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WO2012048013A2 - Phosphorodiamidate derivatives of guanosine nucleoside compounds for treatment of viral injections - Google Patents

Phosphorodiamidate derivatives of guanosine nucleoside compounds for treatment of viral injections Download PDF

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Publication number
WO2012048013A2
WO2012048013A2 PCT/US2011/054945 US2011054945W WO2012048013A2 WO 2012048013 A2 WO2012048013 A2 WO 2012048013A2 US 2011054945 W US2011054945 W US 2011054945W WO 2012048013 A2 WO2012048013 A2 WO 2012048013A2
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WO
WIPO (PCT)
Prior art keywords
methoxy
amino
methyltetrahydrofuran
purin
bis
Prior art date
Application number
PCT/US2011/054945
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French (fr)
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WO2012048013A3 (en
Inventor
Christopher Mcguigan
Mohamed ALJARAH
Karolina MADELA
Claire Bourdin
Sarah Jones
Stanley Chamberlain
Original Assignee
Inhibitex, Inc.
University College Of Cardiff Consultants Limited
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Publication of WO2012048013A2 publication Critical patent/WO2012048013A2/en
Publication of WO2012048013A3 publication Critical patent/WO2012048013A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • This application relates to novel nucleoside phosphorodiamidates and their use as agents for treating viral diseases.
  • Such compounds are inhibitors of RNA- dependant RNA viral replication and specifically, inhibitors of HCV NS5B polymerase.
  • As inhibitors of HCV replication such compounds are useful for treatment of hepatitis C infection in mammals.
  • HCV is a member of the Flaviviridae family of RNA viruses that affect animals and humans.
  • the genome is a single 9.6-kilobase strand of RNA, and consists of one open reading frame that encodes for a polyprotein of approximately 3000 amino acids flanked by untranslated regions at both 5' and 3' ends (5'- and 3'-UTR).
  • the polyprotein serves as the precursor to at least 10 separate viral proteins critical for replication and assembly of progeny viral particles.
  • 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 population. There are an estimated 4.5 million infected people in the United States alone, according to the U.S. Center for Disease control. According to the World Health Organization, 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 remainder can harbor HCV for the rest of their lives. [0008] 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
  • interferon alpha interferon alpha
  • ribavirin interferon alpha
  • adverse side effects such as fatigue, fever, chills, headache, myalgias, arthralgias, mild alopecia, psychiatric effects and associated disorders, autoimmune phenomena and associated disorders and thyroid dysfunction.
  • Ribavirin an inhibitor of inosine 5 '-monophosphate dehydrogenase (IMPDH)
  • Ribavirin causes significant hemolysis in 10-20% of patients treated at currently recommended doses, and the drug is both teratogenic and embryotoxic. Even with recent improvements, a substantial fraction of patients do not respond with a sustained reduction in viral load and there is a clear need for more effective antiviral therapy of HCV infection.
  • a number of other approaches are being pursued to combat the virus. They include, for example, application of antisense oligonucleotides or ribozymes for inhibiting HCV replication. Furthermore, low-molecular weight compounds that directly inhibit HCV proteins and interfere with viral replication are considered as attractive strategies to control HCV infection.
  • the NS3/4A protease/helicase and the NS5B RNA-dependent RNA polymerase are considered the most promising viral targets for new drugs.
  • WO 2003010140 relates to specific inhibitors of RNA dependent RNA polymerases, particularly viral polymerases within the Flaviviridae family, more particularly to HCV polymerase.
  • WO 200204425 incorporated by reference herein, relates to specific inhibitors of RNA dependent RNA polymerases, particularly viral polymerases within the Flaviviridae family, and more particularly the NS5B polymerase of HCV.
  • WO 200147883, incorporated by reference herein relates to specific fused-ring compounds or the like or pharmaceutically acceptable salts thereof. Such compounds and salts exhibit an anti-HCV (hepatitis C virus) activity by virtue of their inhibitory activity against HCV polymerase, thus being useful as therapeutic or preventive agents for hepatitis C.
  • HCV hepatitis C virus
  • Flaviviridae virus family and in view of the limited treatment options, there is a strong need for new effective drugs for treating infections caused by these viruses.
  • This invention is directed to novel compounds that are useful in the treatment of viral infections in mammals mediated, at least in part, by a virus in the Flaviviridae family of viruses.
  • the present invention provides for novel compounds of formula (I) having the structure:
  • R 2 , R 3 , and R 4 are each independently
  • R 7' and R 8° are each independently
  • R 9 is independently
  • Ci-Cgalkyl optionally substituted by C 3 -C 6 cycloalkyl
  • R 1 and R 2 when taken together with the N atom to which they are attached, may form
  • R 3 and R 4 when taken together with the N atom to which they are attached, may form
  • R 6 is selected from H, Ci-Cgalkyl optionally substituted by Ce-Qoaryl, and
  • R 10 and R 11 are independently selected from H, C Csalkyl optionally substituted by C6-C 10 aryl, and C -C 8 cycloalkyl;
  • the present invention includes other forms of all of the disclosed compounds including solvates, hydrates and polymorphs.
  • Such compounds are inhibitors of RNA-dependant RNA viral replication and inhibitors of HCV NS5B polymerase and thus may be utilized in methods wherein such inhibition is desired.
  • the present invention extends to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more compounds of formula I and a pharmaceutically acceptable carrier, excipient or diluent.
  • the pharmaceutically acceptable carrier, excipient or diluent may be pure sterile water, phosphate buffered saline or an aqueous glucose, solution.
  • an instant method comprises administering to a mammal that has been diagnosed with said viral infection a pharmaceutical composition comprising compounds of formula I..
  • an instant method comprises administering to a human or animal patient in need thereof an effective amount of a pharmaceutical composition comprising compounds of formula I.
  • the virus is hepatitis C virus (or HCV).
  • the present methods further extend to combination treatment comprising administration of a therapeutically effective amount of one or more agents active against hepatitis C virus.
  • agents active against hepatitis C virus may include interferon- alpha or pegylated interferon-alpha alone or in combination with ribavirin or levovirin.
  • the present invention relates to chemical compounds, their preparation and their use in the treatment of viral infections particularly in mammals. Particularly, although not exclusively, the present invention relates to chemical compounds useful as anti-hepatitis C virus (HCV) agents.
  • HCV anti-hepatitis C virus
  • the present invention describes certain nucleoside phosphorodiamidates, their synthesis, and their use as inhibitors of RNA-dependent RNA viral polymerase, particularly their use as inhibitors of hepatitis C virus (HCV) NS5-B polymerase, as inhibitors of HCV replication, and for the treatment of hepatitis C infection.
  • the compounds can also be precursors for such inhibitors.
  • the present invention relates to novel compounds of formula (I) having the structure:
  • R 2 , R 3 , and R 4 are each independently
  • R 7' and R 8° are each independently
  • Ci-Cgalkyl Ci-C 3 alkylaryl
  • R 9 is independently
  • R 1 and R 2 when taken together with the N atom to which they are attached, may form
  • R 3 and R 4 when taken together with the N atom to which they are attached, may form
  • R 5 is selected from CI, X'R 6 and NR 10 R U ,
  • X' is O or S
  • R 6 is selected from H, CrCsalkyl optionally substituted by C6-C 10 aryl, and
  • R 10 and R 11 are independently selected from H, CrCgalkyl optionally substituted by C6-C 10 aryl, and Cs-Cgcycloalkyl;
  • the present invention includes other forms of all of the disclosed compounds including solvates, hydrates and polymorphs.
  • the above compounds are provided along with their phosphorus diastereomers.
  • phosphorous diastereomers may be possible in cases where Rl and R2 are not identical to R3 and R4.
  • (2S,2'S)-bis(3,3-dimethylbutyl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
  • compounds in accordance with the present invention include:
  • alkyl refers to a straight or branched saturated
  • cyclic or acyclic hydrocarbon radical having the number of carbon atoms as indicated (or where not indicated, an acyclic alkyl group preferably has 1-20, more preferably 1-6, more preferably 1-4 carbon atoms and a cyclic alkyl group preferably has 3-20, preferably 3-10, more preferably 3-7 carbon atoms), optionally substituted with one, two, three or more substituents independently selected from the group set out above.
  • suitable alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, isopropyl, 2-butyl, cyclopropyl, cyclohexyl, cyclopentyl and dodecyl.
  • C3-C 8 cycloalkyl refers to cyclic alkyl group comprising from about 3 to about 8 C atoms.
  • Cs-Cscycloalkyl-alkyl refers to an acyclic alkyl group substituted by a cyclic alkyl group comprising from about 3 to about 8 C atoms.
  • suitable alkenyl groups include vinyl, propenyl, butenyl, pentenyl and hexenyl.
  • alkynyl refers to a straight or branched unsaturated monovalent acyclic or cyclic hydrocarbon radical having one or more triple C/C bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic alkynyl group preferably has 2-20, more preferably 2-6, more preferably 2-4 carbon atoms and a cyclic alkynyl group preferably has 7-20, more preferably 8-20 carbon atoms), optionally substituted with one, two, three or more substituents independently selected from the group set out above.
  • alkoxy refers to the group alkyl- 0-, where alkyl is as defined above and where the alkyl moiety may optionally be substituted by one, two, three or more substituents as set out above for alkyl.
  • suitable alkoxy groups include methoxy, ethoxy, n- propoxy, isopropoxy, n- butoxy, ie/t-butoxy, sec-butoxy, w-pentoxy, w-hexoxy and 1,2-dimethylbutoxy.
  • cycloalkyloxy refers to the group cyclicalkyl - 0-, where cyclicalkyl is as defined above and where the cyclicalkyl moiety may be optionally substituted by one, two, three or more substituents as set out above for alkyl.
  • alkylthio refers the group alkyl-S-, where alkyl is as defined above and where the alkyl moiety may optionally be substituted by one, two, three or more substituents as set out above for alkyl.
  • suitable alkylthio groups include methylthio, ethylthio, n- propylthio, isopropylthio, w-butylthio, iert-butylthio, sec-butylthio, w-pentylthio, w-hexoxy and 1,2-dimethylbutylthio.
  • aryloxy refers to the group aryl-O-, where aryl is as defined below and where the aryl moiety may optionally be substituted by one, two, three or more substituents as set out above with respect to the group Ar.
  • alkoxyalkyl refers to an alkyl group having an alkoxy substituent. Binding is through the alkyl group.
  • the alkyl moiety and the alkoxy moiety are as defined herein with respect to the definitions of alkyl and alkoxy, respectively.
  • the alkoxy and alkyl moieties may each be substituted by one, two, three or more substituents as set out above with regard to the definition of alkyl.
  • alkylthioalkyl refers to an alkyl group having an
  • alkylthio substituent Binding is through the alkyl group.
  • the alkyl moiety and the alkylthio moiety are as defined herein with respect to the definitions of alkyl and alkylthio, respectively.
  • the alkylthio and alkyl moieties may each be substituted by one, two, three or more substituents as set out above with regard to the definition of alkyl.
  • alkoxyaryl refers to an aryl group having an alkoxy substituent. Binding is through the aryl group.
  • the alkoxy moiety and the aryl moiety are as defined herein with respect to the definitions of alkoxy and aryl, respectively.
  • the alkoxy and aryl moieties may each be substituted by one, two, three or more substituents, as defined herein with regard to the definitions of alkoxy and aryl, respectively.
  • cycloalkylaryl refers to an aryl group having a cyclic alkyl substituent. Binding is through the aryl group.
  • the cycloalkyl moiety and the aryl moiety are as defined herein with respect to the definitions of cycloalkyl and aryl, respectively.
  • aryl(C 1 -C 6 )alkyl- refers to a CrC 6 alkyl group substituted at any carbon by an aryl group. Binding is through the alkyl group.
  • the aryl moiety and the alkyl moiety are as defined herein with respect to the definitions of aryl and alkyl.
  • the aryl group may be substituted.
  • suitable aryl(C 1 -C 6 )alkyl- groups include benzyl, 1- phenylethyl, 3-phenylpropyl, 4-chlorobenzyl, 4-fluorobenzyl, 2,4-difluorobenzyl, and the like.
  • the alkyl moiety is as defined hereinabove.
  • a cycloalkyl moiety and the aryl moiety may each be optionally substituted by one, two, three or more substituents as set out herein with regard to the definitions of alkyl and aryl, respectively.
  • aryl refers to a monovalent unsaturated aromatic
  • aryl group may optionally be substituted by one, two, three or more substituents as set out above with respect to optional substituents that may be present on the group Ar.
  • Preferred aryl groups are: an aromatic monocyclic ring containing 6 carbon atoms; an aromatic bicyclic or fused ring system containing 7, 8, 9 or 10 carbon atoms; or an aromatic tricyclic ring system containing 10, 11, 12, 13 or 14 carbon atoms.
  • Non-limiting examples of aryl include phenyl and naphthyl.
  • heterocycloalkyl refers to a saturated or partially
  • unsaturated heterocyclic ring system having one, two, three, four, five or six rings, preferably one, two or three rings, which may be fused or bicyclic, and having contained within the ring or rings at least one member selected from the group consisting of N, O and S.
  • C5-C 2 o" or “C5-C used before heterocycloalkyl means, respectively, a five to twenty or a five to ten-membered ring system at least one of which members is selected from the group consisting of N, O and S.
  • Preferred heterocycloalkyl systems are: a monocyclic ring system having five members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; a monocyclic ring having six members of which one, two or three members are a N or O atom; a bicyclic ring system having nine members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms; or a bicyclic ring system having ten members of which one, two or three members are a N atom.
  • suitable heterocycloalkyl groups include tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothiopyranyl, thiomorpholinyl, and piperidinyl
  • indanyl refers to the fused bicyclic radical of structure, point of attachment of the radical to the rest of the molecule is on any available non-aromatic carbon atom.
  • heteroatom substituents are selected from oxygen, nitrogen, sulphur and halogen (F, CI, Br and I).
  • halogen F, CI, Br and I
  • the ring(s) is substituted with one or more heteroatoms, preferably there are 1, 2, 3 or 4 heteroatom substituents selected from the group consisting of oxygen, nitrogen and/or halogen.
  • Preferred substituent groups are independently selected from hydroxy, acyl, acyloxy, nitro, amino, S0 3 H, SH, SR', wherein R' is independently selected from the same groups as R; carboxyl, cyano, (C 1 -C 6 )alkylamino, (C 1 -C 6 )dialkylamino, thiol, chloro, bromo, fluoro and iodo.
  • the compounds of this invention contain one or more chiral centers.
  • stereoisomers i.e., as individual enantiomers or diastereomers, or as stereoisomer- enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
  • the compounds of this invention may contain one or more asymmetric centers, depending upon the location and nature of the various substituents desired.
  • Asymmetric carbon atoms or phosphorous atoms may be present in the (R) or (S) configuration or (R,S) configuration. In certain instances, asymmetry may also be present due to restricted rotation about a given bond, for example, the central bond adjoining two substituted aromatic rings of the specified compounds.
  • Substituents on a ring may also be present in either cis or trans form, and a substituent on a double bond may be present in either Z or E form. It is intended that all such configurations (including enantiomers and diastereomers) are included within the scope of the present invention.
  • Preferred compounds are those with the absolute configuration of the compound of this invention which produces the more desirable biological activity.
  • Separated, pure or partially purified isomers or racemic mixtures of the compounds of this invention are also included within the scope of the present invention.
  • Such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention.
  • Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art.
  • racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like, also well-known in the art and exemplified the experimental examples below.
  • pharmaceutically acceptable salt refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
  • “pharmaceutically acceptable salts” refers to compounds having a substituent capable of having more than one group form a salt but less than the maximum amount of such groups actually form a salt.
  • a diphospho group can form a plurality of salts and, if only partially ionized, the resulting group is sometimes referred to herein as a partial salt.
  • polymers arrived at by defining substituents with further substituents to themselves e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, etc.
  • the maximum number of such substituents is three.
  • each of the above definitions is constrained by a limitation that, for example, substituted aryl groups are limited to -substituted aryl- (substituted aryl)-substituted aryl.
  • the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 fluoro groups or a hydroxyl group pendent to a carbon atom of an ethenylic or acetylenic unsaturation). Such impermissible substitution patterns are well known to the skilled artisan.
  • the present compounds include other known forms of the compounds including solvates, hydrates and polymorphs.
  • the compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.
  • the starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof.
  • many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce or Sigma (St. Louis, Mo., USA).
  • Reaction Scheme 1 illustrates a general method of preparation of symmetrical phosphorodiamidates.
  • Application PCT/US 10/20632 is dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent and cooled to below ambient temperature, preferably to 0-5°C.
  • Phosphorus oxychloride phosphoryl chloride
  • the reaction is stirred for 1-48 h at temperatures from -20 °C to 20 °C and optimally for 24 h at 5 °C forming compound (III).
  • the solution is diluted with an aprotic solvent, preferably DCM, and a primary or secondary amine, such as the HC1 or tosylate salt of an amino acid ester, is added at reduced temperatures -78 °C to 5 °C and preferably at 0 °C.
  • a primary or secondary amine such as the HC1 or tosylate salt of an amino acid ester
  • a non-nucleophilic base such as a tertiary amine such as triethylamine, or preferably diisopropylethylamine.
  • the solution is stirred for 1 h to 10 days at reduced temperatures and preferably at 5 °C for 5 days, forming phosphorodiamidate (I).
  • the nucleoside (II) can be dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF, and a non- nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added and stirred for a period of 5 min to 1 h, preferably 30 min.
  • a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF
  • a non- nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added and stirred for a period of 5 min to 1 h, preferably 30 min.
  • the solution is then cooled to -100 °C to rt, or preferably -78 °C, and phosphorus oxychloride (phosphoryl chloride) of high quality is added
  • the reaction is stirred for 5 min to 2 h at temperatures from -100 °C to 0°C and optimally for 30 min at -78 °C, then warmed to ambient temperature for 5 min to 2 h, preferably 30 min forming compound (III).
  • the solution is further diluted with an aprotic solvent, preferably DCM, and a primary or secondary amine, such as the HC1 or tosylate salt of an amino acid ester, is added followed by the addition of a non-nucleophilic base such as a tertiary amine preferably triethylamine at reduced temperatures -78 °C to 5 °C and preferably at -78 °C.
  • reaction Scheme 2 illustrates a general method of preparation of asymmetrical phosphoramidates.
  • nucleoside (II) can be dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF, and a non-nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added and stirred for a period of 5 min to 1 h, preferably 30 min.
  • a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF
  • a non-nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added and stirred for a period of 5 min to 1 h, preferably 30 min.
  • the solution is then cooled to -100 °C to rt, or preferably -78 °C, and phosphorus oxychloride (phosphoryl chloride) of high quality is added slowly to the solution with careful protection from moisture.
  • the reaction is stirred for 5 min to 2 h at temperatures ranging from -100 °C to 0 °C and optimally for 30 min at -78 °C, then warmed to ambient temperature for 5 min to 2 h, preferably 30 min forming compound (III).
  • the solution is further diluted with an aprotic solvent, preferably DCM, and one equivalent of a primary or secondary amine of formula (V), such as the HC1 or tosylate salt of an amino acid ester, is added followed by the addition of a non-nucleophilic base such as a tertiary amine preferably triethylamine at reduced temperatures -78 °C to 5 °C and preferably at -78°C.
  • a tertiary amine preferably triethylamine at reduced temperatures -78 °C to 5 °C and preferably at -78°C.
  • the solution is warmed to ambient temperature and stirred for 1 h to 48 h, preferably 24 h, forming phosphorodiamidate (IV).
  • a phosphorus NMR can be acquired to determine the status of the reaction.
  • the solution is then cooled to -100 °C to rt, or preferably -78 °C.
  • a primary or secondary amine of formula (VI) such as the HCl or tosylate salt of an amino acid ester
  • a non-nucleophilic base such as a tertiary amine, preferably triethylamine (5-10 equivalents) at reduced temperatures -78 °C to 5 °C and preferably at -78 °C.
  • the solution is warmed to ambient temperature and stirred for 1 h to 48 h, preferably 24 h, forming phosphorodiamidate (I).
  • reaction Scheme 3 illustrates an alternative method for synthesizing asymmetrical phosphorodiamidates.
  • This scheme describes a second general method for synthesizing asymmetrical phosphorodiamidates.
  • the nucleoside (II) can be dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF.
  • a non-nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added in excess, preferably 1.2 equivalents.
  • the solution can be stirred at ambient temperature and 1 to 3 equivalents of an amino acid ester dichloridate (VII), can be added.
  • Compounds of the general structure (VII) can be synthesized using techniques familiar to one skilled in the art.
  • a phosphorus NMR can be acquired to determine the status of the formation of the compound of formula (IV)
  • the solution is then cooled to -100 °C to rt, or preferably -78 °C, and 1 to 10 equivalents, preferably 5 equivalents, of a primary or secondary amine of formula (VI) are added.
  • the solution is warmed to ambient temperature and stirred for 1 h to 48 h, preferably 24 h, forming phosphorodiamidate (I).
  • the reaction is worked up using standard methods familiar to one skilled in the art, for example extraction with a sodium chloride solution, drying with sodium sulfate and purification by silica gel chromatography. Changes to this procedure including solvent switches and optimization of the temperature, familiar to those skilled in the art of organic chemistry would be anticipated.
  • Suitable solvents include hydrocarbon solvents such as benzene and toluene; ether type solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene; halogenated hydrocarbon solvents such as methylene chloride, chloroform and chlorobenzene; ketone type solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone; alcohol type solvents such as methanol, ethanol, propanol, isopropanol, w-butyl alcohol and iert-butyl alcohol; nitrile type solvents such as acetonitMRI, propionitrile and benzonitrile; ester type solvents such as ethyl acetate and butyl acetate; carbonate type solvents such as ethylene carbonate and propylene carbonate; and
  • inert solvent means a solvent inert under the conditions of the reaction being described in conjunction therewith including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride (or dichloromethane), diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, ie/t-butanol, dioxane, pyridine, and the like.
  • inert solvent means a solvent inert under the conditions of the reaction being described in conjunction therewith including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride (or dichloromethane), diethyl ether, ethyl acetate, acetone, methyleth
  • the effective amount will be that amount of the compound of this invention that would be understood by one skilled in the art to provide therapeutic benefits, i.e., the active ingredient, and will thus depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors.
  • the drug can be administered more than once a day, and in the preferred mode the drug is administered once or twice a day. As indicated above, all of the factors to be considered in determining the effective amount will be well within the skill of the attending clinician or other health care professional..
  • therapeutically effective amounts of compounds of Formula I may range from approximately 0.05 to 50 mg per kilogram body weight of the recipient per day; preferably about 0.1-25 mg/kg/day, more preferably from about 0.5 to 10 mg/kg/day. Thus, for administration to a 70 kg person, the dosage range would most preferably be about 35-700 mg per day.
  • compounds of this invention will be administered as pharmaceutical compositions by any one of the following routes: oral, systemic (e.g., transdermal, intranasal or by suppository), or parenteral (e.g., intramuscular, intravenous or subcutaneous) administration.
  • the preferred manner of administration is oral using a convenient daily dosage regimen that can be adjusted according to the degree of affliction.
  • compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions.
  • Another preferred manner for administering compounds of this invention is inhalation. This is an effective method for delivering a therapeutic agent directly to the respiratory tract (see U.S. Pat. No. 5,607,915, said patent incorporated herein by reference).
  • the choice of formulation depends on various factors such as the mode of drug administration and bioavailability of the drug substance.
  • the compound can be formulated as liquid solution, suspensions, aerosol propellants or dry powder and loaded into a suitable dispenser for administration.
  • suitable dispenser for administration There are several types of pharmaceutical inhalation devices- nebulizer inhalers, metered dose inhalers (MDI) and dry powder inhalers (DPI).
  • MDI metered dose inhalers
  • DPI dry powder inhalers
  • Nebulizer devices produce a stream of high velocity air that causes the therapeutic agents (which are formulated in a liquid form) to spray as a mist that is carried into the patient's respiratory tract.
  • MDI's typically are formulation packaged with a compressed gas.
  • the device Upon actuation, the device discharges a measured amount of therapeutic agent by compressed gas, thus affording a reliable method of administering a set amount of agent.
  • DPI dispenses therapeutic agents in the form of a free flowing powder that can be dispersed in the patient's inspiratory air- stream during breathing by the device.
  • the therapeutic agent In order to achieve a free flowing powder, the therapeutic agent is formulated with an excipient such as lactose.
  • a measured amount of the therapeutic agent is stored in a capsule form and is dispensed with each actuation.
  • compositions in accordance with the invention generally comprise a compound of formulas (I- VI) in combination with at least one pharmaceutically acceptable carrier, excipient or diluent.
  • excipients are those that are non-toxic, will aid administration, and do not adversely affect the therapeutic benefit of the compound of the invention.
  • excipient may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.
  • Solid pharmaceutical excipients useful in the invention may include starch,
  • Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • Preferred liquid carriers, particularly for injectable solutions include water, saline, aqueous dextrose, and glycols.
  • Compressed gases may be used to disperse a compound of this invention in aerosol form.
  • Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc.
  • Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).
  • the amount of the compound in a formulation can vary within the full range employed by those skilled in the art.
  • the formulation will contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt % wherein the compound is a compound of formula I based on the total formulation, with the balance being one or more suitable pharmaceutical excipients.
  • the compound is present at a level of about 1-80 wt %.
  • the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention in combination with a therapeutically effective amount of another active agent against RNA-dependent RNA virus and, in particular, against HCV.
  • Agents active against HCV include, but are not limited to, ribavirin, levovirin, viramidine, thymosin alpha- 1, an inhibitor of HCV NS3 serine protease, interferon-a, pegylated interferon-a (peginterferon-a), a combination of interferon-a and ribavirin, a combination of peginterferon-a and ribavirin, a combination of interferon-a and levovirin, and a combination of peginterferon- ⁇ and levovirin.
  • Interferon- ⁇ includes, but is not limited to, recombinant interferon-a2a (such as Roferon interferon available from Hoffman-LaRoche, Nutley, N.J.), interferon- a2b (such as Intron-A interferon available from Schering Corp., Kenilworth, N.J., USA), a consensus interferon, and a purified interferon- ⁇ product.
  • interferon-a2a such as Roferon interferon available from Hoffman-LaRoche, Nutley, N.J.
  • interferon-a2b such as Intron-A interferon available from Schering Corp., Kenilworth, N.J., USA
  • a consensus interferon such as Intron-A interferon available from Schering Corp., Kenilworth, N.J., USA
  • the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention in combination with a therapeutically effective amount of another agent active against hepatitis C virus.
  • agents include those that inhibit HCV proteases, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and inosine 5'-monophosphate dehydrogenase.
  • Other agents include nucleoside analogs for the treatment of an HCV infection.
  • Still other compounds include those disclosed in WO 2004/014313 and WO 2004/014852 and in the references cited therein.
  • Specific antiviral agents include Omega IFN (BioMedicines Inc.), BILN-2061 (Boehringer Ingelheim), Summetrel (Endo Pharmaceuticals Holdings Inc.), Roferon A (F. Hoffman-La Roche), Pegasys (F. Hoffman-La Roche), Pegasys/Ribaravin (F. Hoffman-La Roche), CellCept (F.
  • compositions and methods of the present invention contain a compound of formula I- VI and interferon.
  • the interferon is selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
  • compositions and methods of the present invention utilize a combination of a compound of formula I- VI and a compound having anti- HCV activity such as those selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti- sense RNA, Imiqimod, ribavirin, an inosine 5' monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
  • a compound having anti- HCV activity such as those selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti- sense RNA, Imiqimod, ribavirin, an inosine 5' monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
  • WO 97/12033 relates to HCV polymerase assay that can be used to evaluate the activity of the of the compounds described herein.
  • Another HCV polymerase assay has been reported by Bartholomeusz, et al., Hepatitis C Virus (HCV) RNA polymerase assay using cloned HCV non- structural proteins; Antiviral Therapy 1996: 1 (Supp 4) 18-24.
  • HCV Hepatitis C Virus
  • dichloromethane were purchased from Aldrich and used directly.
  • the sugar derivative (2 l S',3R,4R,5R)-5-(benzoyloxymethyl)-3-methyltetrahydrofuran-2,3,4- triyl tribenzoate or equivalently: 2,3,4,5-tetra-0-benzoyl-2-C-methyl- ?-D- ribofuranose was purchased from CarboSynth Limited, 8&9 Old Station Business Park, Compton, Berkshire, RG20 6NE, UK.
  • the purine derivative 2-amino-6- chloropurine or equivalently, 6-chloro-9H-purin-2-amine was purchased from Aldrich.
  • 2'-C-Methylguanosine (2-amino-9-((3R,4R,5R)-3,4-dihydroxy-5- (hydroxymethyl)-3-methyltetrahydrofuran-2-yl)- lH-purin-6(9H)-one) is a commercial reagent and was purchased from CarboSynth Limited, 8&9 Old Station Business Park, Compton, Berkshire, RG20 6NE, UK. Salts of amino acid esters were prepared as described in PCT Int. Appl. (2010), WO 2010081082 A2 20100715. Column chromatography refers to flash column chromatography carried out using Merck silica gel 60 (40-60 ⁇ ) as stationary phase.
  • Coupling constants are referred to as J values.
  • Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), broad signal (br), doublet of doublet (dd), doublet of triplet (dt), or multiplet (m).
  • the electrospray source was operated at a temperature of 130 °C with a desolvation temperature of 300 °C, a capillary voltage of 3 kV, and cone voltage of 30 V. Data were collected in the continuum mode over the mass range 100-2000 amu and processed using Masslynx 4.1 software. Accurate mass measurements were facilitated by the introduction of a single lockmass compound of known elemental composition into the source concurrently with sample.
  • the reaction mixture was then heated at 65 °C for 4 to 6 h, allowed to cool down to room temperature, poured into saturated aqueous sodium bicarbonate (300 mL), and extracted with dichloromethane (3x150 mL). The combined organic phase was dried over sodium sulfate and evaporated under reduced pressure. The residue was precipitated from dichloromethane and methanol, filtrated, the solid was washed 2 times with methanol and dried to give the desired compound (8.5 g, 79 %) as a white solid (yields are from 65% (column) up to 90% (precipitation)).
  • Method D Asymmetrical phosphorodiamidates using amino acid ester phosphordichloridate
  • anhyd dichloromethane (4 mL), the hydrochloride salt of propyloxy-L- alanine (673 g, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one.
  • silica gel column chromatography and preparative TLC 36 mg of the prodrug was obtained in 7.2% yield as an off white solid.
  • the phosphorodiamidate prodrug was synthesized using Method B.
  • a suspension of 6-0-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 ⁇ , 0.964 mmole) and phosphoryl chloride (89 ⁇ , 0.964 mmole).
  • Anhydrous dichloromethane (4 mL) the tosylate salt of isobutyloxy-L-alanine (1.27 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added.
  • silica gel column chromatography and preparative HPLC 66.1 mg of the prodrug was obtained in 13% yield as an off white solid.
  • the phosphorodiamidate prodrug was synthesized using Method B.
  • a suspension of 6-0-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 ⁇ , 0.964 mmole) and phosphorus oxychloride (89 ⁇ , 0.964 mmole).
  • Anhydrous dichloromethane (4 mL) the HCl salt of cyclobutyl-L-alanine (722.2 mg, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the previous mixture.
  • silica gel column chromatography and preparative HPLC 65.1 mg of the phosphorodiamidate was obtained in 13% yield as an off white solid.
  • anhyd dichloromethane (4 mL), the hydrochloride salt of cyclohexyloxy-L-alanine (834 mg, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one.
  • 36.0 mg of the prodrug was obtained in 6.4% yield, as an off white solid.
  • anhyd dichloromethane (4 mL), the tosylate salt of (2 l S')-phenylethyloxy-L-alanine (1.46 g, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one.
  • silica gel column chromatography and preparative TLC 27.1 mg of the prodrug was obtained in 4.5% yield as an off white solid.
  • Example 20 [00346]
  • the compound was prepared according to Method B from (2R,3R,4R,5R)-2-(2- amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran- 3,4-diol, (Example 2, 250 mg, 0.80 mmol), POCl 3 (0.07 mL, 0.80 mmol), E t3 N (0.11 mL, 0.80 mmol), L-alanine 2-indanolyl ester tosylate salt (0.91g, 2.41 mmol), and Et 3 N (0.67 mL, 4.82 mmol) in 10 mL of 1 : 1 mixture of dry THF and DCM. Crude product was purified by column chromatography using a gradient of CHC1 3 to CHC1 3 : MeOH 95:5, to give a pure product as a white foam (52 mg, 9%).
  • the phosphorodiamidate prodrug was synthesized using Method C. [00369] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 ⁇ , 0.964 mmole) and phosphorus oxychloride (89 ⁇ , 0.964 mmole). Anhydrous dichloromethane (4 mL), the HCl salt of cyclohexyloxy-L-alanine (167 mg, 0.803 mmol) and triethylamine (224 ⁇ , 1.61 mmole) were added.
  • Example 23 [00374] (25 ieri-Butyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(( l S')-l-(neopentyloxy)- l- oxopropan-2-ylamino)phosphorylamino)propanoate
  • Example 24 [00383] (25 Methyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(( l S , )-l-(neopentyloxy)- l- oxopropan-2-ylamino)phosphorylamino)-3-methylbutanoate
  • Example 25 Methyl l-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(( l S')-l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphoryl)pyrrolidine-2-carboxylate
  • the tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the mixture.
  • 71 mg of the prodrug was obtained in 14% yield as an off white solid.
  • the phosphorodiamidate prodrug was synthesized using Method C.
  • a suspension of 6-0-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 ⁇ , 0.964 mmole) and phosphorus oxychloride (89 ⁇ , 0.964 mmole).
  • aniline 73 ⁇ , 0.803 mmole
  • triethylamine (112 ⁇ , 0.803 mmole
  • HPLC i R 15.08 and 15.31 min (column: Varian Pursuit XRs 5, C 18 , 150x4.6 mm; method: linear gradient of ACN (10% to 100%) in H 2 0 in 30 min).
  • the tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the mixture.
  • silica gel column chromatography and preparative HPLC 9.3 mg of the prodrug was obtained in 2% yield as an off white solid.
  • HPLC t R 23.49 and 23.75 min (column: Varian Pursuit XRs 5, C 18 , 150x4.6 mm; method: linear gradient of CH 3 OH (10% to 100%) in H 2 0 in 30 min).
  • Step 2 The crude mixture from the above step was dissolved in dioxane (500 mL) followed by addition of HC1 (4M in dioxane) (5eq, 612 mL) at 0 °C and heated to 40 °C for 15 hrs. This mixture was cooled to room temperature and the volatiles were removed under reduced pressure. Trace amounts of dioxane was removed by azetroping with toluene (2 X 200 mL). To this solid was added anhydrous diethyl ether (1L) and stirred vigorously for lhr. The solids were filtered off and dried under vacuum to afford 87.3 gm of D-alanine neopentyl ester hydrochloride salt (3 in above scheme).
  • Example 37 [00515] (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- amino-4-methylthiobutanoate
  • anhyd dichloromethane (4 mL), the tosylate salt of neopentyloxy-L-methionine (1.41 g, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one.
  • silica gel column chromatography and preparative TLC 7 mg of the prodrug was obtained in 1.1% yield as an off white solid.
  • Example 42 [00559] (25,2'5)-Dibenzyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-bis-pyrrolidine-2- methanoate
  • anhyd dichloromethane (2.4 mL), the hydrochloride salt of benzyloxy-L-proline (583 mg, 2.41 mmol) and diisopropylethylamine (840 ⁇ , 4.82 mmol) were added to the mixture obtained in step one.
  • silica gel column chromatography and preparative TLC 8.4 mg of the prodrug was obtained in 2.3% yield as an off white solid.
  • HPLC t R 18.57 min (Varian Polaris C18-A (10 ⁇ ) column using a mobile phase of water/acetonitrile ingradient (90/10 to 0/100 v/v in 30 min, System 1)
  • the phosphorodiamidate was prepared according to Method C.
  • a solution of 6-O-methyl-2' -C-methylguanosine (550 mg, 1.76 mmol) in anhydrous tetrahydrofuran (8 mL) was allowed to react with triethylamine (300 ⁇ L, 2.11 mmol) and phosphorus oxychloride (200 ⁇ L, 2.11 mmole).
  • L-alanine cyclohexyl ester tosylate salt 370 mg, 1.76 mmol
  • triethylamine 0.5 mL, 3.52 mmol
  • Pentylamine (0.56 mL, 3.21mmoles) and triethylamine (0.67 mL, 3.21 mmol) were added. After work-up and silica gel column chromatography, 11 mg of phosphorodiamidate was obtained in 3% yield as an off white solid.
  • the phosphorodiamidate was prepared according to Method B.
  • a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol 300 mg, 0.96 mmol
  • anhydrous tetrahydrofuran 5 mL
  • triethylamine 130 ⁇ L, 0.96 mmol
  • phosphorus oxychloride 90 ⁇ L, 0.96 mmol
  • a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol 150 mg, 0.47 mmol
  • anhydrous tetrahydrofuran 2.5 mL
  • triethylamine 80 ⁇ , 0.56 mmol
  • phosphorus oxychloride 50 ⁇ , 0.56 mmol
  • a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol 180 mg, 0.57 mmol
  • anhydrous tetrahydrofuran 3.0 mL
  • triethylamine 100 ⁇ , 0.69 mmol
  • phosphorus oxychloride 70 ⁇ , 0.69 mmol
  • the phosphorodiamidate was prepared according to the Method B. [00722] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (310 mg, 1.01 mmol) in anhydrous tetrahydrofuran (4.3 mL) was reacted with triethylamine (170 ⁇ L, 1.21 mmol) and phosphorus oxychloride (110 ⁇ L, 1.21 mmol).
  • a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol 300 mg, 0.96 mmol
  • anhydrous tetrahydrofuran 5 mL
  • triethylamine 130 ⁇ , 0.96 mmol
  • phosphorus oxychloride 90 ⁇ , 0.96 mmol
  • the phosphorodiamidate was prepared according to Method B. [00750] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 ⁇ , 0.96 mmol) and phosphorus oxychloride (90 ⁇ , 0.96 mmol).
  • a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol 300 mg, 0.96 mmol
  • anhydrous tetrahydrofuran 5 mL
  • triethylamine 130 ⁇ , 0.96 mmol
  • phosphorus oxychloride 90 ⁇ , 0.96 mmol
  • the phosphorodiamidate was prepared according to Method B. [00770] In a first step, a suspension of 6-O-methyl-2' -C-methylguanosine (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 ⁇ L, 0.96 mmol) and phosphorus oxychloride (90 ⁇ L, 0.96 mmol).
  • a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol 300 mg, 0.96 mmol
  • anhydrous tetrahydrofuran 5 mL
  • triethylamine 130 ⁇ , 0.96 mmol
  • phosphorus oxychloride 90 ⁇ , 0.96 mmol
  • the phosphorodiamidate was prepared according to Method B. [00799] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (500 mg, 1.50 mmol) in anhydrous tetrahydrofuran (8 mL) was reacted with triethylamine (0.25 mL, 1.80 mmol) and phosphoryl chloride (0.13mL, 1.50 mmol).

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Abstract

This invention is directed to novel compounds of formula (I) having the structure that are useful in the treatment of viral infections in mammals mediated, at least in part, by a virus in the Flaviviridae family of viruses. Methods of treating viral infections via administration of these compounds are also disclosed.

Description

PHOSPHORODIAMIDATE DERIVATIVES OF GUANOSINE NUCLEOSIDE COMPOUNDS FOR TREATMENT OF VIRAL INFECTIONS
[0001] CROSS REFERENCE TO RELATED APPLICATION
[0002] The present application claims the benefit of U.S. Provisional Application Ser. No.
61/390,357, filed October 6, 2010, the entire disclosure of which is incorporated herein by reference.
[0003] FIELD OF THE INVENTION
[0004] This application relates to novel nucleoside phosphorodiamidates and their use as agents for treating viral diseases. Such compounds are inhibitors of RNA- dependant RNA viral replication and specifically, inhibitors of HCV NS5B polymerase. As inhibitors of HCV replication, such compounds are useful for treatment of hepatitis C infection in mammals.
[0005] BACKGROUND OF THE INVENTION
[0006] HCV is a member of the Flaviviridae family of RNA viruses that affect animals and humans. The genome is a single 9.6-kilobase strand of RNA, and consists of one open reading frame that encodes for a polyprotein of approximately 3000 amino acids flanked by untranslated regions at both 5' and 3' ends (5'- and 3'-UTR). The polyprotein serves as the precursor to at least 10 separate viral proteins critical for replication and assembly of progeny viral particles.
[0007] 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 population. There are an estimated 4.5 million infected people in the United States alone, according to the U.S. Center for Disease control. According to the World Health Organization, 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 remainder can harbor HCV for the rest of their lives. [0008] 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
[0009] At present, the standard treatment for chronic HCV is interferon alpha (IFN-alpha) in combination with ribavirin, which requires at least six (6) months of treatment. However, treatment of HCV with interferon has frequently been associated with adverse side effects such as fatigue, fever, chills, headache, myalgias, arthralgias, mild alopecia, psychiatric effects and associated disorders, autoimmune phenomena and associated disorders and thyroid dysfunction.
[0010] Ribavirin, an inhibitor of inosine 5 '-monophosphate dehydrogenase (IMPDH),
enhances the efficacy of IFN-alpha in the treatment of HCV. Despite the introduction of ribavirin, more than 50% of the patients do not eliminate the virus with the current standard therapy of interferon- alpha and ribavirin. By now, standard therapy of chronic hepatitis C has been changed to the combination of pegylated IFN-alpha plus ribavirin. However, a number of patients still have significant side effects, primarily related to ribavirin.
[0011] Ribavirin causes significant hemolysis in 10-20% of patients treated at currently recommended doses, and the drug is both teratogenic and embryotoxic. Even with recent improvements, a substantial fraction of patients do not respond with a sustained reduction in viral load and there is a clear need for more effective antiviral therapy of HCV infection.
[0012] A number of other approaches are being pursued to combat the virus. They include, for example, application of antisense oligonucleotides or ribozymes for inhibiting HCV replication. Furthermore, low-molecular weight compounds that directly inhibit HCV proteins and interfere with viral replication are considered as attractive strategies to control HCV infection. Among the viral targets, the NS3/4A protease/helicase and the NS5B RNA-dependent RNA polymerase are considered the most promising viral targets for new drugs.
[0013] A number of patents disclose and claim inventions relating to HCV NS5B inhibitors.
For example, WO 2006/046039, WO 2006/046030 and WO 2006/029912, incorporated by reference herein, relate to tetracyclic indole compounds and pharmaceutically acceptable salts thereof, for the treatment or prevention of infection by hepatitis C virus. WO 2005/080399, incorporated by reference herein, relates to fused hetero tetracyclic compounds, pharmaceutically acceptable salts thereof; and their use in aiding to remedy hepatitis C infection as potent (HCV) polymerase inhibitors. WO 2003007945, incorporated by reference herein, relates to HCV NS5B inhibitors. Further, WO 2003010140, incorporated by reference herein, relates to specific inhibitors of RNA dependent RNA polymerases, particularly viral polymerases within the Flaviviridae family, more particularly to HCV polymerase. WO 200204425, incorporated by reference herein, relates to specific inhibitors of RNA dependent RNA polymerases, particularly viral polymerases within the Flaviviridae family, and more particularly the NS5B polymerase of HCV. WO 200147883, incorporated by reference herein, relates to specific fused-ring compounds or the like or pharmaceutically acceptable salts thereof. Such compounds and salts exhibit an anti-HCV (hepatitis C virus) activity by virtue of their inhibitory activity against HCV polymerase, thus being useful as therapeutic or preventive agents for hepatitis C.
[0014] However, in view of the worldwide epidemic level of HCV and other members of the
Flaviviridae virus family, and in view of the limited treatment options, there is a strong need for new effective drugs for treating infections caused by these viruses.
[0015] SUMMARY OF THE INVENTION
[0016] This invention is directed to novel compounds that are useful in the treatment of viral infections in mammals mediated, at least in part, by a virus in the Flaviviridae family of viruses. According to some embodiments, the present invention provides for novel compounds of formula (I) having the structure:
Figure imgf000004_0001
(I)
wherein R2, R3, and R4 are each independently
Figure imgf000005_0001
wherein
R 7' and R 8° are each independently
hydrogen,
Ci-Cgalkyl,
Ci-Csalkylaryl,
CH3XCH2-,
CH3XCH2CH2- ,
R9XC(0)CH2- benzyl,
benzyl optionally substituted by halogen,
benzyl optionally substituted by Q-Cgalkyl benzyl optionally substituted by Q-Cealkoxy phenyl,
phenyl optionally substituted by halogen,
phenyl optionally substituted by Q-Cgalkyl or phenyl optionally substituted by Q-Cealkoxy wherein X is O or S; and
R9 is independently
hydrogen,
Ci-Cgalkyl optionally substituted by C3-C6cycloalkyl,
C4-Cgcycloalkyl,
tetrahydropyranyl,
benzyl,
benzyl optionally substituted by halogen
benzyl optionally substituted by Q-Cgalkyl benzyl optionally substituted by Q-Cealkoxy
2-phenylethyl optionally substituted on the phenyl ring halogen,
or
2-indanyl,
• hydrogen,
• Q-Cgalkyl optionally substituted by C3-C6cycloalkyl,
• R902CCH2CH2-
• Cs-Cgcycloalkyl,
• C6-C10 aryl optionally substituted by halogen,
or
• benzyl optionally substituted by halogen;
or
R 1 and R 2 when taken together with the N atom to which they are attached, may form
• a heterocyclic ring containing from 3 to 5 C atoms,
• a morpholine ring,
• a piperazine ring,
• a thiomorpholine ring, or
• a ring of formula
Figure imgf000006_0001
or
R3 and R 4 when taken together with the N atom to which they are attached, may form
• a heterocyclic ring containing from 3 to 5 C atoms
• a morpholine ring,
• a piperazine ring,
• a thiomorpholine ring, or
• a ring of formula
Figure imgf000006_0002
wherein X' is O or S,
R6 is selected from H, Ci-Cgalkyl optionally substituted by Ce-Qoaryl, and
CrCscycloalkyl, and
R10 and R11 are independently selected from H, C Csalkyl optionally substituted by C6-C10aryl, and C -C8cycloalkyl;
and the pharmaceutically acceptable salts thereof. In addition to salts, the present invention includes other forms of all of the disclosed compounds including solvates, hydrates and polymorphs. Such compounds are inhibitors of RNA-dependant RNA viral replication and inhibitors of HCV NS5B polymerase and thus may be utilized in methods wherein such inhibition is desired.
[0017] According to other embodiments, the present invention extends to a pharmaceutical composition comprising one or more compounds of formula I and a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutically acceptable carrier, excipient or diluent may be pure sterile water, phosphate buffered saline or an aqueous glucose, solution.
[0018] Also provided are methods for treating a viral infection in a mammal mediated at least in part by a virus in the Flaviviridae family wherein an instant method comprises administering to a mammal that has been diagnosed with said viral infection a pharmaceutical composition comprising compounds of formula I..
[0019] Also provided are methods for treating a viral infection in a human or animal patient that is mediated at least in part by a virus in the Flaviviridae family wherein an instant method comprises administering to a human or animal patient in need thereof an effective amount of a pharmaceutical composition comprising compounds of formula I.
[0020] In one embodiment, the virus is hepatitis C virus (or HCV). The present methods further extend to combination treatment comprising administration of a therapeutically effective amount of one or more agents active against hepatitis C virus. Such active agents against hepatitis C virus may include interferon- alpha or pegylated interferon-alpha alone or in combination with ribavirin or levovirin. [0021] DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention relates to chemical compounds, their preparation and their use in the treatment of viral infections particularly in mammals. Particularly, although not exclusively, the present invention relates to chemical compounds useful as anti-hepatitis C virus (HCV) agents.
[0023] Specifically, the present invention describes certain nucleoside phosphorodiamidates, their synthesis, and their use as inhibitors of RNA-dependent RNA viral polymerase, particularly their use as inhibitors of hepatitis C virus (HCV) NS5-B polymerase, as inhibitors of HCV replication, and for the treatment of hepatitis C infection. The compounds can also be precursors for such inhibitors.
[0024] It is an object of the present invention to provide novel chemical compounds useful for treatment of viral infections in mammals, specifically for treatment of hepatitis C infection in mammals.
[0025] The present invention relates to novel compounds of formula (I) having the structure:
Figure imgf000008_0001
(I)
wherein R2, R3, and R4 are each independently
Figure imgf000008_0002
wherein
R 7' and R 8° are each independently
hydrogen,
Ci-Cgalkyl, Ci-C3alkylaryl,
CH3XCH2-,
CH3XCH2CH2- ,
R9XC(0)CH2- benzyl,
benzyl optionally substituted by halogen,
benzyl optionally substituted by Q-Cgalkyl benzyl optionally substituted by Q-Cealkoxy phenyl,
phenyl optionally substituted by halogen,
phenyl optionally substituted by Q-Cgalkyl, or phenyl optionally substituted by Q-Cealkoxy and
R9 is independently
hydrogen,
Q-Cgalkyl optionally substituted by C3-C6cycloalkyl,
C4-Cgcycloalkyl,
tetrahydropyranyl,
benzyl,
benzyl optionally substituted by halogen
benzyl optionally substituted by Q-Cgalkyl benzyl optionally substituted by Q-Cealkoxy
2-phenylethyl optionally substituted on the phenyl ring halogen,
or
2-indanyl,
• hydrogen,
• Q-Cgalkyl optionally substituted by C3-C6cycloalkyl,
• R902CCH2CH2-
• CrCgcycloalkyl,
• C6-C10 aryl optionally substituted by halogen, • benzyl optionally substituted by halogen;
or
R 1 and R 2 when taken together with the N atom to which they are attached, may form
• a heterocyclic ring containing from 3 to 5 C atoms,
• a morpholine ring,
• a piperazine ring,
• a thiomorpholine ring, or
• a ring of formula
Figure imgf000010_0001
or
R3 and R 4 when taken together with the N atom to which they are attached, may form
• a heterocyclic ring containing from 3 to 5 C atoms
• a morpholine ring,
• a piperazine ring,
• a thiomorpholine ring, or
• a ring of formula
Figure imgf000010_0002
and
R5 is selected from CI, X'R6 and NR10RU,
wherein
X' is O or S,
R6 is selected from H, CrCsalkyl optionally substituted by C6-C10aryl, and
CrCgcycloalkyl, and
R10 and R11 are independently selected from H, CrCgalkyl optionally substituted by C6-C10aryl, and Cs-Cgcycloalkyl;
and the pharmaceutically acceptable salts thereof. In addition to salts, the present invention includes other forms of all of the disclosed compounds including solvates, hydrates and polymorphs. [0026] In each case, the above compounds are provided along with their phosphorus diastereomers. For example, phosphorous diastereomers may be possible in cases where Rl and R2 are not identical to R3 and R4.
[0027] In accordance with the present invention there are provided the following specific embodiments of the above compounds:
Figure imgf000011_0001
Figure imgf000012_0001
11
Figure imgf000013_0001
and the pharmaceutically acceptable salts thereof.
[0038] In accordance with the present invention there are provided the following specific embodiments of the above compounds:
[0039] (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)(2,2')-bis-amino- dipropanoate
[0040] (2S,2'S)-Dimethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0041] (25,2'5)-Diethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0042] (25,2 '5)-Dipropyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate
[0043] (25,2'5)-Dibutyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0044] (2S,2'S)-dipentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate [0045] (2S,2'S)-bis(3,3-dimethylbutyl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0046] (25,2'5)-Diisobutyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)(2,2')-bis-amino- dipropanoate
[0047] (2S,2W)-Bis(cyclopropylmethyl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0048] (25,2'5)-Dibenzyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate
[0049] (25,2'5)-Diisopropyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate
[0050] (2S,2'S)-sec- uty\ 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0051] (2S,2'S)-Dicyclobutyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0052] (2S,2'S)-Dicyclopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0053] (25,2'5)-Dicyclohexyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate [0054] (2S,2'S)-Bis(tetrahydro-2H-pyran-4-yl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy- 9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0055] (25,2'5)-(5)-Phenylethyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- amino-dipropanoate
[0056] (2S,2'S)-Bis(2,3-dihydro- lH-inden-2-yl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0057] (2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l- oxopropan-2-ylamino)phosphorylamino)propanoate
[0058] (2S)-Cyclohexyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l- oxopropan-2-ylamino)phosphorylamino)propanoate
[0059] (25 iert-Butyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l- oxopropan-2-ylamino)phosphorylamino)propanoate
[0060] (25 Methyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l- oxopropan-2-ylamino)phosphorylamino)-3-methylbutanoate
[0061] Methyl l-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphoryl)pyrrolidine-2-carboxylate
[0062] (2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(butylamino)phosphorylamino)propanoate [0063] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(butylamino)phosphorylamino)propanoate
[0064] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(benzylamino)phosphorylamino)propanoate
[0065] (2S)-Neopentyl 2-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(diethylamino)phosphorylamino)propanoate
[0066] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) (pyrrolidin- 1 - yl)phosphorylamino)propanoate
[0067] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(phenylamino)phosphorylamino)propanoate
[0068] (2S)-Neopentyl 2-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) (naphthalen- 1 - ylamino)phosphorylamino)propanoate
[0069] (25,2'5)-Dibenzyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- diethanoate
[0070] (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate ethanoate
[0071] (2R,2'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate [0072] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylpentanoate)
[0073] (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- 4-methylthiobutanoate
[0074] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3methyl butanoate)
[0075] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)
bis (azanediyl)bis (3methylbutanoate)
[0076] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(2-phenylacetate)
[0077] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(2-phenylacetate)
[0078] (25,2'5)-Dibenzyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-bis-pyrrolidine-2- methanoate
[0079] ((2R,3R,4R,5R)-5-(2-Amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methyl di-N-butylphosphinate
[0080] ((2R,3R,4R,5R)-5-(2-Amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methyl dimorpholinophosphinate
[0081] (2S,2'S)-2,4-Difluorobenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin- 9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl) bis(azanediyl)dipropanoate [0082] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0083] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0084] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[0085] (2S)-Cyclohexyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((S)- l-(cyclopentyloxy)- l- oxopropan-2-ylamino)phosphorylamino)propanoate
[0086] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(pentylamino)phosphorylamino)propanoate
[0087] (2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(morpholino)phosphorylamino)propanoate
[0088] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(cyclopropylamino)phosphorylamino)propanoate
[0089] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylpentanoate)
[0090] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylpentanoate) [0091] (2S,2'S)-Dibenzyl 2,2'-((((2R,3R,4R,5R)-5- 2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis 4-methylthiobutanoate)
[0092] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R -amino-6-methoxy-9H-purin-9-yl)
3,4-dihydroxy-4-methyltetrahydrofuran-2
yl)methoxy)phosphoryl)bis(azanediyl)bis 4-methylthiobutanoate)
[0093] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5- 2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis 3 -phenylpropanoate)
[0094] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5- 2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis 3 -phenylpropanoate)
[0095] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R -amino-6-methoxy-9H-purin-9-yl)
3,4-dihydroxy-4-methyltetrahydrofuran-2
yl)methoxy)phosphoryl)bis(azanediyl)bis 3 -phenylpropanoate)
[0096] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R -amino-6-methoxy-9H-purin-9-yl)
3,4-dihydroxy-4-methyltetrahydrofuran-2
yl)methoxy)phosphoryl)bis(azanediyl)bis 3-(4-tert-butoxyphenyl)propanoate)
[0097] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5- 2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis 3 -methylbutanoate)
[0098] (2S,2'S,3R,3'R)-Benzyl 2,2'-((((2R,3R,4R,5R -amino-6-methoxy-9H-purin-9-yl)
3,4-dihydroxy-4-methyltetrahydrofuran-2
yl)methoxy)phosphoryl)bis(azanediyl)bis 3 -methylpentano ate)
[0099] (2S,2'S,3R,3'R)-Dimethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylpentanoate) [00100] (2S,2'S,3R,3'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylpentanoate)
[00101] (2S,2'S,3R,3'R)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylpentanoate)
[00102] Neopentyl l,r-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)dipyrrolidine-2- carboxylate
[00103] (25,2'5)-l,4-Dibenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)disuccinate
[00104] (2S,2'S)-Tetramethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)disuccinate
[00105] Benzyl 3,3'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[00106] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylthiopropanoate)
[00107] (2S,2'S)-Cyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylthiopropanoate)
[00108] In accordance with the present invention there are provided the following additional compounds:
Figure imgf000021_0001
Figure imgf000021_0002
[00109] Accordingly, compounds in accordance with the present invention include:
[00110] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4- fluoro-3-hydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate; and
[00111] (2R,2'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4- fluoro-3-hydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate and the pharmaceutically acceptable salts thereof.
[00112] Once again, in each case with regard to the foregoing specific compounds, it is contemplated that the invention will include phosphorus diastereomers thereof.
[00113] Compounds according to the present invention have surprisingly been found to have enhanced anti-viral activity and this can be used in methods of treating Flaviviridae viral infections. In particular, compounds according to the present invention have been found to have enhanced potency with respect to hepatitis C virus. [00114] Definitions
[00115] As used herein, the term "alkyl" refers to a straight or branched saturated
monovalent cyclic or acyclic hydrocarbon radical, having the number of carbon atoms as indicated (or where not indicated, an acyclic alkyl group preferably has 1-20, more preferably 1-6, more preferably 1-4 carbon atoms and a cyclic alkyl group preferably has 3-20, preferably 3-10, more preferably 3-7 carbon atoms), optionally substituted with one, two, three or more substituents independently selected from the group set out above. By way of non- limiting examples, suitable alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, isopropyl, 2-butyl, cyclopropyl, cyclohexyl, cyclopentyl and dodecyl. The term "C3-C8cycloalkyl" refers to cyclic alkyl group comprising from about 3 to about 8 C atoms. The term "Cs-Cscycloalkyl-alkyl" refers to an acyclic alkyl group substituted by a cyclic alkyl group comprising from about 3 to about 8 C atoms.
[00116] As used herein, the term "alkenyl" refers to a straight or branched unsaturated monovalent acyclic or cyclic hydrocarbon radical having one or more C=C double bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic alkenyl group preferably has 2-20, more preferably 2-6, more preferably 2-4 carbon atoms and a cyclic alkenyl group preferably has 4-20, more preferably 4-6 carbon atoms), optionally substituted with one, two, three or more substituents independently selected from the group set out above. By way of non- limiting examples, suitable alkenyl groups include vinyl, propenyl, butenyl, pentenyl and hexenyl.
[00117] As used herein, the term "alkynyl" refers to a straight or branched unsaturated monovalent acyclic or cyclic hydrocarbon radical having one or more triple C/C bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic alkynyl group preferably has 2-20, more preferably 2-6, more preferably 2-4 carbon atoms and a cyclic alkynyl group preferably has 7-20, more preferably 8-20 carbon atoms), optionally substituted with one, two, three or more substituents independently selected from the group set out above.
[00118] As use herein, the term "alkoxy" or the term "alkyloxy" refers to the group alkyl- 0-, where alkyl is as defined above and where the alkyl moiety may optionally be substituted by one, two, three or more substituents as set out above for alkyl. By way of non-limiting examples, suitable alkoxy groups include methoxy, ethoxy, n- propoxy, isopropoxy, n- butoxy, ie/t-butoxy, sec-butoxy, w-pentoxy, w-hexoxy and 1,2-dimethylbutoxy. The term "cycloalkyloxy" refers to the group cyclicalkyl - 0-, where cyclicalkyl is as defined above and where the cyclicalkyl moiety may be optionally substituted by one, two, three or more substituents as set out above for alkyl.
[00119] As used herein, the term "alkylthio" refers the group alkyl-S-, where alkyl is as defined above and where the alkyl moiety may optionally be substituted by one, two, three or more substituents as set out above for alkyl. By way of non-limiting examples, suitable alkylthio groups include methylthio, ethylthio, n- propylthio, isopropylthio, w-butylthio, iert-butylthio, sec-butylthio, w-pentylthio, w-hexoxy and 1,2-dimethylbutylthio.
[00120] As used herein, the term "aryloxy" refers to the group aryl-O-, where aryl is as defined below and where the aryl moiety may optionally be substituted by one, two, three or more substituents as set out above with respect to the group Ar.
[00121] As used herein, the term "alkoxyalkyl" refers to an alkyl group having an alkoxy substituent. Binding is through the alkyl group. The alkyl moiety and the alkoxy moiety are as defined herein with respect to the definitions of alkyl and alkoxy, respectively. The alkoxy and alkyl moieties may each be substituted by one, two, three or more substituents as set out above with regard to the definition of alkyl.
[00122] As used herein, the term "alkylthioalkyl" refers to an alkyl group having an
alkylthio substituent. Binding is through the alkyl group. The alkyl moiety and the alkylthio moiety are as defined herein with respect to the definitions of alkyl and alkylthio, respectively. The alkylthio and alkyl moieties may each be substituted by one, two, three or more substituents as set out above with regard to the definition of alkyl.
[00123] As used herein, the term "alkoxyaryl" refers to an aryl group having an alkoxy substituent. Binding is through the aryl group. The alkoxy moiety and the aryl moiety are as defined herein with respect to the definitions of alkoxy and aryl, respectively. The alkoxy and aryl moieties may each be substituted by one, two, three or more substituents, as defined herein with regard to the definitions of alkoxy and aryl, respectively.
[00124] As used herein, the term "cycloalkylaryl" refers to an aryl group having a cyclic alkyl substituent. Binding is through the aryl group. The cycloalkyl moiety and the aryl moiety are as defined herein with respect to the definitions of cycloalkyl and aryl, respectively.
[00125] As used herein, the term "aryl(C1-C6)alkyl-" refers to a CrC6 alkyl group substituted at any carbon by an aryl group. Binding is through the alkyl group. The aryl moiety and the alkyl moiety are as defined herein with respect to the definitions of aryl and alkyl. The aryl group may be substituted. By way of non- limiting examples, suitable aryl(C1-C6)alkyl- groups include benzyl, 1- phenylethyl, 3-phenylpropyl, 4-chlorobenzyl, 4-fluorobenzyl, 2,4-difluorobenzyl, and the like.
[00126] As used herein, the term "alkylcarboxy(C1-C6)alkyl-" refers to a C -C alkyl group substituted at any carbon by an alkylcarboxy [alkyl-C(=0)0-] group. The alkyl moiety is as defined hereinabove. By way of non-limiting examples, suitable alkylcarboxy(C1-C6)alkyl- groups include acetoxymethyl [CH3C(=0)0-CH2-], propanoyloxyethyl [CH CH2C(=0)0-CH2CH2-], weopentoyloxypropyl [(CH3)3CCH2C(=0)0-CH2 CH2 CH2-] and the like.
[00127] A cycloalkyl moiety and the aryl moiety may each be optionally substituted by one, two, three or more substituents as set out herein with regard to the definitions of alkyl and aryl, respectively.
[00128] As used herein the term "aryl" refers to a monovalent unsaturated aromatic
carbocyclic radical having one, two, three, four, five or six rings, preferably one, two or three rings, which may be fused or bicyclic. An aryl group may optionally be substituted by one, two, three or more substituents as set out above with respect to optional substituents that may be present on the group Ar. Preferred aryl groups are: an aromatic monocyclic ring containing 6 carbon atoms; an aromatic bicyclic or fused ring system containing 7, 8, 9 or 10 carbon atoms; or an aromatic tricyclic ring system containing 10, 11, 12, 13 or 14 carbon atoms. Non-limiting examples of aryl include phenyl and naphthyl. These compounds may include substituent groups, preferably those substituent groups independently selected from hydroxy (-OH), acyl (R'- C(=0)), acyloxy (R'-C(O)-O-), nitro (-N02 ), amino (-NH2), carboxyl (-COOH), cyano (-CN), CrCemonoalkylamino, Q-C 6dialkylamino, thiol, chloro, bromo, fluoro, iodo, SO3H, -SH, -SR', wherein R' is independently selected from halo, Q-Cealkoxy^nd Q-Cealkyl.
[00129] As used herein, the term "heterocycloalkyl " refers to a saturated or partially
unsaturated heterocyclic ring system having one, two, three, four, five or six rings, preferably one, two or three rings, which may be fused or bicyclic, and having contained within the ring or rings at least one member selected from the group consisting of N, O and S. The prefix "C5-C2o" or "C5-C used before heterocycloalkyl means, respectively, a five to twenty or a five to ten-membered ring system at least one of which members is selected from the group consisting of N, O and S. Preferred heterocycloalkyl systems are: a monocyclic ring system having five members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; a monocyclic ring having six members of which one, two or three members are a N or O atom; a bicyclic ring system having nine members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms; or a bicyclic ring system having ten members of which one, two or three members are a N atom. By way of non-limiting examples, suitable heterocycloalkyl groups include tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothiopyranyl, thiomorpholinyl, and piperidinyl
[00130] When a radical is drawn as a structure, e.g.,
Figure imgf000025_0001
[00131] "indanyl" refers to the fused bicyclic radical of structure,
Figure imgf000025_0002
point of attachment of the radical to the rest of the molecule is on any available non-aromatic carbon atom.
[00132] Available carbon atoms and/or heteroatoms of the " heterocycloalkyl " ring
systems described above may be substituted on the ring with one or more heteroatoms. Where the ring(s) is substituted with one or more heteroatoms, heteroatom substituents are selected from oxygen, nitrogen, sulphur and halogen (F, CI, Br and I). Where the ring(s) is substituted with one or more heteroatoms, preferably there are 1, 2, 3 or 4 heteroatom substituents selected from the group consisting of oxygen, nitrogen and/or halogen. Preferred substituent groups are independently selected from hydroxy, acyl, acyloxy, nitro, amino, S03H, SH, SR', wherein R' is independently selected from the same groups as R; carboxyl, cyano, (C1-C6)alkylamino, (C1-C 6)dialkylamino, thiol, chloro, bromo, fluoro and iodo.
[00133] Furthermore, the compounds of this invention contain one or more chiral centers.
Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer- enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
[00134] The compounds of this invention may contain one or more asymmetric centers, depending upon the location and nature of the various substituents desired. Asymmetric carbon atoms or phosphorous atoms may be present in the (R) or (S) configuration or (R,S) configuration. In certain instances, asymmetry may also be present due to restricted rotation about a given bond, for example, the central bond adjoining two substituted aromatic rings of the specified compounds. Substituents on a ring may also be present in either cis or trans form, and a substituent on a double bond may be present in either Z or E form. It is intended that all such configurations (including enantiomers and diastereomers) are included within the scope of the present invention. Preferred compounds are those with the absolute configuration of the compound of this invention which produces the more desirable biological activity. Separated, pure or partially purified isomers or racemic mixtures of the compounds of this invention are also included within the scope of the present invention. Such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like, also well-known in the art and exemplified the experimental examples below.
[00135] The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
[00136] The term "pharmaceutically acceptable partial salts" in included in the term
"pharmaceutically acceptable salts" and refers to compounds having a substituent capable of having more than one group form a salt but less than the maximum amount of such groups actually form a salt. For example, a diphospho group can form a plurality of salts and, if only partially ionized, the resulting group is sometimes referred to herein as a partial salt. It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substituents is three. That is to say that each of the above definitions is constrained by a limitation that, for example, substituted aryl groups are limited to -substituted aryl- (substituted aryl)-substituted aryl. Similarly, it is understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 fluoro groups or a hydroxyl group pendent to a carbon atom of an ethenylic or acetylenic unsaturation). Such impermissible substitution patterns are well known to the skilled artisan. As also indicated above, the present compounds include other known forms of the compounds including solvates, hydrates and polymorphs. General Synthetic Methods
[00137] The compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
[00138] Additionally, as will be apparent to those skilled in the art, conventional
protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.
[00139] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce or Sigma (St. Louis, Mo., USA). Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Specifically, the compounds of this invention may be prepared by various methods known in the art of organic chemistry in general and nucleoside and nucleotide analogue synthesis in particular. General reviews of the preparation of nucleoside and nucleotide analogues include 1) Michelson A. M., "The Chemistry of Nucleosides and Nucleotides," Academic Press, New York, 1963; 2) Goodman L., "Basic Principles in Nucleic Acid Chemistry," Academic Press, New York, 1974, vol. 1, Ch. 2; and 3) "Synthetic Procedures in Nucleic Acid Chemistry," Eds. Zorbach W. & Tipson R., Wiley, New York, 1973, vol. 1 & 2.
Strategies available for synthesis of compounds of this invention are illustrated in the synthetic schemes below. For example, Reaction Scheme 1 illustrates a general method of preparation of symmetrical phosphorodiamidates.
[00141] Reaction Scheme 1
Figure imgf000030_0001
(I)
[00142] In this scheme, the nucleoside (II), prepared as described in International
Application PCT/US 10/20632 , is dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent and cooled to below ambient temperature, preferably to 0-5°C. Phosphorus oxychloride (phosphoryl chloride) of high quality is added to the solution with careful protection from moisture. The reaction is stirred for 1-48 h at temperatures from -20 °C to 20 °C and optimally for 24 h at 5 °C forming compound (III). The solution is diluted with an aprotic solvent, preferably DCM, and a primary or secondary amine, such as the HC1 or tosylate salt of an amino acid ester, is added at reduced temperatures -78 °C to 5 °C and preferably at 0 °C. This is followed by the addition of a non-nucleophilic base such as a tertiary amine such as triethylamine, or preferably diisopropylethylamine. The solution is stirred for 1 h to 10 days at reduced temperatures and preferably at 5 °C for 5 days, forming phosphorodiamidate (I).
[00143] Alternatively the nucleoside (II) can be dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF, and a non- nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added and stirred for a period of 5 min to 1 h, preferably 30 min. The solution is then cooled to -100 °C to rt, or preferably -78 °C, and phosphorus oxychloride (phosphoryl chloride) of high quality is added slowly to the solution with careful protection from moisture. The reaction is stirred for 5 min to 2 h at temperatures from -100 °C to 0°C and optimally for 30 min at -78 °C, then warmed to ambient temperature for 5 min to 2 h, preferably 30 min forming compound (III). The solution is further diluted with an aprotic solvent, preferably DCM, and a primary or secondary amine, such as the HC1 or tosylate salt of an amino acid ester, is added followed by the addition of a non-nucleophilic base such as a tertiary amine preferably triethylamine at reduced temperatures -78 °C to 5 °C and preferably at -78 °C. The solution is warmed to ambient temperature and stirred for 1 h to 48 h, preferably 24 h, forming phosphorodiamidate (I). The reaction can be worked up using standard methods familiar to one skilled in the art, for example extraction with a sodium chloride solution, drying with sodium sulfate and purification by silica gel chromatography. Changes to this procedure including solvent switches and optimization of the temperature, familiar to those skilled in the art of organic chemistry would be anticipated. Reaction Scheme 2 illustrates a general method of preparation of asymmetrical phosphoramidates.
[00145] Reaction Scheme 2
Figure imgf000032_0001
HNR1 (R2) (V) D I EA or TEA
Figure imgf000032_0002
[00146] In this scheme a general method for synthesizing asymmetrical phosphorodiamidates is described. The nucleoside (II) can be dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF, and a non-nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added and stirred for a period of 5 min to 1 h, preferably 30 min. The solution is then cooled to -100 °C to rt, or preferably -78 °C, and phosphorus oxychloride (phosphoryl chloride) of high quality is added slowly to the solution with careful protection from moisture. The reaction is stirred for 5 min to 2 h at temperatures ranging from -100 °C to 0 °C and optimally for 30 min at -78 °C, then warmed to ambient temperature for 5 min to 2 h, preferably 30 min forming compound (III). The solution is further diluted with an aprotic solvent, preferably DCM, and one equivalent of a primary or secondary amine of formula (V), such as the HC1 or tosylate salt of an amino acid ester, is added followed by the addition of a non-nucleophilic base such as a tertiary amine preferably triethylamine at reduced temperatures -78 °C to 5 °C and preferably at -78°C. The solution is warmed to ambient temperature and stirred for 1 h to 48 h, preferably 24 h, forming phosphorodiamidate (IV). A phosphorus NMR can be acquired to determine the status of the reaction. The solution is then cooled to -100 °C to rt, or preferably -78 °C. One to 10 equivalents, preferably 5 equivalents, of a primary or secondary amine of formula (VI), such as the HCl or tosylate salt of an amino acid ester, can be added. This is followed by the addition of an excess of a non-nucleophilic base such as a tertiary amine, preferably triethylamine (5-10 equivalents) at reduced temperatures -78 °C to 5 °C and preferably at -78 °C. The solution is warmed to ambient temperature and stirred for 1 h to 48 h, preferably 24 h, forming phosphorodiamidate (I). The reaction is worked up using standard methods familiar to one skilled in the art, for example extraction with a sodium chloride solution, drying with sodium sulfate and purification by silica gel chromatography. Changes to this procedure including solvent switches and optimization of the temperature, familiar to those skilled in the art of organic chemistry would be anticipated. Reaction Scheme 3 illustrates an alternative method for synthesizing asymmetrical phosphorodiamidates.
[00148] Reaction Scheme 3
Figure imgf000034_0001
(IV)
HNR3(R4) (VI)
D I EA or TEA
Figure imgf000034_0002
(I)
[00149] This scheme describes a second general method for synthesizing asymmetrical phosphorodiamidates. The nucleoside (II) can be dissolved in a neutral aprotic solvent such as THF or triethyl phosphate or similar solvent, but preferably THF. A non-nucleophilic base such as a tertiary amine or diisopropylethylamine or preferably triethylamine is added in excess, preferably 1.2 equivalents. The solution can be stirred at ambient temperature and 1 to 3 equivalents of an amino acid ester dichloridate (VII), can be added. Compounds of the general structure (VII) can be synthesized using techniques familiar to one skilled in the art. A phosphorus NMR can be acquired to determine the status of the formation of the compound of formula (IV) The solution is then cooled to -100 °C to rt, or preferably -78 °C, and 1 to 10 equivalents, preferably 5 equivalents, of a primary or secondary amine of formula (VI) are added. The solution is warmed to ambient temperature and stirred for 1 h to 48 h, preferably 24 h, forming phosphorodiamidate (I). The reaction is worked up using standard methods familiar to one skilled in the art, for example extraction with a sodium chloride solution, drying with sodium sulfate and purification by silica gel chromatography. Changes to this procedure including solvent switches and optimization of the temperature, familiar to those skilled in the art of organic chemistry would be anticipated.
[00150] The general schemes above are preferably carried out in the presence of a suitable solvent. Suitable solvents include hydrocarbon solvents such as benzene and toluene; ether type solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene; halogenated hydrocarbon solvents such as methylene chloride, chloroform and chlorobenzene; ketone type solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone; alcohol type solvents such as methanol, ethanol, propanol, isopropanol, w-butyl alcohol and iert-butyl alcohol; nitrile type solvents such as acetonitiile, propionitrile and benzonitrile; ester type solvents such as ethyl acetate and butyl acetate; carbonate type solvents such as ethylene carbonate and propylene carbonate; and the like. These may be used singly or two or more of them may be used in admixture. Preferably an inert solvent is used in the process of the present invention. The term "inert solvent" means a solvent inert under the conditions of the reaction being described in conjunction therewith including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride (or dichloromethane), diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, ie/t-butanol, dioxane, pyridine, and the like.
[00151] Dosages and routes of administration.
[00152] In general, the compounds of this invention will be administered in a
therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. The effective amount will be that amount of the compound of this invention that would be understood by one skilled in the art to provide therapeutic benefits, i.e., the active ingredient, and will thus depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors. The drug can be administered more than once a day, and in the preferred mode the drug is administered once or twice a day. As indicated above, all of the factors to be considered in determining the effective amount will be well within the skill of the attending clinician or other health care professional..
[00153] For example, therapeutically effective amounts of compounds of Formula I may range from approximately 0.05 to 50 mg per kilogram body weight of the recipient per day; preferably about 0.1-25 mg/kg/day, more preferably from about 0.5 to 10 mg/kg/day. Thus, for administration to a 70 kg person, the dosage range would most preferably be about 35-700 mg per day. In general, compounds of this invention will be administered as pharmaceutical compositions by any one of the following routes: oral, systemic (e.g., transdermal, intranasal or by suppository), or parenteral (e.g., intramuscular, intravenous or subcutaneous) administration. The preferred manner of administration is oral using a convenient daily dosage regimen that can be adjusted according to the degree of affliction. Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions. Another preferred manner for administering compounds of this invention is inhalation. This is an effective method for delivering a therapeutic agent directly to the respiratory tract (see U.S. Pat. No. 5,607,915, said patent incorporated herein by reference).
[00154] The choice of formulation depends on various factors such as the mode of drug administration and bioavailability of the drug substance. For delivery via inhalation the compound can be formulated as liquid solution, suspensions, aerosol propellants or dry powder and loaded into a suitable dispenser for administration. There are several types of pharmaceutical inhalation devices- nebulizer inhalers, metered dose inhalers (MDI) and dry powder inhalers (DPI). Nebulizer devices produce a stream of high velocity air that causes the therapeutic agents (which are formulated in a liquid form) to spray as a mist that is carried into the patient's respiratory tract. MDI's typically are formulation packaged with a compressed gas. Upon actuation, the device discharges a measured amount of therapeutic agent by compressed gas, thus affording a reliable method of administering a set amount of agent. DPI dispenses therapeutic agents in the form of a free flowing powder that can be dispersed in the patient's inspiratory air- stream during breathing by the device. In order to achieve a free flowing powder, the therapeutic agent is formulated with an excipient such as lactose. A measured amount of the therapeutic agent is stored in a capsule form and is dispensed with each actuation.
[00155] Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a crosslinked matrix of macromolecules. U.S. Pat. No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability. These patents are incorporated herein by reference.
[00156] As indicated above, the compositions in accordance with the invention generally comprise a compound of formulas (I- VI) in combination with at least one pharmaceutically acceptable carrier, excipient or diluent. Some examples of acceptable excipients are those that are non-toxic, will aid administration, and do not adversely affect the therapeutic benefit of the compound of the invention. Such excipient may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.
[00157] Solid pharmaceutical excipients useful in the invention may include starch,
cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. Compressed gases may be used to disperse a compound of this invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). The amount of the compound in a formulation can vary within the full range employed by those skilled in the art. For example, the formulation will contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt % wherein the compound is a compound of formula I based on the total formulation, with the balance being one or more suitable pharmaceutical excipients. Preferably, the compound is present at a level of about 1-80 wt %.
Pharmaceutical formulations containing a compound in accordance with the invention are described further below.
[00158] Additionally, the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention in combination with a therapeutically effective amount of another active agent against RNA-dependent RNA virus and, in particular, against HCV. Agents active against HCV include, but are not limited to, ribavirin, levovirin, viramidine, thymosin alpha- 1, an inhibitor of HCV NS3 serine protease, interferon-a, pegylated interferon-a (peginterferon-a), a combination of interferon-a and ribavirin, a combination of peginterferon-a and ribavirin, a combination of interferon-a and levovirin, and a combination of peginterferon-α and levovirin. Interferon-α includes, but is not limited to, recombinant interferon-a2a (such as Roferon interferon available from Hoffman-LaRoche, Nutley, N.J.), interferon- a2b (such as Intron-A interferon available from Schering Corp., Kenilworth, N.J., USA), a consensus interferon, and a purified interferon-α product. For a discussion of ribavirin and its activity against HCV, see J. O. Saunders and S. A. Raybuck, "Inosine Monophosphate Dehydrogenase: Consideration of Structure, Kinetics and Therapeutic Potential," Am. Rep. Med. Chem., 35:201-210 (2000).
[00159] Even further, the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention in combination with a therapeutically effective amount of another agent active against hepatitis C virus. Such agents include those that inhibit HCV proteases, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and inosine 5'-monophosphate dehydrogenase. Other agents include nucleoside analogs for the treatment of an HCV infection. Still other compounds include those disclosed in WO 2004/014313 and WO 2004/014852 and in the references cited therein. The patent applications WO 2004/014313 and WO 2004/014852 are hereby incorporated by references in their entirety. Specific antiviral agents include Omega IFN (BioMedicines Inc.), BILN-2061 (Boehringer Ingelheim), Summetrel (Endo Pharmaceuticals Holdings Inc.), Roferon A (F. Hoffman-La Roche), Pegasys (F. Hoffman-La Roche), Pegasys/Ribaravin (F. Hoffman-La Roche), CellCept (F. Hoffman-La Roche), Wellferon (GlaxoSmithKline), Albuferon-a (Human Genome Sciences Inc.), Levovirin (ICN Pharmaceuticals), IDN-6556 (Idun Pharmaceuticals), IP-501 (Indevus Pharmaceuticals), Actimmune (InterMune Inc.), Infergen A (InterMune Inc.), ISIS 14803 (ISIS Pharmaceuticals Inc.), JTK- 003 (Japan Tobacco Inc.), Pegasys/Ceplene (Maxim Pharmaceuticals), Ceplene (Maxim Pharmaceuticals), Civacir (Nabi Biopharmaceuticals Inc.), Intron A/Zadaxin (RegeneRx), Levovirin (Ribapharm Inc.), Viramidine(Ribapharm Inc.), Heptazyme (Ribozyme Pharmaceuticals), Intron A (Schering-Plough), PEG- Intron (Schering-Plough), Rebetron (Schering-Plough), Ribavirin (Schering- Plough), PEG-Intron/Ribavirin (Schering-Plough), Zadazim (SciClone), Rebif (Serono), IFN-p/EMZ701 (Transition Therapeutics), T67 (Tularik Inc.), VX-497 (Vertex Pharmaceuticals Inc.), VX-950/LY-5703 10 (Vertex Pharmaceuticals Inc.), Omniferon (Viragen Inc.), XTL-002 (XTL Biopharmaceuticals), SCH 503034 (Schering-Plough), isatoribine and its prodrugs ANA971 and ANA975 (Anadys), R1479 (Roche Biosciences), Valopicitabine (Idenix), NIM811 (Novartis), and Actilon (Coley Pharmaceuticals).
[00160] In some embodiments, the compositions and methods of the present invention contain a compound of formula I- VI and interferon. In some aspects, the interferon is selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
[00161] In other embodiments the compositions and methods of the present invention utilize a combination of a compound of formula I- VI and a compound having anti- HCV activity such as those selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti- sense RNA, Imiqimod, ribavirin, an inosine 5' monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
[00162] Anti-hepatitis C Activity Assays
[00163] Compounds can exhibit anti-hepatitis C activity by inhibiting HCV polymerase, by inhibiting other enzymes needed in the replication cycle, or by other pathways. A number of assays have been published to assess these activities. A general method that assesses the gross increase of HCV virus in culture was disclosed in U.S. Pat. No.5,738, 985 to Miles et al. In vitro assays have been reported in Ferrari et al. J. of Vir., 73: 1649-1654, 1999; Ishii et al., Hepatology, 29:1227- 1235, 1999; Lohmann et al., J. Bio. Chem., 274: 10807-10815, 1999; and Yamashita et al., J. of Bio. Chem., 273:15479-15486, 1998.
[00164] WO 97/12033 relates to HCV polymerase assay that can be used to evaluate the activity of the of the compounds described herein. Another HCV polymerase assay has been reported by Bartholomeusz, et al., Hepatitis C Virus (HCV) RNA polymerase assay using cloned HCV non- structural proteins; Antiviral Therapy 1996: 1 (Supp 4) 18-24.
[00165] Screens that measure reductions in kinase activity from HCV drugs were
disclosed in U.S. Pat. No. 6,030,785, to Katze et al., U.S. Pat. No. 6,228,576, Delvecchio, and U.S. Pat. No.5,759,795 to Jubin et al. Screens that measure the protease inhibiting activity of proposed HCV drugs were disclosed in U.S. Pat. No. 5,861,267 to Su et al., U.S. Pat. No. 5,739,002 to De Francesco et al., and U.S. Pat. No. 5,597,691 to Houghton et al. All of said patents, and all patent and non-patent references disclosed in this application, are incorporated herein by reference. [00166] Examples
[00167] Embodiments of the present invention will now be described by way of example only with respect to the following examples.
[00168] General Procedures
[00169] All experiments involving water-sensitive compounds were conducted under scrupulously dry conditions. Anhydrous tetrahydrofuran (THF) and
dichloromethane were purchased from Aldrich and used directly. The sugar derivative (2lS',3R,4R,5R)-5-(benzoyloxymethyl)-3-methyltetrahydrofuran-2,3,4- triyl tribenzoate or equivalently: 2,3,4,5-tetra-0-benzoyl-2-C-methyl- ?-D- ribofuranose was purchased from CarboSynth Limited, 8&9 Old Station Business Park, Compton, Berkshire, RG20 6NE, UK. The purine derivative 2-amino-6- chloropurine or equivalently, 6-chloro-9H-purin-2-amine, was purchased from Aldrich. 2'-C-Methylguanosine (2-amino-9-((3R,4R,5R)-3,4-dihydroxy-5- (hydroxymethyl)-3-methyltetrahydrofuran-2-yl)- lH-purin-6(9H)-one) is a commercial reagent and was purchased from CarboSynth Limited, 8&9 Old Station Business Park, Compton, Berkshire, RG20 6NE, UK. Salts of amino acid esters were prepared as described in PCT Int. Appl. (2010), WO 2010081082 A2 20100715. Column chromatography refers to flash column chromatography carried out using Merck silica gel 60 (40-60 μνη) as stationary phase. Proton, carbon, and phosphorus nuclear magnetic resonance ( 1 Η, 13 C, 31 P NMR) spectra were recorded on Bruker Avance spectrometers operating either at 500, 125, and 202 MHz or at 300, 75, and 121 MHz or a Varian Unity Inova instrument operating at 400, 100, and 161.9 MHz. The solvents used are indicated for each compound. All 13 C and 31 P spectra were recorded proton decoupled. Chemical shifts for 1 H and 13 C spectra are in parts per million downfield from
tetramethylsilane. Coupling constants are referred to as J values. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), broad signal (br), doublet of doublet (dd), doublet of triplet (dt), or multiplet (m).
Chemical shifts for 31 P spectra are in parts per million relative to an external phosphoric acid standard. Some of the proton and carbon NMR signals were split because of the presence of (phosphate) diastereoisomers in the samples. The mode of ionization for mass spectrometry was fast atom bombardment (FAB) using MNOBA (m-nitrobenzyl alcohol) as matrix for some compounds. Electrospray mass spectra were obtained using a Waters LCT time-of-flight mass spectrometer coupled to a Waters M600 HPLC pump. Samples were dissolved in methanol and injected into the solvent stream via a Rheodyne injector. The mobile phase used was methanol at a flow rate of 200 /zL/min. The electrospray source was operated at a temperature of 130 °C with a desolvation temperature of 300 °C, a capillary voltage of 3 kV, and cone voltage of 30 V. Data were collected in the continuum mode over the mass range 100-2000 amu and processed using Masslynx 4.1 software. Accurate mass measurements were facilitated by the introduction of a single lockmass compound of known elemental composition into the source concurrently with sample.
[00170] Analytical and semipreparative HPLC were conducted by Varian Prostar (LC
Workstation- Varian prostar 335 LC detector) using Varian Polaris C18-A (10 μΜ) as an analytic column and Varian Polaris CI 8- A (10 μΜ) as a semipreparative column; elution was performed using a mobile phase consisting of water/acetonitrile ingradient (System 1, 90/10 to 0/100 v/v in 30 min) or water/methanol (System 2, 90/10 to 0/100 v/v in 30 min).
[00171] Example 1.
[00172] (2R,3R,4R,5R)-2-(2-Amino-6-chloro-9H-purin-9-yl)-5-(benzoyloxymethyl)-3- methyltetrahydrofuran-3,4-diyl dibenzoate
Figure imgf000042_0001
[00173] To a pre-cooled (0 °C) solution of (2S,3R,4R,5R)-5-(benzoyloxymethyl)-3- methyltetrahydrofuran-2,3,4-triyl tribenzoate (or 2,3,4,5-tetra-0-benzoyl-2-C- methyl- ?-D-ribofuranose) (CarboSynth Ltd, 10.0 g, 17.22 mmol), 2-amino-6- chloropurine (Aldrich, 3.2 g, 18.87 mmol), and l,8-diazabicycl[5.4.0]undec-7-ene (DBU) (7.7 mL, 51 mmol) in anhydrous acetonitrile (200 mL), was added trimethysilyl triflate (12.5 mL, 68.8 mmol) dropwise. The reaction mixture was then heated at 65 °C for 4 to 6 h, allowed to cool down to room temperature, poured into saturated aqueous sodium bicarbonate (300 mL), and extracted with dichloromethane (3x150 mL). The combined organic phase was dried over sodium sulfate and evaporated under reduced pressure. The residue was precipitated from dichloromethane and methanol, filtrated, the solid was washed 2 times with methanol and dried to give the desired compound (8.5 g, 79 %) as a white solid (yields are from 65% (column) up to 90% (precipitation)).
[00174] The following are the NMR results analyzing the synthesized compounds:
1H NMR (500 MHz, DC\ d4) δ 8.13 (dd, J = 1.2, 8.3, 2H), 8.02 - 7.94 (m, 5H), 7.65 - 7.60 (m, 1H), 7.58 - 7.45 (m, 4H), 7.35 (q, J = 7.7, 4H), 6.65 (s, 1H), 6.40 (d, J = 6.7, 1H), 5.31 (s, 2H), 5.08 (dd, J = 4.2, 11.6, 1H), 4.79 (dd, J = 6.4, 11.6, 1H), 4.74 (td, J = 4.2, 6.5, 1H), 1.60 (s, 3H).
13C NMR (125 MHz, CDCl3-d4) δ 166.31(C=0), 165.38(C=0), 165.32(C=0), 159.13(C2), 152.87(C6), 152.06(C4), 141.42(C8), 133.77(C-H Bn), 133.69 (C-H Bn), 133.28(C-H Bn), 129.90(C-H Bn), 129.82(C-H Bn), 129.78 (C Bn), 129.70(C-H Bn), 129.41(C Bn), 128.78(C Bn), 128.61(C-H Bn), 128.50(C-H Bn), 128.41(C-H Bn), 126.00(C5), 88.84(C1 '), 85.68(C2'), 79.43(C4'), 76.07(C3'), 63.57(C5'), 17.77(2' -Me).
[00175] Example 2.
[00176] (2R,3R,4R,5R)-2-(2-Amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3- methyltetrahydrofuran-3,4-diol
Figure imgf000043_0001
[00177] To a suspension of (2R,3R,4R,5R)-2-(2-amino-6-chloro-9H-purin-9-yl)-5-
(benzoyloxymethyl)-3-methyltetrahydrofuran-3,4-diyl dibenzoate (3.0 g, 4.78 mmol) in methanol (36 mL) at 0 °C was added NaOMe in methanol (5.4 mL, 25% w/w). The mixture was stirred at room temperature for 24 h then quenched by addition of amberlite (H+). The mixture was then filtrated and methanol was removed under reduced pressure. The resultant residue was dissolved in water (50 mL) and extracted with hexane (50 mL). The organic layer was then extracted with water (50 mL), and the combined water fractions were concentrated under reduced pressure. The residue was purified by silica gel chromatography (CHCl3/MeOH 85: 15) to give the pure compound (1.125 g, 76 %) as a white solid.
[00178] The following are the NMR, HPLC and CHN results analyzing the synthesized compound:
1HNMR (500 MHz, MeOD-J4) δ 8.26 (s, 1H), 5.99 (s, 1H), 4.24 (d, J = 9.1, 1H), 4.08 (s, 3H), 4.04 (ddd, J = 2.3, 5.7, 8.6, 2H), 3.87 (dd, J = 3.0, 12.4, 1H), 0.96 (s, 3H).
13C NMR (125 MHz, MeOD-J4) δ 162.75(C6), 161.86(C2), 154.50(C4), 139.35(C8), 115.36(C5), 93.00(C1'), 84.15(C4'), 80.34(C2'), 73.57(C3'), 61.17(C5'), 54.25(6-OMe), 20.35(2' -Me).
HPLC : tR = 9.00 min; column: Varian Pursuit XRs 5, C18, 150x4.6 mm The method is : Linear gradient H20/ACN : 0% to 100% ACN in 30 min).
Elemental analysis: calculated for C12H17N505+ 0.75 H20 : G44.37,
H:5.74, N:21.56; Found: C:44.24, H:5.49, N:20.83.
[00179] General Methods for Preparation of Diamidates
[00180] Method A: Symmetrical phosphoramidates using triethyl phosphate and phosphoryl chloride
[00181] To a solution of the nucleoside (e.g., Example 2) (1 equiv) in anhydrous triethyl phosphate is added phosphoryl chloride (2 equiv) at 0 °C. The reaction mixture is stirred 24 h at 5 °C. Anhydrous dichloromethane is added to the reaction mixture followed by amino acid ester (5 equiv) and diisopropylethylamine (10 equiv) at 0 °C. After stirring at 5 °C for 5 days, water is added and the layers are separated. The aqueous phase is extracted with dichloromethane and the organic phase washed with brine. The combined organic layers are dried over anhyd sodium sulfate, filtered and evaporated to dryness. The resulting residue is purified by silica gel column chromatography using as eluent a gradient of methanol in dichloromethane. A subsequent repurification, if necessary, is accomplished either by preparative HPLC (gradient of methanol in water) or preparative TLC.
[00182] Method B: Symmetrical phosphoramidates using THF and phosphoryl chloride
[00183] To a suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (e.g., Example 2) (1 equiv) in anhyd tetrahydrofuran is added triethylamine (1.2 equiv). After stirring for 30 min at room temperature, phosphoryl chloride (1.2 equiv) is added dropwise at -78 °C. The reaction mixture is stirred 30 min at -78 °C then allowed to warm to room temperature over 30 min. Anhydrous dichloromethane is added, followed by amino acid ester (5 equiv) and triethylamine (10 equiv) at -78 °C. After stirring at room temperature for 20 h, water is added and the layers are separated. The aqueous phase is extracted with dichloromethane and the organic phase washed with brine. The combined organic layers are dried over anhyd sodium sulfate, filtered and evaporated to dryness. The resulting residue is purified by silica gel column chromatography using as eluent a gradient of methanol in dichloromethane. In some cases, a subsequent repurification is necessary either by preparative HPLC (gradient of methanol in water) or preparative TLC.
[00184] Method C: Asymmetrical phosphorodiamidates with phosphorus chloride
[00185] To a suspension of the nucleoside (e.g., Example 2) (1 equiv) in anhydrous tetrahydrofuran, is added triethylamine (1.2 equiv). After stirring for 30 min at room temperature, phosphoryl chloride (1.2 equiv) is added dropwise at -78 °C. The reaction mixture is stirred 30 min at -78 °C then allowed to warm to room temperature over 30 min. Anhydrous dichloromethane is added, followed by a first amino acid ester or amine (1 equiv) and triethylamine (2 or 1 equiv respectively) at -78 °C. The solution is warmed to room temperature and
31
monitored by P NMR. When NMR indicates completion of the reaction (no starting material, presence of mono-substituted product) a second amino acid ester or amine (5 equiv) is added followed by the addition of triethylamine (10 or 5 equiv respectively) at -78 °C. After stirring at room temperature for 20 h, water is added and the layers are separated. The aqueous phase is extracted with dichloromethane and the organic phase is washed with brine. The combined organic layers are dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The resulting residue is purified by silica gel column chromatography using as eluent a gradient of methanol in chloroform (0-5%).
[00186] Method D: Asymmetrical phosphorodiamidates using amino acid ester phosphordichloridate
[00187] To a suspension of the nucleoside (e.g., Example 2) (1 equiv) in anhydrous tetrahydrofuran is added triethylamine (1.2 equiv). After stirring for 30 min at room temperature an amino acid ester phosphordichloridate (2 equiv) is added. After stirring at room temperature for 20 h, the solution is cooled to -78 °C and a primary amine is added (5 equiv) followed by triethylamine (5 equiv). The solution is warmed to room temperature and stirred for 20 h. Water is added and the layers are separated. The aqueous phase is extracted with dichloromethane and the organic phase washed with brine. The combined organic layers are dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The resulting residue is purified by silica gel column chromatography using as eluent a gradient of methanol in chloroform (0-5%)
[00188] Example 3.
[00189] (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)(2,2')-bis- amino-dipropanoate
Figure imgf000046_0001
[00191] The phosphorodiamidate prodrug was synthesized using Method B. [00192] In the first step, a suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-
9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd tetrahydrofuran (4 mL) was allowed to react with triethylamine (135 μί, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole).
[00193] In the second step, anhyd dichloromethane (4 mL), the tosylate salt of neopentyloxy-L- alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the mixture from step 1.. After work-up and silica gel column chromatography, 151 mg of the prodrug was obtained in 28% yield as an off white solid.
[00194] The following are the NMR results of the synthesized compound:
[00195] 1H NMR (500 MHz, MeOD-J4) δ 7.98 (s, IH), 5.99 (s, IH), 4.41-4.36 (m, 2H),
4.29 (d, IH, J=9.0), 4.22-4.16 (m, IH), 4.07 (s, 3H), 4.04-3.91 (m, 2H), 3.87, 3.85, 3.84, 3.82 (2 AB system, /AB= 10.0 Hz, 2H), 3.75, 3.73, 3.70, 3.68 (2 AB system, /AB= 10.0, 2H), 1.40 and 1.36 (2d, 6H, J=7.1 Hz), 0.99 (s, 3H), 0.93 and 0.94 (2s, 18H).
[00196] 31P NMR (202 MHz, MeOD-J4) δ 14.01.
[00197] HPLC iR= 27.95 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[00198] Example 4.
[00199] (2S,2'S)-Dimethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
[00200]
Figure imgf000047_0001
[00201] The phosphorodiamidate prodrug was synthesized using Method B.
[00202] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the HCl salt of methyloxy-L-alanine (561.1 mg, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the previous mixture. After work-up, silica gel column chromatography and preparative HPLC, 19.8 mg of the prodrug was obtained in 4.4% yield as an off white solid.
[00203] The following are the NMR results of the synthesized compound:
[00204] IH NMR (500 MHz, MeOD-J4) δ 7.99 (s, IH), 5.99 (s, IH), 4.44-4.34 (m, 2H),
4.31 (d, IH, J=8.9 Hz), 4.21-4.16 (m, IH), 4.07 (s, 3H), 3.98-3.89 (m, 2H), 3.70, 3.69 (2s, 6H), 1.33 and 1.32 (2d, 6H, J=7.1 Hz), 1.00 (s, 3H).
[00205] 3 IP NMR (202 MHz, MeOD- d4) δ 14.00.
[00206] HPLC tR = 8.86 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00207] Example 5.
[00208] (2S,2'S)-Diethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000048_0001
[00210] The phosphorodiamidate prodrug was synthesized using Method B.
[00211] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the HCl salt of ethyloxy-L-alanine (645.16 mg, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the previous mixture. After work-up and silica gel column chromatography, 199.8 mg of the prodrug was obtained in 42 % yield as an off white solid.
[00212] The following are the NMR results of the synthesized compound:
[00213] 1H NMR (500 MHz, MeOD-J4) δ 7.99 (s, 1H), 6.00 (s, 1H), 4.44-4.34 (m, 2H),
4.31 (d, 1H, J=8.9 Hz), 4.23-4.08 (m, 5H), 4.07 (s, 3H), 3.98-3.88 (m, 2H), 1.40- 1.20 (m, 12H), 1.00 (s, 3H).
[00214] 3 IP NMR (202 MHz, MeOD-J4) δ 14.02.
[00215] HPLC iR = 10.87 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00216] Example 6.
[00217] (25,2'5)-Dipropyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate
Figure imgf000049_0001
[00219] The phosphorodiamidate prodrug was synthesized using Method A.
[00220] In the first step, a solution of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd triethylphosphate (1 mL) was allowed to react with phosphorus oxychloride (148 \L, 1.61 mmole). In the second step, anhyd dichloromethane (4 mL), the hydrochloride salt of propyloxy-L- alanine (673 g, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one. After work-up, silica gel column chromatography and preparative TLC, 36 mg of the prodrug was obtained in 7.2% yield as an off white solid.
[00221] The following are the NMR results of the synthesized compound:
[00222] 1H NMR (500 MHz, MeOD-J4) δ 7.99 (s, 1H), 6.00 (s, 1H), 4.43-4.33 (m, 2H),
4.30 (d, 1H, J=8.9 Hz), 4.22-4.17 (m, 1H), 4.11-3.90 (m, 9H), 1.68-1.61 (m, 4H), 1.37 and 1.34 (2d, 6H, J=7.1 Hz), 1.00 (s, 3H), 0.96-0.90 (m, 6H).
[00223] 31P NMR (202 MHz, CD3OD) δ 14.00.
[00224] HPLC iR= 13.16 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00225] Example 7.
[00226] (25,2'5)-Dibutyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000050_0001
The compound was prepared according to Method B from (2R,3R,4R,5R)-2-(2- amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-
3,4-diol, (Example 2, 250 mg, 0.80 mmol), POCl3 (0.07 mL, 0.80 mmol), Et3N (0.11 mL, 0.80 mmol), L-alanine butyl ester hydrochloride salt (0.43g, 2.41 mmol), and Et3N (0.67 mL, 4.82 mmol) in 10 mL of 1 : 1 mixture of dry THF and DCM. Crude product was purified by column chromatography using a gradient of CHC13 to CHC13: MeOH 95:5, to give a pure product as a white foam (75 mg, 14%). [00229] 31P NMR (202 MHz, MeOD-J4) δ 14.02. [00230] 1H NMR (500 MHz, MeOD-J4) δ 7.99 (s, 1H, ¾), 6.01 (s, 1H, Hr), 4.40-4.37
(m, 2H, ¾·), 4.29 (d, J= 9.0 Hz, 1H, H3'), 4.21- 4.19 (m, 1H, H4'), 4.15- 4.01 (m, 7H, 60CH3 and 2 x OCH2 ester), 3.94 (q, J=8.0 Hz, 2H, 2 x CHa Ala), 1.63- 1.56 (m, 4 H, 2 x OCH2CH2CH2CH3 ester), 1.41- 1.34 (m, 10H, 2 x OCH2CH2CH2CH3 ester and 2 x CH3 Ala), 1.00 (s, 3H, 2'CCH3), 0.93 (t, J= 7.5 Hz, 6H, 2 x OCH2CH2CH2CHj ester).
[00231] 13C NMR (125 MHz, MeOD-J4) δ 175.72, 175.65 (2 x d, 3J C-C-N-P= 6.30 Hz,
C=0 ester), 162.74 (C6), 161.91 (C2), 154.60 (C4), 139.30 (C8), 115.55 (C5), 93.14 (CI '), 82.35 (d, 3JC-c-o-p = 8.8 Hz, C4'), 80.06 (C2'), 74.81 (C3'), 66.22 (d, 2Jc-o-p = 5.0 Hz, C5'), 66.13 (OCH2CH2CH2CH3 ester), 66.11 (OCH2CH2CH2CH3 ester), 54.26 (60CH3), 51.06 (d, 2/C-N-P = 10.0 Hz, 2 x Ccc Ala), 31.75 (2 x OCH2CH2CH2CH3 ester), 21.03 (d,
Figure imgf000051_0001
5.0 Hz, CH3 Ala), 20.86 (d, 3/C-C-N- P= 5.00 Hz, CH3 Ala), 20.33 (2'CCH3), 20.13 (2 x OCH2CH2CH2CH3 ester), 14.07 (CH3 ester), 14.06 (CH3 ester).
[00232] HPLC tR= 15.23 min. [00233] Example 8. [00234] (2S,2'S)-Dipentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000051_0002
[00236] The phosphorodiamidate prodrug was synthesized using Method B.
[00237] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd tetrahydrofuran (4 mL) was allowed to react with triethylamine (135 μί, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), and the hydrochloride salt of pentoxy-L- alanine (0.79 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added as described in Method B. After work-up and silica gel column chromatography, 260 mg of the prodrug was obtained in 40% yield as an off white solid.
[00238] The following are the NMR results of the synthesized compound:
[00239] 31P NMR (202 MHz, MeOD-J4) δ 14.05.
[00240] 1H NMR (500 MHz, CD3OD) δ 8.00 (s, 1H, ¾), 6.03 (s, 1H, Hr), 4.44-4.35 (m,
2H, ¾·), 4.29 (d, J= 8.5 Hz, 1H, ¾·), 4.22- 4.13 (m, 1H, ¾·), 4.13- 3.93 (m, 9H, 60CH3 and 2 x OCH2 ester and 2 x CHcc Ala), 1.62- 1.59 (m, 4 H, 2 x OCH2CH2CH2CH2CH3 ester), 1.38- 1.31 (m, 14H, 2 x OCH2CH2CH2CH2CH3 ester, 2 x OCH2CH2CH2CH2CH3 ester and 2 x CH3 Ala), 1.00 (s, 3H, 2'CCH3), 0.89 (t, J= 5.00 Hz, 6H, 2 x OCH2CH2CH2CH2CH∑ester).
[00241] 13C NMR (125 MHz, CD3OD) δ 175.72, 175.67 (2 x d,
Figure imgf000052_0001
6.3 Hz, C=0 ester), 162.75 (C6), 161.92 (C2), 154.61 (C4), 139.26 (C8), 115.56 (C5), 93.07 (CI'), 82.34 (d, 3JC-C-0-P = 7.6 Hz, C4'), 80.11 (C2'), 74.81 (C3'), 66.43 (d, 2J c-o-p = 2.5 Hz, C5'), 66.25 (OCH2CH2CH2CH2CH3 ester), 66.22 (OCH2CH2CH2CH2CH3 ester), 54.34 (60CH3), 51.16 (d, 2/C-N-P= 11.3 Hz, Ca Ala), 51.07 (d, 2/C-N-P = 10.0 Hz, Ca Ala), 29.39 (2 x OCH2CH2CH2CH2CH3 ester), 29.16 (2 x OCH2CH2CH2CH2CH3 ester), 23.38 (2 x OCH2CH2CH2CH2CH3 ester), 21.12, 21.01 (2 x d,
Figure imgf000052_0002
6.3 Hz, CH3 Ala), 20.43 (2'CCH3), 14.07 (CH3 ester), 14.42 (OCH2CH2CH2CH2CH3 ester).
[00242] HPLC tR = 18.68 min.
[00243] MS (TOF ES+) m/z: 674.32 (M+H+, 100%).
[00244] HRMS C28H48N7O10Pi Calculated: 674.3279, found: 674.3246.
[00245] Example 9. [00246] (2S,2'S)-Bis(3,3-dimethylbutyl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000053_0001
[00248] The compound was prepared according to Method B from (2R,3R,4R,5R)-2-(2- amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran- 3,4-diol, (Example 2, 250 mg, 0.80 mmol), POCl3 (0.07 mL, 0.80 mmol), Et N (0.11 mL, 0.80 mmol), L-alanine 3,3-dimethylbutyl ester tosylate salt (0.83 g, 2.41 mmol), and Et3N (0.67 mL, 4.82 mmol) in 10 mL of 1: 1 mixture of dry THF and DCM. Crude product was purified by column chromatography using a gradient of CHC13 to CHC13: MeOH 95:5, to give a pure product as a white foam (52 mg, 9%).
[00249] 31P NMR (202 MHz, MeOD-J4) δ 14.03.
[00250] 1H NMR (500 MHz, MeOD-J4) δ 7.99 (s, 1H, ¾), 6.00 (s, 1H, Hr), 4.42-4.36
(m, 2H, Hs , 4.28 (d, J= 9.0 Hz, 1H, ¾·), 4.23- 4.09 (m, 5H, H4' and 2 x OCH2 ester), 4.07 (s, 3H, 60CH3), 3.92 (q, J=7.0 Hz, 2H, 2 x CHcc Ala), 1.60 (t, J= 7.0 Hz, 4H, 2 x OCH2CH2 ester), 1.37 (d, J= 7.0 Hz, 6H, 2 x CH3 Ala), 0.99 (s, 3H, 2'CCH3), 0.97 (s, 9H, 3 x CH3 ester), 0.94 (s, 9H, 3 x CH3 ester).
[00251] 13C NMR (125 MHz, MeOD-J4) δ 175.67, 175.59 (2d,
Figure imgf000053_0002
6.3 Hz, C=0 ester), 162.74 (C6), 161.91 (C2), 154.61 (C4), 139.24 (C8), 115.58 (C5), 93.13 (CI'), 82.35 (d, 3Jc-c-o-p = 8.8 Hz, C4'), 80.06 (C2'), 74.75 (C3'), 66.22 (d, 2J C- O-P = 5.00 Hz, C5'), 66.96 (OCH2 ester), 66.94 (OCH2 ester), 54.24 (60CH3), 51.07 (d, 2Jc-N-p = 10.0 Hz, 2 x Ccc Ala), 42.85 (OCH2CH2 ester), 42.83 (OCH2CH2 ester), 30. 59 (C ester), 30.55 (C ester), 30.03 (6 x CH3 ester), 20.96 (d,
Figure imgf000054_0001
6.3 Hz, CH3 Ala), 20.79 (d, 6.3 Hz, CH3 Ala), 20.34 (2'CCH3).
[00252] HPLC ¾= 20.12 min. [00253] Example 10. [00254] (25,2'5)-Diisobutyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)(2,2')-bi5,-amino- dipropanoate
Figure imgf000054_0002
[00256] The phosphorodiamidate prodrug was synthesized using Method B. [00257] A suspension of 6-0-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the tosylate salt of isobutyloxy-L-alanine (1.27 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After work-up, silica gel column chromatography and preparative HPLC, 66.1 mg of the prodrug was obtained in 13% yield as an off white solid.
[00258] The following are the NMR results of the synthesized compound: [00259] IH NMR (500 MHz, MeOD-J4) δ 7.98 (s, IH), 5.99 (s, IH), 4.41-4.37 (m, 2H),
4.29 (d, IH, J=9.0 Hz), 4.22-4.17 (m, IH), 4.07 (s, 3H), 4.00-3.77 (m, 6H), 1.95- 1.86 (m, 2H), 1.38, 1.35 (2d, 6H, J=7.1 Hz), 0.99 (s, 3H), 0.94-0.89 (m, 12H).
[00260] 3 IP NMR (202 MHz, MeOD-J4) δ 14.00. [00261] HPLC ¾ = 16.36 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method: linear gradient of CH3CN (10% to 100%) in H20 in 30 min).
[00262] Example 11. [00263] (2S,2'S)-Bis (cyclopropylmethyl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-
9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000055_0001
[00265] The phosphorodiamidate prodrug was synthesized using Method B.
[00266] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (0.2881 g, 0.93 mmol) in anhydrous tetrahydrofuran (5 mL) are allowed to react with triethylamine (0.16 mL, 1.12 mmol, 1.2 equiv) and phosphoryl chloride (0.11 mL, 1.12 mmol, 1.2 equiv). Anhydrous dichloromethane (8 mL), and tosylate salt of L-Ala-O- cyclopropylmethyl (1.4695 g, 4.65 mmol) and anhydrous triethylamine (1.30 mL, 9.3 mmol, 10 equiv) are added as describe in Method B. The residue is purified by flash chromatography using CHCl3/MeOH (6%) to give product (0.0336g, 0.063 mmol 18%) as an off white solid.
[00267] The following are the mass and NMR results of the synthesized compound: [00268] 1H NMR (500 MHz, CDC\ d4) δ 7.77 (s, 1H, H8), 5.99 (s, 1H, Hr), 4.54 (m, 1H,
1H CH2 s , 4.43 (m, 1H, ¾·), 4.34-4.31 (m, 1H, 1H CH2 5 , 4.22-4.21 (m, 1H, H4<), 4.03 (s, 3H, OCH3), 3.99-4.86 (m, 6H, 2xO(CH2-cyclopropyl) & 2xNHCH(CH3)CO-0-cyclopropylmethyl), 1.38 (d, 3H, J = 5 Hz, NHCH(CJi)CO-O-cyclopropylmethyl), 1.35 (d, 3H, J = 7 Hz, NHCH(CH3)CO- O-cyclopropylmethyl), 1.08 (m, 2H, 2xCH cyclopropylmethyl), 0.99 (s, 3H, CH3 r), 0.52 & 0.23 (2 broad s, 8H, 4xCH2 cyclopropylmethyl). [00269] C NMR (125 MHz, CDCl3-d4) 5C 174.43 (CO NHCH(CH3)CO-0- cyclopropylmethyl), 174.30 (CO NHCH(CH3)CO-0-cyclopropylmethyl), 161.54 (C6), 159.66 (C2), 152.99 (C4), 137.87 (C8), 115.56 (C5), 91.69 (s, Cr), 81.11 (C4 <), 79.34 (s, Cr), 74.21 (C3 , 64.98 (CH2 5 , 53.80 (OCH3), 49.91 & 49.78 (d, 2xNHCH(CH3)CO-0-cyclopropylmethyl), 20.94 & 20.89 (d, NHCH(CH3)CO-0- cyclopropylmethyl), 20.27 (s, CH 2·), 9.63 (s, 2xCH cyclopropylmethyl), 3.23 & 3.17 (d, 4xCH2 cyclopropylmethyl).
[00270] 31P NMR (202 MHz, CDC13-J4) 13.17.
[00271] MS : 641.61 (M), found 642.2630 (M+H), calculated 642.2653 (M+H) 664.2481
(M+ Na+), 680.2184 (M+ K+).
[00272] Example 12.
[00273] (25,2'5)-Dibenzyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate
Figure imgf000056_0001
[00275] The phosphorodiamidate prodrug was synthesized using Method A.
[00276] In the first step, a solution of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd triethylphosphate (1 mL) was allowed to react with phosphorus oxychloride (148 μί, 1.61 mmole). In the second step, anhyd dichloromethane (4 mL), the tosylate salt of benzyloxy-L- alanine (1.41 g, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one. After work-up, silica gel column chromatography and preparative HPLC, 50.1 mg of the prodrug was obtained in 8.7% yield as an off white solid. [00277] The following are the NMR results of the synthesized compound:
[00278] 1H NMR (500 MHz, MeOD-J4) δ 7.96 (s, IH), 7.34-7.25 (m, 10H), 5.99 (s, IH),
5.16-5.02 (m, 4H), 4.41-4.31 (m, 2H), 4.29 (d, IH, J=9.0 Hz), 4.21-4.15 (m, IH), 4.04 (s, 3H), 4.02-3.94 (m, 2H), 1.33 (d, 6H, J=7.1 Hz), 0.99 (s, 3H).
[00279] 31P NMR (202 MHz, MeOD-J4) δ 13.93.
[00280] HPLC iR= 13.91 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00281] Example 13.
[00282] (25,2'5)-Diisopropyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- amino-dipropanoate
Figure imgf000057_0001
[00284] The phosphorodiamidate prodrug was synthesized using Method B.
[00285] In the first step, a suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-
9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd tetrahydrofuran (4 mL) was allowed to react with triethylamine (135 \L, 0.964 mmole) and phosphorus oxychloride (89 \L, 0.964 mmole). In the second step, anhyd dichloromethane (4 mL), the hydrochloride salt of isopropyloxy-L-alanine (673.8 mg, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the mixture obtained in step one. After work-up and silica gel column chromatography, 55 mg of the prodrug was obtained in 14% yield, as an off white solid.
[00286] The following are the NMR results of the synthesized compound: [00287] 1H NMR (500 MHz, MeOD-J4) δ 7.99 (s, 1H), 5.99 (s, 1H), 5.03-4.89 (m, 2H), 4.44-4.34 (m, 2H), 4.31 (d, 1H, J=8.9 Hz), 4.23-4.16 (m, 1H), 4.07 (s, 3H), 3.94- 3.83 (m, 2H), 1.35 and 1.32 (2d, 6H, J=7.1 Hz), 1.27-1.19 (m, 12H), 1.00 (s, 3H).
[00288] 31P NMR (202 MHz, MeOD-J4) δ 14.07.
[00289] HPLC tR= min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method: linear. [00290] Example 14.
[00291] (2S,2'S)-sec-Butyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000058_0001
[00293] The phosphorodiamidate prodrug was synthesized using Method B.
[00294] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was allowed to react with triethylamine (135 \L, 0.964 mmole) and phosphoryl chloride (89 \L, 0.964 mmole). Anhydrous dichloromethane (4 mL), and the hydrochloride salt of 2- butoxy-L- alanine (0.73 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added as described in Method B. After work-up and silica gel column chromatography, 90 mg of the prodrug was obtained in 17% yield as an off white solid.
[00295] The following are the NMR results of the synthesized compound: [00296] 31P NMR (202 MHz, MeOD-J4) 14.05. [00297] 1H NMR (500 MHz, MeOD-J4) 7.99 (s, IH, H8), 6.00 (s, IH, HI'), 4.80- 4.78 (m, 2H, 2 x CH ester), 4.41- 4.39 (m, 2H, H5'), 4.30 (d, J= 8.50 Hz, IH, H3'), 4.21- 4.20 (m, IH, H4'), 4.07 (s, 3H, 60CH3), 3.92-3.90 (m, 2H, 2 x CHa Ala), 1.63- 1.51 (M, 4H, 2 x CH2 ester), 1.37- 1.33 (m, 6H, 2 x CH3 Ala), 1.24- 1.17 (m, 6H, 2 x CH3p ester), 1.00 (s, 3H, 2'CCH3), 0.89 (t, J= 6.00 Hz, 2 x CH3y ester).
[00298] 13C-NMR (125 MHz, MeOD-J4) 175.30 (d,
Figure imgf000059_0001
3.8 Hz, C=0 ester), 162.74
(C6), 161.89 (C2), 154.58 (C4), 139.37 (C8), 115.58 (C5), 93.19 (CF), 82.38 (d,
7.6 Hz, C4'), 80,06 (C2'), 74.91 (C3'), 74.59, 74.55 (CH ester), 66.40 (d, 2Jc-o-p= 3.8 Hz, C5'), 54.27 (60CH3), 51.19 (d, 2/C-N-P= 7.6 Hz, Ca Ala), 51.14 (d, 2Jc-N-p= 8.8 Hz, Ca Ala), 29.78, 29.74 (CH2 ester), 21.17 (d,
Figure imgf000059_0002
6.2 Hz, CH3 Ala), 21.04 (d,
Figure imgf000059_0003
2.5 Hz, CH3 Ala), 21.01 (CH3 Ala), 20.91(d, 3JC-c- N-P= 5.3 Hz, CH3 Ala), 20.89 (2'CCH3), 19.76, 19.61 (CH3p ester), 10.05 (CH3y ester)..
[00299] HPLC tR = 15.12 min.
[00300] MS (TOF ES+) m/z: 668.28 (M+Na+, 100%).
[00301] HRMS C26H44N7O10Pi calculated: 646.2966 found: 646.2961.
[00302] Example 15.
[00303] (2S,2'S)-Dicyclobutyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000059_0004
[00305] The phosphorodiamidate prodrug was synthesized using Method B. [00306] A suspension of 6-0-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the HCl salt of cyclobutyl-L-alanine (722.2 mg, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the previous mixture. After work-up, silica gel column chromatography and preparative HPLC, 65.1 mg of the phosphorodiamidate was obtained in 13% yield as an off white solid.
[00307] The following are the NMR results of the synthesized compound:
[00308] 1H NMR (500 MHz, MeOD-J4) δ 7.98 (s, 1H), 5.99 (s, 1H), 5.01-4.83 (m, 2H),
4.43-4.32 (m, 2H), 4.30 (d, 1H, J=8.9 Hz), 4.22-4.15 (m, 1H), 4.07 (s, 3H), 3.94- 3.84 (m, 2H), 2.38-2.24 (m, 4H), 2.16- 1.99 (m, 4H), 1.86- 1.74 (m, 2H), 1.71-1.57 (m, 2H), 1.36, 1.33 (2d, 6H, J=7.1 Hz), 1.00 (s, 3H).
[00309] 3 IP NMR (202 MHz, MeOD-J4) δ 14.01.
[00310] HPLC tR = 14.33 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00311] Example 16.
[00312] (2S,2'S)-Dicyclopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000060_0001
[00314] The phosphorodiamidate prodrug was synthesized using Method B. [00315] The compound of Example 2, ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol, 0.3179 g, 1.02 mmol) was dissolved in anhyd THF (5 mL) and to it was added anhyd Et3N (0.17 mL, 1.22 mmol, 1.2 equiv). The mixture was allowed to stir at rt for 30 min, after which the solution was cooled to -78 °C and POCl3 (0.12 mL, 1.22 mmol, 1.2 equiv) was added dropwise. The solution was stirred 30 min at -78 °C and then 30 min at rt. The formation of the intermediate was monitored by phosphorus NMR. Then 5 equiv of L-Ala-O-cyclopentyl pTSA salt (1.6832 g, 5.10 mmol) and anhydrous dichloromethane (8 mL) were added and the reaction was cooled to -78 °C, at which time anhyd Et N (1.42 mL, 10.2 mmol, 10 equiv) was added dropwise. The solution was then allowed to return to rt and stirred overnight, at the of which time phosphorus NMR monitoring suggested the formation of the diamidate. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography using CHCl3/MeOH (up to 4%) to recover the desired phosphorodiamidate which was washed with water to remove the excess Et3N.
[00316] HPLC : tR= 15.35 min
[00317] 1H NMR (500 MHz, CDC13-J4) δ 7.70 (s, 1H, ¾ guanine), 5.89 (s, 1H, Hr), 5.06
(m, 2H, O-CH cyclopentyl), 4.46-4.42 (m, 1H, 1H CH2 5 ), 4.37 (d, 1H, J = 8.5 Hz, ¾·), 4.26-4.22 (m, 1H, 1H CH2 5·), 4.11-4.08 (m, 1H, ¾·), 3.94 (s, 3H, OCH3 guanine), 3.83-3.70 (m, 2H, 2x NHCH(CH3)CO-0-cyclopentyl), 1.73-1.45 (m, 16H, 8 x CH2 cyclopentyl), 1.31 (d, 3H, J = 8 Hz, NHCH(C¾)CO-Q- cyclopentyl), 1.29 (d, 3H, J = 8 Hz, NHCH(CH3)CO-0-cyclopentyl), 1.02 (s, 3H, CH3 r).
[00318] 13C NMR (125 MHz, CDC13-J4) 5C 174.00 (CO NHCH(CH3)CO-0-cyclopentyl),
173.90 (CO NHCH(CH3)CO-0- cyclopentyl), 161.37 (s, C6 guanine), 159.56 (s, C2 guanine), 152.99 (s, C4 guanine), 137.97 (s, Cg guanine), 115.50 (s, C5 guanine), 91.76 (s, Cr), 81.14 (s, C4<), 79.22 (s, C2<), 78.18 & 78.10 (2s, 2 OCH- cyclopentyl), 73.93(s, C3<), 64.95 (s, CH2 5 , 53.70 (s, OCH3 guanine), 49.81 & 49.67 (2s, 2xNHCH(CH3)CO-0-cyclopentyl), 32.56 & 32.34 (2s, 4 CH2 ortho cyclopentyl), 23.55 (s, 4 CH2 meta cyclopentyl), 20.75 & 20.71 (2s, NHCH(C¾)CO-0-cvclopentvl), 20.27 (s, CH3 2<). [00319] 31P NMR (202 MHz, CDCl3-d4) 12.99 ppm (s). [00320] Example 17.
[00321] (25,2'5)-Dicyclohexyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- amino-dipropanoate
Figure imgf000062_0001
[00323] The phosphorodiamidate prodrug was synthesized using Method A.
[00324] In the first step, a solution of(2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd triethylphosphate (1 mL) was allowed to react with phosphorus oxychloride (148 \L, 1.61 mmole). In the second step, anhyd dichloromethane (4 mL), the hydrochloride salt of cyclohexyloxy-L-alanine (834 mg, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one. After work-up and silica gel column chromatography, 36.0 mg of the prodrug was obtained in 6.4% yield, as an off white solid.
[00325] The following are the NMR results of the synthesized compound:
[00326] 1H NMR (500 MHz, CD3OD) δ 7.98 (s, IH), 5.99 (s, IH), 4.76-4.64 (m, 2H),
4.41-4.35 (m, 2H), 4.29 (d, IH, J=9.0), 4.22-4.16 (m, IH), 4.07 (s, 3H), 3.94-3.86 (m, 2H), 1.91-1.25 (m, 26H), 0.99 (s, 3H).
[00327] 31P NMR (202 MHz, CD3OD) δ 14.07. [00328] HPLC iR= 17.60 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method: linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00329] Example 18.
[00330] (2S,2'S)-Bis(tetrahydro-2H-pyran-4-yl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000063_0001
[00332] Prepared according to Method C from (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H- purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol, (Example 2, 250 mg, 0.80 mmol), POCl3 (0.07 mL, 0.80 mmol), Et3N (0.11 mL, 0.80 mmol), L- alanine tetrahydropyranyl ester tosylate salt (1.38g, 4.02 mmol), Et3N (1.12 mL, 8.04 mmol) in 10 mL of 1 : 1 mixture of dry THF and DCM. Crude product was purified by column chromatography using a gradient of CHC13 to CHC13: MeOH 95:5, to give a pure product as a white foam (75 mg, 14%).
[00333] 31P NMR (202 MHz, MeOD-J4) δ 13.94.
[00334] 1H NMR (500 MHz, MeOD-J4) δ 7.99 (s, 1H, ¾), 5.99 (s, 1H, Hr), 4.94- 4.83
(m, 2H, 2 x OCH ester), 4.39-4.38 (m, 2H, ¾·), 4.28 (d, J= 9.0 Hz, 1H, H3'), 4.21- 4.18 (m, 1H, H4'), 4.07 (s, 3H, 60CH3), 3.99- 3.83 (m, 6H, 2 x CHa Ala and 4 x OCH2a ester), 3.56- 3.45 (m, 4H, 4 x OCH2b ester), 1.93- 1.83 (m, 4 H, 4 x OCH2a ester), 1.69- 1.59 (m, 4 H, 4 x OCH2d ester), 1.38 (d, J= 7.0 Hz, 3H, CH3 Ala), 1.36 (d, J= 7.0 Hz, 3H, CH3 Ala), 0.99 (s, 3H, 2'CCH3).
[00335] 13C NMR (125 MHz, MeOD-J4) δ 175.00, 174.92 (2d,
Figure imgf000063_0002
5.00 Hz, C=0 ester), 162.75 (C6), 161.94 (C2), 154.63 (C4), 139.36 (C8), 115.54 (C5), 93.14 (CI '), 82.41 (d, 3J C-C-O-P = 8.8 Hz, C4'), 80.03 (C2'), 74.89 (C3'), 71.27 (OCH ester), 71.23 (OCH ester), 66.47 (d, 2JC-o-p = 5.0 Hz, C5'), 66.18 (4x OCH2 ester), 54.29 (60CH3), 51.15 (d, 2/C-N-P = 6.3 Hz, 2 x Ccc Ala), 32.64 (4x CH2 ester), 20.98 (d, 3J c-c-N-p= 6.3 Hz, CH3 Ala), 20.79 (d,
Figure imgf000064_0001
6.3 Hz, CH3 Ala), 20.31 (2'CCH3).
[00336] HPLC tR= 9.57 min. [00337] Example 19. [00338] (25,2'5)-(5)-Phenylethyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- amino-dipropanoate
[00339]
Figure imgf000064_0002
[00340] The phosphorodiamidate prodrug was synthesized using Method A.
[00341] In the first step, a solution of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd triethylphosphate (1 mL) was allowed to react with phosphorus oxychloride (148 μί, 1.61 mmole). In the second step, anhyd dichloromethane (4 mL), the tosylate salt of (2lS')-phenylethyloxy-L-alanine (1.46 g, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one. After work-up, silica gel column chromatography and preparative TLC, 27.1 mg of the prodrug was obtained in 4.5% yield as an off white solid.
[00342] The following are the NMR results of the synthesized compound: [00343] 1H NMR (500 MHz, MeOD-J4) δ 7.98 (s, 1H), 7.41-7.24 (m, 10H), 5.99 (s, 1H), 5.86-5.80 and 5.78-5.73 (2m, 2H), 4.40-4.32 (m, 2H), 4.29 (d, 1H, J=9. l Hz), 4.21-4.14 (m, 1H), 4.06 (s, 3H), 4.00-3.91 (m, 2H), 1.53 and 1.47 (2d, 6H, J=6.6 Hz), 1.31 and 1.28 (2d, 6H, J=7.1 Hz), 0.99 (s, 3H).
[00344] 31P NMR (202 MHz, MeOD-J4) δ 13.99. [00345] HPLC iR= 18.48 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00346] Example 20. [00347] (2S,2'S)-Bis(2,3-dihydro- lH-inden-2-yl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000065_0001
[00349] The compound was prepared according to Method B from (2R,3R,4R,5R)-2-(2- amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran- 3,4-diol, (Example 2, 250 mg, 0.80 mmol), POCl3 (0.07 mL, 0.80 mmol), Et3N (0.11 mL, 0.80 mmol), L-alanine 2-indanolyl ester tosylate salt (0.91g, 2.41 mmol), and Et3N (0.67 mL, 4.82 mmol) in 10 mL of 1 : 1 mixture of dry THF and DCM. Crude product was purified by column chromatography using a gradient of CHC13 to CHC13: MeOH 95:5, to give a pure product as a white foam (52 mg, 9%).
[00350] 31P NMR (202 MHz, MeOD-J4) δ 13.94. [00351] 1H NMR (500 MHz, MeOD-J4) δ 7.95 (s, IH, ¾), 7.19-7.10 (m, 8H, 8 x CH ester), 5.99 (s, IH, Hr), 5.49- 5.46 (m, IH, OCH ester), 5.42- 5.40 (m, IH, OCH ester), 4.34-4.25 (m, 3H, H5< and H3'), 4.16- 4.14 (m, IH, H4'), 4.03 (s, 3H, 60CH3), 3.83 (q, J=7.0 Hz, 2H, 2 x CHcc Ala), 3.28-3.16 (m, 4H, 2 x CH2 ester), 3.01- 2.90 (m, 4H, 2 x CH2 ester), 1.25 (d, J= 7.0 Hz, 3H, CH3 Ala), 1.24 (d, J= 7.0 Hz, 3H, CH3 Ala), 0.98 (s, 3H, 2'CCH3).
[00352] 13C NMR (125 MHz, MeOD-J4) δ 175.53, 175.48 (2d,
Figure imgf000066_0001
5.00 Hz, C=0 ester), 162.73 (C6), 161.89 (C2), 154.57 (C4), 141.59, 141.51, 141.47 (4 x C ester), 139.30 (C8), 127.84 (4x CH ester), 125.59 (4x CH ester), 115.58 (C5), 93.13 (CI '), 82.35 (d, 3JC-c-o-p = 8.8 Hz, C4'), 80.04 (C2'), 77.70 (OCH ester), 77.67 (OCH ester), 74.81 (C3'), 66.28 (d, 2JC-o-p = 5.0 Hz, C5'), 54.29 (60CH3), 51.11 (d, 2Jc-N-p = 10.0 Hz, 2 x Ccc Ala), 40.42 (2x CH2 ester), 40.36 (2 x CH2 ester), 20.76 (d,
Figure imgf000066_0002
6.3 Hz, CH3 Ala), 20.62 (d, 6.3 Hz, CH3 Ala), 20.38 (2'CCH3).
[00353] Example 21.
[00354] (2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)- l- oxopropan-2-ylamino)phosphorylamino)propanoate
Figure imgf000066_0003
[00356] The phosphorodiamidate prodrug was synthesized using Method C.
[00357] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5-
(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd tetrahydrofuran (5 mL) was allowed to react with triethylamine (110 \L, 0.803 mmole) and phosphoryl chloride (70 \L, 0.803 mmole). The tosylate salt of benzoxy L-alanine (282 mg, 0.803 mmol) and triethylamine (110 μί, 0.803 mmol) were added. Anhydrous dichloromethane (4 mL), and the tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added as described in Method C. After work-up and silica gel column chromatography, 25 mg of the prodrug was obtained in 4% yield as an off white solid.
[00358] The following are the NMR results of the synthesized compound:
[00359] 31P NMR (202 MHz, MeOD-J4) 13.98, 13.94.
[00360] 1H NMR (500 MHz, MeOD-J4) 7.97, 7.96 (2 s, 1H, H8), 7.36-7.30 (m, 5H, Ph),
5.98, 5.97 (2s, 1H, HI'), 5.18- 5.09 (m, 2H, CH2 ester), 4.39- 4.33 (m, 2H, H5'), 4.28 (d, J= 8.0 Hz, 1H, H3'), 4.20- 4.16 (m, 1H, H4'), 4.06, 4.05 (2s, 3H, 60CH3), 4.02- 3.94 (m, 2H, 2 x CHa Ala), 3.84, 3.82, 3.72, 3.67 (2AB, /AB= 10.5 Hz, 2H, CH2 neopentyl ester), 1.39- 1.32 (m, 6H, 2 x CH3 Ala), 0.97 (s, 3H, 2'CCH3), 0.93, 0.91 (2s, 9H, 3 x CH3 neopentyl ester).
[00361] 13C-NMR (125 MHz, MeOD-J4) 175.54, 175.43, 175.39 (C=0 ester), 162.73,
162.71 (C6), 161.93, 161.89 (C2), 154.57, 154.55 (C4), 139.32, 139.08 (C8), 137.39 (ipso Ph), 129.55, 129.35, 129.25, 129.23, 129.20, 129.16, 128.27, 128.00 (Ph), 116.19, 115.54 (C5), 93.34, 93.18 (CI'), 82.39, 82.33 (C4'), 80.01, 79.99 (C2'), 75.34, 75.04 (CH2 neopentyl ester), 74.84, 74.82 (C3'), 67.88, 67.85 (CH2 benzyl ester), 67.86 (d, 2JC-o-p= 3.8 Hz, C5'), 66.36 (d, 2JC-o-p= 5.5 Hz, C5'), 54.18, 54.01 (60CH3), 49.69, 49.64, 49.52, 49.46 (2 x Ca Ala), 32.28, 32.25 (C ester), 26.74, 26.71 (3 x CH3 neopentyl ester), 21.07, 20.90, 20.79, 20.66 (4d, 3JC- C-N-P= 6.3 Hz, 2 x CH3 Ala), 20.39, 20.25 (2'CCH3).
[00362] HPLC tR = 16.11, 16.80 min.
[00363] MS (TOF ES+) m/z: 716.28 (M+Na+, 100%).
[00364] HRMS C30H44N7O10Pi calculated: 694.2966 found: 694.2956.
[00365] Example 22. [00366] (2S)-Cyclohexyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)- l- oxopropan-2-ylamino)phosphorylamino)propanoate
Figure imgf000068_0001
[00368] The phosphorodiamidate prodrug was synthesized using Method C. [00369] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the HCl salt of cyclohexyloxy-L-alanine (167 mg, 0.803 mmol) and triethylamine (224 μί, 1.61 mmole) were added. Finally, the tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After work-up and silica gel column chromatography, 92.6 mg of the prodrug was obtained in 17% yield as an off white solid.
[00370] The following are the NMR results of the synthesized compound: [00371] 1H NMR (500 MHz, MeOD-J4) δ 7.98 (s, 1H), 5.99 (s, 1H), 4.76-4.66 (m, 1H),
4.41-4.36 (m, 2H), 4.29, 4.28 (2d, 1H, J=9.0 Hz), 4.22-4.12 (m, 1H), 4.07 (2s, 3H), 4.01-3.78 (m, 4H), 1.90-1.68, 1.61-1.24 (2m, 16H), 0.99 (s, 3H), 0.94 and 0.92 (2s, 9H).
[00372] 3 IP NMR (202 MHz, MeOD-J4) δ 14.03 and 14.00. [00373] HPLC iR = 27.77 and 28.17 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method: linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[00374] Example 23. [00375] (25 ieri-Butyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)- l- oxopropan-2-ylamino)phosphorylamino)propanoate
Figure imgf000069_0001
[00377] The phosphorodiamidate prodrug was synthesized using Method C.
[00378] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the HCl salt of ie/t-butyloxy-L-alanine (175.2 mg, 0.964 mmol) and triethylamine (269 μί, 1.93 mmole) were added. Finally, the tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After work-up, silica gel column chromatography and preparative HPLC, 26.1 mg of the phosphorodiamidate was obtained in 4% yield as an off white solid.
[00379] The following are the NMR results of the synthesized compound: [00380] 1H NMR (500 MHz, MeOD-J4) δ 7.98 (2s, 1H), 5.98 (2s, 1H), 4.42-4.36 (m, 2H),
4.31, 4.29 (2d, 1H, J=9.1 Hz), 4.22-4.16 (m, 1H), 4.07 (s, 3H), 3.89-3.64 (m, 4H), 1.49- 1.28 (m, 15H), 0.99 (s, 3H), 0.94 and 0.91 (2s, 9H).
[00381] 3 IP NMR (202 MHz, MeOD-J4) δ 14.10, 14.08. [00382] HPLC ¾ = 26.24 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[00383] Example 24. [00384] (25 Methyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((lS,)-l-(neopentyloxy)- l- oxopropan-2-ylamino)phosphorylamino)-3-methylbutanoate
Figure imgf000070_0001
[00386] The phosphorodiamidate was synthesized using Method C. [00387] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the HCl salt of methyloxy-L-valine (161.7 mg, 0.964 mmol) and triethylamine (269 μί, 1.93 mmole) were added. The tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After work-up and silica gel column chromatography, 58.2 mg of the phosphorodiamidate was obtained in 11% yield as an off white solid.
[00388] The following are the NMR results of the synthesized compound: [00389] 1H NMR (500 MHz, MeOD-J4) δ 7.98 (2s, IH), 6.00 (s, IH), 4.45-4.35 (m, 2H),
4.34, 4.31 (2d, IH, J=9.1 Hz), 4.23-4.16 (m, IH), 4.06 (2s, 3H), 4.01-3.93 (m, IH), 3.91, 3.88, 3.84, 3.81 (2AB, 2H, J=10.5 Hz), 3.72-3.67 (m, 4H), 2.10- 1.94 (m, IH), 1.38- 1.35 (2d, 3H, J=7.2 Hz), 1.02-0.83 (m, 18H).
[00390] 31P NMR (202 MHz, MeOD-J4) δ 14.62, 14.49. [00391] HPLC tR = 25.24, 24.81 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm;
method: linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[0001] Example 25. Methyl l-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphoryl)pyrrolidine-2-carboxylate
Figure imgf000071_0001
[00394] The phosphorodiamidate prodrug was synthesized using Method C.
[00395] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the HC1 salt of methyloxy-L-proline (145.9 mg, 0.803 mmol) and triethylamine (224 μί, 1.61 mmole) were added to the mixture. In a third step, the tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the mixture. After work-up and silica gel column chromatography, 71 mg of the prodrug was obtained in 14% yield as an off white solid.
[00396] The following are the NMR results of the synthesized compound:
[00397] 1H NMR (500 MHz, MeOD-J4) δ 7.99, 7.96 (2s, 1H), 6.00, 5.96 (2s, 1H), 4.50- 4.15 (m, 5H), 4.15-3.97 (m, 4H), 3.88, 3.81, 3.79 (3d, 2H, J=10.4 Hz), 3.70, 3.68 (2s, 3H), 3.35-3.22 (m, 2H), 2.30-2.18, 2.12-2.03, 2.00-1.74, 1.74- 1.63 (4m, 4H), 1.42, 1.38 (2d, 3H, J=7.1 Hz), 1.00, 0.99 (s, 3H), 0.96, 0.92 (2s, 9H).
[00398] 3 IP NMR (202 MHz, MeOD-J4) δ 12.82, 12.49.
[00399] HPLC tR = 24.53 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[00400] Example 26. [00401] (2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(butylamino)phosphorylamino)propanoate
Figure imgf000072_0001
[00403] The phosphorodiamidate prodrug was synthesized using Method D. [00404] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 200 mg, 0.642 mmole) in anhyd tetrahydrofuran (5 mL) was allowed to react with triethylamine (105 μί, 0.771 mmole) and benzoxy-L-alaninyl phosphorodichloridate (380 mg, 1.29 mmole). Butylamine (317 μί, 3.21mmol) and triethylamine (447 μί, 3.21 mmol) were added as described in Method D. After work-up and silica gel column chromatography, 43 mg of the prodrug was obtained in 11% yield as an off white solid.
[00405] The following are the NMR results of the synthesized compound: [00406] 31P NMR (202 MHz, MeOD-J4) δ 16.25, 16.09. [00407] 1H NMR (500 MHz, MeOD-J4) δ 7.99, 7.98 (2 x s, 1H, H8), 5.99, 5.98 (2s, 1H,
HI'), 4.35- 4.34 (m, 2H, H5') 4.27 (d, J= 9.0 Hz, 1H, H3'), 4.19- 4.17 (m, 1H, H4'), 4.07 (s, 3H, 60CH3), 3.91- 3.79 (m, 1H, CHa Ala), 3.83, 3.70 (AB, JAB= 12.0 Hz, 2H, CH2 ester), 2.92- 2.87 (m, 2H, NH-CH2CH2CH2CH3), 1.47- 1.43 (m, 2H, NH-CH2CH2CH2CH3), 1.37 (d, J= 7.5 Hz, 3H, CH3 Ala), 1.33- 1.29 (m, 2H, NH-CH2CH2CH2CH3), 0.99, 0.98 (2 x s, 3H, 2'CCH3), 0.94, 0.93 (2s, 9H, 3 x CH3 ester), 0.89 (t, J= 7.5 Hz, 3H, NH-CH2CH2CH2CH3).
[00408] HPLC tR = 13.79 min. [00409] MS (TOF EI+) m/z: 607.25 (MH+, 100%). [00410] HRMS CaeHsgNvOgPi Calculated: 607.2520 found: 607.2504. [00411] Example 27. [00412] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(butylamino)phosphorylamino)propanoate
Figure imgf000073_0001
[00414] The phosphorodiamidate prodrug was synthesized using Method D.
[00415] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 200 mg, 0.642 mmole) in anhyd tetrahydrofuran (5 mL) was allowed to react with triethylamine (105 \L, 0.771 mmole) and 2,2-dimethoxy-L-alaninyl phosphorodichloridate (355 mg, 1.29 mmole). Butylamine (317 \L, 3.21mmol) and triethylamine (447 \L, 3.21 mmol) were added as described in Method D. After work-up and silica gel column chromatography, 11 mg of the prodrug was obtained in 3% yield as an off white solid.
[00416] The following are the NMR results of the synthesized compound: [00417] 3 IP NMR (202 MHz, MeOD-J4) δ 16.25, 16.09. [00418] 1H NMR (500 MHz, MeOD-J4) δ 7.99, 7.98 (2s, 1H, H8), 5.99, 5.98 (2s, 1H,
HI'), 4.35- 4.34 (m, 2H, H5') 4.27 (d, J= 9.0 Hz, 1H, H3'), 4.19- 4.17 (m, 1H, H4'), 4.07 (s, 3H, 60CH3), 3.91- 3.79 (m, 1H, CHcc Ala), 3.83, 3.70 (AB, JAB= 12.0 Hz, 2H, CH2 ester), 2.92- 2.87 (m, 2H, NH-CH2CH2CH2CH3), 1.47- 1.43 (m, 2H, NH-CH2CH2CH2CH3), 1.37 (d, J= 7.5 Hz, 3H, CH3 Ala), 1.33- 1.29 (m, 2H, NH-CH2CH2CH2CH3), 0.99, 0.98 (2s, 3H, 2'CCH3), 0.94, 0.93 (2s, 9H, 3 x CH3 ester), 0.89 (t, J= 7.5 Hz, 3H, NH-CH2CH2CH2CH3). [00419] HPLC iR = 14.71 min (System 1) [00420] MS (TOF EI+) m/z: 587.28 (MH+, 100%). [00421] HRMS C24H42N7O8P1 Calculated: 587.2833 found: 587.2813. [00422] Example 28. [00423] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(benzylamino)phosphorylamino)propanoate
Figure imgf000074_0001
[00425] This phosphorodiamidate was synthesized using Method C. [00426] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), benzylamine (88 μί, 0.803 mmole) and triethylamine (112 μί, 0.803 mmole) were added. Finally, the tosylate salt of neopentyloxy-L- alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After work-up, silica gel column chromatography and preparative HPLC, 22.4 mg of the prodrug was obtained in 5% yield as an off white solid.
[00427] The following are the NMR results of the synthesized compound: [00428] IH NMR (500 MHz, MeOD-J4) δ 7.98, 7.93 (2s, IH), 7.42-7.16 (m, 5H), 5.98 (s,
IH), 4.94-4.82 (m, 2H), 4.42-4.31 (m, 2H), 4.27, 4.21 (2d, IH, J=9.1 Hz), 4.20- 4.12 (m, IH), 4.07 (2s, 3H), 3.97-3.87 (m, IH), 3.85, 3.79, 3.71, 3.65 (2AB, 2H, J=10.6 Hz), 1.34 (pt, 3H), 0.99, 0.96 (2s, 3H), 0.92, 0.90 (2s, 9H).
[00429] 3 IP NMR (202 MHz, MeOD-J4) δ 15.84 and 15.78. [00430] HPLC iR = 21.57 and 21.92 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method: linear gradient of CH3OH (10% to 100%) in H20 in 30 min).
[00431] Example 29.
[00432] (2S)-Neopentyl 2-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(diethylamino)phosphorylamino)propanoate
Figure imgf000075_0001
[00434] The phosphorodiamidate prodrug was synthesized using Method C.
[00435] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2 300 mg, 0.963 mmole) in anhyd tetrahydrofuran (5 mL) was allowed to react with triethylamine (160 \L, 1.16 mmole) and phosphoryl chloride (110 \L, 1.16 mmole). Diethylamine (100 \L g, 0.963 mmol) and triethylamine (134 \L, 0.963 mmol) were added. Anhydrous dichloromethane (4 mL), and the tosylate salt of neopentyloxy-L- alanine (1.56g, 4.82 mmol) and triethylamine (1.34 mL, 9.64 mmol) were added as described in Method C. After work-up and silica gel column chromatography, 15 mg of the prodrug was obtained in 3% yield as an off white solid.
[00436] The following are the NMR results of the synthesized compound:
[00437] J1P NMR (202 MHz, MeOD-J4) δ 16.76, 16.68.
[00438] 1H NMR (500 MHz, MeOD-J4) δ 7.99, 7.95 (2s, 1H, H8), 5.99, 5.97 (2s, 1H,
HI'), 4.41- 4.28 (m, 3H, H5' and H3'), 4.21- 4.17 (m, 1H, H4'), 4.08 (s, 3H, 60CH3), 3.91- 3.79 (m, 3H, CHa Ala and CH2 ester), 3.18- 3.11 (m, 4H, 2 x CH2 amine), 1.40 (d, J= 7.5 Hz, 3H, CH3 Ala), 1.11 (t, J= 7.0 Hz, 6H, 2 x CH3 amine), 0.99 (s, 3H, 2'CCH3), 0.96, 0.93 (2 x s, 9H, 3 x CH3 ester).
[00439] HPLC ¾ = 11.68 min. [00440] MS (TOF EI+) m/z: 587.28 (MH+, 100%). [00441] HRMS C24H42N708P1 Calculated: 587.2833 found: 587.2813. [00442] Example 30. [00443] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) (pyrrolidin- 1 - yl)phosphorylamino)propanoate
[00444]
Figure imgf000076_0001
[00445] The phosphorodiamidate prodrug was synthesized using Method C.
[00446] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd tetrahydrofuran (5 mL) was allowed to react with triethylamine (110 μί, 0.803 mmole) and phosphoryl chloride (70 μί, 0.803 mmole). Pyrrolidine (67 μί, 0.803 mmol) and triethylamine (110 μί, 0.803 mmol) were added. Anhydrous dichloromethane (4 mL), and the tosylate salt of neopentyloxy- L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added as described in Method C. After work-up and silica gel column chromatography, 38 mg of the prodrug was obtained in 8% yield as an off white solid.
[00447] The following are the NMR results of the synthesized compound: [00448] 31P NMR (202 MHz, MeOD-J4) δ 14.54, 14.42. [00449] 1H NMR (500 MHz, MeOD-J4) δ 8.01, 7.99 (2s, IH, H8), 6.00, 5.99 (2s, IH, HI'), 4.38- 4.35 (m, 2H, H5'), 4.31- 4.28 (m, IH, H3'), 4.21- 4.19 (m, IH, H4'), 4.08, 4.07 (2s, 3H, 60CH3), 3.93- 3.91 (m, IH, CHcc Ala), 3.88- 3.71 (m, 2H, CH2 ester), 3.22- 3.19 (m, 4H, 2 x N-CH2 pyrrolidine), 1.87- 1.75 (m, 4H, 2 x CH2 pyrrolidine), 1.40, 1.37 (2 x d, J=7.0 Hz, 3H, CH3 Ala), 1.00, 0.99 (s, 3H, 2'CCH3), 0.95, 0.92 (2s, 9H, 3 x CH3 ester).
[00450] 13C NMR (125 MHz, MeOD-J4) δ 175.71, 175.67 (C=0 ester), 162.75, 162.73
(C6), 161.93 (C2), 154.56, 154.52 (C4), 139.16 (C8), 116.19, 115.57 (C5), 93.32, 93.16 (CI'), 82.44, 82.40 (d, 3JC-c-o-p = 7.6 Hz, C4'), 80.09, 80.03 (C2'), 75.34, 75.29 (CH2 ester), 74.64, 74.60 (C3'), 65.86 (d, 2JC-o-p = 8.8 Hz, C5'), 65.80 (d, 2Jc-o-p = 5.00 Hz, C5'), 54.24 (60CH3), 51.14, 51.02 (Ccc Ala), 47.84 (d, 2/C-N-P = 3.77 Hz, N-CH2 pyrrolidine), 47.84 (d, 2/C-N-P = 3.8 Hz, N-CH2 pyrrolidine), 32.33 (C ester), 27.34, 27.27 (2 x d, Hz, 2 x CH2 pyrrolidine), 26.78, 26.74 (CH3 ester), 21.20, 21.04 (2
Figure imgf000077_0001
6.3 Hz, CH3 Ala), 20.32, 20.26 (2'CCH3).
[00451] HPLC tR = 13.41 min.
[00452] MS (TOF EI+) m/z: 585.27 (MH+, 100%).
[00453] HRMS C24H40N7O8P1 Calculated: 585.2676 found: 585.2662.
[00454] Example 31.
[00455] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(phenylamino)phosphorylamino)propanoate
Figure imgf000077_0002
[00457] The phosphorodiamidate prodrug was synthesized using Method C. [00458] A suspension of 6-0-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), aniline (73 μί, 0.803 mmole) and triethylamine (112 μί, 0.803 mmole) were added. Finally, the tosylate salt of neopentyloxy-L- alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After work-up and silica gel column chromatography, 63.0 mg of the prodrug was obtained in 13% yield as an off white solid.
[00459] The following are the NMR results of the synthesized compound:
[00460] 1H NMR (500 MHz, MeOD-J4) δ 7.98, 7.91 (2s, IH), 7.22-7.05, 6.92-6.81 (2m,
5H), 6.05, 6.01 (2s, IH), 4.56-4.42 (m, 2H), 4.32, 4.28 (2d, IH, J=9.0 Hz), 4.27- 4.20 (m, IH), 4.05 (s, 3H), 4.03-3.91 (m, IH), 3.89, 3.79, 3.78 (3d, 2H, J=10.6 Hz), 1.36 (pt, 3H), 1.00, 0.95 (s, 3H), 0.89, 0.85 (2s, 9H).
[00461] 31P NMR (202 MHz, MeOD-J4) δ 8.76 and 8.67.
[00462] HPLC iR = 15.08 and 15.31 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method: linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00463] Example 32.
[00464] (2S)-Neopentyl 2-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) (naphthalen- 1 - ylamino)phosphorylamino)propanoate
Figure imgf000078_0001
A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), 1-naphthylamine (115 mg, 0.803 mmole) and triethylamine (112 μί, 0.803 mmole) were added to the previous mixture. In a third step, the tosylate salt of neopentyloxy-L-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the mixture. After work-up, silica gel column chromatography and preparative HPLC, 9.3 mg of the prodrug was obtained in 2% yield as an off white solid.
[00467] The following are the NMR results of the synthesized compound:
[00468] 1H NMR (500 MHz, MeOD-J4) δ 8.09-7.93 (m, 2H), 7.83, 7.79 (2d, IH, J=7.7
Hz), 7.57-7.28 (m, 5H), 6.01, 6.00 (2s, IH), 4.62-4.48 (m, 2H), 4.37, 4.31 (2d, IH, J=9.9 Hz), 4.29-4.23 (m, IH), 4.07-3.93 (m, 4H), 3.75, 3.71, 3.58 (3d, 2H, J=10.5), 1.34, 1.24 (2d, 3H, J=7.2 Hz), 1.00, 0.96 (s, 3H), 0.85, 0.84 (2s, 9H).
[00469] 31P NMR (202 MHz, MeOD-J4) δ 10.12 and 9.70.
[00470] HPLC tR = 23.49 and 23.75 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method: linear gradient of CH3OH (10% to 100%) in H20 in 30 min).
[00471] Example 33.
[00472] (25,2'5)-Dibenzyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- diethanoate
Figure imgf000079_0001
[00474] The phosphorodiamidate prodrug was synthesized using Method B.
[00475] In the first step, a suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-
9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd tetrahydrofuran (4 mL) was allowed to react with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). In the second step, anhyd dichloromethane (4 mL), the hydrochloride salt of benzyloxy-L- glycine (1.35 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the mixture obtained in step one. After work-up, silica gel column chromatography and preparative HPLC, 77.1 mg of the prodrug was obtained in 14% yield as an off white solid.
[00476] The following are the NMR results analyzing the synthesized compound:
[00477] 1H NMR (500 MHz, MeOD-J4) δ 8.03 (s, 1H), 7.34-7.24 (m, 10H), 6.01 (s, 1H),
5.10 and 5.09 (2s, 4H), 4.47-4.33 (m, 2H), 4.28 (d, 1H, J=8.9 Hz), 4.21-4.15 (m, 1H), 4.03 (s, 3H), 3.78 and 3.76 (2d, 4H, J=8.0 Hz), 0.98 (s, 3H).
[00478] 31P NMR (202 MHz, MeOD-J4) δ 16.55.
[00479] HPLC tR= 23.89 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[00480] Example 34
[00481] (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- amino-dipropanoate ethanoate
Figure imgf000080_0001
[00483] The phosphorodiamidate prodrug was synthesized using Method B.
[00484] In the first step, a suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-
9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd tetrahydrofuran (4 mL) is allowed to react with triethylamine (135 μί, 0.964 mmole) and phosphorus oxychloride (89 μί, 0.964 mmole). In the second step, anhydrous dichloromethane (4 mL), the tosylate salt of neopentyloxy-L- glycine (1.27 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) are added to the mixture obtained in step one. After work-up and silica gel column chromatography, 84.8 mg of product is obtained in 16% yield as an off white solid.
[00485] The following are the NMR results of the synthesized compound: [00486] 1H NMR (500 MHz, MeOD-J4) δ 8.05 (s, 1H), 6.02 (s, 1H), 4.50-4.35 (m, 2H),
4.28 (d, 1H, J=8.9 Hz), 4.23-4.17 (m, 1H), 4.06 (s, 3H), 3.82-3.73 (m, 8H), 1.00 (s, 3H), 0.91 and 0.90 (2s, 18H).
[00487] 31P NMR (202 MHz, MeOD-J4) δ 16.62. [00488] HPLC ¾= 26.27 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[00489] Example 35. [00490] (2R,2'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000081_0001
[00492] This phosphorodiamidate was synthesized using Method B.
[00493] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the tosylate salt of neopentyloxy-D-alanine (1.33 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After work-up and silica gel column chromatography, 89.9 mg of the phosphorodiamidate was obtained in 17% yield as an off white solid.
[00494] The following are the NMR results of the synthesized compound:
[00495] 1H NMR (500 MHz, MeOD-J4) δ 8.03 (s, 1H), 5.99 (s, 1H), 4.42-4.31 (m, 2H),
4.29 (d, 1H, J=9.0 Hz), 4.19-4.14 (m, 1H), 4.07 (s, 3H), 4.02-3.92 (m, 2H), 3.88, 3.80, 3.73, 3.68 (2AB, 4H, J=10.5 Hz), 1.40 (pt, 6H), 1.00 (s, 3H), 0.92 and 0.91 (2s, 18H).
[00496] 31P NMR (202 MHz, MeOD-J4) δ 14.11.
[00497] HPLC iR = 18.33 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of CH3CN (10% to 100%) in H20 in 30 min).
[00498] The phosphoramidate of Eample 35 ((2R,2'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5- (2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate) optimally could be made by the following three step process.
[00499] Ste l.
Figure imgf000082_0001
[00500] To a solution of N-Boc-D-Alanine, (1 in the above scheme) (lOOgm, 0.529 mol) in CH2C12 (750 mL) was added neopentyl alcohol (44.2 gm, 0.503 mol) at 0 °C followed by addition of EDCI (152 gm, 0.79 mol) in 3 portions. To this mixture a catalytic amount of DMAP (5 mol %) was added at 0 °C and stirred at room temperature for 12 hrs. This mixture was diluted with CH2C12 and washed with water (2 X 1L), 10% aq.NaHS04 (2 X 1L) and brine. The organic layers were dried under anhydrous Na2S04, filtered, concentrated and dried to afford 126.8 gm of crude material (of compound 2) and was taken to next step without further purification.
[00501] Step 2 [00502] The crude mixture from the above step was dissolved in dioxane (500 mL) followed by addition of HC1 (4M in dioxane) (5eq, 612 mL) at 0 °C and heated to 40 °C for 15 hrs. This mixture was cooled to room temperature and the volatiles were removed under reduced pressure. Trace amounts of dioxane was removed by azetroping with toluene (2 X 200 mL). To this solid was added anhydrous diethyl ether (1L) and stirred vigorously for lhr. The solids were filtered off and dried under vacuum to afford 87.3 gm of D-alanine neopentyl ester hydrochloride salt (3 in above scheme).
[00503] Step 3
[00504] A suspension of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (35 gm, 0.112 mol), and POCl3 (10.5 mL, 0.112 mol) in THF (500 mL) was cooled to -78 °C. Et3N (15.7 mL, 0.112 mol) was added slowly, and the solution was stirred at -78 °C for 30
31
min. At this stage P NMR confirms the completion conversion to dichloridate. To this mixture was added the compound from step 2 (D-Alanine neopentyl ester hydrochloride salt) (110 gm, 0.563 mol) dissolved in CH2C12 (1L) at -78 °C. Et3N (125.5 mL, 0.900 mol) was added in dropwise fashion and the solution was stirred at -78 °C for 1 hr. This mixture was slowly warmed to room temperature and stirred for 15 hrs. Then the solids were filtered off and the filtrate was concentrated and purified by column chromatography using a stepwise gradient of methanol (2-5%) in CH2C12 to obtain 36.6 g (0.544 mmol) of the phosphorodiamidate (2R,2'R)-neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate.
[00505] Example 36.
[00506] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylpentanoate)
Figure imgf000084_0001
[00508] The phosphorodiamidate prodrug was synthesized using Method B.
[00509] (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3- methyltetrahydrofuran-3,4-diol (0.3192 g, 1.03 mmol) in anhyd. tetrahydrofuran (5.5 mL) was allowed to react with triethylamine (1.2 equiv, 0.18 mL) and phosphoryl chloride (0.11 mL, 1.2 equiv). Anhydrous dichloromethane (8 mL) and the tosylate salt of H-Leu-OBzl (5 equiv, 5.15 mmol, 2.0356 g) and anh. triethylamine (10.3 mmol, 1.55 mL) were added as describe in Method B. After work-up, the residue was purified by flash chromatography using CHCl3/MeOH (4%) to recover the wanted prodrug (0.2248 g, 0.28 mmol, 28%).
[00510] The following are the mass and NMR results of the synthesized compound:
[00511] 1H NMR (500 MHz, CDC\3-d4) δ 7.72 (s, 1H, H8 guanine), 7.33 (m, 10H, 2 x 5H
Bz), 5.92 (s, 1H, H 1'), 5.15 (m, 4H, 2 x NH-CH(CH2-CH(CH3)2)-CO-0-CH2- Ph), 4.55 (m, 1H, H5'), 4.49 (m, 1H, H3'), 4.22 (m, 1H, H5'), 4.12 (m, 1H, H4'), 4.05 (s, 3H, OCH3 G), 3.59 (t, 2H, 2 x NH-CH(CH2-CH(CH3)2)-CO-0-CH2-Ph), 1.66 (m, 2H, 2 x NH-CH(CH2-CH(CH3)2)-CO-0-CH2-Ph), 1.53 (m, 2H, NH- CH(CH2-CH(CH3)2)-CO-0-CH2-Ph), 1.44 (m, 2H, NH-CH(CH2-CH(CH3)2)-CO- 0-CH2-Ph), 0.97 (s, 3H, CH3 2'C), 0.87 (m, 12H, 4 x NH-CH(CH2-CH(CH3)2)- CO-0-CH2-Ph).
[00512] 13C NMR (CDC13-J4) δ 174.61 (s, CO H-Leu-O-Bz), 174.22 (s, CO H-Leu-O- Bz), 161.61 (s, C6 guanine), 159.62 (s, C2 guanine), 152.86 (s, C4 guanine), 137.94 (s, C8 guanine), 135.45 (d, 2 x Q Bz), 128.56 (m, IOC, 2 x Cpara Bz & 4 x Cmeta Bz & 4 x Cortho Bz), 115.72 (s, C5 guanine), 91.84 (s, C 1'), 81.07 (s, C 4'), 79.41 (s, C 2'), 74.40 (s, C 3'), 67.05 (d, 2 x NH-CH(CH2-CH(CH3)2)-CO-0-CH2- Ph), 65.07 (s, C5'), 53.84 (s, OC¾ G), 50.47 (d, 2 x NH-CH(CH2-CH(CH3)2)- CO-0-CH2-Ph), 43.36 (d, 2 x NH-CH(CH2-CH(CH3)2)-CO-0-CH2-Ph), 24.64 (d, 2 x NH-CH(CH2-CH(CH3)2)-CO-0-CH2-Ph), 22.71 (m, 4 x NH-CH(CH2- CH(CH3)2)-CO-0-CH2-Ph), 20.32 (s, CH3 2'C).
[00513] 31P NMR (CDC\3-d4) δ 13.87. [00514] MS: 797.83 (M), found 798.3611 (M+H), calculated 798.3592 (M+H)
820.3420 (M+Na+), 836.3176 (M+K+).
[00515] Example 37. [00516] (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- amino-4-methylthiobutanoate
[00517]
Figure imgf000085_0001
[00518] The phosphorodiamidate prodrug was synthesized using Method A.
[00519] In the first step, a solution of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 250 mg, 0.803 mmole) in anhyd triethylphosphate (1 mL) was allowed to react with phosphorus oxychloride (148 μί, 1.61 mmole). In the second step, anhyd dichloromethane (4 mL), the tosylate salt of neopentyloxy-L-methionine (1.41 g, 4.02 mmol) and diisopropylethylamine (1.40 mL, 8.03 mmol) were added to the mixture obtained in step one. After work-up, silica gel column chromatography and preparative TLC, 7 mg of the prodrug was obtained in 1.1% yield as an off white solid.
[00520] The following are the NMR results of the synthesized compound: [00521] 1H NMR (500 MHz, MeOD-J4) δ 7.99 (s, 1H), 5.98 (s, 1H), 4.45-4.35 (m, 2H), 4.33 (d, 1H, J=8.8 Hz), 4.25-4.16 (m, 1H), 4.07 (s, 3H), 3.97-3.68 (m, 6H), 2.71- 2.45 (m, 4H), 2.19- 1.80 (m, 10H), 1.00 (s, 3H), 0.95 and 0.93 (2s, 18H).
[00522] 31P NMR (202 MHz, MeOD-J4) δ 14.14. [00523] HPLC ¾= 21.97 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of ACN (10% to 100%) in H20 in 30 min).
[00524] Example 38. [00525] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3methyl butanoate)
Figure imgf000086_0001
[00527] The compound was prepared according to Method B from (2R,3R,4R,5R)-2-(2- amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran- 3,4-diol, (Example 2, 250 mg, 0.80 mmol), POCl3 (0.07 mL, 0.80 mmol), Et N (0.11 mL, 0.80 mmol), L-valine benzyl ester tosylate salt (0.91g, 2.41 mmol), and Et3N (0.67 mL, 4.82 mmol) in 10 mL of 1 : 1 mixture of dry THF and DCM. Crude product was purified by column chromatography in gradient CHC13 to CHC13: MeOH 95:5, to give a pure product as a white foam (64 mg, 10%).
[00528] 31P NMR (202 MHz, MeOD-J4) δ 15.11. [00529] 1H NMR (500 MHz, MeOD-J4) δ 7.94 (s, 1H, H8), 5.98 (s, 1H, Hr), 5.19- 5.06
(m, 4H, 2 x CH2 ester), 4.40-4.34 (m, 2H, ¾' and H3'), 4.20- 4.18 (m, 1H, H4'), 4.04 (s, 3H, 60CH3), 3.72-3.68 (m, 2H, 2 x CHa Val), 2.02- 1.94 (m, 2H, 2 x CH Val), 0.99 (s, 3H, 2'CCH3), 0.87 (d, J= 7.00 Hz, 3H, CH3 Val), 0.84 (d, J= 7.00 Hz, 3H, CH3 Val), 0.79(d, J= 7.0 Hz, 3H, CH3 Val), 0.77 (d, J= 7.0 Hz, 3H, CH3 Val).
[00530] 13C NMR (125 MHz, MeOD-J4) δ 174.53 (C=0 ester), 162.75 (C6), 161.90 (C2),
154.54 (C4), 139.46 (C8), 137.21 (ipso Ph), 137.14 (ipso Ph), 129.61, 129.59, 129.58, 129.55, 129.41, 129.36 (2Ph), 115.68 (C5), 93.29 (CI '), 82.49 (d, 3JC-c-o-p = 8.8 Hz, C4'), 79.94 (C2'), 74.97 (C3'),67.93 (2 x CH2 ester), 66.89 (d, 2JC-o-p = 5.0 Hz, C5'), 61.09 (d, 2/C-N-P = 4.50 Hz, 2 x Ccc Val), 54.27 (60CH3), 33.20 (d, J=6.3 Hz, 2 x C Val), 20.37 (2'CCH3), 19.50 (CH3 Val), 19.42 (CH3 Val), 18.17 (CH3 Val), 18.12 (CH3 Val).
[00531] HPLC tR= 19.63 min.
[00532] Example 39.
[00533] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl) bis(azanediyl)bis(3methylbutanoate)
Figure imgf000087_0001
The compound was prepared according to Method B from (2R,3R,4R,5R)-2-(2- amino-6-methoxy-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-
3,4-diol, (Example 2, 250 mg, 0.80 mmol), POCl3 (0.07 mL, 0.80 mmol), Et3N (0.11 mL, 0.80 mmol), L- valine cyclohexyl ester chloridate salt (0.57g, 2.41 mmol), and Et3N (0.67 mL, 4.82 mmol) in 10 mL of 1 : 1 mixture of dry THF and DCM. Crude product was purified by column chromatography using a gradient of CHC13 to CHC13: MeOH 95:5, to give a pure product as a white foam (32 mg, 5%). [00536] 31P NMR (202 MHz, MeOD-J4) δ 15.28.
[00537] 1H NMR (500 MHz, MeOD-J4) δ 7.96 (s, 1H, ¾), 5.98 (s, 1H, Hr), 4.81-4.76
(m, 2H, 2 x OCH ester), 4.21-4.35 (m, 2H, ¾·), 4.33 (d, J= 9.0 Hz, 1H, H3'), 4.20- 4.17 (m, 1H, H4'), 4.07 (s, 3H, 60CH3), 3.66-3.63 (m, 2H, 2 x CHa Val), 2.07- 1.98 (m, 2H, 2 x CH Val), 1.85- 1.80 (m, 4H, 2 x CH2 ester), 1.75- 1.71 (m, 4H, 2 x CH2 ester), 1.55- 1.29 (m, 12H, 6 x CH2 ester), 1.00 (s, 3H, 2'CCH3), 0.95 (d, J= 6.5 Hz, 3H, CH3 Val), 0.90 (d, J= 6.5 Hz, 3H, CH3 Val), 0.89(d, J= 6.5 Hz, 3H, CH3 Val), 0.82 (d, J= 6.5 Hz, 3H, CH3 Val).
[00538] 13C NMR (125 MHz, CD3OD) δ 174.12 (d,
Figure imgf000088_0001
5.0 Hz, C=0 ester), 162.77
(C6), 161.94 (C2), 154.56 (C4), 139.47 (C8), 115.66 (C5), 93.34 (CI '), 82.55 (d, 3Jc-c-o-p = 6.30 Hz, C4'), 79.91 (C2'), 75.02 (C3'), 74.86 (OCH ester), 74.84 (OCH ester), 67.06 (d, 2JC-o-p = 5.0 Hz, C5'), 61.04 (d, 2/C-N-P = 4.5 Hz, 2 x Ccc Val), 54.22 (60CH3), 33.21 (2 x C Val), 32.60 (4x CH2 ester), 26.43 (CH2 ester), 26.40 (CH2 ester), 24.68 (2 x CH2 ester), 24.64 (2x CH2 ester), 20.27 (2'CCH3), 19.48 (CH3 Val), 19.37 (CH3 Val), 18.08 (CH3 Val), 18.02 (CH3 Val).
[00539] HPLC tR= 22.99 min.
[00540] Example 40.
[00541] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(2-phenylacetate)
Figure imgf000088_0002
[00543] The phosphorodiamidate prodrug was synthesized using Method B. [00544] A suspension of 6-0-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 μί, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the tosylate salt of neopentyloxy-L-phenylglycine (1.58 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added. After workup and silica gel column chromatography, 181.6 mg of the phosphorodiamidate was obtained in 28% yield as an off white solid.
[00545] The following are the NMR results of the synthesized compound:
[00546] 1H NMR (500 MHz, MeOD-J4) δ 7.79 (s, 1H), 7.38-7.19 (m, 10H), 5.97 (s, 1H),
5.02-4.92 (m, 2H), 4.35-4.25 (m, 2H), 4.14-4.01 (m, 5H), 3.88, 3.81, 3.66, 3.56 (4d, 4H, J=10.4 Hz), 0.92 (s, 3H), 0.74 and 0.71 (2s, 18H).
[00547] 31P NMR (202 MHz, MeOD-J4) δ 13.55.
[00548] HPLC tR = 23.51 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of CH3CN (10% to 100%) in H20 in 30 min).
[00549] Example 41.
[00550] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(2-phenylacetate)
Figure imgf000089_0001
[00552] The phosphorodiamidate prodrug was synthesized using Method B.
[00553] A suspension of 6-O-methyl-2'-C-methylguanosine (250 mg, 0.803 mmole) in anhydrous tetrahydrofuran (4 mL) was reacted with triethylamine (135 \L, 0.964 mmole) and phosphoryl chloride (89 μί, 0.964 mmole). Anhydrous dichloromethane (4 mL), the tosylate salt of cyclohexyloxy-L-phenylglycine (1.63 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added to the previous mixture. After work-up, silica gel column chromatography and preparative HPLC, 85.7 mg of the prodrug as a mixture of 3 stereoisomers ((R,R), (S,R), (S,S)), was obtained in 13% yield as an off white solid.
[00554] The following are the NMR results of the synthesized compound: [00555] 1H NMR (500 MHz, MeOD-J4) δ 7.93, 7.80, 7.78 (3s, 1H), 7.41-7.19 (m, 10H),
5.98, 5.95, 5.94 (3s, 1H), 4.96-4.87 (m, 2H), 4.78-4.61 (m, 2H), 4.35-4.25 (m, 2H), 4.15-4.02 (m, 5H), 1.82- 1.16 (m, 20H), 0.99, 0.97, 0.91 (3s, 3H).
[00556] 31P NMR (202 MHz, MeOD-J4) δ 13.62, 13.57, 13.53. [00557] HPLC ¾ = 31.61 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of CH3OH (10% to 100%) in H20 in 30 min).
[00558] Example 42. [00559] (25,2'5)-Dibenzyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-bis-pyrrolidine-2- methanoate
Figure imgf000090_0001
[00561] The phosphorodiamidate prodrug was synthesized using Method A.
[00562] In the first step, a solution of (2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (Example 2, 150 mg, 0.482 mmole) in anhyd triethylphosphate (1 mL) was allowed to react with phosphorus oxychloride (89 μί, 0.964 mmole). In the second step, anhyd dichloromethane (2.4 mL), the hydrochloride salt of benzyloxy-L-proline (583 mg, 2.41 mmol) and diisopropylethylamine (840 μί, 4.82 mmol) were added to the mixture obtained in step one. After work-up, silica gel column chromatography and preparative TLC, 8.4 mg of the prodrug was obtained in 2.3% yield as an off white solid.
[00563] The following are the NMR results of the synthesized compound:
[00564] 1H NMR (500 MHz, MeOD-J4) δ 7.94 (s, 1H), 7.36-7.27 (m, 10H), 5.93 (s, 1H),
5.86-5.80 and 5.78-5.72 (2m, 4H), 4.43-4.32 (m, 3H), 4.27 (q, 2H, J=4.2 Hz), 4.19-4.12 (m, 1H), 4.05 (s, 3H), 3.36-3.09 (m, 4H), 2.24-2.14, 2.04-1.64 and 1.61- 1.52 (3m, 8H), 1.00 (s, 3H).
[00565] 31P NMR (202 MHz, MeOD-J4) δ 11.40.
[00566] HPLC iR=28.05 min (column: Varian Pursuit XRs 5, C18, 150x4.6 mm; method:
linear gradient of MeOH (10% to 100%) in H20 in 30 min).
[00567] Example 43.
[00568] ((2R,3R,4R,5R)-5-(2-Amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methyl di-N- butylphosphinate
Figure imgf000091_0001
[00570] The compound of Example 2, ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol, 1.0428 g, 3.35 mmol) was dissolved in anhyd THF (17 mL), and to it was added anhyd Et3N (0.56 mL, 4.02 mmol, 1.2 equiv). The mixture was allowed to stir at rt for 30 min, after which the solution was cooled to -78 °C, and POCl3 (0.38 mL, 4.08 mmol, 1.2 equiv) was added dropwise. The solution was allowed to return to rt over 1.5 h. The formation of the intermediate was monitored by phosphorus NMR (7.88 ppm). Then 5 equiv of w-butylamine (1.70 mL, 17.2 mmol) was added, followed by anhyd dichloromethane (17 mL) up to total dissolution. The reaction was cooled to -78 °C after which anhyd Et3N (4.7 mL, 33.7 mmol, 10 equiv) was added dropwise. The solution was then allowed to return to rt and stirred for an additional 5 h, at the end of which time phosphorus NMR monitoring suggested the formation of the diamidate (17.06 ppm). The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography using CHCl3/MeOH. The resulting residue was washed several times with water to removed the excess of triethylamine, yielding 0.4172 g (0.83 mmol, 25%) of desired product.
[00571] HPLC : ¾= 12.31 min.
[00572] 1H NMR (CDC13): 7.86 ppm (s, 1H, ¾ guanine), 6.05 ppm (s, 1H, Hr), 5.68 ppm
(broad s, 2H, NH2 guanine), 4.44 - 4.23 ppm (m, 4H, CH2 5· & ¾· & ¾·), 4.04 ppm (s, 3H, OCH3 guanine), 2.88 ppm (m, 4H, 2xNHCH2CH2CH2CH3), 1.41 ppm(m, 4H, 2xNHCH2CH2CH2CH3), 1.27 ppm (m, 4H, 2x NHCH2CH2CH2CH3), 0.99 ppm (s, 3H, CH32<), 0.82 ppm (m, 6H, 2x NHCH2CH2CH2CH3).
[00573] 13C NMR (CDC13) 161.40 ppm (s, C6 guanine), 159.69 ppm (s, C2 guanine),
153.07 ppm (s, C4 guanine), 137.64 ppm (s, CH8 guanine), 115.24 ppm (s, C5 guanine), 91.36 ppm (s, CHI'), 81.11 ppm (s, CH4'), 79.36 ppm (s, C2'), 73.41 ppm (s, CH3'), 64.02 ppm (s, CH25'), 53.75 ppm (s, OCH3), 40.84 ppm (s, 2xNHCH2CH2CH2CH3), 33.91 ppm (s, 2xNHCH2CH2CH2CH3), 20.22 ppm (s, CH32'), 20.22 ppm (s, NHCH2CH2CH2CH3), 19.76 ppm (s, NHCH2CH2CH2CH3), 13.63 ppm (s, NHCH2CH2CH2CH3), 13.45 ppm (s, NHCH2CH2CH2CH3).
[00574] 31P NMR (CDC13) 17.40 ppm (s).
[00575] MS: (M + H+), calculated: 502.2543, found: 502.2562 , 524.2374
(M + H+ + Na), 565.249 (M + H+ + MeCN + Na). [00576] Example 44. [00577] ((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methyl dimorpholinophosphinate
[00578]
Figure imgf000093_0001
[00579] The compound of Example 2, ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol, 0.9088 g, 2.92 mmol) was dissolved in anhyd THF (14 mL) and to it was added anhyd Et3N (0.49 mL, 3.50 mmol, 1.2 equiv). The mixture was allowed to stir at rt for 30 min, after which the solution was cooled to -78 °C and POCl3 (0.33 mL, 3.50 mmol, 1.2 equiv) was added dropwise. The solution was allowed to return to rt over 1.5 h. The formation of the intermediate was monitored by phosphorus NMR (8.54 ppm). Then 5 equiv of morpholine (1.26 mL, 14.6 mmol) was added followed by anhyd dichloromethane (24 mL). The dissolution was not totally complete. The reaction mixture was cooled to -78 °C after which anhyd Et N (4.10 mL, 29.2 mmol, 10 equiv) was added dropwise. The solution was then allowed to return to rt and stirred for an additional 5 h, at the end which time phosphorus NMR monitoring suggested the formation of the diamidate (14.66 ppm). The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography using CHCl3/MeOH (up to 6%). Then the resulting residue was washed several times with water to removed the excess of triethylamine. The triethylamine as well as some of the final product moved to the aqueous layer, so a back wash of the aqueous layer was undertaken with EtOAc to recover the desired phosphorodiamidate product. (Yield: 0.0336 g, 0.063 mmol 18%).
[00580] HPLC : ¾= 3.72 min. [00581] 1H NMR (CDC13) 7.75 ppm (s, 1H, H8 guanine), 6.00 ppm (s, 1H, HI '), 5.51-4.48 ppm (m, 1H, CH2 5'), 4.41 - 4.35 ppm (m, 2H, CH2 5' & H3'), 4.28-4.26 ppm (m, 1H, H4'), 4.07 ppm (s, 3H, OCH3 guanine), 3.66-3.62 ppm (m, 8H, 4xO(CH2CH2)2N morpholine), 3.19-3.12 ppm (m, 8H, 4xO(CH2CH2)2N morpholine), 1.03 ppm (s, 3H, CH3 2').
[00582] 13 C NMR (CDC13) 161.71 ppm (s, C6 guanine), 159.54 ppm (s, C2 guanine),
152.87 ppm (s, C4 guanine), 137.33 ppm (s, C¾ guanine), 115.87 ppm (s, C5 guanine), 91.56 ppm (s, CHr), 81.21 ppm (s, CH4<), 79.36 ppm (s, C2<), 74.43 ppm (s, CH3<), 67.05-67.01 ppm (d, 4xO(CH2CH2)2N morpholine) 64.86-64.82 ppm (d, CH25 , 53.96 ppm (s, OCH3 guanine), 44.72-44.69 ppm (d, 4xO(CH2CH2)2N), 20.35 ppm (s, CH3 v).
[00583] 31P NMR (CDC13) 14.85 ppm (s).
[00584] Example 45
[00585] (2S,2'S)-2,4-Difluorobenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl) bis(azanediyl)dipropanoate
Figure imgf000094_0001
[00586] The phosphorodiamidate was prepared according to the Method B.
[00587] In a first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (0.13 mL, 0.96 mmol) and phosphorus oxychloride (87 \L, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-alanine 2,4-difluorobenzyl ester tosylate salt (1.86 g, 4.80 mmol) and triethylamine (1.30 mL, 9.62 mmol) were added. After work-up, silica gel column chromatography 108 mg of the phosphorodiamidate was obtained in 15% yield as an off white solid [00588] 1H NMR (500MHz, CD3OD) : δ 7.95 (s, IH, ¾), 7.45 (m, 2H, OCH2P/i), 6.9 (m, 4H, OCH2Ph), 5.05-5.2 (m, 4H, 2x OCH2Ph), 4.45-4.37 (m, 2Η, ¾·), 4.35 (d, 1Η, Η3 ), 4.25 (m, 1Η, ¾·), 3.95 (q, 2Η, CHa-Ala), 1.35-1.24 (m, 9H, CH3-Ala and 60CH3), 1.05 (s, 3H, 2'CCH3),
[00589] 13C NMR (125 MHz, CD3OD): δ 175.30 (C=0), 162.76 (C6), 161.63 (C2),
154.54 (C4), 139.21 (C8), 131.60 (ipso Ph), 115.71 (C5), 112.41 (dd, J= 3.8, 21.5 Hz, Ph C-C-F), 104.81 (t, J= 25.4 Hz, Ph F-C-F), 93.08 (CI'), 82.29 (d, 3JP-o-c-c= 7.6 Hz, C4'), 80.13 (C2'), 74.81 (C3'), 66.20 (d, 2JP_0-c= 5.5 Hz, C5'), 61.26 (d, J= 3.7, OCH2 ester), 61.23 (d, J= 3.7 Hz, OCH2 ester), 54.43 (60CH3), 51.07 (2x Ca Ala), 20.92, 20.84 (2d, 2JP_N_C= 5.94 Hz, CH3 Ala), 20.51 (2'CCH3),
[00590] 31P NMR (202 MHz, CD3OD): δ 13.84
[00591] 19F (470 MHz, CD3OD) δ: -113.71, -111.13
[00592] HPLC tR: 18.57 min (Varian Polaris C18-A (10 μΜ) column using a mobile phase of water/acetonitrile ingradient (90/10 to 0/100 v/v in 30 min, System 1)
[00593] MS (ES+): m/z (M + H+) - 786.22; Accurate mass calculated for: C32H37F4N7O10P
- 786.2276
[00594] Example 46
[00595] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000095_0001
[00596] The phosphorodiamidate was synthesized using Method B. [00597] In a first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.92 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (0.12 mL, 0.92 mmol) and phosphorus oxychloride (84 μί, 0.92 mmol). In a second step, anhydrous dichloromethane (4 mL), L-alanine cyclohexyl ester tosylate salt (954 mg, 4.61 mmol) and triethylamine (1.20 mL, 9.22 mmol) were added. After workup and silica gel column chromatography, 43 mg of phosphorodiamidate was obtained in 6.6% yield, as an off white solid.
[00598] 1H NMR (500 MHz, CD3OD): δ 7.95 s (1H, ¾), 6.0 s (1H, Hr), 4.7 m (2H, 2x
OCH cHex), 4.58 (q, 4H, J= 7.0 Hz, 2x CH2 cHex), 4.4 (m, 2H, ¾·), 4.38 (m, 1H, ¾·), 4.2 (m, 1H, H4 , 3.91 (m, 2H, 2x CHa Ala), 1.4- 1.9 (m, 20H, 2x cHex), 1.45 (t, 3H, J= 7.32Hz, 60CH2CH3), 1.34 d of d (6Η, J= 7.5 Hz 2x CH3 Ala), 0.98 s (3H, 2'CMe),
[00599] 13C NMR (125 MHz, CD3OD): δ 175.10 (d, 3JP_N_c-c= 6.0Hz, C=0), 175.04 (d,
3JP_N_c-c= 6.0 Hz, C=0), 162.41 (C6), 161.89 (C2), 154.60 (C4), 139.24 (C8), 115.63 (C5), 93.17 (CI '), 82.36 (d 3JP-0-c-c= 7.67Hz, C4'), 80.04 (C2'), 74.86, 74.80 (2x OCH cHex), 74.59 (C3'), 66.41 (d, 2JP_0-c= 4.86Hz, C5'), 61.57 (OCH2CH3), 51.20 (CHa Ala), 36.32 (CH2 cHex), 32.50 (CH2 cHex), 32.47 (CH2 cHex), 32.43 (CH2 cHex), 26.45 (CH2 cHex), 25.36 (CH2 cHex), 24.69 (CH2 cHex), 24.65 (CH2 cHex), 21.09 (d, J=5.9, 2x CH3 Ala), 20.32 (2'CCH3), 14.52 (OCH2CH3),
[00600] 31P NMR (202 MHz, CD3OD): 514.10
[00601] HPLC = 19.55 min (System 1).
[00602] MS (ES+)m z: 712.34 (M+H+). Accurate mass calculated for C31H51N7O10P - 712.3435
[00603] Example 47
[00604] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000097_0001
[00605] The phosphorodiamidate was synthesized using Method B.
[00606] In a first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.92 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (0.12 mL, 0.92 mmol) and phosphorus oxychloride (84 μί, 0.92 mmol). In a second step, anhydrous dichloromethane (4 mL), L-alanine benzyl ester tosylate salt (1.62 g, 4.61 mmol) and and triethylamine (1.20 mL, 9.22 mmol) were added. After workup and silica gel column chromatography, 96 mg of phosphorodiamidate was obtained in 14% yield, as an off white solid.
[00607] 1H NMR (500 MHz, CD3OD): δ 8.05 (s, IH, ¾), 7.2-7.4 m (10H, OCH2P/i), 6.0
(s, IH, Hr), 5.1 (m, 4H, 2x OCH2Ph), 4.5 (q, 2Η, 60CH2CH3), 4.4 (m, 2H, ¾·), 4.25 (d, IH, ¾·), 4.2 m (IH, ¾·), 3.95 (q, 2H, J= 7.2 Hz, 2x CHa Ala), 1.4 (t, 3H, J=7.3 Hz, 60CH2CH3), 1.3 (d, 3Η, J= 2.4 Hz, CH3 Ala), 0.95 (s, 3H, 2'CCH3).
[00608] 13C NMR (125 MHz, CD3OD): 175.85 (C=0), 175.48 (C=0), 162.23 (C6), 161.78
(C2), 154.10 (C4), 139.12 (C8), 129.79, 129.67, 129.65, 129.62, 129.41, 129.39, 129.34, 129.32, 129.25, 129.24 (OCH2P/i), 114.87 (C5), 93.24 (CI '), 82.51 (d, 3JP_o-c-c= 7.8 Hz, C4'), 80.08 (C2'), 74.75 (C3'), 68.00, 67.97 (OCH2Ph), 66.21 (d, 2JP_o-c= 5.4 Hz, C5'), 63.92 (OCH2CH3), 51.14 (d, J= 4.2 Hz, CHa Ala), 20.98 (d, J= 6.1 Hz, CH3 Ala), 20.93 (d, J= 6.1 Hz, CH3 Ala), 20.49 (2'CCH3), 14.96 (OCH2 H3).
[00609] 3 *P NMR (202 MHz, CD3OD) : δ 13.92. [00610] HPLC tR = 17.59 min (System 1).
[00611] MS (ES+) m/z : 728.28 (M+H+). Accurate mass found for C33H43N7O10P - 728.2809. [00612] Example 48
[00613] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000098_0001
[00614] The phosphorodiamidate was synthesized using Method B.
[00615] In a first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.92 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (0.12 mL, 0.92 mmol) and phosphorus oxychloride (84 \L, 0.92 mmol). In a second step, anhydrous dichloromethane (4 mL), L-alanine 2,2-dimethylpropyl ester tosylate salt (1.52 g, 4.61 mmol) and triethylamine (1.20 mL, 9.22 mmol) were added. After work-up and silica gel column chromatography, 80 mg of phosphorodiamidate was obtained in 12% yield, as an off white solid.
[00616] 1H NMR (500 MHz, MeOD-J4) δ 8.0 (s, IH, ¾), 5.95 (s, IH, Hr), 4.55 (q, 2H,
J= 7.02Hz, OCH2CH3), 4.4 (m, 2H, ¾·), 4.3 (d, IH, J= 9.1 Hz, ¾·), 4.2 (m, IH, H4 , 4.0 (q, 2H, J= 8.4 Hz, Ha Ala), 3.85-3.82 (m, 2H, OCH2 ester), 1.45 (t, 3H, J=7.0 Hz, OCH2CH3), 1.3 (d, 3Η, J= 2.4 Hz, CH3 Ala), 0.96 (s, 3H, 2'CCH3), 0.95 (2s, 18H, 2x OCH2C(CH3)).
[00617] 13C NMR (125 MHz, MeOD-J4) δ 175.68 (C=0), 175.61 (C=0), 162.39 (C6),
161.91 (C2), 154.60 (C4), 139.27 (C8), 115.56 (C5), 93.16 (CI '), 82.35 (d, 3JP_0-c- c= 8.4 Hz, C4'), 80.03 (C2'), 75.37 (2x O H2C(CH3)3), 74.85 (C3'), 66.42 (d, 2JP_ o-c= 4.9 Hz, C5'), 63.52 (OCH2CH3), 51.06 (d, 2JP_N-c= 8.73 Hz, Ccc Ala), 32.27 (2x OCH2C(CH3)3), 26.77 (s, OCH2C(CH3)3), 20.99 (CH3 Ala), 20.28 (2'C H3), 14.87 (OCH2 H3) [00618] 31P NMR (202 MHz, MeOD-J4): δ 14.00 [00619] HPLC iR = 19.65 min (System 1). [00620] MS (ES+)m z: 688.34 (M+H+). Accurate mass found for C^HsiNvOioP - 688.3435
[00621] Example 49 [00622] (2S)-Cyclohexyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)((S)-l-(cyclopentyloxy)-l- oxopropan-2-ylamino)phosphorylamino)propanoate
Figure imgf000099_0001
[00623] The phosphorodiamidate was prepared according to Method C. [00624] In the first step, a solution of 6-O-methyl-2' -C-methylguanosine (550 mg, 1.76 mmol) in anhydrous tetrahydrofuran (8 mL) was allowed to react with triethylamine (300 \L, 2.11 mmol) and phosphorus oxychloride (200 \L, 2.11 mmole). L-alanine cyclohexyl ester tosylate salt (370 mg, 1.76 mmol) and triethylamine (0.5 mL, 3.52 mmol) were added. Anhydrous dichloromethane (2 mL), and the L-alanine cyclopentyl ester tosylate salt (2.90 g, 8.80 mmol) and triethylamine (2.50 mL, 17.60 mmol) were added. After work-up and silica gel column chromatography, 280 mg of the prodrug was obtained in 23% yield as an off white solid.
[00625] 1H NMR (500 MHz, CDC13) 57.74 (s, 1H, ¾), 5.96 (s, 1H, Hr), 5.18 (m, 1H, H
3·), 4.76 (m, 1H, cyclopentyl or cyclohexyl), 4.61 (m, 1H, 1H H5 ), 4.51 (m, 1H, CH cyclopentyl or cyclohexyl), 4.32 (m, 1H, 1H ¾·), 4.23 (m, 1H, ¾·), 4.06 (s, 60CH3), 3.92 (m, 2H, 2x CHcc Ala), 1.86-1.57 (m, 18H, 4x CH2 cyclopentyl and 5x CH2 cyclohexyl), 1.36 (m, 6H, 2x CH3 Ala), 1.00 (s, 3H, 2'CCH3). [00626] 31P NMR (202 MHz, CDC13) δ 13.68, 13.48 [00627] HPLC iR= 14.97 min (System 1). [00628] HPLC tR = 17.37 min (System 2).
[00629] MS (TOF ES+) m/z: 684.32 (M+H+), 706.27 (M+Na+), 722.27 (M+K+); accurate mass: calculated for C29H47N7O10P: 684.3122, found 684.3154.
[00630] Example 50
[00631] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(pentylamino)phosphorylamino)propanoate
Figure imgf000100_0001
[00632] The phosphorodiamidate was synthesized using Method D.
[00633] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (200 mg, 0.642 mmol) in anhydrous tetrahydrofuran (5 mL) was allowed to react with triethylamine (105 μί, 0.771 mmol) and L-alaninyl neopentyl ester phosphorodichloridate (355 mg, 1.29 mmol). Pentylamine (0.56 mL, 3.21mmoles) and triethylamine (0.67 mL, 3.21 mmol) were added. After work-up and silica gel column chromatography, 11 mg of phosphorodiamidate was obtained in 3% yield as an off white solid.
1H NMR (500 MHz, MeOO-d4) δ 8.02, 8.01 (2 s, IH, ¾), 6.00, 5.99 (2 s, IH, Hi , 4.36- 4.33 (m, 2H, ¾■) 4.29, 4.28 (2d, J= 9.0 Hz, IH, ¾■), 4.20- 4.17 (m, IH, H4 , 4.07 (s, 3H, 60CH3), 3.94- 3.90 (m, IH, CHa Ala), 3.87, 3.83, 3.75, 3.70 (2AB, JAB= 10.5 Hz, 2H, CH2 ester), 2.90- 2.85 (m, 2H, NH- CH2CH2CH2CH2CH3), 1.50- 1.43 (m, 2H, NH-CH2CH2CH2CH3), 1.39, 1.37 (2d, J= 6.0 Hz, 3H, CH3 Ala), 1.31-1.21 (m, 4H, NH-CH2CH2CH2CH2CH3 and NH- CH2CH2CH2CH2CH3), 0.99, 0.98 (2s, 3H, 2'CCH3), 0.94, 0.93 (2s, 9Η, OCH2C(CH3)3), 0.87-85 (m, 3Η, NH-CH2CH2CH2CH2CHJ).
[00635] 13C NMR (125 MHz, MeOO-d4) δ 175.75, 175.68 (C=0 ester), 162.72 (C6),
161.92 (C2), 154.61, 154.57 (C4), 139.25, 139.11 (C8), 115.43 (C5), 93.05, 92.94 (CI '), 82.41, 82.34 (2d,
Figure imgf000101_0001
Hz, C4'), 80.08, 80.04 (C2'), 75.34, 75.31 (OCH2C(CH3)3), 74.60, 74.37 (C3'), 65.90, 65.49 (2d, 2JC-o-p=5.0 Hz, C5'), 54.21 (60CH3), 51.27, 51.16 (Ca Ala), 42.06 (NH- H2CH2CH2CH2CH3), 32.76 (d, 3JC- C-N-P=5.0 Hz NH-CH2 H2CH2CH2CH3), 32.72 (d, 3/C-C-N-P=6.3 HZ NH- CH2CH2CH2CH2CH3), 32.27 (O H2C(CH3)3), 30.11, 30.08 (NH- CH2CH2CH2CH2CH3), 26.74, 26.72 (OCH2C(CH3)3), 23.46 (NH- CH2CH2CH2 H2CH3), 21.19, 21.96 (2d, 3/C-C-N-P=6.3 HZ, CH3 Ala), 20.24 (2'CCH3), 14.40 (NH-CH2CH2CH2CH2 H3).
[00636] 31P NMR (202 MHz, MeOD-<¾) δ 16.28, 16.12.
[00637] HPLC ¾ = 17.11 min (System 1).
[00638] MS (TOF EI+) m/z: 624.34 (MNa+, 100%);
[00639] HRMS C25H45N708P1 Calculated: 602.3067 found: 602.3057
[00640] Example 51
[00641] (2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(morpholino)phosphorylamino)propanoate
Figure imgf000101_0002
[00643] The phosphorodiamidate was prepared according to Method C.
[00644] In the first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (250 mg, 0.803 mmol) in anhydrous tetrahydrofuran (5 mL) was allowed to react with triethylamine (110 μί, 0.803 mmol) and phosphorus oxychloride (70μί, 0.803 mmole). The morpholine (70 μί, 0.803 mmol) and triethylamine (110 μί, 0.803 mmol) were added. Anhydrous dichloromethane (4 mL), and the L-alanine benzyl ester tosylate salt (1.41 g, 4.02 mmol) and triethylamine (1.12 mL, 8.03 mmol) were added as described in Method C. After work-up and silica gel column chromatography, 108 mg of phosphorodimaidate was obtained in 22% yield as an off white solid.
[00645] 1H NMR (500 MHz, MeOD-d4) 7.96, 7.95 (2s, IH, H8), 7.37-7.30 (m, 5H,
OCH2Ph), 5.98, 5.97 (2s, IH, HI'), 5.20-5.12 (m, 2H, OCH2Ph), 4.42-4.33 (m, 3Η, Η5' and Η3'), 4.22-4.18 (m, 1Η, Η4'), 4.07, 4.06 (2s, 3Η, 60CH3), 3.96-3.82 (m, IH, CHa Ala), 3.52-3.79 (m, 4H, 2x 0(CH2)2), 3.11-3.08 (m, 4Η, 2x N(CH2)2), 1.38, 1.33 (2d, J=7.5 Hz, 3H, CH3 Ala), 1.01, 1.00 (2s, 3H, 2'CCH3).
[00646] 13C NMR (125 MHz, MeOO-d4) 175.38, 175.34 (2d,
Figure imgf000102_0001
3.8 Hz, C=0 ester), 162.79, 162.75 (C6), 161.91 (C2), 154.52, 154.47 (C4), 139.40, 139.31 (C8), 137.31 (ipso OCH2P/i), 129.60, 129.57, 129.47, 129.38, 129.32 (OCH2P/i), 115.71, 115.60 (C5), 93.46, 93.30 (CI'), 82.39, 82.33 (2d,
Figure imgf000102_0002
3.8 Hz, C4'), 80.04 (C2'), 74.98, 74.82 (C3'), 68.05 (d, 2JC-o-p= 3.8 Hz, C5'), 67.97 (d, 2JC-o-p= 5.0 Hz, C5'), 66.53, 66.49 (O H2Ph and 0( H2)2), 54.23 (60CH3), 51.24, 50.99 (Ca Ala), 45.74 (N(CH2)2), 20.79 (d,
Figure imgf000102_0003
5.0 Hz, CH3 Ala), 20.55 (d, 3/C-C-N- P= 6.3 Hz, CH3 Ala), 20.29, 20.23 (2'CCH3).
[00647] 31P NMR (202 MHz, MeOD-d4) 14.65, 14.30.
[00648] HPLC ¾ = 11.29, 11.60 min (System 1)
[00649] MS (TOF ES+) m/z: 644.22 (M+Na+, 100%);
[00650] HRMS
Figure imgf000102_0004
calculated: 622.2390 found: 622.2381 [00651] Example 52 [00652] (2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(cyclopropylamino)phosphorylamino)propanoate
Figure imgf000103_0001
[00653] The phosphorodiamidate was synthesized using Method D. [00654] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (200 mg, 0.642 mmol) in anhydrous tetrahydrofuran (5 mL) was allowed to react with triethylamine (105 \L, 0.771 mmol) and 2,2-dimethylpropoxy-L-alaninyl phosphorodichloridate (355 mg, 1.29 mmol). Cyclopropylamine (0.33 mL, 3.21mmoles) and triethylamine (0.67 mL, 3.21 mmol) were added. After work-up and silica gel column chromatography, 14 mg of phosphorodiamidate was obtained in 3% yield as an off white solid.
[00655] 1H NMR (500 MHz, MeOD-<¾) δ 8.04, 8.03 (2s, IH, ¾), 6.01, 5.99 (2s, IH, Hr),
4.42- 4.31 (m, 2H, ¾■) 4.29-4.25 (m, IH, ¾■), 4.20- 4.17 (m, IH, ¾■), 4.08, 4.07 (2s, 3H, 60CH3), 3.97- 3.92 (m, IH, CHcc Ala), 3.87, 3.82, 3.75, 3.68 (2AB, /AB= 10.5 Hz, 2H, OCH2C(CH3)3), 2.41-2.36 (m, IH, CH cyclopropylamine), 1.39, 1.38 (2d, J=6.5 Hz, 3H, CH3 Ala), 0.99, 0.98 (2s, 3H, 2'CCH3), 0.94, 0.92 (2s, 9H, OCH2C(CH3)3 ester), 0.58-52 (m, 4Η, 2 x CH2 cyclopropylamine).
[00656] 13C NMR (125 MHz, MeOD-<¾) δ 175.70, 175.67 (C=0 ester), 162.71 (C6),
161.99 (C2), 154.62, 154.56 (C4), 139.24, 139.00 (C8), 115.42, 115.33 (C5), 93.07, 92.82 (CI '), 82.40, 82.24 (2d,
Figure imgf000103_0002
Hz, C4'), 80.10, 80.04 (C2'), 75.35, 75.30 (O H2C(CH3)3). 74.56, 74.23 (C3'), 65.86, 65.23 (2d, 2JC-o-p=5.0Hz, C5'), 54.20 (60CH3), 51.30, 51.14 (Ca Ala), 32.26 (OCH2C(CH3)3), 26.73, 26.70 (OCH2C( H3)3), 23.46 (CH cyclopropylamine), 21.23, 20.99 (2d,
Figure imgf000104_0001
Hz, CH3 Ala), 20.23 (2'CCH3), 7.46, 7.26 (2x H2 cyclopropylamine).
[00657] 31P NMR (202 MHz, MeOD-d4) δ 16.03, 15.77.
[00658] HPLC ¾ = 13.17 min (System 1).
[00659] MS (TOF EI+) m/z: 594.29 (MNa+, 100%);
[00660] HRMS C23H39N708P1 Calculated: 572.2587 found: 572.2598
[00661] Example 53
[00662] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylpentanoate)
Figure imgf000104_0002
[00663] The phosphorodiamidate was prepared according to Method B.
[00664] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (250 mg, 0.80 mmol) in anhydrous tetrahydrofuran (4.3 mL) was reacted with triethylamine (130 μί, 0.96 mmol) and phosphorus oxychloride (90 μί, 0.96 mmol). In a second step, anhydrous dichloromethane (2.3 mL), L-leucine 2,2-dimethylpropyl ester tosylate salt (1.51 g, 4.05 mmol) and triethylamine (1.12 mL, 8.04 mmol) were added. After work-up and silica gel column chromatography, 5 mg of phosphorodiamidate was obtained in 1% yield as an off white solid.
1H NMR (500 MHz, CDC13) δ 7.67 (s, IH, ¾), 5.90 (s, IH, Hr), 4.74 (m, IH, IH HsO, 4.66 (m, IH, ¾·), 4.25 (m, IH, IH ¾·), 4.23 (m, IH, ¾·), 4.06 (s, 3H, 60CH3), 3.97-3.91 (2m, 2H, 2 x CHa Leu), 3.82-3.75 (m, 4H, 2x OCH2C(CH3)3), 1.73- 1.68 (m, 2H, 2x CHy Leu), 1.58- 1.45 (m, 4H, 2x CH2 Leu), 1 1.00 (s, 3H, 2'CCH3), 0.97-0.93 (m, 30H, 4x CH3 Leu, 2x OCH2C(CH3)3).
[00666] 13C NMR (125 MHz, CDC13) δ 175.20 (C=0 Leu), 174.58 (C=0 Leu), 161.61
(C6), 159.63 (C2), 152.86 (C4), 137.05 (C8), 115.96 (C5), 91.25 (CI '), 81.33 (C4'), 79.65 (C2'), 75.19 (C3'), 74.77, 74.65 (2x O H2C(CH3)3), 65.59 (C5'), 53.65 (60CH3), 52.96, 52.82 (2x Ca Leu ), 43.77 (2x <¾β Leu), 31.34, 31.24 (2x OCH2C(CH3)3), 26.52, 26.38 (2x OCH2C(CH3)3), 24.62, 24.56 (2x CHy Leu), 22.68 (2x CH3 Leu), 22.01 (2x CH3 Leu), 20.43 (2'C H3).
[00667] 31P NMR (202 MHz, CDC13) δ 13.76.
[00668] HPLC tR = 25.39 min (System 1).
[00669] MS (TOF ES+) m/z: 758.42 (M+H+), 780.40 (M+Na+); accurate mass: calculated for C34H61N7O10P: 758.4218, found 758.4201.
[00670] Example 54
[00671] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylpentanoate)
Figure imgf000105_0001
[00672] The phosphorodiamidate was prepared according to Method B.
[00673] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (590 mg, 1.89 mmol) in anhydrous tetrahydrofuran (8 mL) was reacted with triethylamine (320 \L, 2.27 mmol) and phosphorus oxychloride (210 \L, 2.27 mmol). In a second step, anhydrous dichloromethane (5.4 mL), L-leucine cyclohexyl ester tosylate salt (3.64 g, 9.45 mmol) and triethylamine (2.67 mL, 18.9 mmol) were added. After work-up and silica gel column chromatography, 100 mg of phosphorodiamidate was obtained in 7% yield as an off white solid.
[00674] 1H NMR (500 MHz, CDC13) δ 7.71 (s, 1H, ¾), 5.95 (s, 1H, Hr), 4.76-4.70 (m,
2H, 2 x OCH cyclohexyl), 4.60 (m, 1H, ¾·), 4.48-4.47 (m, 1H, ¾·), 4.25-4.23 (m, 1H, ¾·), 4.20-4.17 (m, 1H, ¾·), 4.02 (s, 3H, 60CH3), 3.89-3.77 (m, 2H, 2x CHa Leu), 1.79 (m, 4H, 2x CH2 cyclohexyl), 1.67- 1.65 (m, 4H, 2x CH2 cyclohexyl), 1.50- 1.24 (m, 10H, 2x CH2 cyclohexyl, 2x CH2 Leu and 2x CHy Leu), 1.24 (s, 3H, CH3 2'), 0.98-0.84 (m, 12H, 4x CH3 Leu).
[00675] 13C NMR (125 MHz, CDC13-J4) δ 174.25 (C=0 Leu), 173.84 (C=0 Leu), 161.55
(C6), 159.71 (C2), 152.93 (C4), 137.87 (C8), 115.68 (C5), 91.93 (CI '), 81.12 (C4'), 79.41 (C2'), 74.71 (C3'), 73.87, 73.72 (2x OCH cyclohexyl), 65.23 (C5'), 53.43 (60CH3), 52.94, 52.80 (2x Ca Leu), 43.60, 43.54 (2s, 2x <¾β Leu), 31.46 (4x CH2 cyclohexyl), 25.23 (2x CH2 cyclohexyl), 24.56, 24.51 (2x CHj Leu), 23.59 (4x CH2 cyclohexyl), 22.61, 22.03 (2x CH3 Leu), 20.31 (s, 2'CCH3).
[00676] 31P NMR (202 MHz, CDC13-J4) δ 13.78
[00677] HPLC ¾=20.11 min (System 1).
[00678] MS (TOF ES+) m/z: 782.42 (M+ H+), 804.41 (M+Na+); accurate mass: calculated for C36H61N7O10P: 782.4218, found 782.4234.
[00679] Example 55
[00680] (2S,2'S)-Dibenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylthiobutanoate)
[00681]
Figure imgf000107_0001
[00682] The phosphorodiamidate was prepared according to Method B.
[00683] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 μί, 0.96 mmol) and phosphorus oxychloride (90 μί, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-methionine benzyll ester tosylate salt (1.98 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After workup and silica gel column chromatography, 130 mg of phosphorodiamidate was obtained in 16% yield as an off white solid.
[00684] 1H NMR (500 MHz, CD3OD) δ 7.95 (s, 1H, ¾), 7.36-7.30 (2x OCU2Ph) 5.97 (s,
1H, Hr), 5.19-5.05 (m, 2x OCH2Ph), 4.41-4.31 (m, 2Η, ¾< and ¾·), 4.19-4.17 (m, 1Η, ¾·), 4.04 (s, 3Η, 60C¾), 4.07-4.03 (m, 2Η, 2x CHD Met), 2.51-2.39 (m, 4H, 2x CH2y Met), 1.99, 1.95 (2s, 6H, 2x CH3 Met), 1.90-1.80 (m, 4H, 2x CH2D Met), 1.00 (s, 3H, CH3).
[00685] 13C NMR (125 MHz, CD3OD) δ 173.78, 173.75 (2d, 3 J C-C-N-P = 4.8 Hz, 2x C=0
Met), 161.91 (C6), 161.04 (C2), 153.73 (C4), 138.55 (C8), 136.39, 136.35 (2x ipso OCH2Ph), 128.77, 128.66, 128.63, 128.56, 128.53 (2x OCH2Ph), 114.79 (C5), 92.37 (CI '), 81.59 (d, 3JC-c-o-p = 7.3 Hz, C4'), 79.20 (C2'), 74.00 (C3'), 67.25 (d, 2 J c-o-p = 56.3 Hz, C5'), 65.81, 65.77 (2x OCH2Ph), 53.87, 53.72 (2x CH Met), 53.42 (OCH3), 33.62, 33.57 (2x Cy Met), 29.98, 29.93 (2x CD Met), 19.50 (2'CCH3), 14.36, 14.30 (SCH3).
[00686] 31P NMR (202 MHz, CD3OD) δ 14.11
[00687] HPLC tR= 19.05 min (System 1). [00688] MS (TOF ES+) mJ . 856.25 (M+Na+, 100%); [00689] HRMS C34H57N7O10PiS2 calculated: 834.2720 found: 834.2692 [00690] Example 56 [00691] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylthiobutanoate)
[00692]
Figure imgf000108_0001
[00693] The phosphorodiamidate was prepared according to Method B. [00694] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 \L, 0.96 mmol) and phosphorus oxychloride (90 \L, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-methionine cyclohexyl ester tosylate salt (1.94 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After work-up and silica gel column chromatography, 160 mg of phosphorodiamidate was obtained in 20% yield as an off white solid.
[00695] 1H NMR (500 MHz, CD3OD) δ 8.07 (s, 1H, ¾), 6.00 (s, 1H, Hr), 4.79-4.74,
4.73-4.70 (2m, 2H, 2x OCH ester), 4.45-4.36 (m, 2Η, ¾·), 4.30 (d, 1Η, J=8.5 Hz, ¾·), 4.23-4.20 (m, 1H, H4<), 4.08 (s, 3H, 60CH3), 4.02-3.97 (m, 2H, 2x CHD Met), 2.61-2.48 (m, 4Η, 2x CH2y Met), 2.07, 2.05 (2s, 6H, 2x CH3 Met), 2.02- 1.87 (m, 4H, 2x CH2 Met), 1.86- 1.79 (m, 4H, 2x CH2 ester), 1.73- 1.71 (m, 4H, 2x CH2 ester), 1.47- 1.30 (m, 12H, 6x CH2 ester), 1.01 (s, 3H, CH3), 0.95 and 0.93 (2s, 18H, 6x CH3 ester).
[00696] 13C NMR (125 MHz, CD3OD) δ 174.30, 174.21 (2d, 3 J C-C-N-P = 5.3 Hz, 2x C=0),
162.66 (C6), 161.987 (C2), 154.19 (C4), 139.34 (C8), 115.05 (C5), 93.33 (CI '), 82.61 (d, 3Jc-c-o-p = 6.5 Hz, C4'), 80.03 (C2'), 75.07, 75.02 (2x OCH ester), 74.84 (C3'), 66.74 (d, 2 J c-o-p = 5.3 Hz, C5'), 54.87, 54.79 (2x CD Met), 54.41 (OCH3), 34.67, 34.62 (2d, 3 J C-C-N-P = 2.6 Hz, 2x CH2 Met), 32.53 (CH2 ester), 30.95, 30.87 (2x CH2 Met), 26.47 (CH2 ester), 26.44 (CH2 ester), 26.41 (2x CH2y Ile), 24.71 (2 x CH2 ester), 24.68 (2 x CH2 ester), 20.33 (2'CCH3), 15.29, 15.23 (SCH3).
[00697] 31P NMR (202 MHz, CD3OD) δ 14.26.
[00698] HPLC tR = 21.60 min (System 1).
[00699] MS (TOF ES+) m/z: 840.32 (M+Na+, 100%);
[00700] HRMS C34H57N7O10PiS2 calculated: 818.3346 found: 818.3359
[00701] Example 57
[00702] (2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-phenylpropanoate)
Figure imgf000109_0001
[00703] The phosphorodiamidate was prepared according to Method B.
In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (150 mg, 0.47 mmol) in anhydrous tetrahydrofuran (2.5 mL) was reacted with triethylamine (80 μί, 0.56 mmol) and phosphorus oxychloride (50 μί, 0.56 mmol). In a second step, anhydrous dichloromethane (3.6 mL), L-phenylalanine benzyl ester tosylate salt (1.00 g, 2.34 mmol) and triethylamine (0.65 mL, 4.68 mmol) were added. After work-up and silica gel column chromatography, 40 mg of phosphorodiamidate was obtained in 9% yield as an off white solid.
[00705] 1H NMR (500 MHz, CDC13) δ 7.66 (s, 1H, ¾), 7.33-7.26 (m, 10H, OCH2Ph and
Phe), 7.19-1.17 (m, 6H, Phe), 7.02-7.00 (m, 4H, OCH2Ph), 5.91 (s, 1H, Hr), 5.14- 5.02 (m, 4H, 2x OCH2Ph), 4.45 (m, 1Η, ¾·), 4.34 (m, 1Η, ¾·), 4.18 (m, 3Η, 2x CHcc Phe and ¾·), 4.05 (s, 3H, 60CH3), 2.92-2.84 (m, 2H, 2x CH2 Phe), 0.97 (s, 3H, 2'CCH3).
[00706] 13C NMR (125 MHz, CDC13) δ 173.18 (C=0 Phe), 172.69 (C=0 Phe), 161.62
(C6), 159.57 (C2), 152.90 (C4), 137.94 (C8), 135.94, 135.87 (2x ipso Phe), 135.13 (2x ipso OCH2Ph), 129.50, 129.46 (Phe and OCH2Ph), 128.59, 128.49 (Phe and OCH2Ph), 127.04, 127.02 (Phe), 115.86 (C5), 91.82 (CI'), 81.04 (C4'), 79.51 (C2'), 74.94 (C3'), 67.33, 67.19 (2s, 2x O H2Ph), 65.16 (C5'), 55.30, 55.24 (2x Ca Phe), 53.44 (60CH3), 40.15 (2x CH2 Phe), 20.37 (2'CCH3).
[00707] 31P NMR (202 MHz, CDC13) δ 13.20.
[00708] HPLC tR = 21.16 min (System 1).
[00709] MS (TOF ES+) m/z: 866.33 (M+H+), 888.35 (M + Na+); accurate mass: calculated for C44H49N7O10P: 866.3279, found 866.3281.
[00710] Example 58
[00711] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-phenylpropanoate)
Figure imgf000111_0001
[00712] The phosphorodiamidate was prepared according to Method B.
[00713] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (180 mg, 0.57 mmol) in anhydrous tetrahydrofuran (3.1 mL) was reacted with triethylamine (100 μί, 0.69 mmol) and phosphorus oxychloride (70 μί, 0.69 mmol). In a second step, anhydrous dichloromethane (4.4 mL), L-phenylalanine 2,2-dimethylpropyl ester tosylate salt (1.12 g, 2.87 mmol) and triethylamine (0.80 mL, 5.73 mmol) were added. After work-up and silica gel column chromatography, 50 mg of phosphorodiamidate was obtained in 11% yield as an off white solid.
[00714] 1H NMR (500 MHz, CDC13) δΗ 7.68 (s, 1H, ¾), 7.25-7.08 (m, 10H, 2 x H aPhe),
5.92 (s, 1H, Hr), 4.42 (m, 1H, ¾·), 4.35 (m, 1H, 1H ¾·), 4.19 (m, 2H, ¾· and CHa Phe), 4.17-4.08 (m, 2H, Η5· and CHa Phe), 4.04 (s, 3H, 60CH3), 3.79-3.70 (m, 4H, 2x OCH2C(CH3)3), 2.98-2.84 (2m, 4H, 2x CH2 Phe), 0.98 (s, 3H, 2'CCH3)..
[00715] 13C NMR (125 MHz, CDC13) δ 173.50 (C=0 Phe), 173.03 (C=0 Phe), 161.60
(C6), 159.58 (C2), 152.96 (C4), 137.90 (C8), 136.16, 136.03 (2x ipso Phe), 129.48, 129.39 (Phe), 128.55, 128.27 (Phe), 127.05, 127.01 (Phe), 115.77 (C5), 91.82 (CI'), 81.09 (C4'), 79.47 (C2'), 74.89 (C3'), 74.77 (2x O H2C(CH3)3), 65.20 (C5'), 55.22 (2x Ca Phe), 53.80 (60CH3), 45.78 (2x CH2 Phe), 31.21, 31.15 (2x OCH2C(CH3)3), 26.73, 26.37 (2x OCH2C(CH3)3), 20.36 (2'CCH3).
[00716] 31P NMR (202 MHz, CDC13) δ 13.10.
[00717] HPLC ¾ = 24.91 min (System 1). [00718] MS (TOF ES+) m/z: 826.39 (M+H+), 848.50 (M+Na+); accurate mass: calculated for C40H57N7O10P: 826.3905, found 866.3281.
[00719] Example 59 [00720] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-phenylpropanoate)
Figure imgf000112_0001
[00721] The phosphorodiamidate was prepared according to the Method B. [00722] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (310 mg, 1.01 mmol) in anhydrous tetrahydrofuran (4.3 mL) was reacted with triethylamine (170 \L, 1.21 mmol) and phosphorus oxychloride (110 \L, 1.21 mmol). In a second step, anhydrous dichloromethane (4.0 mL), L-phenylalanine cyclohexyl ester tosylate salt (2.13 g, 5.05 mmol) and triethylamine (1.41 mL, 10.1 mmol) were added. After work-up and silica gel column chromatography, 40 mg of the phosphorodiamidate was obtained in 5% yield as an off white solid.
[00723] 1H NMR (500 MHz, CDC13) δ 7.65 (s, 1H, ¾), 7.28-7.11 (m, 12H, Phe), 5.91 (s,
1H, Hr), 4.48 (m, 1H, ¾·), 4.38 (m, 1H, ¾·), 4.13-4.12 (m, 2H, ¾· and ¾·), 4.06 (s, 3H, 6OCH3), 4.02 (m, 1H, CHa Phe), 2.98-2.85 (m, 4H, 2x CH2 Phe), 1.80- 1.67 (m, 8Η, cHex), 1.51 (m, 2H, 2x CH2 cHex), 1.32-1.23 (m, 8H, cHex), 0.98 (s, 3H, 2'CCH3).
Ill [00724] C NMR (125 MHz, CDC13) δ 172.76 (C=0 Phe), 172.28 (C=0 Phe), 161.58 (C6), 159.65 (C2), 152.96 (C4), 137.81 (C8), 136.23, 136.14 (2x ipso Phe), 129.58, 129.52 (Phe), 128.81,128.12 (Phe), 126.97, 126.92 (Phe), 115.72 (C5), 91.76 (CI'), 81.06 (C4'), 79.46 (C2'), 74.81 (C3'), 74.21, 74.06 (2x OCR cHex), 65.17 (C5'), 55.30, 55.26 (2d, 2x CHa Phe), 53.81 (60CH3), 40.40, 40.34 (2d, 2x CH2 Phe), 31.49, 31.40 (4x H2 cHex), 25.22, 23.67 ( H2 cHex), 20.34 (2'CCH3).
[00725] 3 XP NMR (202 MHz, CDC13) δ 13.16 (s). [00726] HPLC tR =20.32 min (System 1).
[00727] MS (TOF ES+) m/z: 850.39 (M+H+), 872.37 (M + Na+); accurate mass: calculated for C4oH57N7010P: 850.3905, found 850.3882.
[00728] Example 60
[00729] (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-(4-tert-butoxyphenyl)propanoate)
Figure imgf000113_0001
[00730] The phosphorodiamidate was prepared according to Method B.
In a first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (260 mg, 0.85 mmol) in anhydrous tetrahydrofuran (4.5 mL) was reacted anhydrous triethylamine (140 μί, 1.02 mmol) with phosphorus oxychloride (90 μί, 1.02 mmol). In a second step, anhydrous dichloromethane (3.4 mL), L-tyrosine i-butyl ether cyclohexyl ester hydrochloride salt (1.07 g, 4.25 mmol) and triethylamine (1.19 mL, 8.5 mmol) were added. After work-up, silica gel column chromatography and preparative TLC, 120 mg of phosphorodiamidate was obtained in 16% yield as an off white solid.
[00732] 1H NMR (500 MHz, CDC13) δ 7.69 (s, 1H, ¾), 7.03-7.02 (d, 2H, J=8.4 Hz, 2x H ortho Tyr), 6.98-6.97 (d, 2H, J=8.4 Hz, 2x H ortho Tyr), 6.87-6.85 (dd, 4H, J=3.0 Hz & 8.40 Hz, 4x H meta Tyr), 5.95 (s, 1H, Hr), 5.45 (bs, 2H, 2x NH Tyr), 4.35- 4.30 (m, 2H, H3< and ¾·), 4.09-4.06 (m, 3H, 2x CH Tyr and ¾·), 4.05 (s, 3H, 60CH3), 4.01-3.96 (m, 1H, ¾·), 3.63, 3.62 (2s, 6H, 2x OCH3), 3.31-3.28 (m, 4H, 2x CH2 Tyr), 1.31, 1.30 (2s, 18H, 6x CH3 iBu), 1.98 (s, 3H, 2'CCH3).
[00733] 13C NMR (125 MHz, CDC13) δ 173.68 (C=0 Tyr), 173.32 (C=0 Tyr), 161.60
(C6), 159.61 (C2), 154.40, 154.15 (2s, 2x C para Tyr), 153.03 (C4), 137.85 (C8), 131.30, 130.80 (2s, 2x ipso Tyr), 130.29, 129.87 (Tyr), 124.20, 124.12 (Tyr), 115.70 (C5), 91.47 (CI '), 80.80 (C4'), 79.36 (C2'), 78.68, 78.42 (2x OC(CH3)3), 74.39 (C3'), 64.77 (C5'), 55.37, 55.22 (2x CH Tyr), 53.81 (60CH3), 52.24, 52.18 (2x OCH3), 39.63 (2x CH2 Tyr), 28.82, 22.77 (2x OC(CH3)3), 20.29 (2'CCH3).
[00734] 31P NMR (202 MHz, CDC13) δ 13.31.
[00735] HPLC ¾ =20.40 min. (System 1).
[00736] MS (TOF ES+) m/z: 858.38 (M+H+), 880.37 (M+Na+); accurate mass: calculated for C4oH57N7Oi2P: 858.3803, found 858.3804.
[00737] Example 61
[00738] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylbutanoate)
Figure imgf000115_0001
[00739] The phosphorodiamidate was prepared according to Method B.
[00740] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 μί, 0.96 mmol) and phosphorus oxychloride (90 μί, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-valine 2,2-dimethylpropyl ester tosylate salt (1.98 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After work-up and silica gel column chromatography 42 mg of phosphorodiamidate was obtained in 6% yield as an off white solid.
[00741] 1H NMR (500 MHz, CD3OD) δ 7.99 (s, 1H, ¾), 5.99 (s, 1H, Hr), 4.41-4.35 (m,
2H, Hs , 4.33 (d, J= 9.0 Hz, 1H, ¾·), 4.20- 4.17 (m, 1H, ¾·), 4.09 (s, 3H, 60CH3), 3.87-3.68 (m,6H, 2x CHa Val and 2x OCH2C(CH3)3), 2.11-1.98 (m, 2H, 2x Οϊβ Val), 0.99, 0.98 (2s, 3H, 2'CCH3), 0.89- 0.83 (m, 12Η, 2x C¾).
[00742] 13C NMR (125 MHz, CD3OD) δ 174.87, 174.80 (2d, 3J C-C-N-P = 2.5 Hz, 2x C=0),
162.77 (C6), 161.92 (C2), 154.54 (C4), 139.49 (C8), 115.68 (C5), 93.35 (CI '), 82.55 (d, 3Jc-c-o-p = 8.8 Hz, C4'), 79.92 (C2'), 75.65, 75.58 (CH2 ester), 75.03 (C3'), 67.11 (d, 2Jc-o-p = 5.0 Hz, C5'), 61.09, 61.03(Ca Val), 54.24 (60CH3), 33.23 (d, 3JC-C-N-P = 6.3 Hz, 2x C Val), 32.14, 32.08 (2x OCH2C(CH3)3), 26.89 (2x OCH2C( H3)3), 20.32 (2'C H3), 18.09, 18.07 (2 x CH3Val).
[00743] 3 *P NMR (202 MHz, CD3OD) 5 15.16
[00744] HPLC ¾ = 22.15 min (System 1).
[00745] MS (TOF ES+) m/z: 752.37 (M+Na+, 100%);
[00746] HRMS C32H56N7O10NaiPi calculated: 752.3724 found: 752.3736. [00747] Examples 62 [00748] (2S,2'S,3R,3'R)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylpentanoate)
Figure imgf000116_0001
[00749] The phosphorodiamidate was prepared according to Method B. [00750] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 μί, 0.96 mmol) and phosphorus oxychloride (90 μί, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-isoleucine benzyl ester tosylate salt (1.89 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After work-up and silica gel column chromatography, 200 mg of phosphorodiamidate was obtained in 16% yield as an off white solid.
[00751] 1H NMR (500 MHz, CD3OD) δ 7.92 (s, 1H, ¾), 7.42-7.28 (m, 10H, 2x OCH2P/i
), 5.99 (s, 1H, Hr), 5.21-5.18 (m, 4H, 2x OCH2Ph), 4.43-4.40 (m, 2Η, ¾·), 4.37 (d, J= 8.5 Hz, 1H, H3<), 4.19- 4.15 (m, 1H, ¾·), 4.03 (s, 3H, 60CH3), 3.74-3.73 (m, 2H, 2x CHcc He), 1.75- 1.73 (m, 2H, 2x ΟΙβ He), 1.37- 1.29 (m, 2H, CH2 He), 1.15- 1.02 (m, 2H, CH2 He), 1.00 (s, 3H, 2'CCH3), 0.83-0.69 (m, 12H, 4x CH3 He).
[00752] 13C NMR (125 MHz, CD3OD) δ 174.47 (2x C=0 ester), 162.77 (C6), 161.90
(C2), 154.58 (C4), 139.42 (C8), 137.19, 137.12 (2x ipso OCH2P/i), 129.93, 129.59, 129.42, 129.39 (2x OCH2P/i), 115.68 (C5), 93.22 (CI '), 82.49 (d, 3J C-c-o- P = 7.5 Hz, C4'), 79.95 (C2'), 74.91 (C3'), 67.91, 67.75 (2x O H2Ph), 66.77 (d, 2J c-o-p = 5.0 Hz, C5'), 60.19, 60.10 (2x Ccc He), 54.31 (60CH3), 40.16 (d, 3/ C-C-N-P = 3.8 Hz C lie), 40.11 (d, 3/ C-C-N-P = 3.8 Hz C He), 25.96, 25.92 (2x CH2y He), 20.42 (2'CCH3), 15.87, 15.74 (2x CH3y He), 11.95, 11.92 (2x H3 lie).
[00753] 31P NMR (202 MHz, CD3OD) δ 14.93
[00754] HPLC ¾ = 20.88 min (System 1).
[00755] MS (TOF ES+) m/z: 820.34 (M+Na+, 100%);
[00756] HRMS C38H52N7O10NaiPi calculated: 820.3411 found: 820.3378
[00757] Example 63
[00758] (2S,2'S,3R,3'R)-Dimethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin- 9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylpentanoate)
Figure imgf000117_0001
[00759] The phosphorodiamidate was prepared according to Method B.
[00760] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 μί, 0.96 mmol) and phosphorus oxychloride (90 μί, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-isoleucine methyl ester hydrochloride salt (1.15 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After work-up and silica gel column chromatography, 135 mg of (37) was obtained in 22% yield as an off white solid.
1H NMR (500 MHz, CD3OD) δ 8.01 (s, 1H, ¾), 5.99 (s, 1H, Hr), 4.47-4.38 (m, 2H, ¾·), 4.34 (d, J= 9.0 Hz, 1H, ¾·), 4.20- 4.18 (m, 1H, ¾·), 4.07 (s, 3H, 60CH3), 3.87-3.83 (m, 2H, 2x CHcc He), 3.72, 3.70 (2s, 6H, OCH3), 1.74- 1.69 (m, 2H, 2x Οϊβ lie), 1.44-1.36 and (m, 2H, CH2 He), 1.09- 1.03 (m, 2H, CH2 lie), 1.00 (s, 3H, 2'CCH3), 0.91 (d, J= 6.5 Hz, 3H, CH3 He), 0.89 (d, J= 6.5 Hz, 3H, CH3 He), 0.84 (d, J= 6.5 Hz, 3H, CH3 He), 0.82 (d, J= 6.5 Hz, 3H, CH3Ile).
[00762] 13C NMR (125 MHz, CD3OD) δ 175.17 (d,
Figure imgf000118_0001
3.8 Hz, 2x C=0 ester),
162.74 (C6), 161.91 (C2), 154.46 (C4), 139.40 (C8), 115.44 (C5), 93.27 (CI '), 82.51 (d, 3J c-c-o-p = 7.5 Hz, C4'), 79.97 (C2'), 74.87 (C3'), 66.72 (d, 2J C-o-p = 5.0 Hz, C5'), 60.02, 60.00 (2d, 2/C-N-P = 4.5 Hz, 2x Ca He), 54.37 (60CH3), 52.54, 52.51 (2x O H3 ester), 33.21 (d, 3/ C-C-N-P = 3.8 Hz C He), 33.21 (d, 3/ C-C-N-P = 7.5 Hz C He), 26.10, 26.08 (2x CH2y He), 20.41 (2'CCH3), 15.91, 15.83 (2x H3y He), 11.93 (CH3 He).
[00763] 31P NMR (202 MHz, CD3OD) δ 14.88 [00764] HPLC tR = 10.87 min (System 1). [00765] MS (TOF ES+) m/z: 668.28 (M+Na+, 100%); [00766] HRMS C26H44N701oNa1P1 calculated: 668.2785 found: 668.2759 [00767] Example 64 [00768] (2S,2'S,3R,3'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylpentanoate)
Figure imgf000118_0002
[00769] The phosphorodiamidate was prepared according to Method B. [00770] In a first step, a suspension of 6-O-methyl-2' -C-methylguanosine (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 \L, 0.96 mmol) and phosphorus oxychloride (90 \L, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-isoleucine 2,2-dimethylpropyl ester tosylate salt (1.80 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After work-up and silica gel column chromatography, 210 mg of (40) was obtained in 29% yield as an off white solid.
[00771] 1H NMR (500 MHz, CD3OD) δ 7.96 (s, 1H, ¾), 6.00 (s, 1H, Hr), , 4.41-4.40 (m,
2H, ¾·), 4.34 (d, J= 8.5 Hz, 1H, ¾·), 4.21- 4.20 (m, 1H, ¾·), 4.07 (s, 3H, 60CH3), 3.84-3.72 (m, 6H, 2x OCH2C(CH3)3 and 2x CHa He), 1.81-1.78 (m, 2H, 2x CH He), 1.46- 1.41, 1.39-1.33 (2m, 2H, CH2 He), 1.16- 1.11, 1.07- 1.04 (2m, 2H, CH2 He), 1.00 (s, 3H, 2'CCH3), 0.95, 0.94 (2s, 2x OCH2C(CH3)3), 0.91-0.81 (m, 12Η, 4x C¾ He).
[00772] 13C NMR (125 MHz, CD3OD) δ 174.79, 174.72 (2d,
Figure imgf000119_0001
3.8 Hz, 2x C=0 ester), 162.79 (C6), 161.90 (C2), 154.58 (C4), 139.46 (C8), 115.70 (C5), 93.28 (CI '), 82.56 (d, 3J c-c-o-p = 6.3 Hz, C4'), 79.97 (C2'), 75.72, 75.64 (2x O H2C(CH3)3), 74.98 (C3'), 67.00 (d, 2J C-o-p = 5.0 Hz, C5'), 60.17, 60.07 (2x Ca He), 54.32 (60CH3), 40.30, 40.25 (2x C He), 32.14, 32.09 (2x OCH2C(CH3)3, 27.08, 26.98 (2x OCH2C( H3)3, 26.04 (2x CH2y He), 20.43 (2'CCH3), 16.14, 16.03 (2x CH3y He), 12.13, 12.09 (2x CH3 He).
[00773] 31P NMR (202 MHz, CD3OD) δ 14.93
[00774] HPLC ¾ = 24.55 min (System 1).
[00775] MS (TOF ES+) m/z: 780.40 (M+Na+, 100%);
[00776] HRMS C34H61N7O10Pi calculated: 758.4218 found: 758.4196
[00777] Example 65
[00778] (2S,2'S,3R,3'R)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylpentanoate)
Figure imgf000120_0001
[00779] The phosphorodiamidate was prepared according to Method B.
In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 μί, 0.96 mmol) and phosphorus oxychloride (90 μί, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), L-isoleucine cyclohexyl ester tosylate salt (1.86 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After work-up and silica gel column chromatography, 140 mg of phosphorodiamidate was obtained in 19% yield as an off white solid.
[00781] 1H NMR (500 MHz, CD3OD) δ 7.97 (s, 1H, ¾), 6.00 (s, 1H, Hr), 4.80-4.74 (m,
2H, 2x OCH ester), 4.43-4.39 (m, 2Η, ¾·), 4.33 (d, J= 9.0 Hz, 1H, ¾·), 4.22- 4.19 (m, 1H, ¾·), 4.07 (s, 3H, 60CH3), 3.74-3.73 (m, 2H, 2x CHa He), 1.82- 1.80 (m, 4H, 2x CH2 ester), 1.75- 1.70 (m, 6H, 2x CH2 ester and CH He), 1.55- 1.29 (m, 14H, 6x CH2 ester and CH2 He), 1.15-1.02 (m, 2H, CH2 He), 1.00 (s, 3H, 2'CCH3), 0.91 (d, J= 6.5 Hz, 3H, CH3 He), 0.88 (d, J= 6.5 Hz, 3H, CH3 He), 0.85 (d, J= 6.5 Hz, 3H, CH3 He), 0.83 (d, J= 6.5 Hz, 3H, CH3Ile).
[00782] 13C NMR (125 MHz, CD3OD) δ 174.05, 174.03 (2d,
Figure imgf000120_0002
3.8 Hz, 2x C=0 ester), 162.79 (C6), 161.91 (C2), 154.59 (C4), 139.44 (C8), 115.69 (C5), 93.26 (CI '), 82.54 (d, 3J c-c-o-p = 7.5 Hz, C4'), 79.97 (C2'), 74.98 (C3'), 74.82 (2x OCH ester), 66.11 (d, 2J C-o-p = 5.0 Hz, C5'), 60.02 (2x Ccc He), 54.33 (60CH3), 40.30 (4x CH2 ester), 32.64 (d, 3J C-C-N-P = 3.8 Hz C He), 32.60 (d, 3J C-C-N-P = 7.5 Hz C He), 26.47 (CH2 ester), 26.44 (CH2 ester), 26.09 (2x OLj Ile), 24.69 (2 x CH2 ester), 24.67 (2 x CH2 ester), 20.42 (2'CCH3), 16.01, 15.91 (2x CH3y He), 12.14, 12.08 (2x CH3 He). [00783] 31P NMR (202 MHz, CD3OD) δ 15.04
[00784] HPLC iR = 25.45 min (System 1).
[00785] MS (TOF ES+) mJz: 804.41 (M+Na+, 100%);
[00786] HRMS C36H6iN7O10Pi calculated: 782.4218 found: 782.4234
[00787] Example 66
[00788] Neopentyl l,l'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)dipyrrolidine-2- carboxylate
Figure imgf000121_0001
[00789] The phosphorodiamidate was prepared according to Method B.
[00790] In a first step, a solution of 6-O-methyl-2'-C-methylguanosine (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (1 mL) was reacted anhydrous triethylamine (130 \L, 0.96 mmol) with phosphorus oxychloride (87 \L, 0.96 mmol). In a second step, anhydrous dichloromethane (3 mL), L-proline 2,2-dimethylpropyl ester tosylate salt (1.71 g, 4.81 mmol) and triethylamine (1.30 mL, 9.61 mmol) were added. After work-up, silica gel column chromatography and preparative TLC, 50 mg of phosporodiamidate was obtained in 7% yield as an off white solid.
[00791] 1H NMR (500 MHz, CD3OD): δ 7.92 (s, 1H, ¾), 5.92 (s, 1H, Hr), 4.48 (m, 1H,
¾·), 4.25 (m, 1H, ¾¾), 4.12 (m, 2H, ¾· and H5<a), 3.8 (m, 4H, 2x OCH2C(CH3)3), 3.45 (m, 2H, 2x CHa Pro), 2.2-2.4 (m, 2H, 2x Pro), 2.2 (s, 3H, 6OCH3), 1.15-2.1 m (m, 10H, 2x Pro), 1.05 (s, 3H, 2'CCH3), 0.95 (2s ,18H, 2xOCH2C(CH3)3).
[00792] 13C NMR (125 MHz, CD3OD): δ 175.91 (C=0), 175.65 (C=0), 162.78 (C6),
161.90 (C2), 154.41 (C4), 139.70 (C8), 115.73 (C5), 93.68 (CI '), 82.55 (d, 3JP_o-c- c= 9.2 Hz, C4'), 79.98 (C2'), 75.18 (C3'), 75.08, 75.06 (2x O H2C(CH3)3), 67.00 (d, 2JP_o-c = 4.3 Hz, C5'), 54.21 (Ca Pro), 48.05 (d, 2JP-N-c = 5.3 Hz, NHCH2), 47.91 (d, 2JP-N-c = 5.3 Hz, NH H2), 32.70 (d, J= 7.5 Hz, CH2 Pro), 32.45 (d, J= 7.5 Hz, CH2 Pro), 26.84, 26.83 (2x OCH2C( H3)3), 26.10 (d, J= 8.4 Hz, CH2 Pro), 26.03 (d, J= 8.4 Hz,CH2 Pro), 20.34 (2'CCH3).
[00793] 31P NMR (202 MHz, CD3OD): δ 11.52.
[00794] HPLC ¾ = 20.57 min (System 1).
[00795] MS (ES+) m/z = (M+H+): 726.35; Accurate mass calculated for C32H53N7O10P - 726.3592
[00796] Examples 67
[00797] (2S,2'S)-1,4-Dibenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)disuccinate
Figure imgf000122_0001
[00798] The phosphorodiamidate was prepared according to Method B. [00799] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (500 mg, 1.50 mmol) in anhydrous tetrahydrofuran (8 mL) was reacted with triethylamine (0.25 mL, 1.80 mmol) and phosphoryl chloride (0.13mL, 1.50 mmol). In a second step, anhydrous dichloromethane (8 mL), L-aspartic acid tetra-benzyl ester tosylate salt (3.6 g, 7.50 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added. After column chromatography (MeOH/ dichloromethane 1 :9), 150 mg of phosphorodiamidate was obtained of the in 10% yield as an off white solid.
[00800] 1H NMR (500 MHz, CD3OD) δ 7.92 (s, 1H, ¾), 7.30-7.20 (m, 20H, Ph), 5.97 (s,
1H, Hr), 5.10-4.89 (m, 8H, CH2Ph), 4.47-4.24 (m, 5H, H3<, 2xCHcc L- Aspartic acid), 4.19-4.1 1 (m, 1H, ¾·), 3.99 (s, 3H, OCH3), 2.91-2.79 (m, 4H, CH2C=0), 0.97 (s, 3H, 2'CH3).
[00801] 13C NMR (125 MHz, CD3OD) 173.3 (C=0), 173.2 (C=0), 172.0 (C=0), 171.9
(C=0), 162.7 (C6), 161.9 (C2), 154.61 (C4), 139.3 (C-8), 137.2(C ipso), 137.1(C ipso), 137.0(C ipso), 136.9 (C ipso), 129.6, 129.5, 129.5, 129.4, 129.4, 129.3, 129.3 129.2, 129.0, 115.6 (C5), 93.1 (CI '), 82.2 (d, J= 7.3 Hz, C4'), 80.0 (C2'), 74.8 (C3'), 68.4 (CH2Ph), 67.7 (CH2Ph), 66.5 (d, J= 4.9 Hz, C5'), 54.2 (CH L- Aspartic acid), 52.2 (60Me), 39.6 (CH2C=0), 39.5 (CH2C=0), 39.5 (CH2C=0), 39.4 (CH2C=0), 20.4 (2'C H3).
[00802] 31P NMR (202 MHz, CD3OD) δ 13.58.
[00803] HPLC tR = 30.88 min (System 2).
[00804] Example 68
[00805] (2S,2'S)-Tetramethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)disuccinate
Figure imgf000124_0001
[00806] The phosphorodiamidate was prepared according to Method B. [00807] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (500 mg, 1.50 mmol) in anhydrous tetrahydrofuran (8 mL) was reacted with triethylamine (0.25 mL, 1.80 mmol) and phosphoryl chloride (0.13mL, 1.50 mole). In a second step, anhydrous dichloromethane (8 mL), L-aspartic acid tetra methyl ester tosylate salt (3.6 g, 7.50 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added. After column chromatography (MeOH/ dichloromethane 1 :9) and TLC preparative, 10 mg of phosphorodiamidate was obtained in 10% yield as an off white solid.
[00808] 1H NMR (500 MHz, CD3OD) δ 7.97 (s, 1H, ¾), 5.99 (s, 1H, Hr), 4.45-4.20 (m,
5H, H3<, 2xCHcc L-aspartic acid), 4.20-4.11 (m, 1H, ¾·), 3.73 (s, 3H, C=OOC¾), 3.71 (s, 3H, C=OOC¾), 3.67 (s, 3H, C=OOC¾), 3.62 (s, 3H, C=OOC¾), 2.89-2.75 (m, 4H, CH2C=0), 1.01 (s, 3Η, 2'C¾).
[00809] 13C NMR (125 MHz, CD3OD) 174.1 (C=0), 172.8 (C=0), 172.7 (C=0), 162.7
(C6), 161.9 (C2), 154.6 (C4), 139.4 (C8), 115.6 (C-5), 93.2 (CI '), 82.2 (d, J= 7.5 Hz, C4'), 80.0 (C2'), 74.7 (C3'), 66.4 (d, J= 5.6 Hz, C-5'), 54.2, 53.08, 52.4, 52.1 (CH AA), 39.3 (d, J= 5.6 Hz, CH2C=0), 39.2 (d, J= 5.6 Hz, CH2C=0), 20.3 (2'CCH3).
[00810] 31P NMR (202 MHz, CD3OD) δ 13.63. [00811] HPLC tR = 15.71 min (System 2). [00812] Example 69 [00813] Benzyl 3,3'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000125_0001
[00814] The phosphorodiamidate was prepared according to Method B.
[00815] In a first step, a suspension of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (300 mg, 0.96 mmol) in anhydrous tetrahydrofuran (5 mL) was reacted with triethylamine (130 μί, 0.96 mmol) and phosphorus oxychloride (90 μί, 0.96 mmol). In a second step, anhydrous dichloromethane (5 mL), β-alanine benzyl ester tosylate salt (1.69 g, 4.82 mmol) and triethylamine (1.34 mL, 9.63 mmol) were added. After work-up and silica gel column chromatography 170 mg of phosphorodiamidate was obtained in 25% yield as an off white solid.
[00816] 1H NMR (500 MHz, CD3OD) δ 8.00 (s, 1H, ¾), 7.32-7.25 (m, 10H, 2x OCH2Ph),
6.00 (s, 1H, Hr), 5.07, 5.06 (2s, 4H, 2x OCH2Ph), 4.33-4.26 (m, 3H, H5' and ¾·), 4.20- 4.17 (m, 1H, ¾·), 4.03 (s, 3H, 60CH3), 3.21-3.15 (m, 4H, 2x NH-CH2 β- Ala), 2.56 (t, J= 6.7 Hz, 2H, CH2 β-Ala), 2.55 (t, J= 6.7 Hz, 2H, CH2 β-Ala), 0.97 (s, 3H, 2'CCH3).
[00817] 13C NMR (125 MHz, CD3OD) δ 173.42 (C=0 ester), 162.72 (C6), 161.89 (C2),
154.63 (C4), 139.10 (C8), 137.52 (2x ipso OCH2P/i), 129.87, 129.55, 129.21, 129.18 (2x OCH2P/i), 115.53 (C5), 92.96 (CI '), 82.28 (d, 3JC-c-o-p = 8.8 Hz, C4'), 79.51 (C2'), 74.44 (C3'), 67.33 (2x OCH2Ph), 65.48 (d, 2JC-o-p = 5.0 Hz, C5'), 54.29 (6OCH3), 38.10 (d, 3/ C-C-N-P =5.0 Hz, NH- H2 β-Ala), 37.99 (d, 3/ C-C-N-P =3.8 Hz, NH- H2 β-Ala), 37.38 (CH2 β-Ala), 37.34 (CH2 β-Ala), 20.36 (2'CCH3).
[00818] 31P NMR (202 MHz, CD3OD) δ 17.45. [00819] HPLC iR =16.12 min.
[00820] MS (TOF ES+) m/z: 736.32 (M+Na+, 100%);
[00821] HRMS C3H4iN7O10Pi calculated: 714.2653 found: 714.2623
[00822] Example 70
[00823] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylthiopropanoate)
Figure imgf000126_0001
[00825] Following the standard procedure B.
In the first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (330 mg, 1.05 mmol) in anhydrous tetrahydrofuran (5.61 mL) was allowed to react with triethylamine (180 μί, 1.25 mmol) and phosphorus oxychloride (Ι ΙΟμί, 0.803 mmole). In the second step L-methylcysteine neopentyl ester tosylate salt (1.98, 5.25 mmol), anhydrous dichloromethane (6 mL) and triethylamine (1.44 mL, 10.5 mmol) were added as described in Method B. After work-up and silica gel column chromatography, 97 mg of phosphorodiamidate was obtained in 12% yield as an off white solid. [00827] 1H NMR (500 MHz, MeOO-d4) δ 7.98 (s, 1H, H8), 5.98 (s, 1H, HI '), 4.47-4.38 (m, 2H, CH2 5'), 4.38 (d, J = 9.0 Hz, 1H, H3'), 4.21-4.14 (m, 2H, 2x CHcc Cys), 4.07 (s, 3H, 60CH3), 3.86-3.73 (m, 5H, H4' and 2x OCH2C(CH3)3), 2.89-2.83 (m, 4H, 2x CH2 Cys), 2.13 , 2.09 (2s, 6H, 2x CH3 SCH3 Cys), 0.99 (s, 3H, 2'CH3), 0.96, 0.94 (2s, 18Η, OCH2C(CH3)3).
[00828] 13C NMR (125 MHz, MeOD-d4) δ 173.72 (C=0), 173.68 (d, 3/C-C-N-P = 6.3 Hz,
C=0), 162.76 (C6), 161.92 (C2), 154.59 (C4), 139.48 (C8), 115.60 (C5), 93.25 (CI '), 82.43 (d, J3 = 7.56 Hz, C4'), 79.99 (C2'), 75.76, 74.96 (2x OCH2C(CH3)3), 74.91 (C3'), 66.88 (C5'), 55.32, 55.00 (2x CHcc Cys), 54.21 (60CH3), 39.43, 39.35 (2x CH2 Cys), 32.25, 32.20 (2x OCH2C(CH3)3), 26.84, 26.57 (2x OCH2C(CH3)3), 20.26 (2'C H3), 16.15, 16.11 (2x SCH3 Cys).
[00829] 31P NMR (202 MHz, MeOD-d4) δ 13.36.
[00830] HPLC tR = 19.89 min (System 1).
[00831] MS (TOF ES+) m/z: 788.28 (M+Na+, 100%);
[00832] HRMS CsoHsiNvOioPiSiNai calculated 788.2852, found: 788.2844 (M+Na+). [00833] Example 71
[00834] (2S,2'S)-Cyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)bis(3-methylthiopropanoate)
Figure imgf000127_0001
[00836] Following the standard procedure B. [00837] In the first step, a solution of ((2R,3R,4R,5R)-2-(2-amino-6-methoxy-9H-purin-9- yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (550 mg, 1.77 mmol) in anhydrous tetrahydrofuran (9.5 mL) was allowed to react with triethylamine (300 μί, 2.12 mmol) and phosphorus oxychloride (200μί, 2.12 mmole). In the second step L-methylcysteine cyclohexyl ester hydrochloride salt (1.98, 5.25 mmol), anhydrous dichloromethane (10 mL) and triethylamine (2.43 mL, 17.7 mmol) were added as described in Method B. After work-up and silica gel column chromatography, 110 mg of phosphorodiamidate was obtained in 8% yield as an off white solid.
[00838] 1H NMR (500 MHz, CDCl3-£¾ δ 7.78 (s, 1H, H8), 5.96 (s, 1H, HI '), 5.52 (broad s, 2H, 2 NH Cys), 4.78-4.73 (m, 2H, 2 CH cHex), 4.57-4.54 (m, 2H, CH2 5'), 4.43 (m, H3'), 4.33-4.11 (m, 3H, 2 CH Cys & H4'), 4.03 (s, 3H, 60CH3), 2.86- 2.06 (m, 4H, 2 CH2 Cys), 2.10 (s, 3H, SCH3), 2.06 (s, 3H, SCH3), 1.79 (m, 4H, 2x CH2 cHex), 1.67 (m, 4H, 2x CH2 cHex), 1.49 (m, 2H, CH2 cHex), 1.40-1.19 (m, 10H, 5x CH2 cHex), 0.97 (s, 3H, 2'CH3).
[00839] 13C NMR (125 MHz, CDCl3-<¾) δ 171.77, 171.60 (2d, 3JC-c-o-p = 5.0 Hz, C=0
Cys), 161.56 (C6), 159.62 (C2), 152.90 (C4), 137.90 (C8), 115.51 (C5), 91.68 (CI '), 80.98 (d, ), 79.33 (C2'), 74.54 (C3'), 74.48, 74.23 (2x OCR cHex),
Figure imgf000128_0001
Hz, C5'), 55.86 & 53.80 (2x CHcc Cys), 53.73 (60CH3), 38.86, 38.78 (2x H2 Cys), 31.41 , 31.37 (CH2 cHex), 25.19 ( H2 cHex), 23.59 (CH2 cHex), 20.25 (2CCH3), 16.17 (2x CH3 Cys).
[00840] 31P NMR (202 MHz, CDCl3-<¾) δ 13.31.
[00841] HPLC ¾ =20.10 min (System 1),
[00842] MS (TOF ES+) m/z: 790.30 (M+H+, 100%);
[00843] HRMS C32H52N7OioPiS2 calculated 790.3033, found; 790.3000
[00844] Example 72
[00845] (2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4- fluoro-3-hydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000129_0001
[00847] The nucleoside (2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4-fluoro-2- (hydroxymethyl)-4-methyltetrahydrofuran-3-ol was prepared using methods familiar to one skilled in the art, for example as described in Clark, J. L.; Mason, J. C; Hollecker, L.; Stuyver, L. J.; Tharnish, P. M.; McBrayer, T. R.; Otto,M. J.; Furman, P. A.; Schinazi, R. F.; Watanabe, K. A. Synthesis and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methyl purine nucleosides as inhibitors of hepatitis C virus RNA replication. Bioorg. Med. Chem. Lett. 2006, 16, 1712-1715.
[00848] The phosphorodiamidate was prepared according to the standard procedure B.
[00849] In a first step, a suspension of (2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9- yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (150 mg, 0.458 mmol) in anhydrous tetrahydrofuran (3 mL) was reacted with triethylamine (60 μί, 0.458 mmol) and phosphorus oxychloride (40 μί, 0.458 mmol). In a second step, anhydrous dichloromethane (3 mL), L-alanine neopentyl ester tosylate salt (760 mg, 2.29 mmol) and triethylamine (640 μί, 4.58 mmol) were added. After work-up and silica gel column chromatography, 30 mg of phosphorodiamidate was obtained in 9% yield as an off white solid.
[00850] 1H NMR (500 MHz, CD3OD) δ 7.97 (s, 1H, H-8), 6.17 (d,
Figure imgf000129_0002
Hz, 1H, H- 1 '), 4.57-4.53 (m, 3H, 60CH2CH3 and H-3'), 4.44-4.36 (m, 2H, H-5'), 4.23-4.19 (m, 1H, H-4'), 3.99-3.95 (m, 2H, 2x CHcc Ala), 3.84, 3.82, 3.72, 3.66 (2AB, J=10.5 Hz, 4H, 2x OCH2C(CH3)3), 1.45 (t, J=7.3 Hz, 3H, 60CH2CH3), 1.39 (d, 3Η, J=7.1 Hz, CH3 Ala), 1.35 (d, 3H, J=7.1 Hz, CH3 Ala), 1.23 (d,
Figure imgf000129_0003
Hz, 1H, 2'CCH3), 0.94 (s, 9Η, OCH2C(CH3)3), 0.92 (s, 9Η, OCH2C(CH3)3). [00851] 13C NMR (125 MHz, CD3OD) δ 175.65 (d,
Figure imgf000130_0001
6.3 Hz, C=0), 175.60 (d,
(C4), 139.42 (C8),
Figure imgf000130_0002
39 Hz, CI '), 81.92 (d, 3J c-c-o-p = 7.5 Hz, C4'), 75.38 (OCH2C(CH3)3), 75.36 (O H2C(CH3)3), 74.04 (d, 2Jc-c-F= 18 Hz, C3'), 66.15 (d, 2JC-o-p = 3.8 Hz, C5'), 63.58 (60CH2CH3), 51.11 (Ca Ala), 51.06 (Ccc Ala), 32.32 (OCH2C(CH3)3), 32.28 (OCH2C(CH3)3), 26.80 (OCH2C( H3)3), 26.77 (OCH2C(CH3)3), 21.11 (CH3 Ala), 20.98 (CH3 Ala), 16.89 (d, 2Jc-c-F= 25 Hz 2'C H3), 14.88 (60CH2CH3).
[00852] 31P NMR (202 MHz, CD3OD) δ 13.98.
[00853] 19F NMR (470 MHz, CD3OD) δ - 162.26.
[00854] HPLC tR = 20.63 min (System 1).
[00855] MS (TOF ES+) m/z: 712.31 (M+Na+, 100%);
[00856] Example 73
[00857] (2R,2'R)-neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl) 4- fluoro-3-hydroxy-4-methyltetrahydrofuran-2- yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
Figure imgf000130_0003
[00858] The phosphorodiamidate was prepared according to the standard procedure B. [00859] A solution of nucleoside (2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4- fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (0.15 g, 0.46 mmol) and anhydrous NEt (0.08 mL, 0.55 mmol) in anhydrous tetrahydrofuran (2.4 mL) was stirred at RT for 30 min, then cooled to -78°C and POCl3 (0.05 mL, 0.55 mmol) was added dropwise. The solution was stirred for 1 hr at -78°C and then at RT for 30 min. The formation of the intermediate was monitored by phosphorus NMR. Then D-Alanine neopentyl ester /?ara-toluene sulfonic acid salt (0.76 g, 2.30 mmol) and anhydrous dichloromethane (3.5 mL) were added and the reaction was cooled to -78°C. Anhydrous NEt3 (0.64 mL, 4.60 mmol, 10 eq) was added dropwise and the solution was returned to RT and stirred overnight. After work up, and silica gel chromatography with methanol (2.5%) in chloroform, 0.022 g (0.03 mmol) of the phosphorodiamidate was obtained as an off white solid.
[00860] 1H NMR (d-CDCls) δ 7.76 (s, 1H, H8 guanine), 6.06, 6.02 (d, 1H, J3 F_H = 18.75
Hz, HI '), 5.31 (broad s, 1H, NH D-Ala), 4.90 (broad s, 1H, NH D-Ala), 4.65-4.61 (m, 1H, H5'), 4.58-4.54 (q, 1H, J = 6.6 Hz, CH2 Et), 4.35-4.31 (m, 1H, H5'), 4.19- 4.18 (m, 1H, H4'), 4.07-3.98 (m, 2H, 2 CH D-Ala), 3.91-3.88 (dd, 2H, J = 4.55 & 10.45 Hz, CH2 neopentyl), 3.73-3.71 (dd, 2H, J = 1.15 & 10.45 Hz, CH2 neopentyl), 1.48- 1.45 (t, 3H, J = 7.1 Hz, CH3 Et), 1.44- 1.42 (t, 6H, J = 6.47 Hz, 2 CH3 D-Ala), 1.29 & 1.25 (d, 3H, , J3 F_H = 22.70 Hz, CH3 2'), 0.94 & 0.92 (2s, 18H, 6 CH3 neopentyl).
[00861] 13C NMR (d-CDCl3) 5C (ppm): 175.15 (s, CO D-Ala), 174.28 (d, J3 P_C= 5.04 Hz,
CO D-Ala), 161.32 (s, C6 guanine), 159.59 (s, C2 guanine), 153.20 (s, C4 guanine), 137.37 (s, C8 guanine), 115.71 (s, C5 guanine), 101.92 & 100.48 (2s, C2'), 89.23 (d, fF_c = 40.32 Hz, CI '), 79.97 (d, J3 P_C = 7.56 Hz, C4'), 74.82 (2s, 2 OCH2C(CH3)3), 71.93 (s, C3'), 63.03 (s, CH2 Et), 62.71 & 62.57 (2d, J2 P_C = 17.64 Hz, C5'), 49.78 & 49.43 (2s, 2 CH D-Ala), 31.36 & 31.23 (2s, 2 OCH2C(CH3)3), 26.33 & 26.25 & 26.14 (3s, 2 OCH^CiCH^ ). 21.35 & 21.00 (2d, J3P_C = 6.30 & 8.87 Hz, 2 CH3 D-Ala), 16.59 (d, fF.c = 25.20 Hz, CH3 2'), 14.47 (s, CH3 Et).
[00862] 31P NMR (d-CDCl3) δΡ (ppm): 13.86 (s).
[00863] MS (TOF ES+): 690.34 (M + H+), 712.33 (M + Na+); accurate mass: calculated for
C29H5oN709FP: 690.3392, found: 690.3406 (M + H+), 712.3275 (M+Na+). [00864] Further to the above Examples, representative compounds, prepared according to the examples were tested for potency in an HCV replicon assay (Genotype lb) for activity against the virus (EC50) and toxicity to the cells (CC50). These results are set forth below.
[00865] Huh7 Replicon Cell Lines and Cell Culture Conditions: A luciferase-reporter genotype lb subgenomic replicon cell line, and a genotype la full-length replicon cell line were obtained from Apath, LLC, Brooklyn, NY: All cell lines were passaged twice a week by splitting 4 or 6 fold. Cells were maintained in DMEM- high glucose medium (HyClone, Logan, UT) supplemented with 9% FBS (HyClone), 2 mM glutamine (Invitrogen, Carlsbad, CA), 100 U/ml PenStrep (Invitrogen). Media also contained 0.25 mg/ml of the antibiotic G-418 to maintain stable expression of the replicon (Invitrogen). Incubation was performed at 37 °C in 5% C02 atmosphere. Replicon cell lines were used until they accumulated 15- to-18 passages, after which cells were restarted from the frozen stock. Seeding cell counts were routinely determined using an automatic Cedex HiRes cell counter (Flownomics Analytical Instruments, Madison, WI) or manually using INCYTO
C-Chip Disposable Hemacytometers (Fisher Scientific, Pittsburg, PA). [00866] The anti-HCV assays were done accordingly:
[00867] Luciferase Genotype lb Replicon Potency Assay. Replicon cells were seeded into white 96- well plates (Nunc/VWR) at a density of 2xl04 cells/well in medium without G-418. A Stacker Multidrop Liquid Dispenser (MTX Lab Systems, Vienna, VA) was employed to ensure uniform and fast cell seeding into multiple plates. 18-24 h after cell plating, inhibitors were added and cells were incubated for additional 24, 48, or 72 h (as indicated). Compounds were tested in triplicates and quadruplicates at 3X or 4X serial dilutions over a range of 0.0001-to-lO μΜ concentrations. HCV replication was monitored by Renilla luciferase reporter activity assay using Renilla luciferase reporter (Promega, Madison, WI) and a Veritas Luminometer (Turner Biosystems, Sunnyvale, CA). 50% and 90% inhibitory concentration (IC50 and IC90) values were calculated as the concentration of compound that results correspondingly in 50% and 90% decreases in the reporter expression as compared to untreated cells. The values were determined by non-linear regression (four-parameter sigmoidal curve fitting) analysis.
[00868] The cell cytotoxicity assay data was obtained as described below:
[00869] Cytotoxicity Assay. Cells were seeded into 96-well plates at a density of 2xl04 cells per well. 24 h after cell plating, 11 serial 2X compound dilutions, starting with 100 μΜ, were applied to the testing plates (3 repeats per compound dilution). Each testing plate was run with a "no-compound" control. Incubation with compounds was continued at 37 °C in a C02 incubator for 72 h. To determine cell viability, the CellTiter-Glo® assay (Promega, Madison, WI) was performed according to the manufacturer's protocol. The compound concentration resulting in 50% luminescent signal was reported as the CC50 concentration.
[00870] The results of the assay in terms of IC50 (μΜ) and CC50 (μΜ) are given in Table 1 below:
[00871] Table 1
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001

Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
] While the invention has been described with reference to particularly preferred embodiments and examples, those skilled in the art recognize that various modifications may be made to the invention without departing from the spirit and scope thereof. [00873] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety.
[00874] Abbreviations and Acronyms
[00875] A number of abbreviations and acronyms are used herein, and a full description of these are provided as follows:
ACN acetonitrile
AIBN azobisisobutryonitrile
anhy anhydrous
Bn benzyl (phenylmethyl)
Boc benzyloxycarbonyl
BSA benzenesulfonic acid
Bu butyl
n-BuOH w-butanol
i-BuOH ie/t-butanol
i-BuOK potassium -iert-butoxide
ie/t-BuMgCl ie/t-butylmagnesium chloride
CDCI3 deuterochloroform
CD3OD methanol-<¾
CI-MS chemical ionization mass spectrometry
13 C NMR carbon- 13 nuclear magnetic resonance spectroscopy cone concentrated
d doublet (NMR)
dd doublet of doublets (NMR)
ddd double doublet of doublets (nmr
DBU diaza(l,3)bicyclo[5.4.0]undecane
DCC dicyclohexylcarbodiimide
DCM dichloromethane
DMAP 4-dimethylaminopyridine
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
dt doublet of triplets (NMR) EDCI 1 -(3-dimethylamnopropyl)-3-ethylcarbodiimide hydrochloride
ee enantiomeric excess
El-MS electron impact mass spectrometry
equiv equivalent(s)
ESI electrospray ionization
ES-MS electrospray mass spectrometry
Et3N triethylamine
Et20 ethyl ether
EtOAc ethyl acetate
EtOH ethanol
g gram(s)
GC-MS gas chromatography-mass spectrometry h hour
HCV hepatitis C virus
1H NMR proton nuclear magnetic resonance spectroscopy
HPLC high performance liquid chromatography
HRMS high resolution mass spectrometry
IMPDH inosine 5 'monophosphate dehydroxgenase
J NMR coupling constant
LC/MS liquid chromatography-mass spectrometry
LG leaving group
LHMDS Lithium hexamethyldisilazide
m multiplet (NMR)
MDI methylenediphenyldisocyanate
Me methyl
MeOH methanol
mg milligram
MHz megahertz
mL milliliter
mmol millimole
mp melting point
m/z mass-to-charge ratio
MTBE methyl t-butyl ether
NaOMe sodium methoxide NMI N-methylimidazole
NMO N-methylmorpholine-N-oxide
NMR nuclear magnetic resonance
31 P NMR phosphorous-31 nuclear magnetic resonance spectroscopy
ppm part per million
q quartet (NMR)
pTSA /7-toluenesulfonic acid
RBV ribavirin
Red-Al® sodium bis(2-methoxyethoxy)aluminumhydride
Rf retention factor (TLC)
rt room temperature
s singlet (NMR)
t triplet (NMR)
TBAF tetra-w-butylammonium fluoride
TBPPS tetra-w-butylphosphonium persulfate
TEA triethylamine
TFA trifluoroacetic acid
THF tetrahydrofuran
TMS tetramethylsilane
TMSOTf trimethylsilyl trifluoromethanesulfonate iR retention time
TLC thin layer chromatography
UV ultraviolet
VCD Vibrational Circular Dichroism

Claims

What is claimed is:
1. A compound of formula (I) having the structure:
Figure imgf000155_0001
wherein R2, R3, and R4 are each independently
Figure imgf000155_0002
wherein
R 7' and R 8° are each independently
hydrogen,
Ci-C8alkyl,
Ci-C3alkylaryl,
CH3XCH2-,
CH3XCH2CH2- ,
R9XC(0)CH2- benzyl,
benzyl optionally substituted by halogen, benzyl optionally substituted by C Csalkyl benzyl optionally substituted by Q-Cealkoxy phenyl,
phenyl optionally substituted by halogen, phenyl optionally substituted by C Csalkyl, or phenyl optionally substituted by Q-Cealkoxy wherein X is O or S; and R9 is independently
hydrogen,
Ci-Cgalkyl optionally substituted by C3-C6cycloalkyl,
C4-C8cycloalkyl,
tetrahydropyranyl,
benzyl,
benzyl optionally substituted by halogen
benzyl optionally substituted by C Csalkyl
benzyl optionally substituted by Q-Cealkoxy
2-phenylethyl optionally substituted on the phenyl ring by halogen,
or
2-indanyl,
• hydrogen,
• Ci-Cgalkyl optionally substituted by C3-C6cycloalkyl,
• R902CCH2CH2-
• CrCgcycloalkyl,
• C6-C10 aryl optionally substituted by halogen,
or
• benzyl optionally substituted by halogen;
or
R 1 and R 2 when taken together with the N atom to which they are attached, may form
• a heterocyclic ring containing from 3 to 5 C atoms,
• a morpholine ring,
• a piperazine ring,
• a thiomorpholine ring, or
• a ring of formula
Figure imgf000156_0001
or
R3 and R 4 when taken together with the N atom to which they are attached, may form • a heterocyclic ring containing from 3 to 5 C atoms a morpholine ring,
a piperazine ring,
a thiomorpholine ring,
a ring of formula
Figure imgf000157_0001
and
R5 is selected from CI, X'R6 and NR10RU,
wherein
X' is O or S,
R6 is selected from H, CrCsalkyl optionally substituted by C6-C10aryl, and
CrCgcycloalkyl, and
R10 and R11 are independently selected from H, CrCgalkyl optionally substituted by
C6-C10aryl, and Cs-Cgcycloalkyl;
and the pharmaceutically acceptable salts thereof.
2. The compound according to claim 1 wherein the compound is selected from the group consisting of the following formulas:
Figure imgf000157_0002
Figure imgf000158_0001
Figure imgf000158_0002
Figure imgf000158_0003
157
Figure imgf000159_0001
Figure imgf000159_0002
Figure imgf000159_0003
Figure imgf000159_0004
and pharmaceutically acceptable salts thereof.
3. The compound of claim 1 wherein the compound is in the form of a solvate or hydrate.
4. The compound of claim 1 wherein the compound is in the form of a polymorph.
5. A pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier, excipient or diluent.
6. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable carrier is pure sterile water, phosphate buffered saline or an aqueous glucose solution.
7. The compound of claim 1 wherein the compound is selected from the group consisting of
(25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)(2,2')-bis-amino-dipropanoate
(25,2'5)-Dimethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(25,2'5)-Diethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(25,2 '5)-Dipropyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino-dipropanoate
(25,2'5)-Dibutyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(25,2'5)-dipentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S,2'S)-bis(3,3-dimethylbutyl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl) dipropanoate
(25,2'5)-Diisobutyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)(2,2')-bis-amino-dipropanoate
(2S,2'S)-Bis(cyclopropylmethyl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9- yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl) dipropanoate (25,2'5)-Dibenzyl (((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino-dipropanoate
(25,2'5)-Diisopropyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino-dipropanoate
(2S,2'S)-sec- uty\ 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S,2'S)-Dicyclobutyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S,2'S)-Dicyclopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(25,2'5)-Dicyclohexyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate
(2S,2'S)-Bis(tetrahydro-2H-pyran-4-yl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl) dipropanoate
(25,2'5)-(5)-Phenylethyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino- dipropanoate
(2S,2'S)-Bis(2,3-dihydro-lH-inden-2-yl) 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H- purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl) dipropanoate
(2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphorylamino)propanoate
(2S)-Cyclohexyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphorylamino)propanoate (25 ieri-Butyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)((lS')-l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphorylamino)propanoate
(25 Methyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)((lS')- l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphorylamino)-3-methylbutanoate
Methyl l-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)((lS')- l-(neopentyloxy)-l-oxopropan-2- ylamino)phosphoryl)pyrrolidine-2-carboxylate
(2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(butylamino)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(butylamino)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(benzylamino)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(diethylamino)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(pyrrolidin- l-yl)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(phenylamino)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(naphthalen- l-ylamino)phosphorylamino)propanoate
(25,2'5)-Dibenzyl (((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino-diethanoate (25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis- dipropanoate ethanoate
(2R,2'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(4-methylpentanoate)
(25,2'5)-Dineopentyl-((((2R,35,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-(2,2')-bis-amino-4- methylthiobutanoate
(2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3methyl butanoate)
(2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)
bis (azanediyl)bis (3methylbutanoate)
(2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(2- phenylacetate)
(2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(2- phenylacetate)
(25,2'5)-Dibenzyl (((2R,35,4R,5R)-5 2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy) phosphoryl)-bis-pyrrolidine-2-methanoate
((2R,3R,4R,5R)-5-(2-Amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methyl di-N-butylphosphinate
((2R,3R,4R,5R)-5-(2-Amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methyl dimorpholinophosphinate (2S,2'S)-2,4-Difluorobenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)
dipropanoate
(2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S)-Cyclohexyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy- 4-methyltetrahydrofuran-2-yl)methoxy)((S)- l-(cyclopentyloxy)- l-oxopropan-2- ylamino)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(pentylamino)phosphorylamino)propanoate
(2S)-Benzyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(morpholino)phosphorylamino)propanoate
(2S)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(cyclopropylamino)phosphorylamino)propanoate
(2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(4- methylpentano ate)
(2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(4- methylpentano ate)
(2S,2'S)-Dibenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(4- methylthiobutanoate) (2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(4- methylthiobutanoate)
(2S,2'S)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- phenylpropanoate)
(2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- phenylpropanoate)
(2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- phenylpropanoate)
(2S,2'S)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3-(4-tert- butoxyphenyl)propanoate)
(2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- methylbutanoate)
(2S,2'S,3R,3'R)-Benzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- methylpentanoate)
(2S,2'S,3R,3'R)-Dimethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- methylpentanoate)
(2S,2'S,3R,3'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- methylpentanoate) (2S,2'S,3R,3'R)-Dicyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- methylpentano ate)
Neopentyl l,r-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)phosphoryl)dipyrrolidine-2-carboxylate
(25,2'5)-l,4-Dibenzyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)disuccinate
(2S,2'S)-Tetramethyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)disuccinate
Benzyl 3,3'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate
(2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- methylthiopropanoate)
(2S,2'S)-Cyclohexyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)bis(3- methylthiopropanoate)
8. A method for treating a viral infection in a mammal mediated at least in part by a virus in the Flaviviridae family of viruses comprising administering to a mammal in need thereof an effective amount of the compound of claim 1.
9. The method according to claim 8 wherein said virus is hepatitis C virus.
10. A method for treating a viral infection in a mammal mediated at least in part by a virus in the Flaviviridae family of viruses comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of claim 5.
11. The method according to claim 10, wherein said virus is hepatitis C virus.
12. A method for treating a hepatitis C viral infection in a mammal comprising administering to a mammal in need thereof an effective amount of the compound of claim 1.
13. The method according to claim 12 wherein the compound is administered in combination with a therapeutically effective amount of one or more agents active against hepatitis C virus.
14. The method of claim 13 wherein said agent active against hepatitis C virus is interferon-alpha or pegylated interferon- alpha alone or in combination with ribavirin or levovirin.
15. The method of claim 13 wherein said agent active against hepatitis C virus is selected from the group consisting of ribavirin, levovirin, viramidine, thymosin alpha- 1, an inhibitor of HCV NS3 serine protease, interferon-a, pegylated interferon-a (peginterferon-a), and combinations thereof
16. The method of claim 15 wherein interferon-α is selected from the group consisting of recombinant interferon- 2a, interferon- 2b, a consensus interferon, and a purified interferon- α product.
17. The method of claim 13 wherein said agent active against hepatitis C virus is an agent that inhibits a material selected from the group consisting of HCV proteases, HCV
polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and inosine 5'-monophosphate dehydrogenase.
17. The method of claim 13 wherein said agent active against hepatitis C virus is a nucleoside analog for the treatment of an HCV infection.
18. The method of claim 13 wherein said agent active against hepatitis C virus is selected from the group consisting of Omega IFN, BILN-2061, Roferon A, Pegasys,
Pegasys/Ribaravin, CellCept, Wellferon, Albuferon-a, Levovirin, IDN-6556, ΓΡ-501, Actimmune, Infergen A, ISIS 14803, JTK-003, Pegasys/Ceplene, Ceplene, Civacir, Intron A/Zadaxin, Levovirin, Viramidine, Heptazyme, Intron A, PEG-Intron, Rebetron, Ribavirin, PEG-Intron/Ribavirin, Zadazim, Rebif, ΙΡΝ-β/ΕΜΖ701, T67, VX-497, VX-950/LY-5703 10, Omniferon, XTL-002, SCH 503034, isatoribine and its prodrugs ANA971 and ANA975, R1479, Valopicitabine, ΝΓΜ811, and Actilon.
19. A method for treating a hepatitis C viral infection in a mammal comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of claim 5.
20. The method according to claim 19 wherein the composition is administered in combination with a therapeutically effective amount of one or more agents active against hepatitis C virus.
21. A method of inhibiting RNA-dependant RNA viral replication comprising
administering an effective amount of the compound of claim 1.
22. A method of inhibiting HCV NS5B polymerase comprising administering an effective amount of the compound of claim 1.
23. The compound according to claim 1 wherein the compound includes different diastereomers around phosphorous in formula I.
24. The compound according to claim 23 wherein the compound includes a mixture of two phosphorous diastereomers in any proportion from 1:99 to 99: 1.
25. A pharmaceutical composition comprising a liquid oral formulation of a compound according to claim 1.
26. The composition according to claim 25 wherein the formulation comprises
DMA/PEG 400/Solutol HS 15/sodium acetate pH 4.0 and Capmul/Tween 80; SEDDS-4; MLM; SE-21.
27. A compound having the formula:
Figure imgf000168_0001
and pharmaceutically acceptable salts thereof.
28. A method for treating a viral infection in a mammal mediated at least in part by a virus in the Flaviviridae family of viruses comprising administering to a mammal in need thereof an effective amount of the compound of claim 27.
29. The method according to claim 28, wherein said virus is hepatitis C virus.
30. A pharmaceutical composition comprising the compound of claim 27 and a pharmaceutically acceptable carrier, excipient or diluent
31. A method for treating a viral infection in a mammal mediated at least in part by a virus in the Flaviviridae family of viruses comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of claim 30.
32. The method according to claim 31, wherein said virus is hepatitis C virus.
33. A method for treating a hepatitis C viral infection in a mammal comprising administering to a mammal in need thereof an effective amount of the compound of claim 27.
34. A method for treating a hepatitis C viral infection in a mammal comprising administering to a mammal in need thereof an effective amount of the composition of claim
30.
35. A compound having a formula selected from the group consisting of the following formulas:
Figure imgf000169_0001
Figure imgf000170_0001
and pharmaceutically acceptable salts thereof.
36. A method for treating a hepatitis C viral infection in a mammal comprising administering to a mammal in need thereof an effective amount of the compound of claim 35.
37. A compound selected from the group consisting of:
(2S,2'S)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4-fluoro-3- hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate; and
(2R,2'R)-Neopentyl 2,2'-((((2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4-fluoro-3- hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate and the pharmaceutically acceptable salts thereof.
38. A pharmaceutical composition comprising the compound of claim 37 and a pharmaceutically acceptable carrier, excipient or diluent.
39. A method for treating a hepatitis C viral infection in a mammal comprising administering to a mammal in need thereof an effective amount of the compound of claim 37.
40. A method for treating a hepatitis C viral infection in a mammal comprising administering to a mammal in need thereof an effective amount of the composition of claim 38.
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