WO2018028666A1 - Dpp-iv长效抑制剂的结晶及其盐 - Google Patents

Dpp-iv长效抑制剂的结晶及其盐 Download PDF

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WO2018028666A1
WO2018028666A1 PCT/CN2017/097046 CN2017097046W WO2018028666A1 WO 2018028666 A1 WO2018028666 A1 WO 2018028666A1 CN 2017097046 W CN2017097046 W CN 2017097046W WO 2018028666 A1 WO2018028666 A1 WO 2018028666A1
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formula
compound
crystallization
weight
phosphate
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PCT/CN2017/097046
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English (en)
French (fr)
Inventor
桑光明
刘林
张爱明
乔家彬
郭晓鹏
张喜全
夏春光
Original Assignee
正大天晴药业集团股份有限公司
连云港润众制药有限公司
北京赛林泰医药技术有限公司
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Application filed by 正大天晴药业集团股份有限公司, 连云港润众制药有限公司, 北京赛林泰医药技术有限公司 filed Critical 正大天晴药业集团股份有限公司
Priority to US16/324,884 priority Critical patent/US10822319B2/en
Priority to CA3033890A priority patent/CA3033890A1/en
Priority to RU2019105352A priority patent/RU2753335C9/ru
Priority to CN201780044045.0A priority patent/CN109874304B/zh
Priority to EP17838796.5A priority patent/EP3498704A4/en
Priority to KR1020197006819A priority patent/KR20190039754A/ko
Priority to JP2019506673A priority patent/JP6997760B2/ja
Priority to AU2017308691A priority patent/AU2017308691B2/en
Publication of WO2018028666A1 publication Critical patent/WO2018028666A1/zh

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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
    • C07D405/02—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 two hetero rings
    • C07D405/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/14—Nitrogen atoms not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • 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

  • the present application relates to (2R,3S,5R)-5-(5-methanesulfonylisoindoline-2-yl)-2-(2,5-difluorophenyl) as a long-acting inhibitor of DPP-IV Crystallization of tetrahydro-2H-pyran-3-amine or a salt thereof or a salt thereof, a pharmaceutical composition thereof, and a medical use.
  • Dipeptidyl peptidase IV is a serine protease that rapidly cleaves the N-terminal proline or alanine protein of the peptide chain and is responsible for certain endogenous peptides in vivo (eg GLP-1 and GIP). Metabolic cleavage and has demonstrated proteolytic activity against a variety of other peptides (GHRH, NPY, G LP-2 and VIP) in vitro.
  • GLP-1 and GIP are rapidly inactivated in vivo, so inhibition of DPP-IV activity can greatly prolong the duration of GLP-1 and GIP physiological activity in vivo, thereby indirectly regulating insulin secretion, and ultimately To control the role of blood sugar.
  • DPP-IV inhibitor As a new type of diabetes treatment, DPP-IV inhibitor has a glucose-dependent stimulation of insulin secretion, is less prone to hypoglycemia in the process of controlling blood glucose, and has the characteristics of retaining islet ⁇ -cell function; It is well tolerated; it can be administered orally without injection; it is equivalent to the existing oral hypoglycemic agents.
  • DPP-IV inhibitors are useful for the prevention and/or treatment of diseases and conditions mediated by DPP-IV, such as diabetes and obesity, especially type II diabetes.
  • WO2016127916 discloses substituted amino six-membered saturated heteroalicyclics as DPP-IV long-acting inhibitors, including the compounds of formula I and processes for their preparation, which are hereby incorporated by reference in its entirety by reference herein in its entirety:
  • the application provides crystallization of a compound of Formula I, a process for the preparation of the crystallization, a crystalline composition comprising the crystallization, a pharmaceutical composition comprising the crystallization or a crystalline composition thereof, and their medical use.
  • the present application provides a phosphate of a compound of Formula I, a process for the preparation of the phosphate, a pharmaceutical composition comprising the phosphate, and their medical use.
  • the present application provides crystallization of a phosphate of a compound of formula I, a process for the preparation of a crystal of the phosphate, a crystalline composition comprising the crystal of the phosphate, a pharmaceutical composition comprising the crystal of the phosphate or a crystalline composition thereof And their medical use.
  • the application provides a fumarate salt of a compound of formula I, a process for the preparation of the fumarate salt, a pharmaceutical composition comprising the fumarate salt, and their medical use.
  • the present application provides a fumarate-containing crystal of a compound of formula I, a method for preparing the fumarate-containing crystal, A crystalline composition comprising the fumarate-containing crystal, a pharmaceutical composition comprising the fumarate crystal or a crystalline composition thereof, and their medical use.
  • references to “an embodiment” or “an embodiment” or “in another embodiment” or “in certain embodiments” throughout this specification are meant to be included in the at least one embodiment.
  • the appearances of the phrase “in one embodiment” or “in an embodiment” or “in another embodiment” or “in some embodiments” are not necessarily all referring to the same embodiment.
  • the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
  • a reaction including a “catalyst” includes a catalyst, or two or more catalysts.
  • the term “or” is generally used in its meaning including “and/or” unless it is specifically defined otherwise.
  • the application provides crystallization of a compound of formula I,
  • XRD X-ray diffraction
  • the crystalline X-ray diffraction peak of the compound of Formula I has the following characteristics:
  • the crystal of the compound of Formula I has an X-ray diffraction pattern as shown in FIG. 1, FIG. 3, FIG. 5, or FIG.
  • the crystals of the compounds of formula I obtained in different crystallization solvents have substantially the same X-ray diffraction pattern and, therefore, they are of the same crystalline form.
  • the crystallization of the compound of Formula I of the present application can also be characterized by DSC: an onset temperature of 193.3 ⁇ 5 °C, and a peak temperature of 195.2 ⁇ 5 °C.
  • the crystal of the compound of Formula I has a DSC pattern as shown in Figure 2 or Figure 4.
  • the application provides a method of preparing a crystal of a compound of formula I, including
  • the crystallization solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, acetone, methyl ethyl ketone, ethyl acetate, acetonitrile, Methyl chloride, toluene, dioxane, n-heptane, n-hexane, methyl tert-butyl ether, isopropyl ether, isopropyl acetate or a mixed solvent thereof.
  • the crystallization solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, ethyl acetate, acetonitrile, dichloromethane, or a mixed solvent thereof; methanol is preferred.
  • the amount of the crystallization solvent added is from 2 to 100 mL, preferably 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL, more preferably 20%, relative to 1 g of the compound of Formula I. 60 mL, 20-40 mL or 30-50 mL.
  • the present application also provides another method of preparing the crystallization of a compound of formula I comprising precipitating a crystal of a compound of formula I from a solvent comprising methanol.
  • the present application also provides crystalline compositions comprising the crystals of the compound of formula I.
  • the crystalline form of the compound of Formula I comprises 50% by weight or more, preferably 80% by weight or more, more preferably 90% by weight or more, and most preferably 95% by weight or more, based on the weight of the crystalline composition.
  • the application also provides a pharmaceutical composition comprising a crystal of the compound of formula I or a crystalline composition comprising the crystal of the compound of formula I.
  • the pharmaceutical compositions may or may not contain pharmaceutically acceptable carriers, excipients and/or vehicles.
  • the present application also provides the use of the crystallization of the compound of Formula I, or a crystalline composition thereof, or a pharmaceutical composition thereof, for the manufacture of a medicament for the treatment or prevention of a disease that benefits from DPP-IV inhibition.
  • the present application also provides a method for treating or preventing a disease that benefits from DPP-IV inhibition, comprising administering to a subject in need thereof a crystal of the compound of formula I or a crystalline composition thereof or a pharmaceutical composition thereof.
  • the present application also provides crystallization of a compound of formula I for use in the treatment or prevention of a disease that would benefit from DPP-IV inhibition, or a crystalline composition thereof, or a pharmaceutical composition thereof.
  • the present application also provides the use of the crystallization of the compound of Formula I, or a crystalline composition thereof, or a pharmaceutical composition thereof, for the treatment or prevention of a disease that benefits from DPP-IV inhibition.
  • the application provides a phosphate of a compound of formula I:
  • the ratio of the molar amount of the compound of formula I to the phosphoric acid in the phosphate of the compound of formula I is from 1:0.5 to 2, preferably from 1:0.5 to 1, more preferably 1:1.
  • the phosphate of the compound of Formula I is in crystalline form.
  • the present application also provides a process for the preparation of a phosphate of a compound of formula I comprising contacting a compound of formula I with phosphoric acid and then separating from the solvent.
  • the solvent is selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, isobutanol, tert-butanol, dichloromethane, acetonitrile, acetone, ethyl acetate, isopropyl acetate 1,4-dioxane, n-heptane, n-hexane, methyl tert-butyl ether, diisopropyl ether, toluene or a mixture of two or more, preferably ethanol.
  • the application provides crystallization of a phosphate of a compound of formula I:
  • XRD X-ray diffraction
  • the diffraction peak of the crystal of the phosphate of the compound of Formula I has the following characteristics:
  • the crystal of the phosphate of the compound of Formula I has an X-ray diffraction pattern as shown in FIG.
  • the crystal of the phosphate of the compound of Formula I has a DSC pattern as shown in FIG.
  • the present application also provides a process for the preparation of a crystallization of a phosphate of a compound of formula I comprising contacting a compound of formula I with phosphoric acid and then crystallization from a solvent.
  • the solvent is selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, isobutanol, tert-butanol, dichloromethane, acetonitrile, acetone, ethyl acetate, isopropyl acetate 1,4-dioxane, n-heptane, n-hexane, methyl tert-butyl ether, diisopropyl ether, toluene or a mixture of two or more, preferably ethanol.
  • the present application also provides crystalline compositions comprising crystals of the phosphate of the compound of formula I.
  • the crystal of the phosphate of the compound of Formula I comprises 50% by weight or more, preferably 80% by weight or more, more preferably 90% by weight or more, and most preferably 95% by weight or more, based on the weight of the crystalline composition.
  • the present application also provides a pharmaceutical composition comprising a phosphate of the compound of formula I or a crystal of a phosphate of said compound of formula I or a crystalline crystalline composition comprising a phosphate of said compound of formula I.
  • the pharmaceutical composition comprises a therapeutically effective amount of a phosphate of the compound of Formula I or a phosphate of the compound of Formula I.
  • the pharmaceutical compositions may or may not contain pharmaceutically acceptable carriers, excipients and/or vehicles.
  • the present application also provides a crystallization of a phosphate of the compound of the formula I or a phosphate of the compound of the formula I or a crystalline composition thereof or a pharmaceutical composition thereof for the treatment or prevention of a disease which benefits from DPP-IV inhibition Use of the drug.
  • the present application also provides a method for treating or preventing a disease that benefits from DPP-IV inhibition, comprising administering to a subject in need thereof a phosphate of the compound of formula I or a crystal of the phosphate of the compound of formula I or crystallizing thereof.
  • Compositions or pharmaceutical compositions thereof comprising administering to a subject in need thereof a phosphate of the compound of formula I or a crystal of the phosphate of the compound of formula I or crystallizing thereof.
  • the present application also provides crystallization of a phosphate of the compound of Formula I or a phosphate of the compound of Formula I, or a crystalline composition thereof, or a pharmaceutical composition thereof, for use in the treatment or prevention of a disease that benefits from DPP-IV inhibition.
  • the present application also provides the use of a phosphate of the compound of the formula I or a phosphate of the compound of the formula I or a crystalline composition thereof or a pharmaceutical composition thereof for the treatment or prevention of a disease which benefits from DPP-IV inhibition .
  • the application provides a fumarate salt of a compound of formula I:
  • the ratio of the molar amount of the compound of formula I in the fumarate salt of the compound of formula I to fumaric acid is from 1:0.5 to 2, preferably from 1:0.5 to 1, more preferably 1:0.5. .
  • the fumarate salt of the compound of Formula I may be in crystalline form.
  • the present application also provides a process for the preparation of a fumarate salt of a compound of formula I comprising contacting a compound of formula I with fumaric acid and then separating from the solvent.
  • the solvent is selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, isobutanol, tert-butanol, dichloromethane, acetonitrile, acetone, ethyl acetate, isopropyl acetate 1,4-dioxane, n-heptane, n-hexane, methyl tert-butyl ether, diisopropyl ether, toluene or a mixture of two or more, preferably ethanol.
  • the application provides a fumarate-containing crystal of a compound of formula I:
  • the fumarate-containing crystal of the compound of Formula I has a DSC pattern as shown in FIG.
  • the present application also provides a process for the preparation of a fumarate-containing crystal of a compound of formula I comprising contacting a compound of formula I with fumaric acid and then precipitating from a solvent.
  • the solvent is selected from the group consisting of methanol, ethanol, isopropanol, n-butanol, isobutanol, tert-butanol, dichloromethane, acetonitrile, acetone, ethyl acetate, isopropyl acetate 1,4-dioxane, n-heptane, n-hexane, methyl tert-butyl ether, diisopropyl ether, toluene or a mixture of two or more, preferably ethanol.
  • the present application also provides crystalline compositions comprising the fumarate-containing crystals of the compound of formula I.
  • the fumarate-containing crystal of the compound of Formula I comprises 50% by weight or more, preferably 80% by weight or more, more preferably 90% by weight or more, and most preferably 95% by weight or more, based on the weight of the crystalline composition. .
  • the application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a fumarate salt of the compound of formula I or a rich form of the compound of formula I Crystallization of the acid salt or crystalline composition of the fumarate-containing crystal of the compound of formula I.
  • the pharmaceutical composition comprises a therapeutically effective amount of a fumarate salt of the compound of Formula I or a fumarate-containing crystal of the compound of Formula I.
  • the pharmaceutical composition may or may not contain a pharmaceutically acceptable carrier, excipient, and/or vehicle.
  • the present application also provides a fumarate salt of the compound of formula I or a fumarate crystal of the compound of formula I or a crystalline composition thereof or a pharmaceutical composition thereof for use in the treatment or prevention of benefit from DPP- Use in drugs for IV-inhibited diseases.
  • the present application also provides a method for treating or preventing a disease that benefits from DPP-IV inhibition, comprising administering to a subject in need thereof a fumarate salt of the compound of Formula I or a fumarate salt of the compound of Formula I. Crystallization or a crystalline composition thereof or a pharmaceutical composition thereof.
  • the present application also provides a fumarate salt of the compound of the formula I or a compound of the compound of the formula I, or a crystalline composition thereof, or a crystalline composition thereof, for use in the treatment or prevention of a disease which benefits from DPP-IV inhibition Pharmaceutical composition.
  • the present application also provides a fumarate salt of the compound of the formula I or a fumarate crystal of the compound of the formula I or a crystalline composition thereof or a pharmaceutical composition thereof for the treatment or prevention of benefit from DPP-IV inhibition Use in disease.
  • the disease that benefits from DPP-IV inhibition is selected from the group consisting of insulin resistance, hyperglycemia, type II diabetes, diabetic lipemia, impaired glucose tolerance (IGT), fasting plasma Glucose reduction (IFG), metabolic acidosis, ketosis, appetite regulation, obesity, various cancers, neurological disorders, or immune system disorders, etc., are preferably type II diabetes or obesity.
  • the X-ray diffraction spectrum is measured by the following method: Instrument: Bruker D8 ADVANCE X-ray diffractometer; Method: Target: Cu: K-Alpha; Wavelength Tube pressure: 40 kV; Current: 40 mA; Scanning range: 4 to 40°; Sample rotation speed: 15 rpm; Scanning speed: 10°/min; or, the following method can be used: Instrument: Bruker D8ADVANCE X-ray diffractometer; method: target: Cu; wavelength Tube pressure: 40 kV; Current: 40 mA; Scanning range: 3 to 40°; Scanning speed: 0.1 second per step, 0.02° per step.
  • DSC differential scanning calorimetry
  • the phosphate or fumarate salt of the compound of formula I described herein can be measured by titration of the ratio of the compound of formula I in the salt to the corresponding acid.
  • Titrator used METTLER T50; titration solution: 0.1 mol/L sodium hydroxide titration solution; titration solvent: water.
  • the diffraction spectrum obtained from the crystalline compound is often characteristic for a specific crystal form, wherein the relative intensity of the band (especially at a low angle) may be due to crystallization conditions.
  • the dominant orientation effect due to the difference in particle size and other measurement conditions varies. Therefore, the relative intensities of the diffraction peaks are not characteristic for the crystal form to be targeted.
  • the position of the peak can be shifted due to changes in temperature during sample analysis, sample movement, or calibration of the instrument, etc., and the measurement error of the 2 ⁇ value is sometimes about ⁇ 0.2°. Therefore, this error should be taken into account when determining each crystal structure.
  • the peak positions of the XRD spectrum have similarities as a whole, and the relative intensity error may be large.
  • the DSC measures the transition temperature when the crystal absorbs or releases heat due to a change in its crystal structure or crystal melting.
  • the thermal transition temperature and melting point error is typically within about 5 ° C in a continuous analysis.
  • a compound has a given DSC peak or melting point, this is Refers to the DSC peak or melting point ⁇ 5 ° C.
  • DSC provides ancillary methods for identifying different crystal forms. Different crystal morphology can be identified based on their different transition temperature characteristics.
  • the term "pharmaceutical composition” refers to a carrier, excipient and/or medium comprising the active compound of the present application and generally accepted in the art for delivery of a biologically active compound into an organism, such as a human. Preparation. Drug combination The purpose of the article is to facilitate administration of the compounds of the present application to an organism.
  • the term “pharmaceutically acceptable carrier” refers to carriers and diluents which have no significant irritation to an organism, such as a human, and which do not impair the biological activity and properties of the active compound.
  • “Pharmaceutically acceptable excipient and/or vehicle” refers to an inert substance that is administered with the active ingredient to facilitate administration of the active ingredient.
  • “Pharmaceutically acceptable carrier, excipient, and/or vehicle” includes, but is not limited to, any carrier, excipient, vehicle, glidant, sweetener, diluent, which can be used in humans or animals, such as livestock.
  • Preservatives dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, disintegrating agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers, and the like.
  • excipients include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols, and the like.
  • Administration of the compounds of the present application or salts thereof or their crystals or their crystalline compositions can be carried out in pure form or in the form of a suitable pharmaceutical composition by any acceptable mode of administration which provides a medicament for similar use.
  • the pharmaceutical composition of the present application can be prepared by combining the compound of the present application or a salt thereof or a crystal thereof or a crystalline composition thereof with a suitable pharmaceutically acceptable carrier, diluent, medium or excipient.
  • compositions of the present application can be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, Gels, microspheres and aerosols, etc.
  • Typical routes of administration of the compounds of the present application or their salts or their crystals or their crystalline compositions or their pharmaceutical compositions include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal Inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration, and the like.
  • a preferred route of administration is oral administration.
  • the pharmaceutical composition of the present application can be produced by a method known to those skilled in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar coating pellet method, a grinding method, an emulsification method, a freeze drying method, and the like.
  • the pharmaceutical composition is in oral form.
  • the pharmaceutical compositions may be formulated by admixing the active compound withpharmaceutically acceptable carriers, excipients and/or vehicles which are well known in the art. These carriers, excipients and vehicles enable the compounds of the present application or their salts or their crystals or their crystalline compositions to be formulated into tablets, pills, troches, dragees, capsules, liquids, gels, Serum, suspension, etc., for oral administration to a patient.
  • Solid oral pharmaceutical compositions can be prepared by conventional methods of mixing, filling or tabletting. For example, it can be obtained by mixing the active compound with a solid excipient, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules, The core of the tablet or dragee is obtained.
  • Suitable excipients include, but are not limited to, fillers such as sugars including lactose, sucrose, mannitol or sorbitol; cellulosics such as microcrystalline cellulose, corn starch, wheat starch, rice starch and potato starch; And other substances, such as gum, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; disintegrants, such as sodium carboxymethyl starch, cross-linking Sodium carboxymethylcellulose, crosslinked polyvinylpyrrolidone, agar or alginic acid may also be used, such as sodium alginate.
  • the core of the dragee may optionally be coated according to methods well known in the ordinary pharmaceutical practice, especially using enteric coatings.
  • reaction is generally carried out under an inert atmosphere such as a nitrogen atmosphere and in an anhydrous solvent.
  • the crystal of the compound of formula I provided herein has the advantages of high purity, high crystallinity, good stability and the like. Moreover, the method for preparing the crystallization of the compound of the formula I provided by the present application has the advantages of simple operation, easy availability of the solvent, mild crystallization conditions, and the like, and is suitable for industrial production.
  • the process for the preparation of a salt of a compound of formula I provided herein is simple to operate, and the obtained compound of formula I has a high salt purity and good pharmacokinetic properties and is suitable for preparation as a desired pharmaceutical composition.
  • Figure 1 shows an XRD spectrum of the crystal of the compound of formula I.
  • Figure 2 shows a DSC spectrum of the crystal of the compound of formula I.
  • Figure 3 shows an XRD spectrum of the crystal of the compound of formula I.
  • Figure 4 shows a DSC spectrum of the crystal of the compound of formula I.
  • Figure 5 shows an XRD spectrum of the crystal of the compound of formula I.
  • Figure 6 shows an XRD spectrum of the crystal of the compound of formula I.
  • Figure 7 shows an XRD pattern of the crystallization of the phosphate of the compound of formula I.
  • Figure 8 shows a DSC spectrum of the crystallization of the phosphate of the compound of formula I.
  • Figure 9 shows an XRD pattern of the fumarate crystal of the compound of formula I.
  • Figure 10 shows a DSC spectrum of the fumarate crystal of the compound of formula I.
  • Figure 11 shows the inhibitory effect of the compound of formula I on serum DPP-IV activity in ob/ob mice.
  • the compound of the formula 3 (22.6 g, 100 mmol) was added to dry THF (250 mL), and the borane-dimethyl sulfide complex (51 mL, 500 mmol) was added dropwise, and then stirred at room temperature for 2 hours and refluxed overnight. After cooling, the methanol was carefully dropped to destroy excess borane. After evaporation and concentration, it was purified by silica gel column chromatography to give 5-bromoiso- porphyrin (10.36 g). MS m/z [ESI]: 198.0 [M + 1].
  • Step 1 (2R,3S,5R)-5-(5-Methanesulfonylisoindoline-2-yl)-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3 - tert-butyl carbamate (8)
  • the compound of formula 7 (150 g, 458.27 mmol) and the compound of formula 2 (117.82 g, 504.09 mmol) were added to 2.25 L of N,N-diisopropylacetamide solvent, stirred uniformly and cooled to -10 ° C, to the reaction system.
  • Acetic acid (26.26 mL, 458.27 mmol) was slowly added dropwise.
  • NaBH(AcO) 3 (194.25 g, 916.54 mmol) was added, and the mixture was stirred and stirred for 1 h.
  • the yield was 95%.
  • FIG. 1 A typical XRD pattern of the crystal prepared by the method 1 using methanol as a crystallization solvent is shown in Fig. 1 and the DSC spectrum is shown in Fig. 2.
  • FIG. 3 Another typical XRD pattern of the crystal prepared by the second method of methanol as a crystallization solvent is shown in FIG. 3 and the DSC spectrum is shown in FIG.
  • Example 3 the crystals of the compound of formula I obtained using different crystallization solvents all belong to the same crystal form.
  • the compound of the formula I (7 g, 17.1 mmol) was added to 350 mL of ethanol solvent, heated to reflux and dissolved, and then 1.0 g of activated carbon was added for decoloration for 10 min, filtered while hot, and then a phosphoric acid solution (1.8 mL, 34.2 mmol) was added dropwise to the filtrate. , a large amount of white solid was precipitated, the reaction system was cooled to room temperature and stirred for 2 h, and the solid was further precipitated, suction filtered, and the solid was blast dried overnight to obtain 8.3 g of the phosphate of the compound of formula I (1:1, titrated by titration) Crystallization, yield 95.8%, purity 99.12%.
  • a typical XRD pattern of the resulting product is shown in Figure 7 and the DSC pattern is shown in Figure 8.
  • the compound of the formula I (7 g, 17.1 mmol) was added to 350 mL of ethanol solvent, heated to reflux and dissolved, and then 1.0 g of activated carbon was added for decoloration for 10 min, and filtered while hot, and fumaric acid (3.97 g, 34.2 mmol) was added to the filtrate. After the addition, the reaction system was cooled to room temperature and stirred in an ice water bath for 2 h to precipitate a solid, which was filtered with suction, and the solid was dried by blasting at 50 ° C for 6 h to obtain 7.2 g of the compound of formula I, fumarate (1:0.5, titrimetric method) As measured, the yield was 80.3% and the purity was 97.7%.
  • a typical XRD pattern of the resulting product is shown in Figure 9 and the DSC pattern is shown in Figure 10.
  • Male Beagle dogs were fasted for approximately 12 h prior to dosing and were given free access to water. Fasting for 4 hours after administration. About 0.8 mL of blood was taken from the forelimb veins at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 30, 48 and 72 h after administration. The cells were stored in an EDTA-K2 centrifuge tube, stored at 4 ° C, and centrifuged at 4000 rpm for 10 min at 4 ° C within 0.5 h after blood collection to separate plasma. After collecting all the plasma, it was stored at -20 ° C for 1 h to be tested.
  • This application uses the following method to determine the inhibitory activity of the compounds of formula I of the present application in plasma DPP-IV enzyme, the inhibitory activity using IC 50 to indicate, compound 50% 50 i.e. DPP-IV enzyme activity IC is inhibited concentration.
  • HEPES buffer 50 ml of 0.5 M HEPES buffer was prepared with 1 M HEPES buffer (Invitrogen, Catalog No. 15630-080): 1 mL HEPES buffer 25 mL, add appropriate amount of ddH 2 O (re-distilled water), with NaOH Adjust the pH to 7.8 and finally add ddH 2 O to 50 ml.
  • Rat plasma Rats were bled with blood, added with heparin, and centrifuged at 4000 rpm for 10 minutes. The supernatant plasma was taken as the source of DPP-IV.
  • DPP-IV enzyme reaction substrate H-Gly-Pro-AMC glycine-valine-7-amino-4-methylcoumarin
  • Test compound positive control compound Omarigliptin and a compound of formula I of the present application
  • test compound Dilute the test compound from 10 mM to 1 mM (100 times the final operating concentration) with DMSO, then perform a 3-fold gradient dilution on a 96-well plate for a total of 11 concentrations, add DMSO to the 12-well as a blank control, then Dilute 25 times with enzyme reaction buffer to 4 times the final working concentration for use;
  • a compound generating enzyme DPP-IV inhibition of the IC 50 IC GraFit6 using 50 values calculated by the software compound.
  • the human liver microsomes frozen at -80 ° C were thawed on ice, and immediately after thawing, 100 ⁇ L to 60 ° C in a constant temperature shaking box was incubated at 100 rpm (1 hour), and the remaining liver microsomes were immediately at -80 Freeze at °C. After 1 hour, 100 ⁇ L of inactivated liver microsomes were taken out, and 400 ⁇ L of phosphate buffer was added to mix into 4 mg/mL of inactivated liver microsome solution; and human liver microsomes frozen at -80 ° C were placed on ice. After melting, just remove 100 ⁇ L and add 400 ⁇ L of phosphate buffer to mix. For the 4 mg/mL liver microsome solution, the positive control and the test article and the negative control group were mixed in parallel according to Table 4 below.
  • the mixture was placed in a constant temperature shaking box at 37 ° C for 5 minutes at 100 rpm.
  • test article or positive control working solution negative control group is added to the test product working solution
  • 91.5 ⁇ L of incubation mixture 6 ⁇ L of NADPH solution and vortex to start the reaction. Place the solution in a constant temperature shaking box at 37 ° C at 100 rpm. Incubation, incubation time is shown in Table 5 below.
  • the reaction was terminated, and the sample for termination of the reaction was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken for injection detection.
  • the quantification of the analyte is defined as the ratio of the peak area of the analyte to the peak area of the internal standard.
  • the analysis was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Parameters such as IC 50 were calculated using Graphpad Prism (version 5.03) software, and the results are shown in Table 6 below.
  • the present application employs the following method to determine the metabolic stability of liver microsomes of a compound of formula I.
  • mice Thirty-six female ob/ob mice were randomly divided into 6 groups, 6 in each group, which were model control group, 1 mg/kg formula I compound group, 3 mg/kg formula I compound group, 10 mg/kg formula I compound group, 30 mg/kg of the compound of formula I and the positive control of 30 mg/kg of the Omarigliptin group.
  • Each group of mice was orally administered with different doses of the compound of formula I or Omarigliptin, and the model control group was orally administered with 0.25% CMC-Na before administration and after administration 2, 4, 10, 24, 34, 48, 58, 72 and Blood was taken at 96 h, serum was separated, and serum DPP-IV activity was measured.
  • Determination of serum DPP-IV activity Take 5 ⁇ L of serum sample, add 45 ⁇ L of 80 mM MgCl 2 buffer, mix, pre-incubate for 5 minutes at room temperature, add 10 ⁇ L of 0.1 mM reaction substrate Gly-Pro-7-AMC and 40 ⁇ L buffer In the dark, mix and measure the fluorescence every 3 minutes (excitation wave 380nm / emission wave 460nm), up to 18 minutes, a total of 6 times, according to the measurement results minus the blank background and then do the time-fluorescence value curve, The slope is the vigor value, and the viability value of serum DPP-IV is 100% at 0 h before administration.

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Abstract

本申请涉及式Ⅰ化合物的结晶、其盐及其盐的结晶，制备所述式Ⅰ化合物的结晶、其盐及其盐的结晶的方法，包含式Ⅰ化合物的结晶或其盐的结晶的结晶组合物、它们的药物组合物以及它们的医药用途。

Description

DPP-IV长效抑制剂的结晶及其盐
相关申请的交叉引用
本申请要求于2016年08月12日向中华人民共和国国家知识产权局提交的第201610665625.9号和第201610666564.8号中国发明专利申请的权益，在此将其全部内容以援引的方式整体并入本文中。
技术领域
本申请涉及作为DPP-IV长效抑制剂的(2R,3S,5R)-5-(5-甲磺酰基异吲哚啉-2-基)-2-(2,5-二氟苯基)四氢-2H-吡喃-3-胺的结晶或其盐或其盐的结晶，它们的药物组合物及医药用途。
背景技术
二肽基肽酶Ⅳ(DPP-IV)是丝氨酸蛋白酶，能迅速将肽链的N端为脯氨酸或丙氨酸的蛋白质裂解，负责体内某些内源性肽(如GLP-1和GIP)的代谢性裂解，并已经证明有体外对抗多种其它肽(GHRH、NPY、G LP-2和VIP)的蛋白分解活性。由于DPP-IV酶的降解，体内GLP-1和GIP迅速失活，因此抑制DPP-IV的活性可以大大延长体内的GLP-1和GIP生理活性的持续时间，从而间接调控胰岛素的分泌，最终起到控制血糖的作用。
DPP-IV抑制剂作为新型的糖尿病治疗手段，具有葡萄糖依赖性的刺激胰岛素分泌的作用，在控制血糖过程中不易发生低血糖的副作用，还具有保留胰岛β细胞功能等特点；胃肠道反应少，耐受性好；可以口服给药而无需注射给药；与现有的口服降糖药疗效相当。
基于以上特点，DPP-IV抑制剂可用于预防和/或治疗由DPP-IV介导的疾病和病症，如糖尿病和肥胖等，尤其是II型糖尿病。
WO2016127916公开了作为DPP-IV长效抑制剂的取代的氨基六元饱和杂脂环类，包括式Ⅰ化合物及其制备方法，在此将其全文整体并入作为参考：
Figure PCTCN2017097046-appb-000001
发明概述
一方面，本申请提供式Ⅰ化合物的结晶、该结晶的制备方法，包含该结晶的结晶组合物，包含该结晶或其结晶组合物的药物组合物，以及它们的医药用途。
另一方面，本申请提供式Ⅰ化合物的磷酸盐、该磷酸盐的制备方法、包含该磷酸盐的药物组合物以及它们的医药用途。
另一方面，本申请提供式Ⅰ化合物的磷酸盐的结晶，该磷酸盐的结晶的制备方法，包含该磷酸盐的结晶的结晶组合物，包含该磷酸盐的结晶或其结晶组合物的药物组合物，以及它们的医药用途。
另一方面，本申请提供式Ⅰ化合物的富马酸盐，该富马酸盐的制备方法、包含该富马酸盐的药物组合物以及它们的医药用途。
另一方面，本申请提供式Ⅰ化合物的富马酸盐的结晶，该富马酸盐的结晶的制备方法、 包含该富马酸盐的结晶的结晶组合物，包含该富马酸盐的结晶或其结晶组合物的药物组合物，以及它们的医药用途。
发明详细说明
在以下的说明中，包括某些具体的细节以对各个公开的实施方案提供全面的理解。然而，相关领域的技术人员会认识到，不采用一个或多个这些具体的细节，而采用其它方法、部件、材料等的情况下可实现实施方案。
除非本申请中另外要求，在整个说明书和其后的权利要求书中，词语“包括(comprise)”及其英文变体例如“包括(comprises)”和“包括(comprising)”应解释为开放式的、含括式的意义，即“包括但不限于”。
在整个本说明书中提到的“一实施方案”或“实施方案”或“在另一实施方案中”或“在某些实施方案中”意指在至少一实施方案中包括与该实施方案所述的相关的具体参考要素、结构或特征。因此，在整个说明书中不同位置出现的短语“在一实施方案中”或“在实施方案中”或“在另一实施方案中”或“在某些实施方案中”不必全部指同一实施方案。此外，具体要素、结构或特征可以任何适当的方式在一个或多个实施方案中结合。
应当理解，在本申请说明书和附加的权利要求书中用到的单数形式的冠词“一”(对应于英文“a”、“an”和“the”)包括复数的对象，除非文中另外明确地规定。因此，例如提到的包括“催化剂”的反应包括一种催化剂，或两种或多种催化剂。还应当理解，术语“或”通常以其包括“和/或”的含义而使用，除非文中另外明确地规定。
一方面，本申请提供了式Ⅰ化合物的结晶，
Figure PCTCN2017097046-appb-000002
在本申请的一些实施方案中，所述式Ⅰ化合物的结晶在X-射线衍射(XRD)图谱中具有2θ＝16.4°、21.8°、25.3°、26.0°±0.2°的衍射峰；典型地，其具有2θ＝16.4°、19.4°、21.2°、21.8°、25.3°、26.0°±0.2°的衍射峰；更典型地，其具有2θ＝13.0°、16.4°、18.5°、19.4°、21.2°、21.8°、25.3°、26.0°±0.2°的衍射峰。
在本申请的一些实施方案中，所述式Ⅰ化合物的结晶的X-射线衍射峰具有如下特征：
Figure PCTCN2017097046-appb-000003
Figure PCTCN2017097046-appb-000004
在本申请的一些实施方案中，所述式Ⅰ化合物的结晶具有如图1、图3、图5或图6所示的X-射线衍射图谱。从图1、图3、图5和图6中可知，在不同的结晶溶剂中获得的式Ⅰ化合物的结晶具有基本上相同的X-射线衍射图谱，因此，它们为同一晶型。
在本申请的一些实施方案中，本申请的式Ⅰ化合物的结晶也可以用DSC进行表征：起始温度为193.3±5℃，以及峰值温度为195.2±5℃。
在本申请的一些实施方案中，所述式Ⅰ化合物的结晶具有如图2或图4所示的DSC图。
本申请提供了制备式Ⅰ化合物的结晶的方法，包括
1)使式Ⅰ化合物溶于结晶溶剂中；
2)冷却析晶，并过滤。
在本申请的一些实施方案中，所述结晶溶剂选自甲醇、乙醇、丙醇、异丙醇、正丁醇、异丁醇、叔丁醇、丙酮、丁酮、乙酸乙酯、乙腈、二氯甲烷、甲苯、二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、乙酸异丙酯或它们的混合溶剂。
在本申请的一些实施方案中，所述结晶溶剂选自甲醇、乙醇、丙醇、异丙醇、乙酸乙酯、乙腈、二氯甲烷或它们的混合溶剂；优选甲醇。
在本申请的一些实施方案中，相对于1g式Ⅰ化合物，加入的结晶溶剂的量为2～100mL，优选20mL、30mL、40mL、50mL、60mL、70mL、80mL、90mL或100mL，更优选20～60mL、20～40mL或30～50mL。
本申请还提供了制备式Ⅰ化合物的结晶的另一方法，包括使式Ⅰ化合物的结晶从包括甲醇的溶剂中析出。
本申请还提供了包含所述式Ⅰ化合物的结晶的结晶组合物。在本申请的一些实施方案中，所述式Ⅰ化合物的结晶占所述结晶组合物的重量的50wt％以上，优选80wt％以上，更优选90wt％以上，最优选95wt％以上。
本申请还提供了药物组合物，其包含所述式Ⅰ化合物的结晶，或者包含含有所述式Ⅰ化合物的结晶的结晶组合物。此外，该药物组合物还可以含有或不含有药学上可接受的载体、赋形剂和/或介质。
本申请还提供了所述式Ⅰ化合物的结晶或其结晶组合物或其药物组合物在制备用于治疗或预防受益于DPP-IV抑制的疾病的药物中的用途。本申请还提供了用于治疗或预防受益于DPP-IV抑制的疾病的方法，包括向有需要的个体给予所述式Ⅰ化合物的结晶或其结晶组合物或其药物组合物。本申请还提供了用于治疗或预防受益于DPP-IV抑制的疾病的所述式Ⅰ化合物的结晶或其结晶组合物或其药物组合物。本申请还提供了所述式Ⅰ化合物的结晶或其结晶组合物或其药物组合物在治疗或预防受益于DPP-IV抑制的疾病中的用途。
另一方面，本申请提供了式Ⅰ化合物的磷酸盐：
Figure PCTCN2017097046-appb-000005
在本申请的一些实施方案中，所述式Ⅰ化合物的磷酸盐中式Ⅰ化合物与磷酸的摩尔量之比为1:0.5～2，优选1:0.5～1，更优选1:1。
在本申请的一些实施方案中，所述式Ⅰ化合物的磷酸盐呈结晶形式。
本申请还提供了制备式Ⅰ化合物的磷酸盐的方法，包括使式Ⅰ化合物与磷酸接触，然后从溶剂中分离。在本申请的一些实施方案中，所述溶剂选自甲醇、乙醇、异丙醇、正丁醇、异丁醇、叔丁醇、二氯甲烷、乙腈、丙酮、乙酸乙酯、乙酸异丙酯、1,4-二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、甲苯或者两种以上的混合物，优选乙醇。
另一方面，本申请提供了式Ⅰ化合物的磷酸盐的结晶：
Figure PCTCN2017097046-appb-000006
在本申请的一些实施方案中，所述式Ⅰ化合物的磷酸盐的结晶在X-射线衍射(XRD)图谱中具有2θ＝6.4°、11.9°、18.2°、21.7°、22.1°、22.9°、23.2°±0.2°的衍射峰；典型地，具有2θ＝6.4°、11.9°、16.5°、17.5°、18.2°、18.6°、21.7°、22.1°、22.9°、23.2°±0.2°的衍射峰；更典型地，具有2θ＝6.4°、10.1°、11.9°、16.5°、17.5°、18.2°、18.6°、19.8°、21.7°、22.1°、22.9°、23.2°、23.8°±0.2°的衍射峰。
在本申请的一些实施方案中，所述式Ⅰ化合物的磷酸盐的结晶的衍射峰具有如下特征：
序号 2θ±0.2(°) 相对强度(％) 序号 2θ±0.2(°) 相对强度(％)
1 6.4 37 9 21.7 100
2 10.1 14 10 22.1 66
3 11.9 39 11 22.9 55
4 16.5 38 12 23.2 56
5 17.5 38 13 23.8 41
6 18.2 70 14 24.0 38
7 18.6 40 15 31.2 35
8 19.8 32 ---- ---- ----
在本申请的一些实施方案中，所述式Ⅰ化合物的磷酸盐的结晶具有如图7所示的X-射线衍射图谱。
在本申请的一些实施方案中，所述式Ⅰ化合物的磷酸盐的结晶具有如图8所示的DSC图谱。
本申请还提供了制备式Ⅰ化合物的磷酸盐的结晶的方法，包括使式Ⅰ化合物与磷酸接触，然后从溶剂中析晶。在本申请的一些实施方案中，所述溶剂选自甲醇、乙醇、异丙醇、正丁醇、异丁醇、叔丁醇、二氯甲烷、乙腈、丙酮、乙酸乙酯、乙酸异丙酯、1,4-二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、甲苯或者两种以上的混合物，优选乙醇。
本申请还提供了包含所述式Ⅰ化合物的磷酸盐的结晶的结晶组合物。在本申请的一些实施方案中，所述式Ⅰ化合物的磷酸盐的结晶占所述结晶组合物的重量的50wt％以上，优选80wt％以上，更优选90wt％以上，最优选95wt％以上。
本申请还提供了药物组合物，其包含所述式Ⅰ化合物的磷酸盐或所述式Ⅰ化合物的磷酸盐的结晶或含有所述式Ⅰ化合物的磷酸盐的结晶的结晶组合物。在本申请的一些实施方案中，所述药物组合物包括治疗有效量的所述式Ⅰ化合物的磷酸盐或所述式Ⅰ化合物的磷酸盐的结晶。此外，该药物组合物还可以含有或不含有药学上可接受的载体、赋形剂和/或介质。
本申请还提供了所述式Ⅰ化合物的磷酸盐或所述式Ⅰ化合物的磷酸盐的结晶或其结晶组合物或它们的药物组合物在制备用于治疗或预防受益于DPP-IV抑制的疾病的药物中的用途。 本申请还提供了用于治疗或预防受益于DPP-IV抑制的疾病的方法，包括向有需要的个体给予所述式Ⅰ化合物的磷酸盐或所述式Ⅰ化合物的磷酸盐的结晶或其结晶组合物或它们的药物组合物。本申请还提供了用于治疗或预防受益于DPP-IV抑制的疾病的所述式Ⅰ化合物的磷酸盐或所述式Ⅰ化合物的磷酸盐的结晶或其结晶组合物或它们的药物组合物。本申请还提供了所述式Ⅰ化合物的磷酸盐或所述式Ⅰ化合物的磷酸盐的结晶或其结晶组合物或它们的药物组合物在治疗或预防受益于DPP-IV抑制的疾病中的用途。
另一方面，本申请提供了式Ⅰ化合物的富马酸盐：
Figure PCTCN2017097046-appb-000007
在本申请的一些实施方案中，所述式Ⅰ化合物的富马酸盐中式Ⅰ化合物与富马酸的摩尔量之比为1:0.5～2，优选1:0.5～1，更优选1:0.5。
在本申请的一些实施方案中，所述式Ⅰ化合物的富马酸盐可以呈结晶形式。
本申请还提供了制备式Ⅰ化合物的富马酸盐的方法，包括使式Ⅰ化合物与富马酸接触，然后从溶剂中分离。在本申请的一些实施方案中，所述溶剂选自甲醇、乙醇、异丙醇、正丁醇、异丁醇、叔丁醇、二氯甲烷、乙腈、丙酮、乙酸乙酯、乙酸异丙酯、1,4-二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、甲苯或者两种以上的混合物，优选乙醇。
另一方面，本申请提供了式Ⅰ化合物的富马酸盐的结晶：
Figure PCTCN2017097046-appb-000008
在本申请的一些实施方案中，所述式Ⅰ化合物的富马酸盐的结晶在X-射线衍射(XRD)图谱中具有2θ＝20.67°±0.2°的衍射峰。在本申请的一些实施方案中，所述式Ⅰ化合物的富马酸盐的结晶具如图9所示的XRD图谱。
在本申请的一些实施方案中，所述式Ⅰ化合物的富马酸盐的结晶具有如图10所示的DSC图谱。
本申请还提供了制备式Ⅰ化合物的富马酸盐的结晶的方法，包括使式Ⅰ化合物与富马酸接触，然后从溶剂中析出。在本申请的一些实施方案中，所述溶剂选自甲醇、乙醇、异丙醇、正丁醇、异丁醇、叔丁醇、二氯甲烷、乙腈、丙酮、乙酸乙酯、乙酸异丙酯、1,4-二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、甲苯或者两种以上的混合物，优选乙醇。
本申请还提供了包含所述式Ⅰ化合物的富马酸盐的结晶的结晶组合物。在本申请的一些实施方案中，所述式Ⅰ化合物的富马酸盐的结晶占所述结晶组合物的重量的50wt％以上，优选80wt％以上，更优选90wt％以上，最优选95wt％以上。
本申请还提供了药物组合物，其包含所述式Ⅰ化合物的富马酸盐或所述式Ⅰ化合物的富 马酸盐的结晶或含有所述式Ⅰ化合物的富马酸盐的结晶的结晶组合物。在本申请的一些实施方案中，该药物组合物中包含治疗有效量的所述式Ⅰ化合物的富马酸盐或所述式Ⅰ化合物的富马酸盐的结晶。此外，在本申请的一些实施方案中，该药物组合物还可以含有或不含有药学上可接受的载体、赋形剂和/或介质。
本申请还提供了所述式Ⅰ化合物的富马酸盐或所述式Ⅰ化合物的富马酸盐的结晶或其结晶组合物或它们的药物组合物在制备用于治疗或预防受益于DPP-IV抑制的疾病的药物中的用途。本申请还提供了用于治疗或预防受益于DPP-IV抑制的疾病的方法，包括向有需要的个体给予所述式Ⅰ化合物的富马酸盐或所述式Ⅰ化合物的富马酸盐的结晶或其结晶组合物或它们的药物组合物。本申请还提供了用于治疗或预防受益于DPP-IV抑制的疾病的所述式Ⅰ化合物的富马酸盐或所述式Ⅰ化合物的富马酸盐的结晶或其结晶组合物或它们的药物组合物。本申请还提供了所述式Ⅰ化合物的富马酸盐或所述式Ⅰ化合物的富马酸盐的结晶或其结晶组合物或它们的药物组合物在治疗或预防受益于DPP-IV抑制的疾病中的用途。
在本申请的一些实施方案中，所述的受益于DPP-IV抑制的疾病选自胰岛素抵抗、高血糖症、Ⅱ型糖尿病、糖尿病性脂血异常、葡萄糖耐量减低(IGT)症、禁食血浆葡萄糖减低(IFG)症、代谢性酸中毒、酮症、食欲调节、肥胖症、各种癌症、神经系统病症或免疫系统病症等，优选Ⅱ型糖尿病或肥胖症。
在本申请中，X-射线衍射光谱采用下述方法测量：仪器：Bruker D8ADVANCE X-射线衍射仪；方法：靶:Cu:K-Alpha；波长
Figure PCTCN2017097046-appb-000009
管压(Voltage):40kV；管流(Current)：40mA；扫描范围：4～40°；样品旋转速度：15rpm；扫描速度：10°/分钟；或者，也可采用下述方法测量：仪器：Bruker D8ADVANCE X-射线衍射仪；方法：靶:Cu；波长
Figure PCTCN2017097046-appb-000010
管压(Voltage):40kV；管流(Current)：40mA；扫描范围：3～40°；扫描速度：每步0.1秒，每步0.02°。
在本申请中，差示扫描量热分析(DSC)采用下述方法测量：仪器：TAQ2000差示扫描量热仪；方法：取样品(～1mg)置于DSC铝锅内进行测量，方法为：25℃-300℃，升温速率10℃/min。
本申请所述的式Ⅰ化合物的磷酸盐或富马酸盐可以通过滴定法测量所述盐中式Ⅰ化合物与对应的酸的比例。所用滴定仪：梅特勒T50；滴定液：0.1mol/L氢氧化钠滴定液；滴定溶剂：水。
需要说明的是，在X-射线衍射光谱中，由结晶化合物得到的衍射谱图对于特定的晶型往往是特征性的，其中谱带(尤其是在低角度)的相对强度可能会因为结晶条件、粒径和其它测定条件的差异而产生的优势取向效果而变化。因此，衍射峰的相对强度对所针对的晶型并非是特征性的，判断是否与已知的晶型相同时，更应该注意的是峰的相对位置而不是它们的相对强度。此外，对任何给定的晶型而言，峰的位置可能存在轻微误差，这在晶体学领域中也是公知的。例如，由于分析样品时温度的变化、样品移动、或仪器的标定等，峰的位置可以移动，2θ值的测定误差有时约为±0.2°。因此，在确定每种晶型结构时，应该将此误差考虑在内。在XRD图谱中通常用2θ角或晶面距d表示峰位置，两者之间具有简单的换算关系：d＝λ/2sinθ，其中d代表晶面距，λ代表入射X-射线的波长，θ为衍射角。对于同种化合物的同种晶型，其XRD谱的峰位置在整体上具有相似性，相对强度误差可能较大。还应指出的是，在混合物的鉴定中，由于含量下降等因素会造成部分衍射线的缺失，此时，无需依赖高纯试样中观察到的全部谱带，甚至一条谱带也可能对给定的晶体是特征性的。
需要说明的是，DSC测量当晶体由于其晶体结构发生变化或晶体熔融而吸收或释放热时的转变温度。对于同种化合物的同种晶型，在连续的分析中，热转变温度和熔点误差典型地在约5℃之内，当我们说一个化合物具有某一给定的DSC峰或熔点时，这是指该DSC峰或熔点±5℃。DSC提供了辨别不同晶型的辅助方法。不同的晶体形态可根据其不同的转变温度特征而加以识别。
本申请中，术语“药物组合物”是指包含本申请的活性化合物以及在本领域中通常接受的用于将生物活性化合物递送至有机体(例如人)内的载体、赋形剂和/或介质的制剂。药物组合 物的目的是有利于对有机体给予本申请的化合物。
本申请中，术语“药学上可接受的载体”是指对有机体(例如人)无明显的刺激作用，而且不会损害活性化合物的生物活性及性能的载体和稀释剂。“药学上可接受的赋形剂和/或介质”是指与活性成分一同给药的、有利于活性成分的给药的惰性物质。“药学上可接受的载体、赋形剂和/或介质”包括但不限于可用于人或动物(如家畜)的任何载体、赋形剂、介质、助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂等。所述赋形剂的非限制性实例包括碳酸钙、磷酸钙、各种糖和各类淀粉、纤维素衍生物、明胶、植物油和聚乙二醇等等。
本申请的化合物或其盐或它们的结晶或它们的结晶组合物的给药可以以纯的形式或以适宜的药物组合物的形式通过提供类似用途的药物的任何可接受的给药方式来进行。本申请的药物组合物可通过将本申请的化合物或其盐或它们的结晶或它们的结晶组合物与适宜的药学上可接受的载体、稀释剂、介质或赋形剂相组合而制备。本申请的药物组合物可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等等。
本申请的化合物或其盐或它们的结晶或它们的结晶组合物或它们的药物组合物的典型的给药途径包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药等。优选的给药途径是口服给药。
本申请的药物组合物可以采用本领域普通技术人员所知悉的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等等。
在优选的实施方案中，药物组合物是口服形式的。对于口服给药，可以通过将活性化合物与本领域熟知的药物可接受的载体、赋形剂和/或介质混合，来配制该药物组合物。这些载体、赋形剂和介质能使本申请的化合物或其盐或它们的结晶或它们的结晶组合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。
可以通过常规的混合、填充或压片方法来制备固体口服药物组合物。例如，可通过下述方法获得：将所述的活性化合物与固体赋形剂混合，任选地碾磨所得的混合物，如果需要则加入其它合适的赋形剂，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的赋形剂包括但不限于：填充剂，如糖，包括乳糖、蔗糖、甘露醇或山梨糖醇；纤维素类，例如微晶纤维素、玉米淀粉、小麦淀粉、大米淀粉和马铃薯淀粉；以及其它物质，如果胶、明胶、黄芪胶、甲基纤维素、羟丙基甲基纤维素、羟甲基纤维素钠和/或聚乙烯吡咯烷酮；崩解剂，如羧甲淀粉钠、交联羧甲基纤维素钠、交联聚乙烯吡咯烷酮、琼脂或藻酸，也可以使用盐，如藻酸钠。可以根据通常药物实践中公知的方法任选地对糖衣剂的核心进行包衣，尤其使用肠溶包衣。
本申请使用的所有溶剂均是市售的，无需进一步纯化即可使用。反应一般是在惰性气氛如氮气气氛下和无水溶剂中进行的。
本申请提供的式Ⅰ化合物的结晶具有纯度高、结晶度高、稳定性好等优点。并且，本申请提供的制备式Ⅰ化合物的结晶的方法具有操作简单，溶剂价廉易得，结晶条件温和等优点，适合工业化生产。本申请提供的制备式Ⅰ化合物的盐的方法操作简单，所获得的式Ⅰ化合物的盐纯度高，并具有良好的药代动力学性质，适合制备成为所期望的药物组合物。
附图说明
图1显示式Ⅰ化合物的结晶的XRD谱图。
图2显示式Ⅰ化合物的结晶的DSC谱图。
图3显示式Ⅰ化合物的结晶的XRD谱图。
图4显示式Ⅰ化合物的结晶的DSC谱图。
图5显示式Ⅰ化合物的结晶的XRD谱图。
图6显示式Ⅰ化合物的结晶的XRD谱图。
图7显示式I化合物的磷酸盐的结晶的XRD谱图。
图8显示式I化合物的磷酸盐的结晶的DSC谱图。
图9显示式I化合物的富马酸盐的结晶的XRD谱图。
图10显示式I化合物的富马酸盐的结晶的DSC谱图。
图11显示式Ⅰ化合物对ob/ob小鼠血清DPP-IV活性的抑制作用。
实施例
下面结合具体的实施例对本申请的内容做示例性说明，但具体的实施例并不是对本申请的范围做任何限制。
实施例1：5-甲磺酰基异吲哚啉盐酸盐(2)
Figure PCTCN2017097046-appb-000011
步骤1：5-溴异吲哚啉(4)
将式3化合物(22.6g,100mmol)加入干燥的四氢呋喃(250mL)中，滴入硼烷-二甲硫醚络合物(51mL，500mmol)，然后在室温下搅拌2小时，再回流过夜。冷却后小心地滴入甲醇以破坏过量的硼烷。蒸发浓缩后用硅胶柱层析纯化，得到5-溴异吲哚啉(10.36g)，收率：52％。MS m/z[ESI]:198.0[M+1]。
步骤2：5-溴-2-叔丁氧羰基异吲哚啉(5)
将式4化合物(10.36g，52.3mmol)溶于80mL二氯甲烷中，冰浴冷却。滴加Boc酸酐(22.8g，104.6mmol)，再加入碳酸钠(16.6g，156.9mmol)和水(150mL)，在冰浴下搅拌4小时。分离出有机相，用盐水洗涤。有机相浓缩后，用硅胶柱层析纯化，得到产物5-溴-2-叔丁氧羰基异吲哚啉(13.3g)，收率：85％。MS m/z[ESI]:298.0[M+1]。1H-NMR(400MHz,CDCl3):δ＝7.37(2H,m)，7.11(1H,m)，4.62(4H,m)，1.51(9H,s)。
步骤3：5-甲磺酰基-2-叔丁氧羰基异吲哚啉(6)
将式5化合物(5.96g,20mmol)、甲基亚磺酸钠(90％，2.94g,26mmol)、碘化亚铜(762mg,4mmol)和L-脯氨酸(920mg,8mmol)加入二甲亚砜(80mL)中，通入氮气以排除空气，并在120℃下搅拌2天。冷却后倾入水中，用乙酸乙酯萃取。将有机相干燥，蒸发浓缩，然后用硅胶柱层析纯化，得到5-甲磺酰基-2-叔丁氧羰基异吲哚啉(5.46g)，收率：92％。MS m/z[ESI]:298.1[M+1]。
步骤4：5-甲磺酰基异吲哚啉盐酸盐(2)
将式6化合物(5.46g，18.4mmol)溶于甲醇/二氯甲烷(1:1,80mL)中，通入氯化氢气体至饱和，并室温搅拌1小时。将反应混合物倾入800mL乙醚中，过滤，收集沉淀物，乙醚洗涤沉淀物，干燥，得到产物5-甲磺酰基异吲哚啉盐酸盐(3.44g)，收率：80％。MS m/z[ESI]:198.0[M+1]。1H-NMR(400MHz,CDCl3):δ＝7.82(1H,s)，7.81(1H,d，J＝8.0Hz)，7.43(1H,d，J＝8.0Hz)，4.31(4H,s)，3.05(3H,s)，2.30(2H,brs)。
实施例2：(2R,3S,5R)-5-(5-甲磺酰基异吲哚啉-2-基)-2-(2,5-二氟苯基)四氢-2H-吡喃-3-胺粗品
步骤1：(2R,3S,5R)-5-(5-甲磺酰基异吲哚啉-2-基)-2-(2,5-二氟苯基)四氢-2H-吡喃-3-基氨基甲酸叔丁酯(8)
Figure PCTCN2017097046-appb-000012
将式7化合物(150g，458.27mmol)和式2化合物(117.82g，504.09mmol)加入到2.25L N,N-二异丙基乙酰胺溶剂中，搅拌均匀并降温至-10℃，向反应体系中缓慢滴加醋酸(26.26mL，458.27mmol)，加毕，加入NaBH(AcO)3(194.25g，916.54mmol)，加毕，保温搅拌反应1h。控温20℃以下，将反应体系用氨水溶液调节pH＝10，搅拌15min，抽滤，滤饼用纯化水打浆洗涤，抽滤，滤饼经60℃鼓风干燥，得223.5g式8化合物，收率95％。
步骤2(2R,3S,5R)-5-(5-甲磺酰基异吲哚啉-2-基)-2-(2,5-二氟苯基)四氢-2H-吡喃-3-胺(Ⅰ)粗品
Figure PCTCN2017097046-appb-000013
将式8化合物(202g，396.44mmol)加入1.2L N,N-二异丙基乙酰胺与纯化水的混合溶剂中(v/v＝1/1)，搅拌均匀，缓慢滴加硫酸溶液(1.1L，5.95mol)，加毕，反应体系加热至40℃并搅拌反应2h，然后，滴加氨水溶液调节溶液的pH＝约10，滴毕，搅拌1h，抽滤，滤饼用纯化水洗涤，并经60℃鼓风干燥，得到式I化合物的粗品133.1g，收率83％。
实施例3：(2R,3S,5R)-5-(5-甲磺酰基异吲哚啉-2-基)-2-(2,5-二氟苯基)四氢-2H-吡喃-3-胺(Ⅰ)的结晶
方法一
将130g粗品加入650mL无水甲醇中，加热溶解得到澄清溶液，用活性炭脱色，趁热抽滤，然后滤液降至室温并析晶2h，抽滤，滤饼经60℃鼓风干燥，得到94.2g结晶，收率72.4％。
方法二
将无水甲醇(26.8L)加热至回流，然后加入粗品(670g)，溶解后过滤，滤液降温至-5℃～5℃并析晶1h，抽滤，滤饼用无水甲醇淋洗，并于50℃～60℃鼓风干燥10～12h，得到528g结晶，收率78％。
甲醇作为结晶溶剂以方法一制备的结晶的典型XRD图谱如图1所示以及DSC图谱如图2所示。
甲醇作为结晶溶剂以方法二制备的结晶的另一典型XRD图谱如图3所示以及DSC图谱如图4所示。
参照实施例3中的方法一或方法二的类似步骤，更换结晶溶剂，所得的对应的结晶如下表。
编号 溶剂 XRD图谱
方法三 乙腈 图5
方法四 乙醇 图6
在实施例3中，采用不同的结晶溶剂得到的式I化合物的结晶均属于同一种晶型。
实施例4：(2R,3S,5R)-5-(5-甲磺酰基异吲哚啉-2-基)-2-(2,5-二氟苯基)四氢-2H-吡喃-3-胺的磷酸盐的结晶
Figure PCTCN2017097046-appb-000014
将式Ⅰ化合物(7g，17.1mmol)加入350mL乙醇溶剂中，加热回流并溶清后，加入1.0g活性炭脱色10min，趁热抽滤，然后向滤液中滴加磷酸溶液(1.8mL，34.2mmol)，析出大量白色固体，滴毕，反应体系降至室温并搅拌2h，继续析出固体，抽滤，固体经鼓风干燥过夜得8.3g式Ⅰ化合物的磷酸盐(1:1，经滴定法测得)的结晶，收率95.8％，纯度99.12％。所得产品的典型XRD图谱如图7所示以及DSC图谱如图8所示。
实施例5：(2R,3S,5R)-5-(5-甲磺酰基异吲哚啉-2-基)-2-(2,5-二氟苯基)四氢-2H-吡喃-3-胺的富马酸盐
Figure PCTCN2017097046-appb-000015
将式Ⅰ化合物(7g，17.1mmol)加入350mL乙醇溶剂中，加热回流并溶清后，加入1.0g活性炭脱色10min，趁热抽滤，向滤液中加入富马酸(3.97g，34.2mmol)，加毕，将反应体系降至室温后在冰水浴中搅拌2h，析出固体，抽滤，固体经50℃鼓风干燥6h，得到7.2g式Ⅰ化合物富马酸盐(1:0.5，经滴定法测得)，收率80.3％，纯度97.7％。所得产品的典型XRD图谱如图9所示以及DSC图谱如图10所示。
实验例1：式Ⅰ化合物的结晶的稳定性试验
依据《原料药与药物制剂稳定性试验指导原则》(中国药典2010版附录XIXC)，评价本申请的式Ⅰ化合物的结晶在40℃、60℃、92.5％高湿和光照条件下的稳定性，分别于第5天或10天取样测试，结果与初始结果进行比较，检测结果如以下表1所示。
表1 式Ⅰ化合物的结晶的稳定性试验结果
Figure PCTCN2017097046-appb-000016
Figure PCTCN2017097046-appb-000017
实验例2：结晶形式的式Ⅰ化合物及其盐的药代动力学
雄性Beagle犬，体重10±1kg。适应7天后，随机分为3组，每组3只，分别给予2mg/kg剂量(以游离形式计)的式Ⅰ化合物的结晶、式Ⅰ化合物的富马酸盐的结晶和式Ⅰ化合物的磷酸盐的结晶。
雄性Beagle犬在给药前禁食约12h，并自由饮水。给药后保持禁食4小时。将受试比格犬分别在给药后0.25、0.5、1、2、4、6、8、10、24、30、48和72h于前肢静脉取血约0.8mL。置于EDTA-K2离心管中，4℃保存，并在取血后0.5h内在4℃下以4000rpm的速度离心10min以分离血浆。收集全部血浆后在1h内存放于-20℃下待测。
分别吸取50μL的待测血浆样品和标准曲线样品，加入300μL含内标的甲醇溶液，振荡混匀5min，以13000rpm离心10min后，取上清液80μL，吸取上清液5μL用于LC/MS/MS测定，记录色谱图。
通过比格犬的体内药物动力学实验评价本申请的式Ⅰ化合物及其盐的口服生物利用度。
式Ⅰ化合物及其盐的药代参数如下表所示。
表2 式Ⅰ化合物及其盐的药代动力学实验结果
Figure PCTCN2017097046-appb-000018
实验例3：对DPP-IV酶抑制活性的测定
本申请采用以下方法来测定本申请的式I化合物对血浆中DPP-IV酶的抑制活性，该抑制活性采用IC50来表示，IC50即DPP-IV酶的活性被抑制50％时的化合物的浓度。
材料和方法：
材料：
a.白色384孔板(Perkin Elmer，Catalog No.607290/99)
b.HEPES缓冲液：用1M HEPES缓冲液(Invitrogen，Catalog No.15630-080)配制50ml的0.5M HEPES缓冲液：取1M HEPES缓冲液25mL，加适量ddH2O(重蒸水)，用NaOH调节pH至7.8，最后加ddH2O至50ml。
c.大鼠血浆：大鼠眼眶取血，加肝素抗凝，以4000rpm离心10分钟，取上清血浆作为DPP-IV的酶源。
d.DPP-IV酶反应底物H-Gly-Pro-AMC(甘氨酸-脯氨酸-7-氨基-4-甲基香豆素)，由本公司自己合成，溶解于DMSO至100mM母液。
e.1M MgCl2
f.1.5M NaCl
g.10％BAS
h.DMSO(二甲基亚砜)
i.ddH2O
j.待测化合物：阳性对照化合物Omarigliptin和本申请的式I化合物
按以下操作顺序进行：
1.配制DPP-IV酶反应缓冲液：50mM HEPES(pH＝7.8)，80mM MgCl2，150mM NaCl，1％BSA，放置于冰上备用；
2.用DMSO将待测化合物从10mM稀释至1mM(终操作浓度的100倍)，然后在96孔板上进行3倍梯度稀释，共11个浓度，第12孔中加入DMSO作为空白对照，然后用酶反应缓冲液稀释25倍至终工作浓度的4倍待用；
3.解冻DPP-IV酶反应底物H-Gly-Pro-AMC，用酶反应缓冲液稀释至160μM(4倍工作浓度)，置于冰上备用；
4.解冻大鼠血浆，用酶反应缓冲液稀释100倍(2倍工作浓度)置于冰上备用；
5.在384孔板中加入5μL的4倍浓度的待测化合物，然后加入10μL的大鼠血浆(2倍工作浓度)，离心混匀；
6.加入5μL酶反应底物H-Gly-Pro-AMC(4倍工作浓度)，离心混匀，用封膜封住384孔板；
7.在孵育箱(22-23℃)中孵育1小时；
8.使用FlexStationI3(Molecular devices)酶标仪读取反应的荧光信号：380nm激发，读取460nm波长的发射光谱；
9.化合物对DPP-IV酶的抑制IC50的生成：使用GraFit6软件计算化合物的IC50值。
表3 式I化合物的DPP-IV酶抑制活性
Figure PCTCN2017097046-appb-000019
实验例4：CYP酶系的抑制IC50的测定
本申请采用以下方法来测定式I化合物对CYP酶系的抑制IC50的测定。
将在-80℃下冻存的人肝微粒体置于冰上融化，刚刚融化后立即取100μL至60℃的恒温振荡箱中以100rpm孵育(1小时)，剩余的肝微粒体立即在-80℃下冻存。1小时后，取出100μL灭活的肝微粒体，加入400μL磷酸盐缓冲液混匀成4mg/mL灭活的肝微粒体溶液；另将在-80℃下冻存的人肝微粒体置于冰上融解，刚刚融解后取出100μL加入400μL磷酸盐缓冲液混匀成 4mg/mL肝微粒体溶液，按照如下表4平行配制阳性对照及测试品和阴性对照组孵育混合液。
表4 阳性对照及测试品和阴性对照组孵育混合液
Figure PCTCN2017097046-appb-000020
将混合液置于37℃的恒温振荡箱中以100rpm孵育5分钟。
取2.5μL测试品或阳性对照工作液(阴性对照组加入测试品工作液)，加入91.5μL孵育混合液、6μL NADPH溶液并涡旋启动反应，将溶液置于37℃的恒温振荡箱中以100rpm孵育，孵育时间如下表5所示。
表5 孵育时间
Figure PCTCN2017097046-appb-000021
孵育相应时间后，加入200μL内标液(CYP2C19的内标液为100ng/mL氯霉素的乙腈溶液、其余内标液为250ng/mL华法林、500ng/mL普萘洛尔的乙腈溶液)终止反应，终止反应的样品于12000rpm离心10分钟，取上清液进样检测。
处理数据用Analyst1.4.2或等同软件。检测积分来确保所有的峰适当积分并且必要时调整积分参数。
分析物的定量被定义为分析物的峰面积与内标的峰面积之比。分析使用液相色谱-串联质谱联用方法(LC-MS/MS)。使用Graphpad Prism(版本5.03)软件计算IC50等参数，结果如下表6所示。
表6 式I化合物对CYP酶系的抑制IC50(μM)
Figure PCTCN2017097046-appb-000022
实验例5：肝微粒体代谢稳定性
本申请采用以下方法来测定式Ⅰ化合物的肝微粒体代谢稳定性。
取8μL人肝微粒体(20mg/mL)、20μL的NADPH和368μL 0.1M的磷酸盐缓冲液混合，在37℃中预孵育5分钟。分别加入4μL分析工作液(测试品或阳性对照)，在37℃下预孵育0、10、20、30、45和60分钟时，取50μL的孵育液加入150μL含内标(0.25M华法林)的乙 腈；取4μL大鼠肝微粒体(20mg/mL)、10μL的NADPH和184μL 0.1M的磷酸盐缓冲液混合，在37℃中预孵育5分钟。分别加入2μL分析工作液(测试品或阳性对照)，在37℃下预孵育0、10、20、30、45和60分钟时，取20μL的孵育液加入180μL含内标(0.25M华法林)的乙腈。所有样品涡旋后于4000rpm离心15min，然后取150μL上清液加入96孔进样板。取5μL进LC/MS/MS系统检测。分析色谱柱为C18 1.7μm 2.1×50mm(Waters)。检测使用三重四极杆质谱(API4000，AB公司)。在正离子模式下检测CT-1225的峰面积与内标峰面积的比值。半衰期值为测试品/内标的峰面积与时间比值。结果数据如下表7所示。
表7 式I化合物及参考化合物的肝微粒体代谢稳定性
Figure PCTCN2017097046-appb-000023
实验例6：单次给药对ob/ob小鼠血清DPP-IV活性的抑制作用
36只雌性ob/ob小鼠，随机分为6组，每组6只，分别为模型对照组、1mg/kg式I化合物组、3mg/kg式I化合物组、10mg/kg式I化合物组、30mg/kg式I化合物组和阳性对照30mg/kg Omarigliptin组。各组小鼠口服给予不同剂量的式I化合物或Omarigliptin，模型对照组口服给予0.25％CMC-Na，于给药前及给药后2、4、10、24、34、48、58、72和96h时取血，分离血清，测定血清DPP-IV的活性。
血清DPP-IV的活性测定方法：取5μL血清样品，加入80mM MgCl2缓冲液45μL，混匀，室温中预孵育5分钟，加入10μL 0.1mM反应底物Gly-Pro-7-AMC及40μL缓冲液，避光，混匀后每间隔3分钟进行一次荧光测定(激发波380nm/发射波460nm)，直到18分钟，共测6次，根据测定结果减去空白本底后做时间-荧光值曲线，斜率为活力值，以给药前0h时血清DPP-IV的活力值为100％，按下列公式计算给药后各时间点的血清DPP-IV的比活力值，比活力值(％)＝给药后活力值/给药前活力值×100％。
试验结果：ob/ob小鼠单次口服给予不同剂量的式I化合物后，血清DPP-IV的活性受到显著抑制，并呈剂量-时间依赖性。1mg/kg式I化合物给药后10h内小鼠血清DPP-IV的活性抑制率高于70％；3mg/kg式I化合物给药后24h内血清DPP-IV的活性抑制率高于70％；10mg/kg式I化合物给药后34h内血清DPP-IV的活性抑制率高于70％；以及30mg/kg式I化合物给药后72h内血清DPP-IV的活性抑制率均维持在70％以上。阳性对照30mg/kg Omarigliptin组小鼠给药后34h内血清DPP-IV的活性抑制率高于70％。
表8 式I化合物及参考化合物对小鼠血清DPP-IV活性的抑制结果
Figure PCTCN2017097046-appb-000024

Claims (19)

  1. 式Ⅰ化合物的结晶，
    Figure PCTCN2017097046-appb-100001
    其在X-射线衍射图谱中具有2θ＝16.4°、21.8°、25.3°、26.0°±0.2°的衍射峰；典型地，其具有2θ＝16.4°、19.4°、21.2°、21.8°、25.3°、26.0°±0.2°的衍射峰；更典型地，其具有2θ＝13.0°、16.4°、18.5°、19.4°、21.2°、21.8°、25.3°、26.0°±0.2°的衍射峰。
  2. 根据权利要求1所述的式Ⅰ化合物的结晶，用DSC表征时，其起始温度为193.3±5℃，以及峰值温度为195.2±5℃。
  3. 根据权利要求1或2所述的式Ⅰ化合物的结晶，其X-射线衍射峰具有如下特征：
    Figure PCTCN2017097046-appb-100002
  4. 制备权利要求1-3中任一项所述的式Ⅰ化合物的结晶的方法，包括
    1)使所述式Ⅰ化合物溶于结晶溶剂中；
    2)冷却析晶、过滤；
    其中，所述结晶溶剂选自甲醇、乙醇、丙醇、异丙醇、正丁醇、异丁醇、叔丁醇、丙酮、丁酮、乙酸乙酯、乙腈、二氯甲烷、甲苯、二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、乙酸异丙酯或它们的混合溶剂；优选甲醇、乙醇、丙醇、异丙醇、乙酸乙酯、乙腈、二氯甲烷或它们的混合溶剂；更优选甲醇。
  5. 根据权利要求4所述的方法，其中相对于1g所述式Ⅰ化合物，加入的所述结晶溶剂的量为2～100mL，优选20mL、30mL、40mL、50mL、60mL、70mL、80mL、90mL或100mL，更优选20～60mL、20～40mL或30～50mL。
  6. 结晶组合物，其包含占所述结晶组合物的重量的50wt％以上，优选80wt％以上，更优选90％wt以上，最优选95wt％以上的权利要求1-3中任一项所述的式Ⅰ化合物的结晶。
  7. 式Ⅰ化合物的磷酸盐，
    Figure PCTCN2017097046-appb-100003
    其中所述式Ⅰ化合物与磷酸的摩尔量之比为1:0.5～2，优选1:0.5～1，更优选1:1。
  8. 权利要求7所述的式Ⅰ化合物的磷酸盐的结晶。
  9. 根据权利要求8所述的式Ⅰ化合物的磷酸盐的结晶，其在X-射线衍射图谱中具有2θ＝6.4°、11.9°、18.2°、21.7°、22.1°、22.9°、23.2°±0.2°的衍射峰；典型地，其具有2θ＝6.4°、11.9°、16.5°、17.5°、18.2°、18.6°、21.7°、22.1°、22.9°、23.2°±0.2°的衍射峰；更典型地，其具有2θ＝6.4°、10.1°、11.9°、16.5°、17.5°、18.2°、18.6°、19.8°、21.7°、22.1°、22.9°、23.2°、23.8°±0.2°的衍射峰。
  10. 结晶组合物，其包含占所述结晶组合物的重量的50wt％以上，优选80wt％以上，更优选90wt％以上，最优选95wt％以上的权利要求8或9所述的式Ⅰ化合物的磷酸盐的结晶。
  11. 制备权利要求8或9所述的式Ⅰ化合物的磷酸盐的结晶的方法，包括使所述式Ⅰ化合物与磷酸接触，然后从溶剂中分离，其中所述溶剂选自甲醇、乙醇、异丙醇、正丁醇、异丁醇、叔丁醇、二氯甲烷、乙腈、丙酮、乙酸乙酯、乙酸异丙酯、1,4-二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、甲苯或者两种以上的混合物，优选乙醇。
  12. 式Ⅰ化合物的富马酸盐，
    Figure PCTCN2017097046-appb-100004
    其中，所述式Ⅰ化合物与富马酸的摩尔量之比为1:0.5～2，优选1:0.5～1，更优选1:0.5。
  13. 权利要求12所述的式Ⅰ化合物的富马酸盐的结晶。
  14. 根据权利要求13所述的式Ⅰ化合物的富马酸盐的结晶，其在X-射线衍射图谱中具有2θ＝20.67°±0.2°的衍射峰。
  15. 结晶组合物，其包含占所述结晶组合物的重量的50wt％以上，优选80wt％以上，更优选90wt％以上，最优选95wt％以上的权利要求13或14所述的式Ⅰ化合物的富马酸盐的结晶。
  16. 制备权利要求13或14所述的式Ⅰ化合物的富马酸盐的结晶的方法，包括使所述式Ⅰ化合物与富马酸接触，然后从溶剂中析出，其中所述溶剂选自甲醇、乙醇、异丙醇、正丁醇、异丁醇、叔丁醇、二氯甲烷、乙腈、丙酮、乙酸乙酯、乙酸异丙酯、1,4-二氧六环、正庚烷、正己烷、甲基叔丁基醚、异丙醚、甲苯或者两种以上的混合物，优选乙醇。
  17. 药物组合物，包含治疗有效量的权利要求1-3中任一项所述的式Ⅰ化合物的结晶，或者权利要求6所述的结晶组合物，或者治疗有效量的权利要求7所述的式Ⅰ化合物的磷酸盐，或者权利要求8或9所述的式Ⅰ化合物的磷酸盐的结晶，或者权利要求10所述的结晶组合物，或者治疗有效量的权利要求12所述的式Ⅰ化合物的富马酸盐，或者权利要求13 或14所述的式Ⅰ化合物的富马酸盐的结晶，或者权利要求15所述的结晶组合物。
  18. 权利要求1-3中任一项所述的式Ⅰ化合物的结晶，或者权利要求6所述的结晶组合物，或者权利要求7所述的式Ⅰ化合物的磷酸盐，或者权利要求8或9所述的式Ⅰ化合物的磷酸盐的结晶，或者权利要求10所述的结晶组合物，或者权利要求12所述的式Ⅰ化合物的富马酸盐，或者权利要求13或14所述的式Ⅰ化合物的富马酸盐的结晶，或者权利要求15所述的结晶组合物，或者权利要求17所述的药物组合物在治疗或预防受益于DPP-IV抑制的疾病中的用途。
  19. 根据权利要求18所述的用途，其中所述受益于DPP-IV抑制的疾病选自胰岛素抵抗、高血糖症、Ⅱ型糖尿病、糖尿病性脂血异常、葡萄糖耐量减低症、禁食血浆葡萄糖减低症、代谢性酸中毒、酮症、食欲调节、肥胖症、各种癌症、神经系统病症或免疫系统病症，优选Ⅱ型糖尿病或肥胖症。
PCT/CN2017/097046 2016-08-12 2017-08-11 Dpp-iv长效抑制剂的结晶及其盐 WO2018028666A1 (zh)

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US16/324,884 US10822319B2 (en) 2016-08-12 2017-08-11 Crystal of DPP-IV long-acting inhibitor and salt
CA3033890A CA3033890A1 (en) 2016-08-12 2017-08-11 Crystal of dpp-iv long-acting inhibitor and salt thereof
RU2019105352A RU2753335C9 (ru) 2016-08-12 2017-08-11 Кристалл ингибитора DPP-IV длительного действия и его соли
CN201780044045.0A CN109874304B (zh) 2016-08-12 2017-08-11 Dpp-iv长效抑制剂的结晶及其盐
EP17838796.5A EP3498704A4 (en) 2016-08-12 2017-08-11 CRYSTAL OF A LONG-TERM DPP-IV INHIBITOR AND SALT THEREOF
KR1020197006819A KR20190039754A (ko) 2016-08-12 2017-08-11 Dpp-iv 지속형 저해제의 결정 및 이의 염
JP2019506673A JP6997760B2 (ja) 2016-08-12 2017-08-11 Ｄｐｐ－ｉｖ長時間作用型阻害剤の結晶及びその塩
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