CN113773305A - Aminopyrimidine derivative and application thereof as EGFR tyrosine kinase inhibitor - Google Patents

Aminopyrimidine derivative and application thereof as EGFR tyrosine kinase inhibitor Download PDF

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CN113773305A
CN113773305A CN202111083628.9A CN202111083628A CN113773305A CN 113773305 A CN113773305 A CN 113773305A CN 202111083628 A CN202111083628 A CN 202111083628A CN 113773305 A CN113773305 A CN 113773305A
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CN113773305B (en
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郑志兵
李鹏运
李松
李行舟
肖军海
周辛波
钟武
曹圣洁
樊士勇
肖典
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Academy of Military Medical Sciences AMMS of PLA
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    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract

The invention provides an aminopyrimidine derivative shown as a formula I, and pharmaceutically acceptable salts, isomers, solvates and pharmaceutical compositions thereof. The invention also provides a preparation method of the compound shown in the formula I. The compound can be used as EGFRT790M/L858RAnd EGFRDel19A mutated EGFR high selectivity ligand molecule for use in the treatment of EGFR kinase mediated related cancers.

Description

Aminopyrimidine derivative and application thereof as EGFR tyrosine kinase inhibitor
Technical Field
The invention relates to the technical field of medicines. Mainly relates to an aminopyrimidine derivative, pharmaceutically acceptable salts, isomers, solvates and pharmaceutical compositions thereof, and also relates to a preparation method and application thereof as a medicament. In particular to the application of the compound as an anti-cancer drug for treating EGFR kinase mediation.
Background
According to the data published by the world health organization, cancer causes huge burden of disease treatment in the world, and new cancer cases and death people in China all live at the first place in the world. Among them, lung cancer, one of the most common malignant tumors, has become the first death caused by malignant tumors in our country. In clinical diagnosis, lung cancer is classified as non-small cell lung cancer (NSCLC) accounting for about 80-85% of all lung cancers, and about 75% of patients are found to be in the middle-advanced stage (stage III or IV), locally advanced or metastatic disease is present at the time of diagnosis, and the 5-year survival rate of advanced non-small cell lung cancer patients is less than 5%, when chemotherapy and radiotherapy are the main treatment for advanced lung cancer patients. Epidermal Growth Factor Receptor (EGFR) is closely related to proliferation, angiogenesis, tumor invasion, metastasis and apoptosis of tumor cells, and is an important target for treating non-small cell lung cancer. Activating mutations in the EGFR tyrosine kinase domain have been identified as an oncogenic driver of NSCLC, with about 30% of patients in our country having EGFR mutations. In recent years, the development of targeted therapies has shifted the treatment strategy of advanced NSCLC patients from traditional chemoradiotherapy to targeted therapies based on the use of Tyrosine Kinase Inhibitors (TKIs).
The representative drugs in the first generation of EGFR TKIs are gefitinib and erlotinib, which are reversibly combined with an EGFR tyrosine kinase region and obtain better clinical response against EGFR-L858R and exon 19 deletion mutation. However, related studies have shown that about 50% of patients treated with first generation EGFR TKIs develop acquired resistance due to the T790M mutation after 12 months. The second generation of irreversible inhibitor afatinib overcomes the resistance due to T790M mutation of EGFR by undergoing an electrophilic michael addition reaction with a cysteine residue (Cys797) near the EGFR-ATP binding site to form a covalently irreversible bound form of receptor and ligand. However, the inhibitor has poor selectivity on wild EGFR, so that clinically serious rash and gastrointestinal side effects occur, the treatment window is narrow, and the application is limited. The third generation EGFR TKI oxitinib (OSImertinib) not only effectively overcomes the drug resistance problem caused by T790M mutation, but also has better inhibition selectivity on wild EGFR, greatly reduces toxic and side effects, and is widely applied clinically. Although the compounds such as the third-generation EGFR inhibitors Osimetinib and CO-1686 have good treatment effect on drug-resistant patients with T790M, the compounds still have inhibition effect on wild-type EGFR, thereby causing skin rash and gastrointestinal adverse reactions on part of patients.
In order to improve the inhibitory activity of the drug on the drug-resistant EGFR T790M mutation and reduce the inhibitory effect of the drug on wild type EGFR, a third generation selective inhibitor targeting EGFR T790M with higher activity, better selectivity and lower toxicity is needed to be further developed.
Disclosure of Invention
The invention aims to provide an aminopyrimidine derivative and a pharmaceutical composition containing the same, which have good EGFR kinase inhibition activity and drug-resistant mutant EGFRT790M/L858R，EGFRdel19The mutations are highly selective and useful for the treatment of EGFR kinase mediated diseases.
The invention provides a compound shown as a formula I, pharmaceutically acceptable salts, isomers, solvates and a pharmaceutical composition containing the derivative.
Figure BSA0000252873150000021
Wherein:
R1is a substituted or unsubstituted heterocyclic group,
R2is a straight chain or branched alkyl, alkenyl, alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic radical of C1-C6
The molecules of formula I according to the invention may also be in the form of their salts, typically with organic or inorganic bases or acids.
Physiologically acceptable salts are preferred in the present invention. Physiologically acceptable salts of the compounds of the invention can be salts of the substances of the invention with inorganic acids, carboxylic acids or sulfonic acids, particularly preferably with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, steroic acid, tannic acid, for example. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be used to prepare salts useful as intermediates to obtain the compounds of the invention and pharmaceutically acceptable salts thereof.
Physiologically acceptable salts can likewise be metal or ammonium salts of the compounds of the invention having free carboxyl groups. Particularly preferred are, for example, sodium, potassium, magnesium or calcium salts, and also ammonium salts from ammonia or organic amines, such as ethylamine, diethylamine, triethylamine, N' -dibenzylethylenediamine, chloroprocaine, choline, N-methylglucamine and procaine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine or ethylenediamine.
The present invention provides methods for the preparation of compounds of formula I, which may be prepared by a variety of synthetic procedures well known to those skilled in the art. Exemplary methods of preparation of these compounds may include, but are not limited to, the schemes described below.
The compounds of formula I of the present invention can be synthesized by the exemplary methods described in the schemes and examples below. In the specific operation process, the steps in the method can be expanded or combined as required.
The scheme is as follows:
Figure BSA0000252873150000031
Reaction conditions：a.AlCl3，dioxane，reflux；b.p-Toluenesulfonic acid monohydrate，1-Butanol，80℃；c.DIPEA，DMF，90℃；d.X-R，K2CO3，DMF；e.Fe，NH4Cl，C2H5OH，H2O，reflux/H2，Pd(C)，CH3OH，r.t；f.Acryloyl chloride，DCM/Acetone，0℃
wherein: r1And R2Is as defined in claim 1.
The compounds of formula I and methods of preparation, pharmaceutical compositions and treatment regimens for the compounds disclosed herein may be suitably modified by those skilled in the art in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included in the invention. While the products, methods, and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The compounds of the invention may exist in tautomeric forms and the invention likewise encompasses such forms.
The compounds of the present invention may also be possible solvates thereof.
The compound of the present invention may also contain a pharmaceutical composition of the compound of the present invention and a pharmaceutically acceptable excipient, which comprises a therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt, isomer, solvate thereof, and one or more other drugs selected from the group consisting of: gefitinib, erlotinib, apatinib, vandetanib, cetuximab, trastuzumab, panitumumab, matuzumab, nimotuzumab, zalutumumab, pertuzumab, axitinib, XL647, CI-1033, BMS-690514, BIBW2992, EKB-569, ARRY-334543, NVP-AEE-788, HK1272, PF00299804, WZ4002, Her2, HSP90 inhibitors, CNF2024, apramycin, tanespimycin, IPI-504, SNX-5422, NVP-AUY922, or a combination thereof. In addition to the compounds of the present invention, pharmaceutically acceptable salts, isomers, solvates thereof, other drugs in the above pharmaceutical compositions are well known to those skilled in the art.
In another aspect, the invention provides methods of treating a condition associated with EGFR-driven cancer (including EGFR mutation-driven cancer, e.g., cancer with a T790M mutation, a L858R mutation, and a L858R/T790M double mutation) in a subject in need thereof, the method comprising: administering to the subject an effective amount of a compound of the invention. In specific embodiments, the EGFR-driven cancer is selected from the group consisting of: non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, nasopharyngeal carcinoma, etc.
The invention also relates to a medicament comprising at least one compound according to the invention, preferably together with one or more pharmacologically acceptable excipients or carriers, and to the use thereof for the above-mentioned purposes. Pharmaceutically acceptable carriers herein include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin.
The active ingredient may have a systemic and/or topical effect and may therefore be administered by a suitable route, such as orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, transdermally, conjunctivally, topically or in the form of an implant.
The active ingredient may also be administered in a form suitable for such administration.
Suitable for oral administration are the known administration forms which deliver the active ingredient rapidly and/or in a modified manner, such as tablets (uncoated or coated, e.g. with enteric or uncoated tablets), capsules, dragees, granules, pellets, powders, emulsions, suspensions and aerosols.
The use of parenteral administration may avoid the absorption step (intravenous, intra-arterial, intracardiac, intraspinal or intralumbar) or involve absorption (intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal). Administration forms suitable for parenteral administration are, in particular, solutions for injection and infusion, suspensions, emulsions, lyophilisates and preparations in the form of sterile powders.
Suitable for other routes of administration are e.g. inhaled (especially powder inhalation, spray), nasal drops/solutions, sprays; tablets or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the ear and eye, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, emulsions, pastes, dusting powders or implants, such as stents.
The active ingredient can be converted into the stated administration forms by methods known per se. This can be achieved with suitable pharmaceutical excipients which are inert and non-toxic. These include, in particular, carriers (for example microcrystalline cellulose), solvents (for example liquid polyethylene glycol), emulsifiers (for example sodium lauryl sulfate), dispersants (for example polyvinylpyrrolidone), synthetic and natural biopolymers (for example proteins), stabilizers (for example antioxidants and ascorbic acid), colorants (for example inorganic pigments such as iron oxide) or flavoring and/or taste masking agents. Where appropriate, the active ingredient may be presented in microencapsulated form in one or more of the above-mentioned carriers.
The above-mentioned pharmaceutical preparations may contain, in addition to the compounds of formula I according to the invention, other pharmaceutically active ingredients.
Other objects and advantages of the present invention will be apparent to those skilled in the art in the detailed description, examples and claims.
Detailed Description
The invention provides a compound shown as a formula I, and pharmaceutically acceptable salts, isomers, solvates and pharmaceutical compositions thereof.
Figure BSA0000252873150000051
Wherein:
R1is composed of
Figure BSA0000252873150000052
Wherein A is methyl or ethyl; b is hydrogen or methyl
R2Is methyl, or is selected from any one of the following groups:
Figure BSA0000252873150000061
the specific structures of preferred compounds are as follows, but they do not represent limitations to the above claims.
Figure BSA0000252873150000062
Compared with the prior art, the invention has the main advantages that: EGFR by the compounds of the present inventionT790M/L858RThe mutant enzymes and cells have high inhibitory activity and low inhibitory activity against EGFR wild-type enzymes and cells, and thus have high selectivity.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are provided to provide those of ordinary skill in the art with a complete disclosure of how to make and evaluate the methods and compounds claimed herein. The present embodiment merely illustrates the present invention and does not limit the scope of the present invention.
A method for synthesizing a compound.
The compounds of the invention may be prepared by conventional methods in the art using appropriate reagents, starting materials and purification procedures known to those skilled in the art.
The process for the preparation of the compounds of formula I according to the invention is described in more detail below, but these particular processes do not limit the invention in any way. The compounds of the present invention may also be conveniently prepared by combining, optionally, various synthetic methods described in the specification or known in the art, such combinations being more readily performed by those skilled in the art to which the invention pertains. The following examples are illustrative of preferred embodiments of the present invention and are not intended to limit the invention in any way.
Example 1 Synthesis of methyl N- (2- ((2-amino-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl N-methylglycinate (ZJT-4-M)
A.3- (2-Chloropyrimidin-4-yl) -1-methyl-1H-indole (ZJT-1) Synthesis
Figure BSA0000252873150000071
Adding 2, 4-dichloropyrimidine (120.0g, 0.81mol) into a 2L reaction bottle, adding 880ml of 1, 4-dioxane, stirring to dissolve, and adding anhydrous AlCl in batches3(120.0g, 0.90mol), stirring rapidly until AlCl is reached3Forming milky uniform dispersion, then pouring 1-methylindole (110.0g, 0.84mol) into the reaction solution, heating to 80 ℃, keeping the temperature and reacting for 3 hours, detecting by TLC that the reaction is complete, the 1-methylindole completely disappears, and a developing agent is petroleum ether and ethyl acetate which are 1: 1. And pouring the reaction solution into 2.5L of purified water while the reaction solution is hot, stirring vigorously to separate out a solid, continuously stirring for 1 hour after pouring, performing suction filtration, and washing the filter cake by using 1L of purified water. The solid was dried overnight under vacuum at 50 ℃ to give 210.0g of crude product. Heating and dissolving the crude product in a mixed solution of 3.2L acetonitrile and 320ml purified water, slowly cooling to 10 ℃ for crystallization for 2 hours, carrying out suction filtration, leaching a filter cake with 200ml acetonitrile, carrying out vacuum drying at 50 ℃ overnight to obtain 162.0g of ZJT-1 product,the yield thereof was found to be 82.3%.1H-NMR(400MHz，DMSO-d6)δppm 8.54(d，J＝5.5Hz，1H)，8.52(s，1H)，8.45-8.39(m，1H)，7.83(d，J＝5.5Hz，1H)，7.61-7.56(m，1H)，7.35-7.26(m，2H)，3.90(s，3H)。ESI-MS m/z：244.06[M+H]+。
Synthesis of N- (4-fluoro-2-methoxy-5-nitrophenyl) -4- (1-methyl-1H-indol-3-yl) pyrimidin-2-amine (ZJT-2)
Figure BSA0000252873150000072
ZJT-1(15.00g, 0.062mol) was charged into a 2L reaction flask, and 700mL of n-butanol, 4-fluoro-2-methoxy-5-nitroaniline (12.63g, 0.068mol), p-toluenesulfonic acid monohydrate (14.15g, 0.074mol) were added and dissolved with stirring, and the reaction was carried out at 110 ℃ for 6 hours, TLC showed that the ZJT-1 reaction was almost complete, and a large amount of solid precipitated from the reaction solution. The mixture was stirred to room temperature and filtered to give a yellow solid, and TLC showed a small amount of SM3 and p-toluenesulfonic acid remained in the solid. Pulping the obtained solid for 1 hour by using 500ml of mixed solution of acetonitrile and 500ml of purified water, carrying out suction filtration, wherein TLC of the obtained solid shows that little SM3 residue exists, the mother solution basically contains no product and contains a large amount of ZJT-1 and SM3, pulping the filter cake for 1 hour by using 500ml of mixed solution of acetonitrile and 500ml of purified water, carrying out suction filtration, and obtaining the product TLC which contains no impurities. The product is dried in vacuum at 50 ℃ overnight to obtain 17.38g of ZJT-2 product with 71.32% yield.1H-NMR(400MHz，Chloroform-d)δppm 8.31(d，J＝5.5Hz，1H)，8.15(s，1H)，8.05(d，J＝8.0Hz，1H)，8.01(d，J＝7.5Hz，1H)，7.69(s，1H)，7.20(t，J＝2.8Hz，1H)，7.18(s，1H)，7.15(d，J＝5.6Hz，1H)，6.84(d，J＝12.5Hz，1H)，6.71(d，J＝12.3Hz，1H)，3.92(s，3H)，3.82(s，3H)。ESI-MS m/z：394.13[M+H]+。
Synthesis of 2-methoxy-N4-methyl-N1- (4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) -N4- (2- (methylamino) ethyl) -5-nitrobenzene-1, 4-diamine (ZJT-3)
Figure BSA0000252873150000081
21.96g of ZJT-2(0.056mol) was placed in a 500ml flask, and 5.91g N, N' -dimethylethylenediamine (0.067mol), 10.86g DIPEA (0.084mol) and 250ml DMF were added thereto, and stirred to be insoluble, and gradually dissolved down after temperature rise, the reaction was carried out at 90 ℃ for 9 hours, TLC showed that the reaction was almost complete with ZJT-2, and the reaction solution turned from yellow to red. And (3) stirring and cooling to room temperature, adding 1L of purified water into the reaction solution, pulping for 1 hour, filtering, and obtaining a product TLC (thin layer chromatography) which does not contain impurities. The product was dried overnight under vacuum at 50 ℃ to give 17.84g, 69.10% yield.1H-NMR(400MHz，DMSO-d6)δppm8.62(s，1H)，8.36(d，J＝8.2Hz，1H)，8.34(s，1H)，8.32(d，J＝5.4Hz，1H)，8.12(s，1H)，7.52(d，J＝8.2Hz，1H)，7.25(ddd，J＝8.2，7.0，1.2Hz，1H)，7.21(d，J＝5.4Hz，1H)，7.12(t，J＝7.0Hz，1H)，6.87(s，1H)，3.96(s，3H)，3.88(s，3H)，3.31(s，1H)，3.28(d，J＝6.4Hz，2H)，2.84(s，3H)，2.73(t，J＝6.5Hz，2H)，2.30(s，3H)。ESI-MS m/z：462.23[M+H]+。
D. Synthesis of methyl N- (2- ((5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) -2-nitrophenyl) (methyl) amino) ethyl) -N-methylglycinate (ZJT-3-M)
Figure BSA0000252873150000091
ZJT-3(0.50g, 1.08mmol) was charged to a 50ml flask, 20ml DMF was added and methyl bromoacetate (0.22g, 1.30mmol) and K were added2CO3(0.22g, 1.62mmol), the reaction was controlled at room temperature for 30min and TLC showed ZJT-3 reaction completion (DCM: MeOH ═ 20: 1). Adding 200ml of purified water and 150ml of EA into the reaction solution, pulping for 10 minutes, then performing layered extraction, separating an organic layer by TLC (thin layer chromatography) to show that a water layer has no target substance, washing with purified water and saturated saline water respectively, drying the obtained product overnight by using anhydrous sodium sulfate, and TLC (thin layer chromatography) to show that the product almost has no impurities, wherein a crude product ZJT-3-M is directly subjected to the next reaction. ESI-MS m/z: 534.24[ M + H]+。
E. Synthesis of methyl N- (2- ((2-amino-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) N-methylglycine ethyl ester (ZJT-4-M)
Figure BSA0000252873150000092
Putting the ZJT-3-M crude product in the last step into a 120ml hydrogenation reaction kettle, using Pd/C as a catalyst to react in a pilot scale, wherein the reaction is incomplete, so Raney Ni is used as the catalyst, adding a proper amount of Raney Ni, and adding 50ml CH3OH, the reaction vessel was sealed and then purged with 10atm of hydrogen overnight at room temperature, and TLC showed completion of ZJT-3-B reaction (DCM: MeOH ═ 10: 1). And (3) paving diatomite on the reaction liquid for suction filtration, sealing and storing the active Raney Ni in the filter cake, decompressing the filtrate, evaporating the solvent to dryness, and then putting the filtrate into a blast box for drying.
Example 2 Synthesis of methyl N- (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) -N-methylglycine ethyl ester (CSJ-I-M)
Figure BSA0000252873150000093
Placing the crude ZJT-4-M in a 100ml flask, adding 20ml DCM to make the product clear, adding K2CO3(0.45g, 3.24mmol), cooling the reaction solution to 0 ℃, stirring for 20min, slowly dropwise adding acryloyl chloride (0.20g, 2.16mmol), keeping the temperature for reaction for 20min under the condition of low temperature, and showing complete ZJT-4-M reaction by TLC (DCM: MeOH: NH)3·H2O is 50: 5: 1). Suction filtering the reaction liquid to remove K2CO3After the solid and the mother liquor were added with 40ml of methanol and stirred at room temperature for 1 hour, the solvent was evaporated to dryness under reduced pressure, and the obtained product was purified by a silica gel preparation plate (developing agent DCM: MeOH: 15: 1) to obtain 0.18g of the final product as an orange-red solid, with a total yield of 29.91% in the three-step reaction.1H-NMR(400MHz，Chloroform-d)δppm 9.87(s，1H)，9.66(s，1H)，9.12(s，1H)，8.38(d，J＝5.3Hz，1H)，8.09-8.04(m，1H)，7.77(s，1H)，7.42-7.39(m，1H)，7.29-7.27(m，1H)，7.26(s，1H)，7.21(d，J＝5.4Hz，1H)，6.79(s，1H)，6.47(s，1H)，6.46(s，1H)，5.76-5.70(m，1H)，4.00(s，3H)，3.89(s，3H)，3.71(s，3H)，3.34(s，2H)，2.93(t，J＝5.8Hz，2H)，2.68(s，3H)，2.56(s，2H)，2.41(s，3H)。ESI-MS m/z：558.2823[M+H]+。
Example 3 Synthesis of N- (2- ((2- (benzyl (methyl) amino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-I-B)
Figure BSA0000252873150000101
The synthesis method is the same as CSJ-I-M, 0.11g of final product is obtained, the yield is light yellow solid, and the total yield of the three steps is 17.74%.1H-NMR(400MHz，Chloroform-d)δppm 9.89(s，1H)，9.84(s，1H)，9.11(s，1H)，8.39(d，J＝5.3Hz，1H)，8.07(dd，J＝7.5，1.8Hz，1H)，7.72(s，1H)，7.40(d，J＝2.3Hz，1H)，7.38(d，J＝1.2Hz，1H)，7.36(s，1H)，7.35(d，J＝1.0Hz，1H)，7.33(d，J＝0.9Hz，1H)，7.32-7.30(m，1H)，7.29-7.27(m，1H)，7.26(s，1H)，7.21(d，J＝5.3Hz，1H)，6.77(s，1H)，6.46-6.37(m，1H)，6.20(s，1H)，5.57(d，J＝10.2Hz，1H)，3.98(s，3H)，3.87(s，3H)，3.58(s，2H)，2.91(s，2H)，2.60(s，3H)，2.40(s，2H)，2.25(s，3H)。ESI-MS m/z：576.31[M+H]+。
Example 4 Synthesis of N- (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -4-bromo-N-methylbenzamide (CSJ-I-C)
Figure BSA0000252873150000102
The synthesis method is the same as CSJ-I-M, 0.18g of final product is obtained, the yield is 24.98% in three steps.1H-NMR(400MHz，Chloroform-d)δppm 9.85(s，1H)，9.05(s，1H)，8.77(s，1H)，8.38(d，J＝5.3Hz，1H)，8.09-8.05(m，1H)，7.76(s，1H)，7.50(d，J＝1.7Hz，1H)，7.48(s，1H)，7.42-7.39(m，1H)，7.36(d，J＝5.1Hz，1H)，7.30(dd，J＝7.1，1.6Hz，1H)，7.29-7.27(m，1H)，7.23(s，1H)，7.21(s，1H)，6.79(s，1H)，6.47(s，1H)，6.39(s，1H)，5.63(s，1H)，3.98(s，3H)，3.89(s，3H)，3.69(s，2H)，3.22(s，2H)，2.87(s，3H)，2.73(s，3H)。ESI-MS m/z：668.20[M+H]+。
Example 5 Synthesis of N- (2- ((2- ((4-bromobenzyl) (methyl) amino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-I-F)
Figure BSA0000252873150000111
The synthesis method is the same as CSJ-I-M, 0.12g of final product is obtained, the yield is light yellow solid, and the total yield of the three steps is 17.02%.1H-NMR(400MHz，Chloroform-d)δppm 9.89(s，1H)，9.68(s，1H)，9.10(s，1H)，8.39(d，J＝5.2Hz，1H)，8.09-8.04(m，1H)，7.73(s，1H)，7.45(s，1H)，7.43(s，1H)，7.41-7.38(m，1H)，7.29-7.27(m，1H)，7.26(d，J＝1.5Hz，1H)，7.24(s，1H)，7.22(s，1H)，7.20(s，1H)，6.76(s，1H)，6.42(dd，J＝16.8，1.8Hz，1H)，6.15(dd，J＝16.9，10.1Hz，1H)，5.61(d，J＝10.4Hz，1H)，3.99(s，3H)，3.87(s，3H)，3.49(d，J＝8.4Hz，2H)，2.91(t，J＝5.8Hz，2H)，2.60(s，3H)，2.38(s，2H)，2.23(s，3H)。ESI-MS m/z：654.2185[M+H]+。
Example 6 Synthesis of (E) -N- (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -N-methylbut-2-enamide (CSJ-I-J)
Figure BSA0000252873150000112
The synthesis method is the same as CSJ-I-M, the final product is 0.18g, light yellow solid is obtained, and the total yield of the three-step reaction is 30.12%.1H-NMR(400MHz，Chloroform-d)δppm 9.85(d，J＝22.2Hz，1H)，9.05(s，1H)，8.80(s，1H)，8.38(t，J＝5.1Hz，1H)，8.08-8.04(m，1H)，7.75(s，1H)，7.42-7.38(m，1H)，7.20(d，J＝5.5Hz，1H)，6.93(dt，J＝14.3，7.1Hz，1H)，6.76(d，J＝11.0Hz，1H)，6.48(d，J＝9.3Hz，1H)，6.42(d，J＝12.6Hz，1H)，6.24(dd，J＝14.7，2.0Hz，1H)，6.01(d，J＝15.2Hz，1H)，5.78(d，J＝9.4Hz，1H)，5.72(dd，J＝9.3，2.5Hz，1H)，3.99(s，3H)，3.89(d，J＝2.7Hz，3H)，3.58(t，J＝6.1Hz，2H)，3.09(t，J＝6.2Hz，2H)，2.96(d，J＝5.5Hz，3H)，2.67(d，J＝9.7Hz，3H)，1.90-1.77(m，3H)。ESI-MS m/z：554.2874[M+H]+。
Example 7 Synthesis of N- (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -N-methylbut-3-enamide (CSJ-I-K)
Figure BSA0000252873150000121
The synthesis method is the same as CSJ-I-M, the final product is 0.27g, light yellow solid is obtained, and the total yield of the three-step reaction is 45.19%.1H-NMR(400MHz，Chloroform-d)δppm 9.85(d，J＝22.1Hz，1H)，9.06(s，1H)，8.79(d，J＝16.1Hz，1H)，8.38(t，J＝5.6Hz，1H)，8.08-8.04(m，1H)，7.77(s，1H)，7.42-7.38(m，1H)，7.30-7.27(m，1H)，7.22-7.19(m，1H)，6.92(tq，J＝14.9，7.1Hz，1H)，6.79-6.74(m，1H)，6.55-6.47(m，1H)，6.47-6.42(m，1H)，6.24(d，J＝14.9Hz，1H)，6.04-5.91(m，1H)，5.74(ddd，J＝12.5，9.2，2.6Hz，1H)，5.20-5.08(m，1H)，3.99(d，J＝1.3Hz，3H)，3.89(d，J＝1.6Hz，3H)，3.55(dt，J＝29.9，6.2Hz，2H)，3.15-3.04(m，3H)，2.97(d，J＝5.5Hz，2H)，2.71-2.66(m，3H)，1.97-1.83(m，2H)。ESI-MS m/z：554.2874[M+H]+。
Example 8 Synthesis of N- (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -N-methacrylamide (CSJ-I-L)
Figure BSA0000252873150000122
The synthesis method is the same as CSJ-I-M, the final product is 0.04g, light yellow solid is obtained, and the total yield of the three-step reaction is 6.87%.1H-NMR(400MHz，Chloroform-d)δppm 9.84(d，J＝16.3Hz，1H)，9.05(s，1H)，8.78(s，1H)，8.52(s，1H)，8.38(t，J＝5.4Hz，1H)，8.09-8.04(m，1H)，7.75(d，J＝6.3Hz，1H)，7.41-7.37(m，1H)，7.29(dd，J＝7.1，1.7Hz，1H)，7.21(dd，J＝7.2，5.2Hz，1H)，6.75(s，1H)，6.55(dd，J＝16.8，10.2Hz，1H)，6.50-6.45(m，1H)，6.44-6.39(m，1H)，6.38-6.33(m，1H)，5.80-5.72(m，1H)，5.72-5.62(m，1H)，3.98(d，J＝1.6Hz，3H)，3.89(s，3H)，3.59(t，J＝6.2Hz，2H)，3.11(t，J＝6.2Hz，2H)，2.97(d，J＝5.7Hz，3H)，2.67(d，J＝18.8Hz，3H)。ESI-MS m/z：540.2719[M+H]+。
Example 9 Synthesis of methyl (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) (methyl) carbamate (CSJ-I-N)
Figure BSA0000252873150000131
The synthesis method is the same as CSJ-I-M, 0.20g of final product is obtained, the yield is pale yellow solid, and the total yield of the three steps is 34.09%.1H-NMR(400MHz，Chloroform-d)δppm 9.74(s，1H)，9.67(s，1H)，8.67(s，1H)，8.49(s，1H)，8.11(s，1H)，8.10(s，1H)，7.58(s，1H)，7.47(s，1H)，7.25(d，J＝2.3Hz，1H)，6.71(s，1H)，6.41(s，1H)，6.16(d，J＝2.2Hz，1H)，5.73(s，1H)，5.06(d，J＝10.9Hz，1H)，4.13(t，J＝6.7Hz，3H)，3.40(s，3H)，3.01(s，3H)，2.88(s，2H)，2.84(s，2H)，2.65(s，3H)，2.38(s，3H)。ESI-MS m/z：544.2667[M+H]+。
Example 10 Synthesis of N- (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -N-methylbutane-2-acetamide (CSJ-I-R)
Figure BSA0000252873150000132
The synthesis method is the same as CSJ-I-M, 0.15g of final product is obtained, the yield is light yellow solid, and the total yield of three steps is 25.19%.1H-NMR(400MHz，Chloroform-d)δppm 9.86(d，J＝12.7Hz，1H)，9.07(s，1H)，8.74(s，1H)，8.61(s，1H)，8.37(dd，J＝5.4，1.6Hz，1H)，8.05(dd，J＝5.9，3.5Hz，1H)，7.78(s，1H)，7.42-7.38(m，1H)，7.29(dd，J＝3.0，1.2Hz，1H)，7.21(t，J＝5.4Hz，1H)，6.76(d，J＝11.6Hz，1H)，6.58(dd，J＝16.8，9.9Hz，1H)，6.46(dd，J＝11.1，1.7Hz，1H)，5.77(ddd，J＝17.8，9.1，2.7Hz，1H)，3.99(d，J＝2.3Hz，3H)，3.90(d，J＝2.0Hz，3H)，3.69-3.55(m，2H)，3.08(q，J＝2.5，1.9Hz，3H)，2.80(s，2H)，2.68(d，J＝7.2Hz，3H)，1.94(d，J＝41.2Hz，3H)。ESI-MS m/z：552.2717[M+H]+。
EXAMPLE 11 Synthesis of N- (4-methoxy-2- (methyl (2-methallyl) amino) ethyl) amino) -5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-I-X)
Figure BSA0000252873150000133
The synthesis method is the same as CSJ-I-M, the final product of 0.22g is obtained, orange red solid is obtained, and the total yield of the three steps of reaction is 37.77 percent.1H-NMR(400MHz，Chloroform-d)δppm 9.91(s，1H)，9.75(s，1H)，9.11(s，1H)，8.38(d，J＝5.3Hz，1H)，8.07(d，J＝7.4Hz，1H)，7.70(s，1H)，7.40(d，J＝7.4Hz，1H)，7.29-7.27(m，1H)，7.26(d，J＝1.2Hz，1H)，7.21(d，J＝5.3Hz，1H)，6.79(s，1H)，6.48(d，J＝17.2Hz，1H)，6.41(d，J＝9.7Hz，1H)，5.71(d，J＝9.8Hz，1H)，4.99(s，1H)，4.92(s，1H)，3.99(s，3H)，3.89(d，J＝1.3Hz，3H)，2.96(s，2H)，2.84(s，2H)，2.67(s，3H)，2.38(s，2H)，2.22(s，3H)，1.84(s，3H)。ESI-MS m/z：540.3081[M+H]+。
Example 12 Synthesis of N- (2- ((2- (but-3-en-1-yl (methyl) amino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-I-Y)
Figure BSA0000252873150000141
The synthesis method is the same as CSJ-I-M, 0.13g of final product and brick red solid are obtained, and the total yield of the three-step reaction is 22.32%.1H-NMR(400MHz，Chloroform-d)δppm 9.87(s，1H)，9.80(s，1H)，9.10(s，1H)，8.38(d，J＝5.2Hz，1H)，8.06(d，J＝7.3Hz，1H)，7.74-7.69(m，1H)，7.42-7.38(m，1H)，7.28(dd，J＝4.6，2.0Hz，1H)，7.26(d，J＝0.9Hz，1H)，7.21(d，J＝5.3Hz，1H)，6.78(s，1H)，6.47(d，J＝16.5Hz，1H)，6.38(s，1H)，5.79(s，1H)，5.72(d，J＝10.3Hz，1H)，5.07(d，J＝17.1Hz，1H)，5.01(d，J＝9.9Hz，1H)，4.00(s，3H)，3.89(s，3H)，2.93(d，J＝18.3Hz，2H)，2.88(s，2H)，2.70(s，3H)，2.51(s，2H)，2.38(s，2H)，2.26(s，3H)。ESI-MS m/z：540.4091[M+H]+。
Example 13 Synthesis of N- (4-methoxy-2- (methyl (prop-2-yn-1-yl) amino) ethyl) amino) -5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-I-z)
Figure BSA0000252873150000142
The synthesis method is the same as CSJ-I-M, 0.15g of final product is obtained, the yield is light yellow solid, and the total yield of the three steps is 26.55%.1H-NMR(400MHz，Chloroform-d)δppm 9.89(s，1H)，9.86(s，1H)，9.12(s，1H)，8.39(d，J＝5.3Hz，1H)，8.10-8.04(m，1H)，7.74(s，1H)，7.43-7.38(m，1H)，7.30(dd，J＝7.1，1.7Hz，1H)，7.28(d，J＝2.8Hz，1H)，7.21(d，J＝5.3Hz，1H)，6.80(s，1H)，6.46(dd，J＝16.9，2.2Hz，1H)，6.37(t，J＝13.0Hz，1H)，5.75-5.66(m，1H)，4.00(s，3H)，3.89(s，3H)，3.41(d，J＝2.4Hz，2H)，2.91(t，J＝5.7Hz，2H)，2.70(s，3H)，2.45(s，2H)，2.34(s，3H)，2.24(t，J＝2.3Hz，1H)。ESI-MS m/z：524.28[M+H]+。
Example 14 Synthesis of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((1-ethyl-1H-pyrazol-4-yl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (CSJ-II-D)
Figure BSA0000252873150000151
Synthesis of 1-ethyl-4-nitro-1H-pyrazole
Figure BSA0000252873150000152
4-Nitropyrazole (1.00g, 8.84mmol) was charged into a 50ml flask, 20ml DMF was added and dissolved with stirring, K was added2CO3(2.44g, 17.68mmol), ethyl iodide (2.51g, 17.68mmol) was added slowly with stirring at room temperature, the reaction was carried out at room temperature for 1 hour, the reaction was complete by TLC, 4-nitropyrazole was completely disappeared without impurity spots, and the ratio of developing solvent was petroleum ether: ethyl acetate 3: 1. Slowly pouring the reaction solution into 200ml of purified water, stirring vigorously, adding 200ml of ethyl acetate, stirring vigorously for 30min, performing layered extraction, and detecting that the target substance still has fluorescence in a water layer by TLC. The aqueous layer was separated, extracted with 150ml ethyl acetate, the aqueous layer was checked by TLC for absence of fluorescence of the target, and the organic layers were combined, washed with purified water and saturated brine, respectively, and dried over anhydrous sodium sulfate overnight. And after the night, performing suction filtration on the product, leaching a filter cake by using ethyl acetate, performing reduced pressure rotary evaporation on the mother liquor to remove the solvent, and performing vacuum drying on the obtained product at 50 ℃ for the night to obtain 1.32g of a 1-ethyl-4-nitro-1H-pyrazole crude product.1H-NMR(400MHz，DMSO-d6)δppm 8.91(d，J＝0.6Hz，1H)，8.26(d，J＝0.8Hz，1H)，4.21(q，J＝7.2Hz，2H)，1.41(t，J＝7.3Hz，3H)。ESI-MS m/z：142.06[M+H]+。
Synthesis of 1-ethyl-1H-pyrazol-4-amine
Figure BSA0000252873150000153
1-Ethyl-4-nitro-1H-pyrazole (1.00g, 7.09mmol) was charged into a 100ml flask, and 15ml of ethanol was added thereto to make it clear; ammonium chloride (0.76g, 14.18mmol) is taken and put into a small flask, 4ml of purified water is added, ammonium chloride aqueous solution is added into the dissolved raw materials, after the temperature is raised to 85 ℃ and the reflux state is reached, reduced iron powder (1.98g, 35.45mmol) is added, the temperature is kept for reaction for 4 hours, TLC detects that the raw materials are completely reacted, and the ratio of developing agent is DCM: MeOH is 10: 1. The crude product was stirred with 1.2equ of silica gel and purified by column chromatography (DCM: MeOH: 150: 1) to give 0.59g of 1-ethyl-1H-pyrazol-4-amine in 74.92% yield.1H-NMR(400MHz，DMSO-d6)δppm 7.02(d，J＝0.9Hz，1H)，6.88(d，J＝0.9Hz，1H)，3.92(q，J＝7.3Hz，2H)，3.75(p，J＝7.3Hz，2H)，1.27(t，J＝7.3Hz，3H)。ESI-MS m/z：112.09[M+H]+。
Synthesis of 2-chloro-N- (1-ethyl-1H-pyrazol-4-yl) pyrimidin-4-amine (inter-1-D)
Figure BSA0000252873150000161
1-Ethyl-1H-pyrazol-4-amine (1.00g, 9.00mmol) was placed in a 50ml flask, 12ml of DMF was added thereto to dissolve it, and K was added2CO3(2.48g, 18.00mmol) and then the reaction solution is cooled to 0 ℃ and stirred for 20min, then 2, 4-dichloropyrimidine (1.60g, 10.80mmol) is slowly added, the reaction is kept for 2H, TLC detection is carried out to ensure that 1-ethyl-1H-pyrazole-4-amine is completely reacted, and the ratio of a developing agent is DCM to MeOH is 10: 1. 1.2equ of silica gel was added to the crude product, and the mixture was purified by column chromatography (DCM: MeOH: 80: 1) to give 1.16g of inter-1-D in 57.78% yield.1H-NMR(400MHz，DMSO-d6)δppm 10.02(s，1H)，8.01(d，J＝5.9Hz，1H)，7.94-7.86(m，1H)，7.54-7.44(m，1H)，6.62-6.55(m，1H)，4.08(q，J＝7.3Hz，2H)，1.32(t，J＝7.3Hz，3H)。ESI-MS m/z：224.07[M+H]+。
Synthesis of D.N- (2- (dimethylamino) ethyl) -5-methoxy-N-methyl-2-nitrobenzene-1, 4-diamine (inter-2)
Figure BSA0000252873150000162
4-fluoro-2-methoxy-5-nitroaniline (1.00g, 5.40mmol), N, N, N' -trimethylethane-1, 2-diamine (0.66g, 6.50mmol), DIPEA (0.90g, 7.00mmol) and 10ml of DMA were added to a 50ml reaction flask and the temperature was raised to 90 ℃ for 6 hours with TLC detection at a developing solvent ratio of DCM: MeOH of 50: 1. The crude product was stirred with 1.2equ of silica gel and purified by column chromatography (DCM: MeOH: 80: 1) to give inter-20.89g, 61.46% yield.1H-NMR(400MHz，Chloroform-d)δppm 7.27(s，1H)，6.62(s，1H)，3.92(d，J＝0.7Hz，3H)，3.76(s，2H)，3.23-3.14(m，2H)，2.81(d，J＝0.5Hz，3H)，2.60-2.51(m，2H)，2.29(d，J＝1.1Hz，6H)。ESI-MS m/z：269.16[M+H]+。
Synthesis of E.N- (4- (((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-nitrophenyl) -N- (1-ethyl-1H-pyrazol-4-yl) pyrimidine-2, 4-diamine (inter-3-D)
Figure BSA0000252873150000163
To a 100ml reaction flask were added inter-1-D (1.16g, 5.20mmol), inter-2(1.53g, 5.72mmol), p-toluenesulfonic acid monohydrate (1.19g, 6.24mmol), and 30ml of n-butanol, TLC showed substantial completion of the reaction between inter-1-D and inter-2, at a ratio of DCM: MeOH: 10: 1 as the developing reagent. Stirring and cooling to room temperature, and adding saturated NaHCO into the reaction liquid3Adjusting the pH of the reaction solution to 7-8, adding 150ml of ethyl acetate, pulping for 1 hour, extracting, detecting by TLC (thin layer chromatography) to show that the water layer has no fluorescence of the target, separating out organic layers, and respectively usingThe purified water and saturated aqueous NaCl solution were washed, and dried over anhydrous sodium sulfate overnight. The crude product was stirred with 1.2equ of silica gel and purified by column chromatography (DCM: MeOH: NH)3·H2O300: 5: 1) to obtain 1.58g of inter-3-D, the yield is 66.95%.1H-NMR(400MHz，DMSO-d6)δppm 9.33(s，1H)，8.46(s，1H)，7.95(d，J＝4.6Hz，1H)，7.90(s，1H)，7.88(s，1H)，7.41(d，J＝0.8Hz，1H)，6.81(s，1H)，6.09(d，J＝5.8Hz，1H)，4.05-3.99(m，2H)，3.93(s，3H)，3.24(t，J＝6.9Hz，2H)，2.82(s，3H)，2.51(q，J＝1.8Hz，3H)，2.46(dd，J＝7.5，6.4Hz，2H)，2.15(s，6H)。ESI-MS m/z：456.25[M+H]+。
Synthesis of F.N- (2- (dimethylamino) ethyl) -N- (4- (((1-ethyl-1H-pyrazol-4-yl) amino) pyrimidin-2-yl) -5-methoxy-N-methylbenzene-1, 2, 4-triamine (inter-4-D)
Figure BSA0000252873150000171
Interer-3-D (1.58g, 3.47mmol) was added to a 100ml flask, and 13ml of ethanol was added to make it clear; taking ammonium chloride (0.56g, 10.41mmol), adding purified water 3ml, dissolving, adding ammonium chloride aqueous solution into the dissolved raw materials, heating to 85 ℃ under reflux, adding reduced iron powder (0.97g, 17.35mmol), keeping the temperature for reaction for 4 hours, detecting that the raw materials are completely reacted by TLC, wherein the ratio of developing solvent is DCM: MeOH is 10: 1. Since the product is easily deteriorated in column chromatography and in solution, the dried crude product is directly subjected to the next reaction. ESI-MS m/z: 426.28[ M + H]+。
Synthesis of G.N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((1-ethyl-1H-pyrazol-4-yl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (CSJ-II-D)
The inter-4-D crude product was placed in a 100ml flask, 20ml acetone was added to make the product clear, K was added2CO3(1.44g, 10.41mmol), cooling the reaction solution to 0 ℃, stirring for 20min, slowly dropwise adding acryloyl chloride (0.63g, 6.94mmol), keeping the temperature under the condition of low temperature for reaction for 30min, and displaying in by TLCthe ter-4-D reaction was complete (DCM: MeOH: NH)3.H2O is 50: 5: 1). Suction filtering the reaction liquid to remove K2CO3After the solid and the mother liquor were added with 40ml of methanol and stirred at room temperature for 1 hour, the solvent was evaporated to dryness under reduced pressure, and the obtained product was purified by a silica gel preparation plate (developing solvent DCM: MeOH: 10: 1) to obtain 0.54g of the final product CSJ-II-D, a tan solid, and the total yield of the two-step reaction was 33.45%.1H-NMR(400MHz，Chloroform-d)δppm 10.10(s，1H)，9.63(s，1H)，7.93(d，J＝5.9Hz，1H)，7.56(s，1H)，7.48(s，1H)，7.43(s，1H)，7.28(s，1H)，6.74(s，1H)，6.51(s，1H)，6.38(dd，J＝16.9，1.9Hz，1H)，6.01(d，J＝5.9Hz，1H)，5.73-5.62(m，1H)，4.11(q，J＝7.4Hz，2H)，3.84(s，3H)，2.94(s，2H)，2.69(s，3H)，2.46(s，2H)，2.36(s，6H)，1.46(t，J＝7.3Hz，3H)。ESI-MS m/z：480.2830[M+H]+。
Example 15 Synthesis of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-II-A)
Figure BSA0000252873150000181
The synthesis method is the same as CSJ-II-D, inter-3-A (0.50g, 1.13mmol) is taken to react, 0.09g of final product CSJ-II-A and light yellow solid are obtained, and the total yield of the two steps is 17.07%.1H-NMR(400MHz，Chloroform-d)δppm 9.12(s，1H)，7.99(d，J＝5.9Hz，1H)，7.32(d，J＝2.4Hz，1H)，6.75(s，1H)，6.73(s，1H)，6.69(s，1H)，6.42(dd，J＝17.0，1.6Hz，1H)，6.25(s，1H)，5.79(dd，J＝10.2，1.6Hz，1H)，4.25(s，3H)，3.91(s，3H)，3.84(s，3H)，3.14(d，J＝6.7Hz，2H)，2.79(s，2H)，2.68(s，3H)，2.55(s，6H)。ESI-MS m/z：466.2673[M+H]+。
Example 16 Synthesis of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- ((1-methyl-1H-pyrazol-4-yl) amino) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-II-B)
Figure BSA0000252873150000182
The synthesis method is the same as CSJ-II-D, inter-3-B (0.50g, 1.13mmol) is taken to react, 0.08g of the final product CSJ-II-B and light yellow solid are obtained, and the total yield of the two steps is 15.17%.1H-NMR(400MHz，Chloroform-d)δppm 7.86(d，J＝6.2Hz，1H)，7.74(d，J＝8.0Hz，1H)，7.67(s，1H)，7.49(s，1H)，6.84(s，1H)，6.76(s，1H)，6.39(d，J＝16.9Hz，1H)，6.09(d，J＝6.1Hz，1H)，5.78(dd，J＝10.2，1.6Hz，1H)，4.16(s，6H)，3.91(s，3H)，3.87(s，3H)，3.24(s，2H)，3.01(s，2H)，2.69(s，3H)，2.67(s，3H)。ESI-MS m/z：466.2673[M+H]+。
Example 17 Synthesis of N- (5- ((4- ((1, 3-dimethyl-1H-pyrazol-4-yl) amino) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide (CSJ-II-C)
Figure BSA0000252873150000191
The synthesis method is the same as CSJ-II-D, inter-3-C (0.50g, 1.10mmol) is taken to react to obtain the final product CSJ-II-C0.21 g, light yellow solid, the total yield of the two steps is 39.89%.1H-NMR(400MHz，Chloroform-d)δppm 9.94(s，1H)，9.78(s，1H)，7.98(d，J＝5.8Hz，1H)，7.47(s，1H)，7.42(s，1H)，6.72(s，1H)，6.58(s，1H)，6.42(d，J＝1.8Hz，1H)，6.37(d，J＝1.8Hz，1H)，5.82(d，J＝5.8Hz，1H)，5.70-5.66(m，1H)，3.85(s，3H)，3.83(s，3H)，2.99(s，2H)，2.70(s，3H)，2.53(s，2H)，2.42(s，6H)，2.18(s，3H)。ESI-MS m/z：480.2829[M+H]+。
Example 18 Synthesis of methyl N- (2- ((2-acrylamido-5-methoxy-4- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -N-methylglycinate (CSJ-III-M)
Figure BSA0000252873150000192
A.Synthesis of 2-chloro-N- (1-methyl-1H-pyrazol-3-yl) pyrimidin-4-amine (med-1)
Figure BSA0000252873150000193
1-methyl-1H-pyrazol-3-amine (23.00g, 0.237mol), 2, 4-dichloropyrimidine (35.06g, 0.237mol), K2CO3(49.13g, 0.355mol) was added to a 500ml reaction flask, and 200ml of 1, 4-dioxane was added as a solvent, and the mixture was heated to 80 ℃ and incubated for 10 hours, and the reaction was almost completed by TLC detection with the ratio of developing solvent DCM to MeOH being 10: 1. And (3) separating out a large amount of solids in the reaction solution, carrying out suction filtration, washing a filter cake by using normal hexane to obtain a light yellow solid, and detecting by TLC without raw materials. The filter cake was washed with 1L of purified water to remove K2CO3And (3) a solid. The product was dried under vacuum at 50 ℃ overnight to give 20.55g, 41.48% yield.1H-NMR(400MHz，DMSO-d6)δppm 10.45(s，1H)，8.17(d，J＝12.3Hz，1H)，7.76(dd，J＝19.6，2.2Hz，1H)，7.63(d，J＝2.2Hz，1H)，6.97(s，1H)，3.49-3.25(m，3H)。ESI-MS m/z：210.06[M+H]+。
Synthesis of N- (4-fluoro-2-methoxy-5-nitrophenyl) -N- (1-methyl-1H-pyrazol-3-yl) pyrimidine-2, 4-diamine (med-2)
Figure BSA0000252873150000201
Med-1(20.00g, 0.095mol), 4-fluoro-2-methoxy-5-nitroaniline (19.58g, 0.105mol), p-toluenesulfonic acid monohydrate (21.68g, 0.114mol) were added to a 1L reaction flask, 480mL of n-butanol was added and dissolved with stirring, the temperature was controlled at 90 ℃ for 8 hours, TLC showed that med-1 was almost completely reacted (DCM: MeOH: 10: 1), and a large amount of solid precipitated from the reaction solution. And (3) cooling to room temperature by stirring, filtering to obtain a yellow solid, and displaying a small amount of 4-fluoro-2-methoxy-5-nitroaniline and p-toluenesulfonic acid remained in the solid by TLC. The obtained solid is put into anhydrous etherWashing and filtering, wherein TLC of the obtained solid shows that the 4-fluoro-2-methoxy-5-nitroaniline is rarely remained, the mother liquor basically contains no product, and TLC of the obtained product shows that the product contains no impurities. The product was dried overnight under vacuum at 50 ℃ to give 29.88g, 87.60% yield.1H-NMR(400MHz，DMSO-d6)δppm 9.82(s，1H)，9.01(d，J＝8.5Hz，1H)，8.05(d，J＝5.8Hz，1H)，7.90(s，1H)，7.55(d，J＝2.2Hz，1H)，7.32(d，J＝13.3Hz，1H)，6.49(s，2H)，4.01(s，3H)，3.76(s，3H)。ESI-MS m/z：360.12[M+H]+。
Synthesis of C.N- (2-methoxy-4- (methyl (2- (methylamino) ethyl) amino) -5-nitrophenyl) -N (1-methyl-1H-pyrazol-3-yl) pyrimidine-2, 4-diamine (med-3)
Figure BSA0000252873150000202
Med-2(28.24g, 0.056mol), N, N' -dimethylethylenediamine (8.32g, 0.094mol), K2CO3(16.17g, 0.117mol) was added to a 500ml flask, and then 250ml of DMF was added as a solvent, and the mixture was stirred at room temperature without dissolution, and gradually cleared after warming, and the reaction was carried out at 90 ℃ for 9 hours, and TLC showed that med-2 was almost completely reacted (DCM: MeOH: 10: 1), and the reaction liquid turned from yellow to red. Stirring and cooling to room temperature, adding 1L of purified water into the reaction solution, pulping for 1 hour, adding 500ml of ethyl acetate, extracting after vigorous stirring, detecting the product in the water layer by TLC, and extracting the water layer again until no product fluorescence exists in the water layer. The organic phases were combined, washed with purified water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (DCM: MeOH: 30: 1) to give med-322.35g, 67.08% yield.1H-NMR(400MHz，DMSO-d6)δppm 9.72(s，1H)，8.55(s，1H)，7.99(d，J＝5.7Hz，1H)，7.73(s，1H)，7.50(d，J＝2.2Hz，1H)，6.82(s，1H)，6.44(s，2H)，3.94(s，3H)，3.75(s，3H)，3.23(t，J＝6.5Hz，2H)，2.80(s，3H)，2.67(t，J＝6.5Hz，2H)，2.51(p，J＝1.8Hz，1H)，2.28(s，3H)。ESI-MS m/z：428.22[M+H]+。
D. Synthesis of methyl N- (2- ((5-methoxy-4- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) -2-nitrophenyl) (methyl) amino) ethyl) -N-methylglycinate (med-3-M)
Figure BSA0000252873150000211
Med-3(0.50g, 1.17mmol) was charged into a 50ml flask, DMF 18ml was added and dissolved, methyl bromoacetate (0.21g, 1.40mmol), CsCO was added3(0.57g, 1.75mmol) at rt for 1h and TLC showed completion of med-3 reaction (DCM: MeOH ═ 20: 1). Adding 200ml of purified water and 150ml of ethyl acetate into the reaction solution, pulping for 10 minutes, detecting no product fluorescence in a water layer by TLC, separating an organic layer, washing with purified water and saturated saline water respectively, drying the obtained product overnight by using anhydrous sodium sulfate to obtain 0.55g of med-3 crude product, wherein the yield is 94.83%, and the crude product is directly subjected to the next reaction. ESI-MS m/z: 500.23[ M + H]+。
E. Synthesis of methyl N- (2- ((2-amino-5-methoxy-4- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -N-methylglycinate (med-4-M)
Figure BSA0000252873150000212
The crude med-3-M (0.48g, 0.90mmol) was placed in a 200ml hydrogenation reactor, 0.05g Pd/C was added as catalyst, and 80ml CH was added3OH, the reaction kettle is sealed and then is aerated with 10atm hydrogen, the temperature is controlled at 30 ℃ for 6 hours, and TLC shows med-3-M reaction is complete (DCM: MeOH is 10: 1). The reaction solution is filtered by spreading kieselguhr, the filtrate is decompressed and the solvent is evaporated to dryness and then is put into a blast box for drying, thus obtaining 0.44g of med-4-M crude product with the yield of 97.78 percent. Since the product is easily deteriorated in column chromatography and in solution, the dried crude product is directly subjected to the next reaction.
F. Synthesis of methyl N- (2- ((2-acrylamido-5-methoxy-4- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) -N-methylglycinate (CSJ-III-M)
The crude med-4-M (0.52g, 1.10mmol) was placed in a 100ml flask, 20ml DCM was added to make it clear, K was added2CO3(0.46g, 3.30mmol), cooling the reaction solution to 0 ℃, stirring for 20min, slowly dropwise adding acryloyl chloride (0.20g, 2.20mmol), keeping the temperature for reaction for 20min under the condition of low temperature, and TLC shows that the med-4-M reaction is complete (DCM: MeOH: NH)3·H2O is 50: 5: 1). Suction filtering the reaction liquid to remove K2CO3After the solid and the mother liquor were added with 40ml of methanol and stirred at room temperature for 1 hour, the solvent was evaporated to dryness under reduced pressure, and the obtained product was purified by a silica gel preparation plate (developing agent DCM: MeOH: 10: 1) to obtain the final product CSJ-III-M0.06g, a pale yellow solid, and the total yield of the three-step reaction was 10.53%.1H-NMR(400MHz，Chloroform-d)δppm 9.79(s，1H)，9.54(s，1H)，8.13(d，J＝5.9Hz，1H)，7.47(s，1H)，7.27(s，1H)，7.25(d，J＝2.3Hz，1H)，6.82(s，1H)，6.74(s，1H)，6.72(d，J＝5.3Hz，1H)，6.42(d，J＝3.1Hz，1H)，6.16(s，1H)，5.71-5.68(m，1H)，3.85(d，J＝0.6Hz，3H)，3.84(s，3H)，3.70(d，J＝0.6Hz，3H)，3.33(s，2H)，2.91(d，J＝5.8Hz，2H)，2.66(s，3H)，2.54(t，J＝5.8Hz，2H)，2.40(s，3H)。ESI-MS m/z：524.2729[M+H]+。
Example 19 Synthesis of methyl (2- ((2-acrylamido-5-methoxy-4- ((4- ((1-methyl-1H-pyrazol 3-yl) amino) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) (methyl) carbamate (CSJ-III-N)
Figure BSA0000252873150000221
The synthesis method is the same as CSJ-III-M, med-3(0.50g, 1.17mmol) is taken to react to obtain the final product CSJ-III-N0.24 g, light yellow solid, and the total yield of three steps is 34.43%.1H-NMR(400MHz，Chloroform-d)δppm 9.75(d，J＝17.1Hz，1H)，8.64(s，1H)，8.46(s，1H)，8.14(d，J＝5.8Hz，1H)，7.42(s，1H)，7.27(s，1H)，7.26(d，J＝2.3Hz，1H)，7.24(s，1H)，6.71(s，1H)，6.41(d，J＝2.9Hz，1H)，6.16(d，J＝2.2Hz，1H)，5.73(s，1H)，3.85(s，3H)，3.84(s，3H)，3.70(s，3H)，3.39(d，J＝18.5Hz，2H)，3.02(s，3H)，2.83(s，2H)，2.65(s，3H)。ESI-MS m/z：510.2572[M+H]+。
EXAMPLE 20 Synthesis of N- (4-methoxy-2- (methyl (2-methallyl) amino) ethyl) amino) -5- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-III-X)
Figure BSA0000252873150000222
The synthesis method is the same as CSJ-III-M, med-3(0.50g, 1.17mmol) is taken to react to obtain the final product CSJ-III-X0.05 g, light yellow solid, and the total yield of three steps is 8.46%.1H-NMR(400MHz，Chloroform-d)δppm 9.71(s，1H)，9.62(s，1H)，8.60(s，1H)，8.33(d，J＝7.5Hz，1H)，8.11(d，J＝6.1Hz，1H)，7.63(s，1H)，7.49(d，J＝13.4Hz，1H)，7.29(s，1H)，7.25(s，1H)，6.70(s，1H)，6.57-6.50(m，1H)，6.42(d，J＝2.8Hz，1H)，6.40(s，1H)，6.18(s，1H)，3.91(d，J＝3.7Hz，2H)，3.84(s，3H)，3.82(s，3H)，3.57(t，J＝6.2Hz，2H)，3.08(q，J＝7.5，6.9Hz，3H)，2.97(s，3H)，2.67(s，3H)，2.62(s，2H)。ESI-MS m/z：506.2623[M+H]+。
Example Synthesis of N- (2- ((2- (but-3-en-1-yl (methyl) amino) ethyl) (methyl) amino) -4-methoxy-5- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-HI-Y)
Figure BSA0000252873150000231
The synthesis method is the same as CSJ-III-M, med-3(0.50g, 1.17mmol) is taken to react to obtain the final product CSJ-III-Y0.10 g, light yellow solid, and the total yield of three steps is 16.91%.1H-NMR(400MHz，Chloroform-d)δppm 9.78(s，1H)，9.72(s，1H)，8.65(s，1H)，8.48(s，1H)，8.14(d，J＝5.8Hz，1H)，7.44(s，1H)，7.26(d，J＝2.4Hz，1H)，7.23(s，1H)，6.71(s，1H)，6.69(s，1H)，6.41(s，1H)，6.16(s，1H)，5.73(s，1H)，5.12(s，1H)，5.06(s，1H)，4.14(t，J＝6.7Hz，3H)，3.85(s，3H)，3.84(s，3H)，3.40(s，2H)，3.01(s，2H)，2.83(s，2H)，2.65(s，3H)，2.38(s，2H)。ESI-MS m/z：506.25[M+H]+。
EXAMPLE 22 Synthesis of N- (4-methoxy-2- (methyl (prop-2-yn-1-yl) amino) ethyl) amino) -5- ((4- ((1-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) amino) phenyl) acrylamide (CSJ-III-Z)
Figure BSA0000252873150000232
The synthesis method is the same as CSJ-III-M, med-3(0.50g, 1.17mmol) is taken to react to obtain the final product CSJ-III-Z0.09 g, light yellow solid, and the total yield of three steps is 15.72%.1H-NMR(400MHz，Chloroform-d)δppm 9.80(s，1H)，9.74(s，1H)，8.12(d，J＝5.9Hz，1H)，7.52(s，1H)，7.43(s，1H)，7.25(d，J＝2.3Hz，1H)，6.75(s，1H)，6.73(s，1H)，6.42(dd，J＝17.0，1.9Hz，1H)，6.31(dd，J＝17.0，10.0Hz，1H)，6.17-6.13(m，1H)，5.68(dd，J＝9.9，1.9Hz，1H)，3.85(s，3H)，3.84(s，3H)，3.41(d，J＝2.4Hz，2H)，2.89(t，J＝5.7Hz，2H)，2.69(s，3H)，2.45(t，J＝5.6Hz，2H)，2.34(s，3H)，2.24(t，J＝2.4Hz，1H)。ESI-MS m/z：490.2672[M+H]+。
EXAMPLE 23 test of kinase inhibitory Activity of Compounds
The inhibitory activity of the 21 compounds listed in the examples on the 19del mutation and the L858R/T790M double mutation EGFR, as well as on the wild-type EGFR, was determined at the enzyme level. The evaluation methods and results are described below.
1. Experimental materials and instruments
Experimental consumable for evaluating biological activity
Figure BSA0000252873150000233
Figure BSA0000252873150000241
2. Experimental procedure
2.1 preparation of stock solutions of Compounds
All compounds were dissolved in DMSO and prepared as 10mM stock solutions. The compounds used were stored in a desiccator at room temperature for three months, others could be stored at-20 ℃ for a long period of time.
Preparing a working solution:
all compounds were diluted in DMSO, 1000nM starting concentration, 3-fold gradient dilutions, 10 concentration points.
The positive reference compound, Osimertinib, was diluted in DMSO at an initial concentration of 1000nM, and was diluted in a 4-fold gradient in EGFR Del19 and EGFR T790M/L858R experiments, at 10 concentration points; in the EGFR WT experiment, 3-fold gradient dilutions were performed, 10 concentration points.
1000X positive control (1mM, Osimetinib) and 1000X vehicle control (100% DMSO) were prepared.
Shake on the shaker for 5 min.
Preparing a buffer solution: 1 volume of enzymic buffer 5X plus 4 volumes of distilled water; 5mM MgCl2；1mM DTT；1mM MnCl2。
2.2 titration of the kinase EGFR del19 (5. mu.l TK-substrate-biotin and ATP)
a) Compound dilutions were transferred to each well of assay plates (784075, Greiner) using Echo 550.
b) The assay plates were sealed and the plates were centrifuged at 1000g for 1 min.
c) 2X EGFR Del19 was prepared in 1X kinase buffer.
d) Mu.l of 2X EGFR Del19 was added to 384 well assay plates (784075, Greiner).
e) Plates were centrifuged at 1000g for 30s and at room temperature for 10 min.
f) A2 XTK-substrate-biotin and ATP mixture was prepared in 1 Xkinase buffer.
g) The reaction was started by adding 5. mu.l TK-substrate-biotin and ATP.
h) Plates were centrifuged at 1000g for 30 seconds. Seal assay plate, RT40 min.
i) 4X Sa-XL 665 was prepared in HTRF detection buffer.
j) Mu.l of Sa-XL 665 and 5. mu.l of TK-antibody-Cryptate were added to each well of the assay plate.
k) Plates were centrifuged at 1000g for 30s, RT 1 h.
l) fluorescence signals at 615nm (cryptate) and 665nm (XL665) were read on an Envision 2104 reader.
2.3 titration of the kinase EGFR T790M/L858R (5. mu.l TK-substrate-biotin and ATP)
a) Compound dilutions were transferred to each well of assay plates (784075, Greiner) using Echo 550.
b) The assay plates were sealed and the plates were centrifuged at 1000g for 1 min.
c) 2X EGFR T790M L858R was prepared in 1X kinase buffer.
d) Mu.l of 2X EGFR T790M L858R was added to 384 well assay plates (784075, Greiner).
e) Plates were centrifuged at 1000g for 30s and at room temperature for 10 min.
f) A2 XTK-substrate-biotin and ATP mixture was prepared in 1 Xkinase buffer.
g) The reaction was started by adding 5. mu.l TK-substrate-biotin and ATP.
h) Plates were centrifuged at 1000g for 30 seconds. Seal assay plate, RT40 min.
i) 4X Sa-XL 665 was prepared in HTRF detection buffer.
j) Mu.l of Sa-XL 665 and 5. mu.l of TK-antibody-Cryptate were added to each well of the assay plate.
k) Plates were centrifuged at 1000g for 30s, RT 1 h.
l) fluorescence signals at 615nm (cryptate) and 665nm (XL665) were read on an Envision 2104 reader.
2.4 EGFRWTTitration (5. mu.l TK-substrate-biotin and ATP)
a) Compound dilutions were transferred to each well of an assay plate (784075, Greiner) using Echo 550
b) The assay plates were sealed and the plates were centrifuged at 1000g for 1 min.
c) 2X EGFR wt was prepared in 1X kinase buffer.
d) Mu.l of 2X EGFR wt was added to 384 well assay plates (784075, Greiner).
e) Plates were centrifuged at 1000g for 30s and at room temperature for 10 min.
f) A2 XTK-substrate-biotin and ATP mixture was prepared in 1 Xkinase buffer.
g) The reaction was started by adding 5. mu.l TK-substrate-biotin and ATP.
h) Plates were centrifuged at 1000g for 30 seconds. Seal assay plate, RT40 min.
i) 4X Sa-XL 665 was prepared in HTRF detection buffer.
j) Mu. lSa-XL 665 and 5. mu.l TK-antibody-Cryptate were added to each well of the assay plate.
k) Plates were centrifuged at 1000g for 30s, RT 1 h.
l) fluorescence signals at 615nm (cryptate) and 665nm (XL665) were read on an Envision 2104 reader.
3. Data analysis
The 665/615nm ratio was calculated for each well
Calculate% Inhibition:
％Inhibition＝100-(Signalcmpd-SignalAve_PC)/(SignalAve_VC-SignalAve_PC)×100.
calculating IC of Compounds50And Plot of the Plot effect:
IC was calculated using GraphPad 6.0 by fitting the logarithm of% Inhibition and compound concentration to a non-linear regression (dose response-variable slope)50The value is obtained.
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))
X：log of inhibitor concentration；Y：％Inhibition
4. Test results
After the% Inhibition is calculated, the logarithm of the concentration of the test compound is plotted as the abscissa and the average value of the enzyme Inhibition ratio is plotted as the ordinateDose response curves, results show IC for 9 compounds50The value is less than 70nM, and is CSJ-I-B, CSJ-I-M, CSJ-I-X, CSJ-I-Y in the first class of target compounds respectively; CSJ-II-A, CSJ-II-B, CSJ-II-C, CSJ-II-D in a second class of compounds of interest; and CSJ-III-Z in a third class of target compounds.
Positive control drug Osimetinib and synthesized compound-to-kinase EGFRdel19EGFR, kinaseT790M/L858REGFR, kinaseWTThe test results are shown below:
IC of the Compound50Value of
Figure BSA0000252873150000261
Figure BSA0000252873150000271
EXAMPLE 24 test of inhibitory Activity of Compounds on relevant tumor cells
Selection of EGFR at cellular level in example 23 at enzyme levelT790M/L858RAnd EGFRdel19Has good inhibitory activity on EGFRWTActive compounds with good selectivity, determined against HCC-827 cells (EGFRdel19) and NCI-H1975 cells (EGFR)T790M/L858R) And on NCI-H1299, A549 (EGFR)WT) The inhibitory activity of (3). The evaluation methods and results are described below.
1. Experimental materials and instruments
1.1 Experimental consumables for bioactivity evaluation
Figure BSA0000252873150000272
Figure BSA0000252873150000281
2 Experimental procedures
2.1 preparation of stock solutions of Compounds
Compounds CSJ-I-B, CSJ-I-F, CSJ-I-C, CSJ-I-R, CSJ-I-Z and CSJ-I-Y were selected and dissolved in DMSO to prepare 10mM stock solutions. The compounds used were stored in a desiccator at room temperature for three months, others could be stored at-20 ℃ for a long period of time.
Preparing a working solution:
the above-mentioned compounds and the positive reference compounds, Osimetinib and Gefitinib, were diluted in DMSO at an initial concentration of 1000nM, and in NCI-H1975, HCC-827, NCI-H1299, A549 cell experiments, 3-fold gradient dilutions were carried out at 10 concentration points. Shake on the shaker for 5 min.
2.2 cell dosing
2.2.1 NCI-H1975, HCC-827, NCI-H1299, A549 cells in logarithmic growth phase were seeded in 96-well plates at 3000-4000 cell mass per well, and the plates were pre-cultured in an incubator for 24 hours (at 37 ℃ C., 5% CO)2Under the conditions of (a).
2.2.2 Medium was changed in the well plates and 100. mu.l of the compound and positive drug at the corresponding concentrations were added to the plates.
2.2.3 incubate the plates in an incubator for 72 hours, add 10. mu.l CCK-8 solution to each well, and incubate the plates in the incubator for 1-4 hours.
2.2.4 Absorbance at 450nm was measured using a microplate reader.
3 data analysis
Calculate% Inhibition:
％Inhibition＝100-(Signalcmpd-SignalAve_PC)/(SignalAve_VC-SignalAve_PC)×100.
calculating IC of Compounds50And Plot of the Plot effect:
IC was calculated using GraphPad 6.0 by fitting the logarithm of% Inhibition and compound concentration to a non-linear regression (dose response-variable slope)50The value is obtained.
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))
X：log of inhibitor concentration；Y：％Inhibition
4 results of the test
After the% Inhibition is calculated, a dose-effect curve is drawn by taking the logarithm of the concentration of the tested compound as the abscissa and the average value of the enzyme Inhibition rate as the ordinate, and the IC is fitted50Values, results show:
CSJ-I-B, CSJ-I-F, CSJ-I-C, CSJ-I-R, CSJ-I-Z, CSJ-I-Y have proliferation inhibitory activity against NCI-H1975, HCC-827, NCI-H1299, A549. Four compounds of the compounds CSJ-I-B, CSJ-I-R, CSJ-I-Z, CSJ-I-Y with AZD9291 (IC)500.0148 μ M) had higher activity than the other. The 6 compounds have higher anti-proliferation activity on NCI-H1975 than A549 or H1299, wherein the compound CSJ-I-Z (IC)500.00258 μ M) showed better NCI-H1975 cells than AZD9291 (IC)50Activity on HCC-827 cells (IC50 ═ 0.00363 μ M) and AZD9291(IC 898 μ M)500.00173 μ M). Inhibitory activity in NCI-H1975 cells compared to the selectivity of compound CSJ-I-Z in A549 and NCI-H1299 by 38760-fold and 27548-fold, respectively, and by 1685-fold and 143-fold over AZD 9291. All target compounds were on EGFR compared to ocitinib and gefitinibWTThe selectivity of the cells is improved, which indicates that the side effect is less.
The test results of the above compound and positive reference compounds Osimertinib and Gefitinib on NCI-H1975, HCC-827, NCI-H1299 and A549 cells are shown as follows:
IC of the Compound50Value of
Figure BSA0000252873150000291

Claims (5)

1. A compound shown as a formula I and pharmaceutically acceptable salts, isomers, solvates and pharmaceutical compositions thereof.
Figure FSA0000252873140000011
Wherein:
R1is composed of
Figure FSA0000252873140000012
Wherein A is methyl or ethyl; b is hydrogen or methyl
R2Is methyl, or is selected from any one of the following groups:
Figure FSA0000252873140000013
2. the compound of claim 1, and pharmaceutically acceptable salts, isomers, solvates and pharmaceutical compositions thereof. Wherein the compound preferably has the following structure.
Figure FSA0000252873140000021
3. A process for preparing a compound according to any one of claims 1 to 2:
Figure FSA0000252873140000022
wherein: r1And R2Is as defined in claim 1.
4. A medicament comprising at least one compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, isomer, solvate thereof, a pharmaceutical composition and at least one further excipient.
5. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, isomer, solvate thereof, for use as an EGFR inhibitor for the treatment of EGFR kinase mediated related cancers.
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