CN111072640A

CN111072640A - Quinazoline derivative and preparation method and application thereof

Info

Publication number: CN111072640A
Application number: CN201911367639.2A
Authority: CN
Inventors: 赵冬梅; 程卯生; 郭靖; 郝晨洲; 朱明月; 庞禹; 殷文博; 吴天啸
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-04-28

Abstract

The invention belongs to the technical field of medicines, and relates to a quinazoline derivative, and a preparation method and application thereof. Compounds of formula (I) and geometric isomers thereof or pharmaceutically acceptable salts, hydrates, solvates thereof and methods for their preparation are provided. Wherein R is₁、R₂、R₃、R₄、R₅As described in the claims and specification. The compound can quickly release proto-type medicines under the action of in vitro and in vivo hydrolytic enzymes, the proto-type medicines have protein kinase inhibitory activity, particularly have inhibitory action on members of PAKs kinase family, and can be used for preparing PAK protein kinase inhibitors.

Description

Quinazoline derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of drug synthesis, relates to quinazoline derivatives, and a preparation method and application thereof, and particularly relates to (R) - (4- ((1H-pyrazol-3-yl) amino) quinazoline-2-yl) - (N-carbamate piperazine-1-yl) -ketone derivatives, pharmaceutically acceptable salts, hydrates, solvates or prodrugs of the derivatives, a preparation method of the derivatives, and application of the derivatives as a therapeutic agent, particularly as a PAK (platelet activating kinase) inhibitor.

Background

The treatment of tumor has become a worldwide problem, and the research and development of high-efficiency and low-toxicity anti-tumor drugs are imminent. The targeted antitumor drug has the characteristics of good specificity, strong effectiveness, low toxic and side effects and the like, and has great success in tumor treatment.

Protein kinases are the largest family of human gene-encoded proteins and are closely associated with tumor development, invasion, metastasis, angiogenesis and chemotherapy resistance. Because part of the kinases are only highly expressed in tumor cells, the inhibition of the kinases does not affect the biological function of normal cells, so that the antitumor drug taking the protein kinases as targets has the advantages of high selectivity and low toxicity. Therefore, protein kinases have become important targets for the development of antitumor drugs.

2001 Bcr-Abl kinase inhibitor imatinib

Is approved by FDA to treat chronic granulocytic leukemia and becomes the first protein kinase inhibitor antineoplastic drug on the market. In the next fifteen short years, 42 protein kinase inhibitors have been marketed, and only 8 FDA-approved small molecule kinase inhibitors for tumor therapy have been marketed since 2013. The research on protein kinase inhibitors with antitumor activity has received a great deal of attention from the biopharmaceutical industry.

PAKs (p 21-activated kinases) are a class of serine/threonine protein kinases belonging to the STE20 family. PAK4 is representative of class II PAKs, and can affect a plurality of downstream proteins related to cell cycle, migration, invasion and apoptosis, thereby causing abnormal differentiation, angiogenesis, proliferation and the like of tumor cells. Therefore, PAK4 is a potential tumor therapy target.

PAKs include six family members (PAK1-PAK6) and are divided into two classes according to their structure and mode of activation: class I PAKs (PAK1, PAK2, PAK3), class II PAKs (PAK4, PAK5, PAK 6). Among these, PAK4 is the most studied of PAKs in class II.

The full length of the PAK4(p21 activated kinase 4) kinase includes 591 amino acid residues, which are divided into three major domains: the protein Kinase comprises a P21 binding Domain (P21-binding Domain, PBD), a self-inhibition Domain (AID) and a Kinase Domain (KD) or a Catalytic Domain (Catalytic Domain), wherein the P21 binding Domain and the self-inhibition Domain are positioned at the N-end of the Kinase, and the Kinase Domain is positioned at the conserved C-end of the Kinase. There is significant structural diversity between class I PAKs and class II PAKs, e.g., the region of class II PAK that does not contain acidic amino acid residues and the PIX/Cool binding domain; the homology of the kinase domains of two subfamilies is only about 50%, the structural difference is large, and the homology in the same subfamily is respectively as high as 92-96% and 79-86%, and the structures are relatively similar.

PAK is a main target protein of guanosine triphosphatase (Rho-GTPases) Cdc42 and Rac1 in the Rho family, and regulates and controls various biological functions such as cytoskeleton recombination, cell migration movement, apoptosis, mitosis, cell differentiation and the like through participating in multiple signal paths in cells. Besides being involved in regulating normal physiological activities of cells, PAK is closely related to the occurrence and development of various diseases, especially tumors. In an RAS-Cdc42/Rac1-PAK signal channel in a cell, Cdc42/Rac1 respectively control the generation of cell filopodia and lamellipodia, and PAK is used as a downstream main effector and plays an important role in the processes of malignant transformation of cells and invasion and metastasis of tumor cells.

The research shows that the PAK kinase, particularly PAK1 and PAK4, has the phenomena of gene amplification, over-expression and abnormal activation in various tumor cells, thereby causing canceration and uncontrollable proliferation, invasion and metastasis of the cells. For example, PAK1 is highly expressed in cell lines of breast, kidney, colon, etc.; in a squamous skin carcinoma tumor-bearing murine model, PAK1 deletion will significantly attenuate tumorigenic development by down-regulating the MAPK and PI3K pathways; PAK1 can also phosphorylate BAD protein, and inhibit apoptosis of tumor cells; phosphorylated DLC1(Dynein light chain 1), promoting cell survival and malignant phenotypes; mediates the expression of mammary epithelial cell cyclinD1, and promotes the generation and development of breast cancer. On the other hand, more than 70% of various human cancer cell lines highly express PAK4, including breast cancer, pancreatic cancer, colon cancer, lung cancer, ovarian cancer and the like, the activation of PAK4 can lead to the anchorage-independent growth of tumor cells, and the PAK4 inactivated mutant can inhibit malignant transformation caused by Ras. The mechanism of upregulation of PAK4 is primarily gene amplification, particularly in pancreatic, ovarian, oral squamous cell, and breast cancers. PAK4 is expressed in a variety of solid tumors, the most prominent feature being its increased expression of mRNA or protein by increased transcription or gene amplification. However, the study also reported that genetic mutation of PAK4 (E329K) was associated with colon cancer.

Researches show that the protein Ras is overexpressed in about 30 percent of tumors, the tumors comprise malignant tumors such as pancreatic cancer, colon cancer and lung cancer, Ras activates Rho family small G protein Rac1/Cdc42 through PI-3k so as to activate PAKs, the activated PAKs further influence downstream cell signal pathway transduction and promote tumor survival, angiogenesis, migration invasion and the like, the researches show that PAK4 can activate a survival signal pathway of tumor cells through NF-kB and promote the survival of the tumor cells, PAK4 can promote the migration of gastric cancer cells through LIMK1-Cofilin signal pathway, PAK4 can also promote the generation of melanin through CREB and Wnt/β -catenin signal pathway and cause diseases such as pigmentation disorder and the like, and PAK4/Raf/MEK/ERK signal pathway exists in the liver cancer cells and can inhibit the proliferation of the liver cancer cells through PAK 4.

The PAK4 is closely related to the occurrence and development of various tumors, inhibits the abnormal function of PAK4, can effectively inhibit the invasion and metastasis and the hyperproliferation of tumor cells, and promotes the apoptosis of the tumor cells. Therefore, the research of the PAK4 inhibitor has important value.

Because of the close association of PAK with tumors, the current study of small molecule PAK inhibitors is in a rapidly progressing stage. Most of the PAK inhibitors found are ATP competitive PAK inhibitors acting on kinase domains, and the ATP competitive inhibitors have the characteristics of high affinity and definite action sites and are the most studied types of kinase inhibitors at present. However, the kinase domain of the kinase catalyzes the same biochemical reaction and has the conservation of structure and sequence, and the realization of the selectivity of the inhibitor among the kinases is a common problem encountered in the current research of ATP competitive inhibitors; meanwhile, the ATP binding cavity of PAK has the individual characteristics of large space and high flexibility, so that the discovery of the PAK subtype selective inhibitor is a remarkably challenging research. However, with the rapid development of related disciplines, more and more high-selectivity ATP competitive kinase inhibitors have been discovered and are the mainstream trend in the current kinase inhibitor research field.

At present, the research on inhibition of PAKs is still in the initial stage, and only PF-3758309 developed by the company Perey enters phase I clinical research. PF-3758309 is a PAKs inhibitor with pyrrolopyrazole structure reported in 2009 by the pfeiffer company, and is the only PAKs inhibitor entering clinical research, and the PAK4 IC thereof₅₀＝19nM，PAK1 IC₅₀14 nM. In an in vitro tumor inhibition experiment and an in vivo model study of tumor-bearing mice, PF-3758309 obviously inhibits tumor cell proliferation and promotes tumor cell apoptosis. However, phase I clinical studies of PF-3758309 were forced to terminate due to poor oral bioavailability (about 1%), adverse gastrointestinal effects, etc. The compound which is reported by gene tack company 2014 and has benzimidazole structure and selectively acts on class II PAKs can generate selective inhibition effect on PAK4 (PAK1 IC)₅₀＝5.4μM，PAK4 IC₅₀7.5 nM). Although such compounds still have the defects of weak cellular activity and poor druggability, the research preliminarily proves the possibility of discovering the subtype selective PAK inhibitor.

In conclusion, targeted antitumor drugs represented by protein kinase inhibitors have become the mainstream of research and development of antitumor drugs at home and abroad. PAK4 is a new target for tumor therapy due to its important role in tumor development, migration and invasion. At present, the research on PAK4 inhibitors is still in the initial stage, and the research and development of PAKs inhibitors with high activity and selectivity and capable of being orally taken are heavyIt has important meaning. The invention designs and synthesizes the compound with the structure shown in the general formula (I), and finds that the compound with the structure can release proto-type medicine under in-vivo and in-vitro conditions, and the proto-type medicine shows better PAK4 inhibition activity and has good PAK4/1 selectivity. The compound with the structure shown in the general formula (I) can be used for water interpretation under the in vivo condition to release proto-drug and CO under the action of carboxylesterase₂And nontoxic alcohols, and the like.

The invention content is as follows:

the invention aims to provide a (R) - (4- ((1H-pyrazol-3-yl) amino) quinazoline-2-yl) - (N-carbamate piperazine-1-yl) -ketone derivative shown as a general formula (I), a geometric isomer thereof or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof;

wherein:

R₁selected from hydrogen, C unsubstituted or substituted by halogen₁-C₆Alkyl, unsubstituted or halogen-substituted C₃-C₆Cycloalkyl radical, C₂-C₄Alkoxy radicals, by hydrogen, hydroxy, amino, C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halogen-substituted 5-to 10-membered heteroaryl, C₃-C₇Heterocycle C₁-C₆An alkyl group, said heteroaryl or heterocycloalkyl group containing 1 to 3 heteroatoms selected from O, N and S, respectively;

R₂、R₃selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), C₁-C₆Alkyl radical, C₁-C₆Alkoxy, hydroxy, halogenated C₁-C₆An alkyl group;

R₄selected from hydrogen, unsubstituted or halogen-substituted C₁-C₆Alkyl radical, C₃-C₆A cycloalkyl group;

R₅selected from hydrogen, 6-to 10-membered aryl, unsubstituted or halogen substituted C₁-C₆Alkyl, - (CR)₆R₇)_n-6-10 membered aryl, - (CR)₆R₇)_n-O-C(＝O)-R₈，-(CR₆R₇)_n-C(＝O)-O-R₈；

Wherein n is an integer of 0 or 1;

R₆、R₇selected from hydrogen, C₁-C₆Alkyl by nitro, hydroxy, amino, C₁-C₆Alkoxy, unsubstituted or halogen-substituted 5-to 10-membered aryl or heteroaryl;

R₈selected from hydrogen, C₁-C₆An alkyl group.

The invention preferably relates to (R) - (4- ((1H-pyrazol-3-yl) amino) quinazoline-2-yl) - (N-carbamate piperazine-1-yl) -ketone derivatives shown in a general formula (I), and geometrical isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof;

R₁selected from hydrogen, C unsubstituted or substituted by halogen₁-C₆Alkyl, unsubstituted or halogen-substituted C₃-C₆Cycloalkyl radical, C₂-C₄Alkoxy radicals, by hydrogen, hydroxy, amino, C₁-C₆Alkyl radical, C₁-C₆An alkoxy group;

R₅selected from hydrogen, unsubstituted or halogen-substituted C₁-C₆Alkyl, - (CR)₆R₇)_n-phenyl, - (CR)₆R₇)_n-O-C(＝O)-R₈，-(CR₆R₇)_n-C(＝O)-O-R₈；

Wherein n is an integer of 0 or 1;

R₆、R₇selected from hydrogen, C₁-C₆Alkyl by nitro, hydroxy, amino, C₁-C₆Alkyl radical, C₁-C₆Alkoxy, unsubstituted or halogen-substituted 5-to 10-membered aryl or heteroaryl;

R₈selected from hydrogen, C₁-C₆An alkyl group.

R₁selected from hydrogen, C unsubstituted or substituted by halogen₁-C₆Alkyl, unsubstituted or halogen-substituted C₃-C₆A cycloalkyl group,

R₅selected from hydrogen, C₁-C₆Alkyl, halogen substituted C₁-C₆Alkyl, - (CR)₆R₇)_n-phenyl, - (CR)₆R₇)_n-O-C(＝O)-R₈，-(CR₆R₇)_n-C(＝O)-O-R₈；

Wherein n is an integer of 0 or 1;

R₆、R₇selected from hydrogen, C₁-C₆Alkyl by nitro, hydroxy, amino, C₁-C₆Alkyl radical, C₁-C₆Alkoxy, unsubstituted or halogen-substituted phenyl;

R₈selected from hydrogen, C₁-C₆An alkyl group.

R₁selected from hydrogen, C unsubstituted or substituted by halogen₃-C₆A cycloalkyl group,

R₂、R₃selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

R₄selected from hydrogen, unsubstituted or halogen-substituted C₁-C₄An alkyl group;

R₅selected from hydrogen, C₁-C₆Alkyl, halogen substituted C₁-C₆Alkyl, - (CR)₆R₇)_n-O-C(＝O)-R₈，-(CR₆R₇)_n-C(＝O)-O-R₈A methyl phenyl group;

the derivatives of formula (I) may exist in different tautomeric forms, all of which are included within the scope of the present invention. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are mutually converted via a low energy barrier.

"halogen" in the present invention means fluoro, chloro, bromo or iodo; "alkyl" refers to straight or branched chain alkyl; "aryl" refers to an organic group derived from an aromatic hydrocarbon by removal of two hydrogen atoms at one or different positions, such as phenyl, naphthyl; "heteroaryl" refers to a monocyclic or polycyclic ring system containing one or more heteroatoms selected from N, O, S, which refers to an organic group having aromatic character and obtained by removing two hydrogen atoms at one or different positions in the ring system, such as thiazolyl, imidazolyl, pyridyl, pyrazolyl, (1,2,3) -and (1,2,4) -triazolyl, furyl, thienyl, pyrrolyl, indolyl, benzothiazolyl, oxazolyl, isoxazolyl, naphthyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, and the like; heterocyclyl means a monocyclic ring system containing one or more heteroatoms selected from N, O, S, such as piperazinyl, tetrahydropyrrolyl, morpholinyl, piperidinyl, tetrahydropyrazolidinyl, tetrahydroimidazolidinyl, thiazolidinyl, and the like.

The invention also discloses a pharmaceutical composition, which contains the derivative of the general formula (I) and pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof as active ingredients, and the pharmaceutical composition is mixed with a pharmaceutically acceptable carrier or excipient to prepare a composition and prepare a clinically acceptable dosage form, wherein the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier which can be used in the pharmaceutical field. The derivatives of the present invention may be used in combination with other active ingredients as long as they do not produce other adverse effects, such as allergic reactions.

The pharmaceutical composition of the present invention can be formulated into several dosage forms containing some excipients commonly used in the pharmaceutical field. The above-mentioned several dosage forms can adopt the dosage forms of injection, tablet, capsule, aerosol, suppository, membrane, dripping pill, external liniment and ointment, etc.

Carriers for the pharmaceutical compositions of the present invention are of the usual type available in the pharmaceutical art, including: binder, lubricant, disintegrating agent, cosolvent, diluent, stabilizer, suspending agent, pigment-free, correctant, antiseptic, solubilizer, matrix, etc. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), and if certain drugs are unstable under gastric conditions, they may be formulated as enteric coated tablets.

The derivatives of the invention comprising formula (I) may be synthesized by methods well known in the art including chemistry, particularly in accordance with the teachings of the present invention.

The starting materials are generally available from commercial sources such as the reagent companies avastin, dary, etc. or are prepared using methods well known to those skilled in the art.

The room temperature in the present invention means an ambient temperature of 10 to 30 ℃.

The examples and preparations provided below further illustrate and exemplify the compounds of the present invention and their methods of preparation. It should be understood that the scope of the following examples and preparations are not intended to limit the scope of the invention in any way. The compounds of formula I according to the invention can be prepared according to the methods of scheme 1, all the variables used in these schemes being as defined in the claims.

Synthesis scheme 1

R₁、R₂、R₃、R₄、R₅As claimed in claim.

As shown in scheme 1, the synthesis of compounds of formula I has several major steps:

step a: the intermediate I-2 is obtained by acylation reaction of the raw material I-1.

Step b: and (3) carrying out oxalyl chloride monoethyl ester acylation reaction on the intermediate I-2 to obtain an intermediate I-3.

Step c: the intermediate I-3 is reacted with sodium ethoxide for cyclization to obtain an intermediate I-4.

Step d: and carrying out alkaline hydrolysis reaction on the intermediate I-4 to obtain an intermediate I-5.

Step e: and performing chlorination reaction on the intermediate I-5 to obtain an intermediate I-6.

Step f: and carrying out selective acylation reaction on the intermediate I-6 and different substituted Boc protected piperazine fragments to obtain an intermediate I-7.

Step g: the intermediate I-7 is subjected to aromatic nucleophilic substitution reaction and reacts with 3 amino pyrazole ring segments with different substitutions at 5-position to obtain an intermediate I-8.

Step h: and removing the Boc protecting group from the intermediate I-8 to obtain an intermediate I-9.

Step i: the intermediate I-9 is reacted with a differently substituted chloroformate to give the final product I.

Preferred conditions are as follows:

in the step a, the reactants are dissolved in anhydrous tetrahydrofuran solvent, triphosgene is added, and the reaction is carried out for 12 to 14 hours under the condition of heating reflux. And cooling to room temperature, adding 1N ammonia water, reacting at 60-65 ℃ for 1-2 hours, cooling to room temperature, separating out a white solid, and performing suction filtration to obtain an intermediate I-2.

In step b, the reactant is dissolved in anhydrous tetrahydrofuran solvent, triethylamine is added at 0-5 ℃, and oxalyl chloride monoethyl ester is added dropwise with stirring. After the dropwise addition, the temperature is raised to room temperature for half an hour, and white solid is separated out. Adding a small amount of water, evaporating to remove tetrahydrofuran, adding ethyl acetate for extraction, and drying the organic layer to obtain an intermediate I-3.

In the step c, the reactant is dissolved in ethanol, sodium ethoxide is added at 0-5 ℃, the temperature is raised to room temperature after the addition is finished, the reaction lasts for 12-14 hours, the pH value is adjusted to be neutral by 2N hydrochloric acid, and the white solid is filtered by suction to obtain an intermediate I-4.

In the step d, the reactant is dissolved in an ethanol-water (1-1) mixed solvent, sodium hydroxide is added, heating reflux is carried out for 2-4 hours, 2N hydrochloric acid is added to adjust the pH value to be acidic, and the white solid is filtered by suction to obtain an intermediate I-5.

In the step e, the reactant is dissolved in chloroform, thionyl chloride is added, heating reflux is carried out for 3-5 hours, and the solvent and the thionyl chloride are evaporated to obtain an intermediate I-6.

In the step f, dissolving the reactant in anhydrous dichloromethane, cooling to-35 ℃, adding the corresponding piperazine fragment, stirring for 1-2 hours, adding water to quench the reaction, extracting with dichloromethane, drying the organic layer, and purifying by silica gel column chromatography to obtain an intermediate I-7.

In step g, the reaction is dissolved in DMF, the corresponding 3-aminopyrazole fragment and potassium iodide are added, the temperature is raised to 65-70 ℃, the mixture is stirred for 10-12 hours and poured into water, and the precipitate is purified by silica gel column chromatography to obtain an intermediate I-8.

In step h, the reaction mass is dissolved in absolute ethanol, trifluoroacetic acid is added, stirring is carried out for 10-12 hours at room temperature, the solvent is evaporated, and the residue is purified by silica gel column chromatography to obtain intermediate I-9.

In the step i, dissolving the reactant in dry tetrahydrofuran, adding 1N sodium hydroxide aqueous solution at room temperature, stirring for 30-45min, reserving the sample, cooling to-20 ℃, slowly dropwise adding different substituted chloroformates, adding water for quenching after 5-10min, extracting with dichloromethane, washing with saturated saline solution, drying with anhydrous sodium sulfate, evaporating to remove the solvent, and carrying out column chromatography to obtain the target compound.

The positive progress effects of the invention are as follows: the invention provides (R) - (4- ((1H-pyrazol-3-yl) amino) quinazoline-2-yl) - (N-carbamate piperazine-1-yl) -ketone derivatives which are completely different from the prior art, and a preparation method, a pharmaceutical composition and application thereof. The compound contained in the invention can release proto-drugs in vivo and in vitro, and the proto-drugs have good selective inhibition effect on PAK4 kinase, and can be used for preventing, treating or adjunctively treating various diseases related to the expression or activity of PAK4 kinase.

The specific implementation mode is as follows:

without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The examples provided below are therefore intended to illustrate but not to limit the scope of the invention.

The starting materials are generally available from commercial sources or may be prepared using methods well known to those skilled in the art or in accordance with the methods described herein. The reagents used are, without particular reference, analytically or chemically pure.

Mass spectra used for compound structure confirmation were determined using an Agilent 1100 LC/MSD. The column chromatography purification product adopts silica gel of 100-200 meshes or 200-300 meshes produced by Qingdao ocean chemical plant.

EXAMPLE 1 preparation of methyl (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate

Step a 2-amino-5-chlorobenzamide (I-2)

2-amino-5-chlorobenzoic acid (I-1,5.0g,29.1mmol) was dissolved in 50mL of dry tetrahydrofuran, triphosgene (2.9g,9.9mmol) was added with stirring, and the temperature was raised to 65 ℃ for reaction for 16 h. TLC monitored the reaction complete. Cooling to room temperature, evaporating under reduced pressure to remove the solvent, adding 1N ammonia water (233mL), heating to 65 ℃ for reaction for 1h, cooling to 0 ℃ to precipitate a white solid, and monitoring the reaction completion by TLC. Cooling to room temperature, filtering, washing (10mL multiplied by 3) the filter cake, drying the filter cake to constant weight to obtain 4.23g of white powdery solid with 85% yield.

Step b ethyl 2- (2-carbamoyl-4-chlorophenyl) aminooxalate (I-3)

2-amino-5-chlorobenzamide (I-2, 3.0g, 17.6mmol) and triethylamine (2.9mL,21.1mmol) were dissolved in 110mL of dry tetrahydrofuran, oxalyl chloride monoethyl ester (2.1mL,19.4mmol) was added dropwise to the reaction mixture with stirring at 0 deg.C, the reaction was warmed to room temperature for 3h after the addition was complete, and the completion of the reaction was monitored by TLC. 300mL of water was added to dilute the mixture to precipitate a white solid. The filter cake was filtered, washed with water (10 mL. times.3) and dried to constant weight to give 4.46g of a white solid with a yield of 94%.

Step c 6-chloro-4-hydroxyquinazoline-2-carboxylic acid ethyl ester (I-4)

Ethyl 2- (2-carbamoyl-4-chloro-phenyl) aminooxalate (I-3,2.70g,10.0mmol) was dissolved in 54mL of ethanol, 8.2mL of 10% ethanolic sodium ethoxide solution (0.82g,12.0mmol) was slowly added dropwise to the solution with stirring at 0 deg.C, the reaction was continued for 3h, and the completion of the reaction was monitored by TLC. The pH was adjusted to 3-4 with 1N hydrochloric acid, a white solid was precipitated, filtered, washed with water (10mL × 3), and the filter cake was dried to a constant weight to obtain 2.17g of a white powdery solid with a yield of 86%.

Step d 6-chloro-4-hydroxyquinazoline-2-carboxylic acid (I-5)

Ethyl 6-chloro-4-hydroxyquinazoline-2-carboxylate (I-4,6.0g,23.8mmol) was dissolved in 80mL of ethanol, 80mL of 10% aqueous sodium hydroxide (7.8g,195mmol) was added to the solution with stirring at room temperature, the reaction was stirred at 78 ℃ for 1h, and the completion of the reaction was monitored by TLC. The pH was adjusted to 2 with 2N hydrochloric acid to precipitate a white solid, which was filtered, washed with water (10mL × 3), and the filter cake was dried to a constant weight to obtain 5.11g of an off-white powdery solid with a yield of 96%.

Step e 4, 6-dichloroquinazoline-2-carbonyl chloride (I-6)

6-chloro-4-hydroxyquinazoline-2-carboxylic acid (I-5,200mg,0.89mmol) was dissolved in 4mL of chloroform, thionyl chloride (0.77mL,10.7mmol) and two drops of N, N-dimethylformamide were added to the solution, the reaction was heated to reflux for 3h, and the reaction was monitored by TLC for completion. The temperature was reduced to room temperature, and the solvent was evaporated under reduced pressure to give an intermediate 4, 6-dichloroquinazoline-2-carbonyl chloride (233mg, quantitative yield), which was immediately subjected to the next reaction.

Step f tert-butyl- (R) -4- (4-chloro-6-chloroquinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate (I-7)

4, 6-dichloroquinazoline-2-carbonyl chloride (I-6,233mg,0.89mmol) was dissolved in 10mL of dry methylene chloride and a solution of triethylamine (0.37mL,2.67mmol) and tert-butyl (R) -2-methylpiperazine-1-carboxylate in methylene chloride (166mg,0.89mmol) was slowly added dropwise to the solution with stirring at-35 ℃ for 0.5h and the reaction was continued and monitored by TLC for completion. Quenched with water (10mL), extracted with dichloromethane (3X 15mL), washed with water, washed with saturated brine and dried over sodium sulfate. The next day the solvent was evaporated and purified by silica gel chromatography (dichloromethane: methanol ═ 50:1) to give 238mg of a white solid in 65% yield.

Step g tert-butyl (R) -4- (4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) -6-chloroquinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate (I-8)

Tert-butyl- (R) -4- (4-chloro-6-chloroquinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate (I-7, 145mg,0.35mmol) was dissolved in 5mL of dry DMF and potassium iodide (67.4mg,0.41mmol), diisopropylethylamine (0.08mL,0.45mmol), 5-methyl-3-aminopyrazole (36.9mg,0.38mmol) were added in that order. Heating to 65 ℃ for reaction for 8h, cooling to room temperature, pouring into ice water (50mL), precipitating light yellow solid, performing suction filtration, drying the filter cake to constant weight, adding ether, stirring, performing suction filtration and drying to obtain a white-like solid 105mg, wherein the yield is 63%.

Step H (R) -6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazolin-2-yl) (3-methylpiperazin-1-yl) methanone hydrochloride (I-9)

Tert-butyl (R) -4- (4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) -6-chloroquinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate (I-8, 145mg,0.35mmol) was dissolved in dichloromethane (5mL), and a 2M ethyl hydrogen chloride solution was added dropwise with stirring. Stirring the reaction solution at a constant temperature for 12h, separating out a solid, performing suction filtration, and drying a filter cake to a constant weight to obtain a white solid.¹H NMR(600MHz,DMSO-d₆)δ9.95(s,2H),9.37(s,1H),8.32(d,J＝8.7Hz,1H),7.75(dd,J＝8.7,3.6Hz,1H),6.43(d,J＝3.7Hz,1H),4.44(t,J＝13.0Hz,1H),4.08(t,J＝13.6Hz,1H),3.61-3.39(m,3H),3.29–3.16(m,1H),3.09(s,1H),2.03(m,1H)1.36(d,J＝6.4Hz,2H),1.14(d,J＝6.2Hz,1H),1.00(d,J＝8.3Hz,2H),0.79(d,J＝2.4Hz,2H).HRMS(ESI,m/z)calcd forC₁₇H₁₉ClN₇O[M+H]⁺,372.1334；found 372.1333.R_f＝0.21(DCM/MeOH,5/1,v/v).Retentiontime:4.82min.

Step I (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid methyl ester (I)

Dissolving (R) -6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazolin-2-yl) (3-methylpiperazin-1-yl) methanone hydrochloride (I-9, 250mg and 0.56mmol) in dry tetrahydrofuran, adding a 1N sodium hydroxide aqueous solution (1.12mL and 1.12mmol) at room temperature, stirring for 30min, reserving a sample, cooling to-20 ℃, slowly dropwise adding methyl chloroformate (0.03mL and 0.33mmol), adding water after 5min for quenching, extracting with dichloromethane, washing with saturated common salt water, drying with anhydrous sodium sulfate, evaporating the solvent, and carrying out column chromatography to obtain the target compound with the yield of 76.2%.¹H NMR(600MHz,DMSO-d₆)δ12.34(d,J＝38.4Hz,1H),10.71(d,J＝48.7Hz,1H),8.88(d,J＝12.3Hz,1H),7.97–7.84(m,1H),7.78(dd,J＝13.9,8.9Hz,1H),6.47(s,1H),4.37(d,J＝11.3Hz,1H),4.30(d,J＝12.8Hz,1H),4.08(s,1H),3.88(d,J＝12.8Hz,1H),3.67(d,J＝11.0Hz,1H),3.61(d,J＝3.3Hz,3H),3.40(d,J＝9.8Hz,1H),3.34(s,4H),3.29(s,1H),3.14(dd,J＝18.6,7.3Hz,1H),3.08–2.99(m,2H),2.92(td,J＝12.7,3.7Hz,1H),1.94–1.87(m,1H),1.19(d,J＝6.7Hz,2H),1.03(d,J＝6.7Hz,1H),0.93(d,J＝8.2Hz,2H),0.70–0.59(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ166.08,158.10,158.06,156.79,156.57,154.88,148.03,147.88,133.78,130.99,129.91,129.82,129.67,122.81,115.14,115.07,94.63,67.27,67.19,52.51,52.47,45.61,44.71,40.06,20.02,15.03,7.74,7.69.HRMS(ESI):m/z Calcd.for C₂₂H₂₅ClN₇O₃[M+H]⁺470.1702,Found 470.1733.

Example 2 Ethyl (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate

Off-white solid, yield 76.8%.¹H NMR(600MHz,DMSO-d₆)δ12.33(d,J＝49.7Hz,1H),10.70(d,J＝50.8Hz,1H),8.87(d,J＝14.2Hz,1H),7.88(ddd,J＝7.1,4.9,2.2Hz,1H),7.78(dd,J＝13.8,8.9Hz,1H),6.47(s,1H),4.34(dd,J＝41.0,12.7Hz,2H),4.11–4.00(m,3H),3.89(d,J＝12.9Hz,1H),3.66(d,J＝9.7Hz,1H),3.40(d,J＝10.1Hz,1H),3.29(d,J＝2.6Hz,1H),3.13(t,J＝11.5Hz,1H),3.09–2.99(m,2H),2.92(td,J＝12.8,4.0Hz,1H),1.90(dtd,J＝13.5,8.4,4.2Hz,1H),1.18(dd,J＝12.6,6.7Hz,5H),1.03(d,J＝6.8Hz,1H),0.93(dd,J＝8.3,1.9Hz,2H),0.71–0.59(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ166.06,158.10,156.79,156.55,154.48,148.03,147.88,133.77,130.98,129.91,129.82,122.82,115.14,115.06,94.61,60.93,60.91,45.60,44.71,40.51,40.06,14.57,7.81,7.74,7.68,7.65.HRMS(ESI):m/z Calcd.for C₂₃H₂₇ClN₇O₃[M+H]⁺484.1858,Found 484.1879.

Example 3 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid isopropyl ester

Off-white solid, yield 78.4%.¹H NMR(600MHz,DMSO-d₆)δ12.34(d,J＝56.9Hz,1H),10.70(d,J＝58.1Hz,1H),8.88(d,J＝15.2Hz,1H),7.91–7.85(m,1H),7.78(dd,J＝13.5,8.9Hz,1H),6.47(d,J＝9.8Hz,1H),4.80(ddd,J＝9.3,8.4,4.2Hz,1H),4.39–4.28(m,2H),4.06(s,1H),3.87(d,J＝13.0Hz,1H),3.64(d,J＝9.8Hz,1H),3.39(d,J＝10.1Hz,1H),3.29(d,J＝2.4Hz,1H),3.08(dd,J＝7.3,4.7Hz,2H),3.05(d,J＝2.7Hz,1H),3.04–3.02(m,1H),3.00(d,J＝3.7Hz,1H),2.91(td,J＝12.8,4.0Hz,1H),1.97–1.85(m,1H),1.19(d,J＝2.3Hz,4H),1.18(s,4H),1.03(d,J＝6.8Hz,1H),0.93(t,J＝6.8Hz,2H),0.71–0.58(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ166.04,158.11,156.79,156.51,154.18,148.03,147.88,147.14,146.97,145.49,145.36,130.97,129.90,129.81,122.83,115.14,95.39,94.51,68.16,45.61,45.56,44.69,40.06,21.97,21.94,8.58,7.80,7.77,7.68,7.65.HRMS(ESI):m/z Calcd.for C₂₄H₂₉ClN₇O₃[M+H]⁺498.2015,Found 498.1946.

Example 4 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid tert-butyl ester

Off-white solid, yield 82.3%.¹H NMR(600MHz,DMSO-d₆)δ12.34(d,J＝63.3Hz,1H),10.70(d,J＝61.6Hz,1H),8.99–8.76(m,1H),7.91–7.84(m,1H),7.78(dd,J＝13.1,8.9Hz,1H),6.55–6.41(m,1H),4.32(dd,J＝34.1,12.7Hz,2H),4.03(s,1H),3.84(t,J＝15.3Hz,1H),3.60(d,J＝10.7Hz,1H),3.43–3.35(m,1H),3.27(s,1H),3.16–2.93(m,3H),2.93–2.86(m,1H),1.98–1.81(m,1H),1.40(d,J＝3.3Hz,12H),1.18(d,J＝6.6Hz,2H),1.01(d,J＝6.7Hz,1H),0.93(d,J＝8.2Hz,2H),0.73–0.57(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ166.99,166.00,158.13,156.48,153.78,148.03,147.87,133.75,131.71,131.63,130.96,129.90,128.68,122.81,115.13,94.41,79.18,67.81,45.61,44.71,40.06,36.39,32.99,31.57,28.01,22.06,19.33,14.23,13.91,7.80,7.68.HRMS(ESI):m/z Calcd.for C₂₅H₃₁ClN₇O₃[M+H]⁺512.2171,Found 512.2159.

Example 5 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid isobutyl ester

Off-white solid, yield 71.4%.¹H NMR(600MHz,DMSO-d₆)δ12.32(s,1H),10.71(d,J＝54.2Hz,1H),8.88(d,J＝16.0Hz,1H),7.88(ddd,J＝6.6,4.5,2.2Hz,1H),7.78(dd,J＝13.7,8.9Hz,1H),6.46(d,J＝15.0Hz,1H),4.34(dd,J＝37.4,12.6Hz,2H),4.12–4.05(m,1H),3.90(d,J＝12.9Hz,1H),3.80(qd,J＝10.3,5.3Hz,2H),3.67(d,J＝8.9Hz,1H),3.41(d,J＝9.7Hz,1H),3.30(s,1H),3.19–3.12(m,1H),3.08–3.00(m,2H),2.94(td,J＝12.8,3.9Hz,1H),1.88(ddt,J＝19.9,13.0,5.7Hz,2H),1.21(d,J＝2.7Hz,3H),0.95–0.91(m,2H),0.87(d,J＝6.5Hz,4H),0.67(ddd,J＝13.5,5.0,1.9Hz,1H),0.61(dd,J＝10.4,5.0Hz,1H).¹³C NMR(150MHz,DMSO-d₆)δ166.05,158.10,156.78,156.52,154.52,148.03,147.88,133.77,130.98,129.91,129.82,129.66,122.80,115.14,94.48,70.82,49.41,45.60,44.69,40.06,29.09,29.04,28.59,27.55,26.56,22.11,18.88,18.86,13.97,7.79,7.76,7.68,7.64.HRMS(ESI):m/z Calcd.for C₂₅H₃₁ClN₇O₃[M+H]⁺512.2171,Found 512.2135.

Example 6 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid 2,2, 2-trichloroethyl ester

Off-white solid, yield 77.2%.¹H NMR(600MHz,DMSO-d₆)δ12.33(d,J＝51.0Hz,1H),10.71(d,J＝58.9Hz,1H),8.89(d,J＝14.5Hz,1H),7.95–7.84(m,1H),7.78(dd,J＝14.7,8.9Hz,1H),6.48(d,J＝8.0Hz,1H),4.87(d,J＝37.7Hz,2H),4.50–4.28(m,2H),4.16(s,1H),3.96(d,J＝10.3Hz,1H),3.73(s,1H),3.48(d,J＝10.0Hz,1H),3.36(d,J＝6.9Hz,1H),3.26(s,1H),3.16–3.06(m,2H),3.00(td,J＝12.8,3.8Hz,1H),1.95–1.84(m,1H),1.27(s,2H),1.11(s,1H),0.93(t,J＝8.5Hz,2H),0.71–0.57(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ166.10,158.00,156.56,152.72,148.02,147.87,147.12,146.97,145.49,145.36,133.76,130.99,129.91,122.82,115.18,95.92,95.90,94.56,74.13,49.21,45.67,45.47,44.56,40.06,7.80,7.79,7.70.HRMS(ESI):m/z Calcd.for C₂₃H₂₄Cl₄N₇O₃[M+H]⁺586.0689,Found586.0639.

EXAMPLE 7 benzyl (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate

Off-white solid, yield 78.3%.¹H NMR(600MHz,DMSO-d₆)δ12.34(d,J＝48.4Hz,1H),10.70(d,J＝52.7Hz,1H),8.88(d,J＝13.1Hz,1H),7.93–7.84(m,1H),7.78(dd,J＝13.8,8.9Hz,1H),7.36(s,4H),7.32(d,J＝6.0Hz,1H),6.48(s,1H),5.24–4.96(m,2H),4.39(dd,J＝15.9,10.1Hz,1H),4.31(d,J＝12.8Hz,1H),4.19–4.09(m,1H),3.92(d,J＝12.8Hz,1H),3.71(d,J＝8.5Hz,1H),3.41(d,J＝8.6Hz,1H),3.17(s,1H),3.10–3.00(m,2H),2.98–2.88(m,2H),1.96–1.83(m,1H),1.20(d,J＝6.7Hz,3H),1.05(d,J＝6.7Hz,2H),0.97–0.87(m,2H),0.72–0.55(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ166.06,158.09,156.55,154.31,148.03,136.78,133.77,130.98,129.91,128.44,127.89,127.55,122.82,115.14,94.62,66.39,49.42,45.67,45.59,44.70,40.06,7.80,7.75,7.68.HRMS(ESI):m/z Calcd.forC₂₈H₂₉ClN₇O₃[M+H]⁺546.2015,Found 546.2007.

Example 8 n-pentyl (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate

Off-white solid, yield 69.9%.¹H NMR(600MHz,DMSO-d₆)δ12.35(d,J＝46.6Hz,1H),10.70(d,J＝50.8Hz,1H),8.88(d,J＝12.0Hz,1H),7.89(d,J＝6.9Hz,1H),7.78(dd,J＝13.9,8.9Hz,1H),6.47(d,J＝8.0Hz,1H),4.37(d,J＝12.0Hz,1H),4.30(d,J＝12.9Hz,1H),4.08(s,1H),3.88(d,J＝12.7Hz,1H),3.66(d,J＝9.4Hz,1H),3.61(d,J＝3.5Hz,3H),3.38(dd,J＝17.2,9.9Hz,2H),3.29(s,1H),3.01(d,J＝3.8Hz,1H),2.92(td,J＝12.9,4.0Hz,1H),1.96–1.85(m,1H),1.23(t,J＝7.2Hz,3H),1.03(d,J＝6.7Hz,1H),0.93(d,J＝8.0Hz,2H),0.71–0.59(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ166.05,158.11,156.78,156.52,154.53,148.03,147.88,133.76,130.97,129.91,129.66,122.81,115.14,115.07,94.53,64.94,49.41,45.60,44.69,40.06,28.18,27.59,26.56,21.78,13.87,7.79,7.75,7.67.HRMS(ESI):m/z Calcd.for C₂₆H₃₃ClN₇O₃[M+H]⁺526.2328,Found 526.2352.

Example 9 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid (isobutyryloxy) methyl ester

Off-white solid, yield 77.2%.¹H NMR(600MHz,DMSO-d₆)δ12.34(s,1H),10.71(d,J＝50.4Hz,1H),8.87(d,J＝13.9Hz,1H),7.93–7.86(m,1H),7.78(dd,J＝13.1,9.0Hz,1H),6.45(d,J＝14.0Hz,1H),5.72(t,J＝18.8Hz,2H),4.34(dd,J＝34.2,12.7Hz,2H),4.07(s,1H),3.84(d,J＝31.9Hz,1H),3.66(s,1H),3.47(d,J＝43.1Hz,1H),3.32(s,1H),3.18(s,1H),3.06(dd,J＝16.9,10.2Hz,2H),2.95(s,1H),2.61–2.53(m,1H),1.90(ddd,J＝16.9,8.6,4.0Hz,1H),1.21–1.18(m,2H),1.16(t,J＝7.3Hz,1H),1.08(d,J＝6.3Hz,5H),1.04(d,J＝6.6Hz,1H),0.96–0.89(m,2H),0.72–0.56(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ175.19,175.16,166.07,158.01,156.58,148.02,133.78,131.01,129.91,122.81,115.16,115.09,94.61,80.28,49.20,45.45,44.55,40.39,40.06,33.05,33.03,18.50,18.46,7.78,7.72,7.66.HRMS(ESI):m/z Calcd.for C₂₆H₃₁ClN₇O₅[M+H]⁺556.2070,Found 556.2075.

Example 10 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid (2-isobutyryloxy) ethyl ester

Off-white solid, yield 53.4%.¹H NMR(600MHz,DMSO-d₆)δ12.33(d,J＝50.0Hz,1H),10.71(d,J＝54.5Hz,1H),8.88(d,J＝15.0Hz,1H),7.88(ddd,J＝6.9,5.0,2.0Hz,1H),7.78(dd,J＝13.5,8.9Hz,1H),6.66(dt,J＝7.4,4.3Hz,1H),6.47(d,J＝11.5Hz,1H),4.35(dd,J＝38.1,12.9Hz,2H),4.05(s,1H),3.86(dd,J＝14.6,7.1Hz,1H),3.64(t,J＝9.0Hz,1H),3.43(d,J＝10.7Hz,1H),3.31(s,1H),3.20–3.12(m,1H),3.06(dd,J＝15.7,11.5Hz,2H),2.98–2.90(m,1H),2.52(s,1H),1.97–1.85(m,1H),1.42(d,J＝4.5Hz,3H),1.20(dd,J＝11.5,6.9Hz,3H),1.05(t,J＝11.6Hz,7H),0.93(d,J＝8.1Hz,2H),0.71–0.59(m,2H).¹³CNMR(150MHz,DMSO-d₆)δ174.50,174.49,166.06,158.02,156.56,152.35,148.02,147.87,133.78,131.01,129.92,122.82,115.16,89.57,89.54,89.48,49.25,45.44,44.55,40.39,40.06,33.09,33.07,19.57,19.55,18.59,18.58,18.39,7.79,7.76,7.72,7.66.HRMS(ESI):m/z Calcd.for C₂₇H₃₃ClN₇O₅[M+H]⁺570.2226,Found 570.2246.

Example 11 (R) -methyl-4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid (pivaloyloxy) methyl ester

Off-white solid, yield 54.6%.¹H NMR(600MHz,DMSO-d₆)δ12.33(d,J＝47.0Hz,1H),10.71(d,J＝53.7Hz,1H),8.88(d,J＝14.4Hz,1H),7.88(ddd,J＝7.2,5.0,2.1Hz,1H),7.78(dd,J＝13.4,8.9Hz,1H),6.47(d,J＝10.4Hz,1H),5.72(dd,J＝23.5,12.8Hz,2H),4.35(dd,J＝34.7,13.0Hz,2H),4.07(s,1H),3.86(s,1H),3.65(s,1H),3.44(d,J＝9.0Hz,1H),3.31(d,J＝3.7Hz,1H),3.18(t,J＝11.2Hz,1H),3.09–3.00(m,2H),1.94–1.85(m,1H),1.25–1.18(m,4H),1.13(s,8H),1.03(d,J＝6.8Hz,1H),0.92(d,J＝8.3Hz,2H),0.87–0.79(m,1H),0.75(s,1H),0.69–0.59(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ176.41,166.07,158.00,156.80,156.57,133.78,131.01,129.91,129.82,122.81,122.77,115.16,115.09,94.58,80.42,49.18,45.47,44.54,40.40,38.24,26.51,7.78,7.73,7.66.HRMS(ESI):m/zCalcd.for C₂₇H₃₃ClN₇O₅[M+H]⁺570.2226,Found 570.2220.

Example 12 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid (2-pivaloyloxy) ethyl ester

Off-white solid, yield 49.8%.¹H NMR(600MHz,DMSO-d₆)δ12.34(d,J＝49.5Hz,1H),10.71(d,J＝56.4Hz,1H),8.88(d,J＝15.9Hz,1H),7.90–7.86(m,1H),7.78(dd,J＝13.2,8.9Hz,1H),6.64(dd,J＝11.4,5.9Hz,1H),6.47(d,J＝14.1Hz,1H),4.36(dd,J＝38.0,12.9Hz,2H),4.05(s,1H),3.86(t,J＝12.6Hz,1H),3.64(t,J＝10.5Hz,1H),3.43(d,J＝10.8Hz,1H),3.31(s,1H),3.21–3.12(m,1H),3.09–2.97(m,2H),2.97–2.90(m,1H),1.96–1.86(m,1H),1.42(d,J＝4.3Hz,3H),1.23–1.17(m,3H),1.11(s,9H),1.03(d,J＝6.4Hz,1H),0.93(d,J＝8.2Hz,2H),0.70–0.59(m,2H).¹³C NMR(150MHz,DMSO-d₆)δ175.76,175.73,175.71,166.05,158.01,157.98,156.79,156.55,152.39,148.01,147.86,133.78,131.01,129.90,129.82,122.82,122.77,115.15,115.08,94.54,89.73,89.70,49.25,45.44,44.53,40.40,40.06,38.14,26.52,19.49,19.45,7.78,7.75,7.73,7.65.HRMS(ESI):m/zCalcd.for C₂₈H₃₅ClN₇O₅[M+H]⁺584.2383,Found 584.2414.

Example 13 phenyl (pivaloyloxy) methylethyl (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylate

White-like solid, yield 78.2%. HRMS (ESI) m/z Calcd₃₃H₃₆ClN₇O₅[M+H]⁺646.2545,Found 646.2551.

Example 14 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid-2- (pivaloyloxy) propyl-2-ethyl ester

White-like solid, yield 80.2%. HRMS (ESI) m/z Calcd₂₉H₃₆ClN₇O₅[M+H]⁺598.2545,Found 598.2550.

Example 15 (R) -4- (6-chloro-4- ((5-cyclopropyl-1H-pyrazol-3-yl) amino) quinazoline-2-carbonyl) -2-methylpiperazine-1-carboxylic acid 2-methyl-1- (pivaloyloxy) propylethyl ester

White-like solid, yield 67.5%. HRMS (ESI) m/z Calcd₃₀H₃₈ClN₇O₅[M+H]⁺612.2701,Found 612.2695.

TABLE 1 structural formulas of examples 1-15

Example 16: in vitro enzyme inhibitory Activity Studies of partial products of the invention

Experimental materials:

Tecan

f500 microplate reader.

KinEASE^TMSTK kit (containing biotinylated polypeptide substrates S2, Eu)³⁺Monoclonal antibodies labeled only for specific phosphorylation sites, Sa-XL665 labeled streptavidin, KinEASE enzyme reaction buffer), 384 shallow well plates, PAK4 full length protein. PAK4 protein concentration 0.0256 ng/. mu.l, MgCl₂Ethylenediaminetetraacetic acid (EDTA), Dithiothreitol (DL-Dithiothreitol, DTT), DMSO.

The experimental method comprises the following steps:

the first step is as follows: and (3) kinase reaction. The compound samples were first prepared as 20mM solutions in DMSO, and then diluted with a kinase reaction buffer solution to concentrations of 50. mu.M, 5.0. mu.M, 0.5. mu.M, etc., as required for the assay. PAK4 kinase (concentration 0.0256 ng/. mu.l), ATP (4. mu.M), biotin-labeled polypeptide substrate S2 (1. mu.M) and compound sample (4. mu.l) were then added to 10. mu.l kinase reaction buffer solution (containing MgCl₂5mM and DTT 1mM) and incubated at room temperature for 40 minutes, the kinase phosphorylates substrate S2. Then, 10. mu.l of an EDTA-containing detection reagent was added to detect the phosphorylated product。

The second step is that: detecting the phosphorylated product. Rare earth element europium (Eu)³⁺) The labelled antibody recognises the phosphorylated substrate and XL665 labelled streptavidin binds to the biotin on the substrate. Eu (Eu)³⁺Is a fluorescence donor, XL665 is a fluorescence acceptor, when Eu³⁺Close to XL665, Eu³⁺The energy is transferred to XL665, which generates the HTRF signal.

And (3) a result evaluation method: the fluorescent signal is formed by Eu³⁺620nm and XL665 nm. The ratio of the HTRF signal (665/620) for each well plate reaction was calculated.

TABLE 2 percentage of PAK4 inhibitory Activity of some example compounds at 50. mu.M, 5.0. mu.M, 0.5. mu.M concentrations in vitro

As shown in the table, the compound of the general formula (I) is a prodrug, has low inhibitory activity on PAK4 kinase, and only releases proto-type drugs under the action of in vitro and in vivo hydrolytic enzymes to inhibit the activity of PAK4 kinase.

Example 17: stability Studies of a portion of the products of the invention

Experimental materials:

1. preparation of phosphate buffer (pH7.4)

Taking 1.36g of monopotassium phosphate, adding 79mL of 0.1mol/L sodium hydroxide solution, and diluting with water to 200mL to obtain the potassium phosphate.

2. Preparation of simulated artificial gastric fluid (SGF pH 1.2)

Taking 16.4mL of dilute hydrochloric acid, adding 800mL of water and 10g of pepsin, shaking up, and adding water to dilute to 1000mL to obtain the compound; wherein the dilute hydrochloric acid is 234mL of concentrated hydrochloric acid, and water is added to dilute the concentrated hydrochloric acid to 1000mL to obtain 9.5-10.5% dilute hydrochloric acid.

3. Preparation of simulated artificial intestinal juice (SIF pH 6.8)

Taking 6.8g of monopotassium phosphate, adding 500mL of water for dissolving, and adjusting the pH value to 6.8 by using 0.1mol/L sodium hydroxide solution; dissolving pancreatin 10g in water, mixing the two solutions, and diluting to 1000 mL.

4. Stability testing in PBS (pH7.4), SGF (pH 1.2), SIF (pH 6.8)

The test compounds were prepared as 1mM stock solutions in DMSO, 1.0. mu.L of which was added to 499. mu.L of the prepared phosphate buffer, simulated artificial gastric fluid, and simulated artificial intestinal fluid, and after high-speed centrifugation of the mixture, the mixture was subjected to shaking table reaction at 37 ℃ for 2.0 hours. After the reaction was completed, 500. mu.L of ice-cold acetonitrile was added, vortexed, centrifuged at high speed, and the supernatant was taken to determine the Peak Area (PAR) of the remaining prodrug by HPLC. % remaining100 ═ PAR assigned time/PAR T0, where PAR ═ prototype peak area/internal standard peak.

5. Stability testing in rat plasma

The compounds to be tested were prepared as 1mM stock solutions in DMSO, from which 1.0. mu.L was added to 499. mu.L of freshly prepared rat plasma, and after high-speed centrifugation of the mixture, the mixture was subjected to a shaking reaction in a water bath at 37 ℃ for 2.0 h. After the reaction, 500. mu.L of ice-cold acetonitrile was added, vortexed, centrifuged at high speed, and the supernatant was taken, and the areas of the peaks of the Remaining prodrug and the prototype-producing drug were measured by HPLC, and the Remaining percentage (% Remaining) and the percentage of Conversion to the prototype-producing drug (% Conversion) were calculated. % Remaining 100 ═ PAR designated time/PART₀) Wherein PAR ═ prototype peak area/internal standard peak. % Conversion 100 ═ PAR assigned time/PART of prototype drug₀)。

6. Stability testing in rat liver S9

Experimental materials: DMSO solution (10. mu.M) of test compound, 3.2mM MgCl2 solution, rat liver S9, quenching solution (100ng/mL propranolol dissolved in cold MeCN), NADPH regeneration system solution.

The experimental method comprises the following steps: mu.L of compound solution and 10. mu.L of MgCl₂The solution, 63. mu.L liver S9, 125. mu.L LPBS were mixed and 50. mu.L was addedNADPH initiated the reaction, incubated in a total volume of 250. mu.L, and incubated at 37 ℃ for 60 min. After 60min, 100. mu.L of sample was taken, 100. mu.L of quenching solution was added to the sample in time to stop the reaction, vortexed for 3min, and centrifuged for 10 min. The supernatant was collected and 5. mu.L was taken for LC-MS/MS analysis.

Table 3 partial examples chemical stability study of compounds

Table 4 part of the examples the compounds were investigated for their stability in rat plasma and liver S9 part

R^aThe remaining rate; c^bConversion of different prodrugs to parent drugs^cAnd is not detected.

Claims

1. A compound of formula I, and pharmaceutically acceptable salts, hydrates, solvates thereof:

wherein:

R₂、R₃selected from hydrogen, halogen, C₁-C₆Alkyl radical, C₁-C₆Alkoxy, hydroxy, halogenated C₁-C₆An alkyl group;

Wherein n is an integer of 0 or 1;

R₈selected from hydrogen, C₁-C₆An alkyl group.

2. The (R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamate piperazin-1-yl) -methanone derivative according to the general formula (I) of claim 1, and geometric isomers thereof or pharmaceutically acceptable salts, hydrates, solvates thereof, wherein:

R₁selected from hydrogen, C unsubstituted or substituted by halogen₁-C₆Alkyl, unsubstituted or halogen-substituted C₃-C₆Cycloalkyl radical, C₂-C₄Alkoxy radicals, by hydrogen, hydroxy, amino, C₁-C₆Alkyl radical, C₁-C₆An alkoxy group.

3. The (R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamate piperazin-1-yl) -methanone derivative according to general formula (I) of claim 1 or 2, and geometric isomers thereof or pharmaceutically acceptable salts, hydrates, solvates thereof, wherein:

R₂、R₃selected from hydrogen and halogen.

4. (R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamate piperazin-1-yl) -methanone derivatives of the general formula (I) according to any one of claims 1 to 3, as well as geometric isomers thereof or pharmaceutically acceptable salts, hydrates, solvates thereof, wherein:

R₅selected from hydrogen, phenyl, unsubstituted or halogen-substituted C₁-C₄Alkyl, - (CR)₆R₇)_n-phenyl, - (CR)₆R₇)_n-O-C(＝O)-R₈，-(CR₆R₇)_n-C(＝O)-O-R₈；

Wherein n is an integer of 0 or 1;

R₆、R₇selected from hydrogen, C₁-C₆An alkyl group;

R₈selected from hydrogen, C₁-C₆An alkyl group.

(R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamate piperazin-1-yl) -methanone derivatives, and geometric isomers thereof, or pharmaceutically acceptable salts, hydrates, solvates thereof, selected from the group consisting of:

6. a pharmaceutical composition comprising as an active ingredient (R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamate piperazin-1-yl) -methanone derivative according to any one of claims 1 to 5, and geometric isomers thereof, or pharmaceutically acceptable salts, hydrates, solvates thereof, and pharmaceutically acceptable excipients.

7. The process for the preparation of (R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamatopiperazin-1-yl) -methanone derivatives, and geometric isomers thereof, or pharmaceutically acceptable salts, hydrates, solvates thereof, according to claim 1,

wherein R is₁、R₂、R₃、R₄、R₅As claimed in claim.

8. Use of the (R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamate piperazin-1-yl) -methanone derivative of claim 1, and geometric isomers thereof or pharmaceutically acceptable salts, hydrates, solvates thereof, or pharmaceutical compositions of claim 6, for the preparation of PAK protein kinase inhibitors.

9. Use of the (R) - (4- ((1H-pyrazol-3-yl) amino) quinazolin-2-yl) - (N-carbamate piperazin-1-yl) -methanone derivative of claim 1, and geometric isomers thereof, or pharmaceutically acceptable salts, hydrates, solvates thereof, or pharmaceutical compositions of claim 6, for the preparation of an antitumor medicament.

10. The use according to claim 9, wherein the tumor is lung cancer, liver cancer, stomach cancer, intestinal cancer, kidney cancer, prostate cancer, uterine cancer, ovarian cancer.