WO2020007329A1 - Hdac6选择性抑制剂的晶型及其应用 - Google Patents

Hdac6选择性抑制剂的晶型及其应用 Download PDF

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WO2020007329A1
WO2020007329A1 PCT/CN2019/094602 CN2019094602W WO2020007329A1 WO 2020007329 A1 WO2020007329 A1 WO 2020007329A1 CN 2019094602 W CN2019094602 W CN 2019094602W WO 2020007329 A1 WO2020007329 A1 WO 2020007329A1
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compound
crystal form
present
formula
pattern
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PCT/CN2019/094602
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English (en)
French (fr)
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郭强
韦昌青
肖瑶
樊莉莉
钱文远
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基石药业（苏州）有限公司
拓石药业（上海）有限公司
基石药业
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Priority to EP19829797.0A priority Critical patent/EP3822267B1/en
Priority to US17/256,670 priority patent/US11345682B2/en
Priority to JP2020573256A priority patent/JP7348214B2/ja
Priority to ES19829797T priority patent/ES2961765T3/es
Priority to CN201980044805.7A priority patent/CN112703190B/zh
Publication of WO2020007329A1 publication Critical patent/WO2020007329A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

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  • the present invention relates to the crystal form A of the compound represented by formula (I) and its application in the preparation of a medicament for treating HDAC6 related diseases.
  • HDAC inhibitors are widely used in a variety of cancers, and can be combined with a variety of drugs to enhance the therapeutic effect of the drug, which is a well-proven antitumor target.
  • HDAC histone deacetylase
  • HAT histone acetyl trans-ferase
  • HDACi Inhibitors
  • HDACi inhibitors can regulate the expression of apoptosis and differentiation-related proteins by inducing histone acetylation, and induce apoptosis and differentiation to become a new class of antitumor drugs.
  • HDAC is also involved in the regulation of many metabolic diseases, such as Alzheimer's disease, Parkinson's disease and other diseases.
  • HDACi inhibitors have shown good results in animal and human experiments.
  • HDAC6 is the only deacetylase subtype in the cytoplasm, while the other 17 HDACs are present in the nucleus.
  • HDAC6 does not directly catalyze histones, but uses tubulin and heat shock protein (Hsp90) as substrates, through which they regulate cell trafficking, adhesion and movement (ie, do not genetically regulate). Therefore, it is believed that it will have fewer effects on the gene-related physiological functions and thus less side effects.
  • Hsp90 tubulin and heat shock protein
  • Current clinical trial results have confirmed that HDAC6 selective inhibitors are safe and effective (POC).
  • the present invention provides the crystal form A of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 11.53 ⁇ 0.2 °, 18.46 ⁇ 0.2 °, 23.60 ⁇ 0.2 °.
  • the X-ray powder diffraction pattern of the above-mentioned Form A has characteristic diffraction peaks at the following 2 ⁇ angles: 11.53 ⁇ 0.2 °, 13.21 ⁇ 0.2 °, 16.33 ⁇ 0.2 °, 17.42 ⁇ 0.2 °, 18.46 ⁇ 0.2 °, 21.43 ⁇ 0.2 °, 22.57 ⁇ 0.2 °, 23.60 ⁇ 0.2 °.
  • the X-ray powder diffraction pattern of the above-mentioned Form A has characteristic diffraction peaks at the following 2 ⁇ angles: 8.898 °, 11.118 °, 11.528 °, 12.652 °, 13.206 °, 13.761 °, 16.325 °, 17.415 ° , 18.067 °, 18.464 °, 19.289 °, 20.697 °, 21.427 °, 22.572 °, 23.226 °, 23.599 °, 25.674 °, 26.619 °, 27.611 °, 29.090 °, 29.879 °, 31.852 °, 33.878 °, 35.252 °, and 36.122 °.
  • the XRPD pattern of the above-mentioned Form A is shown in FIG. 1.
  • the differential scanning calorimetry curve of the above-mentioned Form A has a starting point of an endothermic peak at 135.55 ° C.
  • the DSC spectrum of the above-mentioned Form A is shown in FIG. 2.
  • thermogravimetric analysis curve of the above-mentioned Form A is 0.2115% at 135.40 ° C ⁇ 3 ° C.
  • the TGA spectrum of the above-mentioned Form A is shown in FIG. 3.
  • the invention also provides the application of the above-mentioned Form A to the preparation of a medicament for treating HDAC6 related disorders.
  • the crystal form A of the compound of the formula (I) of the present invention is stable, has little effect on light, heat and humidity, and has good in vivo drug efficacy, and has broad prospects for drug preparation.
  • 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 combining them with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalent alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations: EtOH stands for ethanol; MeOH stands for methanol; TFA stands for trifluoroacetic acid; TsOH stands for p-toluenesulfonic acid; mp stands for melting point; EtSO 3 H stands for ethanesulfonic acid; MeSO 3 H stands for methanesulfonic acid; THF stands for tetrahydrofuran; EtOAc stands for ethyl acetate.
  • XRPD X-ray powder diffraction
  • Test method about 10 ⁇ 20mg sample is used for XRPD detection.
  • Light tube voltage 40kV
  • light tube current 40mA
  • Anti-scattering slit 7.10mm
  • DSC Differential thermal analysis
  • Test method Take a sample ( ⁇ 1mg) in a DSC aluminum pan for testing. Under 50mL / min N 2 condition, heat the sample from 30 °C (room temperature) to 300 °C (or 350) ° C).
  • Thermogravimetric (Analyzer, TGA) method of the present invention is thermogravimetric (Analyzer, TGA) method of the present invention
  • Test method Take a sample (2 ⁇ 5mg) and place it in a TGA platinum pot for testing. Under the condition of 25mL / min N 2 , heat the sample from room temperature to 350 °C or lose weight by 20% at a heating rate of 10 °C / min.
  • FIG. 1 is an XRPD spectrum of Cu-K ⁇ radiation of crystal form A of compound (I);
  • FIG. 2 is a DSC spectrum of a crystal form of the compound of formula (I);
  • FIG. 3 is a TGA spectrum of the crystal form A of the compound (I).
  • step 1
  • the resulting brown-black viscous paste was quenched with a 2M aqueous sodium hydroxide solution at 0 ° C under a nitrogen purge until it became a yellow-brown solid.
  • the reaction and quenching were performed ten times in parallel and combined.
  • a mixed solution of dichloromethane and methanol (volume ratio 10: 1, 20 L) was added to the yellow-brown solid obtained above, and a 2M sodium hydroxide aqueous solution was added dropwise thereto with constant stirring until the suspension became gelatinous and stopped.
  • anhydrous sodium sulfate (5 kg) was added to the system and stirring was continued.
  • the solid-liquid mixture was filtered through diatomaceous earth, and the filtrate was collected.
  • the filtrate was repeatedly stirred with a mixed solution of dichloromethane and methanol (volume ratio 10: 1, 5L ⁇ 4), filtered, and the combined filtrate was concentrated to dryness under reduced pressure.
  • the crude product was purified by silica gel column chromatography (n-heptane: ethyl acetate: dichloromethane ratio of 3: 1: 1) to obtain compound 6.
  • the precipitated white solid was filtered and rinsed with water (2L ⁇ 3).
  • the obtained solid was dispersed in ethyl acetate (21L) and water (10.5L), and under constant stirring, the pH was adjusted to 2-3 with concentrated hydrochloric acid.
  • the white solid was dissolved in the organic phase, and the organic phase was separated.
  • the aqueous phase was ethyl acetate.
  • Extraction with an ester (5L ⁇ 2) the combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the compound of formula (I).
  • the compound of formula (I) (0.21 kg) was stirred with EtOAc (2.1 L) and water (1 L). Concentrated hydrochloric acid (8 mL) was added to dissolve the solid in the organic phase. The organic phase was separated and washed with brine (0.5 L, After washing, the brine has a pH of about 6), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a foamy white solid. Preheated ethyl acetate (2.1L) was added to the solid to keep the internal temperature at 45 ° C, and n-heptane (0.588L) was added dropwise. After the dropwise addition was completed, the internal temperature was slowly reduced to 6-13 ° C and continued. Stir for 12 hours. Filter and collect the cake to get the first recrystallized product. This process was performed 10 times in parallel, and a total of 1.5 kg of a white solid product was obtained.
  • the first recrystallized product (0.25 kg) was stirred with EtOAc (2.5 L) and water (1.25 L). Concentrated hydrochloric acid (about 80 mL) was added to dissolve the solid in the organic phase, the organic phase was separated, and water (0.6 L ⁇ 2), and washed with brine (0.6 L, brine pH is about 6 after washing), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a foamy white solid. Preheated ethyl acetate (2.5L) was added to the solid to keep the internal temperature at 45 ° C, and n-heptane (0.7L) was added dropwise.
  • the second recrystallization product (1.05 kg) was stirred with EtOAc (0.7 L) and water (1.4 L) at 5 ° C for 24 hours. After filtration, the filter cake was rinsed with water (105 mL ⁇ 2), dried, and dried at 30 ° C. for 24 hours in a vacuum drying box with phosphorus pentoxide on the bottom to obtain a white solid.
  • XRPD detects its crystal form state, and thus obtains the crystal form of compound A of formula (I).
  • Example 3 Solid stability test of the crystalline form of compound A of formula (I)
  • the crystal form of the compound (I) has good stability under high temperature, high humidity, high temperature and high humidity, and strong light conditions.
  • mice Female, 6-8 weeks, weighing about 17-20 grams, kept the mice in a special pathogen-free environment and in a single ventilated cage (4 mice per cage). All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory diets. A total of 64 mice purchased from Beijing Weitonglihua were used for the study. 0.2 mL of 5 ⁇ 10 6 MM.1S cells were subcutaneously inoculated into the right back of each mouse for tumor growth. The experiment was started when the average tumor volume reached approximately 100-150 cubic millimeters. experimental method:
  • Antitumor efficacy is determined by dividing the average tumor increase volume of animals treated with a compound by the average tumor increase volume of untreated animals. Experimental results: see Table 3.

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Abstract

本发明公开了式(Ⅰ)所示化合物的A晶型，及其在制备治疗HDAC6相关疾病的药物的应用。

Description

HDAC6选择性抑制剂的晶型及其应用
本申请主张如下优先权：
CN201810726102X，申请日：2018.07.04。
技术领域
本发明涉及式(Ⅰ)所示化合物的A晶型，及其在制备治疗HDAC6相关疾病的药物的应用。
背景技术
WHO专家预测，2020年全球人口将达80亿，癌症发病将达到2000万人，死亡将达到1200万人。癌症将成为新世纪人类的第一杀手，对人类生存构成最严重的威胁。处于工业化进程中的中国，居美国之后成为世界第二癌症高发国，其癌症发病率及死亡率呈现明显上升趋势。城市癌症占全死因的第一位，农村癌症占全死因的第二位。随着我国癌症发病率和死亡率的快速升高，全国每年用于癌症的医疗费用已超过1,500亿元人民币。
HDAC抑制剂广泛应用于多种癌症，并能够与多种药物联合而增强该药物的治疗效果，是一个被充分证实的抗肿瘤靶点。在细胞核内组蛋白去乙酰化酶(histone deacetylase,HDAC)和组蛋白乙酰化转移酶(histone acetyl trans-ferase,HAT)共同调控基因的转录。在癌细胞中，HDAC的过度表达导致去乙酰化作用的增强，从而增加DNA与组蛋白之间的引力,使核小体变得十分紧密，不利于肿瘤抑制基因的表达。抑制剂(HDACi)则可通过提高组蛋白乙酰化，从而调控细胞凋亡及分化相关蛋白的表达，诱导细胞凋亡及分化，成为一类新的抗肿瘤药物。不仅如此，HDAC还参与了很多代谢性疾病的调控，如阿尔兹海默症(Alzheimer),帕金森症(Parkinson)等疾病，HDACi抑制剂在动物和人体试验中都展示了良好的效果。
在总共有18种去乙酰化酶亚型中，HDAC6是唯一在细胞质中的去乙酰化酶亚型，而其他17种HDAC都是存在于细胞核中。HDAC6不直接催化组蛋白，而是以微管蛋白(tubulin)和热休克蛋白(Hsp90)作为底物,通过他们调节细胞贩运，粘附和运动(即不基因调控)。因此，相信它将对基因相关的生理功能产生更少的作用，从而副作用更小。目前的临床实验结果已经证实HDAC6选择性抑制剂安全有效(POC)。第一个HDAC6选择性抑制剂ACY-1215(Acetylon)的临床研究证明，选择性HDAC6抑制剂有更好的安全性，因此有更好的商业前景。
Figure PCTCN2019094602-appb-000001
发明内容
本发明提供了式(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： 11.53±0.2°、18.46±0.2°、23.60±0.2°。
Figure PCTCN2019094602-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：11.53±0.2°、13.21±0.2°、16.33±0.2°、17.42±0.2°、18.46±0.2°、21.43±0.2°、22.57±0.2°、23.60±0.2°。
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：8.898°、11.118°、11.528°、12.652°、13.206°、13.761°、16.325°、17.415°、18.067°、18.464°、19.289°、20.697°、21.427°、22.572°、23.226°、23.599°、25.674°、26.619°、27.611°、29.090°、29.879°、31.852°、33.878°、35.252°和36.122°。
本发明的一些方案中，上述A晶型的XRPD图谱如图1所示。
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：
表1.A晶型的XRPD图谱解析数据
Figure PCTCN2019094602-appb-000003
在本发明的一些方案中，上述A晶型的差示扫描量热曲线在135.55℃处具有吸热峰的起始点。
在本发明的一些方案中，上述A晶型的DSC图谱如图2所示。
在本发明的一些方案中，上述A晶型的热重分析曲线在135.40℃±3℃时失重达0.2115％。
在本发明的一些方案中，上述A晶型的TGA图谱如图3所示。
本发明还提供了上述A晶型在制备治疗HDAC6相关病症的药物上的应用。
技术效果
本发明式(Ⅰ)化合物的A晶型稳定、受光热湿度影响小且具有良好的体内给药药效，成药前景广阔。定义和说明
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。
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。
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。
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。
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。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：EtOH代表乙醇；MeOH代表甲醇；TFA代表三氟乙酸；TsOH代表对甲苯磺酸；mp代表熔点；EtSO 3H代表乙磺酸；MeSO 3H代表甲磺酸；THF代表四氢呋喃；EtOAc代表乙酸乙酯。
本发明X-射线粉末衍射(X-ray powder diffractometer,XRPD)方法
仪器型号：布鲁克D8 advance X-射线衍射仪
测试方法：大约10～20mg样品用于XRPD检测。
详细的XRPD参数如下：
光管：Cu,kα,
Figure PCTCN2019094602-appb-000004
光管电压：40kV，光管电流：40mA
发散狭缝：0.60mm
探测器狭缝：10.50mm
防散射狭缝：7.10mm
扫描范围：4-40deg
步径：0.02deg
步长：0.12秒
样品盘转速：15rpm
本发明差热分析(Differential Scanning Calorimeter,DSC)方法
仪器型号：TA Q2000差示扫描量热仪
测试方法：取样品(～1mg)置于DSC铝锅内进行测试，在50mL/min N 2条件下，以10℃/min的升温速率，加热样品从30℃(室温)到300℃(或350℃)。
本发明热重分析(Thermal Gravimetric Analyzer,TGA)方法
仪器型号：TA Q5000IR热重分析仪
测试方法：取样品(2～5mg)置于TGA铂金锅内进行测试，在25mL/min N 2条件下，以10℃/min的升温速率，加热样品从室温到350℃或失重20％。
附图说明
图1为式(I)化合物A晶型的Cu-Kα辐射的XRPD谱图；
图2为式(I)化合物A晶型的DSC谱图；
图3为式(I)化合物A晶型的TGA谱图。
具体实施方式
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。
实施例1：式(I)化合物的制备
Figure PCTCN2019094602-appb-000005
步骤1：
在N 2保护，将化合物1(1.712kg,8.90mol，1.0当量)，甲苯(17L)依次加入反应釜，干冰乙醇浴调节釜内温度至-70℃。将2.5M正丁基锂(3.92L,9.79mol，1.1当量)的正庚烷溶液用蠕动泵滴加入反 应釜内，控制滴加速度使釜内温度不高于-70℃。滴加完成后继续在该温度下搅拌1小时。将配制好的化合物2(1.785kg,8.90mol，1.0当量)的甲苯(1.8L)溶液用蠕动泵加入到上述体系中，控制加入速度使釜内温度不高于-50℃。然后撤去干冰乙醇浴使反应体系升至15～20℃并继续搅拌14小时。HPLC监测反应完成后，依次加入饱和氯化铵水溶液(2L)及水(10L)，分离有机相，水相用乙酸乙酯(3L×2)萃取。合并的有机相用无水硫酸钠干燥，过滤，滤液减压浓缩干。所得棕黑色油状物用硅胶过滤(正庚烷：乙酸乙酯比例为5:1)，滤液减压浓缩干即得到化合物3。 1H NMR(400MHz,CDCl 3):δ8.26-8.35(m,1H),7.50-7.60(m,1H),7.20-7.30(m,2H),7.13(d,J＝8.0Hz,1H),6.80-6.95(m,2H),3.94(t,J＝6.8Hz,2H),2.36-2.55(m,2H),1.80-1.93(m,2H)；MS ESI计算值C 15H 13ClFNO[M+H]+278,实测值278。
步骤2：
将化合物3(0.6kg,2.16mol，1.0当量)，醋酸钯(0.022kg,0.098mol，0.05当量)，三乙胺(0.6L,4.32mol，2.0当量)，1,3-双二苯基膦丙烷(0.089kg,0.216mol，0.1当量)及甲醇(6L)依次加入10L高压反应釜反应釜，所得混合物用氩气置换三次后，通入2MPa一氧化碳气体并升温至100-105℃搅拌24小时。该反应平行进行四次。合并后进行后处理。合并的反应液过滤，滤液减压浓缩干。然后依次加入乙酸乙酯(10L)及水(5L)，搅拌静置分离有机相，水相用乙酸乙酯(2L×2)萃取。合并的有机相用无水硫酸钠干燥，过滤，滤液减压浓缩干。然后经硅胶过滤(二氯甲烷：乙酸乙酯的比例为5：1)，滤液减压浓缩干得粗品2.15kg。将该固体溶解于乙酸乙酯(21L)并加入活性炭(430g)回流12小时。趁热经硅藻土过滤，滤饼用热的乙酸乙酯(2L×2)润洗。合并的滤液减压浓缩干即得化合物4。 1H NMR(400MHz,CDCl3)δ:8.79(d,J＝1.6Hz,1H),8.05(d,J＝8.4Hz,1H),7.85(dd,J＝8.0,1.6Hz,1H),7.30-7.45(m,2H),6.90-7.05(m,2H),4.00-4.10(m,2H),3.98(s,3H),2.43-2.70(m,2H),1.85-2.05(m,2H)；MS ESI计算值C 17H 16FNO 3[M+H]+302,实测值302。
步骤3：
在氮气吹扫下，室温下向装有化合物4(0.2kg,0.664mol，1.0当量)及化合物5(0.134kg,0.664mol，1.0当量)的四氢呋喃(2L)溶液的5L三口烧瓶中滴加2M三甲基铝甲苯溶液(0.66L,1.32mol，2.0当量)，控制滴加速度使内温不超过50℃，滴加完成后将体系在70℃下加热搅拌20分钟。反应完成后，反应液减压浓缩干。所得棕黑色粘稠膏状物在0℃，氮气吹扫下用2M氢氧化钠水溶液淬灭，直至变为黄褐色固体。该反应及淬灭过程平行进行十次后合并。向上述所得黄褐色固体中加入二氯甲烷及甲醇的混合溶液(体积比10:1,20L)，不断搅拌下向其中滴加2M氢氧化钠水溶液直至悬浊液变为凝胶果冻状停止。此时向体系中加入无水硫酸钠(5kg)继续搅拌。上述固液混合物经硅藻土过滤，收集滤液，滤液反复用二氯甲烷及甲醇的混合溶液(体积比10:1,5L×4)搅拌，过滤，合并的滤液减压浓缩干。粗品经硅胶柱层析(正庚烷：乙酸乙酯：二氯甲烷的比例为3：1：1)纯化即得化合物6。 1H NMR(400MHz,CDCl 3)δ:8.59(s,1H),8.38(brs,1H),8.15(d,J＝8.0Hz,1H),8.00(d,J＝8.4Hz,2H),8.70-8.90(m,1H),7.37-7.45(m,4H),6.94-7.05 (m,2H),4.71(d,J＝6.4Hz,2H),4.07(t,J＝7.2Hz,2H),3.91(s,3H)，2.45-2.65(m,2H),1.90-2.05(m,2H)；MS ESI计算值C 25H 23FN 2O 4[M+H]+435,实测值435。
步骤4：
在50L反应釜中，0℃下向化合物6(2.1kg,4.83mol，1.0当量)的二氯甲烷(4L)及甲醇(20L)溶液中加入50％羟胺水溶液(8L)，当内温为0℃时开始滴加2M氢氧化钠水溶液(3L)并一直保持内温为该温度。滴加完成后缓慢升至6～13℃并继续搅拌12小时。反应完成后，减压除去大部分有机溶剂。加入冰水冷却内温至0℃，不断搅拌下加入浓盐酸调pH为7～8，过滤析出的白色固体，并用水(2L×3)润洗。所得固体分散于乙酸乙酯(21L)及水(10.5L)中，不断搅拌下，用浓盐酸调pH为2～3，此时白色固体溶解于有机相，分离有机相，水相用乙酸乙酯(5L×2)萃取，合并的有机相用盐水荡洗，经无水硫酸钠干燥，过滤，滤液减压浓缩干即得式(I)化合物。 1H NMR(400MHz,CDCl 3)δ:11.15(brs,1H),10.22(brs,1H),9.35(t,J＝6.4Hz,1H),8.72(d,J＝1.5Hz,1H),8.01-8.07(m,1H),7.94-8.00(m,1H),7.68(d,J＝8.3Hz,2H),7.49-7.56(m,2H),7.34(d,J＝8.3Hz,2H),7.15(t,J＝8.9Hz,2H),4.51(d,J＝6.3Hz,2H),3.98(t,J＝7.2Hz,2H),2.62(t,J＝7.2Hz,2H),1.89(qd,J＝7.2,5.1Hz,2H)；MS ESI计算值C 24H 22FN 3O 4[M+H]+436,实测值436。
实施例2：式(I)化合物A晶型的制备
将式(I)化合物(0.21kg)与EtOAc(2.1L)和水(1L)一起搅拌，加入浓盐酸(8mL)使固体溶解于有机相中，分离有机相，用盐水荡洗(0.5L，洗完后盐水pH约为6)，经无水硫酸钠干燥，过滤，滤液减压浓缩干得泡沫状白色固体。向该固体中加入预热的乙酸乙酯(2.1L)保持内温为45℃，并开始滴加正庚烷(0.588L)，滴加完成后使内温缓慢降至6～13℃并继续搅拌12小时。过滤，收集滤饼即得第一次重结晶产品。该过程平行进行10次，共得白色固体产品1.5kg。
将第一次重结晶产品(0.25kg)与EtOAc(2.5L)和水(1.25L)一起搅拌，加入浓盐酸(约80mL)使固体溶解于有机相中，分离有机相，并用水(0.6L×2)及用盐水(0.6L，洗完后盐水pH约为6)荡洗，经无水硫酸钠干燥，过滤，滤液减压浓缩干得泡沫状白色固体。向该固体中加入预热的乙酸乙酯(2.5L)保持内温为45℃，并开始滴加正庚烷(0.7L)，滴加完成后使内温缓慢降至6～13℃并继续搅拌12小时。过滤，收集滤饼即得第二次重结晶产品。该过程平行进行6次，共得产品(白色固体)1.05kg。
将第二次重结晶产品(1.05kg)与EtOAc(0.7L)及水(1.4L)在5℃下一起搅拌24小时。过滤，滤饼用水(105mL×2)润洗，抽干后，在底层放有五氧化二磷的真空干燥箱中30℃下烘干24小时得到白色固体。XRPD检测其晶型状态，即得式(I)化合物A晶型。
实施例3：式(I)化合物A晶型的固体稳定性试验
依据《原料药与制剂稳定性试验指导原则》(中国药典2015版四部通则9001)，考察式(I)化合物A 晶型在高温(60℃，敞口)，高湿(室温/相对湿度92.5％，敞口)，高温高湿(40℃，75％RH；60度，75％RH)及强光照(总照度1.2×10 6Lux·hr/近紫外200w·hr/m 2，密闭)条件下的稳定性。
称取式(I)化合物A晶型15mg，置于玻璃样品瓶的底部，摊成薄薄一层。高温及高湿条件下放置的样品用铝箔纸封瓶口，并在铝箔纸上扎些小孔，保证样品能与环境空气充分接触；强光照条件下放置的样品用螺纹瓶盖密封。不同条件下放置的样品于第5天，10天取样检测(XRPD)，检测结果与0天的初始检测结果进行比较，试验结果见下表2所示：
表2.式(I)化合物A晶型的固体稳定性试验结果
Figure PCTCN2019094602-appb-000006
结论：式(I)化合物A晶型在高温、高湿、高温高湿、强光照条件下具有良好的稳定性。
实验例1：式(I)化合物A晶型在MM.1S异种移植(CDX)模型的体内药效研究
实验材料：
CB-17 SCID小鼠，雌性，6-8周，体重约17-20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(4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64只购于北京维通利华的小鼠用于研究。将0.2mL 5×10 6个MM.1S细胞皮下接种于每只小鼠的右后背，用于肿瘤的生长。当平均肿瘤体积达到约100-150立方毫米时开始实验。实验方法：
在皮下植入人多发性骨髓瘤细胞MM.1S异种移植(CDX)CB-17 SCID小鼠上进行体内选择性实验。将式(I)化合物A晶型用5％二甲基亚砜和95％10％羟丙基-β-环糊精混合溶液配制成制剂以口服给药方式连续给药5天停止2天。伊沙佐米每周第一及第四天各给药一次。肿瘤体积一周两次用二维卡尺测量，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。实验结果：见表3。
表3.体内药效研究结果
Figure PCTCN2019094602-appb-000007
实验结论：
式(I)化合物A晶型(75mg/kg)与伊沙佐米(4mg/kg)联合给药组具有比空白组、式(I)化合物A晶型(75mg/kg)单用组、伊莎佐米(4mg/kg)单用组相比非常好的联合给药药效，小鼠显示出良好的耐受性。

Claims (9)

  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：11.53±0.2°、18.46±0.2°、23.60±0.2°。
    Figure PCTCN2019094602-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：11.53±0.2°、13.21±0.2°、16.33±0.2°、17.42±0.2°、18.46±0.2°、21.43±0.2°、22.57±0.2°、23.60±0.2°。
  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：8.898°、11.118°、11.528°、12.652°、13.206°、13.761°、16.325°、17.415°、18.067°、18.464°、19.289°、20.697°、21.427°、22.572°、23.226°、23.599°、25.674°、26.619°、27.611°、29.090°、29.879°、31.852°、33.878°、35.252°和36.122°。
  4. 根据权利要求3所述的A晶型，其XRPD图谱如图1所示。
  5. 根据权利要求1～4任意一项所述的A晶型，其差示扫描量热曲线在135.55℃处有一个吸热峰的起始点。
  6. 根据权利要求5所述的A晶型，其DSC图谱如图2所示。
  7. 根据权利要求1～4任意一项所述的A晶型，其热重分析曲线在135.40±3℃处失重达0.2115％。
  8. 根据权利要求7所述的A晶型，其TGA图谱如图3所示。
  9. 根据权利要求1～8任意一项所述的A晶型在制备治疗HDAC6相关病症的药物上的应用。
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