Title of Invention

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

Abstract

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) administering 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 tissue in step (c).

Full Text

FORM 2 THE PATENTS ACT 1970 [39 OF 1970] COMPLETE SPECIFICATION ^- [See Section 10] "PRODRUGS OF PHOSPHONATE NUCLEOTIDE ANALOGUES AND METHODS FOR SELECTING AND MAKING SAME" GILEAD SCIENCES, INC., a corporation organized under the laws of the State of Delaware, having a mailing address of 333 Lakeside Drive, Foster City, California 94404, United States of America, GRANTED The following specification particularly describes the nature of the invention and the manner in which it is to be performed:- 9-5-2006 1 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 such prodrugs. Many methoxyphosphonate 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. R (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. 2 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 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 HIV 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, pursuant to these 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 tissue; (c) administering the prodrug to the target tissue and to said at least one non- target tissue; and 25 (d) deterrnirtmg'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), 3 JPCT/US01/23104 (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) 15 20 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 4 5 invention, we provide diastereomerically enriched compounds of this invention having the structure (3) which are substantially free of the diastereomer (4) 15 20 wherein i 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 add bonded to the P atom through an amino group of the amino acid and having each carboxy substituent of 1 2 25 the amino acid optionally esterified, but not both of R andR arehydroxyl; E is -(CH2)2-, -CH(CH3)CH2-, -CH(CH2F)CH2-v -CH(CH20H)CH2-, -CH(CH=CH2)CH2-,-CH(CsCH)CH2-,-CH(CH2N3)CH2-, 30 -CH(R6)OCH(R6K-CH(R9)CH20-or-CH(R8)0-, wherein the right hand bond is 35 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 dialkykmino; 5 R and R are independently H, C1-C6 alkyl, C1-C6 hydroxyalkyl, or C2-C7 alkanoyl; 7 R is independently H, C1-C6 alkyl, or are taken together to form -O- or 8 R is H, CrC6 alkyl, C1-C6 hydroxyalkyl orC1-C6 haloalkyk and 9 10 R is H, hydroxymethyl or acvloxymethyl;_ and their salts, free base, and solvates. The diastereomers of structure (3) are designated the (s) isomers at the phosphorus ehiral center. Preferred embodiments of this invention are the diastereomerically enriched 15 compounds having the structure (5a) R11 (5a) 25 which is substantially free of diastereomer (5b) (5b) 6 5 wherein 5 R is methyl or hydrogen; R independently is H, alkyl, alkenyl, alkynyl, aryl or arylalkyl, or R independently is alkyl, allcenyl, alkynyl, aryl or arylalkyl which is substituted with from 1 to 3 substituents selected from alkykmino, alkylaminoalkyl, 10 dialkykininoalkyl, dialkylarnino, 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 anynatm^ally-occurringorpharmaceutically acceptable amino acid and which, if the side chain comprises carboxyl, the carboxyl 15 group is optionally esterified with an alkyl or aryl group; R is amino, alkykmino, oxo, or dtelkylamino; and 12 R 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), 9-[(R)-2-[[(S)-[[(S)-l- (isopropoxycarbonyl)eihyl]ammo]phenoxyphosphmyl]methoxy]propyl]adenine, also designated herein GS-7340 7 m2 30 CH3 25 35 Another preferred embodiment of this invention is the fumarate salt of structure (5) (structure (7)), 9-[(R)-2-[[(S)-[[(S)-l- 7 PCT/US01/23104 (i^ropoxycarbonyl)ethyl]anuno]phenoxyphosphmyl]methoxy]propyl]adenine fumarate (1:1), also designated herein GS-7340-2 (7) The compounds of structures (1)-(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 9-[2-(phosphonomethoxy)propyl]adenine (hereinafter "PMPA" or 9-[2-(phosphonomethoxy)ethyl] adenine (hereinafter "PMEA") using magnesium allcoxide, which comprises combining 9-(2-hydroxypropyl)adenine or 9-(2-hydroxyethyljaderrine, 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 v 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 O B—E—P—OH OH 8 ! ! 5 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 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 monoamine 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 TU.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 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. 9 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 on the 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 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 amiddtes may be D or L forms, and the phosphonate esters or the amino acid esters can tohtain 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 chirally 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%. 10 PCT/USOl/23104 5 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 latex 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 HIV infection. Lymphoid tissue will be loiown 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 11 WO02/U8241 PCTAJS01/23104 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 untransformed 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 of 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 virally-uninfected cells 35 (corresponding non-target tissue). The same strategy would be employed to select 12 WO 02/08241 PCTAJS01/23104 5 prophylactic prodrugs, i.e., prodrugs metabolized to antivirally active forms incidental to viral infection but which remain substantially unmetabolized in uninfected cells. Similarly, 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. leukemias. 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 35 tissues. 13 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 20 invention. 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 ahtivrally 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 14 . 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 the proper adjustments would be within the ordinary skill. Thus, when step (d) of the method herein calls for deterrnining 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 perse. 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 the hepatic circulation without substantial metabohsm. 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 15 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 synthonp-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 Cx- 3S C6branched or normal alkyl, but is preferably t-butyl (for PMPA) or isopropyl (for 16 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 (usually with bromotrimethylsilane where the tosylate protecting group is alkyl), and the product then recovered by crystallization or other conventional method as will be apparent to the artisan. Heterocyclic Base In the compounds of this invention depicted in structures (3) and (4), the heterocyclic base B is selected from the structures B1S $22 0 518 17 5 -GH2CH20C2H5,-CH2CH20CCH,-CH2CH20CH2CHCH2,and -CH2CH2OC3H7; R2°isNorCH; R^isN,CH,CCN,CCF3,CCsCHorCC(0)NH2; : R22 is H, OH, NH2, SH, SCH3, SCH2CH3, SCH2CCH, SCH2CHCH2, SC3H7, 10 NH(CH3), N(CH3)2, NH(CH2CH3), N(CH2CH3)2, NH(CH2CCH), NH(CH2CHCH2), NH(C3H7), halogen (F, CI, 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 H, 15 Q-C15 alkyl, C2-Q5 alkenyl, C6-Q5 arylalkenyl, C6-C15 arylalkynyl, C2-C15 alkynyl, Ci-C6~aIkylamino-Ci-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroaralkyl, C5-C6 aryl, C2-C6 heterocycloalkyl, C2-C15 alkyl, C3-C15 alkenyl, C6-Q5 arylalkenyl, C3-C15 alkynyl, C7-C15 arylalkynyl, Ci-C6-alkykmino-Ci-C6 alkyl, C5-C15 aralkyl, C6-C15 20 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in the alkyl moiety not adjacent to N6 has been replaced by -0-, 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, a, Br, I, SCH3, SCH2CH3, SCH2CCH, SCH2CHCH2, SC3H7, OR16, NH2, NHRW or R22; and R24isO,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'-dimethoxytaityl (DMT) and the 18 WO 02/08241 PCTAJS01/23104 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, N^-isobutyrylguanine and the like. 10 Protected bases'have the formulas Xa.l, XIa.l, Xlb.l, XIIa.1 or XIIIa.1 (Xa.l) (XIa.1) (Xlb.l) (Xna.l) (XHIa.l) 20 wherein R14, R20, R21, R24 have the meanings previously defined; R2A is R39 or Ra provided that R22 is not NH2; Rm is R39or R^provided that R23 is not NH2; R3' is NHR40, NHC(0)R36 or CR41N(RM)2 wherein R3S is C1-C19 alkyl, C1-C19 alkenyl, €3-C10 aryl, adamanfoyl, alkylanyl, or C3-C10 aryl substituted with 1 or 2 atoms or groups selected from halogen, methyl, ethyl, methoxy, ethoxy, hydroxy and cyano; 25 R38 is C1-ClO alkyl, or both R3a together are lTinorpholino, 1-piperidine or 1-pyrrolidine; R40 is C1-C10 alkyl, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, octyl and decanyl; and R41 is hydrogen or CH3; 39 22 A 23A For bases of structures XIa.l and Xlb.l, if R is present at R orR ,both 39 36 R groups on the same base will generally be the same. Exemplary R are phenyl, 36 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, heptanyl, nonanyl, undecanyl, or undecenyl. Specific bases include hypoxanrhine, guanine, adenine, cytosine, inosine, myrnine, uracil, xanthine, 8-aza derivatives of 2-arninopurine, 2,6-diarninopurine, 35 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza-8-aza 19 WO 02/08241 PCT/US01/23104 5 derivatives of adenine, guanine, 2-aminopurine, 2,6-dianunopurine, 2-ainino-6-dhloropurine, hypoxanthine, inosine and xanthine; 1-tieaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6~cMoropurine, hypoxanthine, inosine and xanthine; 7-deaza derivatives of 2-aminopurine, 2,6-diairunopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 3-deaza derivatives of 2- 10 aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine; 5-bromocytosine; 5-rnethylcytosine; 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil; 5-ethynyluracil and 5-propynyluracil. 15 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-aininopurinyl and 6-thio-2-arninopurinyl, or a B' is a l~pyrimidinyl residue selected from cytosinyl, 5-halocytosinyl, and 5-(C1-C3-alkyl)cytosinyL 20 Preferred B groups have the formula R22 25 wherein R22 independently is halo, oxygen, NH2, X or H, but optionally at least one R22 is X; - X is-(CH2)m(O)n(CH2)mN(R10)2 wherein m is 0-2, n is 0-1, and 30 RlO independently is H, C1-G15 alkyl, C2-C15 alkenyl, C6-C15 arylalkenyl, C6-C15 arylalkynyl, C2-C15 alkynyl, C1-C6^-alkylamino-C1-C6 alkyl, C5-C15 aralkyl, C6 C15 heteroaralkyl, C5-C6 aryl, C2-C6 heterocycloalkyl, 20 C2-C15 alkyl, C3-C15 alkenyl, C6-C15 arylalkenyl, C3-C15 alkynyl, C7-C15 arylalkynyl, C1-C6-alkylamino-C1-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in the alkyl moiety not adjacent to N6 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, or one of the foregoing R10 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 CH, provided that the heterocyclic 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(CHj)CH2- or -CH2CH2-. 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) enantiomer). R1 is an in vivo hydrolyzable oxyester having the structure -OR35 or -OR6 wherein R36 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, ordinarily unsubstituted or para-substituted (as defined in R6) phenoxy. 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. Ra typically has the structure wherein (8) 21 5 n is or 2; R11 is R6 or H; preferably R6 = C3-C9 alkyl; Q-C, 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-C6arylalkyl which is independently substituted with OH, halogen, O or N; 10 R12 independently is H or C3-C9 alkyl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR11 and halogen; C3-C6saryl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR" and halogen; or C3-C9 aryl-alkyl which is unsubstituted or substituted by 15 substituents independently selected from the group consisting of OH, O, N, COOR" and halogen; Rw independently is C(0)-ORn; amino; amide; guanidinyl; imidazolyl; indolyl; sulfoxide; phosphoryl; C3-C6, alkylamino; C1C3 alkyldiamino; C3-C6 alkenykmino; hydroxy; thiol; C1C3 alkoxy; C3-C6 alkthiol; (CH^COOR11;C3-C6 20 alkyl which is unsubstituted or substituted with OH, halogen, SH, NH^, phenyl, hydroxyphenyl or C7-C10, alkoxyphenyl; C2-C6 alkenyl which is unsubstituted or substituted with OH, halogen, SH, NH2, phenyl, hydroxyphenyl orC7-C10 alkoxyphenyl; and C8-C12 aryl which is unsubstituted or substituted with OH, halogen, SH, NH^, phenyl, hydroxyphenyl or Q-C10 alkoxyphenyl; and 25 R" is H or C1-C9 alkyl or Ct-C9 alkyl independentiy substituted with OH, halogen, COOR11,0 or N; C3-C6 aryl; C3-C6 aryl which is independently substituted with OH, halogen, COOR", O or N; or C3-C6 arylalkyl which is independently substituted with OH, halogen, COOR11,0 or N. Preferably, R11 is C3-C6 alkyl, most preferably isopropyl, R13 is the side chain 30 of a naturally occurring amino acid, n = 1, Ru is H and Ru is H. In the compound of structure (2), the invention includes 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 included within the scope of this invention. 35 Aryl and "O" or "N" substitution are defined in column 16, lines 42-58, of 22 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 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, 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: 23 TYW Uii|U014i rci/uaui/zjiuf vacuo at 56°C to yield 20g (65.4%) of 942-(phosphonomethoxy)ethyl]adeiune (PMEA) as a white solid. Mp: > 200°C dec. H NMR (D20) 3.49 (t, 2H); 3.94 (t, 2H); 4.39 (t, 2H); 8.13 (s, 1H); 8.22 (s, 1H). Example lb 10 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 mol) and sodium hydroxide C480g, 0.012 mol). The mixture was heated at 130°C overnight. The reaction was cooled to 100°C and toluene (138 ml) was added 15 followed by methanesulfomc 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- 20 [2-(hydroxy)propyl]adenine (HPA) as an off-white solid. Mp: 188-190°C. 25 (R)-9-[2-(hydroxy)propyl]adeiune (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 followed by diethyl p-toluenesulfonyloxymethylphosphonate (66.0g, 0.205 mol) 24 WO 02/08241 PCT/US01/23104 i * 5 over two hours while the temperature was kept at 78°C. The mixture was stirred at 75°C for 4 hours. After cooling to below 50°C, bromotrrmethylsilane (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 (R-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(R)-9-[2- (phosphonomethpxy)propyl]adenine (PMPA) as an off-white solid. The crude(R)-9-[2-(phosphonornethoxy)propyl]adenine was heated at reflux in water (255 ml) until all 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 (R)-9-[2-(phosphonomemoxy)propyl]adenine (PMPA) as a white solid. Mp: 278-280°C. 25 PCT/US01/23104 Example 2 Preparation of GS-7171 (IE) Scheme 1 r^H2 QH 26 5 A glassrlined reactor was charged with anhydrous PMPA, (I) (14.6 kg, 50.8 mol), phenol (9.6 kg, 102 moD, and l-methyl-2-pyrrohdrnone (39 kg). The mixture was heated to 85°C andjiiethykmine (6.3 kg, 62.3 mol) added. A solution of 13-dicydohexylcarbodiirnide (17.1 kg, 82.9 mol) in l-methyl-2-pyrroKdinone (1.6 kg) was then added over 6 hours at 100°C. 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). Pines 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) 815.0 (decoupled). GS-7171 (III). (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 Memylamine (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 35 (18.2 kg), filtered, rinsed with dichloromethane (28 kg), and concentrated to an oil 27 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. Qean 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 IH as a dark oik *H 15 NMR (CDCL.) 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 (CDC1,) 5 21.0,22.5 (decoupled). 20 Alternate Method for GS-7171(nT) Scheme 2 35 28 5 Monophenyl PMPA (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), N,N-dimethy formamide (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-7X71 OH). Monophenyl PMPA (II) (3 g, 8.3 mmol), tetramethylene sulfone (5 20 mL), and N,N-dimethyHormamide (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 29 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 10 The diastereomers of GS-7171 (III) were resolved by batch elution chromatography using a commercially available Chiralpak AS, 20 urn, 21 x 250 mm semi-preparative HPLC column with a Chiralpak AS, 20 urn, 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 35,919,5,434,298,5,434,299 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 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. 30 The chromatographic conditions were as follows: Mobile Phase(InitiaD (Final) Flow RunTime Detection Temperature Elution Profile GS-7171 - Acetonitrile: Isopropyl Alcohol (90:10) 100% Methyl Alcohol 10 mL/rninute About 45 minute UVat275nm Ambient GS-7339 (diastereomer B) GS-7340 (diastereomer A; (IV)) B. Diastereomer Separation of GS-7171 by SMB chtromatography 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 acetonitrile. Product bearing fractions were concentrated to a 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; XH NMR (CDCL.) 51.15 (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); MP NMR (CDCL.) 8 21.0 (decoupled). C. Diastereomer Separation by C18RP-HPLC GS-7171 (III) was chromatographed by reverse phase HPLC to separate the diastereomers using the following summary protocol. 31 WO 02/08241 PCTAJS01/23104 10 15 Chromatographic column: Phenomenex Luna™ C18(2), 5 urn, 100 A pore size, (Phenomenex, Torrance, CA), or equivalent Guard column: Pellicular C18 (Alltech, Deerfield, IL), or equivalent Mobile Phase: A—0.02% (85%) H3PO4 in water; acetonitrile (95:5) B — 0,02% (85%) H3PO4 in water: acetonitrile (50:50) 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 20 25 Run Time: Equilibration Delay: Flow Rate: Temperature: Detection: Sample Solution: Retention Times: 50 minutes 10 min at 100% mobile phase A 1.2 mL/rnin Ambient UVat260nm . 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 (III) 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% diastereomeric purity (31P NMR): *H NMR (CDCL) 5 35 1.15 (m 12H), 3,7 (t, 1H), 3.95 (m, 2H), 4.05 (m, 2H), 4.2 (m, 2H), 5.0 (m, 1H), 6.4 (s, 2H), 7.1 (m, 5H), 8.0 (s, 1H), 8.2 (s, 1H); 3lP NMR (CDCI3) 819.5 (decoupled). X-ray crystal analysis of a single crystal selected from this product yielded the following data: 32 WO 02/08241 PCT/USO1/23104 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 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; [a]D20 -41.7° (c 1.0, acetic acid). 33 5 10 WO 02/08241 PCT/US01/23104 Example 5 Preparation of GS-7120 (VI) Scheme 3 Nl-U NH2 Wz 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 arninobutyric 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 triemylamine (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 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. 34 WO02/08241 PCT/US01/23104 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 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) Example 6 Diastereomer Separation of GS-7120 by Crystallization AIL 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 mixture was heated to reflux until solids dissolved. Volatiles (0.1 L) were removed by atmospheric distillation 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)-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 Memylamine (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 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 filtrate 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 isolated by filtration and dried under reduced pressure to afford 17.7 g of GS-7342 (Table 5) as a tan powder: aH NMR (CDC1,) 5 0.95 (t, 3H), 1.3 (m, 6H), 1.7, (m, 2H), 35 WO 02/08241 PCT/US01/23104 5 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); 31P NMR (CDCL) 5 21 (decoupled). Example 7 Diastereomer Separation of GS-7097 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 triethylamine 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), Aldrithiol2 (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 ehited 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) Memylamine hydrochloride by HNMR. 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 36 WO 02/08241 PCT/US01/23104 5 sulfate, and concentrating to yield 11.1 g pure GS-7097 as a white solid, 58% yield. The process also is employed to synthesize theiastereomeric mixture of GS-7003a and GS-7003b (the phenylalanyl amidate) and the mixture GS-7119 and GS-7335 (the glycol amidate). These diastereomers are separated using a batch elution procedure such as shown in Example 3A, 6 and 7. 10 Example 9 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 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. 37 WO 02/08241 PCT/US01/23104 After 1 hour, GS-7340 results in lOx and 30x the total intracellular 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 minimum difference expected in vivo. The HPLC 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. 38 PLT/USO1/23104 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, 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 blood. With increasmgtime, 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, IC^ values of tenofoviir, TDF, GS-7340, several nucleosides 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. 39 WO 02/08241 PCT/US01/23104 Table 3. In Vitro Anti-HIV-1 Activities of Antiretroviral Compounds 1. A. S. Mulato and J. M. Cheiringtoa, Antiviral Research 36,91 (1997) Additional studies of the in vitro cell culture anti-HIV-1 activity and CC50 of separated diastereomers of this invention were conducted and the results tabulated below. 40 WO 02/08241 PCTAJS01/23104 5 Table 4. Effect of Diastereomer Assay reference: Arimilli, MN, et al., (1997) Synthesis, in vitro biological evaluation and oral bioavailability of 9-[2-(phosphonomethoxy)propyl]adenine 10 (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. 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. 41 5 The in vitro metabolism and stability of separated diastereomers were determined in PLCE, MT-2 extract and human plasma. A biological sample listed below, 80 JJL, 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 uL, was added to the biological sample and mixed. The reaction 10 rruxture, 20 uL, was immediately sampled and mixed with 60 uL of methanol containing 0.015 mg/mL of 2-hydroxymethymaphthalene 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 JJ.L, was sampled and mixed with 60 u.L of methanol containing the internal standard. The mixture thus obtained was 15 centrifuged at 15/300 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 20 protein/mL) diluted 20fold 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. 25 (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-30 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 dZ-dithiothreitoL pH 7.4 at 37°C. 42 WOU2/U8241 PCT/US01/23104 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 following conditions. Column: Zorbax RX-C8,4.6 x 250 mm, 5 u (MAC-MOD Analytical, Inc. Chadds Ford, PA) Detection: UVat260nm Flow Rate: l.OmL/rnin RunTime: 30min Injection Volume: 20 jjL Column Temperature: Ambient temperature Mobile Phase A: 50 mM potassium phosphate (pH 6.0)/CH3CN = 95/5 (v/v) Mobile Phase B: 50 mM Potassium phosphate (pH 6.OVCH3CN = 50/50 (v/v) Gradient Run: 0 min 100% Mobile Phase A 25 min 100% Mobile Phase B 30 min 100% Mobile Phase B The results are shown below in Table 5 (also including selected ICS0 data from Table 4). 43 44 WUU2/08241 PCT/USOl/23104 Example 10 Plasma and PBMC Exposures Following Oral Administration Of Prodrug Diastereomers to Beagle Dogs The pharmacokinetics of GS 7340 were studied in dogs after oral 10 administration of a 10 mg-eq/kg dose. Formulations. The prodrugs were formulated as solutions in 50 rnM 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: 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 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 45 PCT/US01/23104 5 washed once with PBS. Formed PMBC pellet was reconstituted in 0.5 ml of PBS, cells were resuspended, counted using hemocytometer and maintained 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, R.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 highlyfluorescent 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 precipitate protein. Samples were then evaporated to dryness under reducedpressure at room temperature. Dried samples were reconstituted in 200 ul derivatization cocktail (0.34% chloroacetaldehyde in 100 20 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 rnin. Derivatized samples were then evaporated to dryness and reconstituted in 100 ul of water for HPLC analysis. 25 Before intracellular 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 rhamnose and 30 uL of 0.1M sodium periodate and inaabated at 370Cfor5rriin. Following incubation, 40uLof 4M memylamine and 20 uL of 0.5M inosine were added. After incubation at 37°C for 30 rnin, samples were evaporated to dryness under reduced pressure and 35 reconstituted in water for HPLC analysis. 46 5 No intact prodrug was detected in any PBMC samples. For plasma samples potentially containing intact prodrugs, experiments were performed to verify that no furmer conversion to PMPA occurred during derivatization. Prodrug standards were added to drug-freeplasma and derivatized as 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 autoinjector 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 SmM tetrabutyl ammonium bromide (TBABr), pH 6.0; B, 60% acetonitrile in 25 mM potassium phosphatebuffer with 5 mM TBABr, pH 6.0. The flow rate was 2 ml/rnin 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 run and emission at 420 ran, 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. 47 WU U2/08241 PCT/USO1/23104 Kgure 2. PMPA and Prodrug Concentration in Plasma and PBMCs Following Oral Administration of GS 7340-2 to Dogs at 10 mg-eq/kg. 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. 48 WO 02/08241 Figure 3. Depicts Tenof ovir Exposure in PBMCs and Plasma Upon Administration of 10 mg-eq/kg in dogs 10 AUC(0-24h) for PMPA in PBMC and Plasma Following an Oral Dose of 10 mg-eq/kg PMPA Prodrugs to Dogs. 15 20 25 30 These data establish in vivo that GS 7340 can be delivered orally, xrunirnizes systemic exposure to PMPA and greatly enhances the iatracellular concentration of PMPA in the cells primarily responsible for HIV replication. 49 Table 6 PMPA Exposure In PBMC and Plasma from Oral Prodrugs of PMPA in Doas 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 following oral administratioii to beagle dogs. Two male animals were dosed orally with WC=GS-7340 (8.85 mg-equiv. of PMPA/kg, 33.2 uCi/kg; the 8-carbon of adenine is labeled) in an aqueous solution (50 mM citric add, 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 and tissues removed for analysis. Total radioactivity in tissues was determined fay oxidation and liquid scintillation counting. The biodistribution of PMPA after 24 hours after a single oral dose of radiolabeled 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. 50 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. * Calculated using typical recovery of 15 x 10s cells total, and mean PBMC volume of 0.2 picollters/cell n.s. =s no sample, n.a. = not applicable, n.d. = not determined. 51 We claim : 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) administering 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 tissue in step (c). 2. The method as claimed in claim 1 wherein the activity is antiviral activity or antitumor activity. 3. The method as claimed in claim 2 wherein the activity is anti-HIV or anti-HBV activity. 4. The method as claimed in any preceding claim wherein the prodrug is a prodrug of PMPA or PMEA. 5. The method as claimed in any preceding claim wherein the prodrug is a phosphonoamidate, phosphonoester or mixed phosphonoamidate/phosphonoester. 6. The method as claimed in claim 5 wherein the amidate is an amino acid amidate. 7. The method as claimed in claim 6 wherein the ester is an aryl ester. 8. The method as claimed in any preceding claim wherein said prodrug has relative activity in the target tissue that is greater than 10 times that of the non-target tissue. 9. The method as claimed in any preceding claim 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. 10. The method as claimed in any preceding claim wherein activity in the target and non-target tissues is determined by assaying the amount of at least one metabolite of the prodrug in the tissues. 11. The method as claimed in claim 10 wherein the metabolite is the parental drug. 52 12. The method as claimed in claim 11 wherein the metabolite is the diphosphate of the parental drug. 13. The method as claimed in any preceding claim wherein the target tissue is virally infected tissue and the non-target tissue is the same tissue which is not virally infected. 14. The method as claimed in any one of claims 1 to 12 wherein the target tissue is tympbsrid tissue, 15. The method as claimed in any one of claims 1 to 12 wherein the target tissue is liver and the activity is anti-HBV activity. 16. The method as claimed in any one of claims 1 to 12 wherein the target tissue is hematological and the activity is antitumor activity. 17. The method as claimed in any one of claims 1 to 12 wherein the target tissue is malignant and the non-target tissue is the same tissue but non-malignant. Dated this the 2nd day of January 2003. 7 53

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