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WO2017015439A1 - Forme cristalline d'un inhibiteur du virus de l'hépatite c - Google Patents

Forme cristalline d'un inhibiteur du virus de l'hépatite c Download PDF

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Publication number
WO2017015439A1
WO2017015439A1 PCT/US2016/043285 US2016043285W WO2017015439A1 WO 2017015439 A1 WO2017015439 A1 WO 2017015439A1 US 2016043285 W US2016043285 W US 2016043285W WO 2017015439 A1 WO2017015439 A1 WO 2017015439A1
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Prior art keywords
compound
dihydrochloride
crystalline
crystalline form
inhibitors
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English (en)
Inventor
Xiaojun Huang
Venkat R. Thalladi
Weijiang Zhang
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Theravance Biopharma R&D IP LLC
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Theravance Biopharma R&D IP LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • 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
    • 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

  • the invention is directed to a crystalline salt form of a medicinal compound which is useful as a hepatitis C virus inhibitor.
  • the invention is also directed to pharmaceutical compositions comprising such a crystalline compound, methods of using such a compound to treat HCV infection, and processes and intermediates useful for preparing the crystalline form.
  • HCV hepatitis C virus
  • the virus responsible for HCV infection has been identified as a positive-strand
  • RNA virus belonging to the family Flaviviridae The HCV genome encodes a polyprotein that during the viral lifecycle is cleaved into ten individual proteins, including both structural and non-structural proteins.
  • the six non-structural proteins, denoted as NS2, NS3, NS4A, NS4B, NS5A, and NS5B have been shown to be required for RNA replication.
  • the NS5A protein appears to play a significant role in viral replication, as well as in modulation of the physiology of the host cell. Effects of NS5A on interferon signaling, regulation of cell growth and apoptosis have also been identified. (Macdonald et al, Journal of General Virology (2004), 85, 2485-2502.) Compounds which inhibit the function of the NS5A protein are expected to provide a useful approach to HCV therapy.
  • this compound As a therapeutic agent, it would be desirable to have a solid-state form that can be readily manufactured and that has acceptable chemical and physical stability. For example, it would be highly desirable to have a physical form that is thermally stable at reasonably high temperature, thereby facilitating processing and storage of the material. Crystalline solids are generally preferred over amorphous forms, for enhancing purity and stability of the manufactured product. However, the formation of crystalline forms of organic compounds is highly unpredictable. No reliable methods exist for predicting which, if any, form of an organic compound will be crystalline.
  • the present invention provides a crystalline variable hydrate of [(5)-2-((25',45)-2-
  • the present variable hydrate contains between about 2.3 % and about 2.5% water, and is characterized by a powder x-ray diffraction (PXRD) partem of Figure 1. It has been shown that the crystalline form can incorporate various numbers of water molecules without disrupting the crystalline lattice as evidenced by comparison of PXRD patterns before and after exposure to 95 % relative humidity.
  • PXRD powder x-ray diffraction
  • variable hydrate has been shown to retain solid-state phase stability as well as chemical stability upon storage for 12 months at 25 ⁇ 2 °C and 60 ⁇ 5 % relative humidity and to be thermally stable. No visual changes were observed in the material upon exposure to 150 °C.
  • the crystalline solid form of the invention is expected to be useful for preparing pharmaceutical compositions for treating hepatitis C virus infections. Accordingly, in another of its composition aspects, the invention provides a
  • composition comprising a pharmaceutically-acceptable carrier and the crystalline variable hydrate of [(5 -2-((2 ⁇ ,4S)-2- ⁇ 4-[5'-chloro-4'-( ⁇ 6-[(i?)-4-(2,2- dimethyl-propionyl)-2-methyl-piperazin-l-yl]-pyridine-3-carbonyl ⁇ -amino)-2'- trifluoromethoxy-biphenyl-4-yl]-lH-imidazol-2-yl ⁇ -4-methoxy-pyrrolidin-l-yl)-2-oxo-l- (tetrahydro-pyran-4-yl)-ethyl]-carbamic acid methyl ester (1) dihydrochloride.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a crystalline form of the invention, a pharmaceutically-acceptable carrier and one or more other therapeutic agents useful for treating hepatitis C viral infections.
  • the invention also provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a crystalline form or of a pharmaceutical composition of the invention.
  • the invention provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a crystalline form or a pharmaceutical composition of the invention and one or more other therapeutic agents useful for treating hepatitis C viral infections.
  • the invention provides a method of inhibiting replication of the hepatitis C virus in a mammal, the method comprising administering a crystalline form or a pharmaceutical composition of the invention.
  • the invention also provides a crystalline form of the invention as described herein for use in medical therapy, as well as the use of a crystalline form of the invention in the manufacture of a formulation or medicament for treating a hepatitis C viral infection in a mammal.
  • Figure 1 shows a powder x-ray diffraction (PXRD) partem of the crystalline hydrate of [(S)-2-((2S,4S)-2- ⁇ 4-[5'-chloro-4'-( ⁇ 6-[(R)-4-(2,2-dimethyl-propionyl)-2- methyl-piperazin-l-yl]-pyridine-3-carbonyl ⁇ -amino)-2'-trifluoromethoxy-biphenyl-4-yl]- lH-imidazol-2-y 1 ⁇ -4-methoxy-py rrolidin- 1 -y l)-2-oxo- 1 -(tetrahy dro-py ran-4-y l)-ethy 1] - carbamic acid methyl ester (1) dihydrochloride at about 15 % relative humidity (RH).
  • RH relative humidity
  • Figure 2 shows a powder x-ray diffraction (PXRD) partem of the crystalline hydrate of compound 1 dihydrochloride as a function of relative humidity (RH). Bottom to top 5 %, 15 %, 25 %, 37 %, 45 %, 65 %, 75 %, 85 %, 95 % RH.
  • PXRD powder x-ray diffraction
  • Figure 3 shows a dynamic moisture sorption (DMS) isotherm of the crystalline hydrate of compound 1 dihydrochloride observed at a temperature of about 25 °C.
  • DMS dynamic moisture sorption
  • Figure 4 shows a differential scanning calorimetry (DSC) thermogram of the crystalline hydrate of compound 1 dihydrochloride.
  • Figure 5 shows a thermal gravimetric analysis (TGA) plot of the crystalline hydrate of compound 1 dihydrochloride.
  • the invention provides, inter alia, a crystalline variable hydrate of [( ⁇ S -2- ((25',45)-2- ⁇ 4-[5'-chloro-4'-( ⁇ 6-[(i?)-4-(2,2-dimethyl-propionyl)-2-methyl-piperazin-l-yl]- pyridine-3-carbonyl ⁇ -amino)-2'- ⁇ jifluoromethoxy-biphenyl-4-yl]-lH-imidazol-2-yl ⁇ -4- methoxy-pyrrolidin-l-yl)-2-oxo-l-(tetrahydro-pyran-4-yl)-ethyl]-carbamic acid methyl ester (1) dihydrochloride.
  • Compound 1 may equally be identified as methyl ((5)-2-((25',45 -2-(4-(5'-chloro-4'-(6- ((i?)-2-methyl-4-pivaloylpiperazin-l-yl)nicotinamido)-2'-(trifluoromethoxy)-[l, - bipheny 1] -4-y 1)- lH-imidazol-2-yl)-4-methy lpy rrolidin- 1 -y l)-2-oxo- 1 -(tetrahy dro-2H- pyran-4-yl)ethyl)carbamate, as given by ChemBioDraw Ultra 13.0 (PerkinElmer, Inc., Cambridge, MA) or as carbamic acid, N-[(15 -2-[(2 ⁇ ,4S)-2-[5-[5'-chloro-4'-[[[6-[(2i?)-4- (2,2-dimethyl-
  • compound 1 dihydrochloride has the following structure:
  • Compound 1 has multiple chiral centers. However, it will be understood that minor amounts of other stereoisomers may also be present unless otherwise indicated, provided that the utility of the depicted or named compound is not eliminated by the presence of another stereoisomer.
  • the present application provides a solid form comprising the compound 1 dihydrochloride. In some embodiments, the solid form is crystalline. In some embodiments, the solid form is amorphous. In some embodiments, the solid form is anhydrous. In some embodiments, the solid form is hydrated. In some embodiments, the present application provides a solid form of compound 1 which is a crystalline hydrate.
  • the term “hydrated” is meant to refer to a crystalline form that includes water molecules in the crystalline lattice.
  • Example “hydrated” crystalline forms include monohydrates (e.g., having 1 : 1 molar ratio of water to the compound 1), hemihydrates, dihydrates, trihydrates and the like. Other hydrated forms such as channel hydrates and the like are also included within the meaning of the term.
  • the solid form is a hemihydrate.
  • the solid form is a hydrate having about 1.5 moles of water per about 1 mole of compound 1. Definitions
  • terapéuticaally effective amount means an amount sufficient to effect treatment when administered to a patient in need of treatment.
  • treatment means the treatment of a disease, disorder, or medical condition in a patient (such as hepatitis C viral infection), such as a mammal (particularly a human) which includes one or more of the following:
  • the crystalline form of the invention is a variable hydrate in which the until cell volume changes to accommodate various amounts of water depending upon relative humidity in which the sample is placed. Accordingly, the detailed appearance of the powder x-ray diffraction (PXRD) pattern of the material depends upon the conditions under which the pattern is measured.
  • An X-ray powder diffraction (XRPD) pattern of reflections (peaks) is typically considered a fingerprint of a particular crystalline form.
  • relative intensities of diffraction peaks may vary due to preferred orientation of the crystal structure as well as due to experimental details, such as details of sample preparation and instrument geometry (in some instances, new peaks may be observed or existing peaks may disappear depending on the type of instrument or the settings, for example, whether a Ni filter is used or not), while the angular peak positions are far less affected.
  • peak or characteristic peak refers to a reflection having a relative height/intensity of at least about 3% of the maximum peak height/intensity.
  • instrument variation and other factors can affect the 2-theta values.
  • peak assignments such as those reported herein, can vary by plus or minus about 0.2° (2-theta), and the term “substantially” or “about” as used in the context of XRPD herein is meant to refer to the above-mentioned variations.
  • dihydrochloride is characterized by a PXRD pattern wherein the peak positions are substantially in accordance with those shown in Figure 1 when measured at a temperature of between about 15 °C and about 30 °C, including between about 20 °C and about 25 °C, and at a relative humidity (RH) of about 15 %.
  • the present crystalline hydrate is characterized by a PXRD pattern wherein the peak positions are substantially in accordance with the peak positions shown in at least one of the patterns in Figure 2, when measured at a temperature of between about 20 °C and about 25 °C, and at a corresponding relative humidity.
  • the crystalline hydrate of compound 1 dihydrochloride is characterized by a powder x-ray diffraction (PXRD) pattern having significant diffraction peaks, among other peaks, at 2 ⁇ values of 6.25 ⁇ 0.20, 6.83 ⁇ 0.20, and 9.97 ⁇ 0.20 when the pattern is obtained at a temperature of between about 15 °C and about 30 °C, including between about 20 °C and about 25 °C, and at a relative humidity between about 15 % and about 65 %.
  • PXRD powder x-ray diffraction
  • the crystalline hydrate may be further characterized by two or more additional diffraction peaks at 2 ⁇ values of 12.47 ⁇ 0.20, 18.03 ⁇ 0.20, 18.79 ⁇ 0.20, 19.02 ⁇ 0.20, and 22.01 ⁇ 0.20 when the partem is obtained under the same conditions.
  • the crystalline hydrate of compound 1 dihydrochloride is characterized by a powder x-ray diffraction (PXRD) pattern as shown in the table below: 2-Theta Area A%
  • the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, at about 6.3. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, at about 10.0. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, at about 22.2. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2- theta, at about 23.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, at about 6.3, about 10.0, about 22.2, or about 23.2. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2- theta, at about 6.3, about 22.2, or about 23.2. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least two characteristic XRPD peaks, in terms of 2-theta, at about 6.3, about 10.0, about 22.2, or about 23.2. In some
  • the crystalline hydrate of compound 1 dihydrochloride has at least three characteristic XRPD peaks, in terms of 2-theta, at about 6.3, about 10.0, about 22.2, or about 23.2. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least two characteristic XRPD peaks, in terms of 2-theta, at about 6.3, about 22.2, or about 23.2. In some embodiments, the crystalline hydrate of compound 1
  • the dihydrochloride has at least three characteristic XRPD peaks, in terms of 2-theta, at about 6.3, about 22.2, or about 23.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, selected from about 6.3, about 6.8, and about 10.0.
  • the crystalline hydrate of compound 1 dihydrochloride has at least two characteristic XRPD peaks, in terms of 2- theta, selected from about 6.3, about 6.8, and about 10.0.
  • the crystalline hydrate of compound 1 dihydrochloride has at least three characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, and about 10.0. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least three characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, and about 10.0, and further has at least two characteristic XRPD peaks, in terms of 2-theta, selected from about 12.6, about 18.2, about 18.9, about 19.2, and about 22.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least two characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, and about 10.0, and further has at least two characteristic XRPD peaks, in terms of 2-theta, selected from about 12.6, about 18.2, about 18.9, about 19.2, and about 22.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, selected from about 6.3, about 6.8, about 10.0, about 12.6, about 18.2, about 18.6, about 18.9, about 19.2, about 21.2, about 22.2, about 23.2, about 24.2, and about 25.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least two characteristic XRPD peaks, in terms of 2- theta, selected from about 6.3, about 6.8, about 10.0, about 12.6, about 18.2, about 18.6, about 18.9, about 19.2, about 21.2, about 22.2, about 23.2, about 24.2, and about 25.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least three characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, about 10.0, about 12.6, about 18.2, about 18.6, about 18.9, about 19.2, about 21.2, about 22.2, about 23.2, about 24.2, and about 25.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least four characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, about 10.0, about 12.6, about 18.2, about 18.6, about 18.9, about 19.2, about 21.2, about 22.2, about 23.2, about 24.2, and about 25.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, selected from about 6.3, about 6.8, about 10.0, about 18.2, about 18.9, about 19.2, about 21.2, about 22.2, and about 23.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least two characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, about 10.0, about 18.2, about 18.9, about 19.2, about 21.2, about 22.2, and about 23.2. In some embodiments, the crystalline hydrate of compound 1 dihydrochloride has at least three characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, about 10.0, about 18.2, about 18.9, about 19.2, about 21.2, about 22.2, and about 23.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least four characteristic XRPD peaks, in terms of 2-theta, selected from about 6.3, about 6.8, about 10.0, about 18.2, about 18.9, about 19.2, about 21.2, about 22.2, and about 23.2.
  • the crystalline hydrate of compound 1 dihydrochloride has at least one characteristic XRPD peak, in terms of 2-theta, selected from about 12.6, about 18.2, about 18.9, about 19.2, and about 22.2.
  • the crystalline hydrate of compound 1 dihydrochloride does not have any characteristic XRPD peaks, in terms of 2-theta, from about 14.0 to about 17.0.
  • An indexing solution carried out on the measured peak positions of the PXRD patterns of Figure 2 allowed the unit cell characteristic cell lengths and cell volume to be determined for each pattern as a function of relative humidity.
  • the unit cell volume increases in direct proportion to relative humidity, which is characteristic of variable hydrate behavior.
  • the calculated cell volume increases from approximately
  • a 14.9102(11) A
  • b 14.9102(11) A
  • c 86.306(7)
  • ⁇ 90°
  • ⁇ 120°
  • volume 16,616 (2) A 3 .
  • the crystals contain two-symmetry independent molecules of compound 1 dihydrochloride.
  • variable hydrate The water content of the variable hydrate was determined by the Karl Fisher method using a coulometric titrator to be in the range of about 2.3 % to about 2.5 % by weight when the material is at a relative humidity of about 15 %, which corresponds to about 1.5 moles of water per mole of compound 1.
  • water content of the crystalline hydrate of compound 1 dihydrochloride as determined by the Karl Fisher method is about 2.3%, about 2.4%, about 2.5%, or about 2.6%.
  • the present crystalline hydrate has been demonstrated to have a reversible sorption/desorption profile as illustrated in Figure 3.
  • a weight gain of about 9.5 % is observed at 95 % RH at 25 °C. This corresponds to about 6 moles of water.
  • the moisture sorption-desorption isotherm shows a step-like profile, with steeper slopes in the humidity ranges 0-20 % RH and 65-90 % RH, and a shallower slope in the humidity range 20 -60% RH.
  • the solid absorbs 3.33 % to 5.25 % of moisture in the relative humidity range 25-60 % RH; these amounts correspond to about 2-3 moles of water.
  • the crystalline hydrate of compound 1 dihydrochloride is characterized by its behavior when exposed to high temperature.
  • the differential scanning calorimetry (DSC) thermogram shows two broad endotherms. The onset of the lower endotherm occurs above 0 °C and exhibits a signal maximum between about 70 °C and about 90 °C consistent with the loss of water. The higher temperature endotherm has an onset above 150 °C and exhibits a signal maximum between about 195 °C and about 205 °C consistent with the loss of one mole of hydrogen chloride.
  • the DSC thermogram of the crystalline hydrate of compound 1 dihydrochloride comprises endothermal event at an onset temperature of about 200 °C.
  • the thermal gravimetric analysis (TGA) trace of Figure 5 shows weight loss in the temperature range of about 25 °C to about 120 °C corresponding to the dehydration event of the lower temperature endotherm in the DSC and a weight loss in the temperature range of about 120 °C to about 215 °C corresponding to the event in the DSC identified as the loss of hydrogen chloride.
  • the weight loss above about 215 °C is consistent with loss of an additional mole of hydrogen chloride and decomposition of the compound.
  • the TGA thermogram of the crystalline hydrate of compound 1 dihydrochloride substantially as depicted in Figure 5.
  • the present crystalline form is prepared by dissolving amorphous compound 1 dihydrochloride in a polar diluent to form a crystallization process mixture.
  • a combination of ethanol and acetonitrile in a ratio of ethanol to acetonitrile from about 1 :2 to about 1 : 10 forms a useful polar diluent.
  • the process mixture is held for a period of from a few hours to one or more days at ambient temperature.
  • the crystalline hydrate of compound 1 dihydrochloride is isolated from the process mixture by any conventional means, such as filtration, concentration, centrifugation, and the like.
  • the process of preparing a crystalline form of the invention can optionally include the use of a seed crystal to produce predominately a particular crystalline form.
  • seed crystals are prepared by slow crystallization of a small volume as described, for example, above.
  • dihydrochloride is advantageously prepared directly from the crude amorphous dihydrochloride salt obtained as the product of the final step of the synthesis of compound 1, illustrated in the following scheme.
  • N-(5-chloro-4'-(2-((25',45)-4-methylpyrrolidin- 2-yl)-lH-imidazol-4-yl)-2-(trifluoromethoxy)-[l,r-biphenyl]-4-yl)-6-((i?)-2-methyl-4- pivaloylpiperazin-l-yl)nicotinamide(2) is reacted with (5)-2-((methoxycarbonyl)amino)- 2-(tetrahydro-2H-pyran-4-yl)acetic acid (3).
  • the reaction is typically performed in the presence of an excess of base utilizing an activating agent such as NNN'N'-tetramethyl- 0-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate (HATU).
  • an activating agent such as NNN'N'-tetramethyl- 0-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate (HATU).
  • HATU NNN'N'-tetramethyl- 0-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate
  • the product can be recrystallized by a similar process: the crystalline compound is dissolved in methanol with a trace of water and warmed to provide a clear solution. Acetone and seeds are added, the solution is warmed, and additional acetone is added such that the ratio of methanol: acetone in the mixture is from about 1 : 8 to about 1 : 10. The mixture is stirred for a period of at least 12 hours to provide the crystalline hydrate of compound 1 dihydrochloride, which is recovered
  • the invention provides a process for preparing the crystalline hydate of compound 1 dihydrochloride, the process comprising: (a) dissolving amorphous compound 1 dihydrochloride in a polar diluent comprising ethanol and acetonitrile in a ratio of ethanol to acetonitrile from about 1 :2 to about 1 : 10 to provide a crystallization solution and (b) allowing the solution to evaporate to provide the crystalline hydate of compound 1 dihydrochloride.
  • the invention provides a process for preparing the crystalline hydrate of compound 1 dihydrochloride, the process comprising (a) preparing crude dihydrochloride salt of compound 1 by combining the product of the reaction of compound 2 with compound 3 with HC1, (b) dissolving the product of step (a) in methanol and acetone, (c) adding seeds of the crystalline hydrate of compound 1 dihydrochloride and additional acetone such that the ratio of methanol: acetone is between about 1 : 8 and about 1 : 10 to form a reaction mixture; and (d) stirring the reaction mixture until the crystalline hydrate of compound 1 dihydrochloride is formed.
  • the method can include a recrystallization step comprising: (a) dissolving the crystalline product in methanol with a trace of water, (b) adding acetone and seeds of the crystalline hydrate of compound 1 dihydrochloride, such that the ratio of methanol: acetone is between about 1 : 8 and about 1 : 10 to form a reaction mixture; and (c) stirring the reaction mixture until the crystalline hydrate of compound 1 dihydrochloride is formed.
  • a recrystallization step comprising: (a) dissolving the crystalline product in methanol with a trace of water, (b) adding acetone and seeds of the crystalline hydrate of compound 1 dihydrochloride, such that the ratio of methanol: acetone is between about 1 : 8 and about 1 : 10 to form a reaction mixture; and (c) stirring the reaction mixture until the crystalline hydrate of compound 1 dihydrochloride is formed.
  • the present application provides crystalline hydrate of compound 1 dihydrochloride prepared by any one of the processes described herein.
  • the crystalline form of the invention is typically used in the form of a pharmaceutical composition or formulation.
  • Such pharmaceutical compositions may be administered to a patient by any acceptable route of administration including, but not limited to, oral, rectal, vaginal, nasal, inhaled, topical (including transdermal) and parenteral modes of administration.
  • the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or excipient and a crystalline form of the invention.
  • such pharmaceutical compositions may contain other therapeutic and/or formulating agents if desired.
  • the " crystalline form of the invention” or, alternatively, “solid form of the invention” may also be referred to herein as the "active agent” .
  • compositions of the invention typically contain a
  • a pharmaceutical composition may contain more than a therapeutically effective amount, i.e., bulk compositions, or less than a therapeutically effective amount, i.e., individual unit doses designed for multiple administration to achieve a therapeutically effective amount.
  • such pharmaceutical compositions will contain from about 0.1 to about 95% by weight of the active agent; preferably, from about 5 to about 70% by weight; and more preferably from about 10 to about 60% by weight of the active agent.
  • any conventional carrier or excipient may be used in the pharmaceutical compositions of the invention.
  • the choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the mode of administration being used to treat a particular patient or type of medical condition or disease state. In this regard, the preparation of a suitable pharmaceutical composition for a particular mode of administration is well within the scope of those skilled in the pharmaceutical arts.
  • the carriers or excipients used in the pharmaceutical compositions of this invention are commercially-available.
  • conventional formulation techniques are described in Remington: The Science and Practice of
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, such as microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen
  • compositions are typically prepared by thoroughly and intimately mixing or blending the active agent with a pharmaceutically-acceptable carrier and one or more optional ingredients. The resulting uniformly blended mixture can then be shaped or loaded into tablets, capsules, pills and the like using conventional procedures and equipment.
  • compositions of the invention are preferably packaged in a unit dosage form.
  • unit dosage form refers to a physically discrete unit suitable for dosing a patient, i.e., each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect either alone or in combination with one or more additional units.
  • unit dosage forms may be capsules, tablets, pills, and the like, or unit packages suitable for parenteral administration.
  • the pharmaceutical compositions of the invention are suitable for oral administration.
  • Suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; or as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and the like; each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • the pharmaceutical compositions of the invention When intended for oral administration in a solid dosage form (i.e., as capsules, tablets, pills and the like), the pharmaceutical compositions of the invention will typically comprise the active agent and one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate.
  • such solid dosage forms may also comprise: fillers or extenders, such as starches, microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as
  • absorbents such as kaolin and/or bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof; coloring agents; and buffering agents. Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the
  • antioxidants examples include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite and the like; oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and metal- chelating agents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite and the like
  • oil- soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, lecithin, propyl gallate, alpha-tocop
  • Coating agents for tablets, capsules, pills and like include those used for enteric coatings, such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid, methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like.
  • enteric coatings such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid, methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like.
  • compositions of the invention may also be formulated to provide slow or controlled release of the active agent using, by way of example, hydroxypropyl methyl cellulose in varying proportions; or other polymer matrices, liposomes and/or microspheres.
  • the pharmaceutical compositions of the invention may optionally contain opacifying agents and may be formulated so that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active agent can also be in microencapsulated form, if appropriate, with one or more of the above-described excipients.
  • Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • Liquid dosage forms typically comprise the active agent and an inert diluent, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • Suspensions in addition to the active ingredient, may contain suspending agents such as, for example, ethoxyl
  • microcrystalline cellulose aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • the crystalline form of this invention can also be administered parenterally (e.g. by intravenous, subcutaneous, intramuscular or intraperitoneal injection).
  • parenteral administration the active agent is typically admixed with a suitable vehicle for parenteral administration including, by way of example, sterile aqueous solutions, saline, low molecular weight alcohols such as propylene glycol, polyethylene glycol, vegetable oils, gelatin, fatty acid esters such as ethyl oleate, and the like.
  • Parenteral formulations may also contain one or more anti-oxidants, solubilizers, stabilizers, preservatives, wetting agents, emulsifiers, buffering agents, or dispersing agents. These formulations may be rendered sterile by use of a sterile inj ectable medium, a sterilizing agent, filtration, irradiation, or heat.
  • compositions of the invention are formulated for administration by inhalation.
  • Suitable pharmaceutical compositions for administration by inhalation will typically be in the form of an aerosol or a powder.
  • Such compositions are generally administered using well-known delivery devices, such as a metered-dose inhaler, a dry powder inhaler, a nebulizer or a similar delivery device.
  • compositions of the invention will typically comprise the active ingredient and a suitable propellant, such as dichlorodifluoromethane,
  • the pharmaceutical composition may be in the form of a capsule or cartridge (made, for example, from gelatin) comprising a compound of the invention and a powder suitable for use in a powder inhaler.
  • Suitable powder bases include, by way of example, lactose or starch.
  • the crystalline form of the invention can also be administered transdermally using known transdermal delivery systems and excipients.
  • the active agent can be admixed with permeation enhancers, such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch or similar delivery system.
  • permeation enhancers such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like
  • Additional excipients including gelling agents, emulsifiers and buffers, may be used in such transdermal compositions if desired.
  • the present compound [(S)-2-((25',45 -2- ⁇ 4-[5'-chloro-4'-( ⁇ 6-[(i?)-4-(2,2- dimethyl-propionyl)-2-methyl-piperazin-l-yl]-pyridine-3-carbonyl ⁇ -amino)-2'- trifluoromethoxy-biphenyl-4-yl]-lH-imidazol-2-yl ⁇ -4-methoxy-pyrrolidin-l-yl)-2-oxo-l - (tetrahydro-pyran-4-yl)-ethyl]-carbamic acid methyl ester (1) has been shown to inhibit viral replication in HCV replicon assays and therefore the crystalline forms of the invention are expected to be useful for the treatment of hepatitis C viral infections.
  • the invention provides a method of inhibiting replication of the hepatitis C virus in a mammal (e.g., a human), the method comprising
  • a therapeutically-effective amount of a crystalline form of the invention or of a pharmaceutical composition comprising a pharmaceutically - acceptable carrier and a crystalline form of the invention.
  • the invention further provides a method of treating hepatitis C viral infections in a mammal (e.g., a human), the method comprising administering to the mammal a therapeutically-effective amount of crystalline form of the invention or of a mammal (e.g., a human), the method comprising administering to the mammal a therapeutically-effective amount of crystalline form of the invention or of a mammal (e.g., a human), the method comprising administering to the mammal a therapeutically-effective amount of crystalline form of the invention or of a mammal (e.g., a human), the method comprising administering to the mammal a therapeutically-effective amount of crystalline form of the invention or of a mammal (e.g., a human), the method comprising administering to the mammal a therapeutically-effective amount of crystalline form of the invention or of a mammal (e.g., a human), the method comprising administering to the mammal
  • composition comprising a pharmaceutically-acceptable carrier and a crystalline form of the invention.
  • the crystalline form of the invention may inhibit viral replication by inhibiting the function of the NS5A protein encoded by the HCV genome.
  • the invention provides a method of inhibiting the NS5A protein of HCV in a mammal, the method comprising administering to the mammal, a crystalline form or a composition of the invention.
  • the crystalline forms of the invention When used to treat HCV infections, the crystalline forms of the invention will typically be administered orally in a single daily dose or in multiple doses per day, although other forms of administration may be used.
  • the amount of active agent administered per dose or the total amount administered per day will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • Suitable doses for treating HCV infections will range from about 30 to about 500 mg/day of active agent, including from about 45 to about 300 mg/day and from about 60 to about 240 mg per day of active agent for an average 70 kg human.
  • the crystalline forms of the invention may also be used in combination with one or more agents which act by the same mechanism or by different mechanisms to effect treatment of HCV.
  • agents for combination therapy include, but are not limited to, HCV NS3 protease inhibitors, HCV NS5B nucleoside and non-nucleoside polymerase inhibitors, helicase inhibitors, NS4B protein inhibitors, HCV viral entry inhibitors, cyclophyllin inhibitors, toll-like receptor agonists, inhibitors of heat shock proteins, interfering RNA, antisense RNA, HCV internal ribosome entry site (IRES) inhibitors, thiazolides, nucleoside analogs such as ribavirin and related compounds, interferons and other immunomodulatory agents, inosine 5 '-monophosphate
  • IMPDH dehydrogenase
  • HCV NS3 protease inhibitors which may be used in combination therapy include, but are not limited to, Incivek® (telaprevir,VX-950), boceprevir (SCH-503034), simeprevir (TMC-435), narlaprevir (SCH-900518), vaniprevir (MK-7009), danoprevir (ITMN-191, R-7227), faldaprevir (BI-201335), ABT-450/r, ABT-493, asunaprevir (BMS-650032), GS-9256, GS-9857, GS-9451, sovaprevir (ACH-1625), ACH-2684, BMS-605339, VX-985, PHX-1766, BMS-791325, IDX-320, and grazoprevir (MK-5172).
  • Incivek® telaprevir,VX-950
  • boceprevir SCH-503034
  • simeprevir TMC-435
  • narlaprevir
  • HCV NS5B nucleoside polymerase inhibitors include, but are not limited to, mericitabine (RG7128), IDX-184, sofosbuvir (GS-7977, PSI-7977),
  • non-nucleoside HCV NS5B polymerase inhibitors include but are not limited to, filibuvir (PF-8685540), tegobuvir (GS-9190), VX-222, VX-759, setrobuvir (ANA- 598), ABT-072, ABT-333, BI-207127, BMS-791325, MK-3281, IDX-37, BMS-824393, TMC-647055, and GSK2878175.
  • interferons and pegylated interferons including alpha, beta, omega, and gamma interferons, having antiviral, antiproliferative or immunomodulatory effects, can be combined with the present compounds.
  • Representative examples include, but are not limited to, Intron® A (interferon-alpha2b), Actimmune® (interferon-gamma- lb), Alferon N, Advaferon®, Roferon-A (interferon alpha-2a) Peglntron® (peginterferon- alpha 2b), Alfaferone, Pegasys® (peginterferon alpha-2a), Alfanative (interferon alpha), ZalbinTM (albinterferon alpha-2b), Infergon® (interferon alfacon-1), Omega DUROS® (omega interferon), LocteronTM (interferon alpha), PEG-rIL-29 (pegylated interferon lambda), and Rebif®
  • Nucleoside analog antiviral agents include, but are not limited to, ribavirin (Copegus®, Rebetol®,Virazole®) and Viramidine (taribavirin). Interferons and ribavirin are also provided in the form of kits which include, for example, but are not limited to, Rebetron® (interferon alpha-2b/ribavirin) and Pegetron® (Peginterferon alpha- 2b/ribavirin)
  • Useful compounds acting by other mechanisms include, but are not limited to: cyclophilin inhibitors, such as alisporivir (DEB-025), SCY-635, NIM-811, and cyclosporine and derivatives; toll-like receptor agonists, such as resiquimod, IMO-2125, and ANA-773, HCV viral entry inhibitors, such as civacir, thiazolides, such as nitazoxanide, and broad-spectrum viral inhibitors, such as, inosine-5 '-monophosphate dehydrogenase (IMPDH) inhibitors.
  • cyclophilin inhibitors such as alisporivir (DEB-025), SCY-635, NIM-811, and cyclosporine and derivatives
  • toll-like receptor agonists such as resiquimod, IMO-2125, and ANA-773
  • HCV viral entry inhibitors such as civacir, thiazolides, such as nitazoxanide
  • crystalline forms of the invention may be combined with an NS5A inhibitor, for example, daclatasvir (BMS-790052), AZD-7295, PPI-461, PPI-1301, GS- 5885, GSK2336805, ABT-267, ACH-2928, ACH-3102, EDP-239, IDX-719, MK-8742, or PPI-668.
  • an NS5A inhibitor for example, daclatasvir (BMS-790052), AZD-7295, PPI-461, PPI-1301, GS- 5885, GSK2336805, ABT-267, ACH-2928, ACH-3102, EDP-239, IDX-719, MK-8742, or PPI-668.
  • the invention provides a therapeutic combination for use in the treatment of hepatitis C viral infections, the combination comprising a crystalline form of the invention and one or more other therapeutic agents useful for treating HCV.
  • the invention provides a combination comprising a crystalline form of the invention and one or more agents selected from HCV NS3 protease inhibitors, HCV NS5B nucleoside and non-nucleoside polymerase inhibitors, interferons and pegylated interferons, cyclophilin inhibitors, HCV NS5A inhibitors, and ribavirin and nucleoside analogs related ro ribavirin.
  • a pharmaceutical composition comprising a crystalline form of the invention and one or more other therapeutic agents useful for treating HCV.
  • a crystalline form of the invention and one or more other therapeutic agents selected from HCV NS3 protease inhibitors, HCV NS5B nucleoside inhibitors, and cyclophilin inhibitors.
  • the invention provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a crystalline form of the invention and one or more other therapeutic agents useful for treating HCV.
  • the invention provides a method of inhibiting replication of the hepatitis C virus in a mammal, the method comprising administering to the mammal a crystalline form of the invention and one or more other therapeutic agents useful for inhibiting replication of the hepatitis C virus.
  • the invention provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a crystalline form of the invention and an HCV NS3 protease inhibitor.
  • the protease inhibitor used in the combination method is selected from simeprevir, grazoprevir, ABT-450/ritonavir, ABT-493 and feldaprevir.
  • the protease inhibitor used in the combination method is feldaprevir.
  • the invention provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a crystalline form of the invention and an HCV NS5B nucleoside polymerase inhibitor.
  • the polymerase inhibitor used in the combination method is selected from sofosbuvir, VX-135, ACH-3422, and IDX21437.
  • the invention provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a crystalline form of the invention, an HCV NS3 protease inhibitor, and an HCV NS5B nucleoside polymerase inhibitor.
  • the method comprises administering a crystalline form of the invention, feldaprevir and VX-222.
  • the invention provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a crystalline form of the invention and a cyclophilin inhibitor, for example alisporivir.
  • the invention provides a method of treating a hepatitis C viral infection in a mammal, the method comprising administering to the mammal a crystalline form of the invention and one or more other therapeutic agents selected from HCV NS3 protease inhibitors, HCV NS5B nucleoside inhibitors, and cyclophilin inhibitors.
  • the invention provides a method of inhibiting replication of the hepatitis C virus in a mammal, using a crystalline form of the invention in combination with other agents, as described above.
  • combination therapy including the crystalline form of the invention may additionally include antiretroviral treatment (ART) used to treat HIV.
  • ART agents that potentially may be used in combination with the current solid form include, but are not limited to, atazanavir and darunavir, both in combination with ritonavir, efavirenz, raltegravir, rilpilvirine, and tenofovir in combination with emtricitabine.
  • the agents When used in combination therapy, the agents may be formulated in a single pharmaceutical composition, as disclosed above, or the agents may be provided in separate compositions that are administered simultaneously or at separate times, by the same or by different routes of administration. When administered separately, the agents are administered sufficiently close in time so as to provide a desired therapeutic effect.
  • Such compositions can be packaged separately or may be packaged together as a kit. The two or more therapeutic agents in the kit may be administered by the same route of administration or by different routes of administration.
  • Compound 1 has been demonstrated to be a potent inhibitor of HCV replication in HCV replicon assays, as described in the following biological examples.
  • HCTU 2-(6-chloro-lH-benzotriazole-l- yl)-l, 1,3,3- tetramethylaminium hexafluorophosphate
  • Reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka, Sigma, etc.), and used without further purification. Reactions were run under nitrogen atmosphere, unless noted otherwise. Progress of reaction mixtures was monitored by thin layer chromatography (TLC), analytical high performance liquid chromatography (anal. HPLC), and mass spectrometry. Reaction mixtures were worked up as described specifically in each reaction; commonly they were purified by extraction and other purification methods such as temperature-, and solvent-dependent crystallization, and precipitation.
  • TLC thin layer chromatography
  • HPLC analytical high performance liquid chromatography
  • mass spectrometry mass spectrometry
  • the reaction mixture was degassed for 5 min, then heated at 70 °C for 4.5 h, and cooled to 25 °C.
  • Brine (10 % w/vol) 120 mL was added. The next day, the bottom aqueous layer was drained. The organic layer was evaporated and distilled in an azeotrope with methanol. Methanol (100 mL) was added and the mixture was distilled to about 60 mL. Methanol (200 mL) was added and the mixture was distilled to about 120 mL.
  • 3-Mercaptopropyl-functionalized silica gel (7 g, 9.87 mmol) was added. The reaction mixture was stirred at 25 °C overnight, filtered and rinsed with methanol (20 mL).
  • Trifluoroacetic acid (15.05 mL) was added to the product of the previous step in DCM (50 mL) at 10-25 °C. The mixture was stirred at 22 °C overnight and then
  • Example 1 Crystalline hydrate of [(S)-2-((2 ⁇ ,4S)-2- ⁇ 4-[5'-Chloro-4'-( ⁇ 6-[(i?)- 4-(2,2-dimethyl-propionyl)-2-methyl-piperazin-l-yl]-pyridine-3-carbonyl ⁇ -amino)- 2'-trifluoromethoxy-biphenyl-4-yl]-lH-imidazol-2-yl ⁇ -4-methyl-pyrrolidin-l-yl)--2- oxo— l-(tetrahydro-pyran-4-yl)-ethyl]-carbamic acid methyl ester (compound 1) dihydrochloride
  • Example 2 Recrystallization of hydrate of [( 1 S)-2-((2 ⁇ ,4.S)-2- ⁇ 4-[5'-Chloro-4'- ( ⁇ 6-[(i?)-4-(2,2-dimethyl-propionyl)-2-methyl-piperazin-l-yl]-pyridine-3-carbonyl ⁇ - amino)-2'-trifluoromethoxy-biphenyl-4-yl]-lH-imidazol-2-yl ⁇ -4-methyl-pyrrolidin-l- yl) ⁇ 2-oxo— l-(tetrahydro-pyran-4-yl)-ethyl]-carbamic acid methyl ester
  • Example 2 To a flask was added the product of Example 1 (9.1 g, 9.13 mmol) and methanol (27.3 mL). Water (0.823 mL, 45.7 mmol) was added and the mixture was warmed to 50 °C to provide a clear solution. Acetone (82 mL) was added followed by seeds from Example 6 and the reaction mixture was stirred at 50 °C for 30 min. Acetone (164 mL) was added slowly over 60 min.
  • Amorphous compound 1 dihydrochloride salt (-50 mg) was dissolved in approximately 1 :2 ethanofacetonitrile (2 mL) and allowed to slowly evaporate overnight to produce seeds of the title crystalline solid.
  • Compound 1 was prepared by the reaction of compound 2 with compound 3 according to the process described in Example 1 without isolation. To a solution of the crude product ( ⁇ 1.9 g, 2.07 mmol) was added ethanol (3 mL) and acetonitrile (3 mL), followed by cone. HCl (0.52 mL, 6.2 mmol). Acetonitrile (27 mL) was added slowly; seeds from Example 4 were added and the mixture was stirred overnight, filtered, and washed with ethyl acetate (10 mL) to provide the title compound (1.67 g). HPLC Method A Retention time 13.09 min.
  • dihydrochloride prepared according to the process of Examples 1 and 2 were analyzed by x-ray powder diffraction (XRPD), differential scanning calorimetry (DSC),
  • thermogravimetric analysis TGA
  • DMS dynamic moisture sorption
  • the instrument was operated in Bragg-Brentano geometry with incident, divergence, and scattering slits set to maximize the intensity at the sample.
  • a small amount of powder (5-25 mg) was gently pressed onto a sample holder to form a smooth surface and subjected to X-ray exposure.
  • the samples were scanned in 2 ⁇ -2 ⁇ mode from 2° to 40° in 2 ⁇ with a step size of 0.03° and a scan speed of 2.0° per minute.
  • Thermo ARL measurement software (Version 1.2.0.0) and analyzed by Jade software (version 7.5.1).
  • the instrument was calibrated with a corundum standard, within ⁇ 0.02° two-theta angle. Observed PXRD two-theta peak positions and d-spacings are shown in Tablel (only peaks having a relative area percent (A%) of aboutl2.5 % or greater are listed).
  • X-ray powder diffraction patterns of Figure 2 were collected with a PANalytical X'Pert PRO MPD diffractometer using an incident beam of Cu radiation produced using an Optix long, fine-focus source.
  • An elliptically graded multilayer mirror was used to focus Cu Ka X-rays through the specimen and onto the detector.
  • a silicon specimen NIST SRM 640d was analyzed to verify the observed position of the Si 111 peak is consistent with the NIST-certified position.
  • a specimen of the sample was sandwiched between 3 ⁇ m-thick films and analyzed in transmission geometry.
  • a beam- stop, short antiscatter extension, and antiscatter knife edge were used to minimize the background generated by air.
  • DSC Differential scanning calorimetry
  • Thermogravimetric analysis (TGA) measurements were performed using a TA Instruments Model Q-50 module equipped with high resolution capability. Data were collected using TA Instruments Thermal Analyst controller and analyzed using TA Instruments Universal Analysis software. A weighed sample was placed onto a platinum pan and scanned with a heating rate of 10 °C from ambient temperature to 300 °C. The balance and furnace chambers were purged with nitrogen flow during use.
  • TGA Thermogravimetric analysis
  • DMS Dynamic moisture sorption
  • Chloride ion content of the crystalline hydrate of compound 1 dihydrochloride was analyzed by ion chromatography with conductivity detection using a Dionex ICS- 2000 system. The sample was determined to have a chloride ion content of 7.0%, which may be compared with a theoretical value for a hydrate containing 1.5 moles of water of a dihydrochloride salt of 6.9 %.
  • the water content of the crystalline hydrate of compound 1 dihydrochloride was determined by the Karl Fischer method using a coulometric titrator to be 2.42 %, which may be compared with a theoretical value for a hydrate containing 1.5 moles of water of of 2.6 %.
  • the HCV genotype lb replicon cell line was obtained from Apath LLC
  • This subgenomic replicon contains the N-terminus of the HCV core protein fused to the neomycin-resistance selectable marker.
  • the EMCV IRES lies downstream and drives expression of Renilla luciferase fused to the non-structural proteins NS3-NS5B. This cell line was used to determine compound potency using the luciferase activity readout as a measurement of compound inhibition of replicon levels.
  • luciferase activity was plotted vs. the compound concentration, and EC50 values were determined from a 4-parameter robust fit model with the GraphPad Prism software package (GraphPad Software, Inc., San Diego, CA).
  • Results are expressed as the negative decadic logarithm of the EC50 value, pECso.
  • Test compounds having a higher pECso value in this assay show greater inhibition of HCV genotype lb replication.
  • Compound 1 exhibited a pECso value of at least 1 1 in this assay.
  • the HCV genotype l a replicon cell line was obtained from Apath LLC (APC89;
  • This subgenomic replicon contains the N-terminus of the HCV core protein fused to the neomycin-resistance selectable marker.
  • the EMCV IRES lies downstream and drives expression of the non-structural proteins NS3-NS5B. Compound potencies were determined using the NS3-specific protease activity in lysates as a measurement of compound inhibition of replicon levels.
  • the cells were lysed at room temperature in 50 ⁇ 11 of 50mM Hepes pH 7.5, 150mM NaCl, 15% Glycerol, 0.15% Triton X-100, lOmM DTT for 20 minutes with shaking. 50 ⁇ , of an NS3/4a protease-specific FRET substrate (Anaspec RET S I Cat#22991) was then added to the wells at a final concentration of 15 ⁇ . The plates were incubated at 37°C for 20 minutes, which corresponds to a timepoint at which the protease activity is still in the linear phase. Protease activity was determined by measuring fluorescence (Excitation: 340 nm;
  • the mutation was first introduced into the parental plasmid by site-directed mutagenesis. Mutations in genotype lb included L31V and Y93H. Mutations in genotype la included Q30R and L31V. The replicon plasmid was then linearized and in vitro transcribed to RNA. The RNA was used to stably transfect Huh7 cells by electroporation, and new cell lines were selected with 50C ⁇ g/mL G418. Potencies of test compounds against these mutant cell lines were determined as previously described above for the HCV Genotype lb and la replicon assays. Compound 1 exhibited a pECso value of at least 8 in these assays.
  • Compound 1 exhibited a pECio value of at least 8 in these assays.

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Abstract

L'invention concerne l'hydrate cristallin de dichlorhydrate de méthylester d'acide [(S)-2-((2S,4S)-2-{4-[5'-chloro-4'-({6-[(R)-4-(2,2-diméthyl-propionyl)-2-méthyl-pipérazin-1-yl]-pyridine-3-carbonyl}-amino)-2'-trifluorométhoxy-biphényl-4-yl]-1H-imidazol-2-yl}-4-méthoxy-pyrrolidin-1-yl)-2-oxo-1-(tétrahydro-pyran-4-yl)-éthyl]-carbamique. L'invention concerne également des compositions pharmaceutiques comprenant la forme cristalline, des procédés d'utilisation de la forme cristalline pour traiter une infection par le virus de l'hépatite C, et des procédés utiles pour la préparation de la forme cristalline.
PCT/US2016/043285 2015-07-21 2016-07-21 Forme cristalline d'un inhibiteur du virus de l'hépatite c Ceased WO2017015439A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013067267A1 (fr) * 2011-11-03 2013-05-10 Theravance, Inc. Inhibiteurs du virus de l'hépatite c à structure en bâtonnet contenant le fragment {2-[4-(biphényl-4-yl)-1h-imidazo-2-yl]pyrrolidine-1-carbonylméthyl}amine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013067267A1 (fr) * 2011-11-03 2013-05-10 Theravance, Inc. Inhibiteurs du virus de l'hépatite c à structure en bâtonnet contenant le fragment {2-[4-(biphényl-4-yl)-1h-imidazo-2-yl]pyrrolidine-1-carbonylméthyl}amine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CAIRA: "Crystalline Polymorphism of Organic Compounds", TOPICS IN CURRENT CHEMISTRY, SPRINGER, BERLIN, DE, vol. 198, 1 January 1998 (1998-01-01), pages 163 - 208, XP008166276, ISSN: 0340-1022 *

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