WO2022242563A1 - 一种cdk4/6抑制剂的医药用途 - Google Patents

一种cdk4/6抑制剂的医药用途 Download PDF

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WO2022242563A1
WO2022242563A1 PCT/CN2022/092706 CN2022092706W WO2022242563A1 WO 2022242563 A1 WO2022242563 A1 WO 2022242563A1 CN 2022092706 W CN2022092706 W CN 2022092706W WO 2022242563 A1 WO2022242563 A1 WO 2022242563A1
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compound
acid
tumor
cancer
lung cancer
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French (fr)
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尹磊
姚郑林
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Gan and Lee Pharmaceuticals Co Ltd
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Gan and Lee Pharmaceuticals Co Ltd
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Priority to CN202280033854.2A priority Critical patent/CN117337284A/zh
Priority to US18/561,161 priority patent/US20250332167A1/en
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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
    • A61K31/495—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the application belongs to the field of medicine and relates to the medical use of a CDK4/6 inhibitor.
  • glioma is a tumor originating from glial cells, also known as glioma, and is the most common primary intracranial tumor.
  • the WHO classification of tumors of the central nervous system divides gliomas into WHO grades I-IV, where grades I and II are low-grade gliomas, and grades III and IV are high-grade gliomas.
  • Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, accounting for 54% of gliomas.
  • Glioblastoma is the most lethal brain tumor, with only one-third of patients surviving 1 year and ⁇ 5% surviving beyond 5 years.
  • glioblastoma The treatment of glioblastoma is mainly based on surgical resection of the tumor, combined with comprehensive treatment methods such as radiotherapy and chemotherapy.
  • the main chemotherapy drugs are temozolomide, nitrosourea, procarbazine, platinum, vinblastine and camptothecin. Temozolomide concurrent radiotherapy combined with adjuvant chemotherapy after surgery has become the standard treatment for newly diagnosed GBM.
  • Non-small cell lung cancer includes squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, and large cell carcinoma. Compared with small cell carcinoma, its cancer cells grow and divide more slowly, and spread and metastasize relatively later. Non-small cell lung cancer accounts for about 80% of all lung cancers, and about 75% of the patients are in the advanced stage when they are discovered, and the 5-year survival rate is very low.
  • 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)-nitrogen-(5-(1-methylpiperidine -4-yl)pyridin-2-yl)pyrimidin-2-amine is a highly selective CDK4/6 inhibitor.
  • the structural formula of the drug is:
  • the present invention provides 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl New medical application of )-nitrogen-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine.
  • One aspect of the present invention provides a method for treating cancer, the method comprising administering a therapeutically effective amount of compound (I) or a pharmaceutically acceptable salt thereof to a subject in need, wherein the cancer is glioblastoma tumor or non-small cell lung cancer.
  • the pharmaceutically acceptable salt of the compound of formula (I) is the fumarate salt of the compound of formula (I).
  • Another aspect of the present invention provides a use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer, wherein the cancer is glioblastoma or non-small cell lung cancer.
  • the pharmaceutically acceptable salt of the compound of formula (I) is the fumarate salt of the compound of formula (I).
  • Another aspect of the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the cancer is glioblastoma or non-small cell lung cancer.
  • the pharmaceutically acceptable salt of the compound of formula (I) is the fumarate salt of the compound of formula (I).
  • compound of the present invention refers to the compound of formula (I) and its salt, including the pharmaceutically acceptable salt of the compound and all stereoisomers (including but not limited to diastereoisomers) and enantiomers), tautomers, isotopic compounds, prodrugs, solvates, and hydrates thereof.
  • pharmaceutically acceptable salt refers to a salt that retains the biological effectiveness of the free acids and bases of the specified compound without adverse biological effects.
  • pharmaceutically acceptable salts include, but are not limited to: salts formed between compound (I) and any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, Heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, formic acid Sulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-
  • treatment generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be therapeutic in terms of partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • treatment encompasses any treatment of a disease in a patient, including: (a) inhibiting the symptoms of the disease, ie arresting its development; or (b) relieving the symptoms of the disease, ie causing regression of the disease or symptoms.
  • subject is a mammal, preferably a human.
  • the compound of the present invention has a significant inhibitory effect on glioblastoma, and compared with the positive control drugs Palbociclib and Abemaciclib, it shows a better tumor inhibitory effect.
  • the compound of the present invention has a significant inhibitory effect on non-small cell lung cancer, and compared with the positive control drugs Palbociclib and Abemaciclib, it shows a better tumor inhibitory effect.
  • the compound of the present invention does not show obvious drug toxicity and side effects, and has good tolerance.
  • Figure 1 Changes in body weight of each administration group in the human glioblastoma xenograft mouse model.
  • Figure 2 Changes in body weight of each administration group in the human non-small cell lung cancer NCI-H2228 xenograft mouse model.
  • Figure 3 Changes in body weight of each administration group in the human non-small cell lung cancer NCI-H1975 xenograft mouse model.
  • FBS fetal bovine serum
  • NEAA non-essential amino acids
  • HEC Hydroxyethylcellulose
  • PBS Phosphate buffered saline
  • T/C relative tumor proliferation rate
  • the above solution was filtered through a microporous filter, and transferred to the reactor, stirred, dichloromethane and ethanol were evaporated at normal pressure, and then the temperature of the reactor was kept at 80 ⁇ 5 degrees Celsius, and the fumaric acid ( 1.0eq) of ethanol solution (12 volumes) was slowly dropped into the reaction kettle through a microporous filter, and kept stirring overnight. Cool down to 20-30 degrees Celsius, continue to stir for at least 1 hour, centrifuge, and collect the filter cake.
  • Example 2 Determination of the inhibition of proliferation of the compound of the present invention on the human glioblastoma U118MG cell line
  • Human glioblastoma U118 MG cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.; the positive control drug Abemaciclib was prepared by the inventor according to the synthesis method recorded in WO2010075074A1; the cell detection equipment was In Cell Analyzer 2200 (GE Healthcare); The reagents or consumables used are shown in the table below:
  • Abemaciclib and Compound A were dissolved in DMSO to prepare a 10 mM stock solution. And put it in a -80°C refrigerator for long-term storage; take 5 ⁇ L of 10 mM Abemaciclib and Compound A stock solutions, and dilute them into 60 ⁇ M working solutions respectively, starting at 60 ⁇ M, and diluting 10 points five times.
  • test results are shown in Table 2. The results show that compound A has obvious growth inhibitory activity on U118MG cell line, and compound A has higher growth inhibitory activity than the positive control drug Abemaciclib.
  • the human glioblastoma model (BN2289, Crown Biotechnology Co., Ltd.) was cut into small pieces with a diameter of 2-3mm and inoculated into the right side of BALB/C nude mice, and the tumor growth was observed regularly , when the tumor grew to an average volume of about 100-200mm 3 , they were randomly divided into groups according to the size of the tumor.
  • the positive control drugs were Palbociclib and Abemaciclib, Palbociclib was prepared by the inventor according to the synthesis method described in WO2003062236A1, and Abemaciclib was prepared by the inventor according to the synthesis method described in WO2010075074A1.
  • test is divided into compound A 5.0mg/kg, 10.0mg/kg, 25.0mg/kg and 50.0mg/kg groups, Palbociclib 25.0mg/kg group, Abemaciclib 25.0mg/kg group, and Vehicle group, the above test products and control
  • Palbociclib 25.0mg/kg group Palbociclib 25.0mg/kg group
  • Abemaciclib 25.0mg/kg group Vehicle group
  • Vehicle group the preparation of the product is shown in Table 3.
  • mice in each group were administered orally orally, once a day, for a total of 28 days. Changes in tumor volume and body weight were observed regularly, and curative effect was evaluated based on relative tumor proliferation rate (T/C) and relative tumor inhibition rate (TGI).
  • T/C relative tumor proliferation rate
  • TGI relative tumor inhibition rate
  • T/C Relative tumor proliferation rate
  • TGI relative tumor inhibition rate
  • T/C tumor proliferation rate
  • Compound A has obvious tumor inhibitory effect in the human glioblastoma xenograft mouse model, and the compound has no obvious toxic and side effects, and has good clinical application in the treatment of glioblastoma prospect.
  • P value is to compare each group with Vehicle group by T test.
  • Example 4 Determination of the inhibition of proliferation of non-small cell lung cancer cell lines by the compounds of the present invention
  • the positive control drug Abemaciclib is prepared by the inventor according to the synthesis method described in WO2010075074A1, cell detection equipment For In Cell Analyzer 2200 (GE Healthcare), the reagents or consumables used in the experiment are shown in the table below:
  • Abemaciclib and Compound A were dissolved in DMSO to prepare a 10 mM stock solution. And put it in a -80°C refrigerator for long-term storage; take 5 ⁇ L of 10 mM Abemaciclib and Compound A stock solutions, and dilute them into 60 ⁇ M working solutions respectively, starting at 60 ⁇ M, and diluting 10 points five times.
  • test results are shown in Table 2. The results show that compound A has obvious growth inhibitory activity on 16 non-small cell lung cancer cell lines, and compound A has higher growth inhibitory activity than the positive control drug Abemaciclib.
  • Example 5 Pharmacodynamic experiment of the compound of the present invention in human non-small cell lung cancer NCI-H2228 xenograft mouse model
  • NCI-H2228 cells Human non-small cell lung cancer NCI-H2228 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. NCI-H2228 cells in the exponential growth phase were collected, resuspended in PBS to an appropriate concentration and mixed with Matrigel at a ratio of 1:1 for subcutaneous tumor inoculation in mice. NCI-H2228 cells were subcutaneously inoculated on the right side of 20 female mice, and the cells were resuspended in PBS and mixed evenly with Matrigel at a ratio of 1:1 (0.1ml/mouse). When the average tumor volume reached 143mm 3 , they were randomly divided into groups according to tumor size.
  • Example 3 For the calculation methods of tumor volume, relative tumor proliferation rate (T/C), and tumor growth inhibition rate (TGI).
  • mice in the Vehicle group and Compound A (50mg/kg) treatment group were 766.64mm 3 and 506.53mm 3 on the 50th day after inoculation, respectively.
  • Most mice in the treatment group experienced slight weight loss and were well tolerated during the treatment period.
  • Example 1 has shown obvious tumor inhibitory effect in the human non-small cell lung cancer NCI-H2228 xenograft mouse model, and the compound has no obvious toxic and side effects, and has a good therapeutic effect on non-small cell lung cancer. Prospects for clinical application.
  • P value is to compare each group with Vehicle group by T test.
  • NCI-H1975 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. NCI-H1975 cells in exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in Balb/c nude mice. Female mice were subcutaneously inoculated with NCI-H1975 cells, 0.1ml per mouse. When the average tumor volume was 171 mm 3 , they were randomly divided into groups according to tumor size.
  • the positive control drugs were Palbociclib and Abemaciclib, Palbociclib was prepared by the inventor according to the synthesis method described in WO2003062236A1, and Abemaciclib was prepared by the inventor according to the synthesis method described in WO2010075074A1.
  • test is divided into compound A 5.0mg/kg, 10.0mg/kg, 25.0mg/kg and 50.0mg/kg groups, Palbociclib 25.0mg/kg group, Abemaciclib 25.0mg/kg group, and Vehicle group, the above test products and control
  • Palbociclib 25.0mg/kg group Palbociclib 25.0mg/kg group
  • Abemaciclib 25.0mg/kg group Vehicle group
  • Vehicle group the preparation of the product is shown in Table 8.
  • T/C relative tumor proliferation rate
  • TGI relative tumor inhibition rate
  • the results are shown in Table 9 and Figure 3.
  • Each dose group of compound A showed better tumor inhibition effect than the positive drug Abemaciclib (25mg/kg) group.
  • mice there was no decrease in the body weight of the mice in each administration group 16 days after the start of administration and before, and the body weight of the three groups of compound A 25 mg/kg, 50 mg/kg and the positive control drug Palbociclib on the 20th day after the start of administration Slightly lower, the overall experimental mice have good drug resistance to the test drug.
  • compound A exhibited obvious tumor inhibitory effect in the human non-small cell lung cancer NCI-H1975 xenograft mouse model, and the compound has no obvious toxic and side effects, and has a good clinical application prospect in the treatment of non-small cell lung cancer .
  • P value is to compare each group with Vehicle group by T test.

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Abstract

提供了一种CDK4/6抑制剂的治疗癌症的方法以及相应的制药用途，其中，所述癌症为胶质母细胞瘤或非小细胞肺癌，与阳性对照药Palbociclib和Abemaciclib相比，表现出了更好的肿瘤抑制效果。

Description

一种CDK4/6抑制剂的医药用途 技术领域
本申请属于医药领域，涉及一种CDK4/6抑制剂的医药用途。
背景技术
癌症是世界许多地区的主要公共健康问题。其中，胶质瘤是起源于神经胶质细胞的肿瘤，也称为神经胶质瘤，是最常见的原发性颅内肿瘤。WHO中枢神经系统肿瘤分类将胶质瘤分为WHO I-IV级，I、II级为低级别胶质瘤，III、IV级为高级别胶质瘤。胶质母细胞瘤(Glioblastoma，GBM)是最常见的成人原发性恶性脑肿瘤，占胶质瘤的54％。胶质母细胞瘤是致死性最强的脑肿瘤，只有三分之一的患者生存期为1年，<5％的患者生存期超过5年。胶质母细胞瘤治疗以手术切除肿瘤为主，结合放疗、化疗等综合治疗方法。主要化疗药物有替莫唑胺、亚硝脲类、丙卡巴肼、铂类、长春碱类及喜树碱类药物等。其中手术后替莫唑胺同步放疗联合辅助化疗已成为新诊断GBM的标准治疗方案。
肺癌由于发病率和死亡率高，成为全世界癌症死亡的主要原因。肺癌是男性常见的癌症致死病因，在女性则仅次于乳腺癌，列名第二。近年来，我国肺癌的发病率和死亡呈快速增长。目前基于肺癌的生物学特性、治疗预后，世界卫生组织(WHO)将其分为两大类：非小细胞肺癌(non-small cell lung cancer，NSCLC)和小细胞肺癌(small cell lung cancer，SCLC)。非小细胞型肺癌包括鳞状细胞癌(鳞癌)、腺癌、大细胞癌，与小细胞癌相比其癌细胞生长分裂较慢，扩散转移相对较晚。非小细胞肺癌约占所有肺癌的80％，约75％的患者发现时已处于中晚期，5年生存率很低。
5-氟-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-氮-(5-(1-甲基哌啶-4-基)吡啶-2-基)嘧啶-2-胺是一款高选择性的CDK4/6抑制剂。该药物的结构式为：
Figure PCTCN2022092706-appb-000001
其合成方法具体描述于WO2017/092635A1中。
因此，仍然需要寻找药效显著的治疗胶质母细胞瘤和非小细胞肺癌的药物。
发明内容
针对现有技术的不足及实际的需求，本发明提供5-氟-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-氮-(5-(1-甲基哌啶-4-基)吡啶-2-基)嘧啶-2-胺的医药新用途。
本发明一方面提供一种治疗癌症的方法，所述方法包括向需要的受试者给予治疗有效量的化合物(I)或其药学上可接受的盐，其中，所述癌症为胶质母细胞瘤或非小细胞肺癌。
Figure PCTCN2022092706-appb-000002
在一个实施方案中，所述式(I)化合物的药学上可接受的盐为式(I)化合物的富马酸盐。
本发明另一方面提供一种式(I)化合物或其药学上可接受的盐在制备治疗癌症的药物中的用途，其中，所述癌症为胶质母细胞瘤或非小细胞肺癌。
在一个实施方案中，所述式(I)化合物的药学上可接受的盐为式(I)化合物的富马酸盐。
本发明的再一方面提供一种式(I)化合物或其药学上可接受的盐，其用于治疗癌症，其中，所述癌症为胶质母细胞瘤或非小细胞肺癌。
在一个实施方案中，所述式(I)化合物的药学上可接受的盐为式(I)化 合物的富马酸盐。
如无特别说明，术语“本发明的化合物”，是指式(I)化合物及其盐，包括化合物的药学上可接受的盐以及所有立体异构体(包括但不限于非对映异构体和对映异构体)、互变异构体、同位素化合物、其前药、溶剂合物、和水合物。
术语“药学上可接受的盐”指保留了特定化合物的游离酸和碱的生物学效力而没有生物学不良作用的盐。药学上可接受的盐的例子包括但不限于：化合物(I)与任意如下酸所形成的盐：盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、三氟乙酸、丙酸、己酸、庚酸、环戊烷丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1,2-乙二磺酸、2-羟基乙磺酸、苯磺酸、对氯苯磺酸、对甲苯磺酸、3-苯基丙酸、三甲基乙酸、叔丁基乙酸、十二烷基硫酸、葡糖酸、谷氨酸、羟基萘甲酸、水杨酸、和硬脂酸；优选为富马酸盐。
术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)抑制疾病的症状，即阻止其发展；或(b)缓解疾病的症状，即，导致疾病或症状退化。
术语“受试者”为哺乳动物，优选为人。
本发明的有益效果：
1)本发明的化合物对胶质母细胞瘤具有显著的抑制作用，且与阳性对照药Palbociclib和Abemaciclib相比，表现出了更好的肿瘤抑制效果。
2)本发明的化合物对非小细胞肺癌具有显著的抑制作用，且与阳性对照药Palbociclib和Abemaciclib相比，表现出了更好的肿瘤抑制效果。
3)本发明化合物没有表现出明显的药物毒副作用，具有良好的耐受性。
附图说明
图1：人源胶质母细胞瘤异种移植小鼠模型中各给药组的体重变化。
图2：人源非小细胞肺癌NCI-H2228异种移植小鼠模型中各给药组的体重变化。
图3：人源非小细胞肺癌NCI-H1975异种移植小鼠模型中各给药组的体 重变化。
具体实施方式
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的化学原料、试剂等，如无特殊说明，均为市售购买产品。
缩略语
DLT：剂量限制性毒性
DAPI：4',6-二脒基-2-苯基吲哚
DMSO:二甲基亚砜
FBS：胎牛血清
MEM:最低必需培养基
NEAA:非必需氨基酸
HEC：羟乙基纤维素
HPC：羟丙基纤维素
PBS：磷酸盐缓冲液
RTV：相对肿瘤体积
SEM：标准误差
T/C：相对肿瘤增殖率
TGI：相对肿瘤抑制率
TV：肿瘤体积
Vehicle:溶媒
实施例1 5-氟-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-氮-(5-(1-甲基哌啶-4-基)吡啶-2-基)嘧啶-2-胺富马酸盐(化合物A)
Figure PCTCN2022092706-appb-000003
首先，参照WO2017/092635A1中的合成方法制备得到5-氟-4-(7'-氟-2'-甲 基螺[环戊烷-1,3'-吲哚]-5'-基)-氮-(5-(1-甲基哌啶-4-基)吡啶-2-基)嘧啶-2-胺。
然后，氮气保护下，室温，向反应釜中加入二氯甲烷(22.0体积)和乙醇(22.0体积)，边搅拌边加入5-氟-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-氮-(5-(1-甲基哌啶-4-基)吡啶-2-基)嘧啶-2-胺(2.070kg)，升温至30-40摄氏度，搅拌直至全部溶解，降温至室温，将溶液转移至溶剂桶中备用。在氮气保护下，将上述溶液通过微孔过滤器过滤，并转移至反应釜中，搅拌，常压蒸出二氯甲烷和乙醇，然后保持反应釜温度至80±5摄氏度，将富马酸(1.0eq)的乙醇溶液(12体积)通过微孔过滤器缓慢滴入反应釜中，保温搅拌过夜。降温至20-30摄氏度，继续搅拌至少1个小时，离心，收集滤饼。将滤饼置于真空干燥箱中，干燥过夜，得到1.605kg 5-氟-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-氮-(5-(1-甲基哌啶-4-基)吡啶-2-基)嘧啶-2-胺富马酸盐(化合物A)，收率63.6％。
实施例2 本发明化合物对人脑胶质母细胞瘤U118MG细胞系的增殖抑制测定
人脑胶质母细胞瘤U118 MG细胞购于南京科佰生物科技有限公司；阳性对照药Abemaciclib为发明人根据WO2010075074A1记载的合成方法制备得到；细胞检测设备为In Cell Analyzer 2200(GE Healthcare)；实验用到的试剂或耗材如下表所示：
表1 试剂或耗材
Figure PCTCN2022092706-appb-000004
Figure PCTCN2022092706-appb-000005
将Abemaciclib、化合物A溶解于DMSO中，配制成10mM的贮存液。并放置于-80℃冰箱中长期保存；取5μL的10mM Abemaciclib、化合物A贮存溶液，分别稀释成为60μM的工作溶液，以60μM为起始浓度，五倍稀释10个点。
U118 MG培养基为MEM(低糖)+10％FBS+1％青霉素/链霉素+1％丙酮酸钠+1％MEM NEAA；U118 MG细胞以4000cells/100μl/well，接种至黑色透明底96孔板，37℃培养过夜；取出96孔板，加入20μl稀释好的样品到96孔板，37℃处理72h；72h后取出96孔板，加入中性甲醛固定液(甲醛:PBS＝1:9)，50μl/well，室温固定10-30min；1x PBS洗2次，100μl/well，0.2％Triton TM-X100透化处理5-10min；1x PBS洗2次，100μl/well，DAPI染色(PBS 1:5000稀释)，50μl/well，室温避光孵育20min；1x PBS洗3次，100μl/well，最后加入1x PBS 100μl/well；In Cell Analyzer扫描，分析每孔细胞数目。按如下公式计算各化合物在各浓度点的抑制率，并通过软件Graphpad Prism 6.0进行曲线拟合得到IC 50值。
Figure PCTCN2022092706-appb-000006
试验结果见表2，该结果表明：化合物A对U118MG细胞系具有明显的增殖抑制活性，相对于阳性对照药Abemaciclib，化合物A具有更高的增殖抑制活性。
表2 测试物对U118MG细胞系增殖的抑制活性
Figure PCTCN2022092706-appb-000007
实施例3 本发明化合物在人源胶质母细胞瘤异种移植小鼠模型中的药 效实验
将人源胶质母细胞瘤模型(BN2289，中美冠科生物技术有限公司)瘤块切成直径为2-3mm的小块接种到BALB/C裸鼠的右侧皮下，定期观察肿瘤生长情况，待肿瘤生长至平均体积100-200mm 3左右时，根据肿瘤大小随机分组。
阳性对照药为Palbociclib和Abemaciclib，Palbociclib为发明人根据WO2003062236A1记载的合成方法制备得到,Abemaciclib为发明人根据WO2010075074A1记载的合成方法制备得到。
试验分为化合物A 5.0mg/kg、10.0mg/kg、25.0mg/kg和50.0mg/kg组，Palbociclib 25.0mg/kg组，Abemaciclib 25.0mg/kg组，以及Vehicle组，上述供试品和对照品的配制见表3。
表3 供试品和对照品的配制
Figure PCTCN2022092706-appb-000008
每组8只小鼠，口服灌胃给药，每天给药一次，共给药28天。定期观测肿瘤体积及体重变化，根据相对肿瘤增殖率(T/C)和相对肿瘤抑制率(TGI)进行疗效评价。
肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。
相对肿瘤增殖率(T/C)，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C(％)＝T RTV/C RTV×100％(T RTV：治疗组平均RTV；C RTV：Vehicle组平均RTV；RTV＝V t/V 0，V 0为分组时该动物的瘤体积，V t为治疗后该动物的瘤体积)；
相对肿瘤抑制率(TGI)，计算公式如下：TGI(％)＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW))。
结果见表4、图1，该结果表明：化合物A(5mg/kg、10mg/kg、25mg/kg、50mg/kg)治疗组在第21天时均表现出肿瘤抑制的作用，相对肿瘤增殖率(T/C)为95.08％、88.24％、60.61％、43.56％，相对Vehicle组，统计学上有显著性差异(p值均<0.001)，且具有剂量依赖关系。
化合物A(25mg/kg、50mg/kg)治疗组在第21天时的相对肿瘤增殖率(T/C)分别为60.61％和43.56％，比阳性药Palbociclib(25mg/kg)和Abemaciclib(25mg/kg)组表现出了更好的肿瘤抑制效果。
实验过程中动物整体体重良好，各给药组均没有表现明显的药物毒性，测试药表现出良好的耐受性。
由此可见，化合物A在人源胶质母细胞瘤异种移植小鼠模型中表现出了明显的肿瘤抑制作用，且该化合物无明显的毒副作用，具有良好的治疗胶质母细胞瘤的临床应用前景。
表4 测试物在胶质母细胞瘤异体移植模型中的抑制活性
Figure PCTCN2022092706-appb-000009
Figure PCTCN2022092706-appb-000010
注：P值是采用T检验将各组与Vehicle组比较。
实施例4 本发明化合物对非小细胞肺癌细胞系的增殖抑制测定
采用16种非小细胞肺癌细胞系NCI-H1792、NCI-H1703、NCI-H441、SNU-761、NCI-H1975、NCI-H358、NCI-H1838、NCI-H1915、A549、SK-MES-1、NCI-H292、PC-9、NCI-H460、NCI-H23、NCI-H1581、HLF用于本发明化合物的增殖抑制测定试验，阳性对照药Abemaciclib为发明人根据WO2010075074A1记载的合成方法制备得到，细胞检测设备为In Cell Analyzer 2200(GE Healthcare)，实验用到的试剂或耗材如下表所示：
表5 试剂或耗材
试剂或耗材 货号 厂家
DMSO D2650 Sigma
PBS 20012-027 Gibco
DAPI D8417 Sigma
甲醛 47608 Sigma
Triton TM X-100 T9284 Sigma
96孔黑色透明底细胞板 6005182 PE
将Abemaciclib、化合物A溶解于DMSO中，配制成10mM的贮存液。并放置于-80℃冰箱中长期保存；取5μL的10mM Abemaciclib、化合物A贮存溶液，分别稀释成为60μM的工作溶液，以60μM为起始浓度，五倍稀释10个点。
将对数生长期的每种细胞以4000cells/100μl/well，接种至黑色透明底96孔板，37℃培养过夜；取出96孔板，加入20μl稀释好的样品到96孔板，37℃处理72h；72h后取出96孔板，加入中性甲醛固定液(甲醛:PBS＝1:9)，50μl/well，室温固定10-30min；1x PBS洗2次，100μl/well，0.2％Triton TM-X100透化处理5-10min；1x PBS洗2次，100μl/well，DAPI染色(PBS 1:5000稀释)，50μl/well，室温避光孵育20min；1x PBS洗3次，100μl/well，最后加入1x PBS 100μl/well；In Cell Analyzer扫描，分析每孔细胞数目。按如下公式计算各化合物在各浓度点的抑制率，并通过软件Graphpad Prism 6.0进行曲线拟合得到IC 50值。
Figure PCTCN2022092706-appb-000011
试验结果见表2，该结果表明：化合物A对16种非小细胞肺癌细胞系均具有明显的增殖抑制活性，相对于阳性对照药Abemaciclib，化合物A具有更高的增殖抑制活性。
表6 测试物对不同非小细胞肺癌细胞系增殖的抑制活性
Figure PCTCN2022092706-appb-000012
Figure PCTCN2022092706-appb-000013
实施例5 本发明化合物在人源非小细胞肺癌NCI-H2228异种移植小鼠模型中的药效实验
人源非小细胞肺癌NCI-H2228细胞培养在含10％胎牛血清的RPMI-1640培养液中。收集指数生长期的NCI-H2228细胞，PBS重悬至适合浓度并与基质胶按1：1的比例混合均匀，用于小鼠皮下肿瘤接种。20只雌性小鼠右侧皮下接种NCI-H2228细胞，细胞重悬在PBS中并与基质胶按1：1的比例均匀混合(0.1ml/只)。待肿瘤平均体积达143mm 3时，根据肿瘤大小随机分组。分别给予溶剂、实施例1化合物A(50mg/kg)，一天一次，口服灌胃给药28天。定期观测肿瘤体积及体重变化。所有动物于接种细胞后第50天安乐，取瘤称重并拍照。肿瘤体积、相对肿瘤增殖率(T/C)、肿瘤生长抑制率(TGI)的计算方法参见实施例3。
结果见表7、图2，该结果表明：Vehicle组与化合物A(50mg/kg)治疗组小鼠在接种后第50天平均肿瘤体积分别为766.64mm 3和506.53mm 3，相较Vehicle组，治疗组对NCI-H2228人源非小细胞肺癌有显著抑制作用(p＝0.003)，相对肿瘤抑制率(TGI)为36％。治疗组大多数小鼠出现略微的体重下降，治疗期间耐受性良好。
可见，实施例1化合物A在人源非小细胞肺癌NCI-H2228异种移植小鼠模型中表现出了明显的肿瘤抑制作用，且该化合物无明显的毒副作用，具有良好的治疗非小细胞肺癌的临床应用前景。
表7 测试物在治疗非小细胞肺癌NCI-H2228异体移植模型中的抑制活性
Figure PCTCN2022092706-appb-000014
Figure PCTCN2022092706-appb-000015
注：P值是采用T检验将各组与Vehicle组比较。
实施例6 本发明化合物在人源非小细胞肺癌NCI-H1975异种移植小鼠模型中的药效实验
NCI-H1975细胞培养在含10％胎牛血清的RPMI1640培养液中，收集指数生长期的NCI-H1975细胞，PBS重悬至适合浓度用于Balb/c nude小鼠皮下肿瘤接种。雌性小鼠皮下接种NCI-H1975细胞，每只小鼠接种0.1ml。待肿瘤平均体积171mm 3时，根据肿瘤大小随机分组。
阳性对照药为Palbociclib和Abemaciclib，Palbociclib为发明人根据WO2003062236A1记载的合成方法制备得到,Abemaciclib为发明人根据WO2010075074A1记载的合成方法制备得到。
试验分为化合物A 5.0mg/kg、10.0mg/kg、25.0mg/kg和50.0mg/kg组，Palbociclib 25.0mg/kg组，Abemaciclib 25.0mg/kg组，以及Vehicle组，上述供试品和对照品的配制见表8。
表8 供试品和对照品的配制
Figure PCTCN2022092706-appb-000016
Figure PCTCN2022092706-appb-000017
每组8只小鼠，口服灌胃给药，每天给药一次，共给药21天。定期观测肿瘤体积及体重变化，根据相对肿瘤增殖率(T/C)和相对肿瘤抑制率(TGI)进行疗效评价。肿瘤体积、T/C、TGI的计算方法参见实施例3。
结果见表9、图3，该结果表明：开始给药后16天时，化合物A 25mg/kg和50mg/kg组均产生了极显著的抗肿瘤作用(p值均<0.001)，肿瘤生长抑制率(TGI)分别为50％和69％，平均肿瘤体积分别为1141mm 3和709mm 3；在给药剂量为10mg/kg时也产生了统计学上差异显著的抗肿瘤效果(p＝0.025)，TGI为28％，平均肿瘤体积为1647mm 3；在给药剂量为5mg/kg时产生了少量的抗肿瘤效果(p＝0.333)，TGI为15％，平均肿瘤体积为1889mm 3；化合物A各剂量之间呈现出良好的量效关系。
阳性对照药Palbociclib 25mg/kg组也产生了统计学上差异显著的抗肿瘤效果(p＝0.002)，TGI为34％，平均肿瘤体积为1469mm 3；化合物A(25mg/kg、50mg/kg)治疗组比阳性药Palbociclib(25mg/kg)组表现出了更好的肿瘤抑制效果。阳性对照药Abemaciclib 25mg/kg组未产生明显的抗肿瘤效果(p＝0.911)，TGI为7％，平均肿瘤体积为2071mm 3。化合物A各剂量组比阳性药Abemaciclib(25mg/kg)组表现出了更好的肿瘤抑制效果。
实验过程中各给药组在开始给药后16天及之前未出现小鼠体重降低的情况，在开始给药后第20天化合物A 25mg/kg、50mg/kg及阳性对照药Palbociclib三组体重略有降低，整体实验小鼠对测试药耐药性良好。
可见，化合物A在人源非小细胞肺癌NCI-H1975异种移植小鼠模型中表现出了明显的肿瘤抑制作用，且该化合物无明显的毒副作用，具有良好的治疗非小细胞肺癌的临床应用前景。
表9 测试物在治疗非小细胞肺癌NCI-H1975异体移植模型中的抑制活性
Figure PCTCN2022092706-appb-000018
Figure PCTCN2022092706-appb-000019
注：P值是采用T检验将各组与Vehicle组比较。
本发明已经通过上述实施例进行了说明，但应当理解的是，上述实施例只是用于举例和说明的目的，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明并不局限于上述实施例，根据本发明的教导还可以做出更多种的变型和修改，这些变型和修改均落在本发明所要求保护的范围以内。本发明的保护范围由附属的权利要求书及其等效范围所界定。

Claims (6)

  1. 式(I)化合物或其药学上可接受的盐在制备治疗癌症的药物中的用途，
    Figure PCTCN2022092706-appb-100001
    其中，所述癌症为胶质母细胞瘤或非小细胞肺癌。
  2. 根据权利要求1所述的用途，其特征在于，其中所述式(I)化合物的药学上可接受的盐为式(I)化合物的富马酸盐。
  3. 治疗癌症的方法，所述方法包括向需要的受试者给予治疗有效量的化合物(I)或其药学上可接受的盐，
    Figure PCTCN2022092706-appb-100002
    其中，所述癌症为胶质母细胞瘤或非小细胞肺癌。
  4. 根据权利要求3所述的方法，其特征在于，其中所述式(I)化合物的药学上可接受的盐为式(I)化合物的富马酸盐。
  5. 式(I)化合物或其药学上可接受的盐，其用于治疗癌症，其中，所述癌症为胶质母细胞瘤或非小细胞肺癌。
    Figure PCTCN2022092706-appb-100003
  6. 根据权利要求5的用于所述用途的化合物或其药学上可接受的盐，其特征在于，其中所述式(I)化合物的药学上可接受的盐为式(I)化合物的富 马酸盐。
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