WO2020078170A1 - 化合物A的晶型α及其制备方法和含有该晶型的药物组合物 - Google Patents

化合物A的晶型α及其制备方法和含有该晶型的药物组合物 Download PDF

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WO2020078170A1
WO2020078170A1 PCT/CN2019/107211 CN2019107211W WO2020078170A1 WO 2020078170 A1 WO2020078170 A1 WO 2020078170A1 CN 2019107211 W CN2019107211 W CN 2019107211W WO 2020078170 A1 WO2020078170 A1 WO 2020078170A1
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compound
crystal form
formula
pharmaceutical composition
error
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PCT/CN2019/107211
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English (en)
French (fr)
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许文杰
孙晶超
于涛
毛庆华
张盛彬
李宁
吴成德
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深圳信立泰药业股份有限公司
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Priority to CN201980064845.8A priority Critical patent/CN112955445B/zh
Publication of WO2020078170A1 publication Critical patent/WO2020078170A1/zh

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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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4462—Non condensed piperidines, e.g. piperocaine only substituted in position 3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

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  • the invention belongs to the field of chemical drug preparation, and in particular relates to a crystal form ⁇ of PCSK9 inhibitor compound A, a preparation method thereof, and a pharmaceutical composition containing the crystal form.
  • Plasma low-density lipoprotein cholesterol (LDL-C) levels are closely related to the risk of cardiovascular disease.
  • Low-density lipoprotein receptors (LDL-R) expressed on the surface of liver cells are the primary factors that determine LDL-C levels.
  • PCSK9 in the blood can specifically bind to LDL-R on the cell surface to form a complex and transport it to the lysosome, resulting in accelerated degradation of LDL-R and increased LDL-C levels.
  • a large number of basic research and clinical trial results show that after exogenous interventions inhibit PCSK9 activity, it can accelerate the removal of low-density lipoprotein (LDL) in plasma, resulting in a good lipid-lowering effect. Therefore, PCSK9 inhibitors are likely to be a new generation of drugs for the treatment of dyslipidemia and related cardiovascular diseases.
  • WO2014170786 reports the compound of the following formula PF-06446846 and similar compounds.
  • One of the objects of the present invention is to provide a stable crystal form ⁇ of a stable PCSK9 inhibitor compound A.
  • the chemical structure of the compound A is as follows,
  • the crystalline form ⁇ of the above-mentioned PCSK9 inhibitor compound A shows the characteristic peaks at 7.81 °, 12.26 ° and 16.94 ° with an angle of 2 ⁇ in the X-ray diffraction pattern, and the error is ⁇ 0.2 °.
  • the characteristic peak of the displacement is the strongest characteristic peak of the X-ray of the crystal form, and can sufficiently represent the present crystal form.
  • the crystal form ⁇ of Compound A has characteristic peaks at 7.81 °, 11.61 °, 12.26 °, 15.52 °, 16.94 °, 18.90 °, and 27.19 ° at an angle of 2 ⁇ in the X-ray diffraction pattern, with an error of ⁇ 0.2 ° .
  • the characteristic peak of the displacement is a relatively strong characteristic peak of the X-ray of the crystal form, which can represent the crystal form more completely.
  • the crystal form ⁇ of Compound A is represented by the 2 ⁇ angle at 7.81 °, 9.83 °, 11.61 °, 12.26 °, 15.30 °, 15.52 °, 16.13 °, 16.94 °, 17.87 °, 18.90 in the X-ray diffraction pattern.
  • the characteristic peak of the displacement is a characteristic peak detectable by the X-ray of the crystal form, and can completely represent the present crystal form.
  • the error of each 2 ⁇ angle is usually within ⁇ 0.2 ° (the error of each 2 ⁇ angle is usually within ⁇ 0.2 °
  • the meaning of refers to most of the characteristic peaks, such as more than 80% of the characteristic peak errors are within this range, and occasionally there are a few characteristic peak errors that exceed this range, all should be considered to belong to the same crystal form XRPD spectrum) ;
  • the characteristic peaks of the shifts are medium-intensity absorption peaks, while other weak absorption peaks may change significantly due to experimental operation errors. For those skilled in the art, other absorption peaks are unnecessary for characterizing the crystalline form. Absorption peak.
  • FIG. 1 More specifically, the X-ray diffraction pattern of the crystal form ⁇ of the compound A is shown in FIG. 1 or FIG. 4.
  • the differential scanning calorimetry curve of the crystalline form ⁇ of the above compound A has an endothermic peak at 206.10 ° C ⁇ 3 ° C.
  • the DSC pattern of the crystalline form ⁇ of the above compound A is shown in FIG. 2.
  • Thermogravimetric analysis curve of the crystalline form ⁇ of the above compound A The thermogravimetric analysis curve has a weight loss of 0.09024% at 120 ° C ⁇ 3 ° C.
  • the TGA spectrum of the above crystalline form a of the compound A is shown in FIG. Does not contain crystal water.
  • the compound A is prepared by the preparation method described in PCT / CN2018 / 083380 to obtain compound A (WX002, the aforementioned compound of formula II), and the synthetic route is as follows:
  • the preparation method of the crystal form ⁇ of compound A includes the following steps:
  • Still another object of the present invention is to provide a pharmaceutical composition containing the above-mentioned crystalline form ⁇ of Compound A, and one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier refers to any formulation carrier or medium capable of delivering an effective amount of the active substance of the present invention without interfering with the biological activity of the active substance and having no toxic or side effects on the host or patient.
  • Representative carriers include water and oil , Vegetables and minerals, cream base, lotion base, ointment base, etc. These bases include suspending agents, tackifiers, penetration enhancers and the like. Their formulation is well known to those skilled in the cosmetics field or topical pharmaceutical field. For other information about the carrier, please refer to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), and the contents of this document are incorporated herein by reference.
  • the pharmaceutical composition can be used for the preparation of drugs for PCSK causing related diseases, especially hypercholesterolemia.
  • the present invention has the following outstanding advantages and beneficial effects:
  • the crystal form ⁇ of the compound A of the present invention has high purity, is a solid form with stable quality, and is easier to arrange and use the pharmaceutical composition.
  • the crystal form ⁇ of the compound A of the present invention is more conducive to ensuring the compound A's stable clinical effect and safe use of the flocculent solid obtained from PCT / CN2018 / 083380.
  • the method for preparing the crystal form ⁇ of Compound A in the present invention is simple, fast, and can be prepared under normal temperature conditions, and is easier to industrialize.
  • Example 1 is an XRPD spectrum of Cu-K ⁇ radiation of crystal form ⁇ of compound A of formula (II) in Example 1;
  • Fig. 5 is the DVS spectrum of the crystal form ⁇ of compound A of formula (II) in Example 1, wherein curve (1) is the weight gain of the sample when the humidity is increased; curve (2) is the loss of the sample when the humidity is further reduced Serious situation.
  • the synthetic route is as follows:
  • the specific preparation method is as follows:
  • WXBB-3-1 (10.00g, 49.93mmol, 1.00eq)
  • WX002-1 (8.59g, 49.93mmol, 5.58mL, 1.00eq)
  • sodium tert-butoxide (9.60g, 99.86mmol, 2.00eq)
  • ( ⁇ ) -2,2'-bis- (diphenylphosphino) -1,1'-binaphthalene (4.66g, 7.49mmol, 0.15eq)
  • tris (dibenzylideneacetone) dipalladium (4.57g, 4.99 mmol, 0.10eq) was placed in toluene (20.00mL), replaced with nitrogen three times, the mixture was protected under nitrogen and stirred at 90 degrees for 16 hours.
  • a sufficient amount of compound A of formula (II) is prepared multiple times, about 50 mg of compound A is weighed into a sample bottle, and anhydrous ethanol solvent 0.3 mL is added to prepare a suspension. After the prepared suspension is continuously shaken at 40 °C and 700 rpm for 60 hours, the residual solid is placed in a vacuum drying oven after centrifugation, and the residual solvent is vacuum dried at 30 °C overnight to remove the residual solvent to obtain the compound of formula (II) Of crystal form A.
  • the X-ray diffraction diagram shows the characteristic peaks at 7.81 °, 12.26 °, and 16.94 ° with an angle of 2 ⁇ , and the error is ⁇ 0.2 °.
  • the characteristic peak of the displacement is the strongest characteristic peak of the X-ray of the crystal form, and can sufficiently represent the present crystal form.
  • the crystal form ⁇ of Compound A has characteristic peaks at 7.81 °, 11.61 °, 12.26 °, 15.52 °, 16.94 °, 18.90 °, and 27.19 ° at an angle of 2 ⁇ in the X-ray diffraction pattern, with an error of ⁇ 0.2 ° .
  • the characteristic peak of the displacement is a relatively strong characteristic peak of the X-ray of the crystal form, and can more completely represent the present crystal form.
  • the crystal form ⁇ of Compound A is represented by the 2 ⁇ angle at 7.81 °, 9.83 °, 11.61 °, 12.26 °, 15.30 °, 15.52 °, 16.13 °, 16.94 °, 17.87 °, 18.90 in the X-ray diffraction pattern.
  • the characteristic peak of the displacement is a characteristic peak detectable by the X-ray of the crystal form, and can completely represent the present crystal form.
  • the crystal form ⁇ of Compound A is detected by other detection means, such as a differential scanning calorimetry curve having an endothermic peak at 206.10 ° C ⁇ 3 ° C.
  • DSC is shown in Figure 2.
  • thermogravimetric analysis curve of the crystalline form ⁇ of Compound A has a thermogravimetric analysis curve whose weight loss reaches 0.09024% at 120 ° C ⁇ 3 ° C. More specifically, TGA is specifically shown in FIG. 3, indicating that the compound does not contain crystal water.
  • Test method about 10 ⁇ 20mg sample is used for XRPD detection.
  • Light tube voltage 40kV
  • light tube current 40mA
  • Anti-scattering slit 7.10mm
  • Test method Take the sample (0.5 ⁇ 1mg) in the DSC aluminum pan for testing, under 50mL / min N 2 conditions, with a heating rate of 10 °C / min, heat the sample from 25 °C to 300 °C (or 350 °C) .
  • Thermogravimetric analysis (Thermal Gravimetric Analyzer, TGA) method
  • Test method Take a sample (2 ⁇ 5mg) and place it in a TGA platinum pot for testing. Under 25mL / min N 2 conditions, at a rate of 10 °C / min, heat the sample from room temperature to 300 °C or 350 °C or lose weight 20 %.
  • Test conditions Take a sample (10-15 mg) and place it in the DVS sample tray for testing.
  • ⁇ W% represents the weight gain of the test article at 25 ° C / 80% RH.
  • the 2 ⁇ angle indicates that there are characteristic peaks at 7.70 °, 12.16 °, and 16.82 °, and the error is ⁇ 0.2 °.
  • the characteristic peak of the displacement is the strongest characteristic peak of the X-ray of the crystal form, and can sufficiently represent the present crystal form.
  • the crystal form ⁇ of Compound A has characteristic peaks at 7.70 °, 11.52 °, 12.16 °, 15.40 °, 16.82 °, 18.78 °, and 27.06 ° with an angle of 2 ⁇ in the X-ray diffraction pattern, and the error is ⁇ 0.2 ° .
  • the characteristic peak of the displacement is a relatively strong characteristic peak of the X-ray of the crystal form, and can more completely represent the present crystal form.
  • the crystal form ⁇ of Compound A is represented by the 2 ⁇ angle at 7.70 °, 9.70 °, 11.52 °, 12.16 °, 15.20 °, 15.40 °, 16.02 °, 16.82 °, 17.72 °, 18.78 in the X-ray diffraction pattern.
  • the error is ⁇ 0.2 °.
  • the characteristic peak of the displacement is a characteristic peak detectable by the X-ray of the crystal form, and can completely represent the present crystal form.
  • Test method about 200-300mg sample is used for XRPD detection.
  • Light tube voltage 40kV
  • light tube current 30mA
  • Anti-scattering slit 1.00mm
  • Step size 0.5 seconds.
  • SMSDVSAdvantage dynamic steam adsorption instrument for details, please refer to the aforementioned dynamic steam adsorption analysis (Dynamic Vapor Sorption, DVS) method
  • the crystal form ⁇ of the compound A of formula (II) has a hygroscopic weight gain of 0.1293% at 25 ° C and 80% RH, and has no or almost no hygroscopicity.
  • Example 6 Study on the pharmacodynamics of compound A of formula (II) in crystal form ⁇ in diet-induced hypercholesterolemia hamster model
  • Golden hamsters purchased from Beijing Vital Lihua Laboratory Animal Technology Co., Ltd., males, were kept in a special pathogen-free environment for 7 weeks in a single ventilated cage (4 per cage). All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory diets.
  • the data is expressed as mean ⁇ standard error.
  • the crystal form ⁇ of compound A of formula (II) can reduce low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) in all dosage groups.
  • LDL-C low-density lipoprotein cholesterol
  • TC total cholesterol
  • the purpose of this study was to evaluate the PK properties of the test compound and calculate its bioavailability in male SD rats.
  • the project used four male SD rats, randomly divided into two groups. Two rats were administered intravenously at 1 mg / kg, respectively. Whole blood was collected and plasma samples were prepared at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24h after administration. The other two rats were given intragastrically The drug was 5 mg / kg, and whole blood was collected and plasma samples were prepared at 0.25, 0.5, 1, 2, 4, 6, 8, and 24h after administration.
  • the concentration of the test compound in plasma was determined by LC / MS / MS method, and the relevant pharmacokinetic parameters were calculated using Phoenix WinNonlin 6.2.1 software.
  • the compound of the present invention has good pharmacokinetic properties.

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Abstract

公开了一种PCSK9抑制剂化合物A的晶型α及其制备方法，以及含有该晶型的药物组合物。

Description

化合物A的晶型α及其制备方法和含有该晶型的药物组合物 技术领域
本发明属于化学药物制备领域，具体涉及一种PCSK9抑制剂化合物A的晶型α及其制备方法，以及含有该晶型的药物组合物。
背景技术
血浆低密度脂蛋白胆固醇(LDL-C)水平与心血管疾病的发生风险密切相关肝脏细胞表面表达的低密度脂蛋白受体(LDL-R)是决定LDL-C水平的首要因素。血液中的PCSK9可以与细胞表面的LDL-R发生特异性结合形成复合物并转运至溶酶体，从而导致LDL-R加速降解，使LDL-C水平升高。大量的基础研究和临床试验结果表明，外源性干预措施抑制PCSK9活性后，可加速血浆中低密度脂蛋白(LDL)清除，从而产生良好的降脂效果。因此，PCSK9抑制剂很可能将是治疗血脂紊乱及相关心血管疾病的新一代药物。
WO2014170786报道了下式化合物PF-06446846及相近似化合物。
Figure PCTCN2019107211-appb-000001
申请人之前提交的PCT申请PCT/CN2018/083380，公开了新的一类具有哌啶环结构的PCSK9抑制剂的化合物，具体公开了式(I)所示化合物及具体优选化 合物式(II)-化合物A，所述化合物是通过制备级高效液相色谱纯化得到产品，经干燥后呈絮状固体。
Figure PCTCN2019107211-appb-000002
为了改善该化合物的药用性质，需要进一步研究适宜的物质形态，从而有效地用于药物组合物中，通过抑制PCSK9而治疗病态的患者。
发明内容
本发明的目的之一在于提供一种稳定的PCSK9抑制剂化合物A的稳定晶型α。所述化合物A的化学结构如下，
Figure PCTCN2019107211-appb-000003
上述PCSK9抑制剂化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、12.26°和16.94°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线最强的特征峰，可以充分代表本晶型。
具体的，化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、11.61°、12.26°、15.52°、16.94°、18.90°和27.19°处有特征峰，误差为±0.2°。该位移的特 征峰为该晶型X射线相对较强的特征峰，可以更为完整的代表本晶型。
更为具体的，化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、9.83°、11.61°、12.26°、15.30°、15.52°、16.13°、16.94°、17.87°、18.90°、19.32°、20.65°、22.12°、22.99°、23.38°、23.98°、25.52°、26.34°、27.19°、27.49°、29.81°、30.42°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线可检测的特征峰，可以完整的代表本晶型。
应注意：特定的晶型的不同样品具有同样的主要XRPD峰，但是在粉末图中的小峰可能有变化。此外，当由本领域普通技术人员，采用相应方法得到的同晶型样品采用相同的仪器和检测方法进行检测时，各2θ角误差通常在±0.2°以内(各2θ角误差通常在±0.2°以内的含义指的是大部分特征峰，如超过80％以上的特征峰误差在此范围内，而偶然有个别少数的特征峰的误差超出该范围，均应认为属于相同晶型的XRPD谱图)；而且，所述各位移的特征峰为中等强度吸收峰，而其他弱吸收峰可能由于实验操作误差发生明显变化，对于本领域技术人员来说其他吸收峰均是表征本晶型时不必要的吸收峰。
更为具体地，所述化合物A的晶型α的X射线衍射图如图1或图4所示。
在本发明的一些方案中，上述化合物A的晶型α的差示扫描量热曲线在206.10℃±3℃处具有吸热峰。
在本发明的一些方案中，上述化合物A的晶型α的DSC图谱如图2所示。
水分测定：上述化合物A的晶型α的热重分析曲线其热重分析曲线在120℃±3℃时失重达0.09024％，上述化合物A的晶型α的TGA图谱如图3所示，说明化合物不含有结晶水。
所述化合物A通过PCT/CN2018/083380记载的制备方法制备得到化合物A(WX002，前述式II化合物)，合成路线如下：
Figure PCTCN2019107211-appb-000004
化合物A的晶型α的制备方法包括以下步骤：
室温条件下，
(a)将式(II)化合物A加入无水乙醇溶剂中使其成悬浊液，其中，无水乙醇溶剂的体积(mL)与化合物A质量(g)的比为1：1-8；
(b)悬浊液25～60℃下搅拌16～60小时；
(c)离心后干燥8～16小时；
得式(II)化合物A的晶型α。
本发明的再一目的在于提供一种含有上述的化合物A的晶型α的药物组合物，和一种以上药学上可接受的载体。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂载体或介质，代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。它们的制剂为化妆品领域或局部药物领域的技术人员所周知。关于载体的其他信息，可以参考Remington:The Science and Practice of Pharmacy,21st Ed.,Lippincott,Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。
所述药物组合物可以用于制备PCSK引起相关疾病、特别是高胆固醇血症的药物的应用。
本发明与现有技术相比具有如下突出的优点及有益效果：
1、本发明的化合物A的晶型α的纯度高，为质量稳定的固体形态，更易 于药物组合物的配置和使用。
2、本发明的化合物A的晶型α相对于PCT/CN2018/083380所获得絮状固体更利于保证化合物A稳定的发挥临床疗效和安全用药。
3、本发明制备化合物A的晶型α的方法简单、快捷、在常温条件下即可制备，更易于产业化生产。
附图说明
图1为实施例1式(II)化合物A的晶型α的Cu-Kα辐射的XRPD谱图；
图2为实施例1式(II)化合物A的晶型α的DSC谱图；
图3为实施例1式(II)化合物A的晶型α的TGA谱图；
图4为实施例2式(II)化合物A的晶型α的Cu-Kα辐射的XRPD谱图；
图5为实施例1式(II)化合物A的晶型α的DVS谱图，其中，曲线(1)为增加湿度时，样品的增重情况；曲线(2)为再降低湿度，样品的脱重情况。
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和附图来做进一步的说明，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：化合物A的制备
合成路线如下：
Figure PCTCN2019107211-appb-000005
具体的制备方法如下：
步骤1合成化合物WX002-2：
将WXBB-3-1(10.00g,49.93mmol,1.00eq),WX002-1(8.59g,49.93mmol,5.58mL,1.00eq),叔丁醇钠(9.60g,99.86mmol,2.00eq),(±)-2,2'-双-(二苯膦基)-1,1'-联萘(4.66g,7.49mmol,0.15eq)和三(二亚苄基丙酮)二钯(4.57g,4.99mmol,0.10eq)置于甲苯(20.00mL)中，用氮气置换三次，混合液在氮气保护下，于90度下搅拌16小时。反应完毕，反应液减压浓缩除去甲苯，向残余物中加入水(200mL)，用乙酸乙酯(200mL*3)萃取，合并有机相，用饱和氯化钠溶液200mL洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。残余物用层析柱(石油醚：乙酸乙酯＝20/1-1:1,v/v)纯化，得到化合物WX002-2。 1H NMR(400MHz,CDCl 3)δppm:1.38(s,9H),1.58(br,s,1H),1.90-1.70(m,4H),2.05(s,3H),3.35-3.29(m,1H),3.51(br,2H),3.64-3.61(m,1H),4.14-4.08(m,1H),6.50(t,J＝6Hz,1H),7.20(d,J＝6.8Hz,1H),7.99(d,J＝4.8Hz,1H).MS m/z:292.0[M+H] +。
步骤2化合物WX002-3的合成：
在0度下，向WX002-2(231.57mg,794.69μmol,1.00eq)的四氢呋喃(10.00mL)溶液中滴加入六甲基二硅基胺基锂(1M,2.38mL,3.00eq)，混合液在0度下搅拌1.5小时。随后在0度下加入WXBB-2(200.00mg,794.69μmol,1.00eq)和三乙胺(146.35mg,1.45mmol,200.48μL,1.82eq)的四氢呋喃(5.00mL)溶液。混合液在20度下搅拌14小时。反应完毕，反应液减压浓缩，向残余物中加入水(100mL)，用乙酸乙酯(50mL*3)萃取，合并有机相，用饱和氯化钠溶液(20mL*2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。残余物用制备级层析板(石油醚:乙酸乙酯＝1:1,v/v)纯化，得到化合物WX002-3. 1H NMR(400MHz,CDCl 3)δppm:8.35(br s,1H),7.80(br d,J＝7.8Hz,2H),7.34-7.21(m,3H),7.08-6.98(m,1H),4.44(br d,J＝11.7Hz,1H),4.00(br d,J＝10.8Hz,1H),3.44-3.25(m,1H),2.65-2.21(m,2H),1.95(br d,J＝15.3Hz,3H),1.69(br s,2H),1.62-1.50(m,2H),1.43-1.34(m,15H).MS m/z:507.2[M+H] +。
步骤3化合物WX002的合成：
在0-10℃下，向WX002-3(30.00mg,56.85μmol,1.00eq)的乙酸乙酯(1.00 mL)溶液中加入氯化氢/乙酸乙酯溶液(4M,1.00mL)。混合液在20度下搅拌1小时。反应完毕，反应液减压浓缩，残余物用制备级高效液相色谱纯化得到产品WX002，经干燥后呈絮状固体。
1H NMR(400MHz,CDCl 3)δ:9.33(br s,0.5H),8.97(br s,0.5H),8.38(br s,1H),7.83-7.71(m,2H),7.31(br d,J＝7.3Hz,1H),7.19(s,2H),7.14(br s,1H),5.05(br s,4H),4.02-3.06(m,2.5H),2.82(br s,0.5H),1.98(br s,1H),1.87(br s,3H),1.72(br s,1H),1.35(s,6H).MS m/z:407.1[M+H] +。
实施例2：式(II)化合物A的晶型α的制备
通过前述制备方法，重复多次制备足量的式(II)化合物A，称量大约50mg化合物A于样品瓶中，加入无水乙醇溶剂0.3mL，制备悬浊液。将制备好的悬浊液在40℃，700rmp条件下持续振摇60小时后，离心后将残留固体放入真空干燥箱，在30℃条件下真空干燥过夜去除残留溶剂，得式(II)化合物的A晶型α。
从检测结果可知，在X射线衍射图中以2θ角表示在7.81°、12.26°和16.94°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线最强的特征峰，可以充分代表本晶型。
具体的，化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、11.61°、12.26°、15.52°、16.94°、18.90°和27.19°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线相对较强的特征峰，可以更为完整的代表本晶型。
更为具体的，化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、9.83°、11.61°、12.26°、15.30°、15.52°、16.13°、16.94°、17.87°、18.90°、19.32°、20.65°、22.12°、22.99°、23.38°、23.98°、25.52°、26.34°、27.19°、27.49°、29.81°、30.42°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线可检测的特征峰，可以完整的代表本晶型。
更为具体的，X射线具体如图1所示。
另外，化合物A的晶型α通过其他检测手段检测，如差示扫描量热曲线在 206.10℃±3℃处具有吸热峰。
更为具体的，DSC具体如图2所示。
再者，化合物A的晶型α的热重分析曲线其热重分析曲线在120℃±3℃时失重达0.09024％。更为具体的，TGA具体如图3所示，说明化合物不含有结晶水。
其中，所使用的各测定方法如下：
粉末X-射线衍射(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 PCTCN2019107211-appb-000006
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：3或4-40deg
步径：0.02deg
步长：0.12秒
样品盘转速：15rpm
差热分析(Differential Scanning Calorimeter,DSC)方法
仪器型号：TA DSC Q2000差示扫描量热仪
测试方法：取样品(0.5～1mg)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25℃到300℃(或350℃)。
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300℃或350℃或失重20％。
动态蒸汽吸附分析(Dynamic Vapor Sorption,DVS)方法
仪器型号：SMS DVS Advantage-1动态蒸汽吸附仪
测试条件：取样品(10～15mg)置于DVS样品盘内进行测试。
详细的DVS参数如下：
温度：25℃
平衡：dm/dt＝0.01％/min(最短：10min，最长：180min)
干燥：0％RH下干燥120min
RH(％)测试梯级：10％
RH(％)测试梯级范围：0％-90％-0％
引湿性评价分类如下表1：
吸湿性分类 ΔW％
潮解 吸收足量水分形成液体
极具吸湿性 ΔW％≥15％
有吸湿性 15％>ΔW％≥2％
略有吸湿性 2％>ΔW％≥0.2％
无或几乎无吸湿性 ΔW％<0.2％
注：ΔW％表示受试品在25℃/80％RH下的吸湿增重。
实施例3：式(II)化合物A的晶型α的制备
通过前述制备方法，重复多次制备足量的式(II)化合物A，取163.00g化合物A的粗品，加入无水乙醇(245mL)(溶剂体积为试剂质量的1.5倍)，在40℃下搅拌16h，过滤，滤饼干燥(水泵，50℃)得式(II)化合物的A晶型 α。
在X射线衍射图中以2θ角表示在7.70°、12.16°和16.82°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线最强的特征峰，可以充分代表本晶型。
具体的，化合物A的晶型α在X射线衍射图中以2θ角表示在7.70°、11.52°、12.16°、15.40°、16.82°、18.78°和27.06°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线相对较强的特征峰，可以更为完整的代表本晶型。
更为具体的，化合物A的晶型α在X射线衍射图中以2θ角表示在7.70°、9.70°、11.52°、12.16°、15.20°、15.40°、16.02°、16.82°、17.72°、18.78°、19.18°、20.52°、22.02°、22.86°、23.32°、23.84°、25.36°、26.22°、27.06°、27.36°、29.68°、30.34°处有特征峰，误差为±0.2°。该位移的特征峰为该晶型X射线可检测的特征峰，可以完整的代表本晶型。
更为具体的，X射线具体如图4所示。
其中，所使用的粉末X-射线衍射(X-ray powder diffractometer,XRPD)方法
仪器型号：DX-2700BH X-射线衍射仪
测试方法：大约200～300mg样品用于XRPD检测。
详细的XRPD参数如下：
光管：Cu,kα,
Figure PCTCN2019107211-appb-000007
光管电压：40kV，光管电流：30mA
发散狭缝：1.00mm
探测器狭缝：28.00mm
防散射狭缝：1.00mm
扫描范围：3-40deg
步径：0.02deg
步长：0.5秒。
实施例4：式(II)化合物A的晶型α的吸湿性研究
实验材料：
SMS DVS Advantage动态蒸汽吸附仪，具体参见前述的动态蒸汽吸附分析(Dynamic Vapor Sorption,DVS)方法
实验方法：
取实施例2式(II)化合物A的晶型α10～15mg置于DVS样品盘内进行测试。
实验结果：
式(II)化合物A的晶型α的DVS谱图如图5所示，△W＝0.1293％。
实验结论：
式(II)化合物A的晶型α在25℃和80％RH下的吸湿增重为0.1293％，无或几乎无吸湿性。
实施例5：式(II)化合物A的晶型α的长期固体稳定性试验
取实施例2方法制备的1.5g样品分别装入双层LDPE袋，每层LDPE袋分别扎扣密封，再将LDPE袋子放入铝箔袋中并热封，分别放入25℃/60％RH(10包)，30℃/65％RH(5包)和40℃/75％RH(6包)条件下考察。试验结果见下表2所示：
表1式(II)化合物A的晶型α的固体稳定性试验结果
Figure PCTCN2019107211-appb-000008
结论：式(II)化合物A的晶型α具有良好的稳定性。
实施例6：式(II)化合物A的晶型α在饮食诱导高胆固醇血症地鼠模型药效研究
实验材料：
购自北京维通利华实验动物技术有限公司的金黄地鼠，雄性，7周，将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。
实验方法：
动物适应环境12天后，根据体重随机分为正常组(Normal,n＝12)和模型组(Vehicle,n＝60)。正常组动物给予普通饲料，模型组给予高胆固醇饲料(含0.5％胆固醇，10％棕榈油)开始造模。造模第10天开始至第14天，每天给动物口服灌胃溶媒一次，以训练动物适应给药操作，减少给药操作引起的应激反应。造模第14天后，动物禁食过夜，于第15天眼眶采血，收集血液后分离血清。第16天检测TC、LDL-C水平，根据LDL-C、TC水平及体重进行随机分组。于造模后第17天开始给药。
实验结果：见表3。
表3
Figure PCTCN2019107211-appb-000009
注：
1.数据表示为均值±标准误。
2.运用One Way ANOVA进行统计学分析，与正常组比较，###表示p<0.001；与vehicle比较，*表示p<0.05,**表示p<0.01,***表示p<0.001。
实验结论：
式(II)化合物A的晶型α各个剂量组均可降低低密度脂蛋白胆固醇(LDL-C)和总胆固醇(TC)。
实施例7 SD大鼠药代动力学性质研究
该研究的目的是为了评价受试化合物的PK性质，并计算其在雄性SD大鼠中的生物利用度。该项目使用四只雄性SD大鼠，随机分成两组。两只大鼠分别静脉注射给药1mg/kg，给药后0.0833、0.25、0.5、1、2、4、6、8和24h分别采集全血并制备血浆样品，另外两只大鼠灌胃给药5mg/kg，给药后0.25、0.5、1、2、4、6、8和24h分别采集全血并制备血浆样品。采用LC/MS/MS法测定受试化合物在血浆中的浓度，并用Phoenix WinNonlin 6.2.1软件计算相关药代动力学参数。
实验结果显示，本发明化合物在雄性SD大鼠中具有良好的药代动力学性质。生物利用度数据如表4。
表4生物利用度数据
PF-06446846 WX002
大鼠中F(％) 20％ 68.0％
结论：本发明化合物具有良好的药代动力学性质。
实施例8药物组合物的制备
化合物A(晶型α)     6.78g
糊精                84.00g
按常规方法，将上述物质混合均匀后，分1000等份分别装入普通明胶胶囊， 得到1000颗胶囊。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种化合物A的晶型α，所述化合物A化学结构式为下式(II)，
    Figure PCTCN2019107211-appb-100001
  2. 如权利要求1所述的化合物A的晶型α，其特征在于：所述化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、12.26°和16.94°处有特征峰，误差为±0.2°。
  3. 如权利要求1所述的化合物A的晶型α，其特征在于：所述化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、11.61°、12.26°、15.52°、16.94°、18.90°和27.19°处有特征峰，误差为±0.2°。
  4. 如权利要求1所述的化合物A的晶型α，其特征在于：所述化合物A的晶型α在X射线衍射图中以2θ角表示在7.81°、9.83°、11.61°、12.26°、15.30°、15.52°、16.13°、16.94°、17.87°、18.90°、19.32°、20.65°、22.12°、22.99°、23.38°、23.98°、25.52°、26.34°、27.19°、27.49°、29.81°、30.42°处有特征峰，误差为±0.2°。
  5. 如权利要求1所述的化合物A的晶型α，其特征在于：所述化合物A的晶型α的X射线衍射图如图1或图4所示，误差为±0.2°。
  6. 如权利要求1所述的化合物A的晶型α，其特征在于：化合物A的晶型α的差示扫描量热曲线在206.10℃±3℃处具有吸热峰。
  7. 如权利要求1所述的化合物A的晶型α，其特征在于：化合物A的晶型α的差示扫描量热曲线如图2所示。
  8. 一种如权利要求1～7任意一项所述的化合物A的晶型α的制备方法，其特征在于，包括如下步骤：
    室温条件下，
    (a)将式(II)化合物A加入无水乙醇溶剂中使其成悬浊液，其中，无水乙醇溶剂的体积(mL)与化合物A质量(g)的比为1：1-8；
    (b)悬浊液25～60℃下搅拌16～60小时；
    (c)离心后干燥8～16小时；
    得式(II)化合物A的晶型α。
  9. 一种药物组合物，其特征在于：所述药物组合物中含有如权利要求1～7任意一项所述的化合物A的晶型α，和一种以上药学可接受的载体。
  10. 一种药物用途，其特征在于：权利要求9所述的一种药物组合物在制备用于预防或治疗pcsk9相关疾病药物的应用，优选预防或治疗高胆固醇血症药物的应用。
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Publication number Priority date Publication date Assignee Title
CN105143203A (zh) * 2013-04-17 2015-12-09 辉瑞大药厂 用于治疗心血管疾病的n-哌啶-3-基苯甲酰胺衍生物
WO2018192493A1 (zh) * 2017-04-21 2018-10-25 深圳信立泰药业股份有限公司 作为pcsk9抑制剂的哌啶类化合物

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105143203A (zh) * 2013-04-17 2015-12-09 辉瑞大药厂 用于治疗心血管疾病的n-哌啶-3-基苯甲酰胺衍生物
WO2018192493A1 (zh) * 2017-04-21 2018-10-25 深圳信立泰药业股份有限公司 作为pcsk9抑制剂的哌啶类化合物

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