CN112707902B

CN112707902B - TGF-beta receptor inhibitors

Info

Publication number: CN112707902B
Application number: CN202011579497.9A
Authority: CN
Inventors: 俞智勇; 李刚; 时永强; 赵蒙; 邱庆崇; 季彬
Original assignee: Hangzhou Arnold Biomedical Technology Co ltd
Current assignee: Hangzhou Arnold Biomedical Technology Co ltd
Priority date: 2020-03-23
Filing date: 2020-12-28
Publication date: 2022-04-15
Anticipated expiration: 2040-12-28
Also published as: CN112707902A

Abstract

The invention provides a compound shown in a formula (I) and a pharmaceutical composition thereof. The compounds of formula (I) of the present invention are useful as TGF-beta receptor inhibitors, particularly TGF-beta RI inhibitors, and for example, may be used in the prevention or treatment of various TGF-beta RI (ALK5) -mediated related diseases.

Description

TGF-beta receptor inhibitors

Cross-referencing

This application claims priority from the following patent applications: (1) the priority of chinese patent application 202010207872.0, entitled "TGF-beta receptor inhibitor" filed on 23.3.2020 to the chinese patent office, is incorporated herein by reference in its entirety.

Technical Field

The present invention provides a novel class of heterocyclic compounds, methods for their preparation and their use as TGF-beta receptor (particularly TGF β RI) antagonists.

Background

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor and cytokine, and forms a transforming growth factor-beta superfamily with a plurality of related proteins such as activins, inhibins, Mullerian Inhibitor Substance (MIS) and Bone Morphogenetic Proteins (BMPs). It is involved in the regulation of a variety of biological processes including proliferation, differentiation, development of cells and modification of the extracellular matrix (including tumor stroma and immunosuppression, angiogenesis and fibrohistogenesis).

TGF-. beta.has three major cellular receptors, type I (TGF. beta.RI), type II (TGF. beta.RII), and type III (TGF. beta.RIII). The signaling in the TGF- β signaling pathway is mainly through the binding of TGF- β ligands to the cell surface serine/threonine kinase receptors TGF β RI (ALK5) and TGF β RII to form a heterotetrameric complex, which then phosphorylates the glycine/serine region (GS region) of TGF β RI and activates TGF β RI to phosphorylate the intracellular signaling protein Smad protein molecule Smad2/Smad3 to which it is linked. The complex formed by the association of the phosphorylated Smad2 and Smad3 proteins with Smad4 is transferred to the nucleus of the cell, binds to various transcription factors and transcription coactivators or coactivators to regulate the transcription of target genes, causes various transcription reactions, and thus results in the change of gene expression. Whereas the transforming growth factor-beta type III receptor (TGF-. beta.RIII) is not capable of signaling itself, it indirectly affects signaling by binding TGF-. beta.ligand to the TGF-. beta.type II receptor, and by binding TGF-. beta.and transmitting it to TGF-. beta.RII. In organisms, cytokines, growth factors, microenvironment conditions, hormones, phosphorylation and dephosphorylation kinases, etc., all influence and control signaling in the TGF- β signaling pathway.

Studies have shown that TGF- β plays a key role in the development and progression of fibrosis in different organ systems, such as heart, liver, lung, kidney, etc. Abnormal TGF- β signaling is associated with many human diseases such as cancer, cardiovascular disease, inflammation, organ fibrotic disease, pancreatic disease, and the like. In human tumors, dysregulation of TGF- β signaling is common, promoting tumor cell growth and differentiation, modulating extracellular matrix and epithelial-mesenchymal transition. At present, TGF-beta has been studied with the progress of tumor immunotherapyHas proved to have important function for adjusting anti-tumor immunity. TGF-beta has strong inhibitory effect on T lymphocyte differentiation, and can be used for treating dendritic cell, CD8⁺T cells and NK cells have obvious negative effects, and can enhance immunosuppressive T_regAnd bone Marrow Derived Suppressor Cell (MDSC) activity, thereby providing a favorable tumor microenvironment for tumor growth and metastasis. Therefore, inhibition of signaling in a certain segment of the TGF- β signaling pathway may be one of the effective methods for treating tumors.

TGF beta RI (ALK5) as an important node in a TGF-beta signaling pathway is considered to be an important target point for treating tumors, and by preventing the TGF beta RI from being combined with a ligand, the TGF beta RI is inhibited from phosphorylating a downstream signal protein (Smad2 or Smad3) by ALK5, so that the conduction of TGF-beta signals is influenced or blocked, and various related diseases mediated by ALK5 are prevented and treated.

Currently, international patent application publication nos. WO2000/061576, WO2002/066462, WO2004/111036, WO2004/048382, WO2009/087224, WO2009/013335, WO2012/002680, WO2016/057278, WO2017/015425, WO2017/215506, WO2018/017633, and the like, and chinese patent application publication No. CN107663206A and the like disclose compounds useful as TGF- β receptor antagonists. However, there is still a need for inhibitors with better inhibitory effect on TGF- β receptors, especially on TGF β RI (ALK 5); in particular, antagonists capable of selectively inhibiting ALK5 have important clinical value and therapeutic significance, but few reports are currently made. There is an urgent need in the art for ALK5 inhibitors with better inhibitory effects, in particular for novel antagonist compounds capable of selectively inhibiting ALK 5.

Disclosure of Invention

After long-term and intensive research, a heterocyclic compound with good ALK5 inhibitory activity is discovered.

Based on the above findings, in a first aspect of the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, prodrug, isotopic derivative, isomer, solvate, or metabolite thereof,

wherein:

W₁represents CR^LOr N;

l represents CR^aR^bO or NR^a；

R¹、R²、R^LEach independently represents hydrogen, halogen, nitro, cyano, -OR^a、-NR^aR^bOr C substituted by 0, 1, 2 or 3 substituents₁-C₆Alkyl radical, C₃-C₆A cycloalkyl group; wherein the substituents are selected from: -OR^aCyano, oxo, halogen, -NR^aR^b、-C(O)NR^aR^b、-SO₂R^aand-NR^aC(O)R^b；

R³Represents hydrogen, C₁-C₆Alkyl, halo (C)₁-C₆Alkyl group), C₃-C₈Cycloalkyl, 3-8 membered heterocycloalkyl or C substituted with 0, 1, 2, 3, 4 or 5 substituents₆-C₁₀Aryl or 5-10 membered heteroaryl;

cy represents C₃-C₆Cycloalkyl, 3-6 membered heterocycloalkyl, C₆-C₁₀Aryl or 5-10 membered heteroaryl; and the Cy may be optionally substituted with 0, 1, 2, 3, 4 OR 5 substituents selected from halogen, nitro, cyano, -OR^a-hydroxy (C)₁-C₆Alkyl group), C₁-C₆Alkyl, halo (C)₁-C₆Alkyl group), C₃-C₆Cycloalkyl, - (C)₀-C₆Alkylene) (C₆-C₁₀Aryl), - (C)₀-C₆Alkylene) (5-to 10-membered heteroaryl), -C (O) (C)₁-C₆Alkyl), -C (O) O (C)₁-C₆Alkyl), -C (O) NR^aR^b、-SO₂R^a、-SO₂NR^aR^band-P (O) R^aR^b；

R^a、R^bEach independently represents hydrogen or C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl, -hydroxy (C)₁-C₆Alkyl), - (C)₀-C₆Alkylene) (C₆-C₁₀Aryl), - (C)₀-C₆Alkylene) (5-to 10-membered heteroaryl), - (C)₀-C₆Alkylene) O (C)₁-C₆Alkyl), - (C)₀-C₆Alkylene) O (C)₃-C₆Cycloalkyl), - (C)₀-C₆Alkylene) O (3-6 membered heterocycloalkyl); or R^a、R^bTogether with the atoms to which they are attached form a 5-8 membered ring which may optionally contain 0, 1 or 2 heteroatoms selected from N, O, S;

m is 0, 1 or 2;

n is 0, 1 or 2.

Preferably, the compounds of the present invention have the following structure of formula (II):

wherein R is¹、R²、R³L, Cy, m and n have the meanings given for the compounds of the formula (I).

Preferably, the compounds of the present invention have the following structure of formula (III):

wherein:

R¹、R³and Cy and n have the meanings given for the compounds of the formula (I);

R²represents hydrogen or halogen;

m is 0 or 1.

Preferably, the compounds of the present invention have the following structure of formula (IV):

wherein:

R¹、R²、R³m and n have the meanings given for the compounds of the formula (III).

W₂Represents CR⁵Or N;

R⁴、R⁵each independently represents halogen, nitro, cyano, -OR^a-hydroxy (C)₁-C₆Alkyl group), C₁-C₆Alkyl, halo (C)₁-C₆Alkyl group), C₃-C₆Cycloalkyl, - (C)₀-C₆Alkylene) (C₆-C₁₀Aryl), - (C)₀-C₆Alkylene) (5-to 10-membered heteroaryl), -C (O) (C)₁-C₆Alkyl), -C (O) O (C)₁-C₆Alkyl), -C (O) NR^aR^b、-SO₂R^a、-SO₂NR^aR^bor-P (O) R^aR^b；

R^a、R^bEach independently represents hydrogen or C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl, hydroxy (C)₁-C₆Alkyl), - (C)₀-C₆Alkylene) (C₆-C₁₀Aryl), - (C)₀-C₆Alkylene) (5-to 10-membered heteroaryl), - (C)₀-C₆Alkylene) O (C)₁-C₆Alkyl), - (C)₀-C₆Alkylene) O (C)₃-C₆Cycloalkyl) or- (C)₀-C₆Alkylene) O (3-6 membered heterocycloalkyl); r^a、R^bTogether with the atoms to which they are attached form a 5-8 membered ring which may optionally contain 0, 1 or 2 heteroatoms selected from N, O, S;

wherein o is 0, 1, 2, 3 or 4.

Preferably, the compounds of the present invention are selected from the following structures:

the compounds of the present invention may also be prepared in the form of pharmaceutically acceptable salts, which may be formed using, for example, the following inorganic or organic acids: hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, mandelic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, methanesulfonic, benzenesulfonic or toluenesulfonic acid.

The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example, by dissolving the compound of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol and acetonitrile), adding thereto an excess of an aqueous solution of an organic acid or an inorganic acid to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid therefrom, and separating the precipitated salt.

The compounds of the present invention (or pharmaceutically acceptable salts thereof) may include solvate forms, preferably, the solvates are hydrates.

The invention also provides the use of a compound of the invention in the manufacture of a medicament for the prevention or treatment of a disease that is modulated by the inhibition of ALK5 activity. Preferably, the disease is selected from the group consisting of cancer, tumor, inflammatory disease, autoimmune disease and immune-mediated disease.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, tumor, inflammatory disease, autoimmune disease, neurodegenerative disease, attention-related disease or immune-mediated disease, comprising the compound of formula (I) of the present invention as an active ingredient.

Furthermore, the present invention provides a method for preventing or treating cancer, tumor, inflammatory disease, autoimmune disease, neurodegenerative disease, attention-related disease, or immune-mediated disease, comprising administering the compound of the present invention to a mammal in need thereof.

Representative examples of cancers or tumors can include, but are not limited to, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis carcinoma, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharynx cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, hodgkin lymphoma, non-hodgkin lymphoma, burkitt lymphoma, Acute Lymphatic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder carcinoma, bronchial carcinoma, small-cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, ewing's sarcoma, or plasmacytoma.

The compounds of the present invention or pharmaceutically acceptable salts thereof can provide enhanced anticancer effects when administered in combination with additional anticancer agents or checkpoint inhibitors for the treatment of cancer or tumors.

Representative examples of anticancer agents for the treatment of cancer or tumor may include, but are not limited to, cell signaling inhibitors, chlorambucil, melphalan, cyclophosphamide, ifosfamide, busulfan, carmustine, lomustine, streptozotocin, cisplatin, carboplatin, oxaliplatin, dacarbazine, temozolomide, procarbazine, methotrexate, fluorouracil, cytarabine, gemcitabine, mercaptopurine, fludarabine, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, etoposide, trabectedin, dactinomycin, doxorubicin, epirubicin, daunomycin, mitoxantrone, bleomycin, mitomycin C, ixabepilone, tamoxifene, flutamide, gonadorelin analogs, megestrol, prednisone, dexamethasone, methylprednisolone, thalidomide, doxylamine, fludarabine, dacarbazine, and the like, Interferon alpha, calcium folinate, sirolimus, everolimus, afatinib, alisertib, amuvatinib, apatinib, axitinib, bortezomib, bosutinib, brimonib, cabozantinib, cediranib, crenolanib, crizotinib, dabrafenib, dacomitinib, darnoutilinib, dasatinib, dovitinib, erlotinib, foretinib, ganetespib, gefitinib, ibrutinib, erlotinib, imatinib, inrarib, lapatinib, lentivatinib, linifanib, linsitinib, masitinib, momelotinib, motertinib, lenalitinib, nilapartiib, niraparipariib, oprozolib, paucinib, sergentinib, valtinib, valacitinib, valtinib, valacitinib, valtinib, Vismodegib, volasertib, alemtuzumab, bevacizumab, bernetuzumab, cetuximab, denosumab, gemtuzumab, ipilimumab, nimotuzumab, ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab; checkpoint inhibitors, including but not limited to anti-PD-1 antibodies, anti-PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, and anti-CTLA-4 antibodies, or any combination thereof.

Representative examples of inflammatory, autoimmune and immune-mediated diseases can include, but are not limited to, arthritis, rheumatoid arthritis, spondyloarthritis, gouty arthritis, osteoarthritis, juvenile arthritis, other arthritic conditions, lupus, Systemic Lupus Erythematosus (SLE), skin-related diseases, psoriasis, eczema, dermatitis, allergic dermatitis, pain, lung disease, lung inflammation, Adult Respiratory Distress Syndrome (ARDS), pulmonary sarcoidosis, chronic pulmonary inflammatory disease, Chronic Obstructive Pulmonary Disease (COPD), cardiovascular disease, atherosclerosis, myocardial infarction, congestive heart failure, myocardial ischemia reperfusion injury, inflammatory bowel disease, crohn's disease, ulcerative colitis, irritable bowel syndrome, asthma, sjogren's syndrome, autoimmune thyroid disease, urticaria (rubella), multiple sclerosis, and immune-mediated diseases, Scleroderma, organ transplant rejection, xenografts, Idiopathic Thrombocytopenic Purpura (ITP), parkinson's disease, alzheimer's disease, diabetes-related diseases, inflammation, pelvic inflammatory disease, allergic rhinitis, allergic bronchitis, allergic sinusitis, leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, myeloma, Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), hairy cell leukemia, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferative tumors (MPN), diffuse large B-cell lymphoma, and follicular lymphoma.

The compounds of the present invention or pharmaceutically acceptable salts thereof can provide enhanced therapeutic effects when administered in combination with additional therapeutic agents for the treatment of inflammatory diseases, autoimmune diseases, and immune-mediated diseases.

Representative examples of therapeutic agents for treating inflammatory diseases, autoimmune diseases, and immune-mediated diseases may include, but are not limited to, steroid drugs (e.g., prednisone, prednisolone, methyl prednisolone, cortisone, hydrocortisone, betamethasone, dexamethasone, etc.), methotrexate, leflunomide, anti-TNF α agents (e.g., etanercept, infliximab, adalimumab, etc.), calcineurin inhibitors (e.g., tacrolimus, pimecrolimus, etc.), and antihistamines (e.g., diphenhydramine, hydroxyzine, loratadine, ebastine, ketotifen, cetirizine, levocetirizine, fexofenadine, etc.), and at least one therapeutic agent selected therefrom may be included in the pharmaceutical composition of the present invention.

The compound of the present invention or a pharmaceutically acceptable salt thereof can be administered orally or parenterally as an active ingredient in an effective amount ranging from 0.1 to 2,000mg/kg body weight/day, preferably 1 to 1,000mg/kg body weight/day in the case of mammals including humans (body weight about 70kg), and administered in a single or 4 divided doses per day, or with/without following a predetermined time. The dosage of the active ingredient may be adjusted according to a variety of relevant factors, such as the condition of the subject to be treated, the type and severity of the disease, the rate of administration and the opinion of the physician. In some cases, amounts less than the above dosages may be suitable. Amounts greater than the above doses may be used if they do not cause harmful side effects and may be administered in divided doses per day.

The pharmaceutical composition of the present invention may be formulated into dosage forms for oral administration or parenteral administration (including intramuscular, intravenous and subcutaneous routes) according to any one of conventional methods, such as tablets, granules, powders, capsules, syrups, emulsions, microemulsions, solutions or suspensions.

Pharmaceutical compositions of the invention for oral administration may be prepared by mixing the active ingredient with a carrier such as: cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactants, suspending agents, emulsifying agents, and diluents. Examples of carriers employed in the injectable compositions of the present invention are water, saline solutions, dextrose-like solutions, alcohols, glycols, ethers (e.g., polyethylene glycol 400), oils, fatty acids, fatty acid esters, glycerol esters, surfactants, suspending agents, and emulsifying agents.

Other features of this invention will become apparent in describing exemplary embodiments thereof which are given for illustration of the invention and are not intended to be limiting thereof, the following examples being prepared, isolated and characterized using the disclosed methods of this invention.

The compounds of the present invention can be prepared in a variety of ways known to those skilled in the art of organic synthesis, and can be synthesized using synthetic methods known in the art of organic synthetic chemistry or by variations thereof as would be understood by those skilled in the art. The desired reaction may be carried out in a solvent or solvent mixture suitable for the kit material used and for the transformation to be effected.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

When no preparative route is included, relevant intermediates are commercially available (e.g., from SigmaAldrich, Alfa).

General procedure

Commercial reagents were used without further purification.¹H-NMR spectra were recorded on a Bruker instrument at 500 MHz. Chemical shift values are expressed in parts per million, i.e., delta values. The following abbreviations are used for multiplicity of NMR signals: s is singlet, brs is broad, d is doublet, t is triplet, and m is multiplet. Coupling constants are listed as J values, measured in Hz. NMR and mass spectrometry results were corrected for background peaks. Chromatography refers to column chromatography performed using 100 mesh silica gel and performed under nitrogen pressure (flash chromatography). TLC for monitoring the reaction refers to TLC performed using a specific mobile phase and silica gel F254 from Merck as stationary phase.

The LC-MS experiment was measured under the following conditions:

the instrument comprises the following steps: thermo U3000, ALLtech ELSD, MSQ, UV detector combined ELSD and MSD (outflow ratio 4: 1). Column: waters X-Bridge C-18,3.5 μm, 4.6X50 mm; column temperature: at 30 ℃. Gradient [ time (min)/solvent B in a (%) ]: 0.00/5.0,0.70/95,1.40/95,1.41/5,1.50/5. (solvent a ═ 0.01% trifluoroacetic acid in water; solvent B ═ 0.01% trifluoroacetic acid in acetonitrile). And (4) UV detection: 214/254/280/300 nm; and D, DAD detection: 200-400 nm; flow rate: 4 mL/min; MS: ESI, 100-1500m/z

Preparative HPLC typically used an acidic method (gradient of acetonitrile and water, each containing 0.1% formic acid) with Thermo U3000 AFC-3000; column: globalisil C-1812nm, 250X 20mm,10 μm, or equivalent; flow rate: 20mL/min, separation was performed.

Synthesis of intermediates

Preparation of Compound INT-1:

a solution of 2-pyridinemethanol (500mg,4.6mmol) and 2, 4-dinitrophenylhydroxylamine (912mg,4.6mmol) in dichloromethane (15mL) was stirred overnight at room temperature. Adding diethyl ether into the reaction solution, and filtering; the filter cake was further dried in vacuo to yield INT-1(304mg, yield: 53.0%) as a yellow solid.

Preparation of Compound INT-2:

4-bromopyridine-2-carboxylic acid (5.0g,24.7mmol) was dissolved in ethanol (85mL) and concentrated sulfuric acid (2.69mL) was added. The resulting reaction solution was stirred at 60 ℃ for 6 hours. The reaction mixture was concentrated under reduced pressure, a part of the solvent was removed, and then poured into water (100mL), neutralized with a saturated aqueous solution of sodium hydrogencarbonate, and extracted with ethyl acetate (100 mL. times.3). The extracted organic phase was washed with saturated brine (80mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the resulting crude product was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 1/1) to give compound INT-2a (4.19g, yield: 73.6%) as a yellow oil. MS (ESI) M/z 230.1(M + H)⁺.

Methylmagnesium bromide (3M solution in 2-methyltetrahydrofuran, 3.48mL) was added to a three-necked flask containing anhydrous tetrahydrofuran (18mL), cooled in an ice bath, and a solution of compound INT-2a (1g,4.35mmol) in tetrahydrofuran (5mL) was added thereto under nitrogen. The ice bath was removed and the resulting reaction was stirred at room temperature for 30 minutes, then cooled in an ice bath and quenched by addition of saturated aqueous ammonium chloride and then diluted with ethyl acetate (80 mL). The resulting mixture was transferred to a separatory funnel, and water (50mL) was added for separation. The aqueous phase was extracted with ethyl acetate (60 mL. times.2). The organic phases were combined, washed with saturated brine (80mL), dried over anhydrous sodium sulfate, filtered and the filtrate was reducedConcentrated under reduced pressure, and the resulting crude product was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 2/3) to give compound INT-2b (894mg, yield: 95.2%) as a pale yellow oil.¹H NMR(500MHz,Chloroform-d)δ8.36–8.33(m,1H),7.59(d,J＝1.8Hz,1H),7.38(dd,J＝5.3,1.8Hz,1H),1.54(s,6H).

Compound INT-2b (500mg,2.31mmol), concentrated ammonia (28% w/w,5.5mL) and copper powder (100mg,1.57mmol) were added to a sealed tube, stirred for 30 minutes at room temperature with an open lid, then sealed and allowed to warm to 100 deg.C overnight. The reaction solution was cooled and extracted with 2-methyltetrahydrofuran (15 mL. times.30). The organic phase was concentrated under reduced pressure to give INT-2(252mg, yield: 71.6%) as a white solid. The impurities were used directly in the next step. MS (ESI) M/z 153.3(M + H)⁺.

Synthesis of the Compounds of examples

Example 1:

thionyl chloride (951mg,8.0mmol) was added dropwise to a toluene (10mL) solution in which tetrahydropyran-4-carboxylic acid (520mg,4.0mmol) was dissolved; the resulting mixture was stirred under reflux for 2 hours. The reaction was cooled and concentrated to give a brown oil 1 a. A solution of the compound 1a obtained above and the compound INT-1(173mg,0.56mmol) in pyridine (2mL) was heated at 110 ℃ for 5 hours. The reaction solution was cooled and concentrated, and a saturated sodium hydrogencarbonate solution (30mL) was added to the residue, followed by extraction with ethyl acetate (30 mL. times.2). The combined organic phases are washed by saturated saline solution, dried by anhydrous sodium sulfate and concentrated; the obtained crude product was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 3/1) to give 1b (35mg, yield: 18.9%) as a white solid.¹H NMR(500MHz,Chloroform-d)δ8.28(d,J＝7.1Hz,1H),7.20–7.14(m,1H),7.09–7.02(m,1H),6.73–6.61(m,1H),4.11–4.03(m,4H),3.61–3.47(m,4H),3.06–2.98(m,1H),2.96–2.87(m,1H),2.10–1.96(m,6H),1.89–1.83(m,2H)；MS(ESI):m/z 331.2(M+H)⁺.

An aqueous hydrochloric acid solution (3mL, 10% w/w) containing Compound 1b (35mg,0.11mmol) was stirred under refluxStirring overnight. After cooling to 0 ℃ a saturated solution of sodium bicarbonate (20mL) was carefully added followed by extraction with ethyl acetate (20 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 1c (23mg, yield: 99.5%) as a yellow oil. MS (ESI) M/z 219.1(M + H)⁺.

Potassium tert-butoxide (62mg,0.55mmol) was added to a solution of Compound 1c (23mg,0.11mmol)

A solution of N-methylpyrrolidone (1mL) and stirred at room temperature for 20 minutes; 2-chloro-4-fluoropyridine (36mg,0.27mmol) was then added, and the reaction solution was further stirred under the same conditions for 1 hour. To the reaction mixture was added a saturated ammonium chloride solution (10mL), and the mixture was extracted with ethyl acetate (10 mL. times.2). The combined organic phases are washed by saturated saline solution, dried by anhydrous sodium sulfate and concentrated; the crude product was subjected to preparative thin layer chromatography (petroleum ether/ethyl acetate: 1/1) to give 1d (20mg, yield: 55.2%) as a yellow oil. MS (ESI) M/z 330.1(M + H)⁺.

Compound 1d (20mg, 61. mu. mol), Compound INT-2(16mg, 85. mu. mol), sodium phenolate (11mg, 91. mu. mol), Pd were mixed under a nitrogen atmosphere₂(dba)₃A solution of (5.6mg, 6.1. mu. mol) and XantPhos (7.0mg, 12. mu. mol) in 1, 4-dioxane (1mL) was stirred under reflux overnight. After the reaction solution was cooled to room temperature, a saturated ammonium chloride solution (10mL) was added; and extracted with ethyl acetate (10 mL. times.2). The combined organic phases were concentrated and the crude product was isolated by preparative HPLC to give 1 as a white solid (10mg, yield: 38.1%).¹HNMR(500MHz,DMSO-d6)δ9.36(s,1H),8.63(d,J＝7.1Hz,1H),8.19–8.13(m,2H),7.68–7.62(m,2H),7.33–7.27(m,1H),7.21–7.15(m,1H),6.90–6.84(m,1H),6.64–6.60(m,1H),6.25(d,J＝2.3Hz,1H),5.05(s,1H),3.91–3.82(m,2H),3.41–3.36(m,2H),3.04–2.95(m,1H),1.86–1.72(m,4H),1.37(s,6H)；MS(ESI):m/z 446.6(M+H)⁺.

Example 2:

starting from compound 1d and 4-methanesulfonylaniline, reference compound 1In the last step of the synthesis, sodium phenolate was replaced with cesium carbonate to give compound 2.¹H NMR(500MHz,DMSO-d6)δ9.49(s,1H),8.66–8.60(m,1H),8.15(d,J＝5.8Hz,1H),7.84(d,J＝9.0Hz,2H),7.72(d,J＝9.0Hz,2H),7.34–7.28(m,1H),7.21–7.14(m,1H),6.91–6.84(m,1H),6.67–6.63(m,1H),6.23(d,J＝2.2Hz,1H),3.91–3.83(m,2H),3.43–3.33(m,2H),3.09(s,3H),3.06–2.95(m,1H),1.87–1.73(m,4H)；MS(ESI):m/z 465.2(M+H)⁺.

Example 3:

starting from compound 1d and 4-aminoacetophenone, the synthesis of reference compound 2 gives compound 3.¹HNMR(500MHz,DMSO-d6)δ9.39(s,1H),8.66–8.61(m,1H),8.14(d,J＝5.8Hz,1H),7.82(d,J＝8.8Hz,2H),7.74(d,J＝8.8Hz,2H),7.34–7.28(m,1H),7.21–7.15(m,1H),6.92–6.84(m,1H),6.65–6.61(m,1H),6.22(d,J＝2.3Hz,1H),3.92–3.84(m,2H),3.42–3.35(m,2H),3.06–2.95(m,1H),2.45(s,3H),1.85–1.74(m,4H)；MS(ESI):m/z 429.3(M+H)⁺.

Example 4:

compound 4 was obtained by synthesis of reference compound 2 starting from compound 1d and 4-aminobenzenesulfonamide.¹H NMR(500MHz,DMSO-d6)δ9.34(s,1H),8.65–8.61(m,1H),8.12(d,J＝5.8Hz,1H),7.75(d,J＝8.9Hz,1H),7.63(d,J＝8.9Hz,1H),7.37–7.27(m,1H),7.23–7.14(m,1H),7.09(s,2H),6.90–6.83(m,1H),6.64–6.60(m,1H),6.20(d,J＝2.2Hz,1H),3.93–3.83(m,2H),3.43–3.34(m,2H),3.06–2.94(m,1H),1.87–1.74(m,4H)；MS(ESI):m/z 466.2(M+H)⁺.

Example 5:

starting from compound 1d and 4-aminobenzamide, the synthesis of reference compound 2 yielded compound 5.¹H NMR(500MHz,DMSO-d6)δ9.20(s,1H),8.62(d,J＝7.1Hz,1H),8.11(d,J＝5.8Hz,1H),7.73(d,J＝8.7Hz,2H),7.69(s,1H),7.65(d,J＝8.7Hz,2H),7.31(d,J＝8.9Hz,1H),7.21–7.14(m,1H),7.05(s,1H),6.90–6.83(m,1H),6.62–6.56(m,1H),6.18(d,J＝2.3Hz,1H),3.91–3.83(m,2H),3.44–3.34(m,2H),3.05–2.95(m,1H),1.87–1.73(m,4H)；MS(ESI):m/z 430.2(M+H)⁺.

Example 6:

compound 6 was obtained by reference to the synthesis of compound 2 starting from compound 1d and 4- (dimethylphosphite) aniline.¹H NMR(500MHz,DMSO-d6)δ9.21(s,1H),8.62(d,J＝7.1Hz,1H),8.10(d,J＝5.8Hz,1H),7.76–7.70(m,2H),7.60–7.52(m,2H),7.34–7.28(m,1H),7.20–7.14(m,1H),6.90–6.83(m,1H),6.60–6.56(m,1H),6.19(d,J＝2.2Hz,1H),3.92–3.82(m,2H),3.43–3.34(m,2H),3.06–2.95(m,1H),1.88–1.72(m,4H),1.57(s,3H),1.54(s,3H)；MS(ESI):m/z 463.2(M+H)⁺.

Example 7:

compound 7 was obtained from the synthesis of reference compound 1 starting from cyclopropylcarbonyl chloride.¹HNMR(500MHz,DMSO-d6)δ9.38(s,1H),8.55(d,J＝7.0Hz,1H),8.18–8.14(m,2H),7.67–7.65(m,2H),7.32–7.28(m,1H),7.15(m,1H),6.86–6.78(m,1H),6.65–6.62(m,1H),6.28(d,J＝2.3Hz,1H),5.05(s,1H),1.90–1.84(m,1H),0.96–0.87(m,4H)；MS(ESI):m/z 402.3(M+H)⁺.

Example 8:

potassium nitrate (1.2g,12.2mmol) was added portionwise to concentrated sulphuric acid (4mL) in which 2, 6-lutidine N-oxide (1.0g,8.1mmol) was dissolved at 0 ℃. The resulting reaction mixture was reacted at 100 ℃ overnight. The reaction solution was poured into ice water, adjusted to pH 7-8 with aqueous sodium carbonate (5% w/w), and extracted with ethyl acetate (50 mL. times.2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 8a (1.1g, yield: 82.0%) as a yellow solid. MS (ESI) M/z 169.1(M + H)⁺.

Trifluoroacetic anhydride (3.1g,14.6mmol) was added to a solution of compound 8a (900mg,7.3mmol) in dichloromethane (15mL) under ice-bath conditions; the resulting reaction solution was stirred at 40 ℃ for 18 hours. The reaction solution was washed with saturated sodium bicarbonate solution (50mL) and extracted with ethyl acetate (50 mL. times.2). The combined organic phases are washed by saturated saline solution, dried by anhydrous sodium sulfate and concentrated; the resulting crude product was separated by silica gel column chromatography (dichloromethane/methanol ═ 30/1) to give 8b (340mg, yield: 27.7%) as a yellow solid. MS (ESI) M/z 169.0(M + H)⁺.

Daiss-Martin oxidant (493mg,1.2mmol) was added to a solution of compound 8b (163mg,0.97mmol) in dichloromethane (5mL) at 0 deg.C; the resulting reaction solution was stirred at room temperature for 1 hour. To the reaction solution was added a mixed aqueous solution of saturated sodium bicarbonate (20mL) and sodium sulfite (5% w/w,20mL), and extracted with dichloromethane (30 mL. times.2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 8c (160mg, yield: 99.4%) as a yellow solid.

A methanol solution (3mL) containing potassium hydroxide (148mg,2.5mmol) and a methanol solution (3mL) containing iodine (318mg,1.25mmol) were added to a methanol solution (5mL) containing compound 8c (160mg,0.96mmol) in this order under ice-bath conditions; the reaction solution was stirred under the same conditions for half an hour. To the reaction solution was added aqueous sodium sulfite (5% w/w,30mL) and extracted with ethyl acetate (20 mL. times.2). Mixing organic phases with saturated saltWater washing, drying over anhydrous sodium sulfate, and concentration gave 8d as a yellow solid (60mg, yield: 31.8%).¹HNMR(500MHz,Chloroform-d)δ8.62(d,J＝1.9Hz,1H),8.06(d,J＝1.9Hz,1H),4.06(s,3H),2.83(s,3H).

A methanol solution (5mL) containing compound 8d (60mg,0.31mmol) and palladium on carbon (10% w/w,20mg) was stirred under a hydrogen atmosphere at room temperature for 2 hours. The reaction solution was filtered through celite, the filtrate was concentrated, and the obtained residue was subjected to silica gel column chromatography (dichloromethane/methanol: 20/1) to isolate 8e (25mg, yield: 49.2%) as a white solid. MS (ESI) M/z 167.2(M + H) +.

Under a nitrogen atmosphere, Compound 7a (43mg,0.15mmol), Compound 8e (25mg,0.15mmol), Pd were mixed₂(dba)₃A toluene solution (2mL) of (14mg,0.015mmol), XantPhos (17mg,0.030mmol) and cesium carbonate (98mg,0.30mmol) was stirred at 100 ℃ overnight. Filtering the reaction solution with diatomite, and concentrating the filtrate; the resulting crude product was separated by silica gel column chromatography (dichloromethane/methanol ═ 30/1) to give 8(42mg, yield: 67.2%) as a white solid.¹HNMR(500MHz,DMSO-d6)δ9.59(s,1H),8.57–8.53(m,1H),8.20(d,J＝5.9Hz,1H),8.08(d,J＝2.0Hz,1H),7.73(d,J＝2.0Hz,1H),7.36–7.27(m,1H),7.20–7.11(m,1H),6.87–6.80(m,1H),6.72–6.68(m,1H),6.27(d,J＝2.3Hz,1H),3.81(s,3H),2.40(s,3H),1.89–1.84(m,1H),0.95–0.87(m,4H)；MS(ESI):m/z 416.2(M+H)⁺.

Example 9:

methylmagnesium bromide (3M in tetrahydrofuran, 67 μ L) was added to a solution of compound 8(21mg,51 μmmol) in tetrahydrofuran (2mL) under ice-bath conditions; the resulting reaction solution was stirred under the same conditions for 1 hour. The reaction solution was quenched with water (10mL) and extracted with ethyl acetate (10 mL. times.2). The combined organic phases were concentrated, and the resulting residue was separated by preparative HPLC to give 9(11mg, yield: 52.4%) as a white solid.¹H NMR(500MHz,DMSO-d6)δ9.29(s,1H),8.54(d,J＝7.0Hz,1H),8.15(d,J＝5.8Hz,1H),7.52(d,J＝2.0Hz,1H),7.44(d,J＝2.0Hz,1H),7.34–7.27(m,1H),7.19–7.11(m,1H),6.86–6.78(m,1H),6.64–6.60(m,1H),6.27(d,J＝2.3Hz,1H),5.06(s,1H),2.32(s,3H),1.91–1.82(m,1H),1.35(s,6H),0.98–0.85(m,4H)；MS(ESI):m/z 416.3(M+H)⁺.

Example 10:

starting from compound 7a and 4-aminobenzoate, with reference to the last synthesis of compound 8, cesium carbonate was exchanged for sodium tert-butoxide to give compound 10 a. MS (ESI) M/z 387.2(M + H)⁺.

N, N-dimethylformamide (1mL) mixed with Compound 10a (7.5mg, 19. mu. mol), benzylamine (4.2mg, 38. mu. mol), HATU (11mg, 29. mu. mol) and N, N-diisopropylethylamine (5.0mg, 38. mu. mol) was stirred at room temperature for 1 hour. The reaction mixture was then separated by preparative HPLC to give 10(5.2mg, yield: 56.3%) as a white solid.¹H NMR(500MHz,DMSO-d6)δ9.24(s,1H),8.77(t,J＝6.0Hz,1H),8.54(d,J＝7.0Hz,1H),8.11(d,J＝5.9Hz,1H),7.77(d,J＝8.5Hz,2H),7.68(d,J＝8.5Hz,2H),7.34–7.27(m,4H),7.25–7.19(m,1H),7.15(t,J＝7.8Hz,1H),6.82(t,J＝6.9Hz,1H),6.65–6.56(m,1H),6.22(d,J＝2.2Hz,1H),4.44(d,J＝5.8Hz,2H),1.91–1.83(m,1H),0.97–0.83(m,4H)；MS(ESI):m/z 476.3(M+H)⁺.

Example 11:

compound 11 was synthesized in the last step starting from compound 10a and ethanolamine with reference to compound 10.¹H NMR(500MHz,DMSO-d6)δ9.22(s,1H),8.54(d,J＝7.1Hz,1H),8.16(t,J＝5.6Hz,1H),8.11(d,J＝5.8Hz,1H),7.75(d,J＝8.5Hz,2H),7.67(d,J＝8.5Hz,2H),7.30(d,J＝8.8Hz,1H),7.18–7.12(m,1H),6.86–6.80(m,1H),6.62–6.58(m,1H),6.21(d,J＝2.3Hz,1H),4.69(s,1H),3.51–3.43(m,2H),3.29–3.25(m,2H),1.92–1.83(m,1H),0.99–0.86(m,4H)；MS(ESI):m/z 430.2(M+H)⁺.

Example 12:

compound 12 was obtained by synthesis of reference compound 2 starting from compound 7a and 4-aminobenzenesulfonamide.¹H NMR(500MHz,DMSO-d6)δ9.36(s,1H),8.54(d,J＝7.1Hz,1H),8.12(d,J＝5.8Hz,1H),7.75(d,J＝8.5Hz,2H),7.67(d,J＝8.5Hz,2H),7.33–7.29(m,1H),7.19–7.13(m,1H),7.07(brs,2H),6.87–6.79(m,1H),6.65–6.61(m,1H),6.23(d,J＝2.3Hz,1H),1.92–1.82(m,1H),0.99–0.85(m,4H)；MS(ESI):m/z 422.0(M+H)⁺.

Example 13:

chlorosulfonic acid (4mL) mixed with N-acetyl-3-toluidine (2.0g,13.4mmol) was stirred at room temperature for 20 minutes; the temperature was then raised to 70 ℃ and stirring was continued for 8 hours while maintaining this condition. After the reaction mixture was cooled to room temperature, ice (50g) was added, and then a solid was gradually precipitated.

Dissolving the obtained filter cake in a tetrahydrofuran (50mL) solution, and adding concentrated ammonia water (4mL) under the ice bath condition; the reaction was allowed to warm to room temperature and stirring was continued for 2 hours while maintaining the conditions. The reaction mixture was concentrated, and water (100mL) was added to precipitate a solid.

Dissolving the obtained filter cake in a mixed solution of ethanol (5mL) and hydrochloric acid aqueous solution (6M,5 mL); the reaction solution was stirred at reflux temperature for 1 hour. The reaction mixture was concentrated, adjusted to pH 7-8 with an aqueous sodium hydroxide solution (1M), and extracted with ethyl acetate (50 mL. times.3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated; the obtained crude product was subjected to silica gel column chromatography (dichloromethane/methanol ═ 20/1) to give 13a (500mg, yield: 16.3%) as a white solid.¹H NMR(500MHz,DMSO-d6)δ7.46(d,J＝8.5Hz,1H),6.86(s,2H),6.40(d,J＝2.2Hz,1H),6.36(dd,J＝8.5,2.2Hz,1H),5.66(s,2H),2.40(s,3H)；MS(ESI):m/z 187.4(M+H)⁺.

Compound 13 was obtained from the synthesis of compound 7a and compound 13a with reference to compound 2.¹H NMR(500MHz,DMSO-d6)δ9.26(s,1H),8.59–8.52(m,1H),8.12(d,J＝5.9Hz,1H),7.66(d,J＝8.7Hz,1H),7.64–7.59(m,1H),7.53–7.49(m,1H),7.33–7.27(m,1H),7.18–7.13(m,1H),7.10(s,2H),6.85–6.79(m,1H),6.63–6.59(m,1H),6.22(d,J＝2.3Hz,1H),2.48(s,3H),1.90–1.84(m,1H),0.96–0.88(m,4H)；MS(ESI):m/z 435.9(M+H)⁺.

Example 14:

referring to the synthesis of compound 12, 4-aminobenzenesulfonamide was replaced with 4-aminobenzamide to provide compound 14.¹H NMR(500MHz,DMSO-d6)δ9.24(s,1H),8.56(d,J＝7.1Hz,1H),8.13(d,J＝5.8Hz,1H),7.75(d,J＝8.8Hz,2H),7.72(brs,1H),7.67(d,J＝8.8Hz,2H),7.37–7.29(m,1H),7.21–7.14(m,1H),7.09(s,1H),6.87–6.80(m,1H),6.64–6.60(m,1H),6.23(d,J＝2.3Hz,1H),1.94–1.83(m,1H),1.00–0.88(m,4H)；MS(ESI):m/z 386.2(M+H)⁺.

Example 15:

compound 15 was synthesized in the last step starting from compound 10a and 3-amino-1-propanol with reference to compound 10.¹H NMR(500MHz,DMSO-d6)δ9.24(s,1H),8.61–8.54(m,1H),8.20(t,J＝5.6Hz,1H),8.13(d,J＝5.8Hz,1H),7.73(d,J＝9.0Hz,2H),7.69(d,J＝9.0Hz,2H),7.36–7.31(m,1H),7.22–7.15(m,1H),6.89–6.82(m,1H),6.64–6.60(m,1H),6.23(d,J＝2.3Hz,1H),4.48(t,J＝5.3Hz,1H),3.48–3.44(m,2H),3.32–3.25(m,2H),1.94–1.87(m,1H),1.70–1.63(m,2H),0.99–0.91(m,4H)；MS(ESI):m/z 444.2(M+H)⁺.

Example 16:

compound 16 was synthesized in the last step starting from compound 10a and N-methyl-2-hydroxyethylamine with reference to compound 10.¹H NMR(500MHz,DMSO-d6)δ9.16(s,1H),8.60–8.55(m,1H),8.11(d,J＝5.8Hz,1H),7.67(d,J＝8.6Hz,2H),7.36–7.32(m,1H),7.31(d,J＝8.6Hz,2H),7.21–7.16(m,1H),6.87–6.82(m,1H),6.63–6.60(m,1H),6.22(d,J＝2.3Hz,1H),4.77(t,J＝5.5Hz,1H),3.64–3.35(m,4H),2.97(s,3H),1.94–1.86(m,1H),1.00–0.90(m,4H)；MS(ESI):m/z 444.2(M+H)⁺.

Example 17:

synthesis of reference Compound 13 from 3' -chloroacetanilide gave Compound 17.¹HNMR(500MHz,DMSO-d6)δ9.55(s,1H),8.60–8.56(m,1H),8.20(d,J＝5.9Hz,1H),8.15(d,J＝2.2Hz,1H),7.80(d,J＝8.8Hz,1H),7.55–7.51(m,1H),7.39–7.33(m,1H),7.33(brs,2H),7.22–7.16(m,1H),6.88–6.83(m,1H),6.73–6.70(m,1H),6.25(d,J＝2.3Hz,1H),1.95–1.84(m,1H),0.99–0.90(m,4H)；MS(ESI):m/z 456.1(M+H)⁺.

Example 18:

referring to the synthesis of compound 12, 4-aminobenzenesulfonamide was replaced with 3-aminobenzenesulfonamide to provide compound 18.¹H NMR(500MHz,DMSO-d6)δ9.34(s,1H),8.57(d,J＝7.0Hz,1H),8.17(s,1H),8.11(d,J＝6.0Hz,1H),7.81(d,J＝8.0Hz,1H),7.40(t,J＝8.0Hz,1H),7.37–7.26(m,4H),7.21–7.15(m,1H),6.84(t,J＝7.0Hz,1H),6.66–6.59(m,1H),6.22(s,1H),1.93–1.85(m,1H),0.99–0.87(m,4H)；MS(ESI):m/z 422.2(M+H)⁺.

Example 19:

parafluoronitrobenzene (1.06g,7.51mmol) was added to the reaction flask, followed by chlorosulfonic acid (4 mL). The resulting mixture was stirred at 110 ℃ for 24 hours, and then the reaction solution was cooled to room temperature. The reaction was poured slowly into an ice bath and, after the ice had melted, extracted with ethyl acetate (50 mL. times.3). The organic phase was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give an oil which was dissolved in ethyl acetate (38mL), then cooled to 0 ℃ and concentrated aqueous ammonia (38mL, 28% w/w) was slowly added thereto. The resulting reaction mixture was stirred at room temperature overnight, then water (30mL) was added, and extraction was performed with ethyl acetate (50 mL. times.3). The organic phase was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the resulting crude product was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 1/1) to give compound 19a (830mg, yield: 50.2%) as a tan solid.

Compound 19a (100mg,0.45mmol) was dissolved in methanol (8mL), Pd/C (40mg, 10% w/w) was added, the resulting reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere, the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give compound 19b as a white solid (63mg, yield: 73%).¹HNMR(500MHz,DMSO-d6)δ7.43(s,2H),7.05–6.99(m,1H),6.99–6.94(m,1H),6.74–6.66(m,1H),5.36(s,2H).

Compound 7a (30mg,0.11mmol), compound 19b (24mg,0.13mmol), cesium carbonate (51mg,0.16mmol), XantPhos (12mg, 21. mu. mol) and palladium acetate (2.36mg, 10. mu. mol) were added to a reaction flask followed by 1, 4-dioxane (2 mL). The reaction solution was stirred at 100 ℃ for reaction overnight under nitrogen atmosphere. Cooling the reaction solution, filtering, concentrating the filtrate under reduced pressure to obtain crude product, and purifying with preparative HPLC to obtain white solid compound19(15mg, yield: 32%).¹H NMR(500MHz,DMSO-d6)δ9.26(s,1H),8.57(d,J＝7.0Hz,1H),8.14–8.10(m,1H),8.08(d,J＝5.8Hz,1H),7.93–7.86(m,1H),7.58(s,2H),7.36–7.31(m,1H),7.30–7.23(m,1H),7.21–7.14(m,1H),6.87–6.81(m,1H),6.62–6.58(m,1H),6.15(d,J＝2.2Hz,1H),1.93–1.83(m,1H),0.98–0.89(m,4H)；MS(ESI):m/z 440.0(M+H)⁺.

Example 20:

synthesis of reference compound 19b gave compound 20b starting from p-nitrotoluene.¹H NMR(500MHz,DMSO-d6)δ7.13(d,J＝2.3Hz,1H),7.12(s,2H),6.96(d,J＝8.0Hz,1H),6.62(dd,J＝8.0,2.3Hz,1H),5.27(s,2H),2.38(s,3H).

Synthesis of reference compound 19 yielded compound 20.¹H NMR(500MHz,DMSO-d6)δ9.18(s,1H),8.56(d,J＝7.0Hz,1H),8.10(d,J＝2.4Hz,1H),8.07(d,J＝5.8Hz,1H),7.86–7.82(m,1H),7.35–7.29(m,1H),7.27(brs,2H),7.22–7.18(m,1H),7.17–7.13(m,1H),6.87–6.80(m,1H),6.58–6.54(m,1H),6.18(d,J＝2.3Hz,1H),2.48(s,3H),1.93–1.84(m,1H),0.98–0.87(m,4H)；MS(ESI):m/z 435.8(M+H)⁺.

Example 21:

synthesis of reference compound 19b, starting from o-nitrotoluene, gave compound 21 b.¹H NMR(500MHz,DMSO-d6)δ7.09(s,2H),7.07–7.03(m,2H),6.92–6.87(m,1H),5.28(s,2H),2.08(s,3H).

Synthesis of reference compound 19 yielded compound 21.¹H NMR(500MHz,DMSO-d6)δ8.55(d,J＝7.0Hz,1H),8.27(s,1H),8.23(d,J＝1.8Hz,1H),7.99(d,J＝5.8Hz,1H),7.38–7.28(m,3H),7.24(s,2H),7.20–7.12(m,1H),6.87–6.80(m,1H),6.54–6.50(m,1H),6.36(d,J＝2.3Hz,1H),2.22(s,3H),1.91–1.84(m,1H),0.99–0.88(m,4H)；MS(ESI):m/z 436.3(M+H)⁺.

Example 22:

2-methyl-3-nitroaniline (500mg,3.29mmol) and concentrated hydrochloric acid (2mL) were added to a reaction flask, which was then placed in an ice-salt bath to cool (-5-10 ℃) and an aqueous solution (1.5mL) of sodium nitrite (249mg,3.61mmol) was added with stirring, and stirring was continued for 30 minutes while keeping the reaction temperature below 0 ℃. Water (8mL) was added to the other reaction flask, which was then cooled in an ice salt bath (-5-10 ℃ C.), thionyl chloride (1.1mL,15mmol) was slowly added dropwise to the reaction flask with stirring, the resulting reaction solution was stirred at room temperature for 3 hours and then cooled in an ice salt bath, cuprous chloride (33mg,0.33mmol) was added, and stirring was continued for 30 minutes while maintaining the ice salt bath. The reaction solution in the first reaction flask was added to the second reaction flask, and the resulting reaction solution was stirred at 0 ℃ for 1 hour. The reaction mixture was diluted with water and extracted with ethyl acetate (30 mL. times.5). The organic phase was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 1/1) to give compound 22a (55mg, yield: 7.7%) as a yellow solid.

Starting from compound 22a, reference compound 19 was synthesized to give compound 22.¹HNMR(500MHz,DMSO-d6)δ8.56–8.52(m 1H),8.35(s,1H),7.93(d,J＝5.8Hz,1H),7.73–7.68(m,1H),7.62–7.57(m,1H),7.39(s,2H),7.36–7.31(m,1H),7.27–7.22(m,1H),7.20–7.14(m,1H),6.85–6.79(m,1H),6.48–6.44(m,1H),6.22(d,J＝2.3Hz,1H),2.40(s,3H),1.93–1.83(m,1H),0.99–0.89(m,4H)；MS(ESI):m/z 436.2(M+H)⁺.

Example 23:

a solution (5mL) of ethanolamine (276mg,4.51mmol) and triethylamine (1.89mL,13.5mmol) in methylene chloride was cooled to 0 ℃ and then a solution (5mL) of p-nitrobenzenesulfonyl chloride (1.0g,4.51mmol) in methylene chloride was added dropwise thereto. The resulting reaction solution was stirred at room temperature overnight. Water (50mL) was added, followed by extraction with ethyl acetate (50 mL). The organic phase was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was subjected to silica gel column chromatography (dichloromethane/methanol-20/1) to give compound 23a as a white solid (548mg, yield: 49.3%).

Compound 23a (150mg,0.61mmol) was dissolved in methanol (5mL), Pd/C (30mg, 10% w/w) was added, and the resulting reaction solution was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give compound 23b as a white solid (130mg, yield: 98.7%). MS (ESI) M/z217.4(M + H)⁺.

Synthesis of reference compound 13 from compound 23b gave compound 23.¹H NMR(500MHz,DMSO-d6)δ9.41(s,1H),8.54(d,J＝7.1Hz,1H),8.13(d,J＝5.9Hz,1H),7.79(d,J＝8.5Hz,2H),7.61(d,J＝8.5Hz,2H),7.35–7.25(m,2H),7.19–7.12(m,1H),6.85–6.79(m,1H),6.70–6.62(m,1H),6.24(d,J＝2.4Hz,1H),4.62(t,J＝5.6Hz,1H),3.35–3.32(m,2H),2.74–2.70(m,2H),1.92–1.83(m,1H),0.96–0.87(m,4H)；MS(ESI):m/z 466.0(M+H)⁺.

Example 24:

starting from compound 23b and compound 1d, the synthesis of reference compound 23 yielded compound 24.¹H NMR(500MHz,DMSO-d6)δ9.42(s,1H),8.69–8.63(m,1H),8.15(d,J＝5.9Hz,1H),7.81(d,J＝8.9Hz,2H),7.63(d,J＝8.9Hz,2H),7.36–7.29(m,2H),7.24–7.18(m,1H),6.93–6.87(m,1H),6.68–6.64(m,1H),6.23(d,J＝2.3Hz,1H),4.66(t,J＝5.6Hz,1H),3.93–3.86(m,2H),3.49–3.43(m,4H),3.07–2.99(m,1H),2.78–2.71(m,2H),1.87–1.77(m,4H)；MS(ESI):m/z 510.0(M+H)⁺.

Example 25:

compound 1d (50mg,0.15mmol), methyl p-aminobenzoate (36mg,0.24mmol), cesium carbonate (69mg,0.21mmol), XantPhos (17.6mg, 30. mu. mol) and palladium acetate (3.40mg, 15. mu. mol) were added to a reaction flask followed by 1, 4-dioxane (3 mL). The reaction solution was stirred at 100 ℃ for reaction overnight under nitrogen atmosphere. The reaction solution was cooled and filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was purified by preparative thin layer chromatography (petroleum ether/ethyl acetate: 1/1) to give compound 25a (26mg, yield 38.6%) as a pale yellow solid. MS (ESI) M/z 445.3(M + H)⁺.

Compound 25a (26mg, 58. mu. mol) was dissolved in tetrahydrofuran (4mL) and methanol (1mL), followed by addition of 3N aqueous sodium hydroxide (1 mL). The reaction solution was stirred at 40 ℃ for 4 hours. The reaction mixture was cooled to room temperature, the pH of the reaction mixture was adjusted to 3-4 with 3N aqueous hydrochloric acid, diluted with water (30mL), and extracted with ethyl acetate (30 mL. times.4). The organic phase was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 25b as a pale yellow solid (24.6mg, yield: 97.7%). MS (ESI) M/z 431.1(M + H)⁺.

Starting from compound 25b, reference compound 11 was synthesized to give compound 25.¹H NMR(500MHz,DMSO-d6)δ9.24(s,1H),8.65(d,J＝7.1Hz,1H),8.20(t,J＝5.7Hz,1H),8.13(d,J＝5.8Hz,1H),7.73(d,J＝8.6Hz,2H),7.68(d,J＝8.6Hz,2H),7.32(d,J＝8.9Hz,1H),7.24–7.15(m,1H),6.94–6.86(m,1H),6.64–6.59(m,1H),6.19(d,J＝2.2Hz,1H),4.72(t,J＝5.6Hz,1H),3.96–3.83(m,2H),3.51–3.44(m,2H),3.43–3.36(m,2H),3.32–3.26(m,2H),3.06–2.97(m,1H),1.86–1.76(m,4H)；MS(ESI):m/z 474.5(M+H)⁺.

Example 26:

referring to the synthesis of compound 12, 4-aminobenzenesulfonamide was replaced with 3-aminobenzamide to provide compound 26.¹H NMR(500MHz,DMSO-d6)δ9.10(s,1H),8.56(d,J＝7.0Hz,1H),8.09(d,J＝6.0Hz,1H),8.01(s,1H),7.90–7.81(m,2H),7.37–7.25(m,2H),7.31–7.25(m,2H),7.21–7.15(m,1H),6.84(t,J＝7.0Hz,1H),6.59–6.54(m,1H),6.20(d,J＝1.5Hz,1H),1.93–1.86(m,1H),0.99–0.90(m,4H)；MS(ESI):m/z386.2(M+H)⁺.

Example 27:

compound 27 was obtained from the synthesis of 4, 4-difluorocyclohexanecarboxylic acid with reference to compound 1.¹HNMR(500MHz,DMSO-d6)δ9.38(s,1H),8.64(d,J＝7.0Hz,1H),8.18(d,J＝5.5Hz,1H),8.16(d,J＝5.8Hz,1H),7.70–7.63(m,2H),7.34–7.28(m,1H),7.23–7.16(m,1H),6.93–6.86(m,1H),6.67–6.61(m,1H),6.27(d,J＝2.3Hz,1H),5.07(s,1H),3.01–2.92(m,1H),2.12–2.02(m,2H),2.01–1.93(m,3H),1.92–1.79(m,3H),1.38(s,6H)；MS(ESI):m/z 480.4(M+H)⁺.

Example 28:

compound 28 was obtained from the synthesis of reference compound 1 starting from acetyl chloride.¹H NMR(500MHz,DMSO-d6)δ9.39(s,1H),8.63–8.58(m,1H),8.21–8.16(m,2H),7.71–7.66(m,2H),7.37(d,J＝9.0,1H),7.22–7.17(m,1H),6.91–6.85(m,1H),6.66–6.61(m,1H),6.29(d,J＝2.0Hz,1H),5.08(s,1H),2.27(s,3H),1.40(s,6H)；MS(ESI):m/z376.3(M+H)⁺.

Example 29:

compound 29 was obtained from the synthesis of reference compound 1 starting from cyclobutylformyl chloride.¹H NMR(500MHz,DMSO-d6)δ9.39(s,1H),8.65(d,J＝7.0Hz,1H),8.20(d,J＝5.5Hz,1H),8.16(d,J＝6.0Hz,1H),7.73–7.65(m,2H),7.40–7.32(m,1H),7.25–7.14(m,1H),6.93–6.83(m,1H),6.67–6.60(m,1H),6.26(s,1H),5.08(s,1H),3.64–3.54(m,1H),2.39–2.29(m,2H),2.27–2.18(m,2H),2.03–1.92(m,1H),1.90–1.81(m,1H),1.40(s,6H)；MS(ESI):m/z 416.3(M+H)⁺.

Example 30:

compound 30 was obtained by reference to the synthesis of compound 1 starting from propionyl chloride.¹H NMR(500MHz,DMSO-d6)δ9.40(s,1H),8.62(d,J＝7.0Hz,1H),8.21–8.15(m,2H),7.75–7.65(m,2H),7.36(d,J＝9.0Hz,1H),7.22–7.15(m,1H),6.91–6.84(m,1H),6.69–6.60(m,1H),6.29(d,J＝1.8Hz,1H),5.08(s,1H),2.66(q,J＝7.5Hz,2H),1.39(s,6H),1.22(t,J＝7.5Hz,3H)；MS(ESI):m/z 390.3(M+H)⁺.

Example 31:

compound 31 was obtained from the synthesis of reference compound 1 starting from cyclohexylcarbonyl chloride.¹HNMR(500MHz,DMSO-d6)δ9.39(s,1H),8.62(d,J＝7.0Hz,1H),8.19(d,J＝5.5Hz,1H),8.16(d,J＝6.0Hz,1H),7.75–7.65(m,2H),7.30(d,J＝9.0Hz,1H),7.22–7.14(m,1H),6.90–6.83(m,1H),6.70–6.61(m,1H),6.27(d,J＝2.0Hz,1H),5.08(s,1H),2.79–2.69(m,1H),1.93–1.84(m,2H),1.79–1.71(m,2H),1.68–1.63(m,1H),1.62–1.53(m,2H),1.39(s,6H),1.34–1.16(m,3H)；MS(ESI):m/z 444.4(M+H)⁺.

Example 32:

compound 32 was obtained from the synthesis of reference compound 1 starting from cyclopentylcarbonyl chloride.¹H NMR(500MHz,DMSO-d6)δ9.39(s,1H),8.63(d,J＝7.0Hz,1H),8.19(d,J＝5.5Hz,1H),8.17(d,J＝6.0Hz,1H),7.71–7.66(m,2H),7.32(d,J＝9.0Hz,1H),7.20–7.15(m,1H),6.88–6.84(m,1H),6.65–6.62(m,1H),6.28(d,J＝2.5Hz,1H),5.08(s,1H),3.17–3.09(m,1H),2.00–1.92(m,2H),1.83–1.68(m,4H),1.63–1.55(m,2H),1.40(s,6H)；MS(ESI):m/z 430.2(M+H)⁺.

Example 33:

compound 33 was obtained from the synthesis of reference compound 1 starting from benzoyl chloride.¹H NMR(500MHz,DMSO-d6)δ9.35(s,1H),8.77(d,J＝7.1Hz,1H),8.20–8.15(m,2H),7.93–7.89(m,2H),7.65–7.61(m,2H),7.49–7.42(m,3H),7.41–7.35(m,1H),7.30–7.24(m,1H),7.03–6.98(m,1H),6.75–6.71(m,1H),6.32(d,J＝2.3Hz,1H),5.05(s,1H),1.37(s,6H)；MS(ESI):m/z 438.3(M+H)⁺.

Example 34:

starting from trifluoroacetic anhydride, reference compound 1 was synthesized to give compound 34.¹H NMR(500MHz,DMSO-d6)δ9.43(s,1H),8.90(d,J＝7.0Hz,1H),8.27–8.16(m,2H),7.75–7.67(m,2H),7.64(d,J＝9.0Hz,1H),7.49–7.40(m,1H),7.28–7.20(m,1H),6.75–6.65(m,1H),6.40–6.30(m,1H),5.11(s,1H),1.41(s,6H)；MS(ESI):m/z 430.2(M+H)⁺.

Example 35:

3, 3-Difluorocyclobutylcarboxylic acid (330mg,2.42mmol) was dissolved in dichloromethane (5mL), then cooled to 0 deg.C, to which oxalyl chloride (400mg,3.15mmol) and a drop of DMF were added for catalysis. The resulting reaction solution was stirred at room temperature for 1 hour. The reaction mixture was carefully concentrated under reduced pressure at 30 ℃ to give compound 35a as a yellow oil which was used directly in the next reaction.

Starting from compound 35a, reference compound 1 was synthesized to give compound 35.¹H NMR(500MHz,DMSO-d6)δ9.38(s,1H),8.71(d,J＝7.1Hz,1H),8.24–8.13(m,2H),7.71–7.67(m,1H),7.65(s,1H),7.40(d,J＝8.9Hz,1H),7.28–7.19(m,1H),6.96–6.90(m,1H),6.66–6.60(m,1H),6.28–6.23(m,1H),5.09(s,1H),3.51–3.40(m,1H),3.02–2.85(m,4H),1.38(s,6H)；MS(ESI):m/z 452.4(M+H)⁺.

Example 36:

compound 36a was obtained from (6-methylpyridin-2-yl) methanol, according to the synthesis of intermediate compound INT-1. Compound 36 was obtained by synthesis of reference compound 1 starting from compound 36a and cyclopropanecarbonyl chloride.¹H NMR(500MHz,DMSO-d6)δ9.39(s,1H),8.21–8.12(m,2H),7.70–7.61(m,2H),7.20(d,J＝8.5Hz,1H),7.16–7.08(m,1H),6.80–6.74(m,1H),6.66–6.62(m,1H),6.27(d,J＝2.3Hz,1H),5.06(s,1H),2.61(s,3H),1.96–1.86(m,1H),1.37(s,6H),0.96–0.90(m,4H)；MS(ESI):m/z 416.2(M+H)⁺.

Example 37:

from (3-methylpyridine-2-yl) methanol, reference compound 36 was synthesized to give compound 37.¹H NMR(500MHz,Methanol-d4)δ8.21–8.13(m,3H),7.78(d,J＝2.2Hz,1H),7.59–7.55(m,1H),6.92–6.85(m,1H),6.70(t,J＝7.0Hz,1H),6.63–6.59(m,1H),6.32(d,J＝2.3Hz,1H),2.32(s,3H),1.93–1.84(m,1H),1.50(s,6H),0.98–0.92(m,4H)；MS(ESI):m/z 416.3(M+H)⁺.

Example 38:

synthesis of reference compound 36 from 2-hydroxymethyl-5-methylpyridine gave compound 38.¹HNMR(500MHz,DMSO-d6)δ9.38(s,1H),8.40–8.35(m,1H),8.17(d,J＝5.5Hz,1H),8.14(d,J＝6.0Hz,1H),7.69–7.63(m,2H),7.21(d,J＝9.0Hz,1H),7.05–6.99(m,1H),6.64–6.60(m,1H),6.27(d,J＝2.3Hz,1H),5.06(s,1H),2.25(s,3H),1.90–1.79(m,1H),1.37(s,6H),0.95–0.82(m,4H)；MS(ESI):m/z 416.2(M+H)⁺.Example 39:

synthesis of reference compound 36 from (4-methylpyridin-2-yl) methanol gave compound 39.¹HNMR(500MHz,DMSO-d6)δ9.39(s,1H),8.42(d,J＝7.1Hz,1H),8.17(d,J＝5.5Hz,1H),8.14(d,J＝5.5Hz,1H),7.70–7.65(m,2H),7.07(s,1H),6.68–6.59(m,2H),6.27(d,J＝2.3Hz,1H),5.06(s,1H),2.27(s,3H),1.88–1.79(m,1H),1.37(s,6H),0.94–0.85(m,4H)；MS(ESI):m/z 416.4(M+H)⁺.

Example 40:

compound 40 was obtained from the synthesis of compound 36, reference compound 36, starting from compound 36a and tetrahydropyran-4-carboxylic acid.¹H NMR(500MHz,DMSO-d6)δ9.35(s,1H),8.19–8.13(m,2H),7.66–7.60(m,2H),7.20(d,J＝8.8Hz,1H),7.16–7.11(m,1H),6.82(d,J＝7.0Hz,1H),6.65–6.60(m,1H),6.24(d,J＝2.5Hz,1H),5.04(s,1H),3.92–3.83(m,2H),3.42–3.40(m,2H),3.08–3.00(m,1H),2.66(s,3H),1.90–1.72(m,4H),1.36(s,6H)；MS(ESI):m/z 460.2(M+H)⁺.

Example 41:

synthesis of reference compound 12 from compound 36b gave compound 41.¹H NMR(500MHz,DMSO-d6)δ9.36(s,1H),8.14(d,J＝6.0Hz,1H),7.78(d,J＝9.0Hz,2H),7.65(d,J＝9.0Hz,2H),7.23(d,J＝9.0Hz,1H),7.17–7.11(m,3H),6.82–6.78(m,1H),6.68–6.64(m,1H),6.22(d,J＝2.5Hz,1H),2.63(s,3H),1.97–1.90(m,1H),0.98–0.92(m,4H)；MS(ESI):m/z 436.3(M+H)⁺.

Example 42:

compound 42 was obtained from the synthesis of compound 32a with reference to compound 12.¹H NMR(500MHz,DMSO-d6)δ9.37(s,1H),8.62(d,J＝7.0Hz,1H),8.13(d,J＝5.8Hz,1H),7.77(d,J＝8.6Hz,2H),7.65(d,J＝8.6Hz,2H),7.32(d,J＝9.0Hz,1H),7.21–7.16(m,1H),7.13(s,2H),6.90–6.82(m,1H),6.65–6.61(m,1H),6.20(d,J＝2.2Hz,1H),3.18–3.07(m,1H),2.02–1.90(m,2H),1.83–1.66(m,4H),1.64–1.53(m,2H)；MS(ESI):m/z 450.2(M+H)⁺.

Example 43:

synthesis of reference Compound 12 starting from Compound 34aCompound 43.¹HNMR(500MHz,DMSO-d6)δ9.39(s,1H),8.89(d,J＝7.0Hz,1H),8.17(d,J＝6.0Hz,1H),7.79(d,J＝8.0Hz,2H),7.69(d,J＝8.0Hz,2H),7.66–7.62(m,1H),7.48–7.42(m,1H),7.26–7.22(m,1H),7.14(s,2H),6.70–6.65(m,1H),6.28(d,J＝2.5Hz,1H)；MS(ESI):m/z 450.1(M+H)⁺.

Example 44:

synthesis of reference compound 12 from compound 28a gave compound 44.¹HNMR(500MHz,DMSO-d6)δ9.35(s,1H),8.59(d,J＝7.0Hz,1H),8.14(d,J＝6.0Hz,1H),7.77(d,J＝9.0Hz,2H),7.65(d,J＝9.0Hz,2H),7.37(d,J＝9.0Hz,1H),7.22–7.17(m,1H),7.11(s,2H),6.89–6.84(m,1H),6.66–6.61(m,1H),6.21(d,J＝2.5Hz,1H),2.27(s,3H)；MS(ESI):m/z 396.2(M+H)⁺.

Example 45:

synthesis of reference compound 12 from compound 29a gave compound 45.¹HNMR(500MHz,DMSO-d6)δ9.37(s,1H),8.65(d,J＝7.0Hz,1H),8.13(d,J＝6.0Hz,1H),7.78(d,J＝9.0Hz,2H),7.66(d,J＝9.0Hz,2H),7.35(d,J＝9.0Hz,1H),7.22–7.17(m,1H),7.12(s,2H),6.90–6.85(m,1H),6.64–6.59(m,1H),6.20(d,J＝2.5Hz,1H),3.62–3.53(m,1H),2.38–2.28(m,2H),2.26–2.18(m,2H),2.03–1.92(m,1H),1.91–1.81(m,1H)；MS(ESI):m/z 436.1(M+H)⁺.

Example 46:

reference is made from compound 29a and (trans) -4-aminocyclohexanecarboxylic acid methyl esterSynthesis of compound 12 affords compound 46 a. MS (ESI) M/z 421.3(M + H)⁺.

Compound 46a (40mg, 95. mu. mol) was dissolved in tetrahydrofuran (3mL), followed by addition of an aqueous solution (1mL) of lithium hydroxide (6mg, 250. mu. mol). The resulting reaction solution was stirred at room temperature for 3 hours. Ethyl acetate was added to dilute (100mL), followed by filtration through Celite, and the filtrate was concentrated under reduced pressure to give compound 46b (35mg, yield: 90.5%). MS (ESI) M/z 406.7(M + H)⁺.

To a solution of compound 46b (50mg,0.12mmol) in dichloromethane (3mL) was added ammonia (0.4M solution in 1, 4-dioxane, 0.6mL), HOBT (33mg,0.25 mmol). The resulting reaction was stirred at room temperature for 10 minutes, then N, N-diisopropylethylamine (32mg,0.25mmol) and EDCI (47mg,0.25mmol) were added and the resulting reaction was stirred at room temperature for an additional 5 hours. The crude product obtained by concentrating the reaction solution under reduced pressure was purified by preparative HPLC to give compound 46(10mg, yield: 20.0%) as a white solid.¹H NMR(500MHz,DMSO-d6)δ8.60(d,J＝7.0Hz,1H),7.83(d,J＝6.0Hz,1H),7.29(d,J＝9.0Hz,1H),7.18–7.13(m,2H),6.87–6.81(m,1H),6.65(s,1H),6.30(d,J＝7.5Hz,1H),6.17–7.13(m,1H),5.73(d,J＝2.5Hz,1H),3.60–3.49(m,2H),2.34–2.26(m,2H),2.24–2.17(m,2H),2.04–1.90(m,4H),1.89–1.81(m,1H),1.77–1.70(m,2H),1.42–1.31(m,2H),1.10–1.00(m,2H)；MS(ESI):m/z 406.2(M+H)⁺.

Example 47:

4-amino-3, 5, 6-trichloropyridinecarboxylic acid (5.0g,20.7mmol) was dissolved in methanol (105mL), then cooled to 0 ℃ and thionyl chloride (3.2g,26.9mmol) was slowly added dropwise thereto. The resulting reaction mixture was stirred at 50 ℃ for 4 hours, cooled to room temperature, and then concentrated under reduced pressure to give a crude product, which was then added with ethyl acetate (50mL), transferred to a separatory funnel, washed with water (50mL), and washed with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give white compound 47a (4.9g, yield: 92.6%). MS (ESI):m/z 255.1(M+H)⁺.

Compound 47a (1.0g,3.91mmol) and cyclopropylboronic acid (403mg,4.70mmol) were dissolved in toluene (20mL), followed by addition of water (2mL), tris (cyclohexyl) phosphine (219mg,0.78mmol), potassium phosphate (1.66g,7.83mmol) and palladium acetate (87.9mg,0.39 mmol). The reaction solution thus obtained was stirred and reacted at 100 ℃ for 24 hours in a nitrogen atmosphere. The reaction solution was cooled and then filtered through celite, and the filtrate was concentrated under reduced pressure to give a crude product, which was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 4/1) to give compound 47b (240mg, yield: 23.5%) as a white solid. MS (ESI) M/z 261.3(M + H)⁺.

Compound 47b (240mg,0.92mmol) was dissolved in methanol (5mL) and Pd/C (111mg, 10% w/w) was added. The resulting reaction solution was stirred at room temperature for 2 days under a hydrogen atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a crude product, which was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 4/1) to give compound 47c (24mg, yield: 13.6%) as a white solid.¹H NMR(500MHz,DMSO-d6)δ7.02(d,J＝2.1Hz,1H),6.49(d,J＝2.1Hz,1H),6.21(s,2H),3.79(s,3H),1.94–1.87(m,1H),0.86–0.80(m,4H)；MS(ESI):m/z 193.6.

Starting from compound 47c, reference compound 12 was synthesized to give compound 47 d. MS (ESI) M/z 442.2(M + H)⁺.

Compound 47d (36mg, 81.5. mu. mol) was dissolved in anhydrous tetrahydrofuran (2mL), then cooled to 0 ℃ and methylmagnesium bromide (3M solution in 2-methyltetrahydrofuran, 0.11mL) was added thereto under a nitrogen atmosphere. The resulting reaction solution was further stirred at 0 ℃ for 1 hour. The reaction was quenched by the addition of water (50mL) and extracted with ethyl acetate (50 mL). The organic phase was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and the crude product obtained by concentrating the filtrate under reduced pressure was purified by preparative HPLC to give Compound 47 as a white solid (15mg, yield: 41.7%).¹H NMR(500MHz,DMSO-d6)δ9.30(s,1H),8.58(d,J＝7.0Hz,1H),8.18(d,J＝5.8Hz,1H),7.57(s,1H),7.41(s,1H),7.33(d,J＝8.9Hz,1H),7.20–7.16(m,1H),6.87–6.82(m,1H),6.66–6.63(m,1H),6.29(d,J＝2.2Hz,1H),5.02(s,1H),1.96–1.85(m,2H),1.34(s,6H),0.98–0.91(m,4H),0.88–0.82(m,4H)；MS(ESI):m/z 442.2(M+H)⁺.

Example 48:

compound 48a was obtained from synthesis of reference compound 1 starting from cyclopropylcarbonyl chloride. MS (ESI) M/z175.5(M + H)⁺.

Compound 48a (60mg,0.34mmol) was dissolved in N-methylpyrrolidone (3mL), followed by addition of potassium tert-butoxide (116mg,1.03mmol) and 2- (trimethylsilyl) ethoxymethyl chloride (115mg,0.69 mmol). The resulting reaction mixture was reacted at room temperature for 1 hour, then potassium tert-butoxide (116mg,1.03mmol) and 2- (trimethylsilyl) ethoxymethyl chloride (115mg,0.69mmol) were added, and the reaction was further stirred at room temperature for 2 days. The reaction mixture was poured into water (30mL) and extracted with ethyl acetate (40 mL. times.3). The organic phase was washed with water (30mL), saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by preparative thin layer chromatography (petroleum ether/ethyl acetate: 4/1) to give compound 48b as a yellow oil (77mg, yield: 73.5%). MS (ESI) M/z 305.4(M + H)⁺.

Compound 48b (70mg,0.23mmol) was dissolved in anhydrous tetrahydrofuran (4.5mL), cooled to-78 deg.C, and n-butyllithium (2.5M in n-hexane, 0.14mL) was added under nitrogen. The resulting reaction was stirred at-78 ℃ for 1 hour, and then carbon tetrachloride (0.18mL,1.84mmol) was added. The resulting reaction was stirred for an additional 2 hours at-78 ℃ and then quenched with saturated aqueous ammonium chloride (10 mL). The quenched reaction solution was poured into water (30mL) and extracted with ethyl acetate (40 mL. times.3). The organic phase was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by preparative thin layer chromatography (petroleum ether/ethyl acetate: 4/1) to give compound 48c (13mg, yield: 16.7%) as a brown oil. MS (ESI) M/z 339.3(M + H)⁺.

Hydrochloric acid (3M aqueous solution, 1.28mL) was added to a flask containing compound 48c (13mg, 38. mu. mol), and the mixture was heated to 100 ℃ and stirred for 2 hours. The reaction was cooled to room temperature, diluted with water (30mL) and concentratedExtraction was performed with ethyl acetate (30 mL. times.3). The extracted organic phase was washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude compound 48d (10mg, purity 80%), which was taken to the next step without purification. MS (ESI) M/z 209.3(M + H)⁺.

Crude compound 48d (10mg, 38. mu. mol) was dissolved in N-methylpyrrolidinone (2mL) and potassium tert-butoxide (12.9mg,0.12mmol) and 2-bromo-4-fluoropyridine (6.7mg, 38. mu. mol) were added. The resulting reaction solution was stirred at room temperature overnight, then poured into water (20mL), and extracted with ethyl acetate (30 mL. times.3). The organic phase was washed with water (30mL), saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, and the crude product obtained by concentrating the filtrate under reduced pressure was purified by preparative thin layer chromatography (petroleum ether/ethyl acetate: 4/1) to give compound 48e (5mg, yield: 35.7%) as a yellow oily substance. MS (ESI) M/z 364.0(M + H)⁺.

Compound 48 was obtained by reference to the synthesis of compound 1 starting from compound 48 e.¹H NMR(500MHz,DMSO-d6)δ9.39(s,1H),8.22–8.15(m,2H),7.70–7.64(m,2H),7.42–7.38(m,1H),7.23–7.17(m,2H),6.70–6.66(m,1H),6.28(d,J＝2.2Hz,1H),5.10(s,1H),1.96–1.93(m,1H),1.38(s,6H),0.99–0.95(m,4H)；MS(ESI):m/z 435.8(M+H)⁺.

Example 49:

compound 1b (265mg,0.80mmol) was dissolved in methanol (8mL), palladium dichloride (56.9mg,0.32mmol) was added, and the resulting reaction solution was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a crude product, which was subjected to silica gel column chromatography (petroleum ether/ethyl acetate: 1/1) to give 49a (154mg, yield: 57.4%) as a pale yellow oil.¹H NMR(500MHz,Chloroform-d)δ4.08–3.96(m,6H),3.57–3.40(m,4H),2.86–2.72(m,2H),2.53(t,J＝6.4Hz,2H),2.03–1.94(m,4H),1.94–1.86(m,3H),1.85–1.79(m,3H),1.78–1.70(m,2H)；MS(ESI):m/z 335.4(M+H)⁺.

Starting from compound 49a, seeSynthesis of compound 1 gave compound 49.¹H NMR(500MHz,Chloroform-d)δ8.30(d,J＝5.8Hz,1H),8.16(d,J＝5.8Hz,1H),7.41(d,J＝1.5Hz,1H),7.34–7.30(m,1H),7.03(brs,1H),6.52–6.48(m,1H),6.38(d,J＝2.1Hz,1H),4.08(t,J＝6.1Hz,2H),4.00–3.91(m,2H),3.45–3.37(m,2H),2.83–2.74(m,1H),2.52(t,J＝6.4Hz,2H),2.07–1.98(m,2H),1.90–1.78(m,4H),1.77–1.69(m,2H),1.52(s,6H)；MS(ESI):m/z 450.2(M+H)⁺.

Example 50:

an aqueous solution (1.5mL) containing dissolved sodium nitrite (249mg,3.6mmol) was added in portions to concentrated hydrochloric acid (5mL) containing dissolved 3-methyl-5-nitroaniline (500mg,3.3mmol) at-5 ℃ with stirring; the internal temperature is ensured to be lower than 0 ℃ in the dropping process; the reaction solution was stirred under the same conditions for 30 minutes. At the same time, thionyl chloride (1.1mL) was added dropwise to another flask containing water (8mL) with stirring at-5 ℃; the reaction solution was allowed to warm to room temperature with stirring and stirred at room temperature for 3 hours, after which cuprous chloride (33mg,0.33mmol) was added after the temperature was reduced to-5 ℃ and stirring was continued under these conditions for 30 minutes. The reaction solution in the first round-bottom flask was added in portions to the reaction solution in the second round-bottom flask at 0 ℃ and the resulting reaction solution was stirred under these conditions for 1 hour. Subsequently, concentrated aqueous ammonia (2mL) was added dropwise to the reaction solution at 0 ℃ and stirring was continued for 1 hour. Water (30mL) was added to the reaction solution, and extraction was performed with ethyl acetate (30mLx 5). The combined organic phases were washed with saturated brine (50mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was subjected to silica gel column chromatography (petroleum ether/ethyl acetate 7:3, v/v) to give 50a (455mg, yield: 64%) as a pale yellow solid.

A solution of compound 50a (200mg,0.93mmol) and Pd/C (10% w/w,25mg) in methanol (10mL) was stirred at room temperature under a hydrogen atmosphere for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated to give 50b (167mg, yield: 97%) as a yellow solid.MS(ESI):m/z 187.5(M+H)⁺.

Starting from compound 50b, reference compound 19 was synthesized to give compound 50. 1H NMR (500MHz, DMSO-d6) δ 9.23(s,1H),8.57(d, J ═ 7.0Hz,1H),8.12(d, J ═ 6.0Hz,1H),8.00(s,1H),7.64(s,1H),7.33(d, J ═ 8.5Hz,1H),7.25(s,2H), 7.21-7.12 (m,2H),6.84(t, J ═ 7.0Hz,1H),6.60(d, J ═ 5.0Hz,1H),6.23(s,1H),2.32(s,3H), 1.94-1.85 (m,1H), 1.00-0.88 (m, 4H); MS (ESI) M/z 435.8(M + H)⁺.

Test examples

Determination of inhibitory Effect of Compounds on TGF β RI kinase (ALK5) Activity:

TGF-. beta.RI kinase was purchased from Carna (Cat # 09-141). Mu.l of an enzyme solution (TGF. beta. RI kinase 25nM in the final reaction) in kinase buffer (40mM Tris pH 7.5,20mM MgCl2,1mM DTT,1mg/ml BSA) was added to 384 well plates (Greiner, Cat # 784075). For negative control 2. mu.l kinase buffer was added. Compounds were formulated in DMSO as 10mM stock solutions and were diluted twice with DMSO as required for the initial concentration prior to testing compounds. Mu.l of compound was added to 47.5. mu.l of ddH2O and diluted by 3-fold dilution, with 10 concentration points for each compound. After the compound was diluted, 1. mu.l was added to the enzyme solution. After mixing well, incubation was performed at room temperature for 10min (DMSO concentration in the final reaction system was 1%). For negative and positive controls, 1 μ l of 5% DMSO in water was added. Finally, 2. mu.l of a mixture containing ATP and a TGF-beta RI kinase polypeptide substrate (Signalchem, Cat # T36-58) (final ATP concentration 3.5. mu.M, final substrate concentration 0.1. mu.g/ul) was added, mixed well and then reacted at 28 ℃ for 120 min. Finally, the assay was performed using the ADP-Glo KinaseAssay Kit (Promega, Cat # V9102). The final chemiluminescent signal was read using Molecular Devices, SpectraMax i3x. The resulting data were subjected to 4-parameter curve fitting using XLFIT and IC calculated₅₀。

Determination of the inhibitory effect of compounds on p38 α kinase activity:

p38 alpha kinase was purchased from SignalChem (Cat # M39-10 BG). Mu.l of kinase buffer (40mM Tris pH 7.5,20mM MgCl2,0.05mM DTT,0.1mg/ml B) was added to 384-well plates (Greiner, Cat #784075)SA) (0.8 ng/ul of P38 a kinase in the final reaction system). For negative control 2. mu.l kinase buffer was added. Compounds were formulated in DMSO as 10mM stock solutions and were diluted twice with DMSO as required for the initial concentration prior to testing compounds. Mu.l of compound was added to 47.5. mu.l of ddH2O and diluted by 3-fold dilution, with 10 concentration points for each compound. After the compound was diluted, 1. mu.l was added to the enzyme solution. After mixing well, incubation was performed at room temperature for 10min (DMSO concentration in the final reaction system was 1%). For negative and positive controls, 1 μ l of 5% DMSO in water was added. Finally, 2. mu.l of a mixture containing ATP and P38 kinase polypeptide substrate (Signalchem, Cat # P03-58) (ATP final concentration 50. mu.M, substrate final concentration 0.125. mu.g/. mu.l) was added thereto, mixed well and then reacted at 25 ℃ for 120 min. Finally, the Assay was performed using the ADP-Glo Kinase Assay Kit (Promega, Cat # V9102). The final chemiluminescent signal was read using Molecular Devices, SpectraMax i3x. The resulting data were subjected to 4-parameter curve fitting using XLFIT and IC calculated₅₀。

Results of the kinase activity inhibition of ALK5 and p38 α by the compounds listed in the examples:

reference compound is LY3200882, obtained by internal synthesis

Assay of inhibitory activity of compounds on TGF β RI receptor Smad signaling pathway:

HEK-Blue TGF. beta. cells (Invivogen, Cat # hkb-tgfb) were collected and cell density was adjusted to 1.25X 10 with DMEM 10% FBS⁶cells/ml and added to a 96-well plate at 40. mu.l per well. Compounds were formulated in DMSO as 10mM stock solutions and were diluted twice with DMSO as required for the initial concentration prior to testing compounds. 2.5. mu.l of a compound is combinedThe mixture was added to 1ml of complete medium, pipetted well and 3-fold diluted with DMEM medium containing 0.25% DMSO (DMSO concentration in 100. mu.l system: 0.1%). Mu.l of the prepared compound was applied to a 96-well plate, and 40. mu.l of a medium containing 0.25% DMSO was added to the positive control and the negative control, followed by incubation for 2 hours in a 5% CO2 incubator at 37 ℃. Mu.l of human recombinant TGF β 1(Invivogen, cat # Rcyc-htgfb1) was added to each well at a final concentration of 0.1 ng/ml. Incubated at 37 ℃ in 5% CO2 for 24 hours. After 24 hours 60. mu.l of the supernatant was taken for the secreted alkaline phosphatase (SEAP) assay and the cell plates were supplemented with 60ul/well of medium for cell viability assays. The SEAP Assay was performed using the Great Esecape SEAP cheminescence KIT (Clontech, Cat #631738), and the Cell Viability Assay was performed using the CellTiter-Glo luminescence Cell Viability Assay (Promega, Cat # G7573). The final chemiluminescent signal was read using Molecular Devices, SpectraMax i3x. The resulting data were subjected to 4-parameter curve fitting using XLFIT and IC calculated₅₀。

The results of the inhibitory activity of the compounds listed in the examples on the TGF β RI receptor Smad signaling pathway:

reference compound is LY3200882, obtained by internal synthesis

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, selected from: