Pyridazine compound and use thereof

Abstract

Disclosed in the present invention are a pyridazine compound and a preparation method therefor and a use thereof, and a pharmaceutical composition. The structure of the compound is shown as formula I. Also provided are a stereoisomer, a mesomer, a racemate, a prodrug, a crystal, a pharmaceutically acceptable salt, or a mixture thereof. The compound designed by the present invention has a significant inhibition effect on CD73, has good pharmacokinetic properties, and can realize a treatment effect by means of an immunoregulation effect, so that the compound can be widely applied to preparation of medicines for preventing and / or treating CD73-mediated or dependent diseases or conditions, and and the preparation method for the compound is conducive to structural expansion.

Description

Pyridazine compounds and their applications Technical Field

[0001] The present invention relates to a pyridazine compound, a preparation method, a pharmaceutical composition and an application thereof, and in particular to a pyridazine compound with CD73 inhibitory activity, a preparation method, a pharmaceutical composition and an application thereof. Background Art

[0002] Ecto-5'-nucleotidase (CD73) is a glycoprotein on the cell membrane that participates in the regulation of the adenosine pathway by catalyzing the hydrolysis of extracellular 5'-adenosine monophosphate (5'-AMP) into adenosine. Studies have shown that the CD73-adenosine pathway plays an important role in maintaining the body's immune homeostasis and controlling immune tolerance. Under normal physiological conditions, CD73 is widely expressed at low levels on the cell surface of different tissues, while it is highly expressed in the tumor microenvironment (TME). Under hypoxic conditions in tumor tissue, hypoxia-inducible factor (HIF) produced in tumor cells can be induced to directly bind to the hypoxia response element (HRE) in the CD73 gene promoter, thereby promoting the transcription of CD73. Adenosine, the catalytic hydrolysis product of CD73, can increase the intracellular cAMP level by activating adenosine receptors on the surface of cancer cells. cAMP directly binds to the cAMP response element (CRE) in the CD73 gene promoter, thereby positively feedback-regulating the transcription of the CD73 gene. Other studies have found that estrogen receptor (ER) in breast cancer negatively regulates the expression of CD73, and the loss of ER can significantly enhance the expression of CD73.

[0003] Like cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death receptor 1 (PD-1) and its ligand PD-L1, CD73 plays an important role in tumor immune escape. Tumor cells create an immunosuppressive microenvironment by upregulating the expression of CD73. Clinically, CD73 overexpression and enhanced activity have been found in most cancers with poor prognosis. In the TME, CD73 overexpression leads to ATP consumption and adenosine accumulation, thereby inhibiting effector T cells (Teffs) and promoting the differentiation and proliferation of regulatory T (Tregs) cells, thereby helping tumor cells escape immune attacks.

[0004] The extracellular adenosine formed by CD73 hydrolysis of AMP activates downstream G protein-coupled signals by interacting with cell surface adenosine receptors, thereby exerting its immunomodulatory effect. High concentrations of extracellular adenosine can significantly inhibit infiltrating Teffs, especially CD8 +Teffs proliferation and their cytotoxic effects inhibit the killing ability of natural killer (NK) cells and enhance the immunosuppressive activity of Tregs. Furthermore, extracellular adenosine can regulate the development of dendritic cells, reduce their ability to present tumor antigens, promote the expansion and activation of myeloid-derived suppressor cells (MDSCs), and induce macrophage differentiation toward a tolerant phenotype. It is worth mentioning that CD73 also has an important non-enzymatic hydrolysis function. As a signaling and adhesion molecule that regulates the interaction between cells and extracellular matrix components, it can regulate the adhesion and migration ability of cells and promote the infiltration and metastasis of tumor cells.

[0005] Studies have shown that CD73 is highly expressed in most solid tumors, including breast cancer, colorectal cancer, ovarian cancer, gastric cancer, gallbladder cancer, prostate cancer, etc., and is closely related to tumor stage, pathological type and prognosis. A number of preclinical studies have shown that inhibiting the activity of CD73 can reverse its mediated immune escape, and in clinical trials, the synergistic effect of CD73 inhibitors in combination with immunotherapy (such as immune checkpoint PD-1 / PD-L1 inhibitors) and chemotherapy drugs has begun to appear. However, the currently reported CD73 small molecule inhibitors have a single structure, and most of them are adenosine analogs. Therefore, it is of great significance to develop non-adenosine CD73 small molecule inhibitors with high activity and good drugability. Summary of the Invention

[0006] Objectives of the invention: The first objective of the present invention is to provide a pyridazine compound with high CD73 inhibitory activity, the second objective is to provide a method for preparing the compound, the third objective is to provide a pharmaceutical composition containing the compound, and the fourth objective is to provide a pharmaceutical application of the compound and its pharmaceutical composition.

[0007] Technical solution: The pyridazine compound of the present invention has a structure of formula I, and also includes its stereoisomers, meso-forms, racemates, prodrugs, crystals, pharmaceutically acceptable salts or mixtures thereof.

[0008] Wherein: X is selected from CH2, NH, O, S, or X is absent;

[0009] R1 is selected from hydrogen, C1-C6 alkyl or (CH2) n Ar; wherein n is an integer selected from 0-3, Ar is selected from a 3-8 membered cycloalkyl, a 6-12 membered aryl or a 5-12 membered aromatic heterocycle; the aromatic heterocycle contains 1-3 heteroatoms selected from N, O or S, and the alkyl, cycloalkyl, aryl or aromatic heterocycle is substituted with one or more R3;

[0010] R3 is selected from hydrogen, halogen, nitro, amino, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, phenyl or C1-C6 haloalkyl;

[0011] R2 is selected from hydroxy, amino, halogen, carboxyl, C(O)NR4R5, C1-C6 alkoxy, C3-C8 cycloalkyl, 4-6 membered heterocyclyl, 6-12 membered aryl or 4-12 membered aromatic heterocycle; the heterocycle or aromatic heterocycle contains 1-3 heteroatoms selected from N, O or S, and the carboxyl, alkoxy, aryl, heterocycle or aromatic heterocycle is substituted by one or more R6;

[0012] R6 is selected from hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C1-C6 haloalkyl or C1-C6 haloalkoxy;

[0013] R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or R4, R5 together with the N to which they are attached form a 4-7 membered heterocyclic group.

[0014] Preferably, in the above structure: X is selected from CH2, NH, O, S, or X is absent;

[0015] R1 is selected from hydrogen, C1-C6 alkyl or (CH2) n Ar; wherein n is selected from an integer of 0-3, Ar is selected from a cyclopropyl group, a cyclohexyl group, a phenyl group, a pyridine ring, an indole ring, a morpholine ring, a benzothiophene ring or a thiophene ring; the alkyl group or the phenyl group is substituted by one or more R3 groups;

[0016] R3 is selected from hydrogen, halogen, nitro, C1-C6 alkyl or C1-C6 alkoxy;

[0017] R2 is selected from hydroxy, amino, halogen, CO2CH3, C(O)NR4R5, C1-C6 alkoxy, cyclopropyl, pyrrolidin-1-yl, phenyl or a five-membered aromatic heterocycle; the aromatic heterocycle contains 1-2 heteroatoms selected from N, O or S, and the alkoxy or aromatic heterocycle is substituted with 0-2 halogen, methyl or cyano groups;

[0018] R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or R4, R5 together with the attached N form azetidine or pyrrolidin-1-yl.

[0019] Preferably, in the above structure: X is selected from NH, O, S, or X is absent;

[0020] R1 is selected from methyl, ethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2,2-difluoropropyl, 2,2,3,3-tetrafluoropropyl, 2,2,3,3,3-pentafluoropropyl, 3,3,3-trifluoropropyl, cyclopropyl or (CH2) nAr; wherein n is an integer selected from 2-3, Ar is selected from cyclohexane, phenyl, pyridine ring, indole ring, morpholine ring, benzothiophene ring or thiophene ring, and the phenyl is substituted with 0-2 halogen, nitro, methyl, methoxy or trifluoromethyl groups;

[0021] R2 is selected from hydroxyl, amino, halogen, CO2CH3, C(O)NR4R5, methoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, cyclopropyl, pyrrolidin-1-yl, phenyl, and a five-membered aromatic heterocycle; the aromatic heterocycle is selected from thiophene, furan, pyrazole, or isoxazole, and the aromatic heterocycle is substituted with 0-2 methyl or cyano groups;

[0022] R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or R4, R5 together with the attached N form azetidine or pyrrolidin-1-yl.

[0023] Preferably, -X-R1 is selected from any of the following groups:

[0024] Preferably, R2 is selected from any of the following groups:

[0025] Preferably, the pyridazine compound of the present invention is selected from any of the following compounds:

[0026] Preferably, the pharmaceutically acceptable salt of the present invention is a salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid or ferulic acid.

[0027] The preparation method of the pyridazine compound of the present invention is selected from any of the following methods:

[0028] Method 1: Using 3,6-dichloropyridazin-4-amine as a raw material, the compound of formula I is prepared by nucleophilic substitution, Sandmeyer reaction, nucleophilic substitution, Suzuki coupling, and hydrolysis reaction;

[0029] Method 2: Using 3,4,6-trichloropyridazine as the raw material, the compound of formula I is prepared through substitution, acylation, coupling, and two hydrolysis reactions:

[0030] Method 3: Using 3,4,6-trichloropyridazine as a raw material, the compound of formula I is prepared by nucleophilic substitution, nucleophilic substitution or coupling, coupling, and hydrolysis reaction;

[0031] Method 4: Using 4,6-dichloropyridazine-3-carboxylic acid methyl ester as the starting material, the compound of formula I is prepared by nucleophilic substitution, aminolysis, coupling, and hydrolysis;

[0032] Method 5: Using 3-amino-4-bromo-6-chloropyridazine as the starting material, the compound of formula I is prepared by substitution, acylation, coupling, hydrolysis, substitution, coupling, and hydrolysis;

[0033] Wherein, R1, R2, and X are as defined above; the compound of formula I prepared by the above method is salified with a corresponding acid to obtain a pharmaceutically acceptable salt of the compound.

[0034] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting the compounds with specified substituents with relatively nontoxic acids or bases. When the compound contains relatively acidic functional groups, base addition salts can be obtained by contacting the free form of the compound with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compound contains relatively basic functional groups, acid addition salts can be obtained by contacting the free form of the compound with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, Acids such as citric acid, tartaric acid and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (such as arginine, etc.) and glucuronic acid. When certain specific compounds contain basic and acidic functional groups, they can be converted into either base or acid addition salts. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated to regenerate the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubility in polar solvents.

[0035] "Pharmaceutically acceptable salts" can be synthesized from parent compounds containing acid or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0036] Preferably, the stereoisomer is R 1 、R 2 , isomers introduced by the chiral C and N in X. The tautomers are isomers formed by double bond conjugation, including carbon-carbon double bond tautomerism and carbon-heteroatom double bond tautomerism, such as tautomers formed by double bond tautomerism in pyridazine ring system and pyrimidinone. The prodrug is R 1 、R 2 , ester or amide prodrugs introduced with a carboxyl group, hydroxyl group or amino group in X, more preferably C1-C4 alkyl ester, C1-C4 carboxylate or C1-C4 alkyl amide. The solubilized product is a small molecule binding state formed by the compound and solvent molecules, more preferably a hydrate or alcoholate; the solvate can further form a salt with the corresponding acid to obtain the salt of the solvate. The isotopic compound is a compound in which hydrogen in the compound is replaced by deuterium. The crystal is a specific crystal structure formed by the compound during the crystallization process, including different crystal forms of the compound itself, as well as different crystal forms of its salts, solvates and salts of solvates.

[0037] The pharmaceutical composition of the present invention comprises the pyridazine compound of the present invention and a pharmaceutically acceptable carrier.

[0038] Preferably, the pharmaceutical combination is formulated in the form of a tablet, capsule, powder, pill, granule, injection, oral solution, syrup, inhalant, ointment, patch, or suppository. A "pharmaceutically acceptable carrier" may be any excipient widely used in pharmaceutical production. Excipients are primarily used to provide a safe, stable, and functional pharmaceutical composition. They may also provide methods to ensure that the active ingredient dissolves at a desired rate after administration to a subject, or to promote effective absorption of the active ingredient after administration of the composition. Pharmaceutical excipients may be inert fillers or may provide a specific function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners. The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, according to the disclosure. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or lyophilizing processes can be used. The pharmaceutical compositions of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ophthalmic, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be in a controlled-release or sustained-release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, softgels, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0039] The pyridazine compounds or pharmaceutical compositions thereof described in the present invention are used to prepare CD73 small molecule inhibitor drugs, and are also used to prepare immunomodulator drugs.

[0040] Preferably, the drug is a drug for preventing and / or treating tumors, infectious diseases, inflammatory diseases, organ transplant rejection or autoimmune diseases.

[0041] More preferably, the tumors include, but are not limited to, neuroblastoma, melanoma, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of the head and neck, gastric cancer, glioblastoma, bladder cancer, pancreatic cancer, lymphoma, leukemia, prostate cancer, testicular cancer, kidney cancer, brain cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, thyroid cancer, liver cancer, and esophageal cancer. The inflammatory diseases include, but are not limited to, psoriasis and osteoarthritis. The autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, myasthenia gravis, mixed connective tissue disease, psoriasis, and autoimmune reactions caused by infection.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0043] The compounds designed in this invention have a significant inhibitory effect on CD73 and inhibit IC 50 The concentration value reaches the nanomolar level, even below 10nM, and has good pharmacokinetic properties in vivo, which is conducive to drug development and can achieve effective therapeutic effects through immunomodulatory effects. At the same time, the preparation method of the compound is highly adaptable and can prepare compounds of various structural types. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1 shows the results of the dose-dependent inhibition of the compound of the present invention on the growth of 4T1 breast cancer transplanted tumors in mice;

[0045] FIG2 shows the effects of the compounds of the present invention on cytokines in mouse transplanted tumors;

[0046] FIG3 shows the effects of the compounds of the present invention on immune cells in mouse transplanted tumors. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0048] Reagents and Materials: All reagents required for the experiment are commercially available chemically pure or analytically pure products unless otherwise specified. 1 H NMR was measured using a Bruker AV-300 400 MHz nuclear magnetic resonance instrument. Chemical shifts (δ) are reported in ppm, coupling constants (J) are reported in Hz, and TMS was used as the internal standard. Mass spectrometry (MS) was performed using a Shimadzu LCMS-2020 mass spectrometer. Thin-layer chromatography (TLC) was performed using HG / T2354-92 GF254 thin-layer chromatography silica gel produced by Qingdao Ocean Chemical Co., Ltd., and visualized using a ZF7 triple-UV analyzer at 254 nm. Column chromatography used coarse-pore (ZCX-II) 100–200 mesh silica gel produced by Qingdao Ocean Chemical Plant.

[0049] Example 1: Synthesis of 5-(6-methoxy-5-(phenylethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (1)

[0050] Synthesis of 6-chloro-3-methoxypyridazin-4-amine (1-1)

[0051] 3,6-Dichloropyridazine-4-amine (2.96 g, 5.93 mmol) was placed in a sealed tube, and 5 mL of methanol was added. Sodium methoxide (243 mg, 4.50 mmol) was then added with stirring. The mixture was heated to 100°C and allowed to react for 106 hours. After completion of the reaction, the mixture was concentrated under reduced pressure and crystallized from ethyl acetate to obtain a white solid in an 89% yield. 1 H NMR (300MHz, DMSO-d6) δ6.67 (s, 2H), 6.56 (d, J = 1.8Hz, 1H), 3.97 (s, 3H).

[0052] Synthesis of 4,6-dichloro-3-methoxypyridazine (1-2)

[0053] 1-1 (640 mg, 4.00 mmol) was added to 15 mL of acetonitrile and cuprous chloride (792 mg, 8.00 mmol). Tert-butyl nitrite (1.26 g, 10.80 mmol) was slowly added dropwise under an ice bath. After completion of the addition, the reaction was allowed to proceed for 5 minutes and then at 60°C for 4 hours. The product was then added with 30 mL of water, extracted with ethyl acetate, and purified by column chromatography to obtain a white solid in a 66% yield. 1 H NMR (300MHz, CDCl3) δ7.53 (s, 1H), 4.23–4.22 (m, 3H).

[0054] Synthesis of 6-chloro-3-methoxy-N-phenethylpyridazin-4-amine (1-3)

[0055] 1-2 (179 mg, 1.00 mmol) was added to 15 mL of THF. 2-Chlorophenylethylamine (233 mg, 1.50 mmol) and DIPEA (194 mg, 1.50 mmol) were added with stirring and allowed to react overnight at 70°C. After completion of the reaction, 10 mL of water was added, the mixture was extracted with ethyl acetate, and purified by column chromatography to obtain a white solid in a 49.0% yield. 1H NMR (300MHz, CDCl3) δ7.37 (d, J=1.8Hz, 1H), 7.34 (q, J=1.5Hz, 1H), 7.33–7.29 (m, 1H), 7.26–7. 21(m,2H),6.35(s,1H),5.02(brs,1H),4.09(s,3H),3.46–3.40(m,2H),2.97(t,J=7.2Hz,2H).

[0056] Synthesis of 6-(2,4-dimethoxypyrimidin-5-yl)-3-methoxy-N-phenethylpyridazin-4-amine (1-4)

[0057] 1-3 (190 mg, 0.72 mmol), potassium carbonate (200 mg, 1.44 mmol), 2,4-dimethoxypyrimidine-5-boronic acid (145 mg, 0.79 mmol), and Pd(dppf)Cl2 (7.3 mg, 0.01 mmol) were added sequentially to a dioxane aqueous solution (5 mL) and reacted at 90°C under nitrogen overnight. The product was then added with 10 mL of water, extracted with ethyl acetate, and purified by column chromatography to obtain a white solid in a 75.5% yield. 1 H NMR(300MHz,DMSO-d6)δ8.65(s,1H),7.35–7.24(m,4H),7.23–7.20(m,1H),6.92(s,1H),6.63(t ,J=6.0Hz,1H),4.04(s,3H),3.99(s,3H),3.97(s,3H),3.44–3.36(m,2H),2.87(t,J=6.9Hz,2H).

[0058] Synthesis of 5-(6-methoxy-5-(phenylethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (1)

[0059] 1-4 (200 mg, 0.54 mmol) was added to 1 M hydrochloric acid (4 mL) and 1 mL of methanol and allowed to react overnight at 60°C. After completion of the reaction, saturated potassium carbonate solution was added to adjust the pH to neutral. The product was filtered, dried, and crystallized from methanol to obtain a white solid in a yield of 75.6%. ESI-MS m / z 400.2 [M+H] + . 1 H NMR(300MHz,DMSO-d6)δ11.36(brs,2H),8.11(s,1H),7.34(s,1H),7.32(s,1H),7.29–7.27( m,3H),7.25–7.18(m,1H),6.55(s,1H),4.00(s,3H),3.35–3.31(m,2H)2.86(t,J=7.8Hz,2H).

[0060] Similar operations were performed according to Example 1 to obtain the following compounds:

[0061] Examples 17 and 18: Synthesis of 5-(6-hydroxy-5-(phenylethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (17) and 5-(6-chloro-5-(phenylethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (18)

[0062] Synthesis of 3,6-dichloro-N-phenethylpyridazin-4-amine (17-1)

[0063] 3,4,5-Trichloropyridazine (1.50 g, 8.19 mmol) was added to 15 mL of THF, and phenylethylamine (1.49 g, 12.30 mmol) was added with stirring. The mixture was allowed to react at room temperature for 6 hours. 50 mL of water was added, and the mixture was extracted with ethyl acetate. The mixture was purified by column chromatography to obtain a white solid in a yield of 59.3%. 1 H NMR (300MHz, CDCl3) δ7.42–7.30(m,3H),7.26–7.21(m,2H),6.52(s,1H),5.15(s,1H),3.53–3.46(m,2H),3.01(t,J=6.9Hz,2H).

[0064] Synthesis of tert-butyl (3,6-dichloropyridazin-4-yl)(phenethyl)carbamate (17-2)

[0065] 17-1 (300 mg, 1.35 mmol) and DMAP (329 mg, 2.70 mmol) were added to 5 mL of dichloromethane. Di-tert-butyl dicarbonate (589 mg, 2.70 mmol) was added dropwise with stirring. The mixture was allowed to react at room temperature for 2 hours. 10 mL of water was added, the mixture was extracted with ethyl acetate, and purified by column chromatography to obtain a colorless liquid in a yield of 95%. 1 H NMR (300MHz, CDCl3) δ7.33(t,J=7.2Hz,2H),7.22(d,J=7.5Hz,1H),7.15(d,J=6.9Hz ,2H),6.63(s,1H),3.81(d,J=8.7Hz,3H),2.98(t,J=7.2Hz,2H),1.52–1.48(m,9H).

[0066] Synthesis of tert-butyl (3-chloro-6-(2,4-dimethoxypyrimidin-5-yl)pyridazin-4-yl)(phenethyl)carbamate (17-3)

[0067] A white solid was obtained by the synthesis method of reference compound 1-4 with a yield of 43.4%. 1 H NMR (300MHz, CDCl3) δ9.21 (s, 1H), 8.54 (s, 1H), 7.24 (d, J = 7.2Hz, 2H), 7.18 (d, J = 6.9Hz, 1H), 7.11 (d,J=7.5Hz,2H),4.15(d,J=5.7Hz,6H),4.09(s,3H),4.00(s,2H),2.92–2.88(m,2H),1.41(s,9H).

[0068] Synthesis of 5-(6-chloro-5-(phenylethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (17)

[0069] A white solid was obtained by the synthesis method of reference compound 1 with a yield of 22.2%. ESI-MS m / z 344.2 [M+H] + . 1 H NMR(300MHz,DMSO-d6)δ10.39(s,2H),8.53(s,1H),7.82(s,1H),7.65(s,1H) ,7.32(s,4H),7.22(s,1H),6.99(s,1H),3.26(s,2H),2.84(t,J=7.8Hz,2H).

[0070] Synthesis of 5-(6-hydroxy-5-(phenylethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (18)

[0071] 17-3 (150 mg, 0.32 mmol) was added to sodium acetate (108 mg, 1.59 mmol) and 3 mL of glacial acetic acid and allowed to react overnight at 100°C. After completion of the reaction, the pH was adjusted to neutral with saturated potassium carbonate solution. The mixture was extracted with ethyl acetate and concentrated under reduced pressure to afford crude product 18-1. A light pink solid was obtained by referring to the synthesis method of compound 1 in a yield of 10.5%. ESI-MS m / z 328.1 [M+H] + . 1 H NMR(300MHz,DMSO-d6)δ12.62(s,1H),11.28(d,J=36.9Hz,2H),7.72(s,1H),7.34–7.18(m,5H) ,6.73(s,1H),6.66(t,J=6.0Hz,1H),3.31(dd,J=7.2,4.8Hz,2H),2.87(dd,J=8.7,6.3Hz,2H).

[0072] Similar operations as in Example 1 and 18 were performed to obtain the following compounds:

[0073] Example 21: Synthesis of 5-(6-(2,2-difluoroethoxy)-5-(phenethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (21)

[0074] Synthesis of 6-chloro-3-(2,2-difluoroethoxy)-N-phenethylpyridazin-4-amine (21-1)

[0075] Add 4.00 mmol of methyl parathionine to 5 mL of dioxane, add sodium hydride (160 mg, 4.00 mmol) with stirring in an ice bath, and react at room temperature for 20 minutes. Add 3,6-dichloro-N-phenethylpyridazin-4-amine (1.00 mmol), and react at 100°C overnight. Add 5 mL of water, extract with ethyl acetate, and purify by column chromatography to obtain a colorless liquid in a 78.0% yield. 1 H NMR (300MHz, CDCl3) δ7.40–7.30(m,3H),7.26–7.19(m,2H),6.41(s,1H),6.37–5.95(m,1H),5. 04(brs,1H),4.68(td,J=13.5,3.9Hz,2H),3.45(td,J=7.2,5.7Hz,2H),2.98(t,J=6.9Hz,2H).

[0076] Synthesis of 3-(2,2-difluoroethoxy)-6-(2,4-dimethoxypyrimidin-5-yl)-N-phenethylpyridazin-4-amine (21-2)

[0077] Synthesis method is the same as that of compound 1-4. Colorless liquid; yield 55.8%. 1 H NMR(300MHz, CDCl3)δ8.86(s,1H),7.41–7.30(m,3H),7.28–7.23(m,2H),6.93(s,1H),6.47–6.02(m,1H),5.08( brs,1H),4.75(td,J=13.5,3.9Hz,2H),4.09(s,3H),4.08(s,3H),3.52(q,J=6.9Hz,2H),3.02(t,J=7.2Hz,2H).

[0078] Synthesis of 5-(6-(2,2-difluoroethoxy)-5-(phenethylamino)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (21)

[0079] A white solid was obtained by referring to the synthesis method of compound 1 with a yield of 29.7%. ESI-MS m / z 390.2 [M+H]+ . 1 H NMR(300MHz,DMSO-d6)δ12.18(s,1H),11.87(s,1H),8.64(s,1H),8.39(s,1H),7.67–7.55(m,1H),7.35–7.27(m,4H), 7.23(q,J=4.5Hz,1H),6.64–6.30(m,1H),4.73(td,J=14.4,3.6Hz,2H),3.65(d,J=7.2Hz,2H),2.94(t,J=7.8Hz,2H).

[0080] Similar operations as in Examples 1 and 21 were performed to obtain the following compounds:

[0081] Example 39: Synthesis of 6-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-4-(phenylethylamino)pyridazine-3-carboxamide (39)

[0082] Synthesis of Compound 6-Chloro-4-(phenylethylamino)pyridazine-3-carboxylic Acid Methyl Ester (39-1)

[0083] Methyl 4,6-dichloropyridazine-3-carboxylate (1.04 g, 5.00 mmol) was added to 10 mL of THF and DIPEA (1.21 g, 10.00 mmol). Phenethylamine (1.21 g, 5.00 mmol) was added with stirring and allowed to react at room temperature for 2 h. After completion of the reaction, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain a white solid in an 85.0% yield. 1 H NMR (300MHz, CDCl3) δ8.15 (s, 1H), 7.36 (d, J = 7.2Hz, 2H), 7.27 (t, J = 6.6Hz, 3H) ,6.66(s,1H),4.06–3.98(m,3H),3.48(q,J=6.6Hz,2H),3.01(t,J=7.2Hz,2H).

[0084] Synthesis of 6-chloro-4-(phenylethylamino)pyridazine-3-carboxamide (39-2)

[0085] Compound 39-1 (1.16 g, 4.00 mmol) was added to 6 mL of methanol. With stirring, 6 mL of 30% aqueous ammonia was added, and the temperature was raised to 70°C. The reaction was allowed to proceed overnight. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain a white solid. Add 50 mL of water, filter, and dry to obtain the compound as a white solid in a 95.0% yield. 1H NMR(300MHz, CDCl3)δ8.96(s,1H),7.96(s,1H),7.41–7.33(m,2H),7.33–7.24(m,3 H), 6.62 (s, 1H), 5.57 (brs, 1H), 3.46 (td, J = 7.2, 5.4Hz, 2H), 3.00 (t, J = 7.2Hz, 2H).

[0086] Synthesis of 6-(2,4-dimethoxypyrimidin-5-yl)-4-(phenylethylamino)pyridazine-3-carboxamide (39-3)

[0087] A yellow solid was obtained by the synthesis method of reference 1-4 with a yield of 44.5%. 1 H NMR(300MHz, CDCl3)δ9.08(brs,1H),8.10(brs,1H),7.73–7.65(m,1H),7.50(dd,J=7.8,3.0Hz,1H),7.42–7.33( m,2H),7.31–7.19(m,3H),5.57(brs,1H),4.13(s,3H),4.11(s,3H),3.56(d,J=6.6Hz,2H),3.04(t,J=7.2Hz,2H).

[0088] Synthesis of 6-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-4-(phenylethylamino)pyridazine-3-carboxamide (39)

[0089] A white solid was obtained by referring to the synthesis method of compound 1 with a yield of 38.3%. ESI-MS m / z 353.2 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ11.57(s,2H),8.98(s,1H),8.48(d,J=6.0Hz,1H),8.42–8.34(m,1H),7.83(s,1 H),7.73(s,1H),7.32–7.26(m,4H),7.24(q,J=4.2Hz,1H),3.47(q,J=6.9Hz,2H),2.91(t,J=7.5Hz,2H).

[0090] Similar operations to those in Example 1 and 39 were used to prepare the following compounds:

[0091] Example 52: Synthesis of 5-(5-phenylethoxy-6-(thiophen-2-yl)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (52)

[0092] Synthesis of 6-chloro-4-phenethoxypyridazin-3-amine (52-1)

[0093] Sodium hydride (276 mg, 11.51 mmol) was added to 10 mL of 1,4-dioxane, and phenylethanol (1.41 g, 11.51 mmol) was added dropwise with stirring. The mixture was allowed to react in an ice bath for 0.5 h. 4-Bromo-6-chloropyridazin-3-amine (2 g, 9.6 mmol) was added, and the temperature was raised to 60°C. The reaction was allowed to proceed overnight. After the reaction was complete, 10 mL of water was added, and the mixture was extracted with ethyl acetate. Purification by column chromatography afforded a white solid in an 89.7% yield. 1 H NMR (300MHz, CDCl3) δ7.31–7.22(m,5H),6.63(s,1H),5.13(s,2H),4.29(t,J=6.9Hz,2H),3.19(t,J=6.9Hz,2H).

[0094] Synthesis of 6-chloro-4-phenylethoxypyridazine-3-N,N-di-tert-butoxycarbonyl (52-2)

[0095] 52-1 (2.15 g, 8.6 mmol) was added to 20 mL of dichloromethane and DMAP (4.2 g, 34.44 mmol). Di-tert-butyl dicarbonate (7.51 g, 34.44 mmol) was added dropwise with stirring. The mixture was allowed to react at room temperature for 3 h. After the reaction, 50 mL of water was added, the mixture was extracted with ethyl acetate, and purified by column chromatography to obtain a white solid in a 98.3% yield. 1 H NMR (300MHz, CDCl3) δ7.36–7.29(m,2H),7.25(td,J=7.5,6.9,2.1Hz,3H),6.96(s,1H),4.29(t,J=6.9Hz,2H),3.12(t,J=6.9Hz,2H),1.39(s,18H).

[0096] Synthesis of 6-(2,4-dimethoxypyrimidin-5-yl)-4-phenylethoxypyridazine-3-N,N-di-tert-butoxycarbonyl (52-3)

[0097] The synthesis method of reference compound 1-4 was used to obtain a colorless liquid with a yield of 91%. 1 H NMR (300MHz, CDCl3) δ9.16(s,1H),7.52(s,1H),7.38–7.31(m,2H),7.31–7.25(m,3H),4.33(t,J=6.9Hz,2H),3.15(t,J=6.9Hz,2H),1.43(s,18H).

[0098] Synthesis of 6-(2,4-dimethoxypyrimidin-5-yl)-4-phenylethoxypyridazin-3-amine (52-4)

[0099] Compound 52-3 (2.24 g, 4.05 mmol) was added to 10 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added dropwise with stirring. The mixture was allowed to react at room temperature for 12 hours. After the reaction, saturated potassium carbonate solution was added to adjust the pH to neutral. The mixture was extracted with ethyl acetate and concentrated under reduced pressure to obtain a white solid in a 99.3% yield. 1 H NMR(300MHz, CDCl3)δ8.83(s,1H),7.43–7.30(m,5H),7.14(s,1H),5.62(s, 2H),4.34(t,J=6.9Hz,2H),4.07(s,3H),4.07(s,3H),3.22(t,J=6.9Hz,2H).

[0100] Synthesis of 3-bromo-6-(2,4-dimethoxypyrimidin-5-yl)-4-phenylethoxypyridazine (52-5)

[0101] 52-4 (1.76 g, 4.98 mmol) was added to 10 mL of acetonitrile and cuprous bromide (1.43 g, 9.97 mmol). Tert-butyl nitrite (1.39 g, 13.45 mmol) was added dropwise with stirring. The mixture was allowed to react at room temperature for 5 minutes, then heated to 50°C for 2 hours. After the reaction, 30 mL of water was added, the mixture was extracted with ethyl acetate, and purified by column chromatography to obtain a white solid in a yield of 52.8%. 1 H NMR (300MHz, CDCl3) δ9.06 (s, 1H), 7.39–7.30 (m, 6H), 4.36 (t, J = 6.6Hz, 2H), 4.09 (d, J = 1.5Hz, 6H), 3.25 (t, J = 6.6Hz, 2H).

[0102] Synthesis of 6-(2,4-dimethoxypyrimidin-5-yl)-4-phenylethoxy-3-(thiophen-2-yl)pyridazine (52-6)

[0103] Compound 52-5 (150 mg, 0.36 mmol) was added to 5 mL of 1,4-dioxane and 1 mL of water. Tetrakis(triphenylphosphine)palladium (16.6 mg, 0.014 mmol), sodium carbonate (152 mg, 1.44 mmol), and 2-thiopheneboronic acid (230 mg, 1.80 mmol) were added with stirring. The mixture was reacted at 95°C overnight under N₂ protection. After completion of the reaction, 10 mL of water was added, the mixture was extracted with ethyl acetate, and purified by column chromatography to obtain a white solid in a 68.6% yield. 1H NMR (300MHz, CDCl3) δ9.20(s,1H),7.99(dd,J=3.9,1.2Hz,1H),7.54(s,1H),7.51(dd,J=5.1,1.2Hz,1H),7.38(d,J=2.7Hz, 4H), 7.34 (d, J = 2.1Hz, 1H), 7.15 (dd, J = 5.1, 3.9Hz, 1H), 4.50 (t, J = 6.9Hz, 2H), 4.13 (d, J = 2.1Hz, 6H), 3.35 (t, J = 6.9Hz, 2H).

[0104] Synthesis of 5-(5-phenethoxy-6-(thiophen-2-yl)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (52)

[0105] A white solid was obtained by the synthesis method of reference compound 1 with a yield of 55.63%. ESI-MS m / z 393.1 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ10.40(s,1H),8.75(s,1H),8.32(s,1H),7.73(d,J=3.6Hz,1H),7.63(d,J=5.1Hz,1H),7.42(d,J =7.5Hz,2H),7.34(t,J=7.5Hz,2H),7.29–7.21(m,1H),7.15–7.09(m,1H),4.47(t,J=6.9Hz,2H),3.23(d,J=13.5Hz,2H).

[0106] Similar operations to those in Example 1 and 52 were used to prepare the following compounds:

[0107] Example 102: Synthesis of 5-(5-cyclopropyl-6-(thiophen-2-yl)pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione (102)

[0108] Synthesis of 3,6-dichloro-4-cyclopropylpyridazine (102-1)

[0109] Concentrated sulfuric acid (0.6 mL) was slowly added dropwise to 6 mL of water, followed by the addition of 3,6-dichloropyridazine (0.63 g, 4.23 mmol) and cyclopropanecarboxylic acid (0.36 g, 4.23 mmol). Under nitrogen, the temperature was raised to 70°C and the reaction was allowed to proceed for 0.5 h. Silver trifluoromethanesulfonate (0.54 g, 2.12 mmol) was quickly added, followed by the slow dropwise addition of ammonium persulfate (2.89 g, 12.69 mmol). After the addition was complete, the temperature was raised to 70°C and the reaction was continued for 2 h. After the reaction was complete, 10 mL of water was added, the mixture was extracted with ethyl acetate, and purified by column chromatography to obtain a white solid in a yield of 49.74%. 1 H NMR (300MHz, CDCl3) δ7.45(s,1H),3.39–2.12(m,1H),1.38–1.23(m,2H),0.95–0.84(m,2H).

[0110] Synthesis of 3-chloro-4-cyclopropyl-6-(2,4-dimethoxypyrimidin-5-yl)pyridazine (102-2)

[0111] A white solid was obtained by the synthesis method of reference compound 1-4 with a yield of 34.5%. 1 H NMR (300MHz, CDCl3) δ9.05 (s, 1H), 7.47 (s, 1H), 4.09 (d, J = 3.6Hz, 6H), 2.27–2.12 (m, 1H), 1.31–1.25 (m, 2H), 0.90–0.84 (m, 2H).

[0112] Synthesis of 4-cyclopropyl-6-(2,4-dimethoxypyrimidin-5-yl)-3-(thiophen-2-yl)pyridazine (102-3)

[0113] A white solid was obtained by the synthesis method of reference compound 52-6 with a yield of 74.5%. 1 H NMR (300MHz, CDCl3) δ8.40(d,J=6.0Hz,1H),7.97(s,1H),7.86(d,J=6.3Hz,1H),7.69(s,1H),7. 32–7.21(m,1H),4.12(d,J=3.9Hz,6H),2.27–2.23(m,1H),1.26–1.19(m,2H),0.95–0.84(m,2H).

[0114] Synthesis of 6-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-4-(phenylethylamino)pyridazine-3-carboxamide (102)

[0115] A white solid was obtained by referring to the synthesis method of compound 1 with a yield of 18.3%. ESI-MS m / z 313.0 [M+H]+ . 1 H NMR(300MHz,DMSO-d6)δ11.73(s,1H),11.51(s,1H),8.42(d,J=6.3Hz,1H),8.07(s,1H),7.99–7.85(m,1H), 7.80–7.70(m,1H),7.73(s,1H),7.32–7.21(m,1H),2.28–2.25(m,1H),1.21–1.17(m,2H),0.96–0.80(m,2H).

[0116] Example 103: Evaluation of the inhibitory activity of compounds against recombinant human CD73 enzyme

[0117] The CD73 enzyme is an ecto-5-nucleotidase that converts extracellular nucleoside 5'-monophosphate to adenosine, with AMP as the preferred substrate.

[0118] 1. Experimental Purpose

[0119] Evaluate the inhibitory effect of the compounds of the present invention on the function of human CD73 protein in converting AMP into adenosine.

[0120] 2. Experimental materials and instruments

[0121] Malachite green phosphate detection kit: BioAssay Systems; BioTek multi-function microplate reader: SynergyHTX.

[0122] 3. Experimental methods

[0123] The recombinant CD73 protein was diluted to 10 ng / mL with a buffer solution (150 mM NaCl, 20 mM Tris-HCl, pH = 8.0), 20 μL of CD73 protein dilution was added to a 96-well plate, and then the compound (DMSO content was controlled at 0.5%) was added. After pipetting evenly, the mixture was incubated at room temperature for 15 minutes. At the same time, a positive control group (no compound) and a negative control group (no CD73) were set up. AMP was added to a final concentration of 20 μmol / L, and then placed in a 37 ° C oven for 2 hours. Malachite green reagent was added and placed at room temperature for 30 minutes. After the color stabilized, the absorbance at 620 nm was detected using a BioTek enzyme reader. Three replicates were set for each experimental condition, and the IC values ​​of the compounds were fitted using GraphPad Prism 7.0 software. 50 value.

[0124] 4. Experimental results

[0125] The results are shown in Table 1. The compounds of the present invention have a significant inhibitory effect on the enzymatic activity of recombinant human CD73 protein.

[0126] Table 1 Inhibitory effect of the compounds of the present invention on the protease activity of recombinant human CD73

[0127] Note: The control group LY3475070 is the compound of Example 2 in the WO2019168744A1 patent.

[0128] Example 104: Evaluation of the Inhibition of CD73 Enzyme Activity in Human Breast Cancer MDA-MB-231 Cells by Compounds

[0129] 1. Experimental Purpose

[0130] The inhibitory effect of the compounds of the present invention on the conversion of AMP to adenosine by CD73 protein on the surface of human breast cancer MDA-MB-231 cells was evaluated.

[0131] 2. Experimental materials and instruments

[0132] Malachite green phosphate detection kit: BioAssay Systems; BioTek multi-function microplate reader: SynergyHTX.

[0133] 3. Experimental methods

[0134] When the cell density of MDA-MB-231 reached 90% and the cell viability reached above 95%, trypsin was added for digestion, the cell suspension was transferred to a 15 mL sterile centrifuge tube, centrifuged at 1000 rpm for 3 min, the culture medium was discarded, and the activity test solution (2 mmol / L MgCl2, 125 mmol / L NaCl, 1 mmol / L KCl, 10 mmol / L glucose, 10 mmol / L Hepes pH 7.2) was added to resuspend the cells, and then centrifuged at 1000 rpm for 3 min. The above operation was repeated three times. After cell counting, cells were diluted with activity test solution, and 20 μL (about 2500 cells) of cell suspension was added to each well of a 96-well plate. The compound was then added and pipetted evenly (the DMSO content was controlled at 0.5%). The plate was incubated in a 37°C oven for 10 minutes. The substrate AMP was added to a final concentration of 20 μmol / L. Malachite green reagent was then added to develop color at room temperature for 30 minutes. After the color stabilized, the absorbance at 620 nm was measured using a BioTek microplate reader. Three replicates were set for each experimental condition, and the IC values ​​of the compounds were fitted using GraphPad Prism 8.0 software. 50 value.

[0135] 4. Experimental results

[0136] The results are shown in Table 2. The compounds of the present invention have a significant inhibitory effect on the CD73 enzyme activity on the surface of MDA-MB-231 cells.

[0137] Table 2 Inhibitory activity of the compounds of the present invention on CD73 enzyme in human breast cancer MDA-MB-231 cells

[0138] Example 105: Pharmacokinetics in rats

[0139] 1. Experimental Purpose

[0140] The pharmacokinetic properties of compound 26 of the present invention were evaluated in rats.

[0141] 2. Experimental methods

[0142] Twelve female SD rats were randomly divided into two groups (n=6): Group 1 received a tail vein injection of 2 mg / kg, and Group 2 received an oral gavage of 10 mg / kg. Blood was collected from the retinal venous plexus at 0 min before dosing and at 0.033, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, 10, and 24 h after dosing in Group 1. Blood was collected from the retinal venous plexus at 0 min before dosing and at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, 10, and 24 h after dosing in Group 2. The blood samples were collected into 1.5 mL EP tubes containing 20 μL of heparin. All blood samples were centrifuged at 8000 rpm for 5 min, and the supernatant plasma was stored at -70°C until analysis.

[0143] 3. Experimental results

[0144] The results are shown in Table 3. Compound 26 of the present invention has good pharmacokinetic properties in SD rats, especially a long half-life, a high exposure, and good oral bioavailability.

[0145] Table 3 Pharmacokinetic results of the compounds of the present invention in rats

[0146] Example 106: Pharmacodynamic evaluation of the compounds of the present invention in mice

[0147] 1. Experimental Purpose

[0148] The in vivo efficacy of the compound of the present invention was evaluated in a triple-negative breast cancer 4T1 cell xenograft mouse model.

[0149] 2. Experimental methods

[0150] 4T1 cells were injected into the right mammary pad of 4-6 week old female BALB / c mice. 3The mice were randomly divided into six groups (n = 12 per group), namely the negative control group and the experimental group (10 mg / kg, 50 mg / kg). The body weight and tumor volume of each group of rats were recorded every two days. The volume calculation formula is: V = L × W 2 / 2 (L, longest dimension; W, shortest dimension). The animal experiment was terminated on the 28th day. The mice were weighed, blood was collected from their eyeballs, and the mice were euthanized. The tumor tissues were removed, weighed, and photographed. At the same time, part of the tissue was placed in 10% neutral fixative and sent for paraffin embedding, paraffin tissue sections were prepared, and H&E staining, TUNEL, and immunohistochemistry analysis were performed. The experimental procedures were referred to the instructions of the detection kit.

[0151] 3. Experimental results

[0152] The results are shown in Figures 1-3. Compared with the model group, compound 26 of the present invention inhibited the growth of triple-negative breast cancer xenografts in a dose-dependent manner. At doses of 10 mg / kg and 50 mg / kg, the tumor inhibition rate (TGI) reached 59.0% and 73.6%, respectively, without affecting the body weight of the mice. The results of tumor tissue sections showed that the compound had no obvious toxicity to the liver and kidneys of mice. The results of ELISA experiments showed that the compound could significantly increase the number of CD4 + Helper T cells, CD8 + It can also reduce the number of cytotoxic T cells, effector memory T cells and mature DCs, and reduce the proportion of regulatory T cells, thereby promoting the secretion of TNF-α and IFN-γ, and then stimulating anti-tumor immune response.

Claims

1. A pyridazine compound, characterized in that Having the structure of formula I, also including its stereoisomers, tautomers, solvate salts, crystals, pharmaceutically acceptable salts or mixtures thereof, Wherein: X is selected from CH2, NH, O, S, or X is absent; R1 is selected from hydrogen, C1-C6 alkyl or (CH2) n Ar; wherein n is an integer selected from 0-3, Ar is selected from a 3-8 membered cycloalkyl, a 6-12 membered aryl or a 5-12 membered aromatic heterocycle; the aromatic heterocycle contains 1-3 heteroatoms selected from N, O or S, and the alkyl, cycloalkyl, aryl or aromatic heterocycle is substituted with one or more R3; R3 is selected from hydrogen, halogen, nitro, amino, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, phenyl or C1-C6 haloalkyl; R2 is selected from hydroxy, amino, halogen, carboxyl, C(O)NR4R5, C1-C6 alkoxy, C3-C8 cycloalkyl, 4-6 membered heterocyclyl, 6-12 membered aryl or 4-12 membered aromatic heterocycle; the heterocycle or aromatic heterocycle contains 1-3 heteroatoms selected from N, O or S, and the carboxyl, alkoxy, aryl, heterocycle or aromatic heterocycle is substituted by one or more R6; R6 is selected from hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C1-C6 haloalkyl or C1-C6 haloalkoxy; R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or R4, R5 together with the N to which they are attached form a 4-7 membered heterocyclic group.

2. The pyridazine compound according to claim 1, characterized in that In the structure: X is selected from CH2, NH, O, S, or X is absent; R1 is selected from hydrogen, C1-C6 alkyl or (CH2) n Ar; wherein n is selected from an integer of 0-3, Ar is selected from a cyclopropyl group, a cyclohexyl group, a phenyl group, a pyridine ring, an indole ring, a morpholine ring, a benzothiophene ring or a thiophene ring; the alkyl group or the phenyl group is substituted by one or more R3 groups; R3 is selected from hydrogen, halogen, nitro, C1-C6 alkyl or C1-C6 alkoxy; R2 is selected from hydroxy, amino, halogen, CO2CH3, C(O)NR4R5, C1-C6 alkoxy, cyclopropyl, pyrrolidin-1-yl, phenyl or a five-membered aromatic heterocycle; the aromatic heterocycle contains 1-2 heteroatoms selected from N, O or S, and the alkoxy or aromatic heterocycle is substituted with 0-2 halogen, methyl or cyano groups; R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or R4, R5 together with the attached N form azetidine or pyrrolidin-1-yl.

3. The pyridazine compound according to claim 1, characterized in that In the structure: X is selected from NH, O, S, or X is absent; R1 is selected from methyl, ethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2,2-difluoropropyl, 2,2,3,3-tetrafluoropropyl, 2,2,3,3,3-pentafluoropropyl, 3,3,3-trifluoropropyl, cyclopropyl or (CH2) n Ar; wherein n is an integer selected from 2-3, Ar is selected from cyclohexane, phenyl, pyridine ring, indole ring, morpholine ring, benzothiophene ring or thiophene ring, and the phenyl is substituted with 0-2 halogen, nitro, methyl, methoxy or trifluoromethyl groups; R2 is selected from hydroxyl, amino, halogen, CO2CH3, C(O)NR4R5, methoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, cyclopropyl, pyrrolidin-1-yl, phenyl, and a five-membered aromatic heterocycle; the aromatic heterocycle is selected from thiophene, furan, pyrazole, methylpyrazole, or isoxazole, and the aromatic heterocycle is substituted with 0-2 methyl or cyano groups; R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, or R4, R5 together with the attached N form azetidine or pyrrolidin-1-yl.

4. The pyridazine compound according to claim 1, characterized in that In the structure: -X-R1 is selected from any of the following groups:

5. The pyridazine compound according to claim 1, characterized in that In the structure: Preferably, R2 is selected from any of the following groups:

6. The pyridazine compound according to claim 1, characterized in that A compound selected from any of the following:

7. The pyridazine compound according to claim 1, characterized in that The pharmaceutically acceptable salt is a salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid or ferulic acid.

8. A method for preparing the pyridazine compound according to claim 1, characterized in that: Choose from any of the following methods: Method 1: Using 3,6-dichloropyridazin-4-amine as the starting material, the compound of formula I is prepared by nucleophilic substitution, Sandmeyer reaction, nucleophilic substitution, Suzuki coupling, and hydrolysis reaction; Method 2: Using 3,4,6-trichloropyridazine as the raw material, the compound of formula I is prepared through substitution, acylation, coupling, and two hydrolysis reactions: Method 3: Using 3,4,6-trichloropyridazine as a raw material, the compound of formula I is prepared by nucleophilic substitution, nucleophilic substitution or coupling, coupling, and hydrolysis reaction; Method 4: Using 4,6-dichloropyridazine-3-carboxylic acid methyl ester as the raw material, the compound of formula I is prepared by nucleophilic substitution, aminolysis, coupling, and hydrolysis; Method 5: Using 3-amino-4-bromo-6-chloropyridazine as the starting material, the compound of formula I is prepared by substitution, acylation, coupling, hydrolysis, substitution, coupling, and hydrolysis; Wherein, R1, R2, and X are as defined in claim 1; the compound of formula I prepared by the above method is salified with a corresponding acid to obtain a pharmaceutically acceptable salt of the compound.

9. A pharmaceutical composition, characterized in that Comprising the pyridazines according to claim 1 and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical combination is in the form of tablets, capsules, powders, pills, granules, injections, oral solutions, syrups, inhalants, ointments, patches or suppositories.

11. Use of the pyridazine compound according to claim 1 or the pharmaceutical composition according to claim 9 in the preparation of a CD73 inhibitor drug.

12. Use of the pyridazine compound according to claim 1 or the pharmaceutical composition according to claim 9 in the preparation of an immunomodulatory drug.

13. The use according to claim 11 or 12, characterized in that The medicine is a medicine for preventing and / or treating tumors, infectious diseases, inflammatory diseases, organ transplant rejection or autoimmune diseases.

14. The use according to claim 13, characterized in that The tumors include cell carcinoma, melanoma, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of the head and neck, gastric cancer, glioblastoma, bladder cancer, pancreatic cancer, lymphoma, leukemia, prostate cancer, testicular cancer, kidney cancer, brain cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, thyroid cancer, liver cancer or esophageal cancer.

15. The use according to claim 13, characterized in that The inflammatory disease includes psoriasis or osteoarthritis.

16. The use according to claim 13, characterized in that The autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, myasthenia gravis, mixed connective tissue disease, psoriasis or autoimmune reactions caused by infection.

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