CN112851668A

CN112851668A - ATR inhibitor and application thereof in medicine

Info

Publication number: CN112851668A
Application number: CN202011344488.1A
Authority: CN
Inventors: 吴颢; 沈益飞; 姚智理; 谢晓强; 张瑞鑫; 唐健; 刘奇声; 夏洪峰; 练俊秀; 兰宏; 王家炳; 丁列明
Original assignee: Betta Pharmaceuticals Co Ltd
Current assignee: Betta Pharmaceuticals Co Ltd
Priority date: 2019-11-27
Filing date: 2020-11-26
Publication date: 2021-05-28

Abstract

The present invention relates to a novel compound represented by formula (I) which has ATR-modulating activity. The invention also relates to a preparation method of the compounds and a pharmaceutical composition containing the compounds.

Description

ATR inhibitor and application thereof in medicine

Technical Field

The present invention relates to a novel compound having ATR modulating activity. The invention also relates to a preparation method of the compounds and a pharmaceutical composition containing the compounds.

Background

A highly efficient DNA damage monitoring and repair system in tumor cells is one of the major causes of the development of cancer chemotherapy resistance. DNA Damage response mechanisms (DDRs) have complex signaling pathways that interact with cell cycle checkpoints while activating appropriate DNA repair mechanisms. DDR checkpoint kinases are capable of providing cells with DNA damage repair time prior to replication or mitosis to maintain genomic integrity; if DNA damage cannot be repaired, an apoptotic response may be triggered.

Ataxia telangiectasia mutated gene RAD3-related protein (ATR) is a major regulator of DDR pathway and can be activated by single-stranded DNA. Single-stranded DNA is typically produced by DNA Double Strand Breaks (DSBs) or by replication stress resulting from chemotherapy or oncogene activation. ATR has 2644 amino acid residues, and the N end of the ATR is an ATR interaction protein binding domain which has the function of activating ATR; the C end is a downstream protein phosphorylation kinase domain which can phosphorylate target proteins such as FANCI, WRN, CHK1 and the like.

The invention provides an ATR inhibitor with a novel structure, which can regulate the activity of ATR protein and has good antitumor activity.

Disclosure of Invention

The present invention provides a compound represented by the general formula (I), or a tautomer, a pharmaceutically acceptable salt, a solvate, a chelate, a non-covalent complex, or a prodrug thereof:

wherein the content of the first and second substances,

x is selected from O, S or NR²；

R¹Is selected from

Wherein

Unsubstituted or substituted by C_1-3Alkyl substitution;

R³is selected from

R²、R⁴、R⁵Or R⁶Independently selected from hydrogen, halogen, hydroxy, CN, amino or C_1-6An alkyl group;

and when R is₁Is composed of

When R is₃Is not that

In some embodiments, R in formula (I)¹The substituents being selected from

In some embodiments, X in formula (I) is-NR²，R²Selected from hydrogen or C_1-3An alkyl group.

In some embodiments, R in formula (I)⁴Selected from hydrogen or C_1-3An alkyl group.

In some embodiments, R in formula (I)⁵Or R⁶Independently selected from hydrogen or C_1-3An alkyl group.

In some embodiments, R in formula (I)⁶Is methyl,

Or

The present invention further provides a compound, tautomer or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

1)7- (1-methyl-1H-pyrazol-5-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

2)7- (1-methyl-1H-pyrazol-5-yl) -5- (3-methylmorpholine) -3- (1H-pyrrol-2-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrrol-2-yl) -1H-pyrazolo [4,3-b ] pyridine;

3)3- (1H-imidazol-5-yl) -7- (1-methyl-1H-pyrazol-5-yl) -5- (3-methylmorpholine) -1H-pyrazolo [4,3-b ] pyridine or 3- (1H-imidazol-5-yl) -7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -1H-pyrazolo [4,3-b ] pyridine;

4)7- (1, 5-dimethyl-1H-pyrazol-4-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (1, 5-dimethyl-1H-pyrazol-4-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

5)7- (3-methyl-1H-pyrazol-4-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (3-methyl-1H-pyrazol-4-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

6)7- (1-methyl-1H-imidazol-5-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (1-methyl-1H-imidazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

7)5- (3-methylmorpholine) -7- (3-methylthiophen-2-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 5- ((R) -3-methylmorpholine) -7- (3-methylthiophen-2-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

8)3- (5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) pyridin-2-amine or 3- (5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) pyridin-2-amine;

9)7- (2-fluoropyridin-3-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (2-fluoropyridin-3-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

10)5- (3-methylmorpholine) -7- (6-methylpyridin-3-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 5- ((R) -3-methylmorpholine) -7- (6-methylpyridin-3-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

11)7- (1-ethyl-1H-pyrazol-5-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (1-ethyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

12)7- (1-isopropyl-1H-pyrazol-5-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine or 7- (1-isopropyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine;

13) (1- (5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-yl) methanol or (1- (5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-yl) methanol;

15)7- (1-methyl-1H-pyrazol-5-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) isothiazolo [4,5-b ] pyridine or 7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) isothiazolo [4,5-b ] pyridine; or

16)7- (1-methyl-1H-pyrazol-5-yl) -5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) isoxazolo [4,5-b ] pyridine or (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) isoxazolo [4,5-b ] pyridine; or

17) 1-methyl-4- (1-methyl-5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-ol or (R) -1-methyl-4- (1-methyl-5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-ol; or

18)1- (1-methyl-5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) cyclohexan-1-ol or (R) -1- (1-methyl-5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) cyclohexan-1-ol; or

19)4- (1-methyl-5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-ol or (R) -4- (1-methyl-5- (3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-ol; or

20) 3-methyl-4- (1-methyl-3- (1H-pyrazol-5-yl) -7- (1,2,3, 6-tetrahydropyridin-4-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) morpholine or (R) -3-methyl-4- (1-methyl-3- (1H-pyrazol-5-yl) -7- (1,2,3, 6-tetrahydropyridin-4-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) morpholine; or

21) 3-methyl-4- (1-methyl-7- (1-methyl-1, 2,3, 6-tetrahydropyridin-4-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) morpholine or (R) -3-methyl-4- (1-methyl-7- (1-methyl-1, 2,3, 6-tetrahydropyridin-4-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) morpholine.

The invention also provides a pharmaceutical composition, which is characterized by comprising at least one compound shown as the formula (I) in a therapeutically effective amount and at least one pharmaceutically acceptable auxiliary material.

The invention further provides a pharmaceutical composition, which is characterized in that the mass percentage of the therapeutically effective amount of at least one compound shown in the formula (I) and pharmaceutically acceptable auxiliary materials is 0.0001: 1-10.

The invention provides application of a compound or a pharmaceutical composition shown in a structural formula (I) in preparation of a medicament.

The invention further provides a preferable technical scheme of the application:

preferably, the application is the application in preparing a medicament for treating and/or preventing cancer.

Preferably, the use is for the manufacture of a medicament for the treatment of a disease mediated by ATR. Preferably, the disease is cancer.

Preferably, the cancer is selected from breast cancer, multiple myeloma, bone cancer, bladder cancer, cervical cancer, non-small cell lung cancer, bronchioloalveolar cancer, ovarian cancer, esophageal cancer, colorectal cancer, liver cancer, head and neck tumors, kidney cancer, liver cancer, gastrointestinal tissue cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, testicular cancer, bile duct cancer, or leukemia.

The present invention also provides a method for treating and/or preventing a disease mediated by ATR, comprising administering to a subject a therapeutically effective amount of at least any one compound of formula (I) or a pharmaceutical composition comprising the same.

Unless otherwise indicated, general chemical terms used in the structural formulae have the usual meanings.

For example, the term "halogen" as used herein, unless otherwise specified, refers to fluorine, chlorine, bromine or iodine.

In the present invention, unless otherwise specified, "alkyl" includes straight or branched chain monovalent saturated hydrocarbon groups. For example, alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3- (2-methyl) butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-methylpentyl and the like. Similarly, "1-8" in "alkyl 1-8" refers to a group comprising 1,2,3, 4,5, 6, 7, or 8 carbon atoms arranged in a straight or branched chain.

"alkoxy" refers to the oxygen ether form of the straight or branched chain alkyl group previously described, i.e., -O-alkyl.

In the present invention, "a", "an", "the", "at least one" and "one or more" are used interchangeably. Thus, for example, a composition that includes "a" pharmaceutically acceptable excipient may be interpreted to mean that the composition includes "one or more" pharmaceutically acceptable excipients.

The term "aryl", as used herein, unless otherwise specified, refers to an unsubstituted or substituted monocyclic or fused ring aromatic group comprising carbocyclic atoms. Preferably, aryl is a 6 to 10 membered monocyclic or bicyclic aromatic ring group. Preferably phenyl or naphthyl. Most preferred is phenyl.

The term "heterocyclyl", as used herein, unless otherwise specified, refers to an unsubstituted or substituted 3-8 membered stable monocyclic ring system consisting of carbon atoms and 1-3 heteroatoms selected from N, O or S, wherein the nitrogen or sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatoms may be optionally quaternized. The heterocyclic group may be attached to any heteroatom or carbon atom to form a stable structure. Examples of such heterocyclyl groups include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, tetrahydrofuranyl, dioxolanyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydrooxazolyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, and tetrahydrooxadiazolyl.

The term "heteroaryl", in the present invention, unless otherwise indicated, refers to an unsubstituted or substituted stable 5-or 6-membered monocyclic aromatic ring system or an unsubstituted or substituted 9-or 10-membered benzo-fused heteroaromatic ring system or bicyclic heteroaromatic ring system, consisting of carbon atoms and 1 to 4 heteroatoms selected from N, O or S, and wherein said nitrogen or sulfur heteroatoms may optionally be oxidized and said heteroatoms may optionally be quaternized. The heteroaryl group may be attached at any heteroatom or carbon atom to form a stable structure. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, pyridyl, pyridazinyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzisoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl adenine, quinolinyl, or isoquinolinyl.

The term "cycloalkyl" refers to a cyclic saturated alkyl chain having 3 to 10 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

The term "substituted" means that one or more hydrogen atoms in a group are replaced by the same or different substituents, respectively. Typical substituents include, but are not limited to, halogen (F, Cl, Br or I), C_1-8Alkyl radical, C_3-12Cycloalkyl, -OR₁、-SR₁、＝O、＝S、-C(O)R₁、-C(S)R₁、＝NR₁、-C(O)OR₁、-C(S)OR₁、-NR₁R₂、-C(O)NR₁R₂Cyano, nitro, -S (O)₂R₁、-O-S(O₂)OR₁、-O-S(O)₂R₁、-OP(O)(OR₁)(OR₂) (ii) a Wherein R is₁And R₂Independently selected from-H, C_1-6Alkyl radical, C_1-6A haloalkyl group. In some embodiments, the substituents are independently selected from the group consisting of-F, -Cl, -Br, -I, -OH, trifluoromethoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, -SCH₃、-SC₂H₅Formaldehyde group, -C (OCH)₃) Cyano, nitro, -CF₃、-OCF₃Amino, dimethylamino, methylthio, sulfonyl and acetyl groups.

Examples of substituted alkyl groups include, but are not limited to, 2, 3-dihydroxypropyl, 2-aminoethyl, 2-hydroxyethyl, pentachloroethyl, trifluoromethyl, methoxymethyl, pentafluoroethyl, phenylmethyl, dioxolanylmethyl, and piperazinylmethyl.

Examples of substituted alkoxy groups include, but are not limited to, 2-hydroxyethoxy, 2-fluoroethoxy, 2-difluoroethoxy, 2-methoxyethoxy, 2-aminoethoxy, 2, 3-dihydroxypropoxy, cyclopropylmethoxy, aminomethoxy, trifluoromethoxy, 2-diethylaminoethoxy, 2-ethoxycarbonylethoxy, 3-hydroxypropoxy.

The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids.

When a tautomer exists in the compound of formula (I), the present invention includes any possible tautomer and pharmaceutically acceptable salts thereof, and mixtures thereof, unless otherwise specified.

Certain therapeutic advantages may be provided when compounds of formula (I) are replaced with heavier isotopes such as deuterium, for example, which may be attributed to greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.

When solvates or polymorphs exist of the compounds of formula (I) and pharmaceutically acceptable salts thereof, the present invention includes any possible solvates and polymorphs. The type of solvent forming the solvate is not particularly limited as long as the solvent is pharmaceutically acceptable. For example, water, ethanol, propanol, acetone, and the like can be used.

The term "composition," as used herein, is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Accordingly, pharmaceutical compositions containing the compounds of the invention as active ingredients as well as methods for preparing the compounds of the invention are also part of the invention. In addition, some crystalline forms of the compounds may exist as polymorphs and as such are included in the present invention. In addition, some compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates also fall within the scope of the present invention.

The formulation may also include, as appropriate, one or more additional adjuvant ingredients such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), and the like. Further, other adjuvants may also include penetration enhancers to regulate the osmolarity of the drug with blood. The pharmaceutical composition containing the compound represented by the formula (I), or a pharmaceutically acceptable salt thereof, can be prepared in the form of powder or concentrated solution.

In general, the above-identified conditions or disorders are treated with a dosage level of the drug of about 0.01mg/kg body weight to about 150mg/kg body weight per day, or about 0.5mg to about 7g per patient per day. For example, inflammation, cancer, psoriasis, allergy/asthma, diseases and disorders of the immune system, diseases and disorders of the Central Nervous System (CNS), are effectively treated at a drug dose level of 0.01mg/kg body weight to 50mg/kg body weight per day, or 0.5mg to 3.5g per patient per day.

However, it will be appreciated that lower or higher doses than those described above may be required. The specific dose level and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

Typical compounds of the present invention include, but are not limited to, the compounds shown in table 1:

TABLE 1

Detailed Description

In order to make the above-mentioned contents clearer and clearer, the present invention will be further illustrated by the following examples. The following examples are intended only to illustrate specific embodiments of the present invention so as to enable those skilled in the art to understand the present invention, but not to limit the scope of the present invention. In the embodiments of the present invention, technical means or methods not specifically described are conventional in the art.

All parts and percentages herein are by weight and all temperatures are in degrees Celsius, unless otherwise indicated.

The following abbreviations are used in the examples:

AcOH: acetic acid;

DCM: dichloromethane;

DCE: ethylene dichloride;

DIPEA: n, N-diisopropylethylamine;

LCMS: liquid chromatography-mass spectrometry;

MeOH: methanol;

min: the method comprises the following steps of (1) taking minutes;

Pd/C: a palladium on carbon catalyst;

PE: petroleum ether;

py.: pyridine;

PyBroP: trispyrrolidinylphosphonium bromide hexafluorophosphate;

PMBNH₂: p-methoxybenzylamine;

PE, EtOAc: petroleum ether/ethyl acetate;

POCl₃: phosphorus oxychloride;

PdCl₂(PPh₃)₂: bis (triphenylphosphine) palladium chloride;

TEA: triethylamine;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran.

Preparation of intermediate (R) -3-chloro-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b ] pyridin-7-yl trifluoromethanesulfonate

Step 1: preparation of intermediate M2

The compound 4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (5g) was dissolved in MeOH (10mL), Pd/C (1.24g, 10% purity, with 50% water) was added, hydrogen was purged 3 times, and then the reaction was carried out for 24hrs at room temperature under a hydrogen atmosphere. LCMS check reaction complete. The reaction was filtered directly, washed sequentially with DCM (100ml x 3) and methanol (100ml x 3) and the filtrate was spin dried to give 4.1g of an off-white solid, intermediate M2.

LC-MS[M+H⁺]＝142。

Step 2: preparation of intermediate M3

Under nitrogen, (R) -3-methyl-4-acetylmorpholine (12.78g) was dissolved in anhydrous DCE (10ml), phosphorus oxychloride (16.43g) was added at room temperature, and after stirring and reacting at room temperature for 0.5H, methyl 4-amino-1H-pyrazole-3-carboxylate (4.1g) was added, followed by stirring and reacting at 80 ℃ for 4 hrs. LCMS check reaction complete. Most of phosphorus oxychloride in the reaction system is firstly removed, DCM (200mL) and water (200mL) are added, the mixture is placed in an ice bath, sodium carbonate is slowly added under stirring to adjust the pH value to 8-9, and after DCM (500mL 10) is separated, anhydrous Na is added₂SO₄Drying, concentration under reduced pressure and column chromatography of the residue (DCM: MeOH ═ 20:1) gave 5.5g of a yellow foamy solid, intermediate M3.

LC-MS[M+H⁺]＝267。

And step 3: preparation of intermediate M4

Under nitrogen atmosphere, compound 4- [ (E) -1- [ (3R) -3-methylmorpholin-4-yl]Ethylene amino group]-1H-pyrazole-5-carboxylic acid methyl ester (3.6g) dissolved in anhydrous DMF (10mL), placed in an ice bath, NaH (1.08g, 60% purity) added portionwise, after completion of addition allowed to return to room temperature stirring for thirty minutes, PMBCl (1.57g, 90% purity) added, followed byThe reaction was stirred for 1hr at room temperature. After the reaction was detected by LCMS, the excess NaH was quenched by dropwise addition of water, followed by dilution of the reaction with EA (500mL), multiple washes with water (50 mL. about.3) to remove DMF, and the organic phase was washed with anhydrous Na₂SO₄After drying, concentration under reduced pressure and column chromatography of the residue (DCM: MeOH ═ 100:1-20:1) afforded 4.1g of an off-white solid, intermediate M4.

LC-MS[M+H⁺]＝387。

And 4, step 4: preparation of intermediate M5

The compound (R, E) -methyl 1- (4-methoxybenzyl) -4- ((1- (3-methylmorpholino) ethylidene) amino) -1H-pyrazole-5-carboxylate (4.1g) was dissolved in anhydrous DMF (25mL) under a nitrogen atmosphere, and potassium tert-butoxide (2.98g) was added to react at 50 ℃ for 1 hr. LCMS detection reaction is completed, 1N diluted hydrochloric acid is added to adjust pH to 6-7, mixed system is diluted by DCM (200mL), DMF is removed by washing with water (25mL x 3) for multiple times, and organic phase anhydrous Na₂SO₄Drying, and concentrating under reduced pressure. The crude product was obtained and directly used in the next step.

LC-MS[M+H⁺]＝355。

And 5: preparation of intermediate M6

The compound ethyl (R) -1- (4-methoxybenzyl) -5- (3-methylmorpholino) -1H-pyrazolo [4,3-b ] pyridin-7-ol (crude product from step 4, not isolated) was dissolved in DCM (50ml), 1,1, 1-trifluoro-N-phenyl-N- (trifluoromethylsulfonyl) methanesulfonamide (5.35g), DIPEA (3.87g) was added at rt, stirred at rt for 2hrs, LCMS checked for completion, most of the DCM was removed by rotation, and the residue was purified by column chromatography (PE: EtOAc ═ 20:1-1:1) to give 2.6g of a yellow solid, intermediate M6.

LC-MS[M+H⁺]＝487。

Step 6: preparation of intermediate M7

Reacting (R) -1- (4-methoxybenzyl) -5- (3-methylmorpholino) -1H-pyrazolo [4,3-b]Dissolving pyridine-7-yl trifluoromethanesulfonate (2.6g) in TFA (15mL), refluxing at 80 deg.C for 3hrs, detecting by LCMS, removing most of TFA, diluting the reaction residue with DCM (100mL), adjusting pH to 8-9 under reduced pressure with saturated sodium bicarbonate, separating, combining the organic phases, and adding anhydrous Na₂SO₄Drying, and concentrating under reduced pressure to obtain 2.5g yellow solid, i.e. mediumIntermediate M7.

LC-MS[M+H⁺]＝377。

And 7: preparation of intermediate M8

Reacting (R) -5- (3-methylmorpholino) -1H-pyrazolo [4,3-b]Pyridin-7-yl trifluoromethanesulfonate (0.77g) was dissolved in DMF (10mL), and NCS (0.34g) was added to conduct a reaction at 50 ℃ for 1.5 hrs. LCMS check reaction complete. The reaction was diluted with EtOAc (200mL), washed multiple times with water (25 mL. times.3) to remove DMF, and the organic phase was Na anhydrous₂SO₄Drying, and concentrating under reduced pressure. The residue was purified by column chromatography (PE: EtOAc 10:1) to give 590mg of a yellow solid, intermediate M8.

LC-MS[M+H⁺]＝347。

Preparation of intermediate (R) -4- (3-chloro-1-methyl-7- (1-methyl-1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) -3-methylmorpholine

Step 1: preparation of intermediate S1

Under nitrogen, (R) -3-methyl-4-acetylmorpholine (1.85g) was dissolved in anhydrous DCE (10ml), phosphorus oxychloride (3.56g) was added at room temperature, and after stirring at room temperature for 0.5hr, methyl 4-amino-1H-pyrazole-3-carboxylate (1.0g) was added, followed by stirring at 80 ℃ for 4 hr. LCMS check reaction complete. Most of phosphorus oxychloride in the reaction system is firstly removed, DCM (200mL) and water (50mL) are added, the mixture is placed in an ice bath, sodium carbonate is slowly added under stirring to adjust the pH value to 8-9, and after DCM (200 mL. multidot.5) is separated, anhydrous Na is added₂SO₄Drying and concentration under reduced pressure gave 2.4g of crude product, intermediate S1, which was used directly in the next step.

LC-MS[M+H⁺]＝281。

Step 2: preparation of intermediate S2

The crude methyl (R, E) -1-methyl-4- ((1- (3-methylmorpholino) ethylidene) amino) -1H-pyrazole-5-carboxylate (crude from step 1, not isolated) from the previous step was dissolved in anhydrous DMF (25mL) under nitrogen and potassium tert-butoxide (1.68g) was added and reacted at 50 ℃ for 1 hr. L isAfter the CMS detection reaction, 1N diluted hydrochloric acid was added to adjust pH to 6-7, the mixture was diluted with DCM (300mL), washed with water (25 mL. times.3) several times to remove DMF, and the organic phase was washed with anhydrous Na₂SO₄Drying, and concentrating under reduced pressure. The crude product was obtained and directly used in the next step.

LC-MS[M+H⁺]＝249。

And step 3: preparation of intermediate S3

The compound (R) -1-methyl-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b ] pyridin-7-ol (crude product from step 2, not isolated) in the previous step was dissolved in DCM (25ml), 1,1, 1-trifluoro-N-phenyl-N- (trifluoromethylsulfonyl) methanesulfonamide (3.22g), DIPEA (2.33g) was added at rt, the reaction was stirred at rt for 3hrs, TLC checked for completion, most of the DCM was removed by rotation, and the residue was purified by column chromatography (PE EtOAc: 20:1-1:1) to give 0.36g of a yellow solid, intermediate S3.

LC-MS[M+H⁺]＝381。

And 4, step 4: preparation of intermediate S4

Reacting (R) -1-methyl-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b]Pyridin-7-yl trifluoromethanesulfonate (0.25g, 657.30umol) was dissolved in DMF (8mL), NCS (175.5mg) was added, and the reaction was carried out at 50 ℃ for 3 hrs. LCMS check reaction complete. The reaction was diluted with EA (100mL), washed with water (20 mL. times.4) to remove DMF, and the organic phase was anhydrous Na₂SO₄Drying, and concentrating under reduced pressure. Column chromatography of the residue (PE: EtOAc 10:1) afforded 150mg of a yellow solid, intermediate S4.

LC-MS[M+H⁺]＝415。

And 5: preparation of intermediate S5

(R) -3-chloro-1-methyl-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b ] pyridin-7-yl trifluoromethanesulfonate (90mg), SPhos-Pd-G2(15.61mg), potassium phosphate (138.17mg), 1-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrazole (47.40mg) were weighed into 5mLvia, nitrogen was purged three times, Dioxane (3mL) which had been previously bubbled with nitrogen for 15 minutes was added under a nitrogen atmosphere, and the reaction was stirred at 100 ℃ for 3hrs after sealing. LCMS check reaction complete, reaction mixture filtered directly and concentrated by drying. The residue was purified by plate chromatography (PE: EtOAc 1:1) to give 35mg of a yellow foamy solid. I.e. intermediate S5.

LC-MS[M+H⁺]＝347。

Example 1: synthesis of compound 7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Step 1: preparation of intermediate M9

Reacting (R) -3-chloro-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b]Pyridin-7-yl trifluoromethanesulfonate (400mg), SPhos-Pd-G2(72mg), potassium phosphate (0.64G) 1-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrazole (228mg) was weighed into 25mL, nitrogen was purged three times, and Dioxane/H which had been previously bubbled with nitrogen for 15 minutes was added under a nitrogen atmosphere₂O (7 mL: 1mL), sealed and reacted at 100 ℃ for 3hrs with stirring. LCMS check reaction complete, reaction mixture filtered directly and concentrated by drying. The residue was purified by column chromatography (PE: EA: 1) to give 150mg of a yellow solid. Namely intermediate M9.

LC-MS[M+H⁺]＝333。

Step 2: preparation of intermediate M10

The intermediates ((R) -4- (3-chloro-7- (1-methyl-1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) -3-methylmorpholine (120mg),1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester (200.6mg), SPhos-Pd-G2(25.95mg), and K3PO4(229.62mg) were weighed into a reaction tube, nitrogen gas was pumped three times, and Dioxane/water (2mL/0.4mL) was obtained by deoxygenation through nitrogen bubbling in advance, the reaction tube was covered with a lid, placed in an oil bath at 100 ℃ and stirred for 2 hrs.LCMS detection, the reaction solution was concentrated, and the residue was purified by climbing a large plate (PE: EtOAc ═ 1:1) to obtain 110mg of a yellow foamy solid, namely intermediate M10.

LC-MS[M+H⁺]＝449。

And step 3: preparation of Compound 1

(3R) -3-methyl-4- (7- (1-methyl-1H-pyrazol-5-yl) -3- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) morpholine (110mg) was dissolved in (MeOH) (2mL), and 2.5mL of aqueous hydrogen chloride (0.2M) was added. The reaction was stirred at room temperature for 1 hr. Upon completion of the reaction by LCMS, the reaction mixture was concentrated under reduced pressure and the residue was purified by plate-climbing (DCM: MeOH ═ 20:1) to give 35.4mg of a yellow solid, compound 1.

LC-MS[M+H⁺]＝365。

¹H NMR(500MHz,DMSO-d6)δ13.18(br s,1H),7.64(br s,2H),7.09(s,1H),7.07(s,1H),6.70(s,1H),4.53-4.45(m,1H),4.08(d,J＝10.0Hz,1H),4.00(d,J＝10.0Hz,1H),3.90(s,3H),3.80-3.67(m,2H),3.55(t,J＝15.0Hz,1H),3.19(d,J＝10.0Hz,1H),1.20(d,J＝5.0Hz,3H).

EXAMPLE 2 Synthesis of the Compound 7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrrol-2-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above for example 1, starting from intermediate M8 and the commercial reagent (1-tert-butoxycarbonylpyrrol-2-yl) boronic acid.

LC-MS[M+H]+＝364。

Example 3: synthesis of the compound 3- (1H-imidazol-5-yl) -7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above in example 1, starting from intermediate M8 and the commercially available reagent (1-tetrahydropyran-2-yl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) imidazole.

LC-MS[M+H]+＝365。

Example 4: synthesis of the compound 7- (1, 5-dimethyl-1H-pyrazol-4-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above in example 1, starting from intermediate M8 and the commercial reagent 1, 5-dimethyl-1H-pyrazole-4-boronic acid pinacol ester.

LC-MS[M+H]+＝379。

Example 5: synthesis of compound 7- (3-methyl-1H-pyrazol-4-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above in example 1, starting from intermediate M8 and the commercial reagent 3-methylpyrazole-4-boronic acid pinacol ester.

LC-MS[M+H]+＝379。

Example 6: synthesis of the compound 7- (1-methyl-1H-imidazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above in example 1, starting from intermediate M8 and the commercial reagent 1-methyl-1-H-imidazole-5-boronic acid pinacol ester.

LC-MS[M+H]+＝365。

Example 7: synthesis of compound 5- ((R) -3-methylmorpholine) -7- (3-methylthiophen-2-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above for example 13, starting from intermediate M8 and the commercial reagent 3-methylthiophene-2-boronic acid ester.

LC-MS[M+H]+＝381。

Example 8: synthesis of the compound 3- (5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) pyridin-2-amine

Synthesized according to the synthesis procedure described above in example 1, intermediate M8 and the commercially available reagent, 2-aminopyridine-3-boronic acid pinacol ester.

LC-MS[M+H]+＝377。

Example 9: synthesis of compound 7- (2-fluoropyridin-3-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above in example 1, starting from intermediate M8 and the commercial reagent 2-fluoropyrimidine-3-boronic acid pinacol ester.

LC-MS[M+H]+＝380。

Example 10: synthesis of compound 5- ((R) -3-methylmorpholine) -7- (6-methylpyridin-3-yl) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Step 1: preparation of intermediate M11

Reacting (R) -3-chloro-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b]Pyridin-7-yl Trifluoromethanesulfonate (150mg), 2-MethylYl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) pyridine (82mg), sodium carbonate (0.12g) Pd (dppf) Cl₂DCM (30.6mg) was weighed into a 20mL three-necked flask, nitrogen was purged three times, and DME/H pre-sparged with nitrogen for 15 minutes was added under a nitrogen atmosphere₂O (2 mL: 0.5mL), sealed and reacted in an oil bath at 100 ℃ for 1.5 hrs. Upon completion of the reaction by LCMS, the reaction mixture was directly filtered, dried, concentrated and purified by column chromatography (DCM: MeOH ═ 20:1) to afford 125mg of a yellow solid. Namely intermediate M11.

LC-MS[M+H⁺]＝344。

Step 2: preparation of Compound 10

The intermediate ((R) -4- (3-chloro-7- (6-methylpyridin-3-yl) -1H-pyrazolo [4, 3-b)]Pyridin-5-yl) -3-methylmorpholine (100mg), 1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester (88.9mg), SPhos-Pd-G2(20.91mg), K₃PO₄(184.99mg) was weighed into a 10-20mL microwave tube, and after bubbling deoxygenation with nitrogen gas, Dioxane/water (3.5mL/0.5mL) was obtained, which was covered with a lid and then reacted at 100 ℃ for 20min with a microwave. LCMS check reaction complete. The reaction was diluted with ethyl acetate and the insoluble material was removed by filtration, the filtrate was adjusted to pH 3 with 2N dilute hydrochloric acid at which time the THP protecting group was removed, the product was dissolved in the aqueous phase, the product was extracted with ethyl acetate (3 × 20ml) to remove impurities other than the product, the aqueous phase was adjusted to pH 8 with 15% sodium hydroxide solution, the ethyl acetate was extracted (3 × 20ml), the organic phase was separated, dried and concentrated under reduced pressure, and the crude product was slurried with (PE: EtOAc: DCM: 4:1:1) to give 35.3mg of a yellow solid, compound 10.

LC-MS[M+H⁺]＝376。

¹H NMR(500MHz,CDCl₃)δ12.12(s,2H),8.96(d,J＝29.9Hz,1H),7.91(s,1H),7.72(s,1H),7.35–7.27(m,1H),7.04(s,1H),6.86(d,J＝25.6Hz,1H),4.42(d,J＝6.0Hz,1H),4.12(d,J＝11.8Hz,1H),3.99(d,J＝12.9Hz,1H),3.88(s,2H),3.71(t,J＝11.6Hz,1H),3.42(t,J＝11.8Hz,1H),2.58(d,J＝11.9Hz,3H),1.33(t,J＝12.8Hz,3H).

Example 11: synthesis of the compound 7- (1-ethyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above for example 10, intermediate M8 and the commercially available reagent 1-ethylpyrazole-5-boronic acid pinacol ester.

LC-MS[M+H]⁺＝379。

Example 12: synthesis of compound 7- (1-isopropyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Synthesized according to the synthesis procedure described above for example 10, intermediate M8 and the commercial reagent 1-isopropylpyrazole-5-boronic acid pinacol ester.

LC-MS[M+H]⁺＝393。

Example 13: synthesis of the compound (1- (5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-yl) methanol

Step 1: preparation of intermediate M12

(R) -3-chloro-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b ] pyridin-7-yl-trifluoromethanesulfonate (150mg), piperidin-4-ylmethanol (129mg) and the reaction mixture were reacted in a 5mL microwave tube at 100 ℃ for 4hrs in an oil bath. And (3) detecting the reaction completion by LCMS, diluting the reaction solution with ethyl acetate, washing with water, separating an organic phase, drying, and concentrating to obtain an intermediate M12, wherein the crude product is directly used for the next reaction.

Step 2: preparation of Compound 13

Reference synthesis example 11, step 2 synthesis method, compound 13 was synthesized from (R) - (1- (3-chloro-5- (3-methylmorpholino) -1H-pyrazolo [4,3-b ] pyridin-7-yl) piperidin-4-yl) methanol and the commercially available reagent 1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester.

LC-MS[M+H]⁺＝398。

Example 14: synthesis of compound 1-methyl-7- (1-methyl-1H-pyrazol-5-yl) -5- ((R) -3-methylmorpholine) -3- (1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridine

Step 1: preparation of intermediate S6

The intermediate (R) -4- (3-chloro-1-methyl-7- (1-methyl-1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) -3-methylmorpholine (35mg), 1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester (56.0mg), SPhos-Pd-G2(7.2mg), and K3PO4(63.66mg) were weighed into a reaction tube, deoxygenated by nitrogen bubbling in advance after nitrogen gas was added three times to obtain Dioxane (2mL), and the reaction tube was placed in an oil bath at 100 ℃ for 4hrs with a lid closed. LCMS check reaction complete. The reaction mixture was concentrated under reduced pressure, and the residue was directly used in the next step.

LC-MS[M+H⁺]＝463。

Step 2: preparation of Compound 14

(3R) -3-methyl-4- (1-methyl-7- (1-methyl-1H-pyrazol-5-yl) -3- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-5-yl) -1H-pyrazolo [4,3-b ] pyridin-5-yl) morpholine (crude product of the previous step) was dissolved in (MeOH) (5mL), and 2.5mL of aqueous hydrogen chloride (2N) was added. The reaction was stirred at room temperature for 1 h. Upon completion of the reaction by LCMS, the reaction mixture was concentrated under reduced pressure and the residue was purified by plate-climbing (DCM: MeOH ═ 30:1) to afford 22.0mg of a yellow solid, compound 14.

LC-MS[M+H⁺]＝379。

¹H NMR(500MHz,DMSO-d6)δ13.02(br s,1H),7.68(br s,1H),7.64(s,1H),7.06(s,2H),6.06(s,1H),4.54-4.43(m,1H),4.07(d,J＝10.0Hz,1H),3.99(d,J＝10.0Hz,1H),3.75(d,J＝11.0Hz,1H),3.72(s,3H),3.69(d,J＝11.4Hz,1H),3.56(s,3H),3.58-3.51(m,1H),3.18(t,J＝12.0Hz,1H),1.18(d,J＝6.5Hz,3H)。

Example 17: synthesis of Compound 17

Step 1: synthesis of Compound S8

S7B (1.85g) was dissolved in anhydrous DCE (10mL) under a nitrogen atmosphere, phosphorus oxychloride (3.56g) was added at room temperature, and after keeping at room temperature for 0.5 hour, S7A (1.0g) was added, followed by reaction at 80 ℃ for 4 hours. LCMS check reaction complete. And cooling to room temperature, removing most of phosphorus oxychloride in the reaction system, adding DCM (200mL) and water (50mL), dropwise adding sodium carbonate into the reaction solution under ice bath, adjusting the pH to 9, extracting with DCM, drying with organic phase anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 2.4g of a crude product which is directly used in the next step, namely an intermediate S8.

Step 2: synthesis of Compound S9

Compound S8 was dissolved in anhydrous DMF (25mL) under a nitrogen atmosphere, and potassium tert-butoxide (1.68g) was added to react at 50 ℃ for 1 hour. LCMS detects reaction completion, 1N dilute hydrochloric acid is added to adjust pH to 6-7, the mixed system is diluted with DCM (300mL), DMF is removed by multiple water washes, the organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1.3g of crude product which is directly used in the next step, i.e. intermediate S9.

And step 3: synthesis of Compound S10

Compound S9(1.3g) and tribromooxyphosphorus (10g) were charged into a 10mL reaction flask and reacted for 1 h. Reacting at 100 ℃ for 18h, detecting complete reaction by LCMS, cooling to room temperature, adding the reaction liquid into cooled ice water (20mL), dropwise adding sodium carbonate into the reaction liquid, adjusting the pH value to 9, extracting by DCM, and separating a product by column chromatography (DCM/EA is 1:1) to obtain 0.6g of a yellow solid, namely a compound S10.

And 4, step 4: synthesis of Compound 17-1

Compound S10(400mg) was added to a 50mL three-necked flask under a nitrogen atmosphere, and dissolved in anhydrous THF (10mL), n-butyllithium (1.21mL) was added dropwise at-78 ℃ and the mixture was reacted for 0.5 hour under heat preservation, cyclohexanone (190mg) was added and the reaction was stopped after 0.5 hour. And (3) detecting the generation of the product by LCMS, quenching the reaction liquid by using saturated ammonium chloride solution, extracting by using EA (20mL), drying an organic phase by using anhydrous sodium sulfate, concentrating under reduced pressure to obtain a crude product, and separating by using a reverse phase chromatographic column to obtain a product 17-1(65 mg).

And 5: synthesis of Compound 17-2

A25 mL single-necked flask was charged with Compound 17-1(65mg), dissolved in anhydrous acetonitrile (3mL), and NBS (35mg) was added dropwise at room temperature, incubated for 5min and LCMS checked for completion. Extraction was carried out with EA (20mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude 17-2(53mg) which was used directly in the next step.

Step 6: synthesis of Compound 17-3

A25 mL three-necked flask was charged with Compound 17-2(53mg),1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester (72mg), potassium phosphate (82mg), SPhos-Pd-G2(9.2mg), reacted at 100 ℃ for 3 hours, and LCMS showed that the reaction was complete. Extract with EA (20mL), dry the organic phase over anhydrous sodium sulfate, concentrate under reduced pressure to give crude product for column chromatography (DCM: EA ═ 3:1) to give 42mg, compound 17-3.

And 7: synthesis of Compound 17

In a 10mL single vial was added compound 17-3(42mg), dissolved in anhydrous methanol (1mL), added HCl/MeOH (0.2mL), stirred for 1h, and the reaction was checked for completion by LCMS. Spin-drying the solvent at low temperature, separating the product by reverse phase chromatography to obtain white solid 26.8mg, i.e. compound 17(M + H)⁺:397)¹H NMR(500MHz,MeOD)δ7.79(s,1H),7.07(s,1H),6.94(s,1H),4.50(s,3H),4.39–4.38(m 1H),4.12–4.09(m,1H),3.94–3.86(m,3H),3.72–3.71(m,1H),3.57–3.52(m,1H),2.15–1.69(m,9H),1.49–1.37(m,4H)。

Example 18: synthesis of Compound 18

Step 1: synthesis of Compound 18-1

Under a nitrogen atmosphere, compound S10(600mg) was charged into a 50mL three-necked flask, dissolved in anhydrous THF (10mL), and n-butyllithium (1.81mL) was added dropwise at-78 ℃ to conduct a reaction for 0.5 hour, and then 1-benzyloxycarbonyl-4-piperidone (674mg) was added thereto to conduct a reaction for 0.5 hour, followed by stopping the reaction. The product was detected by LCMS and the reaction was quenched with saturated ammonium chloride solution, extracted with EA (20mL), the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude product which was isolated by column chromatography (DCM: MeOH ═ 20:1) to give 18-1(130 mg).

Step 2: synthesis of Compound 18-2

Compound 18-1(130mg) was added to a 25mL single vial, dissolved in anhydrous acetonitrile (5mL), NBS (50mg) was added dropwise at room temperature, incubated for 5min, and LCMS checked for completion. Extraction was performed with EA (20mL), the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product which was isolated by column chromatography (DCM: EA ═ 1:1) to give 18-2(120 mg).

And step 3: synthesis of Compound 18-3

A25 mL three-necked flask was charged with compound 18-2(120mg),1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester (95mg), potassium phosphate (120mg), SPhos-Pd-G2(12mg), reacted at 100 ℃ for 3 hours, and LCMS showed that the reaction was complete. Extract with EA (20mL), dry the organic phase over anhydrous sodium sulfate, concentrate under reduced pressure to give crude product which is chromatographically isolated (DCM: EA 1:1) to give 111 mg.

And 4, step 4: synthesis of Compound 18-4

In a 10mL single vial, compound 18-3(111mg) was added, dissolved in methanol (5mL), 10% palladium on carbon (10mg) was added, stirred for 1h, and the reaction was checked for completion by LCMS. The solvent was spun dry at low temperature to give 86mg of crude product which was used directly in the next step.

And 5: synthesis of Compound 18

In a 10mL single vial was added compound 18-4(15mg), dissolved in anhydrous methanol (1mL), added HCl/MeOH (0.1mL), stirred for 1h, and the reaction was checked for completion by LCMS. Drying solvent at low temperature, separating by reverse phase chromatography to obtain white solid 3.6mg, i.e. compound 253201(M + H)⁺:398)¹H NMR(500MHz,MeOD)δ8.53(s,1H),7.65(s,1H),7.08(s,1H),6.89(s,1H),4.45(s,3H),4.47–4.42(m,1H)4.06–3.96(m,2H),3.85–3.83(m,2H),3.69–3.59(m,3H),3.43–3.40(m,2H),3.35–3.32(m,1H),2.37–2.23(m,4H),1.28–1.25(m,3H)。

Example 19: synthesis of Compound 19

Step 1: synthesis of Compound 19-1

In a 10mL single vial was added compound 18-4(50mg), dissolved in methanol (5mL), paraformaldehyde (9mg) was added, stirred for 1h, and the reaction was checked for completion by addition of sodium cyanoborohydride (32mg) LCMS at low temperature. The solvent was dried by evaporation at low temperature to give the crude product which was subjected to column chromatography (DCM/MeOH. RTM. 20/1) to give 16mg of product.

Step 2: synthesis of Compound 19

In a 10mL single vial was added compound 19-1(16mg), dissolved in anhydrous methanol (1mL), added HCl/MeOH (0.1mL), stirred for 1h, and the reaction was checked for completion by LCMS. Spin-drying the solvent at low temperature, separating the product by reverse phase chromatography to obtain white solid 4.9mg, i.e. compound 19(M + H)⁺:412)¹H NMR(500MHz,MeOD)δ7.72(s,1H),7.09(s,1H),6.89(s,1H),4.45(s,3H),4.47–4.43(m,1H),4.07–3.96(m,2H),3.87–3.82(m,2H),3.70–3.55(m,5H),3.39–3.33(m,1H),2.99(s,3H),2.42–2.35(m,4H),1.28(d,J＝6.7Hz,3H)。

Example 20: synthesis of Compound 20

Step 1: synthesis of Compound 20-1

Under a nitrogen atmosphere, a 25mL three-necked flask was charged with Compound S10(600mg), N-benzyloxycarbonyl-3, 6-dihydro-2H-pyridine-4-boronic acid pinacol ester (1.0mg), potassium phosphate (1.3G), and SPhos-Pd-G2(140mg), reacted at 100 ℃ for 3 hours, and LCMS showed that the reaction was complete. Extraction was performed with EA (20mL), the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product, which was isolated by column chromatography (DCM: MeOH: 98:2) to give 532mg of the product.

Step 2: synthesis of Compound 20-2

A25 mL single vial was charged with Compound 20-1(500mg), dissolved in anhydrous acetonitrile (10mL), NBS (170mg) was added dropwise at room temperature, incubated for 5min, and LCMS checked for completion. Extracting with EA (20mL), drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to obtain crude product, and separating by column chromatography (DCM: EA ═ 1:1) to obtain product 20-2(465mg)

And step 3: synthesis of Compound 20-3

A25 mL three-necked flask was charged with compound 20-2(465mg),1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester (500mg), potassium phosphate (800mg), SPhos-Pd-G2(110mg), reacted at 100 ℃ for 3 hours, and LCMS showed that the reaction was complete. Extract with EA (40mL), dry the organic phase over anhydrous sodium sulfate, concentrate under reduced pressure to give crude product, which is chromatographically isolated (DCM: EA 1:1) to give 310 mg.

And 4, step 4: synthesis of Compound 20

In a 10mL single vial, compound 20-3(15mg) was added, dissolved in anhydrous methanol (1mL), HCl/MeOH (0.1mL) was added, stirred for 1h, and the reaction was checked for completion by LCMS. Spin-drying the solvent at low temperature, separating the product by reverse phase chromatography to obtain white solid 4.2mg, i.e. compound 20(M + H)⁺:380)¹H NMR(500MHz,MeOD)δ8.54(s,1H),7.67(s,1H),7.08(s,1H),6.79(s,1H),5.96(s,1H),4.43(d,J＝6.5Hz,1H),4.05–3.97(m,5H),3.86–3.79(m,4H),3.67–3.62(m,1H),3.49–3.25(m,5H),2.73(s,2H),1.25(d,J＝6.6Hz,3H)。

Example 21: synthesis of Compound 21

Step 1: synthesis of Compound 21-1

In a 10mL single neck flask, compound 20-3(100mg) was added, dissolved in methanol (5mL), paraformaldehyde (12mg) was added, stirred for 1h, and sodium cyanoborohydride (68mg) was added at low temperature and LCMS was added to check completion of the reaction. The solvent was dried by evaporation at low temperature to give the crude product which was subjected to column chromatography (DCM/MeOH. RTM. 20/1) to give 57mg of product.

Step 2: synthesis of Compound 21

In a 10mL single vial was added compound 21-1(57mg), dissolved in anhydrous methanol (1mL), added HCl/MeOH (0.1mL), stirred for 1h, and the reaction was checked for completion by LCMS. Spin-drying the solvent at low temperature, separating the product by reverse phase chromatography to obtain white solid 26.4mg, i.e. compound 21(M + H)⁺:394)¹H NMR(500MHz,MeOD)δ7.94(s,1H),7.20(s,1H),7.14(s,1H),7.06(s,1H),6.11(s,1H),4.45(s,1H),4.21–3.10(m,5H),4.01–3.69(m,6H),3.59–3.53(m,2H),3.09–2.85(m,5H),1.40(d,J＝5.3Hz,3H)。

Pharmacological experiments

Example A: kinase assay

(1) Dilution of Compounds

Compounds were diluted to the required concentration with DMSO, 100 dilutions were transferred to 96-well plates and diluted 3-fold in gradient, requiring a total of 10 concentrations, 100 additional dilutions, and a total of requirements, and two blank wells were added, as controls for compound blank and enzyme blank, respectively. After the dilution was completed, 40 out of each well was transferred to 384-well plates, and then 60nL of each well was transferred to 384-well detection plates.

(2) Kinase reaction

Adding ATR kinase into 1 enzyme kinase buffer solution to prepare 2 enzyme kinase solution, adding 10 solution into the 384-well detection plate, incubating at room temperature for 10min, adding 10 solution, adding room temperature peptide solution, reacting at 28 deg.C for certain time, and adding 40 specific stop buffer to stop kinase reaction.

(3) Data reading and processing

Data were read and collected with a Caliper and converted to percent inhibition:

percent inhibition ═ (max-conversion)/(max-min) × min).

"maximum" is DMSO control; "minimum" indicates a low control group.

Curve fitting was performed using the analytical software GraphPad Prism and IC50 values were obtained.

All the compounds of the examples have good inhibition of ATR, and the data of the compounds of the examples are shown in table 2.

TABLE 2

Number of Compounds	ATR inhibiting IC of compounds₅₀(nM)
		Compound 1	1.3
Compound 2	1.4
		Compound 10	3.3
Compound 11	1.6
		Compound 12	3.1
Compound 14	2.6

Example B: cell proliferation assay (HGC27)

HGC27 cells were plated at 800/well in 13527 96-well plates. 37 ℃ and 5% CO₂Incubate overnight. Preparing compound series gradient concentration solution (controlling DMSO final concentration to be 0.5%), adding 15 μ L test compound solution (compound final concentration of 30000, 10000, 3333.3, 1111.1, 370.4, 123.5, 41.2, 13.7, 4.6, 0nM) into each well cell, 37 deg.C, 5% CO₂And (5) incubating for 96 h. After incubation, adding 50 percent of solution (working solution, shaking and uniformly mixing, incubating at room temperature for 10min, reading the Luminescence value of luminescense by using a multifunctional microplate reader, and converting the Luminescence value reading into inhibition percentage:

percent inhibition is (max-reading)/(max-min) 100.

"maximum" is DMSO control; "minimum" indicates a cell-free control group.

Curve fitting was performed using Graphpad Prism software and IC50 values were obtained.

All the compounds of the examples have good inhibition on HGC27 cells, and the IC50 data of the compounds of the examples on HGC27 cells are shown in Table 2.

TABLE 3

Number of Compounds	Inhibition rate (nM)
		Compound 1	84
Compound 10	101
		Compound 11	75
Compound 12	89
		Compound 14	24
Compound 17	238.8
		Compound 20	182.3
Compound 21	175.5

While the present invention has been fully described by way of embodiments thereof, it is to be noted that various changes and modifications will be apparent to those skilled in the art. Such changes and modifications are intended to be included within the scope of the appended claims.

Claims

1. A compound of formula (I), or a tautomer, pharmaceutically acceptable salt, solvate, chelate, non-covalent complex, or prodrug thereof:

wherein the content of the first and second substances,

x is selected from O, S or NR²；

R¹Is selected from

Wherein

Unsubstituted or substituted by C_1-3Alkyl substitution;

R³is selected from

and when R is₁Is composed of

When R is₃Is not that

2. A compound of claim 1, wherein R is¹The substituents being selected from

3. A compound according to claim 1 or 2, wherein X is-NR²，R²Selected from hydrogen or C_1-3An alkyl group.

4. A compound according to any one of claims 1 to 3, wherein R is⁴Selected from hydrogen or C_1-3An alkyl group.

5. A compound according to any one of claims 1 to 4, wherein R is⁵Or R⁶Independently selected from hydrogen or C_1-3An alkyl group.

6. A compound according to any one of claims 1 to 5, wherein R is⁶Is methyl,

7. A compound, tautomer or pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of:

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 7 and at least one pharmaceutically acceptable excipient.

9. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for the manufacture of a medicament.