WO2023005280A1 - Preparation and application of aminopyrimidine derivative selectively targeting cdk9 - Google Patents
Preparation and application of aminopyrimidine derivative selectively targeting cdk9 Download PDFInfo
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- WO2023005280A1 WO2023005280A1 PCT/CN2022/088154 CN2022088154W WO2023005280A1 WO 2023005280 A1 WO2023005280 A1 WO 2023005280A1 CN 2022088154 W CN2022088154 W CN 2022088154W WO 2023005280 A1 WO2023005280 A1 WO 2023005280A1
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- pharmaceutically acceptable
- cancer
- acceptable salt
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- C07D251/22—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom to two ring carbon atoms
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/20—Antivirals for DNA viruses
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- A61P31/22—Antivirals for DNA viruses for herpes viruses
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- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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- C07D401/12—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 two hetero rings linked by a chain containing hetero atoms as chain links
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- C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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Definitions
- the invention relates to the field of medicinal chemistry, and specifically includes aminopyrimidine derivatives capable of effectively inhibiting CDK9 protein activity, a preparation method and application thereof.
- CDKs Cyclin-dependent kinases
- CDKs are key protein kinases in cell cycle regulation, which can effectively regulate DNA synthesis and mitosis. Combining with the corresponding chaperones to form a complex is a necessary condition for CDK protein function.
- CDKs can be divided into periodic CDKs (including CDK1, CDK2, CDK3, CDK4, and CDK6) and transcriptional CDKs (including CDK7, CDK8, CDK9, CDK11, CDK12, CDK13, CDK19, etc.).
- CDK9 protein is widely involved in the initiation, elongation and termination stages of transcription.
- CDK9 can effectively promote RNA transcription elongation by phosphorylating the CTD end of RNA polymerase II, and is a key factor in regulating RNA transcription. Effectively regulates downstream protein levels, including anti-apoptotic proteins MCL-1 and MYC.
- Many studies have shown that the imbalance of CDK9 protein appears in the occurrence and development of various tumors, including various types of leukemia, prostate cancer, lung cancer, liver cancer, gastric cancer, breast cancer and so on.
- an article published in the journal Cell pointed out that the inhibition of CDK9 protein can activate genes that are suppressed by tumors, and promote the expression of tumor suppressor genes and cell differentiation.
- the above studies show that finding small molecule drugs that target and inhibit CDK9 may be an effective strategy for the development of anti-tumor drugs.
- CDK9 inhibitors are in the clinical research stage, but most of them are non-selective, which leads to many unpredictable toxic and side effects. Even the clinical trials of some drugs were terminated because of this. Due to the high homology of CDK family members, it is difficult to obtain highly selective CDK9 inhibitors. However, in view of the current clinical research situation and the need to further study the biological role of CDK9, the development of selective CDK9 inhibitors is very important. Therefore, in the past two years, selective CDK9 inhibitors have continuously entered clinical research, and BAY-1143572 is the first selective CDK9 inhibitor developed by Bayer to enter clinical trials.
- BAY-1251152 is the second-generation selective CDK9 inhibitor launched by Bayer. At present, the drug has been transferred to a pharmaceutical company named Vincerx, and the compound is in the clinical research center. At the same time, around 2017, AZD-4573 developed by AstraZeneca also entered clinical research. This is a highly selective CDK9 inhibitor, which has extremely significant in vivo anti-tumor effects in the MV4-11 mouse xenograft model active.
- the main purpose of the present invention is to search for CDK9 small molecule inhibitors with aminopyrimidine or aminotriazine structure, high selectivity and good druggability.
- the present invention provides a compound as shown in general formula I or formula II or a pharmaceutically acceptable salt thereof:
- Ar is selected from the following groups:
- X is selected from hydrogen, deuterium, halogen, cyano, methyl or trifluoromethyl
- M and Y are independently selected from nitrogen or carbon
- L is a bond or CH2 ;
- R 1 is selected from the following groups:
- the present invention also provides a compound represented by general formula III or a pharmaceutically acceptable salt thereof,
- the pharmaceutically acceptable salt forms in the present invention refer to the salts formed by the compounds represented by general formulas I, II and III and pharmaceutically acceptable acids, including inorganic acid salts and organic acid salts.
- Inorganic acid salts include: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, bicarbonate, nitric acid, monohydrogen phosphate, dihydrogen phosphate, hydrobromic acid or hydroiodic acid; organic acids include: maleic acid, tartaric acid, citric acid , Methanesulfonic acid, succinic acid, acetic acid, p-toluenesulfonic acid, mandelic acid, isobutyric acid, malonic acid, etc.
- the present invention provides any of the following compounds or pharmaceutically acceptable salts thereof:
- the present invention also discloses a synthetic method of the compound of the present invention, which is selected from any of the following:
- the present invention also provides a pharmaceutical composition, comprising the compound of the present invention, or a pharmaceutically acceptable salt or prodrug thereof.
- the present invention also provides the application of the compound of the present invention or its pharmaceutically acceptable salt form in the preparation of CDK9 inhibitor drugs.
- the inventors found that the compound of the present invention or its pharmaceutically acceptable salt form can effectively inhibit CDK9 protein Activity or application in the discovery of small molecule drugs targeting CDK9.
- the present invention also provides the application of the compound of the present invention or a pharmaceutically acceptable salt form thereof in the preparation of antiviral drugs or antitumor drugs.
- the viruses include: HIV virus, cytomegalovirus, Epstein-Barr virus, adenovirus, herpes, and human T-cell lymphoblastic virus.
- the tumors include glioma, various types of leukemia, lymphoma, liver cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer, and lung cancer.
- the compound described in the present invention is a seed compound with better CDK9 inhibitory activity.
- the evaluation of biological activity shows that the designed compound has significant CDK9 inhibitory activity, good selectivity to CDK family members, and good anti-proliferation activity on MV4-11, MCF7, MOLM13 and other tumor cells.
- Examples 1-17 can be prepared by the method of Route 1. Unless otherwise specified, the synthetic steps of the intermediates will not be repeated.
- Embodiment 5 Compound LW-005
- Embodiment 6 Compound LW-006
- Embodiment 7 Compound LW-007
- Embodiment 10 Compound LW-010
- the preparation of the compounds described in Examples 24-29 can refer to the synthetic method of route 1, only need to replace the p-aminobenzoic acid (intermediate 2) of route one with p-aminophenylacetic acid (embodiment 24) or 3-aminobenzene Formic acid (Example 25) or 3-aminophenylacetic acid (Examples 26-29) will suffice.
- Example 30 The preparation of the compound described in Example 30 can refer to the synthesis method of Route 1, only need to replace 4-fluoro-2-methoxyphenylboronic acid in Route 1 with 5-fluoro-2-methoxyphenylboronic acid.
- Examples 31-40 can refer to the synthesis method of Route 2.
- the synthesis of the compound described in Example 41 can refer to the synthesis method of Route 1.
- the synthesis of intermediate 12 can refer to the preparation method provided in Scheme 2.
- the synthesis of intermediate 13 can be prepared by referring to the synthesis method provided by Waleed Minzel et al. (doi:10.1016/j.cell.2018.07.045).
- the synthesis of LW-046 uses intermediates 6 and 13 as starting materials, and the synthesis method can refer to the route provided in the route.
- the preparation of the compounds described in Examples 50-53 can refer to the synthesis method of Route 2, only need to replace p-aminobenzoic acid in Route 2 with 6-aminonicotinic acid.
- the experimental method is as follows:
- the cytotoxicity of the compound to tumor cells with high expression of CDK9 was determined by MTT method: the cells were seeded in 96-well plate at 2000 cells/well, after the cells adhered to the wall, the culture medium was aspirated and added 200uL of the diluted drug was incubated at 37°C with 5% CO2 for 72h, and then 10uL/well of MTT was added. Incubate at 37°C for 4 hours, remove the supernatant, add 150uL of DMSO to each well, and detect the absorbance at 492nm with a multi-functional microplate reader. IC50 was calculated with GraphPad and the cell growth curve was plotted.
- the experimental method is as follows:
- the kinase reaction process is as follows:
- test compound test concentration is 5nM, 20nM or 100nM, and repeated well detection.
- Compounds were prepared at 100-fold final concentration in a 384-well plate. Then use Echo550 to transfer 250nl to 384 reaction plate for later use. Add 250 nl of 100% DMSO to negative control wells and positive control wells respectively.
- Conversion%_sample is the conversion rate reading of the sample
- Conversion%_min the average value of negative control wells, representing the conversion rate readings of wells without enzyme activity
- Conversion%_max the average value of positive control wells, representing the conversion rate readings of wells without compound inhibition.
- the concentration of the compound is indicated in the brackets, and the unit is nM, such as 90 (20) indicates that the inhibition rate of the compound to the enzyme is 90% at a concentration of 20 nM.
- the compounds of the present invention show effective inhibitory activity to CDK9, and most of the compounds have an inhibitory rate of more than 70% to CDK9 at a concentration of 20 nM.
- the compound of the present invention shows better CDK9 inhibitory activity and cell activity, such as compound LW-010.
- compounds LW-008 and LW-030 also showed better or similar selectivity than positive drugs while maintaining CDK9 activity.
Abstract
Disclosed in the present invention are preparation and application of an aminopyrimidine derivative selectively targeting CDK9. Also disclosed in the present invention are a preparation method for the compound and an application of the compound in prevention and/or treatment of tumor-related diseases including glioma, various leukemia, lymphoma, liver cancer, stomach cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer and the like. The compound can effectively and selectively inhibit the activity of CDK9 protein, and meanwhile, the compound shows remarkable anti-tumor activity in cancer cells such as MV4-11, MCF-7 and MOLM-13.
Description
本发明涉及到药物化学领域，具体包括可有效抑制CDK9蛋白活性的氨基嘧啶类衍生物及其制备方法和应用。The invention relates to the field of medicinal chemistry, and specifically includes aminopyrimidine derivatives capable of effectively inhibiting CDK9 protein activity, a preparation method and application thereof.
细胞周期依赖性蛋白激酶(Cyclin-dependent kinases,CDKs)是细胞周期调控的关键的蛋白激酶，可有效调节DNA合成和有丝分裂。与相应的伴侣蛋白结合形成复合物是发挥CDK蛋白功能的必要条件。根据其主要功能，可将CDK分为周期性CDK(包括CDK1，CDK2，CDK3，CDK4和CDK6)和转录性CDK(包括CDK7，CDK8，CDK9，CDK11，CDK12，CDK13，CDK19等)。研究表明很多癌症存在“转录成瘾性”，因此，转录性CDK近来备受关注。其中，CDK9蛋白广泛参与了转录的起始、延伸和终止阶段，特别的是，CDK9可通过磷酸化RNA聚合酶II的CTD端进而有效地促使RNA转录延伸，是调节RNA转录的关键因子，可以有效调节下游蛋白水平，包括抗凋亡蛋白MCL-1和MYC。多项研究表明，CDK9蛋白的失调出现在多种肿瘤的发生发展中，包括各类白血病，前列腺癌、肺癌、肝癌、胃癌、乳腺癌等等。2018年，Cell期刊上发表文章指出，CDK9蛋白的抑制可激活被肿瘤抑制的基因，促进抑癌基因的表达和细胞分化。上述研究表明，寻找靶向抑制CDK9的小分子药物可能是一种有效的研发抗肿瘤药物的策略。Cyclin-dependent kinases (CDKs) are key protein kinases in cell cycle regulation, which can effectively regulate DNA synthesis and mitosis. Combining with the corresponding chaperones to form a complex is a necessary condition for CDK protein function. According to their main functions, CDKs can be divided into periodic CDKs (including CDK1, CDK2, CDK3, CDK4, and CDK6) and transcriptional CDKs (including CDK7, CDK8, CDK9, CDK11, CDK12, CDK13, CDK19, etc.). Studies have shown that many cancers have "transcriptional addiction", so transcriptional CDKs have recently attracted attention. Among them, CDK9 protein is widely involved in the initiation, elongation and termination stages of transcription. In particular, CDK9 can effectively promote RNA transcription elongation by phosphorylating the CTD end of RNA polymerase II, and is a key factor in regulating RNA transcription. Effectively regulates downstream protein levels, including anti-apoptotic proteins MCL-1 and MYC. Many studies have shown that the imbalance of CDK9 protein appears in the occurrence and development of various tumors, including various types of leukemia, prostate cancer, lung cancer, liver cancer, gastric cancer, breast cancer and so on. In 2018, an article published in the journal Cell pointed out that the inhibition of CDK9 protein can activate genes that are suppressed by tumors, and promote the expression of tumor suppressor genes and cell differentiation. The above studies show that finding small molecule drugs that target and inhibit CDK9 may be an effective strategy for the development of anti-tumor drugs.
目前已有十余个CDK9抑制剂处于临床研究阶段，但其中大部分为非选择性的，进而导致很多难以预料的毒副作用。甚至有部分药物的临床试验因此被终止。由于CDK家族成员的高度同源性，想获得高选择性的CDK9抑制剂有一定困难。但鉴于目前的临床研究情况和进一步深入研究CDK9生物学作用的需求，选择性CDK9抑制剂的开发至关重要。也因此，近两年不断有选择性CDK9抑制剂进入临床研究，BAY-1143572是拜耳公司研发的第一个进入临床的选择性CDK9抑制剂。但由于嗜中性白血球减少症，BAY-1143572的临床试验已经被终止，导致这一副作用的原因暂没有相关报道。BAY-1251152是拜耳公司推出的第二代选择性CDK9抑制剂，目前该药已被转让给一家名Vincerx的制药公司，该化合物正处于临床研究中心。同时，在2017年左右，阿斯利康研发的AZD-4573 也进入临床研究，这是有一个高选择性的CDK9抑制剂，在MV4-11小鼠异种移植模型中，有着极为显著的体内抗肿瘤活性。值得注意的是，近两年，国外BioTheryX公司的BTX-A51和Kronos Bio的KB-072，国内石药集团和千红药业均推出了自己的选择性CDK9抑制剂，由此可见，选择性CDK9抑制剂的开发已获得制药人的广泛关注。At present, more than ten CDK9 inhibitors are in the clinical research stage, but most of them are non-selective, which leads to many unpredictable toxic and side effects. Even the clinical trials of some drugs were terminated because of this. Due to the high homology of CDK family members, it is difficult to obtain highly selective CDK9 inhibitors. However, in view of the current clinical research situation and the need to further study the biological role of CDK9, the development of selective CDK9 inhibitors is very important. Therefore, in the past two years, selective CDK9 inhibitors have continuously entered clinical research, and BAY-1143572 is the first selective CDK9 inhibitor developed by Bayer to enter clinical trials. However, due to neutropenia, the clinical trial of BAY-1143572 has been terminated, and the cause of this side effect has not been reported yet. BAY-1251152 is the second-generation selective CDK9 inhibitor launched by Bayer. At present, the drug has been transferred to a pharmaceutical company named Vincerx, and the compound is in the clinical research center. At the same time, around 2017, AZD-4573 developed by AstraZeneca also entered clinical research. This is a highly selective CDK9 inhibitor, which has extremely significant in vivo anti-tumor effects in the MV4-11 mouse xenograft model active. It is worth noting that in the past two years, BTX-A51 of foreign BioTheryX and KB-072 of Kronos Bio, domestic CSPC and Qianhong Pharmaceutical have all launched their own selective CDK9 inhibitors. The development of CDK9 inhibitors has received extensive attention from pharmaceutical people.
发明内容Contents of the invention
本发明的主要目的是寻找具有氨基嘧啶或氨基三嗪结构的，高选择性，良好的成药性质的CDK9小分子抑制剂。The main purpose of the present invention is to search for CDK9 small molecule inhibitors with aminopyrimidine or aminotriazine structure, high selectivity and good druggability.
本发明提供一种如通式I或式II所示的化合物或其药学上可接受的盐型：The present invention provides a compound as shown in general formula I or formula II or a pharmaceutically acceptable salt thereof:
其中，Ar选自如下基团：Wherein, Ar is selected from the following groups:
X选自氢，氘，卤素，氰基，甲基或三氟甲基；X is selected from hydrogen, deuterium, halogen, cyano, methyl or trifluoromethyl;
M和Y分别独立选自氮或碳；M and Y are independently selected from nitrogen or carbon;
L是价键或CH
2；
L is a bond or CH2 ;
R
1选自如下基团：
R 1 is selected from the following groups:
本发明还提供一种如通式III所示的化合物或其药学上可接受的盐型，The present invention also provides a compound represented by general formula III or a pharmaceutically acceptable salt thereof,
其中：Ar，X，M，Y和R
1如上所述。
Where: Ar, X, M, Y and R 1 are as described above.
本发明所述的药学上可接受的盐型是指通式I，II和III所示的化合物和药学上可接受的酸形成的盐，包括无机酸盐和有机酸盐。其中无机酸盐包括：盐酸、硫酸、磷酸、碳酸、碳酸氢根、硝酸、磷酸一氢根、磷酸二氢根，氢溴酸或氢碘酸；有机酸包括：马来酸、酒石酸、柠檬酸、甲磺酸、琥珀酸、乙酸、对甲苯磺酸、扁桃酸、异丁酸、丙二酸等。The pharmaceutically acceptable salt forms in the present invention refer to the salts formed by the compounds represented by general formulas I, II and III and pharmaceutically acceptable acids, including inorganic acid salts and organic acid salts. Inorganic acid salts include: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, bicarbonate, nitric acid, monohydrogen phosphate, dihydrogen phosphate, hydrobromic acid or hydroiodic acid; organic acids include: maleic acid, tartaric acid, citric acid , Methanesulfonic acid, succinic acid, acetic acid, p-toluenesulfonic acid, mandelic acid, isobutyric acid, malonic acid, etc.
在一些具体的实例中，本发明提供如下任一所述的化合物或其药学上可接受的盐：In some specific examples, the present invention provides any of the following compounds or pharmaceutically acceptable salts thereof:
本发明还公开本发明所述化合物的合成方法，选自以下任一：The present invention also discloses a synthetic method of the compound of the present invention, which is selected from any of the following:
本发明还提供一种药物组合物，包括本发明所述的化合物，或其药学上可接受的盐型或者前药。The present invention also provides a pharmaceutical composition, comprising the compound of the present invention, or a pharmaceutically acceptable salt or prodrug thereof.
本发明还提供本发明所述化合物或其药学上可接受的盐型在制备CDK9抑制剂药物中的应用，发明人发现本发明所述化合物或其药学上可接受的盐型可有效抑制CDK9蛋白活性或应用于以CDK9为靶点的小分子药物的发现。The present invention also provides the application of the compound of the present invention or its pharmaceutically acceptable salt form in the preparation of CDK9 inhibitor drugs. The inventors found that the compound of the present invention or its pharmaceutically acceptable salt form can effectively inhibit CDK9 protein Activity or application in the discovery of small molecule drugs targeting CDK9.
本发明还提供本发明所述化合物或其药学上可接受的盐型在制备抗病毒药物或者抗肿瘤药物中的应用。The present invention also provides the application of the compound of the present invention or a pharmaceutically acceptable salt form thereof in the preparation of antiviral drugs or antitumor drugs.
在本发明的一些实施例中，所述的病毒包括：HIV病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒。In some embodiments of the present invention, the viruses include: HIV virus, cytomegalovirus, Epstein-Barr virus, adenovirus, herpes, and human T-cell lymphoblastic virus.
本发明的一些实施例中，所述的肿瘤包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌。In some embodiments of the present invention, the tumors include glioma, various types of leukemia, lymphoma, liver cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer, and lung cancer.
本发明所述的化合物为具有较好的CDK9抑制活性的苗头化合物。生物活性评价显示，所设计化合物具有显著的CDK9抑制活性，对CDK家族成员具有较好的选择性，同时对MV4-11，MCF7，MOLM13等肿瘤细胞具有良好的抗增殖活性。The compound described in the present invention is a seed compound with better CDK9 inhibitory activity. The evaluation of biological activity shows that the designed compound has significant CDK9 inhibitory activity, good selectivity to CDK family members, and good anti-proliferation activity on MV4-11, MCF7, MOLM13 and other tumor cells.
下面通过具体的实施例对本发明做进一步的详细说明，但并不将本发明限制在所举的实施例范围之中。The present invention will be further described in detail through specific examples below, but the present invention is not limited to the scope of the given examples.
除非做出说明，实施列中的化合物可通过以下路线进行合成，具体如下：Unless otherwise stated, the compounds in the examples can be synthesized by the following routes, specifically as follows:
路线1：Route 1:
中间体3的合成：Synthesis of intermediate 3:
将2,4-二氯三嗪(1,3.28g,0.024mol)溶于35ml四氢呋喃和异丙醇的混合溶剂中(1:1)，在氮气保护下降温至-40℃，然后缓慢滴加DIPEA(17.85mol,0.088mol)，再将对氨基苯甲酸(2,3g,0.022mol)溶解于35ml四氢呋喃和异丙醇的混合溶剂中(1:1)并缓慢滴加至反应液中，上述过程均控制温度在-40℃以下。滴加完毕后，保持该温度继续反映约2小时，待TLC检测原料2反应完全，停止反应，将反应液浓缩，使用乙酸乙酯与甲醇混合溶剂(30:1)重结晶，可得约2.5g淡黄色固体(3)，收率45％。HRMS[ESI]
+,calcd for C
10H
7ClN
4O
2[M-H]
-,249.0258,found 249.0250.
Dissolve 2,4-dichlorotriazine (1,3.28g, 0.024mol) in a mixed solvent of 35ml tetrahydrofuran and isopropanol (1:1), lower the temperature to -40°C under the protection of nitrogen, and then slowly drop DIPEA (17.85mol, 0.088mol), then p-aminobenzoic acid (2,3g, 0.022mol) was dissolved in a mixed solvent of 35ml tetrahydrofuran and isopropanol (1:1) and slowly added dropwise to the reaction solution, the above The process temperature is controlled below -40°C. After the dropwise addition, keep the temperature and continue to reflect for about 2 hours. After TLC detects that the reaction of raw material 2 is complete, stop the reaction, concentrate the reaction solution, and use a mixed solvent of ethyl acetate and methanol (30:1) for recrystallization to obtain about 2.5 g pale yellow solid (3), yield 45%. HRMS[ESI] + ,calcd for C 10 H 7 ClN 4 O 2 [MH] - ,249.0258,found 249.0250.
中间体4的合成：Synthesis of intermediate 4:
将4-氟-2-甲氧基苯硼酸(2g,8mmol)，中间体3(1.63g,9.6mmol)溶解于20ml二氧六环中，然后加入Pd(PPh
3)
2Cl
2(230mg,0.32mmol),碳酸钠水溶液(20ml,2N)，使用氮气置换空气三次后升温至100℃反应，约10小时后，TLC检测中间体3反应完全，停止反应，待反应液冷却后，使用稀盐酸调节PH至酸性，然后使用乙酸乙酯萃取三次，饱和氯化钠水溶液洗涤1次，无水硫酸钠干燥，抽滤，浓缩，柱层析分离后得1.69g白色固体(4)，收率53％。HRMS[ESI]
+,calcd for C
17H
13FN
4O
3[M-H]
-,339.0972,found 339.0961.
4-fluoro-2-methoxyphenylboronic acid (2g, 8mmol), intermediate 3 (1.63g, 9.6mmol) was dissolved in 20ml of dioxane, and then Pd(PPh 3 ) 2 Cl 2 (230mg, 0.32mmol), sodium carbonate aqueous solution (20ml, 2N), use nitrogen to replace the air three times, then heat up to 100°C for reaction, after about 10 hours, TLC detects that the reaction of intermediate 3 is complete, stop the reaction, after the reaction solution is cooled, use dilute hydrochloric acid Adjust the pH to acidic, then extract three times with ethyl acetate, wash once with saturated aqueous sodium chloride, dry over anhydrous sodium sulfate, filter with suction, concentrate, and separate by column chromatography to obtain 1.69 g of white solid (4), yield 53 %. HRMS[ESI] + ,calcd for C 17 H 13 FN 4 O 3 [MH] - ,339.0972,found 339.0961.
化合物X1的合成：Synthesis of Compound X1:
将中间体4(0.5mmol)，HATU(1.75mmol)溶解在4ml的四氢呋喃中，然后加入DIPEA(1mmol)，室温搅约15分钟，然后加入含裸露氨基的基团侧链(R1，0.5mmol)，室温搅拌反应1-3小时，待TLC检测中间体4反应完全，停止反应，柱层析分离纯化后可得化合物X1。Dissolve intermediate 4 (0.5mmol), HATU (1.75mmol) in 4ml of tetrahydrofuran, then add DIPEA (1mmol), stir at room temperature for about 15 minutes, and then add a side chain containing a naked amino group (R1, 0.5mmol) , stirring and reacting at room temperature for 1-3 hours, after the completion of the reaction of intermediate 4 detected by TLC, the reaction was stopped, and the compound X1 could be obtained after separation and purification by column chromatography.
路线2：Route 2:
中间体8的合成：Synthesis of Intermediate 8:
将2,4-二氯-5-氟嘧啶(6,11mmol)，中间体7(10mmol)溶解于20ml二氧六环中，然后加入Pd(PPh
3)
2Cl
2(3mmol),碳酸钠水溶液(20ml,2N)，使用氮气置换空气三次后升温至100℃反应，约2小时后，TLC检测中间体6反应完全，停止反应，待反应液冷却后，加入适量水，然后使用乙酸乙酯萃取三次，饱和氯化钠水溶液洗涤1次，无水硫酸钠干燥，抽滤，浓缩，最后使用柱层析分离纯化得中间体8。
Dissolve 2,4-dichloro-5-fluoropyrimidine (6,11mmol), intermediate 7 (10mmol) in 20ml dioxane, then add Pd(PPh 3 ) 2 Cl 2 (3mmol), sodium carbonate aqueous solution (20ml, 2N), use nitrogen to replace the air three times, then heat up to 100°C for reaction, after about 2 hours, TLC detects that the reaction of intermediate 6 is complete, stop the reaction, after the reaction liquid is cooled, add an appropriate amount of water, and then use ethyl acetate to extract Three times, washed once with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered with suction, concentrated, and finally separated and purified by column chromatography to obtain intermediate 8.
中间体9的合成：Synthesis of Intermediate 9:
将醋酸钯(0.05mmol)和XPhos(0.06mmol)溶解于6ml二氧六环中，氮气置换空气三次后，于室温搅拌约15分钟备用。将中间体8(1mmol)，对氨基苯甲酸(1mmol)，碳酸铯(3mmol)加入100ml三颈瓶中，然后计入上述新制的催化剂，氮气置换空气三次，然后升温至110℃回流反应，反应2-4小时后，TLC检测反应物消失，停止反应，冷却，使用稀盐酸调节pH至酸性，加入适量水，使用二氯甲烷萃取三次，饱和氯化钠洗涤1次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体9。Palladium acetate (0.05 mmol) and XPhos (0.06 mmol) were dissolved in 6 ml of dioxane, and the air was replaced with nitrogen three times, then stirred at room temperature for about 15 minutes for later use. Add intermediate 8 (1mmol), p-aminobenzoic acid (1mmol), and cesium carbonate (3mmol) into a 100ml three-necked flask, then add the above-mentioned newly prepared catalyst, replace the air with nitrogen three times, then heat up to 110°C for reflux reaction, and react After 2-4 hours, TLC detected that the reactant disappeared, stopped the reaction, cooled, adjusted the pH to acidic with dilute hydrochloric acid, added an appropriate amount of water, extracted three times with dichloromethane, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, Suction filtration, concentration, separation and purification by column chromatography to obtain intermediate 9.
化合物X2的合成：Synthesis of compound X2:
化合物X2的合成以中间体9和含裸露氨基的基团侧链为原料，合成方法同化合物X1的合成。The synthesis of compound X2 uses intermediate 9 and the side chain of a group containing a naked amino group as raw materials, and the synthesis method is the same as that of compound X1.
实施例1-17所述化合物的制备可以通过路线1的方法进行。除作特殊说明，其中间体合成步骤不再赘述。The compounds described in Examples 1-17 can be prepared by the method of Route 1. Unless otherwise specified, the synthetic steps of the intermediates will not be repeated.
实施例1：化合物LW-001Example 1: Compound LW-001
1H NMR(300MHz,CDCL
3):δ(ppm)＝8.83(s,1H),8.00(s,1H),7.89-7.81(m,4H),7.65(s,1H),6.82-6.76(m,2H),3.96(s,3H),3.83(s,4H),3.65(d,J＝6.0Hz,2H),2.75(s,2H),2.67-2.50(m,4H).
1 H NMR (300MHz, CDCL 3 ): δ (ppm) = 8.83 (s, 1H), 8.00 (s, 1H), 7.89-7.81 (m, 4H), 7.65 (s, 1H), 6.82-6.76 (m ,2H),3.96(s,3H),3.83(s,4H),3.65(d,J=6.0Hz,2H),2.75(s,2H),2.67-2.50(m,4H).
实施例2：化合物LW-002Example 2: Compound LW-002
1H NMR(300MHz,CDCL
3):δ(ppm)＝9.04(s,1H),8.67(s,1H),7.86-7.80(m,5H),7.65(s,1H),6.89-6.74(m,2H),3.98(s,3H),3.65(d,J＝6.0Hz,2H),3.01(s,4H),2.51-2.45(m,6H),2.14(s,3H),1.65-1.62(m,2H).
1 H NMR (300MHz, CDCL 3 ): δ (ppm) = 9.04 (s, 1H), 8.67 (s, 1H), 7.86-7.80 (m, 5H), 7.65 (s, 1H), 6.89-6.74 (m ,2H),3.98(s,3H),3.65(d,J=6.0Hz,2H),3.01(s,4H),2.51-2.45(m,6H),2.14(s,3H),1.65-1.62( m,2H).
实施列3：化合物LW-003Example 3: Compound LW-003
1H NMR(300MHz,CDCL
3):δ(ppm)＝8.83(s,1H),8.02(s,1H),7.88-7.81(m,4H),7.73(s,1H),7.12(s,1H),6.82-6.76(m,2H),3.96(s,3H),3.65-3.59(m,2H),2.76-2.65(m,10H),2.39(s,3H).
1 H NMR (300MHz, CDCL 3 ): δ (ppm) = 8.83 (s, 1H), 8.02 (s, 1H), 7.88-7.81 (m, 4H), 7.73 (s, 1H), 7.12 (s, 1H ),6.82-6.76(m,2H),3.96(s,3H),3.65-3.59(m,2H),2.76-2.65(m,10H),2.39(s,3H).
实施列4：化合物LW-004Example 4: Compound LW-004
1H NMR(300MHz,CDCL
3):δ(ppm)＝8.91(s,1H),8.07(s,1H),7.87-7.80(m,4H),7.71(s,1H),7.15(s,1H),6.83-6.78(m,2H),3.96(s,3H),3.60-3.53(m,2H),2.34-2.30(m,3H),2.18(s,3H),2.02-1.96(m,6H).
1 H NMR (300MHz, CDCL 3 ): δ (ppm) = 8.91 (s, 1H), 8.07 (s, 1H), 7.87-7.80 (m, 4H), 7.71 (s, 1H), 7.15 (s, 1H ),6.83-6.78(m,2H),3.96(s,3H),3.60-3.53(m,2H),2.34-2.30(m,3H),2.18(s,3H),2.02-1.96(m,6H ).
实施列5：化合物LW-005Embodiment 5: Compound LW-005
1H NMR(300MHz,CDCL
3):δ(ppm)＝8.98(s,1H),8.03(s,1H),7.87-7.82(m,4H),7.73(s,1H),7.12(s,1H),6.83-6.77(m,2H),3.96(s,3H),3.60-3.54(m,2H),2.51-2.47(m,2H),2.42(s,4H),1.49-1.39(m,6H).
1 H NMR (300MHz, CDCL 3 ): δ (ppm) = 8.98 (s, 1H), 8.03 (s, 1H), 7.87-7.82 (m, 4H), 7.73 (s, 1H), 7.12 (s, 1H ),6.83-6.77(m,2H),3.96(s,3H),3.60-3.54(m,2H),2.51-2.47(m,2H),2.42(s,4H),1.49-1.39(m,6H ).
实施列6：化合物LW-006Embodiment 6: Compound LW-006
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.6(s,1H),9.93(s,1H),8.88(s,1H),8.02--7.95(m,5H),7.21-7.12(m,4H),6.97-6.90(m,1H),6.50-6.46(m,1H),3.95(s,3H),2.90(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ(ppm)=10.6(s,1H),9.93(s,1H),8.88(s,1H),8.02--7.95(m,5H),7.21- 7.12(m,4H),6.97-6.90(m,1H),6.50-6.46(m,1H),3.95(s,3H),2.90(s,6H).
实施列7：化合物LW-007Embodiment 7: Compound LW-007
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.67(s,1H),9.00(s,1H),8.90(s,1H),8.76(s,1H),8.60(d,J＝6.0Hz,1H),8.14-7.95(m,7H),7.65-7.61(m,1H),7.18-7.14(d,J＝12.0Hz,1H),6.98-6.92(m,1H),3.96(s,3H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.67 (s, 1H), 9.00 (s, 1H), 8.90 (s, 1H), 8.76 (s, 1H), 8.60 (d, J =6.0Hz,1H),8.14-7.95(m,7H),7.65-7.61(m,1H),7.18-7.14(d,J=12.0Hz,1H),6.98-6.92(m,1H),3.96( s,3H).
实施列8：化合物LW-008Example 8: Compound LW-008
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝12.67(s,1H),10.53(s,1H),8.85(s,1H),8.24(d,J＝6.0Hz,2H),7.96(br,3H),7.52-7.44(m,2H),7.27-7.10(m,3H),6.96-6.89(m,1H),3.92(s,3H),3.71(s,1H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 12.67 (s, 1H), 10.53 (s, 1H), 8.85 (s, 1H), 8.24 (d, J = 6.0Hz, 2H), 7.96(br,3H),7.52-7.44(m,2H),7.27-7.10(m,3H),6.96-6.89(m,1H),3.92(s,3H),3.71(s,1H).
实施列9：化合物LW-009Example 9: Compound LW-009
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.57(s,1H),8.89(s,1H),8.85(s,1H),8.35(s,1H),8.00(br,2H),7.88(d,J＝9.0Hz,2H),7.19(d,J＝9.0Hz,1H),6.99-6.93(m,1H),3.97(s,3H),2.63-2.54(m,8H),1.04(t,J＝6Hz,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.57 (s, 1H), 8.89 (s, 1H), 8.85 (s, 1H), 8.35 (s, 1H), 8.00 (br, 2H ),7.88(d,J=9.0Hz,2H),7.19(d,J=9.0Hz,1H),6.99-6.93(m,1H),3.97(s,3H),2.63-2.54(m,8H) ,1.04(t,J=6Hz,6H).
实施列10：化合物LW-010Embodiment 10: Compound LW-010
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.64(s,1H),9.26(s,1H),8.91(s,1H),8.66(t,J＝6.0Hz,1H),8.02(br,2H),7.92(d,J＝9.0Hz,3H),7.21(d,J＝9.0Hz,1H),7.00-6.94(m,1H),3.97(s,3H),3.64(d,J＝6.0Hz,2H),3.42(s,2H),3.30(d,J＝6.0Hz,2H),2.89(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.64 (s, 1H), 9.26 (s, 1H), 8.91 (s, 1H), 8.66 (t, J = 6.0Hz, 1H), 8.02(br,2H),7.92(d,J=9.0Hz,3H),7.21(d,J=9.0Hz,1H),7.00-6.94(m,1H),3.97(s,3H),3.64(d ,J=6.0Hz,2H),3.42(s,2H),3.30(d,J=6.0Hz,2H),2.89(s,6H).
实施例11：化合物LW-011Example 11: Compound LW-011
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.85(s,1H),8.68(s,1H),8.64(t,J＝6.0Hz,1H),7.98-7.88(m,5H),7.16(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.98(s,3H),3.72(m,2H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.85 (s, 1H), 8.68 (s, 1H), 8.64 (t, J = 6.0Hz, 1H), 7.98-7.88 (m, 5H ),7.16(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.98(s,3H),3.72(m,2H)
实施例12：化合物LW-012Example 12: Compound LW-012
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.86(s,1H),8.68(s,1H),8.65(t,J＝6.0Hz,1H),7.99-7.89(m,5H),7.18(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99(s,3H),3.75(m,2H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.86 (s, 1H), 8.68 (s, 1H), 8.65 (t, J = 6.0Hz, 1H), 7.99-7.89 (m, 5H ),7.18(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99(s,3H),3.75(m,2H)
实施例13：化合物LW-013Example 13: Compound LW-013
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.85(s,1H),8.68(s,1H),8.65(t,J＝6.0Hz,1H),7.98-7.89(m,5H),7.17(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99,(m,1H)3.97(s,3H),3.52(d,J＝12Hz,2H),1.27(m,1H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.85 (s, 1H), 8.68 (s, 1H), 8.65 (t, J = 6.0Hz, 1H), 7.98-7.89 (m, 5H ),7.17(d,J=12.0Hz,1H),6.97-6.92(m,1H),3.99,(m,1H)3.97(s,3H),3.52(d,J=12Hz,2H),1.27( m,1H)
实施例14：化合物LW-014Example 14: Compound LW-014
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.84(s,1H),8.68(s,1H),8.69(t,J＝6.0Hz,1H),7.98-7.88(m,5H),7.13(d,J＝12.0Hz,1H),6.98-6.92(m,1H),3.98(s,3H),3.72(t,J＝6.0Hz,2H),3.28(s,3H),3.26(m,2H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.84 (s, 1H), 8.68 (s, 1H), 8.69 (t, J = 6.0Hz, 1H), 7.98-7.88 (m, 5H ), 7.13(d, J=12.0Hz, 1H), 6.98-6.92(m, 1H), 3.98(s, 3H), 3.72(t, J=6.0Hz, 2H), 3.28(s, 3H), 3.26 (m,2H)
实施例15：化合物LW-015Example 15: Compound LW-015
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.87(s,1H),8.67(s,1H),8.66(t,J＝6.0Hz,1H),7.98-7.89(m,5H),7.18(d,J＝12.0Hz,1H),6.99-6.92(m,1H),3.94(s,3H),3.83(s,2H),3.52(s,3H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.87 (s, 1H), 8.67 (s, 1H), 8.66 (t, J = 6.0Hz, 1H), 7.98-7.89 (m, 5H ),7.18(d,J＝12.0Hz,1H),6.99-6.92(m,1H),3.94(s,3H),3.83(s,2H),3.52(s,3H)
实施例16：化合物LW-016Example 16: Compound LW-016
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.87(s,1H),8.69(s,1H),8.67(t,J＝6.0Hz,1H),7.99-7.87(m,5H),7.14(d,J＝12.0Hz,1H),6.99-6.92(s,1H),5.32(s,1H),4.94(s,1H),4.21(m,1H),3.59(t,J＝9.0Hz,2H),3.52(t,J＝9.0Hz,2H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.87 (s, 1H), 8.69 (s, 1H), 8.67 (t, J = 6.0Hz, 1H), 7.99-7.87 (m, 5H ), 7.14(d, J=12.0Hz, 1H), 6.99-6.92(s, 1H), 5.32(s, 1H), 4.94(s, 1H), 4.21(m, 1H), 3.59(t, J= 9.0Hz, 2H), 3.52(t, J＝9.0Hz, 2H)
实施列17：化合物LW-017Example 17: Compound LW-017
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.89(s,1H),8.66(s,1H),8.64(t,J＝6.0Hz,1H),7.93-7.89(m,5H),7.19(d,J＝12.0Hz,1H),6.97-6.93(m,1H),3.97(s,3H),3.70-3.67(m,5H),1.99(t,J＝9.0Hz,2H),1.74(t,J＝9.0Hz,2H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.89 (s, 1H), 8.66 (s, 1H), 8.64 (t, J = 6.0Hz, 1H), 7.93-7.89 (m, 5H ),7.19(d,J=12.0Hz,1H),6.97-6.93(m,1H),3.97(s,3H),3.70-3.67(m,5H),1.99(t,J=9.0Hz,2H) ,1.74(t,J=9.0Hz,2H)
实施列18：化合物LW-018Example 18: Compound LW-018
化合物LW-018的合成：Synthesis of compound LW-018:
(1)LW-018-1的合成：LW-018-1的合成可参考路线一的合成方法。(1) Synthesis of LW-018-1: The synthesis of LW-018-1 can refer to the synthesis method of Route 1.
(2)将上述得到的LW-018-1(0.35mmol)溶解在2ml二氯甲烷中，然后加入三氟乙酸(5ml)，氮气保护，常温下反应约2小时，TLC检测反应完全，停止反应，使用2N的碳酸钠水溶液调节pH至碱性，并加入适量水和二氯甲烷搅拌约15分钟，分离有机层，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化，得约105mg固体(LW-018)，收率63％
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.86(s,1H),8.68(s,1H),8.65(t,J＝6.0Hz,1H),7.98-7.87(m,5H),7.15(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99(s,3H),3.62(m,1H),2.89-2.78(m,4H),2.0(s,1H),1.89(m,2H),1.64(m,2H)
(2) Dissolve the above-obtained LW-018-1 (0.35mmol) in 2ml of dichloromethane, then add trifluoroacetic acid (5ml), under nitrogen protection, react at room temperature for about 2 hours, TLC detects that the reaction is complete, stop the reaction , use 2N sodium carbonate aqueous solution to adjust the pH to alkaline, add appropriate amount of water and methylene chloride and stir for about 15 minutes, separate the organic layer, dry over anhydrous sodium sulfate, filter with suction, concentrate, use column chromatography to separate and purify, and obtain about 105mg solid (LW-018), yield 63% 1 H NMR (300MHz, DMSO-d 6 ): δ(ppm)=8.86(s, 1H), 8.68(s, 1H), 8.65(t, J=6.0 Hz,1H),7.98-7.87(m,5H),7.15(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99(s,3H),3.62(m,1H),2.89 -2.78(m,4H),2.0(s,1H),1.89(m,2H),1.64(m,2H)
实施例19-20所述化合物的制备可参考实施例18中的路线和方法进行。The preparation of the compounds described in Examples 19-20 can be carried out with reference to the route and method in Example 18.
实施列19：化合物LW-019Example 19: Compound LW-019
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.89(s,1H),8.66(s,1H),8.64(t,J＝6.0Hz,1H),7.98-7.86(m,5H),7.18(d,J＝12.0Hz,1H),6.97-6.93(m,1H),3.98(s,3H),3.64(m,1H)，2.54(m,4H)1.44-1.73(m,7H)
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.89 (s, 1H), 8.66 (s, 1H), 8.64 (t, J = 6.0Hz, 1H), 7.98-7.86 (m, 5H ), 7.18(d, J=12.0Hz, 1H), 6.97-6.93(m, 1H), 3.98(s, 3H), 3.64(m, 1H), 2.54(m, 4H), 1.44-1.73(m, 7H )
实施列20：化合物LW-020Example 20: Compound LW-020
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.88(s,1H),8.70(s,1H),8.67(d,J＝6.0Hz,1H),8.50(s,1H),7.89-7.78(m,5H),7.36(d,J＝12Hz,1H),7.05-6.98(m,1H),3.90(s,3H),3.82(t,J＝7.8Hz,1H),2.83-2.69(m,4H),1.79-1.67(m,3H),1.67-1.57(m,5H),1.48-1.36(m,2H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.88 (s, 1H), 8.70 (s, 1H), 8.67 (d, J = 6.0Hz, 1H), 8.50 (s, 1H), 7.89-7.78(m,5H),7.36(d,J=12Hz,1H),7.05-6.98(m,1H),3.90(s,3H),3.82(t,J=7.8Hz,1H),2.83- 2.69(m,4H),1.79-1.67(m,3H),1.67-1.57(m,5H),1.48-1.36(m,2H).
实施例21-22所述化合物的制备可参考路线一的合成方法。The preparation of the compounds described in Examples 21-22 can refer to the synthesis method of Route 1.
实施列21：化合物LW-021Example 21: Compound LW-021
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.88(s,1H),8.78(s,1H),8.60(t,J＝5.9Hz,1H),8.02-7.89(m,5H),7.05(d,J＝11.5Hz,1H),6.95-6.83(m,1H),3.90(s,3H),3.85(m,1H),3.59(t,J＝8.5Hz,4H),2.55-2.42(m,5H),1.88–1.70(m,4H),1.69-1.54(m,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.88 (s, 1H), 8.78 (s, 1H), 8.60 (t, J = 5.9Hz, 1H), 8.02-7.89 (m, 5H ),7.05(d,J=11.5Hz,1H),6.95-6.83(m,1H),3.90(s,3H),3.85(m,1H),3.59(t,J=8.5Hz,4H),2.55 -2.42(m,5H),1.88–1.70(m,4H),1.69-1.54(m,4H).
实施列22：化合物LW-022Example 22: Compound LW-022
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.5(s,1H),8.90(s,1H),8.75(s,1H),8.63(t,J＝5.8Hz,1H),7.99-7.86(m,5H),7.10(d,J＝11.8Hz,1H),6.90-6.81(m,1H),3.99(s,3H),3.01-2.89(m,1H),2.75-2.61(m,1H),1.77-1.64(m,4H),1.61-1.45(m,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.5 (s, 1H), 8.90 (s, 1H), 8.75 (s, 1H), 8.63 (t, J = 5.8Hz, 1H), 7.99-7.86(m,5H),7.10(d,J＝11.8Hz,1H),6.90-6.81(m,1H),3.99(s,3H),3.01-2.89(m,1H),2.75-2.61( m,1H),1.77-1.64(m,4H),1.61-1.45(m,4H).
实施列23：化合物LW-023Example 23: Compound LW-023
化合物LW-023的合成：Synthesis of compound LW-023:
(1)化合物LW-023-1的合成可参考路线一的合成方法。(1) The synthesis of compound LW-023-1 can refer to the synthesis method of Route 1.
(2)将上述所得的LW-023-1(0.42mmol)溶解在5ml的甲醇中，然后加入50％的氢氧化钠水溶液(3ml)，室温搅拌2小时，TLC检测反应完全，停止反应，使用稀盐酸调节pH至酸性，析出白色固体，抽滤后，滤饼干燥，使用柱层析分离得化合物LW-017。
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.35(s,1H),8.85(s,1H),8.70(s,1H),8.66(t,J＝6.0Hz,1H),7.98-7.89(m,5H),7.15(d,J＝12.0Hz,1H),6.98-6.92(m,1H),3.9-3.82(m,4H),2.50-2.41(m,1H),1.91-1.68(m,6H),1.65-1.54(m,2H).
(2) Dissolve the LW-023-1 (0.42mmol) obtained above in 5ml of methanol, then add 50% aqueous sodium hydroxide solution (3ml), stir at room temperature for 2 hours, TLC detects that the reaction is complete, stop the reaction, and use The pH was adjusted to acidic with dilute hydrochloric acid, and a white solid was precipitated. After suction filtration, the filter cake was dried and separated by column chromatography to obtain compound LW-017. 1 H NMR (300MHz, DMSO-d 6 ): δ(ppm)=10.35(s,1H),8.85(s,1H),8.70(s,1H),8.66(t,J=6.0Hz,1H), 7.98-7.89(m,5H),7.15(d,J=12.0Hz,1H),6.98-6.92(m,1H),3.9-3.82(m,4H),2.50-2.41(m,1H),1.91- 1.68(m,6H),1.65-1.54(m,2H).
实施例24-29所述化合物的制备可参考路线1的合成方法，只需将路线一种的对氨基苯甲酸(中间体2)替换成对氨基苯乙酸(实施例24)或3-氨基苯甲酸(实施例25)或3-氨基苯乙酸(实施例26-29)即可。The preparation of the compounds described in Examples 24-29 can refer to the synthetic method of route 1, only need to replace the p-aminobenzoic acid (intermediate 2) of route one with p-aminophenylacetic acid (embodiment 24) or 3-aminobenzene Formic acid (Example 25) or 3-aminophenylacetic acid (Examples 26-29) will suffice.
实施列24：化合物LW-024Example 24: Compound LW-024
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.86(s,1H),8.71(s,1H),8.67(t,J＝6.0Hz,1H),7.95-7.82(m,5H),7.10(d,J＝12.0Hz,1H),6.87-6.78(m,1H),3.91(s,3H),3.56-3.43(m,5H),3.40(s,2H),2.01-1.91(m,2H),1.88-1.76(m,2H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.86 (s, 1H), 8.71 (s, 1H), 8.67 (t, J = 6.0Hz, 1H), 7.95-7.82 (m, 5H ),7.10(d,J＝12.0Hz,1H),6.87-6.78(m,1H),3.91(s,3H),3.56-3.43(m,5H),3.40(s,2H),2.01-1.91( m,2H),1.88-1.76(m,2H).
实施列25：化合物LW-025Example 25: Compound LW-025
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.85(s,1H),8.71(s,1H),8.11(d,J＝2.4Hz,1H),8.05(s,1H),7.65-7.48(m,2H),6.89(t,J＝6.0Hz,1H),6.75-6.64(m,3H),3.89(s,3H),3.55(d,J＝6.0Hz,2H),3.28(d,J＝5.7Hz,2H),2.84(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.85 (s, 1H), 8.71 (s, 1H), 8.11 (d, J = 2.4Hz, 1H), 8.05 (s, 1H), 7.65-7.48(m,2H),6.89(t,J=6.0Hz,1H),6.75-6.64(m,3H),3.89(s,3H),3.55(d,J=6.0Hz,2H),3.28 (d,J=5.7Hz,2H),2.84(s,6H).
实施列26：化合物LW-026Example 26: Compound LW-026
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.0(s,1H),8.86(s,1H),8.23(d,J＝2.5Hz,1H),8.02(s,1H),7.63-7.46(m,2H),6.89(t,J＝5.0Hz,1H),6.85-6.75(m,3H),3.91(s,3H),3.64(d,J＝6.0Hz,2H),3.52(s,2H),3.29(d,J＝6.0Hz,2H),2.86(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.0 (s, 1H), 8.86 (s, 1H), 8.23 (d, J = 2.5Hz, 1H), 8.02 (s, 1H), 7.63-7.46(m,2H),6.89(t,J=5.0Hz,1H),6.85-6.75(m,3H),3.91(s,3H),3.64(d,J=6.0Hz,2H),3.52 (s,2H),3.29(d,J=6.0Hz,2H),2.86(s,6H).
实施列27：化合物LW-027Example 27: Compound LW-027
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.97(s,1H)8.82(s,1H),8.11(d,J＝2.4Hz,1H),7.96(s,1H),7.52-7.37(m,2H),6.88(t,J＝5.0Hz,1H),6.83-6.70(m,3H),3.92(s,3H),3.80(s,2H),3.72(t,J＝4.6Hz,4H),3.59(t,J＝5.6Hz,2H),2.61(t,J＝6.0Hz,2H),2.51(t,J＝4.6Hz,4H).
1H NMR (300MHz, DMSO-d 6 ): δ(ppm)=8.97(s,1H)8.82(s,1H),8.11(d,J=2.4Hz,1H),7.96(s,1H),7.52- 7.37(m,2H),6.88(t,J=5.0Hz,1H),6.83-6.70(m,3H),3.92(s,3H),3.80(s,2H),3.72(t,J=4.6Hz ,4H),3.59(t,J=5.6Hz,2H),2.61(t,J=6.0Hz,2H),2.51(t,J=4.6Hz,4H).
实施列28：化合物LW-028Example 28: Compound LW-028
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.01(s,1H),8.81(s,1H),8.11(d,J＝2.7Hz,1H),7.90(s,1H),7.54-7.38(m,2H),6.89(t,J＝6.0Hz,1H),6.86-6.77(m,3H),3.88(s,3H),3.60(s,2H),3.45(t,J＝6.0Hz,4H),2.82(t,J＝6.7Hz,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.01 (s, 1H), 8.81 (s, 1H), 8.11 (d, J = 2.7Hz, 1H), 7.90 (s, 1H), 7.54-7.38(m,2H),6.89(t,J=6.0Hz,1H),6.86-6.77(m,3H),3.88(s,3H),3.60(s,2H),3.45(t,J= 6.0Hz, 4H), 2.82(t, J=6.7Hz, 4H).
实施列29：化合物LW-029Example 29: Compound LW-029
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝8.90(s,1H),8.82(s,1H),8.07(d,J＝2.5Hz,1H),7.97(s,1H),7.53-7.37(m,2H),6.90(t,J＝5.7Hz,1H),6.85-6.79(m,3H),3.90(s,3H),3.65-3.57(m,5H),3.53(s,2H),1.87-1.72(m,2H),1.55-1.42(m,2H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 8.90 (s, 1H), 8.82 (s, 1H), 8.07 (d, J = 2.5Hz, 1H), 7.97 (s, 1H), 7.53-7.37(m,2H),6.90(t,J＝5.7Hz,1H),6.85-6.79(m,3H),3.90(s,3H),3.65-3.57(m,5H),3.53(s, 2H),1.87-1.72(m,2H),1.55-1.42(m,2H).
实施例30所述化合物的制备可参考路线一的合成方法，只需将路线一种的4-氟-2甲氧基苯硼酸替换成5-氟-2-甲氧基苯硼酸即可。The preparation of the compound described in Example 30 can refer to the synthesis method of Route 1, only need to replace 4-fluoro-2-methoxyphenylboronic acid in Route 1 with 5-fluoro-2-methoxyphenylboronic acid.
实施列30：化合物LW-030Example 30: Compound LW-030
实施例31-40的合成可参考路线2的合成方法。The synthesis of Examples 31-40 can refer to the synthesis method of Route 2.
实施列31：化合物LW-031Example 31: Compound LW-031
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.41(s,1H),8.81(s,1H),8.51(s,1H),7.65-7.78(m,5H),7.35(s,1H),6.99(t,J＝6.0Hz,1H),3.80(s,3H),3.62(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.20(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.41 (s, 1H), 8.81 (s, 1H), 8.51 (s, 1H), 7.65-7.78 (m, 5H), 7.35 (s ,1H),6.99(t,J=6.0Hz,1H),3.80(s,3H),3.62(t,J=6.0Hz,2H),2.47(t,J=6.0Hz,2H),2.20(s ,6H).
实施列32：化合物LW-032Example 32: Compound LW-032
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.38(s,1H),8.86(s,1H),7.90(s,1H),7.62-7.80(m,5H),7.35(d,J＝6.0Hz,1H),6.99(t,J＝12.0Hz,1H),3.76(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.20(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.38 (s, 1H), 8.86 (s, 1H), 7.90 (s, 1H), 7.62-7.80 (m, 5H), 7.35 (d ,J=6.0Hz,1H),6.99(t,J=12.0Hz,1H),3.76(s,3H),3.63(t,J=6.0Hz,2H),2.47(t,J=6.0Hz,2H ),2.20(s,6H).
实施列33：化合物LW-033Example 33: Compound LW-033
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.01(s,1H),8.51(s,1H),8.21(s,1H),7.62-7.58(m,5H),7.31(d,J＝6.0Hz,1H),6.97(t,J＝12.0Hz,1H),3.83(s,3H),3.61(t,J＝6.0Hz,2H),2.46(t,J＝6.0Hz,2H),2.38(s,3H),2.22(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.01 (s, 1H), 8.51 (s, 1H), 8.21 (s, 1H), 7.62-7.58 (m, 5H), 7.31 (d ,J=6.0Hz,1H),6.97(t,J=12.0Hz,1H),3.83(s,3H),3.61(t,J=6.0Hz,2H),2.46(t,J=6.0Hz,2H ),2.38(s,3H),2.22(s,6H).
实施列34：化合物LW-034Example 34: Compound LW-034
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.44(s,1H),8.86(s,1H),8.83(s,1H),7.78-7.60(m,5H),7.33(d,J＝6.0Hz,1H),6.96(t,J＝12.0Hz,1H),3.80(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.22(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.44 (s, 1H), 8.86 (s, 1H), 8.83 (s, 1H), 7.78-7.60 (m, 5H), 7.33 (d ,J=6.0Hz,1H),6.96(t,J=12.0Hz,1H),3.80(s,3H),3.63(t,J=6.0Hz,2H),2.47(t,J=6.0Hz,2H ),2.22(s,6H).
实施列35：化合物LW-035Example 35: Compound LW-035
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.40(s,1H),8.81(s,1H),8.53(s,1H),7.71-7.65(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.80(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.22(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.40 (s, 1H), 8.81 (s, 1H), 8.53 (s, 1H), 7.71-7.65 (m, 4H), 7.52 (s ,1H),7.30(d,J=6.0Hz,1H),7.11(d,J=6.0Hz,1H),3.80(s,3H),3.63(t,J=6.0Hz,2H),2.47(t ,J＝6.0Hz,2H),2.22(s,6H).
实施列36：化合物LW-036Example 36: Compound LW-036
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.46(s,1H),8.53-8.48(m,2H),7.62-7.70(m,4H),7.52(s,1H),7.32(t,J＝6.0Hz,1H),7.11(t,J＝6.0Hz,1H),3.82(s,3H),3.50(t,J＝12.0Hz,2H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.46 (s, 1H), 8.53-8.48 (m, 2H), 7.62-7.70 (m, 4H), 7.52 (s, 1H), 7.32 (t,J=6.0Hz,1H),7.11(t,J=6.0Hz,1H),3.82(s,3H),3.50(t,J=12.0Hz,2H).
实施列37：化合物LW-037Example 37: Compound LW-037
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.40(s,1H),8.86(s,1H),8.53(s,1H),7.71-7.62(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.83(s,3H),3.63-3.51(m,6H),2.52-2.41(m,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.40 (s, 1H), 8.86 (s, 1H), 8.53 (s, 1H), 7.71-7.62 (m, 4H), 7.52 (s ,1H),7.30(d,J=6.0Hz,1H),7.11(d,J=6.0Hz,1H),3.83(s,3H),3.63-3.51(m,6H),2.52-2.41(m, 6H).
实施列38：化合物LW-038Example 38: Compound LW-038
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.42(s,1H),8.81(s,1H),8.40(s,1H),7.73-7.66(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.80(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.40(s,3H),2.18(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.42 (s, 1H), 8.81 (s, 1H), 8.40 (s, 1H), 7.73-7.66 (m, 4H), 7.52 (s ,1H),7.30(d,J=6.0Hz,1H),7.11(d,J=6.0Hz,1H),3.80(s,3H),3.63(t,J=6.0Hz,2H),2.47(t ,J＝6.0Hz,2H),2.40(s,3H),2.18(s,6H).
实施列39：化合物LW-039Example 39: Compound LW-039
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.37(s,1H),8.81(s,1H),8.40(s,1H),7.69-7.65(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.83(s,3H),3.62(t,J＝6.0Hz,2H),3.50(t,J＝6.0Hz,4H)2.52(t,J＝6.0Hz,2H),2.43-2.40(m,7H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.37 (s, 1H), 8.81 (s, 1H), 8.40 (s, 1H), 7.69-7.65 (m, 4H), 7.52 (s ,1H),7.30(d,J=6.0Hz,1H),7.11(d,J=6.0Hz,1H),3.83(s,3H),3.62(t,J=6.0Hz,2H),3.50(t ,J=6.0Hz,4H)2.52(t,J=6.0Hz,2H),2.43-2.40(m,7H).
实施列40：化合物LW-040Example 40: Compound LW-040
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝9.40(s,1H),8.40(s,1H),7.80(s,1H),7.72-7.62(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.83(s,3H),3.57-3.70(m,5H),2.40(s,3H),1.97-1.74(m,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 9.40 (s, 1H), 8.40 (s, 1H), 7.80 (s, 1H), 7.72-7.62 (m, 4H), 7.52 (s ,1H),7.30(d,J=6.0Hz,1H),7.11(d,J=6.0Hz,1H),3.83(s,3H),3.57-3.70(m,5H),2.40(s,3H) ,1.97-1.74(m,4H).
实施例41所述化合物的合成可以参考路线一的合成方法。The synthesis of the compound described in Example 41 can refer to the synthesis method of Route 1.
实施列41：化合物LW-041Example 41: Compound LW-041
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.67(s,1H),9.26(s,1H),8.93(s,1H),7.91(d,J＝9.0Hz,2H),7.68(d,J＝6.0Hz,1H),7.47(t,J＝9.0Hz,1H),7.40(s,1H),7.23(s,2H),3.92(s,3H),3.63(d,J＝6.0Hz,2H),3.30(d,J＝6.0Hz,2H),2.88(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.67 (s, 1H), 9.26 (s, 1H), 8.93 (s, 1H), 7.91 (d, J = 9.0Hz, 2H), 7.68(d,J=6.0Hz,1H),7.47(t,J=9.0Hz,1H),7.40(s,1H),7.23(s,2H),3.92(s,3H),3.63(d,J =6.0Hz,2H),3.30(d,J=6.0Hz,2H),2.88(s,6H).
实施例42-44所述化合物的合成可参考路线2的合成方法。For the synthesis of the compounds described in Examples 42-44, refer to the synthesis method of Route 2.
实施列42：化合物LW-042Example 42: Compound LW-042
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.51(s,1H),9.16(s,1H),8.53(s,1H),7.81(d,J＝9.0Hz,2H),7.63(d,J＝6.0Hz,1H),7.50(t,J＝9.0Hz,1H),7.41(s,1H),7.25(d,J＝9.0Hz,2H),3.95(s,3H),3.65(d,J＝6.0Hz,2H),3.23(d,J＝6.0Hz,2H),2.89(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.51 (s, 1H), 9.16 (s, 1H), 8.53 (s, 1H), 7.81 (d, J = 9.0Hz, 2H), 7.63(d, J=6.0Hz, 1H), 7.50(t, J=9.0Hz, 1H), 7.41(s, 1H), 7.25(d, J=9.0Hz, 2H), 3.95(s, 3H), 3.65(d,J=6.0Hz,2H),3.23(d,J=6.0Hz,2H),2.89(s,6H).
实施列43：化合物LW-043Example 43: Compound LW-043
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.50(s,1H),9.11(s,1H),8.54(s,1H),7.88(d,J＝9.0Hz,2H),7.62(d,J＝6.0Hz,1H),7.51(t,J＝9.0Hz,1H),7.38(s,1H),7.15(d,J＝9.0Hz,2H),3.76(t,J＝6.0Hz,4H),3.70-3.59(m,2H),2.64(t,J＝6.0Hz,2H),2.54(s,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.50 (s, 1H), 9.11 (s, 1H), 8.54 (s, 1H), 7.88 (d, J = 9.0Hz, 2H), 7.62(d, J=6.0Hz, 1H), 7.51(t, J=9.0Hz, 1H), 7.38(s, 1H), 7.15(d, J=9.0Hz, 2H), 3.76(t, J=6.0 Hz,4H),3.70-3.59(m,2H),2.64(t,J＝6.0Hz,2H),2.54(s,4H).
实施列44：化合物LW-044Example 44: Compound LW-044
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.50(s,1H),9.11(s,1H),8.54(s,1H),7.88(d,J＝9.0Hz,2H),7.62(d,J＝6.0Hz,1H),7.51(t,J＝9.0Hz,1H),7.38(s, 1H),7.15(d,J＝9.0Hz,2H),4.25(q,J＝3.0Hz,1H),4.01(dt,J
1＝12.0Hz,J
2＝3.0Hz,2H),3.61(t,J＝9.0Hz,2H),2.11-1.96(m,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.50 (s, 1H), 9.11 (s, 1H), 8.54 (s, 1H), 7.88 (d, J = 9.0Hz, 2H), 7.62(d, J=6.0Hz, 1H), 7.51(t, J=9.0Hz, 1H), 7.38(s, 1H), 7.15(d, J=9.0Hz, 2H), 4.25(q, J=3.0 Hz,1H),4.01(dt,J 1 =12.0Hz,J 2 =3.0Hz,2H),3.61(t,J=9.0Hz,2H),2.11-1.96(m,4H).
实施列45：化合物LW-045Example 45: Compound LW-045
化合物LW-045的合成路线如下：The synthetic route of compound LW-045 is as follows:
中间体11的合成：Synthesis of intermediate 11:
将2,4-二氯-5-氟嘧啶(6,11mmol)，中间体10(10mmol)溶解于20ml的DMF中，降温至0℃以下，然后分三次加入钠氢(15mmol)，加完后移至室温搅拌反应约3小时，TLC检测中间体10反应完全，停止反应，使用适量冰水淬灭反应，然后使用乙酸乙酯萃取3次，饱和氯化钠水溶液洗涤2次，无水硫酸钠干燥，抽滤，浓缩，最后使用柱层析分离纯化得中间体11。HRMS[ESI]
+,calcd for C
11H
6ClFN
4[M+H]
+,249.0265,found 249.0251.
Dissolve 2,4-dichloro-5-fluoropyrimidine (6,11mmol) and intermediate 10 (10mmol) in 20ml of DMF, lower the temperature to below 0°C, and then add sodium hydrogen (15mmol) three times. Move to room temperature and stir for about 3 hours. TLC detects that the reaction of intermediate 10 is complete. Stop the reaction. Use an appropriate amount of ice water to quench the reaction, then use ethyl acetate to extract 3 times, wash with saturated sodium chloride aqueous solution 2 times, anhydrous sodium sulfate Dry, filter with suction, concentrate, and finally use column chromatography to separate and purify to obtain intermediate 11. HRMS[ESI] + ,calcd for C 11 H 6 ClFN 4 [M+H] + ,249.0265,found 249.0251.
中间体12的合成可参考路线2中提供的制备方法。The synthesis of intermediate 12 can refer to the preparation method provided in Scheme 2.
化合物LW-046的合成以中间体12原料，具体合成方法可参考路线2提供的制备方法，收率45％。
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.68(s,1H),8.98(s,1H),8.67(d,J＝6.0Hz,1H),8.21(s,1H),8.09(s,1H),7.88(d,J＝9.0Hz,2H),7.62-7.59(m,2H),7.51(t,J＝9.0Hz,1H),7.25(t,J＝9.0Hz,2H),3.79(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.64(t,J＝6.0Hz,2H),2.54(s,4H).
Compound LW-046 is synthesized from intermediate 12, and the specific synthesis method can refer to the preparation method provided in Scheme 2, with a yield of 45%. 1 H NMR (300MHz, DMSO-d 6 ): δ(ppm)=10.68(s,1H),8.98(s,1H),8.67(d,J=6.0Hz,1H),8.21(s,1H), 8.09(s,1H),7.88(d,J=9.0Hz,2H),7.62-7.59(m,2H),7.51(t,J=9.0Hz,1H),7.25(t,J=9.0Hz,2H ), 3.79(t, J=6.0Hz, 4H), 3.69-3.61(m, 2H), 2.64(t, J=6.0Hz, 2H), 2.54(s, 4H).
实施列46：化合物LW-046Example 46: Compound LW-046
化合物LW-046的合成路线如下：The synthetic route of compound LW-046 is as follows:
中间体13的合成可参考Waleed Minzel等人(doi:10.1016/j.cell.2018.07.045)提供的合成方法进行制备。LW-046的合成以中间体6和13为起始原料，合成方 法参考路线提供的路线即可。
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.58(s,1H),8.98(s,1H),8.09(s,2H),7.88(d,J＝9.0Hz,2H),7.62-7.59(m,2H),3.81(s,3H),3.70(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.64(t,J＝6.0Hz,2H),2.54-2.51(m,6H),0.81-0.77(m,1H),0.55-0.50(m,4H).
The synthesis of intermediate 13 can be prepared by referring to the synthesis method provided by Waleed Minzel et al. (doi:10.1016/j.cell.2018.07.045). The synthesis of LW-046 uses intermediates 6 and 13 as starting materials, and the synthesis method can refer to the route provided in the route. 1 H NMR (300MHz, DMSO-d 6 ): δ(ppm)=10.58(s,1H),8.98(s,1H),8.09(s,2H),7.88(d,J=9.0Hz,2H), 7.62-7.59(m,2H),3.81(s,3H),3.70(t,J=6.0Hz,4H),3.69-3.61(m,2H),2.64(t,J=6.0Hz,2H),2.54 -2.51(m,6H),0.81-0.77(m,1H),0.55-0.50(m,4H).
实施例47-49所述化合物的合成可参考实施例46所述的合成方法与路线。For the synthesis of the compounds described in Examples 47-49, refer to the synthesis method and route described in Example 46.
实施列47：化合物LW-047Example 47: Compound LW-047
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.57(s,1H),8.88(s,1H),8.05(s,2H),7.88(d,J＝9.0Hz,2H),7.63-7.59(m,2H),4.25(q,J＝3.0Hz,1H),4.01(dt,J
1＝12.0Hz,J
2＝3.0Hz,2H),3.80(s,3H),3.61(t,J＝9.0Hz,2H),2.55(d,J＝6.0Hz,2H),2.11-1.96(m,4H),0.80-0.75(m,1H),0.55-0.50(m,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.57 (s, 1H), 8.88 (s, 1H), 8.05 (s, 2H), 7.88 (d, J = 9.0Hz, 2H), 7.63-7.59 (m, 2H), 4.25 (q, J = 3.0Hz, 1H), 4.01 (dt, J 1 = 12.0Hz, J 2 = 3.0Hz, 2H), 3.80 (s, 3H), 3.61 (t ,J=9.0Hz,2H),2.55(d,J=6.0Hz,2H),2.11-1.96(m,4H),0.80-0.75(m,1H),0.55-0.50(m,4H).
实施列48：化合物LW-048Example 48: Compound LW-048
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.56(s,1H),8.81(s,1H),8.03(s,2H),7.89(d,J＝9.0Hz,2H),7.63-7.57(m,2H),3.81(s,3H),3.72-3.69(m,4H),3.64(t,J＝6.0Hz,2H),2.64(t,J＝6.0Hz,2H),2.54-2.51(m,6H),2.15(s,3H),0.80-0.75(m,1H),0.55-0.50(m,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.56 (s, 1H), 8.81 (s, 1H), 8.03 (s, 2H), 7.89 (d, J = 9.0Hz, 2H), 7.63-7.57(m,2H),3.81(s,3H),3.72-3.69(m,4H),3.64(t,J=6.0Hz,2H),2.64(t,J=6.0Hz,2H),2.54 -2.51(m,6H),2.15(s,3H),0.80-0.75(m,1H),0.55-0.50(m,4H).
实施列49：化合物LW-049Example 49: Compound LW-049
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.57(s,1H),8.91(s,1H),8.09(s,1H),7.99(s,1H),7.90(s,1H),7.80(d,J＝9.0Hz,2H),7.63-7.58(m,2H),3.80(s,3H),3.72(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.64(t,J＝6.0Hz,2H),2.54(t,J＝6.0Hz,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ(ppm)=10.57(s,1H),8.91(s,1H),8.09(s,1H),7.99(s,1H),7.90(s,1H ),7.80(d,J=9.0Hz,2H),7.63-7.58(m,2H),3.80(s,3H),3.72(t,J=6.0Hz,4H),3.69-3.61(m,2H) ,2.64(t,J=6.0Hz,2H),2.54(t,J=6.0Hz,4H).
实施例50-53所述化合物的制备可参考路线二的合成方法，只需要路线二中的对氨基苯甲酸替换成6-氨基烟酸即可。The preparation of the compounds described in Examples 50-53 can refer to the synthesis method of Route 2, only need to replace p-aminobenzoic acid in Route 2 with 6-aminonicotinic acid.
实施列50：化合物LW-050Example 50: Compound LW-050
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.47(s,1H),8.85(d,J＝6.0Hz,1H),8.67(s,1H),8.12(s,1H),7.98(t,J＝9.0Hz,1H),7.78(t,J＝9.0Hz,1H),7.25(d,J＝12.0Hz,1H),6.98-6.92(m,2H),3.95(s,3H),3.65(d,J＝6.0Hz,2H),3.30(d,J＝6.0Hz,2H),2.88(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.47 (s, 1H), 8.85 (d, J = 6.0Hz, 1H), 8.67 (s, 1H), 8.12 (s, 1H), 7.98(t, J=9.0Hz, 1H), 7.78(t, J=9.0Hz, 1H), 7.25(d, J=12.0Hz, 1H), 6.98-6.92(m, 2H), 3.95(s, 3H ),3.65(d,J=6.0Hz,2H),3.30(d,J=6.0Hz,2H),2.88(s,6H).
实施列51：化合物LW-051Example 51: Compound LW-051
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.51(s,1H),9.16(s,1H),8.53(s,1H),8.12(s,1H),7.98(t,J＝9.0Hz,1H),7.78(t,J＝9.0Hz,1H),7.26(d,J＝12.0Hz,1H),6.95-6.91(m,2H),3.91(s,3H),3.61(d,J＝6.0Hz,2H),3.33(d,J＝6.0Hz,2H),2.89(s,6H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.51 (s, 1H), 9.16 (s, 1H), 8.53 (s, 1H), 8.12 (s, 1H), 7.98 (t, J =9.0Hz, 1H), 7.78(t, J=9.0Hz, 1H), 7.26(d, J=12.0Hz, 1H), 6.95-6.91(m, 2H), 3.91(s, 3H), 3.61(d ,J=6.0Hz,2H),3.33(d,J=6.0Hz,2H),2.89(s,6H).
实施列52：化合物LW-052Example 52: Compound LW-052
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.53(s,1H),9.10(s,1H),8.55(s,1H),8.10(s,1H),7.99(t,J＝9.0Hz,1H),7.77(t,J＝9.0Hz,1H),7.36(d,J＝12.0Hz,1H),6.99-6.92(m,2H),3.91(s,3H),3.39(d,J＝6.0Hz,2H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.53 (s, 1H), 9.10 (s, 1H), 8.55 (s, 1H), 8.10 (s, 1H), 7.99 (t, J =9.0Hz, 1H), 7.77(t, J=9.0Hz, 1H), 7.36(d, J=12.0Hz, 1H), 6.99-6.92(m, 2H), 3.91(s, 3H), 3.39(d ,J＝6.0Hz,2H).
实施列53：化合物LW-053Example 53: Compound LW-053
1H NMR(300MHz,DMSO-d
6):δ(ppm)＝10.57(s,1H),8.79(d,J＝6.0Hz,1H),8.66(s,1H),8.10(s,1H),7.99(t,J＝9.0Hz,1H),7.88(t,J＝9.0Hz,1H),7.21(d,J ＝12.0Hz,1H),6.97-6.92(m,2H),3.95(s,3H),3.72(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.71-2.66(m,2H),2.54(t,J＝6.0Hz,4H).
1 H NMR (300MHz, DMSO-d 6 ): δ (ppm) = 10.57 (s, 1H), 8.79 (d, J = 6.0Hz, 1H), 8.66 (s, 1H), 8.10 (s, 1H), 7.99(t, J=9.0Hz, 1H), 7.88(t, J=9.0Hz, 1H), 7.21(d, J=12.0Hz, 1H), 6.97-6.92(m, 2H), 3.95(s, 3H ),3.72(t,J=6.0Hz,4H),3.69-3.61(m,2H),2.71-2.66(m,2H),2.54(t,J=6.0Hz,4H).
以下为此发明部分化合物的生物活性评价，主要包括细胞水平与酶水平两个方面。The following is the biological activity evaluation of some compounds of this invention, which mainly includes two aspects of cell level and enzyme level.
一、本发明部分化合物对多种癌细胞的增殖抑制活性1. Proliferation inhibitory activity of some compounds of the present invention on various cancer cells
实验方法如下：The experimental method is as follows:
以MTT法测定化合物对CDK9高表达的肿瘤细胞(MV4-11，MCF7以及MOLM13)的细胞毒性：细胞以2000个/孔接种于96孔板中，待细胞贴壁后，吸除培养基，加入200uL稀释好的药物，37度，5％CO2培养72h，之后加入10uL/孔的MTT。37度孵育4个小时，吸去上清，每孔加入150uL的DMSO，多功能酶标仪492nm处检测吸光值。用GraphPad计算IC
50并绘制细胞生长曲线。
The cytotoxicity of the compound to tumor cells with high expression of CDK9 (MV4-11, MCF7 and MOLM13) was determined by MTT method: the cells were seeded in 96-well plate at 2000 cells/well, after the cells adhered to the wall, the culture medium was aspirated and added 200uL of the diluted drug was incubated at 37°C with 5% CO2 for 72h, and then 10uL/well of MTT was added. Incubate at 37°C for 4 hours, remove the supernatant, add 150uL of DMSO to each well, and detect the absorbance at 492nm with a multi-functional microplate reader. IC50 was calculated with GraphPad and the cell growth curve was plotted.
二、本发明部分化合物对CDK9和CDK2蛋白的抑制活性2. Inhibitory activity of some compounds of the present invention on CDK9 and CDK2 proteins
实验方法如下：The experimental method is as follows:
首先将化合物用100％DMSO配制成10mM储存液于低温下避光保存。激酶反应过程如下：Firstly, the compound was prepared into a 10 mM stock solution with 100% DMSO and stored at low temperature in the dark. The kinase reaction process is as follows:
1)配制1×Kinase buffer。1) Prepare 1×Kinase buffer.
2)化合物浓度梯度的配制：受试化合物测试浓度为5nM,20nM或100nM，复孔检测。在384孔板中配置成100倍终浓度的化合物。然后用Echo550转移250nl到384反应板中备用。阴性对照孔和阳性对照孔中分别加250nl的100％DMSO。2) Preparation of compound concentration gradient: test compound test concentration is 5nM, 20nM or 100nM, and repeated well detection. Compounds were prepared at 100-fold final concentration in a 384-well plate. Then use Echo550 to transfer 250nl to 384 reaction plate for later use. Add 250 nl of 100% DMSO to negative control wells and positive control wells respectively.
3)用1×Kinase buffer配制2.5倍终浓度的激酶溶液。3) Use 1×Kinase buffer to prepare a kinase solution with a final concentration of 2.5 times.
4)在化合物孔和阳性对照孔分别加10μl的2.5倍终浓度的激酶溶液；在阴性对照孔中加10μl的1×Kinase buffer。4) Add 10 μl of 2.5-fold final concentration of kinase solution to compound wells and positive control wells; add 10 μl of 1×Kinase buffer to negative control wells.
5)1000rpm离心30秒，振荡混匀后室温孵育10分钟。5) Centrifuge at 1000 rpm for 30 seconds, shake and mix well, and incubate at room temperature for 10 minutes.
6)用1×Kinase buffer配制25/15倍终浓度的ATP和Kinase substrate的混合溶液。6) Use 1×Kinase buffer to prepare a mixed solution of ATP and Kinase substrate at 25/15 times the final concentration.
7)加入15μl的25/15倍终浓度的ATP和底物的混合溶液，起始反应。7) Add 15 μl of a mixed solution of ATP and substrate at 25/15 times the final concentration to initiate the reaction.
8)将384孔板1000rpm离心30秒，振荡混匀后室温孵育相应的时间。8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake and mix well, and incubate at room temperature for a corresponding period of time.
9)加入30μl终止检测液停止激酶反应，1000rpm离心30秒，振荡混匀。9) Add 30 μl of stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, shake and mix well.
10)用Caliper EZ Reader读取转化率。10) Read the conversion rate with Caliper EZ Reader.
最后进行数据计算和分析：Finally, perform data calculation and analysis:
1)抑制率计算公式：1) Inhibition rate calculation formula:
其中：Conversion％_sample是样品的转化率读数；Conversion％_min：阴性对照孔均值，代表没有酶活孔的转化率读数；Conversion％_max：阳性对照孔均值，代表没有化合物抑制孔的转化率读数。Among them: Conversion%_sample is the conversion rate reading of the sample; Conversion%_min: the average value of negative control wells, representing the conversion rate readings of wells without enzyme activity; Conversion%_max: the average value of positive control wells, representing the conversion rate readings of wells without compound inhibition.
2)拟合量效曲线2) Fitting the dose-effect curve
以浓度的log值作为X轴，百分比抑制率为Y轴，采用分析软件GraphPad Prism 5拟合量效曲线，从而得出各个化合物对酶活性的IC
50值。
Taking the log value of the concentration as the X-axis and the percent inhibition rate as the Y-axis, the analysis software GraphPad Prism 5 was used to fit the dose-effect curve to obtain the IC 50 value of each compound on the enzyme activity.
公式如下：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))The formula is as follows: Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))
本发明中部分化合物对CDK9和CDK2的抑制活性和其对MV4‐11，MCF‐7以及MOLM13细胞的抗增殖活性如下表1所示：The inhibitory activity of some compounds of the present invention on CDK9 and CDK2 and their antiproliferative activity on MV4-11, MCF-7 and MOLM13 cells are shown in Table 1 below:
表1.本发明中部分化合物的酶活性和细胞活性Table 1. Enzyme activity and cell activity of some compounds in the present invention
a：抑制率一栏中，括号内表示化合物浓度，单位为nM，如90(20)表示化合物在20nM浓度时对酶的抑制率为90％。
a : In the column of inhibition rate, the concentration of the compound is indicated in the brackets, and the unit is nM, such as 90 (20) indicates that the inhibition rate of the compound to the enzyme is 90% at a concentration of 20 nM.
BAY-1143572的结构式如下所示：The structural formula of BAY-1143572 is as follows:
根据表1所示数据，本发明所述的化合物对CDK9显示出有效的抑制活性，大部分化合物在20nM浓度下对CDK9的抑制率在70％以上，同时，与第一个进入临床的选择性CDK9抑制剂BAY-1143572相比，本发明所述化合物显示更优的CDK9抑制活性与细胞活性，比如化合物LW-010。相比于BAY1143572，LW-010的CDK9抑制活性获得一定改善的同时，其对MV4-11和MOML13细胞毒性显著提升(IC
50＝20-40nM)。选择性上，化合物LW-008和LW-030在保持CDK9活性的同时，也显示出比阳性药更优或相似的选择性。其中化合物LW-008对CDK9和CDK2的IC
50值分别为5.8nM和>5000nM,选择性超过800倍，显著优于BAY1143572(IC
50:CDK9＝8.2nM，CDK2>1000nM,约121倍)。
According to the data shown in Table 1, the compounds of the present invention show effective inhibitory activity to CDK9, and most of the compounds have an inhibitory rate of more than 70% to CDK9 at a concentration of 20 nM. Compared with CDK9 inhibitor BAY-1143572, the compound of the present invention shows better CDK9 inhibitory activity and cell activity, such as compound LW-010. Compared with BAY1143572, while the CDK9 inhibitory activity of LW-010 has been improved to some extent, its cytotoxicity to MV4-11 and MOML13 has been significantly improved (IC 50 =20-40nM). In terms of selectivity, compounds LW-008 and LW-030 also showed better or similar selectivity than positive drugs while maintaining CDK9 activity. Among them, the IC 50 values of compound LW-008 for CDK9 and CDK2 are 5.8nM and >5000nM, respectively, and the selectivity is more than 800 times, which is significantly better than BAY1143572 (IC 50 : CDK9=8.2nM, CDK2>1000nM, about 121 times).
Claims (10)
- 一种如通式I或式II所示的化合物或其药学上可接受的盐型：A compound as shown in general formula I or formula II or a pharmaceutically acceptable salt thereof:
其中，Ar选自如下基团：Wherein, Ar is selected from the following groups:
X选自氢，氘，卤素，氰基，甲基或三氟甲基；X is selected from hydrogen, deuterium, halogen, cyano, methyl or trifluoromethyl;M和Y分别独立选自氮或碳；M and Y are independently selected from nitrogen or carbon;L是价键或CH 2； L is a bond or CH2 ;R 1选自如下基团： R 1 is selected from the following groups:
- 一种如式III所示的化合物或其药学上可接受的盐型，：A compound as shown in formula III or a pharmaceutically acceptable salt thereof,:
其中Ar，X，M，Y和R 1同权利要求1中所述。 Wherein Ar, X, M, Y and R 1 are as described in claim 1. - 如下任一所述的化合物或其药学上可接受的盐：A compound or a pharmaceutically acceptable salt thereof as described in any of the following:
- 根据权利要求1～3任一项所述的化合物或其药学上可接受的盐型，其特征在于药学上可接受的盐型是指通式I，II或III所示的化合物和药学上可接受的酸形成的盐，包括无机酸盐或有机酸盐；优选的无机酸盐包括：盐酸、硫酸、磷酸、碳酸、碳酸氢根、硝酸、磷酸一氢根、磷酸二氢根，氢溴酸或氢碘酸；优选的，有机酸包括：马来酸、酒石酸、柠檬酸、甲磺酸、琥珀酸、乙酸、对甲苯磺酸、扁桃酸、异丁酸或丙二酸。According to the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, it is characterized in that the pharmaceutically acceptable salt refers to the compound shown in general formula I, II or III and pharmaceutically acceptable Salts of acids are accepted, including inorganic or organic acid salts; preferred inorganic acid salts include: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, bicarbonate, nitric acid, monohydrogen phosphate, dihydrogen phosphate, hydrobromic acid or hydroiodic acid; preferably, organic acids include: maleic acid, tartaric acid, citric acid, methanesulfonic acid, succinic acid, acetic acid, p-toluenesulfonic acid, mandelic acid, isobutyric acid or malonic acid.
- 一种制备权利要求1所述化合物的制备方法，，选自以下任一：A preparation method for preparing the compound described in claim 1, selected from any of the following:
- 一种药物组合物，其特征在于，包括权利要求1-4任一项所述的化合物，或其药学上可接受的盐型，及药学上可接受的载体。A pharmaceutical composition, characterized by comprising the compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
- 权利要求1-4任一项所述的化合物，或其药学上可接受的盐型或权利要求6所述的组合物在制备CDK9抑制剂药物中的应用。Use of the compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or the composition according to claim 6 in the preparation of a CDK9 inhibitor drug.
- 权利要求1-4任一项所述的化合物，或其药学上可接受的盐型或权利要求6所述的组合物在制备抗病毒药物或者抗肿瘤药物中的应用。Use of the compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or the composition according to claim 6 in the preparation of antiviral drugs or antitumor drugs.
- 根据权利要求8所述的应用，其特征在于：所述的病毒包括：HIV病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒。The application according to claim 8, characterized in that: the viruses include: HIV virus, cytomegalovirus, Epstein-Barr virus, adenovirus, herpes, human T cell lymphoblastic virus.
- 根据权利要求8所述的应用，其特征在于：所述的肿瘤包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌。The application according to claim 8, characterized in that: said tumors include glioma, various types of leukemia, lymphoma, liver cancer, stomach cancer, prostate cancer, ovarian cancer, breast cancer, and lung cancer.
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