WO2018192576A1 - Parp抑制化合物的制备方法、其中间体、非晶形、溶剂合物、药物组合物及应用 - Google Patents

Parp抑制化合物的制备方法、其中间体、非晶形、溶剂合物、药物组合物及应用 Download PDF

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WO2018192576A1
WO2018192576A1 PCT/CN2018/083969 CN2018083969W WO2018192576A1 WO 2018192576 A1 WO2018192576 A1 WO 2018192576A1 CN 2018083969 W CN2018083969 W CN 2018083969W WO 2018192576 A1 WO2018192576 A1 WO 2018192576A1
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compound
formula
solvent
solvate crystal
reaction
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PCT/CN2018/083969
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English (en)
French (fr)
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蔡鸿飞
焦育红
金远锋
翁玉芳
赵坤
祝盼虎
蔡群芳
李必文
钟万德
汪晶
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上海迪诺医药科技有限公司
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Priority claimed from CN201810354691.3A external-priority patent/CN108727391A/zh
Priority claimed from CN201810354222.1A external-priority patent/CN110386939B/zh
Application filed by 上海迪诺医药科技有限公司 filed Critical 上海迪诺医药科技有限公司
Publication of WO2018192576A1 publication Critical patent/WO2018192576A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings

Definitions

  • the present invention relates to a process for the preparation of a PARP inhibiting compound, an intermediate thereof, an amorphous form, a solvate, a pharmaceutical composition and use thereof.
  • PARP is an abbreviation for "poly(ADP-ribose) polymerase”. Tumor cells repair with PARP enzymes including DNA damage caused by chemotherapy. researchers are investigating whether drugs that inhibit PARP enzymes can also weaken this self-repair mechanism and make tumor cells more sensitive to treatment and accelerate tumor cell death.
  • PARP inhibitors are a family of pharmacological inhibitors of poly ADP ribose polymerase that are important for promoting DNA repair, controlling RNA transcription, regulating cell death, and immune response. Therefore, there are many indications for the development of PARP inhibitors, the most important indication being for the treatment of tumors. Several forms of BRCA-deficient tumor cells are more dependent on the repair function of PARP than normal cells, thus making PARP an attractive target for tumor therapy.
  • CN103237799A relates to a series of pyridazine-1(2H)-one derivatives which have an inhibitory effect on PARP enzymes, and its disclosure of paragraph [0105] of Reaction Scheme 3 mentions its disclosed pyridazine-1(2H)-one derivatives.
  • CN103237799A also discloses a compound of the formula A-5:
  • the preparation method of the compound is also provided, and the obtained compound is a viscous substance which is inferior in fluidity, is unfavorable for transportation and storage, or has poor stability, and its preparation method is not suitable for efficient separation of large production.
  • the present invention provides a process for the preparation of a PARP inhibiting compound, an intermediate thereof, an amorphous form, a solvate, a pharmaceutical composition and use thereof.
  • the preparation method of the PARP inhibitor compound of the invention has high purity and yield of the target compound, and is more suitable for industrial production.
  • the present invention has for the first time found an amorphous form of the compound of the formula A-5 which has good fluidity and is advantageous for transportation and storage. At the same time, the amorphous solubility, bioavailability and the like of the compound of the formula A-5 of the present invention are also good.
  • the present invention provides crystals of a solvate of a compound of formula A-5 which is fluid and stable and substantially non-wetting.
  • the present invention also provides a pharmaceutical composition comprising an amorphous or solvate crystal of a compound of the formula A-5, and the amorphous or solvate crystal and a pharmaceutical composition thereof for preparing a disease improved by inhibiting PARP activity The application of the drug.
  • the invention provides a preparation method of the pharmaceutical compound A for inhibiting PARP activity and improving disease, and the reaction process is as follows:
  • R 1 and R 2 are each independently hydrogen or halogen
  • R is methyl, trifluoromethyl, (tert-butyl), (cyclohexyl), (2-furyl) or (phenyl);
  • the R 1 is preferably fluorine or chlorine.
  • the R 2 is preferably hydrogen.
  • Said R 1 is preferably located in the para position of the methylene group of the benzyl group.
  • the preparation method of the compound A of the present invention comprises the following steps:
  • the compound of formula I is present in solid form in the reaction mixture and is readily separated by a porous medium;
  • the reaction produces Compound A; wherein M is hydrogen, potassium, lithium, sodium or ammonium, preferably sodium.
  • step (1) comprises the steps of reacting a compound of formula II with HCl in an organic solvent to produce a compound of formula I.
  • the compound of formula II is preferably mixed with an organic solvent and then mixed with HCl at 10-40 ° C (for example 10 ° C, 15 ° C or 25 ° C) to carry out the reaction to give a compound of formula I.
  • the amount of the substance to which HCl is added may be 5 to 20 times, preferably 8 to 15 times, more preferably 8 to 12 times the amount of the substance of the compound of the formula II.
  • the post-treatment operation may be further included.
  • the post-treatment operation can be a conventional post-treatment method for such reactions in the art.
  • the present invention preferably comprises the steps of: subjecting the reaction liquid after completion of the reaction to solid-liquid separation (the operation of the solid-liquid separation is preferably filtration), and drying the filter cake (preferably drying at a constant temperature of 50 ° C to constant weight) to obtain a compound of the formula I. .
  • the operation of the solid-liquid separation it is preferred to further include an operation of washing (the washing solvent, preferably a reaction solvent).
  • step (1) comprises the steps of: dissolving the compound of formula II in an organic solvent, and adding HCl in an amount of 5-20 times the amount of the compound of formula II at 10-40 ° C, stirring. The reaction was carried out for 2-24 hours, filtered and dried to give a solid.
  • the organic solvent in the step (1) may be a conventional organic solvent for such a reaction in the art, preferably a C 1-5 alcohol (for example, methanol, ethanol, n-propanol and isopropanol).
  • a C 1-5 alcohol for example, methanol, ethanol, n-propanol and isopropanol.
  • the amount of the organic solvent to be used may not be specifically limited as long as the compound of the formula II can be completely dissolved.
  • the amount of the substance to which HCl is added is 8 to 15 times the amount of the substance of the compound of the formula II; further preferably 8 to 12 times.
  • the HCl may be a hydrogen chloride gas or an aqueous hydrochloric acid solution.
  • the molar concentration of the aqueous hydrochloric acid solution may be a conventional molar concentration for such a reaction in the art, preferably 2 to 12 mol/L (2 to 12 N), and more preferably 6 to 12 mol/L (6 to 12 N).
  • the reaction temperature of the step (1) may be a conventional temperature for such a reaction in the art, preferably 10 to 40 ° C (for example, 20 ° C, 25 ° C, 40 ° C, etc.), more preferably 25 ° C to 40 ° C.
  • the progress of the reaction of the step (1) can be carried out by a conventional detection method in the art, and is generally used as a reaction end point when the compound of the formula II disappears.
  • the reaction time is preferably 2 to 24 hours, for example 2 hours, 10 hours or 20 hours.
  • step (2) comprises the steps of: reacting a compound of formula I with compound B under the action of a condensing agent in an organic solvent to produce compound A.
  • the organic solvent may be a conventional organic solvent of such a reaction in the art, preferably a halogenated alkane solvent such as dichloromethane.
  • the amount of the organic solvent to be used may not be specifically limited as long as it does not affect the progress of the reaction.
  • the amount of the compound of the formula I and the amount of the compound B may be a conventional amount for such a reaction in the art, and the ratio of the two substances is preferably 1:1 to 2:1.
  • the temperature of the reaction may be a conventional temperature for such a reaction in the art, preferably 10 to 30 ° C, more preferably 15 to 25 ° C.
  • the progress of the reaction can be monitored by conventional detection methods in the art, generally as the end of the reaction when the compound of formula I disappears.
  • the time of the reaction is preferably from 2 to 8 hours, for example 2, 4, 5 or 8 hours.
  • step (2) comprises the steps of: mixing a compound of formula I with an organic solvent, adding a condensing agent and compound B, and reacting to produce a compound of formula A.
  • the following operations may further be included: mixing the reaction liquid after the completion of the reaction with water, filtering, and post-treatment (for example, the filtrate is allowed to stand for stratification, the organic phase is washed and dried), and concentrated (preferably reduced). Concentration by pressure) gave Compound A.
  • the reaction step (2) comprises the steps of: adding the solid obtained in the step (1) (the compound of the formula I) to a reaction vessel containing a dichloromethane, adding a condensing agent and a compound B, and reacting 2-8 After a few hours, it was filtered, worked up, and concentrated to give Compound A.
  • the condensing agent may be a conventional condensing agent for such reactions in the art, preferably HOBT (1-hydroxybenzotriazole), EDCI (1-(3-dimethylaminopropyl)- 3-ethyl-carbodiimide hydrochloride), or a mixed system of HOBT and EDCI.
  • the amount of the condensing agent may be a conventional amount for such a reaction in the art, and it is preferably a ratio of the substance to the compound of the formula I in an amount of from 1:1 to 5:1.
  • the condensing agent is a mixed system of HOBT and EDCI, and the ratio of the amount of the HOBT to the EDCI is 1:0.8-1:1.5.
  • the compound A is preferably one of the following structures:
  • the invention also provides a compound of formula I (wherein the compound of formula I may be an intermediate for the manufacture of a medicament for the inhibition of PARP activity to improve disease):
  • R 1 , R 2 and heterocyclic group The definition is as described above.
  • the invention also provides a process for the preparation of a compound of formula I, which comprises the steps of:
  • the compound of formula I is present in solid form in the reaction mixture and is readily separated by a porous medium;
  • R 1 , R 2 and heterocyclic group The definition is as described above.
  • the compound of the formula II of the present invention can be prepared by the method described in the example of CN103237799A, using HATU/DIPEA/DMF, but this method produces a small amount of by-product tetramethylurea which is difficult to separate or remove.
  • the preparation of the compound of the formula II of the present invention preferably comprises the steps of: reacting a compound of the formula III and a compound C under the action of a condensing agent and an acid binding agent to obtain a compound of the formula II; wherein the condensing agent is HOBT (1-hydroxybenzotriazole), or a mixed system of HOBT and EDCI (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride);
  • the compound C is R 1 , R 2 and heterocyclic groups
  • the definition is as described above.
  • the preparation of the compound of the formula II is preferably carried out in the presence of a solvent.
  • the solvent may be a conventional solvent for such a reaction in the art, preferably a halogenated alkane solvent such as dichloromethane.
  • the amount of the solvent to be used may not be specifically limited as long as it does not affect the progress of the reaction.
  • the condensing agent may be used in an amount conventionally used in the field of such a reaction condensing agent, preferably in a ratio of 1.5 to 1-3:1 to the amount of the compound of the compound of the formula III.
  • the condensing agent is a mixed system of HOBT and EDCI
  • the ratio of the amounts of the substances of HOBT and EDCI is preferably 1:0.8-1:1.5, for example 1:1.
  • the acid scavenger can be a conventional acid scavenger for such reactions in the art, such as ethylenediamine and/or N,N-diisopropylethylamine.
  • the amount of the acid scavenger may be a conventional amount for such a reaction in the art, and the ratio of the amount of the acid to the amount of the condensing agent is preferably from 0.5:1 to 1.5:1.
  • the temperature of the reaction is preferably from 10 to 40 °C.
  • the progress of the reaction can be monitored by conventional detection methods in the art, for example, by TLC, when the compound of formula III disappears, the end point of the reaction.
  • the time of the reaction is preferably from 2 to 24 hours, for example 2 hours, 4 hours or 24 hours.
  • the compound of formula III, compound B and a condensing agent are mixed, and then an acid binding agent is added to carry out the reaction.
  • the post-treatment operation may be a conventional method and condition for such post-treatment in the art, and the present invention preferably includes the following steps: separating and concentrating the reaction liquid after completion of the reaction (for example, concentration under reduced pressure).
  • the reaction liquid after completion of the reaction is subjected to extraction (the organic solvent for extraction is preferably a halogenated alkane solvent, and after extraction, the organic phase may be dried, for example, dried over anhydrous magnesium sulfate or anhydrous sodium sulfate), and then the organic phase is subjected to an organic phase. concentrate.
  • the method for preparing the compound of formula II comprises the steps of: adding formula III, compound C to the reaction vessel in a certain ratio, adding 1-hydroxybenzotriazole, 1-(3-dimethylamino) Propyl)-3-ethylcarbodiimide hydrochloride, temperature control 10-40 ° C, dropwise addition of triethylamine or N, N-diisopropylethylamine, temperature control 10-40 ° C, stirring reaction, 2-24 hours, separated and concentrated to obtain a solid;
  • the compound C is R 1 , R 2 and heterocyclic groups
  • the definition is as described above.
  • reaction vessel Before adding the compound of the formula III and the compound C to the reaction vessel in a certain ratio, it is preferred to first add a reaction solvent to the reaction vessel.
  • HOBT and EDCI may be simultaneously added to the reaction system, or may be sequentially added to the reaction system.
  • the amount ratio of the compound of the formula III to the substance of the compound C is from 1:1 to 1:3.
  • the formula III may be 2-fluoro-5-((4-oxo-3,4-dihydropyridazin-1-yl)methyl)benzoic acid.
  • the invention also provides a compound of Formula I-a, or a pharmaceutically acceptable salt thereof,
  • R 1 , R 2 and a heterocyclic group The definition is as described above.
  • the pharmaceutically acceptable salt is hydrochloride, p-toluenesulfonate, tartrate, maleate, lactate, methanesulfonate, sulfate, phosphate, lemon
  • the acid salt or acetate is further preferably a tosylate salt, a hydrochloride salt or a tartrate salt; the pharmaceutically acceptable salt of the present invention can be synthesized by a conventional chemical method.
  • formula I-a is of the following formula I-a-1:
  • the pharmaceutically acceptable salt of the compound of formula I-a-1 is the hydrochloride salt.
  • the compound of formula Ia can be used not only for the preparation of a pharmaceutical compound A which inhibits PARP enzymatic activity, but the applicant demonstrates, by in vitro experiments, a compound of formula Ia (e.g., formula Ia-1) or a pharmaceutically acceptable salt thereof itself It also has good PARP enzyme inhibitory activity.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I-a, or a pharmaceutically acceptable salt thereof, of the invention, together with a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition of the present invention may be formulated into a pharmaceutical preparation together with some commonly used excipients in the pharmaceutical field, and the preparation may be a tablet, a capsule, an injection, an aerosol, a suppository, a film, a pill, or a topical preparation. Agent, ointment, etc.
  • the invention also provides the use of a compound of formula I-a (e.g., a compound of formula I-a-1), or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease ameliorated by inhibition of PARP activity.
  • a compound of formula I-a e.g., a compound of formula I-a-1
  • a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a disease ameliorated by inhibition of PARP activity.
  • the invention also provides a method of inhibiting a disease that is ameliorated by PARP activity comprising administering to a mammal in need thereof an effective amount of a compound of formula Ia (e.g., a compound of formula Ia-1) of the invention or a pharmaceutically acceptable thereof Salt.
  • a compound of formula Ia e.g., a compound of formula Ia-1 of the invention or a pharmaceutically acceptable thereof Salt.
  • the present invention also provides an amorphous form of the compound of the formula A-5, which has no crystal diffraction peak in the XRPD pattern.
  • the content of the amorphous formula A-5 compound of the compound of the formula A-5 is preferably between 98.0% and 102.0%, further preferably between 99.0 and 100.0%;
  • the HPLC purity of the crystal form is not less than 99.5%, and the total amount is not more than 0.5%.
  • the present invention also provides a method for preparing an amorphous form of the PARP inhibitor compound of the formula A-5, which comprises the steps of: mixing a mixed solution of the compound of the formula A-5 and a benign solvent with a non-degradable solvent;
  • the volume-to-mass ratio of the benign solvent to the compound of the formula A-5 is from 1 mL/g to 10 mL/g, and the volume-to-mass ratio of the poor organic solvent to the compound of the formula A-5 is from 10 mL/g to 100 mL/g.
  • the benign solvent generally means that the compound of the formula A-5 has a good solubility in the benign solvent, that is, when the volume ratio of the benign solvent to the compound of the formula A-5 is 1 mL/g to 10 mL. At /g, the compound of formula A-5 is completely soluble in the benign solvent.
  • the poor solvent generally means a mixture of the compound of the formula A-5 and a benign solvent, and after mixing with the poor solvent, the compound of the formula A-5 can be precipitated as a solid, that is, when the defect When the volume-mass ratio of the solvent to the compound of the formula A-5 is more than 10 mL/g, the compound of the formula A-5 cannot be completely dissolved in the defective solvent.
  • the benign solvent is one of ethanol, methanol, acetone, methyl ethyl ketone, ethyl acetate, isobutyl acetate, isopropanol, n-propanol, tetrahydrofuran, toluene and dichloromethane or A variety.
  • the undesirable solvent is one of isopropyl ether, n-hexane, n-pentane, n-heptane, n-octane, methyl tert-butyl ether, petroleum ether and cyclohexane or A variety.
  • the temperature of the mixing is preferably -10 to 40 ° C (for example, 20 to 40 ° C, or 25 to 30 ° C).
  • the mixing operation may be further included at the same time or after the end of the mixing operation.
  • the temperature of the mixed liquid at the time of stirring is preferably -10 to 40 ° C, more preferably 20 to 40 ° C, for example, 25 to 35 ° C.
  • the time for continuing the stirring may not be specifically limited, so that no solid precipitation is allowed.
  • the post-treatment operation may be further included.
  • the post-treatment operation may be a conventional operation of such post-treatment in the art, and preferably includes the following steps: the mixture obtained after the stirring is continued, subjected to solid-liquid separation, and dried.
  • the operation of the solid-liquid separation is preferably filtration.
  • the filter cake obtained after the solid-liquid separation is preferably washed.
  • the washing solvent is preferably the above-mentioned poor solvent.
  • the drying is preferably dried under reduced pressure.
  • a mixed solution of the compound of the formula A-5 and a benign solvent is preferably added to the poor solvent.
  • the manner of addition is preferably added dropwise.
  • the speed of the dropwise addition may not be specifically limited as long as it does not affect the precipitation of the compound of the formula A-5.
  • the amorphous form of the compound of the formula A-5 comprises the steps of: dissolving the compound of the formula A-5 with a benign solvent, dropping into a poor solvent, stirring, precipitating the solid, and filtering.
  • the volume ratio of the benign solvent to the compound of the formula A-5 is from 1 mL/g to 10 mL/g, and the volume-to-mass ratio of the poor solvent to the compound of the formula A-5 is from 10 mL/g to 100 mL/g.
  • the temperature of the precipitated solid is from -10 to 40 ° C, preferably from 20 to 40 ° C, for example from 25 to 35 ° C.
  • the HPLC purity of the compound of the formula A-5 may be 70% or more, preferably 70% to 99%.
  • the HPLC purity of the compound of the formula A-5 is 100%, the specific amorphous form of the present invention can also be obtained by the production method of the present invention.
  • the invention also provides an amorphous pharmaceutical composition comprising the compound of formula A-5.
  • the pharmaceutical composition preferably comprises an amorphous and pharmaceutically acceptable adjuvant of the compound of formula A-5.
  • the present invention also provides the use of an amorphous form of the compound of the formula A-5 or a pharmaceutical composition thereof for the preparation of a medicament for amelioration of a disease by inhibiting PARP activity.
  • the invention also provides a method of inhibiting a disease that is ameliorated by PARP activity, the method comprising administering to a patient in need thereof a therapeutically effective amount of an amorphous or pharmaceutical composition of a compound of formula A-5.
  • the invention also provides a solvate crystal of a compound of formula A-5:
  • the solvate crystal is an ester solvate crystal of the compound of formula A-5; preferably an ethyl acetate solvate crystal of the compound of formula A-5, formic acid of a compound of formula A-5 Ethyl ester solvate crystals, methyl acetate solvate crystals of the compound of formula A-5 or n-propyl formate solvate crystals of the compound of formula A-5.
  • the infrared absorption spectrum of the solvate crystal has an absorption band, expressed as a reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2 cm -1 ), which has an absorption peak at 1668, 1636, 1496, 1449, and 804.
  • the solvate crystal is ethyl acetate solvate crystal A of the compound of formula A-5, in the X-ray powder diffraction pattern of crystal A, at a diffraction angle of 2 ⁇ ⁇ 0.2° of 8.193. , having characteristic peaks at 14.137, 17.799, 19.483, 20.062, 20.861, and 23.837; preferably at diffraction angles 2 ⁇ ⁇ 0.2° of 8.193, 9.507, 11.141, 13.547, 14.137, 15.935, 16.732, 17.799, 19.483, 20.062, 20.861, and 23.837 Has a characteristic peak.
  • the data of the X-ray diffraction powder diffraction pattern of the ethyl acetate solvate crystal A of the compound of the formula A-5 is shown in Table 1 below:
  • the ethyl acetate solvate crystal A of the compound of formula A-5 has an X-diffraction powder diffraction pattern as shown in FIG.
  • the infrared absorption spectrum of the ethyl acetate solvate crystal A of the compound of the formula A-5 has an absorption band expressed by the reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2 cm -1 ), It has absorption peaks at the following positions: 1166.67, 1635.93, 1 495.51, 1450.38, and 805.05, and further preferably has absorption peaks at the following positions: 3345.84, 3204.48, 1173.55, 1166.67, 1635.93, 1495.51, 1450.38, 800.55, and 682.88.
  • the ethyl acetate solvate crystal A of the compound of formula A-5 has an infrared spectrum as shown in FIG.
  • the ethyl acetate solvate crystal A of the compound of the formula A-5 is preferably subjected to a gas phase detection of ethyl acetate in an amount of from 12 to 20%.
  • the solvate crystal is ethyl formate solvate crystal B of the compound of formula A-5, in the X-ray powder diffraction pattern of crystal B, the diffraction angle is 2 ⁇ ⁇ 0.2° is 8.471.
  • the data of the X-ray diffraction powder diffraction pattern of the ethyl formate solvate crystal B of the compound of the formula A-5 is shown in Table 2 below:
  • the ethyl formate solvate crystal B of the compound of formula A-5 has an X-diffraction powder diffraction pattern as shown in FIG.
  • the infrared absorption spectrum of the ethyl formate solvate crystal B of the formula A-5 has an absorption band represented by a reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2 cm -1 ), which There are absorption peaks at the following positions: 1667.77, 1336.87, 1 946.32, 1448.70, and 804.45; and further preferably have absorption peaks at the following positions: 3447.16, 3190.04, 1723.60, 1667.77, 1336.87, 1946.32, 1448.70, 804.45, and 771.41.
  • the ethyl formate solvate crystal B of the compound of formula A-5 has an infrared spectrum as shown in FIG.
  • the ethyl formate solvate crystal B of the formula A-5 is preferably subjected to gas phase detection of ethyl formate in an amount of from 5 to 15%.
  • the solvate crystal is a methyl acetate solvate crystal C of the compound of formula A-5, in the X-ray powder diffraction pattern of the crystal C, at a diffraction angle of 2 ⁇ ⁇ 0.2° of 8.454.
  • the methyl acetate solvate crystal C of the compound of formula A-5 has an X-diffraction powder diffraction pattern as shown in FIG.
  • the infrared absorption spectrum of the methyl acetate solvate crystal C of the formula A-5 has an absorption band, expressed as a reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2 cm -1 ), which There are absorption peaks at the following positions: 1166.61, 16307.02, 1946.34, 1449.40, and 804.71; further preferably, there are absorption peaks at the following positions: 3447.25, 3189.85, 1744.78, 1668.61, 1670.02, 1492.34, 1449.40, 804.71, and 771.49.
  • the methyl acetate solvate crystal C of the compound of formula A-5 has an infrared spectrum as shown in FIG.
  • the methyl acetate solvate crystal C of the formula A-5 is preferably subjected to a gas phase detection of methyl acetate in an amount of from 5 to 15%.
  • the solvate crystal is a n-propyl formate solvate crystal D of the compound of formula A-5, in the X-ray powder diffraction pattern of the crystal D, at a diffraction angle of 2 ⁇ ⁇ 0.2° of 8.152.
  • the n-propyl formate solvate crystal D of the compound of formula A-5 has an X-diffraction powder diffraction pattern as shown in FIG.
  • the infrared absorption spectrum of the n-propyl formate solvate crystal D of the formula A-5 has an absorption band, expressed as a reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2 cm -1 ), It has absorption peaks at the following positions: 1166.42, 1163.53, 1496.39, 1449.31, and 804.41, and further preferably has absorption peaks at 344.7.01, 3203.32, 1726.16, 1668.42, 1163.53, 1946.39, 1449.31, 804.41, and 772.26.
  • the n-propyl formate solvate crystal D of the compound of formula A-5 has an infrared spectrum as shown in FIG.
  • the n-propyl formate solvate crystal D of the compound of the formula A-5 is preferably subjected to a gas phase detection of a content of n-propyl formate of from 12 to 20%.
  • the present invention also provides a process for the preparation of a solvate crystal of the PARP inhibitor of the formula A-5, which comprises the steps of: precipitating a compound of the formula A-5 from a solvent or a solvent-containing solution.
  • the compound of formula A-5 is amorphous (i.e., amorphous).
  • the HPLC purity of the compound of the formula A-5 may be 70% or more, preferably 99% or more.
  • the compound of formula A-5 when the compound of formula A-5 is amorphous (i.e., amorphous), it is preferably prepared by the amorphous preparation of the compound of formula A-5 above.
  • the solute in the solvent-containing solution, is a solvent in the solvate, preferably an ester solvent, and the solvent is an organic solvent other than the solvent in the solvate.
  • the organic solvent is preferably an alcohol solvent (for example, methanol), a nitrile solvent (for example, acetonitrile), an ether solvent (for example, tetrahydrofuran and/or 1,4-dioxane), and a halogenated alkane solvent (for example, dichloromethane).
  • an alcohol solvent for example, methanol
  • a nitrile solvent for example, acetonitrile
  • an ether solvent for example, tetrahydrofuran and/or 1,4-dioxane
  • a halogenated alkane solvent for example, dichloromethane
  • the solvent in the solvent-containing solution i.e., the solvent in the solvate
  • a process for the preparation of an ester solvate crystal of the compound of the formula A-5 which comprises the steps of: precipitating a compound of the formula A-5 from an ester solvent or a solution containing an ester solvent .
  • a process for the preparation of an ester solvate crystal of a compound of formula A-5 which comprises the steps of: mixing a compound of formula A-5 with an ester solvent or a solution containing an ester solvent, at 0 The crystal was stirred and stirred at ⁇ 40 ° C to obtain an ester solvate crystal of the compound of the formula A-5; and the volume ratio of the ester solvent to the compound of the formula A-5 was from 4 mL/g to 30 mL/g.
  • the ester solvent may be a conventional ester solvent in the art, preferably ethyl formate, methyl acetate, n-propyl formate, methyl propionate, ethyl acetate, n-propyl acetate. , ethyl propionate, n-butyl formate, methyl butyrate, n-butyl acetate, ethyl butyrate, n-butyl propionate or propyl butyrate; more preferably ethyl acetate, ethyl formate, methyl acetate Or n-propyl formate.
  • a conventional ester solvent in the art preferably ethyl formate, methyl acetate, n-propyl formate, methyl propionate, ethyl acetate, n-propyl acetate.
  • the ester solvent in the solution containing the ester solvent, is the same as described above; the solvent in the solution is preferably an organic solvent, further preferably an alcohol solvent (for example, methanol), a nitrile One or more of a solvent such as acetonitrile, an ether solvent such as tetrahydrofuran and/or 1,4-dioxane, and a halogenated alkane solvent such as dichloromethane.
  • an organic solvent for example, methanol
  • a nitrile One or more of a solvent such as acetonitrile, an ether solvent such as tetrahydrofuran and/or 1,4-dioxane, and a halogenated alkane solvent such as dichloromethane.
  • the ester solvent-containing solution has a mass fraction of the ester solvent of not less than 50%; further preferably not less than 85%; still more preferably not less than 95%.
  • the ester solvent-containing solution is preferably 195% (w/w) ethyl acetate and 5% (w/w) tetrahydrofuran; 290% (w/w) ethyl acetate and 10% (w/w) acetonitrile; %(w/w) ethyl acetate and 15% (w/w) tetrahydrofuran; 450% (w/w) ethyl acetate and 50% (w/w) acetonitrile; 590% w/w methyl acetate and 10% w/w acetonitrile or 650% (w/w) n-propyl formate and 50% (w/w) acetonitrile.
  • the volume ratio of the ester solvent or the ester solvent-containing solution to the compound of the formula A-5 is from 4 mL/g to 30 mL/g, more preferably from 6 mL/g to 10 mL/g.
  • the crystallization is carried out at a temperature of from 15 to 25 °C.
  • the filtration, washing and drying operations may be further included.
  • the organic solvent for washing is preferably an ester solvent in the ester solvent of the compound of the formula A-5.
  • the drying is usually carried out at 30-70 ° C, further preferably 30-60 ° C.
  • the drying time can be a dry time for the products of the art, generally until the weight of the product is substantially constant.
  • the drying operation may be blast dry or dried under reduced pressure as needed.
  • the method for preparing an ester solvate crystal of the compound of the formula A-5 comprising the steps of: dissolving the compound of the formula A-5 in an ester solvent or a solution containing an ester solvent, The mixture is stirred and crystallized at 0 to 40 ° C, filtered, and dried to obtain an ester solvate crystal of the formula A-5; the volume ratio of the ester solvent to the compound of the formula A-5 is 4 mL/g to 30 mL/ g.
  • the invention also provides a pharmaceutical composition comprising a solvate crystal of the compound of formula A-5.
  • the pharmaceutical composition preferably comprises a solvate crystal of the compound of formula A-5 and a pharmaceutically acceptable adjuvant.
  • the invention also provides a method of inhibiting a disease that is ameliorated by PARP activity, the method comprising administering to a patient in need thereof a therapeutically effective amount of a solvate crystal of the compound of Formula A-5 or the pharmaceutical composition.
  • the present invention also provides the use of the solvate crystal of the formula A-5 or a pharmaceutical composition thereof for the preparation of a medicament for amelioration of a disease by inhibiting PARP activity.
  • the disease which is improved by inhibiting PARP activity is selected from the group consisting of cancer, vascular disease, inflammatory disease, rejection reaction, diabetes, Parkinson's disease, septic shock, ischemic injury, neurotoxicity, hemorrhagic disease. Shock and viral infections.
  • the cancer is defective in a homologous recombination-dependent DNA double-strand break repair pathway, or the cancer cell is defective in BRCA1 or BRCA2.
  • the compound Ia of the present invention or a pharmaceutically acceptable salt thereof, the compound Ia-1 or a pharmaceutically acceptable salt thereof, the amorphous form of the formula A-5 or the solvent of the above formula A-5 The crystal of the compound can be prepared for preventing the formation of poly(ADP-ribose) chains by inhibiting the activity of the cellular PARP enzyme.
  • Diseases treated include: vascular disease, septic shock, ischemic injury, brain and cardiovascular reperfusion injury, neurotoxicity (eg treatment of acute and/or chronic stroke and Parkinson's disease), hemorrhagic shock Eye-related oxidative damage; transplant rejection; inflammatory diseases such as arthritis, inflammatory bowel disease, ulcerative colitis or Crohn's disease; multiple sclerosis; secondary effects of diabetes; and cells after cardiovascular surgery
  • Acute treatment of toxicity, pancreatitis; atherosclerosis or inhibition of PARP activity improves disease;
  • the compound of the formula I is reacted with the compound B in a condensing agent, such as in a HOBT/EDCI mixed condensation system, and the by-products produced are relatively easy to separate and remove, and are simply extracted, washed, and separated by liquid separation during the post-treatment. It can be removed so that the resulting compound A is very high in purity and yield.
  • a condensing agent such as in a HOBT/EDCI mixed condensation system
  • HOBT/EDCI/TEA is used, and the by-products produced are easily separated and removed, and can be removed by simple extraction, washing and liquid separation in the post-treatment process, so that the purity of the formula II is obtained. And the yield is higher.
  • the present invention provides a compound of the formula I, which is a high-purity inhibitor of PARP activity and improves the disease.
  • the compound of the formula I is a novel compound which is used in the preparation of a high-purity drug compound A for inhibiting PARP activity and improving disease. Aspects have positive social significance.
  • the method for preparing compound A provided by the invention is simple in operation and is advantageous for industrial production.
  • the purity and the yield are high, the molar conversion rate per step is above 90%, and the process economy is high.
  • the compound I-a of the present invention or a pharmaceutically acceptable salt thereof can be used for the preparation of the pharmaceutical compound A which inhibits the activity of PARP enzyme, and in vitro experiments have confirmed that I-a or a pharmaceutically acceptable salt thereof also has good PARP inhibitory activity.
  • the compound of the formula A-5 of the present invention has good amorphous solubility, and a high dissolution rate can lead to supersaturation, thereby making the bioavailability high, which is beneficial to the absorption and utilization of the drug.
  • the amorphous form of the compound of the formula A-5 of the present invention has high stability and is advantageous for the preparation and use of the pharmaceutical composition.
  • the method for preparing the amorphous form of the compound of the formula A-5 of the present invention is simple, rapid, and easy to industrialize.
  • the amorphous form of the compound of the formula A-5 of the invention has good fluidity, good uniformity and easy preparation of the preparation.
  • the solvate of the compound of the formula A-5 of the present invention is a crystalline solid, and the XRPD characteristic peak is sharp, indicating that the crystal has good crystallinity, high purity and is not easy to coalesce.
  • the ester solvate of the compound of the formula A-5 of the present invention has good crystal stability, and the experimental results show that the solvate crystal of the compound of the formula A-5 provided by the present invention is under the condition of 75% high humidity bare discharge for 10 days.
  • the wettability is less than 1%, and it is considered that there is substantially no wettability, and the wettability is much smaller than that of the amorphous (ie, amorphous), so that the ester solvate crystal of the compound of the formula A-5 of the present invention is easy to store, thereby packaging Low requirements.
  • the ester solvate of the compound of the formula A-5 has a good fluidity and is easy to industrially produce.
  • the method for preparing a solvate crystal of the formula A-5 of the present invention is very suitable for large-scale production.
  • Solvate means a complex formed by combining a compound of formula A-5 with a solvent.
  • administering refers to the introduction of a medicament into a patient.
  • the therapeutic amount can be administered, which can be determined by a treating physician or the like.
  • an oral route of administration is preferred.
  • administering or administration of when used in connection with a compound or pharmaceutical composition (and grammatical equivalents), refers to direct administration and/or indirect administration, said direct Administration can be administered to the patient by the medical professional or by the patient himself, which can be the act of prescribing the drug.
  • the physician instructs the patient to administer the drug himself and/or to provide the patient with a prescription for administering the drug to the patient. In any event, administration requires delivery of the drug to the patient.
  • Excipient refers to an inert or inactive material used in the manufacture of pharmaceuticals, including, without limitation, any use as a binder, disintegrant, coating, compression/encapsulation aid, emulsion or lotion. , a lubricant, a parenteral injection, a sweetener or flavoring agent, a suspending/gelling agent or a moist granulating agent.
  • “Therapeutically effective amount” or “therapeutic amount” refers to the amount of a drug or agent that, when administered to a patient having a condition, will have an expected therapeutic effect, such as reducing, ameliorating, alleviating or eliminating the condition of the patient. One or more clinical manifestations.
  • the therapeutically effective amount will vary depending on the individual or condition being treated, the weight and age of the individual, the severity of the condition, the particular composition or excipient selected, the dosage regimen to be followed, All of these factors can be readily determined by those skilled in the art, such as time of administration, mode of administration, and the like.
  • the overall therapeutic effect does not have to be produced by administering one dose, but may only be produced after a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations.
  • a therapeutically effective amount in the context of treating anemia refers to an amount of a medicament that reduces, ameliorates, alleviates or eliminates one or more anemia symptoms of the patient.
  • Treatment are defined as acting on a disease, disorder, or condition with an agent to reduce or ameliorate the disease, disorder, or condition and/or its symptoms. Harmful or any other undesirable effect.
  • the treatments used herein include treatment of a human patient and include: (a) reducing the risk of a patient developing a condition that is determined to be predisposed to a disease but not yet diagnosed as having a condition, and (b) preventing the progression of the condition, And/or (c) alleviating the condition, ie causing regression of the condition and/or alleviating one or more symptoms of the condition.
  • An "XRPD pattern” is an x-y pattern having a diffraction angle (i.e., ° 2 ⁇ ) on the x-axis and intensity on the y-axis.
  • the peaks in this pattern can be used to characterize the solid state of the crystal.
  • variability in the XRPD data is often uniquely represented by the diffraction angle of the peak, and does not include the intensity of the peak, as the intensity of the peak may be particularly sensitive to the preparation of the sample (eg, particle size, moisture content, solvent content, and preferred orientation effects affect sensitivity)
  • samples of the same material prepared under different conditions may produce slightly different patterns; this variability is typically greater than the variability of the diffraction angle.
  • the variability in diffraction angle can also be sensitive to the preparation of the sample.
  • Other sources of variability come from instrument parameters and processing of raw X-ray data: different X-ray instrument operations use different parameters and these may result in XRPD patterns that are slightly different from the same solid morphology, and similarly, different software The packet processes the X-ray data in different ways and this also leads to variability.
  • These and other sources of variability are known to those skilled in the pharmaceutical arts. Due to the source of the variability, the diffraction angle in the XRPD pattern is assigned a variability of ⁇ 0.2° 2 ⁇ , that is, the invention includes not only crystals having exactly the same diffraction angle in X-ray powder diffraction, but also an error range of ⁇ 0.2°. Crystals with uniform internal diffraction angles.
  • Figure 1 is an XRPD pattern of the ethyl acetate solvate crystal A of the compound of the formula A-5 in a preferred embodiment of the present invention.
  • Figure 2 is an infrared spectrum of the ethyl acetate solvate crystal A of the compound of formula A-5 in a preferred embodiment of the invention.
  • Figure 3 is an amorphous XRPD pattern of a compound of formula A-5 in a preferred embodiment of the invention.
  • Figure 4 is an XRPD pattern of the ethyl formate solvate crystal B of the compound of formula A-5 in a preferred embodiment of the invention.
  • Figure 5 is an infrared absorption spectrum of ethyl formate solvate crystal B of the compound of formula A-5 in a preferred embodiment of the invention.
  • Figure 6 is an XRPD pattern of the methyl acetate solvate crystal C of the compound of formula A-5 in a preferred embodiment of the invention.
  • Figure 7 is an infrared absorption spectrum of a methyl acetate solvate crystal C of the compound of the formula A-5 in a preferred embodiment of the present invention.
  • Figure 8 is an XRPD pattern of crystalline D of n-propyl formate solvate of a compound of formula A-5 in a preferred embodiment of the invention.
  • Figure 9 is an infrared absorption spectrum of crystal D of n-propyl formate solvate of a compound of formula A-5 in a preferred embodiment of the invention.
  • the structures of all compounds of the invention can be identified by nuclear magnetic resonance ( 1 H NMR) and/or mass spectrometry (MS).
  • the 1 H NMR chemical shift ( ⁇ ) was recorded in PPM (10 -6 ).
  • NMR was performed on a Bruker AVANCE-400 spectrometer.
  • a suitable solvent is deuterated chloroform (CDCl 3 ) tetramethylsilane as an internal standard (TMS).
  • MS mass spectra
  • X-ray powder diffraction patterns were acquired on a Bruker D8 diffractometer using Cu-K ⁇ radiation (40 kV, 40 mA), ⁇ -2 ⁇ goniometer and Lynxeye detector. Check the performance of the instrument using the certified Corundum standard (NIST 1976). The software used for data collection is Diffrac Plus XRD Commander V6, and data is analyzed and presented using DIFFRAC.EVA V4.2.
  • the sample was run as a flat specimen under ambient conditions.
  • the sample was lightly packaged in a cavity that was cut into polished, zero background wafers. The details of the data collection are:
  • N,N-diisopropylethylamine (70.0 g, 0.54 mol) was added dropwise, and the mixture was stirred at room temperature for 2 hr. After adding 700 ml of water, stirring, liquid separation, organic phase washing, dried over anhydrous sodium sulfate, filtered,
  • the temperature was controlled at 10 ° C, and N,N-diisopropylethylamine (37.6 g, 0.29 mol) was added dropwise. After the completion of the dropwise addition, the reaction was stirred at a temperature of 40 ° C for about 24 hours, and the reaction of the starting material was completely monitored by TLC. After adding 500 ml of water, stirring, liquid separation, washing with an organic phase, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure at 40 ° C to give a white solid.
  • Example I-1 The oil obtained in Example I-1 (theoretical 112.3 g, 0.24 mol) was dissolved in 1.7 L of ethyl acetate. The temperature was adjusted to 15 ° C, concentrated hydrochloric acid (160 ml, 1.92 mol) was added dropwise, and the temperature was controlled at 20 ° C for 2 h. The TLC monitors the reaction of the starting material completely, and a large amount of solid precipitates; it is filtered, washed with a small amount of ethyl acetate, and the filter cake is dried at 50 ° C to a constant weight to obtain a solid of 82.8 g, a purity of 99.90%, a two-step yield of 85.0%;
  • Example I-2 The solid obtained in Example I-2 (theoretical 112.3 g, 0.24 mol) was completely dissolved in 1.2 L of methyl ethyl ketone, the temperature was controlled at 10 ° C, concentrated hydrochloric acid (300 ml, 3.6 mol) was added dropwise, and the temperature was controlled at 40 ° C for 10 h. The raw material reaction is complete, a large amount of solids are precipitated; filtered, rinsed with a small amount of methyl ethyl ketone, and the filter cake is dried to constant weight at 50 ° C to obtain a solid 81.0 g, purity 100.0%, two-step yield 83.1%, and nuclear magnetic and mass spectrometry data are implemented simultaneously.
  • Example I-4 The raw material reaction is complete, a large amount of solids are precipitated; filtered, rinsed with a small amount of methyl ethyl ketone, and the filter cake is dried to constant weight at 50 ° C to obtain a solid 81.0 g, purity
  • Example I-3 The solid obtained in Example I-3 (theoretical 112.3 g, 0.24 mol) was completely dissolved in 1.2 L of isopropanol, and the temperature was controlled at 25 ° C, 6 N concentrated hydrochloric acid (576 ml, 2.9 mol) was added dropwise, and the temperature was controlled at 25 ° C for 20 h. TLC monitored the reaction of the starting material completely, a large amount of solid precipitated; filtered, rinsed with a small amount of isopropanol, and the filter cake was dried to constant weight at 50 ° C to obtain a solid 90.6 g, purity 99.99%, two-step yield 93.0%, nuclear magnetic, Mass spectral data was identical to Example I-4.
  • the difference from the embodiment I-4 is that the dropwise addition of concentrated hydrochloric acid (160 mL, 1.92 mol) is replaced by 1.92 mol of hydrogen chloride gas to obtain a solid 90.1 g, a purity of 99.35%, a two-step yield of 92.5%, and a nuclear magnetic field.
  • the mass spectrometry data is the same as in Example I-4.
  • the difference from the embodiment I-5 is that the dropwise addition of concentrated hydrochloric acid (300 mL, 3.6 mol) is replaced by introducing 1.2 mol of hydrogen chloride gas to obtain a solid of 89.6 g, a purity of 99.15%, a two-step yield of 91.8%, and a nuclear magnetic field.
  • the mass spectrometry data is the same as in Example I-4.
  • Example I-6 Applicants replaced 6N concentrated hydrochloric acid (576 ml, 2.9 mol) in Example I-6 with 2N concentrated hydrochloric acid (1450 ml, 2.9 mol) to obtain a solid 90.1 g, purity 99.35%, two-step yield 92.5%, NMR, MS data. Same as Example I-4.
  • Example I-10 1-Cyclopropyl-2-((1S,4S)-5-(2-fluoro-5-((4-oxo-3,4-dihydropyridazin-1-yl)methyl) Preparation of benzoyl)-2,5-diazabicyclo[2,2,1]heptan-2-yl)ethane-1,2-dione (Formula A-5)
  • Example I-4 The solid obtained in Example I-4 (80.0 g, 0.19 mol) was placed in a reaction vessel containing 800 ml of dichloromethane and stirred, and HOBT (39.2 g, 0.29 mol), EDCI (82.4 g, 0.43 mol) and 2-ring were added.
  • Example I-11 1-Cyclopropyl-2-((1S,4S)-5-(2-fluoro-5-((4-oxo-3,4-dihydropyridazin-1-yl)methyl) Preparation of benzoyl)-2,5-diazabicyclo[2,2,1]heptan-2-yl)ethane-1,2-dione (Formula A-5)
  • Example I-5 The solid obtained in Example I-5 (80.0 g, 0.19 mol) was placed in a reaction vessel containing 800 ml of dichloromethane, stirred, and HOBT (31.1 g, 0.23 mol), EDCI (44.1 g, 0.23 mol) and 2-ring were added.
  • Example I-12 1-Cyclopropyl-2-((1S,4S)-5-(2-fluoro-5-((4-oxo-3,4-dihydropyridazin-1-yl)methyl) Preparation of benzoyl)-2,5-diazabicyclo[2,2,1]heptan-2-yl)ethane-1,2-dione (Formula A-5)
  • Example I-6 The solid obtained in Example I-6 (80.0 g, 0.19 mol) was placed in a reaction vessel containing 800 ml of dichloromethane, stirred, and HOBT (31.1 g, 0.23 mol), EDCI (35.2 g, 0.18 mol) and 2-ring were added.
  • Example I-13 1-Cyclopropyl-2-((1S,4S)-5-(2-fluoro-5-((4-oxo-3,4-dihydropyridazin-1-yl)methyl) Preparation of benzoyl)-2,5-diazabicyclo[2,2,1]heptan-2-yl)ethane-1,2-dione (Formula A-5)
  • Example I-8 The solid obtained in Example I-8 (80.0 g, 0.19 mol) was placed in a reaction vessel containing 800 ml of dichloromethane, stirred, and EDCI (44.1 g, 0.23 mol) and sodium 2-cyclopropyl-2-oxoacetate were added.
  • Example I-1 obtained an oily substance containing a small amount of by-product tetramethylurea
  • Examples I-2 and I-3 obtained a solid; but whether the formula II is present in the reaction mixture as an oil or a solid, as long as the formula I (for example, formula I-5) is formed in the subsequent step as a solid formed in the reaction mixture, and then simply filtered and dried.
  • a high purity compound of formula I e.g., formula I-5) is obtained.
  • a compound of the formula I (for example, formula I-5) of high purity is reacted with the compound B to produce a compound A of high purity with a purity of 99.9% or more and a yield of 90% or more.
  • the yield of the compound obtained in the process of the examples in CN103237799 is less than 60%, such as CN103237799A Example 2 and the process of the invention (for example: Examples 10 to 13), the same compound I-5, CN103237799A, Example 2, Formula I- The yield of 5 was 55%, which was much lower than the yield of the process of the invention.
  • Example I-14 PARP inhibition efficacy can be determined by the following experiment
  • test compound information is as follows:
  • Positive control AZD2281 chemical name: 1-(cyclopropylcarbonyl)-4-[5-[(3,4-dihydro-4-oxo-1-pyridazine)methyl]-2-fluorobenzamide Acyl] piperazine.
  • the PARP enzyme reaction system was added to a 96-well plate pre-coated with a histone-labeled substrate, double-welld, and incubated for 1 hour at room temperature.
  • the reaction system consisted of: 50 ⁇ l of reaction buffer containing NAD + (Tris ⁇ HCl, pH 8.0), biotinylated NAD + (see 3), activated DNA (see 3), a PARP enzyme (see 3) and Test compound (see 2). After completion of the reaction when the enzyme incubation, each well was added 50 ⁇ l of a horseradish peroxidase-labeled streptavidin biotin room temperature for 30 minutes before addition of 100 ⁇ l developer using BioTek Synergy TM 2 plate reader reads the light emission value.
  • Enzyme activity % [(LL b ) / (L t - L b )] ⁇ 100.
  • L luminescence value of the test compound well added
  • L b luminescence value of no PARP or TNKS pore
  • L t luminescence value of the test compound not added
  • enzyme activity inhibition ratio % 100 - enzyme activity %.
  • the enzyme inhibitory activity of the compound is expressed as IC 50 and the inhibition curve is represented by a plot with standard deviation.
  • Example I-15 DNA binding activity test of recombinant human PARP1 enzyme
  • test compound information is as follows:
  • Positive control AZD2281 chemical name: 1-(cyclopropylcarbonyl)-4-[5-[(3,4-dihydro-4-oxo-1-pyridazine)methyl]-2-fluorobenzamide Acyl] piperazine.
  • the enzymatic reaction was carried out in a 96-well plate with double replicate wells at room temperature. 45 ml of nick DNA containing 5 nM Alexa488 label, PARP1 buffer of PARP1 enzyme (see 3) and test compound (see 2) were incubated for 30 minutes at room temperature, then 5 ml of NAD was added to initiate PARP1 enzymatic reaction and incubated at room temperature 45 minute. Fluorescence intensities at 485 nM and 528 nM were read using a Tecan Infinite M1000 plate reader. Fluorescence intensity was converted to fluorescence polarization using Tecan Magellan 6 software and data processing was performed using Graphpad Prism software.
  • Fluorescence polarization (FP o ) without test compound in all data was defined as 0% activity
  • fluorescence polarization (FP t ) without NAD and test compound was defined as 100% activity
  • no PARP and fluorescence polarization (FP b) test the test compound was defined as background activity.
  • test compound of the present invention is calculated according to the following formula:
  • % activity [(FP-FP b )-(FP o -FP b )] / [(FP t -FP b )-(FP o -FP b )]*100.
  • Example II-1 50 g of the compound of the formula A-5 obtained in Example II-1 was dissolved in 100 ml of acetone to form an acetone solution of the compound of the formula A-5;
  • the XRPD pattern has no crystal diffraction peak, so the white powder is amorphous.
  • Example II-1 50 g of the compound of the formula A-5 obtained in Example II-1 was dissolved in 500 ml of ethanol to form an ethanol solution of the compound of the formula A-5;
  • Example II-1 50 g of the compound of the formula A-5 obtained in Example II-1 was dissolved in 250 ml of isobutyl acetate to form an isobutyl acetate solution of the compound of the formula A-5;
  • the mixture was stirred at 30 ° C; filtered, and the amount of n-pentane was rinsed; the filter cake was dried under reduced pressure to give 48 g of the compound of formula A-5 as a white powder.
  • Example II-1 50 g of the compound of the formula A-5 obtained in Example II-1 was dissolved in 50 ml of methanol to form a methanol solution of the compound of the formula A-5; the reaction flask was charged with isopropyl ether (0.5 L), stirred, and slowly dropped into the system at 20 ° C. a methanol solution of the compound of the formula A-5, after completion, a large amount of white solid precipitated;
  • Example II-1 50 g of the compound of the formula A-5 obtained in Example II-1 was dissolved in 100 ml of ethyl acetate to form an ethyl acetate solution of the compound of the formula A-5;
  • Example II-1 50 g of the compound of the formula A-5 obtained in Example II-1 was dissolved in 200 ml of methyl ethyl ketone to form a methyl ethyl ketone solution of the compound of the formula A-5;
  • the XRPD patterns of the white powders obtained in Examples II-3 to II-7 were determined to be substantially the same as those in Example II-2.
  • sample The white powder (hereinafter referred to as: sample) obtained in Examples II-2 and II-5 was simulated in a commercial package (medicinal low-density polyethylene bag/medicinal composite film bag), and placed in a drug stability test chamber at a temperature of 40 At °C ⁇ 2°C, the relative humidity was 75% ⁇ 5% for 6 months, and samples were taken at 1, 2, 3, and 6 months. The following items were tested. The results are as follows:
  • the amorphous form of the compound of formula A-5 of the present invention has a temperature of 40 ° C ⁇ 2 ° C and a humidity of RH 75% ⁇ 5%. After being placed for 6 months, the results of various indexes have no significant change compared with the initial results of 0 days.
  • the amorphous form of the compound of the formula A-5 is preferred.
  • the applicant also tested the amorphous forms obtained in Examples II-3, II-4, II-6, and II-7, and the HPLC purity thereof was not less than 99.5%, and the total amount was not more than 0.5%.
  • the solubility test was carried out at 25 °C ⁇ 2 °C (according to the pre-test results, the appropriate amount of the test sample was weighed, the solvent was gradually added, shaken, observed to dissolve completely, and the solubility was judged according to the pharmacopoeia standard).
  • the test results of the amorphous sample of the compound 5 are shown in the following table.
  • each saturated solution is diluted with a 10% acetonitrile aqueous solution to a solution containing 0.05 mg of the main drug per 1 ml, that is, Each test solution is obtained; another appropriate amount of the reference substance is accurately weighed, dissolved in an appropriate amount of acetonitrile, and diluted with 10% acetonitrile aqueous solution to prepare a reference substance containing 0.05 mg of the main drug per 1 ml (sample obtained in Example II-1) ) solution.
  • the saturated solubility of the amorphous sample of the compound of the formula A-5 of the present invention in aqueous solutions of different pH values was examined by external standard method in terms of peak area. The results are shown in the following table.
  • solubility of the amorphous form of the compound of the formula A-5 of the present invention in an aqueous solution of pH 1.0 to 10 is about 1.0 mg/ml, indicating that the solubility of the compound is not pH-dependent.
  • III-1 to III-5 are preparations of ethyl acetate solvate crystal A of formula A-5
  • Example II-1 100 g of the target product obtained in Example II-1 was added to 600 ml of 100% (w/w) ethyl acetate to dissolve completely, and the mixture was stirred and crystallized at a controlled temperature of 10 °C. Filter and rinse with an appropriate amount of ethyl acetate. The filter cake was dried under reduced pressure to a constant weight at 55 ° C to give 92.0 g of ethyl acetate solvate crystals of formula A-5, yield 92.0%.
  • Example II-1 100 g of the target product obtained in Example II-1 was added to 2000 ml of an ethyl acetate solution (95% (w/w) ethyl acetate, 5% (w/w) tetrahydrofuran) to dissolve completely, and the mixture was stirred and crystallized at a controlled temperature of 20 °C. Filter and rinse with an appropriate amount of ethyl acetate. The filter cake was dried under reduced pressure at 40 ° C to a constant weight to obtain 91.5 g of ethyl acetate solvate crystals of formula A-5, yield 91.5%.
  • an ethyl acetate solution 95% (w/w) ethyl acetate, 5% (w/w) tetrahydrofuran
  • Example II-1 100 g of the target product obtained in Example II-1 was added to 1000 ml of an ethyl acetate solution (90% (w/w) ethyl acetate, 10% (w/w) acetonitrile) to dissolve completely, and the mixture was stirred and crystallized at a controlled temperature of 30 °C. Filter and rinse with an appropriate amount of ethyl acetate. The filter cake was dried under reduced pressure at 60 ° C to a constant weight to obtain 92.0 g of ethyl acetate solvate crystals of formula A-5, yield 92.0%.
  • an ethyl acetate solution 90% (w/w) ethyl acetate, 10% (w/w) acetonitrile
  • Example II-1 100 g of the target product obtained in Example II-1 was added to 400 ml of an ethyl acetate solution (85% (w/w) ethyl acetate, 15% (w/w) tetrahydrofuran) to dissolve completely, and the mixture was stirred and crystallized at a controlled temperature of 40 °C. Filter and rinse with an appropriate amount of ethyl acetate. The filter cake was air-dried to a constant weight at 30 ° C to obtain 90.4 g of ethyl acetate solvate crystal of formula A-5 in a yield of 90.4%.
  • an ethyl acetate solution 85% (w/w) ethyl acetate, 15% (w/w) tetrahydrofuran
  • Example II-1 100 g of the target product obtained in Example II-1 was added to 3000 ml of an ethyl acetate solution (50% (w/w) ethyl acetate, 50% (w/w) acetonitrile) to dissolve completely, and the mixture was stirred and crystallized at a controlled temperature of 0 °C. Filter and rinse with an appropriate amount of ethyl acetate. The filter cake was air-dried to constant weight at 70 ° C to obtain 90.9 g of ethyl acetate solvate crystal of formula A-5 in a yield of 90.9%.
  • an ethyl acetate solution 50% (w/w) ethyl acetate, 50% (w/w) acetonitrile
  • Fig. 1 The XRPD pattern of the ethyl acetate solvate crystal A of the compound of the formula A-5 is shown in Fig. 1.
  • the diffraction angle 2? and the relative intensity of each peak in Fig. 1 are shown in the following table.
  • the diffraction angles 2 ⁇ ⁇ 0.2° were 8.193, 14.137, 17.799, 19.483, 20.062, 20.861 and 23.837.
  • the infrared absorption spectrum of the ethyl acetate solvate crystal A of the compound of the formula A-5 is shown in Fig. 2 . Characterized by the following important bands, expressed as the reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2cm -1 ), which has absorption peaks at the following positions: 3345.84, 3204.48, 3077.83, 3009.85, 2984.15, 1173.55, 1693.68, 1166.67, 1653.93 1495.51, 145.38, 1405.77, 1372.64, 1331.30, 1241.81, 1109.35, 1155.83, 1106.21, 1081.60, 1042.61, 1000.42, 947.75, 929.26, 836.47, 805.05, 772.26, 750.17, 723.25, 682.88, 621.94, 577.80, 528.40, 486.00.
  • Example II-1 100 g of the target product obtained in Example II-1 was added to 2000 ml of ethyl formate solution (95% w/w ethyl formate, 5% w/w tetrahydrofuran) to dissolve completely, and the mixture was stirred and crystallized at a controlled temperature of 20 ° C. Filter and dilute the appropriate amount of ethyl formate. The filter cake was dried under reduced pressure at 40 ° C to a constant weight to obtain the ethyl formate solvate crystals of the compound of formula A-5 (92.4 g, yield 92.4%).
  • the XRPD pattern of the ethyl acrylate solvate crystal B of the compound of the formula A-5 is shown in Fig. 4, and the diffraction angles and relative intensities in the XRPD pattern are shown in the following table.
  • the X-ray powder diffraction pattern of the ethyl formate solvate crystal B of the compound of the formula A-5 has a characteristic peak at diffraction angles 2 ⁇ 0.2° of 8.471, 14.034, 16.131 and 19.679; It has characteristic peaks at diffraction angles 2 ⁇ 0.2° of 8.471, 9.776, 11.491, 14.034, 16.131, 19.679, 22.621, and 24.532; more preferably, the diffraction angles 2 ⁇ 0.2° are 8.471, 9.776, 11.151, 11.491, 14.034, 16.131, There are characteristic peaks at 16.790, 17.755, 18.626, 19.679, 22.621, 23.268, and 24.532.
  • the infrared absorption spectrum pattern of the ethyl formate solvate crystal B of the formula A-5 is shown in Fig. 5. Characterized by the following important bands, expressed as the reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2cm -1 ), which has absorption peaks at the following positions: 3447.16, 3190.04, 3076.26, 3010.93, 2893.18, 1723.60, 1667.77, 1366.87, 1559.39 , 1476.32, 1448.70, 1406.16, 1137.35, 1329.87, 1258.92, 1239.50, 1180.47, 1155.24, 1107.13, 1081.35, 1042.77, 1027.06, 1001.43, 948.22, 927.67, 899.76, 836.95, 804.45, 771.41, 750.66, 723.72, 683.19, 655.68, 621.35 578.30, 540.65 and 486.11.
  • Example II-1 100 g of the target product obtained in Example II-1 was added to 1000 ml of methyl acetate solution (90% w/w methyl acetate, 10% w/w acetonitrile) to dissolve completely, and the mixture was stirred and crystallized at a controlled temperature of 30 °C. Filter and dilute the appropriate amount of methyl acetate. The filter cake was dried under reduced pressure at 60 ° C to a constant weight to obtain a crystals of methyl acetate solvate C 92.2 g of the compound of formula A-5, yield 92.2%.
  • methyl acetate solution 90% w/w methyl acetate, 10% w/w acetonitrile
  • the XRPD pattern of the methyl acetate solvate crystal C of the compound of the formula A-5 is shown in Fig. 6, and the diffraction angle 2? and its relative intensity in the XRPD pattern are shown in the following table.
  • the X-ray powder diffraction pattern of the methyl acetate solvate crystal C of the compound of the formula A-5 has a characteristic peak at a diffraction angle of 2 ⁇ ⁇ 0.2° of 8.454, 14.057, 16.11 and 19.681; Characteristic peaks at diffraction angles 2 ⁇ 0.2° of 8.454, 9.748, 11.548, 13.740, 14.057, 16.111, 19.681, and 22.619; more preferably, diffraction angles 2 ⁇ 0.2° are 8.454, 9.748, 11.144, 11.548, 13.740, 14.057, There are characteristic peaks at 16.111, 16.779, 17.778, 18.596, 19.681, 22.619 and 23.256.
  • the infrared absorption spectrum of the methyl acetate solvate crystal C of the compound of the formula A-5 is shown in Fig. 7, and is characterized by the following important bands, expressed by the reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2 cm -1 ), It has absorption peaks at the following positions: 3447.25, 319.985, 3090.97, 3010.12, 2896.69, 1744.78, 1668.61, 1617.02, 1549.53, 1946.34, 1449.40, 1406.28, 1370.92, 1333.37, 1240.52, 1213.97, 1191.31, 1155.98, 1106.65, 1081.38, 1042.71. 1001.04, 947.78, 928.16, 899.53, 836.47, 804.71, 771.49, 723.86, 683.14, 621.65, 578.05, 540.88 and 485.99.
  • Example II-1 100 g of the target product obtained in Example II-1 was added with 3000 ml of n-propyl formate solution (50% (w/w) n-propyl formate, 50% (w/w) acetonitrile) to dissolve completely, and controlled to stand at 0 ° C for stirring and crystallization. . Filter and dilute the proper amount of n-propyl formate. The filter cake was air-dried to constant weight at 70 ° C to obtain 107.8 g of n-propyl formate solvate crystal of the compound of formula A-5 in a yield of 91%.
  • n-propyl formate solution 50% (w/w) n-propyl formate, 50% (w/w) acetonitrile
  • the XRPD pattern of the compound A-5 compound n-propyl formate solvate crystal D is shown in Fig. 8, and the diffraction angles and relative intensities in the XRPD pattern are shown in the following table.
  • the X-ray powder diffraction pattern of the n-propyl formate solvate crystal D of the compound of the formula A-5 has characteristics at diffraction angles 2 ⁇ ⁇ 0.2° of 8.152, 13.989, 15.924, 19.459 and 22.237.
  • a peak preferably having a characteristic peak at a diffraction angle of 2 ⁇ ⁇ 0.2° of 8.152, 13.526, 13.989, 15.924, 16.720, 19.048, 19.459, 22.237, and 22.840; more preferably at a diffraction angle of 2 ⁇ ⁇ 0.2° of 8.152, 9.459, 11.647, 13.526 Characteristic peaks at 13.989, 15.407, 15.924, 16.720, 17.692, 18.423, 19.058, 19.459, 22.237, 22.840, 23.795 and 24.751.
  • the infrared absorption spectrum of the n-propyl formate solvate crystal of the compound of the formula A-5 is shown in Fig. 9, and is characterized by the following important bands, expressed by the reciprocal of the absorption wavelength (cm -1 ) ( ⁇ 2 cm -1 ) It has absorption peaks at the following positions: 34470.11, 3203.33, 3074.46, 3010.34, 2966.46, 2891.74, 1726.16, 1668.42, 1163.53, 1 496.39, 1449.31, 1406.54, 1372.26, 1330.16, 1258.84, 1239.49, 1168.01, 1154.17, 1106.97, 1081.28, 1044.04 1027.37, 1002.12, 948.21, 928.04, 899.53, 836.47, 804.41, 772.26, 724.08, 682.94, 655.68, 621.26, 577.89, 540.67 and 485.97.
  • Examples III-9 to III-13 are preparations of solvate crystals of the compound of formula A-5
  • Example III-1 The difference from Examples III-1, III-3, III-6, III-7, and III-8 was that the "target product obtained in Example II-1" was replaced with the "solid obtained in Example II-3 (The compound of the formula A-5 is amorphous (i.e., amorphous)), and the yield of the product is as follows:
  • the raw material is amorphous (i.e., amorphous) of the compound of the formula A-5 (Examples III-9 to III-13) It is easier to precipitate a solid from a solvent with respect to the compound of the formula A-5 (Examples III-1, III-3, III-6, III-7, III-8) of the target sample obtained in Example II-1, respectively. Moreover, the yield is significantly improved.
  • Samples (crystals obtained in Examples III-1 and III-9) were sampled, and commercial packages (medicinal low-density polyethylene bags/medicinal composite film bags) were simulated and placed in a drug stability test chamber at a temperature of 40 ° C ⁇ 2 ° C, relative humidity 75% ⁇ 5% conditions for 3 months, sampling in the first, second, and third months, according to the following items to test, the results are shown in the table below.
  • Example III-1 Sample (ethyl acetate solvate crystal A of the compound of formula A-5) accelerated test results
  • Example III-9 Sample (ethyl acetate solvate crystal A of the compound of formula A-5) accelerated test results
  • the product has a temperature of 40 ° C ⁇ 2 ° C, humidity RH75% ⁇ 5%, after standing for 3 months, the results of the various indicators have no significant change compared with the initial results of 0 days, indicating the compound of formula A-5 of the present invention
  • the solvate crystals e.g., ethyl acetate solvate crystal A of the compound of formula A-5) are more stable.
  • the solvate crystal of the compound of the formula A-5 is substantially free of hygroscopicity, and the amorphous (i.e., amorphous) compound of the formula A-5 has a certain wettability, and the solvate crystal is relatively easy to store. Avoid packaging problems caused by moisture absorption of raw materials.

Abstract

本发明公开了一种 PARP 抑制化合物的制备方法、其中间体、非晶形、溶剂合物、药物组合物及应用。本发明的 PARP 抑制剂化合物的制备方法，目标化合物的纯度和收率都较高，更适用于工业化生产。另外，本发明首次发现式 A-5 化合物的非晶形，该非晶形流动性较好，利于运输和保存。本发明还公开了流动性和稳定性较好且基本无引湿性的式 A-5 化合物的溶剂合物的晶体。

Description

PARP抑制化合物的制备方法、其中间体、非晶形、溶剂合物、药物组合物及应用
本申请要求申请日为2017年4月21日的中国专利申请CN201710263433.X，申请日为2017年4月19日的中国专利申请CN201810354319.2，CN201810354691.3和CN201810354222.1的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种PARP抑制化合物的制备方法、其中间体、非晶形、溶剂合物、药物组合物及应用。
背景技术
PARP是“多聚(ADP-核糖)聚合酶”的缩写。肿瘤细胞用PARP酶修复包括由化疗引起的DNA损伤。研究者正在研究药物在抑制PARP酶的同时是否也能使这种自身修复机制变弱并使肿瘤细胞对于治疗更敏感，加速肿瘤细胞死亡。
PARP抑制剂是多聚ADP核糖聚合酶的药理学抑制剂族，其对于促进DNA修复、控制RNA转录、调节细胞死亡和免疫应答比较重要。因此，PARP抑制剂发展有多种适应症，最重要的适应症是用于***。有几种形式的BRCA缺陷型的肿瘤细胞比正常细胞更依赖于PARP的修复功能，因此使得PARP成为肿瘤治疗的一个有吸引力的靶点。
CN103237799A涉及一系列对PARP酶具有抑制作用的酞嗪-1(2H)-酮衍生物，并且其公开文本第[0105]段反应式3提到了其公开的酞嗪-1(2H)-酮衍生物的合成方法：采用三氟乙酸脱保护制得式(II)化合物，其为油状物，较难纯化及质量控制，使得最后所得式(B)化合物的纯度和收率较低，并且不利用工业化生产。
同时，CN103237799A还公开了式A-5化合物：
Figure PCTCN2018083969-appb-000001
也提供了该化合物的制备方法，得到的化合物是粘状物，流动性较差，不利于运输 及保存或稳定性差，而且其制备方法不适于大生产的有效分离。
发明内容
为了克服现有技术的不足，本发明提供了一种PARP抑制化合物的制备方法、其中间体、非晶形、溶剂合物、药物组合物及应用。本发明的PARP抑制剂化合物的制备方法，目标化合物的纯度和收率都较高，更适用于工业化生产。另外，本发明首次发现式A-5化合物的非晶形，该非晶形流动性较好，利于运输和保存。同时本发明式A-5化合物的非晶形溶解度、生物利用度等方面也较好。本发明提供流动性和稳定性较好且基本无引湿性的式A-5化合物的溶剂合物的晶体。本发明还提供了包含式A-5化合物的非晶形或溶剂合物晶体的药物组合物，以及所述非晶形或溶剂合物晶体及其药物组合物在制备通过抑制PARP活性而改善的疾病方面的药物中的应用。
本发明提供了一种抑制PARP活性而改善疾病方面的药物化合物A的制备方法，反应流程如下：
Figure PCTCN2018083969-appb-000002
本发明所述化合物A的制备方法，
Figure PCTCN2018083969-appb-000003
其中，
R 1和R 2各自独立地为氢或卤素；
R为甲基、三氟甲基、
Figure PCTCN2018083969-appb-000004
(叔丁基)、
Figure PCTCN2018083969-appb-000005
(环己基)、
Figure PCTCN2018083969-appb-000006
(2-呋喃基)或
Figure PCTCN2018083969-appb-000007
(苯基)；
杂环基
Figure PCTCN2018083969-appb-000008
为
Figure PCTCN2018083969-appb-000009
所述R 1优选为氟或氯。
所述R 2优选为氢。
所述R 1优选位于苄基的亚甲基的对位。
本发明所述化合物A的制备方法，包含以下步骤：
步骤(1)将式II化合物
Figure PCTCN2018083969-appb-000010
与HCl反应生成式I化合物；
Figure PCTCN2018083969-appb-000011
所述式I化合物以固体形式存在于反应混合物中，且易通过多孔性介质分离；
步骤(2)将所得式I化合物与化合物B
Figure PCTCN2018083969-appb-000012
反应生成化合物A；其中M为氢、钾、锂、钠或铵，优选钠。
式I化合物或式II化合物中，R 1、R 2和杂环基
Figure PCTCN2018083969-appb-000013
的定义同前所述。
在一优选实施方案中，步骤(1)包括下列步骤：有机溶剂中，将式II化合物和HCl进行反应，制得式I化合物。优选地将式II化合物与有机溶剂混合，然后在10-40℃(例如10℃，15℃或25℃)下，再与HCl混合，进行反应，得到式I化合物。其中，加入HCl的物质的量可为式II化合物物质的量的5-20倍，优选8-15倍；进一步优选为8-12倍。
步骤(1)中，所述反应结束后，还可进一步包含后处理的操作。所述后处理的操作可为本领域此类反应常规的后处理方法。本发明优选包括下列步骤：将反应结束后的反应液，进行固液分离(所述固液分离的操作优选过滤)，滤饼干燥(优选在恒温50℃干燥至恒重)，得到式I化合物。在所述固液分离的操作之后，优选进一步包括洗涤(所述洗涤溶剂优选反应溶剂)的操作。
在另一优选实施方案中，步骤(1)包括下列步骤：将式II化合物溶于有机溶剂中，控温在10-40℃下加入式II化合物的5-20倍物质的量的HCl，搅拌，反应2-24小时，过滤，干燥得到固体。
在一优选实施方案中，所述步骤(1)中的有机溶剂可为本领域此类反应常规的有机溶剂，优选为C 1-5醇(例如：甲醇、乙醇、正丙醇和异丙醇中的一种或多种)、乙酸乙酯、乙酸异丙酯、C 3-6酮(例如：丙酮和/或丁酮)、二氧六环和甲苯中的一种或多种，更优选为乙酸乙酯、丁酮或异丙醇。所述有机溶剂的用量可不作具体限定，只要能够使式II化合物完全溶解即可。
在一优选实施方案中，加入HCl的物质的量为式II化合物物质的量的8-15倍；进一步优选为8-12倍。
所述HCl可以为氯化氢气体或盐酸水溶液。
所述盐酸水溶液的摩尔浓度可为本领域此类反应常规的摩尔浓度，优选为2～12mol/L(2～12N)，进一步优选为6～12mol/L(6～12N)。
所述步骤(1)的反应温度可为本领域此类反应常规的温度，优选10-40℃(例如20℃、25℃、40℃等)，更优选为25℃-40℃。
所述步骤(1)的反应进程可采用本领域常规的检测方法进行检测，一般以式II化合物消失时作为反应终点。所述反应时间优选2-24小时，例如2小时、10小时或20小时。
在一优选实施方案中，步骤(2)包括下列步骤：有机溶剂中，在缩合剂的作用下，将式I化合物和化合物B进行反应，制得化合物A。
步骤(2)中，所述有机溶剂可为本领域此类反应常规有机溶剂，优选卤代烷烃类溶剂，例如二氯甲烷。所述有机溶剂的用量可不作具体限定，只要不影响反应进行即可。式I化合物和化合物B的用量可为本领域此类反应常规的用量，二者物质的量比优选1:1-2:1。所述反应的温度可为本领域此类反应常规的温度，优选10-30℃，更优选为15-25℃。所述反应的进程可采用本领域常规的检测方法进行监测，一般以式I化合物消失时作为反应的终点。所述反应的时间优选2-8小时，例如2、4、5或8小时。
在一优选实施方案中，步骤(2)包括下列步骤：将式I化合物和有机溶剂混合后，再加入缩合剂和化合物B，进行反应，制得式A化合物。
步骤(2)反应结束后，还可进一步包括下列操作：将反应结束后的反应液与水混合，过滤，后处理(例如将滤液静置分层，将有机相洗涤干燥)，浓缩(优选减压浓缩)得到化合物A。
在另一优选实施方案中，反应步骤(2)包括下列步骤：将步骤(1)所得固体(式I化合物)加入装有二氯甲烷反应容器中，加入缩合剂及化合物B，反应2-8小时，过滤，后处理，浓缩得到化合物A。
在一优选实施方案中，所述缩合剂可为本领域此类反应常规的缩合剂，优选为HOBT(1-羟基苯并***)、EDCI(1-(3-二甲氨基丙基)-3-乙基-碳二亚胺盐酸盐)、或者HOBT与EDCI组成的混合体系。所述缩合剂的用量可为本领域此类反应常规的用量，优选其与式I化合物的物质的量比为1:1-5:1。
在一优选实施方案中，所述缩合剂为HOBT与EDCI组成的混合体系，所述HOBT与EDCI的物质的量比为1:0.8-1:1.5。
所述化合物A最佳地为如下任一结构：
Figure PCTCN2018083969-appb-000014
本发明还提供了一种式I化合物(其中式I化合物可以为用于制备抑制PARP活性而改善疾病方面的药物的中间体)：
Figure PCTCN2018083969-appb-000015
其中，R 1、R 2和杂环基
Figure PCTCN2018083969-appb-000016
的定义同前所述。
所述式I最佳地为如下任一结构：
Figure PCTCN2018083969-appb-000017
本发明还提供了一种式I化合物的制备方法，其包括下列步骤：
将式II
Figure PCTCN2018083969-appb-000018
与盐酸反应生成式I化合物；
Figure PCTCN2018083969-appb-000019
所述式I化合物以固体形式存在于反应混合物中，且易通过多孔性介质分离；
其中，R 1、R 2和杂环基 的定义同前所述。
其中，所述式I化合物的制备方法中的各条件均同前所述。
本发明式II化合物可参考CN103237799A实施例所述方法，采用HATU/DIPEA/DMF来制备，但是该方法会产生少量的副产物四甲基脲且较难分离或去除。为了克服该问题，本发明中式II化合物的制备优选包括下列步骤：在缩合剂和缚酸剂的作用下，将式III化合物和化合物C进行反应，制得式II化合物；其中所述缩合剂为HOBT(1-羟基苯并***)、或者HOBT与EDCI(1-(3-二甲氨基丙基)-3-乙基-碳二亚胺盐酸盐)组成的混合体系；
所述式III化学结构式为：
Figure PCTCN2018083969-appb-000021
所述化合物C为
Figure PCTCN2018083969-appb-000022
R 1、R 2和杂环基
Figure PCTCN2018083969-appb-000023
的定义同前所述。
式II化合物的制备方法优选在溶剂的存在下进行。所述溶剂可为本领域此类反应常规的溶剂，优选卤代烷烃类溶剂，例如二氯甲烷。所述溶剂的用量可不作具体限定，只要不影响反应的进行即可。
所述缩合剂的用量可为本领域此类反应缩合剂常规的用量，优选其与式III化合物的 物质的量的比为1.5:1-3:1。当所述缩合剂为HOBT与EDCI组成的混合体系时，HOBT和EDCI的物质的量的比优选为1:0.8-1:1.5，例如1:1。所述的缚酸剂可为本领域此类反应常规的缚酸剂，例如乙二胺和/或N,N-二异丙基乙胺。所述缚酸剂的用量可为本领域此类反应常规的用量，其与缩合剂的物质的量的比优选为0.5:1-1.5:1。所述反应的温度优选10-40℃。所述反应的进程可采用本领域常规的检测方法进行监测，例如以TLC检测式III化合物消失时作为反应的终点。所述反应的时间优选2-24小时，例如2小时、4小时或24小时。
在一优选实施方案中，式II化合物的制备方法中，将式III化合物、化合物B和缩合剂混合，然后再加入缚酸剂，进行所述反应。
在一优选实施方案中，式II化合物的制备方法中，所述反应结束后，优选还进一步包括后处理的操作。所述后处理的操作可为本领域此类反应后处理常规的方法和条件，本发明优选包括下列步骤：将反应结束后的反应液进行分离浓缩(例如减压浓缩)即可。优选将反应结束后的反应液进行萃取(萃取用有机溶剂优选卤代烷烃类溶剂，萃取后，还可对有机相进行干燥，例如使用无水硫酸镁或无水硫酸钠干燥)后，有机相进行浓缩。
在另一优选实施方案中，式II化合物的制备方法包括下列步骤：将式III、化合物C按一定比例加入到反应容器中，加入1-羟基苯并***、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，控温10-40℃，滴加三乙胺或N,N-二异丙基乙胺，控温10-40℃，搅拌反应，2-24小时，分离浓缩得到固体；
所述式III化学结构式为：
Figure PCTCN2018083969-appb-000024
所述化合物C为
Figure PCTCN2018083969-appb-000025
R 1、R 2和杂环基
Figure PCTCN2018083969-appb-000026
的定义同前所述。
在将式III、化合物C按一定比例加入到反应容器中之前，优选先向反应容器中加入反应溶剂。
当缩合剂为HOBT与EDCI组成的混合体系时，HOBT与EDCI可同时加入到反应 体系中，也可依次加入到反应体系中。
其中一更优选的实施方案，所述式III化合物与化合物C的物质的量比为1:1-1:3。所述式III可以为2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酸。
本发明还提供式I-a化合物或其药学上可接受的盐，
Figure PCTCN2018083969-appb-000027
其中，所述R 1、R 2和杂环基
Figure PCTCN2018083969-appb-000028
的定义同前所述。
其中一个优选的实施方案，所述药学上可接受的盐为盐酸盐、对甲苯磺酸盐、酒石酸盐、马来酸盐、乳酸盐、甲磺酸盐、硫酸盐、磷酸盐、柠檬酸盐或醋酸盐，进一步优选为甲苯磺酸盐、盐酸盐或酒石酸盐；本发明的药学上可接受的盐可以通过常规化学方法合成。
其中一个优选的实施方案，所述式I-a为下式I-a-1：
Figure PCTCN2018083969-appb-000029
其中一个更为优选的实施方案，所述如式I-a-1所示化合物的药学上可接受的盐为盐酸盐。
式I-a化合物或其药学上可接受的盐不仅可用于制备抑制PARP酶活性的药物化合物A，申请人通过体外实验证明，式I-a化合物(例如式I-a-1)或其药学上可接受的盐本身也具有良好的PARP酶抑制活性。
本发明还提供一种药物组合物，它包含本发明所述的式I-a化合物或其药学上可接受的盐，以及药学上可接受的载体或赋形剂。
本发明的药物组合物可与药物领域中一些常用的赋形剂配制成药物制剂，所述的制剂可以为片剂、胶囊剂、注射剂、气雾剂、栓剂、膜剂、滴丸剂、外用搽剂、软膏剂等。
本发明还提供了式I-a化合物(例如式I-a-1化合物)或其药学上可接受的盐在制备通过抑制PARP活性而改善的疾病方面的药物中的应用。
本发明还提供了一种抑制PARP活性而改善的疾病的方法，其包括给予需要治疗的哺乳动物有效量的本发明所述的式I-a化合物(例如式I-a-1化合物)或其药学上可接受的盐。
本发明还提供了式A-5化合物的非晶形，其在XRPD图中无晶体衍射峰
Figure PCTCN2018083969-appb-000030
本发明中，所述式A-5化合物的非晶形中式A-5化合物的含量优选为98.0％-102.0％之间，进一步优选为99.0-100.0％之间；所述式A-5化合物的非晶形的HPLC纯度不低于99.5％，总杂不高于0.5％。
本发明还提供了所述的PARP抑制剂式A-5化合物的非晶形的制备方法，其包含如下步骤：将式A-5化合物和良性溶剂的混合溶液，与不良性溶剂混合，即可；所述良性溶剂与式A-5化合物的体积质量比为1mL/g-10mL/g，所述不良性有机溶剂与式A-5化合物的体积质量比为10mL/g-100mL/g。
本发明中，所述良性溶剂一般是指式A-5化合物在所述良性溶剂中具有较好的溶解度，即当所述良性溶剂与式A-5化合物的体积质量比在1mL/g-10mL/g时，式A-5化合物能够完全溶解于所述良性溶剂中。
本发明中，所述不良性溶剂一般是指当式A-5化合物和良性溶剂的混合液，与所述不良溶剂混合后，式A-5化合物能够以固体形式析出，即当所述不良性溶剂与式A-5化合物的体积质量比大于10mL/g时，式A-5化合物不能够完全溶解于所述不良性溶剂中。
在一优选实施方案中，所述良性溶剂为乙醇、甲醇、丙酮、丁酮、乙酸乙酯、乙酸异丁酯、异丙醇、正丙醇、四氢呋喃、甲苯和二氯甲烷中的一种或多种。
在一优选实施方案中，所述不良性溶剂为异丙醚、正己烷、正戊烷、正庚烷、正辛烷、甲基叔丁基醚、石油醚和环己烷中的一种或多种。
所述式A-5化合物的非晶形的制备方法中，所述混合的温度优选地在-10～40℃(例如20-40℃，或25-30℃)。所述混合的操作的同时或结束后，还可进一步包含搅拌的操作。所述搅拌时混合液的温度优选-10～40℃，更优选20-40℃，例如25-35℃。所述继续搅拌的时间可不作具体限定，以不再有固体析出为准。
所述式A-5化合物的非晶形的制备方法中，所述搅拌的操作结束后，还可进一步包括后处理的操作。所述的后处理的操作可为本领域此类反应后处理常规的操作，优选包括下列步骤：将继续搅拌后得到的混合物，进行固液分离，干燥即可。所述固液分离的操作优选过滤。所述固液分离后得到的滤饼优选进行洗涤。所述洗涤溶剂优选上述不良溶剂。所述干燥优选减压干燥。
所述式A-5化合物的非晶形的制备方法中，优选将式A-5化合物和良性溶剂的混合溶液加入到不良性溶剂中。所述的加入的方式优选滴加。所述的滴加的速度可不作具体限定，只要不影响式A-5化合物析出即可。
在一优选实施方案中，所述式A-5化合物的非晶形的制备方法，其包含如下步骤：用良性溶剂溶解式A-5化合物，滴入到不良性溶剂中搅拌，析出固体，过滤，干燥；所述良性溶剂与式A-5化合物的体积质量比为1mL/g-10mL/g，所述不良性溶剂与式A-5化合物的体积质量比为10mL/g-100mL/g。
在一优选实施方案中，所述析出固体温度为-10～40℃，优选20-40℃，例如25-35℃。
本发明中，式A-5化合物的HPLC纯度在70％以上即可，优选在70％-99％之间。当式A-5化合物的HPLC纯度为100％时，采用本发明的制备方法也可以制备得到本发明特定的非晶形。
本发明还提供一种含有所述式A-5化合物的非晶形的药物组合物。所述的药物组合物优选包含所述式A-5化合物的非晶形和药学上可接受的辅料。
本发明还提供了了一种所述式A-5化合物的非晶形或其药物组合物在制备通过抑制PARP活性而改善的疾病方面的药物中的应用。
本发明还提供了一种抑制PARP活性而改善的疾病的方法，所述方法包括给予需要的患者治疗施用治疗有效量的式A-5化合物的非晶形或所述药物组合物。
本发明还提供了一种式A-5化合物的溶剂合物晶体：
Figure PCTCN2018083969-appb-000031
在一优选实施方案中，所述溶剂合物晶体为式A-5化合物的酯类溶剂合物晶体；优选为式A-5化合物的乙酸乙酯溶剂合物晶体、式A-5化合物的甲酸乙酯溶剂合物晶体、式A-5化合物的乙酸甲酯溶剂合物晶体或式A-5化合物的甲酸正丙酯溶剂合物晶体。
在一优选实施方案中，所述溶剂合物晶体的红外吸收光谱具有吸收光带，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置具有吸收峰：1668、1636、1496、1449和804。
在一优选实施方案中，所述溶剂合物晶体为式A-5化合物的乙酸乙酯溶剂合物晶体A，在该晶体A的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.193、14.137、17.799、19.483、20.062、20.861和23.837处具有特征峰；优选在衍射角度2θ±0.2°为8.193、9.507、11.141、13.547、14.137、15.935、16.732、17.799、19.483、20.062、20.861和23.837处具有特征峰。
在另一优选实施方案中，所述式A-5化合物的乙酸乙酯溶剂合物晶体A的X衍射粉末衍射图的数据如下表1所示：
表1
Figure PCTCN2018083969-appb-000032
Figure PCTCN2018083969-appb-000033
Figure PCTCN2018083969-appb-000034
在一更优选实施方案中，所述式A-5化合物的乙酸乙酯溶剂合物晶体A具有如图1所示的X-衍射粉末衍射图。
在一优选实施方案中，所述式A-5化合物的乙酸乙酯溶剂合物晶体A的红外吸收光谱具有吸收光带，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置具有吸收峰：1666.67、1635.93、1495.51、1450.38和805.05，进一步优选在以下位置具有吸收峰：3435.84、3204.48、1737.55、1666.67、1635.93、1495.51、1450.38、805.05和682.88。
在一优选实施方案中，所述式A-5化合物的乙酸乙酯溶剂合物晶体A具有如图2所示的红外图谱。
在一优选实施方案中，所述式A-5化合物的乙酸乙酯溶剂合物晶体A经气相检测乙酸乙酯的含量优选为12～20％。
在一优选实施方案中，所述溶剂合物晶体为式A-5化合物的甲酸乙酯溶剂合物晶体B，在该晶体B的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.471、14.034、16.131和19.679处具有特征峰；优选在衍射角度2θ±0.2°为8.471、9.776、11.491、14.034、16.131、19.679、22.621和24.532处具有特征峰；更优选在衍射角度2θ±0.2°为8.471、9.776、11.151、 11.491、14.034、16.131、16.790、17.755、18.626、19.679、22.621、23.268和24.532处具有特征峰。
在另一优选实施方案中，所述式A-5化合物的甲酸乙酯溶剂合物晶体B的X衍射粉末衍射图的数据如下表2所示：
表2
Figure PCTCN2018083969-appb-000035
Figure PCTCN2018083969-appb-000036
在一更优选实施方案中，所述式A-5化合物的甲酸乙酯溶剂合物晶体B具有如图4所示的X-衍射粉末衍射图。
在一优选实施方案中，所述式A-5的甲酸乙酯溶剂合物晶体B的红外吸收光谱具有吸收光带，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置具有吸收峰：1667.77、1336.87、1496.32、1448.70和804.45；进一步优选在以下位置具有吸收峰：3447.16、3199.04、1723.60、1667.77、1336.87、1496.32、1448.70、804.45和771.41。
在一更优选实施方案中，所述式A-5化合物的甲酸乙酯溶剂合物晶体B具有如图5所示的红外图谱。
在一优选实施方案中，所述式A-5的甲酸乙酯溶剂合物晶体B经气相检测甲酸乙酯的含量优选为5～15％。
在一优选实施方案中，所述溶剂合物晶体为式A-5化合物的乙酸甲酯溶剂合物晶体C，在该晶体C的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.454、14.057、16.111和19.681处具有特征峰；优选在衍射角度2θ±0.2°为8.454、9.748、11.548、13.740、14.057、16.111、19.681和22.619处具有特征峰；更优选在衍射角度2θ±0.2°为8.454、9.748、11.144、11.548、13.740、14.057、16.111、16.779、17.778、18.596、19.681、22.619和23.256处具有特征峰。
在另一优选实施方案中，所述式A-5化合物的乙酸甲酯溶剂合物晶体C的X衍射粉末衍射图的数据如下表3所示：
表3
Figure PCTCN2018083969-appb-000037
Figure PCTCN2018083969-appb-000038
在一更优选实施方案中，所述式A-5化合物的乙酸甲酯溶剂合物晶体C具有如图6所示的X-衍射粉末衍射图。
在一优选实施方案中，所述式A-5的乙酸甲酯溶剂合物晶体C的红外吸收光谱具有吸收光带，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置具有吸收峰：1668.61、1637.02、1496.34、1449.40和804.71；进一步优选在以下位置具有吸收峰：3447.25、3199.85、1744.78、1668.61、1637.02、1496.34、1449.40、804.71和771.49。
在一更优选实施方案中，所述式A-5化合物的乙酸甲酯溶剂合物晶体C具有如图7所示的红外图谱。
在一优选实施方案中，所述式A-5的乙酸甲酯溶剂合物晶体C经气相检测乙酸甲酯的含量优选为5～15％。
在一优选实施方案中，所述溶剂合物晶体为式A-5化合物甲酸正丙酯溶剂合物晶体D，在该晶体D的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.152、13.989、15.924、19.459和22.237处具有特征峰；优选在衍射角度2θ±0.2°为8.152、13.526、13.989、15.924、16.720、19.058、19.459、22.237和22.840处具有特征峰；更优选在衍射角度2θ±0.2°为8.152、9.459、11.647、13.526、13.989、15.407、15.924、16.720、17.692、18.423、19.058、19.459、22.237、22.840、23.795和24.751处具有特征峰。
在另一优选实施方案中，所述式A-5化合物的甲酸正丙酯溶剂合物晶体D的X衍射粉末衍射图的数据如下表4所示：
表4
Figure PCTCN2018083969-appb-000039
Figure PCTCN2018083969-appb-000040
Figure PCTCN2018083969-appb-000041
在一更优选实施方案中，所述式A-5化合物的甲酸正丙酯溶剂合物晶体D具有如图8所示的X-衍射粉末衍射图。
在一优选实施方案中，所述式A-5的甲酸正丙酯溶剂合物晶体D的红外吸收光谱具有吸收光带，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置具有吸收峰：1668.42、1636.53、1496.39、1449.31和804.41，进一步优选在以下位置具有吸收峰：3447.01、3203.32、1726.16、1668.42、1636.53、1496.39、1449.31、804.41和772.26。
在一更优选实施方案中，所述式A-5化合物的甲酸正丙酯溶剂合物晶体D具有如图9所示的红外光谱图。
在一优选实施方案中，所述式A-5化合物的甲酸正丙酯溶剂合物晶体D经气相检测甲酸正丙酯的含量优选为12～20％。
本发明还提供所述的PARP抑制剂式A-5化合物的溶剂合物晶体的制备方法，包含下列步骤：将式A-5化合物从溶剂或含溶剂的溶液中析出。
在一优选实施方案中，所述式A-5化合物为非晶形(即无定形)。本发明中，式A-5化合物的HPLC纯度在70％以上即可，优选在99％以上。
在一优选实施方案中，当所述式A-5化合物为非晶形(即无定形)时，其优选采用前述式A-5化合物的非晶形的制备方法制备得到。
在一优选实施方案中，所述含溶剂的溶液中，溶质为所述溶剂合物中的溶剂，优选酯类溶剂，溶剂为所述溶剂合物中的溶剂以外的有机溶剂。所述有机溶剂优选为醇类溶剂(例如甲醇)、腈类溶剂(例如乙腈)、醚类溶剂(例如四氢呋喃和/或1,4-二氧六环)和卤代烷烃类溶剂(例如二氯甲烷)中的一种或多种。
在一优选实施方案中，所述含溶剂的溶液中的溶剂(即所述溶剂合物中的溶剂)质量分数不小于50％；进一步优选不小于85％；再进一步优选不小于95％。
在另一优选实施方案中，所述式A-5化合物的酯类溶剂合物晶体的制备方法，其包含下列步骤：将式A-5化合物从酯类溶剂或含酯类溶剂的溶液中析出。
在一更优选实施方案中，式A-5化合物的酯类溶剂合物晶体的制备方法，其包含下列步骤：将式A-5化合物和酯类溶剂或含酯类溶剂的溶液混合，在0～40℃条件下搅拌析 晶，制得式A-5化合物的酯类溶剂合物晶体；所述酯类溶剂和式A-5化合物的体积质量比为4mL/g～30mL/g。
在一优选实施方案中，所述酯类溶剂可为本领域常规的酯类溶剂，优选为甲酸乙酯、乙酸甲酯、甲酸正丙酯、丙酸甲酯、乙酸乙酯、乙酸正丙酯、丙酸乙酯、甲酸正丁酯、丁酸甲酯、乙酸正丁酯、丁酸乙酯、丙酸正丁酯或丁酸丙酯；更优选乙酸乙酯、甲酸乙酯、乙酸甲酯或甲酸正丙酯。
在一优选实施方案中，所述含酯类溶剂的溶液中，所述酯类溶剂同前所述；所述的溶液中的溶剂优选为有机溶剂，进一步优选醇类溶剂(例如甲醇)、腈类溶剂(例如乙腈)、醚类溶剂(例如四氢呋喃和/或1,4-二氧六环)和卤代烷烃类溶剂(例如二氯甲烷)中的一种或多种。
在一优选实施方案中，所述含酯类溶剂的溶液中，所述酯类溶剂的质量分数不小于50％；进一步优选不小于85％；再进一步优选不小于95％。所述含酯类溶剂的溶液优选①95％(w/w)乙酸乙酯和5％(w/w)四氢呋喃；②90％(w/w)乙酸乙酯和10％(w/w)乙腈；③85％(w/w)乙酸乙酯和15％(w/w)四氢呋喃；④50％(w/w)乙酸乙酯和50％(w/w)乙腈；⑤90％w/w乙酸甲酯和10％w/w乙腈或⑥50％(w/w)甲酸正丙酯和50％(w/w)乙腈。
在另一优选实施方案中，所述酯类溶剂或含酯类溶剂的溶液分别和式A-5化合物的体积质量比为4mL/g～30mL/g，进一步优选6mL/g～10mL/g。
在一优选实施方案中，所述析晶的温度为15～25℃。
在一优选实施方案中，所述析晶的操作结束后，还可进一步包含过滤、洗涤和干燥的操作。所述洗涤用有机溶剂优选所述式A-5化合物的酯类溶剂中的酯类溶剂。
在一优选实施方案中，所述干燥通常在30-70℃下进行，进一步优选30-60℃。所述干燥的时间可为本领域产品干燥常规的时间，一般至产品重量基本恒定。干燥的操作根据需要可以为鼓风干燥，或减压干燥。
在一优选实施方案中，所述式A-5化合物的酯类溶剂合物晶体的制备方法，其包括下列步骤：将式A-5化合物溶解于酯类溶剂或含酯类溶剂的溶液中，在0～40℃条件下搅拌析晶，过滤，干燥，得到式A-5的酯类溶剂合物晶体；所述酯类溶剂和式A-5化合物的体积质量比为4mL/g～30mL/g。
本发明还提供一种含有所述式A-5化合物的溶剂合物晶体的药物组合物。所述药物组合物优选包含所述式A-5化合物的溶剂合物晶体和药学上可接受的辅料。
本发明还提供了一种抑制PARP活性而改善的疾病的方法，所述方法包括给予需要的患者治疗施用治疗有效量的所述式A-5化合物的溶剂合物晶体或所述药物组合物。
本发明还提供所述式A-5的溶剂合物晶体或其药物组合物在制备通过抑制PARP活性而改善的疾病方面的药物中的应用。
本发明中，所述通过抑制PARP活性而改善的疾病选自癌症、血管疾病、炎症性疾病、排异反应、糖尿病、帕金森病、感染性休克、缺血性损伤、神经性毒性、失血性休克和病毒感染。
本发明中，所述癌症是同源重组依赖性DNA双链断裂修复途径有缺陷的，或癌细胞是BRCA1或BRCA2有缺陷。
本发明所述化合物I-a或其药学上可接受的盐、所述化合物I-a-1或其药学上可接受的盐、所述的式A-5的非晶形或所述的式A-5的溶剂合物晶体可制备用于通过抑制细胞PARP酶的活性防止多聚(ADP-核糖)链形成的药物。
(a)治疗的疾病包括：血管疾病、感染性休克、缺血性损伤、脑和心血管再灌注损伤、神经毒性(例如对急性和/或慢性中风和帕金森氏病的治疗)、出血休克；眼睛相关的氧化损伤；移植排斥反应；炎症性疾病如关节炎、炎性肠病、溃疡性结肠炎或克罗恩病；多发性硬化症；糖尿病继发效应；以及心血管手术后的细胞毒性的急性治疗、胰腺炎；动脉粥样硬化或抑制PARP活性改善疾病；
(b)作为在治疗癌症方面的辅助剂或用于增强电离放射或化疗药物对肿瘤细胞的作用。
本发明的有益技术效果：
1、本发明提供的抑制PARP酶活性而改善疾病方面的化合物A的制备方法，通过式II与HCl(HCl为盐酸或氯化氢气体，优选盐酸)反应生成对应的盐酸盐，该盐酸盐以稳定的固体形式存在于反应混合物中，易通过多孔性介质分离，如常规的过滤，即可得到高纯度的式I化合物。另一方面，所采用盐酸成本低，易获得，适合工业化生产。
进一步，式I化合物与化合物B在缩合剂中反应，如在HOBT/EDCI混合缩合体系中反应，所产生的副产物较易分离去除，在后处理过程中通过简单的萃取、洗涤、分液便可除去，从而使得到的化合物A纯度和收率都非常高。
再进一步，本发明式II的制备过程，采用HOBT/EDCI/TEA，所产生副产物较易分离去除，在后处理过程中通过简单的萃取、洗涤、分液便可除去，使得式II的纯度和收率较高。
2、本发明提供的高纯度抑制PARP活性而改善疾病方面的药物化合物A的中间体式I化合物，是一种新的化合物，在用于制备高纯度的抑制PARP活性而改善疾病方面的药物化合物A方面，具有积极的社会意义。
3、本发明所提供的制备化合物A方法操作简单，有利于工业化生产。纯度和收率较高，每步的摩尔转化率在90％以上，工艺经济性较高。
4、本发明所述的化合物I-a或其药学上可接受的盐可用于制备抑制PARP酶活性的药物化合物A，体外实验证明，I-a或其药学上可接受的盐也具有良好的PARP抑制活性。
5、本发明的式A-5化合物的非晶形溶解性好，较高的溶出速率可导致过饱和，从而使得生物利用度高，有利于药物的吸收及利用。
6、本发明的式A-5化合物的非晶形的稳定性高，有利于药物组合物的制备和使用。
7、本发明制备式A-5化合物的非晶形的方法简单、快捷，易于产业化生产。
8、本发明制备式A-5化合物的非晶形流动性较好，均匀性较好，易于制剂生产。
9、本发明式A-5化合物的溶剂合物呈明显晶状固体，XRPD特征峰尖锐，表明该晶体结晶性较好，纯度高，不易聚结。
进一步，本发明的式A-5化合物的酯类溶剂合物晶体稳定性好，实验结果表明，75％高湿裸放条件下，本发明提供的式A-5化合物溶剂合物晶体10天的引湿性小于1％，可认为基本没有引湿性，相比非晶形(即无定形)的引湿性小很多，从而使得本发明式A-5化合物的酯类溶剂合物晶体便于储存，从而对包装要求低。而且式A-5化合物的酯类溶剂合物晶体流动性较好，易于工业化生产。
10、本发明的式A-5的溶剂合物晶体的制备方法，非常适合规模化生产。
术语：
“溶剂合物”指的是由式A-5化合物和溶剂结合形成的络合物。
“施用”指的是将药剂引入患者体内。可以施用治疗量，其可以由治疗医生等来决定。对于此处所描述的晶体形态的式A-5化合物，优选口服给药途径。相关术语和短语“施用(administrating)”或“施用(administration of)”当与化合物或药物组合物(和语法上的等同物)联用时，指的是直接施用和/或间接施用，所述直接施用可以通过医疗专业人员给患者施用或患者自己施用，所述间接施用可以是开药物处方的行为。例如，医师指示患者自己施用药物和/或给患者提供药物的处方为给患者施用所述药物。在任何情况下，施用需要将药物递送给患者。
如此处所用的“赋形剂”指的是药品生产中所用的惰性或非活性物质，包括不限于任何用作粘合剂、崩解剂、包衣、压缩/封装助剂、乳剂或洗剂、润滑剂、胃肠外注射物、甜味剂或调味剂、悬浮/胶凝剂或湿润造粒剂的物质。
“治疗有效量”或“治疗量”指的是当施用于患有病症的患者时的药物或药剂的量，其将具有预料的治疗效果，例如减轻、改善、缓解或消除患者所患病症的一种或多种临床表现。治疗有效量会取决于下述因素而变化：被治疗的个体或病症，个体的重量和年龄，病症的严重性，所选的具体组合物或赋形剂，待遵守的剂量给药方案，给药时间，给药方式等，其所有这些因素都可容易地由本领域技术人员确定。总体的治疗效果不必通过施用一个剂量产生，而是可能只在服用一系列剂量后才产生。因此，治疗有效量可能以一次或多次给药来施用。例如但不限于，在治疗贫血症的情况下的治疗有效数量指的是减轻、改善、缓解或消除患者的一个或多个贫血症状的药剂量。
“治疗(treatment)”、“治疗(treating)”以及“治疗(treat)”被定义为用药剂作用于疾病、失调或病症，以减少或改善所述疾病、失调或病症和/或其症状的有害或任何其他不期望的作用。此处使用的治疗包括对人类患者的治疗，并包括：(a)减少患者产生病症的风险，所述患者被确定为倾向患病但还没有诊断为患有病症，(b)阻止病症的发展，和/或(c)减轻病症，即引起病症的消退和/或减轻病症的一种或多种症状。
“XRPD图样”为在x-轴上具有衍射角(即°2θ)并在y-轴上具有强度的x-y图。这种图样中的峰可以用于表征晶体固态形态。在任何数据测量结果的情况下，XRPD数据存在可变性。数据经常唯一地由峰的衍射角来表示，而不包括峰的强度，因为峰的强度可能对样品的制备特别敏感(例如，粒径、水分含量、溶剂含量和优选的取向效果影响敏感性)，因此在不同条件下制备的相同材料的样品可能产生略微不同的图样；这一可变性通常大于衍射角的可变性。衍射角的可变性也可以对样品的制备敏感。其他可变性的来源来自仪器参数和对原始X-射线数据的处理：不同的X-射线仪器操作使用不同的参数并且这些可能导致与相同的固体形态略微不同的XRPD图样，并且类似地，不同软件包以不同方式处理X-射线数据并且这也导致可变性。可变性的这些和其他的来源对药学领域中的技术人员来说是已知的。由于该可变性的来源，对XRPD图样中的衍射角分配±0.2°2θ的可变性，即本发明不仅包括X射线粉末衍射中的衍射角完全一致的结晶，还包括在±0.2°的误差范围内衍射角一致的结晶。
附图说明
图1为本发明一优选实施方案中式A-5化合物的乙酸乙酯溶剂合物晶体A的XRPD图。
图2为本发明一优选实施方案中式A-5化合物的乙酸乙酯溶剂合物晶体A的红外图谱。
图3为本发明一优选实施方案中式A-5化合物的非晶形的XRPD图。
图4为本发明一优选实施方案中式A-5化合物的甲酸乙酯溶剂合物晶体B的XRPD图。
图5为本发明一优选实施方案中式A-5化合物的甲酸乙酯溶剂合物晶体B的红外吸收图谱。
图6为本发明一优选实施方案中式A-5化合物的乙酸甲酯溶剂合物晶体C的XRPD图。
图7为本发明一优选实施方案中式A-5化合物的乙酸甲酯溶剂合物晶体C的红外吸收图谱。
图8为本发明一优选实施方案中式A-5化合物的甲酸正丙酯溶剂合物晶体D的XRPD图。
图9为本发明一优选实施方案中式A-5化合物的甲酸正丙酯溶剂合物晶体D的红外吸收图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件进行选择。
本发明所有化合物的结构可通过核磁共振( 1H NMR)和/或质谱检测(MS)鉴定。 1H NMR化学位移(δ)以PPM记录(10 -6)。NMR通过Bruker AVANCE-400光谱仪进行。合适的溶剂是氘代氯仿(CDCl 3)四甲基硅烷作为内标(TMS)。
低分辨率质谱(MS)由Utimate 3000HPLC-MSQ Plus MS质谱仪测定。
化合物纯度通过高效液相色谱(HPLC)测定。
HPLC型号Waters e2695/2489。
X射线粉末衍射(XRPD)
使用Cu-Kα辐射(40kV，40mA)、θ-2θ测角仪和Lynxeye检测器，在Bruker D8衍射仪上采集X射线粉末衍射图。使用认证的刚玉(Corundum)标准(NIST1976)检查仪器的性 能。用于数据收集的软件是Diffrac Plus XRD Commander V6，且使用DIFFRAC.EVA V4.2分析并呈现数据。
使用所接收的粉末，样品在环境条件下作为平板标本运行。将样品轻轻包装在被切成抛光、零背景硅片的腔中。数据收集的详情是：
角范围：2至40°2θ
步长(step size)：0.02p si
收集时间：0.5s/步
分析持续时间：17min
红外吸收光谱的测定
根据中国药典2015年版四部通则中记载的红外分光光度法(压片法)，利用FT-IR Nicolet is5(Thermo Fisher Scientific)，在4000～400cm -1的测定范围内，测定晶体的红外吸收光谱。
第I部分实施例
实施例I-1 (1S,4S)-5-(2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酰)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸叔丁酯的制备
依次向反应瓶中加入二氯甲烷(700ml)，2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酸(70g，0.24mol)，(1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸叔丁酯(71.4g，0.36mol)和2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(136.5g，0.36mol)，搅拌。滴入N,N-二异丙基乙胺(70.0g，0.54mol)，室温搅拌2小时，TLC监控原料反应完全。加入700ml水，搅拌，分液，有机相洗涤，无水硫酸钠干燥，过滤，浓缩得褐色油状物，质量157.2g。
实施例I-2 (1S,4S)-5-(2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酰)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸叔丁酯的制备
依次向反应瓶中加入500ml二氯甲烷，2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酸(70g，0.24mol)、(1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸叔丁酯(71.4g，0.36mol)，搅拌。依次加入1-羟基苯并***(HOBT)(48.6g，0.36mol)、1-(3-二甲氨基丙基)-3-乙基-碳二亚胺盐酸盐(EDCI)(69.0g，0.36mol)。控温10℃，滴加三乙胺(36.4g，0.36mol)，滴毕，控温10℃搅拌反应约4h，TLC监控原料反应完全。加500ml水，搅拌，分液，有机相洗涤，无水硫酸钠干燥，过滤，滤液于40℃减压浓缩，得到白色固体，质量148g。
实施例I-3 (1S,4S)-5-(2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酰)2,5-二氮杂双环[2,2,1]庚烷-2-甲酸叔丁酯的制备
依次向反应瓶中加入500ml二氯甲烷，2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酸(70g，0.24mol)、(1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸叔丁酯(47.6g，0.24mol)，搅拌。依次加入HOBT(39.2g，0.29mol)、EDCI(55.6g，0.29mol)。控温10℃，滴加N，N-二异丙基乙胺(37.6g，0.29mol)。滴毕，控温40℃搅拌反应约24h，TLC监控原料反应完全。加入500ml水，搅拌，分液，有机相洗涤，无水硫酸钠干燥，过滤，滤液于40℃减压浓缩，得到白色固体，质量152g。
实施例I-4 4-(3-((1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸)-4-氟苄基)酞嗪-1(2H)-酮盐酸盐的制备(式I-5)
将实施例I-1所得油状物(理论112.3g，0.24mol)采用1.7L乙酸乙酯溶解完全，控温15℃，滴入浓盐酸(160ml，1.92mol)，控温在20℃，反应2h，TLC监控原料反应完全，有大量固体析出；过滤，用少量乙酸乙酯淋洗，滤饼在50℃干燥至恒重，得固体82.8g，纯度99.90％，两步收率85.0％；
1H NMR(CDCl 3)：δ12.63-9.64(3H,s,brs)，8.28-7.22(7H,m)，4.86～4.17(2H,m)，4.36(2H,s)，3.64-3.17(4H,m)，2.10-1.78(2H,m)；m/z[M+1] +379.1。
实施例I-5 4-(3-((1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸)-4-氟苄基)酞嗪-1(2H)-酮盐酸盐的制备(式I-5)
将实施例I-2所得固体(理论112.3g，0.24mol)采用1.2L丁酮溶解完全，控温10℃，滴入浓盐酸(300ml，3.6mol)，控温40℃，反应10h，TLC监控原料反应完全，有大量固体析出；过滤，用少量丁酮淋洗，滤饼在50℃干燥至恒重，得固体81.0g，纯度100.0％，两步收率83.1％，核磁、质谱数据同实施例I-4。
实施例I-6 4-(3-((1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸)-4-氟苄基)酞嗪-1(2H)-酮盐酸盐的制备(式I-5)
将实施例I-3所得固体(理论112.3g，0.24mol)采用1.2L异丙醇溶解完全，控温25℃，滴入6N浓盐酸(576ml，2.9mol)，控温在25℃，反应20h，TLC监控原料反应完全，有大量固体析出；过滤，用少量异丙醇淋洗，滤饼在50℃干燥至恒重，得固体90.6g，纯度99.99％，两步收率93.0％，核磁、质谱数据同实施例I-4。
实施例I-7 4-(3-((1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸)-4-氟苄基)酞嗪-1(2H)-酮盐酸盐的制备(式I-5)
与实施例I-4的不同之处在于，所述滴入浓盐酸(160mL，1.92mol)用通入1.92mol氯化氢气体代替，得固体90.1g，纯度99.35％，两步收率92.5％，核磁、质谱数据同实施例I-4。
实施例I-8 4-(3-((1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸)-4-氟苄基)酞嗪-1(2H)-酮盐酸盐的制备(式I-5)
与实施例I-5的不同之处在于，所述滴入浓盐酸(300mL，3.6mol)用通入1.2mol氯化氢气体代替，得固体89.6g，纯度99.15％，两步收率91.8％，核磁、质谱数据同实施例I-4。
实施例I-9 4-(3-((1S,4S)-2,5-二氮杂双环[2,2,1]庚烷-2-甲酸)-4-氟苄基)酞嗪-1(2H)-酮盐酸盐的制备(式I-5)
与实施例I-6的不同之处在于，所述滴入6N浓盐酸(576ml，2.9mol)用通入4.8mol氯化氢气体代替，得固体88.8g，纯度99.10％，两步收率91.2％，核磁、质谱数据同实施例I-4。
申请人将实施例I-6中6N浓盐酸(576ml，2.9mol)替换为2N浓盐酸(1450ml，2.9mol)，得到固体90.1g，纯度99.35％，两步收率92.5％，核磁、质谱数据同实施例I-4。
申请人还将实施例I-4、I-5、I-6、I-7、I-8、I-9中的有机溶剂用甲醇、乙醇、正丁醇、乙酸异丙酯、丙酮等方案替换及多种组合实验，所得式I-5固体纯度均在99％以上，两步收率均在80％以上。
实施例I-10 1-环丙基-2-((1S,4S)-5-(2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酰)-2,5-二氮杂双环[2,2,1]庚烷-2-基)乙烷-1,2-二酮的制备(式A-5)
将实施例I-4所得固体(80.0g，0.19mol)加入装有800ml二氯甲烷反应容器中，搅拌，加入HOBT(39.2g，0.29mol)、EDCI(82.4g，0.43mol)和2-环丙基-2-氧代乙酸钠(39.5g，0.29mol)，加毕，控温15℃，搅拌，反应5h，TLC监控原料反应完全；加入水，搅拌，过滤，滤液静置分层，有机相洗涤，用无水硫酸钠干燥，滤液于40℃减压浓缩，得到化合物A-5，质量89.7g，收率98.1％，纯度99.92％；
m/z[M+1] +475.4； 1H NMR(CDCl 3)：δ10.57-10.30(1H，brs)，8.44(1H，m)，7.77-7.63(3H，m)，7.50-7.23(2H，m)，7.05-6.92(1H，m)，5.25-4.85(2H，m)，4.50-4.48(2H，d)，3.91-3.30(4H，m)，2.94-2.68(1H，m)，1.97-1.85(2H，m)，1.20-0.96(4H，m)。
实施例I-11 1-环丙基-2-((1S,4S)-5-(2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酰)-2,5-二氮杂双环[2,2,1]庚烷-2-基)乙烷-1,2-二酮的制备(式A-5)
将实施例I-5所得固体(80.0g，0.19mol)加入装有800ml二氯甲烷反应容器中，搅拌，加入HOBT(31.1g，0.23mol)、EDCI(44.1g，0.23mol)和2-环丙基-2-氧代乙酸钠(35.0g，0.23mol)，加毕，控温25℃，搅拌，反应2h，TLC监控原料反应完全；加入水， 搅拌，过滤，滤液静置分层，有机相洗涤，用无水硫酸钠干燥，滤液于50℃减压浓缩，得到化合物A-5，质量86.8g，收率95.0％，纯度99.96％，核磁、质谱数据同实施例I-10。
实施例I-12 1-环丙基-2-((1S,4S)-5-(2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酰)-2,5-二氮杂双环[2,2,1]庚烷-2-基)乙烷-1,2-二酮的制备(式A-5)
将实施例I-6所得固体(80.0g，0.19mol)加入装有800ml二氯甲烷反应容器中，搅拌，加入HOBT(31.1g，0.23mol)、EDCI(35.2g，0.18mol)和2-环丙基-2-氧代乙酸钠(35.0g，0.23mol)，加毕，控温25℃，搅拌，反应8h，TLC监控原料反应完全；加入水，搅拌，过滤，滤液静置分层，有机相洗涤，用无水硫酸钠干燥，滤液于50℃减压浓缩，得到化合物A-5，质量84.1g，收率92.0％，纯度99.93％，核磁、质谱数据同实施例I-10。
实施例I-13 1-环丙基-2-((1S,4S)-5-(2-氟-5-((4-氧-3,4-二氢酞嗪-1-基)甲基)苯甲酰)-2,5-二氮杂双环[2,2,1]庚烷-2-基)乙烷-1,2-二酮的制备(式A-5)
将实施例I-8所得固体(80.0g，0.19mol)加入装有800ml二氯甲烷反应容器中，搅拌，加入EDCI(44.1g，0.23mol)和2-环丙基-2-氧代乙酸钠(35.0g，0.23mol)，加毕，控温25℃，搅拌，反应4h，TLC监控原料反应完全；加入水，搅拌，过滤，滤液静置分层，有机相洗涤，用无水硫酸钠干燥，滤液于50℃减压浓缩，得到化合物A-5，质量85.0g，收率93.0％，纯度99.95％，核磁、质谱数据同实施例I-10。
通过比较实施例I-1、I-2、I-3，实施例I-1得到的是油状物，其中含有少量副产物四甲基脲，而实施例I-2、I-3得到的是固体；但无论式II以油状物还是固体形式存在于反应混合物中，只要在后续步骤生成式I(例如式I-5)以固体形成存在于反应混合物中，再通过简单地过滤、干燥就可以得到高纯度的式I(例如式I-5)化合物。
进一步，由高纯度的式I(例如式I-5)化合物与化合物B反应，生成高纯度的化合物A，纯度达99.9％以上，收率达90％以上。
CN103237799中实施例方法中获得的化合物收率不到60％，如CN103237799A实施例2与本发明方法(例如：实施例10至13)制备相同的化合物式I-5，CN103237799A实施例2中式I-5的收率为55％，远低于本发明方法的收率。
实施例I-14 PARP抑制功效可通过以下实验来测定
重组人PARP酶活性测试：
本实验参照BPS公司的PARP酶分析试剂盒(PARP1和PARP2)的方法进行，具体的PARP酶活性检测方法如下：
1、实验材料：本实验均采用BPS公司的PARP分析试剂盒进行。
2、受试化合物信息如下：
1-a-1，来自实施例I-4所得产物样品。
式A-5，来自实施例I-10所得产物样品。
阳性对照AZD2281，化学名称为：1-(环丙基羰基)-4-[5-[(3,4-二氢-4-氧代-1-酞嗪)甲基]-2-氟苯甲酰]哌嗪。
化合物 性状 储备液浓度 稀释溶剂 浓度范围 中间稀释液
1-a-1 固体 10mM DMSO 0.03nM–100μM 10％DMSO
式A-5 固体 10mM DMSO 0.03nM–100μM 10％DMSO
AZD2281 固体 10mM DMSO 0.03nM–100μM 10％DMSO
3、酶和底物
Figure PCTCN2018083969-appb-000042
4、具体测试方法
将PARP酶反应体系加入预先包被组蛋白标记底物的96孔板中，双复孔，室温孵育1小时。反应体系包含：50μl含NAD +的反应缓冲液(Tris·HCl,pH 8.0)，生物素化的NAD +(见3)，活化的DNA(见3)，一种PARP酶(见3)以及受试化合物(见2)。当酶反应结束后，每孔加入50μl辣根过氧化物酶标记的链霉亲和素室温孵育30分钟，之后加入100μl显影剂并使用BioTek Synergy TM 2读板仪读取发光值。PARP酶活性分析采用双复孔，并使用Graphpad Prism软件进行数据处理。不加入受试化合物孔的发光值(L t)被定义为100％活性，不加PARP酶孔的发光值(L b)被定义为0％活性。则受试化合物的酶活性计算公式如下：
酶活性％＝[(L-L b)/(L t-L b)]×100。
L＝加入受试化合物孔的发光值，L b＝不加PARP或TNKS孔的发光值，L t＝不加入受试化合物孔的发光值，酶活性抑制比％＝100–酶活性％。化合物的酶抑制活性以IC 50表示，抑制曲线以带标准差的图表示。
以受试化合物的一系列浓度与相应的酶活性作图，并通过“Sigmoidal剂量反应曲线”进行非线性回归，公式如下：Y＝B+(T-B)/1+10 ((LogEC50-X)×Hill Slope)，Y＝酶活性％，B＝最 小酶活性％，T＝最大酶活性％，X＝化合物对数，Hill Slope＝斜率因子或Hill系数。IC 50值为最大酶活性抑制50％的药物浓度。结果如下：
Figure PCTCN2018083969-appb-000043
实施例I-15 重组人PARP1酶的DNA结合活性测试
本实验参照BPS公司的PARPtrap分析试剂盒的方法进行，具体检测方法如下：
1、实验材料：本实验采用BPS公司的PARPtrap分析试剂盒进行。
2、受试化合物信息如下：
1-a-1，来自实施例I-4所得产物样品。
式A-5，来自实施例I-10所得产物样品。
阳性对照AZD2281，化学名称为：1-(环丙基羰基)-4-[5-[(3,4-二氢-4-氧代-1-酞嗪)甲基]-2-氟苯甲酰]哌嗪。
化合物编号 储备液浓度 稀释溶剂 浓度范围 中间稀释液
1-a-1 10mM DMSO 1nM–10mM 10％DMSO
式A-5 10mM DMSO 1nM–10mM 10％DMSO
AZD2281 10mM DMSO 0.1nM–1mM 10％DMSO
3、酶和底物
Figure PCTCN2018083969-appb-000044
4、具体测试方法
酶反应是在96孔板中，双复孔，室温下进行。将45ml含有5nM Alexa488标记的切口DNA，PARP1酶(见3)的PARPtrap缓冲液和待测化合物(见2)在室温下孵育30分钟，然后加入5ml NAD引发PARP1酶反应，并且在室温下孵育45分钟。用Tecan Infinite M1000酶标仪读取485nM和528nM处的荧光强度。用Tecan Magellan6软件将荧光强度转化成荧光偏振，并使用Graphpad Prism软件进行数据处理。所有数据中不加受试测化合物的荧光偏振(FP o)被定义为0％活性，不加NAD和受试测化合物的荧光偏 振(FP t)被定义为100％活性，不加PARP和受试测化合物的荧光偏振(FP b)被定义为背景活性。
本发明受试化合物按以下公式计算：
％活性＝[(FP-FP b)-(FP o-FP b)]/[(FP t-FP b)-(FP o-FP b)]*100。
FB＝加受试测化合物的荧光偏振。
以受试化合物的一系列浓度与相应的酶活性作图，并通过“Sigmoidal剂量反应曲线”进行非线性回归，公式如下：Y＝B+(T-B)/1+10 ((LogEC50-X)×Hill Slope)，Y＝酶活性％，B＝最小酶活性％，T＝最大酶活性％，X＝化合物对数，Hill Slope＝斜率因子或Hill系数。IC 50值为最大酶活性抑制50％的药物浓度，结果如下：
Figure PCTCN2018083969-appb-000045
第II部分实施例
实施例II-1
1-环丙基-2-((1S，4S)-5-(2-氟-5-((4-氧-3，4-二氢酞嗪-1-基)甲基)苯甲酰)-2，5-二氮杂双环[2，2，1]庚烷-2-基)乙烷-1，2-二酮的制备
1)将2-氟-5-((4-氧-3，4-二氢酞嗪-1-基)甲基)苯甲酸(400mg，1.32mmol)，2，5-二氮杂双环[2，2，1]庚烷-2-甲酸(1S，4S)-叔丁酯(665mg，3.35mmol)，2-(7-偶氮苯并三氮唑)-N，N，N’，N’-四甲基脲六氟磷酸酯(1.02g，2.68mmol)和二异丙基乙胺(1.2ml，6.7mmol)溶于N，N-二甲基甲酰胺(25ml)中，室温搅拌72小时。反应液用二氯甲烷和水(各50ml)稀释，有机相用碳酸氢钠水溶液、饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩得粗品(1.23g)，为褐色油状物。
2)向上一步得到的粗品的二氯甲烷(50ml)的溶液中加入三氟乙酸(4ml)，室温搅拌16小时。反应液减压浓缩得黄色油状粗品(2.64g)。
3)将上一步得到的粗品，2-环丙基-2-氧代乙酸(917mg，8.04mmol)，二异丙基乙胺(1.5ml，12.06mmol)和2-(7-偶氮苯并三氮唑)-N，N，N’，N’-四甲基脲六氟磷酸酯(1.02g，2.68mmol)溶于N，N-二甲基甲酰胺(30ml)中，室温搅拌24小时，反应液用二氯甲烷和水(各100ml)稀释，水层用二氯甲烷萃取(100ml)，合并有机相，用水、碳酸氢钠饱和溶液、饱和食盐水洗涤，硫酸钠干燥，过滤，浓缩得粗品，用硅胶柱层析(二氯甲烷∶甲醇＝40∶1)纯化，得到式A-5化合物(350mg，收率55％，基于2-氟-5-((4-氧-3，4-二氢酞嗪-1-基)甲基)苯甲酸(400mg))，m/z[M+1] +475.4； 1H NMR(CDCl 3)：δ10.57-10.30(1H，brs)， 8.44(1H，m)，7.77-7.63(3H，m)，7.50-7.23(2H，m)，7.05-6.92(1H，m)，5.25-4.85(2H，m)，4.50-4.48(2H，d)，3.91-3.30(4H，m)，2.94-2.68(1H，m)，1.97-1.85(2H，m)，1.20-0.96(4H，m)。
实施例II-2
将50g实施例II-1所得式A-5化合物溶于100ml丙酮形成式A-5化合物的丙酮溶液；
向反应瓶中加入异丙醚(1L)，搅拌，5℃下向体系中缓慢滴入式A-5化合物的丙酮溶液，滴毕，有大量白色固体析出；
25℃条件下搅拌；过滤，适量异丙醚淋洗；滤饼减压干燥得到固体式A-5化合物45g，收率90％，为白色粉末。
从图3可以看出，XRPD图谱无晶体衍射峰，所以该白色粉末为非晶形。
实施例II-3
将50g实施例II-1所得式A-5化合物溶于500ml乙醇形成式A-5化合物的乙醇溶液；
向反应瓶中加入异丙醚(5L)，搅拌，40℃下向体系中缓慢滴入式A-5化合物的乙醇溶液，滴毕，有大量白色固体析出；
35℃条件下搅拌；过滤，适量异丙醚淋洗；滤饼减压干燥得到固体式A-5化合物46g，收率92％，为白色粉末。
实施例II-4
将50g实施例II-1所得式A-5化合物溶于250ml乙酸异丁酯形成式A-5化合物的乙酸异丁酯溶液；
向反应瓶器加入正戊烷(2.5L)，搅拌，30℃下向体系中缓慢滴入式A-5化合物的乙酸异丁酯溶液，滴毕，有大量白色固体析出；
30℃条件下搅拌；过滤，适量正戊烷淋洗；滤饼减压干燥得到固体式A-5化合物48g，收率96％，为白色粉末。
实施例II-5
将50g实施例II-1所得式A-5化合物溶于50ml甲醇形成式A-5化合物的甲醇溶液；反应瓶器加入异丙醚(0.5L)，搅拌，20℃下向体系中缓慢滴入式A-5化合物的甲醇溶液，滴毕，有大量白色固体析出；
25℃条件下搅拌；过滤，适量异丙醚淋洗。滤饼减压干燥得到固体式A-5化合物46g。收率92％，为白色粉末。
实施例II-6
将50g实施例II-1所得式A-5化合物溶于100ml乙酸乙酯形成式A-5化合物的乙酸乙酯溶液；
向反应瓶器加入正戊烷(1L)，搅拌，25℃下向体系中缓慢滴入式A-5化合物的乙酸乙酯溶液，滴毕，有大量白色固体析出；
25℃条件下搅拌；过滤，适量正戊烷淋洗。滤饼减压干燥得到固体式A-5化合物47g。收率93％，为白色粉末。
实施例II-7
将50g实施例II-1所得式A-5化合物溶于200ml丁酮形成式A-5化合物的丁酮溶液；
向反应瓶器加入环己烷(2L)，搅拌，30℃下向体系中缓慢滴入式A-5化合物的丁酮溶液，滴毕，有大量白色固体析出；
30℃条件下搅拌；过滤，适量环己烷淋洗。滤饼减压干燥得到固体式A-5化合物45.5g。收率91％，为白色粉末。
经检测，实施例II-3至II-7所得白色粉末的XRPD图谱基本同实施例II-2。
实施例II-8 稳定性试验
实施例II-2、II-5所得白色粉末(下称：样品)模拟市售包装(药用低密度聚乙烯袋/药用复合膜袋)，放在药品稳定性试验箱内，于温度40℃±2℃，相对湿度75％±5％的条件下放置6个月，分别于第1、2、3、6月取样，按下述考察项目进行检测，结果如下：
Figure PCTCN2018083969-appb-000046
Figure PCTCN2018083969-appb-000047
Figure PCTCN2018083969-appb-000048
本发明式A-5化合物的非晶形在温度40℃±2℃，湿度RH75％±5％，放置6个月后，各项指标检测结果与0天初始结果相比，均无明显变化，说明式A-5化合物的非晶形稳定性较好。
申请人还针对实施例II-3、II-4、II-6、II-7所得的非晶形进行检测，其HPLC纯度均不低于99.5％，总杂不高于0.5％。
实施例II-9 溶解度测定
取本发明式A-5化合物的非晶形(实施例II-2至II-7所得样品)，按《中国药典》2010年版二部凡例十五(2)项下进行溶解度检查。
方法：在25℃±2℃条件下进行溶解度试验(根据预试验结果，称取供试品适量，逐步加入溶剂，振摇，观察至全部溶解，根据药典标准判断溶解度)，本发明式A-5化合物的非晶形样品的试验结果见下表。
Figure PCTCN2018083969-appb-000049
Figure PCTCN2018083969-appb-000050
上表检测结果表明，本发明式A-5化合物的非晶形在甲醇、乙腈中极易溶解，在乙醇中易溶，溶解性较好。
实施例II-10 在不同pH值水溶液中的饱和溶解度测定
考察本发明式A-5化合物的非晶形(实施例II-2至II-7所得样品)在不同pH值水溶液中的饱和溶解度，按照高效液相色谱法(《中国药典》2010年版二部附录ⅤD)进行测定。
取不同pH值的水溶液各约10ml，分别置不同的50ml具塞西林瓶中，在各西林瓶中分别加入适量的样品粉末，使其成为过饱和溶液，于37℃水浴振荡筛中震摇16小时，放置至室温，过滤，即得本品在各pH值水溶液中的饱和溶液，根据预实验结果，将各饱和溶液用10％乙腈水溶液稀释至每1ml含主药为0.05mg的溶液，即得各供试品溶液；另取对照品适量，精密称定，加入适量乙腈溶解，再用10％乙腈水溶液稀释制成每1ml含主药为0.05mg的对照品(实施例II-1所得样品)溶液。按外标法以峰面积计，考察本发明式A-5化合物的非晶形样品在不同pH值水溶液中的饱和溶解度，结果见下表。
实施例II-2 样品在不同pH值水溶液中的饱和溶解度
pH值 1.0 2.0 3.0 4.0 5.0 6.0
溶解度(mg/ml) 1.31 1.10 1.07 1.04 1.05 1.06
pH值 7.0 8.0 9.0 10.0 11.0 12.0
溶解度(mg/ml) 0.98 0.95 0.98 0.96 0.61 0.06
实施例II-3 样品在不同pH值水溶液中的饱和溶解度
pH值 1.0 2.0 3.0 4.0 5.0 6.0
溶解度(mg/ml) 1.22 1.19 1.10 1.06 1.02 1.00
pH值 7.0 8.0 9.0 10.0 11.0 12.0
溶解度(mg/ml) 0.95 0.92 0.93 0.95 0.78 0.04
实施例II-4 样品在不同pH值水溶液中的饱和溶解度
pH值 1.0 2.0 3.0 4.0 5.0 6.0
溶解度(mg/ml) 1.18 1.15 1.09 1.01 1.02 1.02
pH值 7.0 8.0 9.0 10.0 11.0 12.0
溶解度(mg/ml) 0.91 0.90 0.91 0.92 0.80 0.09
实施例II-5 样品在不同pH值水溶液中的饱和溶解度
pH值 1.0 2.0 3.0 4.0 5.0 6.0
溶解度(mg/ml) 1.23 1.08 1.05 1.06 1.07 1.06
pH值 7.0 8.0 9.0 10.0 11.0 12.0
溶解度(mg/ml) 0.94 0.91 0.96 0.95 0.66 0.02
实施例II-6 样品在不同pH值水溶液中的饱和溶解度
pH值 1.0 2.0 3.0 4.0 5.0 6.0
溶解度(mg/ml) 1.25 1.09 1.04 1.05 1.08 1.07
pH值 7.0 8.0 9.0 10.0 11.0 12.0
溶解度(mg/ml) 0.96 0.93 0.96 0.92 0.72 0.05
实施例II-7 样品在不同pH值水溶液中的饱和溶解度
pH值 1.0 2.0 3.0 4.0 5.0 6.0
溶解度(mg/ml) 1.28 1.10 1.04 1.06 1.08 1.08
pH值 7.0 8.0 9.0 10.0 11.0 12.0
溶解度(mg/ml) 0.95 0.90 0.94 0.93 0.79 0.04
由结果可知，本发明式A-5化合物的非晶形在pH值1.0～10水溶液中的饱和溶解度均在1.0mg/ml左右，表明该化合物的溶解度不具有pH值依赖性。
实施例II-11 式A-5化合物的含量测定
采用高效液相(HPLC)检测，测得实施例II-1至II-7所得样品中式A-5化合物的含量(HPLC纯度)如下表：
样品 含量
实施II-1 72％
实施II-2 99.3％
实施II-3 98.0％
实施II-4 100％
实施II-5 99.2％
实施II-6 99.9％
实施例II-7 102.0％
第III部分实施例
实施例III-1至III-5为式A-5的乙酸乙酯溶剂合物晶体A的制备
实施例III-1
取100g实施例II-1所得目标产品中加入600ml的100％(w/w)乙酸乙酯溶解完全，控温10℃搅拌析晶。过滤，适量乙酸乙酯淋洗。滤饼于55℃下减压干燥至恒重，得到式A-5的乙酸乙酯溶剂合物晶体A 92.0g，收率92.0％。
实施例III-2
取100g实施例II-1所得目标产品加入2000ml乙酸乙酯溶液(95％(w/w)乙酸乙酯，5％(w/w)四氢呋喃)溶解完全，控温20℃搅拌析晶。过滤，适量乙酸乙酯淋洗。滤饼于40℃下减压干燥至恒重，得到式A-5的乙酸乙酯溶剂合物晶体A 91.5g，收率91.5％。
实施例III-3
取100g实施例II-1所得目标产品加入1000ml乙酸乙酯溶液(90％(w/w)乙酸乙酯，10％(w/w)乙腈)溶解完全，控温30℃搅拌析晶。过滤，适量乙酸乙酯淋洗。滤饼于60℃下减压干燥至恒重，得到式A-5的乙酸乙酯溶剂合物晶体A 92.0g，收率92.0％。
实施例III-4
取100g实施例II-1所得目标产品中加入400ml乙酸乙酯溶液(85％(w/w)乙酸乙酯，15％(w/w)四氢呋喃)溶解完全，控温40℃搅拌析晶。过滤，适量乙酸乙酯淋洗。滤饼于30℃条件下鼓风干燥至恒重，得到式A-5的乙酸乙酯溶剂合物晶体A 90.4g，收率90.4％。
实施例III-5
取100g实施例II-1所得目标产品中加入3000ml乙酸乙酯溶液(50％(w/w)乙酸乙酯，50％(w/w)乙腈)溶解完全，控温0℃搅拌析晶。过滤，适量乙酸乙酯淋洗。滤饼于70℃条件下鼓风干燥至恒重，得到式A-5的乙酸乙酯溶剂合物晶体A 90.9g，收率90.9％。
经实验验证，实施例III-2至III-5所得白色粉末测得的XRPD图谱基本同实施例III-1。
申请人将实施例III-1至III-5中乙酸乙酯溶液中的溶剂分别替换成1,4-二氧六环、二氯甲烷、甲醇，均能得到式A-5化合物的乙酸乙酯溶剂合物晶体A，本发明方法的重复性很好，可获得稳定的式A-5化合物的乙酸乙酯溶剂合物晶体A。
式A-5化合物的乙酸乙酯溶剂合物晶体A的XRPD图如图1所示，图1中各峰的衍射角度2θ和相对强度如下表所示。
Angle(衍射角2θ) Rel.Intensity(相对强度)
5.578° 1.0％
8.193° 61.6％
9.507° 57.1％
10.003° 0.4％
11.141° 35.1％
11.220° 15.2％
11.841° 21.0％
12.751° 0.6％
13.547° 81.3％
14.137° 95.6％
14.932° 9.4％
15.415° 20.8％
15.935° 56.6％
16.197° 45.3％
16.732° 52.0％
17.799° 83.0％
18.432° 15.5％
19.046° 33.2％
19.483° 86.5％
19.682° 64.3％
20.062° 100.0％
20.832° 71.3％
20.861° 65.7％
21.785° 4.7％
22.344° 28.7％
23.153° 42.8％
23.837° 94.3％
24.570° 15.8％
25.012° 66.1％
25.624° 12.7％
25.860° 14.9％
26.546° 11.0％
26.902° 11.0％
27.272° 29.6％
28.027° 8.4％
28.438° 11.2％
28.823° 6.0％
29.137° 6.1％
29.628° 9.1％
30.443° 9.2％
31.032° 10.8％
31.412° 7.4％
31.890° 2.9％
32.675° 9.2％
33.054° 4.9％
33.393° 4.1％
34.212° 8.5％
35.142° 1.3％
35.976° 13.6％
36.761° 2.3％
36.977° 2.9％
37.904° 2.2％
38.547° 3.8％
38.966° 1.5％
39.321° 0.5％
由图1可以看出，式A-5化合物的乙酸乙酯溶剂合物晶体A的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.193、14.137、17.799、19.483、20.062、20.861和23.837处具有特征峰；优选在衍射角度2θ±0.2°为8.193、9.507、11.141、13.547、14.137、15.935、16.732、17.799、19.483、20.062、20.861和23.837处具有特征峰。
式A-5化合物的乙酸乙酯溶剂合物晶体A的红外吸收光谱图如图2所示。采用以下重要波段进行定性，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置具有吸收峰：3435.84、3204.48、3077.83、3009.85、2894.15、1737.55、1693.68、1666.67、1653.93、1495.51、1450.38、1405.77、1372.64、1331.30、1241.81、1190.35、1155.83、1106.21、1081.60、1042.61、1000.42、947.75、929.26、836.47、805.05、772.26、750.17、723.25、682.88、621.94、577.80、528.40、486.00。
实施例III-6 式A-5甲酸乙酯溶剂合物晶体B的制备
取100g实施例II-1所得目标产品加入2000ml甲酸乙酯溶液(95％w/w甲酸乙酯，5％w/w四氢呋喃)溶解完全，控温20℃搅拌析晶。过滤，适量甲酸乙酯淋洗。滤饼于40℃下减压干燥至恒重，得到式A-5化合物的甲酸乙酯溶剂合物晶体B92.4g，收率92.4％。
式A-5化合物甲酸乙酯溶剂合物晶体B的XRPD图样如图4所示，且在XRPD图样中的衍射角及其相对强度如下表所示。
Angle(衍射角2θ) Rel.Intensity(相对强度)
5.520° 0.7％
8.471° 68.2％
9.776° 27.1％
11.151° 5.4％
11.491° 23.7％
13.777° 23.4％
14.034° 53.8％
14.865° 4.3％
16.131° 89.2％
16.790° 19.0％
17.046° 3.5％
17.755° 18.6％
18.626° 12.1％
19.679° 100.0％
20.319° 3.2％
21.400° 6.0％
21.778° 1.2％
22.621° 42.6％
23.268° 11.0％
23.904° 7.0％
24.165° 14.6％
24.532° 26.5％
25.399° 5.4％
25.949° 1.3％
26.476° 10.1％
26.888° 6.0％
27.314° 3.8％
27.792° 8.4％
28.091° 4.9％
29.014° 2.4％
29.429° 6.3％
29.597° 6.5％
30.028° 3.8％
31.102° 4.8％
31.737° 1.1％
32.659° 1.6％
33.128° 2.7％
33.414° 3.4％
33.781° 2.2％
34.611° 1.6％
35.046° 3.7％
36.000° 1.9％
36.871° 0.7％
37.200° 1.0％
37.944° 2.9％
38.983° 0.9％
39.549° 0.8％
由图4可以看出，式A-5化合物的甲酸乙酯溶剂合物晶体B的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.471、14.034、16.131和19.679处具有特征峰；优选在衍射角度2θ±0.2°为8.471、9.776、11.491、14.034、16.131、19.679、22.621和24.532处具有特征峰；更优选在衍射角度2θ±0.2°为8.471、9.776、11.151、11.491、14.034、16.131、16.790、17.755、18.626、19.679、22.621、23.268和24.532处具有特征峰。
式A-5的甲酸乙酯溶剂合物晶体B的红外吸收光谱图样如图5所示。采用以下重要波段进行定性，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置有吸收峰：3447.16、3199.04、3076.26、3010.93、2893.18、1723.60、1667.77、1636.87、1559.39、1496.32、1448.70、1406.16、1371.35、1329.87、1258.92、1239.50、1180.47、1155.24、1107.13、1081.35、1042.77、1027.06、1001.43、948.22、927.67、899.76、836.95、804.45、771.41、750.66、723.72、683.19、655.68、621.35、578.30、540.65和486.11。
实施例III-7 式A-5化合物乙酸甲酯溶剂合物晶体C的制备
取100g实施例II-1所得目标产品加入1000ml乙酸甲酯溶液(90％w/w乙酸甲酯，10％w/w乙腈)溶解完全，控温30℃搅拌析晶。过滤，适量乙酸甲酯淋洗。滤饼于60℃下减压干燥至恒重，得到式A-5化合物的乙酸甲酯溶剂合物晶体C 92.2g，收率92.2％。
式A-5化合物的乙酸甲酯溶剂合物晶体C的XRPD图样如图6所示，且在XRPD图样中的衍射角2θ及其相对强度如下表所示。
Angle(衍射角2θ) Rel.Intensity(相对强度)
5.468° 0.7％
8.454° 91.9％
9.748° 24.4％
11.144° 13.3％
11.548° 23.7％
13.740° 23.7％
14.057° 54.2％
14.865° 5.5％
16.111° 91.0％
16.779° 17.4％
16.882° 6.7％
17.778° 15.2％
18.596° 18.9％
19.681° 100.0％
20.371° 5.5％
21.345° 4.6％
21.777° 1.1％
22.619° 40.2％
23.256° 15.1％
24.140° 17.8％
24.601° 20.2％
25.433° 7.5％
25.861° 2.1％
26.522° 7.3％
26.806° 8.4％
27.251° 5.0％
27.884° 8.5％
29.020° 2.1％
29.472° 9.3％
30.058° 4.7％
35.151° 4.2％
31.689° 1.9％
32.615° 1.4％
33.347° 3.2％
33.778° 1.9％
34.329° 1.6％
34.545° 2.3％
35.173° 2.9％
36.028° 2.1％
37.280° 0.9％
37.923° 2.0％
39.573° 1.1％
由图6可以看出，式A-5化合物的乙酸甲酯溶剂合物晶体C的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.454、14.057、16.111和19.681处具有特征峰；优选在衍射角度2θ±0.2°为8.454、9.748、11.548、13.740、14.057、16.111、19.681和22.619处具有特征峰；更优选在衍射角度2θ±0.2°为8.454、9.748、11.144、11.548、13.740、14.057、16.111、16.779、17.778、18.596、19.681、22.619和23.256处具有特征峰。
式A-5化合物的乙酸甲酯溶剂合物晶体C的红外吸收光谱图样如图7所示，采用以下重要波段进行定性，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置有吸收峰：3447.25、3199.85、3080.97、3011.02、2890.69、1744.78、1668.61、1637.02、1559.53、1496.34、1449.40、1406.28、1370.92、1330.37、1240.52、1213.97、1191.31、1155.98、1106.65、1081.38、1042.71、1001.04、947.78、928.16、899.53、836.47、804.71、771.49、723.86、683.14、621.65、578.05、540.88和485.99。
实施例III-8 式A-5化合物甲酸正丙酯溶剂合物晶体D的制备
取100g实施例II-1所得目标产品中加入3000ml甲酸正丙酯溶液(50％(w/w)甲酸正丙酯，50％(w/w)乙腈)溶解完全，控温0℃搅拌析晶。过滤，适量甲酸正丙酯淋洗。滤饼于70℃条件下鼓风干燥至恒重，得到式A-5化合物的甲酸正丙酯溶剂合物晶体D107.8g，收率91％。
式A-5化合物甲酸正丙酯溶剂合物晶体D的XRPD图样如图8所示，且在XRPD图样中的衍射角及其相对强度如下表所示。
Angle(衍射角2θ) Rel.Intensity(相对强度)
5.489° 4.4％
8.152° 100.0％
9.459° 20.9％
11.119° 6.4％
11.335° 9.4％
11.647° 17.2％
12.602° 1.8％
13.526° 36.7％
13.989° 66.8％
14.773° 3.9％
15.407° 24.5％
15.924° 61.8％
16.720° 30.0％
17.692° 22.8％
18.423° 15.6％
19.058° 32.3％
19.459° 87.2％
19.986° 6.7％
20.861° 7.2％
21.667° 2.4％
22.237° 75.4％
22.840° 50.9％
23.460° 16.8％
23.795° 35.3％
24.751° 44.4％
24.862° 19.1％
25.563° 11.2％
26.078° 13.3％
26.567° 20.6％
27.258° 2.8％
27.652° 10.0％
28.277° 5.6％
28.764° 18.3％
29.111° 2.5％
29.590° 5.5％
30.194° 1.2％
30.500° 1.2％
31.211° 10.5％
32.391° 2.5％
33.292° 4.6％
33.848° 1.6％
34.193° 3.3％
34.534° 0.8％
35.197° 1.5％
35.585° 7.0％
35.796° 2.3％
36.498° 2.3％
36.536° 1.9％
37.390° 0.8％
37.779° 3.5％
38.525° 1.6％
由图8可以看出，式A-5化合物的甲酸正丙酯溶剂合物晶体D的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.152、13.989、15.924、19.459和22.237处具有特征峰；优选在衍射角度2θ±0.2°为8.152、13.526、13.989、15.924、16.720、19.058、19.459、22.237和22.840处具有特征峰；更优选在衍射角度2θ±0.2°为8.152、9.459、11.647、13.526、 13.989、15.407、15.924、16.720、17.692、18.423、19.058、19.459、22.237、22.840、23.795和24.751处具有特征峰。
式A-5化合物的甲酸正丙酯溶剂合物晶体D的红外吸收光谱图样如图9所示，采用以下重要波段进行定性，以吸收波长的倒数表示(cm -1)(±2cm -1)，其在以下位置有吸收峰：3447.01、3203.32、3074.46、3010.34、2966.46、2891.74、1726.16、1668.42、1636.53、1496.39、1449.31、1406.54、1372.26、1330.16、1258.84、1239.49、1178.01、1154.17、1106.97、1081.28、1044.04、1027.37、1002.12、948.21、928.04、899.53、836.47、804.41、772.26、724.08、682.94、655.68、621.26、577.89、540.67和485.97。
实施例III-9至III-13为式A-5化合物溶剂合物晶体的制备
与实施例III-1、III-3、III-6、III-7、III-8的不同之处在于，将“实施例II-1所得目标产品”更换为“实施例II-3所得固体(式A-5化合物非晶形(即无定形))”，其产品的收率如下：
实施例 收率
实施例III-9 95.5％
实施例III-10 95.2％
实施例III-11 94.3％
实施例III-12 95.0％
实施例III-13 94.8％
经实验验证，实施例III-9、III-10所得乙酸乙酯溶剂合物晶体A测得的XRPD的图谱基本同实施例III-1，实施例III-11、III-12、III-13所得溶剂合物晶体分别与实施例III-6、III-7、III-8测得的XRPD的图谱基本相同。
对比上述式A-5化合物的酯类溶剂合物晶体的制备实施例，在同等工艺条件下，原料采用式A-5化合物的非晶形(即无定形)(实施例III-9至III-13)分别相对于采用实施例II-1所得目标样品式A-5化合物(实施例III-1、III-3、III-6、III-7、III-8)，更易于从溶剂中析出固体，而且收率显著提高。
实施例III-14 式A-5化合物的酯类溶剂合物晶体的检测
1、式A-5化合物的酯类溶剂合物晶体中酯类溶剂的含量测定
方法：采用安捷伦7890B型气相色谱仪顶空进样检测。
结果：在实施例III-1、III-9、III-11、III-12、III-13所得的酯类溶剂合物晶体中，酯类溶剂的含量如下表：
样品 含量
实施例III-1 12～20％
实施例III-9 12～20％
实施例III-11 5～15％
实施例III-12 5～15％
实施例III-13 12～20％
2、稳定性试验
取样品(实施例III-1、III-9所得晶体)，模拟市售包装(药用低密度聚乙烯袋/药用复合膜袋)，放在药品稳定性试验箱内，于温度40℃±2℃，相对湿度75％±5％的条件下放置3个月，分别于第1、2、3月取样，按下述考察项目进行检测，结果见下表。
实施例III-1 样品(式A-5化合物的乙酸乙酯溶剂合物晶体A)加速试验结果
Figure PCTCN2018083969-appb-000051
实施例III-9 样品(式A-5化合物的乙酸乙酯溶剂合物晶体A)加速试验结果
Figure PCTCN2018083969-appb-000052
Figure PCTCN2018083969-appb-000053
本品在温度40℃±2℃，湿度RH75％±5％，放置3个月后，各项指标检测结果与0天初始结果相比，均无明显变化，说明本发明式A-5化合物的溶剂合物晶体(如式A-5化合物的乙酸乙酯溶剂合物晶体A)稳定性较好。
3、高湿实验(RH92.5％,25±2℃)
根据《中国药典》2010版第二部附录XIXC《原料药于药物制剂的稳定性试验指导原则》，对实施例III-1、III-3、III-6、III-7、III-8、III-9所得溶剂合物晶体(下表中称：样品)与实施例II-3、II-6的非晶形(即无定形)进行引湿性考察，结果如下：
Figure PCTCN2018083969-appb-000054
根据上述结果表明式A-5化合物的溶剂合物晶体基本无引湿性，而式A-5化合物非晶形(即无定形)具有一定引湿性，相对而言，溶剂合物晶体更易于储存，可以避免由于原料药吸湿所带来的包装问题。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (28)

  1. 一种抑制PARP活性的化合物A的制备方法，
    Figure PCTCN2018083969-appb-100001
    其中，该方法包括如下步骤：
    (1)将式II化合物
    Figure PCTCN2018083969-appb-100002
    与HCl反应生成式I化合物
    Figure PCTCN2018083969-appb-100003
    (2)将所得式I化合物与化合物B
    Figure PCTCN2018083969-appb-100004
    反应生成化合物A；其中M为氢、钾、锂、钠或铵；
    式I化合物、式II化合物和化合物A中，
    R 1和R 2各自独立地为氢或卤素；
    R为甲基、三氟甲基、
    Figure PCTCN2018083969-appb-100005
    杂环基
    Figure PCTCN2018083969-appb-100006
    为
    Figure PCTCN2018083969-appb-100007
  2. 根据权利要求1所述的制备方法，其特征在于，
    步骤(1)包括下列步骤：有机溶剂中，将式II化合物和HCl进行反应，制得式I化合物；步骤(1)优选包括下列步骤：将式II化合物与有机溶剂混合，然后在10-40℃下，再与HCl混合，进行反应，得到式I化合物；
    或者，步骤(2)包括下列步骤：有机溶剂中，在缩合剂的作用下，将式I化合物和化合物B进行反应，制得化合物A；步骤(2)优选包括下列步骤：将式I化合物和有机溶剂混合后，再加入缩合剂和化合物B，进行反应，制得式A化合物。
  3. 根据权利要求2所述的制备方法，其特征在于，
    步骤(1)中，加入HCl的物质的量为式II化合物物质的量的5-20倍、8-15倍或8-12倍；
    和/或，步骤(1)中，所述有机溶剂为C 1-5醇、乙酸乙酯、乙酸异丙酯、C 3-6酮、二氧六环和甲苯中的一种或多种，优选乙酸乙酯、丁酮或异丙醇；
    和/或，步骤(1)中，所述HCl为氯化氢气体或盐酸水溶液；所述盐酸水溶液的摩尔浓度优选为2～12mol/L，进一步优选为6～12mol/L；
    和/或，步骤(1)中的反应温度为10-40℃；
    和/或，步骤(1)中，所述反应结束后，还可进一步包含后处理的操作，所述后处理的操作包括下列步骤：将反应结束后的反应液，进行固液分离，滤饼干燥，得到式I化合物；
    和/或，步骤(2)中，所述有机溶剂为卤代烷烃类溶剂，例如二氯甲烷；
    和/或，步骤(2)中，式I化合物和化合物B的物质的量比为1:1-2:1；
    和/或，步骤(2)中，当存在缩合剂时，所述缩合剂为HOBT、EDCI、或者HOBT与EDCI组成的混合体系；当所述缩合剂为HOBT与EDCI组成的混合体系，所述HOBT与EDCI的物质的量比为1:0.8-1:1.5；
    和/或，步骤(2)中，当存在缩合剂时，所述缩合剂与式I化合物的物质的量比为1:1-5:1；
    和/或，步骤(2)中，所述反应的温度为10-30℃；
    和/或，步骤(2)中，步骤(2)反应结束后，还进一步包括下列操作：将反应结束后的反应液与水混合，过滤，后处理，浓缩得到化合物A。
  4. 根据权利要求1-3中至少一项所述的制备方法，其特征在于，
    所述步骤(1)包括下列步骤：将式II化合物溶于有机溶剂中，控温在10-40℃下加入式II化合物的5-20倍物质的量的HCl，搅拌，反应2-24小时，过滤，干燥得到固体；
    或者，所述步骤(2)包括下列步骤：将步骤(1)所得固体加入装有二氯甲烷反应容器中，加入缩合剂及化合物B，反应2-8h，过滤，后处理，浓缩得到化合物A。
  5. 根据权利要求4所述的制备方法，其特征在于，步骤(1)中，所述有机溶剂选自C 1- 5醇、乙酸乙酯、乙酸异丙酯、C 3-6酮、二氧六环和甲苯中的一种或多种；或者，步骤(1)中，加入HCl的物质的量为式II化合物的8-15倍。
  6. 根据权利要求1-5中至少一项所述的制备方法，其特征在于，所述化合物A的制备方法还进一步包括下列步骤：在缩合剂和缚酸剂的作用下，将式III化合物和化合物C进行反应，制得式II化合物；其中所述缩合剂为HOBT、或者HOBT与EDCI组成的混合体系；
    所述式III化学结构式为：
    Figure PCTCN2018083969-appb-100008
    所述化合物C为
    Figure PCTCN2018083969-appb-100009
    R 1、R 2和杂环基
    Figure PCTCN2018083969-appb-100010
    的定义同权利要求1所述。
  7. 根据权利要求6所述的制备方法，其特征在于，
    式II化合物的制备方法在溶剂的存在下进行；所述溶剂为卤代烷烃类溶剂，例如二氯甲烷；
    和/或，式II化合物的制备方法中，所述式III化合物与化合物C的物质的量比为1：1-1：3；
    和/或，式II化合物的制备方法中，所述缩合剂与式III化合物的物质的量比为1.5:1-3:1；
    和/或，式II化合物的制备方法中，当所述缩合剂为HOBT与EDCI组成的混合体系时，HOBT和EDCI的物质的量的比优选为1:0.8-1:1.5；
    和/或，式II化合物的制备方法中，所述的缚酸剂为乙二胺和/或N,N-二异丙基乙胺；
    和/或，式II化合物的制备方法中，所述缚酸剂与缩合剂的物质的量比为0.5:1-1.5:1；
    和/或，式II化合物的制备方法中，所述反应的温度为10-40℃；
    和/或，II化合物的制备方法中，将式III化合物、化合物B和缩合剂混合，然后再加入缚酸剂，进行所述反应；
    和/或，II化合物的制备方法中，所述反应结束后，还进一步包括后处理的操作；所述后处理的操作包括下列步骤：将反应结束后的反应液进行分离浓缩即可。
  8. 根据权利要求1-7种至少一项所述的制备方法，其特征在于，所述式II化合物的制备方法包括下列步骤：将式III、化合物C加入到反应容器中，加入1-羟基苯并***、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，控温10-40℃，滴加三乙胺或N,N-二异丙基乙胺，控温10-40℃，搅拌反应，2-24小时，分离浓缩得到固体。
  9. 一种式I化合物
    Figure PCTCN2018083969-appb-100011
    其中R 1、R 2和杂环基
    Figure PCTCN2018083969-appb-100012
    的定义同权利要求1。
  10. 一种式I化合物的制备方法，其特征在于，其包含如下步骤：
    将式II化合物
    Figure PCTCN2018083969-appb-100013
    与盐酸反应生成
    Figure PCTCN2018083969-appb-100014
    其中R 1、R 2和杂环基
    Figure PCTCN2018083969-appb-100015
    的定义同权利要求1；其中式I化合物的制备方法中的各条件均同权利要求1-5中至少一项所述的制备方法中的式I化合物的制备。
  11. 一种化合物I-a或其药学上可接受的盐，
    Figure PCTCN2018083969-appb-100016
    其中，所述R 1、R 2和杂环基
    Figure PCTCN2018083969-appb-100017
    的定义同权利要求1。
  12. 如权利要求11所述的化合物I-a或其药学上可接受的盐，所述化合物I-a为化合物I-a-1，
    Figure PCTCN2018083969-appb-100018
    其中药学上可接受的盐优选为盐酸盐。
  13. 一种式A-5化合物的非晶形：
    Figure PCTCN2018083969-appb-100019
  14. 根据权利要求13所述式A-5化合物的非晶形，其特征在于，所述式A-5化合物的非晶形中式A-5化合物的含量为98.0％-102.0％之间。
  15. 根据权利要求13或14所述式A-5化合物的非晶形制备方法，其特征在于，其包含以下步骤：将式A-5化合物和良性溶剂的混合溶液，与不良性溶剂混合，即可；所述良性溶剂与式A-5化合物的体积质量比为1mL/g-10mL/g，所述不良性有机溶剂与式A-5化合物的体积质量比为10mL/g-100mL/g。
  16. 根据权利要求15所述式A-5化合物的非晶形的制备方法，其特征在于，
    所述良性溶剂为乙醇、甲醇、丙酮、丁酮、乙酸乙酯、乙酸异丁酯、异丙醇、正丙醇、四氢呋喃、甲苯和二氯甲烷中的一种或多种；
    和/或，所述不良性溶剂为异丙醚、正己烷、正戊烷、正庚烷、正辛烷、甲基叔丁基醚、石油醚和环己烷中的一种或多种；
    和/或，所述混合的温度为-10～40℃；
    和/或，将式A-5化合物和良性溶剂的混合溶液加入到不良性溶剂中；
    和/或，所述混合的操作的同时或结束后，还进一步包括搅拌的操作。
  17. 根据权利要求15或16所述式A-5化合物的非晶形的制备方法，其特征在于，
    当将式A-5化合物和良性溶剂的混合溶液加入到不良性溶剂中时，所述加入的方式为滴加；
    和/或，当进行搅拌的操作时，所述搅拌时混合液的温度为-10～40℃；
    和/或，所述搅拌的操作结束后，还进一步包括后处理的操作；所述的后处理包括下列步骤：将继续搅拌后得到的混合液，进行固液分离，干燥即可。
  18. 根据权利要求13-17中至少一项所述式A-5化合物的非晶形的制备方法，其特征在于，用良性溶剂溶解式A-5化合物，滴入到不良性溶剂中搅拌，析出固体，过滤，干燥；所述良性溶剂与式A-5化合物的体积质量比为1mL/g-10mL/g，所述不良性溶剂与式A-5化合物的体积质量比为10mL/g-100mL/g。
  19. 一种药物组合物，其含有如权利要求13或14所述式A-5化合物的非晶形。
  20. 一种式A-5化合物的溶剂合物晶体，
    Figure PCTCN2018083969-appb-100020
  21. 根据权利要求20所述的溶剂合物晶体，其特征在于，
    所述溶剂合物晶体为酯类溶剂合物晶体；优选为式A-5化合物的乙酸乙酯溶剂合物晶体、式A-5化合物的甲酸乙酯溶剂合物晶体、式A-5化合物的乙酸甲酯溶剂合物晶体或式A-5化合物的甲酸正丙酯溶剂合物晶体；
    和/或，所述溶剂合物晶体的红外吸收光谱具有吸收光带，以吸收波长的倒数表示±2cm -1，其在以下位置具有吸收峰：1668、1636、1496、1449和804。
  22. 根据权利要求21所述的溶剂合物晶体，其特征在于，
    所述溶剂合物晶体为式A-5化合物的乙酸乙酯溶剂合物晶体A，在该晶体A的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.193、14.137、17.799、19.483、20.062、20.861和23.837处具有特征峰；优选在衍射角度2θ±0.2°为8.193、9.507、11.141、13.547、14.137、15.935、16.732、17.799、19.483、20.062、20.861和23.837处具有特征峰；
    和/或，所述溶剂合物晶体为式A-5化合物的甲酸乙酯溶剂合物晶体B，在该晶体B的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.471、14.034、16.131和19.679处具有特征峰；优选在衍射角度2θ±0.2°为8.471、9.776、11.491、14.034、16.131、19.679、22.621和24.532处具有特征峰；更优选在衍射角度2θ±0.2°为8.471、9.776、11.151、11.491、14.034、16.131、16.790、17.755、18.626、19.679、22.621、23.268和24.532处具有特征峰；
    和/或，所述溶剂合物晶体为式A-5化合物的乙酸甲酯溶剂合物晶体C，在该晶体C的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.454、14.057、16.111和19.681处具有特征峰；优选在衍射角度2θ±0.2°为8.454、9.748、11.548、13.740、14.057、16.111、19.681和22.619处具有特征峰；更优选在衍射角度2θ±0.2°为8.454、9.748、11.144、11.548、 13.740、14.057、16.111、16.779、17.778、18.596、19.681、22.619和23.256处具有特征峰；
    和/或，所述溶剂合物晶体为式A-5化合物的甲酸正丙酯溶剂合物晶体D，在该晶体D的X射线粉末衍射图中，在衍射角度2θ±0.2°为8.152、13.989、15.924、19.459和22.237处具有特征峰；优选在衍射角度2θ±0.2°为8.152、13.526、13.989、15.924、16.720、19.058、19.459、22.237和22.840处具有特征峰；更优选在衍射角度2θ±0.2°为8.152、9.459、11.647、13.526、13.989、15.407、15.924、16.720、17.692、18.423、19.058、19.459、22.237、22.840、23.795和24.751处具有特征峰。
  23. 根据权利要求21所述的溶剂合物晶体，其特征在于，
    当所述溶剂合物晶体为式A-5化合物的乙酸乙酯溶剂合物晶体A时，所述式A-5化合物的乙酸乙酯溶剂合物晶体A的X衍射粉末衍射图的数据如下表1所示：
    表1
    衍射角2θ 相对强度 5.578° 1.0％ 8.193° 61.6％ 9.507° 57.1％ 10.003° 0.4％ 11.141° 35.1％ 11.220° 15.2％ 11.841° 21.0％ 12.751° 0.6％ 13.547° 81.3％ 14.137° 95.6％ 14.932° 9.4％ 15.415° 20.8％ 15.935° 56.6％ 16.197° 45.3％ 16.732° 52.0％ 17.799° 83.0％ 18.432° 15.5％ 19.046° 33.2％
    19.483° 86.5％ 19.682° 64.3％ 20.062° 100.0％ 20.832° 71.3％ 20.861° 65.7％ 21.785° 4.7％ 22.344° 28.7％ 23.153° 42.8％ 23.837° 94.3％ 24.570° 15.8％ 25.012° 66.1％ 25.624° 12.7％ 25.860° 14.9％ 26.546° 11.0％ 26.902° 11.0％ 27.272° 29.6％ 28.027° 8.4％ 28.438° 11.2％ 28.823° 6.0％ 29.137° 6.1％ 29.628° 9.1％ 30.443° 9.2％ 31.032° 10.8％ 31.412° 7.4％ 31.890° 2.9％ 32.675° 9.2％ 33.054° 4.9％ 33.393° 4.1％ 34.212° 8.5％ 35.142° 1.3％
    35.976° 13.6％ 36.761° 2.3％ 36.977° 2.9％ 37.904° 2.2％ 38.547° 3.8％ 38.966° 1.5％ 39.321° 0.5％
    优选地所述式A-5化合物的乙酸乙酯溶剂合物晶体A具有如图1所示的X-衍射粉末衍射图；
    和/或，当所述溶剂合物晶体为式A-5化合物的甲酸乙酯溶剂合物晶体B，所述式A-5化合物的甲酸乙酯溶剂合物晶体B的X衍射粉末衍射图的数据如下表2所示：
    表2
    衍射角2θ 相对强度 5.520° 0.7％ 8.471° 68.2％ 9.776° 27.1％ 11.151° 5.4％ 11.491° 23.7％ 13.777° 23.4％ 14.034° 53.8％ 14.865° 4.3％ 16.131° 89.2％ 16.790° 19.0％ 17.046° 3.5％ 17.755° 18.6％ 18.626° 12.1％ 19.679° 100.0％ 20.319° 3.2％ 21.400° 6.0％ 21.778° 1.2％ 22.621° 42.6％
    23.268° 11.0％ 23.904° 7.0％ 24.165° 14.6％ 24.532° 26.5％ 25.399° 5.4％ 25.949° 1.3％ 26.476° 10.1％ 26.888° 6.0％ 27.314° 3.8％ 27.792° 8.4％ 28.091° 4.9％ 29.014° 2.4％ 29.429° 6.3％ 29.597° 6.5％ 30.028° 3.8％ 31.102° 4.8％ 31.737° 1.1％ 32.659° 1.6％ 33.128° 2.7％ 33.414° 3.4％ 33.781° 2.2％ 34.611° 1.6％ 35.046° 3.7％ 36.000° 1.9％ 36.871° 0.7％ 37.200° 1.0％ 37.944° 2.9％ 38.983° 0.9％ 39.549° 0.8％
    优选地所述式A-5化合物的甲酸乙酯溶剂合物晶体B具有如图4所示的X-衍射粉末衍射图；
    和/或，当所述溶剂合物晶体为式A-5化合物的乙酸甲酯溶剂合物晶体C时，所述式A-5化合物的乙酸甲酯溶剂合物晶体C的X衍射粉末衍射图的数据如下表3所示：
    表3
    衍射角2θ 相对强度 5.468° 0.7％ 8.454° 91.9％ 9.748° 24.4％ 11.144° 13.3％ 11.548° 23.7％ 13.740° 23.7％ 14.057° 54.2％ 14.865° 5.5％ 16.111° 91.0％ 16.779° 17.4％ 16.882° 6.7％ 17.778° 15.2％ 18.596° 18.9％ 19.681° 100.0％ 20.371° 5.5％ 21.345° 4.6％ 21.777° 1.1％ 22.619° 40.2％ 23.256° 15.1％ 24.140° 17.8％ 24.601° 20.2％ 25.433° 7.5％ 25.861° 2.1％ 26.522° 7.3％ 26.806° 8.4％
    27.251° 5.0％ 27.884° 8.5％ 29.020° 2.1％ 29.472° 9.3％ 30.058° 4.7％ 35.151° 4.2％ 31.689° 1.9％ 32.615° 1.4％ 33.347° 3.2％ 33.778° 1.9％ 34.329° 1.6％ 34.545° 2.3％ 35.173° 2.9％ 36.028° 2.1％ 37.280° 0.9％ 37.923° 2.0％ 39.573° 1.1％
    优选地所述式A-5化合物的乙酸甲酯溶剂合物晶体C具有如图6所示的X-衍射粉末衍射图；
    和/或，所述溶剂合物晶体为式A-5化合物的甲酸正丙酯溶剂合物晶体D，所述式A-5化合物的甲酸正丙酯溶剂合物晶体D的X衍射粉末衍射图的数据如下表4所示：
    表4
    衍射角2θ 相对强度 5.489° 4.4％ 8.152° 100.0％ 9.459° 20.9％ 11.119° 6.4％ 11.335° 9.4％ 11.647° 17.2％ 12.602° 1.8％ 13.526° 36.7％
    13.989° 66.8％ 14.773° 3.9％ 15.407° 24.5％ 15.924° 61.8％ 16.720° 30.0％ 17.692° 22.8％ 18.423° 15.6％ 19.058° 32.3％ 19.459° 87.2％ 19.986° 6.7％ 20.861° 7.2％ 21.667° 2.4％ 22.237° 75.4％ 22.840° 50.9％ 23.460° 16.8％ 23.795° 35.3％ 24.751° 44.4％ 24.862° 19.1％ 25.563° 11.2％ 26.078° 13.3％ 26.567° 20.6％ 27.258° 2.8％ 27.652° 10.0％ 28.277° 5.6％ 28.764° 18.3％ 29.111° 2.5％ 29.590° 5.5％ 30.194° 1.2％ 30.500° 1.2％ 31.211° 10.5％
    32.391° 2.5％ 33.292° 4.6％ 33.848° 1.6％ 34.193° 3.3％ 34.534° 0.8％ 35.197° 1.5％ 35.585° 7.0％ 35.796° 2.3％ 36.498° 2.3％ 36.536° 1.9％ 37.390° 0.8％ 37.779° 3.5％ 38.525° 1.6％
    优选地所述式A-5化合物的甲酸正丙酯溶剂合物晶体D具有如图8所示的X-衍射粉末衍射图。
  24. 根据权利要求21-23中至少一项所述的溶剂合物晶体的制备方法，其特征在于，其包括下列步骤：将式A-5化合物从酯类溶剂或含酯类溶剂的溶液中析出；优选包括下列步骤：将式A-5化合物和酯类溶剂或含酯类溶剂的溶液混合，在0～40℃条件下搅拌析晶，制得式A-5化合物的酯类溶剂合物晶体；所述酯类溶剂和式A-5化合物的体积质量比为4mL/g～30mL/g；所述酯类溶剂优选为乙酸乙酯、甲酸乙酯、乙酸甲酯或甲酸正丙酯。
  25. 根据权利要求24所述的溶剂合物晶体的制备方法，其特征在于，
    所述含酯类溶剂的溶液中，所述的溶液中的溶剂为醇类溶剂、腈类溶剂、醚类溶剂和卤代烷烃类溶剂中的一种或多种；优选甲醇、乙腈、四氢呋喃、1,4-二氧六环和二氯甲烷中的一种或多种；
    和/或，所述酯类溶剂的质量分数不小于50％；进一步优选不小于85％；再进一步优选不小于95％；
    和/或，所述酯类溶剂或含酯类溶剂的溶液分别和式A-5化合物的体积质量比为4mL/g～30mL/g，进一步优选6mL/g～10mL/g；
    和/或，所述析晶的温度为15～25℃；
    和/或，所述析晶的操作结束后，还可进一步包含过滤、洗涤和干燥的操作；所述干燥优选在30-70℃下进行，进一步优选30-60℃。
  26. 根据权利要求21-26中至少一项所述溶剂合物晶体的制备方法，其特征在于，其包含如下步骤：将式A-5化合物溶解于酯类溶剂或含酯类溶剂的溶液中，在0～40℃条件下搅拌析晶，过滤，干燥，得到式A-5的酯类溶剂合物晶体；所述酯类溶剂和式A-5化合物的体积质量比为4mL/g～30mL/g。
  27. 一种药物组合物，其特征在于，包含如权利要求20-23中至少一项所述的溶剂合物晶体。
  28. 根据权利要求11或12所述化合物或其药学上可接受的盐、根据权利要求13和14所述式A-5所示化合物的非晶形、根据权利要求20-23中至少一项所述式A-5所示化合物的溶剂合物晶体、根据权利要求19或27所述的药物组合物在制备通过抑制PARP活性而改善的疾病方面的药物中的应用；所述通过抑制PARP活性而改善的疾病优选选自癌症、血管疾病、炎症性疾病、排异反应、糖尿病、帕金森病、感染性休克、缺血性损伤、神经性毒性、失血性休克和病毒感染；所述癌症优选是同源重组依赖性DNA双链断裂修复途径有缺陷的，或癌细胞优选是BRCA1或BRCA2有缺陷。
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