Novel pyrazolopyrimidine compounds and their compositions, methods for producing them, and uses

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] This invention belongs to the pharmaceutical field and relates to novel pyrazolopyrimidine compounds and their compositions, methods for producing them, and their use in the production of anticancer agents. [Background technology]

[0002] Cancer threatens human health and life. In recent years, research into anticancer drugs has shifted towards the development and research of specific molecularly targeted therapies.

[0003] ATR, also known as ataxia telangiectasia and Rad3-related kinase, is a crucial kinase consisting of 2644 amino acids that can activate cellular responses after DNA damage. It also inhibits cell cycle progression, stabilizes replication forks, and repairs DNA, thereby preventing apoptosis. When DNA replication pressure or DNA damage occurs within a cell, ATR is recruited to the DNA damage site, and various proteins are involved in regulating ATR activation. After activation, ATR can regulate cellular biological processes, including cell cycle blocking, inhibition of replication origins, promotion of deoxyribonucleotide synthesis, replication fork activation, and repair of DNA double-strand breaks, through various signaling pathways.

[0004] Because tumor cells have defects in various DNA repair pathways, they are more dependent on the ATR repair pathway and more sensitive to ATR inhibitors than normal cells. "Synthetic lethality" refers to the phenomenon in tumor cells where a defect caused by a mutation in one pathway leads to the tumor cell's dependence on another complementary pathway more than normal cells. Therefore, if this complementary pathway is inhibited, it results in synthetic lethality in the tumor cell. In contrast, normal cells have another pathway that is functioning normally, so they are not killed by drug inhibition. Studies have found that partially mutated ATR is a target of synthetic lethality, and that, for example, ATM-deficient tumor cells are more sensitive to ATR inhibitors. It has also been found that tumor cells are more sensitive to ATR inhibitory effects when X-rays intersect and complementarily repair the gene I defect. For this reason, ATR inhibitors are promising as excellent potential drugs for treating tumors because they selectively affect tumor cells but have less interference with normal cells.

[0005] As the next most promising "synthetic lethal" therapy after PARP inhibitors, ATR inhibitors have attracted the attention of some giant multinational corporations, including Merck, Bayer, and AstraZeneca. Among these, Merck's Berzosertib is leading the clinical development and is currently in Phase II clinical trials. While few companies in China are developing ATR inhibitors, IMPACT Therapeutics' IMP9064 received FDA Phase I / II clinical trial approval on October 29, 2021. ATR inhibitors approved by the China National Medical Products Administration (NMPA) for clinical trials include Merck's Berzosertib, Bayer's BAY1895344, and Shijiazhuang Zhikang Hongren New Drug's ATR inhibitor, all of which are in Phase I clinical trials. One of the drugs currently under clinical research is AstraZeneca's AZD6738, whose structure is as follows: TIFF0007854505000001.tif33170In the experimental part of the present invention, it serves as a positive control for the ATR kinase activity test. However, to date, there is no approved and commercially available ATR inhibitor. Therefore, the development of new ATR inhibitors with stronger selectivity and better activity is still required.

Summary of the Invention

[0006] One object of the present invention is to provide a novel ATR inhibitor having excellent tumor inhibitory activity, strong selectivity, good water solubility, low toxicity, and applicable to oral administration or intravenous injection.

[0007] According to one form, the present invention provides a compound represented by the general formula (I), its stereoisomer or its pharmaceutically acceptable salt: TIFF0007854505000002.tif36170Here, R1 is TIFF0007854505000003.tif111170selected from R

[0009] , , is selected from H, a 5- to 6-member heteroaryl group or a 5- to 6-member heterocyclic group, where the 5- to 6-member heteroaryl group or 5- to 6-member heterocyclic group may be optionally substituted with R a and R a is selected from H, halogen, -CN, -NH2, C 1-4 alkyl group, C 1-4 alkoxy group or C 1-4 halogenated alkyl group, and R y is selected from H, halogen, NH2, C 1-4 alkyl group or C 1-4 alkoxy group.

[0008] In one embodiment, in the general formula (I), R1 is selected from the following groups: TIFF0007854505000004.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, where the 6-membered heteroaryl group or 6-membered heterocyclic group is R a It may be arbitrarily substituted with R a H, halogen, -CN, -NH2, C 1-4 Alkyl alkyl group, C 1-4 Alkoxy group or C 1-4 Selected from alkyl halogens.

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

[0011] In one embodiment, the compound has the structure of general formula (II): TIFF0007854505000005.tif40170 Here, R1 is selected from the following bases: TIFF0007854505000006.tif36170R a H, halogen, -CN, -NH2, C 1-4 Alkyl alkyl group, C 1-4 Alkoxy group or C 1-4 Selected from halogenated alkyl groups, R y It is selected from H or a methyl group.

[0012] In one embodiment, the compound of the present invention is selected from the following compounds: TIFF0007854505000007.tif250170TIFF0007854505000008.tif212170TIFF0007854505000009.tif105170

[0013] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of general formula (I) or (II) of the present invention, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

[0014] In one embodiment, the pharmaceutical composition further comprises other active agents applicable to the treatment of cancer.

[0015] In one embodiment, the pharmaceutical composition may be prepared as an injectable preparation such as a sterile aqueous solution, a non-aqueous solution, a dispersion, a suspension, or an emulsion; a solid oral preparation such as a tablet, capsule, powder, granule, or pill; or a liquid oral preparation such as a solution, emulsion, suspension, or syrup.

[0016] In yet another embodiment, the present invention provides the use of a compound of the general formula (I) or (II) of the present invention, its stereoisomer, or a pharmaceutically acceptable salt thereof in the manufacture of a drug for treating cancer. The present invention further provides the use of the pharmaceutical composition of the present invention in the manufacture of a drug for treating cancer.

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

[0018] The present invention offers 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 in inhibiting ATR kinase, and this activity is equivalent to or greater than that of the clinical research drug AZD6738, demonstrating that the compounds of the present invention are applicable as ATR inhibitors for the treatment of ATR-mediated diseases.

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

[0021] 4. The pyrazolopyrimidine compounds provided by the present invention have been shown to have very 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 very high in vivo exposure levels after oral administration to mice, and are expected to produce excellent antitumor effects at clinically lower doses.

[0023] 6. The pyrazolopyrimidine compounds provided by the present invention have high selectivity for ATR and low inhibitory activity against other kinases in the family, thereby providing the benefit of greater safety. [Modes for carrying out the invention]

[0024] Unless otherwise defined, all technical and scientific terms of this invention have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

[0025] The above brief description and the following detailed description are for illustrative purposes only and do not limit the subject matter of the present invention.

[0026] definition The term "C" as used herein 1-4 An "alkyl group" refers to a linear or branched 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] The term "C" as used herein 1-4 An "alkoxy group" is defined by the formula -OC. 1-4This refers to a group having an alkyl group, where C 1-4 Alkyl groups are defined as described above. C 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] The term "C" as used herein 1-4 A "halogenated alkyl group" is a C atom substituted with one or more halogen atoms. 1-4 This refers to alkyl groups. Examples include, but are not limited to, fluoromethyl groups, difluoromethyl groups, trifluoromethyl groups, fluoroethyl groups, 1,1-difluoroethyl groups, chloromethyl groups, chloroethyl groups, dichloromethyl groups, and 1,2-dichloroethyl groups, with difluoromethyl and trifluoromethyl groups being preferred.

[0030] As used herein, the term "5-6 membered heteroaryl group" refers to a stable 5-6 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 heteroatoms at their positions or at carbon atoms 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] As used herein, the term "5-6 membered heterocyclic group" refers to a stable 5-6 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 completely saturated. The heteroatoms may be bonded to the heterocyclic group by their positions or by carbon atoms of the heterocycle. Examples of 5-6 membered heterocyclic groups include dihydrofuranyl, dihydrothienyl, 3-pyrrolinyl, 2-pyrrolinyl, 2-imidazolinyl, 2-pyrazolidinyl, dihydrooxazolyl, dihydrothiazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydro-1,2,3-triazolyl, dihydro-1,2,4-triazolyl, and dihydro-1,2,5-oxadiazolyl (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 (thiadiazolyl), dihydro-1,2,3-thiadiazolyl group, dihydro-1,2,4-thiadiazolyl group, dihydro-1,3,4-thiadiazolyl group, tetrahydrofuranyl group, tetrahydrothienyl L group, pyrrolidinyl group, imidazolidinyl group, pyrazolidinyl group, oxazolidinyl group, thiazolidinyl group, isoxazolidinyl group, isothiazolidinyl group, 1,2,3-triazolidinyl group, 1,2,4-triazolidinyl group, 1,2,5-oxadiazolidinyl group, 1,2,3-oxadiazolidinyl group, 1, This includes, but is 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] A "stereoisomer" refers to a compound that consists of the same atoms and is bonded by the same bonds, but has a different three-dimensional structure. This invention includes various stereoisomers and mixtures thereof.

[0033] As used herein, the term “subject” includes mammals and non-mammals. Examples of mammals include, but are not limited to, all members of the class Mammalia, including humans, non-human primates (e.g., chimpanzees, other monkeys and apes), domestic animals such as cattle, horses, sheep, goats and pigs, domestic animals such as rabbits, dogs and cats, and experimental animals such as rodents such as rats, mice and guinea pigs. In some embodiment, the subject is human.

[0034] As used herein, “treatment” and other similar synonyms include alleviating, reducing or improving the symptoms of a disease or condition, and suppressing a disease or condition, for example, preventing the development of a disease or condition, alleviating a disease or condition, improving a disease or condition, alleviating or discontinuing symptoms caused by a disease or condition, preventing other symptoms, and improving or preventing potential metabolic factors that lead to symptoms, wherein the term includes the purpose of prevention. The term further includes obtaining a therapeutic effect and / or preventive effect. The therapeutic effect means curing or improving the potential disease being treated. However, a cure or improvement of one or more physiological symptoms associated with the potential disease is also a therapeutic effect, for example, an improvement in the subject's condition is observed, although the subject may still be affected by the potential disease. Regarding preventive effects, the compound or composition of the present invention can be administered to subjects at risk of contracting a specific disease, or to subjects who have not yet been diagnosed with a disease but exhibit one or more physiological symptoms of the disease.

[0035] As used herein, “therapeutic dose” means the amount of at least one active substance (e.g., the compounds 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 a reduction and / or remission of signs, symptoms or etiology, or any other required change in the biological system. For example, “therapeutic dose” is the amount of a composition containing the compounds disclosed herein that is necessary to provide a clinically significant symptomatic relief effect. For example, the optimal therapeutic dose for any individual case can be determined using the technique of dose escalation experiments.

[0036] As used herein, terms such as “administration” and “dosage” mean a method by which a compound or composition can be delivered to a site where a biological effect is required. These methods include, but are not limited to, oral administration, duodenal administration, extraintestinal injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial injection or infusion), topical application, and rectal administration. The administration techniques used for the compounds and methods described herein are well known to those skilled in the art.

[0037] As used herein, the term "pharmaceutically acceptable adjuvant" means a substance that does not affect the biological activity or properties of the compound of the present invention, and that is relatively non-toxic, i.e., administered to an organism in such a way that it does not cause an adverse biological reaction or interact with any component of the composition in an adverse manner. The pharmaceutically acceptable adjuvant includes, but is not limited to, carriers, stabilizers, diluents, powders, suspending agents, thickeners and / or excipients.

[0038] As used herein, the terms “optional,” “optional,” or “optionally” indicate that the event or situation described later may or may not occur, and that such description includes both cases in which the event or situation occurs and cases in which it does not occur. For example, “R a "May be arbitrarily substituted with " means that it is not substituted, or R a This indicates that it has been replaced with, and that the description has not been replaced and Ra This also includes cases where substitution has occurred.

[0039] Compounds of general formula (I), their stereoisomers, or pharmaceutically acceptable salts thereof This invention provides compounds for treating, improving, or preventing cancer. As described herein, inhibiting ATR kinase can selectively affect tumor cells with minimal interference to normal cells. The compounds of this invention can effectively inhibit the activity of ATR kinase, exhibiting activity equivalent to or greater than that of the clinical research drug AZD6738, and are promising as excellent potential agents for treating tumors.

[0040] In one embodiment, the present invention provides a compound of general formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof: TIFF0007854505000010.tif36170 Here, R1 is Selected from TIFF0007854505000011.tif111170; R x is selected from H, a 5-6 membered heteroaryl group, or a 5-6 membered heterocyclic group, where the 5-6 membered heteroaryl group or 5-6 membered heterocyclic group is R a It may be arbitrarily replaced with, R a H, halogen, -CN, -NH2, C 1-4 Alkyl alkyl group, C 1-4 Alkoxy group or C 1-4 Selected from halogenated alkyl groups, R y H, halogen, NH2, C 1-4 Alkyl alkyl group or C 1-4 Selected from alkoxy groups.

[0041] In one embodiment, R1 is selected from the following groups: TIFF0007854505000012.tif73170

[0042] In one embodiment, R1 is selected from the following groups or their stereoisomers (when applied): TIFF0007854505000013.tif36170

[0043] In one embodiment, R1 is selected from the following groups or their stereoisomers (when applied): TIFF0007854505000014.tif36170

[0044] In one embodiment, R1 is Selected from TIFF0007854505000015.tif35170.

[0045] In one embodiment, R1 is The filename is TIFF0007854505000016.tif34170.

[0046] In one embodiment, R x is selected from H, a 5-6 membered heteroaryl group, or a 5-6 membered heterocyclic group, where the 5-6 membered heteroaryl group or 5-6 membered heterocyclic group is R a It may be replaced as needed.

[0047] In one embodiment, R x H is H.

[0048] In one embodiment, R x is a 5-6 member heteroaryl group. In one embodiment, R x R is a 5-6 membered heteroaryl group containing one or two heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, R x R is a 5-6 membered heteroaryl group containing one or two heteroatoms selected from nitrogen and oxygen. In some embodiments, R x R is a six-membered heteroaryl group containing one or two nitrogen atoms. In some embodiments, R x The group is selected from a pyridyl group, a pyrimidinyl group, or an isoxazolyl group. In some embodiments, the heteroaryl group is R aIt may be replaced as needed.

[0049] In one embodiment, R x is a 5-6 membered heterocyclic group. In one embodiment, R x R is a 5-6 membered heterocyclic group containing one or two heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, R x R is a six-membered heterocyclic group containing one or two heteroatoms selected from nitrogen and oxygen. In one embodiment, R x R is a six-membered heterocyclic group containing one or two nitrogen atoms. In some embodiments, R x R is a six-membered heterocyclic group containing one nitrogen atom. In one embodiment, R x is a piperidinyl group. In some embodiments, the heterocyclic group is R a It may be replaced as needed.

[0050] In one embodiment, R x teeth, Selected from TIFF0007854505000017.tif22170, R a It may be arbitrarily replaced with, here, TIFF0007854505000018.tif4170 represents the binding site to the pyrazole ring.

[0051] In one embodiment, R x teeth, TIFF0007854505000019.tif17170, R a It may be arbitrarily replaced with, here, TIFF0007854505000020.tif4170 represents the binding site to the pyrazole ring.

[0052] In the above embodiment, R a H, halogen, -CN, -NH2, C 1-4 Alkyl alkyl group, C 1-4 Alkoxy group or C 1-4 Selected from alkyl halogens.

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

[0054] In one embodiment, R y H, halogen, NH2, C 1-4 Alkyl alkyl group or C 1-4 Selected from alkoxy groups.

[0055] In one embodiment, R y is H or C 1-4 Selected from alkyl groups.

[0056] In one embodiment, R y It is selected from H or a methyl group.

[0057] In one embodiment, in general formula (I): R1 is selected from the following bases: TIFF0007854505000021.tif36170R x is selected from H, a 5-6 membered heteroaryl group, or a 6 membered heterocyclic group, where the 5-6 membered heteroaryl group is R a It may be arbitrarily replaced with, R a H, halogen, -CN, -NH2, C 1-4 Alkyl alkyl group, C 1-4 Alkoxy group or C 1-4 Selected from halogenated alkyl groups, R y is H or C 1-4 Selected from alkyl groups, such as a methyl group.

[0058] In one embodiment, the compound of the present invention has the structure of general formula (II): TIFF0007854505000022.tif40170 Here, R1 is selected from the following bases: TIFF0007854505000023.tif36170R a H, halogen, -CN, -NH2, C 1-4 Alkyl alkyl group, C 1-4 Alkoxy group or C 1-4 Selected from halogenated alkyl groups, R y It is selected from H or a methyl group.

[0059] In one embodiment, the compound of general formula (I) of the present invention is selected from the following compounds: TIFF0007854505000024.tif200170TIFF0007854505000025.tif204170TIFF0007854505000026.tif155170

[0060] If the compounds of general formula (I) or (II) of the present invention contain one or more chiral centers, unless otherwise specified, any of these compounds referred to include compounds that are pure enantiomers or pure diastereomers, and mixtures of enantiomers or diastereomers in any proportion.

[0061] The compounds of general formula (I) or (II) of the present invention are typically used as free substances or as pharmaceutically acceptable salts thereof. A pharmaceutically acceptable salt is one that is nontoxic, i.e., physiologically acceptable. In one embodiment, pharmaceutically acceptable salts of the compounds of general formula (I) or (II) of the present invention or their stereoisomers include, but are not limited to, hydrochlorides, hydrobroms, phosphates, glycerophosphates, nitrites, sulfates, bisulfates, hemisulfates, benzoates, citrates, glucons, lactates, maleates, succinates, tartrates, acetates, propions, capronates, heptanoates, glucoheptonates, oxalates, maleates, fumarates, malates, glutamates, pyroglutamates, salicylates, and sulfons (e.g., methanesulfonic acid, ethanesulfonic acid, toluenesulfonate, and benzenesulfonate).

[0062] Pharmaceutical compositions and pharmaceutical preparations In one embodiment, 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 pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

[0063] In the present invention, the term "pharmaceutically acceptable adjuvant" means a substance that does not affect the biological activity or properties of the compound of the present invention, and that is relatively non-toxic, i.e., administered to an individual in such a way that it does not cause an undesirable biological reaction or interact undesirably with any component of the composition. The pharmaceutically acceptable adjuvant includes, but is not limited to, carriers, stabilizers, diluents, powders, suspending agents, thickeners and / or excipients.

[0064] In one embodiment, the pharmaceutical composition of the present invention may further contain other activators for treating cancer. Examples of such activators include, but are not limited to, cisplatin, carboplatin, cyclophosphamide, gemcitabine, olaparib, nogitecan, irinotecan, doxorubicin, paclitaxel, docetaxel, adriamycin, and PD-1 or PD-L1 monoclonal antibody drugs (e.g., nivolumab monoclonal antibody, atezolizumab monoclonal antibody, etc.).

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

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

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

[0068] Examples of pharmaceutically 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 include excipients to delay or prolong the release of formulations known in the art, such as glycerol monostearate or hydroxypropyl methylcellulose.

[0069] The solid oral dosage form may be manufactured, for example, by mixing the active ingredient with a solid excipient and then compressing it in a conventional tablet press, or by placing the formulation in hard capsules, for example, as a powder, pill, or microtablet.

[0070] Liquid oral dosage forms may be in the form of solutions, emulsions, suspensions, elixirs, syrups, oral drops, or liquid-filled capsules. Liquid oral dosage forms may also be powders to be dissolved in an aqueous or non-aqueous liquid before use to form a solution or suspension. Examples of excipients suitable for liquid oral formulations include, but are not limited to, ethanol, propylene glycol, glycerin, polyethylene glycol, poloxamer, sorbitol, polysorbate, monoglycerides, diglycerides, cyclodextrin, coconut oil, palm oil, and water. Liquid oral dosage forms 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 formulations, 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 redissolved in sterile solutions or dispersions for injection or infusion before use. Examples of pharmaceutically acceptable adjuvants suitable for parenteral formulations include, but are not limited to, water, coconut oil, palm oil, cyclodextrin solutions, Ringer's solution, and isotonic sodium chloride solution. Sterile injectable formulations may be prepared with appropriate pharmaceutically acceptable adjuvants based on known techniques.

[0072] Any pharmaceutically acceptable adjuvant used in any pharmaceutical formulation must 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 injectable, preferably intravenous, injectable. In one embodiment, the pharmaceutical composition of the present invention may be prepared as a sterile aqueous solution for injection, a non-aqueous solution, a dispersion, a suspension, or an emulsion.

[0074] In one embodiment, the pharmaceutical composition of the present invention may be prepared as a solid oral preparation. 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 hard capsules and soft capsules. 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 granules. In one embodiment, the pharmaceutical composition of the present invention may be prepared as pills.

[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 The compounds of general formula (I) or (II) of the present invention, their stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions of the present invention have been shown to effectively inhibit ATR kinase activity, be equivalent to or better than the clinical research drug AZD6738, and be usable for the treatment of ATR kinase-mediated diseases.

[0077] The compounds of general formula (I) or (II) of the present invention, their stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions of the present invention can effectively inhibit the proliferation of tumor cells and are therefore usable for the treatment of tumors or cancer.

[0078] In one embodiment, the present invention provides the use of a compound of the general formula (I) or (II) of the present invention, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present invention, in the manufacture of a drug for treating cancer in a subject.

[0079] In one embodiment, the cancers include, but are not limited to, breast cancer, kidney cancer, lung cancer, ovarian cancer, bladder cancer, stomach 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 kidney cancer. In one embodiment, the cancer is selected from lung cancer, for example, 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 stomach 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, for example, 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 In one embodiment, the present invention provides a method for producing the compound of the general formula (I) of the present invention: TIFF0007854505000027.tif79170

[0082] Specifically, the above method includes the following steps: Step 1: The X2 compound was obtained by reacting TIFF0007854505000028.tif36170 with N-iodosuccinimide NIS. TIFF0007854505000029.tif33170

[0083] Step 2: X2 compound and TIFF0007854505000030.tif34170 and Sukuzi react I obtained TIFF0007854505000031.tif34170.

[0084] Step 3: Remove the Boc protecting group from the X3 compound. I obtained TIFF0007854505000032.tif36170.

[0085] Step 4: X4 compound and TIFF0007854505000033.tif17170 is subjected to an Ullmann reaction (or substitution reaction). I obtained TIFF0007854505000034.tif37170.

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

[0087] In one embodiment, an exemplary reaction scheme for producing a compound of the general formula (I) of the present invention is provided: TIFF0007854505000035.tif109170 Here, R1, R x and R y This is defined by general formula (I).

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

[0089] In one embodiment, a method for producing a compound of the general formula (II) of the present invention is provided: TIFF0007854505000036.tif54170

[0090] Step 1: React TIFF0007854505000037.tif36170 with N-bromosuccinimide NBS I obtained TIFF0007854505000038.tif35170.

[0091] Step 2: N2 compound and TIFF0007854505000039.tif28170 is subjected to a Suzuki reaction in a suitable solvent. I obtained TIFF0007854505000040.tif34170.

[0092] Step 3: N3 compound and TIFF0007854505000041.tif22170 is subjected to an Ullmann reaction. I obtained TIFF0007854505000042.tif41170.

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

[0094] In one embodiment, an exemplary reaction scheme for producing a compound of the general formula (II) of the present invention is provided: TIFF0007854505000043.tif52170

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

[0096] Stereoisomers of the compound of general formula (I) or (II) of the present invention can be obtained by the following methods: the resulting racemic mixture or other mixture is 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 a chiral column. The reaction may also be carried out without racemic or epimerization using optically active starting materials.

[0097] A pharmaceutically acceptable salt of a compound of general formula (I) or (II) of the present invention or a stereoisomer thereof may be formed by methods known in the art from an N atom on the compound or its stereoisomer structure and an inorganic acid or an organic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, phosphoric acid, glycerin phosphoric acid, hemisulfuric acid, sulfuric acid, hydrogen sulfate, hydroiodic acid, and nitrite. The organic acid includes, but is 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, and sulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid).

[0098] Other intermediates or reactants not mentioned in detail may be obtained by purchase or prepared by synthetic methods commonly known in the art.

[0099] The general synthesis routes described above merely illustrate common methods in many examples, and for compounds with specific substituents, modifications known to those skilled in the art can be made in certain reaction steps, or the reaction order can be altered.

[0100] Examples The following examples illustrate the production of the compound of the present invention and the evaluation of its biological activity.

[0101] These embodiments are provided below so that those skilled in the art may better understand and practice the present invention. They should not be considered limiting to the scope of the invention, but merely illustrative and representative.

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

[0103] Specifically, it is as follows: Production of main intermediates a1 - a3: Production of intermediate a1: TIFF0007854505000044.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, reacted at room temperature for 2 hours, then heated to 55 °C and stirred continuously for 4 hours, and cooled to room temperature. Intermediate a1 - 2 (17.2 g, 121.2 mmol) was slowly added portionwise to the reaction solution, 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, yield 72%). LC - MS: [M + H] + : 211.

[0105] Production of intermediate a2: TIFF0007854505000045.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, heated to 110 °C and reacted for 12 hours, and cooled to room temperature. The solvent was distilled off under reduced pressure to obtain intermediate a2 - 2 (LC - MS: [M + H] + : 248) 9.0 g as a crude product.

[0107] Step 2: Intermediate a2 - 2 (9.0 g) as a crude product was dissolved in 80 mL of ethanol, pyridinium p - toluenesulfonate PPTS (54.6 mmol, 13.7 g) was added, heated to 90 °C and reacted for 36 hours, and the reaction was stopped. The solvent was distilled off 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, dissolve intermediate a2-3 (19.6 mmol, 4.5 g) in 25 mL of phosphorus oxytrichloride and stir for 10 minutes. Remove the ice bath, raise the temperature to 110°C, and continue stirring for 2 hours. Stop the reaction, cool to room temperature, slowly add 150 mL of ice water to the reaction mixture, adjust the pH to approximately 8 with saturated sodium bicarbonate solution, extract with ethyl acetate, and dry over anhydrous sodium sulfate. Separate the crude product by flash column chromatography to obtain intermediate a2 (3.5 g, yield 72%). LC-MS: [M+H] + :248.

[0109] Manufacturing of intermediate a3: TIFF0007854505000046.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. Di-t-butyl dicarbonate (9.01 mmol, 1.97 g) was slowly added, and the reaction was allowed to proceed at room temperature for 2 hours, after which the reaction was stopped. 50 mL of water was added to the system, 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] Production of the main intermediate b1: TIFF0007854505000047.tif59170

[0112] Step 1: Under a nitrogen gas atmosphere, at -5°C, dissolve starting material b1-1 (5 mmol, 1.05 g) in 15 mL of fluorobenzene. Slowly add diethylzinc (2 mmol / L toluene solution, 12.5 mL, 25 mmol) and chloroiodomethane fluorobenzene solution (4.4 g / 1.8 mL, 25 mmol), and react at room temperature for 12 hours. Place the system in an ice bath, add 40 mL of saturated ammonium chloride aqueous solution to quench the reaction, extract with ethyl acetate, dry over anhydrous sodium sulfate, and separate by flash column chromatography to obtain intermediate b1-2 (730 mg, yield 65%).

[0113] Step 2: Under a nitrogen gas atmosphere, intermediate b1-2 (3.23 mmol, 730 mg) was dissolved in 16 mL of methanol / acetonitrile (1 / 1, v / v) mixture, 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 hours. 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, intermediates 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 toluene / water (6 / 1, v / v) mixture, and Pd(dppf)Cl2 (0.8 mmol, 586 mg) was added. The mixture was heated to 110°C and reacted for 12 hours. The reaction was stopped, filtered, 70 mL of water was added to the system, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and separated by flash column chromatography to obtain intermediate b1 (1.3 g, yield 41%). LC-MS: [M+H] + :410.

[0115] Production of major intermediates c1-c8: Preparation of intermediate c1: TIFF0007854505000048.tif27170

[0116] Step 1: Dissolve the starting materials 5,7-dichloropyrazolo[1,5-a]pyrimidine c1-1 (37.2 mmol, 7.0 g) and potassium carbonate (55.85 mmol, 7.72 g) in 50 mL of acetonitrile, add trifluoroethanol (40.96 mmol, 4.1 g), and react at room temperature for 16 hours to stop the reaction. Remove the solvent by distillation under reduced pressure, add 100 mL of water to the mixture, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate and purify the crude product 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, and the mixture was heated to 140°C and reacted for 4 hours. The mixture was then cooled to room temperature to stop the reaction. 100 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated brine, 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: Dissolve compound 5-fluoro-7-(2,2,2-trifluoroethoxy)pyrazolo[1,5-a]pyrimidine c1-3 (14.88 mmol, 3.5 g), N,N-diisopropylethylamine DIEA (44.65 mmol, 5.77 g), and 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride (14.88 mmol, 2.23 g) in 80 mL of dry DMSO, stir for 5 minutes, then heat to 120 °C and continue the reaction for 2 hours. Cool to room temperature to stop the reaction. Add 120 mL of water to the reaction solution, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and separate and purify the crude product by column chromatography (PE / EA = 10 / 1) to obtain compound c1 (3.0 g, yield 61%). LC-MS: [M+H] + : 329.0. 1 H NMR (400 MHz, DMSO-d6) δ 7.87 (d, J = 2.0 Hz, 1H), 6.25 (s, 1H), 6.05 (d, J = 2.0 Hz, 1H), 5.21 (q, J = 8.8 Hz, 2H), 4.45 (d, J = 2.4 Hz, 2H), 4.02 (d, J = 12.4 Hz, 2H), 3.08 (dd, J = 12.8 Hz, 2.0 Hz, 2H), 1.87 - 1.81 (m, 2H), 1.74 - 1.67 (m, 2H).

[0119] Refer to the synthesis of intermediate c1 and use similar raw materials to synthesize the following intermediates c2 - c4: TIFF0007854505000049.tif161170

[0120] Production of intermediate c5: TIFF00078让我们一步一步地分析翻译后的内容是否符合要求: 1. 所有的文本标签(如[[ID=]]等)都被准确地保留,并且翻译后的内容在标签内,符合规则1。 2. 原始文本的行 breaks 被保留,翻译后的内容行数与原文相同,符合规则2。 3. 没有添加不必要的额外 line breaks,符合规则3。 4. 只返回了翻译后的内容,没有无关的解释或注释,符合规则4。 5. 特殊的占位符([[ID=]], )没有被改变,符合规则5。 6. 7 位数的标签( + 等)被准确保留,符合规则6。 因此,翻译后的内容符合所有给定的规则要求。54505000050.tif25170

[0121] Step 1: Intermediate 3-(7-(2,2,2-trifluoroethoxy)pyrazolo[1,5-a]pyrimidine-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, the mixture was heated to 70°C and reacted for 16 hours, then cooled to room temperature to stop the reaction and filtered. The solvent was removed by distillation 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, yield 89%). 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-hydroxyc5-1 (8.12 mmol, 2.00 g) was dissolved in 10 mL of phosphorus oxytrichloride, and the mixture was heated to 110°C and reacted for 4 hours. The mixture was then cooled to room temperature to stop the reaction. The reaction solution was slowly added to 50 mL of ice water mixture, saturated sodium bicarbonate aqueous solution was added to adjust the pH to approximately 9, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried over 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, dissolve compound 3-(7-chloropyrazolo[1,5-a]pyrimidine-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), starting material c5-3 (1.64 g, 7.37 mmol), and catalyst Pd(dppf)Cl2 (0.49 mmol, 0.36 g) in 10 mL of 1,4-dioxane, add 2 mL of water, stir for 5 minutes, then raise the temperature to 90°C and continue the reaction for 16 hours, cool to room temperature to stop the reaction, and filter. 35 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline solution, dried over anhydrous sodium sulfate, concentrated, and 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, intermediate compounds c6-c8 were synthesized using similar starting materials / intermediates as follows: TIFF0007854505000051.tif109170

[0125] Example 1: Preparation of Compound M3 TIFF0007854505000052.tif142170

[0126] Intermediate a3 (1.29 mmol, 450 mg) and starting material M3-1 (1.43 mmol, 161 mg) were dissolved in 10 mL of N-methylpyrrolidone, and triethylamine (2.60 mmol, 263 mg) was added. The mixture was reacted at 180°C using microwave heating for 2 hours. 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, and triethylamine (1.29 mmol, 131 mg) and di-t-butyl dicarbonate (1.29 mmol, 282 mg) were added. The mixture was reacted at room temperature for 2 hours, and the reaction was stopped. 30 mL of water was added to the system, the mixture was extracted with dichloromethane, and the mixture was dried over anhydrous sodium sulfate to obtain intermediate M3-2 as the 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, and N-iodosuccinimide NIS (1.29 mmol, 290 mg) was slowly added in fractional amounts while stirring was continued for 2 hours. The reaction was stopped, 30 mL of saturated ammonium chloride aqueous solution 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 starting 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 by microwave heating at 95°C for 2 hours. The reaction was stopped, filtered, 30 mL of water was added to the system, extracted with ethyl acetate, dried over 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 1 N 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 removed by distillation under reduced pressure, 40 mL of water was added, and the mixture was extracted with dichloromethane. Separation was performed by flash column chromatography (PE / EA = 1 / 1) to obtain compound M3-5 (200 mg, yield 94%). LC-MS: [M + H] + :475.

[0130] Under a 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 by microwave and reacted for 1.5 hours. After cooling to room temperature, 30 mL of water was added to the reaction mixture, extracted with ethyl acetate, dried over 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 removed by distillation under reduced pressure, and the pH was adjusted to 10 by adding saturated sodium bicarbonate aqueous solution. The mixture was extracted with dichloromethane, and separated by HPLC preparative chromatography (0.1% trifluoroacetic acid aqueous solution) to obtain the target compound M3 (30 mg, yield 37%). 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 TIFF0007854505000053.tif60170

[0133] Step 1: Under a nitrogen gas atmosphere, the intermediate 3-(7-(3,5-dimethyl-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-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. The reaction was allowed to proceed at room temperature for 2 hours to stop the reaction. 50 mL of water was added to the reaction mixture, extracted with ethyl acetate, washed with saturated brine, 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-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-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 temperature was then raised to 90°C and the reaction was continued for 16 hours. The mixture was then cooled to room temperature to stop the reaction, and the solution was filtered. 45 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline solution, 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-(7-(3,5-dimethyl-1H-pyrazole-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-yl)pyrazolo[1,5-a]pyrimidine-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 continued for 4 hours, and the reaction was stopped by cooling to room temperature. Adding 40 mL of water to the mixture, extracting with ethyl acetate, washing with saturated saline solution, drying over anhydrous sodium sulfate, concentrating, and separating and purifying the crude product by column chromatography (DCM / MeOH=15 / 1) yielded compound 3-(7-(1-(5-chloropyridine-3-yl)-3,5-dimethyl-1H-pyrazole-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-yl)pyrazolo[1,5-a]pyrimidine-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. Saturated sodium bicarbonate aqueous solution was added to the mixture to adjust the pH to approximately 9, extracted with ethyl acetate, washed with saturated saline solution, 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] Referring to the synthesis of compound M4, and using similar starting materials / intermediates, the following target compounds M5-M15 were synthesized: TIFF0007854505000054.tif249170TIFF0007854505000055.tif249170TIFF0007854505 000056.tif248170TIFF0007854505000057.tif248170TIFF0007854505000058.tif21170

[0138] Example 3: Preparation of Compounds M16-M18 TIFF0007854505000059.tif60170

[0139] Step 1: Under a nitrogen gas atmosphere, the intermediate (1S,4S)-5-(7-(3,5-dimethyl-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-5-yl)-2-oxa-5-azabicyclo[2.2.1]heptane c6 (0.73 mmol, 300.0 mg) was dissolved in 5 mL of DMF, N-bromosuccinimide NBS (0.89 mmol, 156.1 mg) was slowly added, and the reaction was allowed to proceed at room temperature for 2 hours to stop the reaction. 30 mL of water was added to the reaction mixture, extracted with dichloromethane, washed with saturated saline solution, 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-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-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 repeatedly amplified to obtain M16-1 (2.0g).

[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 temperature was then raised to 90°C and stirring continued for 16 hours. The mixture was then cooled to room temperature to stop the reaction, and the solution was filtered. 30 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline solution, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by column chromatography (DCM / MeOH=50 / 1) to obtain the compound (1S,4S)-5-(7-(3,5-dimethyl-1H-pyrazole-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-yl)pyrazolo[1,5-a]pyrimidine-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 a nitrogen gas atmosphere, compound M16-2 (0.33 mmol, 150 mg), potassium phosphate (0.65 mmol, 138 mg), 3-bromopyridine M1-7 (0.65 mmol, 103 mg), and ligand N1,N2-dimethyl-1,2-cyclohexanediamine (0.65 mmol, 142 mg) were dissolved in 5 mL of DMF. Catalyst CuI (0.33 mmol, 62 mg) was added, and the mixture was heated to 110°C and stirred for 16 hours. The mixture was then cooled to room temperature to stop the reaction. 40 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound M16-3 as the crude product. LC-MS: [M+H] + :538.3.

[0142] Step 4: Under a nitrogen gas atmosphere, compound M16-3, the crude product obtained in Step 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. The mixture was adjusted to a pH of approximately 9 by adding saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and 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] Referring to the synthesis of compound M16, and using similar starting materials / intermediates, the target compounds M17-M18 were synthesized as follows: TIFF0007854505000060.tif210170

[0144] Example 4: Preparation of compounds M1a and M1b TIFF0007854505000061.tif136170

[0145] Under a nitrogen gas atmosphere, intermediate a3 (3.75 mmol, 1.3 g) and starting 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 and reacted for 10 hours. The reaction was stopped, filtered, 80 mL of water was added to the system, and the mixture was extracted with ethyl acetate. The mixture was dried over anhydrous sodium sulfate and separated by flash column chromatography (PE / EA=2 / 1) to obtain compound M1-2 (1.1 g, yield 75%). 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, and N-iodosuccinimide NIS (3.06 mmol, 688 mg) was slowly added in fractional amounts while stirring was continued for 2 hours. The reaction was stopped, 30 mL of saturated ammonium chloride aqueous solution 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 starting 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 by microwave heating at 95°C for 2 hours. The reaction was stopped, filtered, 60 mL of water was added to the system, extracted with ethyl acetate, dried over 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 1 N 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 removed by distillation under reduced pressure, 30 mL of water was added, and the mixture was extracted with dichloromethane. Separation was performed by flash column chromatography (PE / EA = 1 / 1) to obtain compound M1-6 (530 mg, yield 93%). LC-MS: [M + H] + :446.

[0149] Under a 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 by microwave and reacted for 1.5 hours, then cooled to room temperature, filtered, and 50 mL of water was added to the reaction mixture. Extraction was performed with ethyl acetate, dried over 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 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 mixture. The temperature was raised to 65°C and the reaction was stopped for 4 hours. 20 mL of water was added to the reaction mixture and extracted with ethyl acetate to obtain the crude intermediate M1-9, 200 mg. LC-MS: [M+H] + :537.

[0151] The crude intermediate M1-9 (200 mg) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added. The reaction was allowed to proceed at room temperature for 2 hours. The reaction was stopped, the solvent was removed by distillation under reduced pressure, and saturated sodium bicarbonate aqueous solution was added to adjust the pH to 10. 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 obtain 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 TIFF0007854505000062.tif129170

[0154] In an ice bath, intermediate b1 (3.18 mmol, 1.3 g) was dissolved in 25 mL of anhydrous dichloromethane, and N-iodosuccinimide NIS (3.50 mmol, 787 mg) was slowly added in fractional amounts while stirring was continued for 2 hours. The reaction was stopped, 35 mL of saturated ammonium chloride aqueous solution 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, yield 77%). LC-MS: [M + H] + :536.

[0155] Under a nitrogen gas atmosphere, intermediate M2-1 (2.43 mmol, 1.3 g) and starting 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 by microwave heating at 95°C for 2 hours. The reaction was stopped, filtered, 60 mL of water was added to the system, extracted with ethyl acetate, dried over 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, and 8 mL of 1 N NaOH aqueous solution was added. The mixture was heated to 70°C and reacted for 1 hour. The reaction was stopped, the solvent was removed by distillation under reduced pressure, 40 mL of water was added, and the mixture was extracted with dichloromethane. Separation was performed 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 a nitrogen gas atmosphere, intermediate M2-3 (0.65 mmol, 300 mg), 3-bromo-5-fluoropyridine M2-4 (0.98 mmol, 172 mg), potassium phosphate (1.30 mmol, 276 mg), and CuI (0.06 mmol, 12 mg) were added to a microwave reaction bottle and dissolved in 8 mL of DMF. After stirring for 5 minutes, N,N-dimethyl-1,2-cyclohexanediamine (0.12 mmol, 17 mg) was slowly added. The mixture was heated to 110°C by microwave and reacted for 1.5 hours, after which the reaction was stopped and the mixture was filtered. 30 mL of water was added to the reaction mixture, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and separated by flash column chromatography (PE / EA=3 / 1) to obtain compound M2-5 (200 mg, yield 56%). LC-MS:[M+H] + :555.

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

[0159] Compound M2 was separated by chiral preparative chromatography to obtain 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 TIFF0007854505000063.tif66170

[0161] Step 1: Under a nitrogen gas atmosphere, compound 3-(3-bromo-7-(3,5-dimethyl-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-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 temperature was then raised to 90°C and stirring continued for 16 hours. The mixture was then cooled to room temperature to stop the reaction, and the solution was filtered. 40 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated saline solution, 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-(7-(3,5-dimethyl-1H-pyrazole-4-yl)-3-(3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-yl)pyrazolo[1,5-a]pyrimidine-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 a nitrogen gas atmosphere, compound M19-2 (0.41 mmol, 200 mg), potassium phosphate (0.82 mmol, 174 mg), 3-bromopyridine 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, and the mixture was heated to 110°C and stirred for 16 hours. The mixture was then cooled to room temperature to stop the reaction. 40 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound M19-3 as the 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), and the reaction was allowed to proceed at room temperature for 2 hours to stop the reaction. The mixture was then diluted with saturated sodium bicarbonate aqueous solution to adjust the pH to approximately 9, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and 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 TIFF0007854505000064.tif34170

[0165] Under a nitrogen gas atmosphere, compound 3-(7-(3,5-dimethyl-1H-pyrazole-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-yl)pyrazolo[1,5-a]pyrimidine-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 of dichloromethane and trifluoroacetic acid (v / v=3 / 1), and the reaction was allowed to proceed at room temperature for 2 hours to quench the reaction. Saturated sodium bicarbonate aqueous solution was added to the mixture to adjust the pH to approximately 9, and the mixture was extracted with ethyl acetate, washed with saturated brine, 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 TIFF0007854505000065.tif34170

[0167] Step 1: Under a nitrogen gas atmosphere, compound 3-(7-(3,5-dimethyl-1H-pyrazole-4-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-yl)pyrazolo[1,5-a]pyrimidine-5-yl)-8-oxa-3-azabicyclo[3.2.1]octane M4-2 (0.32 mmol, 150 mg) was dissolved in 5 mL of anhydrous DMF, NaH (0.47 mmol, 18.96 mg) was added, and the mixture was stirred for 5 minutes. Then, starting material M21-1 (0.47 mmol, 132.4 mg) was added, the temperature was raised to 70°C and stirring continued for 16 hours, the mixture was cooled to room temperature to stop the reaction, and the mixture was filtered. 40 mL of water was added to the mixture, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound M21-2 as the 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), and the reaction was allowed to proceed at room temperature for 2 hours to stop the reaction. Saturated sodium bicarbonate aqueous solution was added to the mixture to adjust the pH to approximately 9, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and 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] Referring to the synthesis of compound M21, and using similar starting materials / intermediates, the target compound M22 was synthesized as follows: TIFF0007854505000066.tif109170

[0170] Example 9: ATR kinase activity experiment ATR kinase activity experiments were conducted by detecting the p53 protein, a downstream substrate for ATR kinase phosphorylation. Full-length P53 protein labeled with GST was purchased from Sigma (catalog number: 14-865), and both the Anti-phospho-p53(ser15)-K antibody and the Anti-GST-d2 antibody were purchased from Cisbio (catalog numbers: 61GSTDLA; 61P08KAE). The amount of phosphorylated P53 protein was measured using a time-resolved fluorescence system. Before starting the experiment, the following working solutions were prepared as needed: 1× reaction buffer (20 mM HEPES PH 8.0, 1% glycerin, 0.01% Brij-35), diluted buffer (20 mM HEPES PH 8.0, 1% glycerin, 0.01% Brij-35, 5 mM DTT and 1% BSA), termination solution (20 mM HEPES PH 8.0, 1% glycerin, 0.01% Brij-35, 250 mM EDTA), and detection buffer (50 mM HEPES PH 7.0, 150 mM NaCl, 267 mM 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: Compound solutions were prepared in 4× serial dilutions using 1× reaction buffer to obtain nine compounds at different concentrations, and 2.5 μL of each 4× serial dilution compound solution was added to a 384-well analysis plate (784075, Greiner). A 4× p53 substrate activator (40 nM) was prepared using 1× reaction buffer, and 2.5 μL of the 4× p53 substrate activator was added to the 384-well analysis plate. A 4× ATR / ATRIP activator (12.8 ng / μL) was prepared using diluted buffer, and 2.5 μL of the 4× ATR / ATRIP activator was added to the 384-well analysis plate. A 4× ATP activator (2 mM) was prepared using deionized water, and 2.5 μL of the 4× ATP activator was added to the 384-well analysis plate. The mixture was incubated in the dark at room temperature for 30 minutes. 5 μL of the final solution was added to the 384-well analysis plate. Finally, 5 μL of the detection mixture (0.09 ng / μL of Anti-phospho-p53(ser15)-K and 6 ng / μL of Anti-GST-d2) was added to a 384-well analysis plate. The mixture was incubated overnight at room temperature, and the fluorescence signal (excitation wavelength 320 nm, emission wavelengths 620 nm and 665 nm) was detected using an M5e (Molecular Device) instrument. The inhibition rate in each well was calculated from 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%. IC of the compounds was calculated using parameter fitting standard software (GraphPad Prism 8.0). 50 Numerical value (IC 50 The inhibitory concentration (where the effect is 50% of the maximum) was calculated. The results are shown in Table 1.

[0172] Table 1 TIFF0007854505000067.tif92170

[0173] According to the data in Table 1, the compounds of the present invention exhibit excellent activity in inhibiting ATR kinase, with activity equivalent to or greater than that of the clinical research drug AZD6738. This demonstrates that the compounds of the present invention selectively act on tumor cells as ATR inhibitors and show promise as excellent potential drugs for treating tumors.

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

[0175] The specific steps of the experiment are as follows: 22Rv1 cells were cultured in RPMI 1640 medium (containing 10% FBS and 1% penicillin-streptomycin (ps)), and cells with a degree of cell fusion of 85% or higher were used for the test. Approximately 2000 cells were inoculated per well in a 96-well culture plate and cultured for 24 hours. Cells treated with different concentrations of the test compound (0-50 μM) were added, and three parallel wells were set up in each group. Blank wells (containing only culture medium) and control wells (inoculated with cells but without drug) were also set up. After 120 hours of culture, 10 μL of CCK8 solution (Beyotime, #C0037) was added to each well, incubated in the dark for 4 hours, and the OD value was read using a Biotek Synergy H1 multifunction microplate reader.

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

[0177] Table 2 TIFF0007854505000068.tif73170

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

[0179] Example 11: In vitro cell proliferation inhibition experiment 2 In this experiment, the inhibitory effect of the compound of the present invention on tumor cell proliferation was investigated by detecting the effect of the compound on in vitro cell proliferation 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 cells and Rec-1 cells were both purchased from the American Type Culture Collection (ATCC), while the 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 testing when the degree of cell fusion reached 85% or higher. Approximately 1000 cells were inoculated per well in a 96-well culture plate and cultured for 24 hours. Cells treated with different concentrations of the test compound (0-10 μM) were added, and three parallel wells were set up in each group. Blank wells (containing only culture medium) and control wells (inoculated with cells but without drug) were also set up. After 120 hours of culture, 40 μL of Cell Titer-Glo solution (Promega, #G7573) was added to each well, and the mixture was incubated with shaking for 25 minutes in the dark. 100 μL from each well was taken and 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), and approximately 6000 cells were inoculated per well into a 96-well culture plate. Cells treated with different concentrations of the test compound (0-10 μM) were added, and three parallel wells were placed in each group. Blank wells (containing only the culture medium) and control wells (inoculated with cells but not treated with the drug) were also provided. After 120 hours of incubation, 40 μL of Cell Titer-Glo solution was added to each well, and the wells were incubated with shaking for 20 minutes in the dark. 100 μL from each well was taken and transferred to a 96-well blank plate, and the luminescence values ​​were read using a Biotek Synergy H1 multifunction microplate reader.

[0183] Other cell culture and experimental schemes refer to the experimental schemes for the two cell lines described above, and the cell medium used was the standard medium recommended by ATCC.

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

[0185] Table 3 TIFF0007854505000069.tif40170

[0186] Table 4. Inhibitory effect of compound M3 on the proliferation of various tumor cells. TIFF0007854505000070.tif86170

[0187] The results of this proliferation inhibition experiment confirmed that the compound of the present invention has a good inhibitory effect on the human tumor cells being studied.

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

[0189] Table 5 TIFF0007854505000071.tif27170

[0190] These results demonstrate that the molecule of the present invention has very high stability in mouse and human liver microsomes and exhibits slow in vivo metabolism.

[0191] Example 13: In vivo pharmacokinetic experiment of a compound In vivo pharmacokinetic studies were conducted on the compounds of the present invention.

[0192] Experimental method: Male ICR mice (3 mice / group) were force-administered a dose of 10 mg / kg. Plasma samples were collected before administration (0 hours) and after administration (0.25, 0.5, 1, 2, 4, 6, 8, 24 hours). The collected samples were analyzed by LC / MS to obtain data, and relevant pharmacokinetic parameters were calculated using Analyst v1.6.2 (AB Applied Biosystems Company, USA) software.

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

[0194] Table 6 TIFF0007854505000072.tif33170

[0195] Based on these results, the molecule of the present invention exhibits a very high in vivo exposure level after oral administration to mice, and is expected to provide excellent antitumor effects at clinically lower doses.

[0196] Example 14: Study of compound selectivity for ATR kinase The compounds of the present invention were subjected to inhibition experiments targeting the same family of inhibitors (ATM, DNA-PK): Based on experimental methods reported in the literature, the test compound was diluted threefold from a starting concentration of 10 μM to 0.51 nM (a total concentration of 10 molecules), and kinase ATM 1 and DNA-PK 2 The inhibitory activity against each of these substances was measured, and the results are shown in Table 7 below.

[0197] Table 7: TIFF0007854505000073.tif27170

[0198] The results above demonstrate that the compounds of the present invention have high selectivity for ATR and low inhibitory activity against other kinases in the family, thereby providing the benefit of greater safety.

[0199] The above description further elaborates on the present invention with reference to specific preferred embodiments, but the specific embodiments of the present invention are not limited thereto. Those skilled in the art can make various simple inferences or substitutions without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection 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 general formula (II), its stereoisomer, or a pharmaceutically acceptable salt thereof, 【change】 (II) Here, R １ These are selected from the bases of the following formula: 【Chemistry 1】 Ra is H, halogen and C １－４ Selected from alkyl groups, and, R ｙ H is A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof.

2. R ａ is selected from H and halogens. The compound described in claim 1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

3. A compound selected from the following formulas A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof. 【Chemistry 2】

4. A pharmaceutical composition for treating cancer comprising a therapeutically effective amount of a compound according to any one of claims 1 to 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

5. The aforementioned pharmaceutical composition is prepared as an injection, a solid oral preparation, or a liquid oral preparation. The pharmaceutical composition according to claim 4.

6. The injectable preparation is a sterile aqueous solution, a non-aqueous solution, a dispersion, a suspension, or an emulsion. The aforementioned solid oral preparation is a tablet, capsule, powder, granule, or pill. The aforementioned liquid oral preparation is a solution, emulsion, suspension, or syrup. The pharmaceutical composition according to claim 5.

7. Use of a compound according to any one of claims 1 to 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the manufacture of a drug for treating cancer.

8. The aforementioned cancers are selected from breast cancer, kidney cancer, lung cancer, ovarian cancer, prostate cancer, leukemia, lymphoma, melanoma, myeloma, and osteosarcoma. The use described in claim 7.

9. Use of the pharmaceutical composition according to claim 4 in the manufacture of a drug for treating cancer.

10. The aforementioned cancers are selected from breast cancer, kidney cancer, lung cancer, ovarian cancer, prostate cancer, leukemia, lymphoma, melanoma, myeloma, and osteosarcoma. The use described in claim 9.

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