WO2022111527A1 - 哌嗪-2,3-二酮衍生物及其在医药上的应用 - Google Patents

哌嗪-2,3-二酮衍生物及其在医药上的应用 Download PDF

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WO2022111527A1
WO2022111527A1 PCT/CN2021/132804 CN2021132804W WO2022111527A1 WO 2022111527 A1 WO2022111527 A1 WO 2022111527A1 CN 2021132804 W CN2021132804 W CN 2021132804W WO 2022111527 A1 WO2022111527 A1 WO 2022111527A1
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compound
isopropyl
methylpyridin
methyl
kras
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PCT/CN2021/132804
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French (fr)
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张靖
魏用刚
杨科
孙毅
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成都百裕制药股份有限公司
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Priority to CN202180046901.2A priority Critical patent/CN115803328A/zh
Publication of WO2022111527A1 publication Critical patent/WO2022111527A1/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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/499—Spiro-condensed pyrazines or piperazines
    • 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
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention relates to piperazine-2,3-dione derivatives or their pharmaceutically acceptable salts or stereoisomers and their application in medicine.
  • the RAS gene is one of the important human proto-oncogenes and plays a key role in regulating cell signal transduction and the occurrence and development of tumors; it is also the most frequently mutated oncogene in human cancers, and the RAS gene is present in about 30% of cancers. mutation.
  • the human RAS superprotein family contains three genes (KRAS, NRAS, and HRAS, respectively) that encode four proteins (KRAS-4A, KRAS-4B, NRAS, and HRAS).
  • KRAS is the most common subtype in the RAS family and the most easily mutated gene; 86% of RAS mutations are KRAS mutations, 86-96% of pancreatic cancer, 40-54% of colorectal cancer and 20-37% of KRAS gene mutations can be detected in lung cancers of 2000, and KRAS mutations are also found in cancers such as cholangiocarcinoma, cervical cancer, bladder cancer, liver cancer, and breast cancer (Kessler et al., 2019).
  • KARS proteins belong to the small GTPase family of proteins. Under normal physiological conditions, KRAS proteins regulate signaling pathways by transitioning between inactive (binding to GDP) and activated (binding to GTP) states (Downward, 2003): when KRAS binds to guanosine diphosphate (GDP) Inactive state; active state can activate downstream signaling pathways when combined with guanosine triphosphate (GTP).
  • GDP guanosine diphosphate
  • GTP guanosine triphosphate
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase activating/accelerating proteins
  • KRAS has a stronger binding ability to GDP, so KRAS in cells is generally inactive; when KRAS is activated by binding to GTP, it can be promoted through multiple downstream signaling pathways such as MAPK, PI3K, and Ral-GEFs. Cell survival, proliferation and cytokine release, etc. (Liu et al., 2019).
  • KRAS When KRAS is mutated or its conformation changes, its GTP hydrolysis activity decreases, its binding stability to GDP increases, and its interaction with GAP is blocked, so that KRAS protein is in a long-term activated state, and by stimulating a large number of downstream cell signals, it continues to promote cell growth and growth. Divide, eventually leading to cancer. Current studies have shown that the most common mutation of KRAS is single-point mutations at codons 12, 13 and 61, of which codon 12 mutations account for about 82% of KRAS mutations.
  • KRAS mutant cells In addition to KRAS gene alterations that directly lead to tumorigenesis, KRAS mutant cells also have a wide range of effects on the microenvironment that causes tumor progression through paracrine action, which can promote the secretion of various cytokines, chemokines and growth factors to fibroblasts and immune cells. It also affects the tumor microenvironment, and can also remodel and reprogram stromal cells.
  • KRAS inhibitors have been carried out in recent decades, and it was generally believed that this target is difficult to be drugged. The main reasons are as follows: (1) The affinity of KRAS protein to guanosine phosphate (GDP or GTP) is as low as picomolar (pM) ), the concentration of guanosine phosphate in cells can reach the millimolar (mM) level, which makes it difficult for general drugs (analogs of guanosine phosphate) to compete with KRAS protein binding (Waters & Der, 2018); (2) KRAS protein The guanosine phosphate binding site also lacks a region suitable for the binding of other small molecule compounds.
  • small-molecule compounds can inhibit tumors with KRAS-G12C mutation. Because the 12th codon of KRAS protein is close to the nucleoside binding domain and catalytic center of the protein, after cysteine mutation at this site, small molecule compounds can affect KRAS through irreversible covalent binding to cysteine The function of the protein, and the small molecule compound is more inclined to bind to the KRAS-GDP protein, which reduces the affinity of GTP to the protein (Lito, Solomon, Li, Hansen, & Rosen, 2016).
  • ARS-853 an early developed small-molecule compound targeting KRAS-G12C tumors, showed good activity in vitro and effectively inhibited the conversion of GDP and GTP, but lacked in vivo activity (Patricelli et al., 2016). Subsequently, the structure was further optimized to obtain ARS-1620, which showed good pharmacological activity against KRAS-G12C tumor cells in vitro and in vivo, but had no significant effect on other KRAS mutant cells (Janes et al., 2018). At present, a variety of KRAS inhibitor drugs have entered the stage of clinical research, but according to the data published so far, the clinical efficacy in tumor patients still needs to be further improved. Therefore, there is still a need to develop a new generation of efficient and safe KRAS-G12C mutation inhibitors to better meet clinical needs.
  • the purpose of this application is to provide novel piperazine-2,3-dione derivatives or their pharmaceutically acceptable salts or stereoisomers, their pharmaceutical compositions and their applications in the preparation of KRAS G12C inhibitors.
  • the compounds of one or more embodiments of the present application can improve therapeutic efficacy while reducing toxic side effects.
  • One or more embodiments of the present application provide a compound, or a pharmaceutically acceptable salt or stereoisomer thereof, that is:
  • One or more embodiments of the present application provide the use of the compound of the present application or a pharmaceutically acceptable salt or stereoisomer thereof or the pharmaceutical composition of the present application in the preparation of an antitumor drug.
  • One or more embodiments of the present application provide a compound of the present application, or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition of the present application, for use as a medicament.
  • One or more embodiments of the present application provide a compound of the present application, or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition of the present application, for use in the treatment of tumors.
  • One or more embodiments of the present application provide methods of treating tumors comprising administering a compound of the present application, or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition of the present application to a subject in need thereof.
  • the carbon, hydrogen, oxygen, sulfur, nitrogen or F, Cl, Br, I involved in the groups and compounds of the present invention all include their isotopic conditions, and the carbons involved in the groups and compounds of the present invention , hydrogen, oxygen, sulfur or nitrogen are optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include 12 C, 13 C and 14 C, and isotopes of hydrogen include protium (H), deuterium (D, Also known as heavy hydrogen), tritium (T, also known as super-heavy hydrogen), the isotopes of oxygen include 16 O, 17 O and 18 O, the isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, and the isotopes of nitrogen include 14 N and 15 N, fluorine isotopes include 17 F and 19 F, chlorine isotopes include 35 Cl and 37 Cl, and bromine isotopes include 79 Br and 81 Br.
  • isotopes of carbon include 12 C, 13 C
  • “Pharmaceutical composition” refers to a mixture of one or more of the compounds of the present invention, pharmaceutically acceptable salts or prodrugs thereof and other chemical components, wherein “other chemical components” refers to pharmaceutically acceptable Accepted carriers, excipients and/or one or more other therapeutic agents.
  • Carrier refers to a material that is not appreciably irritating to the organism and that does not abrogate the biological activity and properties of the administered compound.
  • Excipient refers to an inert substance added to a pharmaceutical composition to facilitate administration of a compound.
  • Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binding agents agent and disintegrant.
  • Steps refer to isomers resulting from different arrangements of atoms in a molecule in space, including cis-trans isomers, enantiomers and conformational isomers.
  • heterocyclyl optionally substituted with an alkyl group means that the alkyl group may, but need not, be present, and the description includes instances where the heterocyclyl group is substituted with an alkyl group, as well as where the heterocyclyl group is not substituted with an alkyl group happening.
  • IC50 refers to the concentration of the compound at which the activity of KRAS is inhibited by 50%.
  • NBT nitrotetrazolium blue
  • reaction temperature is room temperature
  • most suitable reaction temperature at room temperature is 20°C-30°C.
  • the target product compound 2 (white solid, 30 mg, yield 19.3%) was prepared and isolated.
  • the target product compound 3 (white solid, 30 mg, yield 19.3%) was prepared and isolated.
  • the target product compound 4 (white solid, 62 mg, yield 24%) was prepared and isolated.
  • the target product compound 5 (white solid, 48 mg, yield 27%) was prepared and isolated.
  • the target product compound 6 (white solid, 47 mg, yield 14%) was prepared and isolated.
  • the target product compound 7 (white solid, 52 mg, yield 17%) was prepared and isolated.
  • the target product compound 8 (white solid, 43 mg, yield 23%) was prepared and isolated.
  • the target product compound 9 (white solid, 32 mg, yield 34%) was prepared and isolated.
  • the target product compound 10 (white solid, 32 mg, yield 34%) was prepared and isolated.
  • the target product compound 11 was prepared and isolated (white solid, 27 mg, yield 14%).
  • the target product compound 12 (white solid, 36 mg, yield 17%) was prepared and isolated.
  • the target product compound 13 was prepared and isolated (white solid, 36 mg, yield 17%).
  • the target product compound 14 was prepared and isolated (white solid, 27 mg, yield 23%).
  • the target product compound 15 was prepared and isolated (white solid, 14 mg, yield 17%).
  • the target product compound 16 was prepared and isolated (white solid, 19 mg, yield 27%).
  • the target product compound 17 was prepared and isolated (white solid, 23 mg, yield 24%).
  • the target product compound 18 (white solid, 24 mg, yield 27%) was prepared and isolated.
  • the target product compound 19 (white solid, 37 mg, yield 14%) was prepared and isolated.
  • the target product compound 20 (white solid, 31 mg, yield 17%) was prepared and isolated.
  • test compound was prepared into a 10 mM stock solution, doubling dilution (1:5) with growth medium, diluted 8-10 concentration gradients (initial concentration 10 ⁇ M), and added with 96 containing the upper layer of agarose-cells well (50 ⁇ L/well); set solvent control wells; each concentration was replicated twice, placed in a carbon dioxide incubator for 10-14 days.

Abstract

本申请公开了新型哌嗪-2,3-二酮衍生物或者其药物可接受的盐或立体异构体、其药物组合物以及其在制备KRAS G12C抑制剂中的应用，该化合物可改善疗效，同时降低毒副作用。

Description

哌嗪-2,3-二酮衍生物及其在医药上的应用 技术领域
本发明涉及哌嗪-2,3-二酮衍生物或者其药物可接受的盐或立体异构体及其在医药上的应用。
背景技术
RAS基因是人类重要的原癌基因之一，在调控细胞信号转导与肿瘤的发生发展中起着关键作用；也是人类癌症中最常出现突变的致癌基因，大约30％的癌症中存在RAS基因突变。人类RAS超蛋白家族含有三种基因(分别是KRAS、NRAS和HRAS)，编码四种蛋白(KRAS-4A、KRAS-4B、NRAS和HRAS)。其中KRAS是RAS家族中最常见的亚型，也是最容易突变的基因；RAS突变中有86％为KRAS突变，86-96％的胰腺癌、40-54％的结直肠癌和20-37％的肺癌中可以检测到KRAS基因突变，在胆管癌、宫颈癌、膀胱癌、肝癌和乳腺癌等癌症中也出现KRAS突变(Kessler et al.,2019)。
KARS蛋白属于小GTP酶(small GTPase)蛋白家族。在正常生理状态下，KRAS蛋白通过在失活(结合GDP)和激活(结合GTP)状态之间转变实现对信号通路的调控(Downward,2003)：当KRAS与二磷酸鸟苷(GDP)结合时处于失活状态；当与三磷酸鸟苷(GTP)结合时处于激活状态可以激活下游信号通路。这两种状态之间的转换和平衡受到以下两类蛋白的调控：(1)鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factors；GEF)，能促使KRAS与GTP的结合，提高GDP从KRAS中的解离效率，促进KRAS的激活；(2)GTP酶激活蛋白(GTPase activating/accelerating proteins；GAPs)，能提高KRAS蛋白本身相对较弱的GTPase活性，促使GTP水解为GDP，降低KRAS对下游信号通路的影响。生理条件下，KRAS与GDP有着更强的结合能力，因此细胞中的KRAS一般处于失活状态；当KRAS与GTP结合后被激活，可以通过MAPK、PI3K和Ral-GEFs等多条下游信号通路促进细胞生存、增殖和细胞因子释放等(Liu et al.,2019)。
当KRAS发生突变或者构象改变，它的GTP水解活性下降、与GDP结合稳定性增加、与GAP相互作用受阻，使得KRAS蛋白长期处于激活状态，通过刺激大量的下游细胞信号，持续促使细胞继续生长和分裂，最终导致癌症发生。目前研究表明，KRAS最常见的突变方式是在密码子的第12、13和61位发生的单点突变(single-point mutations)，其中第12位密码子突变约占KRAS突变的82％。体细胞中这些位点的错义突变会干扰KRAS本 身的GTPase活性：如第12位(G12)突变通过干扰GAP蛋白结合和GTP水解来持续激活KRAS；第13位(G13)突变造成GAP结合能力降低；第61位(Q61)突变影响GTP水解中间态的稳定性(Ostrem&Shokat,2016)。除了KRAS基因改变直接导致肿瘤的发生外，KRAS突变细胞通过旁分泌作用对引起肿瘤恶化的微环境也影响广泛，可以促进分泌多种细胞因子、趋化因子和生长因子对纤维母细胞和免疫细胞等影响肿瘤微环境，也能对基质细胞进行改造和重编程。这些研究充分证实了KRAS基因可以作为重要的抗肿瘤药物靶点。
研究KRAS抑制剂已经开展了近几十年，之前普遍认为该靶点难以成药，主要原因有以下两点：(1)KRAS蛋白与磷酸鸟苷(GDP或GTP)的亲和力低至皮摩尔(pM)，细胞中磷酸鸟苷的浓度可以达到毫摩尔(mM)级别，这使得一般药物(磷酸鸟苷的类似物)难以与KRAS蛋白进行竞争性结合(Waters&Der,2018)；(2)KRAS蛋白的磷酸鸟苷结合部位也缺乏适合其他小分子化合物结合的区域。最新研究发现，与野生型KRAS蛋白相比，小分子化合物可以针对具有KRAS-G12C突变的肿瘤有抑制作用。因为KRAS蛋白的第12位密码子靠近该蛋白的核苷结合域和催化中心，该位点发生半胱氨酸突变后，小分子化合物可以通过与半胱氨酸不可逆的共价结合来影响KRAS蛋白的功能，同时小分子化合物更倾向于与KRAS-GDP蛋白结合，降低了GTP与蛋白的亲和力(Lito,Solomon,Li,Hansen,&Rosen,2016)。
早期研发的针对KRAS-G12C肿瘤的小分子化合物ARS-853在体外表现出良好的活性，有效抑制了GDP与GTP转换，但是缺乏体内活性(Patricelli et al.,2016)。随后进一步优化结构得到ARS-1620，在体内外均表现出对KRAS-G12C肿瘤细胞良好的药理活性，而对其他KRAS基因突变细胞无明显影响(Janes et al.,2018)。目前已有多种KRAS抑制剂药物进入了临床研究的阶段，但根据目前公布的数据，在肿瘤患者中的临床药效仍有待进一步提高。因此，目前仍需要开发新一代高效安全的KRAS-G12C突变抑制剂，以更好的满足临床需求。
发明内容
本申请的目的在于提供新型哌嗪-2,3-二酮衍生物或者其药物可接受的盐或立体异构体、其药物组合物以及其在制备KRAS G12C抑制剂中的应用。
本申请的一个或多个实施方式中的化合物可改善疗效，同时降低毒副作用。
本申请的一个或多个实施方式提供了化合物或者其药物可接受的盐或立体异构体，所述化合物为：
Figure PCTCN2021132804-appb-000001
Figure PCTCN2021132804-appb-000002
Figure PCTCN2021132804-appb-000003
本申请的一个或多个实施方式提供了药物组合物，其包含
(1)权利要求1所述的化合物或者其药物可接受的盐或立体异构体；
(2)任选的一种或者多种其他活性成分；以及
(3)药学上可接受的载体和/或赋形剂。
本申请的一个或多个实施方式提供了本申请的化合物或者其药物可接受的盐或立体异构体或者本申请的药物组合物在制备抗肿瘤药物中的用途。
本申请的一个或多个实施方式提供了本申请的化合物或者其药物可接受的盐或立体异构体或者本申请的药物组合物，其用作用药物。
本申请的一个或多个实施方式提供了本申请的化合物或者其药物可接受的盐或立体异构体或者本申请的药物组合物，其用于治疗肿瘤。
本申请的一个或多个实施方式提供了治疗肿瘤的方法，其包括将本申请的化合物或者其药物可接受的盐或立体异构体或者本申请的药物组合物给予有此需要的对象。
发明详述
除非有相反的陈述，在说明书和权利要求书中使用的术语具有下述含义。
本发明所述基团和化合物中所涉及的碳、氢、氧、硫、氮或F、Cl、Br、I均包括它们的同位素情况，及本发明所述基团和化合物中所涉及的碳、氢、氧、硫或氮任选进一步被一个或多个它们对应的同位素所替代，其中碳的同位素包括 12C、 13C和 14C，氢的同位素包括氕(H)、氘(D，又叫重氢)、氚(T，又叫超重氢)，氧的同位素包括 16O、 17O和 18O，硫的同位素包括 32S、 33S、 34S和 36S，氮的同位素包括 14N和 15N，氟的同位素包括 17F和 19F，氯的同位素包括 35Cl和 37Cl，溴的同位素包括 79Br和 81Br。
“药物组合物”是指一种或多种本发明所述化合物、其药学上可接受的盐或前药和其它化学组分形成的混合物，其中，“其它化学组分”是指药学上可接受的载体、赋形剂和/或一 种或多种其它治疗剂。
“载体”是指不会对生物体产生明显刺激且不会消除所给予化合物的生物活性和特性的材料。
“赋形剂”是指加入到药物组合物中以促进化合物给药的惰性物质。非限制性实施例包括碳酸钙、磷酸钙、糖、淀粉、纤维素衍生物(包括微晶纤维素)、明胶、植物油、聚乙二醇类、稀释剂、成粒剂、润滑剂、粘合剂和崩解剂。
“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体，包括顺反异构体、对映异构体和构象异构体。
“任选”或“任选地”或“选择性的”或“选择性地”是指随后所述的事件或状况可以但未必发生，该描述包括其中发生该事件或状况的情况及其中未发生的情况。例如，“选择性地被烷基取代的杂环基”是指该烷基可以但未必存在，该描述包括其中杂环基被烷基取代的情况，及其中杂环基未被烷基取代的情况。
具体实施方式
以下实施例详细说明本发明的技术方案，但本发明的保护范围包括但是不限于此。
IC 50：是指KRAS的活性受到50％抑制时化合物的浓度。
NBT：硝基四氮唑蓝；
实施例中无特殊说明，反应温度为室温，室温最适宜的反应温度为20℃–30℃。
实施例1
1-(1-丙烯酰亚胺-4-基)-7-氯-6-(2-氟-6-羟基苯基)-4-(2-异丙基-4-甲基吡啶-3-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物1
1-(1-acryloylpiperidin-4-yl)-7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(2-isopropyl-4-methylpyridin-3-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000004
Figure PCTCN2021132804-appb-000005
第一步：
5,6-二氯-N-(2-异丙基-4-甲基吡啶-3-基)-3-硝基吡啶-2-胺1c
5,6-dichloro-N-(2-isopropyl-4-methylpyridin-3-yl)-3-nitropyridin-2-amine
将2,3,6-三氯-5-硝基吡啶1a(购自华捷明生物科技，30g,131.9mmol)、2-异丙基-4-甲基吡啶-3-胺1b(购自成都爱斯特化学技术有限公司，39.6g,263.85mmol)加入到烧瓶中，在氮气的保护下，加热至85℃下搅拌反应，24h后反应完毕，柱层析分离(PE:EA＝10:1)，得到目标产物1c(黄色固体，20.8g，产率51％)。
1H NMR(400MHz,DMSO-d 6)δ9.94(s,1H),8.75(s,1H),8.39(d,1H),7.20(d,1H),3.20-3.08(m,1H),2.14(s,3H),1.44(s,3H),1.08(s,3H)。
LC-MS m/z(ESI)＝341.19[M+1]。
第二步：
5,6-二氯-氮2-(2-异丙基-4-甲基吡啶-3-基)吡啶-2,3-二胺1d
5,6-dichloro-N2-(2-isopropyl-4-methylpyridin-3-yl)pyridine-2,3-diamine
将化合物1c(7.8g，18.93mmol)溶解于40mL的乙酸乙酯和40mL冰醋酸中，再加入铁粉(购自安耐吉化学，6.35g，113.6mmol)，在室温下搅拌反应，2h后反应完毕，得到化合物1d(白色固体，5.28g，产率90％)。
1H NMR(400MHz,CDCl3)δ8.36(d,1H),7.05(t,2H),5.88(s,1H),3.48(s,2H),3.18(m,1H),2.18(s,3H),1.23(d,6H)。
LC-MS m/z(ESI)＝311.21[M+1]。
第三步：
6,7-二氯-4-(2-异丙基-4-甲基吡啶-3-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮1e
6,7-dichloro-4-(2-isopropyl-4-methylpyridin-3-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
在反应瓶中加入1d(1g，3.22mmol)、草酸二乙酯(购自上海麦克林生化科技有限公司，4.7g，32.2mmol)和冰醋酸(购自上海阿拉丁生化科技股份有限公司，2mg，0.0322mmol)，在160℃下搅拌反应，4h后反应完毕，柱层析分离(PE:EA＝2:1)，得到目标产物1e(淡黄色固体，927mg，产率89％)。
1H NMR(400MHz,DMSO-d6)δ12.55(s,1H),8.54(d,1H),7.75(s,1H),7.29(d,1H),2.85(s,1H),1.99(s,3H),1.03(d,3H),0.98(d,3H)。
LC-MS m/z(ESI)＝365.21[M+1]。
第四步：
4-(6,7-二氯-4-(2-异丙基-4-甲基吡啶-3-基-2,3-二氧-3,4-二氢吡啶[2,3-b]吡嗪-1(2H)-基)哌啶-1-羧酸叔丁酯1f
tert-butyl 4-(6,7-dichloro-4-(2-isopropyl-4-methylpyridin-3-yl)-2,3-dioxo-3,4-dihydropyrido[2,3-b]pyrazin-1(2H)-yl)piperidine-1-carboxylate
将化合物1e(1.5g，4.12mmol)、4-羟基哌啶-1-羧酸叔丁酯(购自江苏艾康生物医药研发有限公司，0.83g，4.12mmol)和三苯基膦(购自上海阿拉丁生化科技股份有限公司，2.4g，9.07mmol)溶解于20mL四氢呋喃中，在0℃下滴加偶氮二甲酸二异丙酯(购自上海麦克林生化科技有限公司，1.75g，8.65mmol)，滴加完毕恢复到室温搅拌反应，4h后反应完毕，加入乙酸乙酯(3×30mL)萃取，旋干有机相，柱层析分离，得到目标产物1f(淡黄色油状，2.2g，产率98％)。
LC-MS m/z(ESI)＝548.47[M+1]。
第五步：
4-(7-氯-6-(2-氟-6-羟基苯基)-4-(2-异丙基-4-甲基吡啶-3-基)-2,3-二氧基-3,4-二氢吡啶[2, 3-b]吡嗪-1(2氢)-哌啶-1-羧酸叔丁酯1g
tert-butyl 4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(2-isopropyl-4-methylpyridin-3-yl)-2,3-dioxo-3,4-dihydropyrido[2,3-b]pyrazin-1(2H)-yl)piperidine-1-carboxylate中间体10
将化合物1f(400mg，0.73mmol)、(2-氟-6-羟基苯基)硼酸(购自成都安斯利生物医药有限公司，456mg，2.925mmol)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(购自上海贤鼎生物科技有限公司，53.5mg，0.07mmol)和醋酸钾(购自上海阿拉丁生化科技股份有限公司，287mg，2.925mmol)溶解于4mL 1,4-二氧六环和1mL水中，在氮气的保护下，90℃搅拌反应，5h后反应完毕，加入20mL水和乙酸乙酯(3×30mL)萃取三次，收集有机相，旋干，柱层析分析(PE:EA＝5:1)，得到目标产物1g(淡黄色油状物，400mg，产率87％)。
LC-MS m/z(ESI)＝624.11[M+1]。
第六步：
7-氯-6-(2-氟-6-羟基苯基)-4-(2-异丙基-4-甲基吡啶-3-基)-1-(哌啶-4-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮1h
7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(2-isopropyl-4-methylpyridin-3-yl)-1-(piperidin-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
将化合物1g(400mg，0.64mmol)溶解于4mL二氯甲烷中，在0℃下滴加1mL三氟乙酸，1h后反应完毕，之后加入2mL甲醇和碳酸氢钠固体粉末调节pH至中性，再在反应体系中加入5mL二氯甲烷，过滤，旋干滤液，得到目标产物1h(淡黄色固体，280mg，产率86％)。
LC-MS m/z(ESI)＝524.99[M+1]。
第七步：
1-(1-丙烯酰哌啶-4-基)-7-氯-6-(2-氟-6-羟基苯基)-4-(2-异丙基-4-甲基吡啶-3-基)-1,4-二氢吡啶基[2,3-b]吡嗪-2,3-二酮化合物1
1-(1-acryloylpiperidin-4-yl)-7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(2-isopropyl-4-methylpyridin-3-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
将化合物1h(280mg，0.53mmol)溶解于2mL二氯甲烷中，在0℃下加入三乙胺(购自上海阿拉丁生化科技股份有限公司，324mg，3.2mmol)，10min后加入丙烯酰氯(购自上海阿拉丁生化科技股份有限公司，70mg，0.76mmol)，滴加完毕恢复到室温搅拌反应，1.5h后反应完毕，旋干，制备分离得到目标产物化合物1(淡黄色固体，40mg，产率13.1％)。
1H NMR(400MHz,DMSO-d6)δ9.99(d,1H),8.46(d,1H),8.11(s,1H),7.28-7.16(m,2H),6.72-6.60(m,2H),6.35(dd,1H),6.11(dd,1H),5.67(dd,1H),4.69(ddd,2H),4.41(t,1H),4.22-4.03(m,2H),3.85-3.45(m,2H),3.14(m,1H),2.68(d,2H),1.92(d,3H),1.15-0.79(m,6H)。
LC-MS m/z(ESI)＝578.04[M+1]
实施例2
1-((1-丙烯酰哌啶-4-基)甲基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1H-吲唑-4-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物2
1-((1-acryloylpiperidin-4-yl)methyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000006
参考化合物1的合成方法，制备分离得到目标产物化合物2(白色固体，30mg，产率19.3％)。
1H NMR(400MHz,Chloroform-d)δ13.03(s,1H),8.47(ddd,1H),8.22-8.16(m,1H),7.41(dd,2H),7.25-7.20(m,1H),7.14-7.01(m,1H),6.61(dd,1H),6.29(dd,1H),5.70(dd,1H),4.77(d,1H),4.45(d,2H),4.10(dd,1H),3.15(t,1H),2.74(t,1H),2.68-2.60(m,1H),2.37-2.29(m,1H),2.10(s,2H),2.03-1.97(m,6H),1.38(s,2H),1.03-0.85(m,6H)。
LC-MS m/z(ESI)＝612.13[M+1]。
实施例3
1-((1-丙烯酰氮杂环丁烷-3-基)甲基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1H-吲唑-4-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物3
1-((1-acryloylazetidin-3-yl)methyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000007
参考化合物1的合成方法，制备分离得到目标产物化合物3(白色固体，30mg，产率19.3％)。
1H NMR(400MHz,Chloroform-d)δ13.01(s,1H),8.52-8.42(m,1H),8.23-8.14(m,1H),7.41(dd,2H),7.25-7.20(m,1H),7.14-7.02(m,1H),6.61(dd,1H),6.29(dd,1H),5.70(dd,1H),4.77(d,1H),4.45(d,2H),4.10(dd,1H),3.15(t,1H),2.74(t,1H),2.66-2.58(m,1H),2.36-2.30(m,1H),2.22(t,1H),2.10(s,2H),2.02(s,3H),1.06-0.93(m,3H),0.93-0.88(m,3H)。
LC-MS m/z(ESI)＝584.08[M+1]。
实施例4
1-(1-(1-丙烯酰氮杂环丁烷-3-基)乙基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1氢-吲唑-4-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物4
1-(1-(1-acryloylazetidin-3-yl)ethyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000008
参考化合物1的合成方法，制备分离得到目标产物化合物4(白色固体，62mg，产率24％)。
1H NMR(400MHz,Chloroform-d)δ13.05(s,1H),8.45(dd,1H),8.19(dd,1H),7.47-7.31(m,2H),7.21(dd,1H),7.04(dt,1H),6.42-6.30(m,1H),6.27-6.13(m,1H),5.69(ddt,1H),4.39(dd,1H),4.27(t,1H),4.15(ddt,1H),3.98(ddd,1H),2.63(dq,1H),2.11-2.02(m,3H),2.01-1.96(m,3H),1.59-1.48(m,2H),1.19(t,3H),0.96-0.87(m,6H)。
LC-MS m/z(ESI)＝598.10[M+1]。
实施例5
1-(1-(1-丙烯酰氮杂环丁烷-3-基)丙基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1H-吲唑-4-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物5
1-(1-(1-acryloylazetidin-3-yl)propyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000009
参考化合物1的合成方法，制备分离得到目标产物化合物5(白色固体，48mg，产率27％)。
1H NMR(400MHz,Chloroform-d)δ13.04(s,1H),8.52-8.42(m,1H),8.23-8.14(m,1H),7.41(dd,2H),7.25-7.20(m,1H),7.14-7.02(m,1H),6.61(dd,1H),6.29(dd,1H),5.70(dd,1H),4.77(d,1H),4.45(d,2H),4.10(dd,1H),3.15(t,1H),2.74(t,1H),2.66-2.58(m,1H),2.36-2.30(m,1H),2.22(t,1H),2.10(s,3H),1.80(td,3H),1.06-0.93(m,3H),0.93-0.88(m,6H)。
LC-MS m/z(ESI)＝612.13[M+1]。
实施例6
1-((1-丙烯酰氮杂环丁烷-3-基)甲基)-7-氯-6-(2-氟-6-羟基苯基)-4-(2-异丙基-4-甲基吡啶-3-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物6
1-((1-acryloylazetidin-3-yl)methyl)-7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(2-isopropyl-4-methylpyridin-3-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000010
参考化合物1的合成方法，制备分离得到目标产物化合物6(白色固体，47mg，产率14％)。
1H NMR(400MHz,DMSO-d6)δ9.99(d,1H),8.46(d,1H),7.42-7.37(m,1H),7.28-7.16(m,2H),6.72-6.60(m,2H),6.35(dd,1H),6.11(dd,1H),5.67(dd,1H),4.69(ddd,2H),4.41(t,1H),4.22-4.03(m,2H),3.85(dd,1H),3.28-3.14(m,1H),2.68(ddd,1H),1.92(d,3H),1.15-0.79(m,6H)。
LC-MS m/z(ESI)＝564.01[M+1]。
实施例7
N-(2-(7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1H-吲唑-4-基)-2,3-二氧基-3,4-二氢吡啶[2,3-b]吡嗪-1(2氢)-基)乙基)丙烯酰胺化合物7
N-(2-(7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-2,3-dioxo-3,4-dihydropyrido[2,3-b]pyrazin-1(2H)-yl)ethyl)acrylamide
Figure PCTCN2021132804-appb-000011
参考化合物1的合成方法，制备分离得到目标产物化合物7(白色固体，52mg，产率17％)。
1H NMR(400MHz,Methanol-d4)δ13.20(s,1H),8.40(d,1H),8.32(dd,1H),7.48-7.32(m,1H),7.27-7.12(m,2H),6.21-6.09(m,2H),5.67-5.61(m,1H),5.48(s,1H),4.54(t,2H),3.81(t,2H),3.14(t,1H),2.94(dt,1H),2.14-1.97(m,6H),1.12(t,3H),0.91(dd,3H)。
LC-MS m/z(ESI)＝558.04[M+1]。
实施例8
1-((1-丙烯酰吡咯烷-3-基)甲基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1H-吲唑-4-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物8
1-((1-acryloylpyrrolidin-3-yl)methyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000012
参考化合物1的合成方法，制备分离得到目标产物化合物8(白色固体，43mg，产率23％)。
1H NMR(400MHz,DMSO-d6)δ13.10(s,1H),8.42-8.31(m,2H),7.45(d,1H),7.34(t,1H),7.26-7.20(m,1H),7.19(d,1H),7.14(d,1H),6.61(dd,1H),6.15(dt,1H),5.68(dd,1H),4.36(d,2H),3.99-3.67(m,2H),3.69-3.41(m,2H),3.18(t,1H),3.02-2.70(m,3H),2.26-2.11(m,1H),1.23(s,1H),1.09-0.95(m,3H),0.93-0.74(m,6H)。
LC-MS m/z(ESI)＝598.10[M+1]
实施例9
1-(1-(1-丙烯酰氮杂环丁烷-3-基)-2-甲基丙基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1氢-吲唑-4-基)-1,4-二氢吡啶基[2,3-b]吡嗪-2,3-二酮化合物9
1-(1-(1-acryloylazetidin-3-yl)-2-methylpropyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000013
参考化合物1的合成方法，制备分离得到目标产物化合物9(白色固体，32mg，产率34％)。
1H NMR(400MHz,Chloroform-d)δ13.12(d,1H),8.52-8.42(m,1H),8.23-8.14(m,1H),7.41(dd,2H),7.25-7.20(m,1H),7.14-7.02(m,1H),6.61(dd,1H),6.29(dd,1H),5.70(dd,1H),4.77(d,1H),4.45(d,2H),3.15(t,1H),2.74(t,1H),2.66-2.58(m,1H),2.36-2.30(m,1H),2.22(t,1H),2.10(s,3H),2.02(s,3H),1.80(td,3H),1.06-0.93(m,3H),0.93-0.88(m,6H)。
LC-MS m/z(ESI)＝626.16[M+1]。
实施例10
1-(1-丙烯酰吡咯烷-3-基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1H-吲唑-4-基)-1,4-二氢吡啶基[2,3-b]吡嗪-2,3-二酮化合物10
1-(1-acryloylpyrrolidin-3-yl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000014
参考化合物1的合成方法，制备分离得到目标产物化合物10(白色固体，32mg，产率34％)。
1H NMR(400MHz,DMSO-d6)δ13.10(d,1H),8.47(s,1H),8.41-8.36(m,1H),7.47(d,1H),7.38-7.33(m,1H),7.23(d,2H),6.61(dd,1H),5.70(dd,1H),5.68(s,1H),4.08-3.93(m,1H),3.84-3.69(m,2H),3.64(d,1H),3.52(s,2H),2.33(s,2H),2.00-1.94(m,6H),,1.05-0.97(m,3H),0.88-0.83(m,3H)。
LC-MS m/z(ESI)＝584.08[M+1]。
实施例11
N-(3-(7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1氢-吲唑-4-基)-2,3-二氧基-3,4-二氢吡啶[2,3-b]吡嗪-1(2氢)-基)丙基)丙烯酰胺化合物11
N-(3-(7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-2,3-dioxo-3,4-dihydropyrido[2,3-b]pyrazin-1(2H)-yl)propyl)acrylamide
Figure PCTCN2021132804-appb-000015
参考化合物1的合成方法，制备分离得到目标产物化合物11(白色固体，27mg，产率14％)。
1H NMR(400MHz,Methanol-d4)δ13.17(s,1H),8.40(d,1H),8.32(dd,1H),7.48-7.32(m,2H),7.27-7.12(m,2H),6.21-6.09(m,2H),5.67-5.61(m,1H),4.54(t,2H),3.81(t,2H),2.94(dt,1H),2.14-1.97(m,7H),2.06(m,2H),1.12(t,3H),0.91(dd,3H)。
LC-MS m/z(ESI)＝572.07[M+1]。
实施例12
1-((1-丙烯酰氮杂环丁烷-3-基)甲基)-6-(6-氨基-4-甲基吡啶-2-基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-1,4-二氢吡啶并[2,3-b]吡嗪-2,3-二酮化合物12
1-((1-acryloylazetidin-3-yl)methyl)-6-(6-amino-4-methylpyridin-2-yl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000016
参考化合物1的合成方法，制备分离得到目标产物化合物12(白色固体，36mg，产率17％)。
1H NMR(400MHz,DMSO-d6)δ8.50(d,1H),8.28(s,1H),7.27(d,1H),6.38-6.31(m,1H),6.25(s,1H),6.19(s,1H),6.11(dd,1H),5.91(s,2H),5.67(dd,1H),4.78–4.60(m,2H),4.39(d,1H),4.23–4.04(m,2H),3.84(dd,1H),3.27–3.11(m,1H),2.76–2.62(m,1H),2.07(s,3H),1.92(s,3H),1.05(d,3H),0.96(d,3H)。
LCMS m/z(ESI)＝560.2[M+1]。
实施例13
1-((1-丙烯酰氮杂环丁烷-3-基)甲基)-6-(6-氨基-4-甲基-3-(三氟甲基)吡啶-2-基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-1,4-二氢吡啶并[2,3-b]吡嗪-2,3-二酮化合物13
1-((1-acryloylazetidin-3-yl)methyl)-6-(6-amino-4-methyl-3-(trifluoromethyl)pyridin-2-yl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000017
参考化合物1的合成方法，制备分离得到目标产物化合物13(白色固体，36mg，产率17％)。
1H NMR(400MHz,DMSO-d6)δ8.47(d,1H),8.34(s,1H),7.26(dd,1H),6.80(d,J＝29.9Hz,2H),6.46-6.29(m,2H),6.11(dd,1H),5.67(dd,1H),4.68(d,2H),4.39(t,1H),4.20-4.05(m,2H),3.91-3.77(m,1H),3.26-3.11(m,1H),2.67-2.65(m,1H),2.25(s,3H),1.88(d,3H),1.04(dd,3H),0.89(dd,3H)。
LCMS m/z(ESI)＝628.2[M+1]。
实施例14
5-((1-丙烯酰氮杂环丁烷-3-基)甲基)-8-(2-异丙基-4-甲基吡啶-3-基)-2-(5-甲基-1H-吲唑-4-基)-5,8-二氢蝶啶-6,7-二酮化合物14
5-((1-acryloylazetidin-3-yl)methyl)-8-(2-isopropyl-4-methylpyridin-3-yl)-2-(5-methyl-1H-indazol-4-yl)-5,8-dihydropteridine-6,7-dione
Figure PCTCN2021132804-appb-000018
参考化合物1的合成方法，制备分离得到目标产物化合物14(白色固体，27mg，产率23％)。
1H NMR(400MHz,DMSO-d6)δ13.02(s,1H),9.23(s,1H),8.55(d,1H),7.92(s,1H),7.48(d,1H),7.32(d,1H),7.20(d,1H),6.41-6.27(m,1H),6.16–6.05(m,1H),5.67(dd,1H),4.74-4.68(m,2H),4.41(t,1H),4.21-4.06(m,2H),3.88-3.84(m,1H),3.23-3.18(m,1H),2.83-2.73(m,1H),2.21(s,3H),2.00(s,3H),1.04(d,3H),0.86(d,3H)。
LCMS m/z(ESI)＝551.2[M+1]。
实施例15
1-(7-丙烯酰基-7-氮杂螺[3.5]壬烷-2-基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1氢-吲唑)-4-基)-1,4-二氢吡啶并[2,3-b]吡嗪-2,3-二酮化合物15
1-(7-acryloyl-7-azaspiro[3.5]nonan-2-yl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000019
参考化合物1的合成方法，制备分离得到目标产物化合物15(白色固体，14mg，产率17％)。
1H NMR(400MHz,DMSO-d6)δ13.01(s,1H),8.43(dd,1H),8.20(d,1H),7.71-7.52(m,1H),7.46(s,1H),7.37(d,2H),7.21-7.18(m,1H),7.03(dd,1H),5.48-5.40(m,1H),4.15-4.07(m,1H),3.47-3.44(m,2H),3.37-3.34(m,2H),3.30-3.27(m,1H),2.23(s,3H),2.10-2.05(m,4H),2.01(s,3H),1.71-1.68(m,2H),1.65-1.62(m,2H),1.06(d,3H),0.88(d,3H)。
LCMS m/z(ESI)＝638.3[M+1]。
实施例16
1-(2-丙烯酰基-2-氮杂螺[3.3]庚烷-6-基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1H-吲唑)-4-基)-1,4-二氢吡啶并[2,3-b]吡嗪-2,3-二酮化合物16
1-(2-acryloyl-2-azaspiro[3.3]heptan-6-yl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000020
参考化合物1的合成方法，制备分离得到目标产物化合物16(白色固体，19mg，产率27％)。
1H NMR(400MHz,DMSO-d6)δ12.99(s,1H),8.41(dd,1H),8.22(d,1H),7.70-7.55(m,1H),7.43(s,1H),7.39(d,2H),7.25-7.19(m,1H),7.06-7.03(m,1H),5.46-5.41(m,1H), 4.11-4.05(m,1H),3.72-3.55(m,4H),3.30-3.27(m,1H),2.25(s,3H),1.98(s,3H),1.73-1.66(m,2H),1.64-1.58(m,2H),1.06(d,3H),0.88(d,3H)。
LCMS m/z(ESI)＝610.2[M+1]。
实施例17
5-((1-丙烯酰氮杂环丁烷-3-基)甲基)-2-(2-氟-6-羟基苯基)-8-(2-异丙基-4-甲基吡啶-3-基)-5,8-二氢蝶啶-6,7-二酮化合物17
5-((1-acryloylazetidin-3-yl)methyl)-2-(2-fluoro-6-hydroxyphenyl)-8-(2-isopropyl-4-methylpyridin-3-yl)-5,8-dihydropteridine-6,7-dione
Figure PCTCN2021132804-appb-000021
参考化合物1的合成方法，制备分离得到目标产物化合物17(白色固体，23mg，产率24％)。
1H NMR(400MHz,DMSO-d6)δ11.61(s,1H),9.18(s,1H),8.56(d,1H),7.28(ddd,2H),6.80-6.54(m,2H),6.47-6.29(m,1H),6.11(dd,1H),5.67(dd,,1H),4.85-4.58(m,2H),4.41(t,1H),4.28-4.04(m,2H),3.86(dd,1H),3.25-3.19(m,1H),2.77(q,1H),1.97(s,3H),1.06(d,3H),0.93(d,3H)。
LCMS m/z(ESI)＝531.21[M+1]。
实施例18
1-(((S)-1-丙烯酰氮杂环丁烷-2-基)甲基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1氢-吲唑-4-基)-1,4-二氢吡啶并[2,3-b]吡嗪-2,3-二酮化合物18
1-(((S)-1-acryloylazetidin-2-yl)methyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000022
参考化合物1的合成方法，制备分离得到目标产物化合物18(白色固体，24mg，产率27％)。
1H NMR(400MHz,DMSO-d6)δ11.81(s,1H),9.28(s,1H),8.53(d,1H),7.21(ddd,2H),6.70-6.51(m,2H),6.43-6.29(m,1H),6.11(dd,1H),5.67(dd,,1H),4.85-4.58(m,2H),4.41(t,1H),4.28-4.04(m,2H),3.25-3.17(m,3H),2.12(s,3H),1.93(s,3H),1.00(d,3H),0.92(d,3H)。
LCMS m/z(ESI)＝584.21[M+1]。
实施例19
1-((R)-1-丙烯酰咪唑-2-基)甲基)-7-氯-4-(2-异丙基-4-甲基吡啶-3-基)-6-(5-甲基-1氢-吲唑-4-基)-1,4-二氢吡啶[2,3-b]吡嗪-2,3-二酮化合物19
1-(((R)-1-acryloylazetidin-2-yl)methyl)-7-chloro-4-(2-isopropyl-4-methylpyridin-3-yl)-6-(5-methyl-1H-indazol-4-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000023
参考化合物1的合成方法，制备分离得到目标产物化合物19(白色固体，37mg，产率14％)。
1H NMR(400MHz,DMSO-d6)δ11.81(s,1H),9.28(s,1H),8.53(d,1H),7.21(ddd,2H),6.70-6.51(m,2H),6.43-6.29(m,1H),6.11(dd,1H),5.67(dd,,1H),4.85-4.58(m,2H),4.41(t,1H),4.28-4.04(m,2H),3.25-3.17(m,3H),2.12(s,3H),1.93(s,3H),1.00(d,3H),0.92(d,3H)。
LCMS m/z(ESI)＝584.21[M+1]。
实施例20
1-((1-丙烯酰氮杂环丁烷-3-基)甲基)-7-氯-6-((2,3-二氢-1氢-茚-4-基)氨基)-4-(2-异丙基-4-甲基吡啶-3-基)-1,4-二氢吡啶并[2,3-b]吡嗪-2,3-二酮化合物20
1-((1-acryloylazetidin-3-yl)methyl)-7-chloro-6-((2,3-dihydro-1H-inden-4-yl)amino)-4-(2-isopropyl-4-methylpyridin-3-yl)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
Figure PCTCN2021132804-appb-000024
参考化合物1的合成方法，制备分离得到目标产物化合物20(白色固体，31mg，产率17％)。
1H NMR(400MHz,DMSO-d6)δ9.42(s,1H),8.60(d,1H),8.21(s,1H),7.74(d,1H),7.33(d,1H),7.12(t,1H),7.03(dd,1H),6.76(dd,1H),6.34-6.22(m,1H),5.91(dd,2H),4.83(s,1H),4.46(s,1H),4.22-4.13(m,2H),3.91(dd,1H),3.33-3.21(m,1H),3.01(t,2H),2.84(s,1H),2.41-2.32(m,2H),1.98(s,3H),1.81-1.72(m,2H),1.13(d,3H),0.91(d,3H)。
LCMS m/z(ESI)＝585.23[M+1]。
生物学试验
软琼脂凝胶克隆形成试验
(1)底层高浓度琼脂糖凝胶层：将高浓度琼脂糖凝胶铺板于96孔板中(50μL/孔)。
(2)上层低浓度琼脂糖凝胶层：将低浓度的琼脂糖和含细胞的培养基按1：1混合后铺于底层琼脂糖凝胶层上(100μL/孔；所用细胞为NCI-H358和MiaPaca细胞，细胞量为10000细胞/孔)；冷却凝固后置于37℃培养过夜。
(3)将受试化合物配制成10mM母液，用生长培养基进行倍比稀释(1：5)，稀释8-10个浓度梯度(起始浓度10μM)，加入含有上层琼脂凝胶-细胞的96孔中(50μL/孔)；设置溶剂对照孔；每个浓度2个重复，置于二氧化碳培养箱培养10-14天。
(4)第7天更换一次含待选化合物的培养基，观察细胞克隆生长情况。
(5)培养结束后，用NBT对细胞进行染色，计算克隆形成数量，GraphPad Prism 8软件拟合IC 50值。
化合物 NCI-H358Cell-IC 50 化合物 NCI-H358Cell-IC 50
化合物1 0.5μM 化合物9 4.9μM
化合物2 1.0μM 化合物10 2.4μM
化合物3 0.2μM 化合物11 8.4μM
化合物4 0.4μM 化合物13 0.9μM
化合物6 0.5μM 化合物17 0.4μM
化合物8 1.6μM    
结果表明，所有化合物对细胞克隆形成的IC 50＜10μM，对KRAS突变肿瘤细胞具有良好的抑制效果。
本发明说明书对具体实施方案进行了详细描述，本领域技术人员应认识到，上述实施方案是示例性的，不能理解为对本发明的限制，对于本领域技术人员来说，在不脱离本发明原理的前提下，通过对本发明进行若干改进和修饰，这些改进和修饰获得技术方案也落在本发明的权利要求书的保护范围内。

Claims (3)

  1. 化合物或者其药物可接受的盐或立体异构体，所述化合物为：
    Figure PCTCN2021132804-appb-100001
    Figure PCTCN2021132804-appb-100002
    Figure PCTCN2021132804-appb-100003
  2. 药物组合物，其包含
    (1)权利要求1所述的化合物或者其药物可接受的盐或立体异构体；
    (2)任选的一种或者多种其他活性成分；以及
    (3)药学上可接受的载体和/或赋形剂。
  3. 权利要求1所述的化合物或者其药物可接受的盐或立体异构体或者权利要求2所述的药物组合物在制备抗肿瘤药物中的用途。
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WO2022266206A1 (en) 2021-06-16 2022-12-22 Erasca, Inc. Kras inhibitor conjugates
US11845761B2 (en) 2020-12-18 2023-12-19 Erasca, Inc. Tricyclic pyridones and pyrimidones

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US20180273523A1 (en) * 2015-09-28 2018-09-27 Araxes Pharma Llc Inhibitors of kras g12c mutant proteins
CN110267957A (zh) * 2017-02-02 2019-09-20 安斯泰来制药株式会社 喹唑啉化合物
WO2019213516A1 (en) * 2018-05-04 2019-11-07 Amgen Inc. Kras g12c inhibitors and methods of using the same
WO2019217307A1 (en) * 2018-05-07 2019-11-14 Mirati Therapeutics, Inc. Kras g12c inhibitors
WO2021219091A2 (zh) * 2020-04-29 2021-11-04 北京泰德制药股份有限公司 喹喔啉酮衍生物作为kras g12c突变蛋白的不可逆抑制剂

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US20180273523A1 (en) * 2015-09-28 2018-09-27 Araxes Pharma Llc Inhibitors of kras g12c mutant proteins
CN110267957A (zh) * 2017-02-02 2019-09-20 安斯泰来制药株式会社 喹唑啉化合物
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US11845761B2 (en) 2020-12-18 2023-12-19 Erasca, Inc. Tricyclic pyridones and pyrimidones
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