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WO2013066991A1 - Solvates cristallins de nucléoside phosphoroamidates, leur préparation stéréosélective, nouveaux intermédiaires de ceux-ci et leur utilisation dans le traitement d'une maladie virale - Google Patents

Solvates cristallins de nucléoside phosphoroamidates, leur préparation stéréosélective, nouveaux intermédiaires de ceux-ci et leur utilisation dans le traitement d'une maladie virale Download PDF

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Publication number
WO2013066991A1
WO2013066991A1 PCT/US2012/062772 US2012062772W WO2013066991A1 WO 2013066991 A1 WO2013066991 A1 WO 2013066991A1 US 2012062772 W US2012062772 W US 2012062772W WO 2013066991 A1 WO2013066991 A1 WO 2013066991A1
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Prior art keywords
compound
formula
crystalline
diastereomeric
solvate
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Inventor
Stanley Chamberlain
David Igo
Joanna Bis
Senthil Kumar Kusalakumari SUKUMAR
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Inhibitex Inc
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Inhibitex Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/205—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring the aromatic ring being a non-condensed ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/242—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyaryl compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • This application relates to novel crystalline solvates of the (S)-P diastereomer of the anti- HCV nucleoside phosphoroamidate, (25)-neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(naphthalen- l-yloxy)phosphorylamino)propanoate (also referred to as ⁇ -08189 or ⁇ -189), and to a recrystallization method for separating the two diastereomers of ⁇ -08189.
  • this application relates to a novel stereoselective method for the preparation of either of the two diastereomers, and to the novel synthetic intermediates used in this stereoselective synthesis.
  • This application also relates to the use of the crystalline solvates to increase the liver exposure of 2'-C-methyl guanosine triphosphate from an oral dose.
  • WO 2010/081082 is designated herein as compound 1, or ⁇ -08189, or ⁇ -189 or (25)-neopentyl 2-((((2R,3R,4R,5R)-5-(2- amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(naphthalen- 1 -yloxy)phosphorylamino)propanoate
  • This invention relates to novel crystalline solvates of formula (la), i.e., crystalline solvates of the (5)-P diastereomer (la), an anti-HCV nucleoside phosphoroamidate, (25)-neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)phosphorylamino)propanoate (1), (also referred to as ⁇ -08189 or ⁇ -189), and to a recrystallization method for separating the two diastereomers la and lb which are present in 1 ( ⁇ -08189).
  • formula (la) i.e., crystalline solvates of the (5)-P diastereomer (la), an anti-HCV nucleoside phosphoroami
  • this invention relates to a novel stereoselective method for the preparation of either of the two diastereomers, and to the novel synthetic intermediates used in this stereoselective synthesis.
  • this application relates to the use of crystalline solvates of la to increase the liver exposure of 2'-C-methyl guanosine triphosphate from an oral dose.
  • the crystalline solvates of the pure diastereomer la provide an unexpected advantage when used as a medicament, as compared to the amorphous mixture of diastereomers 1 ( ⁇ -08189) or as compared to either separated, amorphous diastereomers.
  • An aspect of the present invention provides for a crystalline solvate compound represented by formula (la)
  • X represents a molecule of solvation, selected from p-xylene, anisole, 1.4-dioxane, cyclohexane, l-methoxy-2-propanol, MTB, 1-propanol, toluene, 2-butanone, 2- methoxyethanol, 4-methyl-2-pentanone, pentane, acetone, acetonitrile, hexane, chlorobenzene, chloroform, dichloromethane, ethanol, ethyl acetate, isopropyl acetate, methanol, methyl acetate, nitromethane, tetrahydrofuran, heptane, or combinations thereof, and n represents a numerical value ranging from about 0.01 to about 3.00.
  • Another aspect of the invention provides for a method isolating the diastereomeric compounds la and lb, each substantially free of other diastereomeric forms, from the diastereomeric mixture 1, by fractional crystallization using a suitable solvent.
  • aspects of the invention provide for a stereoselective method for preparation of the compounds la and lb, and for the novel chiral intermediates (,S)-neopentyl 2-((S)- (naphthalen-l -yloxy)(4-nitrophenoxy)phosphorylamino)propanoate 2a, and (5)-neopentyl 2-((R)-(naphthalen-l -yloxy)(4-nitrophenoxy)phosphorylamino)propanoate 2b used therein.
  • An additional aspect of the invention provides for is the use of the compound of formula (la) in the treatment of a viral disease such as a virus from the family Flaviviridae e.g., the hepatitis C virus (HCV).
  • a viral disease such as a virus from the family Flaviviridae e.g., the hepatitis C virus (HCV).
  • Figure 1 shows the Vibrational Spectra of Materials 1,1a and lb
  • Figure 2 shows the Polarized Light Microscopy and XRPD of Materials 1,1a and lb
  • Figure 3 shows the Thermal Analysis of Materials 1,1a and lb
  • FIG. 4 shows the Moisture Sorption Data of Materials 1,1a and lb
  • FIG. 5 shows the Chromatographic Data of Materials 1,1a and lb
  • Figure 6 shows the FT-Raman Spectra and XRPD Patterns of Selected Solvates of formula (la) Discovered in the Studies
  • FIG 10 shows the Moisture Sorption Data for the Anisole Solvate [formula (la), X
  • Figure 15 shows the FT Raman of Compound 2
  • Figure 16 shows the HPLC of Compound 2
  • Figure 17 shows the PLM of Compound 2
  • Figure 18 shows the TGA of Compound 2
  • Figure 19 shows the 31 P NMR of Compound 2
  • Figure 20 shows the XRPD and DSC profiles of Compound 2, Crystalline Form A, assigned structure 2b.
  • Figure 21 shows the XRPD, DSC/TGA and 13 P-NMR profiles of Compound 2, Crystalline Form AB (Mixture of structures 2b and 2a)
  • Figure 22 shows the XRPD, DSC/TGA and 13 P-NMR profiles of Compound 2, Crystalline Form B, assigned structure 2a
  • Figure 23 shows XRPD and DSC overlaid profiles of Compound 2: Crystalline Form A, Crystalline Forms A+ B and Crystalline Form B
  • Figure 24 shows the 31 P NMR of Compound lb
  • FIG. 25 shows the 31 P NMR of Compound la
  • Figure 26 shows the 31 P NMR of the Compound from Example 19
  • Figure 27 shows the 31 P NMR of an Admixture of Authentic Compound lb with the
  • Figure 28 shows the 31 P NMR of the Compound Isolated from Example 19 (Recrystallized)
  • Figure 29 shows the 31 P NMR of an Admixture of Authentic Compound la with the
  • Figure 30 shows the HPLC of the Diastereomeric Mixture, Compound 1
  • An aspect of the present invention provides for a crystalline solvate compound represented by formula (la)
  • nX represents n molecules of solvation of (la) with a suitable solvent X.
  • suitable solvate refers to pharmaceutically acceptable solvates of a compound, which solvates are derived from a variety of solvents well known in the art and include, by way of example only, tetrahydrofuran, anisole, toluene, acetonitrile, ethyl acetate, benzonitrile, 1,2-dichloroethane, 1,2-dimethoxyethane, 1,4-dioxane, methyl ethyl ketone, methyl isobutyl ketone, methyl-i-butyl ketone, nitrobenzene, nitromethane, ⁇ -xylene, tetrachloroethylene, trichloroethene, cyclohexane, heptane, hexane, methylcyclohexane, pentane, acetone, butyronitrile, chlorobenzene, chloroform, cyclohexanone,
  • nX represents n molecules of solvation of (la) with a suitable solvent X; wherein X is selected from p-xylene, anisole, 1.4-dioxane, cyclohexane, l-methoxy-2- propanol, MTB, 1-propanol, toluene, 2-butanone, 2-methoxyethanol, 4-methyl-2-pentanone, pentane, acetone, acetonitrile, hexane, chlorobenzene, chloroform, dichloromethane, ethanol, ethyl acetate, isopropyl acetate, methanol, methyl acetate, nitromethane, tetrahydrofuran, heptane, or combinations thereof, and n represents a numerical value ranging from about 0.01 to about 3.00.
  • X is selected from 2-butanone/toluene, acetone, acetone/MTBE, acetone/pentane, acetone/toluene, anisole, ethyl acetate/cyclohexane, ethyl acetate/heptane, ethyl acetate/hexane, methyl acetate/cyclohexane, nitromethane/MTBE, -xylene, tetrahydrofuran, tetrahydrofuran/heptane, or tetrahydrofuran/p-xylene.
  • the crystalline forms are characterized by their Power Diffraction X-ray Crystallography, which produces a fingerprint that is unique to the crystalline form which distinguishes it from amorphous forms of compound la and exhibit physical, pharmaceutical, physiological and biological characteristics that may be significantly different from amorphous forms of compound la.
  • the solvates can generally prepared by suspending the diastereomeric mixture, compound 1 or the amorphous single diastereomer, compound la, in suitable solvent or solvent mixture and stirring the slurry for a sufficient amount of time, such as from about 12 to 48 hours to cause dissolution of one isomer while promoting crystallization of the other isomer as solvate of compound la, i.e., a compound of formula (la).
  • the crystalline solvates of formula (la) are characterized by their X- ray Powder Diffraction patterns.
  • the crystalline characteristics of the formula (la) compounds can also be observed using other techniques such as thermogravimetric analysis (TGA) and Polarized Light Microscopy (PLM).
  • TGA thermogravimetric analysis
  • PLM Polarized Light Microscopy
  • the stereochemical purity and absolute configuration can be confirmed by comparison of isolated material to standards samples of la and lb obtained by HPLC separation methods, by 31 P-NMR spectroscopy, and by single crystal X-Ray diffraction.
  • the crystalline solvates of formula (la) have surprisingly unexpected enhanced properties, such as enhanced or greater stability, enhanced storage stability and enhanced stability against chemical degradation, as compared to the amorphous material la.
  • the crystalline solvates of formula (la) may have surprisingly unexpected enhanced biological properties, such as enhanced oral bioavailability from a solid dose.
  • Another aspect of the invention provides for a method isolating the diastereomeric compounds la and lb, each substantially free of other diastereomeric forms, from the diastereomeric mixture 1, by fractional crystallization using a suitable solvent.
  • this aspect of the invention provides for a method isolating the diastereomeric compounds la and lb, each substantially free of other diastereomeric forms, said method comprising the steps of
  • the crystals A and the residue B represent samples of the diastereomers la and lb, respectively, which are substantially free of each other, and, whose identity can be confirmed by standard spectroscopic methods.
  • substantially free refers to samples of a compound, e.g., la or lb, which preferably contain less than 10% of other diastereomers, and more particularly, approximately 5% or less of other diastereomers.
  • An embodiment of this aspect comprises the optional addition of seed crystals in step (2)
  • a second embodiment of this aspect comprises the optional warming of the mixture prepared in step (2), followed by cooling prior to step (3).
  • a third embodiment of this aspect comprises the optional repetition of steps (1-4) using the residue obtained in step (4) as the sample containing the compound 1.
  • a fourth embodiment comprises the optional step of recrystallization of the crystals (A) isolated in step 3 with the suitable solvent or solvent mixture used in step (1).
  • the crystalline A is a crystalline solvate of formula (la).
  • the invention provides for a method isolating the diastereomeric compound la substantially free of the diastereomeric compound lb and any other diastereomeric forms, from the diastereomeric mixture 1, by fractional crystallization using a suitable solvent.
  • this aspect of the invention provides for a method isolating crystalline forms of the diastereomeric compound la, substantially free of the diastereomeric compound lb and any other diastereomeric forms, said method comprising the steps of
  • An embodiment of this aspect comprises the optional addition of seed crystals in step (2)
  • a second embodiment of this aspect comprises the optional warming of the mixture prepared in step (2), followed by cooling prior to step (3).
  • ⁇ A third embodiment comprises the optional step of recrystallization of the crystals isolated in step 3 with the suitable solvent or solvent mixture used in step (1).
  • the crystalline material isolated in step 3 is a crystalline solvate of compound la represented by formula (la).
  • Non-limiting examples of suitable solvents useful in carrying out the method of separation of the diastereomeric compounds la and lb from the diastereomeric mixture 1 by fractional crystallization are ⁇ -xylene, toluene, pentane, heptane, 1.4-dioxane, 1-butanol, 1 -propanol, 2- butanone, 2-propanol, acetone, acetonitrile, chloroform, dichloromethane, dimethyl carbonate, ethyl acetate, methyl acetate, tetrahydrofuran, and anisole or combinations thereof.
  • the solvent mixtures may be in any proportion.
  • the preferred solvents or solvent combinations are those that allow full dissolution of one of the diastereomers, therefore facilitating the filtration/isolation of the other diastereomer.
  • a further embodiment of this aspect is a solvent or solvent combination that allows full dissolution of the (R)-P diastereomer lb and crystallization of the (S)-P diastereomer la.
  • the crystalline diastereomer la isolated by filtration may be a solvate formed from one of the crystallizing solvents, as described hereinabove for the compound of formula (la).
  • solvent for crystallization techniques that employ solvent, the choice of solvent or solvents is typically dependent upon one or more factors, such as solubility of the compound, the crystallization technique, and the vapor pressure of the solvent.
  • Combinations of solvents may be employed, for example, the compound may be solubilized into a first solvent to afford a solution, followed by the addition of an antisolvent to decrease the solubility of the compound in the solution and to afford the formation of crystals .
  • An antisolvent is a solvent in which the compound has low solubility.
  • Suitable solvents for preparing crystals include polar and nonpolar solvents.
  • a compound is suspended and/or stirred in a suitable solvent to afford a slurry, which may be heated to promote dissolution.
  • the term "slurry", as used herein, means a saturated solution of the compound, which may also contain an additional amount of the compound to afford a heterogeneous mixture of the compound and a solvent at a given temperature.
  • Seed crystals may be added to any crystallization mixture to promote crystallization. Seeding may be employed to control growth of a particular polymorph or to control the particle size distribution of the crystalline product. Accordingly, calculation of the amount of seeds needed depends on the size of the seed available and the desired size of an average product particle as described, for example, in "Programmed Cooling of Batch Crystallizers ,” J. W. Mullin and J. Nyvlt, Chemical Engineering Science, 1971, 26, 369-377. In general, seeds of small size are needed to control effectively the growth of crystals in the batch. Seed of small size may be generated by sieving, milling, or micronizing of large crystals , or by micro- crystallization of solutions. Care should be taken that milling or micronizing of crystals does not result in any change in crystallinity from the desired crystal form (i.e., change to amorphous or to another polymorph).
  • a cooled crystallization mixture may be filtered under vacuum, and the isolated solids may be washed with a suitable solvent, such as cold recrystallization solvent, and dried under a nitrogen purge to afford the desired crystalline form.
  • the isolated solids may be analyzed by a suitable spectroscopic or analytical technique, such as solid state nuclear magnetic resonance, differential scanning calorimetry, x-ray powder diffraction, or the like, to assure formation of the preferred crystalline form of the product.
  • the resulting crystalline form is typically produced in an amount of greater than about 70 weight % isolated yield, preferably greater than 90 weight % isolated yield, based on the weight of the compound originally employed in the crystallization procedure.
  • the product may be comilled or passed through a mesh screen to delump the product, if necessary.
  • the invention provides a stereoselective method for the preparation of compound of formula (la) comprising the steps of
  • the product (la) may be further purified by recrystallization using solvent(s) and conditions described hereinabove for la.
  • the invention provides a stereoselective method for the preparation of compound of formula la comprising the steps of
  • the product la may be further purified by recrystallization using solvent(s) and conditions described hereinabove for la.
  • the product la may be further converted to a crystalline solvate of formula (la), by recrystallization of la produced by the process using a solvent in which said solvate forms.
  • the present invention provides for a stereoselective method of preparation of the compound lb, comprising the steps of
  • the products lb may be further purified by recrystallization using solvent(s) and conditions described hereinabove.
  • Another aspect of the invention provides the novel compounds 2a and 2b, useful as intermediates in the preparation of compounds la and lb.
  • the present invention provides for a method of preparation of the compounds 2a and 2b in substantially diastereomerically pure form comprising the steps of (1) reaction of a chlorophosphosphoroamidate intermediate 4 with ntirophenol 5 in the presence of a base
  • the chlorophosphosphoroamidate ester of formula 4 is allowed to react with 4-nitrophenol (compound 5) in the presence of a base such as triethylamine in an inert solvent such as THF to produce the phosphoramidate of formula 2 as a mixture of diastereomers.
  • a base such as triethylamine
  • an inert solvent such as THF
  • the crude material isolated from the reaction is purified by column chromatographic methods in order to provide material which is more amendable to fractional recrystallization.
  • the diastereomeric phosphoramidate mixture of formula 2 is separated into pure diastereomers of formulae 2a and 2b by fractional crystallization using a suitable solvent or solvent mixture.
  • suitable solvent or “suitable solvent mixtures” refer to a solvent or solvent mixtures which is capable of dissolving a compound, or useful in the purification of a mixture of compounds by fractional crystallization.
  • suitable solvents for this step are methyl cyclohexane, isobutyl acetate and mixtures thereof.
  • Crystallization is optionally initiated by scratching the surface of the solution-containing vessel or by addition of a seed crystal of the desired compound to the supersaturated solution.
  • a mixture of solvents may also be used to control the solubility properties of the solvent.
  • Typical acceptable solvents which are suitable for this purpose, may be used alone or in a mixture include tetrahydrofuran, anisole, ethanol, methanol, isopropanol, toluene, acetonitrile, ethyl acetate, benzonitrile, 1,2-dichloroethane, 1,2-dimethoxyethane, 1,4- dioxane, methyl ethyl ketone, methyl isobutyl ketone, methyl-i-butyl ketone, nitrobenzene, nitromethane, ⁇ -xylene, tetrachloroethylene, trichloroethene, cyclohexane, heptane, hexane, methylcyclohexane, pentane, acetone, butyronitrile, chlorobenzene, chloroform, cyclohexanone, cyclopentyl methyl ether, 3-pentan
  • Acceptable mixtures include isopropyl ether/methyl cyclohexane, diethyl ether/hexane, methanol/anisole, ethanol/anisole, isopropanol/anisole, n- butanol/anisole, acetone/anisole, isobutyl acetate/methyl cyclohexane, 2-methyl- tetrahydrodfuran/hexane, ethyl acetate/heptane, tetrachloroethylene/ methylcyclohexane and the like.
  • the mixture of solvents may be in any proportion. Non-limiting examples of such proportions are 50/50, 70/30, 60/40, and 95/5 volume %.
  • Preferred solvents and "suitable solvent or solvent mixtures” include isobutyl acetate/methyl cyclohexane, 2-methyl-tetrahydrofuran/methyl cyclohexane, ethyl acetate/heptane, isopropyl ether/methyl cyclohexane and tetrachloroethylene/methyl cyclohexane.
  • the preferred solvents for the formation of crystalline 2a may differ from the preferred solvents for the formation of crystalline 2b.
  • one embodiment of this aspect of the invention is the optional repetition of the recrystallization using the crystalline material isolated in step (2), using a suitable solvent/solvent mixture.
  • the recrystallization may be carried out at a temperature suitable for controlling the rate of crystallization. Preferred temperatures are from about 5 °C to about 50 °C.
  • the compounds are substantially pure diastereomers, i.e., are samples of compound 2a or 2b which preferably contain less than 10% of other diastereomers, and more particularly, approximately 5% or less of other diastereomers.
  • the present invention provides for the use of the crystalline solvent compound of formula (la) in the treatment of viral diseases and for the preparation of medicaments useful in the treatment of viral diseases.
  • the starting materials used are known in the art or are prepared by well-known general methods and procedures. 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).
  • reaction temperatures i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.
  • 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 crystalline solvates of formula (la) prepared by the methods described above may be used to prepare formulated compositions and medicaments, useful for administration of therapeutically acceptable amounts of compound.
  • the crystalline solvates provide advantageous properties such as enhanced or greater stability, enhanced storage stability and enhanced stability against chemical degradation, as compared to the amorphous material of formula (la).
  • the crystalline forms of (la) may have surprisingly unexpected enhanced biological properties, such as enhanced biological activity, enhanced metabolic behavior in vivo, and a reduction in undesirable side effects, including toxicity.
  • the crystalline solvates of formula (la) 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.
  • therapeutically effective amounts of compounds of formula (la) 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 formula (la) in combination with at least one pharmaceutically acceptable vehicle, carrier, excipient or diluent.
  • 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.
  • 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, 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.
  • 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 (la) 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 %.
  • Pharmaceutical formulations containing a compound in accordance with the invention are described further below.
  • 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- ⁇ 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-a 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 (la) 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 (la) 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 of formula (la) 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 rimanta
  • 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.
  • 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
  • the compounds 1, la, lb and the solvates of Compound la [Formula (la) compounds] are studied in oral bioavailability experiments in cynomolgus monkeys.
  • the compounds are dosed as solids in capsules (25 mg/kg) and the resulting plasma concentrations of a selection of metabolites are measured. These results are compared to values obtained when compounds 1, la, lb, and the solvates of Compound la are formulated in 95:5 CapmukTween and dosed orally.
  • 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.
  • cm resolution 64-128 scans, using Happ-Genzel apodization function and 2 level zero- filling.
  • Mass Spectrometry The mode of ionization 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 ⁇ L/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.
  • FAB fast atom bombardment
  • MNOBA
  • PLM Polarized-light Microscopy
  • XRPD Powder X-Ray Diffraction
  • XRPD diffractograms were acquired using PANalytical X'Pert Pro diffractometer on Si zero-background wafers. All diffractograms were collected using a monochromatic Cu Ka (45 kV/40 mA) radiation and a step size of 0.02 ° 2 ⁇ .
  • Differential Scanning Calorimetry (DSC) DSC was conducted with a TA Instruments Q100 differential scanning calorimeter equipped with an autosampler and a refrigerated cooling system under 40 mL/min 2 purge. DSC thermograms were obtained at 15 °C/min in Pt or Al pans.
  • TGA-IR Thermogravimetric Analysis with IR off-gas detection
  • HPLC analyses were conducted with an HP 1 100 system equipped with a Gl 131 Quad pump, G1367A autosampler, and G1315B diode array detector. Column: Luna CI 8(2) (50 x 2.0 mm, 3 ⁇ ). Mobile phase: 100% water (0.05%TFA) to 95% ACN (0.05% TFA) over 8 min and 2 min re-equilibration. Flow rate: 1 HPLC. HPLC analyses were conducted with an HP 1 100 system equipped with a Gl 131 Quad pump, G1367A autosampler, and G1315B diode array detector. Column: Luna CI 8(2) (50 x 2.0 mm, 3 ⁇ ). Mobile phase: 100% water (0.05% TFA) to 95% ACN (0.05% TFA) over 8 min and 2 min re-equilibration. Flow rate: 1 mL/min. Detection: 254 nm.
  • Dynamic Vapor Sorption DVS experiments were conducted on a Surface Measurement Systems DVS-HT at 25 °C. The instrument was operated in step mode and the relative humidity was increased in 10% RH increments from 40% RH to 90% RH, then decreased from 90% RH to 0% RH, then increased from 0% RH to 90% RH, then decreased from 90% RH to 0% RH. An extra step at 75% RH was included in each cycle.
  • the mass equilibrium criterion was set at 0.005% change in mass over time (dm/dt) prior to each humidity level. A minimum step time of 10 minutes and a maximum step time of 240 minutes were specified. mL/min. Detection: 254 nm.
  • the compound is prepared as described in WO 2010/081082.
  • 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)).
  • the compound is prepared as described in WO 2010/081082.
  • the compound can be prepared as described in WO 2010/081082.
  • the compound can be prepared as follows:
  • Step 1 (25)-neopentyl 2-(chloro(naphthalen-l-yloxy)phosphorylamino)-propanoate
  • Step 2 (25)-neopentyl 2-((naphthalen-l-yloxy)(4-nitrophenoxy)phosphorylamino)propanoate (Compound 2)
  • Step 3 (25)-Neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4- dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(naphthalen- 1 - yloxy)phosphorylamino)propanoate
  • a 1 : 1 mixture of diastereomers of 2,2-dimethylpropyl 2(5")-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4- methyltetrahydrofuran-2-yl)methoxy(naphthalene- 1 -yloxy) phosphorylamino)propanoate (10.0 g, Compound 1, Example 3) was taken up in 1 : 1 ethanol : hexanes and loaded onto a Chiral Pak AD chiral column and eluted with the same solvent. UV detection was done at 300 nm.
  • Peak 1 (Compound lb) with a retention time of 6 min on the analytical column, and provided 4.75 g, (95.9% yield) of a single diastereomer with 99.9% ee (enantiomeric excess).
  • Peak 2 (Compound la), with a retention time of 10.7 min on the analytical column, provided 5.29 g (100% yield) of a single diastereomer with 99.8% diastereomeric excess. (95.9% yield) of a single diastereomer with 99.9% ee (enantiomeric excess).
  • the three materials: Compound 1, Compound lb and Compound la were analyzed by the following analytical techniques: FT-Raman spectroscopy, FT-IR spectroscopy, conventional and modulated differential scanning calorimetry (DSC and mDSC, respectively), thermogravimetric analysis (TGA), TGA-IR, high-performance liquid chromatography (HPLC), dynamic vapor sorption (DVS), polarized light microscopy (PLM), and X-ray Powder Diffraction (XRPD).
  • TGA thermogravimetric analysis
  • HPLC high-performance liquid chromatography
  • DVD dynamic vapor sorption
  • PLM polarized light microscopy
  • XRPD X-ray Powder Diffraction
  • Modulated DSC confirmed that these transitions closely correlate to the glass transition temperatures: 70.5 °C, 69.3 °C, and 86.3 °C, respectively.
  • DVS analysis revealed that the amorphous batches are hygroscopic, absorbing up to 7% wt, 6.5% wt, and 4.5% wt of water between 0-90% RH, respectively.
  • HPLC analyses indicated high purity of all three materials (>99%) and highlighted the ability of the method to resolve the two diastereomers present in Compound 1
  • Solvates are highly crystalline by PLM and XRPD.
  • Desolvation on-set occurs above 100°C, even for solvates containing low boiling point solvents (e.g. pentane or acetone; analysis based on DSC measurement of non-hermetically sealed pans)
  • low boiling point solvents e.g. pentane or acetone; analysis based on DSC measurement of non-hermetically sealed pans
  • Desolvation onsets are highest for solvates containing high-boiling solvents (e.g., anisole, p- xylene, and toluene)
  • high-boiling solvents e.g., anisole, p- xylene, and toluene
  • Solvates may contain a single solvent (e.g., acetone solvate) or a mixture of solvents (e.g., acetone/pentane solvate) as confirmed by IR analysis of the gas evolved upon heating (TGA- IR).
  • a single solvent e.g., acetone solvate
  • a mixture of solvents e.g., acetone/pentane solvate
  • loss of solvent on heating proceeds via two steps, as indicated by TGA traces. Whereas the first weight loss step is relatively distinct and can be integrated, the second step is gradual and overlaps with the decomposition of 1. While such desolvation mechanism did not allow for the determination of the exact solvent content for majority of solvates, evidence suggest that the included solvent amounts are sub-stoichiometric ( ⁇ 0.5 molar equivalent)
  • Solvates melt upon desolvation via heating and remain amorphous after the subsequent cooling, as confirmed by variable temperature XRPD analysis or ⁇ -xylene and toluene solvates Solvates are physically stable (no change in FT-Raman and/or XRPD data over time) for at least four weeks of storage in an enclosed container or on drying under vacuum and elevated temperatures (e.g., 40 °C)
  • Crystallization attempts of compound 1, (Example 3) resulted in optical resolution of the R- P and S-P diastereomers. Specifically, the separation was based on precipitation of S-P diastereomer as solvated crystalline solid and dissolution of the amorphous R-P diastereomer. The optical separation was confirmed by HPLC analysis of the produced solids and indicated absence of R-P diastereomer. The optical purity of the isolated S-P diastereomer can be achieved using a suitable crystallization solvent system that allows full dissolution of the amorphous R-P diastereomer, therefore facilitating the filtration/isolation of S-P diastereomer. A list of solvents that facilitated crystallization is provided in Table 5. Table 5. Solvents That Yielded Crystalline Products (la) in Experiments Involving Compound 1 (Example 3).
  • This solvate was isolated from a wide range of (-xylene containing solvent systems. Selected physicochemical data of the (-xylene solvate are presented in Figure 7.
  • a sample of amorphous la 160 mg was weighed into a 2-mL vial and combined with p- xylene (1.0 mL). The suspension was stirred for 15 min at 25 °C.
  • 1-Propanol (0.45 mL) was added. The initially free-flowing powder turned into a gum. The gum was subjected to stirring at 25 °C for 20 h, over which time a white powder formed. The obtained powder was isolated by vacuum filtration, air-dried for 1 h, and dried at 50 °C under vacuum for 6 h.
  • the compound 1 (from Example 3) (160 mg) was weighed into a 2 mL vial and combined p- xylene (1.0 mL). The suspension was stirred for 15 min at 25 °C. 1-Propanol (0.45 mL) was added. The initially free-flowing powder turned into a gum. The gum was subjected to stirring at 25 °C for 20 h, over which time a white powder formed. The obtained powder was isolated on by vacuum filtration and air-dried for 4 h.
  • a sample of amorphous (la) (80 mg) was weighed into a 2 mL vial and combined with p- xylene (1.0 mL). The suspension was stirred for 15 min at 25 °C. Ethyl acetate (0.3 mL) was added. The initially free-flowing powder turned into a gum. The gum was subjected to stirring at 25 °C for 48 h. The suspension was filtered using a syringe equipped with 0.22 ⁇ filter disk. The solution was cooled to 22 °C and held for 24 h. The obtained single crystals, immersed in the mother liquor, were submitted for SCXRD analysis.
  • ⁇ -xylene solvate obtained in Examples 6-8 is a white solid that is crystalline, as indicated by XRPD and PLM. (see Figure 6)
  • the presence of -xylene was confirmed by IR analysis of the gas evolved upon heating. DSC analysis indicates desolvation endotherm between 130-145 °C, followed by decomposition.
  • variable-temperature XRPD variable-temperature XRPD
  • the ⁇ -xylene solvate is physically stable for at least four weeks when stored in an enclosed container. Drying studies conducted at 50 °C and under vacuum for 72 h led to a ⁇ 1% solvent loss; however this operation did not induce a change in the crystal-form. Washing the ⁇ -xylene solvate with pentane did not remove ⁇ -xylene from the compound nor lead to a form change.
  • Suitable crystals of ⁇ -xylene solvate were grown using la as the input material and characterized by single crystal X-ray diffraction.
  • the single crystal structure of this solvate confirmed the following:
  • a sample of la (1.02 g) was weighed into a 20-mL vial and combined with anisole (2.0 mL).
  • the initially free-flowing powder turned into a gum.
  • the gum was seeded with anisole solvate ( ⁇ 2 mg) and was subjected to stirring at 20 °C for 48 h, over which time a white powder formed.
  • the obtained free-flowing suspension was cooled to 5 °C and stirred for 2 h.
  • the white solid was isolated on Buchner funnel by filtration, washed with pre-cooled anisole (5 °C; 3 mL), air-dried for 1 h, and dried at 40 °C under vacuum for 24 h. Yield was approximately 77%.
  • the sample of 1 (0.83 g) was weighed into a 20-mL vial and combined with anisole (3.0 mL).
  • the initially free-flowing powder turned into a gum.
  • the gum was seeded with anisole solvate ( ⁇ 2 mg) and was subjected to stirring at 20 °C for 5 days, over which time a white powder formed.
  • the obtained powder was isolated on Buchner funnel by filtration, air-dried for 1 h, and dried at 40 °C under vacuum for 12 h. Yield was approximately 25%.
  • HPLC analysis of the isolated solid confirmed absence of (5)-P diastereomer.
  • the white solid was isolated on Buchner funnel by filtration, washed with pre- cooled heptane (5 °C; 5 niL), air-dried for 1 h, and dried at 40 °C under vacuum for 12 h. Yield was approximately 75%.
  • the sample of 1 (0.96 g;) was weighed into a 20-mL vial and combined with heptane (4.0 mL). The suspension was stirred for 15 min at 25 °C. Ethyl acetate (8.0 mL) was added. The initially free-flowing powder turned into a gum. The gum was seeded with ethyl acetate/heptane solvate ( ⁇ 2 mg) and was subjected to stirring at 25 °C for 5 days, over which time a white powder formed. The obtained free-flowing suspension was cooled to 5 °C and stirred for 3 h. The white solid was isolated on Buchner funnel by filtration, air-dried for 1 h, and dried at 40 °C under vacuum for 12 h. Yield was approximately 25%. HPLC analysis of the isolated solid confirmed absence of the (S)-P diastereomer.
  • Ethyl acetate/heptane solvate obtained in either of the Examples 13 or 14 is a white solid that is crystalline, as indicated by XRPD and PLM.
  • the presence of both solvents was confirmed by IR analysis of the gas evolved upon heating.
  • DSC analysis indicated desolvation endotherm between 1 10-125 °C, followed by decomposition.
  • the sample of la (1.01 g) was weighed into a 20-mL vial and combined with pentane (4.5 mL). The suspension was stirred for 15 min at 25 °C. Acetone (0.5 mL) was added. The initially free-flowing powder turned into a gum. The gum was seeded with acetone/pentane solvate ( ⁇ 2 mg) and subjected to stirring at 25 °C for 72 h, over which time a white powder formed. The obtained suspension was cooled to 5 °C and stirred for 3 h. The white solid was isolated on Buchner funnel by filtration, washed with pre-cooled pentane (5 °C; 5 mL), air- dried for 1 h, and dried at 40 °C under vacuum for 72 h. Yield was approximately 78%.
  • the sample of 1 (1.01 g) was weighed into a 20-mL vial and combined with pentane (4.5 mL). The suspension was stirred for 15 min at 25 °C. Acetone (5.5 mL) was added. The initially free-flowing powder turned into a gum. The gum was seeded with acetone/pentane solvate ( ⁇ 2 mg) and was subjected to stirring at 20 °C for 5 days, over which time a white powder formed. The obtained free-flowing suspension was cooled to 5 °C and stirred for 3 h. The white solid was isolated on Buchner funnel by filtration, air-dried for 1 h, and then dried at 40 °C under vacuum for 12 h. Yield was approximately 27%. HPLC analysis of the isolated solid confirmed absence of the (5)-P diastereomer.
  • Acetone/pentane solvate obtained from either of the Examples 15 or 16 is a white solid that is crystalline, as indicated by XRPD and PLM.
  • the presence of both solvents was confirmed by IR analysis of the gas evolved upon heating. DSC analysis indicates desolvation endotherm between 100-125 °C, followed by decomposition.
  • Dynamic vapor sorption analysis indicates the solvate is non-hygroscopic (-0.9% water uptake between 0-90% RH).
  • Acetone/pentane solvate maintains its integrity and crystallinity after drying at 40 °C under vacuum for 72 h and in aqueous suspension at 25 °C for 24 h. Grinding the solvate material for 15 min using mortar and pestle causes a partial loss of solvent and crystallinity, as indicated by TGA and XRPD analyses of the ground material.
  • Impure 2 was purified again by column chromatography using a gradient mixture of hexane and ethyl acetate (Hexane:EtOAc, 90: 10) to (Hexane :EtOAc, 85: 15) to give 5.8 g (0.012 mol) of pure Compound 2.
  • Methyl cyclohexane 500 ⁇ was added to amorphous 2 obtained in by the purification procedure described in Examplel5 (20 mg) and the suspension was stirred at RT for 5 min. Isobutyl acetate (25 ⁇ ,) was added. The suspension was stirred while cycling the temperature between 25 °C and 5 °C for 12 h. The suspension was cooled to 5 °C and stored at 5 °C for 48 h. The solids were isolated by vacuum filtration and air dried for 15 min to provide a sample of Crystalline Form A.
  • Crystalline Form A is a crystalline solid form of 2 as confirmed by XRPD. It is mixture of diastereomers 2a and 2b. PLM image shows crystalline, rod-like habit. DSC curve exhibits two endothermic events having maxima at 86 °C and 93 °C. Characterizing data for Form A are provided in Figure 20.
  • Methyl cyclohexane (10 mL) was added to the amorphous Compound 2 (995.7 mg) followed by addition of seed crystals ( ⁇ 10 mg). The suspension was stirred at RT for 5 min followed by addition of isobutyl acetate (0.5 mL). The suspension was stirred at RT during which time precipitation occurred (within 30 min). The suspension was stirred at RT for 12 h, heated to 50 °C, and stirred for 2 h during which time partial dissolution of the solids occurred. The suspension was cooled to 5 °C (0.1 °C/min) and stirred overnight. The solids were isolated by vacuum filtration and air dried for 45 min. The yield of crystallization was -74%.
  • the product is a mixture of diastereomers 2a and 2b and is a crystalline solid as confirmed by XRPD.
  • PLM data indicate a crystalline rod-like habit.
  • DSC data show two endothermic events with a major event occurring at 73.8 °C.
  • TGA data shows no significant weight loss below 200 °C, indicating that the products are not solvated. Characterizing data for Crystalline Form AB are provided in Figure 21.
  • Methylcyclohexane (56.55 mL) was added to the liquid compound 2 (5.8 g) followed by addition of seed crystals (10 mg). The suspension was stirred at RT for 5 min followed by addition of isobutyl acetate (2.88 mL). The suspension was stirred at RT during which precipitation occurred and stirring was continued at RT overnight, then heated to 45 °C for 2 h. The suspension was cooled to 5 °C and stirred overnight. The solids were filtered and dried under vacuum to obtain 4.6 g (79%) of Crystalline Form AB.
  • Crystallinity of Crystalline Form B is shown by XRPD in Figure 22.
  • the 31 P NMR of Crystalline Form B shows that only one diastereomer is present.
  • the absolute stereochemistry was not determined directly; however experiments with this material, described below, demonstrate that it is the phosphorous diastereomer 2a.
  • the DSC curve for Crystalline Form B shows a sharp single endothermic event occurring at 104.2 °C (onset value) with a peak at 107.0 °C. TGA data for the material shows no significant weight loss below 200 °C, indicating that it is non-solvated. Characterizing data for Crystalline Form B of Compound 2a are provided in Figure 22.
  • Crystalline Form B displays a single sharp endotherm at 107 °C, which appears advantageous over Crystalline Forms A or AB which show multiple, broad endotherms.
  • the XRPD data for Crystalline Form B shows that it is peaks are a subset of the Crystalline Form AB peaks and that they are substantially different from Crystalline Form A.
  • Step 1 The crude material la (3.2 g) was stirred with isobutyl acetate/methylcyclohexane (5:95, 40 mL) at RT overnight. The solution was decanted and the residue dried in high vacuum to obtain 2.6 g (246%) of crude la.
  • Step 2 The above residue (2.6 g) was again stirred with isobutyl acetate/methylcyclohexane (9:91, 40 mL) at 40 °C overnight. The solution was decanted and the residue dried in high vacuum to obtain 1.5 g (142%) of brown solid la.
  • Step 3 The above brown solid material (2.6 g, from step 2) was dissolved in anisole (25 mL) and heated to 80 °C for 15 min; the solution was allowed to cool to RT and left overnight at RT. The precipitated solid was filtered, dried in high vacuum to obtain 780 mg of the anisole solvate of Compound la as a white solid and single diastereomer.
  • Step 1 The crude (2S neopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)- 3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(naphthalen-l- yloxy)phosphorylamino)propanoate (la) (1.78 g) was stirred with isobutyl acetate/methylcyclohexane (9:91, 40 mL) at 40 °C overnight.
  • Step 2 The above residue (0.9 g) was again stirred with isobutyl acetate/methylcyclohexane (9:91, 25 mL) at 40 °C for 5 h. The solution was decanted and the residue dried in high vacuum to obtain 0.825 g (130%) of la.
  • Step 3 The above light yellow solid material (0.825 g, from step 2) was dissolved in anisole (5 mL) and heated to 80 °C for 15 min; the solution was allowed to cool to RT and left overnight at RT. The precipitated solid was filtered, washed with anisole (2 mL), dried in high vacuum to obtain 530 mg (0.80 mmol, 84%) single diastereomer as the crystalline anisole solvate of Compound la.
  • the 31 P NMR is shown in Figure 28. The ability of this material to crystallize is consistent with its structural assignment as the anisole solvate of Compound la, since no crystalline solvates of Compound lb had been observed up to this time.
  • a luciferase-reporter genotype lb subgenomic replicon cell line was obtained from Apath, LLC, Brooklyn, NY: Cells were passaged twice a week by splitting 3 or 4 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% CO 2 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-ChipTM Disposable Hemacytometers (Fisher Scientific, Pittsburg, PA). The anti-HCV assays were done accordingly:
  • Luciferase Genotype lb Replicon Potency Assay Replicon cells were seeded into white 96-well plates (Nunc/VWR) at a density of 2x10 4 cells/well in medium without G-418. 18-24 h after cell plating, inhibitors were added and cells were incubated for additional 72 hours. Compounds were tested in triplicates and quadruplicates at 3X or 4X serial dilutions over a range of 0.0001 -to- 100 ⁇ 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% effective concentration (EC 50 ) values were calculated as the concentration of compound that resulted in a 50% decrease in the reporter expression as compared to untreated cells. The values were determined by non-linear regression (four-parameter sigmoidal curve fitting) analysis.
  • Cytotoxicity Assay Cells were seeded into 96-well plates at a density of 2x10 4 cells per well. 24 h after cell plating, serial 2X or 3X compound dilutions, starting with 100 ⁇ , 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 5% CO 2 atmosphere for 72 hours. 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 loss was reported as the CC5 0 concentration.
  • Plasma samples were stored on wet ice and samples were centrifuged at 5 ⁇ 3°C within 30 minutes of collection. The resultant plasma was recovered and split into two 0.5 mL aliquots and placed in polypropylene tubes. All plasma samples were quick frozen over dry ice and stored under conditions set to maintain -75 ⁇ 15°C until analysis.
  • Plasma samples were prepared for analysis as follows. 400 ⁇ ⁇ of 50 mM ammonium acetate containing 2 ng/niL of internal standard Nl-Methyl-2'-deoxyguanosine; Berry &Associates, Dexter, MI; Cat# PR3748) was added to 100 of each sample. Calibration curves were prepared by serial dilution of a stock solution of Metabolite A (2'-C-methyl guanosine) or Metabolite B (6-O-methyl 2'-C-methyl guanosine) into blank media. Solid phase extraction of the samples was performed with H ⁇ O-Philic DVB speed disk columns (J.T.
  • Liquid chromatography was performed with an Agilent 1100 Series HPLC system equipped with a Betasil C18 2.1 x 100 mm 5 ⁇ column (Thermo Fisher Scientific Waltham, MA). Samples were eluted using a linear gradient from 100% Solvent A (0.1% formic acid in H 2 O) to 50% Solvent B (0.1% formic acid in acetonitrile) applied over 6min at 0.5 mL/min flow rate.
  • the HPLC system was coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Framingham, MA). Mass spectrometry was performed in positive ion mode and data was analyzed using Analyst® vl.4.2 software (Applied Biosystems, Framingham, MA).
  • Plasma samples were prepared for analysis as follows. 50 ⁇ of each test sample were distributed in a 96-well V-bottom plate. 200 ⁇ of acetonitrile containing 10 ng/mL Internal Standard was added to each Plasma sample. The precipitated samples were centrifuged at 3000 rpm, 4 °C for 20 minutes in a Sorvall RT6000S centrifuge (Thermo Scientific, Waltham MA) and 50 of Plasma from each sample was transferred into a 96 deep well plate followed by the addition of 50 ⁇ 50:50 acetonitrile:H 2 0 to each sample. Samples were covered, mixed well by vortexing and maintained at 2-8 °C before and during analysis.
  • Calibration curves were constructed by spiking varying concentrations of analyzing protide (Compounds 1,1a or lb) into blank media. 15 ⁇ of each test sample was analyzed for analyte concentrations by liquid chromatography coupled to tandem mass spectrometry (LC- MS/MS). Liquid chromatography was performed with an Agilent 1100 Series HPLC system equipped with a Synergi 4 ⁇ Polar-RP, 30x2.0 mm column (Phenomenex, Torrance, CA). Samples were eluted using a linear gradient from 100% Solvent A (0.1% formic acid in H 2 O) to 100% Solvent B (0.1% formic acid in acetonitrile) applied over 3 min at 1.0 mL/min flow rate. The HPLC system was as described above.
  • each test sample 50 ⁇ 1 of each test sample were distributed in a 96-well V-bottom plate. 200 ⁇ ⁇ of methanol containing 25 ng/mL of Internal Standard was added to each sample. The precipitated samples were centrifuged at 3000 rpm, 4°C for 20 minutes in a Sorvall RT6000S centrifuge (Thermo Scientific, Waltham MA) and 50 ⁇ ⁇ of Plasma from each sample was transferred into a 96 deep well plate followed by the addition of 50 ⁇ H 2 0 to each sample. Samples were covered, mixed well by vortexing and maintained at 2-8°C before and during analysis. Calibration curves were constructed by spiking varying concentrations of analytes into blank media.
  • each test sample 50 ⁇ 1 of each test sample were distributed in a 96-well V-bottom plate. 200 ⁇ ⁇ of methanol containing 50 ng/mL Internal Standard was added to each sample. The precipitated samples were centrifuged at 3000rpm, 4°C for 20 minutes in a Sorvall RT6000S centrifuge (Thermo Scientific, Waltham MA), 100 ⁇ ⁇ of Plasma from each sample was transferred into a 96 deep well plate, and samples were dried under N 2 , overnight at room temperature. The dried samples were reconstituted in 50 ⁇ ammonium acetate in 3 ⁇ 40 per sample. Samples were covered, mixed well by vortexing and maintained at 2-8 °C before and during analysis.
  • Calibration curves were constructed by spiking varying concentrations of analytes into blank media. 15 of each test sample was analyzed for analyte concentrations by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Liquid chromatography was performed with an Agilent 1 100 Series HPLC system equipped with a Agilent/ Varian Polaris 5 C18-A 50x2.0mm; Part# A2000050X020. Samples were eluted using a linear gradient from 100% Solvent A ( ⁇ ammonium acetate in H 2 0) to 60% Solvent B (lOnM ammonium acetate in acetonitrile) applied over 6 min at 0.5 mL/min flow rate. The HPLC system was as described above.
  • the cynomolgus monkey PK data demonstrates that amorphous compound lb shows 3 -fold better oral bioavailability as powder in capsule, than amorphous la.

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Abstract

Cette invention concerne de nouveaux solvates cristallins du (S)-P diastéréoisomère du nucléoside phosphoroamidate anti-HCV, (2S)-néopentyl 2-((((2R,3R,4R,5R)-5-(2-amino-6-méthoxy-9H-purin-9-yl)-3,4-dihydroxy-4- méthyltétrahydrofuran-2-yl)méthoxy)(naphtalén-1-yloxy)phosphorylamino)- propanoate (également désigné comme INX-08189 ou IΝΧ-189), et un procédé de recristallisation pour séparer les deux diastéréoisomères de INX-08189. De plus, cette invention concerne une nouvelle méthode stéréosélective pour la préparation de l'un ou l'autre des deux diastéréoisomères, et les nouveaux intermédiaires de synthèse utilisés dans cette synthèse stéréosélective. L'invention concerne également l'utilisation des solvates cristallins pour augmenter l'exposition du foie au guanosine triphosphate de 2'-C-méthyle à partir d'une dose orale.
PCT/US2012/062772 2011-10-31 2012-10-31 Solvates cristallins de nucléoside phosphoroamidates, leur préparation stéréosélective, nouveaux intermédiaires de ceux-ci et leur utilisation dans le traitement d'une maladie virale Ceased WO2013066991A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9447132B2 (en) 2013-04-12 2016-09-20 Achillion Pharmaceuticals, Inc. Highly active nucleoside derivative for the treatment of HCV

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040167096A1 (en) * 2003-02-19 2004-08-26 Yung-Chi Cheng Anti-viral nucleoside analogs and methods for treating viral infections, especially HIV infections
US20080286230A1 (en) * 2006-12-28 2008-11-20 Idenix Pharmaceuticals, Inc. Compounds and pharmaceutical compositions for the treatment of viral infections
WO2010081082A2 (fr) * 2009-01-09 2010-07-15 University College Of Cardiff Consultants Limited Dérivés de phosphoramidate de composés guanosine nucléoside pour le traitement d'infections virales

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040167096A1 (en) * 2003-02-19 2004-08-26 Yung-Chi Cheng Anti-viral nucleoside analogs and methods for treating viral infections, especially HIV infections
US20080286230A1 (en) * 2006-12-28 2008-11-20 Idenix Pharmaceuticals, Inc. Compounds and pharmaceutical compositions for the treatment of viral infections
WO2010081082A2 (fr) * 2009-01-09 2010-07-15 University College Of Cardiff Consultants Limited Dérivés de phosphoramidate de composés guanosine nucléoside pour le traitement d'infections virales

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Title
DOUILLET, J. D. ET AL.: "Development of a solvate as an active pharamceutical ingredient: Developability, crystallisation and isolation challenges", JOURNAL OF CRYSTAL GROWTH, vol. 342, 1 March 2012 (2012-03-01), pages 2 - 8, XP055068245 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9447132B2 (en) 2013-04-12 2016-09-20 Achillion Pharmaceuticals, Inc. Highly active nucleoside derivative for the treatment of HCV

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