WO2023185468A1 - 一类用作激酶抑制剂的化合物及其应用 - Google Patents

一类用作激酶抑制剂的化合物及其应用 Download PDF

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WO2023185468A1
WO2023185468A1 PCT/CN2023/081557 CN2023081557W WO2023185468A1 WO 2023185468 A1 WO2023185468 A1 WO 2023185468A1 CN 2023081557 W CN2023081557 W CN 2023081557W WO 2023185468 A1 WO2023185468 A1 WO 2023185468A1
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compound
crystal form
egfr
free base
kinase inhibitor
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PCT/CN2023/081557
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French (fr)
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WO2023185468A9 (zh
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李钧
吴豫生
牛成山
郭中伟
梁阿朋
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浙江同源康医药股份有限公司
郑州同源康医药有限公司
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Priority to CN202380013526.0A priority Critical patent/CN117940421A/zh
Publication of WO2023185468A1 publication Critical patent/WO2023185468A1/zh
Publication of WO2023185468A9 publication Critical patent/WO2023185468A9/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the invention belongs to the field of drug synthesis of heterocyclic compounds using nitrogen atoms as heterocyclic atoms, and specifically relates to a class of compounds used as kinase inhibitors and their applications.
  • Epidermal growth factor receptor belongs to the receptor tyrosine kinase (RTK) family, which includes EGFR/ERBB1, HER2/ERBB2/NEU, HER3/ERBB3 and HER4/ERBB4.
  • RTK receptor tyrosine kinase
  • the epidermal growth factor receptor activates its tyrosine kinase activity through homodimerization or heterodimerization, and then phosphorylates its substrate, thereby activating multiple downstream pathways related to it in the cell, such as those involved in cell survival.
  • Mutation or amplification of the epidermal growth factor receptor can lead to the activation of epidermal growth factor receptor kinase, leading to the occurrence of various human diseases, such as malignant tumors.
  • various human diseases such as malignant tumors.
  • EGFR mutations for example, among patients with non-small cell lung cancer, more than 10% of American patients have EGFR mutations, while the proportion of EGFR mutations among Asian patients can reach nearly 50%. Meanwhile, among patients with non-small cell lung cancer, the incidence of HER2 mutations is approximately 2-4%.
  • EGFR mutations mainly include deletions, insertions, and point mutations. Among them, exon 19 deletion and exon 21 L858R point mutation account for nearly 90% of EGFR mutations.
  • the EGFR-TKIs currently on the market include the first-generation Iressa, Tarceva, and Kemena, the second-generation afatinib and dacomitinib, and the third-generation osimertinib.
  • the other 10% of EGFR mutations mainly involve exons 18 and 20 of EGFR, and insertion mutations in EGFR exon 20 account for about 9% of the total EGFR mutations.
  • the most common HER2 mutation is an insertion mutation in HER2 exon 20.
  • TAK-788 has a therapeutic effect on exon 20 insertion mutations of EGFR and HER2.
  • the compound has been marketed in the United States. Judging from its reported clinical trial results, the objective response rate is 43%.
  • DZD9008 is effective in the treatment of advanced non-small cell lung cancer with EGFR or HER2 mutations.
  • the objective response rate of EGFR exon 20ins is 40%, and the efficacy is not satisfactory.
  • WO2021180238 disclosed a series of EGFR and HER2 exon 20 insertion mutation and EGFR exon 19 insertion mutations with excellent activity.
  • Compounds with deletions and L858R point mutations in exon 21 have great potential to be developed into drugs for the treatment of related diseases. It involves a class of compounds with a tripentaamine structure. Further research shows that the stereoconfiguration of the tripentaamine structure is very important. When it is converted from trans to cis, its in vivo drug efficacy will be better. effective, and in Different mutations in related diseases also have promising therapeutic effects.
  • the present invention conducted polymorphic research and screening of free base compounds.
  • the object of the present invention is to provide a class of compounds used as kinase inhibitors, which belong to a cis-tripentaamine structure and are effective against EGFR, HER2 exon 20 insertion mutations, EGFR exon 19 deletions and exon 21 mutations.
  • the mutation has good inhibitory activity.
  • the compound of the present invention has better medicinal efficacy and wider applicability than the compound with the corresponding trans structure in patent WO2021180238.
  • the second object of the present invention is to provide the use of the above compounds in the preparation of drugs for the treatment of related diseases caused by EGFR mutations and/or HER2 mutations.
  • the present invention also relates to the preparation of the free base crystal form of the above compound, and polymorphic research is conducted to determine a practical crystal form with high stability and higher bioavailability.
  • a class of compounds used as kinase inhibitors are compounds represented by Formula 1, or deuterated products thereof, or pharmaceutically acceptable salts, solvates or prodrugs:
  • X is selected from CH and N;
  • R 1 is selected from R 5 is H, C1-C3 alkyl group, C1-C3 fluoroalkyl group;
  • R 20 , R 21 , and R 22 are each independently selected from methyl or deuterated methyl;
  • R 3 is selected from C1-C3 alkyl and C1-C3 haloalkyl
  • R 40 , R 41 , and R 42 are each independently selected from H, D, and F.
  • the above compounds have been confirmed through biological activity experiments to have good inhibitory activity against EGFR and HER2 mutations, and can be used as the original drug of related drugs.
  • the "pharmaceutically acceptable salt” of the original drug refers to a salt that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.
  • Such salts include:
  • Acid addition salt formed with inorganic acid is, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc.
  • Typical inorganic acid salts are selected from hydrochloride, hydrobromide, and hydrogen iodide. Acid, sulfate, bisulfate, nitrate, phosphate, acid phosphate.
  • Acid addition salts formed with organic acids such as formic acid, acetic acid, propionic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, malic acid, Lenic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonate Acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylene bis -(3-hydroxy2-en-1-carboxylic acid), 3-pheny
  • Typical organic acid salts are selected from the group consisting of formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, and marnate. Leate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, isethionate, benzenesulfonate, salicylate, picrate, glutamate Amate, ascorbate, camphorate, camphorsulfonate. It will be readily appreciated that the pharmaceutically acceptable salt is non-toxic.
  • Solvates are compounds containing solvents, such as hydrates, dimethyl sulfoxide, etc.
  • a prodrug refers to a compound that undergoes chemical transformation through metabolism or chemical processes to produce the compound, salt, or solvate of the present invention when treating related diseases.
  • the above-mentioned compound used as a kinase inhibitor is a compound represented by formula 2, or a deuterated product thereof, or a pharmaceutically acceptable salt, solvate or prodrug:
  • the above-mentioned compound used as a kinase inhibitor is a compound represented by formula 3, or a deuterated product thereof, or a pharmaceutically acceptable salt, solvate or prodrug:
  • the compound represented by Formula 1 is:
  • the above-mentioned compounds used as kinase inhibitors are used in medicines for treating related diseases caused by EGFR mutations and/or HER2 mutations.
  • the above compounds have good inhibitory activity against exon 20 insertion mutations of EGFR and HER2, and also have good inhibitory activity against EGFR exon 19 deletions and exon 21 point mutations.
  • the EGFR mutation and/or HER2 mutation includes EGFR exon 20 insertion mutation, HER2 exon 20 insertion mutation, EGFR exon 19 deletion, EGFR exon 20 point mutation, EGFR exon 21 One or a combination of two or more point mutations. It has been confirmed by biological activity experiments that the above compounds have a good inhibitory effect on the above-mentioned mutation types.
  • the EGFR mutation and/or HER2 mutation is selected from the group consisting of EGFR Del 19/T790M/C797S mutation and EGFR L858R/T790M/C797S mutation, and the above compounds have a good inhibitory effect on the above mutation types.
  • the disease is cancer caused by the EGFR mutation and/or HER2 mutation.
  • the above compounds can also be used in combination with other drugs for the treatment of cancer.
  • Other drugs used in combination can be ERK inhibitors or MEK inhibitors.
  • the compound is in crystalline form, amorphous form or the solvate;
  • the solvent contained in the solvate is a non-aqueous solvent or a mixed solvent composed of a non-aqueous solvent and water.
  • Example 1 In order to better improve the stability and activity of the compound, selected parts of the compound represented by general formula 1 were studied for crystal form. Further, polymorphic screening research is conducted on the compound of Example 1 of the present invention to find a stable and reliable crystal form, which can not only ensure the stability of the quality of the compound, but also promote the drug to play a better role in clinical treatment. It was found that the product of Example 1 was a crystal with good crystallinity and was in an acrylic form, and was named the free base crystal form I.
  • the compound of Example 1 has a compound represented by formula A:
  • the crystal form is free base crystal form I.
  • the X-ray powder diffraction pattern of this crystal form is 9.76° ⁇ 0.2°, 10.45° ⁇ 0.2°, 16.54° ⁇ 0.2°, 18.66° ⁇ 0.2°, 20.07° ⁇ 0.2°. There is a characteristic diffraction peak at 25.90° ⁇ 0.2°.
  • the free base crystal form I has X-ray powder diffraction patterns at 9.07° ⁇ 0.2°, 9.76° ⁇ 0.2°, 10.45° ⁇ 0.2°, 11.53° ⁇ 0.2°, 11.80° ⁇ 0.2°, 12.91° ⁇ 0.2°, 13.79° ⁇ 0.2°, 14.67° ⁇ 0.2°, 15.08° ⁇ 0.2°, 15.63° ⁇ 0.2°, 16.54° ⁇ 0.2°, 17.50° ⁇ 0.2°, 18.66° ⁇ 0.2°, 20.07° ⁇ 0.2 °, there are characteristic diffraction peaks at 21.10° ⁇ 0.2°, 23.29° ⁇ 0.2°, 24.16° ⁇ 0.2°, and 25.90° ⁇ 0.2°.
  • the free base crystal form I of the compound of Example 1 can be prepared in most solvents by suspension crystallization, anti-solvent precipitation, high and low temperature cycling, evaporation crystallization and other methods.
  • Solvents here include but are not limited to methylene chloride, 1,4-dioxane, dichloroethane, methyl tert-butyl ether, N-methylpyrrolidone, ethyl acetate, acetone, methanol, dimethyl sulfide
  • the present invention found that the compound of Example 1 has two anhydrous crystal forms (free base crystal form I and free base crystal form V) and three solvates.
  • the free base crystal form II is an ethanol solvate
  • the free base crystal form III is Isopropyl alcohol solvate
  • free base crystal form IV is a solvate of dichloroethane and water, and these free base crystal forms have been characterized.
  • Competitive beating experiments were conducted on two non-crystalline forms of free base crystal form I and free base crystal form V. From the results, it was inferred that free base crystal form I may be a thermodynamically stable crystal form and is more suitable for subsequent development.
  • the crystal form evaluation of the free base crystal form I was carried out, including dry grinding, wet grinding, tableting (30MPa), stability and hygroscopicity studies. After dry grinding for 5 minutes, it became amorphous; after wet grinding with water for 5 minutes, the crystal form basically did not change; after wet grinding with ethanol for 5 minutes, the crystallinity decreased slightly; when the pressure was 30MPa in the tableting test, the crystallinity increased.
  • the free base crystal form I was relatively stable under different temperatures and humidity, and the liquid phase detection results did not change; a dynamic water vapor adsorption (DVS) test was performed, and the results showed that the weight gain after absorbing water at 80% RH 0.66%, free base crystal form I is slightly hygroscopic.
  • the free base crystal form I is a relatively stable acrystalline form, has stable solid-state properties, and is slightly hygroscopic, and can be used for subsequent drug development.
  • Figure 1 shows the tumor volume changes in the LU0387 model (mm 3 );
  • Figure 2 shows the tumor volume changes in the Ba/F3 EGFR D770_N771 ins SVD model (mm 3 );
  • Figure 3 is a single crystal diffraction pattern of the single crystal of Example 1;
  • Figure 4 is the XRPD pattern of the free base crystal form I
  • Figure 5 is the DSC and TGA overlay spectrum of the free base crystal form I;
  • Figure 6 shows the XRPD pattern of the amorphous sample obtained by dry grinding method
  • Figure 7 is the XRPD pattern of the free base crystal form II
  • Figure 8 is the DSC and TGA overlay spectrum of the free base crystal form II
  • Figure 9 is the XRPD pattern of the free base crystal form III
  • Figure 10 is the DSC and TGA overlay spectrum of the free base crystal form III
  • Figure 11 is the XRPD pattern of the free base crystal form IV
  • Figure 12 is the DSC and TGA overlay spectrum of the free base crystal form IV;
  • Figure 13 is the XRPD overlay pattern of the free base crystal form V
  • Figure 14 is the DSC and TGA overlay spectrum of the free base crystal form V.
  • the present invention confirms that the cis-tripentaamine structure has better in vivo medicinal efficacy and also has a good therapeutic effect on different mutations of related diseases.
  • R 1 is And when R 5 is -CF 3 , the compound represented by Formula 1 can also be synthesized according to the following reaction route:
  • compound A' was synthesized with reference to WO2021180238; compound B' was obtained by Friedel-Crafts reaction.
  • Synthesis of compound 8 Add compound 7 (7.7g, 41.6mmol) and palladium on carbon (2.4g, wet palladium on carbon 55%) into a mixed solvent of 100 mL methanol and 100 mL ethyl acetate, and react at room temperature for 4 hours under a hydrogen atmosphere. , spot board monitoring, after the raw material reaction is completed, process it to obtain 7.0g of product.
  • Example 1 Synthesis of Example 1: Compound 8 (7.00g, 12.6mmol) and triethylamine (3.82g, 37.8mmol) were dissolved in methylene chloride, and the temperature was lowered to 0°C under nitrogen protection, and then acryloyl chloride (1.72g) was added dropwise , 18.9 mmol) dichloromethane solution, keep the reaction at 0°C for 1 hour, monitor with a spot plate, and process the raw materials after the reaction is completed. That is, add sodium bicarbonate aqueous solution and methylene chloride, stir and separate the layers, extract the aqueous phase once more with methylene chloride, combine the organic phases, dry, spin dry, and pass through the column.
  • the crude product obtained was first dissolved with a little methylene chloride, and then petroleum ether was added dropwise to precipitate a large amount of solid. It was filtered, and the filter cake was directly added to the methanol for pulping. After drying, 3.5g of the product was finally obtained.
  • Example 1 three deuterated products of Examples 2, 3, and 4 were synthesized, as shown in Table 1 below.
  • Synthesis of compound 5 Add compound 3 (30.0g, 130mmol) to a 2000mL single-neck bottle, dissolve it completely with 1500mL 1,2-dichloroethane, add anhydrous aluminum trichloride (29.3g, 220mmol), and stir at room temperature for 30 minutes. . Cool the temperature to 0°C, add compound 4 (22.1g, 169mmol) dropwise, stir at low temperature for 30 minutes, then raise the temperature to 60°C for reaction, and monitor with a spot plate. After reacting for 4 hours, the temperature was lowered and 20.0 g of the product was obtained.
  • Synthesis of compound 7 Add compound 5 (2g, 6.15mmol), compound 6 (1.37g, 7.38mmol), and p-toluenesulfonic acid (2.11g, 12.3mmol) into 100 mL of dioxane, and raise the temperature under nitrogen protection to 80°C and react overnight. Spot plate monitoring, reaction completion, processing, to obtain 0.65g product.
  • Synthesis of compound 10 Add compound 9 (1.0g, 1.72mmol) and palladium on carbon (500 mg, wet palladium on carbon 55%) into 50 mL of methanol, and react at room temperature for 4 hours under a hydrogen atmosphere. Spot board monitoring, after the raw materials are reacted, are processed to obtain 900mg of product.
  • Example 5 Synthesis of Example 5: Compound 10 (900 mg, 1.63 mmol) and triethylamine (495 mg, 4.90 mmol) were dissolved in methylene chloride, and the temperature was lowered to 0°C in a nitrogen atmosphere, and then acryloyl chloride (223 mg, 2.45 mmol) was added dropwise. ) in methylene chloride, keep the reaction at 0°C for 2 hours. Spot board monitoring, after the raw materials have reacted, are processed to obtain 200mg of product. [M+H] + : 606.8.
  • Example 5 three deuterated products of Examples 6, 7, and 8 were synthesized, as shown in Table 2 below.
  • Synthesis of compound 4 Add compound 3 (6.17g, 11.08mmol) and lithium hydroxide monohydrate (2.33g, 55.39mmol) into a mixed solvent with a volume ratio of tetrahydrofuran/methanol/water of 6:3:1, 40°C Reaction time is 16 hours. Monitor the point board and process the raw materials after the reaction is completed to obtain 6.02g of product.
  • Example 9 Compound 8 (412 mg, 0.68 mmol) and triethylamine (206 mg, 2.04 mmol) were dissolved in 15 mL of methylene chloride, and the temperature was lowered to 0°C under nitrogen, and then acryloyl chloride (93 mg, 1.02 mmol) was added dropwise. ) in methylene chloride, keep the reaction at 0°C for 2 hours. Spot board monitoring, after the raw materials have reacted, are processed to obtain 122 mg of product.
  • Example 9 three deuterated products of Examples 10, 11, and 12 were synthesized, as shown in Table 3 below.
  • Example 13 Synthesis of Example 13: Compound 9 (520 mg, 0.83 mmol) and triethylamine (253 mg, 2.50 mmol) were dissolved in methylene chloride, and the temperature was lowered to 0°C in a nitrogen atmosphere, and then acryloyl chloride (114 mg, 1.25 mmol) was added dropwise. ) in methylene chloride, keep the reaction at 0°C for 2 hours. Spot board monitoring, after the raw materials have reacted, are processed to obtain 170mg of product. [M+H] + : 606.8.
  • Example 14 was synthesized, as shown in Table 4 below.
  • Example 15 In a 50 mL single-neck bottle, add compound 2 (112 mg, 1.24 mmol), 20 ml of methylene chloride, and oxalyl chloride (142 mg, 1.12 mmol), and stir for 2 hours under nitrogen protection. Cool the temperature to 0°C, add compound 1 (438mg, 78.92mmol) dichloromethane solution and triethylamine (152mg, 1.50mmol) dropwise, and keep the reaction at 0°C for 1 hour. Spot board monitoring, after the raw materials are reacted, are processed, and finally 160mg of product is obtained.
  • Example 16 Synthesis of Example 16: Compound 8 (300 mg, 0.48 mmol) and triethylamine (150 mg, 1.48 mmol) were dissolved in methylene chloride, and the temperature was lowered to 0°C in a nitrogen atmosphere, and then acryloyl chloride (70 mg, 0.77 mmol) was added dropwise. ) in methylene chloride, keep the reaction at 0°C for 2 hours. Spot board monitoring, after the raw materials have reacted, are processed to obtain 118mg of product. [M+H] + : 688.8.
  • Example 17 Compound 5 (12 mg, 0.024 mmol), compound 6 (6 mg, 0.018 mmol), and p-toluenesulfonic acid (8 mg, 0.042 mmol) were dissolved in 1 mL of N-methylpyrrolidone and 2 mL of ethylene glycol. In monomethyl ether, the temperature was raised to 100°C under nitrogen atmosphere and the reaction was carried out for 4 hours. Spot board monitoring, after the raw materials have reacted, are processed to obtain 3 mg of product. [M+H] + : 675.8.
  • the engineering cells selected in the experiment Ba/F3-FL-EGFR-V769-D770ins ASV, Ba/F3-FL-EGFR-D770-N771 ins SVD, Ba/F3-FL-EGFR-H773-V774 ins NPH, Ba/ F3-FL-EGFR-A763-Y764 ins FQEA, Ba/F3-HER2-A775-G776 ins YVMA were provided by Hefei Zhongke Presheng Biomedical Technology Co., Ltd. and were validated before use in this study.
  • Inhibition Rate 100-(RLU compound-RLU blank)/(RLU control-RLU blank)*100%. Calculate the cell viability corresponding to different concentrations of compounds in EXCEL, and then use GraphPad Prism software to draw a cell viability curve and calculate the IC 50 value. Table 5 shows the name and structure of the reference substance.
  • Table 6 Table 7 and Table 8, they are divided into 3 categories according to the IC 50 value, including A ⁇ 30nM, 30nM ⁇ B ⁇ 100nM, and C>100nM.
  • the compounds in the examples have good inhibitory capabilities against EGFR exon 20 insertion mutations. Better than DZD9008 and equivalent in activity to TAK-788. As can be seen from Table 7, the cell activity of Example 5 in the HER2exon 20 insertion mutation is equivalent to that of TAK-788 and better than DZD9008.
  • the cell lines NCI-H1975 and PC9 selected in the experiment were provided by CrownBiotech (Beijing) Co., Ltd. and were verified before being used in this study.
  • the compounds of the present invention have a good inhibitory effect on the cell proliferation of cell lines NCI-H975 and PC9, and are better than DZD9008.
  • the lung cancer LU0387PDX model is a commonly used xenograft tumor model of Crown Branch.
  • the experimental conditions of this model are as follows: After the tumor-bearing mice are euthanized, the tumor mass is dissected under aseptic conditions, and the tumor mass is cleaned to remove blood stains and connective tissue. And the necrotic part, the tumor was cut into small pieces of 2*2* 2mm3 . Use an adapter needle to inoculate sterilized experimental mice subcutaneously on the right back. Observe the tumor growth regularly. When the tumor grows to an average volume of 100-200mm3 , the mice will be randomly divided into groups for administration according to the tumor size and body weight.
  • mice were randomly divided into groups according to tumor volume to ensure that tumor volumes were similar between different groups. There were 3 mice in each group, and the drug was administered orally once a day. After administration, the inoculation site was observed. Tumor volume and mouse body weight were measured twice weekly.
  • TGI tumor volume inhibition rate
  • Example 1 At the same dose in the LU0387 model, the efficacy of Example 1 is better than that of TAK-788 and DZD9008.
  • the efficacy of Example 5 is similar to that of TAK-788. As the dosage of Example 1 increases, the drug efficacy also increases significantly.
  • the cell line Ba/F3EGFR D770_N771 ins SVD was cultured with RPMI1640+10% fetal calf serum+1% double antibody at 37°C and 5% CO2 , and was passaged 2-3 times a week. When the cell saturation is 80-90% and the number reaches the requirement, collect the cells. Inoculate 0.2ml (1*106) cells subcutaneously into the right back of female nude mice aged 6-8 weeks and weighing 18-22g. When the average tumor volume reaches about 150-200mm3 , drug administration in groups begins. Before the start of dosing, all animals were weighed and tumor volumes were measured using vernier calipers. Mice were randomly divided into groups according to tumor volume to ensure that tumor volumes were similar between different groups. There were 3 mice in each group, and the drug was administered orally once a day. After administration, the inoculation site was observed. Tumor volume and mouse body weight were measured twice weekly.
  • Example 18 and Example 40 in the table are compounds with a trans-tripentaamine structure in patent WO2021180238. The name and structure are shown in Table 11:
  • the compounds in this patent have a good inhibitory effect on exon 20 insertion mutations of EGFR or HER2, and also have a good inhibitory effect on cell proliferation of cell lines NCI-H1975 and PC9; they also have a good inhibitory effect on EGFR exon 20 insertion mutations.
  • one or more combinations of HER2 exon 20 insertion mutations, EGFR exon 19 deletions, EGFR exon 20 point mutations, and EGFR exon 21 point mutations, and its drugs are expected to have an effect on related diseases. Better therapeutic effect.
  • Example 1 The compound obtained in Example 1 was screened for polymorphism to search for its potential crystal form, in order to find an excellent crystal form suitable for subsequent development. Using the synthesized Example 1 as the starting material, it was detected that the compound was a crystal with good crystallinity and in an amorphous form, which was named the free base crystal form I. The amorphous free base compound was then prepared by dry grinding. Use the above two types as starting materials to carry out crystal form screening experiments.
  • the starting materials for the free base form I of the compound of Example 1 were fully characterized. It was seen through polarized light microscopy (PLM) that the raw material was irregularly shaped crystals with good crystallinity.
  • Figure 4 is the XRPD pattern of the free base crystal form I
  • Figure 5 is the DSC and TGA overlay pattern of the crystal form.
  • DSC differential scanning calorimetry
  • TGA thermogravimetric analysis
  • the solubility of the free base crystal form I of Compound Example 1 at room temperature was roughly measured by visual inspection in 18 solvents. The results are shown in Table 13.
  • the free base crystal form I has high solubility only in dichloroethane and dichloromethane, and has low solubility in most of the tested solvents.
  • XRPD diffraction patterns were collected using a Bruker D2 Phaser. Place the sample to be tested on a smooth, background-free silicon wafer for testing. The measurement parameters are shown in Table 14.
  • PLM analysis was performed using an Otter optical microscope BK-Pol. Take a small amount of sample, place it on a glass slide, disperse it with a drop of silicone oil, cover it with a cover slip, and observe it under a microscope.
  • the DSC curve was collected by the DSC 250 model of TA Instruments.
  • the test method of DSC 250 instrument is as follows: Precisely weigh an appropriate amount of sample into a perforated aluminum crucible, raise the temperature from 25°C to a final temperature of 300°C at a heating rate of 10°C/min, and purge with nitrogen at a flow rate of 50mL/min. .
  • TGA data were collected by the TGA 550 model of TA Instruments. Put an appropriate amount of sample into an aluminum crucible that has been peeled in advance, and heat it from room temperature to 300°C at a heating rate of 10°C/min. Purge the balance chamber with 40mL/min nitrogen and the sample chamber with 25mL/min nitrogen. .
  • Suspension crystallization is based on the visual solubility results of the compound in Example 1.
  • the free base crystal form I and the amorphous crystal form were used as starting materials, respectively, in the selected single solvent (solvent Selected from methylene chloride, 1,4-dioxane, dichloroethane, methyl tert-butyl ether, N-methylpyrrolidone, ethyl acetate, acetone, methanol, dimethyl sulfoxide, isopropyl acetate , butanone, cyclohexane, tetrahydrofuran, water, acetonitrile, isopropyl alcohol, ethanol, n-heptane) or mixed solvents to prepare the crystal form.
  • solvent S selected from methylene chloride, 1,4-dioxane, dichloroethane, methyl tert-butyl ether, N-methylpyrrolidone, ethyl acetate,
  • Antisolvent precipitation is based on the visual solubility results of the compound in Example 1. Dichloromethane and dichloroethane are used as good solvents, and then different antisolvents are added to them under stirring conditions at room temperature ( ⁇ 25°C) to form the crystal form. preparation. Two crystal forms were obtained, free base crystal form I and free base crystal form III.
  • the high and low temperature cycle is to add a solvent to the compound (the solvent is selected from dichloromethane, 1,4-dioxane, dichloroethane, methyl tert-butyl ether, N-methylpyrrolidone, ethyl acetate, acetone, Methanol, dimethyl sulfoxide, isopropyl acetate, butanone, cyclohexane, tetrahydrofuran, water, acetonitrile, isopropyl alcohol, ethanol, n-heptane), and then stirred under temperature cycling conditions of 50°C to 5°C Crystal form preparation. Two crystal forms were obtained, free base crystal form I and free base crystal form II.
  • the solvent is selected from dichloromethane, 1,4-dioxane, dichloroethane, methyl tert-butyl ether, N-methylpyrrolidone, ethyl acetate, acetone, Methanol, dimethyl
  • Evaporative crystallization involves adding a solvent to the compound. After filtering the prepared solvent, the filtrate is added dropwise to the sample bottle. The sealing film covering the bottle mouth is punctured and placed in a fume hood to slowly evaporate. Two crystal forms were obtained, free base crystal form I and free base crystal form IV. The free base crystal form IV was heated to 150°C to remove the solvent and a new crystal form was prepared, which was named free base crystal form V.
  • the free base crystal form I can be obtained in most solvents, and the compound obtained in Example 1 is the free base crystal form I.
  • the characterization results are shown in Figures 4 and 5.
  • Free base crystal form II can be obtained in some solvent systems containing ethanol. Using amorphous compounds as raw materials, free base crystal form II can be obtained by suspension and crystallization in a single solvent ethanol. The characterization results are shown in Figures 7 and 8. The DSC curve of the free base crystal form II has two endothermic peaks at 124°C and 230°C, and the TGA curve has a weight loss of 4.48% at 65-150°C. Form II is obtained in ethanol solvent system. In summary, it can be concluded that the free base crystal form II should be a solvate of ethanol. The endothermic peak at 124°C on the DSC curve should be the desolvation peak.
  • the free base crystalline Form III can be obtained in certain solvent systems containing isopropanol. When the crystalline form is prepared by antisolvent precipitation, it is found in the dichloromethane/isopropanol or dichloroethane/isopropanol system. The free base crystal form III was obtained. The characterization results are shown in Figures 9 and 10.
  • the DSC curve of the free base crystal form III has two endothermic peaks at 121°C and 230°C.
  • the TGA curve has a weight loss of 9.57% at 75-145°C, and the free base crystal form III has a weight loss of 9.57%.
  • Form III is transformed into the free base crystal form I by XRPD after heating to 150°C to remove the solvent.
  • the free base crystal form III is obtained in the solvent system of isopropyl alcohol. Based on the above inference, the free base crystal form III should be a solvate of isopropyl alcohol, and the endothermic peak at 121°C on the DSC curve should be the desolvation peak. .
  • Free base crystalline Form IV can be obtained in certain solvent systems containing dichloroethane. Weigh 92.28 mg of the raw material, add it to a sample bottle, add 5.0 mL of dichloroethane to dissolve, cover the sample bottle with a sealing film, prick a hole and place it in a fume hood to slowly evaporate, thus preparing crystal form IV through evaporation and crystallization.
  • the characterization results are shown in Figures 11 and 12.
  • the DSC curve of the free base crystal form IV has two endothermic peaks at 108°C and 231°C, and the TGA curve has a weight loss of 10.41% at 25-150°C.
  • the free base crystal form V After heating to 150°C to desolvate the free base crystal form IV, it was characterized by XRPD and showed that it transformed into a new crystal form, named the free base crystal form V.
  • the characterization results are shown in Figures 13 and 14.
  • the DSC curve of the free base crystal form V has an endothermic peak at 231°C, and the TGA curve shows no obvious weight loss before decomposition. In summary, it can be concluded that the free base crystal form V should be an acrylic form.
  • free base crystal form I may be the thermodynamically stable crystal form of the compound.
  • free base crystal form V was not directly obtained in the screening experiment, but was only obtained through the desolvation of the free base crystal form IV. Therefore, only the free base crystal form I was evaluated for crystal form, including dry grinding, wet grinding, tableting (30 MPa), stability and hygroscopicity studies.
  • a dynamic water vapor adsorption (DVS) test was conducted on the free base crystal form I.
  • the results showed that the water absorption and weight gain at 80% RH were 0.66%, and the free base crystal form I was slightly hygroscopic.
  • the spatial configuration of this type of compound shown in Formula 1 has been determined, especially the spatial structure of Example 1 has been accurately determined.
  • a total of 2 anhydrous crystal forms (free base crystal form I and free base crystal form V) and 3 solvent compounds (free base crystal form II, free base crystal form III and free base crystal form IV).
  • the free base crystal form I is a relatively stable acrystalline form, has stable solid-state properties, and is slightly hygroscopic, and can be used for subsequent drug development.

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Abstract

本发明属于以氮原子作为杂环原子的杂环化合物药物合成领域，具体涉及一类用作激酶抑制剂的化合物及其应用，还涉及该化合物的游离碱晶型的制备。该用作激酶抑制剂的化合物为如式1所示的化合物，或其氘代物，或药学上可以接受的盐、溶剂化物或前药。以上化合物通过生物活性实验证实，对EGFR和Her2的外显子20***突变、EGFR的外显子19缺失、外显子21的点突变具有良好的抑制活性，可作为相关药物的原药使用。

Description

一类用作激酶抑制剂的化合物及其应用 技术领域
本发明属于以氮原子作为杂环原子的杂环化合物的药物合成领域，具体涉及一类用作激酶抑制剂的化合物及其应用。
背景技术
表皮生长因子受体属于受体酪氨酸激酶(RTK)家族，其包括EGFR/ERBB1、HER2/ERBB2/NEU、HER3/ERBB3和HER4/ERBB4。表皮生长因子受体通过同源二聚或异源二聚激活其酪氨酸激酶活性，接着使它的底物磷酸化，从而激活细胞内与它相关的多个下游通路，如涉及细胞存活的PI3K-AKT-mTOR通路和涉及细胞增殖的RAS-RAF-MEK-ERK通路等。表皮生长因子受体的突变或扩增等都会导致表皮生长因子受体激酶的激活，从而导致人类多种疾病的发生，如恶性肿瘤。如在非小细胞肺癌患者中，美国患者中大约有10％以上的患者具有EGFR突变，而亚洲患者中EGFR突变的患者比例能达到近50％。同时，在非小细胞肺癌患者中，具有HER2突变的发病率大约在2-4％。
EGFR突变主要包括缺失、***和点突变等，其中，外显子19缺失和外显子21的L858R点突变占到EGFR突变的近90％。对于具有这些EGFR突变的肿瘤患者，目前已经上市的EGFR-TKI包括一代的易瑞沙、特罗凯、凯美纳，二代的阿法替尼和达克替尼以及三代的奥西替尼。其他的10％的EGFR突变主要涉及EGFR的外显子18和20，并且，EGFR外显子20的***突变占到整个EGFR突变的9％左右。具有HER2突变的肿瘤患者，最常见的HER2突变是HER2外显子20的***突变。
TAK-788对于EGFR和HER2的外显子20***突变有治疗效果，该化合物已在美国上市，从其已经报道的临床实验结果来看，客观缓解率有43％。最近有报道DZD9008对于用于治疗EGFR或HER2突变的晚期非小细胞肺癌有效果，EGFR外显子20ins的客观缓解率40％，疗效不尽如人意。
发明内容
为了满足临床上EGFR和HER2突变患者，尤其是EGFR、HER2外显子20***突变患者的用药需求，WO2021180238披露了一系列具有优良的EGFR和HER2外显子20***突变活性及EGFR外显子19缺失和外显子21的L858R点突变的化合物，极有潜力开发成治疗相关疾病的药物。其中涉及一类具有三五并胺结构的化合物，经进一步研究表明，该三五并胺结构的立体构型非常重要，当由反式转为顺式时，其体内药效会有更好的效力，且在 相关疾病的不同突变中也有很好的治疗作用。在后续的研发中，为了找到更加稳定且生物利用度更高的游离碱化合物，本发明进行了游离碱化合物的多晶型研究和筛选。
本发明的目的是提供一类用作激酶抑制剂的化合物，其属于顺式三五并胺结构，对EGFR、HER2外显子20***突变、EGFR外显子19缺失和外显子21的点突变具有良好的抑制活性。本发明的化合物比专利WO2021180238中相应反式结构的化合物的药效更好，适用性更广。
本发明的第二个目的在于提供上述化合物在制备用于治疗由EGFR突变和/或HER2突变导致的相关疾病的药物中的应用。
本发明还涉及上述化合物游离碱晶型的制备，进行了多晶型研究，从而确定稳定性高且生物利用度更高的实用晶型。
为了实现以上目的，本发明所采用的技术方案是：
一类用作激酶抑制剂的化合物，所述用作激酶抑制剂的化合物为如式1所示的化合物，或其氘代物，或药学上可以接受的盐、溶剂化物或前药：
式1中，X选自CH、N；
R1选自R5为H、C1-C3的烷基、C1-C3的氟代烷基；
R20、R21、R22各自独立地选自甲基或氘代甲基；
R3选自C1-C3的烷基、C1-C3的卤代烷基；
R40、R41、R42各自独立地选自H、D、F。
以上化合物通过生物活性实验证实，对EGFR和HER2的突变具有良好的抑制活性，可作为相关药物的原药使用。
在以上原药的基础上，原药的“药学上可接受的盐”指的是一种药学上可接受的并且拥有母体化合物的所希望的药理学活性的盐。此类盐包括：
与无机酸形成的酸加成盐，该无机盐例如是盐酸、氢溴酸、氢碘酸、硫酸、硝酸、磷酸等，典型的无机酸盐选自盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、硝酸盐、磷酸盐、酸式磷酸盐。与有机酸形成的酸加成盐，该有机酸例如是甲酸、乙酸、丙酸、己酸、环戊烷丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、3-(4-羟基苯甲酰基)苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1,2-乙烷二磺酸、2-羟基乙磺酸、苯磺酸、4-氯苯磺酸、2-萘磺酸、4-甲苯磺酸、樟脑磺酸、葡庚糖酸、4,4’-亚甲基双-(3-羟基2-烯-1-羧酸)、3-苯丙酸、三甲基乙酸、叔丁基乙酸、十二烷基硫酸、葡糖酸、谷氨酸、羟萘甲酸、水杨酸、硬脂酸、粘康酸等；或存在于母体化合物中的酸性质子与一种有机碱(例如乙醇胺、二乙醇胺、三乙醇胺、氨丁三醇、N-甲葡糖胺等)配位形成的盐。典型的有机酸盐选自甲酸盐、乙酸盐、三氟乙酸盐、丙酸盐、丙酮酸盐、羟乙酸盐、乙二酸盐、丙二酸盐、富马酸盐、马来酸盐、乳酸盐、苹果酸盐、柠檬酸盐、酒石酸盐、甲磺酸盐、乙磺酸盐，羟乙磺酸盐、苯磺酸盐、水杨酸盐、苦味酸盐、谷氨酸盐、抗坏血酸盐、樟脑酸盐、樟脑磺酸盐。容易理解，该药学上可接受的盐是无毒的。
溶剂化物为含有溶剂的化合物，如水合物、二甲基亚砜合物等等。
前药是指一种化合物，在治疗相关疾病时，经过代谢或化学过程的化学转化而产生本发明中的化合物、盐、或溶剂化物。
优选地，上述用作激酶抑制剂的化合物为如式2所示的化合物，或其氘代物，或药学上可以接受的盐、溶剂化物或前药：
式2。
进一步优选地，式2中，X选自CH、N；R3选自-CH3、-CH2CH3、-CH2CF3；R40、R41均为H，R42选自H或F。
优选地，上述用作激酶抑制剂的化合物为如式3所示的化合物，或其氘代物，或药学上可以接受的盐、溶剂化物或前药：

式3；
式3中，X选自CH、N；R3为-CH3、-CH2CH3、-CH2CF3；R40、R41均为H，R42选自H或F；R5选自-CH3、-CF3。
优选地，式1所示的化合物为：

上述用作激酶抑制剂的化合物用于治疗由EGFR突变和/或HER2突变导致的相关疾病的药物中的应用。
上述化合物对EGFR、HER2的外显子20***突变的良好抑制活性，对EGFR外显子19缺失、外显子21的点突变也有良好抑制活性。
优选地，所述EGFR突变和/或HER2突变包括EGFR外显子20***突变、HER2的外显子20***突变、EGFR外显子19缺失、EGFR外显子20点突变、EGFR外显子21的点突变中的一种或两种以上组合。经生物活性实验证实，上述化合物对上述突变类型具有较好的抑制效果。
进一步优选地，所述EGFR突变和/或HER2突变选自EGFR Del 19/T790M/C797S突变、EGFR L858R/T790M/C797S突变，上述化合物对上述突变类型具有较好的抑制效果。
优选地，所述疾病为所述EGFR突变和/或HER2突变导致的癌症。上述化合物也可以和其他药物联用，用于癌症的治疗。其他联用的药物可以是ERK抑制剂或MEK抑制剂。
优选地，所述化合物为晶型、无定型或所述溶剂化物；所述溶剂化物所含溶剂为非水溶剂或非水溶剂和水组成的混合溶剂。
为了更好的提高化合物的稳定性和活性，对通式1所示的化合物，选取部分进行晶型研究。进一步的，对本发明中的实施例1的化合物进行多晶型筛选研究，找到稳定可靠的晶型，既能保证化合物质量的稳定性，也会促使药物在临床治疗中发挥更好的作用。发现实施例1的产品为结晶度较好的晶体，且为无水晶型，命名为游离碱晶型Ⅰ。实施例1的化合物具有如式A所示的化合物：
晶型为游离碱晶型Ⅰ，该晶型的X-射线粉末衍射图谱在9.76°±0.2°，10.45°±0.2°，16.54°±0.2°，18.66°±0.2°，20.07°±0.2°，25.90°±0.2°处有特征衍射峰。
更进一步的，游离碱晶型Ⅰ，其X-射线粉末衍射图谱在9.07°±0.2°，9.76°±0.2°，10.45°±0.2°，11.53°±0.2°，11.80°±0.2°，12.91°±0.2°，13.79°±0.2°，14.67°±0.2°，15.08°±0.2°，15.63°±0.2°，16.54°±0.2°，17.50°±0.2°，18.66°±0.2°，20.07°±0.2°，21.10°±0.2°，23.29°±0.2°，24.16°±0.2°，25.90°±0.2°处有特征衍射峰。
实施例1化合物的游离碱晶型Ⅰ可以在大部分溶剂中通过混悬转晶、反溶剂沉淀、高低温循环和蒸发结晶等方法制备。此处的溶剂包括但不限于二氯甲烷、1,4-二氧六环、二氯乙烷、甲基叔丁基醚、N-甲基吡咯烷酮、乙酸乙酯、丙酮、甲醇、二甲亚砜、乙酸异丙酯、丁酮、环己烷、四氢呋喃、水、乙腈、异丙醇、乙醇、正庚烷中的一种或两种及以上的混合溶剂等。
本发明发现实施例1化合物有两种无水晶型(游离碱晶型Ⅰ和游离碱晶型Ⅴ)和三种溶剂合物，游离碱晶型Ⅱ是乙醇溶剂合物，游离碱晶型Ⅲ是异丙醇溶剂合物，游离碱晶型Ⅳ是二氯乙烷和水的溶剂合物，并对这几种游离碱晶型都进行表征。对游离碱晶型Ⅰ和游离碱晶型Ⅴ两种无水晶型做了竞争打浆实验，从结果推断对游离碱晶型Ⅰ可能为热力学稳定晶型，更适合后续的开发。
对游离碱晶型Ⅰ进行了晶型评价，包括干法研磨、湿法研磨、压片(30MPa)、稳定性和引湿性等研究。干法研磨5min后就变成了无定型；加水的湿法研磨5min晶型基本没变化，加乙醇的湿法研磨5min后结晶度轻微下降；压片测试中压力为30MPa时，结晶度有所 下降；稳定性考察中，不同温度和湿度下，游离碱晶型Ⅰ比较稳定，液相检测结果没有变化；做了动态水蒸汽吸附(DVS)测试，结果表明，在80％RH时吸水增重0.66％，游离碱晶型Ⅰ略有引湿性。总之，游离碱晶型Ⅰ是较稳定的无水晶型，具有稳定的固态性质，且略有引湿性，可以用作后续药物开发。
附图说明
图1为LU0387模型中肿瘤体积变化(mm3)；
图2为Ba/F3 EGFR D770_N771 ins SVD模型肿瘤体积变化(mm3)；
图3为实施例1的单晶的单晶衍射图；
图4为游离碱晶型Ⅰ的XRPD图谱；
图5为游离碱晶型Ⅰ的DSC和TGA叠加图谱；
图6为干法研磨法所得无定型样品的XRPD图谱；
图7为游离碱晶型Ⅱ的XRPD图；
图8为游离碱晶型Ⅱ的DSC和TGA叠加图谱；
图9为游离碱晶型Ⅲ的XRPD图；
图10为游离碱晶型Ⅲ的DSC和TGA叠加图谱；
图11为游离碱晶型Ⅳ的XRPD图；
图12为游离碱晶型Ⅳ的DSC和TGA叠加图谱；
图13为游离碱晶型Ⅴ的XRPD叠加图谱；
图14为游离碱晶型Ⅴ的DSC和TGA叠加图谱。
具体实施方式
相对于专利WO2021180238中的具有反式三五并胺结构的化合物，本发明证实了顺式三五并胺结构的体内药效更好，且在相关疾病的不同突变中也有很好的治疗作用。
本发明的化合物当X为CH时的制备方法，反应路线如下所示：
包括以下步骤：
(1)化合物A和化合物B在有机溶剂中进行傅克反应，得到化合物C；
(2)化合物C和化合物D在有机溶剂中，酸催化下进行取代反应，得到化合物E；
(3)化合物E和化合物F在有机溶剂中，碱催化下进行取代反应，得到化合物G；
(4)化合物G还原后得到化合物H；
(5)化合物H在碱催化剂下进行缩合反应，得到产品。
R1为且R5为-CF3时，式1所示的化合物的合成还可按照以下反应路线：
包括以下步骤：
(1)化合物a和化合物b在有机溶剂中进行取代反应，得到化合物c；
(2)化合物c水解，得到化合物d；
(3)化合物d进行缩合反应，得到化合物e；
(4)化合物e脱保护，得到化合物f；
(5)化合物f在碱催化剂下和三氟乙酸酐进行关环反应，得到化合物g；
(6)化合物g还原后得到化合物h；
(7)化合物h在碱催化剂下进行缩合反应，得到产品。
本发明的化合物当X为N时的制备方法，反应路线如下所示：
包括以下步骤：化合物A′和化合物B′进行取代反应得到产品。
其中，化合物A′参考WO2021180238合成得到；化合物B′由傅克反应得到。
下面结合具体实施例对本发明的实施过程均进行详细说明。
一、用作激酶抑制剂的化合物合成的具体实施例
实施例1
本实施例的用作激酶抑制剂的化合物，结构式如下所示：
本实施例的化合物的合成路线如下：
化合物3的合成：250mL三口瓶，氮气保护，加入化合物1(2.01g,8.5mmol)，用100mL四氢呋喃全溶，加入无水三氯化铝(2.26g,17mmol)，70℃搅拌1小时。滴加化合物2(1.34g,10.2mmol)，滴毕，继续70℃反应，点板监控反应，4小时后基本反应完毕。处理，得到产品1.31g，收率46.4％。
化合物5的合成：将化合物3(1.30g,3.9mmol)，化合物4(0.88g,4.68mmol)，和对甲苯磺酸(1.35g,7.8mmol)加入到65mL的1,4-二氧六环中，N2保护，升温到80℃，反应过夜。点板监控反应，反应完毕，处理，得到产品1.50g，收率79.7％。
化合物7的合成：将化合物5(6.0g,12.5mmol)、化合物6(6.3g,100mmol)和N,N-二异丙基乙胺(3.2g,50mmol)加入到N,N-二甲基乙酰胺120mL中，90℃反应过夜，点板监控，原料反应完后，处理，得到产品8.0g。1H NMR(400MHz,Chloroform-d)δ9.53(s,1H),8.97–8.75(m,1H),7.96(s,1H),7.84(s,1H),7.79(d,J＝8.0Hz,1H),7.38(d,J＝8.2Hz,1H),7.29(d,J＝7.2Hz,1H),7.20(t,J＝7.6Hz,1H),6.81(s,1H),5.09(p,J＝6.4Hz,1H),4.02(s,3H),3.92(s,3H),3.84–3.63(m,1H),3.02(t,J＝6.4Hz,1H),2.84(s,3H),2.70(s,3H),2.43(s,2H),2.32(s,1H),1.27(d,J＝7.2Hz,2H),1.15(d,J＝6.4Hz,6H).
化合物8的合成：将化合物7(7.7g,41.6mmol)和钯碳(2.4g,湿钯碳55％)加入到100mL甲醇和100mL乙酸乙酯的混合溶剂中，氢气气氛下，室温反应4小时，点板监控，原料反应完后，处理，得到产品7.0g。
实施例1的合成：将化合物8(7.00g,12.6mmol)和三乙胺(3.82g,37.8mmol)溶于二氯甲烷中，氮气保护下降温至0℃，随后滴加丙烯酰氯(1.72g,18.9mmol)的二氯甲烷溶液，保持0℃反应1小时，点板监控，原料反应完后，处理。即加入碳酸氢钠水溶液和二氯甲烷，搅拌分层，水相再用二氯甲烷萃取一次，合并有机相，干燥，旋干，过柱。得到的粗产品先用少许二氯甲烷溶解，后面滴加石油醚析出大量固体，过滤，滤饼直接加入甲醇打浆，干燥后最终得到3.5g产品。[M+H]+：610.8；1H NMR(400MHz,Chloroform-d)δ10.38(s,1H),9.60(s,1H),8.87(s,1H),8.61(s,1H),7.89(s,1H),7.58(s,1H),7.32(d,J＝8.2Hz,1H),7.20(t,J＝7.6Hz,1H),7.13(t,J＝7.6Hz,1H),6.80(s,1H),6.44(m,2H),6.32(m,1H),5.68(m,1H),5.00(p,J＝6.2Hz,1H),3.94(s,3H),3.88(s,3H),3.06(s,2H),2.66(s,3H),2.61(m,3H),2.36(s,3H),1.67(s,2H),1.03(d,J＝6.4Hz,6H)。
参照实施例1，合成了实施例2、3、4三个氘代物，具体如下表1所示。
表1实施例2-4的化合物结构及表征

实施例5
本实施例的用作激酶抑制剂的化合物，结构式如下所示：
本实施例的化合物的合成路线如下：
合成过程如下：
化合物2的合成：2000mL的单口瓶中，加入化合物1(54.6g，303mmol)，乙酰肼(26.64g，395mmol)，加入1000mL的1N的氢氧化钠水溶液，80℃下反应4小时，有大量固体析出。降温，加入浓盐酸80mL，0℃左右低温搅拌30分钟，抽滤，滤饼用水搅洗一次，55℃真空干燥过夜，得到32g产品。1H NMR(400MHz,DMSO-d6)δ11.75(s,1H),11.59(s,1H),8.13(s,1H),2.52(s,3H)。
化合物3的合成：2000mL的单口瓶中，加入化合物2(50.0g，258mmol)，室温下加入甲苯1500mL，三氯氧磷(237g,1550mmol)，N,N-二异丙基乙胺(133g,1031mmol)，有白雾生成。加热到80℃，氮气保护，搅拌过夜。次日，降温处理，得到30g产品。1H NMR(400MHz,Chloroform-d)δ9.19(s,1H),2.71(s,3H)。
化合物5的合成：2000mL单口瓶，加入化合物3(30.0g,130mmol)，用1500mL1,2-二氯乙烷全溶，加入无水三氯化铝(29.3g,220mmol)，先室温搅拌30分钟。降温到0℃，滴加化合物4(22.1g,169mmol)，低温先搅拌30分钟，之后升温到60℃反应，点板监控。反应4小时后，降温，处理得到产品20.0g。1H NMR(400MHz,Chloroform-d)δ8.84(s,1H),8.02(dt,J＝7.8,1.0Hz,1H),7.97(s,1H),7.40-7.27(m,3H),3.86(s,3H),2.50(s,3H)。
化合物7的合成：将化合物5(2g,6.15mmol)，化合物6(1.37g,7.38mmol)，和对甲苯磺酸(2.11g,12.3mmol)加入到100mL的二氧六环中，氮气保护升温到80℃，反应过夜。点板监控，反应完毕，处理，得到0.65g产品。
化合物9的合成：将化合物7(950mg,2mmol)、化合物8(1.0g,8mmol)和N,N-二异丙基乙胺(516m g,4mmol)加入到N,N-二甲基乙酰胺20mL中，100℃反应过夜。点板监控，原料反应完后，处理，得到1.0g产品。1H NMR(400MHz,Chloroform-d)δ9.56(s,1H),8.88(s,1H),7.92(d,J＝14.6Hz,2H),7.54(s,1H),7.40(d,J＝8.2Hz,1H),7.32(s,1H),7.15(t,J＝7.6Hz,1H),6.84(s,1H),4.06(s,3H),3.93(s,3H),3.04(t,J＝6.0Hz,1H),2.88(s,3H),2.72(s,3H),2.44(s,2H),2.38(s,3H),1.28(s,4H)。
化合物10的合成：将化合物9(1.0g,1.72mmol)和钯碳(500mg,湿钯碳55％)加入到50mL甲醇中，氢气气氛下，室温反应4小时。点板监控，原料反应完后，处理，得到900mg产品。
实施例5的合成：将化合物10(900mg,1.63mmol)和三乙胺(495mg,4.90mmol)溶于二氯甲烷中，氮气气氛下降温至0℃，随后滴加丙烯酰氯(223mg,2.45mmol)的二氯甲烷溶液，保持0℃反应2小时。点板监控，原料反应完后，处理，得到200mg产品。[M+H]+：606.8。
参照实施例5，合成了实施例6、7、8三个氘代物，具体如下表2所示。
表2实施例6-8的化合物结构及表征
实施例9
本实施例的用作激酶抑制剂的化合物，结构式如下所示：
本实施例的化合物的合成路线如下：
本实施例的化合物的合成过程如下：
化合物3的合成：将化合物1(6.0g,13.30mmol)、化合物2(5.03g,39.91mmol)和N,N-二异丙基乙胺(3.43g,26.6mmol)加入到N,N-二甲基乙酰胺60mL中，80℃反应过夜。点板监控，原料反应完后，处理，得到6.17g产品。1H NMR(400MHz,Chloroform-d)-δ9.50(s,1H),8.90(s,1H),8.24-7.99(m,1H),7.80(s,1H),7.67(s,1H),7.38(dt,J＝8.2,1.0Hz,1H),7.31-7.23(m,1H),7.23-7.16(m,1H),6.65(s,1H),3.95(s,3H),3.94(s,3H),3.70(s,3H),2.95(s,3H),2.89(t,J＝6.6Hz,1H),2.85-2.76(m,2H),2.27(m,2H),2.15(s,3H),1.91–1.81(m,2H)。
化合物4的合成：将化合物3(6.17g,11.08mmol)、一水合氢氧化锂(2.33g,55.39mmol)加入到四氢呋喃/甲醇/水体积比为6：3：1的混合溶剂中，40℃反应16小时。点板监控，原料反应完后，处理，得到6.02g产品。
化合物5的合成：将化合物4(6.02g,11.08mmol)、肼基甲酸叔丁酯(4.39g,33.26mmol)，N,N-二异丙基乙胺(8.58g,66.48mmol)加入到160mL N,N-二甲基甲酰胺中，搅拌15min后再加入1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐(6.90g,13.30mmol)，25℃反应16小时。点板监控，原料反应完后，处理，得到1.67g产品。
化合物6的合成：将化合物5(1.67g,2.54mmol)溶于15mL四氢呋喃中，缓慢加入到15mL的4M的氯化氢二氧六环溶液中，氮气保护下40℃反应5小时。液质监控，原料反应完后，处理，得到1.20g产品。
化合物7的合成：将化合物6(1.0g,1.68mmol)、三乙胺(679mg,6.72mmol)加入到20mL二氯甲烷中，搅拌15分钟后，分批加入三氟乙酸酐(1.59g,7.58mmol)，40℃反应8小时，反应完毕。处理，得到521mg产品。
化合物8的合成：将化合物7(521mg,0.82mmol)和钯碳(156mg,10％)溶解20mL甲醇中，氢气下，25℃搅拌反应3小时，液质监控，原料反应完后，处理，得到412g产品。
实施例9的合成：将化合物8(412mg,0.68mmol)和三乙胺(206mg,2.04mmol)溶于15mL二氯甲烷中，氮气下降温至0℃，随后滴加丙烯酰氯(93mg,1.02mmol)的二氯甲烷溶液，保持0℃反应2小时。点板监控，原料反应完后，处理，得到产品122mg。[M+H]+：660.5；1H NMR(400MHz,Chloroform-d)δ11.36(m,1H),9.87(m,1H),9.09(m,1H),8.92(s,1H),8.37(m,1H),7.95(m,1H),7.33(m,1H),7.18(m,1H),7.01(m,2H),6.80(m,1H),6.46(d,J＝16.6Hz,1H),5.74(d,J＝10.2Hz,1H),3.92(d,J＝13.5Hz,6H),3.29(m,2H),3.04(m,1H),2.78(m,8H),2.21(m,2H)。
参照实施例9，合成了实施例10、11、12三个氘代物，具体如下表3所示。
表3实施例10-12的化合物结构及表征
实施例13
本实施例的用作激酶抑制剂的化合物，结构式如下所示：
本实施例的化合物的合成路线如下：
化合物2的合成：500mL单口瓶，加入化合物1(10.00g,62.88mmol)，用100mL四氢呋喃全溶，加入碳酸铯(20.50g,62.88mmol)，三氟乙醇(6.29g,62.87mmol)，加毕，氮气保护。点板监控，23℃反应6小时基本反应完毕。处理，得到产品14.35g，收率95.5％。1H NMR(400MHz,Chloroform-d)δ8.01(dd,J＝9.1,5.8Hz,1H),7.09–6.72(m,2H),4.50(q,J＝7.8Hz,2H)。
化合物3的合成：将化合物2(14.00g,58.57mmol)溶于60mL乙醇中，加入15mL水，氯化铵(9.60g,179.44mmol)，还原铁粉(20.00g,357.14mmol)，加毕，升温到80℃，反应过夜。点板监控反应，反应完毕，处理，得到产品10.50g，收率85.78％。1H NMR(400MHz,Chloroform-d)δ6.89–6.34(m,3H),4.34(q,J＝8.0Hz,2H),3.84–3.27(m,2H)。
化合物4的合成：250mL三口瓶，加入化合物3(10.00g,47.83mmol)，搅拌下加入50mL浓硫酸，降温到0℃，分批加入硝酸钾固体(6.10g，60.34mmol),加毕，氮气保护。室温下反应4小时后，点板反应完毕，处理，得到产品8.26g，收率67.98％。1H NMR(400MHz,Chloroform-d)δ7.44(d,J＝7.2Hz,1H),6.66(d,J＝11.5Hz,1H),4.46(q,J＝7.7Hz,2H),4.01(m,2H)。
化合物6的合成：将化合物4(2.00g,6.08mmol)和化合物5(2.00g,7.87mmol)加入到50mL乙腈中，再加入对甲苯磺酸的一水合物(0.81g,4.26mmol)，80℃下搅拌过夜。次日，点板监控，原料基本反应完毕。降温，反应液中析出固体，过滤，滤饼旋干后加入甲醇打浆，得到1.67g产品。1H NMR(400MHz,Chloroform-d)δ9.89(s,1H),9.33(d,J＝8.0Hz,1H),8.77(s,1H),8.29(s,1H),7.67(d,J＝8.0Hz,1H),7.39(m,1H),7.33(m,1H),7.19(m,1H),6.89(d,J＝11.3Hz,1H),5.14(p,J＝6.2Hz,1H),4.59(q,J＝7.7Hz,2H),3.95(s,3H),1.22(d,J＝6.2Hz,6H)。
化合物8的合成：将化合物6(1.52g,2.78mmol)、化合物7(1.05g,8.33mmol)和N,N-二异丙基乙胺(1.05g,8.14mmol)加入到N,N-二甲基乙酰胺25mL中，90℃反应过夜。点板监控，原料反应完后，处理，得到1.16g产品。
化合物9的合成：将化合物8(0.59g,0.90mmol)和钯碳(400mg,湿钯碳55％)加入到20mL甲醇和20mL乙酸乙酯的混合溶剂中，氢气气氛下，室温反应3小时。点板监控，原料反应完后，处理，得到520mg产品。
实施例13的合成：将化合物9(520mg,0.83mmol)和三乙胺(253mg,2.50mmol)溶于二氯甲烷中，氮气气氛下降温至0℃，随后滴加丙烯酰氯(114mg,1.25mmol)的二氯甲烷溶液，保持0℃反应2小时。点板监控，原料反应完后，处理，得到170mg产品。[M+H]+：606.8。1H NMR(400MHz,Chloroform-d)δ10.63(s,1H),10.11(s,1H),8.82(s,1H),8.16(s,1H),7.66(m,2H),7.29(d,J＝8.1Hz,1H),7.22–7.16(m,1H),7.15–7.09(m,1H),6.80(s,1H),6.37(dd,J＝16.9,2.0Hz,1H),5.68(dd,J＝10.1,2.0Hz,1H),5.00(p,J＝6.2Hz,1H),4.44(q,J＝8.2Hz,2H),3.85(s,3H),3.27–2.99(m,2H),2.87–2.51(m,6H),2.11(m,2H),1.18–0.91(m,6H)。
参照实施例13，合成了实施例14，具体如下表4所示。
表4实施例14的化合物结构及表征

实施例15
本实施例的用作激酶抑制剂的化合物，结构式如下所示：
本实施例的化合物的合成路线如下：
实施例15的合成：50mL单口瓶中，加入化合物2(112mg，1.24mmol)，二氯甲烷20ml，草酰氯(142mg，1.12mmol)，氮气保护下搅拌2小时。降温到0℃，滴加化合物1(438mg,78.92mmol)的二氯甲烷溶液和三乙胺(152mg,1.50mmol)，保持0℃反应1小时， 点板监控，原料反应完后，处理，最终得到160mg产品。[M+H]+：628.8；1H NMR(400MHz,Chloroform-d)δ9.53(m,1H),9.12(m,1H),8.89(s,1H),8.35(m,1H),7.92(s,1H),7.69(s,1H),7.35(d,J＝8.1Hz,1H),7.23(t,J＝7.5Hz,1H),7.15(t,J＝7.5Hz,1H),6.84(s,1H),5.84(dd,J＝48.2,3.4Hz,1H),5.28(dd,J＝15.5,3.4Hz,1H),5.04(p,J＝6.2Hz,1H),3.92(s,6H),3.49(m,2H),2.64(m,9H),2.19(m,2H),1.09(m,6H)。
实施例16
本实施例的用作激酶抑制剂的化合物，结构式如下所示：
本实施例的化合物的合成路线如下：
化合物3的合成：100mL单口瓶，加入化合物1(313mg,1mmol)，加入5mL乙腈，再加入化合物2的粗品(441mg),加毕，氮气保护。点板监控，80℃反应过夜基本反应完毕。处理，得到产品300mg。
化合物4的合成：将化合物3(1.83g,4.54mmol)溶于60mL甲醇中，加入1.2mL浓盐酸，加毕，升温到60℃，反应过夜。点板监控反应，反应完毕，处理，得到产品0.89g。1H NMR(400MHz,Chloroform-d)δ7.61(s,1H),4.80(q,J＝8.4Hz,2H),3.57(m,2H),3.10(s,3H),3.02(t,J＝6.9Hz,1H),2.59–2.33(m,4H),2.06(s,3H),1.76–1.51(m,2H)。
化合物6的合成：100mL单口瓶，加入化合物4(73.8mg,0.21mmol)，加入化合物5(56mg,0.17mmol),对甲苯磺酸(48mg,0.25mmol)，然后加入二氧六环5mL，80℃搅拌过夜， 氮气保护。点板监控，反应完毕，处理，得到产品50mg。1H NMR(400MHz,Chloroform-d)δ9.76(s,1H),8.90(s,1H),7.94(s,1H),7.80(d,J＝8.0Hz,1H),7.56(s,1H),7.39(d,J＝8.2Hz,1H),7.33–7.27(m,1H),7.24–7.17(m,1H),5.10(p,J＝6.2Hz,1H),4.94(s,2H),3.93(s,3H),3.78(m,2H),3.18(t,J＝6.5Hz,1H),3.01(m,3H),2.57(m,5H),2.31(m,2H),1.16(d,J＝6.2Hz,6H)。
化合物7的合成：将化合物6(98mg,0.15mmol)和钯碳(80mg,湿钯碳55％)加入到3mL甲醇和2mL乙酸乙酯的混合溶剂中，氢气气氛下，室温反应2小时。点板监控，原料反应完后，处理，得到98mg产品。
实施例16的合成：将化合物8(300mg,0.48mmol)和三乙胺(150mg,1.48mmol)溶于二氯甲烷中，氮气气氛下降温至0℃，随后滴加丙烯酰氯(70mg,0.77mmol)的二氯甲烷溶液，保持0℃反应2小时。点板监控，原料反应完后，处理，得到118mg产品。[M+H]+：688.8。1H NMR(400MHz,Chloroform-d)δ10.39(s,1H),9.97(s,1H),8.89(s,1H),8.59(m,1H),7.59(m,2H),7.33(m,1H),7.23(m,1H),7.14(m,1H),6.45(m,1H),6.31(m,1H),5.72(d,J＝10.0Hz,1H),5.01(p,J＝6.5Hz,1H),4.83(q,J＝8.5Hz,2H),3.96(s,3H),3.14–2.88(m,2H),2.85–2.52(m,6H),2.33(s,3H),1.69(m,2H),1.04(d,J＝6.3Hz,6H)。
实施例17
本实施例的用作激酶抑制剂的化合物，结构式如下所示：
本实施例的化合物的合成路线如下：
化合物2的合成：100mL三口瓶，加入化合物1(153mg,0.38mmol)，加入5mL四氢呋喃，再加入4-二甲氨基吡啶(17.8mg),加毕，氮气保护，滴加二碳酸二叔丁酯(1.06g，4.86mmol)。点板监控，80℃反应2小时，基本反应完毕。处理，得到产品104mg。
化合物3的合成：将化合物2(104mg,0.21mmol)溶于2ml甲醇中，加入5.4mol/L的甲醇钠的甲醇溶液0.05mL，加毕，反应30分钟。点板监控反应，反应完毕，处理，得到产品30mg。
化合物4的合成：将化合物3(30mg,0.065mmol)和钯碳(15mg,湿钯碳55％)加入到1mL甲醇和1mL乙酸乙酯的混合溶剂中，氢气气氛下，室温反应2小时。点板监控，原料反应完后，处理，得到30mg产品。
化合物5的合成:将化合物4(30mg,0.069mmol)和三乙胺(13mg,0.14mmol)溶于二氯甲烷中，氮气气氛下降温至0℃，随后滴加丙烯酰氯(7mg,0.077mmol)的二氯甲烷溶液，保持0℃反应2小时。点板监控，原料反应完后，处理，得到12mg产品。
实施例17的合成：将化合物5(12mg,0.024mmol)和化合物6(6mg,0.018mmol)、对甲苯磺酸(8mg,0.042mmol)溶于1mL的N-甲基吡咯烷酮和2mL的乙二醇单甲醚中，氮气气氛下升温至100℃，反应4小时。点板监控，原料反应完后，处理，得到3mg产品。[M+H]+：675.8。1H NMR(400MHz,Chloroform-d)δ10.42(s,0H),9.97(s,0H),8.91(s,0H),8.70(m,1H),7.68(s,1H),7.33(d,J＝8.2Hz,0H),7.18(m,1H),6.99(m,1H),6.45(m,1H),6.33(m,1H),5.73(m,1H),5.35(m,1H),4.85(m,2H),3.99(s,3H),3.05(m,2H),2.78(m,1H),2.62(m,2H),2.35(s,3H),2.22(s,3H),2.10–1.95(m,2H)。
二.化合物生物学测试评价
1.化合物对细胞增殖抑制作用的测试
1.1 EGFR和HER2 exon 20***突变的细胞增殖抑制活性测试实验：
实验中所选取的工程细胞Ba/F3-FL-EGFR-V769-D770ins ASV、Ba/F3-FL-EGFR-D770-N771 ins SVD、Ba/F3-FL-EGFR-H773-V774 ins NPH、Ba/F3-FL-EGFR-A763-Y764 ins FQEA、Ba/F3-HER2-A775-G776 ins YVMA由合肥中科普瑞昇生物医药科技有限公司提供， 并且在用于本研究之前经过验证。
配制20×的待测化合物母液备用，9个浓度，3倍梯度稀释，从1μM起稀释。
取对数生长期细胞悬液，接种于96孔白色细胞培养板，每孔体积为95μL(2000个细胞/孔)。取5μL 20×待测化合物按照铺板图分别加入上述含有95μL细胞悬液的培养板中，混匀。37℃、5％CO2培养箱中孵育72小时。CellTiter-Glo法测定化合物的增殖抑制活性。SpectraMax Paradigm读数得出对应的每孔荧光值RLU。细胞增殖抑制率(Inhibition Rate)数据采用下列公式来处理：Inhibition Rate(Inh％)＝100-(RLU compound-RLU blank)/(RLU control-RLU blank)*100％。在EXCEL中计算不同浓度化合物对应的细胞活力，然后用GraphPad Prism软件作细胞活力曲线图并计算IC50值。表5为对照品的名称和结构。
表5对照品的名称及结构
表6、表7和表8的实验结果中，依据IC50值大小分为3类，其中A≤30nM，30nM＜B≤100nM，C>100nM。
表6不同化合物的EGFR exon 20***突变的细胞增殖抑制活性测试结果

表7不同化合物的HER2 exon 20***突变的细胞增殖抑制活性测试结果
从表6可知，实施例中的化合物对EGFR exon 20***突变均有很好的抑制能力。优于DZD9008，与TAK-788的活性相当。从表7可知，实施例5在HER2exon 20***突变中的细胞活性和TAK-788相当，优于DZD9008。
1.2化合物对细胞系NCI-H1975和PC9的细胞增殖抑制活性测试实验：
实验中所选取的细胞系NCI-H1975和PC9由中美冠科生物技术(北京)有限公司提供，并且在用于本研究之前经过验证。
配制10×的待测化合物母液备用，9个浓度，4倍梯度稀释，从10μM起稀释。
取对数生长期细胞悬液，接种于96孔培养板，每孔体积为90μL(2000个细胞/孔)。取10μL 10×待测化合物按照铺板图分别加入培养板中，混匀。37℃、5％CO2培养箱中孵育。CellTiter-Glo法测定化合物的增殖抑制活性。细胞增殖抑制率(Inhibition Rate)数据采用GraphPad Prism8.0软件作细胞活力曲线图并计算IC50值。结果如表8所示：
表8不同化合物相关突变的细胞增殖抑制活性测试结果
从表8可知，本发明的化合物对细胞系NCI-H975和PC9的细胞增殖有很好的抑制作用，且优于DZD9008。
2.化合物的体内药效实验
2.1对肺癌LU0387PDX模型的药效学研究
肺癌LU0387PDX模型是冠科的一种常用的异种移植瘤模型，该模型的实验情况说明如下：荷瘤鼠安乐死后，无菌状态下剖出瘤块，将瘤块清洗干净，去除血污及***和坏死部分，肿瘤切成2*2*2mm3的小块。用转接针接种于经消毒的实验小鼠于右侧背部皮下。 定期观察肿瘤生长情况，待肿瘤生长至平均体积100～200mm3时根据肿瘤大小和小鼠体重随机分组给药。在给药开始前，称量所有动物的体重，并用游标卡尺测量肿瘤体积。根据鼠的肿瘤体积随机分组，以保证不同组别间的肿瘤体积相似。每组3只小鼠，每日口服给药一次，给药后对接种部位进行观察。每周两次测量肿瘤体积和小鼠体重。
实验设计及结果如表9所示，其中，P.O是口服给药；QD:每天一次；TGI(肿瘤体积抑制率)＝(1-治疗组瘤重量/对照组瘤重量)*100％。
表9 LU0387模型设计及结果
实验中，肿瘤体积随着给药天数发生变化，结果如图1所示。
由图1看出，在LU0387模型中相同剂量下，实施例1的药效优于TAK-788和DZD9008。实施例5的药效和TAK-788类似。随着实施例1剂量的增加，则药效也明显增强。
2.2对Ba/F3-EGFR-D770_N771 ins SVD工程细胞株皮下移植瘤模型的体内药效学研究
细胞株Ba/F3EGFR D770_N771 ins SVD采用RPMI1640+10％胎牛血清+1％双抗,37℃、5％CO2培养，一周2-3次传代处理。当细胞饱和度为80-90％，数量达到要求时，收取细胞。将0.2ml(1*106个)细胞皮下接种于6-8周龄，体重18-22g的雌性裸鼠的右后背。肿瘤平均体积达到约150-200mm3时开始分组给药。在给药开始前，称量所有动物的体重，并用游标卡尺测量肿瘤体积。根据鼠的肿瘤体积随机分组，以保证不同组别间的肿瘤体积相似。每组3只小鼠，每日口服给药一次，给药后对接种部位进行观察。每周两次测量肿瘤体积和小鼠体重。
表10 Ba/F3 EGFR D770_N771 ins SVD模型分组

表中实施例18和实施例40是专利WO2021180238中的具有反式三五并胺结构的化合物。名称及结构见表11：
表11反式结构化合物的名称及结构
实验中，肿瘤体积随着给药天数发生变化，结果如图2所示。
由图2看出，在Ba/F3-EGFR-D770_N771 ins SVD细胞移植模型中，相同剂量下，实施例1和实施例5的体内药效都远优于专利WO2021180238中的具有反式三五并胺结构的化合物，肿瘤抑制率提高达到1.6倍以上。
综上，本专利中的化合物对EGFR或HER2的外显子20***突变有良好抑制效果，对细胞系NCI-H1975和PC9的细胞增殖也有很好的抑制作用；对EGFR外显子20***突变、HER2的外显子20***突变、EGFR外显子19缺失、EGFR外显子20点突变、EGFR外显子21的点突变中的一种或两种以上组合，其药物预期对相关疾病具有较好的治疗效果。
三.实施例1的结构确认和游离碱晶型研究。
1.结构确认
取实施例1的化合物214mg，加入0.5mL的二氯甲烷和0.5mL的乙腈，溶清过滤，滤液装入有封口膜的样品瓶中，扎孔后置于通风橱缓慢挥发，得到粒状晶体(游离碱晶型Ⅰ)。单晶衍射进行结构确认，结果如图3所示，确认化合物构型。
2.实施例1的游离碱晶型研究
对实施例1得到的化合物进行多晶型筛选，寻找其潜在晶型，以期找到适合后续开发的优良的晶体形式。以合成得到的实施例1为起始原料，对其检测发现，化合物为结晶度较好的晶体，且为无水晶型，命名为游离碱晶型Ⅰ。随后用干法研磨的方式制备了无定型的游离碱化合物。用以上两种类型为起始原料，开展晶型筛选实验。
2.1初始原料的制备和表征。
2.1.1实施例1化合物的游离碱晶型Ⅰ的表征
对实施例1化合物的游离碱晶型Ⅰ的原料进行了全面表征。通过偏光显微镜(PLM)看到，原料为结晶度较好的不规则形状晶体。图4为游离碱晶型Ⅰ的XRPD图谱，图5为该晶型的DSC和TGA叠加图谱。差示扫描量热(DSC)曲线在230℃有1个吸热峰，应为熔融峰。热重分析(TGA)曲线在分解前无明显失重，为无水晶型。
表12游离碱晶型Ⅰ的X-射线粉末衍射峰数据

2.1.2实施例1游离碱晶型Ⅰ的溶解度测试
在18种溶剂中通过目测法粗略的测定了化合物实施例1游离碱晶型Ⅰ在室温条件下的溶解度。结果如表13所示。游离碱晶型Ⅰ仅在二氯乙烷和二氯甲烷中溶解度较高，在大部分所测试溶剂中溶解度较低。
表13实施例1的游离碱晶型Ⅰ的溶解度目测结果
2.1.3无定型原料的制备和表征
称取736mg实施例1化合物(游离碱晶型Ⅰ)进行干法研磨5小时，成功制备得到669.86mg无定型产品。做了XRPD检测，结果如图6所示。
2.2.表征中物理化学检测仪器的使用方法和参数
2.2.1 X射线粉末衍射(XRPD)
XRPD衍射图由Bruker D2 Phaser型采集获得。将待测样品放置于光滑无背景的硅片上进行测样。测量参数如表14所示。
表14 XRPD方法参数

2.2.2偏光显微镜(PLM)
使用奥特光学显微镜BK-Pol进行PLM分析。取少量样品，置于载玻片上，滴一滴硅油分散，随后盖上盖玻片，在显微镜下观察。
2.2.3差示扫描量热分析(DSC)
DSC曲线由TA仪器的DSC 250型采集获得。DSC 250型仪器测试方法为：精密称取适量的样品至扎孔的铝坩埚中，以10℃/min的升温速率从25℃升温至最终温度300℃，用流速为50mL/min的氮气吹扫。
2.2.4热重分析(TGA)
TGA数据由TA仪器的TGA 550型采集获得。取适量的样品放入提前去皮的铝坩埚内，以10℃/min的升温速率从室温升温至300℃，天平室用40mL/min的氮气吹扫，样品室用25mL/min的氮气吹扫。
2.3实施例1化合物的游离碱多晶型的研究。
主要采用混悬转晶、反溶剂沉淀、高低温循环和蒸发结晶等方法来制备。
混悬转晶是根据实施例1化合物的目测溶解度结果，在25℃和50℃条件下，分别以游离碱晶型Ⅰ和无定型晶型为起始原料，分别在所选的单一溶剂(溶剂选自二氯甲烷，1,4-二氧六环，二氯乙烷，甲基叔丁基醚，N-甲基吡咯烷酮，乙酸乙酯，丙酮，甲醇，二甲亚砜，乙酸异丙酯，丁酮，环己烷，四氢呋喃，水，乙腈，异丙醇，乙醇，正庚烷)或混合溶剂中进行晶型的制备。共得到两种晶型，游离碱晶型Ⅰ和游离碱晶型Ⅱ。
反溶剂沉淀是根据实施例1化合物的目测溶解度结果，以二氯甲烷和二氯乙烷作为良溶剂，然后于室温(～25℃)搅拌条件下分别向其中加入不同的反溶剂来进行晶型制备。共得到两种晶型，游离碱晶型Ⅰ和游离碱晶型Ⅲ。
高低温循环是向化合物中加入溶剂(溶剂选自二氯甲烷，1,4-二氧六环，二氯乙烷，甲基叔丁基醚，N-甲基吡咯烷酮，乙酸乙酯，丙酮，甲醇，二甲亚砜，乙酸异丙酯，丁酮，环己烷，四氢呋喃，水，乙腈，异丙醇，乙醇，正庚烷)，然后于50℃～5℃温度循环条件下搅拌来进行晶型制备。共得到了两种晶型，游离碱晶型Ⅰ和游离碱晶型Ⅱ。
蒸发结晶是向化合物中加入溶剂，制成的溶剂过滤后将滤液滴加到样品瓶中，瓶口覆盖的封口膜扎孔后，置于通风橱缓慢挥发。得到了两种晶型，游离碱晶型Ⅰ和游离碱晶型Ⅳ。将游离碱晶型Ⅳ加热至150℃脱溶剂后制备得到新晶型，命名为游离碱晶型Ⅴ。
2.3.1游离碱晶型Ⅰ的制备和表征
游离碱晶型Ⅰ可以在大部分溶剂中得到，实施例1所得到的化合物为游离碱晶型Ⅰ。其表征结果如图4和图5所示。
2.3.2游离碱晶型Ⅱ的制备和表征
游离碱晶型Ⅱ可以在某些含有乙醇的溶剂体系中得到，以无定形的化合物为原料，通过单一溶剂乙醇的混悬转晶能得到游离碱晶型Ⅱ。其表征结果如图7和图8所示，游离碱晶型Ⅱ的DSC曲线在124℃和230℃有两个吸热峰，TGA曲线在65-150℃有4.48％的失重，且游离碱晶型Ⅱ是在乙醇的溶剂体系中得到。综上推断，游离碱晶型Ⅱ应为乙醇的溶剂合物。DSC曲线上124℃处的吸热峰应为脱溶剂峰。
表15游离碱晶型Ⅱ的X-射线粉末衍射峰数据
2.3.3游离碱晶型Ⅲ的制备和表征
游离碱晶型Ⅲ可以在某些含有异丙醇的溶剂体系中得到，在通过反溶剂沉淀来制备晶型时，在二氯甲烷/异丙醇或二氯乙烷/异丙醇体系中发现了游离碱晶型Ⅲ。其表征结果如图9和图10所示，游离碱晶型Ⅲ的DSC曲线在121℃和230℃有两个吸热峰，TGA曲线在75-145℃有9.57％的失重，且游离碱晶型Ⅲ在加热至150℃脱溶剂后，XRPD转变为游离碱晶型Ⅰ。游离碱晶型Ⅲ是在异丙醇的溶剂体系中得到，综上推断，游离碱晶型Ⅲ应为异丙醇的溶剂合物，DSC曲线上121℃处的吸热峰应为脱溶剂峰。
表16游离碱晶型Ⅲ的X-射线粉末衍射峰数据

2.3.4游离碱晶型Ⅳ的制备和表征
游离碱晶型Ⅳ可以在某些含有二氯乙烷的溶剂体系中得到。称取92.28mg原料，加入到样品瓶中，加入5.0mL的二氯乙烷溶解，样品瓶上面覆盖封口膜，扎孔后置于通风橱缓慢挥发，这样通过蒸发结晶来制备晶型Ⅳ。其表征结果如图11和图12所示，游离碱晶型Ⅳ的DSC曲线在108℃和231℃有两个吸热峰，TGA曲线在25-150℃有10.41％的失重。1H NMR结果显示只含6.14％二氯乙烷溶剂残留，由此推测该样品可能含有4.27％的水分。综上推断，游离碱晶型Ⅳ可能为二氯乙烷和水混合的溶剂合物。
表17游离碱晶型Ⅳ的X-射线粉末衍射峰数据
2.3.5游离碱晶型Ⅴ的制备和表征
游离碱晶型Ⅳ在加热至150℃脱溶剂后，通过XRPD表征，显示转变为新晶型，命名为游离碱晶型Ⅴ。其表征结果如图13和图14所示，游离碱晶型Ⅴ的DSC曲线在231℃有一个吸热峰，TGA曲线在分解前无明显失重。综上推断，游离碱晶型Ⅴ应为无水晶型。
表18游离碱晶型Ⅴ的X-射线粉末衍射峰数据
3.游离碱晶型Ⅰ的晶型评价
在本次的筛选过程中仅发现了游离碱晶型Ⅰ和游离碱晶型Ⅴ两种无水晶型，用丙酮和乙腈这两种溶剂，分别配制饱和溶液，然后在25℃或50℃，对两者进行了竞争打浆，从结果推断游离碱晶型I可能为化合物的热力学稳定晶型。并且游离碱晶型Ⅴ未在筛选实验中直接获得，仅通过游离碱晶型Ⅳ脱溶剂得到。因此，仅对游离碱晶型I进行了晶型评价，包括干法研磨、湿法研磨、压片(30MPa)、稳定性和引湿性研究。
通过研磨实验发现，游离碱晶型Ⅰ经过干法研磨5分钟后，就转为无定形状态；加水湿法研磨5分钟后，结晶度未见明显变化；加乙醇湿法研磨5分钟后，结晶度轻微下降。
压片实验中，当压力为30MPa时，结晶度轻微下降。
稳定性实验中发现，当游离碱晶型Ⅰ敞口放置在80℃下3天、60℃下7天、25℃/60％RH下7天、40℃/75％RH下7天和25℃/90±5％RH下7天，分别取样进行XRPD检测。结果发现，游离碱晶型Ⅰ没有明显变化，药物的液相检测结果也没有变化。
对游离碱晶型Ⅰ做了动态水蒸汽吸附(DVS)测试，结果表明，在80％RH时吸水增重0.66％，游离碱晶型Ⅰ略有引湿性。
综上，确定了如式1所示的这类化合物的空间构型，尤其精准确定了实施例1的空间结构。在对实施例1化合物进行的晶型筛选研究中，共发现2个无水晶型(游离碱晶型Ⅰ和游离碱晶型Ⅴ)和3个溶剂化合物(游离碱晶型Ⅱ、游离碱晶型Ⅲ和游离碱晶型Ⅳ)。在实验中发现，游离碱晶型Ⅰ为较稳定的无水晶型，具有稳定的固态性质，且略有引湿性，可以用作后续药物开发。

Claims (13)

  1. 一类用作激酶抑制剂的化合物，其特征在于，所述用作激酶抑制剂的化合物为如式1所示的化合物，或其氘代物，或药学上可以接受的盐、溶剂化物或前药：
    式1中，X选自CH、N；
    R1选自R5为H、C1-C3的烷基、C1-C3的氟代烷基；
    R20、R21、R22各自独立地选自甲基或氘代甲基；
    R3选自C1-C3的烷基、C1-C3的卤代烷基；
    R40、R41、R42各自独立地选自H、D、F。
  2. 如权利要求1所述的用作激酶抑制剂的化合物，其特征在于，为如式2所示的化合物，或其氘代物，或药学上可以接受的盐、溶剂化物或前药：
  3. 如权利要求2所述的用作激酶抑制剂的化合物，其特征在于，式2中，X选自CH、N；R3选自-CH3、-CH2CH3、-CH2CF3；R40、R41均为H，R42选自H或F。
  4. 如权利要求1所述的用作激酶抑制剂的化合物，其特征在于，为如式3所示的化合物，或其氘代物，或药学上可以接受的盐、溶剂化物或前药：
  5. 如权利要求4所述的用作激酶抑制剂的化合物，其特征在于，式3中，X选自CH、N；R3为-CH3、-CH2CH3、-CH2CF3；R40、R41均为H，R42选自H或F，R5选自-CH3、-CF3。
  6. 如权利要求1所述的用作激酶抑制剂的化合物，其特征在于，式1所示的化合物为：

  7. 如权利要求1～6中任一项所述的用作激酶抑制剂的化合物，其特征在于，所述化合物为晶型、无定型或所述溶剂化物；所述溶剂化物所含溶剂为非水溶剂或非水溶剂和水组成的混合溶剂。
  8. 如权利要求7所述的用作激酶抑制剂的化合物，其特征在于，其结构如式A所示的化合物：
    晶型为游离碱晶型Ⅰ，该晶型的X-射线粉末衍射图谱中2θ为9.76°±0.2°，10.45°±0.2°，16.54°±0.2°，18.66°±0.2°，20.07°±0.2°，25.90°±0.2°具有特征衍射峰。
  9. 如权利要求8所述的用作激酶抑制剂的化合物，其特征在于，所述游离碱晶型Ⅰ的X-射线粉末衍射图谱中2θ为9.07°±0.2°，9.76°±0.2°，10.45°±0.2°，11.53°±0.2°，11.80°±0.2°，12.91°±0.2°，13.79°±0.2°，14.67°±0.2°，15.08°±0.2°，15.63°±0.2°，16.54°±0.2°，17.50°±0.2°，18.66°±0.2°，20.07°±0.2°，21.10°±0.2°，23.29°±0.2°，24.16°±0.2°，25.90°±0.2°具有特征衍射峰。
  10. 一种如权利要求1～9中任一项所述的用作激酶抑制剂的化合物在制备用于治疗由EGFR突变和/或HER2突变导致的相关疾病的药物中的应用。
  11. 如权利要求10所述的应用，其特征在于，所述EGFR突变和/或HER2突变包括EGFR外显子20***突变、HER2外显子20***突变、EGFR外显子19缺失、EGFR外显子21的点突变、EGFR外显子20的点突变中的一种或两种以上组合。
  12. 如权利要求11所述的应用，其特征在于，所述的EGFR突变和/或HER2突变选自EGFR Del 19/T790M/C797S突变，EGFR L858R/T790M/C797S突变。
  13. 如权利要求10、11或12所述的应用，其特征在于，所述疾病为所述EGFR突变和/或HER2突变导致的癌症。
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CN111777620A (zh) * 2019-04-04 2020-10-16 山东轩竹医药科技有限公司 酪氨酸激酶抑制剂的新用途
CN113278012A (zh) * 2020-02-19 2021-08-20 郑州泰基鸿诺医药股份有限公司 用作激酶抑制剂的化合物及其应用
WO2021180238A1 (zh) * 2020-03-13 2021-09-16 郑州同源康医药有限公司 一类用作激酶抑制剂的化合物及其应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111777620A (zh) * 2019-04-04 2020-10-16 山东轩竹医药科技有限公司 酪氨酸激酶抑制剂的新用途
CN113278012A (zh) * 2020-02-19 2021-08-20 郑州泰基鸿诺医药股份有限公司 用作激酶抑制剂的化合物及其应用
WO2021180238A1 (zh) * 2020-03-13 2021-09-16 郑州同源康医药有限公司 一类用作激酶抑制剂的化合物及其应用

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