Pyrinidines, processes for their preparation, pharmaceutical compositions and uses thereof

Abstract

This invention discloses a pyrazine compound, its preparation method, pharmaceutical composition, and applications. The structure of this class of compounds is shown in Formula I, and also includes its stereoisomers, meso compounds, racemic compounds, prodrugs, crystals, pharmaceutically acceptable salts, or mixtures thereof. The compounds designed in this invention exhibit significant SHP2 protein inhibitory activity at the molecular level in vitro, can efficiently inhibit tumor cell proliferation at the cellular level, and can also significantly inhibit tumor growth in vivo. They can be widely used in the preparation of drugs for the prevention and / or treatment of SHP2-mediated or dependent diseases or conditions. Furthermore, the preparation method of the compounds is widely applicable, facilitating the preparation of compounds with various structural types.

Description

Technical Field

[0001] This invention relates to a pyrazine compound, its preparation method, pharmaceutical composition, and application, specifically to a pyrazine compound with SHP2 inhibitory activity, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] Protein tyrosine phosphatase 2 (SHP2), containing the Src homology 2 domain, belongs to the protein tyrosine phosphatase family. Encoded by the proto-oncogene protein tyrosine phosphatase non-receptor type 11 (PTPN11), it is widely expressed in the cytoplasm of various tissues and specifically catalyzes the dephosphorylation of phosphotyrosine in the cytoplasm. Studies have shown that protein tyrosine phosphatases and protein tyrosine kinases work together to maintain the balance of tyrosine protein phosphorylation, participating in cell signal transduction and regulating cell growth, differentiation, and metabolism. SHP2 is a crucial hub connecting multiple intracellular signaling pathways, including RAS / RAF / MEK / ERK, PI3K / AKT, JAK / STAT, and PD-1 / PD-L1 pathways, thereby influencing life processes such as cell proliferation, differentiation, migration, senescence, and apoptosis. SHP2 mutations and overactivation are closely associated with a variety of diseases, including Noonan syndrome, cheetah syndrome, leukemia, and various solid tumors such as lung cancer, colon cancer, neuroblastoma, glioblastoma, melanoma, liver cancer, pancreatic cancer, gastric cancer, ovarian cancer, prostate cancer, and breast cancer. SHP2 plays a crucial role in receptor tyrosine kinase (RTK)-driven oncogenesis and controls various aspects of tumorigenesis and development, including cell growth and proliferation, invasion and metastasis, the tumor microenvironment, and immune responses.

[0003] Studies have shown that SHP2 is not only a downstream signaling molecule of multiple immune checkpoint co-inhibitory receptors such as PD-1, CTLA-4, BTLA, and TIGIT, but also a key regulator of T cell receptor and CD28 receptor signaling. Abnormal SHP2 activation is associated with tumor immune escape. Furthermore, SHP2 affects TGF-β signaling, promoting cancer development by enhancing immunosuppression during epithelial-mesenchymal transition (EMT) and in the tumor microenvironment. Inhibiting SHP2 can improve the efficacy of immune checkpoint inhibitors by modulating the tumor microenvironment, enhancing immune surveillance, and promoting cancer cell clearance. SHP2 also regulates key signaling pathways involved in antigen-presenting cell (APC) activation and antigen presentation (such as PI3K-AKT and JAK-STAT). Inhibiting SHP2 can enhance the antigen-presenting capacity of APCs, thereby improving the ability of T cells to recognize tumor antigens.

[0004] Studies have shown that SHP2 overexpression or aberrant activation mediates drug resistance in various cancers. SHP2 can induce drug resistance in cancer cells by upregulating the AKT and ERK pathways downstream of multiple RTKs, including cytotoxic antitumor drugs, protein kinase inhibitors, and immune checkpoint inhibitors. SHP2 inhibitors, by blocking the activation of compensatory signaling pathways, hold promise for overcoming resistance to these drugs. SHP2 inhibitors have demonstrated significant antitumor activity in preclinical studies and exhibit synergistic antitumor effects when combined with other antitumor drugs; several SHP2 inhibitors are currently in clinical development.

[0005] Furthermore, SHP2 also plays a crucial role in the inflammatory response. SHP2 promotes the inflammatory response by stimulating IL-1 / tumor necrosis factor-stimulated NF-κB activation and IL-6 production. Inhibiting SHP2 activity can block the inflammatory response in an NF-κB-dependent manner. In addition, inhibiting PD-1 or the interaction between SHP2 and NF-κB pathway activators can block the release of NF-κB-dependent cytokines, preventing the inflammatory response. Recent studies have shown that SHP2 expression is significantly elevated in the synovium of osteoarthritis patients, mediating macrophage polarization. SHP2 inhibitors can attenuate the TLR pathway and its downstream signaling pathways, including the NF-κB and PI3K-AKT pathways, thereby inhibiting the release of inflammatory factors and mitigating the progression of osteoarthritis.

[0006] It is worth mentioning that although SHP2 has become a popular target for new drug development, no SHP2 inhibitor has been approved for marketing globally so far. This indicates that developing selective SHP2 inhibitors with good drug-like properties is extremely challenging. Therefore, it is still necessary to develop novel SHP2 inhibitors with high activity and good drug-like properties. Summary of the Invention

[0007] Objectives of the Invention: The first objective of this invention is to provide a pyrazine compound; 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 the pharmaceutical composition thereof.

[0008] Technical solution: The pyrazine compounds of this invention have the structure of Formula I, and further comprise their stereoisomers, meso compounds, racemic compounds, prodrugs, crystals, pharmaceutically acceptable salts, or mixtures thereof.

[0009]

[0010] in:

[0011] X is selected from carbonyl or sulfonyl groups;

[0012] R 1R 2 Each is independently selected from hydrogen, halogen, hydroxyl, amino, carboxyl, C1-C6 alkyl, C3-C6 cycloalkyl, or R 1 R 2 The carbon atoms attached to them together form 4-8 membered rings; the hydroxyl, amino, carboxyl, alkyl, and cycloalkyl groups are optionally substituted by one or more Z groups; the 4-8 membered rings are optionally fused with 5-7 membered rings or not fused with any ring system; the rings optionally contain 0-4 heteroatoms selected from N, O, or S, and are optionally substituted by one or more W groups.

[0013] W is selected from hydrogen, halogen, hydroxyl, amino, carboxyl, C1-C6 alkyl, C1-C6 alkoxy or C3-C6 cycloalkyl;

[0014] R 3 Selected from hydrogen, amino, cyano, halogen, or C1-C6 alkyl;

[0015] R 4 The ring is selected from amino, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylamino, C2-C8 alkenyl, C3-C8 cycloalkyl or 5-7 membered ring; wherein the amino, alkyl, alkoxy, alkylamino, alkenyl or cycloalkyl are optionally substituted by one or more Y groups; wherein the ring optionally contains 0-4 heteroatoms selected from N, O or S, and is optionally substituted by one or more Y groups;

[0016] Y is selected from hydrogen, halogen, hydroxyl, carboxyl, amino, sulfonyl, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, or a 5-7 membered ring; the hydroxyl, carboxyl, amino, sulfonyl, alkyl, or alkylamino groups are optionally substituted by one or more Z groups; the ring optionally contains 0-4 heteroatoms selected from N, O, or S, and is optionally substituted by one or more Z groups;

[0017] Z is selected from hydrogen, hydroxyl, amino, halogen, C1-C6 alkyl, C1-C6 alkoxy or 5-7 membered ring; the ring optionally contains 0-4 heteroatoms selected from N, O or S.

[0018] Preferably, in the structure:

[0019] R 1 R 2 Independently selected from hydrogen, methyl, amino, or R 1 R 2 Piperidine rings connected together form

[0020] R 3 Selected from hydrogen or amino;

[0021] R 4 The group is selected from amino, C1-C4 alkylamine, C2-C8 alkenyl, C3-C8 cycloalkyl or 5-7 membered ring; the amino, alkylamine, alkenyl or cycloalkyl group is optionally substituted by one or more Y groups; the ring optionally contains 0-2 heteroatoms selected from N, O or S, and the ring is optionally substituted by one or more Y groups;

[0022] Y is selected from halogen, hydroxyl, carboxyl, CONH2, amino, sulfonyl, nitro, cyano, C1-C4 alkyl, C1-C4 alkoxy, or 5-7 membered ring; the hydroxyl, carboxyl, amino, sulfonyl, alkyl, or alkoxy group is optionally substituted by one or more Z groups; the ring optionally contains 0-2 heteroatoms selected from N, O, or S;

[0023] Z is selected from hydroxyl, oxo, amino, halogen, C1-C6 alkyl, C1-C6 alkoxy or 5-7 membered ring; the ring optionally contains 0-2 heteroatoms selected from N, O or S.

[0024] Preferably, in the structure, R 4 Selected from:

[0025]

[0026] Preferably, the pyrazine compounds of the present invention are selected from any of the following compounds:

[0027]

[0028]

[0029]

[0030] Preferably, the pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following:

[0031] 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, ferulic acid.

[0032] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or 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 carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic 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 (e.g., arginine) and glucuronic acid. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating 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 solubilities in polar solvents.

[0033] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0034] Preferably, the stereoisomer is R 1 R 2 R 4 Isomers introduced by chiral C and N, or with R 1 R 2 Isomers introduced by the chiral C of the connection.

[0035] Preferably, the prodrug is R 4 The esters and amides introduced by the carboxyl, hydroxyl, and amino groups in the drug are more preferably C1-C4 alkyl esters, C1-C4 carboxylic esters, or C1-C4 alkylamides.

[0036] The method for preparing the pyrazine compounds of the present invention is selected from any of the following methods:

[0037] Method 1: Compound a is prepared into compound I by nucleophilic substitution, hydrolysis, coupling, nucleophilic substitution, condensation, and deBoc.

[0038]

[0039] Method 2: Compound d is synthesized by condensation and deBoc to obtain compound I;

[0040]

[0041] Method 3: Compound e is synthesized into compound I by condensation, nucleophilic substitution, and deBoc.

[0042]

[0043] Method 4: Compound d is synthesized into compound I by condensation, two-step nucleophilic substitution, and deBoc.

[0044]

[0045] Among them, R 1 R 2 R 3 R 4 The definition of X is as described above;

[0046] The compound of formula I obtained by the above method is salted with the corresponding acid to obtain a pharmaceutically acceptable salt of the compound.

[0047] The pharmaceutical composition of the present invention contains the pyrazine compound of the present invention and a pharmaceutically acceptable carrier.

[0048] Preferably, the formulation of the drug combination is tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalers, ointments, patches, or suppositories.

[0049] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0050] The pharmaceutical compositions described in this invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0051] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, 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 preparations. Examples of parenteral formulations 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 tablets.

[0052] The pyrazine compounds or pharmaceutical compositions thereof described in this invention are used in the preparation of SHP2 inhibitor drugs.

[0053] The pyrazine compounds or pharmaceutical compositions thereof described in this invention are used in the preparation of medicaments for SHP2-mediated or dependent diseases or conditions.

[0054] Preferably, the drug is a drug for the prevention and / or treatment of tumors, inflammatory diseases, or autoimmune diseases.

[0055] Further preferred, the tumors include, but are not limited to, Noonan syndrome, leopard syndrome, juvenile myeloid monocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of the head and neck, gastric cancer, anaplastic large cell lymphoma, glioblastoma, etc.

[0056] Further preferably, the inflammatory diseases include, but are not limited to, psoriasis, osteoarthritis, etc.

[0057] Further preferred, 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.

[0058] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0059] The compound designed in this invention exhibits significant SHP2 protein inhibitory activity (inhibition of IC50) at the molecular level in vitro. 50 All values ​​reached nanomolar concentration levels, with the optimal value reaching picomolar concentration levels. At the cellular level, it can effectively inhibit the proliferation of tumor cells (inhibiting IC50). 50 With a concentration below 20 nM, it can significantly inhibit tumor growth in vivo (tumor inhibition rate exceeding 60%), and can be widely used in the preparation of drugs for the prevention and / or treatment of SHP2-mediated or dependent diseases or conditions. Furthermore, the compound's preparation method is highly adaptable, facilitating the preparation of compounds with various structural types. Attached Figure Description

[0060] Figure 1 The therapeutic effect of compound 24 on a mouse model of human myeloid monocytic leukemia MV-4-11 cell xenografts; A: tumor volume, B: tumor weight, C: mouse body weight;

[0061] Figure 2 Immunohistochemical staining and flow cytometry experiments of tumor tissue after treatment of MV-4-11 heterotopic xenograft mice with compound 24; AB: H&E and Ki67 immunohistochemical staining experiments, CD: flow cytometry experiments, E: ELI SA experiments, FG: immunofluorescence staining experiments. Detailed Implementation

[0062] The technical solution of the present invention will be further described below with reference to the embodiments.

[0063] Example 1: Preparation of compound N-(3-((5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)pyridinecarboxamide (1)

[0064]

[0065] Step (i): Synthesis of 3-(tert-butylthio)-2-chloroaniline (b)

[0066] 2-Chloro-3-fluoroaniline (5.00 g, 34.35 mmol), tert-butylthiol (10.84 g, 0.12 mol), and cesium carbonate (22.38 g, 68.70 mmol) were added to N,N-dimethylformamide (15 mL), and the mixture was sealed and reacted at 120 °C for 18 h. The mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 7.05 g of a yellow oily liquid (95% yield). MS (ESI) m / z 216 [M+H] + ; 1 H NMR (300MHz, Chloroform-d) δ (p pm) 7.05 (t, J = 7.5 Hz, 1H), 6.53 (dd, J = 7.5, 2.4 Hz, 1H), 6.13 (dd, J = 7.5, 2.4 Hz, 1H), 1.61 (s, 9H).

[0067] Step (ii): Synthesis of 3-amino-2-chlorobenzylthiol (c)

[0068] Add b (7.05 g, 32.68 mmol) to concentrated hydrochloric acid (25 mL) and react at 80 °C for 24 h under a nitrogen atmosphere. Cool to room temperature, filter, and dry to give 5.10 g of white solid, yield 80%. MS (ESI) m / z 160 [M+H] + ; 1 H NMR (300MHz, Chloroform-d) δ (ppm) 7.30 (t, J = 7.2 Hz, 1H), 7.06 (dd, J = 7.2, 1.2 Hz, 1H), 6.71 (dd, J = 7.2, 1.2 Hz, 1H).

[0069] Step (iii): Synthesis of 2-chloro-3-((5-chloropyrazin-2-yl)thio)aniline (d)

[0070] Add c (3.50 g, 17.85 mmol) to 1,4-dioxane (15 mL), then add N,N-diisopropylethylamine (15.55 mL, 89.21 mmol), 2-bromo-5-chloropyrazine (3.45 g, 17.84 mmol), Xantphos (0.21 g, 0.36 mmol), and Pd2(dba)3 (0.16 g, 0.18 mmol). React at 95 °C for 4 h under a nitrogen atmosphere. Cool to room temperature, extract with ethyl acetate, and purify by column chromatography to give 2.6 g of a yellow solid, yield 54%. MS (ESI) m / z 272 [M+H] + ; 1 HNMR (300MHz, Chloroform-d) δ (ppm) 8.10 (s, 1H), 7.85 (s, 1H), 7.62 (dd, J = 7.2, 1.2H z, 1H), 7.23 (t, J = 7.2Hz, 1H), 6.57 (dd, J = 6.9, 1.2Hz, 1H).

[0071] Step (iv): Synthesis of tert-butyl (1-(5-((3-amino-2-chlorophenyl)thio)pyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate (e)

[0072] Dissolve d (695 mg, 2.55 mmol) in DMSO (4 mL), add tert-butyl (4-methylpiperidin-4-yl)carbamate (0.82 mg, 3.83 mmol) and N,N-diisopropylethylamine (0.99 g, 7.76 mmol), and react at 100 °C for 3 h under a nitrogen atmosphere. Extract with ethyl acetate, combine the organic phases, wash with saturated brine, and purify by column chromatography to give 1.0 g of yellow solid, yield 86%. MS (ESI) m / z 450 [M+H] + ; 1 H NMR(300MHz,Chlorofor md)δ(ppm)8.21(s,1H),7.89(s,1H),7.57(dd,J=7.1,1.2Hz,1H),7.33(t,J=7.2Hz,1H),6.53(dd ,J=7.2,1.2Hz,1H)4.03(s,1H),3.62-3.56(m,4H),2.33-2.25(m,4H),1.56(s,9H),1.23(s,3H).

[0073] Step (v): Synthesis of tert-butyl 1-(5-((2-chloro-3-(pyridinino)phenyl)thio)pyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate (f)

[0074] Add e (140 mg, 0.31 mmol) to 3 mL of acetonitrile, then add N-methylimidazolium (76 mg, 0.93 mmol), 2-pyridinecarboxylic acid (115 mg, 0.93 mmol), and N,N,N',N'-tetramethylchloroformamidin hexafluorophosphate (245 mg, 0.93 mmol). React at room temperature for 2 h under a nitrogen atmosphere. Extract with ethyl acetate, wash with 1 M HCl, and purify by column chromatography to give 148 mg of a pale yellow solid, yield 86%. MS (ESI) m / z 555 [M+H] + ; 1 H NMR(300MHz,Chloroform-d)δ(ppm)10.82(s,1H),8.70(dd,J=8.4,1.5Hz,1H),8.54(dd,J=8.4,1.5Hz,1H),8.42(s,1H),8.31(s,1H),8.19(t,J=7 .5Hz,1H),7.49-7.46(m,2H),7.45-7.41(m,2H),4.10(s,1H),4.46-4.41 (m,2H),4.33-4.27(m,2H),2,41-2.35(m,4H),2.01(s,9H),1.46(s,3H).

[0075] Step (vi): Synthesis of N-(3-((5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)pyridinecarboxamide (1)

[0076] 85 mg (0.15 mmol) and concentrated hydrochloric acid (0.2 mL) were added to dichloromethane (2 mL), and the reaction was carried out at room temperature for 2 h. The organic phase was washed with water, and the pH of the aqueous phase was adjusted to 7-8 with saturated sodium bicarbonate solution. The mixture was filtered and dried to give 56 mg of a white solid, with a yield of 80%. MS (ESI) m / z 455 [M+H] + ; 1 H NMR(300MHz,DMSO-d6)δ(ppm)10.70(s,1H),8.77(d,J=4.8Hz,1H),8.47(s,1H),8.29(s,1H),8.20(d,J=8.1Hz,2H),8.11(d,J=8.1Hz,1H ),7.77-7.71(m,1H),7.32(t,J=8.1Hz,1H),6.74(d,J=8.1Hz,1H),4.15-4.00(m,2H),3.52-3.46(m,2H),1.77-1.71(m,4H),1.38(s,3H).

[0077] The following compounds were prepared using a procedure similar to that in Example 1:

[0078]

[0079]

[0080]

[0081] Example 22: Preparation of compound (S)-N-(3-(((5-(1-amino-1,3-dihydrospirocyclic [indene-2,4'-piperidin]-1'-yl)pyrazin-2-yl)thio)-2-chlorophenyl)pyridineamide (22)

[0082]

[0083] Step (i): Synthesis of N-(2-chloro-3-((5-chloropyrazin-2-yl)thio)phenyl)pyridine amide (b)

[0084] Add 100 mg (0.37 mmol) of a to 2 mL of acetonitrile, then add N-methylimidazolium (121 mg, 1.47 mmol), 2-pyridinecarboxylic acid (136 mg, 1.10 mmol), and N,N,N',N'-tetramethylchloromethanesulfonyl hexafluorophosphate (309.30 mg, 1.10 μmol). React at room temperature for 2 h under a nitrogen atmosphere. Extract with ethyl acetate, wash the organic phase with 1 M HCl, and purify by column chromatography to give 106 mg of a pale yellow solid, yield 69%. MS (ESI) m / z 377 [M+H] + ; 1 H NMR(300MHz,Chloroform-d)δ(ppm)9.06(s,1H),8.62(dd,J=4.8,1.5Hz,1H),8.21(s,1H),7.98-7.85(m,4H),7.66(dd,J=6.9,1.5Hz,1H),7.50-7.42(m,1H).

[0085] Step (ii): Synthesis of (S)-N-(3-(((5-(1-amino-1,3-dihydrospirocyclic [indene-2,4'-piperidin]-1'-yl)pyrazin-2-yl)thio)-2-chlorophenyl)pyridine amide (22)

[0086] C (60 mg, 0.16 mmol) was added to 2 mL of N,N-dimethylacetamide, followed by K₂CO₃ (137 mg, 0.99 mmol) and (S)-1,3-dihydrospiro[indene-2,4'-piperidine]-1-amine (40 mg, 0.20 mmol). The reaction was carried out at 90 °C for 4 h under a nitrogen atmosphere. 5 mL of water was added, the mixture was filtered, and dried to give 42 mg of a pale yellow solid, yielding 51%. MS (ESI) m / z 543 [M+H]+ ; 1 H NMR (300MHz, DMSO-d6) δ (ppm) 9.43 (s, 1H), 8.83 (d, J = 5.7Hz, 1H), 8.15 (s, 1H), 8.01 (t ,J=5.7Hz,1H),7.86(dd,J=8.1,1.2Hz,1H),7.74(s,1H),7.60(d,J=7.2Hz,1H),7.46-7 .38(m,3H),7.38-7.27(m,3H),7.16(d,J=7.5,1H),5.19(d,J=7.5Hz,1H),5.13(d,J=7. 5,1H),4.29-4.23(m,1H),3.64-3.59(m,4H),2.76(d,J=1.2Hz,2H),1.97-1.94(m,4H).

[0087] The following compounds were prepared using a similar procedure to that in Example 22:

[0088]

[0089]

[0090] Example 38: Preparation of compound tert-butyl(S)-(tert-butoxycarbonyl)(6-(1-((tert-butoxycarbonyl)amino)-1,3-dihydrospiro[indene-2,4'-piperidin]-1'-yl)-3-((2-chloro-3-(4-hydroxynicotinamide)phenyl)thio)pyrazin-2-yl)carbamate (38)

[0091]

[0092] Step (i): Synthesis of (Z)-2-(3-(3-amino-2-chlorophenyl)thio)-6-chloropyrazin-2-yl)diazeline-1-carboxylic acid tert-butyl ester (b)

[0093] Add α (109 mg, 0.68 mmol) to 1,4-dioxane (3 mL), then add N,N-diisopropylethylamine (241 mg, 1.87 mmol), (Z)-2-(3-bromo-6-chloropyrazin-2-yl)diazepine-1-carboxylic acid tert-butyl ester (200 mg, 0.62 mmol), Xantphos (36 mg, 0.62 mmol), and Pd2(dba)3 (28 mg, 0.31 mmol). React at 95 °C for 4 h under a nitrogen atmosphere. Extract with ethyl acetate and purify by column chromatography to give 140 mg of a yellow solid, yield 56%. MS (ESI) m / z 487 [M+H] + ; 1HNMR(300MHz,Chloroform-d)δ8.02(s,1H),7.67(dd,J=7.5,1.5Hz,1H),7.31(t,J=7.5Hz,1H),6.88(dd,J=7.5,1.5Hz,1H),1.26(s,18H).

[0094] Step (ii): Synthesis of (S, Z)-2-(3-((3-amino-2-chlorophenyl)thio)-6-(1-(((tert-butoxycarbonyl)amino)-1,3-dihydrospiro[indene-2,4'-piperidin]-1'-yl)pyrazin-2-yl)diazeline-1-carboxylic acid tert-butyl ester (c)

[0095] Dissolve b (155 mg, 0.32 mmol) in DMSO (3 mL), add (S)-(1,3-dihydrospiro[indene-2,4'-piperidin]-1-yl)tert-butyl carbamate (80 mg, 0.79 mmol) and N,N-diisopropylethylamine (103 g, 0.26 mmol), and react at 100 °C for 3 h under a nitrogen atmosphere. Extract with ethyl acetate, wash with saturated brine, and purify by column chromatography to give 110 mg of a yellow solid, yield 55%. MS (ESI) m / z 753 [M+H] + ; 1 H NMR(300MHz,Chloroform-d)δ(ppm) 1 HNMR(500MHz,Chloroform-d)δ7.91(s,1H),7.88(dd,J=7.2,1.2Hz,1H),7.54-7.49(m,1H),7.36(d,J=7.2Hz,1H),7.33-7.20(m,4H),6.68(dd,J=7.2, 1.2Hz,1H),4.62(s,1H),3.72(dd,J=9.0,6.6Hz,2H),3.64(dd,J=9.0,6.6H z,2H),2.73(d,J=1.2Hz,2H),2.03-1.93(m,4H),,1.50(s,9H),1.45(s,9H).

[0096] Step (iii): Synthesis of (S, Z)-2-(6-(1-((tert-butoxycarbonyl)amino)-1,3-dihydrospiro[indene-2,4'-piperidin]-1'-yl)-3-((2-chloro-3-(4-hydroxynicotinamide)phenyl)thio)pyrazin-2-yl)diazepine-1-carboxylic acid tert-butyl ester (d)

[0097] Add c (100 mg, 0.13 mmol) to 2 mL of acetonitrile, then add N-methylimidazolium (33 mg, 0.40 mmol), 4-hydroxynicotinic acid (37 mg, 0.27 mmol), and N,N,N',N'-tetramethylchloromethanemidane hexafluorophosphate (104 mg, 0.40 mmol). React at room temperature for 2 h under a nitrogen atmosphere. Extract with ethyl acetate, wash the organic phase with 1 M HCl to give 85 mg of a pale yellow solid, yield 73%. MS (ESI) m / z 874 [M+H] + ; 1 H NMR(300MHz,Chloroform-d)δ(ppm) 1 H NMR(300MHz,Chloroform-d)δ8.92(d,J=1.2Hz,1H),8.36(dd,J=5.7,1.2Hz,1H),7.83(dd,J=8.1,1.2Hz, 1H),7.74(s,1H),7.69(dd,J=7.2,1.2Hz,1H),7.54-7.48(m,1H),7.37-7.31(m,2H),7.24-7.29(m,1H),7 .13-7.06(m,2H),6.94(d,J=5.7Hz,1H),5.31(d,J=8.7Hz,1H),4.75(d,J=4.5Hz,1H),3.78-3.69(m,4H), 2.73(d,J=1.2Hz,2H),1.98(dd,J=9.0,6.3Hz,2H),1.90(dd,J=9.0,6.3Hz,2H),1.56(s,9H),1.47(s,9H).

[0098] Step (iv): Synthesis of tert-butyl(S)-(tert-butoxycarbonyl)(6-(1-((tert-butoxycarbonyl)amino)-1,3-dihydrospiro[indene-2,4'-piperidin]-1'-yl)-3-((2-chloro-3-(4-hydroxynicotinamide)phenyl)thio)pyrazin-2-yl)carbamate (38)

[0099] d (80 mg, 0.10 mmol) and concentrated hydrochloric acid (0.2 mL) were added to dichloromethane (2 mL), and the reaction was carried out at room temperature for 2 h. The organic phase was washed with water, and the pH of the aqueous phase was adjusted to 7-8 with saturated sodium bicarbonate solution. The mixture was filtered and dried to give 56 mg of a white solid, with a yield of 80%. MS (ESI) m / z 574 [M+H] + ; 1H NMR (300MHz, DMSO-d6) δ (ppm) 9.44 (s, 1H), 8.93 (d, J = 1.2Hz, 1H), 8.36 (dd, J = 5.7, 1.2Hz, 1H), 7.85 ( dd,J=8.1,1.2Hz,1H),7.68(dd,J=7.2,1.2Hz,1H),7.60(s,1H),7.42(dt,J=7.2,1.2Hz,1H),7.38-7. 30(m,3H),7.19(dd,J=6.6,2.7Hz,1H),6.95(d,J=5.7Hz,1H),6.83(d,J=9.3Hz,1H),6.16(d,J=9.3Hz ,1H),5.21-5.13(m,2H),4.29-4.23(m,1H),3.77-3.69(m,4H),2.83-2.76(m,2H),1.56-1.49(m,4H).

[0100] The following compounds were prepared using a similar procedure to that in Example 38:

[0101]

[0102] Example 42: Preparation of compound N-(3-((5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)morpholine-4-carboxamide (42)

[0103]

[0104] Step (i): Synthesis of tert-butyl carbamate (b)

[0105] Add α (100 mg, 0.22 mmol), triethylamine (67 mg, 0.67 mmol), and phenyl chloroformate (69 mg, 0.44 mmol) to 2 mL of tetrahydrofuran and react at room temperature for 2 h. Extract with ethyl acetate and purify by column chromatography to give 105 mg of white solid, yield 83%. MS (ESI) m / z 570 [M+H] + ; 1HNMR(300MHz,Chloroform-d)δ(ppm)8.97(s,1H),8.07(s,1H),7.89(dd,J=7.8,4.2Hz,1H),7.67(s,1H),7.67(t,J=7.8,1H),7. 06(dd,J=7.8,4.2Hz,1H),6.88-6.81(m,4H),3.98-3.93(m,2H),3.72-3.67(m,2H),2.12-2.06(m,4H),1.46(s,9H),1.34(s,3H).

[0106] Step (ii): Synthesis of (1-(5-((2-chloro-3-(morpholino-4-carbamate)phenyl)thio)pyrazin-2-yl)-4-methylpiperidin-4-yl)tert-butyl carbamate (c)

[0107] 100 mg (0.18 mmol) of triethylamine (35 mg, 0.35 mmol) and morpholine (45 mg, 0.53 mmol) were added to 2 mL of tetrahydrofuran and reacted at room temperature for 2 h. The mixture was extracted with ethyl acetate and purified by column chromatography to give 75 mg of a white solid, with a yield of 74%. MS (ESI) m / z 563 [M+H] + ; 1 H NMR (300MHz, Chloroform-d) δ (ppm) 9.18 (s, 1H), 8.87 (s, 1H), 7.91-7.85 (m, 2H), 7.46 (d, J = 8.1Hz, 1H), 6.77 (t, J = 5.7Hz, 1H ),3.92-3.86(m,4H),3.59-3.54(m,2H),2.87-2.82(m,4H),2.46-2.40(m,2H),1.89-1.82(m,4H),1.42(s,9H),1.17(s,3H).

[0108] Step (iii): Synthesis of N-(3-((5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)morpholine-4-carboxamide (42)

[0109] c (70 mg, 0.12 mmol) and concentrated hydrochloric acid (0.2 mL) were added to dichloromethane (2 mL), and the reaction was carried out at room temperature for 2 h. The organic phase was washed with water, and the pH of the aqueous phase was adjusted to 7-8 with saturated sodium bicarbonate solution. The mixture was filtered and dried to give 45 mg of a white solid, with a yield of 79%. MS (ESI) m / z 463 [M+H] + ; 1H NMR (300MHz, DMSO-d6) δ (ppm) 9.38 (s, 1H), 8.93 (s, 1H), 8.61-8.55 (m, 2H), 8.06 (d, J = 7.8Hz, 1H), 7.67 (t, J = 5.4Hz ,1H),3.83-3.78(m,4H),3.63-3.56(m,2H),2.93-2.86(m,4H),2.39-2.30(m,2H),1.96-1.87(m,4H),1.12(s,3H).

[0110] The following compounds were prepared using a similar procedure to that in Example 42:

[0111]

[0112]

[0113] Example 63: Preparation of compound N-(3-((5-((S)-1-amino-1,3-dihydrospirocyclic[indene-2,4'-piperidine]-1'-yl)pyrazin-2-yl)thio)-2-chlorophenyl)-3-hydroxypiperidine-1-carboxamide (63)

[0114]

[0115] Step (i): Synthesis of N-(2-chloro-3-((5-chloropyrazin-2-yl)thio)phenyl)-3-hydroxypiperidine-1-carboxamide (b)

[0116] Add α (100 mg, 0.25 mmol), triethylamine (77 mg, 0.76 mmol), and 3-hydroxypiperidine (51 mg, 0.51 mmol) to 2 mL of tetrahydrofuran and react at room temperature for 2 h. Extract with ethyl acetate and purify by column chromatography to give 103 mg of white solid, yield 88%. MS (ESI) m / z 399 [M+H] + ; 1 HNMR (300MHz, Chloroform-d) δ (ppm) 8.70 (s, 1H), 8.38 (s, 1H), 7.89-7.80 (m, 2H), 7.75 (d, J = 6.9Hz, 1H), 7.22 (s, 1H), 6. 38(dd,J=8.1,2.7Hz,1H),4.07-3.95(m,2H),3.72-3.69(m,1H),3.50-3.39(m,2H),1.90-1.74(m,2H),1.74-1.62(m,2H).

[0117] Step (ii): Synthesis of N-(3-((5-((S)-1-amino-1,3-dihydrospirocyclic[indene-2,4'-piperidine]-1'-yl)pyrazin-2-yl)thio)-2-chlorophenyl)-3-hydroxypiperidine-1-carboxamide (63)

[0118] β (55 mg, 0.14 mmol), K₂CO₃ (120 mg, 0.87 mmol), and (S)-1,3-dihydrospiro[indene-2,4'-piperidine]-1-amine (35 mg, 0.17 mmol) were added to 2 mL of N,N-dimethylacetamide and reacted at 90 °C for 4 h under a nitrogen atmosphere. 5 mL of water was added, the mixture was filtered, and dried to give 55 mg of a white solid, yield 56%. MS (ESI) m / z 565 [M+H] + ; 1 H NMR (300MHz, DMSO-d6) δ (ppm) 8.45 (s, 1H), 8.23 ​​(d, J = 1.5Hz, 1H), 8.11 (s, 1H), 7.32(dt,J=7.2,1.5Hz,3H),7.16-7.08(m,4H),6.64-6.59(m,1H),4.26-2.17( m,4H),3.86(d,J=4.8Hz,2H),3.21(d,J=12.3Hz,2H),3.08(d,J=4.8Hz,2H),2. 09-2.00(m,4H),1.69(d,J=12.3Hz,2H),1.50-1.45(m,2H),1.18-1.13(m,2H).

[0119] The following compounds were prepared using a similar procedure to that in Example 63:

[0120]

[0121]

[0122] Example 71: Preparation of compound N-(3-((5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)benzenesulfonamide (71)

[0123]

[0124] Step (i): Synthesis of 1-(5-((2-chloro-3-(phenylsulfonamido)phenyl)thio)pyrazin-2-yl)-4-methylpiperidin-4-yl)tert-butyl carbamate (b)

[0125] Add α (100 mg, 0.22 mmol) and 4-fluorobenzenesulfonyl chloride (47 mg, 0.27 mmol) to 2 mL of pyridine and react at room temperature for 2 h. Extract with ethyl acetate and purify by column chromatography to give 110 mg of white solid, yield 84%. MS (ESI) m / z 590 [M+H] + ; 1 HNMR(300MHz,Chloroform-d)δ(ppm)9.05(s,1H),8.75(s,1H),8.25(s,1H),7.92-7.86(dd,J=7.8,1.5Hz 1H),7.19(t,J=7.8Hz,1H),7.47-7.35(m,5H),7.19(dd,J=7.8,1.5Hz,1H),3.6 1-3.54(m,2H),3.46-3.40(m,2H),2.08-2.03(m,4H),1.32(s,9H),1.19(s,3H).

[0126] Step (ii): Synthesis of N-(3-((5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)benzenesulfonamide (71)

[0127] Add b (100 mg, 0.17 mmol) and concentrated hydrochloric acid (0.2 mL) to dichloromethane (2 mL) and react at room temperature for 2 h. Wash the organic phase with water, adjust the pH of the aqueous phase to 7-8 with saturated sodium bicarbonate solution, filter, and dry to give 72 mg of white solid, yield 87%. MS (ESI) m / z 490 [M+H] + ; 1 HNMR(300MHz,DMSO-d6)δ(ppm)9.11(s,1H),8.85(s,1H),8.35(s,1H),8.02(dd,J=7.8,1.5Hz 1H),7.89(t,J=7.8Hz,1H),7.67-7.59(m,5H),7.45(dd,J=7.8,1.5Hz,1H ),3.98-3.91(m,2H),3.66-3.59(m,2H),2.23-2.15(m,4H),1.21(s,3H).

[0128] The following compounds were prepared using a similar procedure to that in Example 71:

[0129]

[0130]

[0131] Example 82: Preparation of compound (S)-N-(3-((5-(1-amino-1,3-dihydrospirocyclic[indene-2,4'-piperidine]-1'-yl)pyrazin-2-yl)thio)-2-chlorophenyl)-4-fluorobenzenesulfonamide (82)

[0132]

[0133] Step (i): Synthesis of N-(3-((5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)benzenesulfonamide (b)

[0134] Add α (100 mg, 0.37 mmol) and 4-fluorobenzenesulfonyl chloride (85 mg, 0.44 mmol) to pyridine (2 mL) and react at room temperature for 2 h. Extract with ethyl acetate and purify by column chromatography to give 120 mg of white solid, yield 76%. MS (ESI) m / z 429 [M+H] + ; 1 HNMR(300MHz,Chloroform-d)δ(ppm).8.73(s,1H),8.27(s,1H),7.93-7.82(m,3H),7.65(dd,J =6.9,1.2Hz,1H),7.43(dd,J=7.5,6.9Hz,1H),7.24-7.16(m,2H),7.02(dd,J=7.5,1.2Hz,1H).

[0135] Step (ii): Synthesis of (S)-N-(3-((5-(1-amino-1,3-dihydrospirocyclic [indene-2,4'-piperidin]-1'-yl)pyrazin-2-yl)thio)-2-chlorophenyl)-4-fluorobenzenesulfonamide (82)

[0136] Add 89 mg (0.21 mmol) of b, 120 mg (0.87 mmol) of K₂CO₃, and (S)-1,3-dihydrospiro[indene-2,4'-piperidine]-1-amine (35 mg, 0.17 mmol) to 2 mL of N,N-dimethylacetamide, and react at 90 °C for 4 h under a nitrogen atmosphere. Add 5 mL of water, filter, and dry to give 65 mg of white solid, yield 63%. MS (ESI) m / z 596 [M+H] + ; 1H NMR(300MHz,DMSO-d6)δ(ppm)8.90(s,1H),8.25(s,1H),7.91-7.83(m,2H),7.70 (s,1H),7.57(dd,J=6.9,1.2Hz,1H),7.31-7.26(m,3H),7.02(d,J=7.5,1.2Hz,1 H),7.30-7.24(m,4H),4.59(dd,J=7.5,6.3Hz,1H),4.23(dd,J=7.5,6.3Hz,1H), 4.35-4.07(m,1H),3.74-3.66(m,4H),2.76(d,J=1.2Hz,2H),1.87-1.79(m,2H).

[0137] The following compounds were prepared using a similar procedure to that in Example 82:

[0138]

[0139] Example 86: In vitro bioactivity assay of compounds

[0140] 1. SHP2 Inhibition Experiment

[0141] 1) Experimental Principle

[0142] SHP2 is allosterically activated by binding to its SH2 domain via a diphosphorylated tyrosine (pTyr) peptide. This activation leads to a self-inhibitory conformational change in the SHP2 protein, thereby separating the N-SH2 domain from the PTP domain and exposing the catalytic site. The exposed catalytic site can recognize the substrate and catalyze its dephosphorylation. The catalytic activity of SHP2 can be monitored using a rapid fluorescence assay by observing the degree of dephosphorylation of the alternative substrate DiFMUP.

[0143] 2) Experimental instruments and reagents

[0144] 384-well polystyrene NBS microplates (Corning), multi-functional microplate reader (Tecan), DiFMUP (Invitrogen), IRS1-pY1172 (Qiangyao Biotechnology), HEPES (Sigma-Aldrich), DMSO (Sigma-Aldrich), DTT (Shanghai Maclean Biotechnology Co., Ltd.), P-20 (Nanjing Chemical Reagent Co., Ltd.), EDTA (Sinopharm Chemical Reagent Co., Ltd.).

[0145] 3) Experimental steps

[0146] a) Preparation of reaction solution: The phosphatase reaction was carried out at 37°C on a 384-well black polystyrene plate with a flat bottom, low flange, and non-bound surface. The final reaction volume was 100 μL. The analytical buffer conditions were 60 mM HEPES (pH = 7.2), 75 nM NaCl, 75 nM KCl, 1 nM EDTA, 0.05% P-20, and 5 mM DTT.

[0147] b) Preparation of test compound concentrations: The test compound was prepared into a series of concentration gradient solutions using 100% DMSO. The final test concentrations of the compound were 10 μM, 3.33 μM, 1.111 μM, 0.370 μM, 0.123 μM, 0.041 μM, 0.014 μM, 0.0046 μM, and 0.0015 μM.

[0148] c) Experimental grouping: The experiment was set up with the largest group (SHP2+IRS1+DiFMUP), the smallest group (SHP2+DiFMUP), the test group (SHP2+IRS1+DiFMUP+test compound), and the control group (SHP2+DiFMUP+test compound). The positive control drug was TNO155.

[0149] d) Enzymatic reaction: The test compound was incubated with 0.5 nM SHP2 and 1 μM activating peptide IRS1-pY1172 in the reaction solution at room temperature for 1 hour. Then, 10 μM of the alternative substrate DiFMUP was added to the reaction solution and incubated at room temperature for another 30 minutes.

[0150] e) Data monitoring: The fluorescence signal of the reaction solution was monitored using an ELISA reader with excitation wavelengths of 340nm and 450nm.

[0151] 4) Data processing

[0152] The inhibitory rate of the compound on SHP2 protein was calculated using the following formula:

[0153]

[0154] The results were processed using the analytical software GraphPad Prism 8.3 to fit the curve of SHP2 inhibitory activity as a function of compound concentration, and the IC50 of the compound against SHP2 was calculated. 50 value.

[0155] 5) Experimental Results

[0156] The experimental results are shown in Table 1. The results indicate that the compounds of this invention have a significant inhibitory effect on the activity of SHP2. Wherein, A represents IC... 50 <20nM; B indicates IC 50≥20nM to <100nM; C represents IC 50 ≥100nM to <200nM; D indicates IC 50 ≥200nM.

[0157] Table 1. Inhibitory activity of the compounds of the present invention against SHP2

[0158]

[0159]

[0160] 2. MV-4-11 cell proliferation experiment

[0161] 1) Experimental methods

[0162] Human myeloid monocytic leukemia MV-4-11 cells (20,000 cells / well) were seeded in 96-well Corning plates and cultured in 80 μL / well IMDM medium containing 10% bovine serum (FBS). After 24 h of culture, 20 μL of the compound of the present invention (DMSO final concentration 0.1% (V / V)) was added. On day 4, 20 μL of CCK8 was added to each well, and after 3 hours, the absorbance of each well was measured at 450 nm using a microplate reader. The inhibition rate and IC50 were calculated. 50 value.

[0163] 2) Experimental Results

[0164] The experimental results are shown in Table 2, indicating that the compound of the present invention has a significant inhibitory effect on the proliferation of human myeloid monocytic leukemia MV-4-11 cells.

[0165] Table 2. Inhibitory effect of the compounds of the present invention on the proliferation of MV-4-11 cells.

[0166] Compound numbering <![CDATA[MV-4-11 IC 50 (nM)]]> 24 3.5 32 16.7 TNO155 39.0

[0167] 3. MV-4-11 xenograft tumor experiment

[0168] The therapeutic effects of the compounds of this invention on a mouse model of human myeloid monocytic leukemia MV-4-11 cell xenograft tumor were evaluated.

[0169] 1) Experimental materials

[0170] Cell line: Human myeloid monocytic leukemia MV-4-11 cells.

[0171] Experimental animals: 4-6 week old BALB / c female nude mice.

[0172] 2) Experimental methods

[0173] Approximately 2×10 7One MV-4-11 cell was resuspended in 100 μL of serum-free high-glucose medium and subcutaneously implanted into the right axillary region of BALB / c nude mice. When the tumor volume reached approximately 50 mm... 3 Oral administration was initiated and continued for 18 consecutive days. The treatment group received 10 mg / kg / day and 30 mg / kg / day, while the control group received an equal volume of physiological saline. Tumor size and mouse weight were measured daily during treatment. The animal experiment was terminated once the tumor reached a certain size. Mice were weighed, blood was collected from their eyes, and the mice were euthanized. Tumor tissue was harvested, weighed, and photographed. Simultaneously, a portion of the tissue was placed in 10% neutral fixative for paraffin embedding, paraffin sectioning, and immunohistochemical staining analysis.

[0174] Formula for calculating tumor volume:

[0175] V(mm 3 = Length (mm) × Width 2 (mm 2 ) / 2;

[0176] Formula for calculating Tumor Growth Inhibition Index (TGI):

[0177] TGI% = (1 - mean tumor weight in the treatment group / mean tumor weight in the control group) × 100%.

[0178] 3) Experimental Results

[0179] Experimental results are as follows Figure 1 As shown in the figure. The results indicate that, compared with the model group, compound 24 of the present invention significantly inhibited the growth of human myeloid monocytic leukemia MV-4-11 xenografts at doses of 10 mg / kg / d and 30 mg / kg / d in a dose-dependent manner, without affecting the body weight of mice. Specifically, compound 24 showed a tumor inhibition rate of 67% at a dose of 30 mg / kg / d, indicating that the compound of the present invention has good in vivo antitumor activity and safety.

[0180] H&E and Ki67 histological staining results are as follows: Figure 2 As shown in Figures A and 2B, the results indicate that compound 24 significantly inhibited tumor cell proliferation compared to the model group. Flow cytometry results are as follows... Figure 2 As shown in C and 2D, the results indicate that compound 24 significantly reduces CD206. + / ly6C + The number of M2-like tumor-associated macrophages (TAMs). ELISA results of ground tumor tissue are shown below. Figure 2 As shown in Figure E, compared with the model group, compound 24 significantly reduced the expression level of IL-10 in tumor tissue. Furthermore, the results of immunofluorescence staining experiments are as follows: Figure 2 As shown in F and 2G, compound 24 significantly reduced F480 compared to the model group. + / CD206 + The same inhibition of M2-like TAM was observed in lymph nodes, indicating that compound 24 can effectively inhibit the activation and infiltration of M2-like TAM.

Claims

1. A pyrazine compound, characterized in that, The pyrazine compounds are selected from any of the following compounds, or pharmaceutically acceptable salts thereof. 。 2. The pyrazine compound according to claim 1, characterized by The pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following: 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, ferulic acid.

3. A pharmaceutical composition, characterized by, It contains the pyrazine compound of claim 1 and a pharmaceutically acceptable carrier.

4. The use of a pyrazine compound of claim 1 or a pharmaceutical composition of claim 3 in the preparation of an SHP2 inhibitor drug.

5. The use of a pyrazine compound of claim 1 or a pharmaceutical composition of claim 3 in the preparation of a medicament for the prevention and / or treatment of SHP2-mediated or dependent diseases or conditions.

6. Use according to claim 4 or 5, characterized in that, The drug is a drug for the prevention and / or treatment of tumors, inflammatory diseases, or autoimmune diseases.

7. Use according to claim 6, characterized in that, The tumors mentioned are Noonan syndrome, leopard syndrome, juvenile myeloid monocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of the head and neck, gastric cancer, anaplastic large cell lymphoma, and glioblastoma.

8. Use according to claim 6, characterized in that, The inflammatory diseases mentioned are psoriasis and osteoarthritis.

9. Use according to claim 6, characterized in that, The autoimmune diseases mentioned are systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, myasthenia gravis, mixed connective tissue disease, and autoimmune reactions caused by infection.

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