Novel pyrazolopyrimidine compounds and compositions, methods of manufacture and uses thereof

Abstract

The present invention relates to a novel pyrazolopyrimidine compound, its composition, its preparation method, and its use as an anticancer agent due to its antitumor activity. The novel pyrazolopyrimidine compound has the following general formula (I), and as an ATR inhibitor, it has excellent tumor inhibitory activity, high selectivity, good water solubility, low toxicity, and is applicable to oral or intravenous administration. TIFF2025503771000083.tif36170

Description

[Technical field]

[0001] The present invention belongs to the pharmaceutical field and relates to novel pyrazolopyrimidine compounds and their compositions, preparation methods and use in the preparation of anticancer drugs. [Background technology]

[0002] Cancer threatens human health and life. In recent years, anti-cancer drug research has shifted to the research and development of specific molecular targeted therapeutic drugs.

[0003] ATR, ataxia telangiectasia and Rad3-related kinase, is a 2644 amino acid kinase that can activate cellular responses after DNA damage, and is an important kinase that prevents apoptosis by blocking cell cycle progression, stabilizing replication forks, and repairing DNA. When DNA replication pressure or DNA damage occurs in cells, ATR is recruited to the site of DNA damage, and various proteins are involved in regulating ATR activation. After ATR is activated, various signals can regulate cellular biological processes, including cell cycle block, inhibition of replication origins, promotion of deoxynucleotide synthesis, replication fork initiation, and repair of DNA double-strand breaks.

[0004] Tumor cells have defects in various DNA repair pathways, so they are more dependent on the ATR repair pathway than normal cells and more sensitive to ATR inhibitors. "Synthetic lethality" refers to the fact that in tumor cells, defects due to mutations in one pathway cause tumor cells to depend on another complementary pathway than normal cells, so when the complementary pathway is inhibited, it causes "synthetic lethality" in tumor cells. In contrast, normal cells have another pathway that is normal, so they are not inhibited by drugs and die. Research has found that ATR is a partially mutated synthetic lethal target, and that, for example, ATM-deficient tumor cells are more sensitive to ATR inhibitors, and that tumor cells are also more sensitive to ATR inhibitors by X-rays crossing to complementarily repair the defect in gene I. For this reason, ATR inhibitors are promising as excellent potential drugs for treating tumors due to their characteristics of selectively affecting tumor cells but causing little interference with normal cells.

[0005] As the most promising "synthetic lethal" therapy after PARP inhibitors, ATR inhibitors have attracted the layout of some large multinational companies, including Merck, Bayer, AstraZeneca, etc., among which Merck's Berzosertib has made clinical progress and is currently in Phase II clinical stage. Although there are few companies that layout ATR inhibitors in China, IMPACT Therapeutics' IMP9064 obtained FDA approval for Phase I / II clinical research on October 29, 2021, and ATR inhibitors that have obtained approval from the China National Medical Products Administration (NMPA) to conduct clinical research include Merck's Berzosertib, Bayer's BAY1895344, and Shijiazhuang Zhikang Hongren New Pharmaceutical's ATR inhibitors, all of which are in Phase I clinical stage. One of the current clinical research drugs is AstraZeneca's AZD6738, whose structure is as follows: TIFF2025503771000002.tif33170 In the experimental part of the present invention, it is used as the positive control for ATR kinase activity test. However, up to now, there is no approved and commercially available ATR inhibitor. Therefore, there is still a need to develop new ATR inhibitors with stronger selectivity and better activity. Summary of the Invention

[0006] One object of the present invention is to provide a novel ATR inhibitor which has excellent tumor-inhibiting activity, high selectivity, good water solubility, low toxicity, and is applicable to oral or intravenous administration.

[0007] According to one aspect, the present invention provides a compound, a stereoisomer or a pharma- ceutically acceptable salt thereof, of general formula (I): TIFF2025503771000003.tif36170, where R1 is, Selected from TIFF2025503771000004.tif111170, R x is selected from H, a 5-6 membered heteroaryl group, or a 5-6 membered heterocyclic group, wherein the 5-6 membered heteroaryl group or the 5-6 membered heterocyclic group is selected from R a and optionally substituted with R a is H, halogen, -CN, -NH2, C 1-4 Alkyl group, C 1-4 Alkoxy group or C 1-4 halogenated alkyl groups, and R y H, halogen, NH2, C 1-4 Alkyl group or C 1-4 The alkoxy group is selected from the group consisting of alkoxy groups.

[0008] In one embodiment, in general formula (I), R1 is selected from the following groups: TIFF2025503771000005.tif73170

[0009] In one embodiment, in general formula (I), R x is selected from H, a 6-membered heteroaryl group, or a 6-membered heterocyclic group, wherein the 6-membered heteroaryl group or the 6-membered heterocyclic group is selected from R a and R a is H, halogen, -CN, -NH2, C 1-4 Alkyl group, C 1-4 Alkoxy group or C 1-4 The alkyl group is selected from halogenated alkyl groups.

[0010] In one embodiment, in general formula (I), R y is H or C 1-4 It is selected from alkyl groups.

[0011] In one embodiment, the compound has the structure of general formula (II): TIFF2025503771000006.tif40170, where R1 is selected from the following groups: TIFF2025503771000007.tif36170R a is H, halogen, -CN, -NH2, C 1-4 Alkyl group, C 1-4 Alkoxy group or C 1-4 halogenated alkyl groups, and R y is selected from H or a methyl group.

[0012] In one embodiment, the compound of the invention is selected from the following compounds: TIFF2025503771000008.tif250170TIFF2025503771000009.tif212170TIFF2025503771000010.tif105170

[0013] According to another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention of general formula (I) or (II), its stereoisomer or a pharma- ceutically acceptable salt thereof and a pharma-ceutically acceptable adjuvant.

[0014] In one embodiment, the pharmaceutical composition further comprises another active agent applicable in the treatment of cancer.

[0015] In one embodiment, the pharmaceutical compositions may be prepared as injectables, e.g., sterile aqueous solutions, non-aqueous solutions, dispersions, suspensions or emulsions; solid oral dosage forms, e.g., tablets, capsules, powders, granules or pills; or liquid oral dosage forms, e.g., solutions, emulsions, suspensions or syrups.

[0016] According to yet another aspect, the present invention provides the use of the compound of general formula (I) or (II) of the present invention, its stereoisomer or its pharma- ceutically acceptable salt in the manufacture of a medicament for treating cancer.The present invention further provides the use of the pharmaceutical composition of the present invention in the manufacture of a medicament for treating cancer.

[0017] In one embodiment, the cancer is selected from breast cancer, renal cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, lymphoma, melanoma, myeloma, and osteosarcoma.

[0018] The present invention has the following beneficial effects: 1. The present invention provides novel pyrazolopyrimidine compounds.

[0019] 2. The pyrazolopyrimidine compounds provided by the present invention have excellent activity of inhibiting ATR kinase, and the activity is equal to or greater than that of the clinical research drug AZD6738, indicating that the compounds of the present invention are applicable as ATR inhibitors to the treatment of ATR-mediated diseases.

[0020] 3. The pyrazolopyrimidine compounds provided by the present invention have excellent effects of inhibiting the proliferation of various types of cancer cells and are applicable to the treatment or prevention of various types of cancer.

[0021] 4. The pyrazolopyrimidine compounds provided by the present invention have been shown to have extremely high stability in mouse and human liver microsomes and to be slowly metabolized in vivo.

[0022] 5. The pyrazolopyrimidine compounds provided by the present invention have extremely high in vivo exposure after oral administration to mice, and are expected to provide excellent antitumor effects at lower doses in clinical practice.

[0023] 6. The pyrazolopyrimidine compounds provided by the present invention have a high degree of selectivity for ATR and low inhibitory activity against other kinases in the family, thereby providing the advantage of higher safety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Unless otherwise defined, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] It should be noted that the above brief description and the following detailed description are used for illustrative purposes only and are not intended to limit the subject matter of the present invention.

[0026] definition As used herein, the term "C 1-4 The term "alkyl group" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms. Examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and t-butyl groups, with methyl being preferred.

[0027] As used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine, with fluorine, chlorine and bromine being preferred.

[0028] As used herein, the term "C 1-4 An "alkoxy group" is an alkoxy group represented by the formula -OC 1-4refers to a group having an alkyl group, where C 1-4 The alkyl group is as defined above. 1-4 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, n-butoxy, isobutoxy, and t-butoxy groups, with methoxy and ethoxy groups being preferred.

[0029] As used herein, the term "C 1-4 "Halogenated alkyl group" refers to a C alkyl group substituted with one or more halogen atoms. 1-4 It refers to an alkyl group. Examples of the alkyl group include, but are not limited to, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a fluoroethyl group, a 1,1-difluoroethyl group, a chloromethyl group, a chloroethyl group, a dichloromethyl group, and a 1,2-dichloroethyl group, and the like, with a difluoromethyl group and a trifluoromethyl group being preferred.

[0030] The term "5- to 6-membered heteroaryl group" as used herein refers to a stable 5- to 6-membered aromatic monocyclic group containing 1 to 3, preferably 1 to 2, heteroatoms selected from nitrogen, oxygen, and sulfur. The heteroaryl group may be bonded to the heteroaryl group via the position of the heteroatom or a carbon atom of the heterocycle. Examples of 5-6 membered heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-thiadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, pyridyl, pyrazinyl, triazinyl, pyrimidinyl and pyridazinyl groups.

[0031] The term "5- to 6-membered heterocyclic group" as used herein refers to a stable 5- to 6-membered non-aromatic monocyclic group containing 1 to 3, preferably 1 to 2, heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocyclic group may be partially or fully saturated. The heterocyclic group may be bonded to the heterocyclic group through the position of the heteroatom or a carbon atom of the heterocycle. Examples of 5- to 6-membered heterocyclic groups include dihydrofuranyl group, dihydrothienyl group, 3-pyrrolinyl group, 2-pyrrolinyl group, 2-imidazolinyl group, 2-pyrazolidinyl group, dihydrooxazolyl group, dihydrothiazolyl group, dihydroisoxazolyl group, dihydroisothiazolyl group, dihydro-1,2,3-triazolyl group, dihydro-1,2,4-triazolyl group, and dihydro-1,2,5-oxadiazolyl group. dihydro-1,2,3-oxadiazolyl group, dihydro-1,2,4-oxadiazolyl group, dihydro-1,3,4-oxadiazolyl group, dihydro-1,2,5-thiadiazolyl group, dihydro-1,2,3-thiadiazolyl group, dihydro-1,2,4-thiadiazolyl group, dihydro-1,3,4-thiadiazolyl group, tetrahydrofuranyl group, tetrahydrothienyl group a pyrrolidinyl group, an imidazolidinyl group, a pyrazolidinyl group, an oxazolidinyl group, a thiazolidinyl group, an isoxazolidinyl group, an isothiazolidinyl group, a 1,2,3-triazolidinyl group, a 1,2,4-triazolidinyl group, a 1,2,5-oxadiazolidinyl group, a 1,2,3-oxadiazolidinyl group, a 1,3,4-oxadiazolidinyl group, a 1, Examples of such groups include, but are not limited to, 2,5-thiadiazolidinyl, 1,2,3-thiadiazolidinyl, 1,3,4-thiadiazolidinyl, 1,2-oxathiacyclopentyl, 1,3-oxathiacyclopentyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, thiomorpholinyl, and piperazinyl groups.

[0032] "Stereoisomers" means compounds composed of the same atoms and bonded by the same bonds, but having different three dimensional structures. The present invention includes various stereoisomers and mixtures thereof.

[0033] As used herein, the term "subject" includes mammals and non-mammals. Examples of mammals include any member of the class Mammalia, including, but not limited to, humans, non-human primates (e.g., chimpanzees, other apes and monkeys), farm animals, such as cows, horses, sheep, goats, pigs, domestic animals, such as rabbits, dogs, cats, and laboratory animals, including rodents, such as rats, mice, guinea pigs, and the like. In some embodiments, the subject is a human.

[0034] As used herein, the term "treatment" and other similar synonyms include alleviating, reducing or improving symptoms of a disease or illness, and inhibiting a disease or illness, for example, preventing the development of a disease or illness, alleviating a disease or illness, improving a disease or illness, alleviating symptoms of a disease or illness, or halting symptoms of a disease or illness, preventing other symptoms, and improving or preventing underlying metabolic factors leading to symptoms, where the term includes preventive purposes. The term further includes obtaining a therapeutic effect and / or a preventive effect. The therapeutic effect means curing or improving the underlying illness being treated. It should be noted that the therapeutic effect also includes curing or improving one or more physiological symptoms associated with the underlying illness, for example, an improvement in the subject's condition, although the subject may still be affected by the underlying illness. For prophylactic effect, the compounds or compositions of the invention can be administered to a subject who is at risk of contracting a particular disease, or to a subject who has not yet been diagnosed with the disease but who experiences one or more physiological symptoms of that disease.

[0035] The term "therapeutically effective amount" as used herein means an amount of at least one active substance (e.g., the compound of the present invention) sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated after administration. The result may be reduction and / or alleviation of signs, symptoms, or pathology, or any other desired change in a biological system. For example, a "therapeutically effective amount" is the amount of a composition containing a compound disclosed herein that is required to provide a clinically significant disease-altering effect. The optimal therapeutically effective amount can be determined for any individual case, for example, using techniques of dose escalation experiments.

[0036] As used herein, the terms "application", "administration" and the like refer to methods by which a compound or composition can be delivered to the site where a biological effect is required. These methods include, but are not limited to, oral administration, intraduodenal administration, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical application, and rectal administration. The application techniques used in the compounds and methods described herein are well known to those skilled in the art.

[0037] As used herein, the term "pharmaceutical acceptable adjuvant" refers to a substance that does not affect the biological activity or properties of the compounds of the present invention, and is relatively non-toxic, i.e., the substance is administered to an individual without causing an adverse biological reaction or interacting in an adverse manner with any of the components contained in the composition. The pharmaceutical acceptable adjuvant includes, but is not limited to, carriers, stabilizers, diluents, powders, suspending agents, thickeners, and / or excipients.

[0038] As used herein, the terms "any," "optional," or "optionally" indicate that the subsequently described event or circumstance may or may not occur, and the description includes both cases in which the event or circumstance occurs and cases in which the event or circumstance does not occur. For example, "R a "Optionally substituted with" means that it is not substituted or that it is a If the description is not substituted, or Ra This also includes the case where it is replaced with.

[0039] A compound of the general formula (I), its stereoisomer or a pharma- ceutically acceptable salt thereof The present invention provides compounds for treating, improving or preventing cancer. As described herein, inhibiting ATR kinase can selectively affect tumor cells, but with less interference with normal cells. The compounds of the present invention can effectively inhibit the activity of ATR kinase, and are equivalent to or greater than the activity of clinical research drug AZD6738, making them promising potential drugs for treating tumors.

[0040] According to one aspect, the present invention provides a compound of general formula (I), a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: TIFF2025503771000011.tif36170, where R1 is, Selected from TIFF2025503771000012.tif111170; R x is selected from H, a 5-6 membered heteroaryl group, or a 5-6 membered heterocyclic group, wherein the 5-6 membered heteroaryl group or the 5-6 membered heterocyclic group is selected from R a and optionally substituted with R a is H, halogen, -CN, -NH2, C 1-4 Alkyl group, C 1-4 Alkoxy group or C 1-4 halogenated alkyl groups, and R y H, halogen, NH2, C 1-4 Alkyl group or C 1-4 The alkoxy group is selected from the group consisting of alkoxy groups.

[0041] In one embodiment, R1 is selected from the following groups: TIFF2025503771000013.tif73170

[0042] In one embodiment, R1 is selected from the following groups or stereoisomeric forms thereof (when applicable): TIFF2025503771000014.tif36170

[0043] In one embodiment, R1 is selected from the following groups or stereoisomeric forms thereof (when applicable): TIFF2025503771000015.tif36170

[0044] In one embodiment, R1 is Selected from TIFF2025503771000016.tif35170.

[0045] In one embodiment, R1 is The file is TIFF2025503771000017.tif34170.

[0046] In one embodiment, R x is selected from H, a 5-6 membered heteroaryl group, or a 5-6 membered heterocyclic group, wherein the 5-6 membered heteroaryl group or the 5-6 membered heterocyclic group is selected from R a may be optionally substituted with

[0047] In some embodiments, R x is H.

[0048] In some embodiments, R x is a 5-6 membered heteroaryl group. In some embodiments, R x is a 5-6 membered heteroaryl group containing 1 or 2 heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, R x is a 5-6 membered heteroaryl group containing 1 or 2 heteroatoms selected from nitrogen and oxygen. In some embodiments, R x is a 6-membered heteroaryl group containing 1 or 2 nitrogen atoms. In some embodiments, R x is selected from a pyridyl group, a pyrimidinyl group, or an isoxazolyl group. In some embodiments, the heteroaryl group is amay be optionally substituted with

[0049] In some embodiments, R x is a 5-6 membered heterocyclic group. In some embodiments, R x is a 5-6 membered heterocyclic group containing 1 or 2 heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, R x is a 6-membered heterocyclic group containing 1 or 2 heteroatoms selected from nitrogen and oxygen. In some embodiments, R x is a 6-membered heterocyclic group containing 1 or 2 nitrogen atoms. In some embodiments, R x is a 6-membered heterocyclic group containing one nitrogen atom. In some embodiments, R x In some embodiments, the heterocyclic group is R a may be optionally substituted with

[0050] In some embodiments, R x teeth, Selected from TIFF2025503771000018.tif22170, R a and optionally substituted with, TIFF2025503771000019.tif4170Represents the bonding position with the pyrazole ring.

[0051] In some embodiments, R x teeth, TIFF2025503771000020.tif17170, R a and optionally substituted with, TIFF2025503771000021.tif4170Represents the bonding position with the pyrazole ring.

[0052] In the above embodiment, R a is H, halogen, -CN, -NH2, C 1-4 Alkyl group, C 1-4 Alkoxy group or C 1-4 The alkyl group is selected from halogenated alkyl groups.

[0053] In the above embodiment, R a is selected from H, F, Cl, Br, -CN, -NH2, a methyl group, a methoxy group, an ethoxy group, a difluoromethyl group, or a difluoroethyl group.

[0054] In some embodiments, R y H, halogen, NH2, C 1-4 Alkyl group or C 1-4 The alkoxy group is selected from the group consisting of alkoxy groups.

[0055] In some embodiments, R y is H or C 1-4 It is selected from alkyl groups.

[0056] In some embodiments, R y is selected from H or a methyl group.

[0057] In one embodiment, in general formula (I): R1 is selected from the following groups: TIFF2025503771000022.tif36170R x is selected from H, a 5- to 6-membered heteroaryl group, or a 6-membered heterocyclic group, wherein the 5- to 6-membered heteroaryl group is R a and optionally substituted with R a is H, halogen, -CN, -NH2, C 1-4 Alkyl group, C 1-4 Alkoxy group or C 1-4 halogenated alkyl groups, and R y is H or C 1-4 It is selected from alkyl groups, for example a methyl group.

[0058] In one embodiment, the compound of the invention has the structure of general formula (II): TIFF2025503771000023.tif40170, where R1 is selected from the following groups: TIFF2025503771000024.tif36170R a is H, halogen, -CN, -NH2, C 1-4 Alkyl group, C 1-4 Alkoxy group or C 1-4 halogenated alkyl groups, and R y is selected from H or a methyl group.

[0059] In one embodiment, the compound of the present invention having general formula (I) is selected from the following compounds: TIFF2025503771000025.tif200170TIFF2025503771000026.tif204170TIFF2025503771000027.tif155170

[0060] When the compounds of the above general formula (I) or (II) of the present invention contain one or more chiral centers, unless otherwise specified, any reference to these compounds includes compounds which are pure enantiomers or pure diastereomers, as well as mixtures of enantiomers or diastereomers in any ratio.

[0061] The compounds of the general formula (I) or (II) of the present invention are generally utilized as free substances or as their pharma- ceutically acceptable salts. Pharmaceutically acceptable salts refer to salts that are non-toxic, i.e., physiologically acceptable. In one embodiment, the pharma-ceutically acceptable salts of the compounds of the general formula (I) or (II) of the present invention or their stereoisomers include, but are not limited to, hydrochloride, hydrobromide, phosphate, glycerophosphate, nitrite, sulfate, hydrogen sulfate, hemisulfate, benzoate, citrate, gluconate, lactate, maleate, succinate, tartrate, acetate, propionate, caproate, heptanoate, glucoheptonate, oxalate, maleate, fumarate, malate, glutamate, pyroglutamate, salicylate, sulfonate (e.g., methanesulfonate, ethanesulfonate, toluenesulfonate and benzenesulfonate), etc.

[0062] Pharmaceutical Compositions and Pharmaceutical Formulations According to one aspect, the present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of general formula (I) or (II) as defined above, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable adjuvant.

[0063] In the present invention, the term "pharmaceutical acceptable adjuvant" refers to a substance that does not affect the biological activity or properties of the compound of the present invention, and is relatively non-toxic, i.e., the substance is administered to an individual without causing an adverse biological reaction or interacting with any component contained in the composition in an adverse manner. The pharmaceutical acceptable adjuvant includes, but is not limited to, a carrier, a stabilizer, a diluent, a powder, a suspending agent, a thickener, and / or an excipient.

[0064] In one embodiment, the pharmaceutical composition of the present invention may further comprise other active agents for treating cancer, examples of which include, but are not limited to, cisplatin, carboplatin, cyclophosphamide, gemcitabine, olaparib, nogitecan, irinotecan, doxorubicin, paclitaxel, docetaxel, adriamycin, PD-1 or PD-L1 monoclonal antibody drugs (e.g., Nivolumab monoclonal antibody, Atezolizumab monoclonal antibody, etc.).

[0065] In one embodiment, the pharmaceutical compositions of the present invention may be prepared as a formulation to be administered by any suitable route, including, but not limited to, oral administration, intraduodenal administration, injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical application, and rectal administration.

[0066] Pharmaceutical compositions used for oral administration include solid oral dosage forms such as, for example, tablets, capsules, powders, granules, liquid oral dosage forms such as, for example, solutions, emulsions, suspensions, syrups, and powders and granules that are dissolved or suspended in a suitable liquid.

[0067] The solid oral dosage forms may be in the form of discrete units (e.g., tablets or hard or soft capsules), each containing a predetermined amount of the active ingredient, and preferably contain one or more suitable pharma- ceutically acceptable adjuvants. When applied, these solid dosage forms may be prepared with coatings, such as enteric coatings, or may be prepared to provide modified release of the active ingredient, such as delayed or extended release, according to methods known in the art.

[0068] Examples of pharma- ceutically acceptable adjuvants suitable for solid oral dosage forms include, but are not limited to, microcrystalline cellulose, corn starch, lactose, mannitol, polyvinylpyrrolidone, croscarmellose sodium, sucrose, cyclodextrin, talc, gelatin, pectin, magnesium stearate, stearic acid, and lower alkyl ethers of cellulose.Similarly, solid formulations may contain excipients for delaying or extending the release of the formulation known in the art, such as, for example, glycerol monostearate or hydroxypropylmethylcellulose.

[0069] Solid oral dosage forms may, for example, be prepared by mixing the active ingredient with solid excipients and then compressing in a conventional tablet machine, or may be prepared, for example, by placing the formulation, for example as a powder, pill or microtablet, into a hard capsule.

[0070] The liquid oral dosage form may be in the form of, for example, a solution, emulsion, suspension, elixir, syrup, oral drops or liquid-filled capsules. The liquid oral dosage form may be in the form of a powder for dissolving in an aqueous or non-aqueous liquid to form a solution or suspension before use. Examples of excipients suitable for liquid oral preparations include, but are not limited to, ethanol, propylene glycol, glycerin, polyethylene glycol, poloxamer, sorbitol, polysorbate, monoglycerides, diglycerides, cyclodextrin, coconut oil, palm oil and water. The liquid oral dosage form may be prepared, for example, by dissolving or suspending the active ingredient in an aqueous or non-aqueous liquid or by incorporating the active ingredient into an oil-in-water or water-in-oil liquid emulsion.

[0071] Pharmaceutical compositions for parenteral administration may be in the form of sterile injectable preparations, such as sterile aqueous and non-aqueous solutions, dispersions, suspensions or emulsions for injection or infusion, concentrates for injection or infusion, and sterile powders to be reconstituted in sterile solutions or dispersions for injection or infusion before use. Examples of pharmaceutical acceptable adjuvants suitable for parenteral preparations include, but are not limited to, water, coconut oil, palm oil, cyclodextrin solutions, Ringer's solutions, and isotonic sodium chloride solutions. Sterile injectable preparations may be prepared with suitable pharmaceutical acceptable adjuvants based on known techniques.

[0072] Pharmaceutically acceptable adjuvants used in any pharmaceutical formulation should meet the expected route of administration and be compatible with the active ingredient.

[0073] In one embodiment, the pharmaceutical composition of the present invention may be prepared as an injection, preferably an intravenous injection, In one embodiment, the pharmaceutical composition of the present invention may be prepared as an injectable sterile aqueous solution, non-aqueous solution, dispersion, suspension or emulsion.

[0074] In one embodiment, the pharmaceutical composition of the present invention may be prepared as a solid oral dosage form. In one embodiment, the pharmaceutical composition of the present invention may be prepared as a tablet. In one embodiment, the pharmaceutical composition of the present invention may be prepared as a capsule, including a hard capsule and a soft capsule. In one embodiment, the pharmaceutical composition of the present invention may be prepared as a powder. In one embodiment, the pharmaceutical composition of the present invention may be prepared as a granule. In one embodiment, the pharmaceutical composition of the present invention may be prepared as a pill.

[0075] In one embodiment, the pharmaceutical composition of the present invention may be prepared as a liquid oral formulation. In one embodiment, the pharmaceutical composition of the present invention may be prepared as an oral administration solution, emulsion, suspension or syrup.

[0076] Purpose It has been shown that the compound of the present invention represented by general formula (I) or (II), its stereoisomer or its pharma- ceutically acceptable salt, or the pharmaceutical composition of the present invention can effectively inhibit ATR kinase activity, which is equal to or greater than that of clinical research drug AZD6738, and can be used for the treatment of ATR kinase-mediated diseases.

[0077] The compound of the present invention represented by general formula (I) or (II), its stereoisomer or its pharma- ceutically acceptable salt, or the pharmaceutical composition of the present invention can effectively inhibit the proliferation of tumor cells, and therefore can be used for the treatment of tumors or cancer.

[0078] According to one aspect, the present invention provides the use of a compound of general formula (I) or (II) of the present invention, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition of the present invention in the manufacture of a medicament for treating cancer in a subject.

[0079] In one embodiment, the cancer includes, but is not limited to, breast cancer, renal cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, lymphoma, melanoma, myeloma, and osteosarcoma.

[0080] In one embodiment, the cancer is selected from breast cancer. In one embodiment, the cancer is selected from renal cancer. In one embodiment, the cancer is selected from lung cancer, e.g., non-small cell lung cancer. In one embodiment, the cancer is selected from ovarian cancer. In one embodiment, the cancer is selected from bladder cancer. In one embodiment, the cancer is selected from gastric cancer. In one embodiment, the cancer is selected from colorectal cancer. In one embodiment, the cancer is selected from liver cancer. In one embodiment, the cancer is selected from pancreatic cancer. In one embodiment, the cancer is selected from prostate cancer. In one embodiment, the cancer is selected from leukemia. In one embodiment, the cancer is selected from lymphoma, e.g., mantle cell lymphoma. In one embodiment, the cancer is selected from melanoma. In one embodiment, the cancer is selected from myeloma. In one embodiment, the cancer is selected from osteosarcoma.

[0081] Manufacturing method According to one aspect, the present invention provides a method for preparing a compound of general formula (I) of the present invention, comprising: TIFF2025503771000028.tif79170

[0082] Specifically, the method includes the following steps: Step 1: TIFF2025503771000029.tif36170 was reacted with N-iodosuccinimide (NIS) to obtain compound X2. TIFF2025503771000030.tif33170

[0083] Step 2: X2 compound and React TIFF2025503771000031.tif34170 with Sukuzi I got TIFF2025503771000032.tif34170.

[0084] Step 3: Removal of the Boc protecting group from the X3 compound I got TIFF2025503771000033.tif36170.

[0085] Step 4: X4 compound and TIFF2025503771000034.tif17170 and Ullmann reaction (or substitution reaction) I got TIFF2025503771000035.tif37170.

[0086] Step 5: The THP protecting group was removed from compound X6 to obtain the compound of the above general formula (I) of the present invention.

[0087] In one embodiment, an exemplary reaction scheme for preparing compounds of general formula (I) of the present invention is provided: TIFF2025503771000036.tif109170, where R1, R x and R y is defined as in general formula (I).

[0088] According to the above scheme, the X1 compound is used as a starting material and reacted with N-iodosuccinimide NIS in a suitable solvent such as dichloromethane to obtain the X2 compound, the X2 compound and 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester analogue N6 are reacted with potassium phosphate and Xphos-Pd-G3 in a suitable solvent such as a mixed solution of 1,4-dioxane / water to obtain the X3 compound, the X3 compound is reacted with an alkali such as NaOH in a suitable solvent such as tetrahydrofuran to obtain the X4 compound, the X4 compound and the X5 compound are reacted in the presence of potassium phosphate, CuI and N,N-dimethyl-1,2-cyclohexanediamine to obtain the X6 compound, and the X6 compound is deprotected under acidic conditions to obtain the compound of general formula (I).

[0089] In one embodiment, there is provided a method for preparing a compound of general formula (II) of the present invention: TIFF2025503771000037.tif54170

[0090] Step 1: TIFF2025503771000038.tif36170 reacted with N-bromosuccinimide (NBS) I got TIFF2025503771000039.tif35170.

[0091] Step 2: N2 compound and TIFF2025503771000040.tif28170 and Suzuki react in an appropriate solvent. I got TIFF2025503771000041.tif34170.

[0092] Step 3: N3 compound and TIFF2025503771000042.tif22170 and Ullmann reaction I got TIFF2025503771000043.tif41170.

[0093] Step 4: The THP protecting group was removed from the N4 compound to obtain the compound of the above general formula (II) of the present invention.

[0094] In one embodiment, an exemplary reaction scheme for preparing a compound of general formula (II) of the present invention is provided: TIFF2025503771000044.tif52170

[0095] According to the above scheme, the N1 compound was used as a starting material and reacted with N-bromosuccinimide NBS in a suitable solvent, such as DMF, to obtain the N2 compound, the N2 compound and the 1H-pyrazole-5-boronic acid pinacol ester analogue N6 were reacted with potassium phosphate and Pd(dtbpf)Cl2 in a suitable solvent, such as a mixed solution of 1,4-dioxane / water, to obtain the N3 compound, the N3 compound and the N5 compound were reacted in the presence of potassium phosphate, CuI and N,N-dimethyl-1,2-cyclohexanediamine to obtain the N4 compound, and the N4 compound was deprotected under acidic conditions to obtain the compound of general formula (II).

[0096] The stereoisomers of the compounds of the general formula (I) or (II) of the present invention can be obtained in the following manner: the obtained racemic or other mixtures are separated by known means, such as fractional crystallization or HPLC techniques, based on their different physicochemical properties. Enantiomers can also be obtained by separation using chiral columns. Optically active raw materials can also be reacted without causing racemic or epimerization.

[0097] The pharma- ceutically acceptable salts of the compounds of general formula (I) or (II) of the present invention or their stereoisomers may be formed from the N atom on the compound or its stereoisomer structure and an inorganic or organic acid by methods known in the art. The inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, phosphoric acid, glycerin phosphoric acid, hemisulfuric acid, sulfuric acid, hydrogen sulfate, hydroiodic acid, nitrous acid, etc. The organic acids include, but are not limited to, benzoic acid, citric acid, gluconic acid, lactic acid, maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, caproic acid, heptanoic acid, glucoheptonic acid, oxalic acid, maleic acid, fumaric acid, malic acid, glutamic acid, pyroglutamic acid, salicylic acid, sulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid), etc.

[0098] Other intermediates or reactants not specifically mentioned may be obtained commercially or may be prepared by synthetic methods generally known in the art.

[0099] The above general synthetic routes are merely illustrative of the general methods in many examples, and in the case of compounds having special substituents, the reactions of certain steps may be modified or the reaction sequence may be changed as known to those skilled in the art.

[0100] Working Example The following examples illustrate the preparation and evaluation of the biological activity of the compounds of the present invention.

[0101] These examples which follow are provided to enable those skilled in the art to more clearly understand and to practice the present invention, and are not to be construed as limiting the scope of the invention, but merely as being illustrative and representative thereof.

[0102] The materials or reagents used herein may be obtained commercially or may be prepared by synthetic methods generally known in the art. The following reaction schemes illustrate specific methods for synthesizing the compounds of the present invention.

[0103] Specifically: Preparation of Key Intermediates A1-A3: Preparation of intermediate a1: TIFF2025503771000045.tif20170

[0104] Intermediate a1-1 (60.6 mmol, 8.5 g) and carbonyldiimidazole (CDI, 19.6 g, 121.2 mmol) were dissolved in 100 mL of anhydrous tetrahydrofuran and reacted at room temperature for 2 hours, then heated to 55 ° C. and continued stirring for 4 hours, and cooled to room temperature. Intermediate a1-2 (17.2 g, 121.2 mmol) was slowly added in portions to the reaction liquid, and the temperature was raised to 55 ° C. and the reaction was continued for 40 hours. The reaction was stopped, filtered, the solvent was distilled off under reduced pressure, 50 mL of water was added, extracted with ethyl acetate, and separated by flash column chromatography to obtain intermediate a1 (9.1 g, 72% yield). LC-MS: [M + H] + :211.

[0105] Preparation of intermediate a2: TIFF2025503771000046.tif52170

[0106] Step 1: Intermediate a1 (43.3 mmol, 9.1 g) was dissolved in 60 mL of pyridine, 3-aminopyrazole a2-1 (34.0 mmol, 2.82 g) was added, and the mixture was heated to 110° C. and reacted for 12 hours, then cooled to room temperature. The solvent was distilled off under reduced pressure to give intermediate a2-2 (LC-MS: [M+H] + :248) 9.0g was obtained.

[0107] Step 2: The crude intermediate a2-2 (9.0 g) was dissolved in 80 mL of ethanol, pyridinium p-toluenesulfonate PPTS (54.6 mmol, 13.7 g) was added, and the temperature was raised to 90° C. for 36 hours to react, after which the reaction was stopped. The solvent was removed by distillation under reduced pressure, 100 mL of water was added to the system, extracted with ethyl acetate, and separated by flash column chromatography to obtain intermediate a2-3 (7.5 g, two-step yield 76%). LC-MS: [M+H] +:230.

[0108] Step 3: In an ice bath, intermediate a2-3 (19.6 mmol, 4.5 g) was dissolved in 25 mL of phosphorus oxytrichloride, stirred for 10 minutes, then the ice bath was removed, the temperature was raised to 110°C, and stirring was continued for 2 hours. The reaction was stopped, cooled to room temperature, and 150 mL of ice water was slowly added to the reaction solution, the pH was adjusted to about 8 with saturated sodium bicarbonate solution, extracted with ethyl acetate, and dried with anhydrous sodium sulfate. The crude product was separated by flash column chromatography to obtain intermediate a2 (3.5 g, 72% yield). LC-MS: [M+H] + :248.

[0109] Preparation of intermediate a3: TIFF2025503771000047.tif23170

[0110] Intermediate a2 (6.01 mmol, 1.5 g) and 4-dimethylaminopyridine DMAP (0.6 mmol, 73 mg) were dissolved in 20 mL of tetrahydrofuran, and di-t-butyl dicarbonate (9.01 mmol, 1.97 g) was slowly added. The reaction was allowed to proceed at room temperature for 2 hours, and then the reaction was stopped. 50 mL of water was added to the system, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate to obtain intermediate a3 (1.9 g, 91% yield). LC-MS: [M+H] + :348.

[0111] Preparation of key intermediate b1: TIFF2025503771000048.tif59170

[0112] Step 1: Under a nitrogen gas atmosphere, at -5°C, raw material b1-1 (5mmol, 1.05g) was dissolved in 15mL of fluorobenzene, diethylzinc (2mmol / L toluene solution, 12.5mL, 25mmol) and fluorobenzene solution of chloroiodomethane (4.4g / 1.8mL, 25mmol) were slowly added, and the reaction was carried out at room temperature for 12 hours. The system was placed in an ice bath, and 40mL of saturated aqueous ammonium chloride was added to quench the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and separated by flash column chromatography to obtain intermediate b1-2 (730mg, yield 65%).

[0113] Step 2: Under nitrogen gas atmosphere, intermediate b1-2 (3.23 mmol, 730 mg) was dissolved in 16 mL of a mixture of methanol / acetonitrile (1 / 1, v / v), and potassium fluoride aqueous solution (749 mg / 3 mL) was slowly added. After stirring the mixture for 10 minutes, L-tartaric acid (6.46 mmol, 968 mg) and tetrahydrofuran (350 μL) were added, and the reaction was continued at room temperature for 1.5 h. The reaction was stopped, filtered, and the filtrate was concentrated to obtain intermediate b1-3 (600 mg, crude product).

[0114] Step 3: Under a nitrogen gas atmosphere, intermediate b1-3 (7.9 mmol, 1.6 g), intermediate a3 (7.9 mmol, 2.75 g) and cesium carbonate (23.7 mmol, 7.7 g) were dissolved in 30 mL of a mixture of toluene / water (6 / 1, v / v), and Pd(dppf)Cl2 (0.8 mmol, 586 mg) was added. The temperature was raised to 110 ° C and the reaction was carried out for 12 hours, the reaction was stopped, filtered, 70 mL of water was added to the system, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography to obtain intermediate b1 (1.3 g, 41% yield). LC-MS: [M + H] + :410.

[0115] Preparation of Key Intermediates C1-C8: Preparation of intermediate c1: TIFF2025503771000049.tif27170

[0116] Step 1: The raw material 5,7-dichloropyrazolo[1,5-a]pyrimidine c1-1 (37.2 mmol, 7.0 g) and potassium carbonate (55.85 mmol, 7.72 g) were dissolved in 50 mL of acetonitrile, trifluoroethanol (40.96 mmol, 4.1 g) was added, and the mixture was reacted at room temperature for 16 hours to terminate the reaction. The solvent was distilled off under reduced pressure, 100 mL of water was added to the mixture, extracted with ethyl acetate, dried with anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (PE / EA=6 / 1) to obtain compound c1-2 (7.90 g, yield 84%). LC-MS: [M+H] + :252.0. 1 H NMR (400MHz, DMSO-d6) δ8.28(d,J=2.0Hz,1H),7.01(s,1H),6.72-6.70(m,1H),5.36(q,J=8.4Hz,2H).

[0117] Step 2: Compound 5-chloro-7-(2,2,2-trifluoroethoxy)pyrazolo[1,5-a]pyrimidine c1-2 (31.4, 79.0 g) and potassium fluoride (157.0 mmol, 9.12 g) were dissolved in 80 mL of dry DMSO, heated to 140 °C and reacted for 4 hours, cooled to room temperature, and the reaction was stopped. 100 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (PE / EA=10 / 1) to obtain compound c1-3 (3.5 g, yield 47%). LC-MS: [M+H] + :236.1.

[0118] Step 3: Compound 5-fluoro-7-(2,2,2-trifluoroethoxy)pyrazolo[1,5-a]pyrimidine c1-3 (14.88mmol, 3.5g), N,N-diisopropylethylamine DIEA (44.65mmol, 5.77g) and 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride (14.88mmol, 2.23g) were dissolved in 80mL of dry DMSO, stirred for 5 minutes, heated to 120°C and reacted for 2 hours, cooled to room temperature, and the reaction was stopped. 120mL of water was added to the reaction solution, extracted with ethyl acetate, washed with saturated saline, dried with anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (PE / EA=10 / 1) to obtain compound c1 (3.0g, yield 61%). LC-MS: [M+H] + :329.0. 1 H NMR(400MHz,DMSO-d6)δ7.87(d,J=2.0Hz,1H),6.25(s,1H),6.05(d,J=2.0Hz,1H),5.21(q,J=8.8Hz,2H),4.45( d,J=2.4Hz,2H),4.02(d,J=12.4Hz,2H),3.08(dd,J=12.8Hz,2.0Hz,2H),1.87-1.81(m,2H),1.74-1.67(m,2H).

[0119] Referring to the synthesis of intermediate c1, the following intermediates c2 to c4 were synthesized using similar raw materials: TIFF2025503771000050.tif161170

[0120] Preparation of intermediate c5: TIFF2025503771000051.tif25170

[0121] Step 1: Intermediate 3-(7-(2,2,2-trifluoroethoxy)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane c1 (9.14 mmol, 3.0 g) and sodium hydroxide (18.28 mmol, 0.73 g) were dissolved in 20 mL of tetrahydrofuran, heated to 70° C. and reacted for 16 hours, cooled to room temperature, stopped the reaction, and filtered. The solvent was distilled off under reduced pressure, and the crude product was separated and purified by flash reverse phase column chromatography (acetonitrile / water) to obtain compound c5-1 (2.0 g, 89% yield). LC-MS: [M+H] + :247.2. 1 H NMR(400MHz,DMSO-d6)δ7.48(d,J=2.0Hz,1H),5.65(d,J=1.6Hz,1H),4.89(s,1H),4. 35(s,2H),3.71(d,J=12.4Hz,2H),2.83(dd,J=12.0Hz,1.6Hz,2H),1.81-1.68(m,4H).

[0122] Step 2: Compound 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-7-hydroxy c5-1 (8.12 mmol, 2.00 g) was dissolved in 10 mL of phosphorus trichloride, heated to 110 °C and reacted for 4 hours, cooled to room temperature, and the reaction was stopped. The reaction solution was slowly added to 50 mL of ice-water mixture, and the pH was adjusted to about 9 by adding saturated aqueous sodium bicarbonate, extracted with ethyl acetate, washed with saturated saline, dried with anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (PE / EA=6 / 1) to obtain compound c5-2 (1.3 g, yield 61%). LC-MS: [M+H] + :265.1. 1H NMR(400MHz,DMSO-d6)δ7.98(d,J=2.4Hz,1H),7.03(s,1H),6.19(d,J=2.4Hz,1H),4.44(d,J=2.0Hz ,2H),4.00(d,J=12.4Hz,2H),3.10(dd,J=12.4Hz,2.0Hz,2H),1.87-1.81(m,2H),1.74-1.66(m,2H).

[0123] Step 3: Under a nitrogen gas atmosphere, compound 3-(7-chloropyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane c5-2 (4.91 mmol, 1.30 g), sodium carbonate (14.73 mmol, 1.56 g), raw material c5-3 (1.64 g, 7.37 mmol) and catalyst Pd(dppf)Cl2 (0.49 mmol, 0.36 g) were dissolved in 10 mL of 1,4-dioxane, 2 mL of water was added, and the mixture was stirred for 5 minutes. The mixture was then heated to 90 ° C. and reacted for 16 hours, cooled to room temperature, and the reaction was stopped and filtered. The mixture was added with 35 mL of water, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by column chromatography (DCM / MeOH=10 / 1) to obtain compound c5 (0.7 g, yield 44%). LC-MS: [M+H] + :325.3. 1 H NMR(400MHz,DMSO-d6)δ12.63(s,1H),7.84(d,J=2.4Hz,1H),6.50(s,1H),6.09(d,J=2.0Hz,1H),4.43(s,2H),4.0 1(d,J=13.2Hz,2H),3.08(dd,J=12.4Hz,2.0Hz,2H),2.18(d,J=28.0Hz,6H),1.89-1.81(m,2H),1.77-1.69(m,2H).

[0124] Referring to the synthesis of intermediate c5, the following intermediate compounds c6-c8 were synthesized using similar raw materials / intermediates: TIFF2025503771000052.tif109170

[0125] Example 1: Preparation of compound M3 TIFF2025503771000053.tif142170

[0126] Intermediate a3 (1.29 mmol, 450 mg) and raw material M3-1 (1.43 mmol, 161 mg) were dissolved in 10 mL of N-methylpyrrolidone, triethylamine (2.60 mmol, 263 mg) was added, and the mixture was reacted for 2 hours by microwave heating at 180 ° C. The reaction was stopped, 40 mL of water was added to the system, and the mixture was extracted with dichloromethane. This mixture and 4-dimethylaminopyridine DMAP (0.06 mmol, 8 mg) were dissolved in 12 mL of dichloromethane, triethylamine (1.29 mmol, 131 mg) and di-t-butyl dicarbonate (1.29 mmol, 282 mg) were added, and the mixture was reacted for 2 hours at room temperature, after which the reaction was stopped. 30 mL of water was added to the system, and the mixture was extracted with dichloromethane and dried with anhydrous sodium sulfate to obtain intermediate M3-2 as a crude product. LC-MS: [M + H] + :425.

[0127] In an ice bath, the crude intermediate M3-2 obtained in the previous step was dissolved in 12 mL of anhydrous dichloromethane, N-iodosuccinimide NIS (1.29 mmol, 290 mg) was slowly added in portions, and stirring was continued for 2 hours. The reaction was stopped, 30 mL of saturated aqueous ammonium chloride was added, and the mixture was extracted with dichloromethane. The mixture was dried over anhydrous sodium sulfate and separated by flash column chromatography (PE / EA=2 / 1) to obtain compound M3-3 (450 mg, two-step yield 64%). LC-MS: [M+H] + :551.

[0128] Under a nitrogen gas atmosphere, intermediate M3-3 (0.82 mmol, 450 mg) and raw material M1-4 (1.23 mmol, 342 mg) were dissolved in 10 mL of a mixture of 1,4-dioxane and water (v / v: 9 / 1), and potassium phosphate (1.64 mmol, 348 mg) and Xphos-Pd-G3 (0.08 mmol, 68 mg) were added. The reaction was carried out for 2 hours by microwave heating at 95 ° C. The reaction was stopped, filtered, 30 mL of water was added to the system, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography (PE / EA = 2 / 1) to obtain compound M3-4 (260 mg, yield 56%). LC-MS: [M + H] + :575.

[0129] Intermediate M3-4 (260 mg) was dissolved in 10 mL of tetrahydrofuran, 5 mL of 1N NaOH aqueous solution was added, and the mixture was heated to 70° C. and reacted for 1 hour. The reaction was stopped, the solvent was distilled off under reduced pressure, 40 mL of water was added, and the mixture was extracted with dichloromethane and separated by flash column chromatography (PE / EA=1 / 1) to obtain compound M3-5 (200 mg, yield 94%). LC-MS: [M+H] + :475.

[0130] Under nitrogen gas atmosphere, intermediate M3-5 (0.42 mmol, 200 mg), 3-bromopyridine M1-7 (0.63 mmol, 100 mg), potassium phosphate (1.26 mmol, 268 mg) and CuI (0.08 mmol, 15 mg) were added to a microwave reaction bottle and dissolved in 6 mL of DMF. After stirring for 5 minutes, N,N-dimethyl-1,2-cyclohexanediamine (0.08 mmol, 12 mg) was slowly added. The mixture was heated to 110°C in a microwave and reacted for 1.5 hours, cooled to room temperature, 30 mL of water was added to the reaction solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography (PE / EA=2 / 1) to obtain compound M3-6 (100 mg, yield 44%). LC-MS: [M+H] + :552.

[0131] The intermediate M3-6 (100 mg) obtained in the previous step was dissolved in 4 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 2 hours. The reaction was stopped, the solvent was distilled off under reduced pressure, and the pH was adjusted to 10 by adding saturated aqueous sodium bicarbonate solution. The mixture was extracted with dichloromethane and separated by HPLC preparative chromatography (aqueous solution of 0.1% trifluoroacetic acid) to obtain the target compound M3 (30 mg, 37% yield). LC-MS: [M+H] + :468. M3: 1 H NMR(400MHz,DMSO-d6)δ12.71(s,1H),8.83(d,J=2.6Hz,1H),8.68(d,J=4.7Hz,1H),8.27(s,1H),8.09-8.02(m,1H),7.63(dd,J=8.2,4.8Hz ,2H),6.72-6.76(m,2H),4.49(s,2H),4.17(s,2H),3.20(d,J=12.6Hz ,2H),2.34(s,3H),2.24(s,3H),1.86-1.89(m,2H),1.76-1.81(m,2H).

[0132] Example 2: Preparation of Compounds M4-M15 TIFF2025503771000054.tif60170

[0133] Step 1: Under a nitrogen gas atmosphere, the intermediate 3-(7-(3,5-dimethyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane c5 (1.85 mmol, 600.0 mg) was dissolved in 10 mL of DMF, and N-bromosuccinimide NBS (2.22 mmol, 395.1 mg) was slowly added and reacted at room temperature for 2 hours to quench the reaction. 50 mL of water was added to the reaction solution, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (DCM / MeOH=10 / 1) to obtain compound 3-(3-bromo-7-(3,5-dimethyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M4-1 (600 mg, yield 80%). LC-MS: [M+H] + :403.1. 1 H NMR(400MHz,DMSO-d6)δ12.67(s,1H),7.95(s,1H),6.58(s,1H),4.46(s,2H),4.09(d,J=12.4Hz,2H) ,3.18-3.07(dd,J=12.4Hz,1.2Hz,2H),2.17(d,J=21.2Hz,7H),1.90-1.81(m,2H),1.77-1.67(m,2H).

[0134] Step 2: Under a nitrogen gas atmosphere, compound M4-1 (1.24 mmol, 500 mg), potassium phosphate (3.72 mmol, 789.5 mg), compound M1-4 (1.86 mmol, 571.3 mg) and catalyst Pd(dtbpf)Cl2 (0.24 mmol, 155 mg) were dissolved in 10 mL of 1,4-dioxane, 2 mL of water was added, and the mixture was stirred for 5 minutes. The mixture was then heated to 90°C and reacted for 16 hours, cooled to room temperature, and the reaction was stopped and filtered. The mixture was added with 45 mL of water, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by column chromatography (DCM / MeOH=10 / 1) to obtain compound 3-(7-(3,5-dimethyl-1H-pyrazol-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M4-2 (420 mg, yield 71%). LC-MS: [M+H] + :475.5. 1 H NMR (400MHz, DMSO-d6) δ8.61(s,1H),7.89(d,J=8.0Hz,1H),4.23(q,J=7.2Hz,3H),1.29(t,J=6.8Hz,3H),1.20-1.14(m,2H),1.06-0.98(m,2H).

[0135] Step 3: Under a nitrogen gas atmosphere, compound M4-2 (0.32 mmol, 150 mg), potassium phosphate (0.63 mmol, 131 mg), 3-bromo-5-chloropyridine M4-3 (0.63 mmol, 122 mg) and ligand N1,N2-dimethyl-1,2-cyclohexanediamine (0.063 mmol, 8.99 mg) were dissolved in 5 mL of DMF, catalyst CuI (0.03 mmol, 6.02 mg) was added, the temperature was raised to 110 °C and stirring was continued for 4 hours, and the reaction was stopped by cooling to room temperature. 40 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (DCM / MeOH=15 / 1) to obtain compound 3-(7-(1-(5-chloropyridin-3-yl)-3,5-dimethyl-1H-pyrazol-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M4-4 (80 mg, yield 44%). LC-MS: [M+H] + :586.3. 1 H NMR (400MHz, DMSO-d6) δ8.61(s,1H),7.89(d,J=8.0Hz,1H),4.23(q,J=7.2Hz,3H),1.29(t,J=6.8Hz,3H),1.20-1.14(m,2H),1.06-0.98(m,2H).

[0136] Step 3: Under a nitrogen gas atmosphere, compound M4-4 (0.14 mmol, 80 mg) was dissolved in 4 mL of a mixed solvent of dichloromethane and trifluoroacetic acid (v / v=3 / 1) and reacted at room temperature for 2 hours. A saturated aqueous solution of sodium bicarbonate was added to the mixture to adjust the pH to about 9, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by preparative chromatography HPLC to obtain compound M4 (13.1 mg). LC-MS: [M+H] + :502.2. 1H NMR(400MHz,DMSO-d6)δ12.79(br s,1H),8.83(d,J=2.4Hz,1H),8.75(d,J=2.0Hz,1H),8.27(t,J=2.4Hz,2H),7.59(br s,1H),6.75(br s,1H),6.69(s,1H),4.50(s,2H),4.15(s,2H),3.20(d,J=11.6Hz,2H),2.38(s,3H),2.24(s,3H),1.90-1.74(m,4H).

[0137] With reference to the synthesis of compound M4, the following target compounds M5 to M15 were synthesized using similar raw materials / intermediates: TIFF2025503771000055.tif249170TIFF2025503771000056.tif249170TIFF2025503771 000057.tif248170TIFF2025503771000058.tif248170TIFF2025503771000059.tif21170

[0138] Example 3: Preparation of Compounds M16-M18 TIFF2025503771000060.tif60170

[0139] Step 1: Under a nitrogen gas atmosphere, the intermediate (1S,4S)-5-(7-(3,5-dimethyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-2-oxa-5-azabicyclo[2.2.1]heptane c6 (0.73 mmol, 300.0 mg) was dissolved in 5 mL of DMF, and N-bromosuccinimide NBS (0.89 mmol, 156.1 mg) was slowly added and reacted at room temperature for 2 hours to quench the reaction. 30 mL of water was added to the reaction solution, extracted with dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by flash reverse phase column chromatography (acetonitrile / water) to obtain compound (1S,4S)-5-(3-bromo-7-(3,5-dimethyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-2-oxa-5-azabicyclo[2.2.1]heptane M16-1 (5.0 mg, yield 6%). LC-MS: [M+H] + :309.9. This reaction was amplified repeatedly to give M16-1 (2.0 g).

[0140] Step 2: Under a nitrogen gas atmosphere, compound M16-1 (0.59 mmol, 230 mg), potassium phosphate (1.77 mmol, 376 mg), compound M1-4 (1.18 mmol, 329 mg) and catalyst Pd(dtbpf)Cl2 (0.12 mmol, 76 mg) were dissolved in 4 mL of 1,4-dioxane, 0.8 mL of water was added, and the mixture was stirred for 5 minutes. The mixture was then heated to 90°C and stirred for 16 hours, cooled to room temperature, and the reaction was stopped and filtered. 30 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (DCM / MeOH=50 / 1) to obtain compound (1S,4S)-5-(7-(3,5-dimethyl-1H-pyrazol-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-2-oxa-5-azabicyclo[2.2.1]heptane M16-2 (150 mg, yield 55%). LC-MS: [M+H] + :461.1.

[0141] Step 3: Under nitrogen gas atmosphere, compound M16-2 (0.33mmol, 150mg), potassium phosphate (0.65mmol, 138mg), 3-bromopyridine M1-7 (0.65mmol, 103mg) and ligand N1,N2-dimethyl 1,2-cyclohexanediamine (0.65mmol, 142mg) were dissolved in 5mL of DMF, catalyst CuI (0.33mmol, 62mg) was added, the temperature was raised to 110°C and stirring was continued for 16 hours, and the reaction was stopped by cooling to room temperature. 40mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain compound M16-3 as a crude product. LC-MS: [M+H] + :538.3.

[0142] Step 4: Under a nitrogen gas atmosphere, the crude product obtained in step 3, compound M16-3, was dissolved in 4 mL of a mixed solvent of dichloromethane and trifluoroacetic acid (v / v=3 / 1) and reacted at room temperature for 2 hours. A saturated aqueous solution of sodium bicarbonate was added to the mixture to adjust the pH to about 9, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by preparative chromatography HPLC to obtain compound M16 (13.8 mg). LC-MS: [M+H] + :454.2. 1 H NMR(400MHz,DMSO-d6)δ12.64(s,1H),8.83(d,J=2.4Hz,1H),8.67(d,J=4.8Hz ,1H),8.25(s,1H),8.08-8.02(m,1H),7.63(dd,J=8.0Hz,4.8Hz,2H),6.64(br s,2H),5.06(s,1H),4.75(s,1H),3.83(d,J=15.6Hz,2H),3.63(d,J=10.0Hz,2H),2.34(s,3H),2.24(s,3H),1.97(dd,J=25.2,9.2Hz,2H).

[0143] With reference to the synthesis of compound M16, the following target compounds M17 to M18 were synthesized using similar raw materials / intermediates: TIFF2025503771000061.tif210170

[0144] Example 4: Preparation of Compounds M1a and M1b TIFF2025503771000062.tif136170

[0145] Under a nitrogen gas atmosphere, intermediate a3 (3.75 mmol, 1.3 g) and raw material M1-1 (5.63 mmol, 1.18 g) were dissolved in 30 mL of a mixture of 1,4-dioxane and water (v / v: 9 / 1), potassium carbonate (11.25 mmol, 1.55 g) and Pd(dppf)Cl2 (0.4 mmol, 292 mg) were added, and the mixture was heated at 100 ° C. for 10 hours. The reaction was stopped, filtered, 80 mL of water was added to the system, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography (PE / EA = 2 / 1) to obtain compound M1-2 (1.1 g, 75% yield). LC-MS: [M + H] + :396.

[0146] In an ice bath, intermediate M1-2 (2.78 mmol, 1.1 g) was dissolved in 25 mL of anhydrous dichloromethane, N-iodosuccinimide NIS (3.06 mmol, 688 mg) was slowly added in portions, and stirring was continued for 2 hours. The reaction was stopped, 30 mL of saturated aqueous ammonium chloride was added, and the mixture was extracted with dichloromethane. The mixture was dried over anhydrous sodium sulfate and separated by flash column chromatography (PE / EA=2 / 1) to obtain compound M1-3 (1.4 g, 97% yield). LC-MS: [M+H] + :522.

[0147] Under a nitrogen gas atmosphere, intermediate M1-3 (2.69 mmol, 1.4 g) and raw material M1-4 (4.03 mmol, 1.1 g) were dissolved in 20 mL of a mixture of 1,4-dioxane and water (v / v: 9 / 1), and potassium phosphate (5.38 mmol, 1.1 g) and Xphos-Pd-G3 (0.27 mmol, 229 mg) were added. The reaction was carried out for 2 hours by microwave heating at 95 ° C. The reaction was stopped, filtered, 60 mL of water was added to the system, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography (PE / EA = 4 / 1) to obtain compound M1-5 (700 mg, yield 48%). LC-MS: [M + H] + :546.

[0148] Intermediate M1-5 (700 mg) was dissolved in 15 mL of tetrahydrofuran, 6 mL of 1N NaOH aqueous solution was added, and the mixture was heated to 70° C. and reacted for 1 hour. The reaction was stopped, the solvent was distilled off under reduced pressure, 30 mL of water was added, and the mixture was extracted with dichloromethane and separated by flash column chromatography (PE / EA=1 / 1) to obtain compound M1-6 (530 mg, yield 93%). LC-MS: [M+H] + :446.

[0149] Under nitrogen gas atmosphere, intermediate M1-6 (1.19 mmol, 530 mg), 3-bromopyridine M1-7 (1.78 mmol, 282 mg), potassium phosphate (2.38 mmol, 505 mg) and CuI (0.12 mmol, 23 mg) were added to a microwave reaction bottle and dissolved in 10 mL of DMF. After stirring for 5 minutes, N,N-dimethyl-1,2-cyclohexanediamine (0.24 mmol, 34 mg) was slowly added. The mixture was heated to 110°C in a microwave and reacted for 1.5 hours, cooled to room temperature, filtered, 50 mL of water was added to the reaction solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography to obtain compound M1-8 (240 mg, yield 39%). LC-MS: [M+H] + :523.

[0150] Under a nitrogen gas atmosphere, NaH (1.66 mmol, 60%, 40 mg) and trimethylsulfoxonium chloride (1.0 mmol, 129 mg) were added to the reaction bottle, and 1 mL of anhydrous DMSO was slowly added to the system and stirred for 20 minutes. Intermediate M1-8 (0.46 mmol, 240 mg) was dissolved in 1 mL of DMSO, and this mixture was slowly poured into the reaction solution. The temperature was raised to 65°C and the reaction was carried out for 4 hours, and the reaction was stopped. 20 mL of water was added to the reaction solution, and the solution was extracted with ethyl acetate to obtain 200 mg of intermediate M1-9 as a crude product. LC-MS: [M+H] + :537.

[0151] The crude intermediate M1-9 (200 mg) was dissolved in 4 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 2 hours. The reaction was stopped, the solvent was distilled off under reduced pressure, and the pH was adjusted to 10 by adding a saturated aqueous solution of sodium bicarbonate. The mixture was extracted with dichloromethane and separated by HPLC preparative chromatography to obtain compound M1 (70 mg, two-step yield 34%). LC-MS: [M+H] + :453.

[0152] Compound M1 was separated by chiral preparative chromatography to give the target compounds M1a and M1b. M1b: 1 H NMR(400MHz,DMSO-d6)δ12.91(d,J=111.8Hz,1H),8.85(d,J=2.5Hz,1H),8.68(d,J=4.7Hz,1 H),8.50(s,1H),8.10-8.03(m,1H),7.77(s,1H),7.64(dd,J=8.2,4.8Hz,1H),6.92(s,2H),3 .90-3.97(m,2H),3.65-3.70(m,1H),3.38-3.41(m,1H),2.91-2.97(m,1H),2.34(s,3H),2.2 4(s,3H),1.99-2.01(m,1H),1.91(s,1H),1.55(dd,J=9.3,4.2Hz,1H),1.23(d,J=5.5Hz,1H).

[0153] Example 5: Preparation of Compounds M2a and M2b TIFF2025503771000063.tif129170

[0154] In an ice bath, intermediate b1 (3.18 mmol, 1.3 g) was dissolved in 25 mL of anhydrous dichloromethane, N-iodosuccinimide NIS (3.50 mmol, 787 mg) was slowly added in portions, and stirring was continued for 2 hours. The reaction was stopped, 35 mL of saturated aqueous ammonium chloride was added, and the mixture was extracted with dichloromethane. The mixture was dried over anhydrous sodium sulfate and separated by flash column chromatography (PE / EA=2 / 1) to obtain compound M2-1 (1.3 g, 77% yield). LC-MS: [M+H] + :536.

[0155] Under a nitrogen gas atmosphere, intermediate M2-1 (2.43 mmol, 1.3 g) and raw material M1-4 (3.65 mmol, 1.01 g) were dissolved in 20 mL of a mixture of 1,4-dioxane and water (v / v: 9 / 1), and potassium phosphate (4.86 mmol, 1.03 g) and Xphos-Pd-G3 (0.24 mmol, 203 mg) were added. The reaction was carried out for 2 hours by microwave heating at 95 ° C. The reaction was stopped, filtered, 60 mL of water was added to the system, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography (PE / EA = 4 / 1) to obtain compound M2-2 (1.3 g, yield 96%). LC-MS: [M + H] + :560.

[0156] Intermediate M2-2 (1300 mg) was dissolved in 20 mL of tetrahydrofuran, 8 mL of 1N NaOH aqueous solution was added, and the mixture was heated to 70° C. and reacted for 1 hour. The reaction was stopped, the solvent was distilled off under reduced pressure, 40 mL of water was added, and the mixture was extracted with dichloromethane and separated by flash column chromatography (PE / EA=1 / 1) to obtain compound M2-3 (1.0 g, yield 94%). LC-MS: [M+H] + :460.

[0157] Under nitrogen gas atmosphere, intermediate M2-3 (0.65mmol, 300mg), 3-bromo-5-fluoro-pyridine M2-4 (0.98mmol, 172mg), potassium phosphate (1.30mmol, 276mg) and CuI (0.06mmol, 12mg) were added to a microwave reaction bottle and dissolved in 8mL of DMF. After stirring for 5 minutes, N,N-dimethyl-1,2-cyclohexanediamine (0.12mmol, 17mg) was slowly added. After heating to 110°C in a microwave and reacting for 1.5 hours, the reaction was stopped and filtered. 30mL of water was added to the reaction solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and separated by flash column chromatography (PE / EA=3 / 1) to obtain compound M2-5 (200mg, yield 56%). LC-MS: [M+H] + :555.

[0158] Intermediate M2-5 (0.36 mmol, 200 mg) was dissolved in 4 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 2 hours. The reaction was stopped, the solvent was distilled off under reduced pressure, and the pH was adjusted to 10 by adding saturated aqueous sodium bicarbonate solution. The mixture was extracted with dichloromethane and separated by HPLC preparative chromatography to obtain compound M2 (80 mg, 48% yield). LC-MS: [M+H] + :471.

[0159] Compound M2 was separated by chiral preparative chromatography to give the target compounds M2a and M2b. M2b: 1H NMR(400MHz,DMSO-d6)δ12.91(d,J=117.3Hz,1H),8.77(s,1H),8.73(d,J=2.6Hz,1H),8.4 9(s,1H),8.14(dd,J=9.6,2.7Hz,1H),7.69(d,J=79.0Hz,1H),6.90(s,2H),3.90-3.97(m, 2H),3.65-3.70(m,1H),3.35-3.41(m,1H),2.94(dt,J=14.4,4.6Hz,1H),2.38(s,3H),2.2 4(s,3H),2.01-2.08(m,1H),1.91(s,1H),1.54(dd,J=9.2,4.2Hz,1H),1.22-1.26(m,1H).

[0160] Example 6: Preparation of compound M19 TIFF2025503771000064.tif66170

[0161] Step 1: Under a nitrogen gas atmosphere, compound 3-(3-bromo-7-(3,5-dimethyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M4-1 (1.24 mmol, 500 mg), potassium phosphate (3.72 mmol, 789.5 mg), compound M19-1 (1.49 mmol, 434.7 mg) and catalyst Pd(dtbpf)Cl2 (0.12 mmol, 80 mg) were dissolved in 10 mL of 1,4-dioxane, 2 mL of water was added, and the mixture was stirred for 5 minutes. The mixture was then heated to 90 ° C. and stirred for 16 hours, cooled to room temperature, and the reaction was stopped and filtered. The mixture was added with 40 mL of water, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by column chromatography (DCM / MeOH=10 / 1) to obtain compound 3-(7-(3,5-dimethyl-1H-pyrazol-4-yl)-3-(3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M19-2 (460 mg, yield 76%). LC-MS: [M+H] + :489.3.

[0162] Step 2: Under nitrogen gas atmosphere, compound M19-2 (0.41 mmol, 200 mg), potassium phosphate (0.82 mmol, 174 mg), 3-bromo-pyridine M1-7 (0.82 mmol, 130 mg) and ligand N1,N2-dimethyl 1,2-cyclohexanediamine (0.41 mmol, 58 mg) were dissolved in 5 mL of DMF, catalyst CuI (0.20 mmol, 39 mg) was added, the temperature was raised to 110 ° C and stirring was continued for 16 hours, and the reaction was stopped by cooling to room temperature. 40 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain compound M19-3 as a crude product. LC-MS: [M + H] + :566.3.

[0163] Step 3: Under a nitrogen gas atmosphere, the crude product M19-3 obtained in step 2 was dissolved in 6 mL of a mixed solvent of dichloromethane and trifluoroacetic acid (v / v=3 / 1), reacted at room temperature for 2 hours, and the reaction was stopped. A saturated aqueous solution of sodium bicarbonate was added to the mixture to adjust the pH to about 9, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by preparative chromatography HPLC to obtain compound M19 (79.5 mg). LC-MS: [M+H] + :482.3. 1 H NMR(400MHz,DMSO-d6)δ12.30(s,1H),8.83(d,J=2.4Hz,1H),8.67(dd,J=4.8Hz,1.2Hz,1H),8.23(s,1H),8.07-8.02(m,1H),7.65-7.60(m,1H) ,6.70(s,1H),6.51(s,1H),4.50(d,J=2.0Hz,2H),4.16(d,J=12.4Hz,2H ),3.19(d,J=11.2Hz,2H),2.33(s,3H),2.23(s,6H),1.92-1.73(m,4H).

[0164] Example 7: Preparation of compound M20 TIFF2025503771000065.tif34170

[0165] Under a nitrogen gas atmosphere, compound 3-(7-(3,5-dimethyl-1H-pyrazol-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M4-2 (0.11 mmol, 50 mg) was dissolved in 4 mL of a mixed solvent (v / v=3 / 1) of dichloromethane and trifluoroacetic acid, reacted at room temperature for 2 hours, and the reaction was stopped. A saturated aqueous solution of sodium bicarbonate was added to the mixture to adjust the pH to about 9, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by preparative chromatography HPLC to obtain compound M20 (18.0 mg). LC-MS: [M+H] + :391.2. 1 H NMR(400MHz,DMSO-d6)δ12.67(s,2H),8.23(s,1H),7.58(s,1H),6.73(s,1H),6.56(s, 1H),4.48(s,2H),4.14(s,2H),3.16(d,J=10.8Hz,2H),2.20(s,6H),1.90-1.73(m,4H).

[0166] Example 8: Preparation of Compounds M21-M22 TIFF2025503771000066.tif34170

[0167] Step 1: Under a nitrogen gas atmosphere, compound 3-(7-(3,5-dimethyl-1H-pyrazol-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M4-2 (0.32mmol, 150mg) was dissolved in 5mL of anhydrous DMF, NaH (0.47mmol, 18.96mg) was added, and the mixture was stirred for 5 minutes. After that, raw material M21-1 (0.47mmol, 132.4mg) was added, and the mixture was heated to 70°C and stirred for 16 hours. The mixture was cooled to room temperature, and the reaction was stopped and filtered. 40mL of water was added to the mixture, which was then extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain compound M21-2 as a crude product. LC-MS: [M+H] + :658.5.

[0168] Step 2: Under a nitrogen gas atmosphere, the crude product M21-2 obtained in step 1 was dissolved in 4 mL of a mixed solvent of dichloromethane and trifluoroacetic acid (v / v=3 / 1), reacted at room temperature for 2 hours, and the reaction was stopped. A saturated aqueous solution of sodium bicarbonate was added to the mixture to adjust the pH to about 9, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by preparative chromatography HPLC to obtain compound M21 (38.89 mg). LC-MS: [M+H] + :474.3. 1 H NMR(400MHz,CDCl3)δ8.34(s,1H),8.23(s,1H),7.59(s,1H),6.74(s,1H),6.57(s,1H),4.48(s,2H),4.44-4.35(m,1H),4.15(d,J=12.0Hz,2H) ,3.25(d,J=11.2Hz,2H),3.16(d,J=11.6Hz,2H),2.87(t,J=11.6Hz,2H) ,2.25(s,3H),2.15-2.03(m,5H),1.94-1.82(m,4H),1.80-1.72(m,2H).

[0169] With reference to the synthesis of compound M21, the target compound M22 was synthesized using similar raw materials / intermediates as follows: TIFF2025503771000067.tif109170

[0170] Example 9: ATR kinase activity experiments ATR kinase activity experiments were performed by detecting the downstream substrate p53 protein phosphorylation of ATR kinase. GST-tagged full-length p53 protein was purchased from Sigma (catalog number: 14-865), and anti-phospho-p53(ser15)-K antibody and anti-GST-d2 antibody were both purchased from Cisbio (catalog numbers: 61GSTDLA; 61P08KAE). The amount of phosphorylated p53 protein was measured by a time-resolved fluorescence system. Before starting the experiment, the following working solutions were prepared as needed: 1x reaction buffer (20mM HEPES PH8.0, 1% glycerin, 0.01% Brij-35), diluted buffer (20mM HEPES PH8.0, 1% glycerin, 0.01% Brij-35, 5mM DTT and 1% BSA), termination solution (20mM HEPES PH8.0, 1% glycerin, 0.01% Brij-35, 250mM EDTA), detection buffer (50mM HEPES PH7.0, 150mM NaCl, 267mM KF, 0.1% sodium cholate, 0.01% Tween-20, 0.0125% sodium azide). Clinical research drug AZD6738 (purchased from selleck) was used as a positive control.

[0171] The specific steps of the experiment are as follows: 4x serially diluted compound solutions were prepared in 1x reaction buffer to obtain 9 compounds with different concentrations, and 2.5 μL of 4x serially diluted compound solutions were added to a 384-well assay plate (784075, Greiner). 4x p53 substrate working solution (40 nM) was prepared in 1x reaction buffer, and 2.5 μL of 4x p53 substrate working solution was added to a 384-well assay plate. 4x ATR / ATRIP working solution (12.8 ng / μL) was prepared in diluted buffer, and 2.5 μL of 4x ATR / ATRIP working solution was added to a 384-well assay plate. 4x ATP working solution (2 mM) was prepared in deionized water, and 2.5 μL of 4x ATP working solution was added to a 384-well assay plate and incubated for 30 min at room temperature, protected from light. 5 μL of the final solution was added to a 384-well assay plate. Finally, 5 μL of detection mixture (0.09 ng / μL Anti-phospho-p53(ser15)-K and 6 ng / μL Anti-GST-d2) was added to the 384-well assay plate. After overnight incubation at room temperature, the fluorescence signals (excitation wavelength 320 nm, emission wavelength 620 nm and 665 nm) were detected by M5e (Molecular Device) instrument. The inhibition rate in each well was calculated according to the fluorescence intensity value of each well: ER (Emission Ratio) = (fluorescence intensity at 665 nm / fluorescence intensity at 620 nm); inhibition rate = (ER positive-ER test compound) / (ER positive-ER negative) × 100%. The IC of the compound was calculated by parameter fitting standard software (GraphPad Prism 8.0). 50 Number (IC 50 The inhibitory concentration at which the effect is 50% of the maximum was calculated. The results are shown in Table 1.

[0172] Table 1 TIFF2025503771000068.tif92170

[0173] According to the data in Table 1, the compounds of the present invention have excellent activity in inhibiting ATR kinase, and the activity is equal to or greater than that of the clinical investigational drug AZD6738, indicating that the compounds of the present invention act selectively on tumor cells as ATR inhibitors and are promising as excellent potential drugs for treating tumors.

[0174] Example 10: In vitro cell proliferation inhibition experiment 1 22Rv1 cells were purchased from the American Type Culture Collection (ATCC).

[0175] The specific operation steps of the experiment are as follows: 22Rv1 cells were cultured in RPMI 1640 medium (containing 10% FBS and 1% penicillin-streptomycin (ps)) and used for the test when the cell confluence reached 85% or more. Approximately 2000 cells were seeded per well in a 96-well culture plate and cultured for 24 hours, and cells treated with different concentrations of test compounds (0-50 μM) were added, and three parallel wells were set up for each group. Blank wells (containing only medium) and control wells (cells seeded, no drug) were set up. After culturing for 120 hours, 10 μL of CCK8 solution (Beyotime, #C0037) was added to each well, incubated for 4 h in the dark, and the OD value was read with a Biotek Synergy H1 multifunction microplate reader.

[0176] Inhibition rate (%) = 100% x (control wells - experimental wells) / (control wells - blank wells) The IC of the compounds was calculated using standard parameter fitting software (GraphPad Prism 8.0). 50 The values ​​were calculated and the results are shown in Table 2.

[0177] Table 2 TIFF2025503771000069.tif73170

[0178] The results of this growth inhibition experiment confirmed that the compounds of the present invention have good inhibitory effects on ATM-mutated human prostate cancer cells 22Rv1, and some IC 50 The value can be less than 1 μM or even less than 0.3 μM.

[0179] Example 11: In vitro cell proliferation inhibition experiment 2 In this experiment, the inhibitory effect of the compounds of the present invention on tumor cell proliferation was investigated by detecting the effect of the compounds of the present invention on cell proliferation in vitro in tumor cell lines TOV21G, SK-OV-3 (ovarian cancer), Rec-1 (lymphoma), HCT-116 (colon cancer), MDA-MB-231 (breast cancer), NCI-H23 (non-small cell lung cancer), SNU216 (gastric cancer), OS-RC-2 (renal cancer), T24 (bladder cancer), AsPC-1 (pancreatic cancer), M14 (melanoma) and U2OS (osteosarcoma).

[0180] TOV21G and Rec-1 cells were both purchased from the American Type Culture Collection (ATCC), and other cells were purchased from Shanghai Medicilon Biopharmaceutical Co., Ltd.

[0181] TOV21G cells were cultured in MCDB105 / M199 medium (containing 15% FBS and 1% ps) and used for the test when the cell confluence reached 85% or more. Approximately 1000 cells were seeded per well in a 96-well culture plate and cultured for 24 hours, and cells treated with different concentrations of test compounds (0-10 μM) were added, and three parallel wells were set up for each group. Blank wells (containing only medium) and control wells (cells seeded, no drug) were set up. After culturing for 120 hours, 40 μL of Cell Titer-Glo solution (Promega, #G7573) was added to each well, and incubated with shaking for 25 min in the dark, and 100 μL per well was transferred to a 96-well blank plate (Corning, #3917), and the luminescence value was read using a Biotek Synergy H1 multifunction microplate reader.

[0182] Rec-1 cells were cultured in RPMI1640 medium (containing 10% FBS and 1% ps), seeded at about 6000 cells per well in a 96-well culture plate, and treated with different concentrations of test compounds (0-10 μM) were added, with three parallel wells in each group. Blank wells (containing only medium) and control wells (cells seeded and not treated with drugs) were set up. After 120 hours of culture, 40 μL of Cell Titer-Glo solution was added to each well, and incubated with shaking for 20 min in the dark, and 100 μL per well was transferred to a 96-well blank plate, and the luminescence value was read using a Biotek Synergy H1 multifunction microplate reader.

[0183] Other cell culture and experimental schemes refer to those of the two cell lines mentioned above, and the cell culture medium used was the normal medium recommended by ATCC.

[0184] Inhibition rate (%) = 100% x (control wells - experimental wells) / (control wells - blank wells) The IC of the compounds was calculated using standard parameter fitting software (GraphPad Prism 8.0). 50 The values ​​were calculated and the results are shown in Tables 3 and 4.

[0185] Table 3 TIFF2025503771000070.tif40170

[0186] Table 4. Antiproliferative effects of compound M3 on various tumor cells. TIFF2025503771000071.tif86170

[0187] The results of this growth inhibition experiment confirmed that the compounds of the present invention have good inhibitory effects on the human tumor cells studied.

[0188] Example 12: In vitro liver microsomal stability studies of compounds A liver microsome stability experiment study was carried out on the compounds of the present invention. The test compounds (final concentration 2.0 nM) were co-incubated with human / mouse liver microsomes with or without the addition of NADPH, and the concentration of the compound in the incubation supernatant was detected within 60 minutes. The results of a representative compound are shown in Table 5 below.

[0189] Table 5 TIFF2025503771000072.tif27170

[0190] These results demonstrate that the molecules of the present invention have extremely high stability in mouse and human liver microsomes and are slowly metabolized in vivo.

[0191] Example 13: In vivo pharmacokinetic studies of compounds In vivo pharmacokinetic studies were carried out on compounds of the present invention.

[0192] Testing Method: Male ICR mice (3 mice / group) were orally administered with 10 mg / kg of the compound by gavage. Plasma samples were collected before (0 h) and after (0.25, 0.5, 1, 2, 4, 6, 8, and 24 h) administration, and the collected samples were analyzed by LC / MS to obtain data. The collected analytical data was used to calculate the relevant parameters of pharmacokinetics using Analyst v1.6.2 (AB Applied Biosystems Company, USA) software.

[0193] Results for representative compounds are shown in Table 6 below.

[0194] Table 6 TIFF2025503771000073.tif33170

[0195] From the above results, the molecule of the present invention is expected to have a very high in vivo exposure after oral administration to mice and to have an excellent antitumor effect at a lower dose in clinical practice.

[0196] Example 14: Study of compound selectivity against ATR kinase The same family (ATM, DNA-PK) inhibition experiments were carried out for the compounds of the present invention: Based on the experimental method reported in the literature, the test compounds were diluted 3-fold from 10 μM to 0.51 nM (total of 10 concentrations) and used to stimulate the kinase ATM. 1 and DNA-PK 2 The inhibitory activity against each of these compounds was measured, and the results are shown in Table 7 below.

[0197] Table 7: TIFF2025503771000074.tif27170

[0198] The above results indicate that the compounds of the present invention have high selectivity for ATR and low inhibitory activity against other kinases in the family, which provides the advantage of higher safety.

[0199] The above content has specifically described the present invention in more detail with reference to preferred embodiments, but the specific examples of the present invention are not limited thereto. A person skilled in the art may make various simple inferences or substitutions without departing from the concept of the present invention, and all of them should be considered as falling within the protection scope of the present invention.

[0200] References: [1]Discovery of Novel 3-Quinoline Carboxamides as Potent,Selective and Orally Bioavailable Inhibitors of Ataxia Telangiectasia Mutated(ATM)Kinase,J. Med. Chem. 2016,56,6281-6292; [2]The Discovery of 7-Methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]- 9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one(AZD7648),a Potent and Selective DNA-Dependent Protein Kinase(DNA-PK)Inhibitor,J. Med. Chem. 2020,63,3461-3471。

Claims

1. A compound represented by the general formula (I), a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: Where: R １ teeth, Selected from R ｘ is selected from H, a 5-6 membered heteroaryl group, or a 5-6 membered heterocyclic group, wherein the 5-6 membered heteroaryl group or the 5-6 membered heterocyclic group is R ａ and optionally substituted with R ａ is H, halogen, -CN, -NH ２ , C １－４ Alkyl group, C １－４ Alkoxy group or C １－４ halogenated alkyl groups, and R ｙ is H, halogen, NH ２ , C １－４ Alkyl group or C １－４ The alkoxy group is selected from the group consisting of alkoxy groups.

2. R １ teeth, Selected from 2. The compound according to claim 1, its stereoisomer or a pharma- ceutically acceptable salt thereof.

3. R ｘ is selected from H, a 6-membered heteroaryl group, or a 6-membered heterocyclic group, wherein the 6-membered heteroaryl group or the 6-membered heterocyclic group is R ａ and optionally substituted with R ａ is H, halogen, -CN, -NH ２ , C １－４ Alkyl group, C １－４ Alkoxy group or C １－４ Selected from halogenated alkyl groups 3. The compound according to claim 1, its stereoisomer or a pharma- ceutically acceptable salt thereof.

4. R ｙ is H or C １－４ Selected from alkyl groups A compound according to any one of claims 1 to 3, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof.

5. A compound having the structure of general formula (II), a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, Where: R １ teeth, Selected from R ａ is H, halogen, -CN, -NH ２ , C １－４ Alkyl group, C １－４ Alkoxy group or C １－４ halogenated alkyl groups, and R ｙ is selected from H or a methyl group 2. The compound according to claim 1, its stereoisomer or a pharma- ceutically acceptable salt thereof.

6. The compound is Selected from 5. A compound according to any one of claims 1 to 4, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof.

7. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 6, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable adjuvant.

8. Further comprising other active agents applicable for the treatment of cancer. The pharmaceutical composition according to claim 7.

9. The pharmaceutical compositions may be prepared as injectables, e.g., sterile aqueous solutions, non-aqueous solutions, dispersions, suspensions, or emulsions; solid oral preparations, e.g., tablets, capsules, powders, granules, or pills; or liquid oral preparations, e.g., solutions, emulsions, suspensions, or syrups. A pharmaceutical composition according to claim 7 or 8.

10. 10. Use of a compound according to any one of claims 1 to 6, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 7 to 9 in the manufacture of a medicament for treating cancer.

11. The cancer is selected from breast cancer, renal cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, lymphoma, melanoma, myeloma, and osteosarcoma. The use according to claim 10.

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