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

Abstract

The present application relates to a novel pyrazolopyrimidine compound, its composition, preparation method and use as anticancer drugs due to its antitumor activity. The novel pyrazolopyrimidine compound has the structural general formula (I) as shown below, as an ATR inhibitor, has excellent tumor inhibition activity, strong selectivity, good water solubility, low toxicity, and can be used for oral or intravenous injection administration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to a novel pyrazolopyrimidine compound and a composition thereof, a preparation method and a use thereof in preparing anticancer drugs. BACKGROUND

[0002] Cancer is a threat to human health and life. In recent years, the research of anticancer drugs has shifted to the development of specific molecular targeted therapeutic drugs.

[0003] ATR, which stands for ataxia telangiectasia and Rad3-related kinase, is composed of 2644 amino acids, and is an important kinase that can activate cell response after DNA damage, and then block cell cycle progression, stabilize replication forks and repair DNA, thereby avoiding apoptosis. When DNA replication pressure and DNA damage occur in cells, ATR is recruited to the DNA damage site, and various proteins are involved in the regulation of ATR activation. When ATR is activated, it can regulate cell biological processes through various signals, including cell cycle arrest, inhibition of replication origins, promotion of deoxynucleotide synthesis, initiation of replication forks, and repair of DNA double-strand breaks.

[0004] Because of the defects in various DNA repair pathways of tumor cells, tumor cells are more dependent on the ATR repair pathway and more sensitive to ATR inhibitors than normal cells. "Synthetic lethality" refers to the defects caused by mutations in one pathway in tumor cells, which makes tumor cells more dependent on another complementary pathway than normal cells, so that inhibition of the complementary pathway will cause "synthetic lethality" to tumor cells. Normal cells will not die under drug inhibition because there is another normal pathway. Studies have found that ATR is a synthetic lethal target of partial mutations, such as ATM-deficient tumor cells that are more sensitive to ATR inhibitors, and the deletion of X-ray cross complementation repair gene I also leads to tumor cells that are more sensitive to ATR inhibition. Therefore, ATR inhibitors, which selectively affect tumor cells and have less interference to normal cells, are expected to be an excellent potential drug for tumor treatment.

[0005] As the most promising "synthetic lethal" therapy after PARP inhibitors, ATR inhibitors have attracted a group of multinational giants, including Merck, Bayer, AstraZeneca, etc. Among them, Merck's Berzosertib is in the forefront of clinical progress, and is currently in clinical phase II. There are fewer domestic enterprises that layout ATR inhibitors. On October 29, 2021, Yingpai Pharmaceutical's IMP9064 was granted I / II phase clinical research permission by FDA. ATR inhibitors approved by NMPA for clinical development include Merck's Berzosertib, Bayer's BAY1895344 and a new drug ATR inhibitor from Shijiazhuang Zhikang Hongren, all of which are in clinical phase I. One of the currently researched drugs is AstraZeneca's AZD6738, the structure of which is shown below:

[0006] It is a positive control for ATR kinase activity test in the experimental part of the present application. However, as of now, no ATR inhibitor has been approved for marketing. Therefore, there is still a need to develop new ATR inhibitors with stronger selectivity and better activity. SUMMARY

[0007] An object of the present application is to provide a new ATR inhibitor which has excellent tumor inhibitory activity, strong selectivity, good water solubility, low toxicity, and can be used for oral or intravenous administration.

[0008] In one aspect, the present application provides a compound represented by general formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0009]

[0010] wherein:

[0011] R1is selected from

[0012] R x is selected from H, 5-6 membered heteroaryl or 5-6 membered heterocyclyl, wherein the 5-6 membered heteroaryl or 5-6 membered heterocyclyl is optionally substituted with R a ;

[0013] R a is selected from H, halogen, -CN, -NH2, C 1-4 alkyl, C 1-4 alkoxy or C 1-4 haloalkyl; and

[0014] R y is selected from H, halogen, -NH2, C 1-4 alkyl or C 1-4 alkoxy.

[0015] In one embodiment, in the general formula (I), R1is selected from the group consisting of:

[0016]

[0017] In one embodiment, in the general formula (I), R x selected from H, 6-membered heteroaryl or 6-membered heterocyclyl, wherein the 6- membered heteroaryl or 6-membered heterocyclyl is optionally substituted with R a ; and R a is selected from H, halogen, -CN, -NH2, C 1-4 alkyl, C 1-4 alkoxy or C 1-4 haloalkyl.

[0018] In one embodiment, in the general formula (I), R y is selected from H or C 1-4 alkyl.

[0019] In one embodiment, the compound has the structure of general formula (II):

[0020]

[0021] wherein:

[0022] R1is selected from the group consisting of:

[0023]

[0024] R a is selected from H, halogen, -CN, -NH2, C 1-4 alkyl, C 1-4 alkoxy or C 1-4 haloalkyl; and

[0025] R y is selected from H or methyl.

[0026] In one embodiment, the compound of the present application is selected from the group consisting of:

[0027]

[0028]

[0029] In another aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of general formula (I) or (II) of the present application, a stereoisomer thereof or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant.

[0030] In one embodiment, the pharmaceutical composition further comprises other active agents useful for treating cancer.

[0031] In one embodiment, the pharmaceutical composition can be formulated as an injectable, such as sterile aqueous and non-aqueous solutions, dispersions, suspensions or emulsions; solid oral formulations such as tablets, capsules, powders, granules or pills; liquid oral formulations such as solutions, emulsions, suspensions or syrups.

[0032] In yet another aspect, the present application provides use of the compound of general formula (I) or (II) as described herein, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer. The present application also provides use of the pharmaceutical composition as described herein in the manufacture of a medicament for the treatment of cancer.

[0033] In one embodiment, the cancer is selected from breast cancer, kidney cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, lymphoma, melanoma, myeloma and osteosarcoma.

[0034] The technical solution of the present application has the following beneficial effects:

[0035] 1. The present application provides a novel pyrazolopyrimidine compound.

[0036] 2. The pyrazolopyrimidine compound provided by the present application has excellent activity in inhibiting ATR kinase, and the activity is comparable to or even better than that of the clinically researched drug AZD6738, indicating that the compound of the present application can be used as an ATR inhibitor for treating ATR-mediated diseases.

[0037] 3. The pyrazolopyrimidine compound provided by the present application has excellent effect in inhibiting the proliferation of various cancer cells, and can be used for treating or preventing various cancers.

[0038] 4. The pyrazolopyrimidine compound provided by the present application has very high stability in mouse and human liver microsomes, indicating slow metabolism in vivo.

[0039] 5. The pyrazolopyrimidine compound provided by the present application has very high in vivo exposure after oral administration in mice, and is expected to have excellent antitumor effect at a lower dose in clinic.

[0040] 6. The pyrazolopyrimidine compound provided by the present application has high selectivity for ATR and low inhibitory activity for other kinases in the family, thereby bringing higher safety benefits. DETAILED DESCRIPTION

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0042] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the inventive subject matter.

[0043] Definitions

[0044] The term "C 1-4 "Alkyl" refers to a straight-chain or branched-chain alkyl group having 1 to 4 carbon atoms. Examples thereof include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and t-butyl, preferably methyl.

[0045] The term "Halogen" as used herein refers to fluorine, chlorine, bromine, or iodine, preferably fluorine, chlorine, bromine.

[0046] The term "C 1-4 "Alkoxy" refers to a group of the formula -O-C 1-4 alkyl group, wherein C 1-4 Alkyl is as defined above. Examples of C 1-4 Alkoxy include, but are not limited to, methoxy, ethoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, and the like, preferably methoxy, ethoxy.

[0047] The term "C 1-4 "Haloalkyl" refers to a C 1-4 alkyl group substituted by one or more halogen atoms. Examples thereof include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, 1,1-difluoroethyl, chloromethyl, chloroethyl, dichloromethyl, 1,2-dichloroethyl, and the like, preferably difluoromethyl, trifluoromethyl.

[0048] The term "5-6 membered heteroaryl" as used herein refers to a stable 5- to 6-membered aromatic monocyclic radical comprising 1 to 3, preferably 1 to 2, heteroatoms selected from nitrogen, oxygen, and sulfur. The attachment to the heteroaryl group can be at the position of the heteroatom or through a carbon atom of the heterocycle. Examples of 5-6 membered heteroaryl 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-oxadiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, 1,2,5-thiadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, pyridyl, pyrazinyl, triazinyl, pyrimidinyl, and pyridazinyl, and the like.

[0049] The term "5-6 membered heterocyclyl" as used herein refers to a stable 5- to 6- membered non-aromatic monocyclic radical comprising 1 to 3, preferably 1 to 2, heteroatoms selected from nitrogen, oxygen and sulfur. The heterocyclyl radical can be partially or fully saturated. The attachment to the heterocyclyl radical can be at the position of the heteroatom or through a carbon atom of the heterocyclic ring. Examples of 5-6 membered heterocyclyl groups include, but are not limited to, dihydrofuranyl, dihydrothienyl, 3-pyrrolinyl, 2-pyrrolinyl, 2-imidazolinyl, 2-pyrazolidinyl, dihydrooxazolyl, dihydrothiazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydro-1,2,3-triazolyl, dihydro-1,2,4-triazolyl, dihydro-1,2,5-oxadiazolyl, dihydro-1,2,3-oxadiazolyl, dihydro-1,2,4-oxadiazolyl, dihydro-1,3,4-oxadiazolyl, dihydro-1,2,5-thiadiazolyl, dihydro-1,2,3-thiadiazolyl, dihydro-1,2,4-thiadiazolyl, dihydro-1,3,4-thiadiazolyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, isothiazolidinyl, 1,2,3-triazolidinyl, 1,2,4-triazolidinyl, 1,2,5-oxadiazolidinyl, 1,2,3-oxadiazolidinyl, 1,3,4-oxadiazolidinyl, 1,2,5-thiadiazolidinyl, 1,2,3-thiadiazolidinyl, 1,3,4-thiadiazolidinyl, 1,2-oxathiolanyl, 1,3-oxathiolanyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, thiomorpholinyl, and piperazinyl, and the like.

[0050] "stereoisomers" refers to compounds which have the same atomic constituents bonded by the same sequence of bonds, but which have a different three-dimensional configuration. The present application encompasses various stereoisomers and mixtures thereof.

[0051] The term "subject" as used herein includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (such as chimpanzees and other apes and monkeys), farm animals (such as cows, horses, sheep, goats, swine), domestic animals (such as rabbits, dogs, and cats) and laboratory animals (including rodents, such as rats, mice and guinea pigs) etc. In some embodiments, the subject is a human.

[0052] The terms "treatment," "treat," and other similar synonymous terms as used herein include alleviating, abating, or ameliorating a disease or condition, inhibiting a disease or condition, e.g., arresting the development of a disease or condition, relieving a disease or condition, causing a regression of a disease or condition, relieving a symptom caused by a disease or condition, or arresting a symptom of a disease or condition, preventing additional symptoms, ameliorating or preventing a metabolic cause of symptoms, and in addition, the term includes the purpose of preventing. The term also includes obtaining a therapeutic effect and / or a prophylactic effect. By therapeutic effect is meant the cure or improvement of a underlying disease being treated. In addition, the cure or improvement of one or more of the physiological symptoms associated with the underlying disease is a therapeutic effect, e.g., the subject is observed to have a reduction in symptoms although the underlying disease is still present. In terms of prophylactic effect, a compound or composition described herein can be administered to a subject at risk of developing a particular disease, or to a subject who is experiencing one or more of the physiological symptoms associated with the disease, even though the subject can not have been diagnosed with the disease.

[0053] The term "therapeutically effective amount," as used herein, refers to the amount of at least one active agent, such as a compound described herein, that when administered is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. For example, a "therapeutically effective amount" is an amount of a composition comprising a compound disclosed herein that is needed to provide clinically significant relief of symptoms of a disorder. Techniques for administering a therapeutically effective amount are known to those of skill in the art, for example, in the form of a dose escalation study.

[0054] The terms "administering," "administered," and the like, as used herein, refer to the methods by which a compound or composition can be delivered to the desired site of biological action. These methods include, but are not limited to, oral routes, transduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injections or infusion), topical, and rectal administration. Administration techniques that can be used with the compounds and methods described herein are well known to those skilled in the art.

[0055] The term "pharmaceutically acceptable excipient," as used herein, refers to a substance that does not interfere with the biological activity of the compounds of this application and that is relatively non-toxic, i.e., the material is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The pharmaceutically acceptable excipients include, but are not limited to, carriers, stabilizers, diluents, dispersing aids, suspending aids, thickening agents, and / or vehicles.

[0056] The terms "optionally," "optional," or "may" as used herein mean that the subsequently described event or circumstance can or can not occur, and, thus, the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" means unsubstituted or substituted with R a substituted" means unsubstituted or substituted with Ra substituted, and the description includes both unsubstituted and substituted cases. a substituted, and the description includes both unsubstituted and substituted cases.

[0057] Compounds of general formula (I), stereoisomers thereof or pharmaceutically acceptable salts thereof

[0058] The present application provides compounds for treating, ameliorating or preventing cancer. As described herein, inhibition of ATR kinase can selectively affect tumor cells with less interference to normal cells. The compounds of the present application can effectively inhibit the activity of ATR kinase, comparable to or even better than the clinical drug AZD6738, and are expected to be an excellent potential drug for tumor treatment.

[0059] In one aspect, the present application provides a compound of general formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0060]

[0061] wherein:

[0062] R1is selected from

[0063] R x H, 5-6 membered heteroaryl or 5-6 membered heterocyclyl, wherein the 5-6 membered heteroaryl or 5-6 membered heterocyclyl is optionally substituted with R a ;

[0064] R a H, halogen, -CN, -NH2, C 1-4 alkyl, C 1-4 alkoxy or C 1-4 haloalkyl; and

[0065] R y H, halogen, -NH2, C 1-4 alkyl or C 1-4 alkoxy.

[0066] In one embodiment, the R1is selected from the following groups:

[0067]

[0068] In one embodiment, the R1is selected from the following groups or stereoisomeric forms thereof (where applicable):

[0069]

[0070] In one embodiment, the R1is selected from the following groups or stereoisomeric forms thereof (where applicable):

[0071]

[0072] In one embodiment, R1is selected from

[0073] In one embodiment, R1is

[0074] In one embodiment, R x is selected from H, 5-6 membered heteroaryl or 5-6 membered heterocyclyl, wherein the 5-6 membered heteroaryl or 5-6 membered heterocyclyl is optionally substituted with R a .

[0075] In some embodiments, R x is H.

[0076] In some embodiments, R x is 5-6 membered heteroaryl. In some embodiments, R x is 5-6 membered heteroaryl containing 1 or 2 heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, R x is 5-6 membered heteroaryl containing 1 or 2 heteroatoms selected from nitrogen and oxygen. In some embodiments, R x is 6 membered heteroaryl containing 1 or 2 nitrogen atoms. In some embodiments, R x is selected from pyridyl, pyrimidinyl or isoxazolyl. In these embodiments, the heteroaryl is optionally substituted with R a .

[0077] In some embodiments, R x is 5-6 membered heterocyclyl. In some embodiments, R x is 5-6 membered heterocyclyl containing 1 or 2 heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, R x is 6 membered heterocyclyl containing 1 or 2 heteroatoms selected from nitrogen and oxygen. In some embodiments, R x is 6 membered heterocyclyl containing 1 or 2 nitrogen atoms. In some embodiments, R x is 6 membered heterocyclyl containing 1 nitrogen atom. In some embodiments, R x is piperidinyl. In these embodiments, the heterocyclyl is optionally substituted with R a .

[0078] In some embodiments, R x is selected from H, optionally substituted with R a , wherein represents the position of attachment to the pyrazole ring.

[0079] In some embodiments, R xfor It is optionally replaced by R a Replace, where Indicates the position of attachment to the pyrazole ring.

[0080] In the aforementioned embodiment, R a Selected from H, halogen, -CN, -NH2, C 1-4 Alkyl, C 1-4 Alkoxy or C 1-4 Halogenated alkyl.

[0081] In the aforementioned embodiment, R a is selected from H, F, Cl, Br, -CN, -NH2, methyl, methoxy, ethoxy, difluoromethyl or difluoroethyl.

[0082] In some embodiments, R y Selected from H, halogen, NH2, C 1-4 Alkyl or C 1-4 Alkoxy.

[0083] In some embodiments, R y Selected from H or C 1-4 alkyl.

[0084] In some embodiments, R y Selected from H or methyl.

[0085] In one embodiment, in the general formula (I):

[0086] R1 is selected from the following groups:

[0087]

[0088] R x is selected from H, 5-6 membered heteroaryl or 6 membered heterocyclyl, wherein the 5-6 membered heteroaryl is optionally replaced by R a replace;

[0089] R a Selected from H, halogen, -CN, -NH2, C 1-4 Alkyl, C 1-4 Alkoxy or C 1-4 haloalkyl; and

[0090] R y Selected from H or C 1-4 Alkyl groups, such as methyl.

[0091] In one embodiment, the compound of the present invention has the structure of formula (II):

[0092]

[0093] wherein:

[0094] R1is selected from the group consisting of:

[0095]

[0096] R a selected from H, halogen, -CN, -NH2, C 1-4 alkyl, C 1-4 alkoxy or C 1-4 haloalkyl; and R y is selected from H or methyl.

[0097] In one embodiment, the compounds of general formula (I) according to the present application are selected from the following compounds:

[0098]

[0099]

[0100] When the compounds of general formula (I) or (II) according to the present application contain one or more chiral centers, the reference to any one of these compounds will encompass the enantiomerically or diastereomerically pure compounds as well as mixtures of enantiomers or diastereomers in any ratio, unless otherwise indicated.

[0101] The compounds of general formula (I) or (II) according to the present application are generally utilized in the form of the free substance or in the form of a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts refer to non-toxic, i.e. physiologically acceptable salts. In one embodiment, the pharmaceutically acceptable salts of the compounds of general formula (I) or (II) according to the present application or of their stereoisomers include, but are not limited to: hydrochlorides, hydrobromides, phosphates, glycerol phosphates, nitrites, sulfates, bisulfates, hemisulfates, benzoates, citrates, gluconates, lactates, maleates, succinates, tartrates, acetates, propionates, hexanoates, heptanoates, gluheptanoates, oxalates, maleates, fumarates, malates, glutamates, pyroglutamates, salicylates, sulfonates (such as methanesulfonates, ethanesulfonates, toluenesulfonates and benzenesulfonates), and the like.

[0102] Pharmaceutical compositions and pharmaceutical formulations

[0103] In one aspect, the present application also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of general formula (I) or (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, as defined above, and a pharmaceutically acceptable adjuvant.

[0104] In the present application, the term "pharmaceutically acceptable excipient" means a substance that does not affect the biological activity or properties of the compounds of the present application, and is relatively nontoxic, i.e., the substance can be administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The pharmaceutically acceptable excipients include, but are not limited to, carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or vehicles.

[0105] In one embodiment, the pharmaceutical composition of the present application can further comprise other active agents for treating cancer. Examples of the active agents include, but are not limited to, cisplatin, carboplatin, cyclophosphamide, gemcitabine, Olaparib, topotecan, irinotecan, doxorubicin, paclitaxel, docetaxel, doxorubicin, PD-1 or PD-L1 monoclonal antibody drugs (such as Nivolumab, Atezolizumab, etc.).

[0106] In one embodiment, the pharmaceutical composition of the present application can be formulated into a preparation for administration by any suitable route. The suitable routes include, but are not limited to, oral route, duodenal route, injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial injection or infusion), external use, and rectal administration, etc.

[0107] The pharmaceutical composition for oral administration includes solid oral dosage forms, such as tablets, capsules, powders, and granules; and liquid oral dosage forms, such as solutions, emulsions, suspensions, and syrups, and powders and granules to be dissolved or suspended in a suitable liquid.

[0108] The solid oral dosage forms can be presented 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 one or more suitable pharmaceutically acceptable excipients. If appropriate, these solid dosage forms can be formulated with coatings, such as enteric coatings, or they can be formulated to provide modified release, such as delayed or extended release, of the active ingredient according to methods well known in the art.

[0109] Examples of the pharmaceutically acceptable excipients suitable for solid oral dosage forms include, but are not limited to, microcrystalline cellulose, corn starch, lactose, mannitol, povidone, croscarmellose sodium, sucrose, cyclodextrin, talc, gelatin, pectin, magnesium stearate, stearic acid, and lower alkyl ethers of cellulose. Similarly, the solid preparations can include excipients known in the art for delayed or extended release formulations, such as glyceryl monostearate or hypromellose.

[0110] The solid oral dosage forms can be prepared, for example, by mixing the active ingredient with a solid excipient and then compressing this mixture in a conventional tabletting machine; or the preparation can be filled into a hard gelatin capsule, for example, in the form of a powder, a pellet, or a microtablet.

[0111] Liquid oral dosage forms can be presented as, for example, solutions, emulsions, suspensions, elixirs, syrups, oral drops, or filled capsules. Liquid oral dosage forms can also be presented as powders for reconstitution with 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, glycerol, polyethylene glycol, poloxamer, sorbitol, polysorbate, glycerol mono- and di-esters, cyclodextrin, coconut oil, palm oil, and water. Liquid oral dosage forms can 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.

[0112] Pharmaceutical compositions for parenteral administration can also be in the form of sterile injectable preparations, such as sterile powders for reconstitution with a sterile vehicle, including water, before use, sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile concentrated solutions or dispersions which can be diluted prior to use. Examples of pharmaceutically acceptable excipients 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 preparations can be formulated according to known techniques using suitable pharmaceutically acceptable excipients.

[0113] Pharmaceutically acceptable excipients for use in any pharmaceutical formulation must be compatible with the intended route of administration and the active ingredient.

[0114] In one embodiment, the pharmaceutical composition of the present application can be formulated as an injection, preferably an intravenous injection. In one embodiment, the pharmaceutical composition of the present application can be formulated as sterile injectable preparations of aqueous or non-aqueous solutions, dispersions, suspensions or emulsions.

[0115] In one embodiment, the pharmaceutical composition of the present application can be formulated as a solid oral formulation. In one embodiment, the pharmaceutical composition of the present application can be formulated as a tablet. In one embodiment, the pharmaceutical composition of the present application can be formulated as a capsule, including a hard or soft capsule. In one embodiment, the pharmaceutical composition of the present application can be formulated as a powder. In one embodiment, the pharmaceutical composition of the present application can be formulated as a granule. In one embodiment, the pharmaceutical composition of the present application can be formulated as a pill.

[0116] In one embodiment, the pharmaceutical composition of the present application can be formulated as a liquid oral formulation. In one embodiment, the pharmaceutical composition of the present application can be formulated as an oral solution, emulsion, suspension or syrup.

[0117] Uses

[0118] The compound of general formula (I) or (II), stereoisomer thereof or pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present application can effectively inhibit the activity of ATR kinase, and the activity is comparable to or even better than the clinically researched drug AZD6738, indicating that it can be used for treating diseases mediated by ATR kinase.

[0119] The compound of general formula (I) or (II), stereoisomer thereof or pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present application can effectively inhibit the proliferation of tumor cells, and thus can be used for treating tumors or cancers.

[0120] In one aspect, the present application provides use of the compound of general formula (I) or (II), stereoisomer thereof or pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present application in the preparation of a medicament for treating cancer in a subject.

[0121] In one embodiment, the cancer includes but is not limited to breast cancer, kidney cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, lymphoma, melanoma, myeloma and osteosarcoma.

[0122] 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 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, 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.

[0123] Methods of preparation

[0124] In one aspect, the present application provides a method for preparing the compound of general formula (I) of the present application:

[0125]

[0126] Specifically, the above method comprises the following steps:

[0127] First step: reacting the X1 compound with N-iodosuccinimide NIS to obtain the X2 compound

[0128] Second step: reacting X2 compound with N6 compound to obtain X3 compound by Sukuzi reaction

[0129] Third step: removing Boc protecting group from X3 compound to obtain X4 compound

[0130] Fourth step: reacting X4 compound with X5 compound (or X7 compound ) to obtain X6 compound by Ullmann reaction (or substitution reaction)

[0131] Fifth step: removing THP protecting group from X6 compound to obtain the compound of general formula (I) according to the present application.

[0132] In one embodiment, an exemplary reaction scheme for preparing the compound of general formula (I) according to the present application is provided:

[0133]

[0134] wherein R1, R x and R y are as defined in general formula (I).

[0135] According to the above scheme, X1 compound is reacted with N-iodosuccinimide NIS in a suitable solvent such as dichloromethane to obtain X2 compound; X2 compound is reacted with 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester analogue N6 in a suitable solvent such as 1,4-dioxane / water mixture solution with potassium phosphate and Xphos-Pd-G3 to obtain X3 compound; X3 compound is reacted with a base such as NaOH in a suitable solvent such as tetrahydrofuran to obtain X4 compound; X4 compound is reacted with X5 compound in the presence of potassium phosphate, CuI and N,N-dimethyl-1,2-cyclohexanediamine to obtain X6 compound; X6 compound is deprotected under acidic condition to obtain the compound of general formula (I).

[0136] In one embodiment, a method for preparing the compound of general formula (II) according to the present application is provided:

[0137]

[0138] First step: reacting N1 compound with N-bromosuccinimide NBS to obtain N2 compound

[0139] Second step: reacting N2 compound with compound N6 Suzuki reaction in a suitable solvent to obtain N3 compound

[0140] Third step: Ullmann reaction of N3 compound with N5 compound

[0141] Fourth step: deprotection of THP protecting group of N4 compound to obtain the compound of general formula (II) according to the present application.

[0142] In one embodiment, an exemplary reaction scheme for preparing the compound of general formula (II) according to the present application is provided:

[0143]

[0144] According to the above scheme, N1 compound is reacted with N-bromosuccinimide NBS in a suitable solvent such as DMF to obtain N2 compound; N2 compound is reacted with 1H-pyrazole-5-boronic acid pinacol analogue N6 in a suitable solvent such as 1,4-dioxane / water mixture with potassium phosphate and Pd(dtbpf)Cl2 to obtain N3 compound; N3 compound is reacted with N5 compound in the presence of potassium phosphate, CuI and N,N-dimethyl-1,2-cyclohexanediamine to obtain N4 compound; N4 compound is deprotected under acidic conditions to obtain the compound of general formula (II).

[0145] The stereoisomers of the compound of general formula (I) or (II) according to the present application can be obtained by separating the obtained racemic mixture or other mixtures, based on their different physico-chemical properties, using well-known means such as, for example, fractional crystallization or HPLC techniques. The enantiomers can also be separated using chiral chromatographic columns. The optically active starting materials can also be used to react under conditions that do not cause racemization or epimerization.

[0146] The pharmaceutically acceptable salts of the compound of general formula (I) or (II) according to the present application or its stereoisomers can be formed by reacting the N atom on the structure of the compound or its stereoisomers with inorganic or organic acids. The inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, phosphoric acid, glycerophosphoric 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, hexanoic acid, heptanoic acid, glucoheptanoic acid, oxalic acid, maleic acid, fumaric acid, malic acid, glutamic acid, pyroglutamic acid, salicylic acid, sulfonic acid (such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid), etc.

[0147] ​Other intermediates or reactants not specifically mentioned can be obtained commercially or prepared by synthetic methods generally known in the art.

[0148] The above general synthetic routes represent general methods for most of the examples. For compounds with special substituents, modifications known to those of ordinary skill in the art or changes in the reaction sequence can be made at certain steps.

[0149] Examples

[0150] The following examples illustrate the preparation and biological activity evaluation of the compounds described in the present application.

[0151] The following examples are provided to enable those of skill in the art to more clearly understand and to practice the present application. They should not be considered as limiting the scope of the application, but merely as being illustrative and representative thereof.

[0152] The materials or reagents used herein are commercially available or prepared by synthetic methods generally known in the art. The following reaction schemes illustrate specific synthetic methods for the compounds of the present application.

[0153] Specifically as follows:

[0154] Preparation of key intermediates a1-a3:

[0155] Preparation of intermediate a1:

[0156]

[0157] 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 the reaction was allowed to proceed at room temperature for 2 hours, then the temperature was raised to 55 °C and stirring was continued for 4 hours, and then the temperature was lowered to room temperature. Intermediate a1-2 (17.2 g, 121.2 mmol) was added to the reaction mixture in portions, and the reaction was allowed to proceed at 55 °C for 40 hours. The reaction was stopped, filtered, and the solvent was removed under reduced pressure. The residue was dissolved in 50 mL of water and extracted with ethyl acetate. The product was isolated by flash column chromatography to obtain intermediate a1 (9.1 g, yield: 72%), LC-MS: [M+H] + : 211.

[0158] Preparation of intermediate a2:

[0159]

[0160] 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 temperature was raised to 110 °C for 12 hours. The reaction was cooled to room temperature, and the solvent was removed under reduced pressure to obtain 9.0 g of crude intermediate a2-2, LC-MS: [M+H] + : 248.

[0161] Step 2: Crude intermediate a2-2 (9.0 g) was dissolved in 80 mL of ethanol, and pyridine p-toluenesulfonate PPTS (54.6 mmol, 13.7 g) was added. The temperature was raised to 90 °C for 36 hours, and the reaction was stopped. The solvent was removed under reduced pressure, 100 mL of water was added to the system, and extraction was performed with ethyl acetate. Flash column chromatography was used to isolate intermediate a2-3 (7.5 g, two-step yield: 76%), LC-MS: [M+H] + : 230.

[0162] Step 3: Intermediate a2-3 (19.6 mmol, 4.5 g) was dissolved in 25 mL of phosphorus oxychloride in an ice bath, and stirring was performed for 10 minutes. The ice bath was removed, the temperature was raised to 110 °C, and stirring was continued for 2 hours. The reaction was stopped, the reaction was cooled to room temperature, 150 mL of ice water was slowly added to the reaction, the pH was adjusted to about 8 with a saturated sodium bicarbonate solution, and extraction was performed with ethyl acetate. The crude product was dried over anhydrous sodium sulfate, and flash column chromatography was used to isolate intermediate a2 (3.5 g, yield: 72%). LC-MS: [M+H] + : 248.

[0163] Preparation of intermediate a3:

[0164]

[0165] 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-tert-butyl dicarbonate (9.01 mmol, 1.97 g) was slowly added. The reaction was stopped after 2 hours of reaction at room temperature. 50 mL of water was added to the system, extraction was performed with ethyl acetate, and anhydrous sodium sulfate was used for drying. This resulted in intermediate a3 (1.9 g, yield: 91%), LC-MS: [M+H] + : 348.

[0166] Preparation of key intermediate b1:

[0167]

[0168] First step: under nitrogen protection, dissolve the starting material b1-1 (5 mmol, 1.05 g) in 15 mL fluorobenzene, slowly add diethylzinc (2 mmol / L in toluene, 12.5 mL, 25 mmol) and chloroiodomethane in fluorobenzene (4.4 g / 1.8 mL, 25 mmol), react at room temperature for 12 hours. Place the system in an ice bath, add 40 mL saturated aqueous ammonium chloride solution to quench the reaction, extract with ethyl acetate, dry over anhydrous sodium sulfate, separate by flash column chromatography to obtain intermediate b1-2 (730 mg, yield: 65%).

[0169] Second step: under nitrogen protection, dissolve intermediate b1-2 (3.23 mmol, 730 mg) in 16 mL of a mixture of methanol / acetonitrile (1 / 1, v / v), slowly add potassium fluoride aqueous solution (749 mg / 3 mL). After stirring the mixture for 10 minutes, add L-tartaric acid (6.46 mmol, 968 mg) and tetrahydrofuran (350 μL), continue to react at room temperature for 1.5 h. Stop the reaction, filter, concentrate the filtrate to obtain intermediate b1-3 (600 mg, crude product).

[0170] Third step: under nitrogen protection, dissolve 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) in 30 mL of a mixture of toluene / water (6 / 1, v / v), add Pd(dppf)Cl2(0.8 mmol, 586 mg). Heat to 110°C and react for 12 hours, stop the reaction, filter, add 70 mL water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, separate by flash column chromatography to obtain intermediate b1 (1.3 g, yield: 41%), LC-MS: [M+H] + :410.

[0171] Preparation of key intermediates c1-c8:

[0172] Preparation of intermediate c1:

[0173]

[0174] First Step: Dissolve the starting material 5,7-dichloropyrazolo[l,5-a]pyrimidine cl-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), react at room temperature for 16 hours, stop the reaction. Remove the solvent under reduced pressure, add 100 mL of water to the mixture, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and purify the crude product by column chromatography (PE / EA = 6 / 1) to obtain compound cl-2 (7.90 g, yield: 84%), LC-MS: [M+H] + : 252.0.

[0175] 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.0 Hz, 1H), 7.01 (s, 1H), 6.72-6.70 (m, 1H), 5.36 (q, J = 8.4 Hz, 2H).

[0176] Second Step: Dissolve compound 5-chloro-7-(2,2,2-trifluoroethoxy)pyrazolo[l,5-a]pyrimidine cl-2 (31.4, 79.0 g) and potassium fluoride (157.0 mmol, 9.12 g) in 80 mL of dry DMSO, warm to 140 °C for 4 hours, cool to room temperature, stop the reaction. Add 100 mL of water to the mixture, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify the crude product by column chromatography (PE / EA = 10 / 1) to obtain compound cl-3 (3.5 g, yield: 47%), LC-MS: [M+H] + : 236.1.

[0177] Third Step: Dissolve compound 5-fluoro-7-(2,2,2-trifluoroethoxy)pyrazolo[l,5-a]pyrimidine cl-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 warm to 120 °C and continue to react for 2 hours, cool to room temperature, 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 purify the crude product by column chromatography (PE / EA = 10 / 1) to obtain compound cl (3.0 g, yield: 61%), LC-MS: [M+H] + : 329.0.

[0178] 1H 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).

[0179] Referring to the synthesis of intermediate cl, using similar starting materials, the following intermediates c2 to c4 were synthesized:

[0180]

[0181]

[0182] Preparation of intermediate c5:

[0183]

[0184] First step: Intermediate 3-(7-(2,2,2-trifluoroethoxy)pyrazolo[l,5-a]pyrimidin-5-yl)-8-oxa-3- azabicyclo[3.2.1]octane cl (9.14 mmol, 3.0 g) and sodium hydroxide (18.28 mmol, 0.73 g) were dissolved in 20 mL of tetrahydrofuran, warmed to 70 °C for 16 hours, cooled to room temperature, the reaction was stopped, filtered. The solvent was evaporated under reduced pressure, the crude was separated and purified by flash reverse column chromatography (acetonitrile / water) to obtain compound c5-1 (2.0 g, yield: 89%), LC-MS: [M+H] + : 247.2.

[0185] 1 H NMR (400 MHz, DMSO-d6) δ 7.48 (d, J = 2.0 Hz, 1H), 5.65 (d, J = 1.6 Hz, 1H), 4.89 (s, 1H), 4.35 (s, 2H), 3.71 (d, J = 12.4 Hz, 2H), 2.83 (dd, J = 12.0 Hz, 1.6 Hz, 2H), 1.81 - 1.68 (m, 4H).

[0186] Second step: compound 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidin-7- yl hydroxyl c5-1 (8.12 mmol, 2.00 g) was dissolved in 10 mL of phosphorus oxychloride, and the temperature was raised to 110 °C for 4 hours, and then cooled to room temperature, and the reaction was stopped. The reaction solution was slowly poured into 50 mL of ice water mixture, and saturated aqueous sodium bicarbonate solution was added to adjust the pH to about 9, and 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.

[0187] 1 H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 2.4 Hz, 1H), 7.03 (s, 1H), 6.19 (d, J = 2.4 Hz, 1H), 4.44 (d, J = 2.0 Hz, 2H), 4.00 (d, J = 12.4 Hz, 2H), 3.10 (dd, J = 12.4 Hz, 2.0 Hz, 2H), 1.87-1.81 (m, 2H), 1.74-1.66 (m, 2H).

[0188] Third step: 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 after stirring for 5 minutes, the temperature was raised to 90 °C and the reaction was continued for 16 hours, and then cooled to room temperature, and the reaction was stopped, and filtered. To the mixture, 35 mL of water was added, and 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 c5 (0.7 g, yield: 44%), LC-MS: [M+H] + : 325.3.

[0189] 1H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 7.84 (d, J = 2.4 Hz, 1H), 6.50 (s, 1H), 6.09 (d, J = 2.0 Hz, 1H), 4.43 (s, 2H), 4.01 (d, J = 13.2 Hz, 2H), 3.08 (dd, J = 12.4 Hz, 2.0 Hz, 2H), 2.18 (d, J = 28.0 Hz, 6H), 1.89 - 1.81 (m, 2H), 1.77 - 1.69 (m, 2H).

[0190] Referring to the synthesis of intermediate c5, using similar starting materials / intermediates, the following intermediate compounds c6 to c8 were synthesized:

[0191]

[0192] Example 1: Preparation of compound M3

[0193]

[0194] 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, triethylamine (2.60 mmol, 263 mg) was added, and the reaction was heated by microwave at 180 °C for 2 hours. The reaction was stopped, 40 mL of water was added to the system, and dichloromethane was extracted. The 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-tert-butyl dicarbonate (1.29 mmol, 282 mg) were added, and the reaction was stopped after 2 hours of reaction at room temperature. 30 mL of water was added to the system, dichloromethane was extracted, and anhydrous sodium sulfate was dried to obtain the crude intermediate M3-2. LC-MS: [M+H] + : 425.

[0195] The crude intermediate M3-2 from the previous step was dissolved in 12 mL of anhydrous dichloromethane under ice bath, N-iodosuccinimide NIS (1.29 mmol, 290 mg) was slowly added in batches, and stirring was continued for 2 hours. The reaction was stopped, 30 mL of saturated aqueous ammonium chloride solution was added, and dichloromethane was extracted. Anhydrous sodium sulfate was dried, and flash column chromatography (PE / EA = 2 / 1) was used to separate to obtain compound M3-3 (450 mg, two-step yield: 64%). LC-MS: [M+H] + : 551.

[0196] Under nitrogen protection, 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), potassium phosphate (1.64 mmol, 348 mg) and Xphos-Pd-G3 (0.08 mmol, 68 mg) were added. The reaction was heated at 95°C for 2 hours in a microwave. 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.

[0197] Intermediate M3-4 (260 mg) was dissolved in 10 mL of tetrahydrofuran, 5 mL of 1N NaOH aqueous solution was added, and the temperature was raised to 70°C for 1 hour. The reaction was stopped, the solvent was evaporated under reduced pressure, 40 mL of water was added, 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.

[0198] Under nitrogen protection, 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 temperature was raised to 110°C for 1.5 hours under microwave, cooled to room temperature, 30 mL of water was added to the reaction, 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.

[0199] The intermediate M3-6 (100 mg) in the above step was dissolved in 4 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 2 hours. The reaction was stopped, the solvent was evaporated under reduced pressure, the pH was adjusted to 10 by adding saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, and separated by HPLC preparative chromatography (0.1% trifluoroacetic acid in water) to obtain the target compound M3 (30 mg, yield: 37%), LC-MS: [M+H] + : 468.

[0200] M3: 1H NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.83 (d, J = 2.6 Hz, 1H), 8.68 (d,

[0201] H NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.83 (d, J = 2.6 Hz, 1H), 8.68 (d,

[0202] Example 2: Preparation of compounds M4-M15

[0203]

[0204] First step: under nitrogen protection, 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 DMF, N-bromosuccinimide NBS (2.22 mmol, 395.1 mg) was slowly added, and the reaction was allowed to proceed at room temperature for 2 hours, and then the reaction was stopped. 50 mL water was added to the reaction solution, and extraction was performed with ethyl acetate, and then saturated brine was washed, 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.

[0205] 1 H NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.83 (d, J = 2.6 Hz, 1H), 8.68 (d,

[0206] Second step: 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, stirred for 5 minutes, then heated to 90°C and continue to react for 16 hours, cooled to room temperature, stop the reaction, filter. Add 45 mL of water to the mixture, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify the crude product 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.

[0207] 1 H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 4.23 (q, J = 7.2 Hz, 3H), 1.29 (t, J = 6.8 Hz, 3H), 1.20-1.14 (m, 2H), 1.06-0.98 (m, 2H).

[0208] Third step: 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, heated to 110°C and continue to stir for 4 hours, cooled to room temperature, stop the reaction. Add 40 mL of water to the mixture, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify the crude product 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.

[0209] 1 H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 4.23 (q, J = 7.2 Hz, 3H), 1.29 (t, J = 6.8 Hz, 3H), 1.20-1.14 (m, 2H), 1.06-0.98 (m, 2H).

[0210] Third step: under nitrogen protection, compound M4-4 (0.14 mmol, 80 mg) was dissolved in 4 mL of mixed solvent of dichloromethane and trifluoroacetic acid (v / v = 3 / 1), and reacted at room temperature for 2 hours. Saturated aqueous sodium bicarbonate solution was added to the mixture to adjust the pH to about 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 HPLC to obtain compound M4 (13.1 mg), LC-MS: [M+H] + : 502.2.

[0211] 1 H NMR (400 MHz, DMSO-d6) δ 12.79 (br s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 8.75 (d, J = 2.0 Hz, 1H), 8.27 (t, J = 2.4 Hz, 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 = 11.6 Hz, 2H), 2.38 (s, 3H), 2.24 (s, 3H), 1.90-1.74 (m, 4H).

[0212] Referring to the synthesis of compound M4, using similar raw materials / intermediates, the following target compounds M5 to M15 were synthesized:

[0213]

[0214]

[0215]

[0216] Example 3: Preparation of compounds M16-M18

[0217]

[0218] Step 1: Under nitrogen protection, 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. The reaction was carried out at room temperature for 2 hours and the reaction was stopped. 30 mL of water was added to the reaction solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by flash reverse 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. The reaction was scaled up repeatedly to afford M16-1 (2.0 g).

[0219] Step 2: Under nitrogen protection, 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 after stirring for 5 minutes, the temperature was raised to 90 ° C and continued to stir for 16 hours. The mixture was cooled to room temperature, the reaction was stopped, and the mixture was filtered. 30 mL of water was added to the mixed solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by column chromatography (DCM / MeOH=50 / 1) to give 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.

[0220] Step 3: Under nitrogen protection, 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, and catalyst CuI (0.33 mmol, 62 mg) was added, and the temperature was raised to 110°C and stirring was continued for 16 hours, and the temperature was cooled to room temperature, and the reaction was stopped. Water 40 mL was added to the mixture, and ethyl acetate was extracted, and saturated brine was washed, and anhydrous sodium sulfate was dried, and concentrated to obtain crude compound M16-3, LC-MS: [M+H] + : 538.3.

[0221] Step 4: Under nitrogen protection, the crude compound M16-3 obtained in step 3 was dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid (v / v = 3 / 1), and the reaction was carried out at room temperature for 2 hours. Saturated aqueous sodium bicarbonate solution was added to the mixture to adjust the pH to about 9, and ethyl acetate was extracted, and saturated brine was washed, and anhydrous sodium sulfate was dried, and concentrated, and the crude product was separated and purified by preparative HPLC to obtain compound M16 (13.8 mg), LC-MS: [M+H] + : 454.2.

[0222] 1 H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 8.67 (d, J = 4.8 Hz, 1H), 8.25 (s, 1H), 8.08-8.02 (m, 1H), 7.63 (dd, J = 8.0 Hz, 4.8 Hz, 2H), 6.64 (br s, 2H), 5.06 (s, 1H), 4.75 (s, 1H), 3.83 (d, J = 15.6 Hz, 2H), 3.63 (d, J = 10.0 Hz, 2H), 2.34 (s, 3H), 2.24 (s, 3H), 1.97 (dd, J = 25.2, 9.2 Hz, 2H).

[0223] Referring to the synthesis of compound M16, using similar raw materials / intermediates, the following target compounds M17 to M18 were synthesized:

[0224]

[0225] Example 4: Preparation of compounds M1a and M1b

[0226]

[0227] Under nitrogen protection, 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 reaction 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 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.

[0228] Under 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 batches, and stirring was continued for 2 hours. The reaction was stopped, 30 mL of saturated aqueous ammonium chloride was added, and dichloromethane was extracted. Dried over anhydrous sodium sulfate, separated by flash column chromatography (PE / EA = 2 / 1) to obtain compound M1-3 (1.4 g, yield: 97%). LC-MS: [M+H] + : 522.

[0229] Under nitrogen protection, 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), potassium phosphate (5.38 mmol, 1.1 g) and Xphos-Pd-G3 (0.27 mmol, 229 mg) were added. The reaction was heated at 95°C for 2 hours under microwave. 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.

[0230] Intermediate M1-5 (700 mg) was dissolved in 15 mL of tetrahydrofuran, 6 mL of 1N NaOH aqueous solution was added, and the reaction was heated to 70°C for 1 hour. The reaction was stopped, the solvent was evaporated under reduced pressure, 30 mL of water was added, dichloromethane was extracted, and separated by flash column chromatography (PE / EA = 1 / 1) to obtain compound M1-6 (530 mg, yield: 93%), LC-MS: [M+H] + : 446.

[0231] Under nitrogen protection, 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 dissolved in 10 mL DMF. After stirring for 5 minutes, N, N-dimethyl-1, 2-cyclohexanediamine (0.24 mmol, 34 mg) was slowly added. The reaction was heated to 110°C under microwave for 1.5 hours, cooled to room temperature, filtered, 50 mL water was added to the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, separated by flash column chromatography to obtain compound M1-8 (240 mg, yield: 39%), LC-MS: [M+H] + :523.

[0232] Under nitrogen protection, NaH (1.66 mmol, 60%, 40 mg) and trimethylsulfoxonium chloride (1.0 mmol, 129 mg) were added to the reaction bottle, 1 mL 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 DMSO, and the mixture was slowly injected into the reaction. The reaction was heated to 65°C for 4 hours, and then stopped. 20 mL water was added to the reaction, extracted with ethyl acetate to obtain 200 mg of crude intermediate M1-9. LC-MS: [M+H] + :537.

[0233] The crude intermediate M1-9 (200 mg) was dissolved in 4 mL dichloromethane, 2 mL trifluoroacetic acid was added, and the reaction was carried out at room temperature for 2 hours. The reaction was stopped, the solvent was removed under reduced pressure, saturated aqueous sodium bicarbonate solution was added to adjust the pH to 10, dichloromethane was extracted, and HPLC preparative chromatography was used to separate to obtain compound M1 (70 mg, two-step yield: 34%), LC-MS: [M+H] + :453.

[0234] Compound M1 was separated by chiral preparative chromatography to obtain target compounds M1a and M1b.

[0235] M1b: 1 H NMR (400 MHz, DMSO-d6) δ 12.91 (d, J = 111.8 Hz, 1H), 8.85 (d, J = 2.5

[0236] Hz, 1H), 8.68 (d, J = 4.7 Hz, 1H), 8.50 (s, 1H), 8.10 - 8.03 (m, 1H), 7.77 (s, 1H), 7.64 (dd, J = 8.2, 4.8 Hz, 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), 2.24 (s, 3H), 1.99 - 2.01 (m, 1H), 1.91 (s, 1H), 1.55 (dd, J = 9.3, 4.2 Hz, 1H), 1.23 (d, J = 5.5 Hz, 1H).

[0237] Example 5: Preparation of compounds M2a and M2b

[0238]

[0239] Intermediate b1 (3.18 mmol, 1.3 g) was dissolved in 25 mL of anhydrous dichloromethane under ice bath, N-iodosuccinimide NIS (3.50 mmol, 787 mg) was added slowly in batches and stirring was continued for 2 hours. The reaction was stopped, 35 mL of saturated aqueous ammonium chloride solution was added, dichloromethane was extracted. Anhydrous sodium sulfate was dried, flash column chromatography was used for separation (PE / EA = 2 / 1) to obtain compound M2-1 (1.3 g, yield: 77%). LC-MS: [M+H] + : 536.

[0240] 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) under nitrogen protection, potassium phosphate (4.86 mmol, 1.03 g) and Xphos-Pd-G3 (0.24 mmol, 203 mg) were added. The reaction was heated under microwave 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 with anhydrous sodium sulfate, and flash column chromatography was used for separation (PE / EA = 4 / 1) to obtain compound M2-2 (1.3 g, yield: 96%). LC-MS: [M+H] + : 560.

[0241] Intermediate M2-2 (1300 mg) was dissolved in 20 mL of tetrahydrofuran, 8 mL of 1N NaOH aqueous solution was added, and the temperature was raised to 70°C for 1 hour. The reaction was stopped, the solvent was evaporated under reduced pressure, 40 mL of water was added, dichloromethane was extracted, and flash column chromatography was used for separation (PE / EA = 1 / 1) to obtain compound M2-3 (1.0 g, yield: 94%), LC-MS: [M+H]+ :460.

[0242] Under nitrogen protection, intermediate M2-3 (0.65 mmol, 300 mg), 3-bromo-5- fluoro-pyridine M2-4 (0.98 mmol, 172 mg), potassium phosphate (1.30 mmol, 276 mg) and Cul (0.06 mmol, 12 mg) were dissolved in 8 mL DMF. After stirring for 5 minutes, N,N-dimethyl-1,2-cyclohexanediamine (0.12 mmol, 17 mg) was slowly added. After the reaction was stopped after being heated to 110°C under microwave for 1.5 hours, the reaction solution was filtered. 30 mL water was added to the reaction solution, which was 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.

[0243] Intermediate M2-5 (0.36 mmol, 200 mg) was dissolved in 4 mL dichloromethane, and 2 mL trifluoroacetic acid was added, and the reaction was carried out at room temperature for 2 hours. The reaction was stopped, the solvent was removed by evaporation under reduced pressure, saturated aqueous sodium bicarbonate solution was added to adjust the pH to 10, dichloromethane was added for extraction, and HPLC preparative chromatography was used for separation to obtain compound M2 (80 mg, yield: 48%), LC-MS: [M+H] + :471.

[0244] Compound M2 was separated by chiral preparative chromatography to obtain target compounds M2a and M2b.

[0245] M2b: 1 H NMR (400 MHz, DMSO-d6) δ 12.91 (d, J = 117.3 Hz, 1H), 8.77 (s, 1H), 8.73

[0246] (d, J = 2.6 Hz, 1H), 8.49 (s, 1H), 8.14 (dd, J = 9.6, 2.7 Hz, 1H), 7.69 (d, J = 79.0 Hz, 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, 4.6 Hz, 1H), 2.38 (s, 3H), 2.24 (s, 3H), 2.01 - 2.08 (m, 1H), 1.91 (s, 1H), 1.54 (dd, J = 9.2, 4.2 Hz, 1H), 1.22 - 1.26 (m, 1H).

[0247] Example 6: Preparation of compound M19

[0248]

[0249] First step: 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, stirred for 5 minutes, then heated to 90 °C and continue to stir for 16 hours, cooled to room temperature, stop the reaction, filter. To the mixture, 40 mL of water was added, 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-(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.

[0250] Second step: 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, heated to 110 °C and continue to stir for 16 hours, cooled to room temperature, stop the reaction. To the mixture, 40 mL of water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to obtain the crude compound M19-3, LC-MS: [M+H] + : 566.3.

[0251] Third step: The crude M19-3 obtained in the second step was dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid (v / v = 3 / 1) under nitrogen protection, and reacted at room temperature for 2 hours, then the reaction was stopped. To the mixture, saturated aqueous sodium bicarbonate solution was added to adjust the pH to about 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 HPLC to obtain compound M19 (79.5 mg), LC-MS: [M+H] + : 482.3.

[0252] 1 H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 8.67 (dd, J = 4.8 Hz, 1.2 Hz, 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 = 2.0 Hz, 2H), 4.16 (d, J = 12.4 Hz, 2H), 3.19 (d, J = 11.2 Hz, 2H), 2.33 (s, 3H), 2.23 (s, 6H), 1.92-1.73 (m, 4H).

[0253] Example 7: Preparation of compound M20

[0254]

[0255] 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 mixed solvent of dichloromethane and trifluoroacetic acid (v / v = 3 / 1) under nitrogen protection, and the reaction was allowed to proceed at room temperature for 2 hours, and then stopped. Saturated aqueous sodium bicarbonate solution was added to the mixture to adjust the pH to about 9, and then extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then the crude product was separated and purified by preparative HPLC to obtain compound M20 (18.0 mg). LC-MS: [M+H] + : 391.2.

[0256] 1 H NMR (400 MHz, DMSO-d6) δ 12.67 (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 = 10.8 Hz, 2H), 2.20 (s, 6H), 1.90-1.73 (m, 4H).

[0257] Example 8: Preparation of compounds M21-M22

[0258]

[0259] Step 1: Under nitrogen protection, 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 (0.32 mmol, 150 mg) was dissolved in 5 mL of anhydrous DMF, NaH (0.47 mmol, 18.96 mg) was added, stirred for 5 minutes, then the raw material M21-1 (0.47 mmol, 132.4 mg) was added, the temperature was raised to 70°C and the stirring was continued for 16 hours, then the temperature was cooled to room temperature, the reaction was stopped, and the mixture was filtered. Add 40 mL of water to the mixture, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate to obtain the crude compound M21-2, LC-MS: [M+H] + 658.5.

[0260] Step 2: Under nitrogen protection, the crude M21-2 obtained in the first step was dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid (v / v = 3 / 1), and the reaction was carried out at room temperature for 2 hours. The reaction was stopped. Add saturated aqueous sodium bicarbonate solution to the mixture to adjust the pH to about 9, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify the crude product by preparative HPLC to obtain compound M21 (38.89 mg), LC-MS: [M+H] + 474.3.

[0261] 1 H NMR (400 MHz, CDCl3) δ 8.34 (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 = 12.0 Hz, 2H), 3.25 (d, J = 11.2 Hz, 2H), 3.16 (d, J = 11.6 Hz, 2H), 2.87 (t, J = 11.6 Hz, 2H), 2.25 (s, 3H), 2.15-2.03 (m, 5H), 1.94-1.82 (m, 4H), 1.80-1.72 (m, 2H).

[0262] Referring to the synthesis of compound M21, using similar raw materials / intermediates, the following target compound M22 was synthesized:

[0263]

[0264] Example 9: ATR kinase activity experiment

[0265] ATR kinase activity test was performed by detecting phosphorylation of downstream substrate p53 protein. GST-tagged full-length P53 protein was purchased from Sigma (Cat# 14-865), Anti-phospho-p53 (ser15)-K antibody and Anti-GST-d2 antibody were purchased from Cisbio (Cat# 61GSTDLA; 61P08KAE). Time-resolved fluorescence system was used to determine the amount of phosphorylated P53 protein. Before starting the experiment, the following working solutions were prepared as needed: 1x reaction buffer (20 mM HEPES pH 8.0, 1% glycerol, 0.01% Brij-35), dilution buffer (20 mM HEPES pH 8.0, 1% glycerol, 0.01% Brij-35, 5 mM DTT and 1% BSA), stop solution (20 mM HEPES pH 8.0, 1% glycerol, 0.01% Brij-35, 250 mM EDTA), 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). The clinical drug in research AZD6738 (purchased from selleck company) was used as a positive control.

[0266] The specific experimental operation steps are as follows:

[0267] The 4x gradient dilution compound solution was prepared with 1x reaction buffer to get 9 different concentrations of compounds, 2.5 μL 4x gradient dilution compound solution was added to the 384-well assay plate (784075, Greiner). The 4x p53 substrate working solution (40 nM) was prepared with 1x reaction buffer, 2.5 μL 4x p53 substrate working solution was added to the 384-well assay plate. The 4x ATR / ATRIP working solution (12.8 ng / μL) was prepared with dilution buffer, 2.5 μL 4x ATR / ATRIP working solution was added to the 384-well assay plate. The 4x ATP working solution (2 mM) was prepared with deionized water, 2.5 μL 4x ATP working solution was added to the 384-well assay plate, and incubated at room temperature for 30 minutes in the dark. 5 μL of stop solution was added to the 384-well assay plate. Finally, 5 μL of detection mixture (0.09 ng / μL of Anti-phospho-p53 (ser15)-K and 6 ng / μL of Anti-GST-d2) was added to the 384-well assay plate. The fluorescence signal (excitation wavelength of 320 nm, emission wavelength of 620 nm and 665 nm) was detected with M5e (Molecular Device) instrument after incubation at room temperature overnight. The inhibition rate in each well was calculated by 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) x 100%, and the IC 50 value (IC 50 value (IC

[0268] Table 1

[0269] Compound No. <![CDATA[ATR / IC 50 / nM]]> M3 7 M7 9 M15 10 M16 445 M18 64 M1a 25 M1b 138 M2a 33 M2b 548 M19 15 M20 41 M21 22 AZD6738 15

[0270] According to the data in Table 1, the compounds of the present application have excellent activity in inhibiting ATR kinase, which is comparable to the clinically studied drug AZD6738, and even better, indicating that the compounds of the present application can be used as ATR inhibitors, selectively acting on tumor cells, and are expected to become excellent potential drugs for tumor treatment.

[0271] Example 10: In vitro cell proliferation inhibition experiment 1

[0272] 22Rv1 cells were purchased from American Type Culture Collection (ATCC).

[0273] The specific operation steps of the experiment are as follows:

[0274] 22Rv1 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin streptomycin (ps), and cells with more than 85% confluence were used for the experiment. About 2000 cells were seeded in each well of a 96-well plate, and the cells were cultured for 24 hours, then different concentrations of the test compound (0-50 μM) were added to treat the cells, and three parallel wells were set for each group. Blank wells (containing only medium) and control wells (seeded with cells without drugs) were set. After 120 hours of culture, 10 μL of CCK8 solution (Beyotime, #C0037) was added to each well, and the OD value was read by Biotek Synergy H1 multifunctional enzyme labeler after 4 hours of incubation in the dark.

[0275] Inhibition rate (%) = 100% x (control well-test well) / (control well-blank well)

[0276] The IC50of the compound was calculated by using the parameter fitting standard software (GraphPad Prism 8.0). 50 Numerical values. The results are shown in Table 2.

[0277] Table 2

[0278] Compound No. 22Rv1 / IC 50 / μM Compound No. 22Rv1 / IC 50 / μM]]> M3 0.21 M13 1.38 M4 0.65 M14 1.40 M5 0.44 M15 0.20 M6 1.33 M16 4.05 M7 0.22 M18 0.33 M8 2.40 M19 0.15 M9 0.97 M20 0.59 M10 0.37 M21 4.33 M11 0.50 M22 4.48 M12 1.71

[0279] The proliferation inhibition experiment results prove that the compound of the present application has good inhibitory effect on 22Rv1 human prostate cancer cells with ATM mutation, and the IC50value of part of the compounds is less than 1 μM, and even less than 0.3 μM. 50

[0280] Example 11: In vitro cell proliferation inhibition experiment 2

[0281] In this experiment, the effect of the compound of the present application on the in vitro cell proliferation in tumor cell lines TOV21G and 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) was detected to study the inhibitory effect of the compound of the present application on tumor cell proliferation.

[0282] TOV21G cells and Rec-1 cells were purchased from American Type Culture Collection (ATCC), and other cells were from Shanghai Medicago Biopharma Co., Ltd.

[0283] ​TOV21G cells were cultured in MCDB105 / M199 medium (containing 15% FBS and 1% ps), and when the cell confluence reached more than 85%, they were used for the experiment. About 1000 cells were inoculated in each well of a 96-well culture plate, and the cells were cultured for 24 hours, then different concentrations of the test compound (0-10 μM) were added to treat the cells, and 3 parallel wells were set for each group. Blank wells (containing only medium) and control wells (inoculated with cells without drug treatment) were set. After 120 hours of culture, 40 μL of Cell Titer-Glo solution (Promega, #G7573) was added to each well, and the light value was read by Biotek Synergy H1 multifunctional microplate reader.

[0284] Rec-1 cells were cultured in RPMI1640 medium (containing 10% FBS and 1% ps), and about 6000 cells were inoculated in each well of a 96-well culture plate, and different concentrations of the test compound (0-10 μM) were added to treat the cells, and 3 parallel wells were set for each group. Blank wells (containing only medium) and control wells (inoculated with cells without drug treatment) were set. After 120 hours of culture, 40 μL of Cell Titer-Glo solution was added to each well, and the light value was read by Biotek Synergy H1 multifunctional microplate reader.

[0285] The rest of the cell culture and experimental scheme refer to the experimental scheme of the above two cell lines, and the cell culture medium uses the conventional medium recommended by ATCC.

[0286] Inhibition rate (%) = 100% x (control well-test well) / (control well-blank well)

[0287] The IC50 of the compound was calculated by using the parameter fitting standard software (GraphPad Prism 8.0). 50 Numerical values. The results are shown in Tables 3 and 4.

[0288] Table 3

[0289]

[0290] Table 4 Anti-proliferation effect of compound M3 on various tumor cells.

[0291]

[0292]

[0293] The proliferation inhibition experiment results prove that the compound of the present application has good inhibition effect in the studied human tumor cells.

[0294] Example 12: In vitro liver microsomal stability experiment of the compounds

[0295] The liver microsomal stability experiment was performed for the compounds of the present application. The test compound (final concentration 2.0 nM) was co-incubated with human / mouse liver microsomes with or without NADPH, and the compound concentration in the incubation supernatant was detected within 60 minutes. The results of representative compounds are shown in Table 5 below.

[0296] Table 5

[0297] The above results show that the molecules of the present application have very high stability in mouse and human liver microsomes, indicating slow metabolism in vivo.

[0298] Example 13: In vivo pharmacokinetic experiment of the compounds

[0299] The in vivo pharmacokinetic experiment was performed for the compounds of the present application.

[0300] Experimental method:

[0301] Male ICR mice (3 per group) were orally administered by gavage at a dose of 10 mg / kg. Plasma samples were collected before administration (0 hour) and after administration (0.25, 0.5, 1, 2, 4, 6, 8, 24 hours), and the collected samples were analyzed by LC / MS and the data were collected. The collected data were analyzed by Analyst v1.6.2 (AB Applied Biosystems Company, USA) software to calculate the relevant pharmacokinetic parameters.

[0302] The results of representative compounds are shown in Table 6 below.

[0303] Table 6

[0304]

[0305] The above results show that the molecules of the present application have very high in vivo exposure after oral administration in mice, and are expected to have excellent anti-tumor effect at a lower dose in clinic.

[0306] Example 14: Study on the selectivity of the compounds for ATR kinase

[0307] The compounds of the present application were tested for inhibition of the same family (ATM, DNA-PK):

[0308] According to the experimental method reported in the literature, the test compound was diluted 3-fold from 10 μM to 0.51 nM (a total of 10 concentrations), and the inhibition of the kinase ATM 1and DNA-PK 2 The results are shown in Table 7 below.

[0309] Table 7:

[0310] The above results indicate that the compounds of the present invention are highly selective for ATR and have low inhibitory activity against other kinases in the family, thereby bringing higher safety benefits.

[0311] The above is a further detailed description of the present invention in conjunction with specific embodiments, and the specific implementation of the present invention cannot be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

[0312] References:

[0313] [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;

[0314] [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 (II), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: ###0001### wherein: R1 is selected from the group consisting of: ###0002### ###0003### ###0004### ###0005### ###0006### ###0007### ###0008### ###0009### ###0010### ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ###0053### ###0054### ###0055### ###0056### ###0057### ###0058### ###0059### ###0060### ###0061### ###0062### ###0063### ###0064### ###0065### ###0066### ###0067### ###0068### ###0069### ###0070### ###0071### ###0072### ###0073### ###0074### ###0075### ###0076### ###0077### ###0078### ###0079### ###0080### ###0081### ###0082### ###0083### ###0084### ###0085### ###0086### ###0087### ###0088### ###0089### ###0090### ###0091### ###0092### ###0093### ###0094### ###0095### ###0096### ###0097### ###0098### ###0099### ###0100### ###0101### ###0102### ###0103### ###0104### ###0105### ###0106### ###0107### ###0108### ###0109### ###0110### ###0111### ###0112### ###0113### ###0114### ###0115### ###0116### ###0117### ###0118### ###0119### ###0120### ###0121### ###0122### ###0123### ###0124### ###0125### ###0126### ###0127### ###0128### ###0129### ###0130### ###0131### ###0132### ###0133### ###0134### ###0135### ###0136### ###0137### ###0138### ###0139### ###0140### ###0141### ###0142### ###0143### ###0144### ###0145### ###0146### ###0147### ###0148### ###0149### ###0150### ###0151### ###0152### ###0153### ###0154### ###0155### ###0156### ###0157### ###0158### ###0159### ###0160### ###0161### ###0162### ###0163### ###0164### ###0165### ###0166### ###0167### ###0168### ###0169### ###0170### ###0171### ###0172### ###0173### ###0174### ###0175### ###0176### ###0177### ###0178### ###0179### ###0180### ###0181### ###0182### ###0183### ###0184### ###0185### ###0186### ###0187### ###0188### ###0189### ###0190### ###0191### ###0192### ###0193### ###0194### ###0195### ###0196### ###0197### ###0198### ###0199### ###0200### ###0201### ###0202### ### ​ ​ R a selected from H, halogen or C 1-4 alkyl; and R y is H.

2. The compound of claim 1, wherein R a is selected from H or halogen.

3. A compound, stereoisomer thereof, or pharmaceutically acceptable salt thereof, wherein, ​ ​ 5. The pharmaceutical composition of claim 4, wherein, ​ ​ ​ ​ ​ ​

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