WO2022135580A1 - Crystal form of pyridopyrrole compound, and preparation method therefor and use thereof - Google Patents

Crystal form of pyridopyrrole compound, and preparation method therefor and use thereof Download PDF

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WO2022135580A1
WO2022135580A1 PCT/CN2021/141302 CN2021141302W WO2022135580A1 WO 2022135580 A1 WO2022135580 A1 WO 2022135580A1 CN 2021141302 W CN2021141302 W CN 2021141302W WO 2022135580 A1 WO2022135580 A1 WO 2022135580A1
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crystal form
compound
formula
following
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PCT/CN2021/141302
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刘迎春
徐招兵
胡利红
丁照中
朱兴训
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南京明德新药研发有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the invention relates to a crystal form of a pyridopyrrole compound, as well as a preparation method and application of the crystal form.
  • CDKs cell cycle-dependent kinases
  • CDK9 is a member of the CDK family and is mainly involved in transcriptional regulation.
  • the heterodimer composed of CDK9 and cyclin (T1, T2a, T2b, K) participates in the formation of positive transcription elongation factor (p-TEFb), of which about 80% of CDK9 binds to cyclinT1.
  • P-TEFb regulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II, primarily Ser-2.
  • Inhibition and transcriptional repression of CDK9 results in rapid depletion of short-lived mRNA transcripts and associated proteins, including Myc and Mcl-1, resulting in the death of cancer cells that are highly dependent on these anti-apoptotic proteins.
  • Targeting CDK9 thus represents a therapeutic strategy for tumor types that are highly dependent on these anti-apoptotic proteins.
  • CDK9 small molecule inhibitors have entered the clinical research stage for the treatment of cancer, namely Bayer's BAY1251152 and AstraZeneca's AZD4573. These patents include WO2012160034, WO2014076091, WO2009047359, WO2011110612, US2016376287.
  • CDK9 inhibitors for the treatment of cancer and other diseases
  • no drugs targeting this target have been marketed so far.
  • the most important clinical grade 3/4 and dose-limiting adverse side effect of BAY1251152 is neutropenia, while AZD4573 has poor kinase selectivity and metabolism, which limits its performance. the medicinal effect. Therefore, there is still an urgent need to develop novel, safer and more effective CDK9 inhibitors that can treat a variety of cancers, including leukemia and lymphoma.
  • the present invention provides crystal form A of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 7.22 ⁇ 0.20°, 17.16 ⁇ 0.20° and 22.34 ⁇ 0.20°;
  • the X-ray powder diffraction pattern of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 7.22 ⁇ 0.20°, 15.24 ⁇ 0.20°, 15.80 ⁇ 0.20°, 17.16 ⁇ 0.20°, 20.70 ⁇ 0.20 °, 22.34 ⁇ 0.20°, 24.46 ⁇ 0.20° and 31.74 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 7.22 ⁇ 0.20°, 8.76 ⁇ 0.20°, 15.24 ⁇ 0.20°, 15.80 ⁇ 0.20°, 17.16 ⁇ 0.20 °, 19.66 ⁇ 0.20°, 20.70 ⁇ 0.20°, 22.34 ⁇ 0.20°, 24.46 ⁇ 0.20°, 25.84 ⁇ 0.20°, 29.76 ⁇ 0.20° and 31.74 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned Form A has characteristic diffraction peaks at the following 2 ⁇ angles: 7.22 ⁇ 0.20°, 17.16 ⁇ 0.20°, and/or 22.34 ⁇ 0.20°, and/or 8.76 ⁇ 0.20°, and/or 9.84 ⁇ 0.20°, and/or 12.24 ⁇ 0.20°, and/or 15.24 ⁇ 0.20°, and/or 15.80 ⁇ 0.20°, and/or 16.22 ⁇ 0.20°, and/or 17.52 ⁇ 0.20° , and/or 18.40 ⁇ 0.20°, and/or 19.26 ⁇ 0.20°, and/or 19.66 ⁇ 0.20°, and/or 20.70 ⁇ 0.20°, and/or 21.46 ⁇ 0.20°, and/or 23.64 ⁇ 0.20°, and /or 24.46 ⁇ 0.20°, and/or 25.84 ⁇ 0.20°, and/or 27.10 ⁇ 0.20°, and/or 27.62 ⁇ 0.20°, and/or 28.02
  • the X-ray powder diffraction pattern of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 7.22°, 8.76°, 9.84°, 12.24°, 15.24°, 15.80°, 16.22°, 17.16° , 17.52°, 18.40°, 19.26°, 19.66°, 20.70°, 21.46°, 22.34°, 23.64°, 24.46°, 25.84°, 27.10°, 27.62°, 28.02°, 29.26°, 29.76°, 30.88°, 31.74 °, 33.38°, 37.10° and 37.68°.
  • the XRPD pattern of the above-mentioned crystal form A is basically as shown in FIG. 1 .
  • the differential scanning calorimetry curve of the differential scanning calorimetry curve has an endothermic peak starting point at 77.71 ⁇ 3°C and 236.85 ⁇ 3°C, respectively.
  • the DSC spectrum of the above-mentioned crystal form A is shown in FIG. 2 .
  • thermogravimetric analysis curve (TGA) of the above crystal form A has a weight loss of 3.420% at 200 ⁇ 3°C.
  • the TGA spectrum of the above-mentioned A crystal form is shown in FIG. 3 .
  • the present invention also provides the B crystal form of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 19.72 ⁇ 0.20°, 21.52 ⁇ 0.20° and 23.20 ⁇ 0.20°;
  • the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 10.74 ⁇ 0.20°, 16.22 ⁇ 0.20°, 19.72 ⁇ 0.20°, 20.58 ⁇ 0.20°, 21.52 ⁇ 0.20 °, 22.30 ⁇ 0.20°, 23.20 ⁇ 0.20° and 28.04 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 7.92 ⁇ 0.20°, 10.74 ⁇ 0.20°, 16.22 ⁇ 0.20°, 17.66 ⁇ 0.20°, 19.72 ⁇ 0.20 °, 20.58 ⁇ 0.20°, 21.52 ⁇ 0.20°, 22.30 ⁇ 0.20°, 23.20 ⁇ 0.20°, 23.88 ⁇ 0.20°, 26.54 ⁇ 0.20°, 27.48 ⁇ 0.20° and 28.04 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 19.72 ⁇ 0.20°, 21.52 ⁇ 0.20°, and/or 23.20 ⁇ 0.20°, and/or 7.92 ⁇ 0.20°, and/or 10.74 ⁇ 0.20°, and/or 11.42 ⁇ 0.20°, and/or 13.52 ⁇ 0.20°, and/or 13.76 ⁇ 0.20°, and/or 15.86 ⁇ 0.20°, and/or 16.22 ⁇ 0.20° , and/or 16.52 ⁇ 0.20°, and/or 17.66 ⁇ 0.20°, and/or 17.90 ⁇ 0.20°, and/or 18.22 ⁇ 0.20°, and/or 18.92 ⁇ 0.20°, and/or 20.58 ⁇ 0.20°, and /or 22.30 ⁇ 0.20°, and/or 23.88 ⁇ 0.20°, and/or 25.32 ⁇ 0.20°, and/or 26.12 ⁇ 0.20°, and/or 26.54
  • the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 7.92°, 10.74°, 11.42°, 13.52°, 13.76°, 15.86°, 16.22°, 16.52° , 17.66°, 17.90°, 18.22°, 18.92°, 19.72°, 20.58°, 21.52°, 22.30°, 23.20°, 23.88°, 25.32°, 26.12°, 26.54°, 27.14°, 27.48°, 27.72°, 28.04 degrees 37.96°, 38.74° and 39.50°.
  • the XRPD pattern of the above-mentioned crystal form B is basically as shown in FIG. 4 .
  • the differential scanning calorimetry curve has an endothermic peak starting point at 257.81 ⁇ 3°C.
  • the DSC spectrum of the above-mentioned crystal form B is shown in FIG. 5 .
  • thermogravimetric analysis curve of the above-mentioned crystal form B loses weight up to 0.326% at 200 ⁇ 3°C.
  • the TGA spectrum of the above-mentioned B crystal form is shown in FIG. 6 .
  • the present invention also provides a method for preparing crystal form A, comprising the following steps:
  • the above reflux temperature is 65°C-80°C, preferably 65°C.
  • the above stirring time is 10-12 hours, preferably 12 hours.
  • the present invention also provides a preparation method of crystal form B, comprising the following steps:
  • the stirring temperature is 20°C;
  • the stirring time is 20-21 hours.
  • the present invention also provides the application of the above-mentioned A crystal form and the above-mentioned B crystal form in the preparation of CDK9 inhibitor drugs.
  • the compound of formula (I) of the present application has good efficacy for in vivo administration, and its crystal form is stable, less affected by light, heat and humidity, and has good solubility.
  • the intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalents, preferred embodiments include, but are not limited to, the examples of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuK ⁇ radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.
  • SXRD single crystal X-ray diffraction method
  • the cultured single crystal is collected by Bruker D8 venture diffractometer
  • the light source is CuK ⁇ radiation
  • the scanning mode is: After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.
  • the solvent used in the present invention is commercially available.
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • DMSO dimethyl sulfoxide
  • EtOH stands for ethanol
  • MeOH stands for methanol
  • ACN stands for acetonitrile
  • THF tetrahydrofuran
  • H 2 O water
  • NCS 1-chloropyrrolidine-2,5-dione
  • NIS for N-iodosuccinimide
  • DMAC for dimethylacetamide
  • Pd(dppf)Cl 2 ⁇ CH 2 Cl 2 represents [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane adduct.
  • the XRPD test used the DX-2700BH X-ray diffractometer of Dandong Haoyuan Company. The test parameters are shown in Table 3.
  • the DSC spectra were collected on a TA 2500 differential scanning calorimeter, and the test parameters are shown in Table 4.
  • TGA was collected on a TA 5500 thermogravimetric analyzer, and the test parameters are shown in Table 5.
  • Figure 1 XRPD pattern of Form A
  • step 1
  • the filter cake was dissolved in 1.50L of water, the organic phase was discarded, and the aqueous phase was transferred into a 5.0-liter there-necked flask, and under stirring, 1 mol/liter aqueous sodium hydroxide solution was added dropwise to pH ⁇ 11, and a large amount of white solid was separated out, and filtered to obtain the formula ( I) Compounds.
  • the A crystal form (50 g, 0.158 mol) of the compound of formula (I) was stirred in 250 mL of ethanol for 21 hours, the mixture was filtered, the filter cake was transferred to an oven, and dried under reduced pressure to obtain the compound of formula (I)
  • the B crystal form, the XRPD detection results are shown in Figure 4, and the TGA and DSC detection results are shown in Figure 5 and Figure 6, respectively.
  • test sample marked S1-condition-time is used for content and related substance detection; the test sample marked S2-condition-time is used as a preparation sample, and the test sample marked S3-condition-time is used for XRPD detection .
  • CDK9/CyclinT1 kinase was purchased from Carna
  • ADP-Glo assay kit was purchased from Promega
  • PKDTide substrate and kinase reaction buffer were purchased from Signalchem. Nivo Multilabel Analyzer (PerkinElmer).
  • the compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 ⁇ M to 0.65 nM, and the DMSO concentration was 5%, and a double-well experiment was set up.
  • Add 1 ⁇ L of each concentration gradient of inhibitor, 2 ⁇ L CDK9/CyclinT1 enzyme (4ng), 2 ⁇ L mixture of substrate and ATP (100 ⁇ M adenosine triphosphate, 0.2 ⁇ g/ ⁇ L substrate) to the microplate, and the final compound concentration gradient is 10 ⁇ M dilution at this time to 0.13 nM.
  • the reaction system was placed at 25°C for 120 minutes.
  • the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode).
  • Table 8 provides the CDK9/CyclinT1 enzymatic inhibitory activity of the compounds of the present invention.
  • the compound of formula (I) has good activity on CDK9 kinase, similar to the activity of reference compounds BAY1251152 and AZD4573.
  • CDK1/CyclinB1 Kinase Assay Kit was purchased from Promega. Nivo Multilabel Analyzer (PerkinElmer).
  • the compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 ⁇ M to 0.65 nM, and the DMSO concentration was 5%, and a double-well experiment was set up.
  • the reaction system was placed at 25°C for 120 minutes.
  • the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode).
  • Table 6 provides the CDK1/CyclinB1 enzymatic inhibitory activity of the compounds of the present invention.
  • the compounds of formula (I) have weak inhibitory activity on CDK1 kinase, so the compounds of the present invention show better selectivity to CDK1 than BAY1251152 and AZD4573.
  • the experimental results are shown in Table 8.
  • CDK2/CyclinE1 Kinase Assay Kit was purchased from Promega. Nivo Multilabel Analyzer (PerkinElmer).
  • the compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 ⁇ M to 0.65 nM, and the DMSO concentration was 5%, and a double-well experiment was set up.
  • Add 1 ⁇ L of each concentration gradient of inhibitor, 2 ⁇ L CDK2/CyclinE1 enzyme (2ng), 2 ⁇ L mixture of substrate and ATP (150 ⁇ M adenosine triphosphate, 0.1 ⁇ g/ ⁇ L substrate) to the microplate, and the final compound concentration gradient is 10 ⁇ M dilution at this time to 0.13 nM.
  • the reaction system was placed at 25°C for 60 minutes.
  • the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode).
  • Table 8 provides the CDK2/CyclinE1 enzymatic inhibitory activity of the compounds of the present invention.
  • the compound of formula (I) does not have strong inhibitory activity on CDK2 kinase, so the compound of the present invention exhibits better selectivity to CDK2 than BAY1251152 and AZD4573.
  • the experimental results are shown in Table 8.
  • IMDM medium fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Promega (Madison, WI).
  • MV-4-11 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo Multilabel Analyzer (PerkinElmer).
  • MV-4-11 cells were seeded in a white 96-well plate, 80 ⁇ L of cell suspension per well, which contained 6000 MV-4-11 cells. Cell plates were incubated overnight in a carbon dioxide incubator.
  • the compounds to be tested were diluted 5-fold to the 8th concentration, that is, from 2 mM to 26 nM, and a double-well experiment was set up. Add 78 ⁇ L of medium to the middle plate, and then transfer 2 ⁇ L of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 ⁇ L of each well to the cell plate. Final compound concentrations ranged from 10 ⁇ M to 0.13 nM.
  • the cell plates were placed in a carbon dioxide incubator for 3 days.
  • the IC 50 value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).
  • Table 8 provides the inhibitory activity of the compounds of the present invention on the proliferation of MV-4-11 cells.
  • mice Female, 6-8 weeks, weighing approximately 18-22 grams, were kept in a special pathogen-free environment in a single ventilated cage (3 mice per cage). All cages, bedding and water were sterilized before use. All animals had free access to standard certified commercial laboratory diets. A total of 36 mice purchased from Shanghai Lingchang biological science and technology Co., LTD. were used for the study. Tumor cells (10 ⁇ 10 6 in 0.2 ml phosphate buffered saline) were implanted subcutaneously in the right flank of each mouse for tumor growth. Dosing was initiated when the mean tumor volume reached approximately 121 cubic millimeters. The test compound was administered weekly by injection at a dose of 10 mg/kg.
  • Antitumor efficacy is determined by dividing the mean tumor increase volume in animals treated with the compound by the mean tumor increase volume in untreated animals.

Abstract

A crystal form of a pyridopyrrole compound, and a preparation method therefor and the use thereof.

Description

吡啶并吡咯类化合物的晶型、制备方法及其应用Crystal form, preparation method and application of pyridopyrrole compounds
本发明主张如下优先权：The present invention claims the following priority:
CN202011563187.8，2020年12月25日。CN202011563187.8, December 25, 2020.
技术领域technical field
本发明涉及了一种吡啶并吡咯类化合物的晶型、以及晶型的制备方法及应用。The invention relates to a crystal form of a pyridopyrrole compound, as well as a preparation method and application of the crystal form.
背景技术Background technique
肿瘤的发生往往伴随着细胞的过度活化和持续性增殖，而CDK(细胞周期依赖性激酶)在细胞内外信号的调节下对细胞周期和转录过程发挥着重要的调控作用。在癌细胞中，CDK-cyclin(周期素)的活性往往是失调的，可能的原因包含：信号传导通路的过度激活、cyclin的过度表达、CDK的异常扩增、内源性抑制因子的失活或缺失，这些启发着人们通过不断寻找新型的CDK抑制剂来发展肿瘤治疗技术。The occurrence of tumors is often accompanied by excessive activation and continuous proliferation of cells, and CDKs (cell cycle-dependent kinases) play an important role in the regulation of cell cycle and transcriptional processes under the regulation of intracellular and extracellular signals. In cancer cells, the activity of CDK-cyclin (cyclin) is often deregulated, and the possible reasons include: excessive activation of signaling pathways, overexpression of cyclin, abnormal amplification of CDK, and inactivation of endogenous inhibitors Or missing, these inspire people to develop tumor treatment technology by constantly searching for new CDK inhibitors.
CDK9是CDK家族成员之一，主要参与转录调控过程，由CDK9和cyclin(T1、T2a、T2b、K)组成的异源二聚体参与组成正性转录延长因子(p-TEFb)，其中约有80％的CDK9与cyclinT1结合。P-TEFb通过使RNA聚合酶II的羧基端结构域磷酸化，主要是Ser-2磷酸化，调节转录延长。CDK9的抑制和转录阻遏导致快速消耗短寿命的mRNA转录物和相关蛋白(包括Myc和Mcl-1)，从而导致高度依赖这些抗凋亡蛋白的癌细胞死亡。因此靶向CDK9代表一种高度依赖这些抗凋亡蛋白的肿瘤类型的治疗策略。CDK9 is a member of the CDK family and is mainly involved in transcriptional regulation. The heterodimer composed of CDK9 and cyclin (T1, T2a, T2b, K) participates in the formation of positive transcription elongation factor (p-TEFb), of which about 80% of CDK9 binds to cyclinT1. P-TEFb regulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II, primarily Ser-2. Inhibition and transcriptional repression of CDK9 results in rapid depletion of short-lived mRNA transcripts and associated proteins, including Myc and Mcl-1, resulting in the death of cancer cells that are highly dependent on these anti-apoptotic proteins. Targeting CDK9 thus represents a therapeutic strategy for tumor types that are highly dependent on these anti-apoptotic proteins.
目前，已经有CDK9小分子抑制剂进入临床研究阶段用于癌症的治疗，即拜耳的BAY1251152和阿斯利康的AZD4573。这些专利包括WO2012160034，WO2014076091，WO2009047359，WO2011110612，US2016376287。At present, CDK9 small molecule inhibitors have entered the clinical research stage for the treatment of cancer, namely Bayer's BAY1251152 and AstraZeneca's AZD4573. These patents include WO2012160034, WO2014076091, WO2009047359, WO2011110612, US2016376287.
Figure PCTCN2021141302-appb-000001
Figure PCTCN2021141302-appb-000001
虽然在开发用于癌症和其他疾病治疗的CDK9抑制剂的道路上已经做了很多努力，但是到目前为止还没有针对该靶点的药物上市。在开展临床研究的这些药物中，BAY1251152的临床上最主要的3/4级和剂量限制性不良副作用为嗜中性白血球减少症，而AZD4573的激酶选择性和代谢不好，限制其发挥更好的药效。因此仍然迫切需要开发新颖的、更加安全有效的、能够治疗多种癌症(包括白血病和淋巴癌)的CDK9抑制剂。Although many efforts have been made to develop CDK9 inhibitors for the treatment of cancer and other diseases, no drugs targeting this target have been marketed so far. Among these drugs in clinical research, the most important clinical grade 3/4 and dose-limiting adverse side effect of BAY1251152 is neutropenia, while AZD4573 has poor kinase selectivity and metabolism, which limits its performance. the medicinal effect. Therefore, there is still an urgent need to develop novel, safer and more effective CDK9 inhibitors that can treat a variety of cancers, including leukemia and lymphoma.
发明内容SUMMARY OF THE INVENTION
本发明提供了式(Ⅰ)化合物的A晶型，其X射线粉末衍射(XRPD)图谱在下列2θ角处具有特征衍射峰：7.22±0.20°、17.16±0.20°和22.34±0.20°；The present invention provides crystal form A of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 17.16±0.20° and 22.34±0.20°;
Figure PCTCN2021141302-appb-000002
Figure PCTCN2021141302-appb-000002
本发明的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22±0.20°、15.24±0.20°、15.80±0.20°、17.16±0.20°、20.70±0.20°、22.34±0.20°、24.46±0.20°和31.74±0.20°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 15.24±0.20°, 15.80±0.20°, 17.16±0.20°, 20.70±0.20 °, 22.34±0.20°, 24.46±0.20° and 31.74±0.20°.
本发明的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22±0.20°、8.76±0.20°、15.24±0.20°、15.80±0.20°、17.16±0.20°、19.66±0.20°、20.70±0.20°、22.34±0.20°、24.46±0.20°、25.84±0.20°、29.76±0.20°和31.74±0.20°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 8.76±0.20°, 15.24±0.20°, 15.80±0.20°, 17.16±0.20 °, 19.66±0.20°, 20.70±0.20°, 22.34±0.20°, 24.46±0.20°, 25.84±0.20°, 29.76±0.20° and 31.74±0.20°.
本发明的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22±0.20°，17.16±0.20°，和/或22.34±0.20°，和/或8.76±0.20°，和/或9.84±0.20°，和/或12.24±0.20°，和/或15.24±0.20°，和/或15.80±0.20°，和/或16.22±0.20°，和/或17.52±0.20°，和/或18.40±0.20°，和/或19.26±0.20°，和/或19.66±0.20°，和/或20.70±0.20°，和/或21.46±0.20°，和/或23.64±0.20°，和/或24.46±0.20°，和/或25.84±0.20°，和/或27.10±0.20°，和/或27.62±0.20°，和/或28.02±0.20°，和/或29.26±0.20°，和/或29.76±0.20°，和/或30.88±0.20°，和/或31.74±0.20°，和/或33.38±0.20°，和/或37.10±0.20°，和/或37.68±0.20°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form A has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 17.16±0.20°, and/or 22.34±0.20°, and/or 8.76± 0.20°, and/or 9.84±0.20°, and/or 12.24±0.20°, and/or 15.24±0.20°, and/or 15.80±0.20°, and/or 16.22±0.20°, and/or 17.52±0.20° , and/or 18.40±0.20°, and/or 19.26±0.20°, and/or 19.66±0.20°, and/or 20.70±0.20°, and/or 21.46±0.20°, and/or 23.64±0.20°, and /or 24.46±0.20°, and/or 25.84±0.20°, and/or 27.10±0.20°, and/or 27.62±0.20°, and/or 28.02±0.20°, and/or 29.26±0.20°, and/or 29.76±0.20°, and/or 30.88±0.20°, and/or 31.74±0.20°, and/or 33.38±0.20°, and/or 37.10±0.20°, and/or 37.68±0.20°.
本发明的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22°、8.76°、9.84°、12.24°、15.24°、15.80°、16.22°、17.16°、17.52°、18.40°、19.26°、19.66°、20.70°、21.46°、22.34°、23.64°、24.46°、25.84°、27.10°、27.62°、28.02°、29.26°、29.76°、30.88°、31.74°、33.38°、37.10°和37.68°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.22°, 8.76°, 9.84°, 12.24°, 15.24°, 15.80°, 16.22°, 17.16° , 17.52°, 18.40°, 19.26°, 19.66°, 20.70°, 21.46°, 22.34°, 23.64°, 24.46°, 25.84°, 27.10°, 27.62°, 28.02°, 29.26°, 29.76°, 30.88°, 31.74 °, 33.38°, 37.10° and 37.68°.
本发明的一些方案中，上述A晶型，其XRPD图谱基本如图1所示。In some solutions of the present invention, the XRPD pattern of the above-mentioned crystal form A is basically as shown in FIG. 1 .
本发明的一些方案中，上述A晶型的XRPD图谱解析数据如表1所示。In some solutions of the present invention, the XRPD pattern analysis data of the above-mentioned A crystal form is shown in Table 1.
表1式(I)化合物A晶型的XRPD解析数据Table 1 XRPD analysis data of the crystal form of compound A of formula (I)
Figure PCTCN2021141302-appb-000003
Figure PCTCN2021141302-appb-000003
Figure PCTCN2021141302-appb-000004
Figure PCTCN2021141302-appb-000004
本发明的一些方案中，上述A晶型，其差示扫描量热曲线在77.71±3℃和236.85±3℃分别有一个吸热峰的起始点。In some embodiments of the present invention, for the above-mentioned crystal form A, the differential scanning calorimetry curve of the differential scanning calorimetry curve has an endothermic peak starting point at 77.71±3°C and 236.85±3°C, respectively.
本发明的一些方案中，上述A晶型，其DSC图谱如图2所示。In some solutions of the present invention, the DSC spectrum of the above-mentioned crystal form A is shown in FIG. 2 .
在本发明的一些方案中，上述A晶型的热重分析曲线(TGA)在200±3℃时失重达3.420％。In some embodiments of the present invention, the thermogravimetric analysis curve (TGA) of the above crystal form A has a weight loss of 3.420% at 200±3°C.
在本发明的一些方案中，上述A晶型的TGA图谱如图3所示。In some embodiments of the present invention, the TGA spectrum of the above-mentioned A crystal form is shown in FIG. 3 .
本发明还提供了式(Ⅰ)化合物的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：19.72±0.20°、21.52±0.20°和23.20±0.20°；The present invention also provides the B crystal form of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 19.72±0.20°, 21.52±0.20° and 23.20±0.20°;
Figure PCTCN2021141302-appb-000005
Figure PCTCN2021141302-appb-000005
本发明的一些方案中，上述B晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：10.74±0.20°、16.22±0.20°、19.72±0.20°、20.58±0.20°、21.52±0.20°、22.30±0.20°、23.20±0.20°和28.04±0.20°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 10.74±0.20°, 16.22±0.20°, 19.72±0.20°, 20.58±0.20°, 21.52±0.20 °, 22.30±0.20°, 23.20±0.20° and 28.04±0.20°.
本发明的一些方案中，上述B晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.92±0.20°、10.74±0.20°、16.22±0.20°、17.66±0.20°、19.72±0.20°、20.58±0.20°、21.52±0.20°、22.30±0.20°、23.20±0.20°、23.88±0.20°、26.54±0.20°、27.48±0.20°和28.04±0.20°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 7.92±0.20°, 10.74±0.20°, 16.22±0.20°, 17.66±0.20°, 19.72±0.20 °, 20.58±0.20°, 21.52±0.20°, 22.30±0.20°, 23.20±0.20°, 23.88±0.20°, 26.54±0.20°, 27.48±0.20° and 28.04±0.20°.
本发明的一些方案中，上述B晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：19.72±0.20°，21.52±0.20°，和/或23.20±0.20°，和/或7.92±0.20°，和/或10.74±0.20°，和/或11.42±0.20°，和/或13.52±0.20°，和/或13.76±0.20°，和/或15.86±0.20°，和/或16.22±0.20°，和/或16.52±0.20°，和/或17.66±0.20°，和/或17.90±0.20°，和/或18.22±0.20°，和/或18.92±0.20°，和/或20.58±0.20°，和/或22.30±0.20°，和/或23.88±0.20°，和/或25.32±0.20°，和/或26.12±0.20°，和/或26.54±0.20°，和/或27.14±0.20°，和/或27.48±0.20°，和/或27.72±0.20°，和/或28.04±0.20°，和/或28.52±0.20°，和/或28.96±0.20°，和/或29.20±0.20°，和/或29.74±0.20°，和/或30.24±0.20°，和/或30.58±0.20°，和/或31.56±0.20°，和/或32.54±0.20°，和/或32.82±0.20°，和/或33.38±0.20°，和/或34.36±0.20°，和/或34.75±0.20°，和/或35.44±0.20°，和/或36.00±0.20°，和/或36.56±0.20°，和/或37.08±0.20°，和/或37.96±0.20°，和/或38.74±0.20°，和/或39.50±0.20°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 19.72±0.20°, 21.52±0.20°, and/or 23.20±0.20°, and/or 7.92± 0.20°, and/or 10.74±0.20°, and/or 11.42±0.20°, and/or 13.52±0.20°, and/or 13.76±0.20°, and/or 15.86±0.20°, and/or 16.22±0.20° , and/or 16.52±0.20°, and/or 17.66±0.20°, and/or 17.90±0.20°, and/or 18.22±0.20°, and/or 18.92±0.20°, and/or 20.58±0.20°, and /or 22.30±0.20°, and/or 23.88±0.20°, and/or 25.32±0.20°, and/or 26.12±0.20°, and/or 26.54±0.20°, and/or 27.14±0.20°, and/or 27.48±0.20°, and/or 27.72±0.20°, and/or 28.04±0.20°, and/or 28.52±0.20°, and/or 28.96±0.20°, and/or 29.20±0.20°, and/or 29.74± 0.20°, and/or 30.24±0.20°, and/or 30.58±0.20°, and/or 31.56±0.20°, and/or 32.54±0.20°, and/or 32.82±0.20°, and/or 33.38±0.20° , and/or 34.36±0.20°, and/or 34.75±0.20°, and/or 35.44±0.20°, and/or 36.00±0.20°, and/or 36.56±0.20°, and/or 37.08±0.20°, and /or 37.96±0.20°, and/or 38.74±0.20°, and/or 39.50±0.20°.
本发明的一些方案中，上述B晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.92°、10.74°、11.42°、13.52°、13.76°、15.86°、16.22°、16.52°、17.66°、17.90°、18.22°、18.92°、19.72°、20.58°、21.52°、22.30°、23.20°、23.88°、25.32°、26.12°、26.54°、27.14°、27.48°、27.72°、28.04°、28.52°、28.96°、29.20°、29.74°、30.24°、30.58°、31.56°、32.54°、32.82°、33.38°、34.36°、34.75°、35.44°、36.00°、36.56°、37.08°、37.96°、38.74°和39.50°。In some embodiments of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 7.92°, 10.74°, 11.42°, 13.52°, 13.76°, 15.86°, 16.22°, 16.52° , 17.66°, 17.90°, 18.22°, 18.92°, 19.72°, 20.58°, 21.52°, 22.30°, 23.20°, 23.88°, 25.32°, 26.12°, 26.54°, 27.14°, 27.48°, 27.72°, 28.04 degrees 37.96°, 38.74° and 39.50°.
本发明的一些方案中，上述B晶型，其XRPD图谱基本如图4所示。In some solutions of the present invention, the XRPD pattern of the above-mentioned crystal form B is basically as shown in FIG. 4 .
本发明的一些方案中，上述B晶型的XRPD图谱解析数据如表2所示。In some solutions of the present invention, the XRPD pattern analysis data of the above-mentioned B crystal form are shown in Table 2.
表2式(I)化合物B晶型的XRPD解析数据Table 2 XRPD analysis data of compound B crystal form of formula (I)
Figure PCTCN2021141302-appb-000006
Figure PCTCN2021141302-appb-000006
本发明的一些方案中，上述B晶型，其差示扫描量热曲线在257.81±3℃有一个吸热峰的起始点。In some embodiments of the present invention, in the above-mentioned crystal form B, the differential scanning calorimetry curve has an endothermic peak starting point at 257.81±3°C.
本发明的一些方案中，上述B晶型，其DSC图谱如图5所示。In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form B is shown in FIG. 5 .
在本发明的一些方案中，上述B晶型的热重分析曲线在200±3℃时失重达0.326％。In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form B loses weight up to 0.326% at 200±3°C.
在本发明的一些方案中，上述B晶型的TGA图谱如图6所示。In some embodiments of the present invention, the TGA spectrum of the above-mentioned B crystal form is shown in FIG. 6 .
本发明还提供了A晶型的制备方法，包含如下步骤：The present invention also provides a method for preparing crystal form A, comprising the following steps:
1)式(I)化合物加入到无水甲醇中回流；1) the compound of formula (I) is added to anhydrous methanol and refluxed;
2)式(I)化合物完全溶解，趁热过滤；2) the compound of formula (I) is completely dissolved, filtered while hot;
3)滤液在回流状态下滴加蒸馏水，析出白色固体，自然降温至室温，室温搅拌；3) The filtrate is dripped with distilled water under reflux state to separate out a white solid, which is naturally cooled to room temperature and stirred at room temperature;
4)混合物过滤，滤饼减压烘干。4) The mixture is filtered, and the filter cake is dried under reduced pressure.
本发明的一些方案中，上述回流温度为65℃-80℃，优选为65℃。In some solutions of the present invention, the above reflux temperature is 65°C-80°C, preferably 65°C.
本发明的一些方案中，上述搅拌时间为10-12小时，优选为12小时。In some solutions of the present invention, the above stirring time is 10-12 hours, preferably 12 hours.
本发明还提供了B晶型的制备方法，包含如下步骤：The present invention also provides a preparation method of crystal form B, comprising the following steps:
1)式(I)化合物的A晶型在乙醇中搅拌，过滤、滤饼减压烘干；1) the A crystal form of the compound of formula (I) is stirred in ethanol, filtered, and the filter cake is dried under reduced pressure;
其中，in,
搅拌温度为20℃；The stirring temperature is 20°C;
搅拌时间为20-21小时。The stirring time is 20-21 hours.
本发明还提供了上述A晶型和上述的B晶型在制备CDK9抑制剂药物中的应用。The present invention also provides the application of the above-mentioned A crystal form and the above-mentioned B crystal form in the preparation of CDK9 inhibitor drugs.
技术效果technical effect
本申请式(Ⅰ)化合物具有良好的体内给药药效且其晶型稳定、受光热湿度影响小、溶解性好，成药前景广阔。The compound of formula (I) of the present application has good efficacy for in vivo administration, and its crystal form is stable, less affected by light, heat and humidity, and has good solubility.
定义和说明Definition and Explanation
除非另有说明，本文所用的下列术语和短语旨在含有下列含义。一个特定的短语或术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文出现商品名时，旨在指代其对应的商品或其活性成分。Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered indeterminate or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.
本发明的中间体化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and those skilled in the art. Well-known equivalents, preferred embodiments include, but are not limited to, the examples of the present invention.
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。The chemical reactions of specific embodiments of the present invention are carried out in suitable solvents suitable for the chemical changes of the present invention and their required reagents and materials. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthetic steps or reaction schemes on the basis of the existing embodiments.
下面会通过实施例具体描述本发明，这些实施例并不意味着对本发明的任何限制。The present invention will be specifically described below through examples, which do not imply any limitation to the present invention.
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。All solvents used in the present invention are commercially available and used without further purification.
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2021141302-appb-000007
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。 The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is:
Figure PCTCN2021141302-appb-000007
After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.
本发明所使用的溶剂可经市售获得。The solvent used in the present invention is commercially available.
本发明采用下述缩略词：DCM代表二氯甲烷；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOH代表乙醇；MeOH代表甲醇；ACN代表乙腈；THF代表四氢呋喃；H 2O代表水；NCS代表1-氯吡咯烷-2,5-二酮；NIS代表N-碘代丁二酰亚胺；DMAC代表二甲基乙酰胺；Pd(dppf)Cl 2·CH 2Cl 2代表[1,1'‐双(二苯基膦基)二茂铁]二氯化钯二氯甲烷加合物。 The following abbreviations are used in the present invention: DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOH stands for ethanol; MeOH stands for methanol; ACN stands for acetonitrile; THF stands for tetrahydrofuran; H 2 O for water; NCS for 1-chloropyrrolidine-2,5-dione; NIS for N-iodosuccinimide; DMAC for dimethylacetamide; Pd(dppf)Cl 2 ·CH 2 Cl 2 represents [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane adduct.
化合物依据本领域常规命名原则或者使用
Figure PCTCN2021141302-appb-000008
软件命名，市售化合物采用供应商目录名称。仪器参数 Compounds are named according to conventional nomenclature in the art or are used
Figure PCTCN2021141302-appb-000008
Software naming, commercially available compounds use supplier catalog names. Instrument parameters
本发明X射线粉末衍射(XRPD)仪器信息和方法X-ray Powder Diffraction (XRPD) Instrument Information and Methods of the Invention
XRPD测试使用丹东浩元公司的DX-2700BH型X射线衍射仪。测试参数如表3所示。The XRPD test used the DX-2700BH X-ray diffractometer of Dandong Haoyuan Company. The test parameters are shown in Table 3.
表3：XRPD测试参数Table 3: XRPD Test Parameters
Figure PCTCN2021141302-appb-000009
Figure PCTCN2021141302-appb-000009
本发明差示扫描量热(DSC)仪器信息及方法Differential Scanning Calorimetry (DSC) Instrument Information and Methods of the Invention
DSC图谱在TA 2500差示扫描量热仪上采集，测试参数如表4所示。The DSC spectra were collected on a TA 2500 differential scanning calorimeter, and the test parameters are shown in Table 4.
表4：DSC测试参数Table 4: DSC test parameters
参数parameter METTLER TOLEDO DSC1METTLER TOLEDO DSC1
样品盘sample tray 高压坩埚High pressure crucible
温度范围temperature range 40～350℃40～350℃
扫描速率(℃/分钟)Scan rate (℃/min) 1010
保护气体Protective gas 氮气nitrogen
本发明热重分析(TGA)仪器信息与方法Thermogravimetric Analysis (TGA) Instrument Information and Methods of the Invention
TGA在TA 5500热重分析仪上采集，测试参数如表5所示。TGA was collected on a TA 5500 thermogravimetric analyzer, and the test parameters are shown in Table 5.
表5：TGA测试参数Table 5: TGA test parameters
参数parameter TA TGA5500TA TGA5500
样品盘sample tray 铝盘，开盖Aluminium tray, open lid
温度范围temperature range 40～500℃40～500℃
扫描速率(℃/分钟)Scan rate (℃/min) 1010
保护气体Protective gas 氮气nitrogen
附图说明Description of drawings
图1：A晶型的XRPD图谱；Figure 1: XRPD pattern of Form A;
图2：A晶型的DSC图谱；Figure 2: DSC spectrum of crystal form A;
图3：A晶型的TGA图谱；Figure 3: TGA spectrum of crystal form A;
图4：B晶型的XRPD图谱；Figure 4: XRPD pattern of Form B;
图5：B晶型的DSC图谱；Figure 5: DSC spectrum of crystal form B;
图6：B晶型的TGA图谱。Figure 6: TGA pattern of Form B.
具体实施方式Detailed ways
为了更好的理解本发明的内容，下面结合具体实施例来做进一步的说明，但具体的实施方式并不是对本发明的内容所做的限制。In order to better understand the content of the present invention, further description will be given below in conjunction with specific embodiments, but the specific embodiments do not limit the content of the present invention.
实施例1：式(I)化合物的制备Example 1: Preparation of compounds of formula (I)
Figure PCTCN2021141302-appb-000010
Figure PCTCN2021141302-appb-000010
第1步：step 1:
在0℃下，向化合物1(900克，5.20摩尔，1当量)的DMF(3.60升)溶液中分批加入NCS(729.37 克，5.46摩尔，1.05当量)，混合物在15℃搅拌反应12小时。LCMS和HPLC监测原料化合物1反应完全；一边搅拌，一边将反应液缓慢倒入w％＝10％的氢氧化钠水溶液(4.0升)中，乙酸乙酯(2.0升*2)萃取，合并有机相，饱和食盐水(2.0升*2)洗涤，分液，有机相浓缩得到残余物。残余物用二氯甲烷(3.0升)打浆2小时。过滤，滤饼烘干得到化合物2。 1H NMR(400MHz,CDCl 3)δ8.05(s,1H),6.79(s,1H),4.57(br s,2H)。LCMS(ESI)m/z:208.9(M+1)。 To a solution of compound 1 (900 g, 5.20 mol, 1 equiv) in DMF (3.60 L) was added NCS (729.37 g, 5.46 mol, 1.05 equiv) portionwise at 0°C, and the mixture was stirred at 15°C for 12 hours. LCMS and HPLC monitored the completion of the reaction of the raw material compound 1; while stirring, the reaction solution was slowly poured into a w%=10% aqueous sodium hydroxide solution (4.0 L), extracted with ethyl acetate (2.0 L*2), and the organic phases were combined. , washed with saturated brine (2.0 L*2), separated, and the organic phase was concentrated to obtain a residue. The residue was slurried with dichloromethane (3.0 L) for 2 hours. Filter and dry the filter cake to obtain compound 2. 1 H NMR (400 MHz, CDCl 3 ) δ 8.05 (s, 1H), 6.79 (s, 1H), 4.57 (br s, 2H). LCMS (ESI) m/z: 208.9 (M+1).
第2步：Step 2:
向化合物2(1870克，9.01摩尔，1当量)的醋酸(5.0升)溶液中加入NIS(2.23千克，9.92摩尔，1.1当量)，混合物氮气置换三次后加热至80℃反应4.0小时。LCMS和HPLC监测原料反应完全；反应液冷却至15℃，一边搅拌一边将反应液缓慢倒入水(2.5升)中，大量固体析出，过滤，滤饼水(1升)洗，抽干得到粗品。粗品用乙醇(2.0升)打浆。混合物过滤，滤饼烘干得到化合物3。LCMS(ESI)m/z:334.8(M+1)。To a solution of compound 2 (1870 g, 9.01 mol, 1 equiv) in acetic acid (5.0 L) was added NIS (2.23 kg, 9.92 mol, 1.1 equiv), the mixture was purged with nitrogen three times and heated to 80°C for 4.0 hours. LCMS and HPLC monitor the complete reaction of the raw materials; the reaction solution was cooled to 15°C, and the reaction solution was slowly poured into water (2.5 liters) while stirring, a large amount of solid was precipitated, filtered, washed with filter cake water (1 liter), and drained to obtain the crude product . The crude product was slurried with ethanol (2.0 L). The mixture was filtered, and the filter cake was dried to obtain compound 3. LCMS (ESI) m/z: 334.8 (M+1).
第3步：Step 3:
向化合物3(2700克，7.48摩尔)的甲苯(16.2升)中加入化合物4(1.88千克，8.98摩尔，1.20当量)、二氯二三苯基膦钯(262.51克，374毫摩尔，0.05当量)、碘化亚铜(142.46克，747.99毫摩尔，0.1当量)、三乙胺(2.27千克，22.44摩尔，3.12升，3当量)；混合物氮气置换三次，加热至110℃反应5小时。混合物冷却至15℃，大量固体析出。混合物过滤，滤饼减压干燥得到粗品；粗品用水(20L)洗涤一次后过滤，滤饼抽干。滤饼用甲苯(12升)重结晶得到化合物5。LCMS(ESI)m/z:416.1(M+1)。To compound 3 (2700 g, 7.48 mol) in toluene (16.2 L) was added compound 4 (1.88 kg, 8.98 mol, 1.20 equiv), dichloroditriphenylphosphine palladium (262.51 g, 374 mmol, 0.05 equiv) , cuprous iodide (142.46 g, 747.99 mmol, 0.1 equiv), triethylamine (2.27 kg, 22.44 mol, 3.12 L, 3 equiv); the mixture was replaced with nitrogen three times, heated to 110° C. for 5 hours. The mixture was cooled to 15°C and a large amount of solid precipitated out. The mixture was filtered, and the filter cake was dried under reduced pressure to obtain the crude product; the crude product was washed once with water (20 L), filtered, and the filter cake was suctioned dry. The filter cake was recrystallized from toluene (12 L) to give compound 5. LCMS (ESI) m/z: 416.1 (M+1).
第4步：Step 4:
向5.0升三口烧瓶中加入DMAC(3.15升)，搅拌下加入化合物5(630克，1.50摩尔，1.0当量)，搅拌得到悬浊液，分批加入叔丁醇钾(252.98克，2.25摩尔，1.50当量)，加毕，15℃搅拌反应16小时得到澄清溶液。一边搅拌，一边将反应液缓慢加入到水(18.9升)中，析出大量固体，搅拌半小时后过滤，滤饼抽干，滤饼用水(12.6升)打浆2小时。过滤，滤饼烘干得到化合物6。DMAC (3.15 liters) was added to a 5.0-liter three-necked flask, compound 5 (630 g, 1.50 mol, 1.0 equiv) was added under stirring, stirred to obtain a suspension, and potassium tert-butoxide (252.98 g, 2.25 mol, 1.50 mol) was added in batches equiv.), the addition was complete and the reaction was stirred at 15°C for 16 hours to obtain a clear solution. While stirring, the reaction solution was slowly added to water (18.9 L), and a large amount of solid was precipitated. After stirring for half an hour, the mixture was filtered, the filter cake was drained, and the filter cake was slurried with water (12.6 L) for 2 hours. Filter and dry the filter cake to obtain compound 6.
第5步：Step 5:
向30升反应釜中加入二氧六环(6.0升)和水(600毫升)，开启搅拌，加入化合物6(600克，1.45摩尔，1当量)、化合物7(361.20克，1.74摩尔，1.20当量)、碳酸铯(942.92克，2.89摩尔，2.0当量)，混合物氮气置换三次，加入Pd(dppf)Cl 2·CH 2Cl 2(29.55克，36.17毫摩尔)，氮气置换三次，升温至105～110℃反应16小时。反应液降温至15℃。反应液分为两批处理，每批约4.0升。一边搅拌，一边将一半反应液缓慢加入到水(15升)中，大量固体析出，过滤，滤饼用乙醇(3.0L×2)洗涤。合并两次的滤饼，用二氯甲烷：甲醇＝5:1(15L)溶解，过滤，滤液加入巯基硅胶(w％＝40％,220克)室温搅拌16小时，垫硅藻土过滤，同样的方法共除钯三次。滤液旋干，残余物用乙醇(3.0升)打浆，过滤，滤饼烘干得到化合物8。LCMS(ESI)m/z:416.2(M+1)。 Dioxane (6.0 L) and water (600 mL) were added to a 30-liter reaction kettle, stirring was turned on, and compound 6 (600 g, 1.45 mol, 1 equiv.), compound 7 (361.20 g, 1.74 mol, 1.20 equiv.) were added. ), cesium carbonate (942.92 g, 2.89 mol, 2.0 equiv), the mixture was replaced with nitrogen three times, Pd(dppf)Cl 2 ·CH 2 Cl 2 (29.55 g, 36.17 mmol) was added, nitrogen replaced three times, and the temperature was raised to 105-110 The reaction was carried out at °C for 16 hours. The reaction solution was cooled to 15°C. The reaction solution was divided into two batches of about 4.0 liters each. While stirring, half of the reaction solution was slowly added to water (15 L), a large amount of solid was precipitated, filtered, and the filter cake was washed with ethanol (3.0 L×2). The two filter cakes were combined, dissolved with dichloromethane:methanol=5:1 (15L), filtered, the filtrate was added with mercapto silica gel (w%=40%, 220 g), stirred at room temperature for 16 hours, filtered through celite, and the same The method removes palladium three times in total. The filtrate was spin-dried, the residue was slurried with ethanol (3.0 L), filtered, and the filter cake was dried to obtain compound 8. LCMS (ESI) m/z: 416.2 (M+1).
第6步：Step 6:
向5.0升三口烧瓶中加入乙酸乙酯(2.35升)，开启搅拌，加入化合物8(470克，1.08摩尔)，固体不能完全溶解得到白色悬浊液，用恒压滴液漏斗滴加盐酸/乙酸乙酯(4摩尔/升，2.35升)，加毕，室温搅拌反应16小时，反应过滤得到式(I)化合物的盐酸盐。式(I)化合物的盐酸盐用乙酸乙酯(2.0升)淋洗后抽干。滤饼用1.50L水溶解，丢弃有机相，水相转入5.0升三口瓶中，搅拌下，滴加1摩尔/升氢氧化钠水溶液至PH～11，大量白色固体析出，过滤，得到式(I)化合物。 1H NMR(400MHz,DMSO-d 6)δ11.76(s,1H),8.29(s,1H),8.15(s,1H),7.95(s,1H),6.30(s,1H),3.97(s,3H),3.02(d,J＝12.0Hz,2H),2.78(ddd,J＝11.6,7.6,3.4Hz,2H),2.58(dd,J＝12.0,10.0Hz,2H),1.93(d,J＝12.2Hz,2H),1.58(qd,J＝12.2,3.8Hz,2H)；LCMS(ESI)m/z:316.2(M+1)。 In a 5.0-liter three-necked flask, add ethyl acetate (2.35 liters), turn on stirring, add compound 8 (470 grams, 1.08 moles), the solid can not be completely dissolved to obtain a white suspension, add hydrochloric acid/acetic acid dropwise with a constant pressure dropping funnel Ethyl ester (4 mol/L, 2.35 L) was added, and the reaction was stirred at room temperature for 16 hours, and the reaction was filtered to obtain the hydrochloride of the compound of formula (I). The hydrochloride salt of the compound of formula (I) was rinsed with ethyl acetate (2.0 L) and drained. The filter cake was dissolved in 1.50L of water, the organic phase was discarded, and the aqueous phase was transferred into a 5.0-liter there-necked flask, and under stirring, 1 mol/liter aqueous sodium hydroxide solution was added dropwise to pH～11, and a large amount of white solid was separated out, and filtered to obtain the formula ( I) Compounds. 1 H NMR (400MHz, DMSO-d 6 )δ11.76(s,1H), 8.29(s,1H), 8.15(s,1H), 7.95(s,1H), 6.30(s,1H), 3.97( s,3H),3.02(d,J＝12.0Hz,2H),2.78(ddd,J＝11.6,7.6,3.4Hz,2H),2.58(dd,J＝12.0,10.0Hz,2H),1.93(d , J=12.2Hz, 2H), 1.58 (qd, J=12.2, 3.8Hz, 2H); LCMS (ESI) m/z: 316.2 (M+1).
实施例2：式(I)化合物A晶型的制备Example 2: Preparation of the crystal form of compound A of formula (I)
向30升高低温夹套反应釜中加入无水甲醇(9.0L)，开启搅拌，加入式(I)化合物(300克，0.92摩尔)，开启加热，外温80℃，内温约65℃，甲醇开始回流，保温65℃，固体完全溶解得到澄清溶液，反应液趁热过滤，滤液转入反应釜，回流下开始滴加蒸馏水(9.0L)，加毕，大量白色固体析出，自然降温至室温，室温搅拌12小时。混合物过滤，滤饼转入烘箱，减压烘干得到式(I)化合物的A晶型，XRPD检测结果如图1，TGA和DSC检测结果分别如图2和图3所示。Add anhydrous methanol (9.0L) to 30 liters of low-temperature jacketed reactor, open stirring, add the compound of formula (I) (300 grams, 0.92 mol), open the heating, the external temperature is 80 ° C, and the internal temperature is about 65 ° C, Methanol began to reflux, kept at 65°C, the solid was completely dissolved to obtain a clear solution, the reaction solution was filtered while hot, the filtrate was transferred to the reaction kettle, and distilled water (9.0L) was added dropwise under reflux. , and stirred at room temperature for 12 hours. The mixture is filtered, the filter cake is transferred to an oven, and dried under reduced pressure to obtain the A crystal form of the compound of formula (I). The XRPD detection results are shown in Figure 1, and the TGA and DSC detection results are shown in Figure 2 and Figure 3, respectively.
实施例3：式(I)化合物B晶型的制备Example 3: Preparation of compound B crystal form of formula (I)
20℃条件下，式(I)化合物的A晶型(50克，0.158摩尔)在250mL的乙醇中搅拌21小时，混合物过滤，滤饼转入烘箱，减压烘干得到得到式(I)化合物的B晶型，XRPD检测结果如图4，TGA和DSC检测结果分别如图5和图6所示。Under the condition of 20°C, the A crystal form (50 g, 0.158 mol) of the compound of formula (I) was stirred in 250 mL of ethanol for 21 hours, the mixture was filtered, the filter cake was transferred to an oven, and dried under reduced pressure to obtain the compound of formula (I) The B crystal form, the XRPD detection results are shown in Figure 4, and the TGA and DSC detection results are shown in Figure 5 and Figure 6, respectively.
实施例4：式(I)化合物A晶型的稳定性实验Example 4: Stability test of the crystal form of compound A of formula (I)
实验目的：Purpose:
对式(I)化合物A晶型进行影响因素(高温、高湿及光照)和加速条件下(40℃/75％RH及30℃/65％RH)稳定性的考察，评估A晶型的固体稳定性。The influence factors (high temperature, high humidity and light) and stability under accelerated conditions (40°C/75%RH and 30°C/65%RH) were investigated for the crystal form of compound A of formula (I), and the solid of crystal form A was evaluated. stability.
实验方法：experimental method:
1)称取式(I)化合物A晶型约1.5g置于干燥洁净的玻璃瓶中，称2份，分别标记为S1-条件-时间和S2-条件-时间，再称取约20mg置于干燥洁净的玻璃瓶中，标记为S3-条件-时间，摊成薄薄一层，作为供试样品，放置于影响因素试验条件下(40℃，60℃，25℃/75％RH，25℃/92.5％RH，光照，光照对照)和加速条件下(40℃/75％RH和30℃/65％RH)，其样品为完全暴露放样。40℃，60℃，25℃/75％RH，25℃/92.5％RH在5天、10天、30天取样；光照对照在5天、10天取样分析；加速条件在1个月、2个月、3个月取样分析，分析方法如表6所示。1) Weigh about 1.5 g of the crystal form of compound A of formula (I) and place it in a dry and clean glass bottle, weigh 2 parts, and mark them as S1-condition-time and S2-condition-time respectively, and then weigh about 20 mg and place it in a dry and clean glass bottle. In a dry and clean glass bottle, marked as S3-condition-time, spread out into a thin layer, as the test sample, placed under the influence factor test conditions (40℃, 60℃, 25℃/75%RH, 25℃ /92.5%RH, light, light control) and accelerated conditions (40°C/75%RH and 30°C/65%RH), the samples were fully exposed. 40°C, 60°C, 25°C/75%RH, 25°C/92.5%RH sampling at 5 days, 10 days and 30 days; light control at 5 days and 10 days sampling and analysis; accelerated conditions at 1 month, 2 Monthly and 3-month sampling analysis, the analysis method is shown in Table 6.
表6Table 6
Figure PCTCN2021141302-appb-000011
Figure PCTCN2021141302-appb-000011
2)在考察时间点，将相应的供试样品取出，用瓶盖盖好，0天的样品从冰箱中取出，待样品恢复至室温后进行分析。标记为S1-条件-时间的供试品用于含量和有关物质检测；标记为S2-条件-时间的供试品用作备样，标记为S3-条件-时间的供试品用于XRPD检测。2) At the inspection time point, take out the corresponding test sample, cover it with a bottle cap, take out the 0-day sample from the refrigerator, and analyze it after the sample has returned to room temperature. The test sample marked S1-condition-time is used for content and related substance detection; the test sample marked S2-condition-time is used as a preparation sample, and the test sample marked S3-condition-time is used for XRPD detection .
实验结果：Experimental results:
A晶型固体稳定性实验结果如表7所示The results of the solid stability test of crystal form A are shown in Table 7.
表7晶型固体稳定性实验结果Table 7 Experiment results of crystal solid stability
Figure PCTCN2021141302-appb-000012
Figure PCTCN2021141302-appb-000012
*光照(总照度可见光＝5000±500lux，紫外90μw/cm 2，敞口)；**与可见光+紫外同时。 *Illumination (total illuminance visible light=5000±500lux, UV 90μw/cm 2 , open); **simultaneous with visible light+UV.
实验结论：式(I)化合物A晶型具有良好的稳定性。Experimental conclusion: The crystal form of compound A of formula (I) has good stability.
生物测试biological test
实验例一：体外CDK9/CyclinT1酶活性测试Experimental example 1: In vitro CDK9/CyclinT1 enzyme activity test
实验材料：Experimental Materials:
CDK9/CyclinT1激酶购自Carna，ADP-Glo检测试剂盒购自Promega，PKDTide底物及激酶反应缓冲液购自Signalchem。Nivo多标记分析仪(PerkinElmer)。CDK9/CyclinT1 kinase was purchased from Carna, ADP-Glo assay kit was purchased from Promega, PKDTide substrate and kinase reaction buffer were purchased from Signalchem. Nivo Multilabel Analyzer (PerkinElmer).
实验方法：experimental method:
使用试剂盒里的激酶缓冲液稀释酶，底物，三磷酸腺苷和抑制剂。Dilute enzyme, substrate, adenosine triphosphate and inhibitor with the kinase buffer included in the kit.
将待测化合物用排枪进行5倍稀释至第8个浓度，即从50μM稀释至0.65nM，DMSO浓度为5％，设置双复孔实验。向微孔板中加入1μL抑制剂各浓度梯度，2μL CDK9/CyclinT1酶(4ng)，2μL底物和ATP的混合物(100μM三磷酸腺苷，0.2μg/μL底物)，此时化合物终浓度梯度为10μM稀释至0.13nM。反应体系置于25℃反应120分钟。反应结束后，每孔加入5μL ADP-Glo试剂，25℃继续反应40分钟，结束反应后每孔加入10μL的激酶检测试剂，25℃反应30分钟后采用多标记分析仪读数化学发光，积分时间0.5秒。数据分析：The compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 μM to 0.65 nM, and the DMSO concentration was 5%, and a double-well experiment was set up. Add 1 μL of each concentration gradient of inhibitor, 2 μL CDK9/CyclinT1 enzyme (4ng), 2 μL mixture of substrate and ATP (100 μM adenosine triphosphate, 0.2 μg/μL substrate) to the microplate, and the final compound concentration gradient is 10 μM dilution at this time to 0.13 nM. The reaction system was placed at 25°C for 120 minutes. After the reaction, 5 μL of ADP-Glo reagent was added to each well, and the reaction was continued for 40 minutes at 25 °C. After the reaction was completed, 10 μL of kinase detection reagent was added to each well. After 30 minutes of reaction at 25 °C, the multi-label analyzer was used to read the chemiluminescence, and the integration time was 0.5 second. data analysis:
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表8提供了本发明的化合物对CDK9/CyclinT1酶学抑制活性。 Using the equation (Sample-Min)/(Max-Min)*100% to convert the raw data into inhibition rate, the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode). Table 8 provides the CDK9/CyclinT1 enzymatic inhibitory activity of the compounds of the present invention.
实验结论：Experimental results:
式(I)化合物对CDK9激酶具有良好的活性，和参照化合物BAY1251152和AZD4573活性类似。The compound of formula (I) has good activity on CDK9 kinase, similar to the activity of reference compounds BAY1251152 and AZD4573.
实验例二：体外CDK1/CyclinB1酶活性测试Experimental Example 2: In Vitro CDK1/CyclinB1 Enzyme Activity Test
实验材料：Experimental Materials:
CDK1/CyclinB1激酶检测试剂盒购自Promega。Nivo多标记分析仪(PerkinElmer)。CDK1/CyclinB1 Kinase Assay Kit was purchased from Promega. Nivo Multilabel Analyzer (PerkinElmer).
实验方法：experimental method:
使用试剂盒里的激酶缓冲液稀释酶，底物，三磷酸腺苷和抑制剂。Dilute enzyme, substrate, adenosine triphosphate and inhibitor with the kinase buffer included in the kit.
将待测化合物用排枪进行5倍稀释至第8个浓度，即从50μM稀释至0.65nM，DMSO浓度为5％，设置双复孔实验。向微孔板中加入1μL抑制剂各浓度梯度，2μL CDK1/CyclinB1酶(12.5ng)，2μL底物和ATP的混合物(25μM三磷酸腺苷，0.2μg/μL底物)，此时化合物终浓度梯度为10μM稀释至0.13nM。反应体系置于25℃反应120分钟。反应结束后，每孔加入5μL ADP-Glo试剂，25℃继续反应40分钟，结束反应后每孔加入10μL的激酶检测试剂，25℃反应30分钟后采用多标记分析仪读数化学发光，积分时间0.5秒。The compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 μM to 0.65 nM, and the DMSO concentration was 5%, and a double-well experiment was set up. Add 1 μL of each concentration gradient of inhibitor, 2 μL CDK1/CyclinB1 enzyme (12.5 ng), 2 μL mixture of substrate and ATP (25 μM adenosine triphosphate, 0.2 μg/μL substrate) to the microplate, and the final compound concentration gradient is 10 μM. Dilute to 0.13nM. The reaction system was placed at 25°C for 120 minutes. After the reaction, 5 μL of ADP-Glo reagent was added to each well, and the reaction was continued for 40 minutes at 25 °C. After the reaction was completed, 10 μL of kinase detection reagent was added to each well. After 30 minutes of reaction at 25 °C, the multi-label analyzer was used to read the chemiluminescence, and the integration time was 0.5 second.
数据分析：data analysis:
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表6提供了本发明的化合物对CDK1/CyclinB1酶学抑制活性。 Using the equation (Sample-Min)/(Max-Min)*100% to convert the raw data into inhibition rate, the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode). Table 6 provides the CDK1/CyclinB1 enzymatic inhibitory activity of the compounds of the present invention.
实验结论：Experimental results:
式(I)化合物对CDK1激酶的抑制活性不强，所以，本发明化合物表现出对CDK1的选择性好于BAY1251152和AZD4573，实验结果如表8所示。The compounds of formula (I) have weak inhibitory activity on CDK1 kinase, so the compounds of the present invention show better selectivity to CDK1 than BAY1251152 and AZD4573. The experimental results are shown in Table 8.
实验例三：体外CDK2/CyclinE1酶活性测试Experimental Example 3: In Vitro CDK2/CyclinE1 Enzyme Activity Test
实验材料：Experimental Materials:
CDK2/CyclinE1激酶检测试剂盒购自Promega。Nivo多标记分析仪(PerkinElmer)。CDK2/CyclinE1 Kinase Assay Kit was purchased from Promega. Nivo Multilabel Analyzer (PerkinElmer).
实验方法：experimental method:
使用试剂盒里的激酶缓冲液稀释酶，底物，三磷酸腺苷和抑制剂。Dilute enzyme, substrate, adenosine triphosphate and inhibitor with the kinase buffer included in the kit.
将待测化合物用排枪进行5倍稀释至第8个浓度，即从50μM稀释至0.65nM，DMSO浓度为5％，设置双复孔实验。向微孔板中加入1μL抑制剂各浓度梯度，2μL CDK2/CyclinE1酶(2ng)，2μL底物和ATP的混合物(150μM三磷酸腺苷，0.1μg/μL底物)，此时化合物终浓度梯度为10μM稀释至0.13nM。反应体系置于25℃反应60分钟。反应结束后，每孔加入5μL ADP-Glo试剂，25℃继续反应40分钟，结束反应后每孔加入10μL的激酶检测试剂，25℃反应30分钟后采用多标记分析仪读数化学发光，积分时间0.5秒。The compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 μM to 0.65 nM, and the DMSO concentration was 5%, and a double-well experiment was set up. Add 1 μL of each concentration gradient of inhibitor, 2 μL CDK2/CyclinE1 enzyme (2ng), 2 μL mixture of substrate and ATP (150 μM adenosine triphosphate, 0.1 μg/μL substrate) to the microplate, and the final compound concentration gradient is 10 μM dilution at this time to 0.13 nM. The reaction system was placed at 25°C for 60 minutes. After the reaction, 5 μL of ADP-Glo reagent was added to each well, and the reaction was continued for 40 minutes at 25 °C. After the reaction was completed, 10 μL of kinase detection reagent was added to each well. After 30 minutes of reaction at 25 °C, the multi-label analyzer was used to read the chemiluminescence, and the integration time was 0.5 second.
数据分析：data analysis:
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表8提供了本发明的化合物对CDK2/CyclinE1酶学抑制活性。 Using the equation (Sample-Min)/(Max-Min)*100% to convert the raw data into inhibition rate, the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode). Table 8 provides the CDK2/CyclinE1 enzymatic inhibitory activity of the compounds of the present invention.
实验结论：Experimental results:
式(I)化合物对CDK2激酶的抑制活性不强，所以，本发明化合物表现出对CDK2的选择性好于BAY1251152和AZD4573，实验结果如表8所示。The compound of formula (I) does not have strong inhibitory activity on CDK2 kinase, so the compound of the present invention exhibits better selectivity to CDK2 than BAY1251152 and AZD4573. The experimental results are shown in Table 8.
实验例四：体外细胞活性测试Experimental Example 4: In Vitro Cell Viability Test
实验材料：Experimental Materials:
IMDM培养基，胎牛血清，盘尼西林/链霉素抗生素购自Promega(Madison,WI)。MV-4-11细胞系购自中国科学院细胞库。Nivo多标记分析仪(PerkinElmer)。IMDM medium, fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Promega (Madison, WI). The MV-4-11 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo Multilabel Analyzer (PerkinElmer).
实验方法：experimental method:
将MV-4-11细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含6000个MV-4-11细胞。细胞板置于二氧化碳培养箱中过夜培养。MV-4-11 cells were seeded in a white 96-well plate, 80 μL of cell suspension per well, which contained 6000 MV-4-11 cells. Cell plates were incubated overnight in a carbon dioxide incubator.
将待测化合物用排枪进行5倍稀释至第8个浓度，即从2mM稀释至26nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。化合物终浓度为10μM至0.13nM。细胞板置于二氧化碳培养箱中培养3天。The compounds to be tested were diluted 5-fold to the 8th concentration, that is, from 2 mM to 26 nM, and a double-well experiment was set up. Add 78 μL of medium to the middle plate, and then transfer 2 μL of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 μL of each well to the cell plate. Final compound concentrations ranged from 10 μM to 0.13 nM. The cell plates were placed in a carbon dioxide incubator for 3 days.
向细胞板中加入每孔25μL的Promega CellTiter-Glo试剂，室温孵育10分钟使发光信号稳定。采用PerkinElmer Nivo多标记分析仪读数。Add 25 μL per well of Promega CellTiter-Glo reagent to the cell plate and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Readings were performed on a PerkinElmer Nivo multi-label analyzer.
数据分析：data analysis:
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表8提供了本发明的化合物对MV-4-11细胞增殖的抑制活性。 Using the equation (Sample-Min)/(Max-Min)*100% to convert the raw data into inhibition rate, the IC 50 value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode). Table 8 provides the inhibitory activity of the compounds of the present invention on the proliferation of MV-4-11 cells.
实验结论：Experimental results:
式(I)化合物对MV4-11具有良好的细胞抗增殖活性。实验结果如表8所示：Compounds of formula (I) have good cellular antiproliferative activity against MV4-11. The experimental results are shown in Table 8:
表8激酶及细胞活性结果Table 8 Kinase and cell activity results
Figure PCTCN2021141302-appb-000013
Figure PCTCN2021141302-appb-000013
实验例五：体内药效研究Experimental example 5: In vivo efficacy study
在皮下植入MV4-11急性髓系白血病患者来源的基于人源肿瘤细胞系的异种移植(CDX)BALB/c裸小鼠上进行体内药效实验。In vivo efficacy experiments were carried out in BALB/c nude mice implanted subcutaneously on MV4-11 acute myeloid leukemia patient-derived xenograft (CDX) BALB/c nude mice.
实验操作：Experimental operation:
BALB/c裸鼠，雌性，6-8周，体重约18-22克，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(3只小鼠每笼)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标准认证的商业实验室饮食。共有36只购于上海灵畅生物科技有限公司(Shanghai Lingchang biological science and technology Co.,LTD.)的小鼠用于研究。每只小鼠在右胁腹皮下植入肿瘤细胞(10×10 6在0.2毫升磷酸盐缓冲液中)，用于肿瘤的生长。当平均肿瘤体积达到约121立方毫米时开始给药。将试验化合物每周注射给药，给药剂量为10毫克/公斤。肿瘤体积每周2次用二维卡尺测量，体积以立方毫米计量，通过以下公式计算：V＝0.5a×b 2，其中a和b分别是肿瘤的长径和短径。抗肿瘤药效是通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定。 BALB/c nude mice, female, 6-8 weeks, weighing approximately 18-22 grams, were kept in a special pathogen-free environment in a single ventilated cage (3 mice per cage). All cages, bedding and water were sterilized before use. All animals had free access to standard certified commercial laboratory diets. A total of 36 mice purchased from Shanghai Lingchang biological science and technology Co., LTD. were used for the study. Tumor cells (10×10 6 in 0.2 ml phosphate buffered saline) were implanted subcutaneously in the right flank of each mouse for tumor growth. Dosing was initiated when the mean tumor volume reached approximately 121 cubic millimeters. The test compound was administered weekly by injection at a dose of 10 mg/kg. Tumor volume was measured twice a week with a two-dimensional caliper, and volume was measured in cubic millimeters and calculated by the following formula: V=0.5a×b 2 , where a and b are the long and short diameters of the tumor, respectively. Antitumor efficacy is determined by dividing the mean tumor increase volume in animals treated with the compound by the mean tumor increase volume in untreated animals.
实验结论：Experimental results:
在MV4-11急性髓系白血病CDX体内药效模型中，式(I)化合物展现良好的药效和安全性。体内药效结果如表9所示：In the MV4-11 acute myeloid leukemia CDX in vivo pharmacodynamic model, the compound of formula (I) exhibited good efficacy and safety. The in vivo efficacy results are shown in Table 9:
表9体内药效结果Table 9 In vivo efficacy results
Figure PCTCN2021141302-appb-000014
Figure PCTCN2021141302-appb-000014

Claims (23)

  1. 式(Ⅰ)化合物的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22±0.20°、17.16±0.20°和22.34±0.20°；Form A of the compound of formula (I), its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 17.16±0.20° and 22.34±0.20°;
    Figure PCTCN2021141302-appb-100001
    Figure PCTCN2021141302-appb-100001
  2. 根据权利要求1所述的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22±0.20°、15.24±0.20°、15.80±0.20°、17.16±0.20°、20.70±0.20°、22.34±0.20°、24.46±0.20°和31.74±0.20°。The crystal form A according to claim 1, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 15.24±0.20°, 15.80±0.20°, 17.16±0.20°, 20.70±0.20 °, 22.34±0.20°, 24.46±0.20° and 31.74±0.20°.
  3. 根据权利要求2所述的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22±0.20°、8.76±0.20°、15.24±0.20°、15.80±0.20°、17.16±0.20°、19.66±0.20°、20.70±0.20°、22.34±0.20°、24.46±0.20°、25.84±0.20°、29.76±0.20°和31.74±0.20°。The crystal form A according to claim 2, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 8.76±0.20°, 15.24±0.20°, 15.80±0.20°, 17.16±0.20 °, 19.66±0.20°, 20.70±0.20°, 22.34±0.20°, 24.46±0.20°, 25.84±0.20°, 29.76±0.20° and 31.74±0.20°.
  4. 根据权利要求3所述的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.22°、8.76°、9.84°、12.24°、15.24°、15.80°、16.22°、17.16°、17.52°、18.40°、19.26°、19.66°、20.70°、21.46°、22.34°、23.64°、24.46°、25.84°、27.10°、27.62°、28.02°、29.26°、29.76°、30.88°、31.74°、33.38°、37.10°和37.68°。The crystal form A according to claim 3, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.22°, 8.76°, 9.84°, 12.24°, 15.24°, 15.80°, 16.22°, 17.16° , 17.52°, 18.40°, 19.26°, 19.66°, 20.70°, 21.46°, 22.34°, 23.64°, 24.46°, 25.84°, 27.10°, 27.62°, 28.02°, 29.26°, 29.76°, 30.88°, 31.74 °, 33.38°, 37.10° and 37.68°.
  5. 根据权利要求4所述的A晶型，其XRPD图谱基本如图1所示。The crystal form A according to claim 4, its XRPD pattern is basically as shown in Figure 1.
  6. 根据权利要求1～5所述的A晶型，其差示扫描量热曲线在77.71±3℃和236.85±3℃分别有一个吸热峰的起始点。The crystal form A according to claims 1 to 5, the differential scanning calorimetry curve has an endothermic peak starting point at 77.71±3°C and 236.85±3°C, respectively.
  7. 根据权利要求6所述的A晶型，其DSC图谱如图2所示。Form A according to claim 6, its DSC spectrum is shown in Figure 2.
  8. 根据权利要求1～5所述的A晶型，其热重分析曲线在200±3℃时失重达3.420％。The crystal form A according to claims 1 to 5 has a thermogravimetric analysis curve of 3.420% weight loss at 200±3°C.
  9. 根据权利要求8所述的A晶型，其TGA图谱如图3所示。Form A according to claim 8, its TGA spectrum is shown in Figure 3.
  10. 式(Ⅰ)化合物的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：19.72±0.20°、21.52±0.20°和23.20±0.20°；Form B of the compound of formula (I), its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 19.72±0.20°, 21.52±0.20° and 23.20±0.20°;
    Figure PCTCN2021141302-appb-100002
    Figure PCTCN2021141302-appb-100002
  11. 根据权利要求10所述的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：10.74±0.20°、16.22±0.20°、19.72±0.20°、20.58±0.20°、21.52±0.20°、22.30±0.20°、23.20±0.20°和28.04±0.20°。The crystal form B according to claim 10, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.74±0.20°, 16.22±0.20°, 19.72±0.20°, 20.58±0.20°, 21.52±0.20 °, 22.30±0.20°, 23.20±0.20° and 28.04±0.20°.
  12. 根据权利要求11所述的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.92±0.20°、10.74±0.20°、16.22±0.20°、17.66±0.20°、19.72±0.20°、20.58±0.20°、21.52±0.20°、22.30±0.20°、23.20±0.20°、23.88±0.20°、26.54±0.20°、27.48±0.20°和28.04±0.20°。The crystal form B according to claim 11, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.92±0.20°, 10.74±0.20°, 16.22±0.20°, 17.66±0.20°, 19.72±0.20 °, 20.58±0.20°, 21.52±0.20°, 22.30±0.20°, 23.20±0.20°, 23.88±0.20°, 26.54±0.20°, 27.48±0.20° and 28.04±0.20°.
  13. 根据权利要求12所述的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.92°、10.74°、11.42°、13.52°、13.76°、15.86°、16.22°、16.52°、17.66°、17.90°、18.22°、18.92°、19.72°、20.58°、21.52°、22.30°、23.20°、23.88°、25.32°、26.12°、26.54°、27.14°、27.48°、27.72°、28.04°、28.52°、28.96°、29.20°、29.74°、30.24°、30.58°、31.56°、32.54°、32.82°、33.38°、34.36°、34.75°、35.44°、36.00°、36.56°、37.08°、37.96°、38.74°和39.50°。The crystal form B according to claim 12, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.92°, 10.74°, 11.42°, 13.52°, 13.76°, 15.86°, 16.22°, 16.52° , 17.66°, 17.90°, 18.22°, 18.92°, 19.72°, 20.58°, 21.52°, 22.30°, 23.20°, 23.88°, 25.32°, 26.12°, 26.54°, 27.14°, 27.48°, 27.72°, 28.04 degrees 37.96°, 38.74° and 39.50°.
  14. 根据权利要求13所述的B晶型，其XRPD图谱基本如图4所示。The crystal form B according to claim 13, its XRPD pattern is basically as shown in FIG. 4 .
  15. 根据权利要求10～14所述的B晶型，其差示扫描量热曲线在257.81±3℃有一个吸热峰的起始点。The crystal form B according to claims 10-14, wherein the differential scanning calorimetry curve has an endothermic peak starting point at 257.81±3°C.
  16. 根据权利要求15所述的B晶型，其DSC图谱如图5所示。The crystal form B according to claim 15, its DSC spectrum is shown in Figure 5.
  17. 根据权利要求10～14所述的B晶型，其热重分析曲线在200±3℃时失重达0.326％。The crystal form B according to claims 10-14 has a weight loss of 0.326% in the thermogravimetric analysis curve at 200±3°C.
  18. 根据权利要求17所述的B晶型，其TGA图谱如图6所示。The crystal form B according to claim 17, its TGA spectrum is shown in Figure 6.
  19. 根据权利要求1-9任意一项所述的式(Ⅰ)化合物A晶型的制备方法，包含如下步骤：The preparation method of compound A crystal form of formula (I) according to any one of claims 1-9, comprising the following steps:
    1)式(I)化合物加入到无水甲醇中回流；1) the compound of formula (I) is added to anhydrous methanol and refluxed;
    2)式(I)化合物完全溶解，趁热过滤；2) the compound of formula (I) is completely dissolved, filtered while hot;
    3)滤液在回流状态下滴加蒸馏水，析出白色固体，自然降温至室温，室温搅拌；3) The filtrate is dripped with distilled water under reflux state to separate out a white solid, which is naturally cooled to room temperature and stirred at room temperature;
    4)混合物过滤，滤饼减压烘干。4) The mixture is filtered, and the filter cake is dried under reduced pressure.
  20. 根据权利要求19所述的制备方法，其中，所述回流温度为65℃-80℃，优选为65℃。The preparation method according to claim 19, wherein the reflux temperature is 65°C-80°C, preferably 65°C.
  21. 根据权利要求19所述的制备方法，其中，搅拌时间为10-12小时，优选为12小时。The preparation method according to claim 19, wherein the stirring time is 10-12 hours, preferably 12 hours.
  22. 根据权利要求10-17任意一项所述的式(Ⅰ)化合物B晶型的制备方法，包含如下步骤：The preparation method of compound B crystal form of formula (I) according to any one of claims 10-17, comprising the following steps:
    1)权利要求1-9任意一项所述的式(Ⅰ)化合物A晶型在乙醇中搅拌，过滤、滤饼减压烘干。1) The crystal form of compound A of formula (I) described in any one of claims 1-9 is stirred in ethanol, filtered, and the filter cake is dried under reduced pressure.
  23. 权利要求1～9任意一项所述的A晶型或权利要求10～18任意一项所述的B晶型在制备CDK9抑制剂药物中的应用。Use of the crystal form A of any one of claims 1 to 9 or the crystal form B of any one of claims 10 to 18 in the preparation of a CDK9 inhibitor drug.
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