CN115403516A

CN115403516A - 3,4, 5-trisubstituted benzene ring-containing aromatic heterocyclic compound, pharmaceutical composition, preparation method and application thereof

Info

Publication number: CN115403516A
Application number: CN202110894982.3A
Authority: CN
Inventors: 段勇涛; 楚亚楠; 苏景天; 孙默然; 杨华; 姚永芳
Original assignee: Henan Childrens Hospital Zhengzhou Childrens Hospital
Current assignee: Henan Childrens Hospital Zhengzhou Childrens Hospital
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2022-11-29
Anticipated expiration: 2041-08-03
Also published as: CN115403516B

Abstract

The invention provides an aromatic heterocyclic compound containing 3,4, 5-trisubstituent benzene ring, a preparation method and application thereof. The compound has obvious inhibition effect on a plurality of tumors including but not limited to A549 (lung cancer cells), hela (cervical cancer cells), MCF-7 (breast cancer cells), MGC-803 (stomach cancer cells), hepG2 (liver cancer cells), U937 (lymphoma cells and leukemia cells), and can be used for preparing antitumor drugs. The compound disclosed by the invention has better biological activity, higher selectivity and lower toxicity. The preparation method has the advantages of strong repeatability, good stability, simpler conditions required by experimental reaction, mild experimental environment and better yield, and can be used for mass production under the condition of less investment.

Description

3,4, 5-trisubstituted benzene ring-containing aromatic heterocyclic compound, pharmaceutical composition, preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to an aromatic heterocyclic compound containing 3,4, 5-trisubstituted benzene ring, a pharmaceutical composition, a preparation method and an application thereof.

Background

Along with the prolonging of the average life span of residents, the enhancement of physical examination consciousness, the improvement of detection level, the change of factors such as living environment, eating habits and the like, malignant tumors become one of the most common diseases with higher death rate all over the world. With the continuous development of medicine, various treatment modes such as chemical drug therapy, endocrine therapy, molecular targeted therapy, immune gene therapy and the like have been derived at present.

Microtubules perform a series of biological functions during cell growth, including cytoskeleton formation, maintenance of cell morphology, participation in substance transport, participation in cell signaling, and regulation of cell mitosis. Especially in tumor cells, the mitosis of the cells is more active than that of normal cells, and in view of the important role played by microtubules in the growth and development of the cells, the treatment of cancer by targeting tubulin has become an effective strategy. There are three major tubulin binding sites known today: a vinblastine binding site, a paclitaxel binding site and a colchicine binding site. Most of the tubulysins that bind to the colchicine binding site contain 3,4,5-trimethoxyphenyl, which is important for the binding and activity of the drug molecule to tubulin. Over the past decade, there have been many successful microtubule inhibitors containing a 3,4, 5-trisubstituted benzene ring, but to date, no such microtubule inhibitors have been FDA approved for cancer therapy and such compounds remain a promising avenue for anticancer drugs.

Disclosure of Invention

In order to improve the technical problems, the invention provides a compound shown as a formula (I), and a racemate, a stereoisomer, a tautomer, an isotopic label, a solvate, a polymorphic substance or pharmaceutically acceptable salts thereof:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Identical or different, independently of one another, from H, halogen, NH ₂ OH, alkyl, alkoxy;

ar is selected from heteroarylene, alkylene-heteroaryl, unsubstituted or optionally substituted with one, two or more Ra;

l is selected from NH, C (O) NH, alkylene-amino-alkyl, unsubstituted or optionally substituted with one, two or more Rb;

each Ra, rb is the same or different and is independently selected from H, OH, NH ₂ Halogen, alkyl, alkoxy, cycloalkyl, heteroalkyl, aryl, heteroaryl.

According to an embodiment of the present invention, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Identical or different, independently of one another, from H, halogen, NH ₂ 、OH、C _1-12 Alkyl radical, C _1-12 An alkoxy group;

ar may be selected from 5-14 membered heteroaryl, C, arylene unsubstituted or optionally substituted with one, two or more Ra _1-12 Alkyl-5-14 membered heteroaryl;

l may be selected from NH, C (O) Y optionally substituted by one, two or more Rb _1-12 alkyl-NH, C _1-12 alkyl-NH-C _1-12 An alkyl group;

each Ra, rb may be the same or different and are independently selected from H, OH, NH ₂ Halogen, C _1-12 Alkyl radical, C _1-12 Alkoxy radical, C _3-12 Cycloalkyl, 3-12 membered heterocyclyl, C _6-14 Aryl, 5-14 membered heteroaryl.

According to an embodiment of the invention, R ₁ 、R ₂ 、R ₃ Can be the same or different and are independently selected from H, halogen, C _1-8 An alkoxy group;

R ₄ 、R ₅ can be identical or different and are selected independently of one another from H, halogen, NH ₂ 、OH、C _1-8 Alkyl radical, C _1-8 An alkoxy group;

ar may be selected from 5-10 membered heteroaryl, C _1-8 Alkyl-5-10 membered heteroaryl;

l may be selected from NH, C (O) O, C (O) sub-C _1-8 alkyl-NH, C _1-8 alkyl-NH-C _1-8 An alkyl group.

According to an embodiment of the present invention, R ₁ 、R ₂ 、R ₃ Can be the same or different and are independently selected from H, F, cl, br, I, methoxy;

R ₄ 、R ₅ can be the same or different and are independently selected from H, F, cl, br, I, NH ₂ OH, methyl, ethyl, propyl, methoxy, ethoxy;

ar can be selected from the group consisting of pyridylene, pyrimidylene, pyrazinylene, pyridazinylene, indolyl, indazolylene, thiazolyl, thiadiazolylene, methylene-indolyl, methylene-indazolyl;

l may be selected from NH, C (O) NH, methylene-NH.

According to an exemplary embodiment of the invention, the compound of formula (I) is selected from the following structures:

the invention also provides a preparation method of the compound shown in the formula (I), which comprises the following steps:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Ar, L independently have the definitions described above and X is selected from halogen or NH ₂ ；

Reacting the compound I-1 with a compound I-2 to obtain a compound shown in a formula (I);

according to an embodiment of the present invention, the reaction may be performed under the action of a base, which may be an inorganic base such as at least one of sodium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, potassium phosphate, or an organic base; the organic base is at least one of triethylamine, N-diisopropylethylamine and N, N-dimethylaminopyridine;

according to an embodiment of the invention, the reaction may be carried out under the action of a catalyst; the catalyst may be a palladium catalyst, for example palladium acetate; when the catalyst is a palladium catalyst, an organic ligand, such as (+/-) -2,2 '-bis- (diphenylphosphino) -1,1' -Binaphthyl (BINAP), can be added into the reaction system;

according to an embodiment of the present invention, the reaction may be performed in the presence of an organic solvent, which may be, for example, at least one of N, N-dimethylformamide, methanol, ethanol, dioxane, tetrahydrofuran, dichloromethane.

According to an embodiment of the invention, the preparation process further comprises at least one of the following schemes:

scheme 1:

wherein Ar has the meaning as defined above, X ₁ And X ₂ Selected from halogen, R is methyl or H;

reacting the compound A1 with 4-methyl phenyl boric acid to obtain a compound A2;

reacting the compound A2 with 3,4, 5-trimethoxyaniline to obtain a compound 1-compound 11 and a compound 17-compound 23.

Scheme 2:

reacting 5-bromo-1, 3, 4-thiadiazole-2-amine with 4-methylphenylboronic acid to obtain a compound M12;

reaction of compound M12 with 3,4, 5-trimethoxybenzoic acid affords compound 12.

Scheme 3:

wherein X ₃ Is selected from N or C;

reacting the compound C1 with 4-methylbenzyl chloride to obtain a compound C2;

the compound C2 reacts with 3,4, 5-trimethoxyaniline to obtain a compound 13-a compound 15.

Scheme 4:

reacting the compound 6-bromo-1H-indazole with 5-bromo-1, 2, 3-trimethoxybenzene to obtain a compound M16;

the compound M16 is reacted with 4-methylphenylboronic acid to obtain a compound 16.

Scheme 5:

wherein Ar has the definitions described above;

reacting 5-bromo-2-methylphenol with benzyl chloride to obtain phenylboronic acid M24-1;

reacting the intermediate M24-1 with pinacol diboron to obtain an intermediate M24-2;

the intermediate M24-2 firstly reacts with the compound A1, and the obtained product then reacts with 3,4, 5-trimethoxyaniline to obtain an intermediate E2;

and removing benzyl from the intermediate E2 to obtain a compound 24-a compound 29.

Scheme 6:

wherein Ar is pyridinylene or pyrimidinylene, R ₁ 、R ₂ 、R ₃ Having the definitions described above;

3-Nitro-4-methylphenylboronic acid is reacted with a compound A1 to give a product which is then reacted with

Reacting to obtain an intermediate M30;

the intermediate M30 is reduced with nitro to obtain compounds 30, 32-35 and 37-39.

Scheme 7:

wherein R is ₁ 、R ₂ 、R ₃ Having the definitions described above;

reacting 3-nitro-4-methylphenyl boric acid with 6-bromoindazole to obtain a product, and reacting the product with

Reacting to obtain an intermediate J;

the intermediate J is then reduced to the nitro group to give compounds 31, 36.

According to an embodiment of the invention, the preparation method further comprises at least one of the following steps:

step 1: 200mg of Compound A1 (1 eq.) are weighed out together with 4-methylphenylboronic acid (1.1 eq.), tetrakis (triphenylphosphine) palladium (0.05 eq.) and base (Na) ₂ CO ₃ Or K ₂ CO ₃ 2 eq.) was dissolved in a mixed solvent of water and THF and heated at reflux temperature overnight. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The volatile solvent in the system was removed in vacuo and the reaction mixture was extracted with ethyl acetate and the combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate A2.

And 2, step: when Ar is pyridylene, palladium acetate (0.04 eq.) and BINAP (0.04 eq.) are weighed and dissolved in anhydrous toluene (or 1, 4-dioxane), and the system is stirred under the protection of argon at normal temperature for 10 minutes. Then add intermediate A (1 eq.), 3,4, 5-trimethoxyaniline (1.2 eq.) and base (t-BuOK or Cs) ₂ CO ₃ 3 eq.) the system was heated to reflux under argon overnight. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The reaction mixture was extracted with ethyl acetate, and the combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed in vacuo. The residue was purified by column chromatography.

And step 3: when Ar is pyrimidylene, the intermediate A2 (1 eq.) and 3,4, 5-trimethoxyaniline (or 3,4, 5-trimethoxybenzylamine, 1.2 eq.) are dissolved in EtOH and H ₂ Concentrated hydrochloric acid (4 eq.) was slowly added dropwise to the mixed solvent of O, and the system was allowed to stand at reflux temperature overnight. With the progress of the reaction, solids are continuously precipitated in the system. The reaction was monitored by TLC, after the starting material had reacted completely, cooled to room temperature, filtered directly to give a solid, and the solid was washed alternately with cooled ethanol solution and DCM and dried.

And 4, step 4:to an anhydrous DCM solution in which intermediate M12 (1 eq.) was dissolved, 3,4,5-trimethoxybenzoic acid (1.2 eq.), EDCI (1.2 eq.) and HOBt (1.2 eq.) were added, and the mixture was heated under reflux for 8 to 10 hours. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The systematic DCM was removed in vacuo, the reaction mixture was extracted with ethyl acetate, the combined organic layers were collected and respectively neutralized with 10% HCl solution, saturated NaHCO ₃ The solution was washed with water, then dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography.

And 5: the substrate (6-bromoindole or 6-bromoindazole, 1 eq.) was dissolved in anhydrous DMF and the resulting mixture was cooled to 0 ℃. Add 60% NaH (1.5 eq.) in portions and stir the resulting mixture at room temperature for 30 minutes. The reaction was then cooled to 0 ℃, 4-methylbenzyl chloride (1.6 eq.) was added, followed by stirring at room temperature overnight. The reaction was monitored by TLC and after the reaction of the starting materials was complete, a large amount of H was added ₂ O, the reaction mixture was extracted with ethyl acetate. The combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate C2.

And 6: to 6-bromo-1H-indazole (1 eq.), 3,4, 5-trimethoxybromobenzene (1 eq.), N' -dimethylethylenediamine (2.5 eq.), and K ₃ PO ₄ (2.2 eq.) CuI (2.5 eq.) was added to an anhydrous DMF solution and the mixture was heated at 80 ℃ for 20 hours. The reaction was monitored by TLC and after the starting material had reacted completely, a large amount of H was added ₂ O, the reaction mixture was extracted with ethyl acetate. The combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate M16.

And 7: to a solution of 5-bromo-2-methylphenol (1) and benzyl chloride (1.5 eq.) in DMF was added potassium carbonate (3 eq.) and the reaction was mixed with stirring at room temperature for 4 hours. The reaction was monitored by TLC and after the starting material had reacted completely, a large amount of H was added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue is purified by column chromatography to giveTo intermediate M24-1.

And 8: dissolving pinacol diboron (1.1 eq.) and catalyst [1,1' -bis (diphenylphosphino) ferrocene ] in argon protection]To an anhydrous 1, 4-dioxane solution of palladium dichloride (0.05 eq.) and potassium acetate (4.4 eq.) was added intermediate M24-1 (1 eq.) and the reaction was stirred at 110 ℃ overnight. The reaction was monitored by TLC and after the starting material had reacted completely, a large amount of H was added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography to give intermediate M24-2.

And step 9: intermediate E2 (1 eq.) was dissolved in the appropriate amount of MeOH/THF (1). The reaction was monitored by TLC, after the starting material had reacted completely, DCM was added to dilute the system, palladium on carbon was removed by filtration through celite, the filtrate was collected and the solvent was removed in vacuo. The residue was purified by column chromatography.

Step 10: stannous chloride dihydrate (5 eq.) and a few drops of concentrated hydrochloric acid were added to the ethanol solution in which intermediate I or J (1 eq.) was dissolved, and the reaction system was allowed to react overnight at reflux temperature. The reaction was monitored by TLC and after the starting material had reacted completely, an appropriate amount of saturated NaHCO was added ₃ After the pH of the solution adjusting system is adjusted to be neutral (pH is approximately equal to 7), a large amount of H is added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography.

Step 11: at an ice bath temperature, zinc powder (4 eq.) and glacial acetic acid (4 eq.) were added to a mixed solution of ethanol and water in which intermediate I or J (1 eq.) was dissolved, and the reaction system was reacted at reflux temperature overnight. The reaction was monitored by TLC and after the reaction of the starting material was complete, an appropriate amount of saturated NaHCO was added ₃ After the pH of the solution adjusting system is adjusted to be neutral (pH is approximately equal to 7), a large amount of H is added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered andthe solvent was removed in vacuo. The residue was purified by column chromatography.

The invention also provides a pharmaceutical composition, which comprises at least one of a therapeutically effective amount of compound shown in formula (I), racemate, stereoisomer, tautomer, isotopic label, solvate, polymorph or pharmaceutically acceptable salt thereof.

According to an embodiment of the invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

According to an embodiment of the invention, the pharmaceutical composition may further comprise one or more additional therapeutic agents.

The present invention also provides a method of treating a neoplastic disease comprising administering to a patient a prophylactically or therapeutically effective amount of at least one compound of formula (I), racemates, stereoisomers, tautomers, isotopic labels, solvates, polymorphs thereof or pharmaceutically acceptable salts thereof.

The present invention also provides a method of treating a neoplastic disease comprising administering to a patient a prophylactically or therapeutically effective amount of the above-described pharmaceutical composition.

The tumor can be leukemia cell, breast cancer cell, liver cancer cell, lung cancer cell, gastric cancer cell, cervical cancer cell, and lymph cancer cell; preferably U937 cells, MCF7 breast cancer cells, hepG2 liver cancer cells, A549 lung cancer cells, MGC-803 stomach cancer cells and HeLa cervical cancer cells.

In some embodiments, the patient is a mammal, preferably a human.

The invention also provides application of at least one of the compounds shown in the formula (I), racemates, stereoisomers, tautomers, isotopic markers, solvates, polymorphs or pharmaceutically acceptable salts thereof in preparing medicines.

According to an embodiment of the invention, the medicament is an antineoplastic medicament, such as a tubulin inhibitor and/or a VEGFR-2 inhibitor.

According to an embodiment of the present invention, the tumor may be leukemia cell, breast cancer cell, liver cancer cell, lung cancer cell, stomach cancer cell, cervical cancer cell, lymph cancer cell; preferably U937 cells, MCF7 breast cancer cells, hepG2 liver cancer cells, A549 lung cancer cells, MGC-803 stomach cancer cells and HeLa cervical cancer cells.

Advantageous effects

The invention provides an aromatic heterocyclic compound containing 3,4, 5-trisubstituted benzene ring and a preparation method and application thereof. The compound of the invention has obvious inhibition effect on a plurality of tumors including but not limited to A549 (lung cancer cells), hela (cervical cancer cells), MCF-7 (breast cancer cells), MGC-803 (stomach cancer cells), hepG2 (liver cancer cells), U937 (lymphoma cells and leukemia cells). Through in vitro evaluation of anti-proliferative activity and evaluation of inhibition of tubulin and VEGFR-2, the inventors have found that the compounds of the present invention, particularly compound 35, are highly desirable dual-target inhibitors of tubulin and VEGFR-2. It can block cell cycle in G2 phase by regulating cycle related protein (Cyclin-B1 and p-Cdc2 protein), and induce apoptosis by regulating apoptosis related protein (Caspase-3/-7/-9 and PARP), reducing mitochondrial membrane potential and generating active oxygen. Immunofluorescence experiments and experiments inhibiting microtubule polymerization indicate that 35 can destroy intracellular microtubule tissue and inhibit tubulin polymerization. The test on the inhibition effect of VEGFR-2 kinase activity shows that the compound 35 has obvious inhibition effect on VEGFR-2.

The compound disclosed by the invention has better biological activity, higher selectivity and lower toxicity. The preparation method has the advantages of strong repeatability, good stability, simple conditions required by experimental reaction, mild experimental environment and good yield, and can be used for mass production under the condition of small investment.

Definition and description of terms

Unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present application, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. Such combinations and definitions of groups and structures of compounds when combined are to be understood as being within the scope of the present description and/or claims.

Unless otherwise indicated, the numerical ranges set forth in the specification and claims are equivalent to at least each specific integer recited therein. For example, a numerical range of "1 to 40" is equivalent to reciting each of the integer values in the numerical range of "1 to 12", i.e., 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, and each of the integer values in the numerical range of "13 to 40", i.e., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40. Further, when certain numerical ranges are defined as "numbers," it should be understood that the two endpoints of the range, each integer within the range, and each decimal within the range are recited. For example, "a number of 0 to 10" should be understood to not only recite each integer of 0, 1,2,3, 4,5, 6, 7, 8, 9, and 10, but also to recite at least the sum of each integer with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, respectively.

It should be understood that in describing one, two or more herein, "more" shall mean an integer greater than 2, such as greater than or equal to 3, e.g., 3,4,5, 6, 7, 8, 9, or 10.

The term "halogen" denotes fluorine, chlorine, bromine and iodine.

The term "alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon radical having from 1 to 40 carbon atoms, for example from 1 to 20 carbon atoms. "C _1-12 Alkyl "is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms. For example, "C _1-10 Alkyl "denotes straight-chain and branched alkyl groups having 1,2,3, 4,5, 6, 7, 8, 9 or 10 carbon atoms," C _1-8 Alkyl "denotes straight and branched chain alkyl groups having 1,2,3, 4,5, 6, 7, or 8 carbon atoms," C _1-6 Alkyl "denotes straight and branched chain alkyl groups having 1,2,3, 4,5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl pentyl, hexyl, isopropyl, isobutyl sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, methyl 1-methylbutyl, 1-ethylpropyl, a,1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, and the like, or isomers thereof.

The term "cycloalkyl" is understood to mean a saturated monocyclic, bicyclic (e.g. fused, bridged, spiro) hydrocarbon or tricyclic alkane ring having from 3 to 20 carbon atoms, preferably "C _3-12 Cycloalkyl ", more preferably" C _3-8 Cycloalkyl groups ". "C _3-12 Cycloalkyl "is understood to mean a saturated monovalent monocyclic, bicyclic (e.g. fused, bridged, spiro) hydrocarbon or tricyclic hydrocarbon ring having 3 to 12 carbon atoms, preferably" C _3-10 Cycloalkyl group ", more preferably" C _3-8 Cycloalkyl groups ". The term "C _3-10 Cycloalkyl "is understood to mean a saturated, monovalent, monocyclic (e.g. bridged, spiro) hydrocarbon or tricyclic hydrocarbon ring having 3,4,5, 6, 7, 8, 9 or 10 carbon atoms. Said C is _3-10 Cycloalkyl can be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or bicyclic, such as bornyl, indolyl, hexahydroindolyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo [2.1.1]Hexyl, bicyclo [2.2.1]Heptyl, bicyclo [2.2.1]Heptenyl, 6-dimethylbicyclo [3.1.1]Heptyl, 2, 6-trimethylbicyclo [3.1.1]Heptyl, bicyclo [2.2.2]Octyl, 2, 7-diazaspiro [3,5 ]]Nonanyl, 2, 6-diazaspiro [3,4 ]]An octyl group, or a tricyclic hydrocarbon group such as an adamantyl group.

Unless otherwise defined, the term "heterocyclyl" refers to a saturated or unsaturated non-aromatic ring or ring system containing one or more heteroatoms independently selected from N, O, and S and having from 3 to 20 total ring atoms (e.g., 3,4,5, 6, 7, 8, 9, 10, etc.). "3-12 membered heterocyclyl" refers to a saturated or unsaturated non-aromatic ring or ring system that is, for example, a 4-, 5-, 6-, or 7-membered monocyclic, 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic (e.g., fused, bridged, spiro) or tricyclic ring system, and contains at least one,e.g., 1,2,3, 4,5 or more heteroatoms selected from O, S and N, wherein N and S may also optionally be oxidized to various oxidation states to form nitroxides, -S (O) -or-S (O) ₂ -state of (c). Preferably, the heterocyclic group may be selected from "3-10 membered heterocyclic group". The term "3-10 membered heterocyclyl" means a saturated or unsaturated non-aromatic ring or ring system and contains at least one heteroatom selected from O, S and N. The heterocyclic group may be attached to the rest of the molecule through any of the carbon atoms or nitrogen atom (if present). The heterocyclic group may include fused or bridged rings as well as spiro rings. In particular, the heterocyclic group may include, but is not limited to: 4-membered rings such as azetidinyl, oxetanyl; 5-membered rings such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclic group may be benzo-fused. The heterocyclic group may be bicyclic, for example but not limited to a5,5 membered ring, such as hexahydrocyclopenta [ c ]]Pyrrole-2 (1H) -cyclic rings, or 5, 6-membered bicyclic rings, e.g. hexahydropyrrolo [1,2-a ]]A pyrazin-2 (1H) -yl ring. The heterocyclyl group may be partially unsaturated, i.e. it may contain one or more double bonds, such as, but not limited to, dihydrofuranyl, dihydropyranyl, 2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4]Thiadiazinyl, 1,2,3, 5-tetrahydrooxazolyl or 4H- [1,4]Thiazinyl, or it can be benzo-fused, such as but not limited to dihydroisoquinolinyl. When the 3-12 membered heterocyclic group is linked to another group to form the compound of the present invention, the carbon atom of the 3-12 membered heterocyclic group may be linked to another group, or the heterocyclic atom of the 3-12 membered heterocyclic ring may be linked to another group. For example, when the 3-12 membered heterocyclic group is selected from piperazinyl, it may be such that the nitrogen atom on the piperazinyl group is attached to another group. Or when the 3-12 membered heterocyclyl group is selected from piperidinyl, it may be that the nitrogen atom on the piperidinyl ring and the carbon atom in the para position are attached to other groups.

The term "aryl" is understood to mean preferably a radical having from 6 to 20 carbon atomsAromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon rings of a subgroup (e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 carbon atoms). "C _6-14 Aryl "is understood as preferably meaning a monocyclic, bicyclic or tricyclic hydrocarbon ring of monovalent or partial aromaticity having 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (" C) _6-14 Aryl "), in particular a ring having 6 carbon atoms (" C ₆ Aryl "), such as phenyl; or biphenyl, or is a ring having 9 carbon atoms ("C ₉ Aryl), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C ₁₀ Aryl radicals), such as tetralinyl, dihydronaphthyl or naphthyl, or rings having 13 carbon atoms ("C ₁₃ Aryl radicals), such as the fluorenyl radical, or a ring having 14 carbon atoms ("C) ₁₄ Aryl), such as anthracenyl. When said C is _6-14 When the aryl group is substituted, it may be mono-or polysubstituted. The substitution site is not limited, and may be, for example, ortho-, para-or meta-substituted.

The term "heteroaryl" is understood to include such monocyclic, bicyclic (e.g., fused, bridged, spiro) or tricyclic aromatic ring systems: which has from 5 to 20 ring atoms and contains one or more (e.g., 1-5) heteroatoms independently selected from N, O, and S, e.g., "5-14 membered heteroaryl. "5-14 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: which has 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and which contains 1 to 5, preferably 1 to 3, heteroatoms each independently selected from the group consisting of N, O and S and, in addition, can be benzo-fused in each case. "heteroaryl" also refers to a group in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclic rings, where the radical or point of attachment is on the heteroaromatic ring. When the 5-14 membered heteroaryl is linked to another group to form a compound of the present invention, the carbon atom on the 5-14 membered heteroaryl ring may be linked to another group, or the heteroatom on the 5-14 membered heteroaryl ring may be linked to another group. When the 5-14 membered heteroaryl group is substituted, it may be mono-or poly-substituted. Also, there is no limitation on the substitution site thereof, and for example, hydrogen attached to a carbon atom on a heteroaryl ring may be substituted, or hydrogen attached to a heteroatom on a heteroaryl ring may be substituted.

The term "spirocyclic" refers to a ring system in which two rings share 1 ring-forming atom.

The term "fused ring" refers to a ring system in which two rings share 2 ring atoms.

The term "bridged ring" refers to a ring system in which two rings share more than 3 ring-forming atoms.

Unless otherwise specified, heterocyclyl, heteroaryl or heteroarylene include all possible isomeric forms thereof, e.g., positional isomers thereof. Thus, for some illustrative non-limiting examples, forms may be included that are substituted at 1,2 or more of their 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-positions, etc. (if present) or bonded to other groups, including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, and pyridin-4-yl; thienyl or thienylene groups include thien-2-yl, thien-3-yl, and thien-3-yl; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

The term "arylene" denotes a divalent group substituted with two bonds.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

The preparation method of the invention is summarized as follows:

step 1: 200mg of a bis-halogen-substituted pyridine or pyrimidine substrate (1 eq.) were weighed, along with 4-methylphenylboronic acid (1.1 eq.), tetrakis (triphenylphosphine) palladium (0.0)5 eq.) and the corresponding base (Na) ₂ CO ₃ Or K ₂ CO ₃ 2 eq.) was dissolved in a mixed solvent of water and THF and heated at reflux temperature overnight. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The volatile solvent in the system was removed in vacuo and the reaction mixture was extracted with ethyl acetate and the combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate A2.

And 2, step: when Ar is pyridine ring, palladium acetate (0.04 eq.) and BINAP (0.04 eq.) are weighed and dissolved in anhydrous toluene (or 1, 4-dioxane), and the system is stirred for 10 minutes at normal temperature under the protection of argon gas. Then the intermediate A2 (1 eq.), 3,4, 5-trimethoxyaniline (1.2 eq.) and the corresponding base (t-BuOK or Cs) were added ₂ CO ₃ 3 eq.) the system was heated to reflux under argon overnight. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The reaction mixture was extracted with ethyl acetate, and the combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed in vacuo. The residue was purified by column chromatography to give the objective compounds 1,4, 5, 7, 8, 10, 11, 17-21.

And 3, step 3: when Ar is a pyrimidine ring, to EtOH and H dissolved with intermediate A (1 eq.) and 3,4, 5-trimethoxyaniline (or 3,4, 5-trimethoxybenzylamine, 1.2 eq.) are added ₂ Concentrated hydrochloric acid (4 eq.) was slowly added dropwise to the mixed solvent of O, and the system was kept at reflux temperature overnight. With the progress of the reaction, solids are continuously precipitated in the system. Monitoring the reaction by TLC, cooling to room temperature after the raw materials completely react, directly filtering to obtain a solid, alternately washing the solid with a cooled ethanol solution and DCM, and drying to obtain the target compounds 2,3, 6, 9, 22 and 23.

And 4, step 4: to a solution of intermediate M12 (1 eq.) in anhydrous DCM was added 3,4,5-trimethoxybenzoic acid (1.2 eq.), EDCI (1.2 eq.) and HOBt (1.2 eq.) and the mixture was heated under reflux for 8-10 hours. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. Removing DCM from the system in vacuo, extracting the reaction mixture with ethyl acetate, collecting the combined organic layers, and separately using 10% HCl solution, saturated NaHCO ₃ The solution was washed with water, then dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give the objective compound 12.

And 5: the substrate (6-bromoindole or 6-bromoindazole, 1 eq.) was dissolved in anhydrous DMF and the resulting mixture was cooled to 0 ℃. Add 60% nah (1.5 eq.) in portions and stir the resulting mixture at room temperature for 30 minutes. The reaction was then cooled to 0 ℃, 4-methylbenzyl chloride (1.6 eq.) was added, followed by stirring at room temperature overnight. The reaction was monitored by TLC and after the starting material had reacted completely, a large amount of H was added ₂ O, the reaction mixture was extracted with ethyl acetate. The combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate C2.

Step 6: to 6-bromo-1H-indazole (1 eq.), 3,4, 5-trimethoxybromobenzene (1 eq.), N' -dimethylethylenediamine (2.5 eq.), and K ₃ PO ₄ (2.2 eq.) to a solution of CuI in anhydrous DMF was added (2.5 eq.) and the mixture was heated at 80 ℃ for 20 h. The reaction was monitored by TLC and after the reaction of the starting materials was complete, a large amount of H was added ₂ O, the reaction mixture was extracted with ethyl acetate. The combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate M16.

And 7: to a solution of 5-bromo-2-methylphenol (1) and benzyl chloride (1.5 eq.) in DMF was added potassium carbonate (3 eq.) and the reaction was mixed with stirring at room temperature for 4 hours. The reaction was monitored by TLC and after the reaction of the starting materials was complete, a large amount of H was added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography to give intermediate M24-1.

And step 8: dissolving pinacol diboron (1.1 eq.) and catalyst [1,1' -bis (diphenylphosphino) ferrocene ] in argon protection]To an anhydrous 1, 4-dioxane solution of palladium dichloride (0.05 eq.) and potassium acetate (4.4 eq.) was added intermediate M24-1 (1 eq.) and the reaction was stirred at 110 ℃ overnight. The reaction was monitored by TLC,after the raw materials are completely reacted, a large amount of H is added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography to give intermediate M24-2.

And step 9: intermediate E2 (1 eq.) was dissolved in the appropriate amount of MeOH/THF (1. The reaction was monitored by TLC, after the starting material had reacted completely, DCM was added to dilute the system, palladium on carbon was removed by filtration through celite, the filtrate was collected and the solvent was removed in vacuo. The residue was purified by column chromatography to give compounds 24-29.

Step 10: stannous chloride dihydrate (5 eq.) and a few drops of concentrated hydrochloric acid were added to an ethanol solution in which intermediate I or J (1 eq.) was dissolved, and the reaction system was allowed to react overnight at reflux temperature. The reaction was monitored by TLC and after the starting material had reacted completely, an appropriate amount of saturated NaHCO was added ₃ After the pH of the solution is adjusted to neutral (pH 7), a large amount of H is added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography to give compounds 30-39.

Step 11: at an ice bath temperature, zinc powder (4 eq.) and glacial acetic acid (4 eq.) were added to a mixed solution of ethanol and water in which intermediate I or J (1 eq.) was dissolved, and the reaction system was reacted at reflux temperature overnight. The reaction was monitored by TLC and after the starting material had reacted completely, an appropriate amount of saturated NaHCO was added ₃ After the pH of the solution adjusting system is adjusted to be neutral (pH is approximately equal to 7), a large amount of H is added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography to give compound 30-39.

Example 1:

preparation of Compound 1

Step 1: 200mg of 2-chloro-5-bromopyridine (1 eq.) was weighed together with 4-methylphenylboronic acid (1.1 eq.), tetrakis (triphenylphosphine) palladium (0.05 eq.) and the corresponding base (Na) ₂ CO ₃ 2 eq.) was dissolved in a mixed solvent of water and THF and heated at reflux temperature overnight. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The volatile solvent in the system was removed in vacuo, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate M1.

Step 2: palladium acetate (0.04 eq.) and BINAP (0.04 eq.) were dissolved in anhydrous toluene (or 1, 4-dioxane) and the system was stirred under argon atmosphere at room temperature for 10 minutes. Then the intermediate M1 (1 eq.), 3,4, 5-trimethoxyaniline (1.2 eq.) and the corresponding base (Cs) were added ₂ CO ₃ 3 eq.) the system was heated to reflux under argon overnight. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The reaction mixture was extracted with ethyl acetate, and the combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed in vacuo. The residue was purified by column chromatography to give the objective compound 1.

Reddish brown solid, yield 61%; m.p.155.1-157.7 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.44(d,J＝2.3Hz,1H),7.72(dd,J＝8.6,2.4Hz,1H),7.43(d,J＝8.0Hz,2H),7.25(d,J＝8.3Hz,2H),6.90(d,J＝8.6Hz,1H),6.63(s,3H),3.85(d,J＝6.7Hz,9H),2.39(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ155.35,153.72,146.41,136.86,136.47,136.22,135.17,129.67,126.13,108.18,98.82,61.0,56.16,21.09.

example 2:

preparation of Compound 2

Step 1: weighing 200mg of 5-bromo-2-chloropyrimidine (1 eq.) and 4-methylphenylboronic acid (1.1 eq.), tetrakis (triphenylphosphine) palladium (0.05 eq.) and the corresponding base (Na) ₂ CO ₃ 2 eq.) was dissolved in a mixed solvent of water and THF and heated at reflux temperature overnight. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. The volatile solvent in the system was removed in vacuo, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate M2.

Step 2: to a solution of intermediate M2 (1 eq.) and 3,4, 5-trimethoxyaniline (1.2 eq.) in EtOH and H ₂ Concentrated hydrochloric acid (4 eq.) was slowly added dropwise to the mixed solvent of O, and the system was allowed to stand at reflux temperature overnight. As the reaction proceeds, solids are continuously precipitated from the system. Monitoring the reaction by TLC, cooling to room temperature after the raw materials are completely reacted, directly filtering to obtain a solid, alternately washing the solid with a cooled ethanol solution and DCM, and drying to obtain a target compound 2, namely a white solid with a yield of 75%; m.p.183.6-185.7 deg.c; ¹ H NMR(400MHz,CDCl ₃ )δ8.65(s,2H),7.46(s,1H),7.41(d,J＝8.1Hz,2H),7.30–7.26(m,2H),6.96(s,2H),3.89(s,6H),3.84(s,3H),2.40(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ155.92,153.41,129.93,125.89,97.53,56.14,21.13.

example 3:

preparation of Compound 3

The preparation method is referred to example 2 except that 3,4, 5-trimethoxyaniline is replaced with 3,4, 5-trimethoxybenzylamine. White solid, yield 42%; m.p.152.0-154.4 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.52(s,2H),7.37(d,J＝8.1Hz,2H),7.25(d,J＝6.4Hz,2H),6.61(s,2H),5.68(t,J＝5.5Hz,1H),4.63(d,J＝5.9Hz,2H),3.84(d,J＝7.2Hz,9H),2.39(s,3H),1.71(s,2H)； ¹³ C NMR(101MHz,CDCl ₃ )δ161.44,156.10,153.41,134.79,132.48,129.85,125.71,124.12,104.45,60.82,56.10,45.92,21.11.

example 4:

preparation of Compound 4

Preparation method reference is made to example 1, with the difference that 2-chloro-5-bromopyridine is replaced by 3-bromo-2-chloropyridine. Yellow solid, yield 52%; m.p.109.8-112.0 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.21(dd,J＝5.0,1.9Hz,1H),7.40(dd,J＝7.3,1.9Hz,1H),7.34(q,J＝8.1Hz,4H),6.82(dd,J＝7.3,5.0Hz,1H),6.79(s,2H),6.44(s,1H),3.84(s,6H),3.80(s,3H),2.44(s,3H)； ¹³ CNMR(101MHz,CDCl ₃ )δ153.26,152.78,146.53,138.10,137.94,136.62,134.38,130.09,128.92,123.70,115.01,97.57,60.94,56.08,21.26.

example 5:

preparation of Compound 5

Preparation method reference is made to example 1, with the difference that 2-chloro-5-bromopyridine is replaced by 2-bromo-3-chloropyridine. Dark brown solid, yield 55%; m.p.142.3-144.6 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.24(dd,J＝4.6,1.3Hz,1H),7.58(d,J＝8.2Hz,3H),7.29(d,J＝7.9Hz,2H),7.13(dd,J＝8.3,4.6Hz,1H),6.29(s,2H),5.70(s,1H),3.82(s,3H),3.80(s,6H),2.41(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ153.97,148.16,141.40,138.48,138.17,133.63,129.64,128.63,123.52,122.59,97.46,61.01,56.11,21.34.

example 6:

preparation of Compound 6

Preparation method referring to example 2, except that 5-bromo-2-chloropyrimidine was replaced with 2, 4-dichloropyrimidine. Yellow solid, yield 70%；m.p.176.6-178.2℃； ¹ H NMR(400MHz,CDCl ₃ )δ9.73(s,1H),8.32(d,J＝6.1Hz,1H),8.05(d,J＝8.3Hz,2H),7.35(d,J＝8.1Hz,2H),7.25(d,J＝6.1Hz,1H),7.04(s,2H),3.90(s,6H),3.86(s,3H),2.46(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ153.34,135.12,132.97,131.77,130.09,128.07,106.61,98.73,61.04,56.20,21.70.

Example 7:

preparation of Compound 7

The preparation is as described in example 1, except that 2-chloro-5-bromopyridine is replaced by 2-bromo-6-chloropyridine. Black-cyan solid, yield 80%; m.p.174.7-176.0 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ7.92(d,J＝8.2Hz,2H),7.55(t,J＝7.9Hz,1H),7.25(d,J＝8.8Hz,3H),7.18(d,J＝7.5Hz,1H),6.81(s,2H),6.69(d,J＝8.2Hz,1H),6.52(s,1H),3.86(d,J＝11.7Hz,9H),2.40(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ155.67,153.55,139.00,138.58,136.61,133.71,129.32,126.68,111.16,107.16,98.12,61.03,56.11,29.70,21.28.

example 8:

preparation of Compound 8

The preparation is as described in example 1, except that 2-chloro-5-bromopyridine is replaced by 2-bromo-4-chloropyridine. Dark brown solid, yield 75%; m.p.97.5-100.7 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.37(d,J＝5.6Hz,1H),7.83–7.77(m,2H),7.25(d,J＝9.0Hz,3H),7.18(d,J＝2.2Hz,1H),6.73(dd,J＝5.7,2.3Hz,1H),6.47(s,2H),6.06(s,1H),3.87(s,3H),3.84(s,6H),2.39(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ158.39,153.83,151.80,150.09,138.81,136.84,135.81,134.81,129.35,126.71,107.91,106.15,99.88,92.65,61.02,56.14,21.24.

example 9:

preparation of Compound 9

Preparation method reference was made to example 2, with the difference that 5-bromo-2-chloropyrimidine was replaced by 4, 6-dichloropyrimidine. Yellow solid, yield 63%; m.p.193.8-197.4 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ10.58(s,1H),8.65(s,1H),7.78(d,J＝8.2Hz,2H),7.45(s,1H),7.16(d,J＝8.0Hz,2H),6.99(s,2H),3.86(s,3H),3.84(s,6H),2.29(s,3H).

example 10:

preparation of Compound 10

Preparation method reference is made to example 1, with the difference that 2-chloro-5-bromopyridine is replaced by 4-bromo-2-chloropyridine. White solid, yield 93%; m.p.164.1-167.7 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.22(d,J＝5.3Hz,1H),7.47(d,J＝8.1Hz,2H),7.26(d,J＝7.9Hz,2H),7.02(s,1H),6.96(dd,J＝5.3,1.5Hz,1H),6.65(d,J＝2.5Hz,3H),3.85(d,J＝2.1Hz,9H),2.40(s,3H).； ¹³ CNMR(101MHz,CDCl ₃ )δ156.96,153.70,148.64,139.01,136.53,129.69,126.69,113.37,105.73,99.00,61.03,56.16,21.21.

example 11:

preparation of Compound 11

The preparation is as described in example 1, except that 2-chloro-5-bromopyridine is replaced by 3-bromo-5-chloropyridine. White solid, yield 80%; m.p.163.0-165.8 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.43(d,J＝2.7Hz,1H),7.82(d,J＝8.1Hz,2H),7.58(d,J＝8.6Hz,1H),7.41(dd,J＝8.6,2.8Hz,1H),7.24(d,J＝8.1Hz,2H),6.32(s,2H),6.05–5.96(m,1H),3.82(s,3H),3.78(s,6H),2.38(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ153.95,149.66,139.37,138.79,138.34,137.95,136.38,133.26,129.45,126.01,124.26,120.41,96.44,61.06,56.08,21.21.

example 12:

preparation of Compound 12

Intermediate M12 was prepared according to step 1 of example 1, except that 2-chloro-5-bromopyridine was replaced with 5-bromo-1, 3, 4-thiadiazole. To a solution of intermediate M12 (1 eq.) in anhydrous DCM was added 3,4,5-trimethoxybenzoic acid (1.2 eq.), EDCI (1.2 eq.) and HOBt (1.2 eq.) and the mixture was heated under reflux for 8-10 hours. The reaction was monitored by TLC and after the starting material had reacted completely, it was cooled to room temperature. Removing DCM from the system in vacuo, extracting the reaction mixture with ethyl acetate, collecting the combined organic layers, and separately using 10% HCl solution, saturated NaHCO ₃ The solution was washed with water, then dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give the title compound 12 as a white solid in 63% yield; m.p.245.2-247.2 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ13.07(d,J＝4.5Hz,1H),7.91–7.81(m,2H),7.53(s,2H),7.36(d,J＝8.0Hz,2H),3.89(s,6H),3.76(s,3H),2.45–2.35(m,3H)； ¹³ C NMR(101MHz,DMSO-d ₆ )δ152.74,129.87,126.81,126.20,105.96,60.13,56.12,20.95.

example 13:

preparation of Compound 13

The substrate 6-bromoindazole (1 eq.) was dissolved in anhydrous DMF and the resulting mixture was cooled to 0 ℃. Add 60% NaH (1.5 eq.) in portions and stir the resulting mixture at room temperature for 30 minutes. The reaction was then cooled to 0 ℃, 4-methylbenzyl chloride (1.6 eq.) was added, followed by stirring at room temperatureOvernight. The reaction was monitored by TLC and after the starting material had reacted completely, a large amount of H was added ₂ O, the reaction mixture was extracted with ethyl acetate. The combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate M13. Title compound 13 was obtained following the procedure of example 1, step 2, except that intermediate M1 was replaced with intermediate M13. Compound 13 was a yellow oily liquid with a yield of 47%; ¹ H NMR(400MHz,CDCl ₃ )δ7.75(s,1H),7.50(d,J＝8.9Hz,1H),7.18(q,J＝8.1Hz,4H),6.84–6.78(m,1H),6.38(s,2H),5.93(s,1H),5.69(s,1H),5.48(s,2H),3.82(s,3H),3.81(s,6H),2.34(s,3H).

example 14:

preparation of Compound 14

Preparation method reference is made to example 13, except that 6-bromoindazole is replaced with 6-bromoindole. Dark brown oily liquid, yield 43%; ¹ H NMR(400MHz,CDCl ₃ )δ7.54(d,J＝8.4Hz,1H),7.11–7.03(m,4H),6.96(d,J＝8.0Hz,2H),6.85(dd,J＝8.4,1.8Hz,1H),6.49(d,J＝3.1Hz,1H),6.21(s,2H),5.60(s,1H),5.20(s,2H),3.78(s,3H),3.66(s,6H),2.30(s,3H)； ¹³ CNMR(101MHz,CDCl ₃ )δ153.85,141.29,137.90,137.21,137.05,134.59,131.54,129.44,127.87,126.51,124.28,121.68,114.33,101.61,100.23,93.91,61.11,55.85,49.75,21.11.

example 15:

preparation of Compound 15

Preparation method reference example 13. White solid, yield 52%; m.p.119.0-121.0 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ7.90(d,J＝0.9Hz,1H),7.57(d,J＝8.6Hz,1H),7.08–6.99(m,4H),6.92(s,1H),6.79(dd,J＝8.6,1.8Hz,1H),6.31(s,2H),5.89(s,1H),5.42(s,2H),3.82(s,3H),3.69(s,6H),2.28(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ153.83,142.94,140.79,138.78,133.97,133.20,129.32,126.88,122.02,119.28,114.48,96.66,94.25,61.02,55.91,52.45,21.03.

example 16:

preparation of Compound 16

Step 1: to 6-bromo-1H-indazole (1 eq.), 3,4, 5-trimethoxybromobenzene (1 eq.), N' -dimethylethylenediamine (2.5 eq.), and K ₃ PO ₄ (2.2 eq.) to a solution of CuI in anhydrous DMF was added (2.5 eq.) and the mixture was heated at 80 ℃ for 20 h. The reaction was monitored by TLC and after the starting material had reacted completely, a large amount of H was added ₂ O, the reaction mixture was extracted with ethyl acetate. The combined organic layers were collected, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by column chromatography to give intermediate M16.

Step 2: compound 16 was obtained according to example 1, step 2, except that intermediate M1 was replaced with M16. Compound 16 was a white solid, yield 57%; m.p.106.8-108.0 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.19(s,1H),7.88–7.81(m,2H),7.54(d,J＝8.1Hz,2H),7.48(dd,J＝8.4,1.3Hz,1H),7.32–7.24(m,2H),6.96(s,2H),3.92(d,J＝1.8Hz,9H),2.41(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ153.81,140.79,139.63,137.53,137.01,135.93,135.02,129.65,127.41,121.80,121.49,108.17,101.04,61.05,56.39,21.11.

example 17:

preparation of Compound 17

Intermediate M17 was obtained following example step 1, except that 4-methylphenylboronic acid was replaced with phenylboronic acid and 2-chloro-5-bromopyridine was replaced with 2-bromo-6-chloropyridine. Obtained by the method of example 1, step 2The target compound 17. White solid, yield 51%; m.p.128.7-131.3 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.06–7.99(m,2H),7.57(t,J＝7.9Hz,1H),7.49–7.35(m,3H),7.20(d,J＝7.5Hz,1H),6.81(s,2H),6.73(t,J＝9.8Hz,2H),3.86(s,6H),3.84(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ155.78,155.55,153.55,139.10,138.58,136.62,133.73,128.95,128.59,126.81,111.46,107.53,98.13,61.04,56.11.

example 18:

preparation of Compound 18

Preparation method reference is made to example 17, with the difference that 2-bromo-6-chloropyridine is replaced by 5-bromo-2-chloropyridine. Brown solid, yield 47%; m.p.146.9-148.5 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.44(d,J＝2.1Hz,1H),7.75(dd,J＝8.7,2.5Hz,1H),7.53(dt,J＝8.1,1.7Hz,2H),7.45(t,J＝7.7Hz,2H),7.37–7.32(m,1H),6.92(d,J＝8.7Hz,1H),6.77(s,1H),6.64(s,2H),3.87(s,6H),3.85(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ155.19,153.76,144.96,137.58,137.20,135.73,134.55,129.05,128.01,127.30,126.26,108.61,99.27,61.04,56.18.

example 19:

preparation of Compound 19

Preparation method referring to example 17, 2-bromo-6-chloropyridine was replaced with 4-bromo-2-chloropyridine. White solid, yield 48%; m.p.134.5-137.8 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.17(d,J＝5.3Hz,1H),7.53–7.45(m,2H),7.42–7.29(m,3H),6.96(s,1H),6.89(dd,J＝5.3,1.5Hz,2H),6.58(s,2H),3.77(s,9H)； ¹³ C NMR(101MHz,CDCl ₃ )δ157.05,153.72,150.35,148.67,138.73,136.51,134.22,128.98,126.86,113.47,106.03,99.07,61.01,56.16,29.70.

example 20:

preparation of Compound 20

Preparation method referring to example 17, 2-bromo-6-chloropyridine was replaced with 2-bromo-4-chloropyridine. Brown solid, yield 46%; m.p.43.5-48.3 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.35(d,J＝5.7Hz,1H),7.87(d,J＝7.7Hz,2H),7.40(dt,J＝12.1,6.6Hz,3H),7.20(d,J＝2.1Hz,1H),6.77(dd,J＝5.7,2.2Hz,2H),6.44(s,2H),3.86(s,3H),3.83–3.78(m,6H).

example 21:

preparation of Compound 21

Preparation method referring to example 17, 2-bromo-6-chloropyridine was replaced with 3-bromo-5-chloropyridine. White solid, yield 43%; m.p.157.6-159.2 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.36(dd,J＝4.4,2.1Hz,2H),7.54(dt,J＝3.9,1.9Hz,3H),7.49–7.37(m,3H),6.40(s,2H),5.98(s,1H),3.84(s,3H),3.82(s,6H)； ¹³ C NMR(101MHz,CDCl ₃ )δ153.98,140.90,137.73,137.56,137.34,133.71,129.08,128.30,127.07,121.57,97.30,61.07,56.13.

example 22:

preparation of Compound 22

Preparation method referring to example 2, except that 4-methylphenylboronic acid was replaced with phenylboronic acid and 5-bromo-2-chloropyrimidine was replaced with 4, 6-dichloropyrimidine. White solid, yield 63%; m.p.175.1-179.1 deg.C; ¹ HNMR(400MHz,CDCl ₃ )δ8.78–8.72(m,1H),8.14(s,1H),7.97–7.90(m,2H),7.49–7.39(m,3H),7.09(s,1H),6.69(s,2H),3.88(s,3H),3.86(s,6H)； ¹³ C NMR(101MHz,CDCl ₃ )δ163.51,162.04,158.66,153.76,143.19,137.34,135.19,134.44,130.42,128.82,126.84,100.35,92.55,61.07,61.02,56.19,55.84.

example 23:

preparation of Compound 23

Preparation method referring to example 22, 4, 6-dichloropyrimidine was replaced with 2, 4-dichloropyrimidine. White solid, yield 67%; m.p.131.7-1337.7 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.47(d,J＝5.2Hz,1H),8.10(dd,J＝7.4,2.3Hz,2H),7.54–7.44(m,3H),7.28(s,2H),7.17(d,J＝5.2Hz,1H),7.07(s,2H),3.91(s,6H),3.84(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ164.85,160.26,158.62,153.30,137.13,135.88,133.35,130.89,128.79,127.06,108.34,96.95,61.05,56.08.

example 24:

preparation of Compound 24

Step 1: to a solution of 5-bromo-2-methylphenol (1 eq.) and benzyl chloride (1.5 eq.) in DMF was added potassium carbonate (3 eq.) and the reaction was stirred and mixed at room temperature for 4 hours. The reaction was monitored by TLC and after the reaction of the starting materials was complete, a large amount of H was added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography to give intermediate M24-1.

Step 2: dissolving pinacol diboron (1.1 eq.) and catalyst [1,1' -bis (diphenylphosphino) ferrocene ] in argon atmosphere]To a solution of palladium dichloride (0.05 eq.) and potassium acetate (4.4 eq.) in anhydrous 1, 4-dioxane was added intermediate M24-1 (1 eq.) and the reaction was stirred at 110 ℃ overnight. The reaction was monitored by TLC and the starting material was allowed to react to completionThereafter, a large amount of H is added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, then dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The residue was purified by column chromatography to give intermediate M24-2.

And 3, step 3: reference example 7 gave intermediate M24-3, except that 4-methylphenylboronic acid was replaced with intermediate M24-2.

And 4, step 4: intermediate M24-3 (1 eq.) was dissolved in the appropriate amount of MeOH/THF (1. The reaction was monitored by TLC, after the starting material had reacted completely, DCM was added to dilute the system, palladium on carbon was removed by filtration through celite, the filtrate was collected and the solvent was removed in vacuo. The residue was purified by column chromatography to give compound 24, a brown solid in 67% yield; m.p.117.2-120.5 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ7.56–7.50(m,2H),7.43(dd,J＝7.8,1.6Hz,1H),7.17(d,J＝7.9Hz,1H),7.12(d,J＝7.4Hz,1H),6.74(s,2H),6.70(d,J＝8.1Hz,1H),6.61(s,1H),3.85(s,6H),3.84(s,3H),2.28(s,3H).

example 25:

preparation of Compound 25

Preparation method referring to example 6, intermediate M25-3 was obtained except that 4-methylphenylboronic acid was replaced with intermediate M24-2, and referring to example 24, step 4, compound 25 was prepared except that M24-3 was replaced with intermediate M25-3. White solid, yield 73%; m.p.154.7-157.9 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.91(s,1H),9.56(s,1H),8.48(s,1H),8.26(s,1H),7.07(d,J＝7.6Hz,1H),6.79(d,J＝1.8Hz,1H),6.69(d,J＝7.5Hz,1H),6.61(s,2H),3.78(s,6H),3.65(s,3H),2.10(s,3H).

example 26:

preparation of Compound 26

Preparation method referring to example 24, except that 2-bromo-6-chloropyridine was replaced with 2-bromo-4-chloropyridine. White solid in powder, yield 76%; m.p.99.7-101.2 deg.c; ¹ H NMR(400MHz,CDCl ₃ )δ8.23(d,J＝5.8Hz,1H),7.54–7.48(m,1H),7.11–7.01(m,2H),6.91(d,J＝2.2Hz,1H),6.69(dd,J＝5.8,2.3Hz,1H),6.55(s,1H),6.39(s,2H),3.85(s,3H),3.76(s,6H),2.18(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ158.50,155.42,153.78,152.11,149.27,138.08,135.56,134.74,131.07,126.08,118.17,114.30,107.72,107.06,99.72,61.03,56.08,15.99.

example 27:

preparation of Compound 27

Preparation method referring to example 24, except that 2-bromo-6-chloropyridine was replaced with 3-bromo-5-chloropyridine. White solid, yield 78%; m.p.214.2-217.0 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.53(s,1H),8.37(s,1H),8.29(d,J＝2.5Hz,1H),8.21(d,J＝1.8Hz,1H),7.57–7.50(m,1H),7.16(d,J＝7.7Hz,1H),7.03–6.97(m,2H),6.45(s,2H),3.76(s,6H),3.63(s,3H),2.15(s,3H)； ¹³ C NMR(101MHz,DMSO-d6)δ156.33,153.91,141.08,138.74,138.34,137.64,136.39,132.50,131.74,124.54,119.52,117.68,113.03,96.35,60.61,56.20,16.19.

example 28:

preparation of Compound 28

Preparation method referring to example 24, except that 2-bromo-6-chloropyridine was replaced with 4-bromo-2-chloropyridine. A tan solid in 73% yield; m.p.199.0-202.6 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.61(s,1H),9.05(s,1H),8.18(d,J＝5.3Hz,1H),7.19(d,J＝7.8Hz,1H),7.09(d,J＝6.1Hz,3H),7.03(dd,J＝7.7,1.7Hz,1H),6.98(s,1H),6.92(dd,J＝5.4,1.5Hz,1H),3.78(s,6H),3.63(s,3H),2.17(s,3H)； ¹³ C NMR(101MHz,DMSO-d6)δ156.62,155.85,152.75,148.37,147.87,137.90,136.50,131.67,131.21,125.07,116.95,112.29,111.91,107.11,96.15,60.11,55.67,15.75.

example 29:

preparation of Compound 29

The preparation process is referred to example 25, with the difference that 2, 4-dichloropyrimidine is replaced by 4, 6-dichloropyrimidine. White solid, yield 68%; m.p.172.2-173.7 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.74(s,2H),8.66(d,J＝0.9Hz,1H),7.54(d,J＝1.6Hz,1H),7.36(dd,J＝7.8,1.6Hz,1H),7.19(d,J＝7.8Hz,2H),7.11(s,2H),3.79(s,6H),3.64(s,3H),2.18(s,3H)； ¹³ C NMR(101MHz,DMSO-d6)δ161.52,161.47,158.57,156.24,153.26,136.47,136.01,133.40,131.40,127.10,117.30,112.80,101.63,98.34,60.61,56.26,16.42.

example 30:

preparation of Compound 30

Step 1: preparation method referring to example 1, except that 4-methylphenylboronic acid is replaced with 3-nitro-4-methylphenylboronic acid, intermediate M30 is obtained.

And 2, step: stannous chloride dihydrate (5 eq.) and a few drops of concentrated hydrochloric acid were added to the ethanol solution in which intermediate M30 (1 eq.) was dissolved, and the reaction system was allowed to react overnight at reflux temperature. The reaction was monitored by TLC and after the reaction of the starting material was complete, an appropriate amount of saturated NaHCO was added ₃ After the pH of the solution is adjusted to neutral (pH 7), a large amount of H is added ₂ O, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic layers were collected, washed 3 times with saturated brine, dried over anhydrous magnesium sulfate, and filteredThe solvent was filtered and removed in vacuo. The residue was purified by column chromatography to give compound 30. Brown solid, yield 86%; m.p.154.6-159 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.41(d,J＝2.0Hz,1H),7.69(dd,J＝8.6,2.4Hz,1H),7.11(d,J＝7.7Hz,1H),6.90–6.82(m,3H),6.67(s,1H),6.62(s,2H),3.85(s,6H),3.84(s,3H),3.70(s,2H),2.20(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ155.25,153.71,146.09,145.04,136.38,131.05,128.32,121.39,116.61,112.60,108.17,98.82,61.02,56.15,17.06.

example 31:

preparation of Compound 31

Preparation method reference example 16, step 1, gave intermediate M31, except that 4-methylphenylboronic acid was replaced with 3-nitro-4-methylphenylboronic acid. Referring to step 2 of example 30 again, compound 30 was obtained as a reddish brown oily liquid in a yield of 46%; ¹ H NMR(400MHz,CDCl ₃ )δ8.19(d,J＝0.8Hz,1H),7.86–7.79(m,2H),7.46(dd,J＝8.4,1.4Hz,1H),7.15(d,J＝7.7Hz,1H),6.95(s,3H),6.34(d,J＝3.9Hz,1H),6.19(s,1H),3.92(s,9H),2.23(s,3H).

example 32:

preparation of Compound 32

Preparation method referring to example 30, the difference is that 2-chloro-5-bromopyridine is replaced with 2-bromo-6-chloropyridine. Grey solid, yield 72%; m.p.160.9-162.4 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ7.48(t,J＝7.9Hz,1H),7.39–7.31(m,2H),7.09(dd,J＝13.6,7.6Hz,2H),7.05(s,1H),6.75(s,2H),6.67(d,J＝8.2Hz,1H),3.82(s,3H),3.78(s,6H),3.64(s,2H),2.16(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ155.83,153.43,144.94,138.27,137.25,133.10,130.65,123.31,117.10,113.21,111.19,107.34,97.60,61.05,56.02,17.25.

example 33:

preparation of Compound 33

Preparation method referring to example 30, the difference is that 2-chloro-5-bromopyridine is replaced with 4-bromo-2-chloropyridine. Orange solid, yield 44%; m.p.137.5-139.6 deg.C; ¹ H NMR(400MHz,CDCl ₃ )δ8.19(d,J＝5.3Hz,1H),7.37(s,1H),7.09(d,J＝7.8Hz,1H),7.00(s,1H),6.90(dq,J＝5.4,1.6Hz,2H),6.84(d,J＝1.7Hz,1H),6.65(s,2H),3.84(s,3H),3.81(s,6H),2.17(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ157.08,153.64,150.48,148.42,145.15,137.48,136.91,133.82,131.01,123.29,116.93,113.29,112.99,105.94,98.75,61.03,56.10,17.20.

example 34:

preparation of Compound 34

Preparation method referring to example 30, the difference is that 2-chloro-5-bromopyridine is replaced with 4, 6-dichloropyrimidine. White solid, yield 40%; m.p.153.7-155.6 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.71(d,J＝1.0Hz,1H),7.71(s,1H),7.34(d,J＝1.6Hz,1H),7.20(dd,J＝7.8,1.7Hz,1H),7.11(d,J＝7.8Hz,1H),7.03(d,J＝1.0Hz,1H),6.65(s,2H),3.88(s,3H),3.86(s,6H),2.20(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ163.81,161.95,158.57,153.82,145.08,136.09,135.49,134.19,130.84,125.06,116.89,113.11,100.72,99.42,61.03,56.24,29.70,17.34.

example 35:

preparation of Compound 35

Preparation method referring to example 30, zoneExcept that 2-chloro-5-bromopyridine was replaced with 2, 4-dichloropyrimidine. Yellow solid, yield 78%; m.p.196.3-197.8 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.80(s,1H),8.59(d,J＝5.3Hz,1H),8.20(d,J＝1.5Hz,1H),8.09(dd,J＝8.0,1.6Hz,1H),7.53(d,J＝8.2Hz,1H),7.33(d,J＝5.3Hz,1H),7.25(s,2H),3.79(s,6H),3.64(s,3H),2.45(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ164.55,157.93,141.29,140.92,140.25,137.74,137.23,137.14,131.46,126.65,112.85,102.25,65.35,60.95,22.40.

example 36:

preparation of Compound 36

The preparation process is referred to example 31 with the difference that 3,4, 5-trimethoxyaniline is replaced by 3,4, 5-trifluoroaniline. White solid, yield 64%; m.p.123.5-126.2 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ8.22(d,J＝0.9Hz,1H),7.86–7.83(m,2H),7.53–7.46(m,3H),7.19(d,J＝7.5Hz,1H),7.03–6.97(m,2H),3.84(s,2H),2.27(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ145.02,142.04,139.89,139.16,136.48,131.06,124.73,122.57,122.25,121.66,117.98,113.92,107.77,106.98,106.91,106.81,106.73,17.12.

example 37:

preparation of Compound 37

The preparation process is referred to example 35, except that 3,4,5-trimethoxyaniline is replaced by aniline. White solid, yield 76%; m.p.168.2-170.1 deg.c; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.60(s,1H),8.48(d,J＝5.2Hz,1H),7.86(dt,J＝8.8,1.6Hz,2H),7.41(d,J＝1.8Hz,1H),7.36–7.29(m,2H),7.27(dd,J＝7.7,1.8Hz,1H),7.20(d,J＝5.3Hz,1H),7.09(d,J＝8.2Hz,1H),6.95(tt,J＝7.5,1.1Hz,1H),5.08(s,2H),2.13(s,3H)； ¹³ C NMR(101MHz,DMSO-d6)δ164.97,160.56,158.94,147.41,141.21,135.54,130.83,128.99,124.85,121.57,119.16,115.35,112.45,108.09,17.95.

example 38:

preparation of Compound 38

The preparation process is referred to example 33, with the difference that 3,4, 5-trimethoxyaniline is replaced by aniline. Brown solid, yield 73%; m.p.116.3-119.4 deg.C; ¹ H NMR(400MHz,CDCl ₃ )δ8.26(dd,J＝5.3,0.7Hz,1H),7.43–7.35(m,5H),7.14(d,J＝7.7Hz,1H),7.11(dd,J＝1.5,0.8Hz,1H),7.11–7.06(m,1H),6.98–6.94(m,2H),6.89(d,J＝1.8Hz,1H),3.74(s,2H),2.23(s,3H)； ¹³ C NMR(101MHz,CDCl ₃ )δ156.69,150.59,148.53,145.10,140.81,137.66,131.02,129.40,123.28,122.73,120.47,117.16,113.61,113.14,105.80,17.24.

example 39:

preparation of Compound 39

The preparation process is referred to in example 35, except that 3,4, 5-trimethoxyaniline is replaced with 3,4, 5-trifluoroaniline. White solid, yield 78%; m.p.217.6-221.0 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.02(s,1H),8.55(d,J＝5.3Hz,1H),7.81(dd,J＝11.2,6.5Hz,2H),7.36(d,J＝1.6Hz,1H),7.30(d,J＝5.3Hz,1H),7.25(dd,J＝7.6,1.7Hz,1H),7.10(d,J＝7.9Hz,1H),5.08(s,2H),2.13(s,3H)； ¹³ C NMR(101MHz,DMSO-d6)δ165.33,159.94,158.99,147.51,135.25,130.90,125.11,115.32,112.49,109.36,102.79,102.54,17.94.

example 40:

SRB method and CCK-8 method are adopted to test the anti-tumor proliferation inhibition activity of the target compound. Wherein A549 (lung cancer cell), hela (cervical cancer cell), MCF-7 (breast cancer cell), MGC-803 (gastric cancer cell), and HepG2 (liver cancer cell) are determined by SRB method, and U937 (lymphoma cell, leukemia cell) is determined by CCK-8 method.

1. Preparation of Experimental solution

Preparation of a culture medium: 500mL LRPMI-1640 (or DMEM) was supplemented with 50mL of special grade BI serum and 10mL of a mixed solution of streptomycin (100X) to prepare a medium containing 10% serum and 1% double antibody.

Preparation of 10 × PBS: separately weighing Na ₂ HPO ₄ ·12H ₂ O 17.907g，KH ₂ PO ₄ 1.2248g, naCl40.03345g, KCl 1.00845g, adding ultrapure water to make the volume to 500mL, and adjusting the pH value to 7.2-7.6. Diluted with ultrapure water to 1X when used. PBS used in cell culture needs 120 ℃, is sterilized under high pressure for 20min and is used after being placed at normal temperature.

Preparation of 20 × TBS: respectively weighing 24.23g of Tris and 80.06g of NaCl, adding pure water for dissolving, fixing the volume to 500mL, and adjusting the pH value to 7.6 by HCl. And storing in a refrigerator at 4 ℃. Diluting with pure water to 1 ×.

TBST:500mL of 1 × TBS plus 1mL of 25% Tween 20.

5 × preparation of catholyte: 30.275g of Tris, 44.8g of tricine and 2.5g of SDS are weighed respectively, and are dissolved by adding pure water, and the volume is determined to be 500mL. And storing in a refrigerator at 4 ℃. When used, diluted to 1 x with purified water.

5 × preparation of anolyte: and adding pure water to dissolve 60.55g of Tris, metering to 500mL, and adjusting the pH value to 8.9.

And storing in a refrigerator at 4 ℃. When used, diluted to 1 x with pure water.

10 × preparation of wet conversion solution: tris 15.2g, glycine 71.3g. Adding pure water to dissolve, and fixing the volume to 500mL. When in use, after being diluted to 1X, 100mL of anhydrous methanol is added into every 400mL of the solution, and the solution is used after precooling in a refrigerator at 4 ℃.

preparing a pH meter electrode protection solution: weighing 22.35g of KCl in a clean beaker, adding a proper amount of ultrapure water to dissolve completely, pouring into a 500mL volumetric flask to fix the volume to obtain the product, and storing in a refrigerator at 4 ℃ for later use.

Ponceau dye liquor: weighing 2.5g of ponceau powder and a proper amount of ultrapure water solution in a reagent bottle, adding 2.5mL of glacial acetic acid, uniformly mixing, pouring the solution into a 500mL volumetric flask, adding ultrapure water to a constant volume, and transferring into the reagent bottle again for room temperature storage for later use.

Preparation of 5% milk: weigh 2g milk into 40ml of LTBST and shake up on a shaker.

Preparation of primary-antibody secondary-antibody rapid eluent: 5g of SDS and 1.875g of glycine were weighed out and the pH was adjusted to 2.0 with HCl.

Preparation of 10% Ammonium Persulfate (AP): 1gAP was dissolved in 1mL of purified water.

10% preparation of TCA: 50g of trichloroacetic acid is weighed, ultrapure water is added to the solution until the volume is reduced to 500mL, and the solution is preserved at 4 ℃.

0.4% preparation of SRB: weighing 2g of SRB, sucking 5mL of glacial acetic acid by a pipette, adding ultrapure water to the volume of 500mL, and preserving at 4 ℃.

Preparation of 10mM Tris: weighing 0.6057g of Tris, diluting with ultrapure water to 500mL, adjusting the pH value to 10.5, and standing at room temperature.

2. Antitumor cell proliferation assay

(1) Cell culture

Human breast cancer MCF-7, human gastric cancer cell MGC-803, human cervical cancer cell HELA, human liver cancer cell HepG-2 are all cultured in DMEM, human lung cancer cell A549 and human leukemia cell U937 are all cultured in RPMI 1640. The culture medium used for the experiment contains 10% fetal bovine serum and 1% penicillin two antibody, and the cells are treated at 37 deg.C with 5% CO ₂ Culturing under the culture condition, replacing fresh culture medium every two days, and transferring generation every three days. Cells in logarithmic growth phase were taken for experiments.

(2) Cell passage

And (5) carrying out cell passage when the cell density is about 80%. For adherent cells such as MCF-7, the supernatant is discarded, PBS is used to wash off residual culture medium, appropriate amount of pancreatin is added, digestion is carried out at 37 deg.C for 1-2min, the cell state is observed under microscope, and fresh culture medium is added to stop digestion after cell rounding. The cell suspension was collected in a sterile ep tube and centrifuged at 800-900r for 4-5min. Discard the supernatant, add fresh medium, adjust cell density, 1/3 or 1/4 passage. For suspension cells, the cell suspension was collected in a sterile ep tube and centrifuged at 800-900r for 4-5min. The supernatant was discarded, and fresh medium was added to adjust the cell density for 1/3 or 1/4 passage.

(3) Cell resuscitation

Firstly, opening a water bath kettle in a cell chamber to 37 ℃, taking out cells from a liquid nitrogen tank, quickly putting the cells into the water bath kettle at 37 ℃ for quick melting, centrifuging for 4min at 900r, discarding the supernatant, adding a fresh culture medium for re-suspension, transferring the mixture into a sterile culture dish for culture, changing the culture solution on the next day, transferring for 2-3 generations, stabilizing the cell state, and carrying out experiments.

(4) Cell cryopreservation

Collecting cells in logarithmic growth phase, and freezing the cells according to the cell suspension: serum: DMSO =7:2, or 6. Placing in a refrigerator of-80 deg.C, and storing in a liquid nitrogen tank the next day.

(5) Cell proliferation inhibition activity assay

Determination of adherent cell proliferation inhibition Activity

Cells in logarithmic growth phase were collected, counted and, according to the formula: c ₁ V ₁ ＝C ₂ V ₂ Calculating the cell suspension amount required by each 96-well plate, adding 100 microliters of cell suspension into each 96-well plate, adhering to the wall overnight, removing the upper culture medium, adding culture media containing different drugs and different concentration gradients, culturing for 72h, and testing IC by an SRB method ₅₀ The value is obtained.

Suspension cell proliferation inhibition activity assay

Cells in logarithmic growth phase were collected, counted and according to the formula: c ₁ V ₁ ＝C ₂ V ₂ Calculating the cell suspension amount required by each 96-well plate, adding 50 microliters of cell suspension into each well, preparing target compound solution with 2 times of different concentration gradients, adding 50 microliters of medicine-containing RPMI-1640 into each well, culturing for 72h, and measuring IC by CCK-8 method ₅₀ The value is obtained.

SRB method

MCF-7, hepG2, MGC-803, hela, A549 cells in logarithmic growth phase were seeded in 96-well plates at 3000-4000/well at 37 ℃ with 5% CO ₂ Incubating the incubator overnight to allow the incubator to adhere to the wall, removing the upper layer culture medium, adding the medicine-containing culture medium with different concentration gradients, culturing for 72h, removing the upper layer culture solution, gently adding 100 μ L10% (w/v) trichloroacetic acid into each hole, fixing at 4 deg.C for 30min, removing, washing the residue with distilled waterDrying the reagent at 65 deg.C, adding 0.4% SRB of 100 μ L per well, shaking and dyeing at room temperature for 20min, discarding the dye solution, washing the excess dye solution with newly prepared 1% glacial acetic acid, and drying at 65 deg.C. The dye was dissolved by applying 150. Mu.L of 10mM unbuffered Trisbase plate per well for 5min with shaking, and the absorbance value (Optical density, OD) was measured at a wavelength of 560nm with a microplate reader. Inhibition (%) = (OD) _{Control well} -OD _{Medicine feeding hole} )/OD _{Control well} X 100%, and the concentration of the compound at which the inhibition rate was 50% was calculated. The experiment was repeated three times and the data are presented as means and standard deviations.

Cell Counting Kit (CCK-8) method

U937 cells in logarithmic growth phase were collected, seeded in 96-well plates at 8000/well, and drug-containing medium and cell suspension were added to the wells together. Three wells were taken and a blank medium was added as a blank group. At 37 ℃,5% CO ₂ Incubate for 72h. Adding 10 mu L CCK-8 dye solution into each hole, continuously incubating for 3-4 h, measuring the absorbance value under the wavelength of 450nm by an enzyme-linked immunosorbent assay, and preferably controlling the OD value of a drug-free control group to be 0.6-1.0. Formula for calculating proliferation inhibition rate: inhibition (%) = OD _{Control well} -OD _{Medicine feeding hole} )/(OD _{Control well} -OD _{Blank hole} )×100％。

Median Inhibitory Concentration (IC) ₅₀ ) Defined as the drug concentration when 50% of the tumor cells survived. A standard curve of the cell growth inhibition rate was prepared from the measured optical density (OD value), and the drug concentration corresponding to the standard curve was determined.

Measured IC ₅₀ See table 1.

TABLE 1

The experimental results were averaged for 3 parallel experiments.

From the above experiments, it can be seen that: the invention has obvious inhibiting effect on A549 (lung cancer cell), hela (cervical cancer cell), MCF-7 (breast cancer cell), MGC-803 (stomach cancer cell), hepG2 (liver cancer cell) and U937 (lymphoma cell and leukemia cell). When the Ar ring is a six-membered ring, the activity is higher when the rings on the left side and the right side are substituted by meta-positions; when the Ar ring is an indazole ring, the activity is higher after the 1-position is substituted, and the activity is improved to a certain extent by introducing methyl and amino into the substituent of the left ring; higher activity when the right ring is substituted by trimethoxy.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A compound of formula (I), racemates, stereoisomers, tautomers, isotopic labels, solvates, polymorphs, or pharmaceutically acceptable salts thereof:

2. A compound of claim 1, wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Identical or different, independently of one another, from H, halogen, NH ₂ 、OH、C _1-12 Alkyl radical, C _1-12 An alkoxy group;

ar is selected from 5-14 membered heteroaryl, C, arylene, unsubstituted or optionally substituted with one, two or more Ra _1-12 Alkyl-5-14 membered heteroaryl;

l is selected from NH, C (O) sub-C optionally substituted by one, two or more Rb _1-12 alkyl-NH, C _1-12 alkyl-NH-C _1-12 An alkyl group;

each Ra, rb is the same or different and is independently selected from H, OH, NH ₂ Halogen, C _1-12 Alkyl radical, C _1-12 Alkoxy radical, C _3-12 Cycloalkyl, 3-12 membered heterocyclyl, C _6-14 Aryl, 5-14 membered heteroaryl.

3. A compound according to claim 1 or 2, wherein R is ₁ 、R ₂ 、R ₃ Identical or different, independently of one another, from H, halogen, C _1-8 An alkoxy group;

R ₄ 、R ₅ identical or different, independently of one another, from H, halogen, NH ₂ 、OH、C _1-8 Alkyl radical, C _1-8 An alkoxy group;

ar is selected from 5-10 membered heteroaryl, C _1-8 Alkyl-5-10 membered heteroaryl;

l is selected from NH, C (O) O, sub-C _1-8 alkyl-NH, C _1-8 alkyl-NH-C _1-8 An alkyl group.

4. A compound according to any one of claims 1 to 3, wherein R is ₁ 、R ₂ 、R ₃ Identical or different, independently of one another, from H, F, cl, br, I, methoxy;

R ₄ 、R ₅ identical or different, independently of one another, from H, F, cl, br, I, NH ₂ OH, methyl, ethyl, propyl, methoxy, ethoxy;

ar is selected from the group consisting of pyridinylene, pyrimidinylene, pyrazinylene, pyridazinylene, indolyl, indazolylene, thiazolyl, thiadiazolylene, methylene-indolyl, methylene-indazolyl;

l is selected from NH, C (O) NH, methylene-NH.

5. The compound of any one of claims 1-4, wherein the compound of formula (I) is selected from the following structures:

6. a process for the preparation of a compound according to any one of claims 1 to 5, comprising the steps of:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Ar, L independently have the definitions as defined in any one of claims 1 to 5, X is selected from halogen or NH ₂ ；

preferably, the reaction is carried out under the action of a base, which is an inorganic base such as at least one of sodium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, potassium phosphate, or an organic base; the organic base is at least one of triethylamine, N-diisopropylethylamine and N, N-dimethylaminopyridine;

preferably, the reaction is carried out under the action of a catalyst; the catalyst is a palladium catalyst, such as palladium acetate; when the catalyst is a palladium catalyst, an organic ligand, such as (+/-) -2,2 '-bis- (diphenylphosphino) -1,1' -binaphthyl, is also added into the reaction system.

7. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound of any one of claims 1-5, racemates, stereoisomers, tautomers, isotopic labels, solvates, polymorphs thereof, or pharmaceutically acceptable salts thereof.

8. Use of at least one compound of any one of claims 1 to 5, racemates, stereoisomers, tautomers, isotopic labels, solvates, polymorphs thereof, or pharmaceutically acceptable salts thereof, for the manufacture of a medicament.

9. Use according to claim 8, wherein the medicament is an antineoplastic medicament, such as a tubulin inhibitor and/or a VEGFR-2 inhibitor.

10. The use according to claim 9, wherein the tumor is a leukemia cell, a breast cancer cell, a liver cancer cell, a lung cancer cell, a stomach cancer cell, a cervical cancer cell, a lymphoma cell; preferably U937 cells, MCF7 breast cancer cells, hepG2 liver cancer cells, A549 lung cancer cells, MGC-803 stomach cancer cells and HeLa cervical cancer cells.