WO2020206603A1 - 2-(2，4，5-取代苯氨基)嘧啶衍生物及其晶形b - Google Patents

2-(2，4，5-取代苯氨基)嘧啶衍生物及其晶形b Download PDF

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WO2020206603A1
WO2020206603A1 PCT/CN2019/081902 CN2019081902W WO2020206603A1 WO 2020206603 A1 WO2020206603 A1 WO 2020206603A1 CN 2019081902 W CN2019081902 W CN 2019081902W WO 2020206603 A1 WO2020206603 A1 WO 2020206603A1
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crystal form
lung cancer
doxitinib
osimertinib
pyrimidine derivative
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PCT/CN2019/081902
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French (fr)
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常俊标
杜锦发
朱凯凯
王凯
李建永
张春霞
戈东旭
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河南真实生物科技有限公司
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Priority to PCT/CN2019/081902 priority Critical patent/WO2020206603A1/zh
Priority to EA202100252A priority patent/EA202100252A1/ru
Priority to EP19924592.9A priority patent/EP3954682A4/en
Publication of WO2020206603A1 publication Critical patent/WO2020206603A1/zh
Priority to US17/497,994 priority patent/US20220024897A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • 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 invention relates to a new type of anti-lung cancer medicine, in particular to 2-(2,4,5-substituted aniline) pyrimidine derivatives and crystal form B thereof, belonging to the field of medicinal chemistry.
  • Non-small cell lung cancer is the “majority” of lung cancer, accounting for about 85% of lung cancer cases; in recent years, the diagnosis and treatment of non-small cell lung cancer (NSCLC) has changed significantly.
  • NSCLC non-small cell lung cancer
  • EGFR is the most common gene mutation in patients with non-small cell lung cancer, and the corresponding targeted drug EGFR-TKI has been on the market for many years. According to some conventions, EGFR-TKI is currently divided into three generations: the first generation is gefitinib, erlotinib and icotinib; the second generation is afatinib and Dacomitinib; the third generation is currently only A osimertinib is on the market. Normally, patients with advanced non-small cell lung cancer will be treated with the first-generation EGFR-TKI after genetic testing confirms EGFR mutations.
  • Osimertinib was developed by AstraZeneca, and the R&D stage code is AZD9291.
  • the State Food and Drug Administration approved the import application of osimertinib, only 1 year and 4 months after the first approval in the world. The approval process for Osimertinib's listing in my country only took 7 months, reflecting the urgency of patients' needs.
  • the indole N-methyl group of osimertinib is easily oxidized by P450 in the body to produce desmethyl-osimertinib (AZ5104, below).
  • the activity of AZ5104 to inhibit wild EGFR is 14.5 times stronger than that of osimertinib, while its anti-cancer activity is only 7.5 times stronger.
  • the inhibitory effect on wild EGFR is believed to be related to the occurrence of side effects such as skin rashes after medication (J.Med.Chem.2014, 57, 8249). Therefore, a new type with stronger anti-cancer activity and less side effects has been found Drugs to treat lung cancer are still necessary.
  • Henan Meitaibao Biopharmaceutical Co., Ltd. has developed a series of new drugs of 2-(2,4,5-substituted aniline) pyrimidine derivatives and applied for a patent "2-(2,4,5-substituted aniline) pyrimidine derivatives , Its preparation method and its application in the preparation of anti-tumor drugs" (2017112825988). In order to find a compound with better biological activity, the applicant made further compound modifications on this basis.
  • the purpose of the present invention is to provide a new type of anti-lung cancer drug-2-(2,4,5-substituted aniline) pyrimidine derivative I and its crystal form B.
  • Compound I is referred to as Dositinib.
  • the inventors found through preclinical animal pharmacokinetic and pharmacodynamic studies that the indole N-demethylated metabolite AZ5104-2D of doxitinib is better than AZ5104 of osimertinib (see Figure 2-5 and Table 1)
  • the production of EGFR was reduced by 98% (male mice) and 83% (female mice), which is beneficial to reduce the toxic side effects caused by inhibiting wild EGFR.
  • crystal form B the dominant crystal form of doxitinib is crystal form B.
  • the crystal form obtained by rapid crystallization or solvent evaporation is A, or a mixed crystal of A and B.
  • AZD9291 obtained a mixture of crystal forms A and B under the same conditions ( Figure 8), which was also a mixture of crystal forms A and B purchased commercially.
  • the anti-cancer activity of osimertinib crystal form B is significantly higher than that of the purchased osimertinib raw material ( Figure 8, a mixture of crystal forms A and B) in the HCC-827 nude mouse model, the former is twice that of the latter ( figure 1).
  • the CuK ⁇ -XRD pattern of the doxitinib crystal form B is shown in FIG. 7.
  • the advantages of the present invention include: 1.
  • the results of pharmacokinetics and pharmacodynamic studies have confirmed that compared with osimertinib, doxitinib or its crystal form B can significantly reduce the toxicity of the more toxic indole N demethylation metabolite Generate and improve efficacy.
  • the bioavailability of doxitinib crystal form B is better. Therefore, Doxitinib is expected to be developed as a safer and more effective treatment for non-small cell lung cancer with EGFR mutations than osimertinib.
  • Figure 1 is a comparison diagram of the anti-cancer activity (HCC-827) of doxitinib crystal form B and osimertinib;
  • Figure 2 is a graph of the average plasma concentration of Doxitinib in male and female mice after intragastric administration of Doxitinib Form B 12mg/kg;
  • Figure 3 is a graph showing the average concentration of osimertinib (AZD9291) in plasma of male and female mice after intragastric administration of 12 mg/kg of osimertinib over time;
  • Figure 4 is a graph showing the change of the average concentration of AZ5104-D2 in plasma in male and female mice at a dose of 12 mg/kg doxitinib crystal B over time;
  • Figure 5 is a graph showing the change of the average concentration of AZ5104 in plasma in male and female mice at a dose of 12 mg/kg osimertinib over time;
  • Figure 6 shows the DSC and TGA spectra of Doxitinib Form B.
  • Figure 7 is the CuK ⁇ -XRD pattern of Doxitinib crystal form B.
  • Figure 8 is a eutectic CuK ⁇ -XRD pattern of commercially available AZD9291.
  • Example 5 in vivo pharmacodynamic study of doxitinib single crystal B and AZD9291 on human non-small cell lung cancer HCC827 cell subcutaneous xenograft tumor BALB/c nude mouse model
  • Cell culture Human non-small cell lung cancer HCC827 (ATCC-CRL-2868) in vitro monolayer culture, culture conditions: RPMI-1640 medium with 10% fetal bovine serum, 100U/mL penicillin and 100 ⁇ g/mL Streptomycin was cultured in a 5% CO 2 incubator at 37°C. Use pancreatin-EDTA for routine digestion and passage twice a week. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.
  • mice BALB/c nude mice, female, 6-8 weeks old, weighing 18-22 grams. A total of 60 (40+50%) are needed. Provided by Shanghai Sipuer-Bike Laboratory Animal Co., Ltd. or other companies.
  • Tumor inoculation 0.2mL (1 ⁇ 10 7 cells) of HCC827 cells (with matrigel, volume ratio 1:1) were subcutaneously inoculated on the right back of each mouse, and the grouping began when the average tumor volume reached 150-200mm 3 Administration.
  • the experimental grouping and dosing schedule are shown in the table below.
  • Dosing volume 10 ⁇ l/g based on mouse body weight. If the weight loss exceeds 15%, the dosage regimen should be adjusted accordingly.
  • the experimental indicators are to investigate whether tumor growth is inhibited, delayed or cured.
  • the tumor diameter was measured with vernier calipers twice a week.
  • TGI (%) reflects the tumor growth inhibition rate.
  • TGI(%) [(1-(Average tumor volume at the end of a certain treatment group-average tumor volume at the beginning of the treatment group))/(Average tumor at the end of treatment in the solvent control group Volume-the average tumor volume at the start of treatment in the solvent control group)] ⁇ 100%.
  • the relative tumor volume (RTV) is calculated according to the results of tumor measurement.
  • T weight and C weight represent the tumor weight of the administration group and the vehicle control group, respectively.
  • T test was used for comparison between two groups.
  • One-way ANOVA is used for comparison between three or more groups. If the F value is significantly different, multiple comparisons should be performed after the ANOVA analysis.
  • SPSS 17.0 for all data analysis. p ⁇ 0.05 considered a significant difference.
  • the pharmacological results are shown in Figure 1.
  • the anti-cancer activity of doxitinib single crystal B (code 90-1408) 0.5 mg/kg in the nude mouse HCC-827 model is equivalent to 1 mg/kg of AZD9291. It shows that the anti-cancer activity of Doxitinib single crystal B in this model is doubled compared with AZD9291.
  • HPMC hydroxypropyl methylcellulose, 4000CP
  • the preparations for intragastric administration are prepared the day before administration, and the preparations are analyzed. When not in use, the preparations are stored in a refrigerator at 2°C to 8°C and used within 8 days.
  • rats (SD) Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., 36 rats (18 males and 18 females), 6-10 weeks old, 200 ⁇ 300g (male); 170 ⁇ 280g( Female).
  • the animals in the first group were given doxitinib single crystal B (code 90-1408) solution via a single injection through the tail vein; the animals in the second, third, and fourth groups were given different oral administration methods.
  • Dositinib single crystal B (code 90-1408) solution the volume of administration is shown in section 4.4; group 5 animals, once a day, for 7 consecutive days, doxitinib single crystal B (code 90- 1408) solution, the administration volume was 10 mL/kg, the animals in group 6 were given a single oral administration of AZD9291 (Mesylate) solution by gavage, the administration volume was 10 mL/kg. 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24h before and after administration.
  • Medication Collect whole blood samples (about 0.15mL for group 5, about 0.23mL for other groups) through jugular vein (or other suitable blood collection site) puncture method at the specified time, wet ice operation throughout, and record in the experimental record Actual blood collection time.
  • the acceptable error of the collection time point is the time point within 1 hour of administration ⁇ 1 minute, and the other time point is the theoretical time ⁇ 5%.
  • Plasma samples The plasma concentrations of doxitinib single crystal B and AZD9291 and their metabolites AZ5104-D2 and AZ5104 in plasma were confirmed by the bioanalytical department of Shanghai WuXi AppTec New Drug Development Co., Ltd. using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) for determination.
  • LC-MS/MS high performance liquid chromatography-tandem mass spectrometry
  • the retention time of compounds and internal standards, chromatogram acquisition and chromatogram integration are processed by the software Analyst (Applied Biosystems), and the data statistics are processed by the software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems).
  • the unit of the analyte concentration in the sample is nM, with 3 significant digits reserved, and all values expressed in percentage (such as% deviation and% coefficient of variation, etc.) are kept to one decimal place.

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Abstract

本发明公开了一种新型抗肺癌药物，具体涉及2-(2，4，5-取代苯氨基)嘧啶衍生物I及其晶形B，属于药物化学领域。其具有如下结构： (I) 据药代和药效研究结果，与奥希替尼相比，哆希替尼显著减少了毒性较大的N-Me代谢产物生成，并保持或提高了药效。哆希替尼可望开发成较奥希替尼更安全有效的治疗具有EGFR突变的非小细胞肺癌新药。

Description

2-(2，4，5-取代苯氨基)嘧啶衍生物及其晶形B 技术领域
本发明涉及一种新型抗肺癌药物，具体涉及2-(2，4，5-取代苯氨基)嘧啶衍生物及其晶形B，属于药物化学领域。
背景技术
肺癌是发病率和死亡率最高的恶性肿瘤之一，全球每年约有160万人死于这类癌症，中国在2017年肺癌病人达到80万。非小细胞肺癌(non-small cell lung cancer，NSCLC)作为肺癌的“绝大多数”，约占肺癌病例的85％；最近几年，非小细胞肺癌(NSCLC)的诊疗已经有了显著改变。随着非小细胞肺癌进入精准医疗时代，根据患者肺癌的基因特征选择对应靶向药物治疗是目前的流行趋势。特别是在晚期非小细胞肺癌的治疗中，靶向药扮演着重要角色。EGFR是非小细胞肺癌患者最常见的基因突变，相应的靶向药物EGFR-TKI也已上市多年。根据一些约定俗成的说法，EGFR-TKI目前分为三代：第一代为吉非替尼、厄洛替尼和埃克替尼；第二代有阿法替尼、Dacomitinib；第三代，目前仅有一款奥希替尼上市。通常情况下，晚期非小细胞肺癌患者经基因检测确认EGFR突变后会先采用第一代EGFR-TKI治疗。但遗憾的是，几乎所有服用EGFR-TKI的患者到最后都会耐药，而导致耐药最重要的原因就是T790M突变。因此，克服T790M突变导致的耐药就是新一代EGFR-TKI的使命，奥希替尼就是其中的佼佼者。奥希替尼由阿斯利康公司研发，研发阶段代号为AZD9291。2017年3月，国家食品药品监管总局批准奥希替尼的进口申请，距全球首次批准时间仅相隔1年零4个月。奥希替尼在我国上市的审批环节仅用时7个月，从侧面体现了患者需求的迫切性。但是，奥希替尼的吲哚N-甲基容易被体内的P450氧化而脱去生成脱甲基-奥希替尼(AZ5104，如下)。AZ5104抑制野生EGFR的活性比奥希替尼的强14.5倍，而抗癌活性仅强7.5倍。而且，对野生EGFR的抑制作用被认为与用药后的皮疹等毒副作用的发生有关(J.Med.Chem.2014，57，8249)，因此，发现抗癌活性更强，毒副作用更小的新型治疗肺癌药仍然十分必要。
Figure PCTCN2019081902-appb-000001
河南美泰宝生物制药有限公司研发了2-(2，4，5-取代苯氨基)嘧啶衍生物一系列新药，并申请了专利“2-(2，4，5-取代苯氨基)嘧啶衍生物、其制备方法及其在制备抗肿瘤药物中的应用”(2017112825988)。为发现生物活性更好的化合物，申请人在此基础上进行了进一步化合物修饰。
发明内容
本发明目的在于提供一种新型抗肺癌药物——2-(2，4，5-取代苯氨基)嘧啶衍生物I及其晶型B，化合物I简称哆希替尼(Dositinib)。
为实现本发明目的，技术方案如下：
哆希替尼中文化学名：N-(2-((2-(二甲胺基)乙基)(甲基)胺基)-4-甲氧基-5-((4-(1-氘代甲基-1H-吲哚-3-)嘧啶-2-)氨基-)氘代乙酰胺基苯)英文化学名：N-(2-{[(2-(Dimethylamino)ethyl)](methyl)amino)-4-methoxy-5-((4-(d3-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)d2-acrylamide-mesylate。该化合物结构式如下：
Figure PCTCN2019081902-appb-000002
本发明人通过临床前动物药代和药效学研究发现，哆希替尼的吲哚N-脱甲基代谢产物AZ5104-2D较奥希替尼的AZ5104(见图2-5和表1)的生成量分别减少了98％(雄鼠)和83％(雌鼠)，这有利于减少因抑制野生EGFR而产生的毒副作用。
Figure PCTCN2019081902-appb-000003
表1 哆希替尼与AZ9291的大鼠药代动力学结果
Figure PCTCN2019081902-appb-000004
本发明人还发现，哆希替尼的优势晶型为晶型B。快速结晶或通过溶剂蒸发所得到的晶型为A，或者是A和B的混晶。而AZD9291在同样条件下，得到晶型A和B的混合物(图8)，商业购买的也是晶型A和B的混合物。哆希替尼晶型B比购买的奥希替尼原料药(图8，晶型A和B的混合物)在HCC-827裸鼠模型的抗癌活性显著提高，前者是后者的2倍(图1)。动物的药代动力学研究结果表明，哆希替尼晶型B的生物利用度较AZD9291混晶的原料药提高了20％(图2-5，表1)。哆希替尼晶型B比AZD9291混晶生物利用度的提高可能是其抗癌活性增强的原因之一。
所述哆希替尼晶型B的CuKα-XRD图谱如附图7所示。
本发明的优点包括：1、经药代和药效研究结果证实，与奥希替尼相比，哆希替尼或其晶形B能显著减少毒性较大的吲哚N脱甲基代谢产物的生成，并提高药效。2、哆希替尼晶型B的生物利用度更好。因此，哆希替尼可望开发成较奥希替尼更安全有效的治疗具有EGFR突变的非小细胞肺癌新药。
附图说明
图1为哆希替尼晶型B与奥希替尼抗癌活性(HCC-827)对比图；
图2为哆希替尼晶型B 12mg/kg剂量灌胃雌雄小鼠后血浆中哆希替尼平均浓度随时间变化图；
图3为奥希替尼(AZD9291)12mg/kg剂量灌胃雌雄小鼠后血浆中奥希替尼平均浓度随时间变化图；
图4为哆希替尼晶型B 12mg/kg剂量灌胃雌雄小鼠后血浆中AZ5104-D2平均浓度随时间变化图；
图5为奥希替尼12mg/kg剂量灌胃雌雄小鼠后血浆中AZ5104平均浓度随时间变化图；
图6为哆希替尼晶型B的DSC和TGA图谱。
图7为哆希替尼晶型B的CuKα-XRD图谱。
图8为商业购买的AZD9291的共晶CuKα-XRD图谱。
具体实施方式
为对本发明进行更好地说明，举实施例如下：
实施例1、制备中间体9
第一步：
Figure PCTCN2019081902-appb-000005
将化合物1(2.00kg,1.71mol)和KOH(1.44kg,25.6mol)加入预冷在0-15℃的DMF(6.0L)中，混合物搅拌0.5h。将化合物2(2.47kg,17.1mol)在0-5℃，4h内滴入上述混合物中，反应混合物在0-10℃继续搅拌2h,然后5-15℃搅拌12h。加入冰水(15.0L)后，用石油醚(10L)和甲基叔丁醚(10.0L)混合液萃取，有机相用盐水洗，Na 2SO 4干燥，过滤，蒸去溶剂得到一油状物(2.60kg，82％纯度，含有溶剂)。 1H NMR(400MHz,DMSO-d 6)δppm6.41-6.48(m,1H)；6.96-7.03(m,1H)；7.03-7.05(m,1H)；7.11-7.17(m,1H)；7.24-7.26(m,1H)；7.55-7.57(m,1H)。
第二步
Figure PCTCN2019081902-appb-000006
将化合物4(2.42kg，16.24mol)，DME(8.0L)和无水FeCl 3(2.64kg，16.3mol)加入反应器中，反应温度控制在10℃，然后加热到60℃。将化合物3(2.18kg，16.2mol)的DME(2.0L)溶液加入，搅拌反应2h。在10-30℃先后将甲醇(5.0L)和水(10L)加入反应混合物，搅拌0.5h,过滤得到红色固体(6kg)。用乙氰(12.0L)和水(24.0L)混合搅拌，过滤得到红色固体(4kg)。再与乙氰(5.0L)混合搅拌1h，过滤，干燥，得到产物5(2.14kg，54％)。 1H NMR(400MHz,DMSO-d 6)δppm 7.20-7.36(m,2H)；7.58(br d,J＝7.70Hz,1H)；7.82(d,J＝5.38Hz,1H)；8.41(br d,J＝7.58Hz,1H)；8.47-8.57(m,2H).
第三步
Figure PCTCN2019081902-appb-000007
将化合物5(1.0kg，3.81mol)，乙氰(8.0L)，对甲苯磺酸单水合物(0.435kg，2.29mol)和化合物6(0.852kg，4.57mol)混合于反应器内，在80℃搅拌12h，冷却至20℃，过滤。所得固体用乙氰(3.0L x 6)洗，在空气中干燥得到一黄色固体7(1.2kg，85％)。 1H NMR(400MHz,DMSO-d 6)δppm 4.00 (s,3H)；7.09-7.17(m,1H)；7.31(t,J＝7.64Hz,1H)；7.40-7.52(m,2H)；7.59(d,J＝8.19Hz,1H)；8.09-8.40(m,2H)；8.49-8.69(m,1H)；8.83(br d,J＝7.83Hz,1H).
第四步
Figure PCTCN2019081902-appb-000008
将化合物7(3.60kg，8.81mol)，DIPEA(1.76kg，13.6mol)，DMA(14.4L)，在15℃加入化合物8(1.11kg，10.9mol)。反应混合物在80℃搅拌12h，加入乙氰(21.6L)，冷却至20℃，析出红色固体，过滤，乙氰(12L)洗，得到固体9(3.45kg，81％)。 1H NMR(400MHz,DMSO-d 6)δppm 2.16(s,6H)2.44-2.48(m,2H)2.86(s,3H)3.26(t,J＝6.85Hz,2H)3.95(s,3H)6.85(s,1H)7.11(t,J＝7.52Hz,1H)7.18-7.29(m,2H)7.52(d,J＝8.07Hz,1H)8.09(s,1H)8.27-8.42(m,3H)8.62(s,1H).
实施例2、制备化合物12
第一步
Figure PCTCN2019081902-appb-000009
将化合物9(2.39g,5mmol)、MeOH(200mL)、甲酸铵(2.39g)和钯碳(200mg,5％)依次加入单口瓶，反应化合物在氢气球下搅拌16h，过滤，滤液浓缩至干，加入水(100mL)，用饱和碳酸氢钠溶液调pH为9，混合物用DCM萃取(3x100mL)，有机相合并，Na 2SO 4干燥，过滤，蒸去溶剂得到白色固体10(2.05g,91％)。 1H-NMR(400MHz,DMSO-d 6)δ2.18(s,6H),2.36(t,J＝6.8Hz,2H),2.64(s,3H),2.89(t,J＝6.8Hz,2H),3.75(s,3H),4.58(br s,2H),6.77(s,1H),7.23-7.27(m,3H),7.50-7.53(m,2H),7.79(s,1H),8.28(d,J＝5.2Hz,1H),8.30(s,1H),8.43(d,J＝8.0Hz,1H)。LCMS[M+1] +:449.3。
第二步
Figure PCTCN2019081902-appb-000010
将化合物10(4.93g,0.011mol)，二乙基磷乙酸(2.35g,0.012mol)和 N,N-二异丙基乙胺(1.68g,0.013mol),溶于四氢呋喃(25mL)，慢慢加入HATU(4.94g,0.013mol)。加入完毕后，25℃继续搅拌5小时。反应液倒入水(50mL)中，并用乙酸乙酯(50mL x 2)萃取，合并有机相。有机相依次用水(50mL x 4)和半饱和食盐水(50mL x 4)洗涤，无水Na 2SO 4干燥，浓缩得到淡黄色固体11(4.93g，收率71.5％)，不用纯化，直接用于下步反应。 1HNMR(400MHz,DMSO-d 6):δ1.26(t,J＝7.2Hz,6H),2.20(s,6H),2.31(m,2H),2.70(s,3H),2.94(m,2H),3.12,3.17(ss,2H),3.84(s,3H),4.11(q,J＝7.2Hz,4H),7.02(s,1H),7.16-7.27(m,3H),7.53(d,J＝7.2Hz,1H),7.92(s,1H),8.23(d,J＝8.0Hz,1H),8.31(d,J＝5.2Hz,1H),8.62(s,1H),8.97(br,1H),9.86(s,1H)。LCMS[M+1] +:627.3
第三步
Figure PCTCN2019081902-appb-000011
将化合物11(100mg,0.16mmol),(CD 2O) n(5mg,0.05mmol),氯化锂(10mg,0.24mmol)，氢氧化钾(27mg,0.48mmol)溶在THF/H 2O(1mL/0.5mL)溶液中，室温搅拌过夜(16h)。向反应液中加入水(5mL)，用乙酸乙酯(10mL)萃取，并依次用水(5mL x 2)，食盐水(10mL x 2)洗涤。有机相用无水硫酸钠干燥，浓缩得到粗品(90mg)，柱层析(洗脱液:二氯甲烷:甲醇＝10:1)分离得到淡黄色固体12(50mg,收率62％)。 1HNMR(400MHz,DMSO-d 6):δ2.24(s,6H),2.30(br,2H),2.72(s,3H),2.89(br,2H),3.87(s,3H),6.43(s,1H),7.05(s,1H),7.16(m,1H),7.24(m,2H)，7.53(d,J＝8.4Hz,1H)， 7.89(s,1H)，8.24(d,J＝7.6Hz,1H),8.33(d,J＝5.2Hz,1H),8.68(s,1H),9.16(s,1H),10.20(br,1H)。LCMS[M+1] +:505.3。
实施例3 化合物I晶体B的制备
Figure PCTCN2019081902-appb-000012
将化合物12(5.04g,10mmol)加入丙酮(55mL)，再在50℃加入水(5mL)，向反应液滴加甲磺酸(0.94g,9.8mmol)，有固体析出，在50℃搅拌1h。冷却至室温，过滤，所得固体用丙酮(5mL)洗，25℃真空干燥得到固体产物(5.5g,91％)。 1HNMR(400MHz,DMSO-d 6):δ2.72,2.75(ss,6H)，2.90(s,6H)，3.29(m,2H)，3.50(m,2H),4.03(s,3H),6.56(s,1H)，6.99(s,1H)，7.21(m,3H)，7.46(d,J＝8.0Hz,1H),8.17(s,1H),8.26(d,J＝5.6Hz,1H),8.35(d,J＝7.6Hz，1H)，8.67(s,1H)。LCMS[M+1] +:505.3。
化合物I的单晶B的表征数据如下表：
2θ 强度(％)
7.15 41.4
8.55 37.3
9.47 40.3
10.31 88
12.65 4.7
14.47 5.6
15.17 100.0
15.61 10.7
16.29 52.4
17.07 38.5
17.31 27.3
17.73 30.1
18.22 12.1
18.74 45.3
19.47 61.3
19.70 57.6
20.24 30.2
20.72 47.8
21.68 19.2
22.08 41.6
22.80 43.1
23.50 22.1
23.99 8.6
24.20 40.6
24.83 19.8
25.65 60.4
26.15 45.5
26.99 4.6
27.69 7.3
28.30 5.8
29.57 5.6
30.74 4.2
32.58 5.0
实施例4、混晶型的制备
将化合物12或者AZD9291(1mmol)溶于乙醇(6mL),在50℃，将甲磺酸(1mmol)的乙氰(2mL)溶液加入，搅拌0.5h，冷却至室温后过滤，石油醚洗，空气干燥得到混晶产物。
实施例5、哆希替尼单晶B和AZD9291对人非小细胞肺癌HCC827细胞皮下异种移植肿瘤BALB/c裸小鼠模型的体内药效学研究
细胞培养：人非小细胞肺癌HCC827(ATCC-CRL-2868)体外单层培养，培养条件为：RPMI-1640培养基中加质量百分含量10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，37℃下，体积百分比5％CO 2孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。
动物：BALB/c裸小鼠，雌性，6-8周龄，体重18-22克。共需60(40+50％)只。由上海西普尔-必凯实验动物有限公司或其他公司提供。
肿瘤接种：将0.2mL(1×10 7个)HCC827细胞(加基质胶，体积比为1:1)皮下接种于每只小鼠的右后背，肿瘤平均体积达到150-200mm 3时开始分组给药。实验分组和给药方案见下表。
动物实验分组和给药方案：
Figure PCTCN2019081902-appb-000013
注：
1.N:每组小鼠数目
2.给药容积：根据小鼠体重10μl/g。如果体重下降超过15％，给药方案应做出相应调整。
实验指标：实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。TGI(％)反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝T RTV/C RTV×100％(T RTV：治疗组RTV；C RTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝V t/V 0，其中V 0是分组给药时(即d0)测量所得平均肿瘤体积，V t为某一次测量时的平均肿瘤体积，T RTV与C RTV取同一天数据。
在实验结束后将检测肿瘤重量，并计算T/C weight百分比，T weight和C weight分别 表示给药组和溶媒对照组的瘤重。
数据分析：T检验用于两组间比较。三组或多组间比较用one-way ANOVA。如果F值有显著性差异，应在ANOVA分析之后再进行多重比较。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。
药学结果如图1所示，哆希替尼单晶B(代号90-1408)0.5毫克/公斤在裸鼠HCC-827模型的抗癌活性相当于AZD9291的1毫克/公斤。表明，哆希替尼单晶B在这种模型的抗癌活性较AZD9291提高了1倍。
实施例5、哆希替尼和AZD9291的药代动力学研究
(一)供试品溶液配制
(1)静注给药制剂的配制：
a.称取适量的化合物粉末到合适的容器中。
b.加入一定体积的D5W(质量百分含量5％葡萄糖)水溶液，涡旋或搅拌直至得到澄清溶液(必要时可采取加热方法助溶)。
c.加入剩余体积的D5W(5％葡萄糖)水溶液，涡旋或搅拌至得到澄清溶液。将配制好的静脉给药制剂用0.22μm微孔滤膜过滤除菌，避光保存于2℃到8℃。静注给药制剂为给药当天配制，并进行制剂分析后给药。
(2)经口给药制剂的配制：
a.称取适量的化合物粉末到合适的容器中。
b.加入适当体积的质量百分含量0.5％HPMC(羟丙基甲基纤维素，4000CP)水溶液，不断搅拌或涡旋至得到均一溶液。
a)加入剩余体积的0.5％HPMC(羟丙基甲基纤维素，4000CP)水溶液至最终体积，不断搅拌或涡旋至得到均一溶液。
灌胃给药制剂为给药前一天配制，并进行制剂分析，制剂不用时避光放在2℃到8℃冰箱中保存，8天内使用。
动物：大鼠(SD)，北京维通利华实验动物技术有限公司，36只大鼠(18只 雄性和18只雌性)，6-10周龄，200～300g(雄性)；170～280g(雌性)，试验第一天，第1组动物通过尾静脉单次注射给予哆希替尼单晶B(代号90-1408)溶液；第2、3、4组动物分别灌胃方式经口给予不同剂量的哆希替尼单晶B(代号90-1408)溶液，给药体积见4.4部分；第5组动物，每天一次，连续7天，灌胃给予哆希替尼单晶B(代号90-1408)溶液，给药体积为10mL/kg，第6组动物通过灌胃方式单次经口给予AZD9291(Mesylate)溶液，给药体积为10mL/kg。给药前及给药后0.083、0.25、0.5、1、2、4、6、8、12、24h。
用药：通过颈静脉(或其他合适的采血位点)穿刺方式在规定的时间采集全血样品(第5组约0.15mL，其它组约0.23mL)，全程湿冰操作，并在实验记录中记录实际采血时间。采集时间点可接受的误差为给药1小时内时间点±1分钟，其他时间点的为理论时间±5％。血样采集以后，立即转移至贴有标签的含K 2-EDTA的商品化离心管中，随后离心处理(3,000g，4℃，15分钟)后取血浆。将血浆转移至离心管，在干冰中速冻，并储存在-60℃或更低的超低温冰箱中，直到进行LC-MS/MS分析(第1-4组及第6组分析母药及其代谢物，第5组只分析母药)。
血浆样品：哆希替尼单晶B和AZD9291及其代谢物AZ5104-D2和AZ5104在血浆中的浓度由上海药明康德新药开发有限公司生物分析部门采用已确证的高效液相色谱-串联质谱(LC-MS/MS)进行测定。
化合物和内标的保留时间、色谱图采集和色谱图的积分采用软件Analyst(Applied Biosystems)进行处理，数据的统计采用软件Watson LIMS(Thermo Fisher Scientific)或Analyst(Applied Biosystems)进行处理。样品中分析物浓度单位为nM，保留3位有效数字，所有以百分数表示的数值(如：％偏差和％变异系数等)均保留到小数点后一位。
数据分析和报告：采用WinNonlin TM Version 6.3(Pharsight,Mountain View,CA)或以上版本的药动学软件的非房室模型处理血药浓度，使用线性对数梯形法方法计算药动学参数。
通过临床前动物药代动力学和药效学的研究，发明人惊奇地发现，晶型B的 哆希替尼的生物利用度较混晶的AZD9291的提高了20％(见表1)，有利于提高哆希替尼的抗肺癌药效。同时，哆希替尼的吲哚N-脱甲基代谢产物AZ5104-2D较奥希替尼的AZ5104(见图2-5和表1)的生成量分别减少了98％(雄鼠)和83％(雌鼠)，这将有利于减少因其抑制野生EGFR而发生的皮疹等毒副作用。与奥希替尼比较，哆希替尼在治疗非小细胞肺癌的疗效和安全性方面可能具有明显的的临床优势。

Claims (5)

  1. 2-(2，4，5-取代苯氨基)嘧啶衍生物I，其特征在于，结构式如下：
    Figure PCTCN2019081902-appb-100001
  2. 如权利要求1所述的2-(2，4，5-取代苯氨基)嘧啶衍生物I的晶型B，其特征在于，其CuKα-XRD图谱2θ在8.55、9.47、10.31、12.65、14.47、15.17、15.61、16.29、17.07、17.31、17.73、18.22、18.74、19.47、19.70、20.24、20.72、21.68、22.08、22.80、23.50、23.99、24.20、24.83、25.65、26.15、26.99、27.69、28.30、29.57、30.74、32.58处有特征峰，其中2θ值误差范围为±0.2。
  3. 如权利要求2所述的2-(2，4，5-取代苯氨基)嘧啶衍生物I晶型B，其特征在于，其CuKα-XRPD图谱如附图7所示。
  4. 如权利要求1-3其中之一所述的2-(2，4，5-取代苯氨基)嘧啶衍生物I或其晶型B在药物制备中的应用，其特征在于，化合物I或其晶型B作为活性成分，用于制备治疗非小细胞肺癌的药物中。
  5. 如权利要求4所述的2-(2，4，5-取代苯氨基)嘧啶衍生物I或其晶型B在药物制备中的应用，其特征在于，化合物I或其晶型B与其它肺癌治疗药合并用于制备治疗非小细胞肺癌的药物中。
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