111111
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111
`US 20100298257Al
`
`(19) United States
`c12) Patent Application Publication
`ROSS et al.
`
`(10) Pub. No.: US 2010/0298257 A1
`Nov. 25, 2010
`(43) Pub. Date:
`
`(54) NUCLEOSIDE PHOSPHORAMIDATES
`
`Related U.S. Application Data
`
`(75)
`
`Inventors:
`
`BRUCE ROSS, PLAINSBORO, NJ
`(US); MICHAEL JOSEPH
`SOFIA, DOYLESTOWN, PA (US);
`GANAPATI REDDY
`PAMULAPATI, PLAINSBORO,
`NJ (US); SUGUNA
`RACHAKONDA, TWINSBURG,
`NJ (US); HAI-REN ZHANG,
`ELLICOTT CITY, MD (US);
`BYOUNG-KWON CHUN,
`ROBBINSVILLE, NJ (US);
`PEIYUAN WANG, GLEN ROCK,
`NJ (US)
`
`Correspondence Address:
`MERCHANT & GOULD PC
`P.O. BOX 2903
`MINNEAPOLIS, MN 55402-0903 (US)
`
`(73) Assignee:
`
`PHARMASSET, INC.,
`PRINCETON, NJ (US)
`
`(21) Appl. No.:
`
`12/783,680
`
`(22) Filed:
`
`May20, 2010
`
`(60) Provisional application No. 61/179,923, filed on May
`20, 2009, provisional application No. 61/319,513,
`filed on Mar. 31, 2010.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`(2006.01)
`A61K 3117072
`(2006.01)
`C07H 19110
`(2006.01)
`C07H 19106
`(2006.01)
`C07F 9124
`(2006.01)
`A61P 31114
`(2006.01)
`A61P 31112
`(2006.01)
`A61P 31116
`(2006.01)
`G01N 30102
`(2006.01)
`G01N 231207
`(52) U.S. Cl. ......... 514/51; 536/26.8; 536/28.2; 558/179;
`73/61.52; 378/73
`
`(57)
`
`ABSTRACT
`
`Disclosed herein are nucleoside phosphoramidates and their
`use as agents for treating viral diseases. These compounds are
`inhibitors of RNA-dependent 5 RNA viral replication and are
`useful as inhibitors of HCV NS5B polymerase, as inhibitors
`ofHCV replication and for treatment of hepatitis C infection
`in mannnals.
`
`F1B
`
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`Patent Application Publication
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`Nov. 25, 2010 Sheet 1 of 21
`
`US 2010/0298257 A1
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`Patent Application Publication Nov. 25, 2010 Sheet 7 of 21
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`Patent Application Publication Nov. 25, 2010 Sheet 8 of 21
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`US 2010/0298257 A1
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`IPR2018-00390
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`Patent Application Publication
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`Nov. 25, 2010 Sheet 9 of 21
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`FIG. 10
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`Patent Application Publication Nov. 25, 2010 Sheet 11 of 21
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`US 2010/0298257 A1
`
`FIG. 11
`
`Cl1
`
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`
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`Patent Application Publication Nov. 25, 2010 Sheet 12 of 21
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`IPR2018-00390
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`Patent Application Publication Nov. 25, 2010 Sheet 13 of 21
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`US 2010/0298257 A1
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`FIG. 14
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`IPR2018-00390
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`FIG. 15
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`FIG. 16
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`FIG. 17
`
`100~,_~----------------~~------------~
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`80
`
`60
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`80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 oc
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`Integral -12.64 mJ
`normalized ~ 11 .40 JgA~ 1
`Onset 58.88 oc
`Peak 63.50 oc
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`FIG.18
`
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`FIG. 19
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`Integral -45.1:3 mJ
`normalized -42. ·13 ,JgA-1
`Onset 93.98 "C
`Peak 104.67 oc
`
`c
`rFJ
`N
`0 ....
`0 ..._
`0
`N
`\0
`QO
`N
`Ul
`-....l
`
`> ....
`
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`1
`
`NUCLEOSIDE PHOSPHORAMIDATES
`
`[0005] HCV belongs to a much larger family of viruses that
`share many common features.
`
`PRIORITY CLAIM
`
`Flaviviridae Viruses
`
`[0001] The right of priority is claimed to U.S. Provisional
`Patent Application Nos. 61/179,923, filed May 20, 2009, and
`61/319,513, filed Mar. 31, 2010, the subject matter of which
`is incorporated by reference in its entirety.
`
`FIELD OF THE INVENTION
`
`[0002] Disclosed herein are nucleoside phosphoramidates
`and their use as agents for treating viral diseases. These com(cid:173)
`pounds are inhibitors of RNA-dependent RNA viral replica(cid:173)
`tion and are useful as inhibitors ofHCV NS5B polymerase, as
`inhibitors ofHCV replication and for treatment of hepatitis C
`infection in mammals.
`
`BACKGROUND
`
`[0003] Hepatitis C virus (HCV) infection is a major health
`problem that leads to chronic liver disease, such as cirrhosis
`and hepatocellular carcinoma, in a substantial number of
`infected individuals, estimated to be 2-15% of the world's
`population. There are an estimated 4.5 million infected
`people in the United States alone, according to the U.S. Cen(cid:173)
`ter for Disease Control. According to the World Health Orga(cid:173)
`nization, there are more than 200 million infected individuals
`worldwide, with at least 3 to 4 million people being infected
`each year. Once infected, about 20% of people clear the virus,
`but the rest can harbor HCV the rest of their lives. Ten to
`twenty percent of chronically infected individuals eventually
`develop liver-destroying cirrhosis or cancer. The viral disease
`is transmitted parenterally by contaminated blood and blood
`products, contaminated needles, or sexually and vertically
`from infected mothers or carrier mothers to their offspring.
`Current treatments for HCV infection, which are restricted to
`immunotherapy with recombinant interferon-a alone or in
`combination with the nucleoside analog ribavirin, are oflim(cid:173)
`ited clinical benefit. Moreover, there is no established vaccine
`for HCV. Consequently, there is an urgent need for improved
`therapeutic agents that effectively combat chronic HCV
`infection.
`[0004] The HCV virion is an enveloped positive-strand
`RNA virus with a single oligoribonucleotide genomic
`sequence of about 9600 bases which encodes a polyprotein of
`about 3,010 amino acids. The protein products of the HCV
`gene consist of the structural proteins C, E1, and E2, and the
`non-structural proteins NS2, NS3, NS4A and NS4B, and
`NS5A and NS5B. The nonstructural (NS) proteins are
`believed to provide the catalytic machinery for viral replica(cid:173)
`tion. The NS3 protease releases NS5B, the RNA-dependent
`RNA polymerase from the polyprotein chain. HCV NS5B
`polymerase is required for the synthesis of a double-stranded
`RNA from a single-stranded viral RNA that serves as a tem(cid:173)
`plate in the replication cycle ofHCV. Therefore, NS5B poly(cid:173)
`merase is considered to be an essential component in the HCV
`replication complex (K. Ishi, et a!, Heptology, 1999, 29:
`1227-1235; V. Lohmann, et a!., Virology, 1998, 249: 108-
`118). Inhibition of HCV NS5B polymerase prevents forma(cid:173)
`tion of the double-stranded HCV RNA and therefore consti(cid:173)
`tutes an attractive approach to the development of HCV(cid:173)
`specific antiviral therapies.
`
`[0006] The Flaviviridae family of viruses comprises at least
`three distinct genera: pestiviruses, which cause disease in
`cattle and pigs;jlavivruses, which are the primary cause of
`diseases such as dengue fever and yellow fever; and hepacivi(cid:173)
`ruses, whose sole member is HCV. The flavivirus genus
`includes more than 68 members separated into groups on the
`basis of serological relatedness (Calisher eta!., J. Gen. Viral,
`1993,70,37-43). Clinical symptoms vary and include fever,
`encephalitis and hemorrhagic fever (Fields Virology, Editors:
`Fields, B. N., Knipe, D. M., and Howley, P.M., Lippincott(cid:173)
`Raven Publishers, Philadelphia, Pa., 1996, Chapter 31, 931-
`959). Flaviviruses of global concern that are associated with
`human disease include the Dengue Hemorrhagic Fever
`viruses (DHF), yellow fever virus, shock syndrome and Japa(cid:173)
`nese encephalitis virus (Halstead, S. B., Rev. Infect. Dis.,
`1984, 6, 251-264; Halstead, S. B., Science, 239:476-481,
`1988; Monath, T. P., New Eng. J. Med, 1988,319, 64 1-643).
`[0007] The pestivirus genus includes bovine viral diarrhea
`virus (BVDV), classical swine fever virus (CSFV, also called
`hog cholera virus) and border disease virus (BDV) of sheep
`(Moennig, V. eta!. Adv. Vir. Res. 1992, 41, 53-98). Pestivirus
`infections of domesticated livestock (cattle, pigs and sheep)
`cause significant economic losses worldwide. BVDV causes
`mucosal disease in cattle and is of significant economic
`importance to the livestock industry (Meyers, G. and Thiel, H.
`J., Advances in Virus Research, 1996, 47, 53-118; Moennig
`V., eta!, Adv. Vir. Res. 1992, 41, 53-98). Humanpestiviruses
`have not been as extensively characterized as the animal
`pestiviruses. However, serological surveys indicate consider(cid:173)
`able pestivirus exposure in humans.
`[0008] Pestiviruses and hepaciviruses are closely related
`virus groups within the Flaviviridae family. Other closely
`related viruses in this family include the GB virus A, GB virus
`A-like agents, GB virus-Band GB virus-C (also called hepa(cid:173)
`titis Gvirus, HGV). The hepacivirus group (hepatitis C virus;
`HCV) consists of a number of closely related but genotypi(cid:173)
`cally distinguishable viruses that infect humans. There are at
`least 6 HCV genotypes and more than 50 subtypes. Due to the
`similarities between pestiviruses and hepaciviruses, com(cid:173)
`bined with the poor ability of hepaciviruses to grow effi(cid:173)
`ciently in cell culture, bovine viral diarrhea virus (BVDV) is
`often used as a surrogate to study the HCV virus.
`[0009] The genetic organization of pestiviruses and hepa(cid:173)
`civiruses is very similar. These positive stranded RNA viruses
`possess a single large open reading frame (ORF) encoding all
`the viral proteins necessary for virus replication. These pro(cid:173)
`teins are expressed as a polyprotein that is co- and post(cid:173)
`translationally processed by both cellular and virus-encoded
`proteinases to yield the mature viral proteins. The viral pro(cid:173)
`teins responsible for the replication of the viral genome RNA
`are located within approximately the carboxy-terminal. Two(cid:173)
`thirds of the 0 RF are termed nonstructural (NS) proteins. The
`genetic organization and polyprotein processing of the non(cid:173)
`structural protein portion of the ORF for pestiviruses and
`hepaciviruses is very similar. For both the pestiviruses and
`hepaciviruses, the mature nonstructural (NS) proteins, in
`sequential order from the amino-terminus of the nonstruc(cid:173)
`tural protein coding region to the carboxy-terminus of the
`ORF, consist of p7, NS2, NS3, NS4A, NS4B, NS5A, and
`NS5B.
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`[0010] The NS proteins of pestiviruses and hepaciviruses
`share sequence domains that are characteristic of specific
`protein functions. For example, the NS3 proteins of viruses in
`both groups possess amino acid sequence motifs characteris(cid:173)
`tic of serine proteinases and of helicases (Gorbalenya et a!.,
`Nature, 1988, 333, 22; Bazan and Fletterick Virology, 1989,
`171, 637-639; Gorbalenya eta!., Nucleic Acid Res., 1989, 17,
`3889-3897). Similarly, the NS5B proteins of pestiviruses and
`hepaciviruses have the motifs characteristic of RNA-directed
`RNA polymerases (Koonin, E. V. and Dolja, V. V., Crir. Rev.
`Biochem. Malec. Biol. 1993, 28, 375-430).
`[0011] The actual roles and functions of the NS proteins of
`pestiviruses and hepaciviruses in the lifecycle of the viruses
`are directly analogous. In both cases, the NS3 serine protein(cid:173)
`ase is responsible for all proteolytic processing of polyprotein
`precursors downstream of its position
`in
`the ORF
`(WiskerchenandCollett, Virology, 1991, 184, 341-350; Bar(cid:173)
`tenschlager eta!., J. Viral. 1993, 67, 3835-3844; Eckart eta!.
`Biochem. Biophys. Res. Comm. 1993,192, 399-406; Grakoui
`etal.,J. Viral. 1993,67, 2832-2843; Grakoui eta!., Proc. Nat!.
`A cad Sci. USA 1993,90, 10583-10587; Hijikata etal.,J. Viral.
`1993, 67, 4665-4675; Tome eta!., J. Viral., 1993, 67,4017-
`4026). The NS4A protein, in both cases, acts as a cofactor
`with the NS3 serine protease (Bartenschlager et a!., J. Viral.
`1994, 68, 5045-5055; Failla eta!., J. Viral. 1994, 68, 3753-
`3760; Xu eta!., J. Viral., 1997, 71:53 12-5322). The NS3
`protein ofboth viruses also functions as a helicase (Kim eta!.,
`Biochem. Biophys. Res. Comm., 1995, 215, 160-166; Jin and
`Peterson, Arch. Biochem. Biophys., 1995, 323, 47-53; Warr(cid:173)
`ener and Collett, J. Viral. 1995, 69,1720-1726). Finally, the
`NS5B proteins of pestiviruses and hepaciviruses have the
`predicted RNA-directed RNA polymerases activity (Behrens
`eta!., EMBO, 1996, 15, 12-22; Lechmann eta!., J. Viral.,
`1997, 71, 8416-8428; Yuan et a!., Biochem. Biophys. Res.
`Comm. 1997, 232, 231-235; Hagedorn, PCT WO 97/12033;
`Zhong eta!, J. Viral., 1998, 72, 9365-9369).
`[0012] Currently, there are limited treatment options for
`individuals infected with hepatitis C virus. The current
`approved therapeutic option is the use of immunotherapy
`with recombinant interferon-a alone or in combination with
`the nucleoside analog ribavirin. This therapy is limited in its
`clinical effectiveness and only 50% of treated patients
`respond to therapy. Therefore, there is significant need for
`more effective and novel therapies to address the unmet medi(cid:173)
`cal need posed by HCV infection.
`[0013] A number of potential molecular targets for drug
`development of direct acting antivirals as anti-HCV therapeu(cid:173)
`tics have now been identified including, but not limited to, the
`NS2-NS3 autoprotease, the N3 protease, the N3 helicase and
`the NS5B polymerase. The RNA-dependent RNA poly(cid:173)
`merase is absolutely essential for replication of the single(cid:173)
`stranded, positive sense, RNA genome and this enzyme has
`elicited significant interest among medicinal chemists.
`[0014]
`Inhibitors of HCV NS5B as potential therapies for
`HCV infection have been reviewed: Tan, S.-L., eta!., Nature
`Rev. Drug Discov., 2002, 1, 867-881; Walker, M.P. eta!., Exp.
`Opin. Investigational Drugs, 2003, 12, 1269-1280; Ni, Z-J.,
`eta!., Current Opinion in Drug Discovery and Development,
`2004, 7, 446-459; Beaulieu, P. L., eta!., Current Opinion in
`Investigational Drugs, 2004, 5, 838-850; Wu, J., eta!., Cur(cid:173)
`rent Drug Targets-Infectious Disorders, 2003, 3, 207-219;
`Griffith, R. C., eta!, Annual Reports in Medicinal Chemistry,
`2004, 39, 223-237; Carrol, S., et a!., Infectious Disorders(cid:173)
`Drug Targets, 2006,6, 17-29. The potential for the emergence
`
`of resistant HCV strains and the need to identify agents with
`broad genotype coverage supports the need for continuing
`efforts to identify novel and more effective nucleosides as
`HCV NS5B inhibitors.
`[0015] Nucleoside inhibitors ofNS5B polymerase can act
`either as a non-natural substrate that results in chain termina(cid:173)
`tion or as a competitive inhibitor which competes with nucle(cid:173)
`otide binding to the polymerase. To function as a chain ter(cid:173)
`minator the nucleoside analog must be taken up by the cell
`and converted in vivo to a triphosphate to compete for the
`polymerase nucleotide binding site. This conversion to the
`triphosphate is commonly mediated by cellular kinases which
`imparts additional structural requirements on a potential
`nucleoside polymerase inhibitor. Unfortunately, this limits
`the direct evaluation of nucleosides as inhibitors of HCV
`replication to cell-based assays capable of in situ phosphory(cid:173)
`lation.
`[0016]
`In some cases, the biological activity of a nucleoside
`is hampered by its poor substrate characteristics for one or
`more of the kinases needed to convert it to the active triphos(cid:173)
`phate form. Formation of the monophosphate by a nucleoside
`kinase is generally viewed as the rate limiting step of the three
`phosphorylation events. To circumvent the need for the initial
`phosphorylation step in the metabolism of a nucleoside to the
`active triphosphate analog, the preparation of stable phos(cid:173)
`phate pro drugs has been reported. Nucleoside phosphorami(cid:173)
`date prodrugs have been shown to be precursors of the active
`nucleoside triphosphate and to inhibit viral replication when
`administered to viral infected whole cells (McGuigan, C., et
`a!., J. Med. Chern., 1996,39, 1748-1753; Valette, G., eta!., J.
`Med. Chern., 1996, 39, 1981-1990; Balzarini, J., eta!., Proc.
`National Acad Sci USA, 1996, 93, 7295-7299; Siddiqui, A.
`Q., eta!., J. Med. Chern., 1999, 42, 4122-4128; Eisenberg, E.
`J., eta!., Nucleosides, Nucleotides and Nucleic Acids, 2001,
`20, 1091-1098; Lee, W. A., eta!., Antimicrobial Agents and
`Chemotherapy, 2005,49, 1898); US 2006/0241064; and WO
`2007/095269.
`[0017] Also limiting the utility of nucleosides as viable
`therapeutic agents is their sometimes poor physicochemical
`and pharmacokinetic properties. These poor properties can
`limit the intestinal absorption of an agent and limit uptake into
`the target tissue or cell. To improve on their properties pro(cid:173)
`drugs of nucleosides have been employed. It has been dem(cid:173)
`onstrated that preparation of nucleoside phosphoramidates
`improves the systemic absorption of a nucleoside and further(cid:173)
`more, the phosphoramidate moiety of these "pronucleotides"
`is masked with neutral lipophilic groups to obtain a suitable
`partition coefficient to optimize uptake and transport into the
`cell dramatically enhancing the intracellular concentration of
`the nucleoside monophosphate analog relative to administer(cid:173)
`ing the parent nucleoside alone. Enzyme-mediated hydroly(cid:173)
`sis of the phosphate ester moiety produces a nucleoside
`monophosphate wherein the rate limiting initial phosphory(cid:173)
`lation is unnecessary. To this end, U.S. patent application Ser.
`No. 12/053,015, which corresponds to WO 2008/121634 and
`US 2010/0016251, discloses a number of phosphoramidate
`nucleoside prodrugs, many of which show activity in an HCV
`assay. Several compounds disclosed in US 2010/0016251
`were tested as a potential clinical candidate for approval by
`the FDA.
`
`SUMMARY OF THE INVENTION
`[0018] Disclosed herein is a compound represented by for(cid:173)
`mula 4 and its respective phosphorus-based diastereomers
`represented by formulas Sp-4 and Rp-4.
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`3
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`4
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0019] FIG. 1. High resolution XRD diffractogram of 4.
`[0020] FIG. 2. HighresolutionXRD diffractogram ofRp-4.
`[0021] FIG. 3. HighresolutionXRD diffractogram ofSp-4
`(Form 1).
`[0022] FIG. 4. High resolution XRD diffractogram of Sp-4
`(Form 1).
`[0023] FIG. 5. HighresolutionXRD diffractogram ofSp-4.
`CH2 Cl2 (Form 2).
`[0024] FIG. 6. HighresolutionXRD diffractogram ofSF-4.
`CHC13 (Form 3).
`[0025] FIG. 7. High resolution XRD diffractogram of Sp-4
`(Form 4).
`[0026] FIG. 8. High resolution XRD diffractogram of Sp-4
`(Form 5).
`[0027] FIG. 9. High resolution XRD diffractogram of Sp-4
`(amorphous).
`[ 0028] FIG. 10. X-Ray Crystal Structure for S p-4 (Form 1)
`[0029] FIG. 11. X-Ray Crystal (Isotropic) Structure for
`Sp-4.CH2 Cl2 (Form 2)
`[0030] FIG. 12. X-Ray Crystal (Anisotropic) Structure for
`Sp-4.CH2 Cl2 (Form 2)
`[0031] FIG. 13. X-Ray Crystal Structure for Sp-4.CHC13
`(Form 3)
`[0032] FIG. 14. FT-IR spectrum of 4.
`[0033] FIG. 15. FT-IR spectrum ofRp-4.
`[0034] FIG. 16. FT-IR spectrum of Sp-4
`[0035] FIG. 17. TGA and DSC analysis of 4.
`[0036] FIG. 18. TGA and DSC analysis ofRp-4.
`
`[0037] FIG. 19. TGA and DSC analysis of Sp-4.
`[0038] FIG. 20A. X-Ray Crystal Structure for 8 (SF-iso(cid:173)
`mer) (molecule no. 1 of the asymmetric unit).
`[0039] FIG. 20B. X-Ray Crystal Structure for 8 (Sp-iso(cid:173)
`mer) (molecule no. 2 of the asymmetric unit).
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Definitions
`[0040] The phrase "a" or "an" entity as used herein refers to
`one or more of that entity; for example, a compound refers to
`one or more compounds or at least one compound. As such,
`the terms "a" (or "an"), "one or more", and "at least one" can
`be used interchangeably herein.
`[0041] The terms "optional" or "optionally" as used herein
`means that a subsequently described event or circumstance
`may but need not occur, and that the description includes
`instances where the event or circumstance occurs and
`instances in which it does not. For example, "optional bond"
`means that the bond may or may not be present, and that the
`description includes single, double, or triple bonds.
`[0042] The term "P*" means that the phosphorus atom is
`chiral and that it has a corresponding Cahn-Ingold-Prelog
`designation of "R" or "S" which have their accepted plain
`meanings.
`[0043] The term "purified," as described herein, refers to
`the purity of a given compound. For example, a compound is
`"purified" when the given compound is a major component of
`the composition, i.e., at least 50% w/w pure. Thus, "purified"
`embraces at least 50% w/w purity, at least 60% w/w purity, at
`least 70% purity, at least 80% purity, at least 85% purity, at
`least 90% purity, at least 92% purity, at least 94% purity, at
`least 96% purity, at least 97% purity, at least 98% purity, at
`least 99% purity, at least 99.5% purity, and at least 99.9%
`purity, wherein "substantially pure" embraces at least 97%
`purity, at least 98% purity, at least 99% purity, at least 99.5%
`purity, and at least 99.9% purity
`[0044] The term "metabolite," as described herein, refers to
`a compound produced in vivo after administration to a subject
`in need thereof.
`[0045] The term "about" (also represented by-) means that
`the recited numerical value is part of a range that varies within
`standard experimental error.
`[0046] The expression "substantially as shown in ... " a
`specified XRPD pattern means that the peak positions shown
`in the XRPD pattern are substantially the same, within visual
`inspection or resort to selected peak listings (±0.2° 28). One
`of ordinary skill understands that the intensities can vary
`depending on the sample.
`[0047] The term "substantially anhydrous" means that a
`substance contains at most 10% by weight of water, prefer(cid:173)
`ably at most 1% by weight of water, more preferably at most
`0.5% byweightofwater, and most preferably atmost0.1% by
`weight of water.
`[0048] A solvent or anti-solvent (as used in reactions, crys(cid:173)
`tallization, etc. or lattice and/or adsorbed solvents) includes at
`least one of a C 1 to C 8 alcohol, a C2 to C 8 ether, a C3 to C7
`ketone, a c3 to c7 ester, a cl to c2 chlorocarbon, a c2 to c7
`nitrile, a miscellaneous solvent, a C5 to C 12 saturated hydro(cid:173)
`carbon, and a c6 to cl2 aromatic hydrocarbon.
`[0049] The C 1 to C 8 alcohol refers to a straight/branched
`and/or cyclic/acyclic alcohol having such number of carbons.
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`The C 1 to C8 alcohol includes, but is not limited to, methanol,
`ethanol, n-propanol, isopropanol, isobutanol, hexanol, and
`cyclohexanol.
`[0050] The C2 to C8 ether refers to a straight/branched and/
`or cyclic/acyclic ether having such number of carbons. The
`C2 to C8 ether includes, but is not limited to, dimethyl ether,
`diethyl ether, di-isopropyl ether, di-n-butyl ether, methyl+
`butyl ether (MTBE), tetrahydrofuran, and dioxane
`[0051] The C3 to C7 ketone refers to a straight/branched
`and/or cyclic/acyclic ketone having such number of carbons.
`The C3 to C7 ketone includes, but is not limited to, acetone,
`methyl ethyl ketone, propanone, butanone, methyl isobutyl
`ketone, methyl butyl ketone, and cyclohexanone.
`[0052] The C3 to C7 ester refers to a straight/branched and/
`or cyclic/acyclic ester having such number of carbons. The C3
`to C7 ester includes, but is not limited to, ethyl acetate, propyl
`acetate, n-butyl acetate, etc.
`[0053] The C 1 to C2 chlorocarbon refers to a chlorocarbon
`having such number of carbons. The c l to c2 chlorocarbon
`includes, but is not limited to, chloroform, methylene chlo(cid:173)
`ride (DCM), carbon tetrachloride, 1 ,2-dichloroethane, and
`tetrachloroethane.
`[0054] A C2 to C7 nitrile refers to a nitrile have such number
`of carbons. The C2 to C7 nitrile includes, but is not limited to,
`acetonitrile, propionitrile, etc.
`[0055] A miscellaneous solvent refers to a solvent com(cid:173)
`monly employed in organic chemistry, which includes, but is
`not limited to, diethylene glycol, diglyme (diethylene glycol
`dimethyl ether), 1,2-dimethoxy-ethane, dimethylformamide,
`dimethylsulfoxide, ethylene glycol, glycerin, hexameth(cid:173)
`ylphsphoramide, hexamethylphosphorous triame, N-methyl-
`2-pyrrolidinone, nitromethane, pyridine, triethyl amine, and
`acetic acid.
`[0056] The term C5 to C 12 saturated hydrocarbon refers to a
`straight/branched and/or cyclic/acyclic hydrocarbon. The C5
`to cl2 saturated hydrocarbon includes, but is not limited to,
`n-pentane, petroleum ether (ligroine), n-hexane, n-heptane,
`cyclohexane, and cycloheptane.
`[0057] The term C6 to C 12 aromatic refers to substituted and
`unsubstituted hydrocarbons having a phenyl group as their
`backbone. Preferred hydrocarbons include benzene, xylene,
`toluene, chlorobenzene, a-xylene, m-xylene, p-xylene,
`xylenes, with toluene being more preferred.
`[0058] The term "halo" or "halogen" as used herein,
`includes chloro, bromo, iodo and fluoro.
`[0059] The term "blocking group" refers to a chemical
`group which exhibits the following characteristics. The
`"group" is derived from a "protecting compound." Groups
`that are selective for primary hydroxyls over secondary
`hydroxy Is that can be put on under conditions consistent with
`the stability of the phosphoramidate (pH 2-8) and impart on
`the resulting product substantially different physical proper(cid:173)
`ties allowing for an easier separation of the 3'-phosphorami(cid:173)
`date-5'-new group product from the unreacted desired com(cid:173)
`pound. The group must react selectively in good yield to give
`a protected substrate that is stable to the projected reactions
`(see Protective Groups in Organic Synthesis, 3nd ed. T. W.
`Greene and P. G. M. Wuts, John Wiley & Sons, New York,
`N.Y., 1999). Examples of groups include, but are not limited
`to: benzoyl, acetyl, phenyl-substituted benzoyl, tetrahydro(cid:173)
`pyranyl,
`trityl, DMT
`(