USOO8324179B2

(12) United States Patent (10) Patent No.: US 8,324,179 B2 Chen et al. (45) Date of Patent: Dec. 4, 2012

(54) NUCLEOSIDE ANALOGS FOR ANTIVIRAL WO WO-96,07666 A1 3/1996 TREATMENT WO WO-03/073989 A2 9, 2003 WO WO-2004/096286 A2 11/2004 WO WO-2006/015261 A2 2, 2006 (75) Inventors: James M. Chen, San Ramon, CA (US); Alan X. Huang, San Mateo, CA (US); OTHER PUBLICATIONS Richard L. Mackman, Millbrae, CA Lera et al. Organic Letters (2000), vol. 2, pp. 3873-3875.* (US); Jay Parrish, Redwood City, CA Abbas et al. (2000) “An Improved procedure for the synthesis of (US); Jason K. Perry, San Francisco, vinylphosphonate-linked nucleic acids.” Tetrahedron Letters AY 41:4513-4517. CA (US); Oliver L. Saunders, San Abbas et al. (2001) “Commercially Available 5'-DMT Mateo, CA (US); David Sperandio, Palo Phosphoramidites as Reagents for the Synthesis of Alto, CA (US) Vinylphosphonate-Linked Oligonucleic Acids.” Organic Letters 3(21):3365-3367. (73) Assignee: Gilead Sciences, Inc., Foster City, CA Abbas, S. etal (1999) “A Novel Palladium-Catalysed Coupling Strat (US) egy for the Rapid Synthesis of Nucleic Acid Analogues Bearing Modified Backbones.” Synlett 7:1124-1126. c Almer et al. (1991) “Synthesis of a Phosphonomethyl Analogue of (*) Notice: Subject to any disclaimer, the term of this 3'-Deoxy-3'-fluorothymidine.” Acta Cheinica Scandinavica 45:766 patent is extended or adjusted under 35 767. U.S.C. 154(b) by 486 days. Bertram et al. (2002) “Vinylphosphonate Internucleotide Linkages inhibit the Activity of Per A DNA Helicase.” Biochemistry 41:7725 7731. (21) Appl. No.: 12/524,545 Freeman et al. (1992) “3'-Azido-3',5'-dideoxythymidine-5'- 1-1. methylphosphonic Acid Disphosphate: Synthesis and HIV-1 Reverse (22) PCT Filed: Feb. 8, 2008 Transcriptase Inhibitors.” J. Med. Chem 35:3192-3196. Garvey et al. (1998) “Nucleotide and Nucleoside Analogues as (86). PCT No.: PCT/US2O08/OO1743 Inhibitors of Cytosolic 5'-Nucleotidase I from Heart.” Biochemistry 17:9043-9051. S371 (c)(1), Hai et al. (1982) "Species- or isozyme-Specific Enzyme Inhibitors. (2), (4) Date: Nov.30, 2009 7. Selective Effects in Inhibitions of Rat Adenylate Kinase isozymes by Adenosine 5'-Phosphate Derivatives.”.J. Med. Chem. 25:806-812. (87) PCT Pub. No.: WO2008/100447 Hampton et al. (1976) “Evidence for the Conformation About the PCT Pub. Date: Aug. 21, 2008 C(5') -O(5') Bond of AMP Complexed to AMP Kinase: Substrate l ale: Aug. All, Properties of a Vinyl Phosphonate Analog of AMP Bioorganic O O Chemistry 5:31-35. (65) Prior Publication Data International Preliminary Report on Patentability for PCT/US2008/ US 2010/O104532 A1 Apr. 29, 2010 00.1743, issued Aug. 11, 2009. International Search Report and Written Opinion for PCT/US2008/ Related U.S. Application Data 00.1743, mailed May 11, 2008. Jones (1968) “The Synthesis of 6'-Deoxyhomonucleoside-6'- (60) Provisional application No. 60/900,692, filed on Feb. phosphonic Acids,” Journal of the American Chemical Society 90:19 9, 2007. pp. 5337-5338. (51) Int. Cl. (Continued) AOIN 43/04 (2006.01) Primary Examiner — Patrick Lewis A6 IK3I/70 (2006.01) 74). Att Agent, or Fi Fitzpatrick, Cella. H & (52) U.S. Cl...... 514/43; 514/44 R: 514/45; 514/49 St. Orney, Agent, or Firm — F1LZpaur1cK, Uella, Harper (58) Field of Classification Search ...... None See application file for complete search history. (57) ABSTRACT (56) References Cited The invention provides unsaturated phosphonates of Formula I or a tautomer orpharmaceutically acceptable salt thereof, as U.S. PATENT DOCUMENTS described herein, as well as pharmaceutical compositions 5,672,697 A * 9/1997 Buhr et al...... 536, 26.7 comprising the compounds, and therapeutic methods com 5,817,647 A 10, 1998 Casara et al. prising administering the compounds. The compounds have 5,892,024 A 4/1999 Chaturvedula et al. anti-viral properties and are useful for treating viral infections

3. A 38. E.aXe ea.s al (e.g. HCV) in animals (e.g. humans). 6,087,490 A 7/2000 Baxter et al. 2003, OOO4345 A1 1/2003 Casara et al. 2004/0023921 A1 2/2004 Hong et al. 5 O (I) 2004/00591.04 A1 3f2004 Cook et al. Ys- B 2006/0074035 A1 4/2006 Hong et al. / \ R1 R6 2 FOREIGN PATENT DOCUMENTS ww. R4 EP O479640 A2 4f1992 R3 R5 EP OS32 423 A1 3, 1993 EP O 618214 A1 10, 1994 EP O 629 633 A2 12, 1994 WO WO-94,22882 A1 10, 1994 22 Claims, No Drawings US 8,324,179 B2 Page 2

OTHER PUBLICATIONS Lera et al. (2001) "An Olefin Cross-Metathesis Approach to Vinylphophonate-Linked Nucleic Acids.” Organis Letters Jung et al. (2000) “Synthesis of Phosphonate Derivatives of Uridine, Cytidine, and Cytosine Arabinoside.” Bioorganic & Medicinal 3(17):2765-2768. Chemistry 8:2501-2509. Montgomery et al. (1979) “Phosphonate Analogue of 2'-Deoxy-5- Kappler et al. (1985) "Use of a Vinyl Phosphonate Analog of ATP as fluorouridylic Acid.” Journal of Medicinal Chemistry 22(1): 109-111. a Rotationally Constrained Probe of the C5'-O5' Torsion Angle in Szabo et al. (1995) “Synthesis and Some Conformational Features of ATP Complexed to Methionine Adenosyl Transferase.” Bioorganic the 5'-Deoxy-5-methylphosphonate Linked Dimmer, 5'-Deoxy-5'-C- Chemistry 13:289-295. (phosphonomethyl)thymidin-3'-yl(Thymidin-5'- Kers et al. (1999) “Preparation of nucleoside 5'-deoxy-5'- yl)methylphosphonate p(CH2)T(CH2)T).” Tetrahedron methylenephosphonates as building blocks for the synthesis of 51(14):4145-4160. methylenephosphonate analogues.” J. Chem. Soc. Perkin Trans I Zhao et al. (1996) “Synthesis and Preliminary Biochemical Studies 2585-1590. with 5'-Deoxy-5'-methylidyne Phosphonate Linked Thymidine Koh et al. (2005) “Design, Synthesis, and Antiviral Activity of Oligonucleotides.” Tetrahedron Letters 37(35):6239-6242. Adenosine 5'-Phosphonate Analogues as Chain Terminators against Hepatitis C Virus.”J Med Chem 48:2867-2875. * cited by examiner US 8,324,179 B2 1. 2 NUCLEOSDE ANALOGS FOR ANTIVIRAL 8, 2501-2509; J. Chem. Soc., Perkins Trans. 1999, 2585 TREATMENT 2590: Synlett 1999, 1124-1126: Biochemistry 1998, 37, 9043-9051; Tetrahedron Lett. 1996, 37, 6239-6242; Tetrahe RELATED APPLICATIONS dron 1995, 51, 4145-4160; Nucleic Acids Research 1995, 23, 893-900; Nucleosides Nucleotides 1995, 14, 871-874; Anti This application claims the benefit of U.S. Provisional viral Chemistry Chemotherapy 1994, 5, 221-228; Tetrahe Application Ser. No. 60/900,692 filed on Feb. 9, 2007, the dron Lett. 1993, 34, 2723-2726; EP 479640; J. Med. Chem. entirety of which is incorporated herein by reference. 1992, 35, 3192-3196: Nucleosides & Nucleotides 1992, 11, 947-956: Acta Chemica Scandinavica 1991, 45, 766–767; FIELD OF THE INVENTION 10 Bioorganic Chemistry 1985, 13, 289-295; J. Med. Chem. The invention relates generally to compounds with antivi 1982, 25, 806-812; J. Med. Chem. 1979, 22, 109-111; and ral activity and more specifically to inhibitors of hepatitis C Bioorganic Chemistry 1976, 5, 31-35. Unsaturated linker virus RNA-dependent RNA polymerase. phosphonate derivatives of purine and pyrimidine com 15 pounds have been reported to be useful as antiviral agents (US BACKGROUND OF THE INVENTION 2003/0004345A1 EP 0532423A1. EP 0618214A1. EP 0701562B1; U.S. Pat. Nos. 5,817,647; 5,922,696; WO The hepatitis C virus (HCV) is the leading cause of chronic 94/22882). liver disease worldwide (Boyer, N. et al. J Hepatol. 32:98 112, 2000). An estimated 170 million persons are infected SUMMARY OF THE INVENTION with HCV worldwide. (Boyer, N. etal, J Hepatol. 32:98-112, 2000). A significant focus of current antiviral research is In one aspect, this invention provides a compound of For directed toward the development of improved methods of mula I: treatment of chronic HCV infections in humans (Di Besceg lie, A. M. and Bacon, B. R., Scientific American, October: 25 80-85, (1999)). A number of HCV treatments are reviewed by Formula I Bymocket al. in Antiviral Chemistry & Chemotherapy, 11:2: 79-95 (2000). Viral serine protease and the RNA-dependent RNA poly merase (RdRp) are the best studied targets for the develop 30 ment of novel HCV therapeutic agents. The NS5B poly merase is a target for inhibitors in early human clinical trials (Sommadossi, J., WO 01/90121 A2). These enzymes have wherein: been extensively characterized at the biochemical and struc A is CR-CR or tural level, with screening assays for identifying selective 35 inhibitors (De Clercq, E. (2001) J. Pharmacol. Exp. Ther. 297:1-10; De Clercq, E. (2001) J. Clin. Virol. 22:73-89). -CEC-: Recent structural work on HCV RdRp has identified catalytic and regulatory nucleotide binding sites (Bressanelli S. et al (2002).J. Virol. 76:3482-92). Since HCV does not replicate in 40 B is a nucleoside base which is optionally substituted; the laboratory, there are difficulties in developing cell-based each R', R. R. R', and R is independently H, OR, assays and preclinical animal systems. N(R), N, CN, NO, SR, halogen, C-C alkyl, C-Cs Currently, there are two primary antiviral compounds, rib Substituted alkyl, C-C alkenyl, C-Cs. Substituted alk avirin and interferon-alpha (C.) (IFN) which are used for the enyl, C-Cs alkynyl, or C-Cs. Substituted alkynyl; or R treatment of chronic HCV infections in humans. Ribavirin 45 and Rare taken together along with the atoms to which alone is not effective in reducing viral RNA levels, has sig they are attached to form a double bond; or R and R. nificant toxicity, and is known to induce anemia. The combi taken together are —O, —NR, or—CRR'; or R and nation of IFN and ribavirin for the treatment of HCV infection R taken together with the carbonatom to which they are has been reported to be effective in the treatment of IFN-naive attached form a 3-7 membered carbocyclic ring wherein patients (Battaglia, A. M. et al., Ann. Pharmacother. 34:487 50 one carbonatom can optionally be replaced with —O—, 494, 2000). Results are promising for this combination treat —S– or —NR' ; ment both before hepatitis develops or when histological R is H, halogen, C-Cs alkyl, C-Cs substituted alkyl, disease is present (Berenguer, M. et al. (1998) Antivir. Ther.3 C-Cs alkenyl, C-Cs. Substituted alkenyl, C-Calkynyl, (Suppl. 3):125-136), but there is a need for improved anti or C-C substituted alkynyl: HCV therapeutic agents, i.e. drugs having improved antiviral 55 each R" is independently H. (C-C)alkyl, (C-C)alkenyl, and pharmacokinetic properties with enhanced activity (C-C)alkynyl or (C-C)alkanoyl: against development of HCV resistance, improved oral bio each R is independently H, (C-C)alkyl, (C-C)alkenyl, availability, greater efficacy, fewer undesirable side effects (C-C)alkynyl, O (C-C)alkyl or OH: and extended effective half-life in vivo (De Francesco, R. etal each R and R is independently H, (C-C)alkyl, (C-C) (2003) Antiviral Research 58:1-16). The instant invention 60 alkenyl, (C-C)alkynyl or halo; provides improved anti-HCV therapeutic agents. wherein each (C-C)alkyl, (C-C)alkenyl, or (C-C) Unsaturated linker phosphonate analogs of nucleotides alkynyl of R R is optionally substituted with one or have been disclosed in U.S. Pat. No. 5,672,697; WO more halo, hydroxy, or (C-C)alkoxy: 9607666; EP 629633; US 2006074035; J. Med. Chem. 2005, Y is O, S, NR, N(O)(R), N(OR), "N(O)(OR), or 48,2867-2875; Biochemistry 2002, 41,7725-7731: Org. Lett. 65 N NR; 2001, 3,3365-3367; Org. Lett. 2001, 3, 2756-2768; Tetrahe each Y is independently O, S, NR, dron. Lett. 2000, 41,4513-4517: Bioorg. & Med. Chem. 2000, N(OR), "N(O)(OR), or N NR; US 8,324,179 B2 3 W' and Ware each independently a group of the formula: provided that when

Yl

R 2 of Formula I is: 10 wherein: each Y is independently a bond, O, CRNR, “N(O)(R), N(OR), "N(O)(OR), N NR, S, S. S. S(O), or S(O); 15 M2 is 0, 1 or 2: each R" is independently H, F, Cl, Br, I, OH, R, C(=Y) R, —C(=Y)OR, C(=Y)N(R), N(R), - "N(R) - SR, S(O)R, S(O).R, S(O)(OR), - S(O),(OR), OC(=Y)R, OC(=Y)OR, OC (=Y)(N(R).), SC(=Y)R, SC(=Y)OR, SC (=Y)(N(R).), N(R)C(=Y)R, N(R)C(=Y)OR, then or N(R)C(=Y)N(R), amino ( NH), ammonium (—NH), alkylamino, dialkylamino, trialkylammo nium, C-C alkyl, C-C alkylhalide, carboxylate, Sul 25 fate, Sulfamate, Sulfonate, C-C alkylsulfonate, C-Cs alkylamino, C-C alkylhydroxyl, C-C alkylthiol, alkylsulfone (—SOR), sulfonamide (—SONR), alkylsulfoxide ( SOR), ester (—C(=O)CR), amido (—C(=O)NR), nitrile (—CN), azido ( N), nitro 30 (—NO), C-C alkoxy (—OR), C-C alkyl, C-Cs Substituted alkyl, C-C alkenyl, C-C substituted alk enyl, C-C alkynyl, C-Cs. Substituted alkynyl, a pro of Formula I is not: tecting group or W.; or when taken together, two R' on the same carbon atom form a carbocyclic ring of 3 to 7 35 carbon atoms; each R is independently R', a protecting group, or the formula: 40

R;

2 2 2 Y afe af2c Y Y afe 45 wherein: M1a, M1 c, and M1d are independently 0 or 1; M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; or 50 when taken together, two R are optionally substituted C-C alkylene thereby forming a phosphorous-contain ing heterocycle; each R is independently H, halogen, C-C alkyl, C-Cs 55 Substituted alkyl, C-C alkenyl, C-Cs. Substituted alk enyl, C-C alkynyl, C-Cs. Substituted alkynyl, Co-Co aryl, Co-Co Substituted aryl, C-Co heterocycle, C-C substituted heterocycle, or a protecting group: W is W or W5; W is R, C(Y)R’, C(Y)Ws, 60 —SOR, or -SOW: and W is a carbocycle or a heterocycle wherein W is independently substituted with 0 to 3 R' groups; or or a pharmaceutically acceptable salt, thereof; 65 provided that W' or W is not an oxygen-linked 2'-deoxy nucleoside; US 8,324,179 B2 5 6 -continued of Formula I is not:

21 O

O N." O B 10 O N O HO OH

wherein B is: 15

NH, NH2 O NH2 NS N(CH3) or N nN. NH or SN ; and - NH N N O N O

O-N-N-N/ \ N NS 25 provided that the compound of Formula I is not:

21 O 21 O or O NH 30 1No-2Sic Sa r 1n SCH O r O R2 O 35 HO OH wherein R is H. For N: O | 21 O. provided that when O11. Ya HCs 40 HG SCH O N." Y’s5 - A O w W. N 45 In another aspect, the present invention provides novel of Formula I is: compounds with activity against infectious viruses. Without wishing to be bound by theory, the compounds of the inven tion may inhibit retroviral RNA-dependent RNA polymerase 50 and thus inhibit the replication of the virus. They may be useful for treating human patients infected with a human s -CH=CH retrovirus, Such as hepatitis C. / M In another aspect, the present invention relates generally to HO OH the accumulation or retention of therapeutic compounds 55 inside cells. The invention is more particularly related to then attaining high concentrations of active metabolite molecules in HCV infected cells. Intracellular targeting may beachieved by methods and compositions which allow accumulation or 60 retention of biologically active agents inside cells. Such O B effective targeting may be applicable to a variety of therapeu tic formulations and procedures. RI R6 R4 In another aspect, the invention provides a pharmaceutical R2 composition comprising an effective amount of a Formula I R3 R5 65 compound, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent or carrier. US 8,324,179 B2 7 8 In another embodiment, the present application provides In one aspect, this invention provides a compound of For for combination pharmaceutical agent comprising: mula II: a) a first pharmaceutical composition comprising a com pound of Formula I; or a pharmaceutically acceptable salt, Solvate, or ester thereof, and 5 Formula II b) a second pharmaceutical composition comprising at least one additional therapeutic agent selected from the group consisting of interferons, ribavirin analogs, NS3 protease inhibitors, alpha-glucosidase 1 inhibitors, hepatoprotectants, non-nucleoside inhibitors of HCV. 10 and other drugs for treating HCV. In another embodiment, the present application provides wherein: for a method of inhibiting HCV polymerase, comprising con A is CR-CR or tacting a cell infected with HCV with an effective amount of a compound of Formula I; or a pharmaceutically acceptable 15 salts, Solvate, and/or ester thereof. -CEC In another embodiment, the present application provides for a method of inhibiting HCV polymerase, comprising con tacting a cell infected with HCV with an effective amount of B is a nucleoside base which is optionally substituted; a compound of Formula I; or a pharmaceutically acceptable each R', R. R. R', and R is independently H, OR, salts, Solvate, and/or ester thereof, and at least one additional N(R'), N, CN, NO, SR, halogen, C-C alkyl, C-Cs therapeutic agent. Substituted alkyl, C-C alkenyl, C-C substituted alk In another embodiment, the present application provides enyl, C-Cs alkynyl, or C-Cs substituted alkynyl; or R for a method of treating HCV in a patient, comprising admin and Rare taken together along with the atoms to which istering to said patientatherapeutically effective amount of a 25 they are attached to form a double bond; or R and R. compound of Formula I; or a pharmaceutically acceptable taken together are =O, =NR, or=CRR'; or R and salt, solvate, and/or ester thereof. R taken together with the carbonatom to which they are In another embodiment, the present application provides attached form a 3-7 membered carbocyclic ring wherein for a method of treating HCV in a patient, comprising admin one carbonatom can optionally be replaced with —O—, istering to said patientatherapeutically effective amount of a 30 —S– or —NR' ; compound of Formula I; or a pharmaceutically acceptable R is H. halogen, C-Cs alkyl, C-Cs substituted alkyl, salt, solvate, and/or ester thereof; and at least one additional C-Cs alkenyl, C-Cs. Substituted alkenyl, C-C alky therapeutic agent. nyl, or C-Cs. Substituted alkynyl: Another aspect of the invention provides a method for the each R" is independently H. (C-C)alkyl, (C-C)alkenyl, treatment or prevention of the symptoms or effects of an HCV 35 (C-C)alkynyl or (C-C)alkanoyl; infection in an infected animal which comprises administer each R is independently H, (C-C)alkyl, (C-C)alkenyl, ing to, i.e. treating, said animal with a pharmaceutical com (C-C)alkynyl, O (C-C)alkyl or OH: bination composition or formulation comprising an effective each R and R is independently H, (C-C)alkyl, (C-C) amount of a Formula I compound, and a second compound alkenyl, (C-C)alkynyl or halo; having anti-HCV properties. 40 wherein each (C-C)alkyl, (C-C)alkenyl, or (C-C) In another aspect, this invention pertains to a method of alkynyl of R R is optionally substituted with one or increasing cellular accumulation and retention of drug com more halo, hydroxy, or (C-C)alkoxy; pounds, thus improving their therapeutic and diagnostic each Y is independently O, S, NR, value. N(OR), "N(O)(OR), or N NR; In another aspect, the invention also provides a method of 45 W' and Ware each independently a group of the formula: inhibiting HCV, comprising administering to a mammal infected with HCV an amount of a Formula I compound, effective to inhibit the growth of said HCV infected cells. Yl In another aspect, the invention also provides processes and novel intermediates disclosed herein which are useful for 50 R Y2-B Y2 preparing Formula I compounds of the invention. In other aspects, novel methods for synthesis, analysis, J. separation, isolation, purification, characterization, and test ing of the compounds of this invention are provided. R 2 55 DETAILED DESCRIPTION OF EXEMPLARY wherein: EMBODIMENTS each Y is independently a bond, O, CRNR, "N(O)(R), N(OR), "N(O)(OR), N NR, S, S-S, S(O), or S(O); Reference will now be made in detail to certain embodi M2 is 0, 1 or 2: ments of the invention, examples of which are illustrated in 60 each R” is independently H, F, Cl, Br, I, OH, R, C(=Y) the accompanying description, structures and formulas. R, —C(=Y)OR, C(=Y)N(R) - N(R), While the invention will be described in conjunction with the - "N(R) - SR, S(O)R, S(O).R, S(O)(OR), enumerated embodiments, it will be understood that they are - S(O)(OR), OC(=Y)R, OC(=Y)OR, OC not intended to limit the invention to those embodiments. On (=Y)(N(R).), SC(=Y)R, SC(=Y)OR, SC the contrary, the invention is intended to coverall alternatives, 65 (=Y)(N(R).), N(R)C(=Y)R, N(R)C(=Y)OR, modifications, and equivalents, which may be included or N(R)C(=Y)N(R), amino ( NH), ammonium within the scope of the present invention. (—NH), alkylamino, dialkylamino, trialkylammo US 8,324,179 B2 9 nium, C-C alkyl, C-C alkylhalide, carboxylate, Sul then fate, Sulfamate, Sulfonate, C-C alkylsulfonate, C-Cs alkylamino, C-Cs alkylhydroxyl, C-C alkylthiol, alkylsulfone (—SOR), sulfonamide (—SONR), alkylsulfoxide ( SOR), ester (—C(=O)CR), amido O B (—C(=O)NR), nitrile (—CN), azido ( N), nitro R1 R6 (—NO), C-C alkoxy (—OR), C-C alkyl, C-Cs R4 Substituted alkyl, C-C alkenyl, C-C substituted alk R2 enyl, C-Cs alkynyl C-Cs. Substituted alkynyl, a protect R3 R5 ing group, or W.; or when taken together, two R' on the 10 same carbon atom forms a carbocyclic ring of 3 to 7 carbon atoms; of Formula II is not: each R is independently R', a protecting group, or the formula: 15

R; 2 2 2 Y afe af2c Y afic Y afe wherein: M1a, M1 c, and M1d are independently 0 or 1; 25 M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; or when taken together, two R are optionally substituted C-C alkylene thereby forming a phosphorous-contain ing heterocycle; 30 each R is independently H, halogen, C-C alkyl, C-Cs O Substituted alkyl, C-C alkenyl, C-C substituted alk enyl, C-C alkynyl, C-Cs. Substituted alkynyl, Co-Co aryl, Co-Co Substituted aryl, C-Co heterocycle, C-C substituted heterocycle or a protecting group: 35 r W is W or W5; W is R, C(Y)R’, C(Y)Ws, —SOR, or -SOW: and W is a carbocycle or a S-N- heterocycle wherein W is independently substituted O with 0 to 3 R' groups; or 40 or a pharmaceutically acceptable salt, thereof; 21 O provided that W' or W is not an oxygen-linked 2'-deoxy nucleoside; 45 y provided that when O

50

of Formula II is: 55

60

O

65 US 8,324,179 B2 11 12 -continued -continued NH2 21 NN O N." O

R2 10

wherein R is H. For N: H provided that when N O or 15 P 21 1N7's1n CH (?s s - A W1 w O HO OH O of Formula II is: l 21 O. o7 HC 25 HO Sc O N." s -CH=CH Cri /N N HO OH 30 In one embodiment of the invention of Formula II, R' is H. then In another embodiment, R' is not H. In another embodiment, R" is CN, N., methyl, OR", ethenyl, or ethynyl. In another embodiment R' is N. 35 In one embodiment of the invention of Formula II, Rand O B R taken together are =O, =NR, or =CR'R''. In another embodiment, RandR taken together with the carbon atom R1 R6 R4 to which they are attached form a 3-7 membered carbocyclic R2 40 ring wherein one carbonatom can optionally be replaced with R3 R5 —O-, - S - or NR—. In another embodiment, Rand Rare both Hand at least one of RandR is not H. In another embodiment, RandR are both Hand at least one of Rand of Formula II is not: R is OH. In another embodiment, R and R are taken 45 together along with the atoms to which they are attached to form a double bond. In another embodiment, each Rand R' is H, R is Hor F and R is OH. In anotherembodiment Rand O Rare H, R is OH and R is F. In one embodiment of the invention of Formula II, R is r methyl, ethenyl, or ethynyl. In another embodiment, R is H. O s NH In one embodiment of the invention of Formula II, A is —CR'—CR wherein each Risindependently H, (C-C) O alkyl, (C-C)alkenyl, (C-C)alkynyl or halo. In another N O HO OH embodiment A is 55

10 wherein B is: -CEC

NH2 O 60 In a preferred embodiment A is CR'—CR and each R" is H or halo. In a preferred embodiment, A is cis N n N O —CH=CH-. In another preferred embodiment, A is trans —CH=CH-. In another preferred embodiment, A is cis —CF=CH-. In another preferred embodiment, A is trans 65 —CF=CH-. In another preferred embodiment, A is cis —CH=CF . In another preferred embodiment, A is trans CH-CF US 8,324,179 B2 13 14 In one embodiment of the invention of Formula II, W or -continued W° is not an oxygen-linked 2'-deoxynucleoside; when 5 r s - A O N N O

W1 VW2 rO 10 of Formula II is: O 21

O N N O CH=CH- , 15 r S. O

then lN O 25 O N /

O B R1 R6 2 R4 30 N(CH3)2: R R3 R 5 e N NH of Formula II is not: 35 O N / \ Ns/

O 21 when 40 O s" O os R2 45 w W.

of Formula II is:

O, 50

O N ->NH s -CH=CH / M HO OH O 55 F ? O, then

O R4 R2 65 R3 R5 O US 8,324,179 B2 15 16 of Formula II is not: wherein: Ro is OH or (C-C)alkoxy that is optionally substituted with one or more R, and R is H or (C-C)alkyl that is optionally substituted with one or more R, or Rio and R together with the nitrogen to which they are r attached form a heterocyclic ring that is optionally Sub sitituted with one or more R. O N NH each R is independently (C-C)alkyl, (C-C)alkylthio. O 10 (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkanoyloxy, N O HO OH (C-C)alkoxycarbonyl, NRR, -C(=O)NRR, aryl, heteroaryl, cyano, halo, hydroxy, nitro, carboxy, or (C-C)cycloalkyl; wherein B is: R and R2 are each independently H. (C-C)alkyl or 15 (C-C)alkanoyl; wherein each aryl or heteroaryl of R is optionally Substi tuted with one or more (C-C)alkyl, (C-C)alkylthio.

s O (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, NRR, -C(=O)NRR, cyano, halo, hydroxy, nitro, carboxy, (C-C)cycloalkyl, trifluoromethoxy, mercapto, or trifluoromethyl; and D, E, E, E, and Fare each independently >Nor>C Rs: 25 each Rs is independently H, cyano, nitro, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, - NHCONH2, C(=O)NRR-7, COORs, hydroxy, (C-C)alkoxy, —NRR-7, halo, 1.3-oxazol-2-yl, 1.3-thiazol-2-yl, imidazol-2-yl, 2-oxo-1.3dithiol-4-yl, furan-2-yl, or 30 2H-1.2.3 triazol-4-yl: each R and R27 is independently H., (C-C)alkyl, (C- C.)alkenyl, (C-C)alkynyl, (C-C)cycloalkyl, aryl, heterocycle, hydroxy, (C-C)alkoxy; or Re and R, together with the nitrogen to which they are attached 35 form a heterocycle; and each Rs is independently H. (C-C)alkyl, (C-C)alk enyl, (C-C)alkynyl, (C-C)cycloalkyl, aryl, or hetero cycle; 40 wherein each (C-C)alkyl, (C-C)alkenyl, (C-C)alky nyl, (C-C)cycloalkyl, aryl, heterocycle, and (C-C) HO OH alkoxy of R and R, is optionally substituted with one or more (C-C)alkyl, (C-C)alkylthio, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxy 45 carbonyl, NH, cyano, halo, hydroxy, nitro, carboxy, (C-C)cycloalkyl, (C-C)cycloalkoxy, trifluo romethoxy, or mercapto. A specific value for Rao is OH. methoxy, or propoxy, and 50 for R is H. A specific value for Rao and R together with the nitrogen In another embodiment of the invention of Formula II, B to which they are attached form a pyrrolidin-1-yl, 1.3.4. has the following formula: 9-tetrahydro-beta-carbolin-2-yl, piperidinyl, azetidinyl, 55 3,6-dihydro-2H-pyridin-1-yl, or 3,4-dihydro-1H-iso quinolin-2-yl ring, which ring is optionally substituted R21 R20 with–C(=O)NH2. In a preferred embodiment, Rand NN1 R taken together are =O, —NR, or =CR'R''. In another preferred embodiment RandR are both Hand E2 2 N 60 one of R or R is OH. In another embodiment, each R and R is H, R is H or F and R is OH. In another R 2F embodiment RandR are H, R is OH and R is F. In N 2 another preferred embodiment, R' is CN, N. methyl, OR', ethenyl, or ethynyl. In another preferred embodi 65 ment, R' is H. In another embodiment R' is N. In -- another embodiment, R is methyl, ethenyl, or ethynyl. In another embodiment, R is H.

US 8,324,179 B2 21 22 In another preferred aspect, X is CH and B is OH. In another embodiment RandR are H, R is OH and R is F. In another preferred embodiment, R' is CN, N, methyl, OR', ethenyl, or ethynyl. In another pre R55 ferred embodiment, R' is H. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethynyl. In another embodiment, R is H. Rs6 r In a preferred aspect, X is N and B of Formula II is O. -- 10 In another preferred aspect X is N. In another preferred aspect, X is CH and B is 15

Rs In another aspect, X is CH. In another aspect X is 's C-R. In another aspect, X is N. In a preferred embodi ment, X is CH and RandR taken togetherare=O. =NR, ---, or=CR'R''. In another preferred embodiment, X is CH and R° and Rare both H and one of R or R is OH. In another embodiment, X is CH and RandR are H, R is OH and R 25 is F. In another embodiment, X is CH, each RandR is H.R is H or F and R is OH. In another preferred embodiment, X In another preferred aspect X is N. is CH and R' is CN, N., methyl, OR", ethenyl, or ethynyl. In In another preferred aspect, B is another embodiment, X is CH and R' is N. In another 30 embodiment, R is methyl, ethenyl, or ethynyl. In another preferred aspect, X is N and B of Formula II is "N'S R58. Na2N 35 c." 40 In another embodiment of the invention of Formula II, B has the following formula: In another aspect, X and X are N. In another aspect X, X, and X are N. In another aspect, X and X are N and X R60 R60 45 is CH or C-R. In another aspect, X and X are N. In a preferred embodiment, Xs and X are N and RandR taken N21 R 6 7 NN together are —O, —NR, or =CR'R''. In another preferred embodiment, X and X are N and R and Rare both Hand O1. 1 X* O X w N 2.2 one of R or R is OH. In another embodiment, X and X are 50 N, each RandR is H, R is Hor F and R is OH. In another embodiment, X and X are N and Rand Rare H, R is OH and R is F. In another preferred embodiment, X and X are N and R' is CN, N., methyl, OR", ethenyl, or ethynyl. In another embodiment, X and X are NandR is N. In another wherein: 55 embodiment, R is methyl, ethenyl, or ethynyl. X, X, and X are each independently N. CH, or C-R. In another embodiment of the invention of Formula II, B Reo Re, and R are each independently H, halo, has the following formula: NR'R'', hydroxyamino, NRNR.R., N, NO, NO, formyl, cyano. —C(=O)NR,R, —C(=S)NRR, —C(=O)CR, R, OR, or SR; and 60 R, and Rare each independently H. (C-C)alkyl, (C- C.)alkenyl, (C-C)alkynyl, aryl, (C-C)alkanoyl, —S(O)(C-C)alkyl oraryl(C-C)alkyl. In a preferred embodiment, RandR taken together are=O. =NR, or =CRR". In another preferred embodiment R and 65 Rare both H and one of R or R is OH. In another embodiment, each RandR is H, R is Hor F and R is US 8,324,179 B2 23 24 wherein: pate in an optional bond represented by a dotted Xs, X, and X7, are each independently N. CH, or C-R. line - - - in the formula, then that W is CH, or N: Ro and R7 are each independently H, halo, NR7R-7, each Ro, is independently H, halo, NRoRo hydroxyamino, NR NR7R-7, N, NO, NO, formyl, hydroxyamino, NRNRoRo N. cyano. —C(=O) cyano, —C(=O)NRR, —C(=S)NRR, 5 NRR —C(=S)NRR —C(=S)NRR, —C(=O)CR, R-7, OR-7 or SR-7, and —C(=NH)OR, R, OR, or SR; R7, and Rare each independently H. (C-C)alkyl, (C- each Ro, is independently H. (C-C)alkyl, (C-C)alk C.)alkenyl, (C-C)alkynyl, aryl, (C-C)alkanoyl, or enyl, (C-C)alkynyl, aryl, (C-C)alkanoyl, or aryl (C- aryl(C-C)alkyl. In a preferred embodiment, RandR C.)alkyl, and taken together are =O, =NR, or=CRR". In another 10 Ro, and Ro are each independently H. (C-C)alkyl, (C- preferred embodiment R and Rare both H and one of C.)alkenyl, (C-C)alkynyl, aryl, (C-C)alkanoyl, or aryl R or R is OH. In another embodiment, each Rand R' (C-C)alkyl. In a preferred embodiment, R and R taken is H, R is H or F and R is OH. In another embodiment together are =O, =NR, or=CR'R''. In another preferred R° and R are H, R is OH and R is F. In another embodiment Rand Rare both Hand one of R or R is OH. preferred embodiment, R' is CN,Ns, methyl, OR", ethe 15 In another embodiment, each RandR is H, R is Hor F and nyl, or ethynyl. In another preferred embodiment, R' is R is OH. In another embodiment R and Rare H, R is OH H. In another embodiment R' is N. In another embodi and R is F. In another preferred embodiment, R' is CN, N. ment, R is methyl, ethenyl, or ethynyl. In another methyl, OR, ethenyl, or ethynyl. In another preferred embodiment, R is H. In a preferred aspect, Xs is CH and embodiment, R' is H. In another embodiment R' is N. In X and X, are N. In a particularly preferred aspect, Xs is another embodiment, R is methyl, ethenyl, or ethynyl. In CH, X and X, are N, and Rio is —C(=O)NR7R-7. another embodiment, R is H. In another embodiment of the invention of Formula II, B Another specific value for B has one of the following has the following formula: formulae: 25

30

wherein: 35 Y=Yis-N=N-,-N=CRs or -CH=CRs : and each Rs is independently H, halo, or (C-C)alkyl. In another embodiment of the invention of Formula II, B 40 has the following formula:

45

50

wherein: each Z is independently N. C. R. O, S, NR, DC=O, 55 >C=S, >C=NR, >S—O, >S(O), or CH R pro vided that if a Z participates in an optional bond repre sented by a dotted line - - - in the formula, then that Z is N or C-Ro, and provided that if a Z1 does not partici pate in an optional bond represented by a dotted 60 line - - - in the formula, then that Z is O, S, NR, >C=O, >C=S, >C=NR, >S—O, >S(O), or CH-R; X is O, S, SO, SO, Se, SeO, SeO, or NR; each Wis C, CH, or N; wherein if a W participates in an 65 optional bond represented by a dotted line - - - in the formula, then that W is C; and if a W does not partici US 8,324,179 B2 25 26 -continued -continued H3CO

10 In another embodiment of the invention of Formula II, B has the following formula:

15

25 wherein: X is CRoo, or N: Xo is O, S, or NRo: Ro and Rare each independently H, halo, hydroxy, (C- 30 Co.)alkoxy, NRooRoi, or heterocycle; Roo, is H. halo, methyl, azido, or amino; Ro is H, or (C-C)alkyl, and Roo, and Ro, are each independently H. (C-C)alkyl, (C-C)cycloalkyl, or aryl(C-C)alkyl; 35 wherein each (C-C)alkyl, (C-C)cycloalkyl, aryl (C- C.)alkyl, and heterocycle of Roo Ro, Ro, and Roo Ro, are optionally Substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, amino, (C-C)alkyl, and (C-C)alkoxy. In a preferred embodiment, R and 40 R taken together are =O, —NR, or =CR'R''. In another preferred embodiment RandR are both Hand one of R or R is OH. In another embodiment, each R and R is H, R is H or F and R is OH. In another preferred embodiment, R' is CN, N., methyl, OR", ethe 45 nyl, or ethynyl. In a preferred embodiment, X is CRoo. In another preferred embodiment, X is N. In another preferred embodiment, X is CRoo, and X is O. In another preferred embodiment, X is CR, and Xois S. In another preferred embodiment, X is CRoo, and Xois 50 NR. In another preferred embodiment, X is N and X is O. In another preferred embodiment, X is N and X is S. In another preferred embodiment, X is N and Xo is NRol. In another embodiment of the invention of Formula II, B 55 has the following formula:

60

65 US 8,324,179 B2 27 28 wherein: each Vso is independently N or CRs, and X is O, S, or NRo: each Rso and Rs is independently H, carbocycle, (C-C) R7s and R7 are each independently H, halo, hydroxy, mer capto, aryl, heterocycle, (C-C)alkyl, (C-C)alkenyl, alkyl, (C-C)alkenyl, (C-C)alkynyl, halo, (C-C) (C-C)alkynyl, (C-C)alkylthio, arylthio. —S(=O) alkoxy, amino, methylamino, dimethylamino, cyano, (C-C)alkyl, -S(=O)(C-C)alkyl, -S(=O) (C-C)alkanoyl, aryl, aryl(C-C)alkyl, an amino acid NRoR (C-C)alkoxy, aryloxy, (heterocycle)oxy: residue (e.g. a naturally-occurring amino acid residue) Ro is H. methyl, ethyl, or acetyl; and or heterocycle; or Rs and Rs taken together with the Rio, and R-7, are each independently H., (C-C)alkyl, atom(s) to which they are attached form a 3-7 membered aryl, aryl(C-C)alkyl, (C-C)alkanoyl, or aryl-C 10 carbocyclic or heterocyclic ring. (=O)—. In another embodiment of the invention of Formula II, B In a preferred embodiment, Rand R taken together are has the following formula: =O, =NR, or=CR'R''. In another preferred embodiment R° and Rare both Hand one of R or R is OH. In another

embodiment, each RandR is H, R is H or F and R is OH. 15 In another preferred embodiment, R' is CN, N. methyl, OR, ethenyl, or ethynyl. In a preferred embodiment, X is O. In another preferred embodiment, X is S. In another preferred embodiment, X is NRio. O In another embodiment of the invention of Formula II, B has the following formula:

Z Y 25 - n N V80 N V80 - N ls - N -N V80 Yso V80 Z80 30

Yso 35 wherein: Ro, is H., hydroxy, mercapto, (C-C)alkyl, (C-C)alkoxy, (C-C)alkenyl, (C-Cs)cycloalkyl, aryl(C-C)alkyl, aryl or (C-C)alkynyl: V Z V80 Y80 . 80 80 40 Ros is H. hydroxy, mercapto, or (C-C)alkyl; Roo is H. halo, azido, cyano, nitro, ORoo, SRoo, NRooRoo, (C-C)alkyl, (C-C)alkenyl, (C-Cs)cy cloalkyl, aryl (C-C)alkyl, aryl or (C-C)alkynyl: each Roo, is independently H, (C-C)alkyl, or (C-C) 45 alkanoyl; R80 Z 80 each Roo, and Roo is independently H. (C-C)alkyl, or (C-C)alkanoyl; Z80 ls R80 ls N N1 wherein each (C-C)alkoxy (C-C)alkenyl, (C-C)alky N N 50 nyl, (C-C)cycloalkyl, aryl, and aryl(C-C)alkyl of R97, Ros. Roo. Roo, Roo, and Roo, is optionally Substi tuted with one or more (C-C)alkyl, (C-C)alkylthio. (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, NH, cyano, azido, halo, 55 hydroxy, nitro, carboxy, trifluoromethoxy, aryl, or mer wherein: capto. In a preferred embodiment, R and R taken Aso, Bso, and Yso are each independently H, halo, OR so. together are —O, =NR, or =CRR. In another pre S(O).R.so, NRsoRs, cyano, trifluoromethyl, C(=W) ferred embodiment Rand Rare both H and one of R' ORs, C(=W)SRs, C(=W)NRRs, nitro, azido, car 60 or R is OH. In another embodiment, each RandR is bocyclic, (C-C)alkyl, (C-C)alkenyl, (C-C)alky H, R is Hor F and R is OH. In another embodiment R nyl, aryl, aryl(C-C)alkyl, or heterocycle; or Aso and and Rare H, R is OH and R is F. In another preferred Bso taken together with the carbon atoms to which they embodiment, R' is CN, N. methyl, OR, ethenyl, or are attached from a 4-7 membered carbocyclic or het ethynyl. In another preferred embodiment, R' is H. In erocyclic ring; 65 another embodiment R' is N. In another embodiment, n is 0, 1, or 2 R is methyl, ethenyl, or ethynyl. In another embodi Zso is O, S, NRso, or CRso Rs: ment, R is H. US 8,324,179 B2 29 30 In a preferred embodiment, B of Formula II is or mercapto. In a preferred embodiment, R and R taken together are —O, —NR, or =CRR. In another preferred

embodiment Rand Rare both Hand one of R or R is OH. In another embodiment, each RandR is H, R is Hor F and R is OH. In another embodiment R and Rare H, R is OH and R is F. In another preferred embodiment, R' is CN, N. methyl, OR, ethenyl, or ethynyl. In another preferred embodiment, R' is H. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethynyl. In 10 another embodiment, R is H. In a preferred embodiment of the invention of Formula II, B is adenine. In another embodiment, B is cytosine. In another embodiment, B is thymine. In another embodiment, 15 B is guanine. In another embodiment, B is uracil. In another embodiment, B is not adenine, guanine, cytosine, thymine or uracil. In another preferred embodiment, B is adenine, gua nine, cytosine, thymine, or uracil and R and R taken together are —O, —NR, or =CRR. In another preferred embodiment, B is adenine, guanine, cytosine, thymine, or uracil and Rand Rare both Hand one of R or R is OH. In another preferred embodiment, B is adenine, guanine, cytosine, thymine, or uracil and each RandR is H, R is H or F and R is OH. In another preferred embodiment, B is 25 adenine, guanine, cytosine, thymine, or uracil and R' is CN, N, methyl, OR, ethenyl, or ethynyl. In a preferred embodiment, Rand R taken together are In another preferred embodiment of the invention of For =O, =NR, or=CR'R''. In another preferred embodiment mula II, B is R° and Rare both Hand one of R or R is OH. In another 30 embodiment, each RandR is H, R is H or F and R is OH. In another embodiment RandR'are H, R is OH andR is F. O NH NH In another preferred embodiment, R' is CN, N. methyl, OR, ethenyl, or ethynyl. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethynyl. 35 In another embodiment of the invention of Formula II, B is NH NN cr) adenine, guanine, cytosine, uracil, thymine, 7-deazaadenine, 1s, Cl { e s 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaad enine, inosine, nebularine, nitropyrrole, nitroindole, 2-ami nopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, 40 hypoxanthine, pseudouridine, pseudocytosine, pseudoisocy tosine, 5-propynylcytosine, isocytosine, isoguanine, 7-deaza guanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, O'-methylguanine, N'-methyladenine, O-me thylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-me 45 thylindole, substituted triazole, or pyrazolo 3,4-Dipyrimi dine. In a preferred embodiment, RandR taken together are =O, =NR, or=CR'R''. In another preferred embodiment R° and Rare both Hand one of R or R is OH. In another O embodiment, each RandR is H, R is H or F and R is OH. 50 In another embodiment RandR are H, R is OH and R is F. NH2 N In another preferred embodiment, R' is CN, N. methyl, OR, O ethenyl, or ethynyl. In anotherpreferred embodiment, R' is H. N N In another embodiment R' is N. In another embodiment, R ^ NN ( NN is methyl, ethenyl, or ethynyl. In another embodiment, R is 55 H. Y as N e In another embodiment of the invention of Formula II, B is olo 22 olo s 6-amino-2-chloro-purin-9-yl; 6-amino-2-iodo-purin-9-yl; 6-amino-2-fluoro-purin-9-yl; 6-amino-2-methylthio-purin 9-yl; 6-amino-purin-9-yl; or 4-amino-7H-pyrrolo2,3-dpy 60 NH2 O rimidin-7-yl. In another embodiment of the invention of Formula II, B is N n N adenine or cytosine, which adenine or cytosine is optionally M N M NH Substituted with one or more (C-C)alkyl, (C-C)alkylthio. \llN 2 \llN 4\s (C-C)alkoxy, (C-C)alkanoyl, optionally Substituted ben 65 Zoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, NH2, cyano, halo, hydroxy, nitro, carboxy, trifluoromethoxy, aryl, US 8,324,179 B2 31 32 -continued together are =O, =NR, or =CRR". In another preferred O NH2 embodiment Rand Rare both Hand one of R or R is OH. In another embodiment, each RandR is H. R. is H or F and R is OH. In another embodiment Rand Rare H, R is OH ( NH ( NN 5 and R is F. In another preferred embodiment, R is CN, N. N es NH2, N es NH2, methyl, OR, ethenyl, or ethynyl. In another preferred embodiment, R' is H. In another embodiment R' is N. In O another embodiment, R is methyl, ethenyl, or ethynyl. In 10 another embodiment, R is H. N In another embodiment of Formula II, R and R' are not / NH2. protecting groups. N-N In another aspect, this invention provides a compound of Formula III or Formula IV: 15

In a preferred embodiment, Rand R taken together are Formula III =O, =NR, or=CR'R''. In another preferred embodiment O R° and Rare both Hand one of R or R is OH. In another os- B embodiment, each RandR is H, R is H or F and R is OH. WIM \,W R' HR6 In another embodiment RandR are H, R is OH and R is F. R2 In another preferred embodiment, R' is CN,Ns, methyl, OR", R ethenyl, or ethynyl. In another preferred embodiment, R is H. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethynyl. In another embodiment, R is 25 Formula IV H. In another embodiment of Formula II, W and W are not os- O B phenoxy or 2-chlorophenoxy. In another embodiment, W. WIA \,,W. R' HR6 and Ware not ethoxy. In another embodiment W' and Ware HO not OH. In another embodiment, W' is not phenoxy and W° is 30 H F not OH. In one embodiment of Formula II, W or Ware selected from a nitrogen-linked naturally-occurring or unnatural wherein: C.-aminoacid or C.-aminoacid ester oran oxygen-linked natu A is CR-CR or rally-occurring or unnatural C-hydroxycarboxylic acid or 35 C-hydroxycarboxylic acid ester. In another embodiment, W. or W is a nitrogen-linked alanine or an alanine ester. In -CEC-: another embodiment, W' and W are each independently selected from a nitrogen-linked naturally-occurring or unnatural C.-aminoacid or O-aminoacid ester or an oxygen 40 B is a nucleoside base which is optionally substituted; linked naturally-occurring or unnatural C-hydroxycarboxylic R" is H, OR, N(R), N, CN, NO, SR, halogen, C-Cs. acid or C-hydroxycarboxylic acid ester. In another embodi alkyl, C-C substituted alkyl, C-C alkenyl, C-C sub ment, W' and Ware each independently a nitrogen-linked stituted alkenyl, C-C alkynyl, or C-Cs. Substituted alanine or analanine ester. In another embodiment, W or W alkynyl: is C-C alkoxy. In another embodiment W' or W is aryloxy 45 or substituted aryloxy. R’ is H or F: In another embodiment of Formula II, W and W° are R is OR, N(R), N, CN, NO, SR, halogen, C-Cs. independently a nitrogen-linked naturally-occurring or alkyl, C-C substituted alkyl, C-C alkenyl, C-C sub unnatural O-aminoacid or C.-aminoacid ester. In a preferred stituted alkenyl, C-C alkynyl, or C-Cs. Substituted embodiment, R and R taken together are =O, =NR, or 50 =CRR". In another preferred embodiment R and R are alkynyl: both H and one of R or R is OH. In another embodiment, R is H, halogen, C-Cs alkyl, C-Cs substituted alkyl, each R and R is H, R is H or F and R is OH. In another C-Cs alkenyl, C-Cs. Substituted alkenyl, C-C alky embodiment RandR are H, R is OH and R is F. In another nyl, or C-Cs. Substituted alkynyl: preferred embodiment, R' is CN, Ns, methyl, OR", ethenyl, 55 each R" is independently H. (C-C)alkyl, (C-C)alkenyl, or ethynyl. In another preferred embodiment, R' is H. In (C-C)alkynyl or (C-C)alkanoyl; another embodiment R' is NIn another embodiment, R is methyl, ethenyl, or ethynyl. In another embodiment, R is H. each R is independently H, (C-C)alkyl, (C-C)alkenyl, In another preferred embodiment, W and Ware each inde (C-C)alkynyl, O (C-C)alkyl or OH: pendently a nitrogen-linked alanine or an alanine ester. In 60 each R and R is independently H, (C-C)alkyl, (C-C) another preferred embodiment, W and Ware each indepen alkenyl, (C-C)alkynyl or halo: dently a nitrogen-linked alanine ester wherein the ester group is C-C alkyl. wherein each (C-C)alkyl, (C-C)alkenyl, or (C-C) In another embodiment of Formula II, W and W are alkynyl of R R is optionally substituted with one or independently an oxygen-linked naturally-occurring or 65 more halo, hydroxy, or (C-C)alkoxy; unnatural C-hydroxycarboxylic acid or C-hydroxycarboxylic each Y is independently O, S, NR, acid ester. In a preferred embodiment, R and R taken N(OR), "N(O)(OR), or N NR; US 8,324,179 B2 33 34 W' and Ware each independently a group of the formula: In one embodiment of the invention of Formula III or Formula IV. R is methyl, ethenyl, or ethynyl. In another embodiment, R is H. Yl In one embodiment of the invention of Formula III or Formula IV. A is –CR-CR - wherein each R is inde Y2-P, pendently H. (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl J. or halo. In another embodiment A is R / 10 -CEC wherein: each Y is independently a bond, O, CRNR, “N(O)(R), In a preferred embodiment A is CR'—CR and each N(OR), "N(O)(OR), N NR, S, S. S. S(O), or S(O); R" is H or halo. In a preferred embodiment, A is cis M2 is 0, 1 or 2: 15 —CH=CH-. In another preferred embodiment, A is trans each R" is independently H, F, Cl, Br, I, OH, R, C(=Y) —CH=CH-. In another preferred embodiment, A is cis R, —C(=Y)OR, C(=Y)N(R), N(R), —CF=CH-. In another preferred embodiment, A is trans - "N(R) - SR, S(O)R, S(O).R, S(O)(OR), —CF=CF . In another preferred embodiment, A is cis - S(O),(OR), OC(=Y)R, OC(=Y)OR, OC —CH=CF . In another preferred embodiment, A is trans (=Y)(N(R).), SC(=Y)R, SC(=Y)OR, SC CH-CF (=Y)(N(R).), N(R)C(=Y)R, N(R)C(=Y)OR, In another embodiment of the invention of Formula III or or N(R)C(=Y)N(R), amino ( NH), ammonium (—NH), alkylamino, dialkylamino, trialkylammo Formula IV, B has the following formula: nium, C-C alkyl, C-C alkylhalide, carboxylate, Sul fate, Sulfamate, Sulfonate, C-C alkylsulfonate, C-Cs 25 R21 R20 alkylamino, C-Cs alkylhydroxyl, C-C alkylthiol, NN1 alkylsulfone ( SOR), sulfonamide ( -SONR), alkylsulfoxide ( SOR), ester (—C(=O)CR), amido A.E2 N (—C(=O)NR), nitrile (—CN), azido ( N), nitro (—NO), C-C alkoxy (—OR), C-C alkyl, C-Cs 30 F. N 2F Substituted alkyl, C-C alkenyl, C-Cs. Substituted alk enyl, C-C alkynyl, C-C substituted alkynyl, a pro tecting group, or W.; or when taken together, two Ron the same carbonatome form a carbocyclic ring of 3 to 7 carbon atoms; 35 each R is independently R', a protecting group, or the wherein: formula: Ro is OH or (C-C)alkoxy that is optionally substituted with one or more R, and R is H or (C-C)alkyl that is optionally Substituted with one or more R, or Ro 40 and R together with the nitrogen to which they are R. R. attached form a heterocyclic ring that is optionally Sub R; sitituted with one or more R. 2 2 2 each R is independently (C-C)alkyl, (C-C)alkylthio. n Y afe af2c Y afic Y afe (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkanoyloxy, 45 (C-C)alkoxycarbonyl, NRR, -C(=O)NRR, wherein: aryl, heteroaryl, cyano, halo, hydroxy, nitro, carboxy, or M1a, M1 c, and M1d are independently 0 or 1; (C-C)cycloalkyl: M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; or R and R2 are each independently H. (C-C)alkyl or when taken together, two R are optionally substituted (C-C)alkanoyl: C-C alkylene thereby forming a phosphorous-contain 50 wherein each aryl or heteroaryl of R is optionally Substi ing heterocycle; tuted with one or more (C-C)alkyl, (C-C)alkylthio. each R is independently H, halogen, C-C alkyl, C-Cs (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkanoyloxy, Substituted alkyl, C-C alkenyl, C-Cs. Substituted alk (C-C)alkoxycarbonyl, NRR, -C(=O)NRR, enyl, C-C alkynyl, C-C substituted alkynyl, Co-Co cyano, halo, hydroxy, nitro, carboxy, (C-C)cycloalkyl, aryl, Co-Co Substituted aryl, C-Co heterocycle, 55 trifluoromethoxy, mercapto, or trifluoromethyl; and C-C substituted heterocycle or a protecting group: D, E, E, E, and Fare each independently >Nor>C Rs: W is W or W.; W is R, C(Y)R’, C(Y)W, each Rs is independently H. cyano, nitro, (C-C)alkyl, —SOR, or -SOW: and W is a carbocycle or a (C-C)alkenyl, (C-C)alkynyl, - NHCONH2, heterocycle wherein W is independently substituted C(=O)NRR-7, COORs, hydroxy, (C-C)alkoxy, with 0 to 3 R' groups; or 60 —NRR-7, halo, 1.3-oxazol-2-yl, 1.3-oxazol-5-yl, 1.3- or a pharmaceutically acceptable salt, thereof. thiazol-2-yl, imidazol-2-yl, 2-oxo-1.3dithiol-4-yl, In another embodiment of Formula III, R is H. In another furan-2-yl, or 2H-1.2.3 triazol-4-yl; embodiment R is F. each R and R, is independently H., (C-C)alkyl, (C- In one embodiment of the invention of Formula III or C.)alkenyl, (C-C)alkynyl, (C-C)cycloalkyl, aryl, Formula IV. R' is H. In another embodiment, R' is not H. In 65 heterocycle, hydroxy, (C-C)alkoxy; or Re and R, another embodiment, R' is CN, N. methyl, OR, ethenyl, or together with the nitrogen to which they are attached ethynyl. In another embodiment R' is N. form a heterocycle; and

US 8,324,179 B2 39 40 In another preferred aspect, X is CH and B is In another aspect, X is CH. In another aspect X is C-R. In another embodiment X is N. In another preferred aspect, X is N and B is 'sRs ---, 10

In another preferred aspect X is N. In another preferred aspect, B is 15 In another aspect, X and X are N. In another aspect X, X, and X are N. In another aspect, X and X are N and X NS R58. is CH or C-R. In another aspect, X and X are N. N 2N In another embodiment of the invention of Formula III or Formula IV, B has the following formula: c." 25 In another embodiment of the invention of Formula III or Formula IV, B has the following formula:

30

wherein: Xs, X, and X7, are each independently N, CH, or C-R.

35 Ro and R7 are each independently H, halo, NR7R-7, hydroxyamino, NRNRR, N, NO, NO, formyl, cyano. —C(=O)NR7R, -C(=S)NR7R-7, —C(=O)CR, R-7, OR-7 or SR-7, and R7, and Rare each independently H. (C-C)alkyl, (C- 40 C.)alkenyl, (C-C)alkynyl, aryl, (C-C)alkanoyl, or wherein: aryl(C-C)alkyl. In one embodiment R' is H. In another X, X, and X are each independently N. CH, or C-R. embodiment, R' is CN, N., methyl, OR", ethenyl, or R. R. and R are each independently H, halo, ethynyl. In another embodiment R' is N. In another NR,R, hydroxyamino, NRNRR, N, NO, NO, embodiment, R is methyl, ethenyl, or ethynyl. In formyl, cyano. —C(=O)NR,R, —C(=S)NRR, 45 —C(=O)CR, R OR or SR; and another embodiment, R is H. In a preferred aspect, Xs is R, and Rare each independently H. (C-C)alkyl, (C- CH and X and X, are N. In a particularly preferred C.)alkenyl, (C-C)alkynyl, aryl, (C-C)alkanoyl. aspect, XS is CH, X and X, are N, and Rio is —C(=O) —S(O)(C-C)alkyl or aryl(C-C)alkyl. In on NR.R. embodiment R' is H. In another embodiment, R' is CN, In another embodiment of the invention of Formula III or N, methyl, OR, ethenyl, or ethynyl. In another 50 embodiment R' is N. In another embodiment, R is Formula IV, B has the following formula: methyl, ethenyl, or ethynyl. In another embodiment, R is H. In a preferred aspect, X is N and B is 55

R60 / \ 60 -2. S.

wherein:

65 Y3 Y4 is - N=N , - CH=N , —N=CRs. . O —CH=CRs—, and each Rs is independently H, halo, or (C-C)alkyl. US 8,324,179 B2 41 42 In another embodiment of the invention of Formula III or -continued Formula IV, B has the following formula: O

ZZ1. Z.Z1. 5 f t Xs A t Xs Z. W 6 Z A 6 A.W6 W.--NW. A. 6Sws2NN W. Z : Z : Y- SN-Zi Y- SN-Zi 10

15 wherein: each Z is independently N. C. R. O, S, NR, DC=O, >C=S, >C=NR, DS—O, >S(O) or CH Re; pro vided that if a Z participates in an optional bond repre sented by a dotted line - - - in the formula, then that Z is N or C-Ro, and provided that if a Z1 does not partici pate in an optional bond represented by a dotted 25 line - - - in the formula, then that Z is O, S, NR, >C=O, >C=S, >C=NR, >S—O, >S(O), or CH-R; X is O, S, SO, SO, Se, SeO, SeO, or NR; each Wis C, CH, or N; wherein if a W participates in an 30 optional bond represented by a dotted line - - - in the formula, then that W is C; and if a W does not partici pate in an optional bond represented by a dotted line - - - in the formula, then that W is CH, or N: 35 each R is independently H, halo, NRR, hydroxyamino, NRNRoRo N. cyano. —C(=O) NRoRo —C(=S)NRoRo —C(=S)NRoRo —C(=NH)OR, R, OR, or SRs: 40 each R, is independently H., (C-C)alkyl, (C-C)alk enyl, (C-C)alkynyl, aryl, (C-C)alkanoyl, or aryl(C- C.)alkyl, and Ro, and Ro are each independently H. (C-C)alkyl, (C- C.)alkenyl, (C-C)alkynyl, aryl, (C-C)alkanoyl, or " aryl(C-C)alkyl. In one embodiment R' is H. In another embodiment, R' is CN, N., methyl, OR", ethenyl, or ethynyl. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethynyl. In another embodiment, R is H. 50 Another specific value for B has one of the following formulae:

55

/ 60 ^ %.Y-S ;4,N1 n 4. US 8,324,179 B2 43 44 -continued Roo Ro, are optionally Substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, amino, (C-C)alkyl, and (C-C)alkoxy. In a preferred embodiment, X is CR. In another pre ferred embodiment, X is N. In another preferred embodi ment, X is CRoo, and Xois O. In another preferred embodi ment, X is CR, and X is S. In another preferred embodiment, X is CRoo, and X is NR. In another pre 10 ferred embodiment, X is N and X is O. In another preferred embodiment, X is N and X is S. In another preferred embodiment, X is N and Xo is NRs. In another embodiment of the invention of Formula III or Formula IV, B has the following formula: 15

25

wherein: X is O, S, or NRo: 30 R7s and R, are each independently H, halo, hydroxy, mer capto, aryl, heterocycle, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, (C-C)alkylthio, arylthio. —S(=O) HCO3 NH ( \M (C-C)alkyl, -SO-O)(C-C)alkyl, -SO-O). NR7oR-71, NR7oR-71 (C-C)alkoxy, aryloxy, (het 35 erocycle)oxy; Ro is H. methyl, ethyl, or acetyl; and Rio, and R-7, are each independently H. (C-C)alkyl, aryl, aryl (C-C)alkyl, (C-C)alkanoyl, or aryl-C 40 (=O)—. In a preferred embodiment, X is O. In another preferred embodiment, X is S. In another pre In another embodiment of the invention of Formula III or ferred embodiment, X is NR7. Formula IV, B has the following formula: In another embodiment of the invention of Formula III or Formula IV, B has the following formula: 45

Z Y

n 50 N V80 N V80 - N ls - N -N V80 Yso V80 Z80

55 wherein: X is CRoo, or N: X is O, S, or NRo: Ro and Rare each independently H, halo, hydroxy, (C- Y80 Co.)alkoxy, NRooRoi, or heterocycle; 60 Roo, is H. halo, methyl, azido, or amino; Ro is H, or (C-C)alkyl; and Roo, and Ro, are each independently H. (C-C)alkyl, (C-C)cycloalkyl, or aryl (C-C)alkyl: 65 wherein each (C-C)alkyl, (C-C)cycloalkyl, aryl(C- C.)alkyl, and heterocycle of Roo Ro, Ro, and US 8,324,179 B2 45 46 -continued embodiment, R' is CN, N., methyl, OR", ethenyl, or ethynyl. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethynyl. In another embodiment, R is H. In a preferred embodiment, B is

10 wherein: Aso, Bso, and Yso are each independently H, halo, OR so. S(O),Rs NRsRs, cyano, trifluoromethyl, C(=W) 15 ORs, C(=W)SRs, C(=W)NRRs, nitro, azido, car bocyclic, (C-C)alkyl, (C-C)alkenyl, (C-C)alky nyl, aryl, aryl(C-C)alkyl, or heterocycle; or Aso and Bso taken together with the carbon atoms to which they are attached from a 4-7 membered carbocyclic or het erocyclic ring; n is 0, 1, or 2 Zso is O, S, NRso, or CRso Rs: each Vso is independently N or CRs, and each Rso and Rs is independently H, carbocycle, (C-C) 25 alkyl, (C-C)alkenyl, (C-C)alkynyl, halo, (C-C) alkoxy, amino, methylamino, dimethylamino, cyano, (C-C)alkanoyl, aryl, aryl(C-C)alkyl, an amino acid residue (e.g. a naturally-occurring amino acid residue) or heterocycle; or Rs and Rs taken together with the 30 atom(s) to which they are attached forma3-7 membered carbocyclic or heterocyclic ring. In another embodiment of the invention of Formula III or In another embodiment of the invention of Formula III or Formula IV, B is adenine, guanine, cytosine, uracil, thymine, Formula IV, B has the following formula: 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 35 7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole, 2-aminopurine, 2-amino-6-chloropurine, 2,6-di aminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isogua nine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 40 4-thiothymine, 4-thiouracil, O-methylguanine, N-methy ladenine, O-methylthymine, 5,6-dihydrothymine, 5,6-dihy drouracil, 4-methylindole, substituted triazole, or pyrazolo3. 4-Dipyrimidine. In one embodiment R' is H. In another 45 embodiment, R' is CN, N., methyl, OR", ethenyl, or ethynyl. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethynyl. In another embodiment, R is wherein: H. R., is H. hydroxy, mercapto, (C-C)alkyl, (C-C)alkoxy, In another embodiment of the invention of Formula III or (C-C)alkenyl, (C-Cs)cycloalkyl, aryl(C-C)alkyl, 50 Formula IV. B is 6-amino-2-chloro-purin-9-yl; 6-amino-2- aryl or (C-C)alkynyl: iodo-purin-9-yl, 6-amino-2-fluoro-purin-9-yl; 6-amino-2- Ros is H. hydroxy, mercapto, or (C-C)alkyl; methylthio-purin-9-yl; 6-amino-purin-9-yl; or 4-amino-7H Roo is H. halo, azido, cyano, nitro, ORoo. SRoo, pyrrolo2,3-dipyrimidin-7-yl. NRoo Roo, (C-C)alkyl, (C-C)alkenyl, (C-Cs)cy In another embodiment of the invention of Formula III or cloalkyl, aryl(C-C)alkyl, aryl or (C-C)alkynyl: 55 Formula IV. B is adenine or cytosine, which adenine or each Roo, is independently H. (C-C)alkyl, or (C-C) cytosine is optionally substituted with one or more (C-C) alkanoyl; alkyl, (C-C)alkylthio, (C-C)alkoxy, (C-C)alkanoyl, each Roo, and Roo is independently H. (C-C)alkyl, or optionally Substituted benzoyl (C-C)alkanoyloxy, (C-C) (C-C)alkanoyl; alkoxycarbonyl, NH, cyano, halo, hydroxy, nitro, carboxy, wherein each (C-C)alkyl, (C-C)alkoxy (C-C)alk 60 trifluoromethoxy, aryl, or mercapto. In one embodiment R' is enyl, (C-C)alkynyl, (C-C)cycloalkyl, aryl, and aryl H. In another embodiment, R is CN, N. methyl, OR, ethe (C-C)alkyl of R97, Ros, Roo. Roo. Root, and Roo, is nyl, or ethynyl. In another embodiment R' is N. In another optionally substituted with one or more (C-C)alkyl, embodiment, R is methyl, ethenyl, or ethynyl. In another (C-C)alkylthio, (C-C)alkoxy, (C-C)alkanoyl, (C- embodiment, R is H. C.)alkanoyloxy, (C-C)alkoxycarbonyl, NH, cyano, 65 In a preferred embodiment of the invention of Formula III azido, halo, hydroxy, nitro, carboxy, trifluoromethoxy, or Formula IV, B is adenine. In another embodiment, B is aryl, or mercapto. In one embodiment R' is H. In another cytosine. In another embodiment, B is thymine. In another US 8,324,179 B2 47 48 embodiment, B is guanine. In another embodiment, B is oxygen-linked optionally Substituted aryloxy. In another uracil. In another embodiment, B is not adenine, guanine, embodiment, W' and Ware independently an oxygen-linked cytosine, thymine or uracil. naturally-occurring or unnatural C-hydroxycarboxylic acid In another preferred embodiment of the invention of For or C-hydroxycarboxylic acid ester. 5 In another embodiment of Formula III or Formula IV. W' mula III or Formula IV, B is and Ware independently a nitrogen-linked naturally-occur ring or unnatural O-aminoacid or CL-aminoacid ester. In one embodiment R' is H. In another embodiment, R' is CN, N. O NH2 NH2 methyl, OR", ethenyl, or ethynyl. In another embodiment R' is N. In another embodiment, R is methyl, ethenyl, or ethy NH NN N NN 10 nyl. In another embodiment, R is H. In another preferred embodiment, W' and Ware each independently a nitrogen lulN O, N - O, (-llN N , linked alanine or an alanine ester. In another preferred embodiment, W' and Ware each independently a nitrogen olo olo olo linked alanine ester wherein the ester group is C-C alkyl. 15 In another embodiment of Formula III or Formula IV. W' NH and W are independently an oxygen-linked naturally-occur | NH ring or unnatural C-hydroxycarboxylic acid or C-hydroxycar boxylic acid ester. In one embodiment R' is H. In another ^ nN. -s N embodiment, R' is CN, N. methyl, OR, ethenyl, or ethynyl. N e f In another embodiment R' is N. In another embodiment, R N N N \ / is methyl, ethenyl, or ethynyl. In another embodiment, R is H. In another embodiment of Formula III or Formula IV, R O 25 and Rare not protecting groups. NH2 ti-- In another preferred aspect, a compound of the invention is N n N-N M N NN 30

35

45 O HG OH NH

N NH2, 50 NN.

55 In one embodiment R' is H. In another embodiment, R is Hó 6H CN. N. methyl, OR, ethenyl, or ethynyl. In another embodi ment R' is N. In another embodiment, R is methyl, ethenyl, NH or ethynyl. In another embodiment, R is H. N In another embodiment of Formula III or Formula IV. W' 60 M. NN. and Ware independently a nitrogen-linked naturally-occur O O O N e2 ring or unnatural O-aminoacid or C.-aminoacid ester. In o N another preferred embodiment, W and Ware each indepen HO1 No1 No1 dently a nitrogen-linked alanine or analanine ester. In another OH OH OH preferred embodiment, W' and Ware each independently a 65 nitrogen-linked alanine ester wherein the estergroup is C-C, H6 oH alkyl. In another embodiment, W' and Ware independently

US 8,324,179 B2 53 54 -continued -continued O

10

30

NH2

n N

Nd

NH2,

US 8,324,179 B2 57 58 -continued -continued

65

US 8,324,179 B2 63 64 -continued have 1 to 20 carbon atoms (i.e. C-C alkyl), 1 to 10 carbon O atoms (i.e., C-C alkyl), or 1 to 6 carbon atoms (i.e., C-C, alkyl). Examples of suitable alkyl groups include, but are not NH limited to, methyl (Me. —CH), ethyl (Et, —CHCH), O 1-propyl (n-Pr, n-propyl. —CHCHCH), 2-propyl (i-Pr. HON-P / -N i-propyl. —CH(CH)), 1-butyl (n-Bu, n-butyl, HO 21 o, N O, —CH2CH2CHCH), 2-methyl-1-propyl (i-Bu, i-butyl, —CHCH(CH)), 2-butyl (s-Bu, s-butyl, -CH(CH) CHCH), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH)), 2 \ 10 Hó OH 1-pentyl (n-pentyl, —CH2CH2CH2CHCH), 2-pentyl O (—CH(CH)CHCHCH), 3-pentyl ( CH(CHCH)), 2-methyl-2-butyl ( C(CH) CHCH), 3-methyl-2-butyl

(-CH(CH)CH(CH)), 3-methyl-1-butyl ( CHCHCH 15 (CH)), 2-methyl-1-butyl ( CHCH(CH)CHCH), 1-hexyl ( CHCH2CH2CH2CHCH), 2-hexyl ( CH (CH)CHCHCHCH), 3-hexyl (-CH(CHCH.) (CHCHCH)), 2-methyl-2-pentyl (—C(CH) CHCHCH), 3-methyl-2-pentyl ( CH(CH)CH(CH) CHCH), 4-methyl-2-pentyl ( CH(CH)CH-CH(CH)), 3-methyl-3-pentyl ( C(CH)(CHCH)), 2-methyl-3-pen SO.Me, M tyl ( CH(CHCH)CH(CH)), 2,3-dimethyl-2-butyl ( C HN (CH),CH(CH)), 3.3-dimethyl-2-butyl ( CH(CH)C V N N (CH), and octyl (—(CH2)CH). 25 "Alkoxy' means a group having the formula—O-alkyl, in HO O t which an alkyl group, as defined above, is attached to the D O N / \ parent molecule via an oxygen atom. The alkyl portion of an HO alkoxy group can have 1 to 20 carbon atoms (i.e., C-Co N-n- NF alkoxy), 1 to 12 carbon atoms(i.e., C-C alkoxy), or 1 to 6 OH 30 carbon atoms(i.e., C-C alkoxy). Examples of suitable SOMe, or alkoxy groups include, but are not limited to, methoxy A HN ( O CH or -OMe), ethoxy ( OCHCH or - OEt), w t-butoxy ( O C(CH), or-OtBu) and the like. N N "Haloalkyl is an alkyl group, as defined above, in which 35 one or more hydrogen atoms of the alkyl group is replaced Ho-1-0--0- O N-/ \ with a halogen atom. The alkyl portion of a haloalkyl group OH OH -ny- N=/ can have 1 to 20 carbonatoms (i.e., C-Cohaloalkyl), 1 to 12 carbon atoms(i.e., C-C haloalkyl), or 1 to 6 carbon atoms OH (i.e., C-C alkyl). Examples of Suitable haloalkyl groups O 40 include, but are not limited to. —CF, —CHCF, and the like. NH Alkenyl is a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsat O N n -N uration, i.e. a carbon-carbon, sp double bond. For example, Hos O N O; 45 an alkenyl group can have 2 to 20 carbon atoms (i.e., C.-Co alkenyl), 2 to 12 carbon atoms (i.e., C-C alkenyl), or 2 to 6 carbon atoms (i.e., C-C alkenyl). Examples of suitable alk enyl groups include, but are not limited to, ethylene or vinyl HG OH (—CH=CH), allyl ( CH-CH=CH-), cyclopentenyl 50 (—OH,), and 5-hexenyl ( CHCHCHCH-CH=CH-). or a pharmaceutical acceptable salt thereof. Alkynyl is a hydrocarbon containing normal, secondary, Definitions tertiary or cyclic carbon atoms with at least one site of unsat Unless stated otherwise, the following terms and phrases as uration, i.e. a carbon-carbon, sp triple bond. For example, an used herein are intended to have the following meanings: alkynyl group can have 2 to 20 carbon atoms (i.e., C.-Co When trade names are used herein, applicants intend to 55 alkynyl), 2 to 12 carbon atoms (i.e., C-C alkyne.), or 2 to 6 independently include the tradename product and the active carbon atoms (i.e., C-C alkynyl). Examples of suitable pharmaceutical ingredient(s) of the tradename product. alkynyl groups include, but are not limited to, acetylenic As used herein, “a compound of the invention” or “a com (—C=CH), propargyl ( CH-C=CH), and the like. pound of Formula I means a compound of Formula I or a Alkylene' refers to a saturated, branched or straight chain pharmaceutically acceptable salt, Solvate, or physiologically 60 or cyclic hydrocarbon radical having two monovalent radical functional derivative thereof. Similarly, with respect to iso centers derived by the removal of two hydrogen atoms from latable intermediates, the phrase “a compound of Formula the same or two different carbonatoms of a parent alkane. For (number) means a compound of that formula and pharma example, an alkylene group can have 1 to 20 carbon atoms, 1 ceutically acceptable salts, Solvates and physiologically func to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkylene tional derivatives thereof. 65 radicals include, but are not limited to, methylene (-CH2—), Alkyl is hydrocarbon containing normal, secondary, ter 1,1-ethyl ( CH(CH)—), 1.2-ethyl ( CHCH ), 1,1- tiary or cyclic carbonatoms. For example, an alkyl group can propyl ( CH(CHCH)—), 1,2-propyl ( CHCH US 8,324,179 B2 65 66 (CH)—), 1,3-propyl (—CH2CH2CH2—), 1,4-butyl atoms, e.g., the alkenyl moiety is 1 to 6 carbon atoms and the ( CHCHCHCH ), and the like. aryl moiety is 6 to 14 carbon atoms. Alkenylene' refers to an unsaturated, branched or straight Arylalkynyl refers to an acyclic alkynyl radical in which chain or cyclic hydrocarbon radical having two monovalent one of the hydrogenatoms bonded to a carbonatom, typically radical centers derived by the removal of two hydrogenatoms a terminal or sp carbon atom, but also an sp carbon atom, is from the same or two different carbon atoms of a parent replaced with an aryl radical. The aryl portion of the aryla alkene. For example, and alkenylene group can have 1 to 20 lkynyl can include, for example, any of the aryl groups dis carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. closed herein, and the alkynyl portion of the arylalkynyl can Typical alkenylene radicals include, but are not limited to, include, for example, any of the alkynyl groups disclosed 10 herein. The arylalkynyl group can comprise 6 to 20 carbon 1.2-ethylene (—CH=CH-). atoms, e.g., the alkynyl moiety is 1 to 6 carbon atoms and the Alkynylene' refers to an unsaturated, branched or straight aryl moiety is 6 to 14 carbon atoms. chain or cyclic hydrocarbon radical having two monovalent The term “substituted in reference to alkyl, alkylene, aryl, radical centers derived by the removal of two hydrogenatoms arylalkyl, alkoxy, heterocyclyl, heteroaryl, carbocyclyl, etc., from the same or two different carbon atoms of a parent 15 for example, “substituted alkyl, “substituted alkylene'. alkyne. For example, an alkynylene group can have 1 to 20 “substituted aryl”, “substituted arylalkyl, “substituted het carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. erocyclyl', and “substituted carbocyclyl means alkyl, alky Typical alkynylene radicals include, but are not limited to, lene, aryl, arylalkyl, heterocyclyl, carbocyclyl respectively, in acetylene ( -C=C- ), propargyl ( CH2C=C- ), and which one or more hydrogen atoms are each independently 4-pentynyl ( CHCH2CH2C=CH-). replaced with a non-hydrogen Substituent. Typical Substitu Amino” refers generally to a nitrogen radical which can be ents include, but are not limited to, —X, —R, —O, —O, considered a derivative of ammonia, having the formula —OR, SR, S, NR - NR =NR, CX-CN, —NCX), where each “X” is independently H, substituted or OCN, SCN, N=C=O, NCS, NO, NO, unsubstituted alkyl, substituted or unsubstituted carbocyclyl, —N - N - NHC(=O)R, C(=O)R, C(=O)NRR substituted or unsubstituted heterocyclyl, etc. The hybridiza 25 S(=O) , S(=O),OH, -S(=O).R, OS(=O),OR, tion of the nitrogen is approximately sp. Nonlimiting types —S(=O)NR, S(=O)R, OP(=O)(OR), -P(=O) of amino include —NH —N(alkyl). —NH(alkyl). (OR), -P(=O)(O) = P(=O)(OH), -P(O)(OR)(O), —N(carbocyclyl) —NH(carbocyclyl), N(heterocyclyl), - C(=O)R, C(=O)x, C(S)R, C(O)OR, —C(O)O, —NH(heterocyclyl), —N(aryl), NH(aryl), —N(alkyl) -C(S)OR, C(O)SR, C(S)SR, C(O)NRR, C(S) (aryl), —N(alkyl)(heterocyclyl), N(carbocyclyl)(hetero 30 NRR, C(=NR)NRR, where each X is independently a cyclyl), —N(aryl)(heteroaryl), —N(alkyl)(heteroaryl), etc. halogen: F, Cl, Br, or I; and each R is independently H, alkyl, The term “alkylamino” refers to an amino group substituted aryl, arylalkyl, a heterocycle, or a protecting group or prodrug with at least one alkyl group. Nonlimiting examples of amino moiety. Alkylene, alkenylene, and alkynylene groups may groups include —NH, NH(CH), N(CH), NH also be similarly substituted. Unless otherwise indicated, (CHCH), N(CHCH), NH(phenyl), N(phenyl), 35 when the term “substituted” is used in conjunction with —NH(benzyl), —N(benzyl), etc. Substituted alkylamino groups such as arylalkyl, which have two or more moieties refers generally to alkylamino groups, as defined above, in capable of substitution, the substituents can be attached to the which at least one substituted alkyl, as defined herein, is aryl moiety, the alkyl moiety, or both. attached to the amino nitrogen atom. Non-limiting examples The terms “phosphonate' and “phosphonate group” mean of substituted alkylamino includes —NH(alkylene-C(O)— 40 a functional group or moiety within a molecule that com OH), —NH(alkylene-C(O)—O-alkyl). —N(alkylene-C prises at least one phosphorus-carbon bond, and at least one (O) OH), N(alkylene-C(O)—O-alkyl), etc. phosphorus-oxygen double bond. The phosphorus atom is Aryl means an aromatic hydrocarbon radical derived by further Substituted with oxygen, Sulfur, or nitrogen Substitu the removal of one hydrogenatom from a single carbonatom ents. These substituents may be part of a prodrug moiety. As of a parent aromatic ring system. For example, an aryl group 45 defined herein, "phosphonate” and “phosphonate group' can have 6 to 20 carbon atoms, 6 to 14 carbonatoms, or 6 to include molecules with phosphonic acid, phosphonic 12 carbon atoms. Typical aryl groups include, but are not monoester, phosphonic diester, phosphonamidate, phosphon limited to, radicals derived from benzene (e.g., phenyl), Sub diamidate and phosphonthioate functional groups. stituted benzene, naphthalene, anthracene, biphenyl, and the The term “prodrug as used herein refers to any compound like. 50 that when administered to a biological system generates the Arylalkyl refers to an acyclic alkyl radical in which one drug Substance, i.e., active ingredient, as a result of sponta of the hydrogen atoms bonded to a carbon atom, typically a neous chemical reaction(s), enzyme catalyzed chemical reac terminal or sp carbon atom, is replaced with an aryl radical. tion(s), photolysis, and/or metabolic chemical reaction(s). A Typical arylalkyl groups include, but are not limited to, ben prodrug is thus a covalently modified analog or latent form of Zyl. 2-phenylethan-1-yl, naphthylmethyl 2-naphthylethan-1- 55 a therapeutically active compound. yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. One skilled in the art will recognize that substituents and The arylalkyl group can comprise 6 to 20 carbon atoms, e.g., other moieties of the compounds of Formula I-IV should be the alkyl moiety is 1 to 6 carbon atoms and the aryl moiety is selected in order to provide a compound which is sufficiently 6 to 14 carbon atoms. stable to provide a pharmaceutically useful compound which Arylalkenyl refers to an acyclic alkenyl radical in which 60 can be formulated into an acceptably stable pharmaceutical one of the hydrogenatoms bonded to a carbonatom, typically composition. Compounds of Formula I-IV which have such a terminal or sp carbon atom, but also an spf carbon atom, is stability are contemplated as falling within the scope of the replaced with an aryl radical. The aryl portion of the arylalk present invention. enyl can include, for example, any of the aryl groups dis “Heteroalkyl refers to an alkyl group where one or more closed herein, and the alkenyl portion of the arylalkenyl can 65 carbon atoms have been replaced with a heteroatom, such as, include, for example, any of the alkenyl groups disclosed O, N, or S. For example, if the carbon atom of the alkyl group herein. The arylalkenyl group can comprise 6 to 20 carbon which is attached to the parent molecule is replaced with a US 8,324,179 B2 67 68 heteroatom (e.g., O, N, or S) the resulting heteroalkyl groups Zotriazolyl, benzisoxazolyl, OXindolyl, benzoxazolinyl, isati are, respectively, an alkoxy group (e.g., —OCH, etc.), an noyl, and bis-tetrahydrofuranyl: amine (e.g., -NHCH, -N(CH), etc.), or a thioalkyl group (e.g., —SCH). If a non-terminal carbon atom of the alkyl group which is not attached to the parent molecule is replaced with a heteroatom (e.g., O, N, or S) the resulting heteroalkyl groups are, respectively, an alkyl ether (e.g., O —CH2CH2—O—CH, etc.), an alkyl amine (e.g., —CH-NHCH, —CHN(CH), etc.), or a thioalkyl ether (e.g., —CH2—S-CH). If a terminal carbon atom of the 10 alkyl group is replaced with a heteroatom (e.g., O, N, or S), By way of example and not limitation, carbon bonded the resulting heteroalkyl groups are, respectively, a hydroxy heterocycles are bonded at position 2, 3, 4, 5, or 6 of a alkyl group (e.g., —CH2CH2—OH), an aminoalkyl group pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, (e.g., —CH-NH), oran alkylthiol group (e.g., —CHCH 15 or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, SH). A heteroalkyl group can have, for example, 1 to 20 position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, carbonatoms, 1 to 10 carbonatoms, or 1 to 6 carbonatoms. A thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of C-C heteroalkyl group means a heteroalkyl group having 1 an oxazole, imidazole or thiazole, position 3, 4, or 5 of an to 6 carbon atoms. isoxazole, pyrazole, or isothiazole, position 2 or 3 of an “Heterocycle' or "heterocyclyl as used herein includes by aziridine, position 2, 3, or 4 of anaZetidine, position 2, 3, 4, 5, way of example and not limitation those heterocycles 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an described in Paquette, Leo A.; Principles of Modern Hetero isoquinoline. Still more typically, carbon bonded hetero cyclic Chemistry (W. A. Benjamin, New York, 1968), particu cycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, larly Chapters 1, 3, 4, 6, 7, and 9: The Chemistry of Hetero 6-pyridyl, 3-pyridazinyl, 4-pyridaZinyl, 5-pyridazinyl, 6-py cyclic Compounds, A Series of Monographs” (John Wiley & 25 ridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-py Sons, New York, 1950 to present), in particular Volumes 13, rimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. In 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl. one specific embodiment of the invention "heterocycle' By way of example and not limitation, nitrogen bonded includes a “carbocycle” as defined herein, wherein one or heterocycles are bonded at position 1 of an aziridine, aZeti more (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with 30 dine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imida a heteroatom (e.g. O, N, or S). The terms "heterocycle' or Zole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, "heterocyclyl' includes saturated rings, partially unsaturated pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, pipera rings, and aromatic rings (i.e., heteroaromatic rings). Substi Zine, indole, indoline, 1H-indazole, position 2 of a isoindole, tuted heterocyclyls include, for example, heterocyclic rings or isoindoline, position 4 of a morpholine, and position 9 of a substituted with any of the substituents disclosed herein 35 carbazole, or 3-carboline. Still more typically, nitrogen including carbonyl groups. A non-limiting example of a car bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrro bonyl substituted heterocyclyl is: lyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl. “Heterocyclylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, 40 typically a terminal or sp carbon atom, is replaced with a heterocyclyl radical (i.e., a heterocyclyl-alkylene-moiety). Typical heterocyclyl alkyl groups include, but are not limited to heterocyclyl-CH , 2-(heterocyclyl)ethan-1-yl, and the like, wherein the "heterocyclyl portion includes any of the X-r 45 heterocyclyl groups described above, including those described in Principles of Modern Heterocyclic Chemistry. Examples of heterocycles include by way of example and One skilled in the art will also understand that the heterocy not limitation pyridyl, dihydroypyridyl, tetrahydropyridyl clyl group can be attached to the alkyl portion of the hetero (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized cyclyl alkyl by means of a carbon-carbon bond or a carbon tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, 50 heteroatom bond, with the proviso that the resulting group is pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphtha chemically stable. The heterocyclyl alkyl group comprises 6 lenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimi to 20 carbon atoms, e.g., the alkyl portion of the arylalkyl dazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolido group is 1 to 6 carbon atoms and the heterocyclyl moiety is 5 nyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, to 14 carbon atoms. Examples of heterocyclylalkyls include tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroiso 55 by way of example and not limitation 5-membered sulfur, quinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, oxygen, and/or nitrogen containing heterocycles Such as thia 2H,6H-1.5.2-dithiazinyl, thienyl, thianthrenyl, pyranyl, Zolylmethyl, 2-thiazolylethan-1-yl, imidazolylmethyl, isobenzofuranyl, chromenyl, Xanthenyl, phenoxathinyl, oxazolylmethyl, thiadiazolylmethyl, etc., 6-membered sul 2H-pyrrolyl, isothiazolyl, isoxazolyl pyrazinyl, pyridazinyl, fur, oxygen, and/or nitrogen containing heterocycles such as indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 60 piperidinylmethyl, piperazinylmethyl, morpholinylmethyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, pyridinylmethyl, pyridizylmethyl, pyrimidylmethyl, pyrazi quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carba nylmethyl, etc. Zolyl, B-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, “Heterocyclylalkenyl refers to an acyclic alkenyl radical phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phe in which one of the hydrogenatoms bonded to a carbon atom, noxazinyl, isochromanyl, chromanyl, imidazolidinyl, imida 65 typically a terminal or sp carbon atom, but also a sp carbon Zolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, atom, is replaced with a heterocyclyl radical (i.e., a heterocy isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, ben clyl-alkenylene-moiety). The heterocyclyl portion of the het US 8,324,179 B2 69 70 erocyclyl alkenyl group includes any of the heterocyclyl -alkylene-S-alkylene-aryl, etc. In addition, any of the alky groups described herein, including those described in Prin lene moieties in the general formulae above can be further ciples of Modern Heterocyclic Chemistry, and the alkenyl substituted with any of the substituents defined or exemplified portion of the heterocyclyl alkenyl group includes any of the herein. alkenyl groups disclosed herein. One skilled in the art will also understand that the heterocyclyl group can be attached to “Heteroarylalkyl refers to an alkyl group, as defined the alkenyl portion of the heterocyclyl alkenyl by means of a herein, in which a hydrogen atom has been replaced with a carbon-carbon bond or a carbon-heteroatom bond, with the heteroaryl group as defined herein. Non-limiting examples of proviso that the resulting group is chemically stable. The heteroaryl alkyl include —CH-pyridinyl, -CH2-pyrrolyl, heterocyclyl alkenyl group comprises 6 to 20 carbon atoms, 10 —CH2-oxazolyl. —CH-indolyl. —CH2-purinyl, -CH2 e.g., the alkenyl portion of the heterocyclyl alkenyl group is 1 furanyl, —CH2-thienyl, —CH-benzofuranyl, —CH-ben to 6 carbon atoms and the heterocyclyl moiety is 5 to 14 Zothiophenyl, —CH-carbazolyl, —CH-imidazolyl, carbon atoms. —CH2-thiazolyl, —CH2-isoxazolyl, —CH2-pyrazolyl, “Heterocyclylalkynyl refers to an acyclic alkynyl radical —CH2-isothiazolyl, -CH2-quinolyl. —CH2-isoquinolyl, in which one of the hydrogenatoms bonded to a carbonatom, 15 —CH-pyridaZyl. —CH-pyrimidyl, —CH-pyrazyl, -CH typically a terminal or sp carbon atom, but also an sp carbon (CH)-pyridinyl, —CH(CH)-pyrrolyl. —CH(CH)-ox atom, is replaced with a heterocyclyl radical (i.e., a heterocy azolyl, —CH(CH)-indolyl, —CH(CH)-isoindolyl, —CH clyl-alkynylene-moiety). The heterocyclyl portion of the het (CH)-purinyl, -CH(CH)-furanyl, —CH(CH)-thienyl, erocyclyl alkynyl group includes any of the heterocyclyl —CH(CH-)-benzofuranyl, —CH(CH-)-benzothiophenyl, groups described herein, including those described in Prin ciples of Modern Heterocyclic Chemistry, and the alkynyl —CH(CH-)-carbazolyl, —CH(CH)-imidazolyl, —CH portion of the heterocyclyl alkynyl group includes any of the (CH)-thiazolyl, -CH(CH)-isoxazolyl, -CH(CH)-pyra alkynyl groups disclosed herein. One skilled in the art will Zolyl, —CH(CH)-isothiazolyl. —CH(CH)-quinolyl. —CH also understand that the heterocyclyl group can be attached to (CH)-isoquinolyl. —CH(CH)-pyridazyl, —CH(CH)- the alkynyl portion of the heterocyclyl alkynyl by means of a 25 pyrimidyl, -CH(CH)-pyrazyl, etc. carbon-carbon bond or a carbon-heteroatom bond, with the The term “optionally substituted in reference to a particu proviso that the resulting group is chemically stable. The lar moiety of the compound of Formula I-IV (e.g., an option heterocyclyl alkynyl group comprises 6 to 20 carbon atoms, ally Substituted aryl group) refers to a moiety having 0, 1, 2, e.g., the alkynyl portion of the heterocyclyl alkynyl group is 1 or more Substituents. to 6 carbon atoms and the heterocyclyl moiety is 5 to 14 30 carbon atoms. "Linker” or “link’ means a chemical moiety comprising a “Heteroaryl” refers to an aromatic heterocyclyl having at covalent bond or a chain of atoms. Linkers include repeating least one heteroatom in the ring. Non-limiting examples of units of alkyloxy (e.g. polyethyleneoxy, PEG, polymethyl suitable heteroatoms which can be included in the aromatic eneoxy) and alkylamino (e.g. polyethyleneamino, Jeffam ring include oxygen, Sulfur, and nitrogen. Non-limiting 35 ineTM); and diacid ester and amides including Succinate. Suc examples of heteroaryl rings include all of those listed in the cinamide, diglycolate, malonate, and caproamide. definition of "heterocyclyl', including pyridinyl, pyrrolyl, The terms such as “oxygen-linked”, “nitrogen-linked'. oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, ben “carbon-linked, “sulfur-linked, or “phosphorous-linked' Zofuranyl, benzothiophenyl, carbazolyl, imidazolyl, thiaz mean that if a bond between two moieties can be formed by olyl, isoxazolyl pyrazolyl, isothiazolyl, quinolyl, iso 40 quinolyl pyridaZyl, pyrimidyl, pyrazyl, etc. using more than one type of atom in a moiety, then the bond “Carbocycle' or “carbocyclyl refers to a saturated (i.e., formed between the moieties is through the atom specified. cycloalkyl), partially unsaturated (e.g., cycloakenyl, For example, a nitrogen-linked amino acid would be bonded cycloalkadienyl, etc.) or aromatic ring having 3 to 7 carbon through a nitrogenatom of the amino acid rather than through atoms as a monocycle, 7 to 12 carbon atoms as a bicycle, and 45 an oxygen or carbon atom of the amino acid. up to about 20 carbon atoms as a polycycle. Monocyclic Certain Y and Y alternatives are nitrogen oxides such as carbocycles have 3 to 6 ring atoms, still more typically 5 or 6 N(O)(R) or "N(O)(OR). These nitrogen oxides, as shown ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g., here attached to a carbon atom, can also be represented by arranged as a bicyclo 4.5, 5.5, 5.6 or 6.6 system, or 9 or charge separated groups such as 10 ring atoms arranged as a bicyclo 5.6 or 6.6 system, or 50 spiro-fused rings. Non-limiting examples of monocyclic car bocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cy clopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclo hex-3-enyl, and phenyl. Non-limiting examples of bicyclo 55 carbocycles includes naphthyl. Arylheteroalkyl refers to a heteroalkyl as defined herein, respectively, and are intended to be equivalent to the afore in which a hydrogen atom (which may be attached either to a mentioned representations for the purposes of describing this carbon atom or a heteroatom) has been replaced with an aryl invention. 60 group as defined herein. The aryl groups may be bonded to a Unless otherwise specified, the carbonatoms of this inven carbon atom of the heteroalkyl group, or to a heteroatom of tion are intended to have a Valence of four. In some chemical the heteroalkyl group, provided that the resulting arylhet structure representations where carbon atoms do not have a eroalkyl group provides a chemically stable moiety. For sufficient number of variables attached to produce a valence example, an arylheteroalkyl group can have the general for 65 of four, the remaining carbon Substitutents needed to provide mulae -alkylene-O-aryl, -alkylene-O-alkylene-aryl, -alky a valence of four should be assumed to be hydrogen. For lene-NH-aryl, -alkylene-NH-alkylene-aryl, -alkylene-S-aryl, example, US 8,324,179 B2 71 72 combinations of the invention. An exemplary acyloxymethyl ester is pivaloyloxymethoxy, (POM) —CHOC(=O)C (CH). An exemplary acyloxymethyl carbonate prodrug 21 O moiety is pivaloyloxymethylcarbonate (POC) —CHOC (=O)CC(CH). The phosphonate group may be a phosphonate prodrug O s" moiety. The prodrug moiety may be sensitive to hydrolysis, O Such as, but not limited to a pivaloyloxymethyl carbonate R2 (POC) or POM group. Alternatively, the prodrug moiety may 10 be sensitive to enzymatic potentiated cleavage, such as a lactate ester or a phosphonamidate-ester group. has the same meaning as Aryl esters of phosphorus groups, especially phenyl esters, are reported to enhance oral bioavailability (DeLambert etal (1994) J. Med. Chem. 37: 498). Phenyl esters containing a CH 15 carboxylic ester ortho to the phosphate have also been described (Khamnei and Torrence, (1996) J. Med. Chem. H 21 O. 39:4109-4115). Benzyl esters are reported to generate the parent phosphonic acid. In some cases, Substituents at the O N NH ortho-or para-position may accelerate the hydrolysis. Benzyl H 1 analogs with an acylated phenol or an alkylated phenol may H. O. generate the phenolic compound through the action of enzymes, e.g. esterases, oxidases, etc., which in turn under R2 H. H. goes cleavage at the benzylic C-O bond to generate the phosphoric acid and the quinone methide intermediate. “Pharmaceutically acceptable prodrug” refers to a com 25 Examples of this class of prodrugs are described by Mitchell pound that is metabolized in the host, for example hydrolyzed et al (1992).J. Chem. Soc. Perkin Trans. I 2345; Brook et al or oxidized, by either enzymatic action or by general acid or WO 91/19721. Still other benzylic prodrugs have been base Solvolysis, to form an active ingredient. Typical described containing a carboxylic ester-containing group examples of prodrugs of the compounds of the invention have attached to the benzylic methylene (Glazier et al WO biologically labile protecting groups on a functional moiety 30 91/19721). Thio-containing prodrugs are reported to be use of the compound. Prodrugs include compounds that can be ful for the intracellular delivery of phosphonate drugs. These oxidized, reduced, aminated, deaminated, esterified, deesteri proesters contain an ethylthio group in which the thiol group fied, alkylated, dealkylated, acylated, deacylated, phosphory is either esterified with an acyl group or combined with lated, dephosphorylated, photolyzed, hydrolyzed, or other another thiol group to form a disulfide. Deesterification or functional group change or conversion involving forming or 35 reduction of the disulfide generates the free thio intermediate breaking chemical bonds on the prodrug. which Subsequently breaks down to the phosphoric acid and “Prodrug moiety' means a labile functional group which episulfide (Puech et al (1993) Antiviral Res., 22: 155-174: separates from the active inhibitory compound during Benzaria et al (1996).J. Med Chem. 39: 4958). Cyclic phos metabolism, systemically, inside a cell, by hydrolysis, enzy phonate esters have also been described as prodrugs of phos matic cleavage, or by some other process (Bundgaard, Hans, 40 phorus-containing compounds (Erion et al., U.S. Pat. No. “Design and Application of Prodrugs” in Textbook of Drug 6,312,662). Design and Development (1991), P. Krogsgaard-Larsen and “Protecting group' refers to a moiety of a compound that H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113 masks or alters the properties of a functional group or the 191). Enzymes which are capable of an enzymatic activation properties of the compound as a whole. The chemical Sub mechanism with the phosphonate prodrug compounds of the 45 structure of a protecting group varies widely. One function of invention include, but are not limited to, amidases, esterases, a protecting group is to serve as an intermediate in the Syn microbial enzymes, phospholipases, cholinesterases, and thesis of the parental drug Substance. Chemical protecting phosphases. Prodrug moieties can serve to enhance solubility, groups and strategies for protection/deprotection are well absorption and lipophilicity to optimize drug delivery, bio known in the art. See: “Protective Groups in Organic Chem availability and efficacy. 50 istry’. Theodora W. Greene (John Wiley & Sons, Inc., New A prodrug moiety may include an active metabolite or drug York, 1991. Protecting groups are often utilized to mask the itself. reactivity of certain functional groups, to assist in the effi Exemplary prodrug moieties include the hydrolytically ciency of desired chemical reactions, e.g. making and break sensitive or labile acyloxymethyl esters —CHOC(=O)R’ ing chemical bonds in an ordered and planned fashion. Pro and acyloxymethyl carbonates —CHOC(=O)CR where 55 tection of functional groups of a compound alters other R is C-C alkyl, C-C substituted alkyl, Co-Co aryl or physical properties besides the reactivity of the protected Co-Co Substituted aryl. The acyloxyalkyl ester was used as a functional group, Such as the polarity, lipophilicity (hydro prodrug strategy for carboxylic acids and then applied to phobicity), and other properties which can be measured by phosphates and phosphonates by Farquhar et al (1983) J. common analytical tools. Chemically protected intermedi Pharm. Sci. 72: 324; also U.S. Pat. Nos. 4,816,570, 4,968, 60 ates may themselves be biologically active or inactive. 788, 5,663,159 and 5,792,756. In certain compounds of the Protected compounds may also exhibit altered, and in some invention, a prodrug moiety is part of a phosphonate group. cases, optimized properties in vitro and in vivo, Such as pas The acyloxyalkyl ester may be used to deliver phosphonic sage through cellular membranes and resistance to enzymatic acids across cell membranes and to enhance oral bioavailabil degradation or sequestration. In this role, protected com ity. A close variant of the acyloxyalkyl ester, the alkoxycar 65 pounds with intended therapeutic effects may be referred to as bonyloxyalkyl ester (carbonate), may also enhance oral bio prodrugs. Another function of a protecting group is to convert availability as a prodrug moiety in the compounds of the the parental drug into a prodrug, whereby the parental drug is US 8,324,179 B2 73 74 released upon conversion of the prodrug in vivo. Because nucleoside bases that can be incorporated into the compounds active prodrugs may be absorbed more effectively than the of this invention are disclosed in United States Patent Appli parental drug, prodrugs may possess greater potency in vivo cation Publication Number 2004/0147464, United States than the parental drug. Protecting groups are removed either Patent Application Publication Number 2005/0215511, Inter in vitro, in the instance of chemical intermediates, or in vivo, national Patent Application Publication Number WO in the case of prodrugs. With chemical intermediates, it is not particularly important that the resulting products after depro 03/061385, International Patent Application Publication tection, e.g. alcohols, be physiologically acceptable, although Number WO 03/062257, International Patent Application in general it is more desirable if the products are pharmaco Publication Number WO 03/072757, International Patent logically innocuous. 10 Application Publication Number WO 03/073989, Interna Any reference to any of the compounds of the invention tional Patent Application Publication Number WO 2005/ also includes a reference to a physiologically acceptable salt 021568, International Patent Application Publication Num thereof Examples of physiologically acceptable salts of the ber WO 2005/123087, International Patent Application compounds of the invention include salts derived from an 15 Publication Number WO 2006/002231, and International appropriate base. Such as an alkali metal (for example, Patent Application Publication Number WO 2006/000922. Sodium), an alkaline earth (for example, magnesium), ammo nium and NX (wherein X is C-C alkyl). Physiologically The term "chiral refers to molecules which have the prop acceptable salts of an hydrogen atom or an amino group erty of non-Superimposability of the mirror image partner, include salts of organic carboxylic acids such as acetic, ben while the term “achiral” refers to molecules which are super Zoic, lactic, fumaric, tartaric, maleic, malonic, malic, imposable on their mirror image partner. isethionic, lactobionic and Succinic acids; organic Sulfonic The term “stereoisomers' refers to compounds which have acids, such as methanesulfonic, ethanesulfonic, benzene identical chemical constitution, but differ with regard to the Sulfonic and p-toluenesulfonic acids; and inorganic acids, arrangement of the atoms or groups in space. Such as hydrochloric, Sulfuric, phosphoric and Sulfamic acids. Physiologically acceptable salts of a compound of an 25 “Diastereomer' refers to a stereoisomer with two or more hydroxy group include the anion of said compound in com centers of chirality and whose molecules are not mirror bination with a suitable cation such as Na" and NX images of one another. Diastereomers have different physical (wherein X is independently selected from Hora C-C alkyl properties, e.g. melting points, boiling points, spectral prop group). 30 erties, and reactivities. Mixtures of diastereomers may sepa For therapeutic use, salts of active ingredients of the com rate under high resolution analytical procedures such as elec pounds of the invention will be physiologically acceptable, trophoresis and chromatography. i.e. they will be salts derived from a physiologically accept "Enantiomers' refer to two stereoisomers of a compound able acid or base. However, salts of acids or bases which are which are non-Superimposable mirror images of one another. not physiologically acceptable may also find use, for example, in the preparation or purification of a physiologi 35 Stereochemical definitions and conventions used herein cally acceptable compound. All salts, whether or not derived generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of form a physiologically acceptable acid or base, are within the Chemical Terms (1984) McGraw-Hill Book Company, New Scope of the present invention. York; and Eliel, E. and Wilen, S., Stereochemistry of Organic “Nucleobase' or “nucleoside base' means any nitrogen Compounds (1994) John Wiley & Sons, Inc., New York. containing heterocyclic moiety capable of forming Watson 40 Many organic compounds exist in optically active forms, i.e., Crick hydrogen bonds in pairing with a complementary they have the ability to rotate the plane of plane-polarized nucleobase or nucleobase analog, e.g. a purine, a 7-deaZapu light. In describing an optically active compound, the prefixes rine, or a pyrimidine. Typical nucleobases are the naturally D and L or R and S are used to denote the absolute configu occurring nucleobases: adenine, guanine, cytosine, uracil, 45 ration of the molecule about its chiral center(s). The prefixes thymine, and analogs of the naturally-occurring nucleobases, d and 1., D and L, or (+) and (-) are employed to designate the e.g. 7-deazaadenine, Substituted 7-deazapurines Such as sign of rotation of plane-polarized light by the compound, 7-alkynyl, 7-cyano, 7-carboxamido, 7-deazaguanine, with S. (-), or 1 meaning that the compound is levorotatory 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine, nebu while a compound prefixed with R. (+), or dis dextrorotatory. larine, nitropyrrole, nitroindole, 2-aminopurine, 2-amino-6- chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouri 50 For a given chemical structure, these stereoisomers are iden dine, pseudocytosine, pseudoisocytosine, tical except that they are mirror images of one another. A 5-propynylcytosine, isocytosine, isoguanine, 7-deazagua specific stereoisomer may also be referred to as an enanti nine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, omer, and a mixture of Such isomers is often called an enan 4-thiouracil, O-methylguanine, N-methyladenine, O-me tiomeric mixture. A 50:50 mixture of enantiomers is referred thylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-me 55 to as a racemic mixture or a racemate, which may occur where thylindole, pyrazine bases including 3-oxo-2-carboxami there has been no stereoselection or stereospecificity in a dopyrazine, 5-fluoro-3-oxo-2-carboxamidopyrazine, chemical reaction or process. The terms “racemic mixture' 6-fluoro-3-oxo-2-carboxamidopyrazine, pyrazolo 3,4-Dpy and "racemate” refer to an equimolar mixture of two enan rimidines (U.S. Pat. Nos. 6,143,877 and 6,127,121; WO 60 tiomeric species, devoid of optical activity. 01/38584), and ethenoadenine (Fasman (1989) in Practical Whenever a compound described herein is substituted with Handbook of Biochemistry and Molecular Biology, pp. 385 more than one of the same designated group, e.g., “R” or 394, CRC Press, Boca Raton, Fla.). “R'', then it will be understood that the groups may be the The invention provides compounds of Formula I-IV same or different, i.e., each group is independently selected. wherein B is a nucleoside base. The compounds may include 65 Wavy lines, M, indicate the site of covalent bond attach any nucleoside base, provided the final compound possesses ments to the adjoining Substructures, groups, moieties, or useful therapeutic (e.g. anti-viral) properties. Additional atOmS. US 8,324,179 B2 75 76 The compounds of the Formula I-IV bear a phosphonate W is W or W.; W is R, C(Y)R’, C(Y)W, group, which may be a prodrug moiety —SOR, or -SOW; and W is a carbocycle or a heterocycle wherein W is independently substituted with 0 to 3 R' groups. W carbocycles and Wheterocycles may be indepen s - A dently substituted with 0 to 3 R' groups. W may be a Saturated, unsaturated or aromatic ring comprising a W1 W. mono- or bicyclic carbocycle or heterocycle. W may have 3 to 10 ring atoms, e.g., 3 to 7 ring atoms. The W wherein W' and W are each independently a group of the 10 rings are Saturated when containing 3 ring atoms, Satu formula: rated or mono-unsaturated when containing 4 ring atoms, Saturated, or mono- or di-unsaturated when con taining 5 ring atoms, and Saturated, mono- or di-unsat Yl urated, or aromatic when containing 6 ring atoms. 15 A Wheterocycle may be a monocycle having 3 to 7 ring R y- Y2 members (2 to 6 carbonatoms and 1 to 3 heteroatoms selected Y2 from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N. R 2 O, P, and S). Wheterocyclic monocycles may have 3 to 6 ring atoms (2 to 5 carbon atoms and 1 to 2 heteroatoms selected from N, O, and S); or 5 or 6 ring atoms (3 to 5 carbon atoms wherein: and 1 to 2 heteroatoms selected from NandS). Wheterocy each Y is independently a bond, O, CRNR, “N(O)(R), clic bicycles have 7 to 10 ring atoms (6 to 9 carbon atoms and N(OR), "N(O)(OR), N NR, S, S. S. S(O), or S(O); 1 to 2 heteroatoms selected from N, O, and S) arranged as a M2 is 0, 1 or 2: 25 bicyclo 4.5, 5.5, 5.6, or 6.6 system; or 9 to 10 ring each R" is independently H, F, Cl, Br, I, OH, R, C(=Y) atoms (8 to 9 carbon atoms and 1 to 2 hetero atoms selected R, —C(=Y)OR, C(=Y)N(R), N(R), from NandS) arranged as a bicyclo[5.6 or 6.6 system. The - "N(R) - SR, S(O)R, S(O).R, S(O)(OR), Wheterocycle may be bonded to Y through a carbon, nitro - S(O),(OR), OC(=Y)R, OC(=Y)OR, OC gen, Sulfur or other atom by a stable covalent bond. (=Y)(N(R).), SC(=Y)R, SC(=Y)OR, SC 30 Wheterocycles include for example, pyridyl, dihydropy (=Y)(N(R).), N(R)C(=Y)R, N(R)C(=Y)OR, ridyl isomers, piperidine, pyridazinyl, pyrimidinyl, pyrazi or N(R)C(=Y)N(R), amino ( NH), ammonium nyl. S-triazinyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, (—NH), alkylamino, dialkylamino, trialkylammo pyrazolyl, isothiazolyl, furanyl, thiofuranyl, thienyl, and pyr nium, C-C alkyl, C-C alkylhalide, carboxylate, Sul rolyl. Walso includes, but is not limited to, examples such fate, Sulfamate, Sulfonate, C-C alkylsulfonate, C-Cs 35 alkylamino, C-Cs alkylhydroxyl, C-C alkylthiol, alkylsulfone (—SOR), sulfonamide (—SONR), alkylsulfoxide ( SOR), ester (—C(=O)CR), amido (—C(=O)NR), nitrile (—CN), azido ( N), nitro 40 (—NO), C-C alkoxy (-OR), C-C alkyl, C-Cs Substituted alkyl, C-C alkenyl, C-Cs. Substituted alk enyl, C-C alkynyl, C-Cs. Substituted alkynyl, a pro N1 N H tecting group or W.; or when taken together, two R' on the same carbon atom form a carbocyclic ring of 3 to 7 45 carbon atoms; each R is independently R', a protecting group, or the formula:

50 R. R. Yl

Y2 Y2 afe M12c (Y),2 afe W carbocycles and heterocycles may be independently 55 substituted with 0 to 3 R groups, as defined above. For wherein: example, substituted W carbocycles include: M1a, M1 c, and M1d are independently 0 or 1; M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; or OH when taken together, two R are optionally substituted 60 C-C alkylene thereby forming a phosphorous-contain ing heterocycle; each R is H. halogen, C-C alkyl, C-C substituted alkyl, C-Cs alkenyl, C-Cs. Substituted alkenyl, C-C alky nyl, C-Cs. Substituted alkynyl, Co-Co aryl, Co-Co Sub 65 stituted aryl, C-Coheterocycle, C-Co Substituted het erocycle, or a protecting group: , , . US 8,324,179 B2 77 78 -continued Embodiments of

On1 S. A

of Formula I-IV compounds include substructures such as: / \, NH 10

R

a A1 l 21 2 15 wherein each Y is, independently, O or N(R'). Another embodiment of

2-O 2-O s 2- v/ \/" 2- \/ \/ O w W. 25 of Formula I-IV includes the substructures:

Examples of substituted phenyl carbocycles include: 30

wherein each Y is, independently, O, N(R') or S. 35 Another embodiment of

40

of Formula I-IV compounds includes the substructures:

45 O | P R A.1. Ny.-w 5 50 wherein W is a carbocycle such as phenyl or substituted phenyl. Such a substructure includes:

55 N H(R).

V -O R 60 A1 P ,- O

65 wherein Y is O or N(R) and the phenyl carbocycle is substituted with 0 to 3 R groups. US 8,324,179 B2 79 80 An embodiment of rally occurring amino acids or naturally occurring amino acid esters. In another preferred embodiment, W and Ware, independently, naturally occurring 2-hydroxy carboxylic acids or naturally occurring 2-hydroxy carboxylic acid esters wherein the acid orester is linked to P through the 2-hydroxy group. Another embodiment of of Formula I-IV includes phenyl phosphonamidate amino acids, e.g. alanate esters and phenyl phosphonate-lactate 10 esters:

15

25 In one preferred embodiment each R is, independently, C-C alkyl. In another preferred embodiment each R is, independently, C-C aryl or C-C substituted aryl. Embodiments of R include esters, carbamates, carbon ates, thioesters, amides, thioamides, and urea groups: OR. 30

The chiral carbon of the amino acid and lactate moieties 35 may be either the R or S configuration or the racemic mixture. af2. R" and Another embodiment of Yl R. R. O 40 Yl - Aal-w y W. af2. --- of Formula I-IV is 45 Cellular Accumulation One aspect of the invention is HCV polymerase inhibitor compounds capable of accumulating in human PBMC (pe R ripheral blood monocyte cells). Optionally, the compounds of the invention demonstrate O 50 improved intracellular half-life of the compounds or intrac A-P Y2 YRy ellular metabolites of the compounds in human PBMC when compared to analogs of the compounds not having the phos n- Y' J. phonate or phosphonate prodrug. Typically, the half-life is improved by at least about 50%, more typically at least in the wherein each Y is, independently, O or S and each Y is, 55 range 50-100%, still more typically at least about 100%, more independently, —O— or —NH-. In a preferred embodi typically yet greater than about 100%. mentY' is O. In anotherpreferredembodiment,Y' is O and R' In one embodiment, the intracellular half-life of a metabo is C-C alkyl, C-Cs. Substituted alkyl, C-C alkenyl, C-Cs lite of the compound in human PBMC is improved when Substituted alkenyl, C-Cs alkynyl or C-Cs. Substituted alky compared to an analog of the compound not having the phos nyl. In another preferred embodiment, Y is O; R is C-Cs 60 phonate or phosphonate prodrug. In such embodiments, the alkyl, C-C substituted alkyl, C-C alkenyl, C-C substi metabolite is typically generated intracellularly, more typi tuted alkenyl, C-C alkynyl or C-Cs. Substituted alkynyl: cally, it is generated within human PBMC. Still more typi and R is CH. In another preferred embodiment,Y' is O: R” is cally, the metabolite is a product of the cleavage of a phos C-Cs alkyl, C-Cs. Substituted alkyl, C-C alkenyl, C-Cs phonate prodrug within human PBMCs. More typically yet, Substituted alkenyl, C-Cs alkynyl or C-Cs. Substituted alky 65 the phosphonate prodrug is cleaved to form a metabolite nyl: R is CH; and each Y is NH-. In a preferred embodi having at least one negative charge at physiological pH. Most ment, W' and Ware, independently, nitrogen-linked, natu typically, the phosphonate prodrug is enzymatically cleaved US 8,324,179 B2 81 82 within human PBMC to form a phosphonate having at least reactions are described in “Protective Groups in Organic one active hydrogen atom of the form P OH. Chemistry’. Theodora W. Greene (John Wiley & Sons, Inc., Recursive Substituents New York, 1991, ISBN 0-471-62301-6) (“Greene'). See also Selected substituents within the compounds of the inven Kocienski, Philip J.; “Protecting Groups” (Georg Thieme tion are present to a recursive degree. In this context, “recur 5 Verlag Stuttgart, New York, 1994), which is incorporated by sive substituent’ means that a substituent may recite another reference in its entirety herein. In particular Chapter 1, Pro instance of itself. Because of the recursive nature of such tecting Groups: An Overview, pages 1-20, Chapter 2, Substituents, theoretically, a large number of compounds may Hydroxyl Protecting Groups, pages 21-94, Chapter 3, Diol be present in any given embodiment. For example, R con Protecting Groups, pages 95-117, Chapter 4, Carboxyl Pro tains a R” substituent. R' can be R. One of ordinary skill in the 10 tecting Groups, pages 118-154, Chapter 5, Carbonyl Protect art of medicinal chemistry understands that the total number of such substituents is reasonably limited by the desired prop ing Groups, pages 155-184. For protecting groups for car erties of the compound intended. Such properties include, by boxylic acid, phosphonic acid, phosphonate, Sulfonic acid of example and not limitation, physical properties Such as and other protecting groups for acids see Greene as set forth molecular weight, solubility or log P. application properties 15 below. Such groups include by way of example and not limi Such as activity against the intended target, and practical tation, esters, amides, hydrazides, and the like. properties such as ease of synthesis. Ether-and Ester-forming Protecting Groups By way of example and not limitation, W and R are Ester-forming groups include: (1) phosphonate ester-form recursive Substituents in certain embodiments. Typically, ing groups, such as phosphonamidate esters, phosphorothio each of these may independently occur 20, 19, 18, 17, 16, 15, ate esters, phosphonate esters, and phosphon-bis-amidates; 14, 13, 12, 11, 10,9,8,7,6, 5, 4, 3, 2, 1, or 0, times in a given (2) carboxyl ester-forming groups, and (3) Sulphur ester embodiment. More typically, each of these may indepen forming groups, such as Sulphonate, Sulfate, and Sulfinate. dently occur 12 or fewer times in a given embodiment. More The phosphonate moieties of the compounds of the inven typically yet, W will occur 0 to 8 times, R” will occur 0 to 6 tion may or may not be prodrug moieties, i.e. they may or may times in a given embodiment. Even more typically, W will 25 not be susceptible to hydrolytic or enzymatic cleavage or occur 0 to 6 times, R” will occur 0 to 4 times and R will occur modification. Certain phosphonate moieties are stable under 0 to 8 times in a given embodiment. most or nearly all metabolic conditions. For example, a Recursive substituents are an intended aspect of the inven dialkylphosphonate, where the alkyl groups are two or more tion. One of ordinary skill in the art of medicinal chemistry carbons, may have appreciable stability in vivo due to a slow understands the versatility of such substituents. To the degree 30 rate of hydrolysis. that recursive Substituents are present in an embodiment of Within the context of phosphonate prodrug moieties, a the invention, the total number will be determined as set forth large number of structurally-diverse prodrugs have been above. described for phosphonic acids (Freeman and Ross in Protecting Groups Progress in Medicinal Chemistry 34:112-147 (1997) and are In the context of the present invention, embodiments of 35 included within the scope of the present invention. An exem protecting groups include prodrug moieties and chemical plary embodiment of a phosphonate ester-forming group is protecting groups. the phenyl carbocycle in a substructure having the formula: Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, i.e. routes or 40 N methods to prepare the compounds of the invention. For the most part the decision as to which groups to protect, when to HR do so, and the nature of the chemical protecting group “PRT 2 will be dependent upon the chemistry of the reaction to be O R protected against (e.g., acidic, basic, oxidative, reductive or 45 W-O other conditions) and the intended direction of the synthesis. The PRT groups do not need to be, and generally are not, the A.A1 <--" same if the compound is substituted with multiple PRT. In O general, PRT will be used to protect functional groups such as carboxyl, hydroxyl or amino groups and to thus prevent side 50 wherein the phenyl carbocycle is substituted with 0 to 3 R reactions or to otherwise facilitate the synthetic efficiency. groups. Also, in this embodiment, where Y' is O, a lactate The order of deprotection to yield free, deprotected groups is ester is formed. Alternatively, where Y' is NR, N OR or dependent upon the intended direction of the synthesis and N N(R), then phosphonamidate esters result. R susbstitu the reaction conditions to be encountered, and may occur in ents include H and C-C alkyl. any order as determined by the artisan. 55 In its ester-forming role, a protecting group typically is Various functional groups of the compounds of the inven bound to any acidic group Such as, by way of example and not tion may be protection. For example, protecting groups for limitation, a COH or —C(S)OH group, thereby resulting —OH groups (whether hydroxyl, carboxylic acid, phospho in —COR where R is defined herein. Also, R for example nic acid, or other functions) are embodiments of “ether- or includes the enumerated ester groups of WO95/07920. ester-forming groups. Ether- or ester-forming groups are 60 Examples of protecting groups include (a)-(): capable of functioning as chemical protecting groups in the (a) C-C heterocycle (described above) or aryl. These synthetic schemes set forth herein. However, some hydroxyl aromatic groups optionally are polycyclic or monocy and thio protecting groups are neither ether- nor ester-form clic. Examples include phenyl, Spiryl, 2-and 3-pyrrolyl, ing groups, as will be understood by those skilled in the art, 2-and 3-thienyl, 2-and 4-imidazolyl, 2-, 4-and 5-OX and are included with amides, discussed below. 65 azolyl, 3-and 4-isoxazolyl, 2-, 4-and 5-thiazolyl, 3-, A very large number of hydroxyl protecting groups and 4-and 5-isothiazolyl, 3-and 4-pyrazolyl, 1-, 2-, 3-and amide-forming groups and corresponding chemical cleavage 4-pyridinyl, and 1-, 2-, 4-and 5-pyrimidinyl. US 8,324,179 B2 83 84 (b) C-C, heterocycle or aryl substituted with halo, R', —(CH2)CH, —(CH2)CH, CHCHF, R'-O-C-C alkylene, C-C alkoxy, CN, NO. —CHCHCl, —CHCF, and —CHCCl), OH, carboxy, carboxyester, thiol, thioester, C-C, haloalkyl (1-6 halogenatoms), C-C alkenyl or C-C, alkynyl. Such groups include, but are not limited to, 2-, 3-and 4-alkoxyphenyl (C-C alkyl); 2-, 3-and 4-meth N-1a/ Y oxyphenyl: 2-, 3-and 4-ethoxyphenyl; 2.3-, 2,4-, 2.5- \ / 2,6-, 3,4-and 3,5-diethoxyphenyl: 2-and 3-carboethoxy 4-hydroxyphenyl: 2-and 3-ethoxy-4-hydroxyphenyl: —N-2-propylmorpholino, 2,3-dihydro-6-hydroxyin 2-and 3-ethoxy-5-hydroxyphenyl: 2-and 3-ethoxy-6- 10 hydroxyphenyl: 2-, 3-and 4-O-acetylphenyl: 2-, 3-and dene, Sesamol, catechol monoester, —CH2—C(O)—N 4-dimethylaminophenyl: 2-, 3-and 4-methylmercap (R'), —CH2 S(O)(R'), —CH2 S(O),(R'), tophenyl: 2-, 3-and 4-halophenyl (including 2-, 3-and CH, CH(OC(O)CHR) CH(OC(O)CHR), 4-fluorophenyl and 2-, 3-and 4-chlorophenyl); 2.3-, enolpyruvate (HOOC C(=CH-)—) or glycerol. 2.4-, 2.5-, 2,6-, 3,4-and 3,5-dimethylphenyl; 2.3-, 2,4-, 15 (e) A 5 or 6 carbon monosaccharide, disaccharide or oli 2.5-, 2,6-, 3,4-and 3,5-biscarboxyethylphenyl; 2.3-, gosaccharide (3 to 9 monosaccharide residues). 2.4-, 2.5-, 2,6-, 3,4-and 3.5-dimethoxyphenyl; 2.3-, 2,4-, (f) Triglycerides such as C.-D-f-diglycerides (wherein the 2.5-, 2,6-, 3,4-and 3,5-dihalophenyl (including 2,4-dif fatty acids composing glyceride lipids generally are luorophenyl and 3,5-difluorophenyl); 2-, 3-and 4-ha naturally-occurring Saturated or unsaturated C-2, Ces loalkylphenyl (1 to 5 halogen atoms, C-C alkyl or Co fatty acids Such as linoleic, lauric, myristic, including 4-trifluoromethylphenyl); 2-, 3-and 4-cy palmitic, Stearic, oleic, palmitoleic, linolenic and the anophenyl: 2-, 3-and 4-nitrophenyl: 2-, 3-and 4-ha like fatty acids) linked to acyl of the parental compounds loalkylbenzyl (1 to 5 halogen atoms, C-C alkyl herein through a glyceryl oxygen of the triglyceride; including 4-trifluoromethylbenzyl and 2-, 3-and (g) Phospholipids linked to the carboxyl group through the 4-trichloromethylphenyl and 2-, 3-and 4-trichlorometh 25 phosphate of the phospholipid. ylphenyl): 4-N-methylpiperidinyl: 3-N-methylpiperidi (h) Phthalidyl (shown in FIG. 1 of Clayton et al., Antimi nyl: 1-ethylpiperazinyl; benzyl; alkylsalicylphenyl (C- crob. Agents Chemo. (1974)5(6):670-671. C alkyl, including 2-, 3-and 4-ethylsalicylphenyl), 2-, (i) Cyclic carbonates such as (5-R-2-oxo-1,3-dioxolen-4- 3-and 4-acetylphenyl: 1,8-dihydroxynaphthyl yl)methyl esters (Sakamoto et al., Chem. Pharm. Bull. (—CoH OH) and aryloxyethyl Co-Co aryl (includ 30 (1984) 32(6)2241-2248) where R is R. R. or aryl. ing phenoxy ethyl), 2,2'-dihydroxybiphenyl, 2-, 3-and () 4-N,N-dialkylaminophenol; —CHCH N(CH): trimethoxybenzyl; triethoxybenzyl; and 2-alkyl pyridi nyl (C. alkyl). (c) 35

The hydroxyl groups of the compounds of this invention optionally are substituted with one of groups III, IV or V 40 disclosed in WO94/21604, or with isopropyl. N As further embodiments, Table Alists examples of protect O ing group ester moieties that for example can be bonded via N oxygen to —C(O)O— and —P(O)(O—) groups. Several amidates also are shown, which are bound directly to 45 —C(O)— or - P(O). Esters of structures 1-5, 8-10 and 16, 17, 19-22 are synthesized by reacting the compound herein having a free hydroxyl with the corresponding halide (chlo ride or acyl chloride and the like) and N,N-dicyclohexyl-N- morpholine carboxamidine (or another base such as DBU, triethylamine, CsCO, N,N-dimethylaniline and the like) in DMF (or other solvent such as acetonitrile or N-methylpyr esters of 2-carboxyphenyl; and C-C alkylene-C-C aryl rolidone). When the compound to be protected is a phospho (including benzyl, —CH-pyrrolyl, -CH2-thienyl, —CH nate, the esters of structures 5-7, 11, 12, 21, and 23-26 are imidazolyl, -CH2-oxazolyl, —CH2-isoxazolyl, -CH2 55 synthesized by reaction of the alcohol or alkoxide salt (or the thiazolyl, -CH2-isothiazolyl, -CH2-pyrazolyl. —CH-py corresponding amines in the case of compounds Such as 13, ridinyl and —CH-pyrimidinyl) substituted in the aryl moiety 14 and 15) with the monochlorophosphonate or dichloro by 3 to 5 halogen atoms or 1 to 2 atoms or groups selected phosphonate (or another activated phosphonate). from halogen, C-C alkoxy (including methoxy and ethoxy), cyano, nitro, OH, C-C haloalkyl (1 to 6 halogen 60 TABLE A atoms; including —CHCCl), C-C alkyl (including methyl and ethyl), C-C alkenyl or C-C alkynyl. (d) Alkoxy ethyl C-C alkyl including —CH2—CH2— O CH (methoxyethyl), alkyl substituted by any of the groups set forth above foraryl, in particular OH or by 65 1 to 3 halo atoms (including —CH, —CH(CH), N-ethylmorpholino —C(CH), —CH2CH, -(CH2)CH, -(CH2)CH, US 8,324,179 B2 86 Protecting groups also includes “double ester forming profunctionalities such as —CHOC(O)OCH,

10 O —CHSCOCH, —CHOCON(CH), or alkyl- or aryl-acy loxyalkyl groups of the structure -CH(R' or W)O((CO) R7) or -CH(R' or W)(CO)OR) (linked to oxygen of the 15 acidic group) wherein R7 and Rare alkyl, aryl, or alkylaryl groups (see U.S. Pat. No. 4,968,788). Frequently R7 and R are bulky groups such as branched alkyl, ortho-Substituted aryl, meta-substituted aryl, or combinations thereof, includ ing normal, secondary, iso-and tertiary alkyls of 1-6 carbon atoms. An example is the pivaloyloxymethyl group. These are of particular use with prodrugs for oral administration. Examples of Such useful protecting groups are alkylacy loxymethyl esters and their derivatives, including —CH 25 (CHCHOCH)OC(O)C(CH),

30 N-1

O s

—CHOC(O)CHs —CHOC(O)C(CH), —CH 35 (CHOCH)OC(O)C(CH), —CH(CH(CH))OC(O)C (CH), —CHOC(O)CH-CH(CH), —CHOC(O)CH, —CHOC(O)CHs —CHOC(O)CHs —CHOC(O) CHCH, -CHOC(O)CH(CH), —CHOC(O)C(CH), 40 and —CHOC(O)CHCHs. For prodrug purposes, the ester typically chosen is one heretofore used for antibiotic drugs, in particular the cyclic carbonates, double esters, or the phthalidyl, aryl or alkyl esterS. 45 In Some embodiments the protected acidic group is an ester of the acidic group and is the residue of a hydroxyl-containing functionality. In other embodiments, an amino compound is used to protect the acid functionality. The residues of suitable hydroxyl or amino-containing functionalities are set forth 50 above or are found in WO95/07920. Of particular interest are the residues of amino acids, amino acid esters, polypeptides, or aryl alcohols. Typical amino acid, polypeptide and car boxyl-esterified amino acid residues are described on pages 11-18 and related text of WO95/07920 as groups L1 or L2. 55 WO95/07920 expressly teaches the amidates of phosphonic acids, but it will be understood that such amidates are formed with any of the acid groups set forth herein and the amino acid residues set forth in WO95/07920. Typical esters for protecting acidic functionalities are also 60 described in WO95/07920, again understanding that the same esters can be formed with the acidic groups herein as with the phosphonate of the 920 publication. Typical ester groups are defined at least on WO95/07920 pages 89-93 (under R' or R), the table on page 105, and pages 21-23 (as 65 R). Of particular interest are esters of unsubstituted aryl such Otheresters that are suitable for use herein are described in as phenyl or arylalkyl such benzyl, or hydroxy-, halo EP 632O48. alkoxy-, carboxy-and/or alkylestercarboxy-Substituted aryl US 8,324,179 B2 87 88 or alkylaryl, especially phenyl, ortho-ethoxyphenyl, or C-C, Esters (Formate, Benzoylformate, Acetate, Choroacetate, alkylestercarboxyphenyl (salicylate C-C alkylesters). Dichloroacetate, Trichloroacetate, Trifluoroacetate, The protected acidic groups, particularly when using the Methoxyacetate, Triphenylmethoxyacetate, Phenoxy esters or amides of WO95/07920, are useful as prodrugs for acetate, p-Chlorophenoxyacetate, p-poly-Phenylac oral administration. However, it is not essential that the acidic etate, 3-Phenylpropionate, 4-Oxopentanoate (Levuli group be protected in order for the compounds of this inven nate), 4.4-(Ethylenedithio)pentanoate, Pivaloate, tion to be effectively administered by the oral route. When the Adamantoate, Crotonate, 4-Methoxycrotonate, Ben compounds of the invention having protected groups, in par Zoate, p-Phenylbenzoate, 2.4.6-Trimethylbenzoate ticular amino acid amidates or Substituted and unsubstituted (Mesitoate)); aryl esters are administered systemically or orally they are 10 capable of hydrolytic cleavage in vivo to yield the free acid. Carbonates (Methyl, 9-Fluorenylmethyl, Ethyl, 2.2.2- A plurality of the acidic hydroxyls may be protected. If Trichloroethyl 2-(Trimethylsilyl)ethyl 2-(Phenylsulfo more than one acidic hydroxyl is protected then the same or a nyl)ethyl, 2-(Triphenylphosphonio)ethyl, Isobutyl, different protecting group is employed, e.g., the esters may be Vinyl, Allyl, p-Nitrophenyl, Benzyl, p-Methoxybenzyl, different or the same, or a mixed amidate and ester may be 15 3,4-Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, used. S-Benzyl Thiocarbonate, 4-Ethoxy-1-naphthyl, Methyl Typical acid hydroxy protecting groups described in Dithiocarbonate); Greene (pages 14-118) include substituted methyl and alkyl Groups With Assisted Cleavage (2-Iodobenzoate, 4-Azi ethers, substituted benzyl ethers, silyl ethers, esters including dobutyrate, 4-Nitro-4-methylpentanoate, o-(Dibro Sulfonic acid esters, and carbonates. For example: Ethers (methyl, t-butyl, allyl); momethyl)benzoate, 2-Formylbenzenesulfonate, Substituted Methyl Ethers (Methoxymethyl, Methylthi 2-(Methylthiomethoxy)ethyl Carbonate, 4-(Methylthi omethyl, t-Butylthiomethyl, (Phenyldimethylsilyl) omethoxy)butyrate, 2-(Methylthiomethoxymethyl)ben methoxymethyl, Benzyloxymethyl, p-Methoxybenzy Zoate); Miscellaneous Esters (2,6-Dichloro-4-meth loxymethyl, (4-Methoxyphenoxy)methyl, 25 ylphenoxyacetate, 2,6-Dichloro-4-(1,1.3.3 Guaiacolmethyl, t-Butoxymethyl, 4-Pentenyloxym tetramethylbutyl)phenoxyacetate, 2,4-Bis(1,1-dimeth ethyl, Siloxymethyl, 2-Methoxyethoxymethyl, 2.2.2- ylpropyl)phenoxyacetate, Chlorodiphenylacetate, Trichloroethoxymethyl, Bis(2-chloroethoxy)methyl, Isobutyrate, Monosuccinate, (E)-2-Methyl-2-butenoate 2-(Trimethylsilyl)ethoxymethyl, Tetrahydropyranyl, (Tigloate), o-(Methoxycarbonyl)benzoate, p-poly-Ben 3-Bromotetrahydropyranyl, Tetrahydropthiopyranyl, 30 Zoate, C.-Naphthoate, Nitrate, Alkyl N,N,N',N'-Tetram 1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl, ethylphosphorodiamidate, N-Phenylcarbamate, Borate, 4-Methoxytetrahydrothiopyranyl, 4-Methoxytetrahy Dimethylphosphinothioyl, 2,4-Dinitrophenyl dropthiopyranyl SS-Dioxido, 1-(2-Chloro-4-methyl) Sulfenate); and phenyl-4-methoxypiperidin-4-yl, 1,4-Dioxan-2-yl, Sulfonates (Sulfate, Methanesulfonate (Mesylate), Ben Tetrahydrofuranyl, Tetrahydrothiofuranyl, 2.3.3a,4,5,6, 35 7.7a-Octahydro-7,8,8-trimethyl-4,7-methanobenzofu Zylsulfonate, Tosylate). ran-2-yl)); Typical 1.2-diol protecting groups (thus, generally where Substituted Ethyl Ethers (1-Ethoxyethyl, 1-(2-Chloroet two OH groups are taken together with the protecting func hoxy)ethyl, 1-Methyl-1-methoxyethyl, 1-Methyl-1- tionality) are described in Greene at pages 118-142 and benzyloxyethyl, 1-Methyl-1-benzyloxy-2-fluoroethyl, 40 include Cyclic Acetals and Ketals (Methylene, Ethylidene, 2.2.2-Trichloroethyl, 2-Trimethylsilylethyl, 2-(Phe 1-t-Butylethylidene, 1-Phenylethylidene, (4-Methoxyphe nylselenyl)ethyl, nyl)ethylidene, 2.2.2-Trichloroethylidene, Acetonide (Iso p-Chlorophenyl, p-Methoxyphenyl, 2,4-Dinitrophenyl, propylidene), Cyclopentylidene, Cyclohexylidene, Cyclo Benzyl); heptylidene, Benzylidene, p-Methoxybenzylidene, 2,4- Substituted Benzyl Ethers (p-Methoxybenzyl, 3,4- 45 Dimethoxybenzylidene, 3,4-Dimethoxybenzylidene, Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, p-Ha 2-Nitrobenzylidene); Cyclic Ortho Esters (Methoxymethyl lobenzyl, 2,6-Dichlorobenzyl, p-Cyanobenzyl, p-Phe ene, Ethoxymethylene, Dimethoxymethylene, 1-Methoxy nylbenzyl, 2-and 4-Picolyl. 3-Methyl-2-picolyl ethylidene, 1-Ethoxyethylidine, 1,2-Dimethoxyethylidene, N-Oxido, Diphenylmethyl, p.p'-Dinitrobenzhydryl, C.-Methoxybenzylidene, 1-(N,N-Dimethylamino)ethylidene 5-Dibenzosuberyl, Triphenylmethyl, C-Naphthyldiphe 50 Derivative, C.-(N,N-Dimethylamino)benzylidene Derivative, nylmethyl, p-methoxyphenyldiphenylmethyl, Di(p- 2-Oxacyclopentylidene); Silyl Derivatives (Di-t-butylsi methoxyphenyl)phenylmethyl, Tri(p-methoxyphenyl) lylene Group, 1,3-(1,1,3,3-Tetraisopropyldisiloxanylidene), methyl, 4-(4-Bromophenacyloxy) and Tetra-t-butoxydisiloxane-1,3-diylidene), Cyclic Carbon phenyldiphenylmethyl, 44'4"-Tris(4,5- ates, Cyclic Boronates, Ethyl Boronate and Phenyl Boronate. dichlorophthalimidophenyl)methyl, 44'4"-Tris 55 (levulinoyloxyphenyl)methyl, 44'4"-Tris More typically, 1,2-diol protecting groups include those (benzoyloxyphenyl)methyl, 3-(Imidazol-1-ylmethyl) shown in Table B, still more typically, epoxides, acetonides, bis(4,4'-dimethoxyphenyl)methyl, 1,1-Bis(4- cyclic ketals and aryl acetals. methoxyphenyl)-1-pyrenylmethyl, 9-Anthryl, 9-(9- Phenyl)xanthenyl, 9-(9-Phenyl-10-oxo)anthryl, 1,3- 60 Benzodithiolan-2-yl, Benzisothiazolyl SS-Dioxido); Silyl Ethers (Trimethylsilyl, Triethylsilyl, Triisopropylsi lyl, Dimethylisopropylsilyl, Diethylisopropylsilyl, Dimethylthexylsilyl, t-Butyldimethylsilyl, t-Butyl ~ y, In 1 diphenylsilyl, Tribenzylsilyl, Tri-p-xylylsilyl, Triph 65 enylsilyl, Diphenylmethylsilyl, t-Butylmethoxyphenyl x silyl);

US 8,324,179 B2 91 92 N-Si Derivatives, N-S Derivatives, and N-Sulfenyl residue. Amino acids are low molecular weight compounds, Derivatives: (N-benzenesulfenyl, N-o-nitrobenzene on the order of less than about 1000 MW and which contain Sulfenyl, N-2,4-dinitrobenzenesulfenyl, N-pentachlo at least one amino or imino group and at least one carboxyl robenzenesulfenyl, N-2-nitro-4-methoxybenzenesulfe group. Generally the amino acids will be found in nature, i.e., nyl, N-triphenylmethylsulfenyl, N-3- can be detected in biological material Such as bacteria or other nitropyridinesulfenyl); and N-sulfonyl Derivatives microbes, plants, animals or man. Suitable amino acids typi (N-p-toluenesulfonyl, N-benzenesulfonyl, N-2,3,6-tri cally are alpha amino acids, i.e. compounds characterized by methyl-4-methoxybenzenesulfonyl, N-2,4,6-tri one amino orimino nitrogenatom separated from the carbon methoxybenzenesulfonyl, N-2,6-dimethyl-4-methoxy atom of one carboxyl group by a single Substituted or unsub benzenesulfonyl, N-pentamethylbenzenesulfonyl, N-2, 10 3,5,6,-tetramethyl-4-methoxybenzenesulfonyl, N-4- stituted alpha carbon atom. Of particular interest are hydro methoxybenzenesulfonyl, N-2,4,6- phobic residues such as mono-ordi-alkyl orarylamino acids, trimethylbenzenesulfonyl, N-2,6-dimethoxy-4- cycloalkylamino acids and the like. These residues contribute methylbenzenesulfonyl, N-2,2,5,7,8- to cell permeability by increasing the partition coefficient of pentamethylchroman-6-sulfonyl, N-methanesulfonyl, 15 the parental drug. Typically, the residue does not contain a N-B-trimethylsilyethanesulfonyl, N-9-anthracenesulfo Sulfhydryl or guanidino Substituent. nyl, N-4-(4,8-dimethoxynaphthylmethyl)benzene Naturally-occurring amino acid residues are those residues sulfonyl, N-benzylsulfonyl, N-trifluoromethylsulfonyl, found naturally in plants, animals or microbes, especially N-phenacylsulfonyl). proteins thereof. Polypeptides most typically will be substan Protected amino groups include carbamates, amides and tially composed of Such naturally-occurring amino acid resi amidines, e.g. -NHC(O)OR', -NHC(O)R' or dues. These amino acids are glycine, alanine, Valine, leucine, —N=CR'N(R'). Another protecting group, also useful as a isoleucine, serine, threonine, cysteine, methionine, glutamic prodrug for amino or -NH(R), is: acid, aspartic acid, lysine, hydroxylysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, asparagine, 25 glutamine and hydroxyproline. Additionally, unnatural O amino acids, for example, Valanine, phenylglycine and homoarginine are also included. Commonly encountered amino acids that are not gene-encoded may also be used in the ... . present invention. All of the amino acids used in the present 30 invention may be either the D- or L-optical isomer. In addi tion, other peptidomimetics are also useful in the present invention. For a general review, see Spatola, A. F., in Chem istry and Biochemistry of Amino Acids, Peptides and Pro See for example Alexander, J. et al (1996) J. Med. Chem. teins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 39:48.0-486. 35 (1983). Amino Acid and Polypeptide Protecting Group and Conju When protecting groups are single amino acid residues or gates polypeptides they optionally are substituted with substitu An amino acid or polypeptide protecting group of a com ents. These conjugates are generally produced by forming an pound of the invention has the structure R'NHCH(R')C amide bond between a carboxyl group of the amino acid (or (O) , where R' is H, an amino acid or polypeptide residue, 40 C-terminal amino acid of a polypeptide for example). Gen or R', and R' is defined below. erally, only one of any site in the scaffold drug-like compound R" is lower alkyl or lower alkyl (C-C) substituted with is amidated with an amino acid as described herein, although amino, carboxyl, amide, carboxyl ester, hydroxyl, C-C, it is within the scope of this invention to introduce amino aryl, guanidinyl, imidazolyl, indolyl, Sulfhydryl, Sulfox acids at more than one permitted site. Usually, a carboxyl ide, and/or alkylphosphate. R'' also is taken together 45 group of R is amidated with an amino acid. In general, the with the amino acid C-N to form a proline residue C.-amino or C-carboxyl group of the amino acid or the termi (R'—CH) ). However, R' is generally the side nal amino or carboxyl group of a polypeptide are bonded to group of a naturally-occurring amino acid such as H. the scaffold, parental functionalities. Carboxyl or amino —CH, —CH(CH), —CH2—CH(CH), groups in the amino acid side chains generally may be used to —CHCH, CH, CH, —CH2—CHs. 50 form the amide bonds with the parental compound or these - CHCH, S CH, CHOH, -CH(OH)—CH, groups may need to be protected during synthesis of the —CH2—SH, —CH2—CHOH, -CH CO. NH2, conjugates as described further below. CH-CH CO. NH-CH-COOH, -CH With respect to the carboxyl-containing side chains of CH-COOH, -(CH), NH and —(CH), NH amino acids or polypeptides it will be understood that the C(NH2) NH. R'' also includes 1-guanidinoprop-3- 55 carboxyl group optionally will be blocked, e.g. esterified or y1, benzyl, 4-hydroxybenzyl, imidazol-4-yl, indol-3-yl, amidated with R. methoxyphenyl and ethoxyphenyl. Such ester or amide bonds with side chain amino or car Another set of protecting groups include the residue of an boxyl groups, like the esters or amides with the parental amino-containing compound, in particular an amino acid, a molecule, optionally are hydrolyzable in vivo or in vitro polypeptide, a protecting group, —NHSOR, NHC(O)R. 60 under acidic (pH-3) or basic (pH>10) conditions. Alterna —N(R), NH, or NH(R)(H), whereby for example a car tively, they are Substantially stable in the gastrointestinal tract boxylic acid is reacted, i.e. coupled, with the amine to forman of humans but are hydrolyzed enzymatically in blood or in amide, as in C(O)NR. A phosphonic acid may be reacted intracellular environments. The esters or amino acid or with the amine to form a phosphonamidate, as in —P(O)(OR) polypeptide amidates also are useful as intermediates for the (NR). 65 preparation of the parental molecule containing free amino or Amino acids have the structure RC(O)CH(R')NH-, carboxyl groups. The free acid or base of the parental com where R'' is -OH, -OR, an amino acid or a polypeptide pound, for example, is readily formed from the esters or US 8,324,179 B2 93 94 amino acid or polypeptide conjugates of this invention by Phenylalanine, tryptophan and ring-Substituted C-amino conventional hydrolysis procedures. acids such as the phenyl- or cyclohexylamino acids C.-ami When an amino acid residue contains one or more chiral nophenylacetic acid, C-aminocyclohexylacetic acid and centers, any of the D. L. meso, threo or erythro (as appropri C.-amino-B-cyclohexylpropionic acid; phenylalanine ana ate) racemates, scalemates or mixtures thereof may be used. logues and derivatives comprising aryl, lower alkyl, hydroxy, In general, if the intermediates are to be hydrolyzed non guanidino, oxyalkylether, nitro, Sulfur or halo-substituted enzymatically (as would be the case where the amides are phenyl (e.g., tyrosine, methyltyrosine and o-chloro-, used as chemical intermediates for the free acids or free p-chloro-3,4-dichloro, o-, m- or p-methyl-, 2,4,6-trimethyl-, amines), D isomers are useful. On the other hand, L isomers 2-ethoxy-5-nitro-, 2-hydroxy-5-nitro-and p-nitro-phenylala are more versatile since they can be susceptible to both non 10 nine); furyl-, thienyl-, pyridyl-, pyrimidinyl-, purinyl- or enzymatic and enzymatic hydrolysis, and are more efficiently naphthyl-alanines; and tryptophan analogues and derivatives transported by amino acid or dipeptidyl transport systems in including kynurenine, 3-hydroxykynurenine, 2-hydroxytryp the gastrointestinal tract. tophan and 4-carboxytryptophan; Examples of Suitable amino acids whose residues are rep 15 C.-Amino Substituted amino acids including sarcosine resented by R or R' include the following: (N-methylglycine), N-benzylglycine, N-methylalanine, Glycine; N-benzylalanine, N-methylphenylalanine, N-ben Aminopolycarboxylic acids, e.g., aspartic acid, B-hydrox Zylphenylalanine, N-methylvaline and N-benzylvaline: yaspartic acid, glutamic acid, B-hydroxyglutamic acid, B-me and thylaspartic acid, B-methylglutamic acid, B.B-dimethylaspar C.-Hydroxy and Substituted C-hydroxy amino acids includ tic acid, Y-hydroxyglutamic acid, B.Y-dihydroxyglutamic ing serine, threonine, allothreonine, phosphoserine and acid, B-phenylglutamic acid, Y-methyleneglutamic acid, phosphothreonine. 3-aminoadipic acid, 2-aminopimelic acid, 2-aminoSuberic Polypeptides are polymers of amino acids in which a car acid and 2-aminosebacic acid; boxyl group of one amino acid monomer is bonded to an Amino acid amides Such as glutamine and asparagine; 25 amino or imino group of the next amino acid monomer by an Polyamino- or polybasic-monocarboxylic acids Such as amide bond. Polypeptides include dipeptides, low molecular arginine, lysine, 3-aminoalanine, Y-aminobutyrine, ornithine, weight polypeptides (about 1500-5000 MW) and proteins. citruline, homoarginine, homocitrulline, hydroxylysine, allo Proteins optionally contain 3, 5, 10, 50, 75, 100 or more hydroxylsine and diaminobutyric acid; residues, and Suitably are substantially sequence-homolo Other basic amino acid residues such as histidine, 30 gous with human, animal, plant or microbial proteins. They Diaminodicarboxylic acids such as C.C.'-diaminosuccinic include enzymes (e.g., hydrogen peroxidase) as well as acid, C.C.'-diaminoglutaric acid, C.C.'-diaminoadipic acid, immunogens such as KLH, or antibodies or proteins of any C.C.'-diaminopimelic acid, C.C.'-diamino-B-hydroxypimelic type against which one wishes to raise an immune response. acid, O.C.'-diaminoSuberic acid, C.C.'-diaminoaZelaic acid, The nature and identity of the polypeptide may vary widely. and C.C.'-diaminosebacic acid; 35 The polypeptide amidates are useful as immunogens in Imino acids such as proline, hydroxyproline, allohydrox raising antibodies against either the polypeptide (if it is not yproline, Y-methylproline, pipecolic acid, 5-hydroxypipe immunogenic in the animal to which it is administered) or colic acid, and azetidine-2-carboxylic acid; against the epitopes on the remainder of the compound of this A mono- or di-alkyl (typically C-C branched or normal) invention. amino acid Such as alanine, Valine, leucine, allylglycine, 40 Antibodies capable of binding to the parental non-peptidyl butyrine, norvaline, norleucine, heptyline, C.-methylserine, compound are used to separate the parental compound from C.-amino-C.-methyl-y-hydroxyvaleric acid, C.-amino-C.-me mixtures, for example in diagnosis or manufacturing of the thyl-6-hydroxyvaleric acid, C.-amino-O-methyl-e-hydroxy parental compound. The conjugates of parental compound caproic acid, isovaline, C.-methylglutamic acid, C.-ami and polypeptide generally are more immunogenic than the noisobutyric acid, C.-aminodiethylacetic acid, 45 polypeptides in closely homologous animals, and therefore C.-aminodiisopropylacetic acid, C.-aminodi-n-propylacetic make the polypeptide more immunogenic for facilitating rais acid, C-aminodiisobutylacetic acid, C.-aminodi-n-butylacetic ing antibodies against it. Accordingly, the polypeptide or acid, C.-aminoethylisopropylacetic acid, C.-amino-n-propy protein may be immunogenic in an animal typically used to lacetic acid, C.-aminodiisoamyacetic acid, C.-methylaspartic raise antibodies, e.g., rabbit, mouse, horse, or rat. The acid, C.-methylglutamic acid, 1-aminocyclopropane-1-car 50 polypeptide optionally contains a peptidolytic enzyme cleav boxylic acid, isoleucine, alloisoleucine, tert-leucine, B-meth age site at the peptide bond between the first and second yltryptophan and C.-amino-B-ethyl-3-phenylpropionic acid; residues adjacent to the acidic heteroatom. Such cleavage 3-phenylserinyl; sites are flanked by enzymatic recognition structures, e.g. a Aliphatic C.-amino-B-hydroxy acids such as serine, B-hy particular sequence of residues recognized by a peptidolytic droxyleucine, B-hydroxynorleucine, B-hydroxynorvaline, 55 enzyme. and C.-amino-B-hydroxy Stearic acid; Peptidolytic enzymes for cleaving the polypeptide conju C.-Amino, Cl-, y-, 6- ore-hydroxy acids such as homoserine, gates of this invention are well known, and in particular Ö-hydroxynorvaline, Y-hydroxynorvaline and e-hydroxynor include carboxypeptidases, which digest polypeptides by leucine residues; canavine and canaline; Y-hydroxyornithine; removing C-terminal residues, and are specific in many 2-hexosaminic acids such as D-glucosaminic acid or D-ga 60 instances for particular C-terminal sequences. Such enzymes lactosaminic acid; and their substrate requirements in general are well known. C.-Amino-B-thiols such as penicillamine, B-thiolnorvaline For example, a dipeptide (having a given pair of residues and or B-thiolbutyrine; a free carboxyl terminus) is covalently bonded through its Other Sulfur containing amino acid residues including cys C.-amino group to the phosphorus or carbon atoms of the teine; homocystine, B-phenylmethionine, methionine, S-al 65 compounds herein. In certain embodiments, a phosphonate lyl-L-cysteine Sulfoxide, 2-thiolhistidine, cystathionine, and group Substituted with an amino acid or peptide will be thiol ethers of cysteine or homocysteine: cleaved by the appropriate peptidolytic enzyme, leaving the US 8,324,179 B2 95 96 carboxyl of the proximal amino acid residue to autocatalyti poorly cleaved polypeptide amidates are immunogens or are cally cleave the phosphonoamidate bond. useful for bonding to proteins in order to prepare immuno Suitable dipeptidyl groups (designated by their single letter genS. code) are AA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL, Intracellular Targeting AK, AM, AF, AP, AS, ATAW, AY, AV, RA, RR, RN, RD, RC, The phosphonate group of Formula I-IV compounds may RE, RO, RG, RH, RI, RL, RK, RM, RF, RP, RS, RT, RW, RY, cleave in vivo in stages after they have reached the desired site RV, NA, NR, NN, ND, NC, NE, NO, NG, NH, NI, NL, NK, of action, i.e. inside a cell. One mechanism of action inside a NM, NF, NP, NS, NT, NW, NY, NV, DA, DR, DN, DD, DC, cell may entail a first cleavage, e.g. by esterase, to provide a DE, DQ, DG, DH, DI, DL, DK, DM, DF, DP, DS, DT, DW, negatively-charged "locked-in' intermediate. Cleavage of a DY, DV, CA, CR, CN, CD, CC, CE, CO, CG, CH, CI, CLCK, 10 terminal ester grouping in Formula I-IV compounds thus CM, CF, CP, CS, CT, CW, CY, CV, EA, ER, EN, ED, EC, EE, affords an unstable intermediate which releases a negatively EQ, EG, EH, EI, EL, EK, EM, EF, EP, ES, ET, EW, EY, EV, charged “locked in intermediate. QA, QR, QN. QD, QC, QE, QC, QG, QH, QI, QL, QK, QM, After passage inside a cell, intracellular enzymatic cleav QF, QP, QS, QT, QW, QY, QV, GA, GR, GN, GD, GC, GE, age or modification of the phosphonate prodrug compound GQ, GG, GH, GI, GL, GK, GM, GF, GP, GS, GT, GW, GY, 15 may result in an intracellular accumulation or retention of the GV, HA, HR, HN, HD, HC, HE, HQ, HG, HH, HI, HL, HK, cleaved or modified compound by a “trapping mechanism. HM, HF, HP, HS, HT, HW, HY, HV, IA, IR, IN, ID, IC, IE, IQ, The cleaved or modified compound, i.e. active metabolite, IG, IH, II, IL, IK, IM, IF, IP, IS, IT, IW, IY, IV, LA, LR, LN, may then be “locked-in' the cell, i.e. accumulate in the cell by LD, LC, LE, LO, LG, LH, LI, LL, LK, LM, LF, LP, LS, LT, a significant change in charge, polarity, or other physical LW, LY, LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH, KI, property change which decreases the rate at which the cleaved KL, KK, KM, KF KP, KS, KT, KW, KY, KV, MA, MR, MN, or modified compound can exit the cell, relative to the rate at MD, MC, ME, MQ, MG, MH, MI, ML, MK, MM, MF, MP, which it entered as the phosphonate prodrug. Other mecha MS, MT, MW, MY, MV, FA, FR, FN, FD, FC, FE, FO, FG, nisms by which atherapeutic effect is achieved may be opera FH, FI, FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN, tive as well. Enzymes which are capable of an enzymatic PD, PC, PE, PQ, PG, PH, PI, PL, PK, PM, PF, PP, PS, PT, PW, 25 activation mechanism with the phosphonate prodrug com PY, PV, SA, SR, SN, SD, SC, SE, SQ, SG, SH, SI, SL, SK, pounds of the invention include, but are not limited to, ami SM, SF, SPSS, ST, SW, SY, SV, TA TR, TN, TD, TC, TE, dases, esterases, microbial enzymes, phospholipases, cho TQ, TG, TH, TI, TL, TK, TM, TF, TP. TS, TT, TW, TY, TV, linesterases, and phosphatases. WA, WR, WN, WD, WC, WE, WQ, WG, WH, WI, WLWK, It is known that the drug is activated in vivo by phospho WM, WF, WPWS, WT, WW, WY, WV, YAYR,YN.YD,YC, 30 rylation. Such activation may occur in the present system by YE,YO, YG, YHYI, YLYK, YM,YF,YPYS, YTYW,YY, enzymatic conversion of the “locked-in' intermediate with YV, VA, VR, VN, VD, VC, VE, VQ, VG, VH, VI, VL, VK, phosphokinase to the active phosphonate diphosphate and/or VM, VF, VP, VS, VT, VW, VY and VV. by phosphorylation of the drug itself after its release from the Tripeptide residues are also useful as protecting groups. “locked-in' intermediate as described above. In either case, When a phosphonate is to be protected, the sequence -X'- 35 the original nucleoside-type drug will be converted, via the pro-'-(where X" is any amino acid residue and X is an derivatives of this invention, to the active phosphorylated amino acid residue, a carboxyl ester of proline, or hydrogen) species. will be cleaved by luminal carboxypeptidase to yield X" with From the foregoing, it will be apparent that many structur a free carboxyl, which in turn is expected to autocatalytically ally different known approved and experimental HCV poly cleave the phosphonoamidate bond. The carboxy group of 40 merase inhibitor drugs can be derivatized in accord with the X" optionally is esterified with benzyl. present invention. Numerous such drugs are specifically men Dipeptide or tripeptide species can be selected on the basis tioned herein. However, it should be understood that the dis of known transport properties and/or Susceptibility to pepti cussion of drug families and their specific members for dases that can affect transport to intestinal mucosal or other derivatization according to this invention is not intended to be cell types. Dipeptides and tripeptides lacking an O-amino 45 exhaustive, but merely illustrative. group are transport Substrates for the peptide transporter As another example, when the selected drug contains mul found in brush border membrane of intestinal mucosal cells tiple reactive hydroxyl functions, a mixture of intermediates (Bai, J. P. F., (1992) Pharm Res. 9:969-978. Transport com and final products may again be obtained. In the unusual case petent peptides can thus be used to enhance bioavailability of in which all hydroxy groups are approximately equally reac the amidate compounds. Di- or tripeptides having one or 50 tive, there is not expected to be a single, predominant product, more amino acids in the D configuration may be compatible as each mono-Substituted product will be obtained inapproxi with peptide transport. Amino acids in the D configuration mate by equal amounts, while a lesser amount of multiply can be used to reduce the susceptibility of a di- or tripeptide to Substituted product will also result. Generally speaking, how hydrolysis by proteases common to the brush border such as ever, one of the hydroxyl groups will be more susceptible to aminopeptidase N. In addition, di- or tripeptides alternatively 55 substitution than the other(s), e.g. a primary hydroxyl will be are selected on the basis of their relative resistance to hydroly more reactive than a secondary hydroxyl, an unhindered sis by proteases found in the lumen of the intestine. For hydroxyl will be more reactive than a hindered one. Conse example, tripeptides or polypeptides lacking asp and/or glu quently, the major product will be a mono-Substituted one in are poor Substrates for aminopeptidase A, di- or tripeptides which the most reactive hydroxyl has been derivatized while lacking amino acid residues on the N-terminal side of hydro 60 other mono-substituted and multiply-substituted products phobic amino acids (leu, tyr, phe, Val, trp) are poor Substrates may be obtained as minor products. for endopeptidase, and peptides lacking a pro residue at the Stereoisomers penultimate position at a free carboxyl terminus are poor The compounds of the invention, exemplified by Formula substrates for carboxypeptidase P. Similar considerations can I, II, III or IV may have chiral centers, e.g. chiral carbon or also be applied to the selection of peptides that are either 65 phosphorus atoms. The compounds of the invention thus relatively resistant or relatively susceptible to hydrolysis by include racemic mixtures of all stereoisomers, including cytosolic, renal, hepatic, serum or other peptidases. Such enantiomers, diastereomers, and atropisomers. In addition, US 8,324,179 B2 97 98 the compounds of the invention include enriched or resolved reversibility. Those compounds binding substantially irre optical isomers at any or all asymmetric, chiral atoms. In versibly are ideal candidates for use in this method of the other words, the chiral centers apparent from the depictions invention. Once labeled, the substantially irreversibly bind are provided as the chiral isomers or racemic mixtures. Both ing compositions are useful as probes for the detection of racemic and diastereomeric mixtures, as well as the indi HCV polymerase. Accordingly, the invention relates to meth vidual optical isomers isolated or synthesized, Substantially ods of detecting HCV polymerase in a sample Suspected of free of their enantiomeric or diastereomeric partners, are all containing HCV polymerase comprising the steps of treating within the scope of the invention. The racemic mixtures are a sample Suspected of containing HCV polymerase with a separated into their individual, Substantially optically pure composition comprising a compound of the invention bound isomers through well-known techniques such as, for example, 10 the separation of diastereomeric salts formed with optically to a label; and observing the effect of the sample on the active adjuncts, e.g., acids or bases followed by conversion activity of the label. Suitable labels are well known in the back to the optically active Substances. In most instances, the diagnostics field and include stable free radicals, fluoro desired optical isomer is synthesized by means of stereospe phores, radioisotopes, enzymes, chemiluminescent groups cific reactions, beginning with the appropriate stereoisomer 15 and chromogens. The compounds herein are labeled in con of the desired starting material. ventional fashion using functional groups such as hydroxyl, The compounds of the invention can also exist as tauto carboxyl, Sulfhydryl or amino. meric isomers in certain cases. Although only one delocalized Within the context of the invention, samples suspected of resonance structure may be depicted, all Such forms are con containing HCV polymerase include natural or man-made templated within the scope of the invention. For example, materials such as living organisms; tissue or cell cultures; ene-amine tautomers can exist for purine, pyrimidine, imida biological samples such as biological material samples Zole, guanidine, amidine, and tetrazole systems and all their (blood, serum, urine, cerebrospinal fluid, tears, sputum, possible tautomeric forms are within the scope of the inven saliva, tissue samples, and the like); laboratory samples; food, tion. water, or air samples; bioproduct samples Such as extracts of Salts and Hydrates 25 cells, particularly recombinant cells synthesizing a desired The compositions of this invention optionally comprise glycoprotein; and the like. Typically the sample will be sus salts of the compounds herein, especially pharmaceutically pected of containing an organism which produces HCV poly acceptable non-toxic salts containing, for example, Na', Li". merase, frequently a pathogenic organism Such as HCV. K", Ca' and Mg". Such salts may include those derived by Samples can be contained in any medium including water and combination of appropriate cations such as alkali and alkaline 30 organic solvent\water mixtures. Samples include living earth metal ions or ammonium and quaternary amino ions with an acid anion moiety, typically a carboxylic acid. organisms such as humans, and man made materials such as Monovalent salts are preferred if a water soluble salt is cell cultures. desired. The treating step of the invention comprises adding the Metal salts typically are prepared by treating a metal 35 composition of the invention to the sample or it comprises hydroxide with a compound of this invention. Examples of adding a precursor of the composition to the sample. The metal salts which are prepared in this way are salts containing addition step comprises any method of administration as Li, Na', and K". Aless soluble metal salt may be precipitated described above. from the solution of a more soluble salt by addition of the If desired, the activity of HCV polymerase after applica Suitable metal compound. 40 tion of the composition can be observed by any method In addition, salts may be formed from acid addition of including direct and indirect methods of detecting HCV poly certain organic and inorganic acids, e.g., HCl, HBr, HSO merase activity. Quantitative, qualitative, and semiquantita HPO or organic Sulfonic acids, to basic centers, typically tive methods of determining HCV polymerase activity are all amines, or to acidic groups. Finally, it is to be understood that contemplated. Typically one of the screening methods the compositions herein comprise compounds of the inven 45 described above are applied, however, any other method such tion in their un-ionized, as well as Zwitterionic form, and as observation of the physiological properties of a living combinations with Stoichiometric amounts of water as in organism are also applicable. hydrates. Organisms that contain HCV polymerase include the HCV Also included within the scope of this invention are the virus. The compounds of this invention are useful in the salts of the parental compounds with one or more amino 50 treatment or prophylaxis of HCV infections in animals or in acids. Any of the amino acids described above are Suitable, a. especially the naturally-occurring amino acids found as pro However, in screening compounds capable of inhibiting tein components, although the amino acid typically is one human immunodeficiency viruses, it should be kept in mind bearing a side chain with a basic or acidic group, e.g., lysine, that the results of enzyme assays may not correlate with cell arginine or glutamic acid, or a neutral group Such as glycine, 55 culture assays. Thus, a cell based assay should be the primary serine, threonine, alanine, isoleucine, or leucine. screening tool. Methods of Inhibition of HCV Polymerase Screens for HCV Polymerase Inhibitors. Another aspect of the invention relates to methods of inhib Compositions of the invention are screened for inhibitory iting the activity of HCV polymerase comprising the step of activity against HCV polymerase by any of the conventional treating a sample Suspected of containing HCV with a com 60 techniques for evaluating enzyme activity. Within the context position of the invention. of the invention, typically compositions are first screened for Compositions of the invention may act as inhibitors of inhibition of HCV polymerase in vitro and compositions HCV polymerase, as intermediates for such inhibitors or have showing inhibitory activity are then screened for activity in other utilities as described below. The inhibitors will bind to vivo. Compositions having in vitro Ki (inhibitory constants) locations on the surface or in a cavity of HCV polymerase 65 ofless then about 5x10M, typically less than about 1x107 having a geometry unique to HCV polymerase. Compositions M and preferably less than about 5x10 Mare preferred for binding HCV polymerase may bind with varying degrees of in vivo use. US 8,324,179 B2 99 100 Useful in vitro screens have been described in detail and employed with either a paraffinic or a water-miscible oint will not be elaborated here. However, the examples describe ment base. Alternatively, the active ingredients may be for Suitable in vitro assays. mulated in a cream with an oil-in-water cream base. Pharmaceutical Formulations If desired, the aqueous phase of the cream base may The compounds of this invention are formulated with con include, for example, at least 30% w/w of a polyhydric alco ventional carriers and excipients, which will be selected in hol, i.e. an alcohol having two or more hydroxyl groups such accord with ordinary practice. Tablets will contain excipients, as propylene glycol, butane 1,3-diol, mannitol, Sorbitol, glyc glidants, fillers, binders and the like. Aqueous formulations erol and polyethylene glycol (including PEG 400) and mix are prepared in sterile form, and when intended for delivery tures thereof. The topical formulations may desirably include by other than oral administration generally will be isotonic. 10 a compound which enhances absorption or penetration of the All formulations will optionally contain excipients such as active ingredient through the skin or other affected areas. those set forth in the “Handbook of Pharmaceutical Excipi Examples of such dermal penetration enhancers include dim ents” (1986). Excipients include ascorbic acid and other anti ethyl Sulphoxide and related analogs. oxidants, chelating agents such as EDTA, carbohydrates Such The oily phase of the emulsions of this invention may be as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellu 15 constituted from known ingredients in a known manner. lose, stearic acid and the like. The pH of the formulations While the phase may comprise merely an emulsifier (other ranges from about 3 to about 11, but is ordinarily about 7 to wise known as an emulgent), it desirably comprises a mixture 10. of at least one emulsifier with a fat or an oil or with both a fat While it is possible for the active ingredients to be admin and an oil. Preferably, a hydrophilic emulsifier is included istered alone it may be preferable to present them as pharma together with a lipophilic emulsifier which acts as a stabilizer. ceutical formulations. The formulations, both for veterinary It is also preferred to include both an oil and a fat. Together, and for human use, of the invention comprise at least one the emulsifier(s) with or without stabilizer(s) make up the active ingredient, as above defined, together with one or more so-called emulsifying wax, and the wax together with the oil acceptable carriers therefor and optionally other therapeutic and fat make up the so-called emulsifying ointment base ingredients. The carrier(s) must be “acceptable' in the sense 25 which forms the oily dispersed phase of the cream formula of being compatible with the other ingredients of the formu tions. lation and physiologically innocuous to the recipient thereof. Emulgents and emulsion stabilizers suitable for use in the The formulations include those suitable for the foregoing formulation of the invention include Tween(R) 60, SpanR 80. administration routes. The formulations may conveniently be cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyc presented in unit dosage form and may be prepared by any of 30 eryl mono-stearate and sodium lauryl Sulfate. the methods well known in the art of pharmacy. Techniques The choice of suitable oils or fats for the formulation is and formulations generally are found in Remington’s Phar based on achieving the desired cosmetic properties. The maceutical Sciences (Mack Publishing Co., Easton, Pa.). cream should preferably be a non-greasy, non-staining and Such methods include the step of bringing into association the washable product with Suitable consistency to avoid leakage active ingredient with the carrier which constitutes one or 35 from tubes or other containers. Straight or branched chain, more accessory ingredients. In general the formulations are mono- or dibasic alkyl esters such as di-isoadipate, isocetyl prepared by uniformly and intimately bringing into associa Stearate, propylene glycol diester of coconut fatty acids, iso tion the active ingredient with liquid carriers or finely divided propyl myristate, decyl oleate, isopropyl palmitate, butyl Solid carriers or both, and then, if necessary, shaping the stearate, 2-ethylhexyl palmitate or a blend of branched chain product. 40 esters known as Crodamol CAP may be used, the last three Formulations of the present invention suitable for oral being preferred esters. These may be used alone or in com administration may be presented as discrete units such as bination depending on the properties required. Alternatively, capsules, cachets or tablets each containing a predetermined high melting point lipids such as white soft paraffin and/or amount of the active ingredient; as a powder or granules; as a liquid paraffin or other mineral oils are used. Solution or a Suspension in an aqueous or non-aqueous liquid; 45 Pharmaceutical formulations according to the present or as an oil-in-water liquid emulsion or a water-in-oil liquid invention comprise a combination according to the invention emulsion. The active ingredient may also be administered as together with one or more pharmaceutically acceptable car a bolus, electuary or paste. riers or excipients and optionally other therapeutic agents. A tablet is made by compression or molding, optionally Pharmaceutical formulations containing the active ingredient with one or more accessory ingredients. Compressed tablets 50 may be in any form suitable for the intended method of may be prepared by compressing in a Suitable machine the administration. When used for oral use for example, tablets, active ingredient in a free-flowing form Such as a powder or troches, lozenges, aqueous or oil Suspensions, dispersible granules, optionally mixed with a binder, lubricant, inert dilu powders or granules, emulsions, hard or soft capsules, syrups ent, preservative, Surface active or dispersing agent. Molded or elixirs may be prepared. Compositions intended for oral tablets may be made by molding in a suitable machine a 55 use may be prepared according to any method known to the mixture of the powdered active ingredient moistened with an art for the manufacture of pharmaceutical compositions and inert liquid diluent. The tablets may optionally be coated or Such compositions may contain one or more agents including scored and optionally are formulated so as to provide slow or Sweetening agents, flavoring agents, coloring agents and pre controlled release of the active ingredient therefrom. serving agents, in order to provide a palatable preparation. For infections of the eye or other external tissues e.g. mouth 60 Tablets containing the active ingredient in admixture with and skin, the formulations are preferably applied as a topical non-toxic pharmaceutically acceptable excipient which are ointment or cream containing the active ingredient(s) in an suitable for manufacture of tablets are acceptable. These amount of for example, 0.075 to 20% w/w (including active excipients may be, for example, inert diluents, such as cal ingredient(s) in a range between 0.1% and 20% in increments cium or Sodium carbonate, lactose, calcium or Sodium phos of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.), preferably 65 phate; granulating and disintegrating agents, such as maize 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When starch, oralginic acid; binding agents, such as starch, gelatin formulated in an ointment, the active ingredients may be or acacia; and lubricating agents, such as magnesium Stearate, US 8,324,179 B2 101 102 Stearic acid or talc. Tablets may be uncoated or may be coated agents which have been mentioned above. The sterile inject by known techniques including microencapsulation to delay able preparation may also be a sterile injectable solution or disintegration and adsorption in the gastrointestinal tract and Suspension in a non-toxic parenterally acceptable diluent or thereby provide a Sustained action over a longer period. For Solvent, such as a solution in 1,3-butane-diol or prepared as a example, a time delay material Such as glyceryl monostearate lyophilized powder. Among the acceptable vehicles and Sol or glyceryl distearate alone or with a wax may be employed. vents that may be employed are water, Ringer's Solution and Formulations for oral use may be also presented as hard isotonic sodium chloride solution. In addition, sterile fixed gelatin capsules where the active ingredient is mixed with an oils may conventionally be employed as a solventor Suspend inert Solid diluent, for example calcium phosphate or kaolin, ing medium. For this purpose any bland fixed oil may be or as Soft gelatin capsules wherein the active ingredient is 10 employed including synthetic mono- or diglycerides. In addi mixed with water oran oil medium, Such as peanut oil, liquid paraffin or olive oil. tion, fatty acids Such as oleic acid may likewise be used in the Aqueous Suspensions of the invention contain the active preparation of injectables. materials in admixture with excipients suitable for the manu The amount of active ingredient that may be combined facture of aqueous Suspensions. Such excipients include a 15 with the carrier material to produce a single dosage form will Suspending agent, such as Sodium carboxymethylcellulose, vary depending upon the host treated and the particular mode methylcellulose, hydroxypropyl methylcellulose, sodium of administration. For example, a time-release formulation alginate, polyvinylpyrrolidone, gum tragacanthandgum aca intended for oral administration to humans may contain cia, and dispersing or wetting agents such as a naturally approximately 1 to 1000 mg of active material compounded occurring phosphatide (e.g., lecithin), a condensation product with an appropriate and convenient amount of carrier material of an alkylene oxide with a fatty acid (e.g., polyoxyethylene which may vary from about 5 to about 95% of the total Stearate), a condensation product of ethylene oxide with a compositions (weight: weight). The pharmaceutical compo long chain aliphatic alcohol (e.g., heptadecaethyleneoxycet sition can be prepared to provide easily measurable amounts anol), a condensation product of ethylene oxide with a partial for administration. For example, an aqueous solution ester derived from a fatty acid and a hexitol anhydride (e.g., 25 intended for intravenous infusion may contain from about 3 to polyoxyethylene Sorbitan monooleate). The aqueous Suspen 500 ug of the active ingredient per milliliter of solution in sion may also contain one or more preservatives such as ethyl order that infusion of a suitable volume at a rate of about 30 or n-propyl p-hydroxy-benzoate, one or more coloring mL/hr can occur. agents, one or more flavoring agents and one or more Sweet Formulations suitable for topical administration to the eye ening agents. Such as Sucrose or saccharin. 30 also include eye drops wherein the active ingredient is dis Oil Suspensions may be formulated by Suspending the Solved or Suspended in a suitable carrier, especially an aque active ingredient in a vegetable oil, such as arachis oil, olive ous solvent for the active ingredient. The active ingredient is oil, Sesame oil or coconut oil, or in a mineral oil such as liquid preferably present in Such formulations in a concentration of paraffin. The oral Suspensions may contain a thickening 0.5 to 20%, advantageously 0.5 to 10%, and particularly agent, such as beeswax, hard paraffin or cetyl alcohol. Sweet 35 about 1.5% w/w. ening agents, such as those set forth above, and flavoring Formulations suitable for topical administration in the agents may be added to provide a palatable oral preparation. mouth include lozenges comprising the active ingredient in a These compositions may be preserved by the addition of an flavored basis, usually Sucrose and acacia or tragacanth; pas antioxidant Such as ascorbic acid. tilles comprising the active ingredient in an inert basis such as Dispersible powders and granules of the invention suitable 40 gelatin and glycerin, or Sucrose and acacia; and mouthwashes for preparation of an aqueous suspension by the addition of comprising the active ingredient in a suitable liquid carrier. water provide the active ingredient in admixture with a dis Formulations for rectal administration may be presented as persing or wetting agent, a suspending agent, and one or more a Suppository with a Suitable base comprising for example preservatives. Suitable dispersing or wetting agents and Sus cocoa butter or a salicylate. pending agents are exemplified by those disclosed above. 45 Formulations Suitable for intrapulmonary or nasal admin Additional excipients, for example Sweetening, flavoring and istration have a particle size for example in the range of 0.1 to coloring agents, may also be present. 500 microns, such as 0.5, 1,30, 35 etc., which is administered The pharmaceutical compositions of the invention may by rapid inhalation through the nasal passage or by inhalation also be in the form of oil-in-water emulsions. The oily phase through the mouth so as to reach the alveolar sacs. Suitable may be a vegetable oil. Such as olive oil or arachis oil, a 50 formulations include aqueous or oily solutions of the active mineral oil. Such as liquid paraffin, or a mixture of these. ingredient. Formulations suitable for aerosol or dry powder Suitable emulsifying agents include naturally-occurring administration may be prepared according to conventional gums, such as gum acacia and gum tragacanth, naturally methods and may be delivered with other therapeutic agents occurring phosphatides, such as soybean lecithin, esters or Such as compounds heretofore used in the treatment or pro partial esters derived from fatty acids and hexitol anhydrides, 55 phylaxis of HCV infections as described below. Such as Sorbitan monooleate, and condensation products of Formulations suitable for vaginal administration may be these partial esters with ethylene oxide. Such as polyoxyeth presented as pessaries, tampons, creams, gels, pastes, foams ylene Sorbitan monooleate. The emulsion may also contain or spray formulations containing in addition to the active Sweetening and flavoring agents. Syrups and elixirs may be ingredient Such carriers as are known in the art to be appro formulated with Sweetening agents, such as glycerol, Sorbitol 60 priate. or Sucrose. Such formulations may also contain a demulcent, Formulations suitable for parenteral administration a preservative, a flavoring or a coloring agent. include aqueous and non-aqueous sterile injection solutions The pharmaceutical compositions of the invention may be which may contain anti-oxidants, buffers, bacteriostats and in the form of a sterile injectable preparation, such as a sterile solutes which render the formulation isotonic with the blood injectable aqueous or oleaginous Suspension. This suspen 65 of the intended recipient; and aqueous and non-aqueous ster sion may be formulated according to the known art using ille Suspensions which may include Suspending agents and those Suitable dispersing or wetting agents and Suspending thickening agents. US 8,324,179 B2 103 104 The formulations are presented in unit-dose or multi-dose Combinations of the compounds of Formula I-IV are typi containers, for example sealed ampoules and vials, and may cally selected based on the condition to be treated, cross be stored in a freeze-dried (lyophilized) condition requiring reactivities of ingredients and pharmaco-properties of the only the addition of the sterile liquid carrier, for example combination. For example, when treating an infection (e.g., water for injection, immediately prior to use. Extemporane HCV), the compositions of the invention are combined with ous injection solutions and Suspensions are prepared from other active therapeutic agents (such as those described sterile powders, granules and tablets of the kind previously herein). described. Preferred unit dosage formulations are those con Suitable active therapeutic agents or ingredients which can taining a daily dose or unit daily Sub-dose, as herein above be combined with the compounds of Formula I-IV can 10 include interferons, e.g., pegylated rFN-alpha2b, pegylated recited, or an appropriate fraction thereof, of the active ingre rIFN-alpha2a, rIFN-alpha2b. IFN alpha-2b XL, rIFN-alpha dient. 2a, consensus IFN alpha, infergen, rebif, locteron, AVI-005, It should be understood that in addition to the ingredients PEG-infergen, pegylated IFN-beta, oral interferon alpha, particularly mentioned above the formulations of this inven feron, reaferon, intermax alpha, r-IFN-beta, infergen--actim tion may include other agents conventional in the art having 15 mune, IFN-omega with DUROS, and albuferon: ribavirin regard to the type of formulation in question, for example analogs, e.g., rebetol, copegus, VX-497, and viramidine those Suitable for oral administration may include flavoring (taribavirin); NS5a inhibitors, e.g., A-831 and A-689; NS5b agents. polymerase inhibitors, e.g., NM-283, valopicitabine, R1626, The invention further provides veterinary compositions PSI-6130 (R1656), HCV-796, BILB 1941, MK-0608, comprising at least one active ingredient as above defined NM-107, R7128, VCH-759, PF-868554, GSK625433, and together with a veterinary carrier therefor. XTL-2125; NS3 protease inhibitors, e.g., SCH-503034 Veterinary carriers are materials useful for the purpose of (SCH-7), VX-950 (Telaprevir), ITMN-191, and BILN-2065; administering the composition and may be solid, liquid or alpha-glucosidase 1 inhibitors, e.g., MX-3253 (celgosivir) gaseous materials which are otherwise inert or acceptable in and UT-231B: hepatoprotectants, e.g., IDN-6556, ME3738, the veterinary art and are compatible with the active ingredi 25 Mito O, and LB-84451; non-nucleoside inhibitors of HCV. ent. These veterinary compositions may be administered e.g., benzimidazole derivatives, benzo-1,2,4-thiadiazine orally, parenterally or by any other desired route. derivatives, and phenylalanine derivatives; and other drugs Compounds of the invention are used to provide controlled for treating HCV, e.g., Zadaxin, nitazoxanide (alinea), BIVN release pharmaceutical formulations containing as active 401 (virostat), DEBIO-025, VGX-410C, EMZ-702, AVI ingredient one or more compounds of the invention ("con 30 4065, bavituximab, oglufanide, PYN-17, KPE02003002, trolled release formulations”) in which the release of the actilon (CPG-10101), KRN-7000, civacir, GI-5005, ANA active ingredient are controlled and regulated to allow less 975, XTL-6865, ANA971, NOV-205, tarvacin, EHC-18, and frequency dosing or to improve the pharmacokinetic or tox NIM811. icity profile of a given active ingredient. In yet another embodiment, the present application dis Effective dose of active ingredient depends at least on the 35 closes pharmaceutical compositions comprising a compound nature of the condition being treated, toxicity, whether the of the present invention, or a pharmaceutically acceptable compound is being used prophylactically (lower doses) or salt, Solvate, and/or ester thereof, in combination with at least against an active viral infection, the method of delivery, and one additional therapeutic agent, and a pharmaceutically the pharmaceutical formulation, and will be determined by acceptable carrier or exipient. the clinicianusing conventional dose escalation studies. It can 40 According to the present invention, the therapeutic agent be expected to be from about 0.0001 to about 100 mg/kg body used in combination with the compound of the present inven weight per day. Typically, from about 0.01 to about 10 mg/kg tion can be any agent having atherapeutic effect when used in body weight per day. More typically, from about 0.01 to about combination with the compound of the present invention. For 5 mg/kg body weight per day. More typically, from about 0.05 example, the therapeutic agent used in combination with the to about 0.5 mg/kg body weight per day. For example, the 45 compound of the present invention can be interferons, ribavi daily candidate dose for an adult human of approximately 70 rin analogs, NS3 protease inhibitors, alpha-glucosidase 1 kg body weight will range from 1 mg to 1000 mg, preferably inhibitors, hepatoprotectants, non-nucleoside inhibitors of between 5 mg and 500 mg, and may take the form of single or HCV, and other drugs for treating HCV. multiple doses. In another embodiment, the present application provides Routes of Administration 50 pharmaceutical compositions comprising a compound of the One or more compounds of the invention (herein referred present invention, or a pharmaceutically acceptable salt, Sol to as the active ingredients) are administered by any route vate, and/or ester thereof, in combination with at least one appropriate to the condition to be treated. Suitable routes additional therapeutic agent selected from the group consist include oral, rectal, nasal, topical (including buccal and Sub ing of pegylated riFN-alpha 2b, pegylated rIFN-alpha 2a, lingual), vaginal and parenteral (including Subcutaneous, 55 rIFN-alpha2b. IFN alpha-2b XL, rFN-alpha 2a, consensus intramuscular, intravenous, intradermal, intrathecal and epi IFN alpha, infergen, rebif, locteron, AVI-005, PEG-infergen, dural), and the like. It will be appreciated that the preferred pegylated IFN-beta, oral interferon alpha, feron, reaferon, route may vary with for example the condition of the recipi intermax alpha, r-IFN-beta, infergen--actimmune, IFN ent. An advantage of the compounds of this invention is that omega with DUROS, albuferon, rebetol, copegus, VX-497, they are orally bioavailable and can be dosed orally. 60 viramidine (taribavirin), A-831, A-689, NM-283, valopicit Combination Therapy abine, R1626, PSI-6130 (R1656), HCV-796, BILB 1941, Compositions of the invention are also used in combination MK-0608, NM-107, R7128, VCH-759, PF-868554, with other active ingredients. Preferably, the other active GSK625433, XTL-2125, SCH-503034 (SCH-7), VX-950 therapeutic ingredients or agents are interferons, ribavirin (Telaprevir), ITMN-191, and BILN-2065, MX-3253 (celgo analogs, NS3 protease inhibitors, alpha-glucosidase 1 inhibi 65 sivir), UT-231B, IDN-6556, ME 3738, MitoC), and tors, hepatoprotectants, non-nucleoside inhibitors of HCV. LB-84451, benzimidazole derivatives, benzo-1,2,4-thiadiaz and other drugs for treating HCV. ine derivatives, and phenylalanine derivatives, Zadaxin, nita US 8,324,179 B2 105 106 Zoxanide (alinea), BIVN-401 (virostat), DEBIO-025, VGX phostin, derivatives of tyrphostin, quercetin, derivatives of 410C, EMZ-702, AVI 4065, bavituximab, oglufanide, PYN quercetin, S-1360, Zintevir (AR-177), L-870812, and 17, KPE02003002, actilon (CPG-10101), KRN-7000, L-870810, MK-0518 (raltegravir), BMS-707035, MK-2048, civacir, GI-5005, ANA-975, XTL-6865, ANA 971, NOV BA-011, BMS-538158, GSK364735C, 6) a gp41 inhibitor, 205, tarvacin, EHC-18, and NIM811 and a pharmaceutically e.g., enfuvirtide, sifuvirtide, FB006M, TRI-1144, SPC3, acceptable carrier or exipient. DES6, Locus gp41, CovX, and REP 9, 7) a CXCR4 inhibitor, In yet another embodiment, the present application pro e.g., AMD-070, 8) an entry inhibitor, e.g., SP01A, TNX-355, vides a combination pharmaceutical agent comprising: 9) a gp120 inhibitor, e.g., BMS-488043 and Block Aide/CR, a) a first pharmaceutical composition comprising a com 10) a G6PD and NADH-oxidase inhibitor, e.g., immunitin, pound of the present invention, or a pharmaceutically 10 10) a CCR5 inhibitor, e.g., aplaviroc, vicriviroc, INCB9471, acceptable salt, Solvate, or ester thereof, and PRO-140, INCB15050, PF-232798, CCR5mAb004, and b) a second pharmaceutical composition comprising at maraviroc. 11) an interferon, e.g., pegylated rFN-alpha2b. least one additional therapeutic agent selected from the pegylated rFN-alpha 2a, rIFN-alpha2b. IFN alpha-2b XL, group consisting of HIV protease inhibiting compounds, rIFN-alpha2a, consensus IFN alpha, infergen, rebif, locteron, HIV non-nucleoside inhibitors of reverse transcriptase, 15 AVI-005, PEG-infergen, pegylated IFN-beta, oral interferon HIV nucleoside inhibitors of reverse transcriptase, HIV alpha, feron, reaferon, intermax alpha, r-IFN-beta, infergen nucleotide inhibitors of reverse transcriptase, HIV inte actimmune. IFN-omega with DUROS, and albuferon, 12) grase inhibitors, gp41 inhibitors, CXCR4 inhibitors, ribavirin analogs, e.g., rebetol, copegus, VX-497, and vira gp120 inhibitors, CCR5 inhibitors, interferons, ribavirin midine (taribavirin) 13) NS5a inhibitors, e.g., A-831 and analogs, NS3 protease inhibitors, alpha-glucosidase 1 A-689, 14) NS5b polymerase inhibitors, e.g., NM-283, inhibitors, hepatoprotectants, non-nucleoside inhibitors valopicitabine, R1626, PSI-6130 (R1656), HCV-796, BILB of HCV, and other drugs for treating HCV, and combi 1941, MK-0608, NM-107, R7128, VCH-759, PF-868554, nations thereof. GSK625433, and XTL-2125, 15) NS3 protease inhibitors, Combinations of the compounds of Formula I-IV and addi e.g., SCH-503034 (SCH-7), VX-950 (Telaprevir), ITMN tional active therapeutic agents may be selected to treat 25 191, and BILN-2065, 16) alpha-glucosidase 1 inhibitors, e.g., patients infected with HCV and other conditions such as HIV MX-3253 (celgosivir) and UT-231B, 17) hepatoprotectants, infections. Accordingly, the compounds of Formula I-IV may e.g., IDN-6556, ME 3738, MitoC), and LB-84451, 18) non be combined with one or more compounds useful in treating nucleoside inhibitors of HCV, e.g., benzimidazole deriva HIV, for example HIV protease inhibiting compounds, HIV tives, benzo-1,2,4-thiadiazine derivatives, and phenylalanine non-nucleoside inhibitors of reverse transcriptase, HIV 30 derivatives, 19) other drugs for treating HCV, e.g., Zadaxin, nucleoside inhibitors of reverse transcriptase, HIV nucleotide nitazoxanide (alinea), BIVN-401 (virostat), DEBIO-025, inhibitors of reverse transcriptase, HIV integrase inhibitors, VGX-410C, EMZ-702, AVI 4065, bavituximab, oglufanide, gp41 inhibitors, CXCR4 inhibitors, gp120 inhibitors, CCR5 PYN-17, KPE02003002, actilon (CPG-10101), KRN-7000, inhibitors, interferons, ribavirin analogs, NS3 protease civacir, GI-5005, ANA-975, XTL-6865, ANA 971, NOV inhibitors, alpha-glucosidase 1 inhibitors, hepatoprotectants, 35 205, tarvacin, EHC-18, and NIM811, 19) pharmacokinetic non-nucleoside inhibitors of HCV, and other drugs for treat enhancers, e.g., BAS-100 and SPI452, 20) RNAse H inhibi ing HCV. tors, e.g., ODN-93 and ODN-112, 21) other anti-HIV agents, More specifically, one or more compounds of the present e.g., VGV-1, PA-457 (bevirimat), ampligen, HRG214, cyto invention may be combined with one or more compounds lin, polymun, VGX-410, KD247, AMZ 0026, CYT 99007, selected from the group consisting of 1) HIV protease inhibi 40 A-221 HIV, BAY 50-4798, MDX010 (iplimumab), PBS119, tors, e.g., amprenavir, atazanavir, fosamprenavir, indinavir, ALG889, and PA-1050040. lopinavir, ritonavir, lopinavir-i-ritonavir, nelfinavir, It is also possible to combine any compound of the inven saquinavir, tipranavir, brecanavir, darunavir, TMC-126. tion with one or more other active therapeutic agents in a TMC-114, mozenavir (DMP-450), JE-2147 (AG1776), unitary dosage form for simultaneous or sequential adminis AG 1859, DG35, L-756423, ROO334649, KNI-272, DPC 45 tration to a patient. The combination therapy may be admin 681, DPC-684, and GW640385X, DG17, PPL-100, 2) a HIV istered as a simultaneous or sequential regimen. When non-nucleoside inhibitor of reverse transcriptase, e.g., administered sequentially, the combination may be adminis capravirine, emivirine, delaviridine, efavirenz, nevirapine, tered in two or more administrations. (+)calanolide A, etravirine, GW5634, DPC-083, DPC-961, Co-administration of a compound of the invention with one DPC-963, MIV-150, and TMC-120, TMC-278 (rilpivirine), 50 or more other active therapeutic agents generally refers to efavirenz, BILR 355 BS, VRX 840773, UK-453,061, simultaneous or sequential administration of a compound of RDEA806, 3) a HIV nucleoside inhibitor of reverse tran the invention and one or more other active therapeutic agents, Scriptase, e.g., Zidovudine, emitricitabine, didanosine, stavu such that therapeutically effective amounts of the compound dine, Zalcitabine, lamivudine, abacavir, amdoxovir, elvucit of the invention and one or more other active therapeutic abine, alovudine, MIV-210, racivir (-t-FTC), D-da-FC, 55 agents are both present in the body of the patient. emtricitabine, phosphazide, fozivudine tidoxil, fosalvudine Co-administration includes administration of unit dosages tidoxil, apricitibine (AVX754), amdoxovir, KP-1461, aba of the compounds of the invention before or after administra cavir-lamivudine, abacavir-lamivudine--zidovudine, tion of unit dosages of one or more other active therapeutic zidovudine--lamivudine, 4) a HIV nucleotide inhibitor of agents, for example, administration of the compounds of the reverse transcriptase, e.g., tenofovir, tenofovir disoproxil 60 invention within seconds, minutes, or hours of the adminis fumarate--emitricitabine, tenofovir disoproxil fumarate-- tration of one or more other active therapeutic agents. For emtricitabine+efavirenz, and adefovir. 5) a HIV integrase example, a unit dose of a compound of the invention can be inhibitor, e.g., curcumin, derivatives of curcumin, chicoric administered first, followed within seconds or minutes by acid, derivatives of chicoric acid, 3.5-dicaffeoylquinic acid, administration of a unit dose of one or more other active derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic 65 therapeutic agents. Alternatively, a unit dose of one or more acid, derivatives of aurintricarboxylic acid, caffeic acid phen other therapeutic agents can be administered first, followed ethyl ester, derivatives of caffeic acid phenethyl ester, tyr by administration of a unit dose of a compound of the inven US 8,324,179 B2 107 108 tion within seconds or minutes. In some cases, it may be cally acceptable salt, Solvate, and/or ester thereof, and at least desirable to administer a unit dose of a compound of the one additional active therapeutic agent selected from the invention first, followed, after a period of hours (e.g., 1-12 group consisting of interferons, ribavirin analogs, NS3 pro hours), by administration of a unit dose of one or more other tease inhibitors, alpha-glucosidase 1 inhibitors, hepatopro active therapeutic agents. In other cases, it may be desirable to 5 tectants, non-nucleoside inhibitors of HCV, and other drugs administer a unit dose of one or more other active therapeutic for treating HCV. agents first, followed, after a period of hours (e.g., 1-12 In still yet another embodiment, the present application hours), by administration of a unit dose of a compound of the provides for the use of a compound of the present invention, invention. or a pharmaceutically acceptable salt, Solvate, and/or ester The combination therapy may provide “synergy' and 'syn 10 thereof, for the preparation of a medicament for treating an ergistic”, i.e. the effect achieved when the active ingredients HCV infection in a patient. used together is greater than the sum of the effects that results Metabolites of the Compounds of the Invention from using the compounds separately. A synergistic effect Also falling within the scope of this invention are the in may be attained when the active ingredients are: (1) co-for vivo metabolic products of the compounds described herein, mulated and administered or delivered simultaneously in a 15 to the extent such products are novel and unobvious over the combined formulation; (2) delivered by alternation or in par prior art. Such products may result for example from the allel as separate formulations; or (3) by Some other regimen. oxidation, reduction, hydrolysis, amidation, esterification When delivered in alternation therapy, a synergistic effect and the like of the administered compound, primarily due to may be attained when the compounds are administered or enzymatic processes. Accordingly, the invention includes delivered sequentially, e.g. in separate tablets, pills or cap noveland unobvious compounds produced by a process com Sules, or by different injections in separate Syringes. In gen prising contacting a compound of this invention with a mam eral, during alternation therapy, an effective dosage of each mal for a period of time sufficient to yield a metabolic product active ingredient is administered sequentially, i.e. serially, thereof. Such products typically are identified by preparing a whereas in combination therapy, effective dosages of two or radiolabelled (e.g. ''C or H) compound of the invention, more active ingredients are administered together. A syner 25 administering it parenterally in a detectable dose (e.g. greater gistic anti-viral effect denotes an antiviral effect which is than about 0.5 mg/kg) to an animal Such as rat, mouse, guinea greater than the predicted purely additive effects of the indi pig, monkey, or to man, allowing Sufficient time for metabo vidual compounds of the combination. lism to occur (typically about 30 seconds to 30 hours) and In still yet another embodiment, the present application isolating its conversion products from the urine, blood or provides for methods of inhibiting HCV polymerase in a cell, 30 other biological samples. These products are easily isolated comprising: contacting a cell infected with HCV with an since they are labeled (others are isolated by the use of anti effective amount of a compound of Formula I-IV, or a phar bodies capable of binding epitopes surviving in the metabo maceutically acceptable salt, Solvate, and/or ester thereof, lite). The metabolite structures are determined in conven whereby HCV polymerase is inhibited. tional fashion, e.g. by MS or NMR analysis. In general, In still yet another embodiment, the present application 35 analysis of metabolites is done in the same way as conven provides for methods of inhibiting HCV polymerase in a cell, tional drug metabolism studies well-known to those skilled in comprising: contacting a cell infected with HCV with an the art. The conversion products, so long as they are not effective amount of a compound of Formula I-IV, or a phar otherwise found in Vivo, are useful in diagnostic assays for maceutically acceptable salt, Solvate, and/or ester thereof, therapeutic dosing of the compounds of the invention even if and at least one additional active therapeutic agent, whereby 40 they possess no HCV polymerase inhibitory activity of their HCV polymerase is inhibited. OW. In still yet another embodiment, the present application Recipes and methods for determining stability of com provides for methods of inhibiting HCV polymerase in a cell, pounds in Surrogate gastrointestinal secretions are known. comprising: contacting a cell infected with HCV with an Compounds are defined hereinas stable in the gastrointestinal effective amount of a compound of Formula I-IV, or a phar 45 tract where less than about 50 mole percent of the protected maceutically acceptable salt, Solvate, and/or ester thereof, groups are deprotected in Surrogate intestinal or gastric juice and at least one additional active therapeutic agent selected upon incubation for 1 hour at 37° C. Simply because the from the group consisting of interferons, ribavirin analogs, compounds are stable to the gastrointestinal tract does not NS3 protease inhibitors, alpha-glucosidase 1 inhibitors, mean that they cannot be hydrolyzed in vivo. The phospho hepatoprotectants, non-nucleoside inhibitors of HCV, and 50 nate prodrugs of the invention typically will be stable in the other drugs for treating HCV. digestive system but may be substantially hydrolyzed to the In still yet another embodiment, the present application parental drug in the digestive lumen, liver or other metabolic provides for methods of treating HCV in a patient, compris organ, or within cells in general. ing: administering to the patient a therapeutically effective Exemplary Methods of Making the Compounds of the Inven amount of a compound of Formula I-IV, or a pharmaceuti 55 tion. cally acceptable salt, Solvate, and/or ester thereof. The invention provides many methods of making the com In still yet another embodiment, the present application positions of the invention. The compositions are prepared by provides for methods of treating HCV in a patient, compris any of the applicable techniques of organic synthesis. Many ing: administering to the patient a therapeutically effective Such techniques are well known in the art, such as those amount of a compound of Formula I-IV, or a pharmaceuti 60 elaborated in “Compendium of Organic Synthetic Methods’ cally acceptable salt, Solvate, and/or ester thereof, and at least (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and one additional active therapeutic agent, whereby HCV poly Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen merase is inhibited. Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, In still yet another embodiment, the present application 1977: Vol. 4, Leroy G. Wade, jr., 1980; Vol. 5, Leroy G. Wade, provides for methods of treating HCV in a patient, compris 65 Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., ing: administering to the patient a therapeutically effective “Advanced Organic Chemistry. Third Edition', (John amount of a compound of Formula I-IV, or a pharmaceuti Wiley & Sons, New York, 1985), “Comprehensive Organic US 8,324,179 B2 109 110 Synthesis. Selectivity, Strategy & Efficiency in Modern including the teachings of Ellis, US 2002/0103378 A1 and Organic Chemistry. In 9 Volumes”. Barry M. Trost, Editor Hajima, U.S. Pat. No. 6,018,049. in-Chief (Pergamon Press, New York, 1993 printing). Schemes and Examples Dialkyl phosphonates may be prepared according to the A number of exemplary methods for the preparation of the methods of: Quast et al (1974) Synthesis 490; Stowell et al compositions of the invention are provided below. These (1990) Tetrahedron Lett. 3261; U.S. Pat. No. 5,663,159. methods are intended to illustrate the nature of Such prepara In general, synthesis of phosphonate esters is achieved by tions are not intended to limit the scope of applicable meth coupling a nucleophile amine or alcohol with the correspond ods. ing activated phosphonate electrophilic precursor. For General aspects of these exemplary methods are described example, chlorophosphonate addition on to 5'-hydroxy of 10 below and in the Examples. Each of the products of the nucleoside is a well known method for preparation of nucleo following processes is optionally separated, isolated, and/or side phosphate monoesters. The activated precursor can be purified prior to its use in Subsequent processes. prepared by several well known methods. Chlorophospho Generally, the reaction conditions such as temperature, nates useful for synthesis of the prodrugs are prepared from 15 reaction time, solvents, work-up procedures, and the like, will the substituted-1,3-propanediol (Wissner, et al. (1992).J. Med be those common in the art for the particular reaction to be Chem. 35: 1650). Chlorophosphonates are made by oxidation performed. The cited reference material, together with mate of the corresponding chlorophospholanes (Anderson, et al. rial cited therein, contains detailed descriptions of such con (1984) J. Org. Chem. 49: 1304) which are obtained by reac ditions. Typically the temperatures will be -100° C. to 200° tion of the substituted diol with phosphorus trichloride. Alter C., solvents will be aprotic or protic, and reaction times will natively, the chlorophosphonate agent is made by treating be 10 seconds to 10 days. Work-up typically consists of substituted-1,3-diols with phosphorusoxychloride (Patois, et quenching any unreacted reagents followed by partition al, (1990).J. Chem. Soc. Perkin Trans. I, 1577). Chlorophos between a water/organic layer system (extraction) and sepa phonate species may also be generated in situ from corre rating the layer containing the product. sponding cyclic phosphites (Silverburg, et al., (1996) Tetra 25 Oxidation and reduction reactions are typically carried out hedron Lett., 37.771-774), which in turn can be either made at temperatures near room temperature (about 20° C.), from chlorophospholane or phosphoramidate intermediate. although for metal hydride reductions frequently the tem Phosphoroflouridate intermediate prepared either from pyro perature is reduced to 0°C. to -100°C., solvents are typically phosphate or phosphoric acid may also act as precursor in aprotic for reductions and may be either protic or aprotic for preparation of cyclic prodrugs (Watanabe et al., (1988) Tet 30 rahedron Lett., 29:5763-66). Caution: fluorophosphonate oxidations. Reaction times are adjusted to achieve desired compounds may be highly toxic conversions. Phosphonate prodrugs of the present invention may also be Condensation reactions are typically carried out at tem prepared from the precursor free acid by Mitsunobu reactions peratures near room temperature, although for non-equili (Mitsunobu, (1981) Synthesis, 1: Campbell, (1992) J. Org. 35 brating, kinetically controlled condensations reduced tem Chem., 52.6331), and other acid coupling reagents including, peratures (0°C. to -100°C.) are also common. Solvents can but not limited to, carbodiimides (Alexander, et al. (1994) be either protic (common in equilibrating reactions) or apro Collect. Czech. Chem. Commun. 59:1853; Casara, et al., tic (common in kinetically controlled reactions). (1992) Bioorg. Med. Chem. Lett., 2:145; Ohashi, et al. (1988) Standard synthetic techniques such as azeotropic removal Tetrahedron Lett., 29:1189), and benzotriazolyloxytris-(dim 40 of reaction by-products and use of anhydrous reaction condi ethylamino)phosphonium salts (Campagne, et al. (1993) Tet tions (e.g. inert gas environments) are common in the art and rahedron Lett., 34:6743). will be applied when applicable. Aryl halides undergo Ni" catalyzed reaction with phos The terms “treated”, “treating, “treatment, and the like, phite derivatives to give aryl phosphonate containing com mean contacting, mixing, reacting, allowing to react, bring pounds (Balthazar, etal (1980).J. Org. Chem. 45:5425). Phos 45 ing into contact, and other terms common in the art for indi phonates may also be prepared from the chlorophosphonate cating that one or more chemical entities is treated in Such a in the presence of a palladium catalyst using aromatic triflates manner as to convert it to one or more other chemical entities. (Petrakis, etal, (1987).J. Am. Chem. Soc. 109:2831; Lu, etal, This means that “treating compound one with compound (1987) Synthesis, 726). In another method, aryl phosphonate two' is synonymous with “allowing compound one to react esters are prepared from aryl phosphates under anionic rear 50 with compound two', 'contacting compound one with com rangement conditions (Melvin (1981) Tetrahedron Lett. pound two”, “reacting compound one with compound two'. 22:3375; Casteel, etal, (1991) Synthesis, 691). N-Alkoxyaryl and other expressions common in the art of organic synthesis salts with alkali metal derivatives of cyclic alkylphosphonate for reasonably indicating that compound one was “treated'. provide general synthesis for heteroaryl-2-phosphonate link “reacted”, “allowed to react', etc., with compound two. ers (Redmore (1970).J. Org. Chem. 35:4114). These above 55 “Treating indicates the reasonable and usual manner in mentioned methods can also be extended to compounds which organic chemicals are allowed to react. Normal con where the W group is a heterocycle. Cyclic-1,3-propanyl centrations (0.01M to 10M, typically 0.1M to 1M), tempera prodrugs of phosphonates are also synthesized from phos tures (-100° C. to 250° C., typically -78°C. to 150° C., more phonic diacids and Substituted propane-1,3-diols using a cou typically -78° C. to 100° C., still more typically 0°C. to 100° pling reagent such as 1,3-dicyclohexylcarbodiimide (DCC) 60 C.), reaction vessels (typically glass, plastic, metal), Solvents, in presence of a base (e.g., pyridine). Other carbodiimide pressures, atmospheres (typically air for oxygen and water based coupling agents like 1,3-disopropylcarbodiimide or insensitive reactions or nitrogen or argon for oxygen or water water soluble reagent, 1-(3-dimethylaminopropyl)-3-ethyl sensitive), etc., are intended unless otherwise indicated. The carbodiimide hydrochloride (EDCI) can also be utilized for knowledge of similar reactions known in the art of organic the synthesis of cyclic phosphonate prodrugs. 65 synthesis are used in selecting the conditions and apparatus The carbamoyl group may be formed by reaction of a for “treating in a given process. In particular, one of ordinary hydroxy group according to the methods known in the art, skill in the art of organic synthesis selects conditions and US 8,324,179 B2 111 112 apparatus reasonably expected to Successfully carry out the Alternatively, by method (2), the substrate to be resolved is chemical reactions of the described processes based on the reacted with one enantiomer of a chiral compound to form a knowledge in the art. diastereomeric pair (Eliel, E. and Wilen, S. (1994) Stere Modifications of each of the exemplary schemes above and ochemistry of Organic Compounds, John Wiley & Sons, Inc., in the examples (hereafter “exemplary schemes”) leads to 5 p. 322). Diastereomeric compounds can be formed by react various analogs of the specific exemplary materials produce. ing asymmetric compounds with enantiomerically pure The above cited citations describing suitable methods of chiral derivatizing reagents, such as menthyl derivatives, fol organic synthesis are applicable to such modifications. lowed by separation of the diastereomers and hydrolysis to In each of the exemplary Schemes it may be advantageous yield the free, enantiomerically enriched xanthene. A method to separate reaction products from one another and/or from 10 starting materials. The desired products of each step or series of determining optical purity involves making chiral esters, of steps is separated and/or purified (hereinafter separated) to Such as a menthyl ester, e.g. (-)menthyl chloroformate in the the desired degree of homogeneity by the techniques com presence of base, or Mosher ester, C.-methoxy-O-(trifluorom mon in the art. Typically such separations involve multiphase ethyl)phenyl acetate (Jacob III. (1982) J. Org. Chem. extraction, crystallization from a solvent or solvent mixture, 15 47:4165), of the racemic mixture, and analyzing the NMR distillation, Sublimation, or chromatography. Chromatogra spectrum for the presence of the two atropisomeric diastere phy can involve any number of methods including, for omers. Stable diastereomers of atropisomeric compounds can example: reverse-phase and normal phase; size exclusion; ion be separated and isolated by normal-and reverse-phase chro exchange; high, medium, and low pressure liquid chromatog matography following methods for separation of atropiso raphy methods and apparatus; Small scale analytical; simu meric naphthyl-isoquinolines (Hoye, T., WO 96/15111). By lated moving bed (SMB) and preparative thin or thick layer method (3), a racemic mixture of two enantiomers can be chromatography, as well as techniques of Small scale thin separated by chromatography using a chiral stationary phase layer and flash chromatography. (Chiral Liquid Chromatography (1989) W. J. Lough, Ed. Another class of separation methods involves treatment of Chapman and Hall, New York: Okamoto, (1990).J. of Chro a mixture with a reagent selected to bind to or render other 25 matogr: 513:375-378). Enriched or purified enantiomers can wise separable a desired product, unreacted starting material, be distinguished by methods used to distinguish other chiral reaction by product, or the like. Such reagents include adsor molecules with asymmetric carbon atoms, such as optical bents or absorbents such as activated carbon, molecular rotation and circular dichroism. sieves, ion exchange media, or the like. Alternatively, the All literature and patent citations above are hereby reagents can be acids in the case of a basic material, bases in 30 expressly incorporated by reference at the locations of their the case of an acidic material, binding reagents such as anti citation. Specifically cited sections or pages of the above cited bodies, binding proteins, selective chelators such as crown works are incorporated by reference with specificity. The ethers, liquid/liquidion extraction reagents (LIX), or the like. invention has been described in detail sufficient to allow one Selection of appropriate methods of separation depends on of ordinary skill in the art to make and use the subject matter the nature of the materials involved. For example, boiling 35 of the following Embodiments. It is apparent that certain point, and molecular weight in distillation and Sublimation, modifications of the methods and compositions of the follow presence or absence of polar functional groups in chromatog ing Embodiments can be made within the scope and spirit of raphy, stability of materials in acidic and basic media in the invention. multiphase extraction, and the like. One skilled in the art will apply techniques most likely to achieve the desired separa 40 tion. A single stereoisomer, e.g. an enantiomer, Substantially Scheme A O O free of its stereoisomer may be obtained by resolution of the M M racemic mixture using a method Such as formation of diaste R-link-P-OR --> R-link-P-OR reomers using optically active resolving agents (“Stere 45 ochemistry of Carbon Compounds.” (1962) by E. L. Eliel, ORI OH 27.1 27.2 McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., O O 113:(3) 283-302). Racemic mixtures of chiral compounds of M M the invention can be separated and isolated by any Suitable R-link-P-OR - - R-link-P-OH method, including: (1) formation of ionic, diastereomeric 50 ORI OH salts with chiral compounds and separation by fractional 27.1 27.3 crystallization or other methods, (2) formation of diastereo O O meric compounds with chiral derivatizing reagents, separa M M tion of the diastereomers, and conversion to the pure stereoi R-link-P-OR --> R-link-P-OH Somers, and (3) separation of the Substantially pure or 55 OH OH enriched Stereoisomers directly under chiral conditions. 27.2 27.3 Under method (1), diastereomeric salts can be formed by O O reaction of enantiomerically pure chiral bases such as bru M M cine, quinine, ephedrine, Strychnine, C.-methyl-3-phenyl R-link-P-OR - T - R-link-P-OR ethylamine (amphetamine), and the like with asymmetric 60 OH ORI compounds bearing acidic functionality. Such as carboxylic 27.2 27.1 acid and Sulfonic acid. The diastereomeric salts may be O O induced to separate by fractional crystallization orionic chro R-link-P'-OHM --> R-link-P-ORM matography. For separation of the optical isomers of amino compounds, addition of chiral carboxylic or Sulfonic acids, 65 OH OH Such as camphorsulfonic acid, tartaric acid, mandelic acid, or 27.3 27.2 lactic acid can result in formation of the diastereomeric salts. US 8,324,179 B2 113 114 -continued ethanol, for example using the procedure described in Helv. O O Chim. Acta., 68:618, 1985. Palladium catalyzed hydro R-link-P-OHM He-6 R-link-P-OR... 4 on genolysis of phosphonate esters 27.1 in which R" is benzyl is described in J. Org. Chem., 24:434, 1959. Platinum-catalyzed OH ORI hydrogenolysis of phosphonate esters 27.1 in which R' is 27.3 27.1 phenyl is described in J. Amer: Chem. Soc., 78:2336, 1956. The conversion of a phosphonate monoester 27.2 into a Scheme A shows the general interconversions of certain phosphonate diester 27.1 (Scheme A. Reaction 4) in which phosphonate compounds: acids —P(O)(OH), mono-esters the newly introduced R' group is alkyl, arylalkyl, orhaloalkyl -P(O)(OR)(OH); and diesters -P(O)(OR), in which the 10 such as chloroethyl, can be effected by a number of reactions R" groups are independently selected, and defined herein in which the substrate 27.2 is reacted with a hydroxy com before, and the phosphorus is attached through a carbon moi pound ROH, in the presence of a coupling agent. Suitable ety (link, i.e. linker), which is attached to the rest of the coupling agents are those employed for the preparation of molecule, e.g. drug or drug intermediate (R). The R' groups 15 carboxylate esters, and include a carbodiimide such as dicy attached to the phosphonate esters in Scheme 1 may be clohexylcarbodiimide, in which case the reaction is prefer changed using established chemical transformations. The ably conducted in a basic organic solvent such as pyridine, or interconversions may be carried out in the precursor com (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluo pounds or the final products using the methods described rophosphate (PYBOP Sigma), in which case the reaction is below. The methods employed for a given phosphonate trans performed in a polar solvent such as dimethylformamide, in formation depend on the nature of the substituent R. The the presence of a tertiary organic base such as diisopropyl preparation and hydrolysis of phosphonate esters is described ethylamine, or Aldrithiol-2 (Aldrich) in which case the reac in Organic Phosphorus Compounds, G. M. Kosolapoff, L. tion is conducted in a basic solvent such as pyridine, in the Maeir, eds, Wiley, 1976, p. 9ff. presence of a triaryl phosphine Such as triphenylphosphine. The conversion of a phosphonate diester 27.1 into the 25 Alternatively, the conversion of the phosphonate monoester corresponding phosphonate monoester 27.2 (Scheme A, 27.1 to the diester 27.1 can be effected by the use of the Reaction 1) can be accomplished by a number of methods. Mitsunobu reaction. The substrate is reacted with the hydroxy For example, the ester 27.1 in which R" is an arylalkyl group compound ROH, in the presence of diethylazodicarboxylate Such as benzyl, can be converted into the monoester com and a triarylphosphine Such as triphenyl phosphine. Alterna pound 27.2 by reaction with a tertiary organic base such as 30 diazabicyclooctane (DABCO) or quinuclidine, as described tively, the phosphonate monoester 27.2 can be transformed in J. Org. Chem., 1995, 60:2946. The reaction is performed in into the phosphonate diester 27.1, in which the introduced R' an inert hydrocarbon solvent such as toluene or Xylene, at group is alkenyl or arylalkyl, by reaction of the monoester about 110°C. The conversion of the diester 27.1 in which R' with the halide R'Br, in which R" is as alkenyl or arylalkyl. is an aryl group Such as phenyl, or an alkenyl group Such as 35 The alkylation reaction is conducted in a polar organic Sol allyl, into the monoester 27.2 can be effected by treatment of vent Such as dimethylformamide or acetonitrile, in the pres the ester 27.1 with a base such as aqueous sodium hydroxide ence of a base Such as cesium carbonate. Alternatively, the in acetonitrile or lithium hydroxide in aqueous tetrahydrofu phosphonate monoester can be transformed into the phospho ran. Phosphonate diesters 27.2 in which one of the groups R' nate diester in a two step procedure. In the first step, the is arylalkyl, such as benzyl, and the other is alkyl, can be 40 phosphonate monoester 27.2 is transformed into the chloro converted into the monoesters 27.2 in which R" is alkyl, by analog-P(O)(OR")C1 by reaction with thionyl chloride or hydrogenation, for example using a palladium on carbon oxalyl chloride and the like, as described in Organic Phos catalyst. Phosphonate diesters in which both of the groups R' phorus Compounds, G. M. Kosolapoff, L. Maeir, eds, Wiley, are alkenyl. Such as allyl, can be converted into the monoester 1976, p. 17, and the thus-obtained product -P(O)(OR")C1 is 27.2 in which R" is alkenyl, by treatment with chlorotris 45 then reacted with the hydroxy compound ROH, in the pres (triphenylphosphine)rhodium (Wilkinson's catalyst) in aque ence of a base Such as triethylamine, to afford the phospho ous ethanol at reflux, optionally in the presence of diazabicy nate diester 27.1. clooctane, for example by using the procedure described in J. A phosphonic acid—P(O)(OH) can be transformed into a Org. Chem., 38:3224 1973 for the cleavage of allyl carboxy phosphonate monoester - P(O)(OR')(OH) (Scheme A, lates. 50 Reaction 5) by means of the methods described above of for The conversion of a phosphonate diester 27.1 or a phos the preparation of the phosphonate diester -P(O)(OR"), phonate monoester 27.2 into the corresponding phosphonic 27.1, except that only one molar proportion of the component acid 27.3 (Scheme A. Reactions 2 and 3) can effected by ROH or R'Br is employed. reaction of the diester or the monoester with trimethylsilyl A phosphonic acid—P(O)(OH), 27.3 can be transformed bromide, as described in J. Chem. Soc., Chem. Comm., 739, 55 into a phosphonate diester -P(O)(OR"), 27.1 (Scheme A, 1979. The reaction is conducted in an inert solvent such as, for Reaction 6) by a coupling reaction with the hydroxy com example, dichloromethane, optionally in the presence of a pound ROH, in the presence of a coupling agent such as silylating agent Such as bis(trimethylsilyl)trifluoroacetamide, Aldrithiol-2 (Aldrich) and triphenylphosphine. The reaction at ambient temperature. A phosphonate monoester 27.2 in is conducted in a basic solvent Such as pyridine. Alternatively, which R" is arylalkylsuch as benzyl, can be converted into the 60 phosphonic acids 27.3 can be transformed into phosphonic corresponding phosphonic acid 27.3 by hydrogenation over a esters 27.1 in which R" is aryl, such as phenyl, by means of a palladium catalyst, or by treatment with hydrogen chloride in coupling reaction employing, for example, phenol and dicy an ethereal Solvent such as dioxane. A phosphonate clohexylcarbodiimide in pyridine at about 70° C. Alterna monoester 27.2 in which R' is alkenyl such as, for example, tively, phosphonic acids 27.3 can be transformed into phos allyl, can be converted into the phosphonic acid 27.3 by 65 phonic esters 27.1 in which R" isalkenyl, by means of an reaction with Wilkinson's catalyst in an aqueous organic Sol alkylation reaction. The phosphonic acid is reacted with the vent, for example in 15% aqueous acetonitrile, or in aqueous alkenyl bromide R'Br in a polar organic solvent such as US 8,324,179 B2 115 116 acetonitrile solution at reflux temperature, in the presence of Nucleosides Nucleotides 1995, 14, 871, and diphenyl chlo a base such as cesium carbonate, to afford the phosphonic rophosphate, as described in J. Med. Chem., 1988, 31, 1305. ester 27.1. Phosphonic acids are converted into amidates and esters by Phosphonate prodrugs of the present invention may also be means of the Mitsonobu reaction, in which the phosphonic prepared from the precursor free acid by Mitsunobu reactions acid and the amine or hydroxy reactant are combined in the (Mitsunobu, (1981) Synthesis, 1: Campbell, (1992) J. Org. presence of a triaryl phosphine and a dialkyl azodicarboxy Chem., 52.6331), and other acid coupling reagents including, late. The procedure is described in Org. Lett., 2001, 3, 643, or but not limited to, carbodiimides (Alexander, et al. (1994) J. Med. Chem., 1997, 40, 3842. Collect. Czech. Chem. Commun. 59:1853; Casara, et al., Phosphonic esters are also obtained by the reaction (1992) Bioorg. Med. Chem. Lett., 2:145; Ohashi, et al. (1988) 10 Tetrahedron Lett., 29:1189), and benzotriazolyloxytris-(dim between phosphonic acids and halo compounds, in the pres ethylamino)phosphonium salts (Campagne, et al. (1993) Tet ence of a suitable base. The method is described, for example, rahedron Lett., 34:6743). in Anal. Chem., 1987, 59, 1056, or J. Chem. Soc. Perkin Preparation of Carboalkoxy-Substituted Phosphonate Bisa Trans., I, 1993, 19, 2303, or J. Med. Chem., 1995, 38, 1372, midates, Monoamidates, Diesters and Monoesters. 15 or Tet. Lett., 2002, 43, 1161. A number of methods are available for the conversion of Schemes 1-4 illustrate the conversion of phosphonate phosphonic acids into amidates and esters. In one group of esters and phosphonic acids into carboalkoxy-Substituted methods, the phosphonic acid is either converted into an phosphorobisamidates (Scheme 1), phosphoroamidates isolated activated intermediate such as a phosphorylchloride, (Scheme 2), phosphonate monoesters (Scheme 3) and phos or the phosphonic acid is activated in situ for reaction with an phonate diesters, (Scheme 4). amine or a hydroxy compound. Scheme 1 illustrates various methods for the conversion of The conversion of phosphonic acids into phosphoryl chlo phosphonate diesters 1.1 into phosphorobisamidates 1.5. The rides is accomplished by reaction with thionyl chloride, for diester 1.1, prepared as described previously, is hydrolyzed, example as described in J. Gen. Chem. USSR, 1983, 53,480, either to the monoester 1.2 or to the phosphonic acid 1.6. The Zh. Obschei Khim., 1958, 28, 1063, or J. Org. Chem., 1994, 25 methods employed for these transformations are described 59, 6144, or by reaction with oxalyl chloride, as described in above. The monoester 1.2 is converted into the monoamidate J. Am. Chem. Soc., 1994, 116, 3251, or J. Org. Chem., 1994, 1.3 by reaction with an aminoester 19, in which the group R 59, 6144, or by reaction with phosphorus pentachloride, as is H or alkyl, the group R is an alkylene moiety such as, for described in J. Org. Chem., 2001, 66, 329, or in J. Med. example, CHCH, CHPr', CH(CHPh), CHCH(CH) and Chem., 1995, 38, 1372. The resultant phosphoryl chlorides 30 the like, or a group present in natural or modified aminoacids, are then reacted with amines or hydroxy compounds in the and the group R is alkyl. The reactants are combined in the presence of a base to afford the amidate or ester products. presence of a coupling agent such as a carbodiimide, for Phosphonic acids are converted into activated imidazolyl example dicyclohexyl carbodiimide, as described in J. Am. derivatives by reaction with carbonyl diimidazole, as Chem. Soc., 1957, 79,3575, optionally in the presence of an described in J. Chem. Soc., Chem. Comm., 1991, 312, or 35 activating agent Such as hydroxybenztriazole, to yield the Nucleosides Nucleotides 2000, 19, 1885. Activated sulfony amidate product 1.3. The amidate-forming reaction is also loxy derivatives are obtained by the reaction of phosphonic effected in the presence of coupling agents such as BOP, as acids with trichloromethylsulfonyl chloride, as described in J. described in J. Org. Chem., 1995, 60, 5214, Aldrithiol, Med. Chem. 1995, 38, 4958, or with triisopropylbenzene PYBOP and similar coupling agents used for the preparation sulfonyl chloride, as described in Tet. Lett., 1996, 7857, or 40 of amides and esters. Alternatively, the reactants 1.2 and 1.9 Bioorg. Med. Chem. Lett., 1998, 8, 663. The activated sulfo are transformed into the monoamidate 1.3 by means of a nyloxy derivatives are then reacted with amines or hydroxy Mitsonobu reaction. The preparation of amidates by means of compounds to afford amidates or esters. the Mitsonobu reaction is described in J. Med. Chem., 1995, Alternatively, the phosphonic acid and the amine or 38, 2742. Equimolar amounts of the reactants are combined hydroxy reactant are combined in the presence of a diimide 45 in an inert Solvent Such as tetrahydrofuran in the presence of coupling agent. The preparation of phosphonic amidates and a triaryl phosphine and a dialkylazodicarboxylate. The thus esters by means of coupling reactions in the presence of obtained monoamidate ester 1.3 is then transformed into ami dicyclohexyl carbodiimide is described, for example, in J. date phosphonic acid 1.4. The conditions used for the Chem. Soc., Chem. Comm., 1991, 312, or J. Med. Chem. hydrolysis reaction depend on the nature of the R' group, as 1980, 23, 1299 or Coll. Czech. Chem. Comm., 1987, 52, 50 described previously. The phosphonic acid amidate 1.4 is 2792. The use of ethyl dimethylaminopropyl carbodiimide then reacted with an aminoester 1.9, as described above, to for activation and coupling of phosphonic acids is described yield the bisamidate product 1.5, in which the amino substitu in Tet. Lett., 2001, 42, 8841, or Nucleosides Nucleotides, ents are the same or different. 2000, 19, 1885. An example of this procedure is shown in Scheme 1, A number of additional coupling reagents have been 55 Example 1. In this procedure, a dibenzylphosphonate 1.14 is described for the preparation of amidates and esters from reacted with diazabicyclooctane (DABCO) in toluene at phosphonic acids. The agents include Aldrithiol-2, and reflux, as described in J. Org. Chem., 1995, 60,2946, to afford PYBOP and BOP, as described in J. Org. Chem., 1995, 60, the monobenzyl phosphonate 1.15. The product is then 5214, and J. Med. Chem., 1997, 40, 3842, mesitylene-2- reacted with equimolar amounts of ethylalaninate 1.16 and sulfonyl-3-nitro-1,2,4-triazole (MSNT), as described in J. 60 dicyclohexyl carbodiimide in pyridine, to yield the amidate Med. Chem., 1996, 39, 4958, diphenylphosphoryl azide, as product 1.17. The benzyl group is then removed, for example described in J. Org. Chem., 1984, 49, 1158, 1-(2,4,6-triiso by hydrogenolysis over a palladium catalyst, to give the propylbenzenesulfonyl-3-nitro-1,2,4-triazole (TPSNT) as monoacid product 1.18. This compound is then reacted in a described in Bioorg. Med. Chem. Lett., 1998, 8, 1013, bro Mitsonobu reaction with ethyl leucinate 1.19, triphenylphos motris(dimethylamino)phosphonium hexafluorophosphate 65 phine and diethylazodicarboxylate, as described in J. Med. (BroP), as described in Tet. Lett., 1996, 37,3997, 2-chloro Chem., 1995, 38, 2742, to produce the bisamidate product 5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane, as described in 120. US 8,324,179 B2 117 118 Using the above procedures, but employing, in place of The product 1.8 is then reacted with an aminoester 1.9 in the ethyl leucinate 1.19 or ethyl alaninate 1.16, different ami presence of a base such as pyridine, to give an intermediate noesters 1.9, the corresponding products 1.5 are obtained. monoamidate product 1.3. The latter compound is then con Alternatively, the phosphonic acid 1.6 is converted into the verted, by removal of the R' group and coupling of the prod bisamidate 1.5 by use of the coupling reactions described uct with the aminoester 1.9, as described above, into the above. The reaction is performed in one step, in which case bisamidate 1.5. the nitrogen-related substituents present in the product 1.5 are An example of this procedure, in which the phosphonic the same, or in two steps, in which case the nitrogen-related acid is activated by conversion to the chloro derivative 1.26, is substituents can be different. An example of the method is shown in Scheme 1, Example 4. In this procedure, the phos shown in Scheme 1. Example 2. In this procedure, a phos 10 phonic monobenzyl ester 1.15 is reacted, in dichloromethane, phonic acid 1.6 is reacted in pyridine solution with excess with thionyl chloride, as described in Tet. Let., 1994, 35, ethyl phenylalaninate 1.21 and dicyclohexylcarbodiimide, 4097, to afford the phosphoryl chloride 1.26. The product is for example as described in J. Chem. Soc., Chem. Comm., then reacted in acetonitrile solution at ambient temperature 1991, 1063, to give the bisamidate product 1.22. with one molar equivalent of ethyl 3-amino-2-methylpropi Using the above procedures, but employing, in place of 15 onate 1.27 to yield the monoamidate product 1.28. The latter ethyl phenylalaninate, different aminoesters 1.9, the corre compound is hydrogenated in ethyl acetate over a 5% palla sponding products 1.5 are obtained. dium on carbon catalyst to produce the monoacid product As a further alternative, the phosphonic acid 1.6 is con 1.29. The product is subjected to a Mitsonobu coupling pro verted into the mono or bis-activated derivative 1.7, in which cedure, with equimolar amounts of butyl alaninate 1.30, LV is a leaving group Such as chloro, imidazolyl, triisopropy triphenyl phosphine, diethylazodicarboxylate and triethy Ibenzenesulfonyloxy etc. The conversion of phosphonic acids lamine in tetrahydrofuran, to give the bisamidate product into chlorides 1.7 (LV-Cl) is effected by reaction with thionyl 131. chloride or oxalyl chloride and the like, as described in Using the above procedures, but employing, in place of Organic Phosphorus Compounds, G. M. Kosolapoff, L. ethyl 3-amino-2-methylpropionate 1.27 or butyl alaninate Maeir, eds, Wiley, 1976, p. 17. The conversion of phosphonic 25 1.30, different aminoesters 1.9, the corresponding products acids into monoimidazolides 1.7 (LV-imidazolyl) is 1.5 are obtained. described in J. Med. Chem., 2002, 45, 1284 and in J. Chem. The activated phosphonic acid derivative 1.7 is also con Soc. Chem. Comm., 1991, 312. Alternatively, the phosphonic verted into the bisamidate 1.5 via the diamino compound acid is activated by reaction with triisopropylbenzenesulfonyl 1.10. The conversion of activated phosphonic acid derivatives chloride, as described in Nucleosides and Nucleotides, 2000, 30 Such as phosphoryl chlorides into the corresponding amino 10, 1885. The activated product is then reacted with the ami analogs 1.10, by reaction with ammonia, is described in noester 1.9, in the presence of a base, to give the bisamidate Organic Phosphorus Compounds, G. M. Kosolapoff, L. 1.5. The reaction is performed in one step, in which case the Maeir, eds. Wiley, 1976. The diamino compound 1.10 is then nitrogen Substituents present in the product 1.5 are the same, reacted at elevated temperature with a haloester 1.12, in a or in two steps, via the intermediate 1.11, in which case the 35 polar organic solvent such as dimethylformamide, in the pres nitrogen substituents can be different. ence of a base Such as dimethylaminopyridine or potassium Examples of these methods are shown in Scheme 1, carbonate, to yield the bisamidate 1.5. Examples 3 and 5. In the procedure illustrated in Scheme 1, An example of this procedure is shown in Scheme 1, Example 3, a phosphonic acid 1.6 is reacted with ten molar Example 6. In this method, a dichlorophosphonate 1.23 is equivalents of thionyl chloride, as described in Zh. Obschei 40 reacted with ammonia to afford the diamide 1.37. The reac Khim., 1958, 28, 1063, to give the dichloro compound 1.23. tion is performed in aqueous, aqueous alcoholic or alcoholic The product is then reacted at reflux temperature in a polar Solution, at reflux temperature. The resulting diamino com aprotic solvent Such as acetonitrile, and in the presence of a pound is then reacted with two molar equivalents of ethyl base such as triethylamine, with butyl serinate 1.24 to afford 2-bromo-3-methylbutyrate 1.38, in a polar organic solvent the bisamidate product 1.25. 45 such as N-methylpyrrolidinone at ca. 150°C., in the presence Using the above procedures, but employing, in place of of a base such as potassium carbonate, and optionally in the butyl serinate 1.24, differentaminoesters 1.9, the correspond presence of a catalytic amount of potassium iodide, to afford ing products 1.5 are obtained. the bisamidate product 1.39. In the procedure illustrated in Scheme 1, Example 5, the Using the above procedures, but employing, in place of phosphonic acid 1.6 is reacted, as described in J. Chem. Soc. 50 ethyl 2-bromo-3-methylbutyrate 1.38, different haloesters Chem. Comm., 1991, 312, with carbonyl diimidazole to give 1.12 the corresponding products 1.5 are obtained. the imidazolide 1.32. The product is then reacted in acetoni The procedures shown in Scheme 1 are also applicable to trile solution at ambient temperature, with one molar equiva the preparation of bisamidates in which the aminoester moi lent of ethyl alaninate 1.33 to yield the monodisplacement ety incorporates different functional groups. Scheme 1, product 1.34. The latter compound is then reacted with car 55 Example 7 illustrates the preparation of bisamidates derived bonyl diimidazole to produce the activated intermediate 1.35, from tyrosine. In this procedure, the monoimidazolide 1.32 is and the product is then reacted, under the same conditions, reacted with propyl tyrosinate 1.40, as described in Example with ethyl N-methylalaninate 1.33a to give the bisamidate 5, to yield the monoamidate 1.41. The product is reacted with product 1.36. carbonyl diimidazole to give the imidazolide 1.42, and this Using the above procedures, but employing, in place of 60 material is reacted with a further molar equivalent of propyl ethylalaninate 1.33 or ethyl N-methylalaninate 1.33a, differ tyrosinate to produce the bisamidate product 1.43. ent aminoesters 1.9, the corresponding products 1.5 are Using the above procedures, but employing, in place of obtained. propyl tyrosinate 1.40, different aminoesters 1.9, the corre The intermediate monoamidate 1.3 is also prepared from sponding products 1.5 are obtained. The aminoesters the monoester 1.2 by first converting the monoester into the 65 employed in the two stages of the above procedure can be the activated derivative 1.8 in which LV is a leaving group such as same or different, so that bisamidates with the same or dif halo, imidazolyl etc, using the procedures described above. ferent amino Substituents are prepared. US 8,324 179 B2 119 120 Scheme 2 illustrates methods for the preparation of phos Alternatively, the phosphonate monoester 1.1 is coupled, phonate monoamidates. In one procedure, a phosphonate monoester 1.1 is converted, as described in Scheme 1, into the as described in Scheme 1, with an aminoester 1.9 to produce activated derivative 1.8. This compound is then reacted, as the amidate 2.1. If necessary, the R' substituent is then described above, with an aminoester 1.9, in the presence of a altered, by initial cleavage to afford the phosphonic acid 2.2. base, to afford the monoamidate product 2.1. The procedure is illustrated in Scheme 2, Example 1. In this The procedures for this transformation depend on the nature method, a monophenyl phosphonate 2.7 is reacted with, for of the R' group, and are described above. The phosphonic example, thionyl chloride, as described in J. Gen. Chem. acid is then transformed into the esteramidate product 2.3, by USSR., 1983, 32, 367, to give the chloro product 2.8. The reaction with the hydroxy compound ROH, in which the product is then reacted, as described in Scheme 1, with ethyl 10 group R is aryl, heteroaryl, alkyl, cycloalkyl, haloalkyl etc. alaninate 2.9, to yield the amidate 2.10. Using the above procedures, but employing, in place of using the same coupling procedures (carbodiimide, Ald ethylalaninate 2.9, differentaminoesters 1.9, the correspond rithiol-2, PYBOP. Mitsonobu reaction etc) described in ing products 2.1 are obtained. Scheme 1 for the coupling of amines and phosphonic acids.

Scheme 1

R-link-P-NH/ Ha(R)COR4 5 O O AO /R 2 V 1.12 M W 19 R-link-P -N NH (RCOR --- R-link-P -NH2 -- R-link-P-Lv He V Ex6 V (Lv or OH) (R) (R) NH2 (Lv or OH) A V COR COR 1.5 1.10 1.7 1.11 N |. O R2 M / O O O R-link-P-N 5 R-link-P-OR.4 on -- R-link-P-OR.4 on -- R-link-P-OH/ -- \N. \,-COR 2 ORI OH OH (R) R V 5 COR 1.1 1.2 1.6 1.5 1 9 1.9 /

O O O R-link-P-OR... 4 on R-link-P-OHA M 2 V 2 R-link-POR - - - N. R. - e. N-R R2NH(R)COR A. M Lw 1.9 (R) R COR COR 1.8 1.3 1.4 Scheme 1 Example 1 H2NCH(Me)COEt O O O / H. M M 1.16 R-link-P-N Me R-link-P-OBn -- R-link-P-OH --> N N. Ho OB OB OB COOEt 1.14 1.15 1.17

O O M H M H R-link-P-N Me R-link-P-N Me HNCH(CHPr)COEt \h Yon 1.19 i COOEt COOEt Pr'HC COOEt 1.18

US 8,324,179 B2 123 124 -continued Scheme 1 Example 6 Pri O ) -COEt O O A R-link-P-CM -- R-link-P/ NH2 -BrCH(Pri)COEt - - R-link-PV -NH 1.38 NH C NH2 Pri COEt 1.23 1.37 1.39 Scheme 1 Example 7 HO O O M M R-link-P-OH Rink re-in NH NH O M HN COPr R-link-P-OH He- He 1.40 COPr COPr He Im.

HO HO 1.32 1.41 1.42 PrCO O M R-link-P-NH V NH

COPr OH

HO 1.43

Examples of this method are shown in Scheme 2, reacted, as described in Scheme 1, with a haloester 2.5, in the Examples and 2 and 3. In the sequence shown in Example 2. presence of a base, to produce the amidate product 2.6. If a monobenzyl phosphonate 2.11 is transformed by reaction 45 appropriate, the nature of the R' group is changed, using the with ethyl alaninate, using one of the methods described procedures described above, to give the product 2.3. The above, into the monoamidate 2.12. The benzyl group is then method is illustrated in Scheme 2. Example 4. In this removed by catalytic hydrogenation in ethyl acetate Solution sequence, the monophenyl phosphoryl chloride 2.18 is over a 5% palladium on carbon catalyst, to afford the phos reacted, as described in Scheme 1, with ammonia, to yield the 50 amino product 2.19. This material is then reacted in N-meth phonic acid amidate 2.13. The product is then reacted in ylpyrrolidinone solution at 170° C. with butyl 2-bromo-3- dichloromethane solution at ambient temperature with phenylpropionate 2.20 and potassium carbonate, to afford the equimolar amounts of 1-(dimethylaminopropyl)-3-ethylcar amidate product 2.21. bodiimide and trifluoroethanol 2.14, for example as described Using these procedures, but employing, in place of butyl in Tet. Lett., 2001, 42,8841, to yield the amidate ester 2.15. 55 2-bromo-3-phenylpropionate 2.20, different haloesters 2.5, In the sequence shown in Scheme 2. Example 3, the the corresponding products 2.6 are obtained. The monoamidate products 2.3 are also prepared from the monoamidate 2.13 is coupled, in tetrahydrofuran solution at doubly activated phosphonate derivatives 1.7. In this proce ambient temperature, with equimolar amounts of dicyclo dure, examples of which are described in Synlett., 1998, 1,73, hexylcarbodiimide and 4-hydroxy-N-methylpiperidine 2.16, the intermediate 1.7 is reacted with a limited amount of the to produce the amidate ester product 2.17. 60 aminoester 1.9 to give the mono-displacement product 1.11. Using the above procedures, but employing, in place of the The latter compound is then reacted with the hydroxy com ethylalaninate product 2.12 different monoacids 2.2, and in pound ROH in a polar organic solvent such as dimethylfor place of trifluoroethanol 2.14 or 4-hydroxy-N-methylpiperi mamide, in the presence of a base such as diisopropylethy dine 2.16, different hydroxy compounds ROH, the corre lamine, to yield the monoamidate ester 2.3. sponding products 2.3 are obtained. 65 The method is illustrated in Scheme 2. Example 5. In this Alternatively, the activated phosphonate ester 1.8 is reacted method, the phosphoryl dichloride 2.22 is reacted in dichlo with ammonia to yield the amidate 2.4. The product is then romethane solution with one molar equivalent of ethyl N-me US 8,324,179 B2 125 126 thyl tyrosinate 2.23 and dimethylaminopyridine, to generate Using these procedures, but employing, in place of ethyl the monoamidate 2.24. The product is then reacted with phe- N-methyl tyrosinate 2.23 or phenol 2.25, the aminoesters 1.9 nol 2.25 in dimethylformamide containing potassium carbon- and/or the hydroxy compounds ROH, the corresponding ate, to yield the ester amidate product 2.26. products 2.3 are obtained.

Scheme 2 O R2 O M / M R-link-P-N R-link-P-Ly - as V V 1.9 Lw (R') Lw A COR 1.7 1.11 ot O O O / R-link-P-ORM R-link-P-OHM R-link-P-ORM Rik?- OR - - N-R -- A. - R -- -R OH (R) (R) (R) COR COR COR 1.1 2.1 2.2 2.3 R2NH(R)COR 1.9 2 O O O R-link-P-ORM R-link-P-ORM -- Rink-R-4 OR —Ha(R'COR - NH Lw NH2 (R) COR 1.8 2.4 2.6 Scheme 2 Example 1 O O O R-link-P-OPhA M M H2NCH(Me)COEt V R-link-P-OP -- R-link-P-OP — —- NH OH C Me COEt 2.7 2.8 2.10 Scheme 2 Example 2 O O O / R-link-P-OBM Rink re-M OH R-link-P-OCHCFA CFCH-OH Rik?- OBn -- NH He- NH ST- NH

COEt COEt COEt 2.11 2.12 2.13 2.15 Scheme 2 Example 3 OH O O M M R-link-P-OH N R-link-P-O N-Me VNH Me1MS -> VNH 2.16

COEt COEt 2.13 2.17 US 8,324,179 B2 127 128 -continued Scheme 2 Example 4 O M O O R-link-P-OP M M BrCH(Bn)COBu R-link-P-OPh - R-link-P-OPh - - - NH C NH2 Bn COBu 2.18 2.19 2.21 Scheme 2 Example 5 HO

O O O Me n R-link-P-CIM R-link-P-OM

R-link 4.a C He-N COEt YM-We He-PhOH YM-We 2.23 2.25 C HO HO COEt COEt 2.22 2.24 2.26

Scheme 3 illustrates methods for the preparation of car compound is then coupled, in pyridine Solution at ambient boalkoxy-substituted phosphonate diesters in which one of temperature, in the presence of dicyclohexyl carbodiimide, the ester groups incorporates a carboalkoxy Substituent. with one molar equivalent of 3-hydroxypyridine 3.16 to yield In one procedure, a phosphonate monoester 1.1, prepared 30 the mixed diester 3.17. as described above, is coupled, using one of the methods Using the above procedures, but employing, in place of the described above, with a hydroxyester 3.1, in which the groups ethyl lactate 3.13 or 3-hydroxypyridine, a different hydrox R" and R are as described in Scheme 1. For example, yester 3.1 and/or a different hydroxy compound ROH, the equimolar amounts of the reactants are coupled in the pres corresponding products 3.4 are obtained. ence of a carbodiimide Such as dicyclohexylcarbodiimide, as 35 The mixed diesters 3.2 are also obtained from the described in Aust. J. Chem., 1963, 609, optionally in the monoesters 1.1 via the intermediacy of the activated presence of dimethylaminopyridine, as described in Tet. monoesters 3.5. In this procedure, the monoester 1.1 is con 1999, 55, 12997. The reaction is conducted in an inert solvent verted into the activated compound 3.5 by reaction with, for at ambient temperature. example, phosphorus pentachloride, as described in J. Org. The procedure is illustrated in Scheme3, Example 1. In this 40 Chem..., 2001, 66, 329, or with thionyl chloride or oxalyl method, a monophenylphosphonate 3.9 is coupled, indichlo chloride (LV-Cl), or with triisopropylbenzenesulfonyl chlo romethane solution in the presence of dicyclohexyl carbodi ride in pyridine, as described in Nucleosides and Nucleotides, imide, with ethyl 3-hydroxy-2-methylpropionate 3.10 to 2000, 19, 1885, or with carbonyl diimidazole, as described in yield the phosphonate mixed diester 3.11. J. Med. Chem., 2002, 45, 1284. The resultant activated Using this procedure, but employing, in place of ethyl 45 monoester is then reacted with the hydroxyester 3.1, as 3-hydroxy-2-methylpropionate 3.10, different hydroxyesters described above, to yield the mixed diester 3.2. 3.1, the corresponding products 3.2 are obtained. The procedure is illustrated in Scheme 3, Example 3. In this The conversion of a phosphonate monoester 1.1 into a sequence, a monophenylphosphonate 3.9 is reacted, in aceto mixed diester 3.2 is also accomplished by means of a Mit nitrile solution at 70° C., with ten equivalents of thionyl Sonobu coupling reaction with the hydroxyester 3.1, as 50 chloride, so as to produce the phosphoryl chloride 3.19. The described in Org. Lett., 2001, 643. In this method, the reac product is then reacted with ethyl 4-carbamoyl-2-hydroxybu tants 1.1 and 3.1 are combined in a polar solvent such as tyrate 3.20 in dichloromethane containing triethylamine, to tetrahydrofuran, in the presence of a triarylphosphine and a give the mixed diester 3.21. dialkyl azodicarboxylate, to give the mixed diester 3.2. The Using the above procedures, but employing, in place of R" substituent is varied by cleavage, using the methods 55 ethyl 4-carbamoyl-2-hydroxybutyrate 3.20, different hydrox described previously, to afford the monoacid product3.3. The yesters 3.1, the corresponding products 3.2 are obtained. product is then coupled, for example using methods described The mixed phosphonate diesters are also obtained by an above, with the hydroxy compound ROH, to give the diester alternative route for incorporation of the RO group into product 3.4. intermediates 3.3 in which the hydroxyester moiety is already The procedure is illustrated in Scheme3, Example 2. In this 60 incorporated. In this procedure, the monoacid intermediate method, a monoallyl phosphonate 3.12 is coupled in tetrahy 3.3 is converted into the activated derivative 3.6 in which LV drofuran Solution, in the presence of triphenylphosphine and is a leaving group Such as chloro, imidazole, and the like, as diethylazodicarboxylate, with ethyl lactate 3.13 to give the previously described. The activated intermediate is then mixed diester 3.14. The product is reacted with tris(triph reacted with the hydroxy compound ROH, in the presence of enylphosphine)rhodium chloride (Wilkinson catalyst) in 65 a base, to yield the mixed diester product 3.4. acetonitrile, as described previously, to remove the allyl The method is illustrated in Scheme 3, Example 4. In this group and produce the monoacid product 3.15. The latter sequence, the phosphonate monoacid 3.22 is reacted with US 8,324,179 B2 129 130 trichloromethanesulfonyl chloride in tetrahydrofuran con diisopropylethylamine, as described in Anal. Chem., 1987, taining collidine, as described in J. Med. Chem., 1995, 38. 59, 1056, or triethylamine, as described in J. Med. Chem. 4648, to produce the trichloromethanesulfonyloxy product 1995, 38, 1372, or in a non-polar solvent such as benzene, in 3.23. This compound is reacted with 3-(morpholinomethyl) the presence of 18-crown-6, as described in Syn. Comm., phenol 3.24 in dichloromethane containing triethylamine, to 5 1995, 25, 3565. yield the mixed diester product 3.25. The method is illustrated in Scheme 3, Example 5. In this Using the above procedures, but employing, in place of procedure, the monoacid 3.26 is reacted with ethyl 2-bromo with 3-(morpholinomethyl)phenol 3.24, different carbinols 3-phenylpropionate 3.27 and diisopropylethylamine in dim ROH, the corresponding products 3.4 are obtained. ethylformamide at 80°C. to afford the mixed diester product The phosphonate esters 3.4 are also obtained by means of 10 3.28. alkylation reactions performed on the monoesters 1.1. The Using the above procedure, but employing, in place of reaction between the monoacid 1.1 and the haloester 3.7 is ethyl 2-bromo-3-phenylpropionate 3.27, different haloesters performed in a polar solvent in the presence of a base Such as 3.7, the corresponding products 3.4 are obtained.

Scheme 3

R-link-P-OR/ O A (R) COR5 3.4 her -COORS 3.7

- P- 5 R-link-P-OR/ HO-R-COOR- R-link-P-OR/ -- R-link-P-OH/ -- R-link-P-OR/ OH O-R-COOR O-R-COOR O-R-COOR 1.1 3.2 3.3 3.4

O O Rink-14-OR/ Rink M is Lw O-R-COOR 3.5 3.6 Scheme 3 Example 1 O M R-link-P-OPh O / HOCH2CH(Me)COE2 e)CO2Et O R-link-P-OP - -e- 3.10 OH COEt Me 3.9 3.11

Scheme 3 Example 2 OH O O N MO R-link-P-OA R-link-P-OHA 2 R-link-P-O HOCH(Me)COEt N V -- O -- O OH 3.13 \ 3.16 \ Me Me COEt COEt 3.12 3.14 3.15 US 8,324,179 B2

-continued O

R-link-link-- 4. O p || N Me 4. COEt 3.17 Scheme 3 Example 3 O M O O EtOCCH(OH)CH2CH2CONH2 R-link-P-OPh M SOCl. M 3.20 O R-link-P-OPh Hibs R-link-P-OPh Ho 3.18 O OH C COEt HN 3.9 3.19 3.21 Scheme 3 Example 4 HO MO MO N MO R-link-P-OH R-link-P-OSOCC1 O Rikro N O Ho- O O ~ 3.24 O Me Me Me COEt COEt COEt 3.22 3.23 3.25 Scheme 3 Example 5 O O R-link-P-OHM —BrCH(Bn)COEt - R-link-P-OCH(Bn)COEtM OCH2CF OCH2CF 3.26 3.28

Scheme 4 illustrates methods for the preparation of phos 40 Using the above procedure, but employing, in place of phonate diesters in which both the ester substituents incorpo ethyl 3-bromo-2-methylpropionate 4.7, different haloesters rate carboalkoxy groups. 4.1, the corresponding products 4.3 are obtained. The compounds are prepared directly or indirectly from the The diesters 4.3 are also obtained by displacement reac phosphonic acids 1.6. In one alternative, the phosphonic acid tions of activated derivatives 1.7 of the phosphonic acid with is coupled with the hydroxyester 4.2, using the conditions 45 the hydroxyesters 4.2. The displacement reaction is per described previously in Schemes 1-3. Such as coupling reac formed in a polar solvent in the presence of a Suitable base, as tions using dicyclohexylcarbodiimide or similar reagents, or described in Scheme 3. The displacement reaction is per under the conditions of the Mitsonobu reaction, to afford the formed in the presence of an excess of the hydroxyester, to diester product 4.3 in which the ester substituents are identi afford the diester product 4.3 in which the ester substituents cal. 50 are identical, or sequentially with limited amounts of differ This method is illustrated in Scheme 4. Example 1. In this ent hydroxyesters, to prepare diesters 4.3 in which the ester procedure, the phosphonic acid 1.6 is reacted with three molar equivalents of butyl lactate 4.5 in the presence of Ald substituents are different. rithiol-2 and triphenyl phosphine in pyridine at ca. 70° C., to The methods are illustrated in Scheme 4, Examples 3 and 4. afford the diester 4.6. 55 As shown in Example 3, the phosphoryl dichloride 2.22 is Using the above procedure, but employing, in place of reacted with three molar equivalents of ethyl 3-hydroxy-2- butyl lactate 4.5, different hydroxyesters 4.2, the correspond (hydroxymethyl)propionate 4.9 in tetrahydrofuran contain ing products 4.3 are obtained. ing potassium carbonate, to obtain the diester product 4.10. Alternatively, the diesters 4.3 are obtained by alkylation of Using the above procedure, but employing, in place of the phosphonic acid 1.6 with a haloester 4.1. The alkylation 60 ethyl 3-hydroxy-2-(hydroxymethyl)propionate 4.9, different reaction is performed as described in Scheme 3 for the prepa hydroxyesters 4.2, the corresponding products 4.3 are ration of the esters 3.4. obtained. This method is illustrated in Scheme 4. Example 2. In this Scheme 4. Example 4 depicts the displacement reaction procedure, the phosphonic acid 1.6 is reacted with excess between equimolar amounts of the phosphoryl dichloride ethyl 3-bromo-2-methylpropionate 4.7 and diisopropylethy 65 2.22 and ethyl 2-methyl-3-hydroxypropionate 4.11, to yield lamine in dimethylformamide at ca. 80° C., as described in the monoester product 4.12. The reaction is conducted in Anal. Chem., 1987, 59, 1056, to produce the diester 4.8. acetonitrile at 70° C. in the presence of diisopropylethy US 8,324,179 B2 133 134 lamine. The product 4.12 is then reacted, under the same -continued conditions, with one molar equivalent of ethyl lactate 4.13, to Scheme 4 Example 4 give the diester product 4.14. O Using the above procedures, but employing, in place of M HOCH2CH(CH)COEt R-link-P-C - He Ho ethyl 2-methyl-3-hydroxypropionate 4.11 and ethyl lactate 4.11 4.13, sequential reactions with different hydroxyesters 4.2, C the corresponding products 4.3 are obtained. 2.22 O 10 M HOCH(CH3)COEt Scheme 4 R-link-P-OCHCHCH3CO.Et - I - O O C / M R-link-P-OH Her R-link-P-Ly 4.12 O O(R)COR O(R)COR 15 M 4.5 4.4 R-link-P-OCHCHCCH)COEt OCH(CH)COEt 1. N . 4.14 2O O HO(R)COR O M 4.2 M One skilled in the art will recognize that nucleobases can R-link-P-OH R R-link-P-O(R)COR exist in tautomeric forms. For example, structures (a) and (b) Ha(R)COR OH O(R)COR can have equivalent tautomeric forms as shown below: 1.6 4.1 4.3 25 (a) 4, t OH O O O N n N M M 30 M N M NH R-link-P-OH - - R-link-P-Ly 2 FK re- 2 FK 8. OH O(R)COR {N 2 N 2 1.7 4.4 Scheme 4 Example 1 35

O O M HOCH(CH3)COBu A R-link-P-OH — - R-link-P-OCHCH3CO-Bu (b) OH OCH(CH3)COBu OH O 40 1.6 4.6 2 2 Scheme 4 Example 2 / s NN s / s NH. N 2 D2 F N 2 D2 F / BrCH-CH(CH3)COEt Rink-4 on P-3892P-4.7 45 OH All possible tautomeric forms of the nucleobases of all of the 1.6 embodiments are within the scope of the invention. O A 50 The compounds of Formula I-IV include all stereoisomers, R-link-P - OCH2CH(CH3)COEt and mixtures thereof. For example and not by way of limita OCH2CH(CH3)COEt tion, the compounds of Formula I include at least the follow 4.8 ing stereoisomers: Scheme 4 Example 3

55 O M (HOCH2)2CHCOEt R-link-P-Cl He 4.9 C 2.22 60 O M R-link-P-OCHCH(CHOH)COEt OCH2CH(CHOH)COEt 4.10 65 US 8,324,179 B2 135 136 -continued X1.X2.X3.L.B. Thus, for example, X1 a.X2c.)x3a.L1.B1 rep resents the following structure: w B

10 amino H

15 TABLE 1

X1 Structures

Code Structure

N ethylenyl H 25 ethynyl

TABLE 2 30

X2 Structures

Code Structure 35

H OH F 40

TABLE 3 In still another embodiment, the compounds of Formula I, Formula II, Formula III or Formula IV are named below in 45 X3 Structures tabular format (Table 6) as compounds of general Formula V: Code Structure Formula V 50 H OH F

55 TABLE 4 wherein X1,X2, X3, and L represent substituents attached to the tetrahydrofuranyl ring as defined in Tables 1-4, below: B L. Structures is a purine defined in Table 5, below; and each W and Ware 60 as previously defined above. Code Structure The point of attachment of the core structure C is indicated in each of the structures of X1, X2, X3, L and B. Each H structure in Tables 1-5 is represented by an alphanumeric CH “code'. Each structure of a compound of Formula V can thus 65 - CH-OH be designated in tabular form by combining the “code” rep resenting each structural moiety using the following syntax:

US 8,324,179 B2 139 140 TABLE 6-continued List of Compounds of Formula V

B4, X B1, X B1, X B1, X B1, X B1, X B1, X B1, X B1, X B1, X B1, X B1, X B1, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X B2, X

30 Phosphonate Embodiments of Compounds of Formula I-IV TABLE 1.1-continued By way of example and not limitation, the phosphonate embodiments of Formula I-IV may be represented by the general formula “MBF': 35

40

Each embodiment of MBF is depicted as a substituted 4s nucleus (Sc). Sc is described in formulae A-G of Table 1.1 below, wherein Sc is a generic formula for a compound of Formula I, Formula II, Formula III or Formula IV and the point of attachment to P(O)Pd*Pd is indicated with a wavy line. 50

TABLE 1.1

55

60

65 US 8,324,179 B2 141 142 TABLE 1.1-continued R is R', R, R or R, provided that when R is bound to a heteroatom, then R is R or R' R" is F, Cl, Br, I, CN, N or - NO; R is Y: R is R', N(R)(R), SR', S(O)R’, S(O),R, S(O)(OR), S(O)(OR), OC(Y)R, OC(Y) OR', OC(Y)(N(R)(R)), SC(Y)R', SCCY) OR', SC(Y)(N(R)(R)), N(R)C(Y)R, N(R) 10 C(Y)OR', or N(R)C(Y)(N(R)(R)); Riis C(Y)R’, C(Y)OR or C(Y)(N(R)(R)); R" is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms, or alkynyl of 2 to 18 carbon atoms; R is R' wherein each R is substituted with 0 to 3 R' 15 groups: R" is independently alkylene of 1 to 18 carbon atoms, alkenylene of 2 to 18 carbon atoms, or alkynylene of 2-18 carbon atoms any one of which alkylene, alk enylene or alkynylene is substituted with 0-3 R groups: W is W or W5; W is R., C(Y)R, C(Y)W, SOR, or SOW: W is carbocycle or heterocycle wherein W is indepen 25 dently substituted with 0 to 3 R' groups;

TABLE 2.0.1 30 Nr's O 1 H . S OH 35 -N-r's OH O F 2

O 40 21 unrise O H - NH . 3

45 OS N/ \ N Ns/ H % -N-r's OH O 4 G 50 ->r O NH Pd' and Pd are each independently selected from species O in Tables 20.1 to 20.37. The definition of the variables used in Tables 20.1-20.37 (e.g., W. R', etc.) pertain 5 only to Tables 20.1-20.37, unless otherwise indicated. 55 Additional phosphonate groups are disclosed in U.S. O patent publication No. 2004/100960, the entirety of ->r NCH, which is incorporated herein by reference. O The variables used in Tables 20.1 to 20.37 have the follow 6 ing definitions and pertain only to those tables, unless other 60 wise indicated: R" is independently H or alkyl of 1 to 18 carbon atoms; R’ is independently H, R, R or R' wherein each R is unrs-" independently substituted with 0 to 3 R groups or taken O together at a carbon atom, two R groups form a ring of 65 7 3 to 8 carbons and the ring may be substituted with 0 to 3 R groups; US 8,324,179 B2 143 144 TABLE 20.1-continued TABLE 20.3-continued CH O ->r N-1\ch, 5 SN O O NH O 8 O 16

10 CH3 TABLE 20.2 O -Yo NCH, c r On 1 N-CH3 15 17 O 9 2O ->--" c r O N CH3 18 O CH3 CH 10 25 O CH -Yo N1\ch, O

c O O CH 30 19

O 11 TABLE 20.4

35 CH3 TABLE 20.3 un-N-" CH O -Yo O Yw3 40 2O O CH 12 SN O CH 45 -Yo N O CH SN O NR5 21 CH CH O 50 O -N 13 -Yo CH O 22

14 TABLE 20.5 H3C CH3 60

SN O On RI SN O On W3

O 15 65 23 US 8,324,179 B2 145 146 TABLE 20.5-continued TABLE 20.6

H3C -Yo O NR5 -Yo ON1-N-CH3 O 24 31 10

H3C

O 15 -Yo NR4 arO CH3 O 32 25 H3C NullsCH3 O -Yo CH3 -Yo NR O 25 O 33 26

HC TABLE 20.7 30

O -Yo N O O -Yo Yw3 27 35

H3C -Yo O NCH, 40 O 28 45

H3C 50 No O N1 CH3

O 29 55

HC 60 No O N1\ch,

65 US 8,324,179 B2 147 148 TABLE 20.7-continued TABLE 20.9-continued

10

15

O 41

25

TABLE 20.8 R4 R4 30 c. O Yw3 c O NR5 O O 42 43 35 R4 R4 c O NR4 c. O NR O O 40 44 45 R1 RI c. O Yw3 c O NR5 45 O O 46 47 RI RI 50

O O c NR4 c - NR TABLE 2010 O O 55 48 49 cr O N-1N1'''CH H O TABLE 20.9 58 60 c 1 O N CH3 H O CH3 65 59 US 8,324,179 B2 149 150 TABLE 20.10-continued TABLE 2.0.11-continued

CH c O N-1\ch, H O 68 10

TABLE 2011 TABLE 2012

CH3 15 CH3 c On 1 N-CH3

H O 61 69 CH3 CH3 25 c 1.r CH 62 h O CH 70

CH3 30 CH CH

& NullsCH 63 35 H O 71 CH3

40 TABLE 2013

45

72

50

O c NCH, 55 73

60

65 74

US 8,324,179 B2 153 154 TABLE 20.16-continued TABLE 2.0.17-continued

RI R1 ch O Yw3 c. O NR5 5 cro H O H O 106 95 96 10

R R TABLE 2.0.18 O O c- YR4 c- NR 15 c ON1-CH3 H O H O 1 97 98 R3 O

107 2O TABLE 20.17 O CH3

O a. c N W3 R3r’s O CH3 25 R3 O 108 99 CH cr O n R5 30 c s O ~sCH R3 O R3 O 100 109

O c N NR4 35 TABLE 20.19 R3 O CH3 101 40 c O

O c N NR R3 O Sw 110 R3 O 45 102 CH3 c On c O NR5 N H 50 R3 O R3 O 111 103 CH3 O cr O n CH 55 c n R 4 R3 O R3 O 104 112 60 CH

65 US 8,324,179 B2 155 TABLE 2.0.19-continued TABLE 2.0.21-continued

HC c O NR5 R3 O 122 10

HC

O 15 c YR4 R3 O 123

HC c O NR 25 R3 O 124

HC 30 O c n H TABLE 20.2O R3 O 125 CH3 35

( N On 1 N-CH3

R3 O HC 118 40 CH3 c O YCH, c N O CH R3 O 45 126 R3 O CH 119

CH3 CH3 50 H3C c N --- CH3 O c O n-1 CH"3 120 55 R3 O 127 TABLE 20.21 H3C HC 60

O n 9N-1N c W3 CH3 R3 O R3 O 121 65 US 8,324,179 B2 157 158 TABLE 20.22 TABLE 20.24

129 10 R4 R4 O 3. O CH3 15 c R3 O is R3 O is R3 O CH3 142 143 130 RI R1 2O & O Yw3 c O NR5 R3 O R3 O 25 144 145

RI R1 131

TABLE 20.23

35 ? O a W3 ( r O NR5 TABLE 20.25 40 c. c. c. 149 150

- O NR4 c O NR 45 c., c. 151 152 153 c - R5 Q o1 R4 50 154 155 156 c O n W3 c O NR5 R1 1 H 3. R3 O c- o1 55 157 158 159

TABLE 20.26 O c O 60 NR4 NR W3 N1

65 160 US 8,324,179 B2 159 160 TABLE 2.0.26-continued TABLE 2.0.28-continued

O

Q O 1. R O CH CH3 CH 182 10 O CH3 &- No CH3 15 183

O &- RaNo

184

25 TABLE 20.27 O O &- RaNo JuC c- RaNo us W3 < o1 rulNo R5 30 185 1 72 1 73

O O TABLE 20.29 35 c- RaNo ls R4 Q o1 rulNo RI O O 1 74 1 75 c --- &-s-s O O 186 187 40 O O c - rullNo H &- rullsn O CH3 1 76 177 c ---. c ---, O O CH 188 189 45 &- RaNo 1such a o1 RaNo ulu O O 178 179 &-s-s c --- 50 190 191

TABLE 20.28 O O CH3 H3C &--- CH3 Q 1No lu 55 &- RaNo J 192 193 18O TABLE 20.30 O 60 H3C O O CH &- J 181 65 194 US 8,324,179 B2 161 162 TABLE 20.30-continued TABLE 20.31-continued

O c --- CH 3 O CH &- No o1 195 205

O 10 O &- CH 3 CH3 &- rullsNo o1 Nchi, CH3 15 2O6 196

O CH3 H3C &- CH3 rullsO D 197 &- No O

O O 25 TABLE 20.32

198 30 &- rullsNo O O rCH3 208

O CH3 35 &- rullsNo O CH3 209 TABLE 20.31 O CH3 O 40 Ra &- RaNo ls o1 W3 &-s-s-ko1 No O CH3 210 200 45 O O &- RaNo ls O1 R5 &- *No - r> -t; 2O1 CH 50 211 O &- RaNo lso1 R4 2O2 55 &- rusNo O O O O 212 Ra ls R1 &- No o1 O 60 2O3 &- RaNo ls O O Q o1 RaNo ls O1 H 65 213 204 US 8,324,179 B2 163 164 TABLE 20.33 TABLE 20.34-continued

O O CH3

&-O O usO -W 5 Q 1No usO k"CH3

O 10 O

3. 1N ls -R Q ls CH 3 O O O 1No r 215 CH O 15 225 &- ls O1 R4 216 2O &-s-sUC O &- ulo1 226 217 2 - 1 O &- O us H &- O1 30 227 218

O < us CH TABLE 20.35 1No o1 3 35 Ra O 219 &- Yw3

O O 228 &-s-s1No o1 Nchi, 40 220 &- Ra r O NR5 H3C 45 O

&- l OD 229S. -R ON 221 50 O R4

O 230 TABLE 20.34 55 Ra O O r o1 NR &- lsO O 222 60

O CH 3. Ria O n &-s-s-s1No O CH Sw 223 65 232 US 8,324,179 B2 165 166 TABLE 20.35-continued TABLE 20.37-continued

&-s-srise 5 V* O N 3. O H O 2 HR s 233 246 247 10

3. N1 Ra On R4 Pd" and Pd 2 of the “Sc structures of Table 1.1 can also be independently selected from Table 30.1, below: H O 234 15 TABLE 30.1

3. Ra O CH3 Y N O CH 2O H O R ch n1": H O 235 67

25 CH TABLE 20.36 3. O N1\ch, c Ra O Sw 3. Ra riseO H O R3 O R3 O 236 237 CH3 35 & O n1n 13CH 3. Y Ra On 4 3. Y Ra On rise Crs, H. O. R3 O R3 O 69 238 239 40 H3C 21 21 N * O-- NX 2 a---O NX 5 45 On-CH R R H O 240 241 78

& N X. c O O N1\ch, 242 243 H O 55

TABLE 20.37 HC

60 & N & N N On 1 N-CH3 --R2 --Rs

244 245 US 8,324,179 B2 167 168 TABLE 30.1-continued TABLE 30.1-continued

c -----"O CH 5 o H O 10 253 248

15 c O O 254 c O N1\ch, 2O H O c -n, 249 255 25 c. 256 c N ON1-CH3 30 c - H O 257 250 Combinations of “Sc” and Pd' and Pd independently 35 selected from table 30.1 can be expressed in the form of CH3 Sc.Pd'.Pd, where Sc is represented by the respective letter A-G from Table 1.1 and Pd' and Pd are represented by the N respective number from Table 30.1. Thus, A.256.256 repre &-N- sents the following compound: H O 40 251 OH H3C NS1SN 45 P H O (N els2 &-N-N HO1 21 N NH.2 H O HO H a 252 50 OH

TABLE 7 List of Compounds of MBF A.254.67, A.254.68, A.254.69, A.254.78, A.254.79, A.254.80, A.254.248, A.254.249, A.254.250, A.254.251, A.254.252, A.254.253, B.254.67, B.254.68, B.254.69, B.254.78, B.254.79, B.254.80, B.254.248, B.254.249, B.254.250, B.254.251, B.254.252, B.254.253, C.254.67, C.254.68, C.254.69, C.254.78, C.254.79, C.254.80, C.254.248, C.254.249, C.254.250, C.254.251, C.254.252, C.254.253, D.254.67, D.254.68, D.254.69, D.254.78, D.254.79, D.254.80, D.254.248, D.254.249, D.254.250, D.254.251, D.254.252, D.254.253, E.254.67, E.254.68, E.254.69, E.254.78, E.254.79, E.254.80, E.254.248, E.254.249, E.254.250, E.254.251, E.254.252, E.254.253, F.254.67, F.254.68, F.254.69, F.254.78, F.254.79, F.254.80, F.254.248, F.254.249, F.254.250, F.254.251, F.254.252, F.254.253, G.254.67, G.254.68, G.254.69, G.254.78, G.254.79, G.254.80, G.254.248, G.254.249, G.254.250, G.254.251, G.254.252, G.254.253, A.255.67, A.255.68, A.255.69, A.255.78, A.255.79, A.255.80, A.255.248, US 8,324,179 B2 169 170 TABLE 7-continued List of Compounds of MBF A.255.249, A.255.250, A.255.251, A.255.252, A.255.253, B.255.67, B.255.68, B.255.69, B.255.78, B.255.79, B.255.80, B.255.248, B.255.249, B.255.250, B.255.251, B.255.252, B.255.253, C.255.67, C.255.68, C.255.69, C.255.78, C.255.79, C.255.80, C.255.248, C.255.249, C.255.250, C.255.251, C.255.252, C.255.253, D.255.67, D.255.68, D.255.69, D.255.78, D.255.79, D.255.80, D.255.248, D.255.249, D.255.250, D.255.251, D.255.252, D.255.253, E.255.67, E.255.68, E.255.69, E.255.78, E.255.79, E.255.80, E.255.248, E.255.249, E.255.250, E.255.251, E.255.252, E.255.253, F.255.67, F.255.68, F.255.69, F.255.78, F.255.79, F.255.80, F.255.248, F.255.249, F.255.250, F.255.251, F.255.252, F.255.253, G.255.67, G.255.68, G.255.69, G.255.78, G.255.79, G.255.80, G.255.248, G.255.249, G.255.250, G.255.251, G.255.252, G.255.253, A.67.67, A.68.68, A.69.69, A.78.78, A.79.79, A.80.80, A.248.248, A.249.249, A.250.250, A.251.251, A252.252, A.253.253, B.67.67, B.68.68, B.69.69, B.78.78, B.79.79, B.80.80, B.248.248, B.249.249, B.250.250, B.251.251, B252.252, B.253.253, C.67.67, C.68.68, C.69.69, C.78.78, C.79.79, C.80.80, C.248.248, C.249.249, C.250.250, C.251.251, C252.252, C.253.253, D.67.67, D.68.68, D.69.69, D. 78.78, D.79.79, D.80.80, D.248.248, D.249.249, D.250.250, D.251.251, D252.252, D.253.253, E.67.67, E.68.68, E.69.69, E.78.78, E.79.79, E.80.80, E.248.248, E.249.249, E.250.250, E.251.251, E252.252, E.253.253, F.67.67, F.68.68, F.69.69, F.78.78, F.79.79, F.80.80, F.248.248, F.249.249, F.250.250, F.251.251, F252.252, F.253.253, G.67.67, G.68.68, G.69.69, G.78.78, G.79.79, G.80.80, G.248.248, G.249.249, G.250.250, G.251.251, G252.252, G.253.253, A.256.257, B.256.257, C.256.257, D.256.257, E.256.257, F.256.257, G.256.257.

EXAMPLES TABLE 1-continued 25

Certain abbreviations and acronyms are used in describing List of abbreviations and acronyms. the experimental details. Although most of these would be understood by one skilled in the art, Table 1 contains a list of Abbreviatireviation Meaning many of these abbreviations and acronyms. 30

TABLE 1. TEAB triethylammonium bicarbonate TFA trifluoroacetic acid List of abbreviations and acronyms. TLC or tic thin layer chromatography Abbreviation Meaning 35 8 parts per million down field from tetramethylsilane AIBN 2,2'-azobis(2-methylpropionitrile) BnBr benzylbromide BSA bis(trimethylsilyl)acetamide BZC. benzoyl chloride CDI carbonyl diimidazole 40 Example 1 DBU 1,5-diazabicyclo[5.4.0]undecene-5 DCA dichloroacetamide DCC dicyclohexylcarbodiimide DMAP 4-dimethylaminopyridine DME 1,2-dimethoxyethane DMTC dimethoxytrityl chloride 45 DMSO dimethylsulfoxide DMF dimethylformamide EtOAC ethyl acetate DMP ESI electrospray ionization N N acetOne HMDS hexamethyldisilaZane HO CHSOH HPLC High pressure liquid chromatography 50 LDA lithium diisopropylamide S LRMS low resolution mass spectrum HG mCPBA meta-chloroperbenzoic acid MeOH methanol MMTC mono methoxytrityl chloride mz or me mass to charge ratio 55 MH mass plus 1 MHT mass minus 1 MSOH methanesulfonic acid MS or ms mass spectrum 1) TMSCI rt or rt. room temperature HO TBAF tetrabutylammonium fluoride 60 TMSC chlorotrimethylsilane TMSBr bromotrimethylsilane TMS iodotrimethylsilane TEA triethylamine TBA tributylamine TBAP tributylammonium pyrophosphate 65 TBSC t-butyldimethylsilyl chloride US 8,324,179 B2 172 -continued -continued NHBZ NH2 N nN. N n O N N 2 5 ( HO N 1) DCC P P P O NN 2 2) diethyl(triphenylphosp HO1 No1 No1 N s horanylidene)methylphosphinate OH OH OH Ö O X 10 6.8 6.3

NHBZ 15 6-(7-Amino-1.2.3 triazolo 4,5-dipyrimidin-3-yl)-2, 2-dimethyl-tetrahydro-furo3,4-d1.3dioxol-4-yl)-

N n N methanol (6.2) ( Compound 6.1 (2g, 7.46 mmol) was dissolved in 83 mL 1 O NN e% TFA/HO 2O EtO1 Her acetone, treated with 2,2-dimethoxypropane 50 mL and OEt CHSOH (0.5 mL, 7.46 mmol). The mixture was stirred for 3 hand then concentrated under reduced pressure. The resi due was partitioned between CHC1 and saturated NaHCO. d 6 The organic layer was separated, dried over MgSO filtered and concentrated under reduced pressure. The residue was X 25 Subjected to a silica gel column chromatography eluting with 6.4 0-5% MeOH in CHCl to yield compound 6.2 (2.2g,96% yield). HNMR (300 MHz, CDC1) & 1.41 (s, 3H), 1.69 (s. 3H), 3.77-4.00 (m, 2H), 4.61 (s, 1H), 5.12 (d. 1H, J=6), 5.25 NHBZ 30 (t, 1H, J=5.4), 6.57 (d. 1H, J–4.5), 8.46 (s, 1H). LRMS M-H CHNO requires 307.1, Found 307.1. N n O M N N-3-(6-Hydroxymethyl-2,2-dimethyl-tetrahydro | N TMSBr N 2 Her furo3,4-d1.3dioxol-4-yl)-3H-1.2.3 triazolo 4.5- 1. O N N 2,6-lutidine 35 dpyrimidin-7-yl-benzamide (6.3) EtO1 OEt Compound 6.2 (2.2g, 7.14 mmol) was dissolved in 30 mL HG OH pyridine and cooled to 0°C. To this was added TMSC1 (2.7 mL, 21.4 mmol). The mixture was stirred at 0°C. for 30 min 6.5 40 and then treated with BZC1 (4.95 mL, 35.7 mmol). The mix ture was stirred at 0°C. for 3 hand then treated with NH-OH (7.3 mL), stirred at 0°C. for another 30 min. The mixture was NHBZ concentrated under reduced pressure and the residue was partitioned between CH2Cl and 1NHC1. The water layer was 45 N n extracted three times with CHC1. The organic layer was O M N combined, dried over MgSO, filtered and concentrated N e NH4OH under reduced pressure. The residue was subjected to a silica 1. O N % gel column chromatography eluting with 80% EtOAc in Hex HO1 ane to yield compound 6.3 (1.84g, 63% yield). HNMR (300 OH 50 MHz, CDC1) & 1.42 (s.3H), 1.70 (s.3H), 3.79-401 (m, 2H), 4.63 (s, 1H), 5.14-5.16 (m, 1H), 5.31-5.35 (m. 1H), 6.68 (d. HG OH 1H, J=4.2), 7.55-7.61 (m, 2H), 7.61-7.69 (m, 1H), 8.08 (d. 6.6 2H, J–72), 8.90 (s, 1H). LRMS (ESI) MH CHNO requires 413.2. Found 412.9. 55 {2-6-(7-Benzoylamino-1,2,3triazolo 4,5-dipyrimi NH2 din-3-yl)-2,2-dimethyl-tetrahydro-furo3,4-d1.3 N n dioxol-4-yl)-vinyl-phosphonic acid diethyl ester M N (6.4) N 60 N 2 Hess N Compound 6.3 (1.2g, 2.91 mmol) was dissolved in 23 mL DMSO, and treated with DCC (3.6 g., 17.5 mmol) and DCA (0.24 mL, 2.91 mmol). The mixture was stirred for 2 h and then treated with 0.3 mL pyridine. Diethyl(triphenylphospho 65 ranylidene)methylphosphonate (2.9 g, 6.98 mmol) was dis solved in 5 mL CH2Cl and it was added to the mixture dropwise and the mixture was stirred for 3 h. The mixture was US 8,324,179 B2 173 174 concentrated under reduced pressure and the residue was 1H, J=4.2), 6.40-6.54 (m, 1H), 8.36 (s, 1H). 'P NMR: 4.39, dissolved in EtOAc. The organic layer was washed with HO 4.19, -10.62, -10.79, -23.31 ppm. three times, dried over MgSO filtered and concentrated down under reduced pressure. The residue was subjected to a Example 2 silica gel column chromatography eluting with 100% EtOAc to 5% MeOH in EtOAc to give compound 6.4 (469 mg, 30% yield). "HNMR (300 MHz, CDOD) & 1.17-1.35 (m, 6H), 1.46 (s, 3H), 1.64 (s.3H), 3.85-3.97 (m, 4H), 4.04-4.12 (m, NHBZ 1H), 5.01 (m, 1H), 5.33-5.45 (m, 2H), 5.81 (d. 1H, J=5.7), 10 6.63-6.77 (m. 1H), 7.57-8.12 (m, 5H), 8.87 (s, 1H). LRMS N (ESI) MHCHNOP requires 545.2, Found 545.0. {2-5-(7-Benzoylamino-1,2,3triazolo 4,5-dipyrimi O (1.N e 1) Dess-Martin din-3-yl)-3,4-dihydroxy-tetrahydro-furan-2-yl)-vi -ny- 2) NaH nyl-phosphonic acid diethyl ester (6.5) 15 s OEt Compound 6.4 (170 mg, 0.31 mmol) was dissolved in 90% d 6 EtO1-N-K TFA in HO (10 mL). The mixture was stirred for 30 min and EtO OEt concentrated down under reduced pressure. The residue was F Subjected to a silica gel column chromatography eluting with 7.1 7.2 10% MeOH in CHCl to yield compound 6.5 (110 mg, 70% yield). "HNMR (300 MHz, CDOD) & 1.24-1.28 (m, 6H), NHBZ 3.96-4.06 (m, 4H), 4.63 (t, 1H, J=5.1), 4.73 (s, 1H), 4.97 (t, N 1H, J–4.2), 5.92-6.04 (m, 1H), 6.5 (d. 1H, J=3.9), 6.81-6.96 n N (m. 1H), 7.55-8.12 (m, 5H), 8.77 (s, 1H). LRMS (ESI) MH" 25 O ( EtO-PM O N N 2 TFAfwater CHNOP requires 505.2. Found 504.9. / He {2-5-(7-Benzoylamino-1,2,3triazolo 4,5-dipyrimi EtO din-3-yl)-3,4-dihydroxy-tetrahydro-furan-2-yl)-vi F nyl-phosphonic acid (6.6) 30 Compound 6.6 (190 mg 0.38 mmol) was synthesized X using the procedure described for the preparation of com pound 1.10 (150 mg, 88% yield). HNMR (300 MHz, DO) & 7.3 4.52-4.55 (m. 1H), 5.03-5.06 (m, 1H), 5.79-5.91 (m, 1H), 35 NHBZ 6.26-6.40 (m, 1H), 6.44 (d. 1H, J=3.9), 743-7.91 (m, 5H), N n N 8.76 (s, 1H). LRMS (ESI) MH" C.H.N.O.P requires O / 449.1. Found 449.0. M ! TMSBr EtO-P O N 4. 2,6-lutidine {2-5-(7-Amino-1,2,3triazolo 4,5-dipyrimidin-3- 40 / yl)-3,4-dihydroxy-tetrahydro-furan-2-yl-ethyl EtO phosphonic acid (6.7) F HG OH Compound 6.6 (50 mg, 0.11 mmol) was dissolved in 15 mL 7.4 45 NHOH and stirred under N2 for overnight. The mixture was NHBZ concentrated under reduced pressure. The residue was Sub jected to reverse phase HPLC eluting with 0-25% CHCN in water to yield product 6.7 (34 mg. 89% yield). HNMR (300 N N1SN MHz, DO) & 4.47-4.51 (m. 1H), 4.59–4.61 (m, 1H), 4.96 / ( NH4OH 50 HO-P O N 4. 4.99 (m. 1H), 5.85 (t, 1H, J–17.1), 6.22-6.37 (m, 2H), 8.20 (s, / 1H). PNMR: 10.98 ppm. LRMS (ESI) MHCHNOP HO requires 345.1. Found 345.8. F Diphosphophosphonate 6.8 H6 OH 55 7.5 Compound 6.7 (15 mg, 0.0436 mmol) was dissolved in 2 NH2 mL anhydrous DMF and treated with nBuN (21 uI, 0.0872 mmol) and CDI (71 mg, 0.436 mmol). The mixture was N n N stirred for 20 min and then treated with MeOH (16 uL, 0.392 mmol), stirred for another 30 min. To this mixture was added 60 /O ( 2 -> 1 mL DMF solution of tetrabutylammonium pyrophosphate (250 mg, 0.436 mmol), stirred for 2 hand concentrated down under reduced pressure. The residue was subjected to ion exchange reverse phase HPLC eluting with 0-40% TEAB in water to yield product (7.9 mg, 36% yield). HNMR (300 65 MHz, DO) & 1.10-1.16 (m, 54H), 3.02-3.09 (m, 36H), 4.53 (t, 1H, J=5.1), 4.99 (t, 1H, J=4.8), 5.98 (t, 1H, J=17.7), 6.32 (d. US 8,324,179 B2 175 176 -continued preparation for compound 6.5. "HNMR (300 MHz, CDCN) NH2 Ö 1.21-1.38 (m, 6H), 4.06-4.24 (m, 4H), 4.47 (s, 1H), 4.92 (s. 1H), 5.00 (s, 1H), 6.08 (d. 1H, J–4.8), 6.31-6.47 (m. 1H), O O N NN 7.57-8.06 (m, 5H), 8.31 (s, 1H), 8.72 (s, 1H). {2-5-(6-Benzoylamino-purin-9-yl)-3,4-dihydroxy HO1 No1-s n / {l 2 tetrahydro-furan-2-yl)-1-fluoro-vinyl-phosphonic OH OH acid (7.5) Compound 7.5 (57 mg, 91% yield) was synthesized from 10 compound 7.4 (70 mg) using the procedure described for the preparation for compound 1.10. "HNMR (300 MHz, DO) & 4.44 (t, 1H, J=4.8), 4.91 (t, 1H, J=4.8), 5.10-5.15 (m, 1H), {2-6-(6-Benzoylamino-purin-9-yl)-2,2-dimethyl 5.67-5.85 (m, 1H), 6.23 (d. 1H, J=5.1), 7.55-8.01 (m, 5H), tetrahydro-furo3,4-d1.3dioxol-4-yl)-1-fluoro 15 8.61 (s, 1H), 8.67 (s, 1H). LRMS (ESI) MHCHFNOP vinyl-phosphonic acid diethyl ester (7.3) requires 466.1. Found 465.8. {2-5-(6-Amino-purin-9-yl)-3,4-dihydroxy-tetrahy Compound 7.1 (500 mg, 1.22 mmol) was dissolved in 5 mL dro-furan-2-yl)-1-fluoro-vinyl-phosphonic acid anhydrous CHCl and treated with Dess-Martin reagent (670 (7.6) mg, 1.59 mmol). The mixture was stirred for 2 h, filtered through a syringe filter, and concentrated down under reduced Compound 7.5 (50 mg 0.11 mmol) was dissolved in 5 mL pressure. The residue was dissolved in EtOAc, filtered, con centrated down under reduced pressure and then dried on high NH-OH and stirred under N for overnight. The mixture was vacuum. 60% NaH (150 mg, 3.66 mmol) was dissolved in 5 concentrated under reduced pressure and the residue was mL anhydrous THF at 0°C. and treated with compound 7.2 25 subjected to reverse phase HPLC eluting with 0-25% CHCN (1.8 g., 3.66 mmol). The mixture was stirred for 30 minatrt to in water to give compound 7.6 (22 mg, 57% yield). "HNMR generate the anion. The generated anion was then added to the (300 MHz, DO) & 4.24-4.27 (m. 1H), 4.69-4.71 (m, 1H), aldehyde formed in the first step, and the mixture was stirred 4.96 (m. 1H), 5.54-5.68 (m, 1H), 5.97 (d. 1H, J=4.8), 8.11 (s, for 2 h. Water was added to quench the reaction, and the 1H), 8.21 (s, 1H). LRMS M-H CHFNOP requires mixture was concentrated under reduced pressure. The resi 30 361.1. Found 361.5. due was subjected to a silica gel column chromatography Diphosphophosphonate 7.7 eluting with 50% EtOAc in Hexane to 100% EtOAc to yield The compound (3.9 mg, 29% yield) was synthesized from compound 7.3 (100 mg, 15% yield). "HNMR (300 MHz, compound 7.6 (9.5 mg) using the procedure described for the CDCN) & 1.22-1.40 (m, 6H), 1.41 (s, 3H), 1.63 (s, 3H), 3.97-4.17 (m, 4H), 5.21-5.29 (m, 2H), 5.64 (d. 1H, J=6), preparation compound 6.8. 5.93-6.12 (m. 1H), 6.30 (s, 1H), 7.55-8.05 (m, 5H), 8.28 (s, 35 'HNMR (300 MHz, DO) & 1.11-1.16 (m, 63H), 3.05-3.22 1H), 8.70 (s, 1H). LRMS (ESI)MHCHFNOP requires (m, 42H), 4.31-4.34 (m. 1H), 4.71-4.74 (m. 1H), 4.98 (s, 1H), 5.78-5.96 (m, 1H), 5.99 (d. 1H, J=5.7), 8.14 (s, 1H), 8.27 (s, 562.2. Found 562.2. 1H). PNMR:-9.31, -9.50, -9.87, -10.04, -10.35, -10.65, {2-5-(6-Benzoylamino-purin-9-yl)-3,4-dihydroxy -10.81, -23.00, -23.56 ppm. LRMS M-H tetrahydro-furan-2-yl)-1-fluoro-vinyl-phosphonic 40 CHFNOP, requires 520.0. Found 519.9. acid diethyl ester (7.4) Compound 7.4 (70 mg, 76% yield) was synthesized from compound 7.3 (100mg) using the procedure described for the Example 3

9.1 9.2

BZO

O S O BuSnH BZO IIIO NaOMe He He S- ABN O°C.-r.t. o

9.3 9.4 US 8,324,179 B2 177 178 -continued O

O 1) DCC, DMSO l O 1 NHCI HO IO ) EtO1 IO () >< 2) BuOK, THF EtO >< 500 C % °o 9.5 EtO1 PIn 1 P YOE 9.6 OEt OEt O Bio-2| O BzCI, Pyridine EtO1 P O OB Z. BSA, SnCl4 EtO 450 C. EtO O OH OBz -- 9.7 9.8 (1. N Nals NHAC

1.8 O 's O

N n N N NH O O {ll --le. {ll EtO171. O N N NHA 2) NaOH HO1/1. O N N NH2 EtO HO OBz OH 9.9 9.10

O O

On-Bu 9.11 9.12

Benzoic acid 6-hydroxy-2,2-dimethyl-tetrahydro compound 9.2 (22.0 g, 71% yield). (Taken from: Johnson, C. furo2,3-d1.3dioxol-5-ylmethyl ester (9.2) R. Bhumralkar, D. R. Nucleosides & Nucleotides 1995, 14, 185.) 1.2-O-Isopropylidene-C-D-xylofuranose (Aldrich, 20 g, Benzoic acid 6-(imidazole-1-carbothioyloxy)-2.2- 100 mmol) in CHCl (300 mL) and TEA (44 mL, 310 mmol) dimethyl-tetrahydro-furoI2,3-d1.3dioxol-5-ylm was cooled to 0°C. The solution was treated with benzoyl 60 chloride (12.8 mL, 110 mmol) in CHCl (10 mL) dropwise ethyl ester (9.3) and stirred for 2h. The solution was diluted with HO (30 mL) Compound 9.2 (3.0 g, 10 mmol) in CHCl (100 mL) was and washed with saturated NaHCO, (2x30 mL). The solution treated with 1,1-thiocarbonyldilimdiazole (3.6 g. 20 mmol) was dried over MgSO, filtered and concentrated in vacuo. 65 and stirred for 2 h. The solution was concentrated in vacuo The residue was treated to flash column chromatography and treated to silica gel column chromatography (SiO2, 4x20 (SiO, 4x20 cm, 0-100% EtOAc-hexanes gradient) to afford cm, 35-65% EtOAc-hexanes gradient) to afford compound US 8,324,179 B2 179 180 9.3 (5.5 g, >100% yield). (Taken from: De Bernardo, S.; and the mixture was stirred at 45° C. for 1 h (significant Tengi, J. P.; Sasso, G.J.; Weigele, M.J. Org. Chem. 1985, 50, exotherm upon BZCl addition). The mixture was cooled to 0° 3457-3462.) C. and EtOH (2 mL) was added dropwise and stirred for 10 minutes. The Solution was concentrated in vacuo and the Benzoic acid 2.2-dimethyl-tetrahydro-furoI2,3-d1, residue was dissolved in EtOAc (100 mL) and washed with 1 3dioxol-5-ylmethyl ester (9.4) N HCl (10 mL) and brine (10 mL). The solution was dried over MgSO, filtered, and concentrated in vacuo. The residue Compound 9.3 (5.5g, 13.6 mmol) in toluene (20 mL) was was treated to silica gel column chromatography (SiO2, 4x20 added dropwise to a refluxing solution of BuSnH (6.7 mL, cm, 50-100% EtOAc-hexanes gradient) to afford compound 24.0 mmol) and AIBN (300 mg, 1.8 mmol) intoluene (80 mL) 10 9.8 (4.2g, 75% yield). under N. The mixture was stirred for 2 h then cooled. The Benzoic acid 2-(2-acetylamino-6-diphenylcarbamoy solution was partitioned between MeCN (300 mL) and hex loxy-purin-9-yl)-5-[2-(diethoxy-phosphoryl)-vinyl anes (3x200 mL). The combined hexanes fractions were extracted with MeCN (200 mL), then the combined MeCN tetrahydro-furan-3-yl ester (9.9) fractions were concentrated in vacuo and the residue was 15 A suspension of N-acetyl-O-(diphenylcarbamoyl)gua treated to silica gel column chromatography (SiO2, 4x20 cm, nine (582 mg, 1.5 mmol) in Mecn (10 mL) was treated with 35-7.5% EtOAc-hexanes gradient) to afford compound 9.4 N.O-bis(trimethylsilyl)acetamide (0.45 mL, 1.84 mmol) and (1.7g, 46% yield). (Taken from: De Bernardo, S.; Tengi, J. P.; heated to 70° C. for 0.5 h. The solution was cooled down to Sasso, G. J.; Weigele, J. Org. Chem. 1985, 50, 3457-3462.) room temperature and compound 9.8 (330 mg. 0.696 mmol) (2,2-Dimethyl-tetrahydro-furo2,3-d1.3dioxol-5- and SnCl4 (2 mL, 1M in CHCl) were added. The mixture yl)-methanol (9.5) was heated to 70° C. for 1.5 h, then cooled to 0° C., and NaHCO (1.5 g) and water (0.5 mL) were added. After stirred Compound 9.4 (11.0 g, 39.6 mmol) in MeOH (100 mL) at at room temperature for 1 h, the mixture was filtered through 0° C. was treated with NaOMe (4.3 M in MeOH, 3 mL. 20 a pad of silica gel and eluted with 50% EtOH in EtOAc. The mmol) and stirred for 30 min. The mixture was warmed to filtrate was concentrated under reduced pressure and the resi ambient temperature and stirred for 1 h. The mixture was 25 due was subjected to chromatography on silica gel (eluted cooled to 0° C. and treated with 1 N HCl (10 mL) then with 5% EtOH in EtOAc) to give compound 9.9 as a colorless concentrated under reduced pressure. The residue was dis foam (128 mg, 24.8% yield). solved in EtOAc (100 mL) and stirred for 30 minutes, dried Benzoic acid 2-(2-acetylamino-6-oxo-1,6-dihydro over MgSO, filtered, and concentrated in vacuo. The residue purin-9-yl)-5-(2-phosphono-vinyl)-tetrahydro-furan was Subjected to a silica gel column chromatography (SiO, 30 3-yl ester (9.10) 4x20 cm, 20-50% EtOAc-hexanes gradient) to afford com pound 9.5 (4.1 g, 60% yield). A suspension of compound 9.9 (128 mg, 0.173 mmol) in MeCN (5 mL) was treated with 2.6-lutidine (0.08 mL, 0.7 2-(2,2-Dimethyl-tetrahydro-furoI2,3-d1.3dioxol mmol) and bromotrimethylsilane (0.3 mL, 2.27 mmol) and 5-yl)-vinyl-phosphonic acid diethyl ester (9.6) 35 stirred at room temperature for 2 h. The mixture was evapo rated and co-evaporated with MeCN. The residue was dis Compound 9.5 (4.0g, 23 mmol) in DMSO (30 mL) was solved in MeOH HO (1:1, 10 mL) and treated with NaOH treated with DCC (7.1 g, 34.5 mmol) and pyridine-TFA (1.1 (1N, 3 mL). The mixture was stirred at room temperature for g, 5.7 mmol) and stirred for 5 h. KCO (780 mg, 5.7 mmol) 16 h then at 45° C. for 1 h, and concentrated under reduced was added and the mixture was stirred for 30 min. The mix 40 pressure. The residue was subjected to reverse phase HPLC ture was treated with a solution of tetraethyl ethylenediphos eluting with 0-25% CHCN in water to give the desired prod phonate (7.3 g, 25.3 mmol) and BuOK (1 M in THF, 26 mL, uct 9.10 (32 mg, 48%). "H NMR (DO) & 2.1 (m. 2H), 4.55 26 mmol) and stirred for 20 min. The mixture was cooled to (m. 1H), 4.8 (m, 1H), 5.7 (d. 1H, J=1.5), 5.9 (m, 1H), 6.1(m, 0° C. and 3 NHCl (6 mL) was added followed by EtOAc (100 1H), 7.7 (s, 1H). PNMR (DO)d 17.17. LRMS (ESI)MH' mL). The organic layer was separated, dried over MgSO, 45 CHNOP requires 344.2. Found 344.0. filtered, and concentrated in vacuo. The residue was subjected Bis-amidate prodrug 9.11 to a silica gel column chromatography (SiO2, 4x20 cm, 50-100% EtOAc-hexanes gradient) afforded compound 9.6 A solution of triphenylphosphine (48 mg, 0.183 mmol) and (3.6 g. 51% yield). Aldrithiol-2 (38 mg, 0.173 mmol) in pyridine was treated 50 with compound 9.10 (9.5 mg, 0.0245 mmol) and L-alanine 2-(4,5-Dihydroxy-tetrahydro-furan-2-yl)-vinyl n-butyl ester hydrochloride (35 mg, 0.193 mmol). The mix phosphonic acid diethyl ester (9.7) ture was stirred at 60° C. for 1.5 h then concentrated under reduced pressure. The residue was subjected to reverse phase Compound 9.6 (3.6 g., 11.76 mmol) in MeCN (40 mL) was HPLC eluting with 20-70% CHCN in water to give com treated with 1 NHCl (10 mL) and stirred at 50° C. for 5 h with 55 pound 9.11 (7.8 mg, 53% yield). H NMR (CDOD) 8 0.95 slow distillation of residual acetone. The solution was cooled (m, 6H), 1.4 (m. 10H), 1.6 (m, 4H), 2.3 (m. 2H), 3.95 (m. 2H), to ambient temperature and treated with NaHCO (1.0 g) then 4.15 (m, 4H), 4.7 (m, 1H), 4.98 (m. 1H), 5.85 (d. 1H, J=2.1), concentrated in vacuo. The residue was lyophilized overnight 6.1 (m. 1H), 6.8 (m, 1H), 7.8 (s, 1H). 'P NMR (CDOD) d and MeOH (20 mL) was added and the solution was filtered. 18.77. LRMS (ESI) MH" C.H.N.O.P requires 598.6. The solution was then co-evaporated with Mecn to afford 60 Found 598.1. crude compound 9.7 (3.5g, D100% yield). Diphosphophosphonate 9.12 Compound (5 mg) was synthesized from compound 9.10 2-(4,5-Dibenzoyl-tetrahydro-furan-2-yl)-vinyl (30 mg) using the procedure described for the preparation of phosphonic acid diethyl ester (9.8) the diphosphophosphonate 6.8. 'H NMR (D.O) & 2.25 (m, 65 2H), 4.60 (m. 1H), 4.91 (m. 1H), 5.80 (d. 1H, J=2.1), 6.05 (m, Compound 9.7 (3.1 g, 11.8 mmol) in pyridine (15 mL) was 1H), 6.52 (m, 1H), 7.78 (s, 1H). LRMS M-H treated with benzoyl chloride (4.0 mL. 35 mmol) dropwise CHNOP requires 502.0. Found 502.0. US 8,324,179 B2 181 182 Example 4

EtO-P O OBZ O OEt % N NH OBz O M

9.8 EtOt()- O Y. als BSA, SnCl4 N NH2 -- -- MeCN, rt, 24h OEt O 2. OBz N M NH 10.1 NCN || 2 N N NH2 1) TMSBr, 2.6-lutidine 2) NH4OH, H2O, 459 C. O

N NH (M als to- O N N NH2 OH (2 Na+) % OH 10.2 / COBu-n Ph3P, Aldrithiol-2 n Py, 60° C., 3 h NH, HCI

O O N COn-Bu N O|| O|| Q ( . " n " HO1S-7's O O-P o, YN Nals NH HNN-P o, YN Nals NH HO OH 2 HN 2 OH ls 2 w CO2n-Bu 2 OH OH 10.4 10.3

Benzoic acid 2-(5-amino-7-oxo-6,7-dihydro-1,2,3 50 {2-5-(5-Amino-7-oxo-6,7-dihydro-1,2,3 triazolo 4, triazolo 4,5-dipyrimidin-3-yl)-5-[2-(diethoxy-phos 5-dpyrimidin-3-yl)-4-hydroxy-tetrahydro-furan-2- phoryl)-vinyl-tetrahydro-furan-3-yl ester (10.1) yl)-vinyl-phosphonic acid (10.2) Compound 10.1 (390 mg, 0.774 mmol) was dissolved in 15 8-AZaguanine was persilylated first using the procedure 55 mL CHCN, treated with 2.6-lutidine (0.3 mL, 2.59 mmol) described for the preparation of compound 9.9. Compound and TMSBr(1.5 mL, 11.4 mmol). The mixture was stirred for 9.8 (490 mg, 1.03 mmol) was dissolved in 20 mL anhydrous 2 h and coevaporated with CHCN under reduced pressure. The residue was treated with NH-OH (10 mL) and stirred at CHCN, and treated with 10 mL CHCN solution of persily 45° C. for 1.5 h. The mixture was treated with 500 mg lated 8-azaguanine base (392.9 mg, 1.03 mmol), followed by 60 NaHCO and concentrated down under reduced pressure. The addition of SnCl (1M solution in CHCl) (3 mL, 3 mmol). residue was subjected to reverse phase HPLC eluting with The mixture was stirred at r.t. for 24h, cooled with ice-water 0-25% CHCN in water to yield compound 10.2 (220 mg. 73% yield). "H NMR (DO) & 2.28 (m. 1H), 2.45 (m. 1H), and NaHCO, filtered, and concentrated down under reduced 4.90 (m, 2H), 5.8 (m, 1H), 6.15 (m, 1H). 'P NMR (DO) d pressure. The residue was subjected to a silica gel column 65 18.77. LRMS (ESI) MH" CHNOP requires 345.2. eluting with 10% EtOH in EtOAc to give compound 10.1 Found 345.0. (430 mg, 83% yield). Bis-amidate prodrug 10.3 US 8,324,179 B2 183 184 Compound 10.3 (19.4 mg, 55% yield) was synthesized {2-5-(4-Amino-2-oxo-2H-pyridin-1-yl)-4-hydroxy from compound 10.2 (23 mg) using the procedure described tetrahydro-furan-2-yl)-vinyl-phosphonic acid (11.2) for the preparation of compound 9.11. "H NMR (CDOD) & 0.95 (m, 6H), 1.35 (m, 10H), 1.60 (m, 4H), 2.35 (m, 1H), 2.62 Compound 11.2 (97 mg, 86.5% yield) was synthesized (m. 1H), 3.95 (m, 2H), 4.10 (m, 4H), 4.88 (m, 1H), 5.02 (m, 5 from compound 11.1 (215 mg) using the procedure for the 1H), 5.95 (m, 1H), 6.14 (m, 1H), 6.75 (m, 1H). 'P NMR preparation of compound 10.2. HNMR (D.O)ö 1.8 (m. 1H), (CDOD) d 18.94. LRMS (ESI)MH" C.H.N.O.P requires 599.6. Found 599.1. 2.05 (m. 1H), 4.3 (d. 1H, J–4.8), 4.8 (m. 1H), 5.69 (s.1H), 6.0 Diphosphophosphonate 10.4 (m. 1H), 6.2 (m, 1H), 7.52 (d. 1H, J–7.8). 'P NMR (DO) d This compound (18 mg, 51.4% yield) was synthesized 9.8. LRMS (ESI) MHCHNOP requires 304.2. Found from compound 10.2 (27 mg, 0.07 mmol) using the procedure 10 304.O. described for the preparation of compound 6.8. 'HNMR (300 Bis-amidate prodrug 11.3 MHz, DO): 8 2.25-2.50 (m, 2H), 4.87-4.95 (m, 2H), 5.95 6.05 (m, 2H), 6.30-6.42 (m, 1H).31PNMR: 4.51, 4.33, -5.33, Compound 11.3 (4.4 mg, 55% yield) was synthesized from -5.50, -21.10, -21.27, -21.44 ppm. LRMS M-H 15 compound 11.2 (5 mg) using the procedure for the prepara CHNOP, requires 503.0. Found 502.9. tion of compound 9.11. HNMR (300 MHz, CDOD) 8 0.95 (m, 6H), 1.4 (m. 10H), 1.65 (m, 4H), 1.85 (m. 1H), 2.1 (m, 1H), 3.95 (m, 2H), 4.15 (m, 4H), 4.32 (d. 1H, J–4.5), 4.98 (m, Example 5 1H), 5.8 (s, 1H), 5.95 (d. 1H, J–7.8), 6.1 (m. 1H), 7.64 (d. 1H,

NHBZ

O NHBZ EO | O OB NN t()- Z. 21 N N SnCl4 O 1) TMSBr OEt -- BSA N O 2) NHOH 2 O N to- O OBZ H OEt OBz 9.8 11.1 NH2 NH2 COBu-n NN n 2 nie SN O -N HeNH, HCI O -N to- O, O PhP, Aldrithiol-2 HNS O. O Py, 60° C., 3 h HN1 OH N %OH w COn-Bu %OH 11.2 11.3 CDI, TBAP TBA, DMSO NH2 r1s.

11.4

Benzoic acid 2-(4-benzoylamino-2-oxo-2H-pyridin 60 J=7.8). 'P NMR (CDOD) d 18.43. LRMS (ESI) MH' 1-yl)-5-[2-(diethoxy-phosphoryl)-vinyl-tetrahydro CHNOP requires 558.6. Found 558.1. furan-3-yl ester (11.1) Diphosphophosphonate of 11.2. (11.4) Compound 11.1 (215 mg, 60% yield) was synthesized as Compound 11.4 (5 mg, 19% yield) was synthesized from from compound 9.8 (430 mg) using the procedure for the compound 11.2 (20 mg 0.058 mmol) using the procedure preparation of compound 9.9. described for the preparation of compound 6.8. 'HNMR (300 US 8,324,179 B2 185 186 MHz, DO): 81.81-2.16 (m, 2H), 4.32 (d. 1H, J–4.8), 4.91 (s, {2-5-(6-Amino-purin-9-yl)-4-hydroxy-tetrahydro 1H), 5.74 (s, 1H), 5.94 (d. 1H, J=7.8), 6.06-6.18 (t, 1H, furan-2-yl)-vinyl-phosphonic acid (12.2) J=18.3), 6.48-6.63 (m, 1H), 7.54 (d. 1H, J=7.5). 31PNMR: 4.40, 4.21, -9.52, -9.69, -22.68, -22.85, -23.03 ppm. LRMS Compound 12.2 (103 mg, 68% yield) was synthesized M-H CHNOPs requires 462.0. Found 461.9. 5 from compound 12.1 (200 mg, 0.41 mmol) using the proce dure for the preparation of compound 10.2. "H NMR (D.O) & Example 6 2.10 (m. 1H), 2.19 (m, 1H), 4.62 (m, 1H), 4.87 (n, 1H), 5.95

NH

NH2 N NN EtO-P O OBz N O ( 2 -- NN SnCl4 | N 2 OEt ( MeCN EtO-P O N s 2. N 22 48% OBz H N OEt OBz 9.8 12.1

1) TMSBr 2) NH4OH NH2

N n ( N N 2 to- O N OH OH 12.2

COBu - n Aldrithiol-2,Py, 659 C. Ph3P NH2 HCI 60%0

NH NH2 O 2 w N n N NN n-BuO / N O ( HN O| 2 P P N 2 N: O. N Ho7 No7 Yo-Y O N HN1 HO HO OH n-BuO . % OH OH O

12.4 12.3

Benzoic acid 2-(6-amino-purin-9-yl)-5-[2-(diethoxy 55 (m. 2H), 6.16 (m, 1H), 8.03 (s, 1H), 8.09 (s, 1H). 'P NMR phosphoryl)-vinyl-tetrahydro-furan-3-yl ester (12.1) (DO) d 10.07. LRMS (ESI) MH CHNOP requires 328.2. Found 328.0. Compound 9.8 (430 mg. 0.907 mmol) and adenine (135 Bis-amidate prodrug 12.3 mg, 1.0 mmol) was dissolved in 10 mL anhydrous CHCN. Compound 12.3 (11.5 mg, 61% yield) was synthesized The mixture was treated with SnCl4 (0.234 mL, 2.0 mmol) 60 from compound 12.2 (12 mg) using the procedure for the and stirred at r.t. for 20 h. The mixture was cooled to 0°C., preparation of compound 9.11. HNMR (CDOD) 80.93 (m, treated with NaHCO (2 g), water (2 mL), and stirred for 2 h. 6H), 1.36 (m. 10H), 1.60 (m, 4H), 2.28 (m, 2H), 3.92 (m, 2H), The mixture was diluted with CHCN, filtered, and concen 4.08 (m, 4H), 4.76 (s, 1H), 5.00 (m, 1H), 6.05 (m, 1H), 6.08 trated under reduced pressure. The residue was subjected to a (m. 1H), 6.82 (m, 1H), 8.22 (s. 2H). 'P NMR (CDOD) d silica gel column chromatography eluting with EtOAc to 20% 65 19.63. LRMS (ESI) MH" C.H.N.O.P requires 582.6. MeOH in EtOAc to yield compound 12.1 (210 mg, 48% Found 582.2. yield). Diphosphophosphonate 12.4 US 8,324,179 B2 187 188 This compound (3 mg, 13.4% yield) was made from com jected to a silica gel column chromatography eluting with pound 12.2 (15 mg, 0.046 mmol) using the procedure EtOAc to yield compound 13.1 (295 mg, 33.5% yield). described for the preparation of compound 6.8. 'HNMR (300 (Taken from: J. Med. Chem 1986, 29, 203-213.) MHz, DO): 8 2.17-2.29 (m, 2H), 4.99 (s, 1H), 6.02-6.16 (m, 2H), 6.43-6.52 (m, 2H), 8.11 (s, 1H), 8.14 (s, 1H).31PNMR: {2-5-(2,6-Diamino-purin-9-yl)-4-hydroxy-tetrahy 4.37, 4.20, -5.41, -5.57, -21.32 ppm. dro-furan-2-yl)-vinyl-phosphonic acid (13.2) Compound 13.1 (147.5 mg 0.283 mmol) was dissolved in Example 7 3 mL CHCN, treated with TMSBr (0.3 mL) and 2.6-lutidine

C

O C N NN O HMDS / EtO-P OBZ N SnCl4 O -- NN TMSC N 2 OEt ( els CH3CN EtO-P O N NH2 2 2.OBZ NH N NH2 N-ny oBz 9.8 13.1

1) TMSBr 2) NH4OH NH2 y n N O ( HO- O N 21 N. 2

OH

OH 13.2

/ N-teNH2 HCI Aldrithiol-2, PhP Py, 659 C. NH NH2 O 2 s N n N n N n-BuO / N O ( O P P N als HN n P|| O N N 2 NH2 Ho? Yo 7 No-1 O N NH2 HN1 HO HO OH n-BuO 2. OH OH O

13.4 13.3 55 Benzoic acid 2-(2-amino-6-chloro-purin-9-yl)-5-2- (0.075 mL). The mixture was stirred at room temperature for (diethoxy-phosphoryl)-vinyl-tetrahydro-furan-3-yl 1.5 h, concentrated and coevaporated with CHCN under ester (13.1) reduced pressure. The residue was dissolved in 30% NH in water, and the mixture was stirred at 100° C. for 1.5 h. The Compound 9.8 (800 mg, 1.69 mmol) and 2-amino-6-chlo 60 mixture was then treated with NaHCO (500 mg) and con ropurine were dissolved in 15 mL anhydrous CHCN. The centrated under reduced pressure. The residue was subjected mixture was treated with HMDS (0.33 mL, 1.6 mmol), to reverse phase HPLC eluting with 0-25% CHCN in water TMSC1 (0.20 mL, 1.6 mmol), and then SnCl4 (0.27 mL, 2.3 to yield compound 13.2 (81 mg, 74.3% yield). H NMR mmol). The mixture was stirred at 75°C. for 0.5 hand cooled (DO) & 2.05-2.25 (m, 2H), 4.58 (m, 1H), 5.8 (d. 1H, J=1.5), to 0°C., treated with NaHCO, (2.4 g. 28.6 mmol), water (2 65 5.95 (m, 1H), 6.15 (m. 1H), 7.8 (s, 1H). 'P NMR (DO) d mL), and stirred at rt for 1 h. The mixture was then filtered, 9.15. LRMS (ESI)MHCHNOP requires 343.3. Found concentrated under reduced pressure. The residue was Sub 3431. US 8,324,179 B2 189 Bis-amidate prodrug 13.3 -continued Compound 13.3 (9 mg, 46.8% yield) was synthesized from O compound 13.2 (12.5 mg, 0.032 mmol) using the procedure for the preparation of compound 9.11. "H NMR (CDOD) & N 5 ( NH O 0.92 (m, 6H), 1.35 (m, 10H), 1.60 (m, 4H), 2.28 (m, 2H), 3.95 (m. 2H), 4.10 (m, 4H), 4.72 (m. 1H), 4.95 (m. 1H), 5.88 (d. O. Nall N iBu Formic 1H, J=1.8), 6.84 (m. 1H), 7.85 (s, 1H). PNMR (CDOD) d H acid 18.76. LRMS (ESI) MH" C.H. N.O.P requires 597.6 Found 597.2. 10 Diphosphophosphonate 13.4 OMe Compound 13.3 (solium salt) (25 mg, 0.073 mmol) was 16.4 dissovled in 2 mL water, treated with 1M HCl (0.2 mL). The O solution was stirred for 10 min and then treated with 0.1 mL 15 ( NH O 1) TFA, DCC, NHOH. It was then lyophilized to give 35 mg residue. The Pyridine residue was dissovled in 2 mL DMSO, treated with TBA (60 N Nall. N mg), stirred for 30 min, then treated with CDI (60 mg), stirred O iBu 2 O for 1 h, the mixture was finally treated with TBAP (250mg) - Ero1 Na 3 in 2 mL DMF solution, and stirred for 2 h. The mixture was 2. OEt OMe then concentrated under reduced pressure. The residue was 16.6 dissolved in water, filtered through a C-18 column eluting 16.5 with 2% NH.H.O.The solvent was removed and the residue O was purified by ion-exhange HPLC to give the desired com N pound (8.5 mg, 23% yield). "H NMR (300 MHz, DO): 8 NH O 2.16-2.25 (m, 2H), 4.67 (s, 1H), 4.93 (s, 1H), 5.83 (s, 1H, 25 ( J=1.8), 6.07 (t, 1H, J=18.3), 6.41-6.56 (m, 1H), 7.82 (s, 1H). N Nals N ls iBu TMSBr 31PNMR: 4.30, 4.12, -5.87, -6.04, 31 21.79 ppm. LRMS H M-H CHNOP, requires 501.0. Found 501.0 30 Bzó oMe Example 8 16.7 O

N 35 (N NH N als 1) TMSCI, Pyridine O N NH2 2) Isobutylrylchloride 40 Bzó OMe ry 16.8 O

N 45 ( NH N 1. He O N NH2 ( NH O O N Nals N ls iBu PyridinePMTC 50 HG OMe -ny 16.9

55

7 NH O 2 ydride O. &lluN N iBu isDMAP try H Pyridine

65 US 8,324,179 B2 191 192 N-9-(4-Hydroxy-5-hydroxymethyl-3-methoxy-tet DCC (1.47g, 7.12 mmol) and pyridine (0.20 mL, 2.49 mmol). rahydro-furan-2-yl)-6-oxo-6,9-dihydro-1H-purin-2- The mixture was stirred for 16 h to form the aldehyde. Com yl)-isobutyramide (16.2) pound 16.6 (1.47g, 3.56 mmol) dissolved in 6 mL anhydrous DMSO was added rapidly to the mixture and stirred for Compound 16.1 (5.0 g, 17.9 mmol) was dissolved in anhy 5 another 12 h. Oxalic acid anhydrate (0.3 g) dissolved in 2 mL drous pyridine 60 mL under nitrogen and cooled to 0° C. MeOH was added to quench excess DCC. The mixture was TMSC1 (9.05 mL, 71.6 mmol) was added dropwise over a period of 10 min. The mixture was allowed to stir at r.t. for 1 stirred for 10 min and concentrated under reduced pressure. hand cooled to 0°C., treated with isobutylryl chloride (2.06 The residue was resuspended in EtOAc (100 mL) and filtered. mL, 19.7 mmol). The mixture was allowed to rise to r.t. and 10 The filtrated was washed with water (200 mL), Brine (200 stirred for 3 h. MeOH (120 mL) was added to the mixture and mL), dried over MgSO4, and concentrated under reduced stirred for 3 h to desilylate the product. The mixture was pressure. The residue was subjected to a silica gel column concentrated under reduced pressure and the residue was chromatography to yield product 16.7 (460 mg, 43% yield). Subjected to a quick plug of silica gel to give product 16.2 15 (6.18 g, 100% yield). Benzoic acid 5-(2-isobutyrylamino-6-oxo-1,6-dihy N-(9-5-Bis-(4-methoxy-phenyl)-phenyl-meth dro-purin-9-yl)-4-methoxy-2-(2-phosphono-vinyl)- oxymethyl-4-hydroxy-3-methoxy-tetrahydro-furan tetrahydro-furan-3-yl ester (16.8) 2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-isobutyra mide (16.3) Compound 16.7 (460 mg, 0.763 mmol) was dissolved in 8 mL anhydrous DMF, treated with 2.6-lutidine (0.443 mL, Compound 16.2 (6.18 g. 17.19 mmol) was dissolved in 15 3.81 mmol) and TMSBr (1.5 mL, 11.4 mmol). The mixture mL anhydrous pyridine, and treated with DMTC1 (6.4 g. was stirred at 80° C. for 4 hand quenched with MeOH. The 18.91 mmol). The mixture was stirred for 12 hand poured on 25 mixture was concentrated under reduced pressure and the to 200 mL ice-water, filtered, and the solid was washed with residue was subjected to reverse phase HPLC to yield product 2x100 mL water. The solid was redissolved in 200 mL ethyl acetate. The organic layer was washed with 3x100 mL water, 16.8 (60 mg, 14.4% yield). dried over MgSO filtered, and concentrated down under reduced pressure. The residue was subjected to a silica gel 30 {2-5-(2-Amino-6-oxo-1,6-dihydro-purin-9-yl)-3- column chromatography to yield product 16.3 (2.6 g. 23% hydroxy-4-methoxy-tetrahydro-furan-2-yl)-vinyl yield). phosphonic acid (16.9) Benzoic acid 2-bis-(4-methoxy-phenyl)-phenyl methoxymethyl-5-(2-isobutyrylamino-6-oxo-1,6- 35 Compound 16.9 (37.4 mg., 91% yield) was synthesized dihydro-purin-9-yl)-4-methoxy-tetrahydro-furan-3-yl from compound 16.8 (60 mg, 0.11 mmol) using the procedure ester (16.4) described for the preparation of compound 2.4. HNMR (D.O,300 MHz) & 7.89 (bs, 1H), 6.45 (m, 1H), 6.07-5.85 (m, Compound 16.3 (2.6 g., 3.9 mmol) was dissolved in 15 mL 2H), 4.55(bs, 1H), 4.41 (m, 2H), 3.32(s.3H). PNMR (DO, anhydrous pyridine, and treated with benzoic anhydride 40 300 MHz) & 11.23 ppm. LRMS (ESI) MH CHNOP (17.62g, 78 mmol), DMAP (95 mg, 0.78 mmol). The mixture requires 374.1. Found 374.0. was stirred for 4 h and poured on to 200 mL ice-water. The solid was collected by filtration and redissolved in 200 mL Diphosphophosphonate 16.95 ethyl acetate. The organic layer was washed with 3x100 mL This compound (1 mg, 7% yield) was synthesized from water, dried over MgSO filtered and concentrated down 45 compound 16.9 (10 mg 0.027 mmol) using the procedure under reduced pressure. The residue was Subjected to a silica described for the preparation of diphosphophosphonate 6.8. gel column chromatography to yield product 16.4 (1.5g, 50% yield). Example 9 Benzoic acid 2-hydroxymethyl-5-(2-isobutyry 50 lamino-6-oxo-1,6-dihydro-purin-9-yl)-4-methoxy tetrahydro-furan-3-yl ester (16.5)

Compound 16.4 (1.5 g, 1.54 mmol) was dissolved in a NHBZ mixture of 8 mL CH2Cl and 8 mL methanol, and treated with 55 formic acid 16 mL. When the reaction was completed, 20 mL n-butanol was added to quench the reaction. The mixture was r 1) Dess-Martin reagent evaporated to dryness under reduced pressure and the byprod O. S. O 2) NaH, THF ucts were removed by washing the residue with hexane 3x200 HO O O mL. Compound 16.5 (840 mg. 92% yield) was obtained. o–E -o Benzoic acid 2-2-(diethoxy-phosphoryl)-vinyl-5- s 2 /N1 v. (2-isobutyrylamino-6-oxo-1,6-dihydro-purin-9-yl)-4- BZO OMe O O methoxy-tetrahydro-furan-3-yl ester (16.7) 17.1 - X 65 Compound 16.5 (840 mg, 1.78 mmol) was dissolved in 6 17.2 mL anhydrous DMSO, treated with TFA (96 uL, 0.25 mmol), US 8,324,179 B2 193 194 -continued {2-5-(4-Amino-2-oxo-2H-pyrimidin-1-yl)-3-hy NHBZ droxy-4-methoxy-tetrahydro-furan-2-yl)-vinyl phosphonic acid (17.5) Compound 17.5 (37 mg, 95% yield) was synthesized from compound 17.4 (62 mg, 0.11 mmol) by treating 17.4 with concentrated NH-OH (10 ml) at 45° C. for 30 min. The reaction mixture was concentrated to dryness and purified by C-18 HPLC to afford compound 17.5. "HNMR (D.O, 300 10 MHz) & 7.65 (d. 1H), 6.29-6.45 (m. 1H), 5.96-6.10 (m, 3H), 5.85 (d. 1H), 4.42 (t, 1H), 4.10 (t, 1H), 3.91 (m, 1H), 3.40 (s, 3H), "PNMR (DO, 300 MHz): 0.90 ppm. LRMS M-H CHNO,P requires 332.1. Found 331.2. 15 Bis-amidate prodrug 17.6 Compound 17.6 (9 mg, 30% yield) was synthesized from compound 17.5 (17 mg, 0.05 mmol) using the procedure described for the preparation of compound 9.11. HNMR (CDCN, 300 MHz) & 7.47 (d. 1H), 6.75 (m. 1H), 5.80-6.2 (m,3H), 4.39 (m. 1H), 4.08 (m, 2H), 4.09 (m, 4H), 3.94 (m, 3H), 3.80 (m. 1H), 3.63 (m, 2H), 3.55 (s.3H), 1.63 (m, 4H), Bzó oMe 1.39 (m, 10H), 0.90 (m, 6H). PNMR (CDCN, 300 MHz): 15.019 ppm. LRMS (ESI) MH" CHNOP requires 17.4 5883. Found 588.0. NH2 25 n N ne Example 10 NH, HCI Hes HO o7 21 O Aldrithiol-2,Py, 659 C. Ph3P 30 HO HG OMe 17.5 35 O NH2 1) TMSCI -e-2) Isobutylryl chloride 1N1N O w NN

HN N O 40 HN DN4N - N-1- . HG OMe 45 O 17.6 ls -as1) TFA, DCC O iBu 2) O Ero1 Na 3 Benzoic acid 5-(4-benzoylamino-2-oxo-2H-pyrimi OEt din-1-yl)-2-[2-(diisopropoxy-phosphoryl)-vinyl-4- methoxy-tetrahydro-furan-3-yl ester (17.3) 18.2 Compound 17.3 (300 mg, 16% yield) was synthesized 55 from compound 17.1 (1.41 g, 2.88 mmol) and compound 17.2 (3.96 g, 11.52 mmol) using the procedure described for the preparation of compound 7.3.

Benzoic acid 5-(4-benzoylamino-2-oxo-2H-pyrimi din-1-yl)-4-methoxy-2-(2-phosphono-vinyl)-tetrahy dro-furan-3-yl ester (17.4)

Compound 17.4 (124 mg. 47.5% yield) was synthesized 65 from compound 17.3 (300 mg, 0.462 mmol) using the proce dure described for the preparation of compound 16.8. US 8,324,179 B2 195 196 {2-3,4-Dihydroxy-5-(2-isobutyrylamino-6-oxo-1,6- -continued dihydro-purin-9-yl)-tetrahydro-furan-2-yl)-vinyl phosphonic acid diethyl ester (18.4) Compound 18.3 (850 mg, 1.62 mmol) was dissolved in 9 P O. H iBu TMSBr mL TFA and 1 mL water mixture, stirred for 1.5 h. The EtO1 mixture was concentrated under reduced pressure, and the OEt residue was Subjected to a silica gel column chromatography eluting with 10% MeOH in CHCl to give compound 18.4 HG OH 10 (680 mg, 86.4% yield). 18.4 {2-3,4-Dihydroxy-5-(2-isobutyrylamino-6-oxo-1,6- O dihydro-purin-9-yl)-tetrahydro-furan-2-yl)-vinyl phosphonic acid (18.5) & NH O 15 Compound 18.5 (400 mg, 67% yield) was synthesized P O. Nall. N iBu Not from compound 18.4 (680 mg, 1.4 mmol) using the procedure HO1 described for the preparation of compound 16.8. OH {2-5-(2-Amino-6-oxo-1,6-dihydro-purin-9-yl)-3,4- dihydroxy-tetrahydro-furan-2-yl)-vinyl-phosphonic HG OH acid (18.8) 18.5 O Compound 18.8 (180 mg, 54% yield) was synthesized from compound 18.5 (400 mg. 0.932 mmol) by treating 18.5 N with concentrated NH-OH (i0 ml) at 45° C. for 30 min. "H 7 NH NMR (DO, 300 MHz) 7.98 (s, 1H), 6.41 (m, 1H), 5.95 (m, 1H), 5.75 (d. J=5.4 Hz, 1H), 4.63 (m, 1H), 4.49 (m. 1H), 4.19 O| O. Nals NH2 (t, J–4.5 Hz, 1H), P NMR (D.O. 121.4 MHz) 9.7; MS (ESI) m/z 360 M+H" Ho1 Bis-amidate prodrug 18.9 OH Compound 18.9 (2 mg, 3% yield) was synthesized from 30 compound 18.8 (400 mg. 0.932 mmol) using the procedure HG OH described for the preparation of compound 9.11. 18.8 Example 11 COBu Aldrithiol-2, Ph3P NHBZ Py, 659 C. 35 NH, HCI N NN O O ( 1) Dess-Martin reagent N 2 2) NaH, THF N O O I I ~. O (y alsNH 40 HNS! O. N. YN1 NH, HN Nay Bzó ÖMe N-1- 21.1 HO OH 45 17.2 O NHBZ 18.9 N N1SN O (N 21e - N-9-(6-Hydroxymethyl-2,2-dimethyl-tetrahydro 50 P O N TMSBr furo3,4-d1.3dioxol-4-yl)-6-oxo-6,9-dihydro-1H o1 purin-2-ylisobutyramide (18.2) - O -> Bzó oMe Compound 18.2 (12.15 g, 100% yield) was synthesized 55 from compound 18.1 (10.0 g, 30.93 mmol) using the proce 21.2 dure described for the preparation of compound 16.2. NHBZ N {2-6-(2-Isobutyrylamino-6-oxo-1,6-dihydro-purin 9-yl)-2,2-dimethyl-tetrahydro-furo3,4-d1.3di 60 O (1.2 NHOH | N He oxol-4-yl)-vinyl-phosphonic acid diethyl ester O N HO11. (18.3) OH

Compound 18.3 (1.7 g. 64% yield) was was synthesized 65 Bzó OMe from compound 18.2 (2.0 g, 5.09 mmol) using the procedure 21.3 described for the preparation of compound 16.7.