WO2020199440A1 - 硝酮嗪晶型、制备方法及应用 - Google Patents

硝酮嗪晶型、制备方法及应用 Download PDF

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WO2020199440A1
WO2020199440A1 PCT/CN2019/098724 CN2019098724W WO2020199440A1 WO 2020199440 A1 WO2020199440 A1 WO 2020199440A1 CN 2019098724 W CN2019098724 W CN 2019098724W WO 2020199440 A1 WO2020199440 A1 WO 2020199440A1
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Prior art keywords
crystal form
nitroxazine
preparation
heptane
dihydrate
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PCT/CN2019/098724
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English (en)
French (fr)
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刘伟
孙业伟
王玉强
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广州喜鹊医药有限公司
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Priority to AU2019439986A priority Critical patent/AU2019439986A1/en
Priority to JP2021558685A priority patent/JP2022540275A/ja
Priority to EP19922697.8A priority patent/EP3950679A4/en
Priority to US17/600,915 priority patent/US20220153707A1/en
Priority to CA3135637A priority patent/CA3135637A1/en
Publication of WO2020199440A1 publication Critical patent/WO2020199440A1/zh
Priority to IL286834A priority patent/IL286834A/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/12—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of medicinal chemistry, in particular to nitroxazine crystal form, preparation method and application thereof.
  • Nitrozine is a nitrone derivative of ligustrazine (TMP). It is a new compound chemically synthesized by adding a nitrone pharmacophore to the structure of ligustrazine. It is called TBN for short and its chemical name is (cis)- 2-Methyl-N-[(3,5,6-trimethylpyrazine-2-)methine]2-propylamine oxide, molecular formula is C 12 H 19 N 3 O, molecular weight is 221.30, with the following The chemical structure shown in the formula:
  • Nitrazine can inhibit the oxidative damage of nerve cells caused by ischemia, thereby exerting nerve cell protection and alleviating the neurological symptoms and dysfunction associated with cerebral embolism. Clinically, it can be used for the treatment of nervous system diseases, cardiovascular and cerebrovascular diseases and degenerative aging diseases.
  • the purpose of the present invention is to provide nitroxazine crystal form, preparation method and application.
  • One of the objectives of the present invention is to provide a crystal form A of nitroxazine, the XRPD pattern of the crystal form at 2 ⁇ angle positions: 10.60 ⁇ 0.2, 11.03 ⁇ 0.2, 15.31 ⁇ 0.2, 15.55 ⁇ 0.2, 17.14 ⁇ There are characteristic diffraction peaks at 0.2, 17.93 ⁇ 0.2, and 23.81 ⁇ 0.2.
  • the crystalline form A of nitroxazine, the XRPD pattern of the crystalline form at 2 ⁇ angle positions is: 10.60 ⁇ 0.2, 11.03 ⁇ 0.2, 13.51 ⁇ 0.2, 15.31 ⁇ 0.2, 15.55 ⁇ 0.2 , 17.14 ⁇ 0.2, 17.93 ⁇ 0.2, 21.22 ⁇ 0.2, 23.81 ⁇ 0.2, 25.23 ⁇ 0.2, 27.08 ⁇ 0.2 have characteristic diffraction peaks.
  • the crystalline form A of nitroxazine has substantially the same XRPD pattern as in Figure 1 of the specification.
  • nitroxazine crystal form A of the present invention has three crystal states: needle, square, and rod, and the micrographs are shown in Fig. 5 and Fig. 6.
  • the melting point of the nitroxazine crystal form A of the present invention is 76°C-78°C, respectively.
  • the present invention provides an infrared spectrum of nitroxazine crystal form A, as shown in FIG. 9.
  • the present invention has gone through a systematic crystal type screening experiment, wherein the screening methods include evaporation crystallization method (single solvent method and mixed solvent method), hot solution cold precipitation method and suspension beating method.
  • the screening includes solvents such as tetrahydrofuran, ethyl acetate, toluene, acetone, dioxane, isopropanol, petroleum ether, n-hexane, isopropyl acetate, isooctane and isobutyl acetate, and the screening results are all crystal form A .
  • the present invention also provides a preparation method of nitroxazine crystal form A, which includes the following steps:
  • step (2) The crystalline solid obtained in step (1) and n-heptane are mixed and heated to dissolve, cooled and crystallized to obtain nitroxazine crystal form A.
  • the organic solvent in step (1) is selected from one or more of ethyl acetate, n-hexane, n-heptane and cyclohexane. More preferably, the organic solvent is The solvent is n-hexane or n-heptane; or the organic solvent is selected from a mixed solvent of n-hexane and ethyl acetate. Applicants have found that selecting a preferred organic solvent can significantly reduce the impurity content.
  • the weight-volume ratio of the crude nitroxazine to the organic solvent in step (1) is 1:5-20, preferably 1:8-12.
  • the temperature of cooling and crystallization in step (1) is selected from 2 to 12°C, more preferably 3 to 10°C, and most preferably 3 to 5°C. The applicant found that the most preferred Within the range, the impurity content can be significantly reduced.
  • the weight-volume ratio of the crude nitroxazine to n-heptane is preferably 1:1 to 5, and most preferably 1:1 to 3.
  • step (2) the mixing heating temperature of the crystalline solid and n-heptane is 60-80°C, preferably 65-75°C.
  • the temperature for cooling and crystallization in step (2) is selected from 2 to 12°C, more preferably 4 to 10°C.
  • the second objective of the present invention is to provide a dihydrate of nitroxazine, the XRPD pattern of which is at 2 ⁇ angle positions: 8.91 ⁇ 0.2, 11.46 ⁇ 0.2, 14.29 ⁇ 0.2, 17.60 ⁇ 0.2, 21.19 ⁇ 0.2, 22.02 ⁇ 0.2 , 23.19 ⁇ 0.2, 24.30 ⁇ 0.2, 24.92 ⁇ 0.2, 29.20 ⁇ 0.2, 31.41 ⁇ 0.2 have characteristic diffraction peaks.
  • the XRPD pattern of the dihydrate of nitroxazine at 2 ⁇ angle positions is: 8.91 ⁇ 0.2, 11.46 ⁇ 0.2, 12.00 ⁇ 0.2, 14.29 ⁇ 0.2, 17.60 ⁇ 0.2, 19.50 ⁇ 0.2, There are characteristic diffraction peaks at 21.19 ⁇ 0.2, 22.02 ⁇ 0.2, 23.19 ⁇ 0.2, 24.30 ⁇ 0.2, 24.92 ⁇ 0.2, 26.70 ⁇ 0.2, 29.20 ⁇ 0.2, 31.41 ⁇ 0.2, 36.20 ⁇ 0.2.
  • the dihydrate of nitroxazine has substantially the same XRPD pattern as in Figure 2 of the specification.
  • the melting point of the dihydrate of nitroxazine in the present invention is 37-40°C.
  • the weight begins to decrease, and the weight loss is 13.67%.
  • the dihydrate of nitroxazine according to the present invention is obtained by cold precipitation of nitroxazine in a saturated ethanol aqueous solution binary system, and the volume percentage of ethanol in the ethanol aqueous solution is preferably 5-50%, more preferably 5-20%.
  • the present invention provides a pharmaceutical composition comprising one or more crystal form A or dihydrate of the present invention.
  • the pharmaceutical composition may also optionally include pharmaceutically acceptable carriers, excipients, fillers, binders, disintegrants, glidants, and/or media.
  • the crystalline form A and/or dihydrate of the present invention, or the pharmaceutical composition of the present invention can be administered by oral or injection or other administration routes.
  • the present invention also provides a dosage form containing the crystal form A and/or dihydrate of the present invention, including but not limited to tablets, capsules, powder injections, dispersions, etc., preferably tablets and powder injections .
  • the present invention also provides the application of the crystal form A and/or dihydrate or pharmaceutical composition of the present invention in the preparation of drugs for the treatment of neurological diseases, cardiovascular and cerebrovascular diseases and degenerative aging diseases.
  • the preparation method of the nitroxazine crystal form A and the nitroxazine dihydrate of the present invention is simple and easy for large-scale industrial production.
  • the nitroxazine crystal form A of the present invention is stable in heat treatment, mechanical treatment and acceleration (50°C, 75% RH) as crystal form A, with low risk of crystal transformation and good stability, which is beneficial to the preparation of preparations. And its stability during storage, effectively guarantee the consistency of the crystal form content in the preparation, safe, effective and controllable quality.
  • the nitroxazine crystal form A of the present invention is easily soluble in most solvents (such as acetonitrile, methanol, ethanol, acetone, ethyl acetate, etc.), is easily soluble in water, and has high biological activity and Good medicinal properties, high bioavailability, rapid drug effect, and high biological activity.
  • solvents such as acetonitrile, methanol, ethanol, acetone, ethyl acetate, etc.
  • Figure 1 is the XRD pattern of nitroxazine crystal form A
  • Figure 2 is the XRD comparison chart of nitroxazine crystal form A and dihydrate
  • Figure 3 is the DSC spectrum of nitroxazine crystal form A
  • Figure 4 is the DSC spectrum of nitroxazine crystal form A and dihydrate
  • Figure 5 is a biological microscope image of nitroxazine crystal form A
  • Figure 6 is the crystalline observation diagram of nitroxazine crystal form A
  • Figure 7 is the TGA spectrum of nitroxazine crystal form A
  • Figure 8 is a comparison chart of nitroxazine crystal form A and dihydrate TGA;
  • Figure 9 is the infrared spectrum of nitroxazine crystal form A
  • Figure 10 shows the XRD pattern of nitroxazine crystal form A after suspension beating for one week
  • Figure 11 is the XRD pattern of nitroxazine crystal form A after grinding for 10 minutes;
  • Figure 12 shows the XRD pattern of nitroxazine dihydrate after grinding for 10 minutes
  • TBN means the nitroxazine of the present invention.
  • Powder X-ray diffraction analyzer (Bruker D2PHASER), the voltage and tube current are 30KV and 10mA, respectively, the 2 ⁇ scanning angle of the sample is from 3°-40°, and the scanning step size is 0.02°.
  • the XRD patterns of nitroxazine crystal form A prepared in Examples 1-7 of the present invention are shown in Fig. 1 and Table 1.
  • the 2 ⁇ angle positions of the crystal form A in the XRD pattern are: 10.60 ⁇ 0.2, 11.03 ⁇ 0.2, There are characteristic diffraction peaks at 15.31 ⁇ 0.2, 15.55 ⁇ 0.2, 17.14 ⁇ 0.2, 17.93 ⁇ 0.2, and 23.81 ⁇ 0.2.
  • the DSC spectrum is shown in Figure 3.
  • the TGA map is shown in Figure 7.
  • the infrared spectrum is shown in Figure 9.
  • Nitrazine is cold-precipitated in a saturated ethanol aqueous solution (9% by volume of ethanol) in a binary system to obtain nitroxazine dihydrate.
  • the XRD patterns of the prepared dihydrate are shown in Figure 2 and Table 1.
  • the 2 ⁇ angle positions are: 8.91 ⁇ 0.2, 11.46 ⁇ 0.2, 14.29 ⁇ 0.2, 17.60 ⁇ 0.2, 21.19 ⁇ 0.2, 22.02 ⁇ 0.2, 23.19 ⁇ 0.2 , 24.30 ⁇ 0.2, 24.92 ⁇ 0.2, 29.20 ⁇ 0.2, 31.41 ⁇ 0.2 have characteristic diffraction peaks.
  • the DSC spectrum is shown in Figure 4, where the melting point of nitroxazine dihydrate is between 37-40°C.
  • the TGA map is shown in Figure 8.
  • nitroxazine crystal form A was slurried in petroleum ether (1.0 mL) at room temperature for 7 days, then the suspension was centrifuged, and solid samples were collected for XRPD characterization.
  • the crystal form A is cooled to -55°C and then heated to 25°C.
  • the crystal form A has not undergone a crystalline transformation, as shown in Figures 15 and 16.
  • SD rats were given intravenous injection of nitroxazine crystal form A at a dose of 30 mg/kg to investigate the distribution of nitroxazine crystal form A in various tissues.
  • Fig. 22 The results of the experiment are shown in Fig. 22.
  • the results show that after intravenous administration of 30 mg/kg of nitroxazine to male and female rats, all tissues reached rapid and widespread distribution almost at the same time as plasma, and were eliminated in vitro at the same time as plasma at 24 hours. There was no nitroxazine in the tested tissue Accumulation tendency.
  • the concentration of nitroxazine in the brain tissue is 0.6-0.7 times the plasma concentration after administration at 0.25h, and the plasma concentration is 0.8-1.1 times after 4h, and it is completely cleared like plasma at 24h.

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Abstract

本公开涉及硝酮嗪晶型、制备方法及其应用，提供一种硝酮嗪的晶型A，其XRPD图谱在2θ角度位置为：10.60±0.2、11.03±0.2、15.31±0.2、15.55±0.2、17.14±0.2、17.93±0.2、23.81±0.2处有特征衍射峰。所述的晶型A转晶风险小，稳定性好，有利于制剂在制备及其储存过程中的稳定性，有效保证制剂中晶型含量的一致性，安全、有效、质量可控，有较高的生物活性和良好的成药性，生物利用度高，药效发挥快，具有较高的生物活性。

Description

硝酮嗪晶型、制备方法及应用 技术领域
本发明涉及医药化学领域，具体涉及硝酮嗪晶型、制备方法及其应用。
背景技术
硝酮嗪是川芎嗪(TMP)的硝酮衍生物，是在川芎嗪的结构基础上增加硝酮药效基团经化学合成的全新化合物，简称为TBN，它的化学名为(顺)-2-甲基-N-[(3，5，6-三甲基吡嗪-2-)次甲基]2-丙胺氧化物，分子式为C 12H 19N 3O，分子量是221.30，具有下式所示的化学结构：
Figure PCTCN2019098724-appb-000001
硝酮嗪可以抑制缺血所致的神经细胞氧化损伤，从而发挥神经细胞保护作用，缓解脑栓塞伴随的神经症状及功能障碍。临床上可用于神经系统疾病、心脑血管疾病及退行性老化疾病等的治疗。
发明内容
本发明目的在于提供硝酮嗪晶型、制备方法及其应用。
本发明的目的之一在于提供一种硝酮嗪的晶型A，所述的晶型其XRPD图谱在2θ角度位置为：10.60±0.2、11.03±0.2、15.31±0.2、15.55±0.2、17.14±0.2、17.93±0.2、23.81±0.2处有特征衍射峰。
在一种具体的实施方案中，硝酮嗪的晶型A，所述的晶型其XRPD图谱在2θ角度位置为：10.60±0.2、11.03±0.2、13.51±0.2、15.31±0.2、15.55±0.2、17.14±0.2、17.93±0.2、21.22±0.2、23.81±0.2、25.23±0.2、27.08±0.2处有特征衍射峰。
在一种具体的实施例中，硝酮嗪的晶型A具有与说明书图1基本上相同的XRPD图谱。
进一步的，本发明所述的硝酮嗪晶型A有针状、方块状、棒状三种晶态，显微照片如图5和图6。
进一步的，本发明所述的硝酮嗪晶型A熔点分别在76℃-78℃。
进一步的，本发明所述的硝酮嗪晶型A的DSC图谱基本如图3。
进一步的，本发明所述的硝酮嗪晶型A的TGA图谱如图7。
进一步的，本发明提供了硝酮嗪晶型A红外图谱，如图9。
本发明经过系统的晶型筛选实验，其中筛选方法包括蒸发结晶法(单一溶剂法和混合溶剂法)，热溶冷析法和混悬打浆法等。筛选包括四氢呋喃、乙酸乙酯、甲苯、丙酮、二氧六环、异丙醇、石油醚、正己烷、乙酸异丙酯、异辛烷和乙酸异丁酯等溶剂，筛选结果均为晶型A。
进一步的，本发明还提供了一种硝酮嗪晶型A的制备方法，包括以下步骤：
(1)将硝酮嗪粗品与有机溶剂混合，水浴加热至60～80℃，搅拌过滤，滤液冷却结晶得晶体状固体；
(2)步骤(1)得到的晶体状固体和正庚烷混合加热溶解，冷却结晶得硝酮嗪晶型A。
进一步的，本发明所述的制备方法，步骤(1)中所述的有机溶剂选自乙酸乙酯、正己烷、正庚烷及环己烷中的一种或几种，进一步优选的，有机溶剂为正己烷或正庚烷；或者所述有机溶剂选自正己烷与乙酸乙酯的混合溶剂，申请人发现，选用优选的有机溶剂可以显著降低杂质含量。
进一步的，本发明所述的制备方法，步骤(1)中硝酮嗪粗品与有机溶剂的重量体积比为1：5～20，优选1：8～12。
进一步的，本发明所述的制备方法，步骤(1)中冷却结晶的温度选自2～12℃，更优选3～10℃，最优选为3～5℃，申请人发现，在最优选的范围内，可以显著降低杂质含量。
进一步的，本发明所述的制备方法，步骤(2)中硝酮嗪粗品与正庚烷的重量体积比优选1：1～5，最优选1:1～3。
进一步的，本发明所述的制备方法，步骤(2)中晶体状固体和正庚烷混合加热温度为60～80℃，优选65～75℃。
进一步的，本发明所述的制备方法，步骤(2)中冷却结晶的温度选自2～12℃，更优选4～10℃。
本发明的目的之二在于提供一种硝酮嗪的二水合物，其XRPD图谱在2θ角度位置为：8.91±0.2、11.46±0.2、14.29±0.2、17.60±0.2、21.19±0.2、22.02±0.2、23.19±0.2、24.30±0.2、24.92±0.2、29.20±0.2、31.41±0.2处有特征衍射峰。
在一种具体的实施方案中，硝酮嗪的二水合物，其XRPD图谱在2θ角度位置为：8.91±0.2、11.46±0.2、12.00±0.2、14.29±0.2、17.60±0.2、19.50±0.2、21.19±0.2、22.02±0.2、23.19±0.2、24.30±0.2、24.92±0.2、26.70±0.2、29.20±0.2、31.41±0.2、36.20±0.2处有特征衍射峰。
在一种具体的实施例中，硝酮嗪的二水合物具有与说明书图2基本上相同的XRPD图谱。
进一步的，本发明所述的硝酮嗪的二水合物熔点在37–40℃。
进一步的，本发明所述的硝酮嗪的二水合物的DSC图谱基本如图4。
进一步的，本发明所述的硝酮嗪的二水合物的TGA图谱如图8。
本发明所述的硝酮嗪二水合物在达到熔点时(约37–40℃)重量开始发生减少，失重13.67％。
本发明所述的硝酮嗪的二水合物是硝酮嗪在饱和的乙醇水溶液二元体系中冷析得到，优选乙醇水溶液中乙醇体积百分比为5～50％，更优选为5～20％。
在另一方面，本发明提供一种药物组合物，其包含一种或多种本发明的晶型A或二水合物。所述药物组合物还可以任选地包含药学上可接受的载体、赋形剂、填充剂、粘合剂、崩解剂、助流剂和/或介质等。
在另一方面，本发明所述的晶型A和/或二水合物，或者本发明所述的药物组合物可以通过口服或注射等给药途径。
在另一方面，本发明还提供一种包含本发明的晶型A和/或二水合物的剂型，包括但不限于片剂、胶囊剂、粉针剂、分散剂等，优选片剂和粉针剂。
在另一方面，本发明还提供本发明所述的晶型A和/或二水合物或药物组合物在用于制备治疗神经系统疾病、心脑血管疾病及退行性老化疾病药物中的应用。
本发明的有益效果：
(1)本发明所述的硝酮嗪晶型A和硝酮嗪二水合物制备方法简单，易于工业化大生产。
(2)本发明所述的硝酮嗪晶型A在热处理、机械处理和加速(50℃，75％RH)稳定的为晶型A，转晶风险小，稳定性好，有利于制剂在制备及其储存过程中的稳定性，有效保证制剂中晶型含量的一致性，安全、有效、质量可控。
(3)本发明所述的硝酮嗪晶型A在大部分溶剂中(如乙腈、甲醇、乙醇、丙酮和乙 酸乙酯等)易溶，在水中极易溶解，有较高的生物活性和良好的成药性，生物利用度高，药效发挥快，具有较高的生物活性。
附图说明
图1为硝酮嗪晶型A的XRD图谱；
图2为硝酮嗪晶型A与二水合物XRD对比图谱；
图3为硝酮嗪晶型A的DSC图谱；
图4为硝酮嗪晶型A与二水合物的DSC图谱；
图5为硝酮嗪晶型A的生物显微镜图；
图6为硝酮嗪晶型A的晶态观察图；
图7为硝酮嗪晶型A的TGA图谱；
图8为硝酮嗪晶型A与二水合物TGA对比图谱；
图9为硝酮嗪晶型A的红外光谱；
图10为硝酮嗪晶形A混悬打浆一周后XRD图谱；
图11为硝酮嗪晶型A研磨10min后XRD图谱；
图12为硝酮嗪二水合物研磨10min后XRD图谱；
图13晶型A压片处理试验XRPD分析图谱；
图14水合物压片处理试验XRPD分析图谱；
图15晶型A冷处理DSC图谱
图16晶型A冷处理XRPD图谱
图17晶型A热处理1DSC图谱
图18晶型A热处理1XRPD图谱；
图19硝酮嗪水合物热处理DSC图谱；
图20硝酮嗪水合物热处理XRPD图谱；
图21晶型A加速7天试验后XRPD图谱；
图22静脉给予SD大鼠硝酮嗪(30mg/kg，n＝6，雌雄各半)后组织分布结果。
具体实施方式
下面结合实施例对本发明做进一步说明，应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，凡在本发明的构思前提下对本发明制备方法的简单改进都属于本发明的保护范围之内。下面实施例未注明具体条件的实验方法，通常按照本领 域的公知手段。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
以下实施例及附图中，若无特别说明，代号TBN即表示本发明所述的硝酮嗪。
以下实施例中硝酮嗪晶型A及硝酮嗪二水合物的XRD图特征衍射峰是在以下实验条件下测得的：
粉末X射线衍射分析仪(Bruker D2PHASER)，电压和管电流分别为30KV和10mA，样品的2θ扫描角度为从3°-40°，扫描步长为0.02°。
实施例1
在250ml圆底烧瓶中，加入硝酮嗪粗品(10g)和环己烷(100ml)及乙酸乙酯(2ml),调节转速140±5rpm，水浴加热至70℃，持续搅拌，然后趁热过滤得橙黄色溶液。滤液静置冷却至环境温度(10℃)，然后再置于4℃下的冷库中静置过滤，滤饼用正己烷洗涤，减压除去溶剂得到浅黄色晶体状固体。
在250ml圆底烧瓶中，加入上述硝酮嗪粗品固体及其2体积的正庚烷，调节转速为140±5rpm，水浴加热至70℃，持续搅拌，得橙黄色澄清溶液。撤去水浴，继续搅拌、冷却至10℃结晶、抽滤，固体用正庚烷洗涤，于38℃下真空干燥24小时至干燥失重<1.0％得到硝酮嗪晶型A。
实施例2
在250ml圆底烧瓶中，加入硝酮嗪粗品(10g)和正己烷(100ml)及乙酸乙酯(2ml),调节转速140±5rpm，水浴加热至70℃，持续搅拌，然后趁热过滤得橙黄色溶液。滤液静置冷却至环境温度(10℃)，然后再置于4℃下的冷库中静置过滤，滤饼用正己烷洗涤，减压除去溶剂得到浅黄色晶体状固体。
在250ml圆底烧瓶中，加入上述硝酮嗪粗品固体及其2体积的正庚烷，调节转速为140±5rpm，水浴加热至70℃，持续搅拌，得橙黄色澄清溶液。撤去水浴，继续搅拌、冷却至10℃结晶、抽滤，固体用正庚烷洗涤，于38℃下真空干燥24小时至干燥失重<1.0％得到硝酮嗪晶型A。
实施例3
在250ml圆底烧瓶中，加入硝酮嗪粗品(10g)和环己烷(100ml)，调节转速140±5rpm，水浴加热至70℃，持续搅拌，然后趁热过滤得橙黄色溶液。滤液静置冷却至环境温度(10℃)，然后再置于4℃下的冷库中静置过滤，滤饼用正己烷洗涤，减压除去溶剂得到 浅黄色晶体状固体。
在250ml圆底烧瓶中，加入上述硝酮嗪粗品固体及其2体积的正庚烷，调节转速为140±5rpm，水浴加热至70℃，持续搅拌，得橙黄色澄清溶液。撤去水浴，继续搅拌、冷却至10℃结晶、抽滤，固体用正庚烷洗涤，于38℃下真空干燥24小时至干燥失重<1.0％得到硝酮嗪晶型A。
实施例4
在250ml圆底烧瓶中，加入硝酮嗪粗品(10g)和正己烷(100ml),调节转速140±5rpm，水浴加热至70℃，持续搅拌，然后趁热过滤得橙黄色溶液。滤液静置冷却至环境温度(10℃)，然后再置于4℃下的冷库中静置过滤，滤饼用正己烷洗涤，减压除去溶剂得到浅黄色晶体状固体。
在250ml圆底烧瓶中，加入上述硝酮嗪粗品固体及其2体积的正庚烷，调节转速为140±5rpm，水浴加热至70℃，持续搅拌，得橙黄色澄清溶液。撤去水浴，继续搅拌、冷却至10℃结晶、抽滤，固体用正庚烷洗涤，于38℃下真空干燥24小时至干燥失重<1.0％得到硝酮嗪晶型A。
实施例5
在250ml圆底烧瓶中，加入硝酮嗪粗品(10g)和正庚烷(100ml)，调节转速140±5rpm，水浴加热至70℃，持续搅拌，然后趁热过滤得橙黄色溶液。滤液静置冷却至环境温度(10℃)，然后再置于4℃下的冷库中静置过滤，滤饼用正己烷洗涤，减压除去溶剂得到浅黄色晶体状固体。
在250ml圆底烧瓶中，加入上述硝酮嗪粗品固体及其2体积的正庚烷，调节转速为140±5rpm，水浴加热至70℃，持续搅拌，得橙黄色澄清溶液。撤去水浴，继续搅拌、冷却至10℃结晶、抽滤，固体用正庚烷洗涤，于38℃下真空干燥24小时至干燥失重<1.0％得到硝酮嗪晶型A。
实施例6
在250ml圆底烧瓶中，加入硝酮嗪粗品(10g)和正己烷(100ml)，调节转速140±5rpm，水浴加热至70℃，持续搅拌，然后趁热过滤得橙黄色溶液。滤液静置冷却至环境温度(10℃)，然后再置于10℃下的冷库中静置过滤，滤饼用正己烷洗涤，减压除去溶剂得到浅黄色晶体状固体。
在250ml圆底烧瓶中，加入上述硝酮嗪粗品固体及其2体积的正庚烷，调节转速为 140±5rpm，水浴加热至70℃，持续搅拌，得橙黄色澄清溶液。撤去水浴，继续搅拌、冷却至10℃结晶、抽滤，固体用正庚烷洗涤，于38℃下真空干燥24小时至干燥失重<1.0％得到硝酮嗪晶型A。
实施例7
在250ml圆底烧瓶中，加入硝酮嗪粗品(10g)和正己烷(100ml),调节转速140±5rpm，水浴加热至70℃，持续搅拌，然后趁热过滤得橙黄色溶液。滤液静置冷却至环境温度(10℃)，然后再置于4℃下的冷库中静置过滤，滤饼用正己烷洗涤，减压除去溶剂得到浅黄色晶体状固体。
在250ml圆底烧瓶中，加入上述硝酮嗪粗品固体及其2体积的正庚烷，调节转速为140±5rpm，水浴加热至70℃，持续搅拌，得橙黄色澄清溶液。撤去水浴，继续搅拌、冷却至10℃结晶、抽滤，固体用正庚烷洗涤，于38℃下真空干燥24小时至干燥失重<1.0％得到硝酮嗪晶型A。
本发明实施例1～7制备的硝酮嗪晶型A，XRD图谱如图1和表1所示，所述的晶型A在XRD图谱的2θ角度位置为：10.60±0.2、11.03±0.2、15.31±0.2、15.55±0.2、17.14±0.2、17.93±0.2、23.81±0.2处有特征衍射峰。DSC图谱如图3所示，其中有1个吸热峰，为熔融峰，熔点在76℃-78℃之间。通过图5和图6的显微照片可见，硝酮嗪有三种晶态，分别为针状、方块状、棒状。TGA图谱如图7所示。红外光谱如图9所示。
实施例8
硝酮嗪在饱和的乙醇水溶液(乙醇体积百分比为9％)二元体系中冷析得到硝酮嗪二水合物。制备的二水合物在XRD图谱如图2和表1所示，在2θ角度位置为：8.91±0.2、11.46±0.2、14.29±0.2、17.60±0.2、21.19±0.2、22.02±0.2、23.19±0.2、24.30±0.2、24.92±0.2、29.20±0.2、31.41±0.2处有特征衍射峰。DSC图谱如图4所示，其中硝酮嗪二水合物熔点在37–40℃之间。TGA图谱如图8所示。
表1 晶型A与二水合物特征峰对比
Figure PCTCN2019098724-appb-000002
Figure PCTCN2019098724-appb-000003
制备方法考察：
1、溶剂对硝酮嗪终产品收率及品质的影响，结果如表2所示：
表2.溶剂对硝酮嗪终产品收率及品质的影响
Figure PCTCN2019098724-appb-000004
2、结晶温度对硝酮嗪终产品收率及品质的影响，如表2所示：
表3 结晶温度对硝酮嗪终产品收率及品质的影响
Figure PCTCN2019098724-appb-000005
Figure PCTCN2019098724-appb-000006
效果考察：
1、稳定性考察
1.1混悬转晶试验
(1)将硝酮嗪晶型A在石油醚中(1.0mL)，室温条件下打浆7天后，对混悬液进行离心，收集固体样品进行XRPD表征。
结果显示：硝酮嗪晶形A在石油醚中混悬打浆一周，依旧为晶形A，XRD图谱见图10。
1.2机械处理稳定性试验
(2)将硝酮嗪晶型A和硝酮嗪二水合物分别研磨10min，对处理后的样品用XRPD加以分析。
结果显示：本发明所述的晶型A研磨10min后XRPD特征峰未发生改变。硝酮嗪二水合物研磨10min后未发生晶型的改变，图谱见图11和图12。
1.3压片处理试验
(3)将晶型A和硝酮嗪二水合物进行压片处理，压力为2tons，压片时间为2min，压制成片状后用XRPD加以分析。
结果显示：晶形A和硝酮嗪二水合物加压2tons，施压2min，均未发生晶形改变(结晶度均变小)见图13和14。
1.4冷热处理稳定性实验
(4)将晶型A冷却至-55℃，再升温至25℃，晶型A未发生晶型的转变，见图15和16。
(5)将晶型A加热至110℃(降解点之前)，后冷却至25℃，得到黄色油状液体，搅拌后有白色固体析出，经过XRPD表征为晶型A，见图17和18。
(6)将硝酮嗪水合物加热至110℃(降解点之前)，后冷却至25℃，得到黄色油状液体，搅拌后有白色固体析出，经过XRPD表征为晶型A，见图19和20。
1.5加速影响实验
晶型A在40℃，RH＝75％环境下加速7天，XRPD表征结果为晶型A，见图21。
2、生物活性考察
在食蟹猴脑卒中动物模型药效学研究的同时，我们也进行了硝酮嗪晶型A在食蟹猴脑脊液中的药代动力学研究。与第二次给药(30mg/kg,第一次静脉给药30mg/kg后6h)后10min、30min、60min、120min各采0.5ml脑脊液进行药代动力学检测。
表4 食蟹猴静脉注射给予30mg/kg硝酮嗪的脑脊液药物浓度-时间数据
Figure PCTCN2019098724-appb-000007
表4的实验结果显示第二次给药后10min，脑脊液中的硝酮嗪浓度为176μM，几乎与血浆中的药物浓度相等(195μM)，超过了硝酮嗪在体外细胞实验中的有效保护浓度30μM。此结果表明硝酮嗪晶型A是可以通过血脑屏障，并且能够达到有效保护浓度。
3、代谢数据清除率
SD大鼠静脉注射给予30mg/kg剂量的硝酮嗪晶型A，考察硝酮嗪晶型A在各组织中的分布情况。
实验结果如图22，结果显示雌雄大鼠静脉给药硝酮嗪30mg/kg后，所有组织几乎与血浆同时达到迅速广泛分布，且24h基本与血浆同时清除体外，硝酮嗪在检测组织中没有蓄积倾向。给药后0.25h硝酮嗪在脑组织中的浓度为血浆浓度的0.6-0.7倍，4h后为血浆浓度0.8-1.1倍，24h同血浆一样完全清除。

Claims (10)

  1. 一种硝酮嗪的晶型A，其特征在于，所述的晶型其XRPD图谱在2θ角度位置为：10.60±0.2、11.03±0.2、15.31±0.2、15.55±0.2、17.14±0.2、17.93±0.2、23.81±0.2处有特征衍射峰；优选的，所述的晶型A其XRPD图谱在2θ角度位置为：10.60±0.2、11.03±0.2、13.51±0.2、15.31±0.2、15.55±0.2、17.14±0.2、17.93±0.2、21.22±0.2、23.81±0.2、25.23±0.2、27.08±0.2处有特征衍射峰。
  2. 根据权利要求1所述的晶型A，其特征在于，具有与图1基本上相同的XRPD图谱。
  3. 根据权利要求1所述的晶型A，其特征在于，A熔点在76℃-78℃。
  4. 根据权利要求1所述的晶型A，其特征在于，DSC图谱基本如图3，TGA图谱基本如图7，红外图谱基本如图9。
  5. 权利要求1～4任一项所述晶型A的制备方法，其特征在于，包括以下步骤：
    (1)将硝酮嗪粗品与有机溶剂混合，水浴加热至60～80℃，搅拌过滤，滤液冷却结晶得晶体状固体；
    (2)步骤(1)得到的晶体状固体和正庚烷混合加热溶解，冷却结晶得硝酮嗪晶型A。
  6. 根据权利要求5所述的制备方法，其特征在于，步骤(1)中所述的有机溶剂选自乙酸乙酯、正己烷、正庚烷及环己烷中的一种或几种，进一步优选的，有机溶剂为正己烷或正庚烷；或者所述有机溶剂选自正己烷与乙酸乙酯的混合溶剂；步骤(1)中硝酮嗪粗品与有机溶剂的重量体积比为1：5～20，优选1：8～12；步骤(1)中冷却结晶的温度选自2～12℃，更优选3～10℃，最优选为3～5℃；步骤(2)中硝酮嗪粗品与正庚烷的重量体积比优选1：1～5，最优选1:1～3；步骤(2)晶体状固体和正庚烷混合加热温度为60～80℃，优选65～75℃；冷却结晶的温度选自2～12℃，更优选4～10℃。
  7. 一种硝酮嗪的二水合物，其特征在于，其XRPD图谱在2θ角度位置为：8.91±0.2、11.46±0.2、14.29±0.2、17.60±0.2、21.19±0.2、22.02±0.2、23.19±0.2、24.30±0.2、24.92±0.2、29.20±0.2、31.41±0.2处有特征衍射峰；优选的，其XRPD图谱在2θ角度位置为：8.91±0.2、11.46±0.2、12.00±0.2、14.29±0.2、17.60±0.2、19.50±0.2、21.19±0.2、22.02±0.2、23.19±0.2、24.30±0.2、24.92±0.2、26.70±0.2、29.20±0.2、31.41±0.2、36.20±0.2处有特征衍射峰。
  8. 根据权利要求7所述的二水合物，其特征在于，熔点在37–40℃。
  9. 一种药物组合物，其特征在于，包含权利要求1～4任一项所述的晶型A或权利要求 7～8任一项所述的二水合物。
  10. 权利要求1～4任一项所述的晶型A或权利要求7～8任一项所述的二水合物在用于制备治疗神经系统疾病、心脑血管疾病及退行性老化疾病药物中的应用。
PCT/CN2019/098724 2019-04-01 2019-07-31 硝酮嗪晶型、制备方法及应用 WO2020199440A1 (zh)

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US17/600,915 US20220153707A1 (en) 2019-04-01 2019-07-31 Crystal form of tetramethylpyrazine nitrone, preparation method and use thereof
CA3135637A CA3135637A1 (en) 2019-04-01 2019-07-31 Crystal form of tetramethylpyrazine nitrone, preparation method and use thereof
IL286834A IL286834A (en) 2019-04-01 2021-09-30 Crystal form of nitro tetramethylpyrazine, method of preparation and use thereof

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