.e., ability to be absorbed from the gastrointestinal tract and 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 HIV infection. The objective of this invention is, among other advantages, to produce less toxicity 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, pursuant to these observations, a screening method is provided for identifying a methoxyphosphonate nucleotide analogue prodrug conferring enhanced activity in a target tissue comprising: (a) providing at least one of said prodrugs; (b) selecting at least one therapeutic target tissue and at least one non-target tissue; (c) administering the prodrug to the target tissue and to said at least one non- target tissue; and (d) deterrnining ' 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 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), (1) 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) (2) and its enriched diasteromers, salts, free base and solvates. 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 invention, we provide diastereomerically enriched compounds of this invention having the structure (3) which are substantially free of the diastereomer (4) wherein R is an oxyester which is hydrolyzable in vivo, or hydroxyl; B is a heterocyclic base; 2 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 1 2 the amino acid optionally esteritied, but not both of R and R are hydroxyl; E is -(CH2)2-, -CH(CH3)CH2-, -CH(CH2F)CH2-, -CH(CH2θH)CH2-, -CH(CH=CH2)CH2-, -CH(C≡CH)CH2-, -CH(CH2N3)CH2-, -CH(R 6 )OCH(R 6' )-, -CH(R 9 )CH2θ- or -CH(R 8 )0-, wherein the right hand bond is linked to the heterocyclic base; the broken line represents an optional double bond; 4 5 R and R are independently hydrogen, hydroxy, halo, amino or a substituent having 1-5 carbon atoms selected from acyloxy, alkyoxy, alkylthio, alkylamino and dialkylamino; hydroxyalkyl, or C 2 -C 7 alkanoyl; 7 R is independently H, C^C g alkyl, or are taken together to form -O- or -CH 2 -; g R is H, C 2 -C 6 alkyl, C^C g hydroxyalkyl or C α -C 6 haloalkyl; and 9 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 compounds having the structure (5a) (5a) which is substantially free of diastereomer (5b) wherein 5 R 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 alkylamino, alkylaminoalkyl, dialkylaminoal yl, dialkylamino, hydroxyl, oxo, halo, amino, alkylthio, alkoxy, alkoxyalkyl, aryloxy, aryloxyalkyl, arylalkoxy, arylalkoxyalkyl, haloalkyl, nitro, nitroalkyl, azido, azidoalkyl, alkylacyl, alkylacylalkyl, carboxyl, or alkylacylamino; 7 R 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; 11 R is amino, alkylamino, oxo, or dialkylamino; and 12 R is amino or H; and its salts, tautomers, free base and solvates. A preferred embodiment of this invention is the compound of structure (6), 9-[(R)-2-[[(S)-[[(S)-l- (isopropoxycarbonyl)ethyl]arru^o]phenoxyphosplunyl]methoxy]propyl]aderdne, also designated herein GS-7340 (6) Another preferred embodiment of this invention is the fumarate salt of structure (5) (structure (7)), 9-[(R)-2-[[(S)-[[(S)-l- 5 (isopropoxycarbonyl)ethyl]arruno]phenoxyphosphinyl]methoxy]propyl]adenine fumarate (1:1), also designated herein GS-7340-2 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 0 9-[2-(phosphonomethoxy)propyl]adenine (hereinafter "PMPA" or 9-[2- (phosphonomethoxy)ethyl] adenine (hereinafter "PMEA") using magnesium alkoxide, which comprises combming 9-(2-hydroxypropyl)adenine or 9-(2- hydroxyethyl)adenine, protected p-toluenesulfonyloxymethylphosphonate and magnesium alkoxide, and recovering PMPA or PMEA, respectively. 25 Detailed Description of the Invention The methoxyphosphonate nucleotide analogue parent drugs for use in this screening method are compounds having the structure A-OH 2 P(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 wherein the group B is defined below and group E 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 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 include substitutions on the base and aglycon. None of these modifications per se is part of this invention and none are to be considered liiniting 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 (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 screening method of this invention are disclosed for example in U.S. Patent No. 5,798,340. 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 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 dispositive of whether or not a prodrug will be shown to be selective in the assay. 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 on the basis of either their stereochemistry or their covalent structure, or these features are varied in combination. Each prodrug tested, however, desirably is structurally and stereochemicaUy substantiaUy pure, else the output of the screening assay wiU 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 typicaUy then one would compare the results with prior studies with other prodrugs. We have found that the 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 weU. Chiral sites also are found on the nucleoside analogue portion of the molecules, but these typicaUy are already dictated by the stereochemistry of the parent drug and wiU 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 wiU be chiraUy enriched if not pure at each site so that the results of the screen wiU be more meaningful. As noted, distinctiveness of stereoisomers is conferred by enriching or purifying the stereoisomer (typicaUy this wiU 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 substantiaUy homogeneous. By substantiaUy homogeneous or chiraUy 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 labeled with a detectable group, e.g. radiolabeled, in order to faciUtate detection later in tissues or ceUs. However, a label is not required since other suitable assays for the prodrug or its metaboUtes (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 more of its metaboUtes, 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 aU cases) that the degree and rate of conversion of prodrug to antiviraUy active parent diphosphate is the same across aU tissues tested. Otherwise, one can test for the diphosphate. The target tissue preferably wiU be lymphoid tissue when screening for prodrugs useful in the treatment of HIV infection. Lymphoid tissue wiU be known to the artisan and includes CD4 ceUs, lymphocytes, lymph nodes, macrophages and macrophage-Uke ceUs including monocytes such as peripheral blood monocytic ceUs (PBMCs) and gUal ceUs. Lymphoid tissue also includes non-lymphoid tissues that are enriched in lymphoid tissues or ceUs, e.g. lung, skin and spleen. Other targets for other antiviral drugs of course wiU be the primary sites of repUcation or latency for the particular virus concerned, e.g., Uver for hepatitis and peripheral nerves for HSV. SimUarly, target tissues for tumors wiU in fact be the tumors themselves. These tissues are aU weU-known to the artisan and would not require undue experimentation to select. When screening for antiviral compounds, target tissue can be infected by the virus. Non-target tissues or ceUs also are screened as part of the method herein. Any number or identity of such tissues or ceUs can be employed in this regard. In general, tissues for which the parent drug is expected to be toxic will be used as non-target tissues. The selection of a non-target tissue is entirely dependent upon the nature of the prodrug and the activity of the parent. For example, non-hepatic tissues would be selected for prodrugs against hepatitis, and untransformed ceUs of the same tissue as the tumor wiU suffice for the antitumor-selective prodrug screen. It should be noted that the method of this invention is distinct from studies typicaUy undertaken to determine oral bioavaUabUity of prodrugs. In oral bioavaUabUity studies, the objective is to identify a prodrug which passes into the systemic circulation substantiaUy 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 of this invention generaUy do not include the smaU intestines or, if the intestines are included, then the tissues also include additional tissues other than the sma'U intestines. The target and non-target tissues used in the screening method of this invention typicaUy wUl be in an intact Uving animal. Prodrugs containing esters are more desirably tested in dogs, monkeys or other animals than rodents; mice 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 ceU fines maintained in various forms of ceU culture, e.g. roUer bottles or zero gravity suspension systems. For example, MT-2 ceUs can be used as a target tissue for selecting HIV prodrugs. Thus, the term "tissue" shaU not be construed to require organized ceUular structures, or the structures of tissues as they may be found in nature, although such would be preferred. Rather, the term "tissue" shaU be construed to be synonymous with ceUs of a particular source, origin or differentiation stage. The target and non-target tissue may in fact be the same tissue, but the tissues wiU be in different biological status. For example, the method herein could be used to select for prodrugs that confer activity in viraUy-infected tissue (target tissue) but which remain substantiaUy inactive in vhaUy-uninfected ceUs (corresponding non-target tissue). The same strategy would be employed to select prophylactic prodrugs, i.e., prodrugs metabolized to antiviraUy active forms incidental to viral infection but which remain substantiaUy unmetaboUzed in uninfected cells. Similarly, prodrugs could be screened in transformed ceUs and the untransformed counterpart tissue. This would be particularly useful in comparative testing to select prodrugs for the treatment of hematological maUgnancies, e.g. leukemias. Without being limited by any particular theory of operation, tissue selective prodrugs are thought to be selectively taken up by target ceUs and/or selectively metabolized within the ceU, as compared to other tissues or ceUs. The unique advantage of the methoxyphosphonate prodrugs herein is that their metaboUsm to the dianion at physiological pH ensures that they will be unable to diffuse back out of the ceU. They therefore remain effective for lengthy periods of time and are maintained at elevated intraceUular concentrations, thereby exhibiting increased potency. The mechanisms for enhanced activity in the target tissue are believed to include enhanced uptake by the target ceUs, enhanced intraceUular retention, or 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 aU of the metaboUc 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; metaboUsm in other tissues may provide intermediates finaUy converted to antiviral forms in the target tissue. The degree of selectivity or enhanced deUvery that is desired wiU 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 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 wiU be on kidney and lymphoid tissues. The step of determining the relative antiviral activity of a prodrug in the selected tissues ordinarily is accompUshed by assaying target and non-target tissues for the relative presence or activity of a metaboUte of the prodrug, which metaboUte is known to have, or is converted to, a metaboUte having antiviral or antitumor activity. Thus, typicaUy one would determine the relative amount of the parent drug in the tissues over substantiaUy the same time course in order to identify prodrugs that are preferentiaUy metabolized in the target tissue to an antiviraUy or antitumor active metaboUte or precursor thereof which in the target tissue ultimately produces the active metaboUte. In the case of antiviral compounds, the active metaboUte is the diphosphate of the phosphonate parent compounds. It is this metaboUte that is incorporated into the viral nucleic acid, thereby truncating the elongating nucleic acid strand and halting viral repUcation. MetaboUtes of the prodrug can be anaboUc metaboUtes, cataboUc metaboUtes, or the product of anaboUsm and cataboUsm together. The manner in which the metaboUte is produced is not important in the practice of the method of this invention. The method of this invention is not limited to assaying a metaboUte which per se possesses antiviral or antitumor activity. Instead, one can assay inactive precursors of the active metaboUtes. Precursors of the antiviraUy active diphosphate metaboUte include the monophosphate of the parent drug, monophosphates of other metaboUtes of the parent drug (e.g., an intermediate modification of a substituent on the heterocycUc base), the parent itself and metaboUtes generated by the ceU in converting the prodrug to the parent prior to phosphorylation. The precursor structures may vary considerably as they are the result of ceUular metaboUsm. However, this information is aheady known or could be readfiy 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 intraceUular processing of the inactive metaboUte to an active metaboUte occurs at different rates among the tissues being tested, the raw assay results with the inactive metaboUte would need to be adjusted to take account of the differences among the ceU types because the relevant parameter is the generation of activity in the target tissue, not accumulation of inactive metaboUtes. However, determining the proper adjustments would be within the ordinary skill. Thus, when step (d) of the method herein caUs for deterrnining the activity, activity can be either measured directly or extrapolated. It does not mean that the method herein is 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. Step (d) of the method of this invention caUs for deterrnining the "relative" activity of the prodrug. It wiU be understood that this does not require that each and every assay or series of assays necessarUy 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 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 optimaUy 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. It wiU be appreciated that some preassessment of prodrug candidates can be undertaken before the practice of the method of this invention. For example, the prodrug wiU need to be capable of passing largely unmetaboUzed through the gastrointestinal tract, it wiU need to be substantiaUy stable in blood, and it should be able to permeate ceUs at least to some degree. In most cases it also wiU need to complete a first pass of the hepatic circulation without substantial metaboUsm. Such prestudies are optional, and are weU-known to those skilled in the art. The same reasoning as is described above for antiviral activity is appUcable to antitumor prodrugs of methoxyphosphonate nucleotide analogues as weU. These include, for example, prodrugs of PMEG, the guanyl analogue of PMEA. In this case, cytotoxic phosphonates such as PMEG are worthwhUe 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 precUnical or clinical program to confirm that the desired objectives have been met. TypicaUy, a prodrug is considered to be selective if the 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 wiU be those resulting in greater than about lOx concentration or activity in the 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 starting materials (parent drugs) of this invention, PMEA and (R)-PMPA. TypicaUy, 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. 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. TypicaUy the tosylate is protected by lower alkyl groups, but other suitable groups wiU be apparent to the artisan. It may be convenient to employ the tosylate presubstituted with the prodrug phosphonate substituents which are capable of acting as protecting groups in the tosylation reaction, thereby aUowing 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 C x - C 6 branched or normal alkyl, but is preferably t-butyl (for PMPA) or isopropyl (for PMEA). The reaction conditions also are not critical, but preferably comprise heating the reaction mixture at about 70-75°C with stirring or other moderate agitation. If there is no interest in retaining the phosphonate substituents, the product is deprotected (usuaUy with bromotrimethylsUane where the tosylate protecting group is alkyl), and the product then recovered by crystallization or other conventional method as wiU be apparent to the artisan. HeterocycUc Base In the compounds of this invention depicted in structures (3) and (4), the heterocycUc base B is selected from the structures wherein R 15 is H, OH, F, Cl, Br, I, OR 16 , SH, SR 16 , NH2, or NHR 17 ; R 16 is C1-C6 alkyl or C2-C6 alkenyl including CH3, CH2CH3, CH2CCH, CH2CHCH2 and C3H7; R 17 is C1-C6 alkyl or C2-C6 alkenyl including CH3, CH2CH3, CH2CCH, CH2CHCH2, and C3H7; R 18 is N, CF, CC1, CBr, CI, CR 19 CSR 19 , or COR 19 ; 19 R is H, C1-C9 alkyl, C2-C9 alkenyl, C2 - C9 alkynyl, C1-C9 alkyl-Cι-C9 19 alkoxy, or C7-C9 aryl-alkyl unsubstituted or substituted by OH, F, Cl, Br or I, R therefore including -CH3, -CH2CH3, -CHCH2, -CHCHBr, -CH2CH2CI, -CH2CH2F, -CH2CCH, -CH2CHCH2, -C3H7, -CH2OH, -CH2OCH3, -CH2OC2H5, -CH2OCCH, -CH2OCH2CHCH2, -CH2C3H7, -CH2CH2OH, -CH2CH2OCH3, -CH2CH2OC2H5, -CH2CH2OCCH, -CH2CH2OCH2CHCH2, and -CH2CH2OC3H7; R 20 is N or CH; R 21 is N, CH, CCN, CCF3, CC≡CH or CC(0)NH2; R 22 is H, OH, NH2, SH, SCH3, SCH2CH3, SCH2CCH, SCH2CHCH2, SC3H7, NH(CH3), N(CH3)2, NH(CH2CH3), N(CH2CH3)2, NH(CH2CCH), NH(CH2CHCH2), NH(C3H7), halogen (F, Cl, Br or I) or X wherein X is -(CH2)m(O)n(CH2)mN(R 10 )2 wherein each m is independently 0-2, n is 0-1, and R 10 independently is H, C1-C15 alkyl, C2- 5 alkenyl, C.6- 5 arylalkenyl, C6- 5 arylalkynyl, C2-C15 alkynyl, Ci-C6-alkylamino-Cι-C6 alkyl, C5- 5 aralkyl, C6- C15 heteroaralkyl, C5-C6 aryl, C2-C6 heterocycloalkyl, C2-C15 alkyl, C3-C15 alkenyl, C6-C15 arylalkenyl, C3-C15 alkynyl, C7-C15 arylalkynyl, Ci-C6-alkylamino-Cι-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in the alkyl moiety not adjacent to ° has been replaced by -O-, optionaUy both R 10 are joined together with N to form a saturated or unsaturated C2-C5 heterocycle containing one or two N heteroatoms and optionaUy an additional O or S heteroatom, or one of the foregoing R 10 groups which is substituted with 1 to 3 halo, CN or N3; but optionaUy at least one R 10 group is not H; R 23 is H, OH, F, Cl, Br, I, SCH3, SCH2CH3, SCH2CCH, SCH2CHCH2, SC3H7, OR 16 , NH2, NHR 17 or R 22 ; and R 24 is O, S or Se. B also includes both protected and unprotected heterocycUc bases, particularly purine and pyrimidine bases. Protecting groups for exocycUc amines and other labUe groups are known (Greene et al. "Protective Groups in Organic Synthesis") and include N-benzoyl, isobutyryl, 4,4'-dimethoxytrityl (DMT) and the like. The selection of protecting group wiU be apparent to the ordinary artisan and wiU depend upon the nature of the labUe 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 N4-benzoylcytosine, N^- benzoyladenine, N^-isobutyrylguanine and the like. Protected bases have the formulas Xa.l, XIa.l, Xlb.l, XIIa.1 or XIIIa.1 (Xa.l) (XIa.l) (Xlb.l) (XIIa.1) (XIIIa.1) wherein R 18 , R 20 , R 21 , R 24 have the meanings previously defined; R^ A is R 39 or R 22 provided that R 22 is not NH2; R 23A is R 39 or R 23 provided that R 23 is not NH2; R 39 is NHR 40 , NHC(0)R 36 or CR 41 N(R 38 )2 wherein R 36 is C1-C19 alkyl, C1- 9 alkenyl, C3- C10 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; R 38 is C1-C10 alkyl, or both R 38 together are 1-morpholino, 1-piperidine or 1- pyrroUdine; R 40 is C j -C la alkyl, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, octyl and decanyl; and R 41 is hydrogen or CH3. 39 22A 23A For bases of structures XIa.l and Xlb.l, if R is present at R or R , both R groups on the same base wiU generaUy be the same. Exemplary R are phenyl, phenyl substituted with one of the foregoing R aryl substituents, -C10H 5 (where C10H15 is 2-adamantoyl), -CH2-C6H5, -C6H5, -CH(CH3)2, -CH2CH3, methyl, butyl, t-butyl, heptanyl, nonanyl, undecanyl, or undecenyl. Specific bases include hypoxanthine, guanine, adenine, cytosine, inosine, thymine, uradl, xanthine, 8-aza derivatives of 2-aminopιιrine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza-8-aza 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-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6- chloropurine, hypoxanthine, inosine and xanthine; 3-deaza derivatives of 2- aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine; 5- bromocytosine; 5-methylcytosine; 5-bromovinyluracU; 5-fluorouracU; 5- chlorouracU; 5-iodouracU; 5-bromouracU; 5-trifluoromethyluracU; 5- methoxymethyluracU; 5-ethynyluracU and 5-propynyluracU. Preferably, B is a 9-purinyl residue selected from guanyl, 3-deazaguanyl, 1- deazaguanyl, 8-azaguanyl, 7-deazaguanyl, adenyl, 3-deazaadenyl, 1-dezazadenyl, 8-azaadenyl, 7-deazaadenyl, 2,6-diaminopurinyl, 2-aminopurinyl, 6-chloro-2- aminopurinyl and 6-t o-2-aminopurinyl, or a B' is a 1-pyrimidinyl residue selected from cytosinyl, 5-halocytosinyl, and 5-(C ι-C3-alkyl)cytosinyl. Preferred B groups have the formula wherein R2 independently is halo, oxygen, NH2, X or H, but optionaUy at least one R 22 is X; X is -(CH2)m(O)n(CH2) m N(R 10 )2 wherein m is 0-2, n is 0-1, and RlO independently is H, C1-C15 alkyl, C2-C15 alkenyl, C6- 5 arylalkenyl, C6-C15 arylalkynyl, C2-C15 alkynyl, Cι-C6-alkylamino-Cι-C6 alkyl, C5-C15 aralkyl, C6- C15 heteroaralkyl, C5-C6 aryl, C2-C heterocycloalkyl, C2-C15 alkyl, C3-C15 alkenyl, C6-C15 arylalkenyl, C3-C15 alkynyl, C7-C15 arylalkynyl, Cι-C6-alkylamino-Cι-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in the alkyl moiety not adjacent to N > has been replaced by -O-, optionaUy both RΪO are joined together with N to form a saturated or unsaturated C2-C5 heterocycϊe containing one or two N heteroatoms and optionaUy an additional O or S heteroatom, or one of the foregoing RlO groups is substituted with 1 to 3 halo, CN or N3; but optionaUy at least one RlO group is not H; and Z is N or CH, provided that the heterocycUc nucleus varies from purine by no more than one Z. E groups represent the aglycons employed in the methoxyphosphonate nucleotide analogues. Preferably, the E group is -CH(CH 3 )CH 2 - or -CH 2 CH 2 -. Also, it is preferred that the side groups at chiral centers in the aglycon be substantiaUy solely in the (R) configuration (except for hydroxymethyl, which is the enriched (S) enantiomer). R 1 is an in vivo hydrolyzable oxyester having the structure -OR 35 or -OR 6 wherein R 35 is defined in column 64, line 49 of U.S. Patent No. 5,798,340, herein incorporated by reference, and R 6 is defined above. Preferably R 1 is aryloxy, ordinarily unsubstituted or para-substituted (as defined in R 6 ) phenoxy. R 2 is an amino acid residue, optionaUy provided that any carboxy group linked by less than about 5 atoms to the amidate N is esterified. R 2 typicaUy has the structure (8) wherein n is 1 or 2; R 11 is R 6 or H; preferably R 6 = C 3 -C 9 alkyl; C 3 -C 9 alkyl substituted independently with OH, halogen, O or N; C 3 -C 6 aryl; C 3 -C 6 aryl which is independently substituted with OH, halogen, O or N; or C 3 -C 6 arylalkyl which is independently substituted with OH, halogen, O or N; R 12 independently is H or -C, alkyl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR 11 and halogen; C 3 -C 6 aryl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR 11 and halogen; or C 3 -C 9 aryl-alkyl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR 11 and halogen; R 13 independently is C(0)-OR π ; amino; amide; guanidinyl; imidazolyl; indolyl; sulfoxide; phosphoryl; -C 3 alkylamino; C C 3 alkyldiamino; - alkenylamino; hydroxy; thiol; C C 3 alkoxy; -C 3 alkthiol; (CH 2 ) n COOR"; C C 6 alkyl which is unsubstituted or substituted with OH, halogen, SH, NH 2 , phenyl, hydroxyphenyl or C 7 -C 1D alkoxyphenyl; C 2 -C 6 alkenyl which is unsubstituted or substituted with OH, halogen, SH, NH 2 , phenyl, hydroxyphenyl or C 7 -C 10 alkoxyphenyl; and C 6 -C 12 aryl which is unsubstituted or substituted with OH, halogen, SH, NH 2 , phenyl, hydroxyphenyl or C 7 -C 10 alkoxyphenyl; and R 14 is H or C j -C 9 alkyl or C C 9 alkyl independently substituted with OH, halogen, COOR 11 , 0 or N; C 3 -C 6 aryl; C 3 -C 6 aryl which is independently substituted with OH, halogen, COOR 11 , 0 or N; or C 3 -C 6 arylalkyl which is independently substituted with OH, halogen, COOR", O or N. Preferably, R 11 is C C 6 alkyl, most preferably isopropyl, R 13 is the side chain of a naturaUy occurring amino acid, n = 1, R 12 is H and R 14 is H. In the compound of structure (2), the invention includes metaboUtes in which the phenoxy and isopropyl esters have been hydrolyzed to -OH. SimUarly, the de-esterified enriched phosphonoamidate metaboUtes of compounds (5a), 5(b) and (6) are included within the scope of this invention. Aryl and "O" or "N" substitution are defined in column 16, lines 42-58, of United States Patent No. 5,798,340. TypicaUy, the amino acids are in the natural or / amino acids. 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, tertiary or cychc hydrocarbon. Unless stated to the contrary alkyl is -C^. Examples are -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH2CH2CH3), -CH CH(CH3)2, -CH(CH )CH2CH3, -C(CH 3 )3, -CH2CH2CH2CH2CH3, -CH(CH3)CH2CH2CH3, -CH(CH2CH3)2, -C(CH3)2CH2CH3), -CH(CH3)CH(CH 3 )2, -CH 2 CH2CH(CH 3 )2), -CH2CH(CH3)CH2CH 3 , -CH2CH2CH2CH2CH2CH3, -CH(CH3)CH2CH2CH2CH3, -CH(CH2CH3)(CH2CH2CH3), -C(CH3)2CH2CH2CH3, -CH(CH3)CH(CH3)CH2CH 3 , -CH(CH3)CH2CH(CH 3 )2, -C(CH3)(CH2CH 3 )2, -CH(CH 2 CH 3 )CH(CH3)2, -C(CH 3 )2CH(CH3)2, and -CH(CH3)C(CH3)3. Alkenyl and alkynyl are defined in the same fashion, but 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 weU. 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 formulated with pharmaceuticaUy acceptable excipients or solvating dUuents for use as pharmaceutical products also as set forth in U. S. Patent No. 5,798,340. These prodrugs have the antiviral and utilities aheady estabUshed 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) 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 wiU be more fuUy understood by reference to the foUowing examples: 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 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, fUtered, and rinsed with toluene (2x). The wet soUd was dried in vacuo at 65°C to yield 20.0g (90%) of 9-(2- hydroxyethyl)adenine as an off-white soUd. Mp: 238-240°C. 9-(2-Hydroxyethyl)adenine (HEA) (20.0g, 0.112 mol) was suspended in DMF (125 ml) and heated to 80°C. Magnesium isopropoxide (11.2g, 0.0784 mol) , or alternatively magnesium t-butoxide, was added to the mixture foUowed by diethyl p-toluenesuhonyloxymethylphosphonate (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 distiUation and the reaction was recharged with 30 ml of fresh DMF. After cooling to room temperature, bromotrimethylsUane (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 hour. The soUds were isolated by filtration, washed with water (2x), and dried in vacuo at 56°C to yield 20g (65.4%) of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) as a white soUd. Mp: > 200°C dec. Η NMR (D z O) • 3.49 (t, 2H); 3.94 (t, 2H); 4.39 (t, 2H); 8.13 (s, IH); 8.22 (s, IH). Example lb Adenine to PMPA using Magnesium t-Butoxide. To a suspension of adenine (40g, 0.296 mol) in DMF (41.9 ml) was added (iD-propylene carbonate (34.5g, 0.338 mol) 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 foUowed by methanesulfonic acid (4.7g, 0.049 mol) while maintaining 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 soUd was isolated by filtration and rinsed with acetone (2x). The wet soUd was dried in vacuo at 80°C to yield 42.6g (75%) of (R)-9- [2-(hydroxy)propyl]adenine (HPA) as an off-white soUd. Mp: 188-190°C. (R)-9-[2-(hydroxy)propyl]adenine (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 the mixture over one hour foUowed by diethylp-toluenesulfonyloxymethylphosphonate (66.0g, 0.205 mol) over two hours whUe the temperature was kept at 78°C. The mixture was stirred at 75°C for 4 hours. After cooling to below 50°C, bromotrimethylsUane (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 volatUes were removed via atmospheric distiUation. The residue was dissolved into water (120 ml) at 50°C and then extracted with ethyl acetate (101 ml). The pH of the aqueous phase was adjusted to pH 1.1 with sodium hydroxide, seeded with authentic (R)-FMPA, 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 soUd was isolated by filtration, washed with water (60 ml), and dried in vacuo at 50°C to yield 18.9g (63.5%) of crade(R)-9-[2- (phosphonomethoxy)propyl]adenine (PMPA) as an off-white soUd. The crude(jR)-9-[2-(phosphonomethoxy)propyl]adenine was heated at reflux in water (255 ml) until aU soUds dissolved. The solution was cooled to room temperature over 4 hours. The resulting slurry was cooled at 4°C for three hours. The soUd 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 (R)-9-[2- (phosphonomethoxy)propyl]adenine (PMPA) as a white soUd. Mp: 278-280°C. Example 2 Preparation of GS-7171 (III) Scheme 1 (anhydrous) I π GS-7171 A glass-lined reactor was charged with anhydrous PMPA, (I) (14.6 kg, 50.8 mol), phenol (9.6 kg, 102 mol), and l-methyl-2-pyrroUdinone (39 kg). The mixture was heated to 85°C and _riethylamine (6.3 kg, 62.3 mol) added. A solution of 1,3- dicyclohexylcarbodiimide (17.1 kg, 82.9 mol) in l-methyl-2-pyrroUdinone (1.6 kg) was then added over 6 hours at 100°C. Heating was continued for 16 hours. The reaction was cooled to 45°C, water (29 kg) added, and cooled to 25°C. SoUds 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) foUowed 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 HCl (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). SoUds were isolated by filtration, washed with methanol (8.5 kg), and dried under reduced pressure to yield 9.33 kg II as a white powder: Η NMR (D z O) δ 1.2 (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, IH), 7.15 (t, 2H), 8.15 (s, IH), 8.2 (s, IH); 31 P NMR (D 2 0) δ 15.0 (decoupled). GS-7171 (III). (Scheme 1) A glass-lined reactor was charged with monophenyl PMPA, (II), (9.12 kg, 25.1 mol) and acetonitrUe (30.7 kg). Thionyl chloride (6.57 kg, 56.7 mol) was added below 50°C. The mixture was heated at 75°C until soUds dissolved. Reaction temperature was increased to 80°C and volatUes (11.4 kg) coUected by atmospheric distiUation under nitrogen. The pot residue was cooled to 25°C, dichloromethane (41 kg) added, and cooled to -29°C. A solution of (L)- alanine isopropyl ester (7.1 kg, 54.4 mol) in dichloromethane (36 kg) was added over 60 minutes at -18°C foUowed by tiiethylamine (7.66 kg, 75.7 mol) 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 (18.2 kg), filtered, rinsed with dichloromethane (28 kg), and concentrated to an oU under reduced pressure. Acetone (20 kg) was charged to the oU and the mixture concentrated under reduced pressure. Acetone (18.8 kg) was charged to the resulting oU. Half the product solution was purified by chromatography over a 38 x 38 cm bed of 22 kg siUca gel 60, 230 to 400 mesh. The column was eluted with 480 kg acetone. The purification was repeated on the second half of the oU using fresh siUca gel and acetone. Clean product bearing fractions were concentrated under reduced pressure to an oU. AcetonitrUe (19.6 kg) was charged to the oU and the mixture concentrated under reduced pressure. AcetonitrUe (66.4 kg) was charged and the solution chilled to 0 to -5°C for 16 hours. SoUds were removed by filtration and the filtrate concentrated under reduced pressure to 5.6 kg III as a dark oU: J H NMR (CDC 3 ) δ 1.1 (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); 31 P NMR (CDCl,) δ 21.0, 22.5 (decoupled). Alternate Method for GS-7171 (ID) Scheme 2 II Monophenyl PMPA (II). A round-bottom flask with reflux condenser and nitrogen inlet was placed in a 70°C oU bath. The flask was charged with anhydrous PMPA (I) (19.2 g, 67 mmol), N, -dimethy_Lformamide (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 solution resulted. PhenoxytrimethylsUane (11.7 g, 70 mmol) was added to the solution over 5 minutes. Heating in the 100°C oU bath continued for two hours more. The reaction was poured into rapidly stirring acetone (400 mL) with cooling at 0°C. SoUds were isolated by filtration, dried under reduced pressure, and dissolved in methanol (75 mL). The solution pH was adjusted to 3.0 with potassium hydroxide solution (45% aq.) with cooling in ice/water. The resulting soUds 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 mL), and N, -dimethyUormamide (1 drop) were combined in a round bottom flask in a 40°C oU bath. Thionyl chloride (1.96 g, 16.5 mmol) was added. After 20 minutes the clear solution was removed from heat, dUuted with dichloromethane (10 ml), and added to a solution of (L)-alanine isopropyl ester (5g, 33 mmol) and dusopropylethylamine (5.33 g, 41 mmol) in dichloromethane (20 mL) at -10°C. The reaction mixture was warmed to room temperature and washed three times with sodium dmydrogenphosphate solution (10% aq., 10 mL each wash). The organic solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to a oU. The oU was combined with fumaric acid (0.77g, 6.6 mmol) and acetonitrUe (40 mL) and heated to reflux to give a homogeneous solution. The solution was cooled in an ice bath and soUds isolated by filtration. The soUd 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 soUd dissolved. The organic layer was isolated, then washed with water (5 mL), dried over anhydrous 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 commerciaUy avaUable Chiralpak AS, 20 μm, 21 x 250 mm semi-preparative HPLC column with a Chiralpak AS, 20 μm, 21 x 50 mm guard column. Chiralpak ® AS is a proprietary packing material manufactured by Diacel and sold in North America by Chiral Technologies, Inc. (U. S. Patent Nos. 5,202,433, RE 35,919, 5,434,298, 5,434,299 and 5,498,752). Chiralpak AS is a chiral stationary phase (CSP) comprised of amylosetris[(S)- -methylbenzyl carbamate] coated onto a silica gel support. The GS-7171 diastereomeric mixture was dissolved in mobUe phase, and approximately 1 g aUquots 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 mobUe phase was immediately switched to 100% methyl alcohol, which caused the desired diastereomer, designated GS-7340 (IV), to elute as a sharp peak from the column with the methyl alcohol solvent front. The methyl alcohol was used to reduce the over-aU cycle time. After the first couple of injections, both diastereomers were coUected as a single large fractions containing one of the purified diastereomers (>99.0% single diastereomer). The mobUe phase solvents were removed in vacuo to yield the purified diastereomer as a friable foam. 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. The chromatographic conditions were as foUows: MobUe Phase(Initial) GS-7171 - AcetonitrUe : Isopropyl Alcohol (90:10) (Final) 100% Methyl Alcohol Flow 10 mL/minute Run Time About 45 minute Detection UV at 275 nm Temperature Ambient Elution Profile GS-7339 (diastereomer B) GS-7340 (diastereomer A; (IV)) 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 (III), 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 acetonitrUe. Product bearing fractions were concentrated to a solution of IV in acetonitrUe (2.48 kg). The solution soUdified to a crystalline mass wet with acetonitrUe 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; Η NMR (CDCI 3 ) δ 1.15 (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, IH); 31 P NMR (CDCI 3 ) δ 21.0 (decoupled). C. Diastereomer Separation by C18 RP-HPLC GS-7171 (III) was chromatographed by reverse phase HPLC to separate the diastereomers using the foUowing summary protocol. Chromatographic column: Phenomenex Luna™ C18(2), 5 μm, 100 A pore size, (Phenomenex, Torrance, CA), or equivalent Guard column: PeUicular C18 (AUtech, Deerfield, IL), or equivalent MobUe Phase: A — 0.02% (85%) H3PO4 in water : acetonitrUe (95:5) B — 0.02% (85%) H3PO4 in water : acetonitrUe (50:50) MobUe Phase Gradient: Run Time: 50 minutes EquiUbration Delay: 10 min at 100% mobUe phase A Flow Rate: 1.2 mL/min Temperature: Ambient Detection: UV at 260 nm Sample Solution: 20 mM sodium phosphate buffer, pH 6 Retention Times: GS-7339, about 25 minutes GS-7340, about 27 minutes D. Diastereomer Separation by Crystallization GS-7340 (IV). A solution of GS-7171 (III) in acetonitrUe was concentrated to an amber foam (14.9g) under reduced pressure. The foam was dissolved in acetonitrUe (20 mL) and seeded with a crystal of IV. The mixture was stirred overnight, cooled to 5°C, and soUds isolated by filtration. The soUds were dried to 2.3 g IV as white crystals, 98% diastereomeric purity ( 31 P NMR): Η NMR (CDCI 3 ) δ 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); 31 P NMR (CDCI 3 ) δ 19.5 (decoupled). X-ray crystal analysis of a single crystal selected from this product yielded the foUowing data: Crystal Color, Habit colorless, column Crystal Diminsions 0.25 X 0.12 X 0.08 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) A 3 Space Group P2.2.2. (#19) Z value 4 calc 1.333 g/αn F 1008.00 μ(MoKα) 1.60 cm "1 Example 4 Preparation of Fumarate Salt of GS-7340 GS-7340-02 (V). (Scheme 1) A glass-lined reactor was charged with GS-7340 (IV), (1.294 kg, 2.71 mol), fumaric acid (284 g, 2.44 mol), and acetonitrUe (24.6 kg). The mixture was heated to reflux to dissolve the soUds, fUtered whUe hot and cooled to 5°C for 16 hours. The product was isolated by filtration, rinsed with acetonitrUe (9.2 kg), and dried to 1329 g (V) as a white powder: mp 119.7 - 121.1°C; [α] D 20 -41.7° (c 1.0, acetic acid). Example 5 Preparation of GS-7120 (VI) Scheme 3 II GS-7120 A 5 L round bottom flask was charged with monophenyl PMPA, (II), (200 g, 0.55 mol) and acetonitrUe (0.629 kg). Thionyl chloride (0.144 kg, 1.21 mol) was added below 27°C. The mixture was heated at 70°C until soUds dissolved. VolatUes (0.45 L) were removed by atmospheric distiUation 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)-α 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 foUowed by 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 sodium sulfate (0.5 kg) and fUtered. The soUds were rinsed with dichloromethane (0.6 kg) and the combined filtrate and rinse was concentrated to an oU under reduced pressure. The oU 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. Example 5a Diastereomer Separation of GS-7120 by Batch Elution Chromatography The diastereomeric mixture was purified using the conditions described for GS- 7171 in Example 3A except for the foUowing: MobUe Phase (Initial) GS-7120 - AcetonitrUe : Isopropyl Alcohol (98:2) (Final) 100% Methyl Alcohol Elution ProfUe GS-7341 (diastereomer B) GS-7342 (diastereomer A) Example 6 Diastereomer Separation of GS-7120 by Crystallization A I L round bottom flask was charged with monophenyl PMPA, (II), (50 g, 0.137 mol) and acetonitrUe (0.2 L). Thionyl chloride (0.036 kg, 0.303 mol) was added with a 10°C exotherm. The mixture was heated to reflux until soUds dissolved. VolatUes (0.1 L) were removed by atmospheric distiUation under nitrogen. The pot residue was cooled to 25°C, dichloromethane (0.2 kg) was added, and the mixture was cooled to -20°C. A solution of (L)- 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 foUowed by friethylamine (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 sodium dUiydrogenphosphate solution (10% aq., 0.075 L each wash). The organic solution was dried with anhydrous sodium sulfate (0.1 kg) and fUtered. The soUds were rinsed with ethyl acetate (0.25 L, and the combined filtrate and rinse was concentrated to an oU under reduced pressure. The oU was dUuted with ethyl acetate (0.25 L), seeded, stirred overnight, and chilled to - 15°C. The soUds were isolated by filtration and dried under reduced pressure to afford 17.7 g of GS-7342 (Table 5) as a tan powder: Η NMR (CDC1,) δ 0.95 (t, 3H), 1.3 (m, 6H), 1.7, (m, 2H), 3.7 (m, 2H), 4.1(m, 6H), 4.4 (dd, IH), 5.8 (s, 2H), 7.1 (m, 5H), 8.0 (s, IH), 8.4 (s, IH); 31 P NMR (CDCI 3 ) δ 21 (decoupled). Example 7 Diastereomer Separation of GS-7097 The diastereomeric mixture was purified using the conditions described for GS-7171 (Example 3 A) except for the foUowing: MobUe Phase (Initial) GS-7120 - AcetonitrUe : Isopropyl Alcohol (95:5) (Final) 100% Methyl Alcohol Elution ProfUe GS-7115 (diastereomer B) GS-7114 (diastereomer A) 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 friethylamine (11.5 mL, 83 mmol) were slurried together in 500 mL pyridine under dry N 2 . This suspension was combined with a solution of friphenylphosphine (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 yeUow paste, 100 g. 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 Uters of 2% methanol in dichloromethane foUowed by a linear gradient over a course of 26 Uters 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 with about 15% (weight) tiiethylamine hydrochloride by H NMR. The contamination was removed by dissolving the product in 350 mL ethyl acetate, extracting with 20 mL water, drying the organic solution over anhydrous sodium sulfate, and concentrating to yield 11.1 g pure GS-7097 as a white soUd, 58% yield. The process also is employed to synthesize the diastereomeric mixture of GS-7003a and GS-7003b (the phenylalanyl amidate) and the mixture 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. Example 9 In Vitro Studies of Prodrug Diastereomers The in vitro anti-HIV-1 activity and cytotoxicity in MT-2 ceUs and stability in human plasma and MT-2 ceU extracts of GS-7340 (ffeebase) and tenofovir disoproxU 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 stabiUty. This greater plasma stabiUty is expected to result in higher circulating levels of GS-7340 than TDF after oral administration. Table 1. In Vitro Activity and Stability In order to estimate the relative intraceUular PMPA resulting from the intraceUular metaboUsm 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 ceUs (RBCs) and peripheral blood mononuclear ceUs (PBMCs) were isolated and analyzed by HPLC with radiometric detection. The results are shown in Table 2. After 1 hour, GS-7340 results in lOx and 30x the total intraceUular concentration of PMPA species in PBMCs as compared to TDF and PMPA, respectively. In plasma after 1 hour, 84% of the radioactivity is due to intact GS- 7340, whereas no TDF is detected at 1 hour. Since no intact TDF is detected in plasma, the lOx difference at 1 hour between TDF and GS-7340 is the nώiimum difference expected in vivo. The HPLC chromatogram for aU 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. Figure 1. HPLC/C-14 Traces of PBMC Extracts from Human Blood Incubated for 1 h at 37°C with TDF, GS-7340 or PMPA. GS-7340/PBMC Met. X and Met Y (metaboUtes X and Y) are shown in Table 5. Lower case "p" designates phosphorylation. These results were obtained after 1 hour in human 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 50 values seen in vitro. In Table 3 below, IC 50 values of tenofovir, TDF, GS-7340, several nucleosides and the protease inhibitor nelfinivir are Usted. As shown, nelfinavir and GS-7340 are 2-3 orders of magnitude more potent than aU other nucleotides or nucleosides. Table 3. In Vitro Anti-HIV-1 Activities of Antiretroviral Compounds 1. A. S. Mulato and J. M. Cherrington, Antiviral Research 36, 91 (1997) Additional studies of the in vitro ceU culture anti-HIV-1 activity and CC 50 of separated diastereomers of this invention were conducted and the results tabulated below. Table 4. Effect of Diastereomer Assay reference: ArimUU, MN, et al., (1997) Synthesis, in vitro biological evaluation and oral bioavaUabUity of 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) prodrugs. Antiviral Chemistry and Chemotherapy 8(6):557-564. "Phe-methylester" is the methylphenylalaninyl monoamidate, phenyl monoester of tenofovir; "gly-methylester" is the methylglycyl monoamidate, phenyl monoester of tenofovir. In each instance above, isomer A is beUeved to have the same absolute stereochemistry as GS-7340 (S), and isomer B is beUeved to have the same absolute stereochemistry that of GS-7339. The in vitro metaboUsm and stability of separated diastereomers were determined in PLCE, MT-2 extract and human plasma. A biological sample Usted below, 80 μL, was transferred into a screw-capped centrifuge tube and incubated at 37°C for 5 min. A solution containing 0.2 mg/mL of the test compound in a suitable buffer, 20 μL, was added to the biological sample and mixed. The reaction mixture, 20 μL, was immediately sampled and mixed with 60 μL 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 μL, was sampled and mixed with 60 μL of methanol contaming 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 foUows. (1) PLCE (porcine Uver carboxyesterase from Sigma, 160 u/mg protein, 21 mg protein/mL) dUuted 20 fold with PBS (phosphated-buffered saline). (2) MT-2 ceU extract was prepared from MT-2 ceUs according to the pubUshed 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 foUows. 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 ceU 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 d/-dithiothreitol. pH 7.4 at 37°C. In the study for human plasma, the test compound was dissolved in TBS. TBS (tris- buffered saline, Sigma) contains 0.05 M Tris, 0.0027 M potassium chloride, and 0.138 M sodium chloride. pH 7.5 at 37°C. The HPLC analysis was carried out under the foUowing conditions. Column: Zorbax Rχ-C8, 4.6 x 250 mm, 5 μ (MAC-MOD Analytical, Inc. Chadds Ford, PA) Detection: UV at 260 nm Flow Rate: 1.0 mL/min Run Time: 30 min Injection Volume: 20 μL Column Temperature: Ambient temperature MobUe Phase A: 50 mM potassium phosphate (pH 6.OVCH3CN = 95/5 (v/v) MobUe Phase B: 50 mM Potassium phosphate (pH 6.OVCH3CN = 50/50 (v/v) Gradient Run: 0 min 100% MobUe Phase A 25 min 100% MobUe Phase B 30 min 100% MobUe Phase B The results are shown below in Table 5 (also including selected IC 50 data from Table 4). Table 5. In Vitro Metabolism of Isomers A and B of PMPA monoamidate at 37°C Example 10 Plasma and PBMC Exposures FoUowing Oral Administration Of Prodrug Diastereomers to Beagle Dogs The pharmacokinetics of GS 7340 were studied in dogs after oral administration 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. AU compounds used in the studies were synthesized by GUead Sciences. The foUowing lots were used: Dose Administration and Sample Collection. The in-life phase of this study was conducted in accordance with the recommendations of the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health pubUcation 86-23) and 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 coUected at 0 (pre-dose), 2,8, and 24 h post-dose. For plasma, blood samples were coUected at 0 (pre-dose), 5, 15, and 30 min, and 1, 2, 3, 4, 6, 8, 12 and 24 h post-dose. Blood (1.0 ml) was processed immediately for 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) drawn at specified time points was mixed in equal proportion with phosphate buffered saline (PBS), layered onto 15 ml of FicoU-Paque solution (Pharmacia Biotech,) and centrifuged at 400 x g for 40 min. PBMC layer was removed and washed once with PBS. Formed PMBC peUet was reconstituted in 0.5 ml of PBS, ceUs were resuspended, counted using hemocytometer and maintained at 70°C until analyzed. The number of ceUs multipUed by the mean single-ceU volume was used in calculation of intraceUular concentrations. A reported value of 200 femtoUters/ceU was used as the resting PBMC volume (B. L. Robins, RN. Srinivas, 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 chloroacetaldehyde to yield a highly fluorescent N 1 , N 6 -ethenoadenine derivative (L. Naesens, J. Balzarini, and E. De Clercq, Clin. Chem. 38, 480 (1992). Briefly, plasma (100 μl) was mixed with 200 μl acetonitrUe to precipitate protein. Samples were then evaporated to dryness under reduced pressure at room temperature. Dried samples were reconstituted in 200 μl derivatization cocktaU (0.34% chloroacetaldehyde inlOO mM sodium acetate, pH 4.5), vortexed, and centrifuged. Supernatant was 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 μl of water for HPLC analysis. Before intraceUular 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 pressure. The dried samples were derivatized as described in the plasma assay. The derivatized samples were mixed with 20 μL of 1M rhamnose and 30 μL of 0.1M sodium periodate and incubated at 37°C for 5 min. FoUowing incubation, 40 μL of 4M methylamine and 20 μL of 0.5M inosine were added. After incubation at 37°C for 30 min, samples were evaporated to dryness under reduced pressure and reconstituted in water for HPLC analysis. No intact prodrug was detected in any PBMC samples. For plasma samples potentiaUy containing intact prodrugs, experiments were performed to verify that no further conversion to PMPA occurred during derivatization. Prodrug standards were added to drug-free plasma and derivatized as described. There were no detectable levels of PMPA present in any of the plasma samples, and the projected % of conversion was less than 1 %. The HPLC system was comprised of a P4000 solvent deUvery system with AS3000 autoinjector and F2000 fluorescence detector (Thermo Separation, San Jose, CA). The column was an InertsU ODS-2 column (4.6 x 150 mm). The mobUe phases used were: A, 5% acetonitrUe in 25 mM potassium phosphate buffer with 5 mM tetrabutyl ammonium bromide (TBABr), pH 6.0; B, 60% acetonitrUe in 25 mM potassium phosphate buffer with 5 mM TBABr, pH 6.0. The flow rate was 2 rnl/min and the column temperature was maintained at 35°C by a column oven. The gradient profUe was 90% A/10% B for 10 min for PMPA and 65%A/35%B for 10 min for the prodrug. Detection was by fluorescence with excitation at 236 nm and emission at 420 nm, and the injection volume was 10 μl. Data was acquired and stored by a laboratory data acquisition system (PeakPro, Beckman, AUendale, NJ). Pharmacokinetic Calculations. PMPA and prodrug exposures were expressed as 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 metaboUsm summary of plasma and PBMC exposures foUowing oral administration of pure diastereoisomers of the PMPA prodrugs. Figure 2. PMPA and Prodrug Concentration in Plasma and PBMCs Following Oral Administration of GS 7340-2 to Dogs at 10 mg-eq/kg. 10 15 20 25 Time Postdose (h) The bar graph in Figure 2 shows the AUC (0-24h) for tenofovir in dog PBMCs and plasma after administration of PMPA s.c, TDF and amidate ester prodrugs. AU 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 Upon Administration of 10 mg-eq/kg in dogs AUC(0-24h) for PMPA in PBMC and Plasma Following an Oral Dose of 10 mg-eq/kg PMPA Prodrugs to Dogs. These data estabUsh in vivo that GS 7340 can be deUvered oraUy, minimizes systemic exposure to PMPA and greatly enhances the intraceUular concentration of PMPA in the ceUs primarily responsible for HIV repUcation. Table 6 PMPA Exposure in PBMC and Plasma from Oral Prodrugs of PMPA in Dogs Example 11 Biodistribution 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 alaninyl monoamidate, phenyl monoester of tenofovir) was examined foUowing oral administration to beagle dogs. Two male animals were dosed oraUy with 14 C=GS- 7340 (8.85 mg-equiv. of PMPA/kg, 33.2 μCi/kg; the 8-carbon of adenine is labeled) in an aqueous solution (50 mM citric acid, pH 2.2). Plasma and peripheral blood mononuclear ceUs (PBMCs) were obtained over the 24-hr period. Urine and feces were cage coUected over 24 hr. At 24 h after the dose, the animals were sacrificed and tissues removed for analysis. Total radioactivity in tissues was determined by oxidation and Uquid scintiUation counting. The biodistribution of PMPA after 24 hours after a single oral dose of radiolabeUed GS 7340 is shown in Table 4 along with the data from a previous study with TDF (GS-4331). In the case of TDF, the prodrug concentration in the 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 bone marrow is probably due to the high percentage of lymphocytes (70%) in this tissue. 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. n.s. = no sample, n.a. = not app icable, n. d. = = not eterm ne .

Claims: CLAIMS: 1. A screening method for identifying a methoxyphosphonate nucleotide analogue prodrug conferring enhanced activity in a target tissue comprising: (a) providing at least one of said prodrugs; (b) selecting at least one therapeutic target tissue and at least one non- target tissue; (c) adrninistering the prodrug to the target tissue and to said at least one non-target tissue; and (d) determining the relative activity conferred by the prodrug in the tissues in step (c). 2. The method of claim 1 wherein the activity is antiviral activity or antitumor activity. 3. The method of claim 2 wherein the activity is antiviral activity. 4. The method of claim 3 wherein the activity is anti-HIV or anti-HBV activity. 5. The method of claim 1 wherein the prodrug is a prodrug of PMPA or PMEA. 6. The method of claim 5 wherein the prodrug is a phosphonoamidate, phosphonoester or mixed phosphonoamidate /phosphonoester. 7. The method of claim 6 wherein the amidate is an amino acid amidate. 8. The method of claim 6 wherein the ester is an aryl ester. 9. The method of claim 1 further comprising selecting a prodrug having a relative activity in the target tissue that is greater than 10 times that of the non- target tissue. 10. The method of claim 1 wherein the target and non-target tissue are in an animal, the prodrug is administered to the animal and the relative activity is determined by analysis of the animal tissues after administration of the prodrug. 11. The method of claim 1 wherein activity in the target and non-target tissues is determined by assaying the amount of at least one metaboUte of the prodrug in the tissues. 12. The method of claim 12 wherein the metaboUte is the parental drug. 13. The method of claim 12 wherein the metaboUte is the diphosphate of the parental drug. 14. The method of claim 1 wherein the target tissue is viraUy infected tissue and the non-target tissue is the same tissue which is not viraUy infected. 15. The method of claim 1 wherein the target tissue is lymphoid tissue and the activity is anti-HIV activity. 16. The method of claim 1 wherein the target tissue is Uver and the activity is anti-HBV activity. 17. The method of claim 1 wherein the target tissue is hematological and the activity is antitumor activity. 18. The method of claim 1 wherein the target tissue is maUgnant and the non- target tissue is the same tissue but non-mahgnant. 19. A compound having the structure (1) (i) where Ra is H or methyl, and chiraUy enriched compositions thereof, salts, their free base and solvates thereof. 20. A compound having the structure (2) (2) and its enriched diasteromers, salts, free base and solvates. 21. A diastereomericaUy enriched compound having the structure (3) which is substantiaUy free of the diastereomer (4) wherein R is an oxyester which is hydrolyzable in vivo, or hydroxyl; B is a heterocycUc base; 2 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 1 2 the amino acid optionaUy esterified, but not both of R and R are hydroxyl; E is -(CH2)2-/ -CH(CH3)CH2-, -CH(CH2F)CH 2 -, -CH(CH2θH)CH2~, -CH(CH=CH2)CH2-, -CH(C≡CH)CH2-, -CH(CH2N3)CH2-, -CH(R 6 )OCH(R 6' )-, -CH(R 9 )CH2θ- or -CH(R 8 )0-, wherein the right hand bond is linked to the heterocycUc base; the broken line represents an optional double bond; 4 5 R and R are independently hydrogen, hydroxy, halo, amino or a substituent having 1-5 carbon atoms selected from acyloxy, alkyoxy, alkylthio, alkylamino and dialkylamino; R and R are independently H, C α -C 6 alkyl, C--C 6 hydroxyalkyl, or C 2 -C 7 alkanoyl; 7 R is independently H, C j -C 6 alkyl, or are taken together to form -O- or -CH 2 -; 8 R is H, C j -C 8 alkyl, C α -C 6 hydroxyalkyl or C^C g haloalkyl; and 9 R is H, hydroxymethyl or acyloxymethyl; and their salts, free base, and solvates. 22. A diastereomericaUy enriched compound having the structure (5a) (5a) which is substantiaUy free of diastereomer (5b) (5b) wherein 5 R is methyl or hydrogen; independently is H, alkyl, alkenyl, alkynyl, aryl or arylalkyl, independently is alkyl, alkenyl, alkynyl, aryl or arylalkyl which is substituted with from 1 to 3 substituents selected from alkylamino, alkylaminoalkyl, dialkylaminoalkyl, dialkylamino, hydroxyl, oxo, halo, amino, alkylthio, alkoxy, alkoxyalkyl, aryloxy, aryloxyalkyl, arylalkoxy, arylalkoxyaUcyl, haloalkyl, nitro, nitroalkyl, azido, azidoalkyl, alkylacyl, alkylacylalkyl, carboxyl, or alkylacylamino; 7 R is the side chain of any naturaUy-occurring or pharmaceuticaUy acceptable amino acid and which, if the side chain comprises carboxyl, the carboxyl group is optionaUy esterified with an alkyl or aryl group; 11 R is amino, alkylamino, oxo, or dialkyla_mino; and 12 R is amino or H; and it salts, tautomers, free base and solvates. 23. A compound of structure (6) and its salts and solvates. 24. A compound of structure (7) 25. A composition comprising a compound of any of claims 19-24 and a pharmaceuticaUy effective excipient. 26. The composition of claim 25 wherein the excipient is a gel. 27. The composition of claim 25 which is suitable for topical administration. 28. A method for antiviral therapy or prophylaxis comprising administering a compound of any of claims 19-24 in a therapeuticaUy or prophylacticaUy effective amount to a subject in need of such therapy or prophylaxis. 29. A method for use of magnesium alkoxide comprising reacting 9-(2- hydroxypropyl)adenine (HPA) or 9-(2-hydroxyethyl)adenine (HEA), magnesium alkoxide, and protected p-toluenesulfonyloxymethylphosphonate. 30. The method of claim 29 further comprising recovering PMPA or PMEA, respectively. 31. The method of claim 29 wherein the phosphonate of the p- toluenesulfonyloxymethylphosphonate is protected by ethyl ester. 32. The method of claim 29 wherein the alkoxide is a C 1 -C 6 alkoxide. 33. The method of claim 32 wherein the alkoxide is t-butyl or isopropyl oxide.

Inventors: BECKER MARK W, CHAPMAN HARLAN H, CIHLAR TOMAS, EISENBERG EUGENE J, HE GONG-XIN, KERNAN MICHAEL R, LEE WILLIAM A, PRISBE ERNEST J, ROHLOFF JOHN C, SPARACINO MARK L, BECKER, MARK, W., CHAPMAN, HARLAN, H., CIHLAR, TOMAS, EISENBERG, EUGENE, J., HE, GONG-XIN, KERNAN, MICHAEL, R., LEE, WILLIAM, A., PRISBE, ERNEST, J., ROHLOFF, JOHN, C., SPARACINO, MARK, L.
Applicants: BECKER MARK W, CHAPMAN HARLAN H, CIHLAR TOMAS, EISENBERG EUGENE J, GILEAD SCIENCES INC, HE GONG XIN, KERNAN MICHAEL R, LEE WILLIAM A, PRISBE ERNEST J, ROHLOFF JOHN C, SPARACINO MARK L, BECKER, MARK, W., CHAPMAN, HARLAN, H., CIHLAR, TOMAS, EISENBERG, EUGENE, J., GILEAD SCIENCES, INC., HE, GONG-XIN, KERNAN, MICHAEL, R., LEE, WILLIAM, A., PRISBE, ERNEST, J., ROHLOFF, JOHN, C., SPARACINO, MARK, L.
Application year: 2001
Main classification: C07H21
IPC codes: A61K31, A61P31, A61P35, C07F9, C07H19, C07H21, C12Q1, G01N33