CN112812111A

CN112812111A - Benzothiazole compound and medical application thereof

Info

Publication number: CN112812111A
Application number: CN202110217243.0A
Authority: CN
Inventors: 孙宏斌; 陈彩萍; 周鑫煜; 刘胜杰; 袁浩亮; 温小安
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-03-03
Filing date: 2021-02-26
Publication date: 2021-05-18
Anticipated expiration: 2041-02-26
Also published as: CN111763201A; CN112812111B; WO2021175234A1

Abstract

The present invention discloses benzothiazole compounds as shown in formula I and their medicinal uses, specifically refers to benzothiazole compounds of USP7 C-terminal domain regulators or pharmaceutically acceptable salts, esters or solvates and preparations thereof Methods and uses, the compounds of the present invention or their pharmaceutically acceptable salts or esters or solvates have strong binding force to the USP7 C-terminal protein, have a regulatory effect on the USP7 C-terminal domain, and have significant anti-tumor cell proliferation. The effect and anti-inflammatory activity can be used to prepare a drug for preventing or treating myelodysplastic syndrome, malignant tumor, inflammation or autoimmune disease.

Description

Benzothiazole compound and medical application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to benzothiazole compounds and application thereof in pharmacy, and particularly relates to benzothiazole compounds serving as a USP 7C-terminal domain regulator and application thereof in preventing and treating myelodysplastic syndrome, malignant tumor, inflammation or autoimmune disease.

Background

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal diseases of myeloid lineage that originate from hematopoietic stem cells and is characterized by abnormal differentiation and development of myeloid lineage cells, manifested by ineffective hematopoiesis, refractory cytopenia, and high risk of transformation into Acute Myeloid Leukemia (AML). The causes of death in patients are mainly complications from the disease itself and death following conversion to AML (Cancer 2010,16: 2174-. Statistically, the disease rate in the United states is about 0.004-0.005%, while the pre-disposed population is older men or people who have previously received chemotherapy (Blood 2008,112: 45-52).

MDS is divided into two types of high-risk and low-risk, and the classification is based on the proportion of immature cells in bone marrow and mutant gene analysis. The results were scored clinically using the International Prognostic Scoring System (IPSS) based on the bone marrow primary cell ratio, Blood cell number and type of genetic mutation (am.J. Hematol.2014,89: 98-108; Blood 1997,89: 2079-. The therapeutic goals for different types of MDS also vary. The therapeutic goals for low-risk MDS are to reduce transfusion demand, delay the process of transforming AML, and increase survival, while the therapeutic goals for high-risk MDS are to increase survival.

Clinically there are different treatment regimens for different types of MDS. For low-risk MDS patients, lenalidomide (N.Engl.J.Med.2006,355: 1456-containing 1465) or blood cell growth factor injection (J.Natl.cancer Inst.2008,100: 1542-containing 1551) is selected clinically as a first choice, and DNMT1 inhibitor is used for treatment if the patients are ineffective. For high-risk MDS patients, the DNMT1 inhibitor is the first standard therapy (Leukemia 2014,28: 1-14).

DNMT1(DNA methyltransferase 1) is an enzyme that transfers a methyl group to a cytosine nucleotide of genomic DNA and consists of 1616 amino acids and is structurally divided into a C-terminal catalytic region, an N-terminal regulatory region and an intermediate KG junction region. The C-terminal mainly plays the catalytic function of methylation, and the N-terminal mainly regulates the activity of a catalytic region through allosteric action, so as to control the interaction of DNMT1 and other proteins (prog.mol.biol.Transl.Sci.2011,101: 221-. The basic function of DNMT1 is to methylate newly synthesized DNA during the S phase of the cell cycle (Nature 2007,447: 396-398). In mammals, the time points of methylation are precise and fixed, while the control of the time points of methylation in humans is achieved by the modulation of the levels of DNMT1 protein: under the control of a series of transcriptions and post-transcriptional modifications, the protein level of DNMT1 varied with cell cycle changes, peaking early in S phase and then declining and culminating in G1 phase (sci. signal.2010,3: ra 80).

The protein level of DNMT1 is controlled by various post-transcriptional modifications: ubiquitination, acetylation (Sci. Signal.2011,4: pe 3; mol. cell biol.2011,31: 4720-. These mechanisms allow DNMT1 to be activated at the correct point in the cell cycle, while receiving the correct instructions to exert its DNA methylation. Among numerous post-transcriptional regulatory mechanisms, ubiquitination plays a crucial role in the stability of DNMT1 protein by directly mediating its degradation.

USP7 is a deubiquitinase, which is one of ubiquitin-specific proteases (USPs), and can efficiently hydrolyze ubiquitin chains on substrate proteins to deubiquitinate and stabilize target substrates. In distribution, USP7 is a nuclear protein which is mainly distributed at nuclear sites (nuclear dots) in the nucleus of a cell to exert its physiological functions. In mammals, USP7 is highly conserved in structure (up to 98.6% structural homology between human and murine USP 7), consisting of 1102 amino acids, with a relative molecular mass of about 135kDa (Cell 2009,138: 389-. USP7 can be divided into N-terminal TRAF-like domain (residual 53-206), catalytic domain (residual 208-560) and C-terminal UBL domain (residual 564-1084) according to the difference of amino acid sequence and function.

USP7 plays an important role in the protein stability, enzyme activity and target DNA recognition of DNMT 1. On the one hand, USP7 is a deubiquitinase of DNMT1, in which an acidic pocket consisting of four amino acid residues Glu736, Asp758, Glu759 and Asp764 in the C-terminal UBL1-2 region interacts with the KG junction region (residual 1109-1119) of DNMT1 (nat. Commun.2015,6:7023-7034) to deubiquitinate and stabilize the DNMT1 protein. USP7 plays an important role in the stability of DNMT1, knocking out USP7 on tool cell HEK293 resulted in a significant decrease in DNMT1 levels (sci. signal.2010,3: ra80), whereas in human colon cancer tissues, DNMT1 levels were positively correlated with USP7 levels (j. cell biochem.2011,112: 439-444). On the other hand, USP7 also had an effect on the enzymatic activity of DNMT 1. In vitro experiments show that the enzyme activity of DNMT1 is improved by two times when USP7 exists, and the effect is independent of the deubiquitinating enzyme activity of USP7 (Nucleic Acids Res.2011,39:8355-8365), which indicates that USP7 can also regulate the enzyme activity of DNMT1 through protein-protein interaction. In addition, USP7 mediates the binding of DNMT1 to the target DNA. DNMT1 by itself does not recognize hemimethylated target DNA sequences and requires first formation of a trimeric complex with USP7 and UHRF1 to effect demethylation. In the trimer complex, the N-terminus of USP7 binds to UHRF1, the C-terminus of USP7 binds to the N-terminal TS region of DNMT1, and UHRF1 binds to the N-terminal RFTS region of DNMT 1. After the trimer complex is formed, the SDR region of UHRF1 can specifically recognize the CpG island of DNA hemimethylation, and the DNMT1-UHRF1-USP7 complex is further drawn to a DNA target region to play a role (Nucleic Acids Res.2011,39: 8355-. Therefore, the inhibition of DNMT1 by interfering the interaction of the C-terminal domain of USP7 and DNMT1 is a potential therapeutic approach for diseases such as myelodysplastic syndrome and malignant tumors.

In addition, the research shows that USP7 can stabilize NF kappa B through deubiquitination, and further promote the transcriptional regulation of NF kappa B. Interestingly, the above-mentioned "acidic pocket" at the C-terminus of USP7 (especially amino acids 757-760) is also important for USP7 to recognize and bind NF-. kappa.B (J.biol.chem.295(33): 11754-11763). Therefore, pharmacological intervention of the C-terminal domain of USP7 can also become a new treatment way for inflammation and autoimmune diseases.

In conclusion, the USP 7C-terminal regulator has potential important medical value. However, no small molecule regulator compound of USP 7C-terminal protein has been reported so far, and the development of the USP 7C-terminal domain small molecule regulator with high activity and small toxic and side effects has important significance.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a benzothiazole compound, which can be used as a USP 7C-terminal regulator, has strong binding force with USP 7C-terminal protein, can reduce the protein level of DNMT1 in tumor cells, has obvious anti-tumor cell proliferation effect, and can inhibit the deubiquitination of the USP7 to NF kappa B.

The invention also provides a preparation method, a pharmaceutical composition and a medical application of the benzothiazole compound and an intermediate thereof.

The technical scheme is as follows: in order to achieve the above objects, the present invention provides a benzothiazole compound represented by formula I or a pharmaceutically acceptable salt, ester or solvate thereof:

x is methylene, carbonyl or sulfonyl;

y is hydrogen or XR¹；

R¹、R²Each independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, or substituted or unsubstituted heterocycloaryl.

R³Is hydrogen, hydroxy, heterocyclyl, alkyl, NH₂、NO₂、COOH、CN、SH、CF₃、SO₃H、SO₂CH₃Or a halogen.

Preferably, in the benzothiazole compound shown in the formula I or the pharmaceutically acceptable salt, ester or solvate thereof, X is methylene, carbonyl or sulfonyl;

y is hydrogen or XR¹；

R¹Is substituted alkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted benzyl, substituted or unsubstituted heteroarylmethyl, substituted or unsubstituted aryl or heteroaryl;

R²is substituted and unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl;

R³is hydrogen, hydroxyl or heterocyclic radical.

Further, in the benzothiazole compound shown in the formula I or the pharmaceutically acceptable salt, ester or solvate thereof, X is sulfonyl;

y is hydrogen or XR¹；

R²is a substituted or unsubstituted heterocycloalkyl;

R³is hydrogen.

In certain preferred embodiments, the benzothiazole compounds of the present invention are compounds represented by formula II or III below or pharmaceutically acceptable salts or solvates thereof:

y is hydrogen or SO₂R¹；

In certain more preferred embodiments, the benzothiazole compounds of the present invention are any of the following compounds in table 1, or a pharmaceutically acceptable salt or ester or solvate thereof:

TABLE 1 partial Compounds of the invention

The invention provides application of benzothiazole compounds shown in formulas I to III or pharmaceutically acceptable salts, esters or solvates thereof in preparation of a USP 7C-terminal regulator. The inventor finds that the benzothiazole compounds shown in formulas I to III can be combined with the C end of USP and cause the conformational change of the C end of the USP, and the benzothiazole compounds are the first USP7C end small molecule modulators disclosed so far.

The invention also provides application of the benzothiazole compounds shown in the formulas I to III or pharmaceutically acceptable salts, esters or solvates thereof in preparing medicaments for preventing or treating inflammation, autoimmune diseases, myelodysplastic syndrome and malignant tumors.

Such inflammatory, autoimmune diseases include, but are not limited to: ulcerative colitis, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, multiple sclerosis or Behcet's disease.

Such tumors include, but are not limited to: bone cancer, hematologic cancer, cancer of the nervous system, gastrointestinal tumor, cancer of the urinary system, lung cancer, liver cancer or skin cancer.

Such bone cancers include, but are not limited to: osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, ewing's sarcoma, malignant lymphoma (reticulosarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (flexible tubular exogenic condyloma), benign chondroma, chondroblastoma, cartilage and tumor-like fibroma, osteoid osteoma, and giant cell tumor. Such hematologic cancers include, but are not limited to: acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma and myelodysplastic syndrome, hodgkin's lymphoma (malignant lymphoma), and waldenstrom's macroglobulinemia. Such cancers of the nervous system include, but are not limited to: meningeal cancers, such as meningioma, meningeal sarcoma, and glioma; brain cancers such as astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; spinal myelomas such as fibroneuroma, meningioma, glioma and sarcoma. Such gastrointestinal tumors include, but are not limited to: esophageal cancers such as squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma; stomach cancers, such as tumors, lymphomas, and leiomyosarcomas; pancreatic cancers such as ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, and vasoactive intestinal peptide tumor; small bowel cancers such as adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma; large bowel cancers such as adenocarcinoma, ductal adenocarcinoma, villous adenoma, hamartoma and leiomyoma. The urological cancers include, but are not limited to: kidney cancers such as adenocarcinoma, wilms' tumor (nephroblastoma), lymphoma, and leukemia; bladder and urinary tract cancers, such as squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma; prostate cancer such as adenocarcinoma and sarcoma; testicular cancer, such as seminoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, and lipoma. The lung cancers include, but are not limited to: bronchial carcinomas such as squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, and adenocarcinoma; bronchioloalveolar carcinoma; bronchial adenoma; a sarcoma; lymphoma; pulmonary chondromatous hamartomas and mesotheliomas. The liver cancer includes but is not limited to: hepatocellular carcinoma, such as hepatocellular carcinoma; bile duct cancer; hepatoblastoma; angiosarcoma; hepatocellular adenoma and hemangioma. Such skin cancers include, but are not limited to: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis.

In certain embodiments, the compounds of the present invention may be used in combination with one or more other types of agents for the prophylaxis or treatment of the above-mentioned diseases, including but not limited to the following combinations:

other types of prophylactic or therapeutic drugs that may be selected for use in combination with the compounds of the present invention may be one or more anticancer drugs, including alkylating agents (e.g., cisplatin, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bendamustine, estramustine, thiotepa, iminoquinone, busulfan, dibromomannitol, lomustine, carmustine, pyrimidine nitrosourea, methylcyclonitrosourea, cimetidine, procarbazine, and the like), antimetabolites (e.g., fluorouracil, cytarabine, fururouracil, difurofluorouracil, mercaptopurine sodium, azathioprine, thioguanine, methotrexate, aminopterin, and the like), antitumor antibiotics (e.g., mitomycin C, bleomycin, actinomycin D, mithramycin, daunorubicin, doxorubicin, chromomycin A3, enramycin, neocarzinone, anticancer, antibiotic, daunomycin, and the like), Natural anticancer drugs (e.g., vincristine, colchicine, camptothecin, hydroxycamptothecin, cantharidin, indirubin, etc.), hormonal drugs (e.g., prednisone, prednisolone, hydrocortisone, dexamethasone, diethylstilbestrol, bromoacetylhexol estrol, testosterone propionate, methyltestosterone, nandrolone phenylpropionate, naproxen, tamoxifen, etc.), immunotherapeutic agents (e.g., PD-1 inhibitors nivolumab and pembrolizumab, etc.; PD-L1 inhibitors atezolizumab, durvalumab, and avelumab, etc.; CTLA-4 inhibitors Iplilimumab, etc.; other immunotherapeutic drugs such as cellular therapeutic agents), antibody drug conjugates (e.g., Kadcyla, etc.), kinase inhibitors (e.g., SHP-2 inhibitors, B-RAF inhibitors, MEK inhibitors, Btk inhibitors, etc.), IDO inhibitors (e.g., Epacadostat), etc.

The invention also provides a pharmaceutical composition for preventing or treating inflammation, autoimmune diseases, myelodysplastic syndrome and tumors, which contains a therapeutically effective amount of any one of benzothiazole compounds shown in formulas I to III or pharmaceutically acceptable salts or solvates thereof as an active ingredient and pharmaceutically acceptable auxiliary materials. Excipients which may be optionally blended may vary depending on the dosage form, administration form, etc., and the carriers include, but are not limited to, excipients, binders, disintegrants, lubricants, flavoring agents, coloring agents, sweetening agents, etc. The pharmaceutical composition can be in the form of ordinary tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, granules, powder, syrup, oral liquid, injection and other preparations which are conventional in pharmaceutics.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the benzothiazole compound or the pharmaceutically acceptable salt, ester or solvate thereof has strong binding force with USP 7C-terminal protein, is a first USP 7C-terminal small molecule regulator disclosed so far, can reduce the protein level of DNMT1 in tumor cells, has obvious anti-tumor cell proliferation effect, and can be used for preparing medicines for preventing or treating myelodysplastic syndrome and malignant tumors. In addition, the USP 7C-terminal small molecule regulator can also block the combination of the USP7C terminal and the NF kappa B, inhibit the deubiquitination of the NF kappa B and have obvious anti-inflammatory activity, so the regulator can also be used for preparing anti-inflammatory drugs and drugs for treating autoimmune diseases.

(2) The benzothiazole compound or the pharmaceutically acceptable salt or ester or solvate thereof has good drug forming property, good metabolic stability in human liver microsomes, and good drug forming property, and can be orally absorbed.

(3) The benzothiazole compound has the advantages of simple structure, ingenious design of a synthetic route, cheap and easily-obtained raw materials, safe and environment-friendly synthetic process and easy large-scale production.

Drawings

FIG. 1 is a structural diagram of the X-ray co-crystal of Compound 25 with the C-terminal protein of USP 7;

FIG. 2 is a graph showing the results of GST Pull-down experiments in which compounds affect the interaction of USP 7C-terminal protein with DNMT 1;

FIG. 3 is a graph showing the results of a Western Blot experiment in which concentration-dependent reduction of DNMT1 levels in NB4 was achieved with compound 55;

FIG. 4 is a graph showing the results of a Western Blot experiment in which concentration-dependent reduction of DNMT1 levels in NB4 was achieved with compound 60;

FIG. 5 is a graph showing the effect of compound 55 on LPS-induced IL-1b and IL-6 expression on Raw264.7 cells.

Detailed Description

The present invention will be described in detail with reference to examples. In the present invention, the following examples are given for better illustration of the present invention and are not intended to limit the scope of the present invention. Various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

Example 1

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) isonicotinamide (Compound 1)

2-amino-6-nitrobenzothiazole (1a,1.0g,5.13mmol) was dissolved in pyridine (20mL), fresh furoyl chloride (758. mu.L, 7.70mmol) was added dropwise, and after the addition was complete, the mixture was warmed to 40 ℃ and stirred for 8 hours. After completion of the reaction monitored by TLC, 1N hydrochloric acid (30mL) was added for neutralization, and a large amount of solid was precipitated. After suction filtration, the solid was washed with a small amount of ethyl acetate (2 mL. times.3) to give the crude intermediate 1b, which was directly used in the next step without purification.

All of the resulting crude intermediate 1b was added to methanol (25mL), 10% palladium on carbon (100mg) was added, and the mixture was stirred at 50 ℃ for 7 hours after passing hydrogen gas. After TLC monitoring reaction, stopping heating, cooling to room temperature, then performing suction filtration on the solution by using kieselguhr, and evaporating the solvent from the filtrate under reduced pressure to obtain a compound 1c (brown solid, 520mg, two-step yield 39%):¹H NMR(300MHz,DMSO-d₆)δ12.37(s,1H),8.00(s,1H),7.65(d,J＝3.6Hz,1H),7.44(d,J＝8.5Hz,1H),7.05(d,J＝2.1Hz,1H),6.92–6.52(m,2H),5.17(s,2H).ESI-MS m/z 258.0[M-H]^-。

compound 1c (100mg,0.39mmol) was added to dichloromethane (3mL) (suspension), and isonicotinic acid (52mg,0.47mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (96mg,0.47mmol), and 4-dimethylaminopyridine (47mg,0.47mmol) were added in this order and stirred at room temperature overnight. A large amount of a grey solid was observed to precipitate, TLC monitored the reaction completed and then stopped stirring, filtered and the filter cake washed with a small amount of dichloromethane (1mL × 2) to give compound 1 (off-white solid, 91mg, 65% yield):¹H NMR(500MHz,DMSO-d₆)δ12.85(s,1H),10.68(s,1H),8.95–8.77(m,2H),8.50(s,1H),8.07(d,J＝1.6Hz,1H),8.01–7.87(m,2H),7.83–7.74(m,3H),6.79(dd,J＝3.6,1.7Hz,1H).ESI-MS m/z 363.1[M-H]^-。

example 2

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) picolinamide (Compound 2)

Referring to the procedure of example 1, substituting isonicotinic acid for picolinic acid, compound 2 (off-white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.80(s,1H),10.80(s,1H),8.77(d,J＝4.7Hz,1H),8.60(s,1H),8.20(d,J＝7.9Hz,1H),8.15–8.01(m,2H),7.95(d,J＝8.9Hz,1H),7.84–7.60(m,3H),6.77(s,1H).ESI-MS m/z 363.1[M-H]^-。

example 3

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) pyrazine-2-carboxamide (Compound 3)

Referring to the procedure of example 1, substituting isonicotinic acid for pyrazine-2-carboxylic acid yielded compound 3 (off-white solid):¹H NMR(300MHz,DMSO-d₆)δ12.83(s,1H),10.88(s,1H),9.33(s,1H),8.95(s,1H),8.83(s,1H),8.57(s,1H),8.04(s,1H),7.94(d,J＝8.9Hz,2H),7.86–7.48(m,2H),6.76(s,1H).ESI-MS m/z 364.1[M-H]^-。

example 4

(±) -N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) piperidine-3-carboxamide (Compound 4)

Compound 1c (20mg,0.08mmol) was dissolved in tetrahydrofuran (2mL), followed by addition of N-t-butoxycarbonylpiperidine-3-carboxylic acid 4a (21mg,0.09mmol), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,35mg,0.09mmol), and N, N-diisopropylethylamine (20. mu.L, 0.12mmol), and stirred at room temperature overnight. TLC (thin layer chromatography) was used to monitor the completion of the reaction, stirring was stopped, the solvent was evaporated under reduced pressure, dichloromethane (10mL) was added to dissolve the solvent, the organic phase was washed with water (5mL), anhydrous sodium sulfate was added to dry the mixture for 30 minutes, and the mixture was filtered to prepare sand. Column chromatography on silica gel (dichloromethane: methanol 300:1) afforded compound 4b (white solid, 20mg, 54% yield).

Compound 4b was placed in an eggplant-shaped bottle, and a dichloromethane solution of trifluoroacetic acid (trifluoroacetic acid: dichloromethane ═ 1:2, 3mL) was added thereto, followed by stirring at room temperature for 0.5 hour. Stopping stirring after TLC monitoring reaction is completed, evaporating the solvent under reduced pressure, adding dichloromethane (2mL), dropwise adding a saturated sodium hydrogen carbonate solution until the solution is neutral, separating out a solid, performing suction filtration, and drying to obtain a compound 4 (a white solid, 10mg, yield 54%);¹H NMR(300MHz,Methanol-d₄)δ8.28(d,J＝2.0Hz,1H),7.84(dd,J＝1.8,0.8Hz,1H),7.71(d,J＝8.7Hz,1H),7.58–7.35(m,2H),6.71(dd,J＝3.6,1.7Hz,1H),3.10–3.30(m,2H),2.95–3.05(m,1H),2.75–2.95(m,1H),2.05–2.15(m,1H),1.85–2.05(m,2H),1.65–1.85(m,1H).ESI-MS m/z 369.1[M-H]^-。

example 5

(±) -N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) piperidine-2-carboxamide (Compound 5)

Referring to the procedure of example 4, N-tert-butoxycarbonylpiperidine-3-carboxylic acid was replaced with N-tert-butoxycarbonylpiperidine-2-carboxylic acid to obtain compound 5 (white solid):¹H NMR(300MHz,Methanol-d₄)δ8.33(d,J＝2.1Hz,1H),7.86(d,J＝1.6Hz,1H),7.75(d,J＝8.7Hz,1H),7.58(dd,J＝8.8,2.1Hz,1H),7.47(d,J＝3.6Hz,1H),6.72(dd,J＝3.6,1.8Hz,1H),4.04(dd,J＝11.6,3.1Hz,1H),3.47(d,J＝13.1Hz,1H),3.23–2.99(m,1H),2.39(d,J＝12.4Hz,1H),2.06–1.70(m,5H).ESI-MS m/z369.1[M-H]^-。

example 6

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) piperidine-4-carboxamide (Compound 6)

Referring to the procedure of example 4, N-tert-butoxycarbonylpiperidine-3-carboxylic acid was replaced with N-tert-butoxycarbonylpiperidine-4-carboxylic acid to obtain compound 6 (white solid):¹H NMR(300MHz,DMSO-d₆)δ10.14(s,1H),8.32(s,1H),8.03(s,1H),7.70(d,J＝7.2Hz,2H),7.55(d,J＝9.0Hz,2H),6.86–6.66(m,1H),3.37–3.4(m,1H),2.99–2.87(m,2H),2.75–2.58(m,2H),1.92–2.05(m,2H),1.75–1.92(m,2H).ESI-MS m/z 369.1[M-H]^-。

example 7

N- (6- (4-aminobenzoyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 7)

P-aminobenzoic acid (7a, 493mg, 3.60mmol) was dissolved in a mixed solution of dioxane and water (dioxane: water ═ 2:1, 10mL), triethylamine (1mL, 7.30mmol) was added dropwise at room temperature, and after stirring for 5 minutes, Boc anhydride (1.6g,7.30mmol) was added, and the mixture was stirred at room temperature for 36 hours. After completion of the reaction monitored by TLC, 2N hydrochloric acid was added to adjust the solution to pH 5-6, and a large amount of solid precipitated. Filtration with suction and drying of the solid gave compound 7b (white solid, 708mg, 83% yield).

Compound 1c (100mg,0.39mmol) was added to dichloromethane (5mL) (as a suspension), and intermediate 7b (103mg,0.47mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (96mg,0.47mmol), and 4-dimethylaminopyridine (47mg,0.47mmol) were added in this order and stirred at room temperature overnight. A large amount of white solid was observed to precipitate, TLC monitored that the reaction was complete and then stopped stirring, suction filtered, and the filter cake was washed with a small amount of dichloromethane/methanol mixed solution (1 mL. times.2) to afford compound 7c (an off-white solid).

All of compound 7c was placed in an eggplant-shaped bottle, and a dichloromethane solution of trifluoroacetic acid (trifluoroacetic acid: dichloromethane ═ 1:2, 6mL) was added thereto, followed by stirring at room temperature for 1 hour. After the reaction was monitored by TLC, a saturated sodium bicarbonate solution was added to the system until neutral, a large amount of solid precipitated, filtered, and the filter cake was washed with ethyl acetate (0.5mL × 3) and dried to give compound 7 (white solid, 78mg, two-step yield 53%):¹H NMR(300MHz,DMSO-d₆)δ12.75(s,1H),9.88(s,1H),8.39(d,J＝2.0Hz,1H),7.99(s,1H),7.89–7.49(m,4H),6.83–6.49(m,3H),5.74(s,2H).ESI-MS m/z 377.1[M-H]^-。

example 8

N- (6- (3-aminobenzoyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 8)

Referring to the procedure of example 7, compound 8 (white solid) was prepared by substituting p-aminobenzoic acid with m-aminobenzoic acid:¹H NMR(300MHz,DMSO-d₆)δ12.78(s,1H),10.23(s,1H),8.46(s,1H),8.05(s,1H),7.93–7.55(m,3H),7.25–7.00(m,3H),6.77(d,J＝5.0Hz,2H),5.31(s,2H).ESI-MS m/z 377.1[M-H]^-。

example 9

N- (6- (4-cyanobenzoyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 9)

Referring to the procedure of example 1, substituting isonicotinic acid for 4-cyanobenzoic acid, compound 9 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.81(s,1H),10.64(s,1H),8.47(s,1H),7.90–8.30(m,5H),7.60–7.90(m,3H),6.76(s,1H).ESI-MS m/z 387.1[M-H]^-。

example 10

N- (6- (4-trifluoromethylbenzoyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 10)

Referring to the procedure of example 1, substituting isonicotinic acid for 4-trifluoromethylbenzoic acid, compound 10 (grey solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.81(s,1H),10.63(s,1H),8.48(s,1H),8.19(d,J＝8.1Hz,2H),8.05(d,J＝1.7Hz,1H),7.94(d,J＝8.1Hz,2H),7.85–7.65(m,3H),6.77(dd,J＝3.7,1.7Hz,1H).ESI-MS m/z 430.1[M-H]^-。

example 11

N- (6- (2, 4-difluorobenzoyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 11)

With reference to the procedure of example 1, isonicotinic acid was replaced with 2, 4-difluorobenzoic acid to obtain compound 11 (white solid):¹H NMR(300MHz,DMSO-d₆)δ12.82(s,1H),10.58(s,1H),8.45(d,J＝2.0Hz,1H),8.06(d,J＝1.7Hz,1H),7.91–7.63(m,4H),7.44(td,J＝9.9,2.4Hz,1H),7.25(td,J＝8.5,2.4Hz,1H),6.77(dd,J＝3.6,1.7Hz,1H).ESI-MS m/z 398.0[M-H]^-。

example 12

N- (6- (3-fluoro-4-chlorobenzoyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 12)

Referring to the procedure of example 1, substituting isonicotinic acid for 3-fluoro-4-chlorobenzoic acid, compound 12 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.72(s,1H),10.56(s,1H),8.47(s,1H),8.05(s,2H),8.00–7.40(m,6H),6.77(s,1H).ESI-MS m/z 414.0[M-H]^-。

example 13

N- (6- (4-piperazin-1-yl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 13)

Ethyl p-fluorobenzoate (13a,100mg,0.59mmol) was dissolved in dry dimethyl sulfoxide (1mL), 1-Boc-piperazine (13b,121mg,0.65mmol) and potassium carbonate (246mg,1.78mmol) were sequentially added, stirred at 120 ℃ for 5h, TLC monitored for completion of the reaction, saturated brine (5mL) was added, and extracted with ethyl acetate (5 mL. times.3). The organic phases were combined, dried over anhydrous sodium sulfate for 30 minutes, then chromatographed on a silica gel column (ethyl acetate: petroleum ether ═ 8:1) to give compound 13c (off-white solid, 141mg, 78% yield).

Compound 13c (100mg,0.30mmol) was added to 2N aqueous sodium hydroxide (5mL), stirred at 80 ℃ for 24 hours, the solution was clarified, TLC monitored for completion of the reaction and then 2N hydrochloric acid was added to adjust the pH to 5-6, and a large amount of solid precipitated. Carrying out suction filtration, and collecting and drying a filter cake; the filtrate was extracted with ethyl acetate (10 mL. times.3), the organic phase was dried over anhydrous sodium sulfate and then dried by rotary drying, and the combined organic phase and dried cake were combined to give compound 13d (white solid, 73mg, yield 79%).

Compound 13 (white solid) was prepared by substituting compound 7b for compound 13d according to the procedure of example 7:¹H NMR(300MHz,DMSO-d₆)δ10.07(s,1H),8.42(s,1H),8.00(s,1H),7.91(d,J＝8.5Hz,2H),7.80–7.51(m,3H),7.02(d,J＝8.6Hz,2H),6.74(s,1H),3.35–3.15(m,5H),2.88(s,4H).ESI-MS m/z 448.2[M+H]⁺。

example 14

N- (6- (4-Methylsulfonylbenzenesulfonyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 14)

Referring to the procedure of example 1, substituting isonicotinic acid for 4-methylsulfonylbenzoic acid, compound 14 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.82(s,1H),10.66(s,1H),8.49(s,1H),8.22(d,J＝8.2Hz,2H),8.11(d,J＝8.2Hz,2H),8.06(d,J＝1.7Hz,1H),7.85–7.65(m,3H),6.77(dd,J＝3.6,1.7Hz,1H),3.31(s,3H).ESI-MS m/z 440.1[M-H]^-。

example 15

N- (6- (4-Methylsulfonylbenzenesulfonyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 15)

Compound 2(50mg,0.14mmol) was dissolved in N, N-dimethylformamide (3mL), methyl iodide (86. mu.L, 1.37mmol) was added dropwise at room temperature, and after the addition was completed, the mixture was stirred at room temperature for 1 hour. TLC monitored the reaction was completed and then the stirring was stopped, and after the solvent was evaporated under reduced pressure, the obtained solid was washed with dichloromethane (2mL × 2) to obtain compound 15 (white solid, 61mg, yield 88%):¹H NMR(300MHz,DMSO-d₆)δ12.75(s,1H),10.92(s,1H),9.45(s,1H),9.05(d,J＝6.1Hz,1H),8.97(d,J＝8.1Hz,1H),8.38(d,J＝2.0Hz,1H),8.22(dd,J＝8.1,6.0Hz,1H),8.07–7.91(m,1H),7.83–7.55(m,3H),6.69(dd,J＝3.6,1.7Hz,1H),4.36(s,3H).ESI-MS m/z 379.1[M-H]^-。

example 16

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) benzo [1,2,5] dioxazole-5-carboxamide (Compound 16)

Referring to the procedure of example 1, substituting isonicotinic acid for 2,1, 3-benzooxadiazole-5-carboxylic acid, compound 16 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.83(s,1H),10.80(s,1H),8.72(s,1H),8.50(s,1H),8.20(d,J＝9.4Hz,1H),8.03(d,J＝9.6Hz,2H),7.60–7.93(m,3H),6.81–6.70(m,1H).ESI-MS m/z 404.0[M-H]^-。

example 17

N- (6- (furan-3-carboxamide) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 17)

Referring to the procedure of example 1, substituting isonicotinic acid for 3-furoic acid, compound 17 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.82(s,1H),10.10(s,1H),8.40(s,2H),8.05(d,J＝1.7Hz,1H),7.91–7.59(m,4H),7.03(dd,J＝2.0,0.8Hz,1H),6.77(dd,J＝3.6,1.7Hz,1H).MS(ESI)m/z 376.1[M+Na]⁺。

example 18

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) isoquinoline-3-carboxamide (Compound 18)

Referring to the procedure of example 1, substituting isonicotinic acid for isoquinoline-3-carboxylic acid, compound 18 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.74(s,1H),10.95(s,1H),8.88(d,J＝8.5Hz,1H),8.78–8.47(m,2H),8.22–7.96(m,3H),7.96–7.53(m,5H),6.87–6.64(m,1H).ESI-MS m/z 413.1[M-H]^-。

example 19

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) quinoline-3-carboxamide (Compound 19)

By substituting isonicotinic acid with quinoline-3-carboxylic acid according to the procedure of example 1, compound 19 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.83(s,1H),11.00(s,1H),9.43(d,J＝2.3Hz,1H),9.12(d,J＝2.2Hz,1H),8.56(d,J＝2.2Hz,1H),8.23–8.08(m,2H),8.05(d,J＝1.6Hz,1H),7.85–7.95(m,2H),7.84–7.68(m,3H),6.76(dd,J＝3.6,1.7Hz,1H).ESI-MS m/z 413.1[M-H]^-。

example 20

N- (6- (4-Nitrophenylacetamido) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 20)

By substituting isonicotinic acid for p-nitrophenylacetic acid, according to the procedure of example 1, compound 20 (yellow solid) was obtained:¹H NMR(300MHz,DMSO-d₆)δ12.83(s,1H),10.52(s,1H),8.32(s,1H),8.22(d,J＝8.2Hz,2H),8.05(s,1H),7.48–7.80(m,4H),6.76(s,1H),3.88(s,2H).ESI-MS m/z 421.1[M-H]^-。

example 21

N- (6- (4-Methylphenylacetamido) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 21)

With reference to the procedure of example 1, isonicotinic acid was substituted for p-methylphenylacetic acid to obtain compound 21 (white solid):¹H NMR(300MHz,DMSO-d₆)δ12.79(s,1H),10.48(s,1H),8.34(d,J＝1.9Hz,1H),8.05(d,J＝1.6Hz,1H),7.83–7.52(m,3H),7.39–7.01(m,4H),6.76(dd,J＝3.6,1.7Hz,1H),3.63(s,2H),2.28(s,3H).ESI-MS m/z 414.1[M+Na]⁺。

example 22

N- (6- ((4-Nitrophenylethyl) amino) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 22)

Compound 1c (50mg,0.19mmol) was dissolved in N, N-dimethylformamide (2mL), p-nitrobenzyl bromide (22a, 46mg,0.21mmol) and potassium carbonate (50mg,0.39mmol) were added in this order, and the mixture was stirred at room temperature overnight. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Ethyl acetate (10mL) was added to the system to dissolve the solid, and the organic layer was washed with saturated brine (5)mL), dried over anhydrous sodium sulfate for 0.5 hour, filtered, and the filtrate was subjected to silica gel column chromatography (dichloromethane: methanol 400:1) to give compound 22 (golden yellow solid, 14mg, yield 19%):¹H NMR(300MHz,DMSO-d₆)δ8.21(d,J＝8.3Hz,2H),7.88(s,1H),7.60(d,J＝8.4Hz,2H),7.41–7.21(m,2H),7.01(d,J＝2.1Hz,1H),6.8–6.52(m,2H),5.74–5.86(m,2H),5.33(s,2H).ESI-MS m/z 417.1[M+Na]⁺。

example 23

N- (6- ((4-Nitrophenyl) sulfonamido) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 23)

Compound 1c (50mg,0.19mmol) was placed in a bottle shaped like a eggplant, methylene chloride (3mL) was added to make a suspension, paranitrobenzoyl chloride (23a,43mg,0.21mmol) was added thereto, triethylamine (32. mu.L, 0.21mmol) was added dropwise to the reaction system under stirring, and after the addition was completed, the reaction was carried out at room temperature overnight, and a large amount of solid was precipitated. TLC monitored the reaction completed and stopped stirring, filtered and the filter cake was washed with a small amount of dichloromethane/methanol to give compound 23 (yellow solid, 39mg, 48% yield):¹H NMR(300MHz,DMSO-d₆)δ12.83(s,1H),10.66(s,1H),8.42–8.30(m,2H),8.08–7.93(m,3H),7.69–7.77(m,2H),7.65(d,J＝8.6Hz,1H),7.15(dd,J＝8.7,2.2Hz,1H),6.76(dd,J＝3.7,1.7Hz,1H).ESI-MS m/z 467.1[M+Na]⁺。

example 24

4- (N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) sulfonamide) benzoic acid (Compound 24)

Compound 1c (100mg,0.38mmol) was placed in a solanaceous flask, tetrahydrofuran (4mL) solution was added to make a suspension, ethyl 4- (chlorosulfonyl) benzoate (24a,105mg,0.42mmol) was added thereto, N-diisopropylethylamine (200 μ L,0.42mmol) was added dropwise to the reaction system under stirring, and after the addition was completed, the reaction was allowed to proceed overnight at room temperature, and a large amount of solid was precipitated. TLC monitored the reaction completion and stopped stirring, suction filtration, filter cake with dichloromethane/methanol mixed solution (1mL dichloromethane with 5 drops of methanol) to obtain 24b crude product.

All the crude products 24b were placed in a 10mL eggplant-shaped bottle, an aqueous solution (3mL) of sodium hydroxide having a mass fraction of 8% was added thereto, and the mixture was stirred at 40 ℃ for 2 hours to clarify the solution. After TLC monitoring reaction is completed, stopping heating, dropwise adding 1N hydrochloric acid into the reaction system to adjust to be neutral, precipitating a large amount of solid, and performing suction filtration to obtain a compound 24 (white solid, 43mg, two-step yield 28%):¹H NMR(300MHz,DMSO-d₆)δ12.87(s,2H),10.47(s,1H),8.06(d,J＝8.6Hz,3H),7.86(d,J＝8.1Hz,2H),7.78–7.58(m,3H),7.15(d,J＝8.7Hz,1H),6.76(s,1H).ESI-MS m/z 442.0[M-H]^-。

example 25

N- (6- (Thienylsulfonylamino) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 25)

Referring to the procedure of example 23, substituting p-nitrobenzoyl chloride for 2-thiophenesulfonyl chloride yielded compound 25 (light yellow solid):¹H NMR(300MHz,DMSO-d₆)δ12.81(s,1H),10.46(s,1H),8.04(d,J＝1.6Hz,1H),7.87(dd,J＝5.0,1.4Hz,1H),7.79–7.59(m,3H),7.53(dd,J＝3.8,1.4Hz,1H),7.20(dd,J＝8.7,2.2Hz,1H),7.10(dd,J＝5.0,3.8Hz,1H),6.75(dd,J＝3.6,1.7Hz,1H).ESI-MS m/z 404.0[M-H]^-。

example 26

N- (6- ((4-Methanesulfonylphenyl) sulfonamido) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 26)

By substituting p-nitrobenzoyl chloride for p-methanesulfonyl benzenesulfonyl chloride according to the procedure of example 23, Compound 26 (I)Light yellow solid):¹H NMR(300MHz,DMSO-d₆)δ10.66(s,1H),8.09(d,J＝8.3Hz,2H),8.05–7.95(m,3H),7.76(d,J＝2.2Hz,1H),7.71(d,J＝3.7Hz,1H),7.63(d,J＝8.6Hz,1H),7.17(dd,J＝8.7,2.2Hz,1H),6.74(dd,J＝3.6,1.7Hz,1H),3.25(s,3H).ESI-MS m/z 478.0[M+H]^-。

example 27

(±) -N- (2- (tetrahydrofuran-2-carboxamide) benzo [ d ] thiazol-6-yl) nicotinamide (Compound 27)

Compound 1a (100mg,0.51mmol) was dissolved in tetrahydrofuran (3mL) and 2-tetrahydrofuranic acid 27a (71mg,0.61mmol), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,232mg,0.61mmol) and N, N-diisopropylethylamine (134. mu.L, 0.77mmol) were added in that order and stirred at 40 ℃ for 6 hours. After the reaction was monitored by TLC, stirring was stopped, the solvent was distilled off under reduced pressure, and then ethyl acetate (3mL) was added for beating, followed by suction filtration to obtain compound 27b (white solid, 154mg, yield 97%):¹H NMR(300MHz,DMSO-d₆)δ12.66(s,1H),9.07(s,1H),8.29(d,J＝8.0Hz,1H),7.92(d,J＝8.5Hz,1H),4.64(t,J＝6.4Hz,1H),4.00(d,J＝7.2Hz,1H),3.85(d,J＝7.2Hz,1H),2.35–2.20(m,1H),2.17–1.77(m,3H)。

compound 27b (250mg,0.85mmol) was dissolved in methanol (25mL), 10% palladium on carbon (25mg) was added, hydrogen was introduced, and the mixture was stirred at 50 ℃ for 9 hours. After the TLC monitoring reaction was completed, heating was stopped, the solution was cooled to room temperature, filtered with celite, and the filtrate was evaporated under reduced pressure to remove the solvent to prepare sand, and silica gel column chromatography (dichloromethane: methanol: 200:1) was performed to obtain compound 27c (gray solid, 100mg, yield 43%):¹H NMR(300MHz,DMSO-d₆)δ11.67(s,1H),7.33(d,J＝8.6Hz,1H),6.92(d,J＝2.2Hz,1H),6.62(dd,J＝8.6,2.3Hz,1H),5.09(s,2H),4.44(dd,J＝8.2,5.1Hz,1H),4.09–3.84(m,1H),3.82–3.58(m,1H),2.20–2.05(m,1H),1.88–1.60(m,3H)。

compound 27c (25mg,0.10mmol) was added to dichloromethane (2mL), followed by nicotinic acid (14mg,0.11mmol)mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (24mg,0.11mmol) and 4-dimethylaminopyridine (14mg,0.11mmol), and stirred at room temperature overnight. After completion of the reaction monitored by TLC, the reaction was stopped, and the mixture was diluted with dichloromethane (5mL), washed once with saturated brine (3mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and subjected to silica gel column chromatography (dichloromethane: methanol: 200:1) to obtain compound 27 (white solid, 31mg, yield 84%):¹H NMR(300MHz,DMSO-d₆)δ11.89(s,1H),10.51(s,1H),9.05(d,J＝2.2Hz,1H),8.69(dd,J＝4.9,1.6Hz,1H),8.38(s,1H),8.23(dt,J＝8.1,1.9Hz,1H),7.67(s,1H),7.50(dd,J＝8.0,4.8Hz,1H),4.50(dd,J＝8.2,5.1Hz,1H),4.00–3.85(m,1H),3.80–3.70(m,1H),2.20–2.05(m,1H),2.00–1.70(m,3H).ESI-MS m/z 367.1[M-H]^-。

example 28

(R) -N- (2- (tetrahydrofuran-2-carboxamide) benzo [ d ] thiazol-6-yl) nicotinamide (Compound 28)

By substituting 2-tetrahydrofurecarboxylic acid with (R) -2-tetrahydrofurecarboxylic acid according to the procedure of example 27, compound 28 (white solid) was obtained:¹H NMR(300MHz,DMSO-d₆)δ12.04(s,1H),10.49(s,1H),9.04(d,J＝2.2Hz,1H),8.68(dd,J＝5.0,1.6Hz,1H),8.37(s,1H),8.31–8.15(m,1H),7.66(d,J＝1.3Hz,2H),7.49(dd,J＝8.0,4.8Hz,1H),4.49(dd,J＝8.2,5.2Hz,1H),4.00–3.85(m,1H),3.82–3.65(m,1H),2.20–2.10(m,1H),2.00–1.70(m,3H).ESI-MS m/z 367.0[M-H]^-。

example 29

(S) -N- (2- (tetrahydrofuran-2-carboxamide) benzo [ d ] thiazol-6-yl) nicotinamide (Compound 29)

Compound (D) was obtained by substituting 2-tetrahydrofurecarboxylic acid with (S) -2-tetrahydrofurecarboxylic acid according to the procedure of example 27Material 29 (white solid):¹H NMR(300MHz,DMSO-d₆)δ12.05(s,1H),10.50(s,1H),9.05(d,J＝2.3Hz,1H),8.69(dd,J＝4.8,1.6Hz,1H),8.37(s,1H),8.31-8.13(m,1H),7.66(s,2H),7.50(dd,J＝7.9,4.8Hz,1H),4.50(dd,J＝8.3,5.2Hz,1H),3.95-3.85(m,1H),3.83-3.70(m,1H),2.20-2.05(m,1H),2.00-1.70(m,3H).ESI-MS m/z 367.1[M-H]^-。

example 30

(±) -N- (6- (4-nitrobenzoyl) benzo [ d ] thiazol-2-yl) tetrahydrofuran-2-carboxamide (Compound 30)

Referring to the procedure of example 24, substituting 24a for p-nitrobenzenesulfonyl chloride and 1c for 27c, compound 30 (yellow solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.16(s,1H),10.63(s,1H),8.35(d,J＝8.9Hz,2H),7.97(d,J＝9.0Hz,2H),7.76–7.52(m,2H),7.13(dd,J＝8.7,2.2Hz,1H),4.56(dd,J＝8.2,5.2Hz,1H),4.00–3.85(m,1H),3.85–3.70(m,1H),2.30–2.14(m,1H),2.06–1.82(m,3H).ESI-MS m/z 447.1[M-H]^-。

example 31

(±) -N- (6- (4-methylbenzoyl) benzo [ d ] thiazol-2-yl) tetrahydrofuran-2-carboxamide (Compound 31)

Referring to the procedure of example 24, substituting 24a for p-methylbenzenesulfonyl chloride and 1c for 27c, compound 31 (yellow solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.09(s,1H),10.22(s,1H),7.84–7.50(m,4H),7.29(d,J＝8.0Hz,2H),7.11(dd,J＝8.6,2.2Hz,1H),4.59–4.44(m,1H),3.95–3.86(m,1H),3.86–3.74(m,1H),2.29(s,3H),2.13–2.25(m,1H),2.04–1.74(m,3H).ESI-MS m/z 416.1[M-H]^-。

example 32

(±) -N- (6-thiophene-2-sulfonylbenzo [ d ] thiazol-2-yl) tetrahydrofuran-2-carboxamide (Compound 32)

Referring to the procedure of example 24, substituting 24a for 2-thiophenesulfonyl chloride and 1c for 27c, compound 32 (yellow solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.15(s,1H),10.45(s,1H),7.88(dd,J＝5.0,1.4Hz,1H),7.73(d,J＝2.2Hz,1H),7.65(d,J＝8.6Hz,1H),7.53(dd,J＝3.7,1.4Hz,1H),7.19(dd,J＝8.7,2.2Hz,1H),7.10(dd,J＝5.0,3.7Hz,1H),4.57(dd,J＝8.2,5.1Hz,1H),4.05–3.95(m,1H),3.95–3.80(m,1H),2.41–2.06(m,1H),2.08–1.80(m,3H).ESI-MS m/z 432.0[M+Na]⁺。

example 33

N- (6- (4-Methylbenzenesulfonamido) benzo [ d ] thiazol-2 yl) -5- (morpholinylmethyl) furan-2-carboxamide (Compound 33)

2-amino-6-nitrobenzothiazole (33a,200mg,1.03mmol) was added to dichloromethane (20mL) (suspension), Boc anhydride (305mg,1.23mmol) and 4-dimethylaminopyridine (150mg,1.23mmol) were added sequentially and stirred at room temperature for 6 hours. After TLC monitoring the reaction was complete, the stirring was stopped, the filter cake was filtered, washed with dichloromethane (2mL) and methanol (1mL) and dried to give compound 33b (white solid, 245mg, 8% yield).

Compound 33b (245mg,0.83mmol) was placed in a 50mL round bottom flask, methanol (15mL) was added, 10% palladium on carbon (25mg) was added, hydrogen gas was introduced, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction monitored by TLC, stirring was stopped, the reaction mixture was filtered with suction through celite, and the solvent was distilled off from the filtrate under reduced pressure to give compound 33c (white solid, 201mg, 91% yield).

Compound 33c (100mg,0.38mmol) was dissolved in pyridine (5mL), p-toluenesulfonyl chloride (80mg,0.42mmol) was added in portions under ice bath, and the mixture was allowed to warm to room temperature naturally, stirred overnight, and a large amount of solid was precipitated. And (3) after the TLC monitoring reaction is completed, dropwise adding 1N hydrochloric acid into the reaction system to be neutral, carrying out suction filtration, washing a filter cake with a small amount of ethyl acetate, and drying to obtain a 33d crude product.

All the crude 33d was dissolved in dichloromethane (4mL), trifluoroacetic acid (2mL) was added dropwise, stirring was carried out at room temperature for 0.5 hour, the reaction was monitored by TLC for completion, a saturated sodium bicarbonate solution was added dropwise to the reaction system to neutrality, suction filtration was carried out, and the filter cake was washed with water and a small amount of ethyl acetate (1mL) to obtain compound 33e (white solid, 77mg, two-step yield 64%):¹H NMR(300MHz,DMSO-d₆)δ10.14(s,1H),8.88(s,2H),7.63–7.41(m,3H),7.30–10(m,3H),6.94(dd,J＝8.7,2.2Hz,1H),2.24(s,3H).ESI-MS m/z 318.1[M-H]^-。

ethyl 5-chloromethyl-2-furancarboxylate (33f,200mg,1.06mmol) was dissolved in acetonitrile (10mL), and morpholine (55mg,1.27mmol), potassium iodide (176mg,1.27mmol) and potassium carbonate (212mg,1.27mmol) were added in this order, followed by stirring at room temperature for 7 hours. After completion of the reaction monitored by TLC, the solvent was evaporated under reduced pressure, and the mixture was subjected to silica gel column chromatography (eluting with petroleum ether, ethyl acetate, 10: 1, and then eluting with a small polar impurity, and then eluting with dichloromethane, methanol, 40: 1) to obtain 33g of compound (white solid, 260mg, 99% yield).

33g (260mg,1.06mmol) of the obtained compound was dissolved in methanol (10mL), sodium hydroxide solid (87mg,2.17mmol) was added, 2 drops of water were added dropwise, the mixture was stirred at 40 ℃ for 10 hours, after completion of the reaction was monitored by TLC, concentrated hydrochloric acid was added dropwise to a solution pH of 4 to 6, the solvent was distilled off, the obtained solid was dissolved in a dichloromethane/methanol mixed solution (dichloromethane: methanol ═ 3:1), suction filtration was performed, and the solvent was distilled off from the filtrate to obtain compound 33h (light brown solid, 220mg, yield 94%).

Compound 33e (40mg,0.13mmol) was dissolved in tetrahydrofuran (2mL), and compound 33h (37mg,0.15mmol), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,57mg,0.15mmol) and N, N-diisopropylethylamine (48. mu.L, 0.15mmol) were added in that order and heated at 40 ℃ for 48 hours. TLC monitoring after reaction completionStirring was stopped, the solvent was evaporated under reduced pressure and dissolved in methanol to prepare sand, and silica gel column chromatography (dichloromethane: methanol 100:1) was performed to obtain compound 33 (white solid, 25mg, yield 38%):¹H NMR(300MHz,DMSO-d₆)δ12.73(s,1H),10.24(s,1H),7.75–7.55(m,5H),7.32(d,J＝7.8Hz,2H),7.15(d,J＝8.8Hz,1H),6.58(d,J＝3.5Hz,1H),3.82–3.49(m,6H),2.50–2.40(m,4H),2.32(s,3H).ESI-MS m/z 511.1[M-H]^-。

example 34

5- (Morpholinylmethyl) -N- (6-thiophene-2-sulfonylaminobenzo [ d ] thiazol-2-yl) -furan-2-carboxamide (Compound 34)

Referring to the procedure of example 33, substituting p-methylbenzenesulfonyl chloride for 2-thiophenesulfonyl chloride produced compound 34 (light yellow solid):¹H NMR(300MHz,DMSO-d₆)δ12.73(s,2H),10.40(s,1H),7.91–7.73(m,1H),7.67(s,1H),7.65–7.51(m,2H),7.45(s,1H),7.11(d,J＝8.7Hz,1H),7.02(d,J＝5.1Hz,1H),6.54(s,1H),3.90–3.45(m,6H),2.50–2.30(m,4H).ESI-MS m/z 505.1[M+H]⁺。

example 35

5-hydroxymethyl-N- (6- (4-methylbenzenesulfonyl) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 35)

By substituting 33f with 5-hydroxymethyl-2-furancarboxylic acid according to the procedure of example 33, compound 35 (white solid) was obtained:¹H NMR(300MHz,DMSO-d₆)δ12.67(s,1H),10.24(s,1H),7.80–7.51(m,5H),7.32(d,J＝8.1Hz,2H),7.15(d,J＝8.1Hz,1H),6.55(d,J＝3.4Hz,1H),5.47(t,J＝5.9Hz,1H),4.50(d,J＝5.8Hz,2H),2.32(s,3H).ESI-MS m/z 442.1[M-H]^-。

example 36

5-hydroxymethyl-N- (6-thiophene-2-sulfonylbenzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 36)

Referring to the procedure of example 33, substituting p-methylbenzenesulfonyl chloride for 2-thiophenesulfonyl chloride and 33f for 5-hydroxymethyl-2-furancarboxylic acid gave compound 36 (white solid):¹H NMR(300MHz,DMSO-d₆)δ12.72(s,1H),10.44(s,1H),7.86(dd,J＝4.9,1.4Hz,1H),7.73(d,J＝2.2Hz,1H),7.64(d,J＝10.4Hz,2H),7.52(dd,J＝3.8,1.4Hz,1H),7.18(dd,J＝8.7,2.2Hz,1H),7.09(dd,J＝5.0,3.7Hz,1H),6.55(d,J＝3.5Hz,1H),5.47(s,1H),4.49(d,J＝3.8Hz,2H).ESI-MS m/z434.0[M-H]^-。

example 37

N- (4-hydroxy-6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 37)

2-amino-5-nitrophenol (37a,5.00g,32.40mmol) was dissolved in N, N-dimethylformamide (50mL), benzyl bromide (3.65mL,30.78mmol) was added dropwise under ice bath, and after the addition was completed, the mixture was stirred at room temperature overnight. After the completion of the reaction was monitored by TLC, water (400mL) was added to the reaction system for dilution, and a large amount of solid was observed to be precipitated, which was then filtered off with suction and dried. The obtained solid was recrystallized from a mixed solution of petroleum ether and ethyl acetate (petroleum ether: ethyl acetate: 1,160mL) to obtain orange yellow crystals (37b,2.08 g). The mother liquor after recrystallization was evaporated under reduced pressure to remove the solvent, ethyl acetate (30mL) was added to the resulting solid, and slurried, followed by suction filtration to give a yellow solid (37b,2.06g), which was combined with the solid obtained by recrystallization and fed to the next step.

The crude 37b fraction (2.08g,8.52mmol) was dissolved in glacial acetic acid (20mL), potassium thiocyanate (3.31g,34.06mmol) was added under ice salt bath, after stirring for 10 min, liquid bromine (873. mu.L, 17.03mmol) was slowly added dropwise, after stirring for 2h under ice salt bath, slowly warmed to room temperature, and stirring was continued for 7 days with solid precipitated. After the completion of the reaction was monitored by TLC,ethyl acetate (150mL) was added for dilution, suction filtered, the filtrate was collected, and the solvent was evaporated off. Ethyl acetate (100mL) was added to the resulting solid to dissolve it, and 15% sodium hydroxide solution was added dropwise thereto to adjust the pH to 6-7, followed by adjusting the pH to 7-8 with saturated sodium bicarbonate solution. And (4) decompressing, steaming and removing ethyl acetate, then carrying out suction filtration, collecting a filter cake, and drying. The filtrate was extracted with ethyl acetate (80mL × 3), the ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate was added for 0.5 hour, filtered to remove the anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure, and the obtained solid was combined with the dried cake to obtain compound 37c (yellow solid, 1.23g, two-step yield 27%):¹H NMR(300MHz,DMSO-d₆)δ8.39(d,J＝2.3Hz,1H),8.21(s,2H),7.75(d,J＝2.3Hz,1H),7.50(d,J＝7.5Hz,2H),7.31–7.47(m,3H),5.30(s,2H)。

compound 37c (660mg,2.19mmol) was dissolved in pyridine (15mL), freshly prepared furan-2-carbonyl chloride (282. mu.L, 2.85mmol) was added dropwise at room temperature, stirred at 60 ℃ for 5 hours and reacted at room temperature overnight, after completion of the reaction monitored by TLC, 2N hydrochloric acid solution was added to adjust the pH to 5-6 and a large amount of grey solid was observed to precipitate. Suction filtration, filter cake drying, sand making, silica gel column chromatography (dichloromethane: methanol 1000:1) to obtain compound 37d (light yellow solid, 737mg, yield 85%):¹H NMR(300MHz,DMSO-d₆)δ13.36(s,1H),8.80–8.75(m,1H),8.12(s,1H),7.97(d,J＝2.2Hz,1H),7.85(d,J＝3.4Hz,1H),7.66–7.40(m,5H),6.82(dd,J＝3.7,1.8Hz,1H),5.44(s,2H)。

compound 37d (512mg,1.29mmol) was dissolved in 95% ethanol (30mL), stannous chloride dihydrate (877mg,3.88mmol) was added at room temperature, after which the temperature was raised to 80 ℃ and stirred for 14 hours, the reaction was carried out overnight at room temperature, and then the reaction was further stirred for 10 hours at 80 ℃. After completion of the reaction, the reaction was monitored by TLC, heating was stopped, the reaction mixture was cooled to room temperature, and the solvent was evaporated, and a saturated sodium bicarbonate solution (20mL) was added to the obtained residue to precipitate a large amount of a white solid. After suction filtration, the filter cake was placed in tetrahydrofuran (suspension), stirred for 0.5h under nitrogen, suction filtered, and after collecting the filtrate, the solvent was evaporated under reduced pressure to give compound 37e (khaki solid, 300mg, 59% yield):¹H NMR(300MHz,DMSO-d₆)δ9.82(s,2H),8.01–7.35(m,7H),6.73(d,J＝2.0Hz,1H),6.62(s,1H),6.34(d,J＝2.0Hz,1H),5.31(s,2H)。

2-thiophenesulfonyl chloride (28mg,0.15mmol) was dissolved in pyridine (1mL), and a solution of compound 37e (50mg,0.28mmol) in pyridine (1mL) was added dropwise under nitrogen protection in ice bath, followed by stirring for 2 hours in ice bath. After the reaction was monitored by TLC, ethyl acetate (20mL), 1N hydrochloric acid (20mL × 3) were added, and the organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to give compound 37f (pale purple solid, 72mg, yield 97%):¹H NMR(300MHz,DMSO-d₆)δ12.89(s,1H),10.47(s,1H),8.03(s,1H),7.89(d,J＝5.0Hz,1H),7.73(d,J＝3.7Hz,1H),7.65–7.37(m,6H),7.31(s,1H),7.09(d,J＝4.5Hz,1H),6.94(s,1H),6.74(s,1H),5.16(s,2H)。

compound 37f (72mg,0.14mmol) and pentamethylbenzene (146mg,1.40mmol) were placed in a jar, dichloromethane (5mL) was added and stirred to give a suspension, the temperature was lowered to-78 ℃ and 1M boron trichloride dichloromethane solution (369. mu.L, 0.36mmol) was slowly added dropwise, and the mixture was stirred at-78 ℃ for 1 hour. After the TLC monitoring reaction is completed, methanol (5 drops) is added dropwise to quench the reaction, and the reaction is naturally warmed to room temperature and stirred for 1 hour. After the reaction solution was diluted with additional dichloromethane (3mL), the reaction solution was transferred to a 15mL centrifuge tube and centrifuged at 2500rpm for 5 minutes, the supernatant was discarded, and additional dichloromethane (3mL) was added and the centrifugation was continued at 2500rpm for 5 minutes. After 4 repetitions compound 37 (white solid, 56mg, 93% yield) was obtained:¹H NMR(300MHz,DMSO-d₆)δ12.82(s,1H),10.33(s,1H),10.07(s,1H),8.02(s,1H),7.87(s,1H),7.72(s,1H),7.54(s,1H),7.12(d,J＝9.0Hz,2H),6.76(d,J＝12.8Hz,2H).ESI-MS m/z 420.0[M-H]^-。

example 38

N- (4-hydroxy-6- (4-methylbenzenesulfonamido) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 38)

Referring to the procedure of example 37, substituting 2-thiophenesulfonyl chloride for p-methylbenzenesulfonyl chloride gave compound 38 (white solid):¹H NMR(300MHz,DMSO-d₆)δ12.72(s,1H),10.44(s,1H),7.86(dd,J＝4.9,1.4Hz,1H),7.73(d,J＝2.2Hz,1H),7.64(d,J＝10.4Hz,2H),7.52(dd,J＝3.8,1.4Hz,1H),7.18(dd,J＝8.7,2.2Hz,1H),7.09(dd,J＝5.0,3.7Hz,1H),6.55(d,J＝3.5Hz,1H),5.47(s,1H),4.49(d,J＝3.8Hz,2H).ESI-MS m/z 434.0[M-H]^-。

example 39

N- (2- (Furan-2-carboxamide) -4-Morpholinylbenzo [ d ] thiazol-6-yl) nicotinamide (Compound 39)

2-fluoro-4-nitrobenzoic acid (39a,10.00g,54.00mmol) was dissolved in dichloromethane (40mL), oxalyl chloride (40mL,65.00mmol) was added dropwise under ice-bath, 3 drops of N, N-dimethylformamide were added dropwise as a catalyst, and then stirred at room temperature for 3 hours. After TLC monitoring reaction is completed, the solvent is evaporated under reduced pressure to obtain acyl chloride intermediate. The intermediate was dissolved in acetonitrile (20mL), and an aqueous ammonia solution (30mL) was added under ice-cooling, and the mixture was allowed to warm to room temperature and stirred at room temperature for 2 hours. After completion of the reaction monitored by TLC, stirring was stopped, the solvent was distilled off under reduced pressure, and the obtained solid was washed with a small amount of ethyl acetate (5mL), suction-filtered and dried to obtain compound 39b (yellow solid, 9.60g, yield 96%).

Compound 39b (3.20g,25.42mmol) was dissolved in DMSO solution (25mL), morpholine (4.42mL,50.84mmol) and potassium carbonate (5.27g,38.18mmol) were added in that order, and after the addition was completed, the mixture was warmed to 90 ℃ and stirred for 4 hours. The heating was stopped after TLC monitored completion of the reaction. After cooling to room temperature, the reaction solution was poured into ice water (50mL), 1N hydrochloric acid was added to adjust the pH to neutral, suction filtration was performed, the filter cake was collected and dried, the filtrate was extracted with ethyl acetate (20mL × 3), the organic phase was dried over anhydrous sodium sulfate, suction filtration was performed, the solid obtained after evaporation of the filtrate solvent was combined with the previously dried filter cake to obtain compound 39c (yellow solid, 3.98g, yield 63%):¹H NMR(300MHz,DMSO-d₆)δ7.98(s,1H),7.77(dd,J＝8.3,2.3Hz,1H),7.75–7.47(m,3H),3.94–3.47(m,4H),3.15–2.75(m,4H)。

compound 39c (1.78g,7.10mmol) was dissolved in dioxane (80mL), sodium hydroxide solid (1.70g,42.60mmol) was added under ice bath, water (40mL) was added to the system, and stirring was carried out for 30 minutes under ice bath. Thereafter, a 5% sodium hypochlorite solution (29mL,42.60mmol) was added dropwise to the system, and the mixture was stirred at room temperature overnight. After the completion of the reaction, TLC monitored and the stirring was stopped, the solvent was distilled off under reduced pressure, and then water (10mL) was added thereto, followed by suction filtration to obtain compound 39d (white solid, 1.44g, yield 91%):¹H NMR(500MHz,DMSO-d₆)δ7.84(dd,J＝8.9,2.6Hz,1H),7.75(d,J＝2.6Hz,1H),6.77(d,J＝8.9Hz,1H),6.39(s,2H),3.81(t,J＝4.5Hz,4H),2.85(t,J＝4.4Hz,4H)。

by substituting 37b for 39d according to the procedure of example 37, compound 39e (brown solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.30(s,1H),8.01(d,J＝1.7Hz,1H),7.69(d,J＝3.7Hz,1H),6.84–6.55(m,2H),6.24(d,J＝2.2Hz,1H),5.12(s,2H),3.95–3.67(m,4H),3.35–3.25(m,4H)。

with reference to the procedure of example 1, substituting 1c for 39e and substituting isonicotinic acid for nicotinic acid, compound 39 (light yellow solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.66(s,1H),10.49(s,1H),9.15(s,1H),8.78(d,J＝5.2Hz,1H),8.33(d,J＝8.0Hz,1H),8.15–8.05(m,2H),7.76(d,J＝3.7Hz,1H),7.60(dd,J＝8.1,4.8Hz,1H),7.30(s,1H),6.77(d,J＝3.7Hz,1H),3.97–3.78(m,4H),3.45–3.35(m,4H).ESI-MS m/z 448.1[M-H]^-。

example 40

N- (6- (2- (thien-2-yl) acetylamino) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 40)

Furoic acid (40a,1.0g,8.9mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (5mL,44.6mmol) was slowly added under ice bath, and after completion of the dropwise addition, N-dimethylformamide (1 drop) was added and reacted at room temperature for 1 hour. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure, redissolved in anhydrous dichloromethane (6mL), slowly added dropwise to anhydrous dichloromethane (10mL) containing 2-amino-6-nitrobenzothiazole (1a,1.58g,8.1mmol), and the reaction was stirred at room temperature for 13 hours. After completion of the reaction, the reaction mixture was diluted with anhydrous dichloromethane (30mL), washed successively with 1N diluted hydrochloric acid (50mL), water (50 mL. times.2) and saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and recrystallized from ethyl acetate (10mL) to give compound 1b (pale yellow solid, 1.7g, 73% yield).

Taking the compound 1b (600mg,2.07mmol), suspending the compound in a mixed solution of acetic acid (8mL) and water (2mL), slowly adding zinc powder (678mg,10.4mmol) under stirring at room temperature, after TLC detection reaction is completed, adding ethyl acetate (40mL) for dilution, adding sodium bicarbonate to adjust the pH to be neutral, washing with saturated sodium bicarbonate aqueous solution (40mL multiplied by 2), drying with anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain a crude product (yellow solid, 420mg, yield 78%) of the compound 1c, and directly putting the crude product into the next step without purification.

Compound 1c (179mg,0.69mmol) was dissolved in anhydrous dichloromethane (5mL) and a solution of triethylamine (419mg,4.14mmol) and 2-thiopheneacetylchloride (133mg,0.83mmol) in anhydrous dichloromethane (10mL) was added slowly. Stirring at room temperature until the reaction is complete by TLC. Anhydrous dichloromethane (20mL) was added for dilution, washed with 1N diluted hydrochloric acid (30mL) and saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 200:1) to give the crude compound 40, which was then slurried with ethyl acetate (5mL) to give the pure compound 40 (off-white solid, 47mg, 15% yield):¹H NMR(300MHz,DMSO-d₆)δ12.76(s,1H),10.36(s,1H),8.32(s,1H),8.11–7.96(m,1H),7.82–7.62(m,2H),7.63–7.49(m,1H),7.39(d,J＝4.6Hz,1H),7.12–6.91(m,2H),6.75(d,J＝1.7Hz,1H),3.91(s,2H).ESI-MS m/z 382.1[M-H]^-。

EXAMPLE 41

N- (6- (2- (thien-2-yl) acetamido) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide hydrochloride (Compound 41)

Referring to the procedure of example 40, N-Boc precursor compound (greenish black solid, 140mg) of compound 41 was prepared by substituting furoic acid for 1-Boc-4-piperidinecarboxylic acid.

The N-Boc precursor compound (140mg,0.28mmol) was dissolved in tetrahydrofuran (5mL), and ethanolic hydrochloride solution (10mL) was added, stirred at room temperature overnight, filtered with suction, and dried to afford compound 41 (off-white solid, 100mg, 82% yield):¹H NMR(300MHz,DMSO-d₆)δ12.43(s,1H),10.60(s,1H),9.37–9.11(m,1H),9.11–8.78(m,1H),8.33(s,1H),7.67(d,J＝8.7Hz,1H),7.56(d,1H),7.38(d,J＝4.7Hz,1H),7.17–6.83(m,2H),3.92(s,2H),3.40–3.22(m,2H),3.09–2.72(m,3H),2.14–61.95(m,2H),1.95–61.75(m,2H).ESI-MS m/z 399.1[M-HCl-H]^-。

example 42

N- (2- (furan-2-carboxamide) benzo [ d ] thiazol-6-yl) tetrahydro-2H-pyran-4-carboxamide (Compound 42)

Referring to the procedure of example 40, substituting furoic acid for tetrahydropyran-4-carboxylic acid, compound 42 (white solid, 103mg) was prepared:¹H NMR(300MHz,CDCl₃)δ12.70(s,1H),10.05(s,1H),8.34(s,1H),8.10–7.93(m,1H),7.82–7.61(m,2H),7.62–7.42(m,1H),6.87–6.63(m,1H),3.99–3.84(m,2H),3.46–3.35(m,2H),2.72–2.55(m,1H),1.81–1.58(m,4H).ESI-MS m/z 372.1[M+H]⁺。

example 43

N- (6- ((5-chlorothiophene) -2-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide hydrochloride (Compound 43)

1-Boc-4-piperidinecarboxylic acid (43a,2.18g,9.5mmol) was dissolved in N, N-dimethylformamide (20mL), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,3.65g,11.85mmol) and triethylamine (1.92g,19.0mmol) were added in this order to react at room temperature for 1 hour. After completion of the TLC detection reaction, 2-amino-6-nitrobenzothiazole (1.0g,5.1mmol) was added, the reaction was stirred at room temperature until completion of the TLC detection reaction, and then ethyl acetate (50mL) was added to dilute the reaction solution, which was washed with water (50mL × 3) and saturated brine (50mL) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 100:1) to obtain compound 43b (pale yellow solid, 1.93g, 93% yield).

Taking the compound 43b (150mg,0.37mmol), suspending the compound in a mixed solution of acetic acid (12mL) and water (3mL), slowly adding zinc powder (121mg,1.85mmol) under stirring at room temperature, stirring at room temperature until TLC detection reaction is completed, adding ethyl acetate (30mL) for dilution, adding sodium bicarbonate to adjust the pH to be neutral, washing with saturated sodium bicarbonate aqueous solution (30mL multiplied by 2), drying with anhydrous sodium sulfate, filtering, concentrating the filtrate, and obtaining the crude compound 43c (yellow brown solid, 197mg, yield 71%) which is directly put into the next step without purification.

Compound 43c (70mg,0.19mmol) was dissolved in a mixed solvent of dichloromethane (4mL) and pyridine (1mL), and 5-chlorothiophene-2-sulfonyl chloride (45mg,0.21mmol) was slowly added. The reaction was stirred at room temperature until completion of the TLC detection. Ethyl acetate (30mL) was added for dilution, and the mixture was washed with 1N diluted hydrochloric acid (30mL × 2) and saturated brine (30mL) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 100:1) to obtain compound 43d (pale yellow solid, 85mg, yield 83%).

Compound 43d (85mg,0.15mmol) was dissolved in ethanol hydrochloride solution (10mL), stirred overnight at room temperature, filtered with suction, and dried to give compound 43 (yellow solid, 52mg, 69% yield):¹H NMR(300MHz,DMSO-d₆)δ12.50(s,1H),10.64(s,1H),9.18–8.98(m,1H),8.94–8.71(m,1H),7.75(s,1H),7.66(d,J＝8.6Hz,1H),7.41(d,J＝4.0Hz,1H),7.25–7.14(m,2H),3.41–3.23(m,2H),3.01–2.77(m,2H),2.13–1.95(m,2H),1.94–1.75(m,2H).ESI-MS m/z 457.0[M-Cl]⁺。

example 44

N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide hydrochloride (Compound 44)

Referring to the procedure of example 43, 5-chlorothiophene-2-sulfonyl chloride was replaced with 2-thiophenesulfonyl chloride to give compound 44 (white solid, 42 mg):¹H NMR(300MHz,DMSO-d₆)δ12.49(s,1H),10.47(s,1H),9.22–9.04(m,1H),9.00–8.77(m,1H),7.86(d,J＝4.8Hz,1H),7.71(s,1H),7.62(d,J＝8.6Hz,1H),7.51(d,J＝3.4Hz,1H),7.18(d,J＝8.6Hz,1H),7.14–7.04(m,1H),3.40–3.23(m,2H),3.00–2.77(m,3H),2.12–1.95(m,2H),1.95–1.73(m,2H).ESI-MS m/z 423.2[M-Cl]⁺。

example 45

N- (6- (morpholine-4-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide hydrochloride (Compound 45)

Referring to the procedure of example 43, substituting 5-chlorothiophene-2-sulfonyl chloride for 4-morpholinesulfonyl chloride, compound 45 (yellow solid, 48mg) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.47(s,1H),10.09(s,1H),9.27–9.01(m,1H),8.99–8.75(m,1H),7.79(s,1H),7.68(d,J＝8.7Hz,1H),7.32(d,J＝8.7Hz,1H),3.65–3.42(m,4H),3.42–3.21(m,2H),3.19–3.00(m,4H),3.03–2.77(m,3H),2.17–1.96(m,2H),1.95–1.73(m,2H).ESI-MS m/z 426.2[M-Cl]⁺。

example 46

N- (6- (morpholine-4-sulfonylamino) benzo [ d ] thiazol-2-yl) furan-2-carboxamide hydrochloride (Compound 46)

Referring to the procedure of example 45, substituting 1-Boc-4-piperidinecarboxylic acid for furoic acid produced compound 46 (pale yellow solid, 60 mg):¹H NMR(300MHz,DMSO-d₆)δ12.79(s,1H),10.07(s,1H),8.04(s,1H),7.86–7.77(m,1H),7.78–7.64(m,2H),7.45–7.25(m,1H),6.86–6.67(m,1H),3.67–3.40(m,4H),3.18–2.95(m,4H).ESI-MS m/z 431.1[M+Na]⁺。

example 47

N- (6- ((5-chlorothiophene) -2-sulfonylamino) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 47)

Referring to the procedure of example 43, substituting 1-Boc-4-piperidinecarboxylic acid for furoic acid produced compound 47 (light yellow solid, 62 mg):¹H NMR(300MHz,DMSO-d₆)δ12.83(s,1H),10.61(s,1H),8.04(s,1H),7.78(d,J＝1.7Hz,1H),7.76–7.63(m,2H),7.42(d,J＝4.0Hz,1H),7.27–7.13(m,2H),6.76(d,J＝1.8Hz,1H).ESI-MS m/z 462.0[M+H]⁺。

example 48

N- (6- (pyridine-3-sulfonylamino) benzo [ d ] thiazol-2-yl) furan-2-carboxamide (Compound 48)

Referring to the procedure of example 47, substituting 5-chlorothiophene-2-sulfonyl chloride for pyridine-3-sulfonyl chloride produced compound 48 (white solid, 109 mg):¹H NMR(300MHz,DMSO-d₆)δ12.81(s,1H),10.54(s,1H),8.88(s,1H),8.78(s,1H),8.11(d,J＝8.0Hz,1H),8.04(s,1H),7.80–7.69(m,2H),7.69–7.51(m,2H),7.20–7.11(m,1H),6.82–6.70(m,1H).ESI-MS m/z 399.2[M-H]^-。

example 49

N- (6- ((4-Nitrophenyl) sulfonamido) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide hydrochloride (Compound 49)

Referring to the procedure of example 43, 5-chlorothiophene-2-sulfonyl chloride was replaced with p-nitrobenzenesulfonyl chloride to give compound 49 (pale yellow solid, 69 mg):¹H NMR(300MHz,DMSO-d₆)δ12.52(s,1H),10.71(s,1H),9.47–9.23(m,1H),9.22–8.96(m,1H),8.87(s,1H),8.77(d,J＝4.4Hz,1H),8.14(d,J＝8.0Hz,1H),7.79–7.67(m,1H),7.60(d,J＝8.7Hz,2H),7.21–7.09(m,1H),3.41–3.15(m,2H),3.00–2.74(m,3H),2.13–1.95(m,2H),1.94–1.72(m,2H).ESI-MS m/z 418.1[M-Cl]⁺。

example 50

N- (6- (pyridine-3-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide hydrochloride (Compound 50)

Referring to the procedure of example 43, 5-chlorothiophene-2-sulfonyl chloride was replaced with pyridine-3-sulfonyl chloride to give compound 50 (light yellow solid, 54 mg):¹H NMR(300MHz,DMSO-d₆)δ12.50(s,1H),10.73(s,1H),9.29–9.04(m,1H),9.05–8.77(m,1H),8.35(d,J＝8.7Hz,2H),7.99(d,J＝8.7Hz,2H),7.78–7.67(m,1H),7.61(d,J＝8.7Hz,1H),7.27–7.07(m,1H),3.40–3.21(m,2H),3.02–2.75(m,3H),2.14–1.94(m,2H),1.94–1.73(m,2H).ESI-MS m/z 462.1[M-Cl]⁺。

example 51

5- (N- (2- (piperidine-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) thiophene-3-carboxylate (Compound 51)

Dissolving 3-thiophenecarboxylic acid (51a,1.0g,7.80mmol) in ethanol (15mL), adding concentrated sulfuric acid (2 drops) dropwise, heating and refluxing at 78 deg.C until TLC detection reaction is completedAfter completion, the reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, ethyl acetate (30mL) was added to redissolve the mixture, and the redissolved mixture was washed with a saturated sodium bicarbonate solution (30mL), water (30mL) and a saturated brine (30mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 51b (pale yellow oily liquid, 1.07g, yield 88%):¹H NMR(300MHz,CDCl₃)δ8.14–8.04(m,1H),7.53(d,J＝4.8Hz,1H),7.34–7.27(m,1H),4.33(q,J＝7.1Hz,2H),1.37(t,J＝7.1Hz,3H)。

compound 51b (500mg,3.20mmol) was dissolved in dry dichloromethane (4mL) and slowly added dropwise with chlorosulfonic acid (746mg,6.40mmol) dissolved in dry dichloromethane (4mL) under argon protection in an ice bath. Naturally raising the temperature to room temperature for reaction until the reaction is completely detected by TLC. The reaction solution was slowly added dropwise to ice water (20mL), extracted with dichloromethane (20mL × 2), and the organic phase was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (ethyl acetate: petroleum ether ═ 30: 1) to obtain compound 51c (light yellow liquid, 404mg, yield 50%):¹H NMR(300MHz,CDCl₃)δ8.47(s,1H),8.26(s,1H),4.38(q,J＝7.1Hz,2H),1.39(t,J＝7.1Hz,3H)。

compound 43c (220mg,0.58mmol) was dissolved in a mixed solvent of dichloromethane (4mL) and pyridine (1mL), and compound 51c (178mg,0.70mmol) was added slowly and stirred at room temperature until the reaction was complete by TLC. After concentration, the mixture was diluted with ethyl acetate (30mL), washed successively with 1N diluted hydrochloric acid (30mL × 3) and saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (dichloromethane: methanol ═ 80:1) to give compound 51d (yellow solid, 316mg, yield 92%).

Compound 51d (200mg,0.336mmol) was dissolved in a mixed solvent of methanol (6mL) and water (2mL), and lithium hydroxide monohydrate (28mg,0.673mmol) was added to the solution to react at room temperature overnight. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure, water (5mL) was added for redissolution, methylene chloride (5 mL. times.2) was washed, the residual methylene chloride was distilled off from the aqueous phase, the pH was adjusted to 4-5 with 1N diluted hydrochloric acid while stirring at room temperature, suction filtration was performed, and the filter cake was dried to obtain compound 51e (142mg of a white solid, 74% yield).

Dissolve compound 51e (142mg,0.25mmol) in EtOAc (hydrochloric acid)Solution (10mL), stirred at rt overnight, after TLC detection reaction was complete, filtered with suction and the filter cake was dried to afford compound 51 (white solid, 77mg, 62% yield):¹H NMR(300MHz,DMSO-d₆)δ12.49(s,1H),10.63(s,1H),9.26–9.06(m,1H),9.03–8.78(m,1H),8.47(s,1H),7.78–7.71(m,1H),7.70–7.60(m,2H),3.40–3.21(m,2H),2.99–2.76(m,3H),2.09–1.95(m,2H),1.94–1.75(m,2H).ESI-MS m/z 467.1[M-Cl]⁺。

example 52

5- (N- (2- (piperidine-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) thiophene-3-carboxylic acid ethyl ester hydrochloride (Compound 52)

Compound 51d (96mg,0.16mmol) was dissolved in ethyl acetate hydrochloride solution (10mL), stirred overnight at room temperature, filtered by TLC and dried to give compound 52 (white solid, 80mg, 93% yield):¹H NMR(300MHz,DMSO-d₆)δ12.52(s,1H),10.64(s,1H),9.26–9.03(m,1H),9.02–8.74(m,1H),8.54(s,1H),7.81–7.70(m,2H),7.66(d,J＝8.6Hz,1H),7.20(d,J＝8.7Hz,1H),4.24(q,J＝7.1Hz,2H),3.43–3.21(m,2H),3.02–2.77(m,3H),2.13–1.96(m,2H),1.96–1.74(m,2H),1.26(t,J＝7.1Hz,3H).ESI-MS m/z 495.2[M-Cl]⁺。

example 53

3- ((5- (N- (2- (piperidine-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) thiophene-3-carboxamido) methyl) benzoic acid hydrochloride (Compound 53)

51e (200mg,0.35mmol) was dissolved in N, N-dimethylformamide (4mL), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,128mg,0.42mmol) and triethylamine (65mg,0.64mmol) were added in this order, followed by stirring at room temperature for 1 hour. At the same time, methyl 3- (aminomethyl) benzoate hydrochloride (65mg,0.32mmol) and triethylamine (65mg,0.64mmol) were added to methylene chloride (4mL) in this order and stirred for 1 hour to release methyl 3- (aminomethyl) benzoate. After completion of the TLC detection reaction, the liberated methyl 3- (aminomethyl) benzoate in methylene chloride was added dropwise to the above N, N-dimethylformamide reaction solution, and potassium carbonate (147mg,1.06mmol) was added thereto. After completion of the TLC reaction by stirring at room temperature, the reaction mixture was diluted with dichloromethane (30mL), washed with 1N diluted hydrochloric acid (30mL) and saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (ethyl acetate: petroleum ether ═ 1:2) to obtain compound 53a (white solid, 116mg, yield 47%).

Compound 53a (80mg,0.112mmol) was dissolved in a mixed solvent of methanol (8mL) and water (2mL), and lithium hydroxide monohydrate (19mg,0.449mmol) was added to the solution to react at room temperature overnight. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure, water (5mL) was added for redissolution, methylene chloride (5 mL. times.2) was washed, the residual methylene chloride was distilled off from the aqueous phase, the pH was adjusted to 4-5 with 1N diluted hydrochloric acid while stirring at room temperature, suction filtration was performed, and the filter cake was dried to obtain compound 53b (57mg of a white solid, 73% yield).

Compound 53b (57mg,0.083mmol) was dissolved in ethyl acetate hydrochloride solution (10mL) and stirred at room temperature overnight, after completion of the TLC reaction, suction filtered and the filter cake was dried to afford compound 53 (white solid, 30mg, 58% yield):¹H NMR(300MHz,DMSO-d₆)δ12.91(s,1H),12.49(s,1H),10.55(s,1H),9.21–9.07(m,1H),9.06–8.88(m,1H),8.83–8.62(m,1H),8.44(s,1H),7.94(s,1H),7.87(s,1H),7.82(d,J＝7.4Hz,1H),7.78–7.71(m,1H),7.66(d,J＝8.6Hz,1H),7.56–7.48(m,1H),7.48–7.37(m,1H),7.25–7.11(m,1H),4.45(d,J＝5.5Hz,2H),3.62–3.21(m,10H),3.03–2.76(m,4H),2.13–1.95(m,2H),1.95–1.76(m,2H).ESI-MS m/z 600.3[M-Cl]⁺。

example 54

3- ((5- (N- (2- (piperidine-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) thiophene-3-carboxamido) methyl) benzoic acid methyl ester hydrochloride (Compound 54)

By substituting compound 51d for compound 53a according to the procedure of example 52, compound 54 (white solid, 10mg) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.48(s,1H),10.56(s,1H),9.22–9.11(m,1H),9.09–8.90(m,1H),8.90–8.66(m,1H),8.43(s,1H),7.95–7.91(m,1H),7.86(s,1H),7.82(d,J＝7.6Hz,1H),7.73(d,J＝1.8Hz,1H),7.67–7.60(m,1H),7.57–7.50(m,1H),7.49–7.40(m,1H),7.17(dd,J＝8.6Hz,1H),4.43(d,J＝5.7Hz,2H),3.82(s,3H),3.36–3.23(m,2H),3.00–2.77(m,3H),2.08–1.92(m,2H),1.92–1.72(m,2H).ESI-MS m/z 614.3[M-Cl]⁺。

example 55

1-methyl-N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide (Compound 55)

55a (500g,3.49mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.59mL,8.98mmol) was added slowly under ice bath, and after completion of the addition, N-dimethylformamide (1 drop) was added and reacted at room temperature for 1 hour. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure to give compound 55b, which was redissolved in anhydrous dichloromethane (10mL), and slowly added dropwise to anhydrous dichloromethane (10mL) in which 2-amino-6-nitrobenzothiazole (681mg,3.49mmol) and triethylamine (968. mu.L, 6.98mmol) were dissolved, and the reaction was stirred at room temperature for 13 hours. After completion of the reaction, the reaction mixture was concentrated, ethyl acetate (30mL) was added, the mixture was neutralized with a saturated aqueous sodium bicarbonate solution (30mL), suction-filtered under reduced pressure, and the cake was washed with water (30mL) and ethyl acetate (10mL) in this order to give compound 55c (brown solid, 1.05g, 94% yield).

Compound 55c (1.05g,3.26mmol) was suspended in a mixed solution of acetic acid (12mL) and water (3mL), and after completion of the reaction, zinc powder (1.07g,16.3mmol) was slowly added with stirring at room temperature, and TLC monitored, the solvent was distilled off under reduced pressure, methylene chloride (20mL) was added, the pH was adjusted to 8-9 with a saturated aqueous sodium bicarbonate solution, and concentrated aqueous ammonia (10mL) was added and stirred at room temperature for 1 hour. Water (10mL) was added for separation, the aqueous phase was extracted with dichloromethane (20mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound 55d (tan solid, 197mg, 71% yield) which was directly used in the next step without purification.

Referring to the procedure of example 43, substituting 43c for 55d and 5-chlorothiophene-2-sulfonyl chloride for 2-thiophenesulfonyl chloride gave compound 55 (white solid, 47 mg):¹H NMR(300MHz,DMSO-d₆)δ12.25(s,1H),10.37(S,1H),7.84(dd,J＝5.0,1.2Hz,1H),7.67(d,J＝2.0Hz,1H),7.60(d,J＝8.7Hz,1H),7.49(dd,J＝3.7,1.2Hz,1H),7.22–7.11(m,1H),7.11–7.02(m,2H),2.48–2.35(m,1H),2.17(S,3H),1.99–1.84(m,2H),1.84–1.73(m,2H),1.73–1.54(m,2H).ESI-MS m/z 435.1[M-H]^-。

example 56

3- ((3- (N- (2- (1-methylpiperidine-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) propyl) carbamoyl) benzoic acid (compound 56)

Monomethyl isophthalate (56a,500mg,2.77mmol), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,1023mg,3.32mmol) and triethylamine (561mg,5.54mmol) were sequentially added to N, N-dimethylformamide (6mL), and after stirring at room temperature for 1 hour, 3-amino-1-propanesulfonic acid (579mg,4.16mmol) was added and the reaction was continued with stirring at room temperature. After completion of the TLC detection, the reaction mixture was diluted with ethyl acetate (20mL), extracted with water (20mL) and the aqueous phase was washed with ethyl acetate (20 mL). The aqueous phase was evaporated under reduced pressure and purified by silica gel column chromatography (dichloromethane: methanol 15:1) to give compound 56b (white solid, 420mg, yield 50%).

Compound 56b (400mg,1.33mmol) was suspended in anhydrous dichloromethane (15mL), phosphorus pentachloride (553mg,2.66mmol) was slowly added under ice bath, after the addition was completed, the reaction was naturally warmed to room temperature until the reaction was complete by TLC detection, water (15mL) was added to quench, dichloromethane (10mL) was added to separate the solution, the organic phase was washed with saturated brine (25mL), dried, filtered, concentrated, and purified by silica gel column chromatography (ethyl acetate: petroleum ether ═ 3:1) to give compound 56c (white solid, 140mg, yield 33%).

Compound 55d (76mg,0.26mmol) was dissolved in a mixed solvent of dichloromethane (4mL) and pyridine (1mL), and compound 56c (100mg,0.31mmol) was added slowly and stirred at room temperature until the reaction was complete by TLC. After concentration, ethyl acetate (30mL) was added to dilute the solution, which was washed with water (30mL × 3) and saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (dichloromethane: methanol ═ 25:1) to give compound 56e (pale yellow solid, 65mg, yield 44%).

Compound 56e (65mg,0.11mmol) was dissolved in a mixed solvent of methanol (4mL) and water (1mL), and lithium hydroxide monohydrate (24mg,0.57mmol) was added to the solution to react at room temperature overnight. After completion of the TLC reaction, the solvent was distilled off under reduced pressure, water (5mL) was added for redissolution, dichloromethane (5mL × 2) was washed, the residual dichloromethane was distilled off from the aqueous phase, pH was adjusted to 6-7 with 1N diluted hydrochloric acid under stirring at room temperature, suction filtration was performed, and the filter cake was dried to obtain compound 56 (34 mg of white solid, 56% yield):¹H NMR(300MHz,DMSO-d₆)δ13.11(s,1H),12.45(s,1H),9.87(s,1H),8.80–8.62(m,1H),8.35(s,1H),8.04(d,J＝7.8Hz,1H),7.95(d,J＝7.7Hz,1H),7.80–7.70(m,1H),7.58(d,J＝8.6Hz,1H),7.52(t,J＝7.8Hz,1H),7.30–7.20(m,1H),3.59–3.41(m,2H),3.40–3.33(m,2H),3.22–3.10(m,2H),3.08–2.88(m,2H),2.76(s,3H),2.16–2.03(m,2H),2.03–1.75(m,4H).ESI-MS m/z 560.3[M+H]⁺。HRMS m/z(ESI):calculated for C₂₅H₃₀N₅O₆S₂[M+H]⁺560.1632,found 560.1632。

example 57

N- (6- (2- (thien-2-yl) acetylamino) benzo [ d ] thiazol-2-yl) morpholine-4-carboxamide (Compound 57)

Dissolving the compound 1a (976mg,5mmol) and triethylamine (2mL,15mmol) in dichloromethane (40mL), adding a dichloromethane solution (10mL) of phenyl chloroformate (1.25mL,10mmol), stirring at room temperature until TLC detection reaction is complete, evaporating the solvent under reduced pressure, pulping with ethyl acetate (20mL), suction-filtering, washing the filter cake with 1N diluted hydrochloric acid (20mL), suction-filtering, and drying to obtain a crude product (yellow solid, 1.168g) of the intermediate 57 a.

The crude intermediate 57a (315mg,1mmol) was dissolved in dimethyl sulfoxide (5mL), morpholine (435 μ L,5mmol) was added, the mixture was stirred at room temperature until the TLC detection reaction was complete, water (50mL) and ethyl acetate (20mL) were added to the reaction mixture, the mixture was separated, the aqueous phase was extracted with ethyl acetate (20mL × 3), the organic phases were combined, the organic phase was washed with saturated brine (25mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 50:1) to obtain compound 57b (yellow solid, 173mg, 56% yield in two steps).

Compound 57b (166mg,0.54mmol) was dissolved in a mixed solution of acetic acid (7.5mL) and water (2.5mL), zinc powder (176mg,2.7mmol) was added, and after stirring at room temperature until the TLC detection reaction was complete, saturated sodium bicarbonate solution (15mL) was added, followed by addition of sodium bicarbonate solid until no bubbles were generated. To the mixture was added ethyl acetate (30mL), the layers were separated, the aqueous phase was extracted with ethyl acetate (30mL × 3), the organic phases were combined, the organic phase was washed with saturated brine (25mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 57c (brown solid, 137mg, 91% yield).

Dissolving 2-thiopheneacetyl chloride (30. mu.L, 0.24mmol) in dry dichloromethane (2mL), slowly dropping triethylamine (166. mu.L, 1.2mmol) and 57c (56mg,0.2mmol) in dry dichloromethane (5mL), stirring at room temperature until TLC detection reaction is completed, adding 1N diluted hydrochloric acid (5mL) and dichloromethane (5mL), separating, extracting the aqueous phase with dichloromethane (5 mL. times.3), combining the organic phases, saturating the organic phaseWashed with brine (10mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 50:1) to give compound 57 (yellow solid, 41mg, yield 51%):¹H NMR(300MHz,DMSO-d₆)δ11.46(s,1H),10.33(s,1H),8.19(s,1H),7.57-7.45(m,1H),7.46-7.40(m,1H),7.12-6.95(m,1H),3.92(s,2H),3.73-3.61(m,4H),3.61-3.46(m,4H).ESI-MS m/z 425.1[M+Na]⁺。

example 58

N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) morpholine-4-carboxamide (Compound 58)

By substituting 37e for 57c, according to the procedure of example 37, compound 58 (yellow solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ11.21(s,1H),10.33(s,1H),7.87(d,J＝4.7Hz,1H),7.57(s,1H),7.50(d,J＝3.0Hz,1H),7.47-7.33(m,1H),7.18-7.04(m,2H),3.71-3.57(m,4H),3.57-3.43(m,4H).ESI-MS m/z 423.1[M-H]^-。

example 59

N- (6- (tetrahydro-2H-pyran-4-carboxamido) benzo [ d ] thiazol-2-yl) piperazine-1-carboxamide hydrochloride (Compound 59)

Crude intermediate 57a (315mg,1mmol) was dissolved in dimethyl sulfoxide (5mL), N-Boc-piperazine (931mg,5mmol) was added, the mixture was stirred at room temperature until the TLC detection reaction was complete, then water (50mL) and ethyl acetate (20mL) were sequentially added to the reaction solution, followed by liquid separation, extraction of the aqueous phase with ethyl acetate (20mL × 3), and the organic phases were combined, washed with saturated saline (25mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 50:1) to obtain compound 59a (pale yellow solid, 213mg, 52% yield in two steps).

Compound 59a (213mg,0.52mmol) was dissolved in a mixed solution of acetic acid (7.5mL) and water (2.5mL), zinc powder (171mg,2.6mmol) was added, and after stirring at room temperature until the TLC detection reaction was complete, a saturated solution of sodium bicarbonate (15mL) was added, followed by addition of solid sodium bicarbonate until no bubbles were produced. To the mixture was added ethyl acetate (30mL), the layers were separated, the aqueous phase was extracted with ethyl acetate (30mL × 3), the organic phases were combined, the organic phase was washed with saturated brine (25mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 50:1) to give compound 59b (yellow solid, 180mg, yield 92%).

Tetrahydropyran-4-carboxylic acid (20mg,0.15mmol) was dissolved in N, N-dimethylformamide (1.5mL), 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate (HATU,69mg,0.23mmol) and triethylamine (43 μ L,0.3mmol) were added, the mixture was stirred at room temperature for 1 hour, compound 59b (57mg,0.15mmol) was added, the mixture was further stirred at room temperature until the TLC detection reaction was complete, and the reaction mixture was directly subjected to silica gel column chromatography (dichloromethane: methanol ═ 20:1) to purify the compound 59c (pale yellow solid, 64mg, yield 87%).

Dissolving the compound 59c (48.9mg,0.1mmol) in tetrahydrofuran (1mL), adding ethanol hydrochloride solution (5mL), stirring at room temperature until the TLC detection reaction is complete, performing suction filtration, and drying a filter cake to obtain a compound 59 (brown solid, 32mg, yield 75%):¹H NMR(300MHz,DMSO-d₆)δ10.09(s,1H),9.39(s,2H),8.16(s,1H),7.54–7.46(m,1H),7.46–7.37(m,1H),3.98–3.86(m,2H),3.86–3.73(m,4H),3.48–3.26(m,2H),3.21–2.99(m,4H),2.75–2.56(m,1H),1.81–1.54(m,4H).ESI-MS m/z 390.2[M-Cl]⁺。

example 60

N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide hydrochloride (Compound 60)

Compound 43c (180mg,0.48mmol) was dissolved in dichloromethane (15mL), triethylamine (291mg,2.88mmol) and 2-thiophenesulfonyl chloride (105mg,0.57mmol) were added slowly, and the mixture was stirred at room temperature until the TLC detection reaction was complete, then dichloromethane (40mL) was added for dilution, washed with 1N diluted hydrochloric acid (40mL × 3) and saturated brine (40mL), dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (dichloromethane: methanol ═ 200:1) to obtain compound 60a (white solid, 80mg, yield 25%).

Compound 60a (80mg,0.12mmol) was dissolved in ethyl acetate hydrochloride solution (10mL), stirred overnight at room temperature, filtered by TLC after completion of the reaction, and the filter cake was dried to give compound 60 (white solid, 40mg, 56% yield):¹H NMR(300MHz,DMSO-d₆)δ12.71(s,1H),9.14–8.87(m,1H),8.87–8.58(m,1H),8.23(d,J＝4.7Hz,2H),7.84(d,J＝1.9Hz,1H),7.74(dd,J＝8.8,5.9Hz,3H),7.35–7.23(m,2H),7.08–6.95(m,1H),3.42–3.26(m,2H),3.06–2.80(m,3H),2.15–1.96(m,2H),1.96–1.76(m,2H).ESI-MS m/z 569.0[M-Cl]⁺。

example 61

1-methyl-N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide (Compound 61)

Referring to the procedure of example 60, substituting 1-Boc-4-piperidinecarboxylic acid for 1-methylpiperidine-4-carboxylic acid, compound 61 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ12.43(s,1H),8.23(d,J＝5.0Hz,2H),7.81(d,J＝2.0Hz,1H),7.77–7.65(m,3H),7.34–7.22(m,2H),7.00(dd,J＝8.6,2.1Hz,1H),2.89–2.75(m,2H),2.49–2.44(m,1H),2.18(s,3H),1.97–1.76(m,4H),1.75–1.55(m,2H).ESI-MS m/z 583.0[M+H]⁺。

example 62

N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) piperazine-1-carboxamide hydrochloride (Compound 62)

By substituting compound 43c for compound 59b according to the procedure of example 60, compound 62 (white solid) was prepared:¹H NMR(300MHz,DMSO-d₆)δ9.29(s,2H),8.29–8.18(m,2H),7.78–7.71(m,2H),7.70–7.65(m,1H),7.55(s,1H),7.36–7.24(m,2H),7.52(s,1H),6.99–6.91(m,1H),3.88–3.68(m,4H),3.21–3.07(m,4H).ESI-MS m/z 570.2[M-Cl]⁺。

example 63

N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclohexanecarboxamide (Compound 63)

2-amino-6-nitrobenzothiazole (500mg,2.56mmol) was suspended in anhydrous dichloromethane (10mL), triethylamine (0.71mL,5.12mmol) was added, 63a (563mg,3.84mmol) was added dropwise slowly and stirred at room temperature until TLC monitored the reaction completion. The reaction solution was concentrated, diluted with ethyl acetate (20mL), washed successively with 1N diluted hydrochloric acid (20mL), water (20mL), saturated sodium bicarbonate (20mL) and saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 100:1) to give compound 63b (pale yellow solid, 513mg, yield 66%).

Taking the compound 63b (500mg,1.64mmol), suspending the compound in a mixed solution of acetic acid (8mL) and water (2mL), slowly adding zinc powder (535mg,8.18mmol) under stirring at room temperature, after TLC monitoring reaction completion, adding water (10mL) and ethyl acetate (20mL) for dilution, adding sodium bicarbonate to adjust pH to be neutral, washing with saturated aqueous sodium bicarbonate solution (20mL multiplied by 2), drying with anhydrous sodium sulfate, filtering, concentrating filtrate to obtain a crude product of the compound 63c (yellow brown solid, 197mg, yield 71%), and directly putting the crude product to the next step without purification.

Compound 63c (165mg,0.60mmol) was suspended in dichloromethane (15mL), triethylamine (250. mu.L, 1.80mmol), 4-dimethylaminopyridine (DMAP,7mg,0.06mmol) and 2-thiophenesulfonyl chloride (274mg,1.50mmol) were added in that order, and after stirring at room temperature until TLC monitored that the reaction was complete, the mixture was addedAfter dilution with dichloromethane (15mL), washing with 1N diluted hydrochloric acid (20mL), water (30mL) and saturated brine (30mL) in this order, drying over anhydrous sodium sulfate, filtration, concentration of the filtrate, and purification by silica gel column chromatography (dichloromethane: methanol ═ 350:1) gave compound 63 (white solid, 69mg, yield 20%). 1H NMR (300MHz, CDCl)₃)δ9.84(s,1H),7.85–7.78(m,2H),7.78–7.73(m,2H),7.71(s,1H),7.56(d,J＝2.0Hz,1H),7.22–7.12(m,3H),2.44–2.29(m,1H),2.02–1.87(m,2H),1.86–1.74(m,2H),1.62–1.46(m,2H),1.36–1.06(m,4H).calculated for C₂₂H₂₂N₃O₅S₅[M+H]⁺568.0158,found 568.0161。

Example 64

N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclopentanecarboxamide (Compound 64)

Referring to the synthetic procedure of example 63, cyclohexanoyl chloride was substituted for cyclopentanoyl chloride to give compound 64 (white solid, 136 mg). 1H NMR (300MHz, DMSO-d)₆)δ12.52(s,1H),8.28–8.18(m,2H),7.82(M,1H),7.78–7.70(m,3H),7.29(t,2H),7.00(dd,J＝8.6,2.1Hz,1H),3.07–2.93(m,1H),2.01–1.85(m,2H),1.83–1.51(m,6H).HRMS m/z(ESI):calculated for C₂₁H₂₀N₃O₅S₅[M+H]⁺554.0001,found 544.0010。

Example 65

4-oxo-N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclohexane-1-carboxamide (Compound 65)

2-amino-6-nitrobenzothiazole (1g,5.12mmol) was suspended in dichloromethane (15mL), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI,2.93g,0.71mL,1.53mmol), 4-dimethylaminopyridine (DMAP,187mg,0.153mmol) and 65a (2.17g, 1.53mmol) were added sequentially, the mixture was stirred at room temperature until the TLC monitoring reaction was complete, dichloromethane (15mL) was added for dilution, the reaction solution was washed with a saturated aqueous sodium bicarbonate solution (35mL), 1N diluted hydrochloric acid (35mL) and a saturated saline solution (35mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated and then slurried with dichloromethane (4mL) and N-hexane (4mL), and compound 65b (a yellow-white solid, 775mg, 48% yield) was obtained by suction filtration.

Referring to the synthetic procedure of example 63, intermediate 63b was replaced with 65b to give compound 65 (white solid, 136 mg). 1H NMR (300MHz, DMSO-d)₆)δ12.66(s,1H),8.22(d,J＝4.9Hz,2H),7.90–7.80(m,1H),7.79–7.66(m,3H),7.43–7.22(m,2H),7.01(dd,J＝8.6,2.0Hz,1H),3.15–2.97(m,1H),2.47–2.38(m,2H),2.37–2.26(m,2H),2.26–2.07(m,2H),2.03–1.80(m,2H).HRMS m/z(ESI):calculated for C₂₂H₂₀N₃O₆S₅[M+H]⁺581.9950,found 581.9949。

Example 66

2- (piperidin-4-yl) -N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) acetamide (Compound 66)

Referring to the synthetic procedure of example 65, starting material 65a was replaced with 1-tert-butoxycarbonyl-4-piperidineacetic acid to give intermediate 66a (white solid, 497 mg).

Compound 66a (497mg,0.73mmol) was dissolved in trifluoroacetic acid (4mL) and dichloromethane (4mL), stirred at room temperature until TLC monitored reaction completion, the reaction was concentrated, adjusted to pH 8-9 with saturated sodium bicarbonate solution, filtered with suction, and dried under infrared to give compound 66 (white solid, 420mg, 99%):¹H NMR(300MHz,DMSO-d₆)δ8.27–8.19(m,2H),7.87–7.80(m,1H),7.78–7.65(m,3H),7.30(t,1H),7.00(dd,J＝8.6,2.1Hz,1H),3.25–3.18(m,3H),2.90–2.78(m,2H),2.16–1.97(m,1H),1.88–1.74(m,2H),1.46–1.25(m,2H).HRMS m/z(ESI):calculated for C₂₂H₂₃N₄O₅S₅[M+H]⁺583.0266,found 583.0280。

example 67

1-methyl-N- (6- (N- (methylsulfonyl) methylsulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide (Compound 67)

Referring to the synthetic procedure of example 61, 2-thiophenesulfonyl chloride was replaced with methanesulfonyl chloride to give compound 67 (white solid, 85 mg). 1H NMR (300MHz, DMSO-d)₆)δ12.46(s,1H),8.22(s,1H),7.87–7.72(m,1H),7.62–7.46(m,1H),3.56(s,6H),2.82(d,J＝9.3Hz,2H),2.17(s,3H),2.06–1.76(m,4H),1.77–1.53(m,2H).HRMS m/z(ESI):calculated for C₁₆H₂₃N₄O₅S₃[M+H]⁺447.0825,found 447.0827。

Example 68

1-methyl-N- (6- (N- (phenylsulfonyl) phenylsulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide (compound 68)

Referring to the synthetic procedure of example 61, 2-thiophenesulfonyl chloride was replaced with benzenesulfonyl chloride to give compound 68 (white solid, 195 mg). 1H NMR (300MHz, DMSO-d)₆)δ12.40(s,1H),7.91–7.76(m,7H),7.75–7.62(m,5H),6.95(d,J＝8.6Hz,1H),3.00–2.79(m,2H),2.57(d,J＝10.9Hz,1H),2.24(s,3H),2.13–1.95(m,2H),1.94–1.79(m,2H),1.79–1.58(m,2H).HRMS m/z(ESI):calculated for C₂₆H₂₇N₄O₅S₃[M+H]⁺571.1138,found 571.1148。

Example 69

1-methyl-N- (6- (((1-methyl-N- ((1-methyl-1H-pyrazol-4-yl) sulfonyl) -1H-pyrazole) -4-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide (Compound 69)

Referring to the synthetic procedure of example 61, 2-thiophenesulfonyl chloride was replaced with 1-methyl-1H-pyrazole-3-sulfonyl chloride to give compound 69 (white solid, 137 mg). 1H NMR (300MHz, DMSO-d)₆)δ12.46(s,1H),8.40(s,2H),7.85(d,J＝2.1Hz,1H),7.79(s,2H),7.73(d,J＝8.6Hz,1H),7.06(dd,J＝8.6,2.1Hz,1H),3.94(s,6H),2.82(d,J＝11.3Hz,2H),2.59–2.53(m,1H),2.17(s,3H),1.96–1.77(m,4H),1.76–1.55(m,2H).HRMS m/z(ESI):calculated for C₂₂H₂₇N₈O₅S₃[M+H]⁺579.1261,found 579.1267。

Example 70

1-methyl-N- (6- (N- (pyridin-3-ylsulfonyl) pyridine-3-sulfonylamino) benzo [ d ] thiazol-2-yl) piperidine-4-carboxamide (Compound 70)

Referring to the synthetic procedure of example 61, 2-thiophenesulfonyl chloride was replaced with 3-pyridinesulfonyl chloride to give compound 70 (white solid, 130 mg). 1H NMR (300MHz, DMSO-d)₆)δ12.43(s,1H),9.02(d,J＝4.0Hz,2H),8.97(d,J＝1.9Hz,2H),8.28(d,J＝8.2Hz,2H),7.98(d,J＝1.9Hz,1H),7.87–7.70(m,3H),7.06(dd,J＝8.6,2.0Hz,1H),3.00–2.81(m,2H),2.64–2.53(m,1H),2.25(s,3H),2.14–1.97(m,2H),1.94–1.81(m,2H),1.78–1.61(m,2H).HRMS m/z(ESI):calculated for C₂₄H₂₅N₆O₅S₃[M+H]⁺573.1043,found 573.10396。

Example 71

2-oxo-2- ((6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonamido) benzo [ d ] thiazol-2-yl) amino) ethane-1-ammonium chloride (Compound 71)

2-amino-6-nitrobenzothiazole (1g,5.12mmol) was suspended in dichloromethane (40mL) and Boc was added sequentially₂O (1.34g,6.15mmol) and 4-dimethylaminopyridine (DMAP,751mg, 6.15mmol) were stirred at room temperature until TLC monitored for reaction completion. The solid was collected by filtration and the filter cake was washed with dichloromethane (4mL) to give compound 71b (white solid, 1.26g, 84% yield).

Compound 71b (1.6mg,5.42mmol) was suspended in methanol (50mL), 10% palladium on carbon (160mg, 10%) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere until the reaction was complete by TLC. After the reaction was completed, suction filtration was performed with celite, and the solvent was distilled off from the filtrate under reduced pressure to obtain intermediate 71c (pink solid, 1.3g, yield 90%).¹H NMR(300MHz,DMSO-d₆)δ11.32(s,1H),7.33(d,J＝8.5Hz,1H),7.03–6.93(m,1H),6.71–6.61(m,1H),5.08(s,2H),1.49(s,9H)。

Referring to the synthetic procedure of example 65, intermediate 65c was replaced with 71c to give intermediate 71d (white solid, 1.2g, 72% yield).¹H NMR(300MHz,DMSO-d₆)δ11.96(s,1H),8.23(d,J＝4.9Hz,2H),7.79–7.64(m,4H),7.29(t,J＝4.3Hz,2H),7.00–6.91(m,1H),1.52(s,9H)。

Reference was made to the synthetic procedure of example 66 substituting intermediate 66a for 71d to give intermediate 71e (white solid, 806mg, 82%).¹H NMR(300MHz,DMSO-d₆)δ8.21(d,J＝4.0Hz,2H),7.78(s,2H),7.72(d,J＝2.8Hz,2H),7.45(d,J＝2.0Hz,1H),7.34–7.22(m,3H),6.75(dd,J＝8.5,2.0Hz,1H)。

Reference example 65 synthetic procedure starting material 65a was replaced with Boc-glycine and 2-amino-6 nitrobenzothiazole was replaced with 71e to afford intermediate 71f (white solid, 240mg, 85%).

Intermediate 71f (240mg,0.39mmol) was dissolved in ethyl acetate hydrochloride solution (6mL), stirred overnight at room temperature, after TLC to monitor completion of the reaction, filtered with suction and the filter cake was dried to afford compound 71 (white solid, 101mg, 47% yield).¹H NMR(300MHz,DMSO-d₆)δ13.07(s,1H),8.44(s,3H),8.24(d,J＝4.9Hz,2H),7.94(d,J＝2.1Hz,1H),7.80(d,J＝8.6Hz,1H),7.74(d,J＝3.8Hz,2H),7.30(t,J＝4.4Hz,2H),7.01(dd,J＝8.6,2.1Hz,1H),4.14–4.00(m,2H).HRMS m/z(ESI):calculated for C₁₇H₁₅N₄O₅S₅[M-Cl]⁺514.9640,found 514.9645。

Example 72

2- ((6- ((1-methyl-N- ((1-methyl-1H-pyrazol-4-yl) sulfonyl) -1H-pyrazole) -4-sulfonylamino) benzo [ d ] thiazol-2-yl) amino) -2-oxoethane-1-ammonium chloride (Compound 72)

Referring to the synthetic route for example 65, starting material 65a was replaced with Boc-glycine and 2-thiophenesulfonyl chloride was replaced with 1-methyl-1H-pyrazole-3-sulfonyl chloride to afford intermediate 72a (yellow solid, 150 mg).

Referring to the synthetic route to example 71, substituting 71f for 72a gave compound 72 (light yellow solid, 81mg, 90%).¹H NMR(300MHz,DMSO-d₆)δ13.03(s,1H),8.45(s,3H),8.42(s,2H),7.98–7.92(m,1H),7.88–7.74(m,3H),7.14–7.04(m,1H),4.06–3.98(m,2H),3.95(s,6H).HRMS m/z(ESI):calculated for C₁₇H₁₉N₈O₅S₅[M-Cl]⁺511.0635,found 511.0646。

Example 73

2- ((6- (N- (cyclopropylsulfonyl) cyclopropanesulfonamido) benzo [ d ] thiazol-2-yl) amino) -2-oxa-1-aminium chloride (Compound 73)

Referring to the synthetic route for example 72, 1-methyl-1H-pyrazole-3-sulfonyl chloride was substituted for cyclopropylsulfonyl chloride to give compound 73 (white solid, 80 mg).¹H NMR(300MHz,DMSO-d₆)δ13.01(s,1H),8.36(s,3H),8.22(d,J＝2.1Hz,1H),7.84(d,J＝8.6Hz,1H),7.51(dd,J＝8.6,2.2Hz,1H),4.05–3.92(m,2H),3.30–3.17(m,2H),1.33–1.15(m,4H),1.12–0.99(m,4H).HRMS m/z(ESI):calculated for C₁₅H₁₉N₄O₅S₃[M-Cl]⁺431.0512,found 431.0518。

Example 74

trans-4-amino-N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclohexane-1-carboxamide (Compound 74)

Referring to the synthetic procedure of example 66, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with trans-4- (Boc-amino) cyclohexanecarboxylic acid to give compound 74 (white solid, 130 mg).¹H NMR(300MHz,DMSO-d₆)δ9.04(s,2H),8.23(d,J＝4.8Hz,2H),7.82(d,J＝1.8Hz,1H),7.78–7.66(m,3H),7.29(t,J＝4.4Hz,2H),7.00(dd,J＝8.6,1.8Hz,1H),3.11–2.94(m,1H),2.62–2.52(m,1H),2.13–1.89(m,4H),1.63–1.46(m,2H),1.44–1.28(m,2H).HRMS m/z(ESI):calculated for C₂₂H₂₃N₄O₅S₅[M+H]⁺583.0266,found 583.0279。

Example 75

3- ((6- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) carbamoyl) azetidine hydrochloride (Compound 75)

Referring to the synthetic procedure of example 71, Boc-glycine was replaced with 1-Boc-azetidine-3-carboxylic acid to give compound 75 (white solid, 120 mg).¹H NMR(300MHz,DMSO-d₆)δ12.79(s,1H),9.47(s,1H),9.06(s,1H),8.24(d,J＝4.8Hz,2H),7.89(s,1H),7.82–7.54(m,3H),7.35–7.20(m,2H),7.07–6.93(m,1H),4.25–4.04(m,4H),4.03–3.99(m,1H).HRMS m/z(ESI):calculated for C₁₉H₁₇N₄O₅S₅[M-Cl]⁺550.9797,found 550.9805。

Example 76

2- ((6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) carbamoyl) pyrrole hydrochloride (Compound 76)

Referring to the synthetic procedure of example 71, Boc-glycine was replaced with N-Boc-DL-proline to give compound 76 (white solid, 140 mg).¹H NMR(300MHz,DMSO-d₆)δ13.23(s,1H),10.20(s,1H),8.93(s,1H),8.29–8.13(m,2H),7.98–7.85(m,1H),7.84–7.77(m,1H),7.77–7.63(m,2H),7.35–7.18(m,2H),7.07–6.94(m,1H),4.69–4.44(m,1H),3.39–3.13(m,3H),2.45–2.32(m,1H),2.14–2.01(m,1H),2.00–1.80(m,2H).HRMS m/z(ESI):calculated for C₂₀H₁₉N₄O₅S₅[M-Cl]⁺554.9953,found 554.9963。

Example 77

1-methyl-N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) -1H-pyrrole-2-carboxamide (Compound 77)

Referring to the synthetic procedure of example 71, Boc-glycine was replaced with N-methyl-2-pyrrolecarboxylic acid to give compound 77 (white solid, 129 mg).¹H NMR(300MHz,CDCl₃)δ9.68(s,1H),7.82(dd,J＝3.8,1.2Hz,2H),7.77(dd,J＝4.9,1.1Hz,2H),7.67(d,J＝8.6Hz,1H),7.56(d,J＝2.1Hz,1H),7.21–7.10(m,3H),6.96–6.89(m,2H),6.26–6.17(m,1H),4.06(s,3H).HRMS m/z(ESI):calculated for C₂₁H₁₇N₄O₅S₅[M+H]⁺564.9797,found 564.97935。

Example 78

2- (dimethylamino) -N- (6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) acetamide (Compound 78)

Referring to the synthetic procedure of example 71, Boc-glycine was replaced with N, N-dimethylglycine to give compound 78 (white solid, 75 mg).¹H NMR(300MHz,CDCl₃)δ7.82(dd,J＝3.8,1.3Hz,2H),7.76(dd,J＝4.8,3.6Hz,3H),7.57(d,J＝2.0Hz,1H),7.22–7.17(m,2H),7.17–7.14(m,1H),3.26(s,2H),2.44(s,6H).HRMS m/z(ESI):calculated for C₁₉H₁₉N₄O₅S₅[M+H]⁺542.99535,found 542.99463。

Example 79

4-oxo-4- ((6- (N- (thien-2-ylsulfonyl) thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) amino) butanoic acid (Compound 79)

Referring to the synthetic procedure of example 71, Boc-glycine was substituted for succinic anhydride to give compound 79 (white solid, 64 mg).¹H NMR(300MHz,DMSO-d₆)δ12.82–12.07(m,2H),8.30–8.15(m,2H),7.82(d,J＝2.1Hz,1H),7.79–7.67(m,3H),7.29(t,2H),6.99(dd,J＝8.6,2.1Hz,1H),2.76(t,J＝6.5Hz,2H),2.60(t,J＝6.3Hz,2H).HRMS m/z(ESI):calculated for C₁₉H₁₆N₃O₇S₅[M+H]⁺557.9586,found 557.9595。

Example 80

2- (piperidin-4-yl) -N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) acetamide (Compound 80)

Referring to the synthetic procedure of example 65, 65a was replaced with 1-tert-butoxycarbonyl-4-piperidineacetic acid to give intermediate 80a (pale red solid, 628 mg).

Intermediate 80a (350mg,0.90mmol) was dissolved in dichloromethane (8mL) and pyridine (1mL), 2-thiophenesulfonyl chloride (196mg,1.08mmol) was slowly added, stirred at room temperature until the reaction was monitored by TLC, diluted by addition of ethyl acetate (20mL), washed successively with 1N diluted hydrochloric acid (20mL × 2) and saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 50:1) to give compound 80b (white solid, 454mg, yield 94%).¹H NMR(300MHz,DMSO-d₆)δ12.29(s,1H),10.41(s,1H),7.87(dd,J＝5.0,1.3Hz,1H),7.69(d,J＝2.0Hz,1H),7.61(d,J＝8.7Hz,1H),7.51(dd,J＝3.7,1.3Hz,1H),7.16(dd,J＝8.7,2.1Hz,1H),7.12–7.06(m,1H),4.00–3.82(m,2H),2.85–2.62(m,2H),2.42(d,J＝7.0Hz,1H),2.04–1.84(m,1H),1.73–1.56(m,2H),1.39(s,9H)。

Referring to the synthetic procedure of example 66, intermediate 66a was replaced with 80b to give compound 80 (light yellow solid, 304mg, 83%).¹H NMR(300MHz,DMSO-d₆)δ7.65(d,J＝4.9Hz,1H),7.52(d,J＝2.0Hz,1H),7.47(d,J＝8.7Hz,1H),7.36(d,J＝3.5Hz,1H),7.04(dd,J＝8.7,2.0Hz,1H),6.99(t,1H),3.14–3.01(m,2H),2.75–2.58(m,2H),2.39(d,J＝7.0Hz,2H),2.04–1.87(m,1H),1.76–1.62(m,2H),1.31–1.16(m,2H).HRMS m/z(ESI):calculated for C₁₈H₂₁N₄O₃S₃[M+H]⁺437.0770,found 437.0780。

Example 81

4-oxo-N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclohexane-1-carboxamide (Compound 81)

Referring to the synthetic procedure of example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with 4-carbonylCyclohexanecarboxylic acid to give compound 81 (white solid, 187 mg).¹H NMR(300MHz,DMSO-d₆)δ12.44(s,1H),10.42(s,1H),7.87(d,J＝4.9Hz,1H),7.70(d,J＝2.0Hz,1H),7.64(d,J＝8.7Hz,1H),7.56–7.48(m,1H),7.18(dd,J＝8.7,2.1Hz,1H),7.09(t,1H),3.06–2.94(m,1H),2.47–2.36(m,2H),2.37–2.25(m,2H),2.26–2.08(m,2H),1.96–1.78(m,2H).HRMS m/z(ESI):calculated for C₁₈H₁₈N₃O₄S₃[M+H]⁺436.0454,found 436.0456。

Example 82

N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) azetidine-3-carboxamide hydrochloride (Compound 82)

Referring to the synthetic route of example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with 1-Boc-azetidine-3-carboxylic acid to give intermediate 82a (white solid, 102 mg).¹H NMR(300MHz,DMSO-d₆)δ12.42(s,1H),10.43(s,1H),7.92–7.84(m,1H),7.77–7.69(m,1H),7.63(d,J＝8.7Hz,1H),7.56–7.49(m,1H),7.17(dd,J＝8.7,2.1Hz,1H),7.12–7.06(m,1H),4.13–3.89(m,4H),3.70–3.59(m,1H),1.39(s,9H)。

Referring to the synthetic procedure of example 72, intermediate 72a was replaced with 82a to give compound 82 (white solid, 37mg, 42% yield).¹H NMR(300MHz,DMSO-d₆)δ12.55(s,1H),10.50(s,1H),9.34(s,1H),8.99(s,1H),7.94–7.85(m,1H),7.79–7.73(m,1H),7.70–7.62(m,1H),7.57–7.51(m,1H),7.24–7.16(m,1H),7.14–7.07(m,1H),4.23–4.02(m,4H),4.02–3.85(m,1H).HRMS m/z(ESI):calculated for C₁₅H₁₅N₄O₃S₃[M-Cl]⁺395.0301,found 395.0310。

Example 83

trans-4-amino-N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclohexane-1-carboxamide (Compound 83)

Referring to the synthetic procedure of example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with trans-4- (Boc-amino) cyclohexanecarboxylic acid to give compound 83 (white solid, 70 mg).¹H NMR(300MHz,DMSO-d₆)δ7.61(d,J＝4.5Hz,1H),7.54–7.38(m,2H),7.38–7.21(m,1H),7.09–6.82(m,2H),2.93–2.74(m,1H),2.46–2.32(m,1H),2.05–1.76(m,4H),1.60–1.33(m,2H),1.34–1.02(m,2H).HRMS m/z(ESI):calculated for C₁₈H₂₁N₄O₃S₃[M+H]⁺437.0770,found 437.0769。

Example 84

4- (dimethylamino) -N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) benzamide (compound 84)

Referring to the synthetic procedure of example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with 4-dimethylaminobenzoic acid to give compound 84 (white solid, 55 mg).¹H NMR(300MHz,DMSO-d₆)δ12.40(s,1H),10.42(s,1H),8.03(d,J＝8.9Hz,2H),7.93–7.84(m,1H),7.71(s,1H),7.64(d,J＝8.6Hz,1H),7.58–7.49(m,1H),7.26–7.15(m,1H),7.15–7.06(m,1H),6.77(d,J＝8.9Hz,2H),3.03(s,6H).HRMS m/z(ESI):calculated for C₂₀H₁₉N₄O₃S₃[M+H]⁺459.0614,found 459.0620。

Example 85

3- (N- (2- (tetrahydro-2H-pyran-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) propionic acid methyl ester (Compound 85)

Referring to the synthetic procedure of example 80, 1-tert-butoxycarbonyl-4-piperazinoPyridine acetic acid was replaced with tetrahydropyran-4-carboxylic acid and 2-thiophenesulfonyl chloride was replaced with methyl 3- (chlorosulfonyl) propionate to give compound 85 (white solid, 160 mg).¹H NMR(300MHz,DMSO-d₆)δ12.32(s,1H),9.96(s,1H),7.78(d,J＝2.0Hz,1H),7.69(d,J＝8.7Hz,1H),7.32–7.22(m,1H),3.91(d,J＝11.3Hz,2H),3.58(s,3H),3.36(t,J＝7.3Hz,4H),2.88–2.69(m,3H),1.86–1.56(m,4H).HRMS m/z(ESI):calculated for C₁₇H₂₂N₃O₆S₂[M+H]⁺428.0945,found 428.0950。

Example 86

3- (N- (2- (tetrahydro-2H-pyran-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) propionic acid (compound 86)

Referring to the synthetic procedure of example 53, substituting 53a for 85, compound 86 (white solid, 50mg) was obtained.¹H NMR(300MHz,DMSO-d₆)δ12.51(s,1H),12.33(s,1H),9.93(s,1H),7.79(d,J＝2.0Hz,1H),7.69(d,J＝8.7Hz,1H),7.34–7.22(m,1H),4.01–3.79(m,2H),3.43–3.31(m,4H),2.87–2.72(m,1H),2.72–2.60(m,2H),1.85–1.56(m,4H).HRMS m/z(ESI):calculated for C₁₆H₂₀N₃O₆S₂[M+H]⁺414.0788,found 414.0791。

Example 87

3- (N- (2- (1-methylpiperidine-4-carboxamido) benzo [ d ] thiazol-6-yl) sulfamoyl) propionic acid (compound 87)

Referring to the synthetic procedure of example 85, tetrahydropyran-4-carboxylic acid was replaced with 1-methylpiperidine-4-carboxylic acid to give a methyl ester of compound 87. Referring again to the synthetic procedure of example 86, compound 85 was replaced with the methyl ester of compound 87 to give compound 87 (white solid, 127 mg).¹H NMR(300MHz,DMSO-d₆)δ12.49(s,1H),10.43(s,1H),9.96(s,1H),7.81(d,J＝2.0Hz,1H),7.70(d,J＝8.7Hz,1H),7.30(dd,J＝8.7,2.0Hz,1H),3.52–3.34(m,4H),3.12–2.91(m,2H),2.89–2.77(m,1H),2.74(s,3H),2.66(t,J＝7.3Hz,2H),2.15–2.04(m,2H),2.03–1.84(m,2H).HRMS m/z(ESI):calculated for C₁₇H₂₃N₄O₅S₂[M+H]⁺427.1104,found 427.1111。

Example 88

4-methyl-N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) piperazine-1-carboxamide (Compound 88)

Referring to the synthetic procedure of example 58, morpholine was substituted for methylpiperazine to give compound 88 (light yellow solid, 105 mg).¹H NMR(300MHz,DMSO-d₆)δ11.39(s,1H),10.45(s,1H),7.87(d,J＝4.8Hz,1H),7.59–7.52(m,1H),7.50(d,J＝3.4Hz,1H),7.43(d,J＝8.6Hz,1H),7.16–7.05(m,1H),3.61–3.45(m,3H),2.38–2.24(m,3H),2.19(s,2H).HRMS m/z(ESI):calculated for C₁₇H₂₀N₅O₃S₃[M+H]⁺438.0723,found 438.0725。

Example 89

2- (dimethylamino) -N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) acetamide (Compound 89)

Referring to the synthetic procedure of example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with N, N-dimethylglycine to give compound 89 (white solid, 65 mg).¹H NMR(300MHz,DMSO-d₆)δ11.71(s,1H),10.56(s,1H),7.92–7.83(m,1H),7.75–7.69(m,1H),7.62(d,J＝8.7Hz,1H),7.57–7.48(m,1H),7.22–7.13(m,1H),7.13–7.06(m,1H),3.28(s,2H),2.29(s,6H).HRMS m/z(ESI):calculated for C₁₅H₁₇N₄O₃S₃[M+H]⁺397.0457,found 397.0461。

Example 90

1-methyl-N- (6- (phenylsulfamoyl) benzothiazol-2-yl) piperidine-4-carboxamide (Compound 90)

Referring to the synthetic procedure of example 55, 2-thiophenesulfonyl chloride was replaced with benzenesulfonyl chloride to give compound 90 (white solid, 132 mg).¹H NMR(300MHz,DMSO-d₆)δ12.05(s,1H),10.45(s,1H),7.81–7.69(m,2H),7.66–7.62(m,1H),7.61–7.44(m,4H),7.11(dd,J＝8.7,2.1Hz,1H),2.88–2.70(m,2H),2.48–2.38(m,1H),2.16(s,3H),1.99–1.73(m,4H),1.72–1.53(m,2H).HRMS m/z(ESI):calculated for C₂₂H₂₃N₄O₃S₂[M+H]⁺431.1206,found 431.1207。

Example 91

N- (6- ((5-chlorothiophene) -2-sulfonamido) benzothiazol-2-yl) -1-methylpiperidine-4-carboxamide (91)

Referring to the synthetic procedure of example 85, 2-thiophenesulfonyl chloride was replaced with 5-chloro-2-thiophenesulfonyl chloride to give compound 91 (pink solid, 84 mg).¹H NMR(300MHz,DMSO-d₆)δ12.23(s,1H),7.71(s,1H),7.63(d,J＝8.6Hz,1H),7.47–7.31(m,1H),7.27–7.08(m,2H),3.04–2.84(m,2H),2.62–2.52(m,1H),2.29(s,3H),2.21–2.03(m,2H),1.95–1.79(m,2H),1.79–1.59(m,2H).HRMS m/z(ESI):calculated for C₁₈H₁₉ClN₄O₃S₃[M+H]⁺471.0381,found 471.0391。

Example 92

N- (6- (thiophene-2-sulfonamido) benzo [ d ] thiazol-2-yl) acetamide (Compound 92)

Reference example 1 Synthesis Experimental procedure substituting furfuryl chloride for acetyl chloride gave N- (6-aminobenzo [ d]Thiazol-2-yl) acetamide. Referring again to the synthetic experimental procedure of example 23, p-nitrobenzoyl chloride was replaced with 2-thiophenesulfonyl chloride and 1c was replaced with N- (6-aminobenzo [ d ]]Thiazol-2-yl) acetamide to afford compound 92 (white solid).¹H NMR(300MHz,DMSO-d₆)δ12.30(s,1H),10.42(s,1H),7.87(d,J＝4.8Hz,1H),7.75–7.67(m,1H),7.62(d,J＝8.6Hz,1H),7.52(d,J＝3.3Hz,1H),7.22–7.12(m,1H),7.12–7.04(m,1H),2.19(s,3H).ESI-MS m/z 352.0[M-H]^-。

Example 93

N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclopentanecarboxamide (Compound 93)

Referring to the synthetic experimental procedure for example 92, acetyl chloride was substituted for cyclopentanoyl chloride to provide compound 93 (yellow solid).¹H NMR(300MHz,DMSO-d₆)δ12.31(s,1H),10.43(s,1H),7.92–7.80(m,1H),7.74–7.65(m,1H),7.65–7.55(m,1H),7.56–7.44(m,1H),7.21–7.12(m,1H),7.11–7.01(m,1H),3.04–2.85(m,1H),1.99–1.80(m,2H),1.80–1.46(m,6H).ESI-MS m/z430.1[M+Na]⁺。

Example 94

N- (6- (thiophene-2-sulfonylamino) benzo [ d ] thiazol-2-yl) cyclohexanecarboxamide (94)

Referring to the synthetic experimental procedure of example 92, acetyl chloride was substituted for cyclohexanecarbonyl chloride to give compound 94 (white solid).¹H NMR(300MHz,DMSO-d₆)δ12.22(s,1H),10.40(s,1H),7.93–7.81(m,1H),7.68(d,J＝2.0Hz,1H),7.61(d,J＝8.7Hz,1H),7.55–7.45(m,1H),7.16(dd,J＝8.7,2.1Hz,1H),7.12–7.03(m,1H),2.62–2.53(m,1H),1.91–1.11(m,10H).ESI-MS m/z 420.1[M-H]^-。

Example 95

Micro-calorimetry (MST) method for determining binding force of compound and USP 7C-terminal protein

1. Purpose of experiment

The binding force of the compound and the USP 7C-terminal protein is measured by adopting an MST method, and the respective equilibrium dissociation constant (K) is calculated_D)。

2. Experimental Material

The compound of the invention (dissolved by dimethyl sulfoxide (DMSO) to prepare mother liquor, and diluted by Tris-HCl buffer solution to proper concentration before use); USP 7C-terminal protein (10 μ M); microcalorimetric phorometers (nt.115); an MST standard capillary; MST protein fluorescence labeling kit (NT-647); Tris-HCl buffer.

3. Experimental methods

(1) Diluting USP 7C-terminal protein to 10 μ M protein solution with PBS (pH7.4), replacing PBS solution with labeling buffer solution (100 μ L) with high labeling efficiency by desalting column in MST protein fluorescence labeling kit, adding 100 μ L labeling buffer solution containing fluorescent dye (NT-647) to make the dye concentration 20 μ M, and incubating at room temperature in dark place; (2) after 1 hour of incubation, separating the marked protein from redundant fluorescent dye by using a separation column in a protein fluorescence labeling kit to obtain 500 mu L of marked USP 7C-terminal protein solution; (3) absorbing the marked protein solution by using an MST standard capillary, detecting the fluorescence intensity of the protein solution, and determining 100% of LED power; (4) preparing a gradient diluent of the small molecule compound in a 0.2mL EP tube by using a Tris-HCl buffer solution, wherein the volume of the small molecule solution in each tube is 10 mu L, and the DMSO content in each tube is kept consistent (1%); (5) adding an equal volume (10 mu L) of protein solution into an EP tube filled with the compound solution of the invention, uniformly mixing, and then incubating at room temperature for 0.5 h; (6) the small molecule-protein mixture solution (one capillary per concentration) was aspirated using MST standard capillaries, tested on a machine (LED power 100%, MST power 40%), and the test results were fitted using the thiophhoresis + T-Jump model.

4. Results of the experiment

Binding force of partial compound to USP 7C-terminal protein (K)_D) The test results are shown in table 1. The experimental result shows that the compound can be combined with the C-terminal protein of USP7, part of the compound has stronger affinity, and other compounds also have better affinity.

TABLE 1 binding force of Compounds 1-39 with USP 7C-terminal protein

Compound (I)	K_D(μM)	Compound (I)	K_D(μM)	Compound (I)	K_D(μM)	Compound (I)	K_D(μM)
								1	85.9	12	68.0	23	0.4	34	>250
2	>250	13	45.3	24	>250	35	>250
								3	25.6	14	25.0	25	2.2	36	49.2
4	>250	15	127.0	26	2.5	37	2.6
								5	105.0	16	108.0	27	76.4	38	2.9
6	>250	17	36.2	28	85.5	39	>250
								7	47.0	18	>250	29	84.5
8	56.7	19	>250	30	63.4
								9	8.4	20	5.4	31	>250
10	>250	21	>250	32	52.1
								11	>250	22	3.3	33	>250

Example 96

Method for measuring binding force of compound and USP 7C-terminal protein by Surface Plasmon Resonance (SPR) method

1. Purpose of experiment

The binding force of the compound and the USP 7C-terminal protein is measured by an SPR method, and the respective equilibrium dissociation constant (K) is calculated_D)。

2. Experimental Material

The compound of the invention (dissolved in DMSO to prepare mother liquor, and diluted to proper concentration by PBST (pH7.4, Tween 20 content 0.05%) buffer solution before use); USP 7C-terminal protein (1 mg/mL); biacore T200(GE Healthcare); CM5 chip (GE Healthcare); amino coupling kit (GE Healthcare); PBST buffer.

3. Experimental methods

(1) The USP 7C-terminal protein is diluted into a protein solution (diluted by 50 times) of 20 mu g/mL by 10mM sodium acetate solutions with pH 4.0, 4.5, 5.0 and 5.5 respectively, and flows through a second channel (the first channel is a reference channel) of a CM5 chip in sequence, and the comparison shows that the pre-enrichment signal is higher and the protein activity is better at pH 4.5, so that USP 7C-terminal protein diluted by 10mM sodium acetate solution with pH 4.5 is used for coupling; (2) setting a program, namely enabling 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to simultaneously flow through a first channel and a second channel (namely activation), enabling diluted protein to flow through the second channel (namely coupling), and finally enabling ethanolamine to simultaneously flow through the two channels (namely sealing), wherein a coupling signal is 16000RU, and the coupling effect is better; (3) the compound of the invention was diluted with 5% DMSO in PBST, the compound solution was flowed through both channels simultaneously, bound for 90 seconds, dissociated for 90 seconds to give binding and dissociation curves of the compound to the protein, and K was obtained by Biacore T200 Evaluation Software fitting_D。

4. Results of the experiment

Binding force of partial compound to USP 7C-terminal protein (K)_D) The test results are shown in table 2. The experimental result shows that the compound can be combined with the C-terminal protein of USP7, the affinity of some compounds is stronger, and the affinity of other compounds is better.

TABLE 2 binding of partial compounds to the C-terminal protein of USP7

Compound (I)	K_D(μM)	Compound (I)	K_D(μM)	Compound (I)	K_D(μM)	Compound (I)	K_D(μM)
								25	4.26	45	61.2	51	9.7	57	27.7
40	17.8	46	39.1	52	187.3	58	17.8
								41	51.4	47	153.0	53	37.5	59	17.9
42	13.6	48	49.7	54	22.7	60	2.80
								43	68.8	49	31.1	55	0.360	61	20.2
44	3.7	50	66.6	56	3.2	62	8.9
								63	9.26	64	1.99	65	1.48	66	37.4
67	19.3	68	9.01	69	>100	70	35.7
								71	21.0	72	3.34	73	7.06	74	1.70
75	15.7	76	4.70	77	>100	78	12.4
								79	13.2	80	23.9	81	>100	82	3.93
83	3.57	84	>100	85	>100	86	11.4
								87	>100	88	14.4	89	2.98	90	1.99
91	22.2	92	85.79	93	86.70	94	42.99

Example 97

Surface Plasmon Resonance (SPR) method for determining binding force of compound to full-length protein and different structural domains of USP7

1. Purpose of experiment

The binding force of the compound with the full-length protein of USP7 and different structural domains is measured by a Surface Plasmon Resonance (SPR) method to examine whether the compound has specificity to the C-terminal structural domain protein of USP 7.

2. Experimental methods

Reference (nat. Commun.2015,6:7023-^ΔUBl2And CTD^Δ666-723A protein.

The binding force of protein to compound was measured by Surface Plasmon Resonance (SPR) method using USP 7C-terminal protein replaced with USP7 full-length protein or different domain protein according to the test method of reference example 96.

3. Results of the experiment

The results of the binding assays for

compounds

25, 55 and 60 with the full-length protein and different domains of USP7 are shown in table 3. The results of the experiments show that compounds 25, 55 and 60 have strong specificity for the C-terminal domain protein of USP7, which does not bind to the N-terminal domain protein of USP7, and although also binds to the catalytic domain protein of USP7, the affinity is inferior to that of the C-terminal domain protein. The binding of the compound 55 to the USP 7C-terminal domain protein is highly dependent on the C-terminal UBl2 domain and the amino acid sequence 666-one 723, and when the UBl2 domain or the amino acid 666-one 723 sequence is deleted, the binding force is reduced sharply; other compounds are also specific for the C-terminal domain protein of USP 7.

TABLE 3 binding of partial compounds to full-length protein of USP7 and different domains

Note: CTD^ΔUBl2USP 7C-terminal protein with deletion of UBL2 domain; CTD^Δ666-723USP7C end protein deleted for the amino acid 666-723 sequence; n.d. indicates not tested.

Example 98

Culture, X-ray diffraction and structure analysis of USP 7C-terminal protein and compound 25 compound eutectic crystal

USP 7C-terminal protein was prepared according to the reference (nat. Commun.2015,6: 7023-. The purified USP 7C-terminal protein is sent to an X-ray crystallography platform of the high-precision center of innovation of structural biology of Qinghua university, and culture and structural analysis of the USP 7C-terminal protein and compound 25 compound eutectic are entrusted to the platform. The results showed that crystals appeared after two days of incubation with a protein concentration of 9mg/ml, protein: compound 1:1.2, crystallization buffer 200mM ammonium iodide, 20% w/v polyethylene glycol 3350, with a good morphology. And fishing out the crystal, carrying out quick freezing by using liquid nitrogen, bringing the crystal to the sea to carry out X-ray diffraction by using a synchrotron radiation light source, and collecting data. The eutectic structure (resolution) of the compound 25 and the USP7C terminal protein is resolved by molecular replacement (PDB ID:4YOC)

)。

As shown in fig. 1, compound 25 was bound to the UBL2 domain of USP 7C-terminal protein, and the binding pocket was composed of key amino acid residues such as Asp666, Tyr706, and Arg 723. The sulfonyl group of the compound 25 forms hydrogen bond interaction with Asp666 residue of UBL2 of USP7C terminal protein, amide group and thiazole ring form hydrogen bond interaction with Tyr706 residue, benzothiazole skeleton has pi-cation interaction with Arg723 residue, and benzene ring also forms pi-pi interaction with Tyr706 residue.

Example 99

GST Pull-down experiments examine the effect of compounds on the interaction of DNMT1 with USP 7C-terminal protein

1. Experimental methods

GST affinity resin was centrifuged at 3000rpm for 2 minutes in a 1.5mL EP tube, the supernatant carefully aspirated and PBS was added, and centrifugation was carried out at 3000rpm for 2 minutes and repeated three times.

PBS was added to the resin from which the supernatant was discarded, and after mixing by pipetting, the PBS solution containing the resin was dispensed in parallel into a number of 1.5mL EP tubes (30. mu.L of resin per tube), and then 4. mu.g of GST-USP 7C-terminal protein was added to the tubes as a control against GST protein, and then different compound solutions were added (470. mu.L of PBS per tube). The EP tubes were incubated in a freezer at 4 ℃ for 30 minutes with a 360 ℃ rotator (speed 10rpm) to bring the GST-USP7C terminal protein, GST protein, compound into intimate contact with GST affinity resin.

Taking the cultured 293T cells highly expressed by flag-DNMT1, washing the cells twice with PBS, adding a cell mild lysis solution and a protease inhibitor (v/v 100:1), carrying out lysis on ice for 1 hour, scraping the lysed cells from a culture dish, adding the cells into a 1.5mL EP tube, centrifuging the tubes at 12000rpm and 4 ℃ for 20 minutes, taking the supernatant, quantifying BCA (30 muL per tube until the final volume is 500 muL per tube, the DMSO content is 0.1 percent and the compound final concentration is 20 muM), adding the tubes into an EP tube incubated with protein, compound and resin, and continuously carrying out rotary incubation at 4 ℃ overnight (the rotation speed is 10 rpm).

The resin was allowed to settle spontaneously, the supernatant carefully aspirated, washed five times with 500. mu.L each of 50mM Tris-HCl (pH7.5), allowed to settle spontaneously for the first three times, and centrifuged twice (3000rpm for 2 minutes). Western Blot was performed by adding 100. mu.L of loading buffer to each tube.

2. Results of the experiment

As shown in FIG. 2, compounds with stronger affinity for the C-terminal protein of USP7 all interfered with the interaction of DNMT1 with the C-terminal protein of USP7, e.g., compound 84 (K) where the effect of

compounds

55 and 60 was very significant and no binding or weaker affinity_D>100μM)、92(K_D86.0 μ M) had no effect on the interaction of DNMT1 with USP 7C-terminal protein.

Example 100

Evaluation of proliferation inhibitory Activity of Compounds on in vitro tumor cells Using CellTiter-Glo method

1. Purpose of experiment

The CellTiter-Glo method is adopted to evaluate the influence of the compound on the in vitro proliferation activity of various tumor cells, and the respective half inhibitory concentration IC is calculated₅₀。

2. Experimental Material

The compound of the invention (dissolved by DMSO to prepare mother liquor, diluted by complete culture medium to proper concentration before use); CellTiter-Glo Luminescent cell viability assay kit (from Promega corporation); media and fetal bovine serum (purchased from Biological Industries); 96-well cell culture plates (from Thermo Fisher Scientific), EnSpire microplate reader (from PerkinElmer).

3. Experimental methods

Selecting cells with living cell proportion of more than 90% for cell plating, namely adding 100 mu L of cell suspension into each hole of a 96-hole plate, wherein each hole of LNCaP cells has 2500 cells and RS 4 cells; 5000 cells per well, 16500 mm.1s cells per well, 2000 NB4, MCF7, Huh7 and HCT-116 cells per well. The 96-well plate was placed at 37 ℃ in 5% CO₂The culture was carried out in an incubator for 24 hours. Diluting the drug with complete culture medium to desired concentration (maximum concentration of 200 μ M, 2-fold gradient dilution to obtain 9 concentrations, i.e. 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μ M), adding 100 μ L of culture medium containing drug into each well, setting 3 multiple wells for each compound, and setting up negativeA positive control group, a vehicle control group. The 96-well plate is placed at 37 ℃ and 5% CO₂Continuously culturing in the incubator, wherein the LNCaP cells are continuously cultured for 6 days, the MM.1S cells are continuously cultured for 5 days, and the RS 4 cells are continuously cultured; 11. NB4, MCF7, Huh7 and HCT-116 cells were cultured for an additional 3 days. The 96-well plate was removed from the incubator and left at room temperature for 30 minutes to allow the plate to equilibrate to room temperature. The CellTiter-Glo reagent was removed from the-20 ℃ freezer and thawed at room temperature, which took about 10 minutes. Adding 50 mu L CellTiter-Glo reagent into each hole of a 96-hole plate, shaking and uniformly mixing for 2-5 minutes to fully crack cells, standing at room temperature for 10 minutes, reading the plate by an EnSpire enzyme labeling instrument Luminescence 96 program, calculating the inhibition rate of compounds with different concentrations on tumor cells, and fitting IC (integrated Circuit) through Graphpad prism 5.0₅₀The value is obtained.

4. Results of the experiment

As shown in table 4, compound 60 of the present invention has significant in vitro proliferation inhibitory activity against the above-mentioned various tumor cells, particularly against LNCaP, RS 4; 11. NB 4. Other compounds in the invention also have an inhibiting effect on the growth of tumor cells, and the result indicates that the compounds can be used for preparing antitumor drugs.

TABLE 4 inhibitory Activity of Compound 60 on the in vitro proliferation of different tumor cells

Cell lines

LNCaP

MCF7

HCT-116

Huh7

MM.1S

RS 4；11

NB4

IC₅₀(μM)

9.9±2.5

25.6±3.2

22.8±3.7

15.5±1.7

25.5±6.6

8.6±1.8

5.6±1.0

Example 101

Western blot evaluation of the Effect of Compounds on DNMT1 levels in tumor cells

1. Purpose of experiment

The effect of compounds on the level of DNMT1 in human promyelocytic leukemia cells NB4 was determined using the Western blot method.

2. Experimental Material

The compound of the invention (dissolved in DMSO to prepare mother liquor, and diluted to the concentration of 1. mu.M, 5. mu.M, 10. mu.M and 20. mu.M by using complete culture medium before use); modified RMPI-1640 medium and fetal bovine serum (purchased from Biological Industries, Inc.); 6 well cell culture plates (from NEST).

3. Experimental methods

And (3) taking cells with the living cell proportion of more than 90% for plating, namely adding 1000 mu L of cell suspension into each hole of a 6-hole plate, and 100 ten thousand NB4 cells into each hole. The drug was diluted to the desired concentration (0. mu.M, 1. mu.M, 5. mu.M, 10. mu.M and 20. mu.M) with the complete medium, and 1000. mu.L of the drug-containing medium was added to each well, while a negative control group was established. Placing the 6-well plate at 37 deg.C and 5% CO₂The culture was carried out in an incubator for 24 hours. Collecting cells, centrifuging, washing cells with PBS, lysing cells with cell lysate, extracting protein, and separating with 8%And (4) gel electrophoresis.

4. Results of the experiment

As shown in fig. 3 and 4, compounds 55 and 60 concentration-dependently reduced DNMT1 levels of NB4 cells. The other compounds in the invention such as 25, 44, 56 and the like have the same effect, which shows that the compounds of the invention inhibit deubiquitination of DNMT1 by USP7 by interfering combination of USP7C terminal and DNMT1, so that DNMT1 ubiquitination degradation is increased, and DNMT1 level is reduced.

Example 102

Study on metabolic stability of compound to human liver microsome

The evaluation of the metabolic stability of human liver microsome is an important means for evaluating the pharmacokinetic properties of candidate compounds in the preclinical process of drug development. This part of the experiment was performed by the medicepia pharmaceutical technology ltd.

An experimental incubation system (the volume is 250 mu L, and n is 3) consists of liver microsomes, a sample working solution and a phosphate buffer solution, the incubation system is incubated for one hour at 37 ℃, timing is started after an NADPH solution is added, the reaction is stopped at each time point by adding a stop solution, and sampling time points are 0, 5, 15, 30 and 60 minutes and are 5 points in total. Negative control was not NADPH added, and sampling time points were 0, 60 minutes. Analysis was performed by LC-MS/MS, and the absolute value of the slope, k, was determined by plotting the natural logarithm of the percentage of the remaining test substance against time, and was calculated as follows: t is_1/2(half-life) ═ ln2/k ═ 0.693/k, Clint ═ 0.693/T1/2) × (1/microsomal protein concentration) × conversion factor, microsomal protein concentration was 0.5mg/mL, conversion factor 1254.2). The results of the experiment are shown in Table 5.

TABLE 5 metabolic stability of some compounds in human liver microsomes

Compound numbering	T_1/2(min)	Cl_int(mL/min/kg)
			55	75.26	72.52
60	12.72	428.88
			61	7.78	701.70

The experimental result shows that the compound 55 has better metabolic stability in human liver microsomes and T_1/2For 75.26 minutes. Other compounds in the invention also have better metabolic stability of liver microsomes.

Example 103

Pharmacokinetics research of compound in Balb/C mouse

This part of the experiment was performed by the medicepia pharmaceutical technology ltd.

1. Laboratory apparatus

Ultra-high performance liquid chromatography system (Waters corporation, acquisition UPLC) comprising Binary Solvent Manager (acquisition UPLC Binary Solvent Manager), Sample Manager (acquisition UPLC Sample Manager), high throughput Sample tissue Manager (acquisition UPLC Sample Organizer), high temperature Column oven (acquisition UPLC Column Heater HT). Mass spectrometer (TQ 6500+, applied biosystems, usa), electrospray ion source (ESI), tandem quadrupole mass analyzer. A microanalysis balance (XP26, mettler-toledo instruments (shanghai) ltd); vortex oscillators (SI-A256, Scientific Industries, Inc.; Multi-TUBE VORTEXER, Fisher Scientific); small bench top high speed refrigerated centrifuge (5417R, Eppendorf); ultra pure water machines (Millipore); pipettors (Eppendorf). The data processing system is Analyst software (american application biosystems, software version number 1.6.3).

2. Experimental reagent

Methanol (Burdick & Jackson, HPLC), acetonitrile (Burdick & Jackson, HPLC), formic acid (J & K), water as ultrapure water.

3. Solvent

Solvent: 5% DMSO + 10% solutol + 85% saline.

4. Laboratory animal

The species are as follows: Balb/C male mice, SPF grade.

The source is as follows: zhejiang vitamin Tonglihua laboratory animal technology Co.

5. Animal administration

Balb/C mice, grouped according to the experiments in Table 6.

TABLE 6 animal groups for IV and PO administration

Note:^*all animals were fasted overnight (10-14 hours) before oral administration and fed 4 hours after administration.

6. Sample collection and processing

Blood is collected through the orbit, about 0.03mL of each sample is collected, heparin sodium is anticoagulated, and the samples are placed on ice after collection.

Blood samples were collected, placed on ice and plasma was centrifuged within 1 hour (centrifugation conditions: 6800g, 6 min, 2-8 ℃). The plasma samples were stored in a-80 ℃ freezer prior to analysis and were analyzed by the laboratory department of analysis using LC-MS/MS.

The blood sampling time points were as follows:

oral group: 0.25h,0.5h,1h,2h,4h,6h,8h and 24h after administration.

Vein group: 0.083h,0.25h,0.5h,1h,2h,4h,8h and 24h after administration.

LC-MS/MS conditions

(1) Liquid phase conditions

A: 0.1% aqueous formic acid solution, B: 0.1% formic acid acetonitrile solution; column temperature: 40 ℃; autosampler temperature: 4 ℃; flow rate: 0.6 ml/min; sample introduction amount: 2 μ l.

Chromatographic column ACQUITY UPLC BEH C181.7 μm (2.1X 50mm)

(2) Conditions of Mass Spectrometry

Scanning mode: a negative ion multi-reaction detection mode; an ion source: an electrospray ion source; atomizing mode: carrying out electric spraying; q1 resolution: a Unit; q3 resolution: a Unit; atomizing Gas (Gas 1): 50 psi; auxiliary heater (Gas 2): 50 psi; air curtain gas (CUR): 40 psi; collision gas (CAD): 10; ion source voltage (IS): -4500 v; ion source Temperature (TEM): at 500 ℃.

8. Preparation of standard curve and quality control sample

Preparation of 400,000ng/mL working solution: stock compound 55 was diluted with methanol to 400,000ng/mL working solution.

Preparation of standard curve and quality control sample: taking a certain amount of 400000ng/mL of working solution, and mixing the working solution according to the weight ratio of 1: 39, to a certain amount of blank plasma, to obtain a plasma sample having a concentration of 10000 ng/mL. 10000ng/mL plasma samples are taken and diluted to 5000, 1000, 500, 100, 50, 10, 5ng/mL standard curve samples and 4000, 800, 15ng/mL quality control samples by blank plasma in sequence.

Preparing an internal standard working solution: sucking a certain amount of tolbutamide internal standard stock solution with the concentration of 1018,000ng/mL into a volumetric flask with a certain volume, using methanol to fix the volume to the scale, and mixing uniformly to prepare the internal standard working solution with the concentration of 200 ng/mL.

9. Sample pretreatment

And (3) taking 10 mu l of plasma sample into a 1.5mL centrifuge tube, adding 400 mu l of internal standard solution (containing 100ng/mL internal standard substance, and adding methanol with the same volume without adding the internal standard substance in the blank), uniformly mixing by vortex, rotating at 18000 r/min, centrifuging for 7 min, taking 200 mu l of supernatant, adding the supernatant into a 96-well sample feeding plate, and performing LC-MS/MS sample injection analysis.

10. Pharmacokinetic analysis

According to the average blood concentration data of each group of medicines at each time point, using pharmacokinetic calculation software Phoenix

7.0 separately calculating the pharmacokinetic parameter AUC of the test sample by the non-compartmental model_0-t、AUC_0-∞、MRT_0-∞、C_max、T_maxAnd T_1/2And the like. In addition, the bioavailability (F) will be calculated by the following formula.

For samples with concentrations below the lower limit of quantitation, when pharmacokinetic parameter calculations are performed, C is reached_maxThe previously sampled samples were calculated to zero when C is reached_maxSamples of the sample points were later counted as non-quantifiable (labeled "BLQ").

(Note: this part of the experiment was carried out by Meidi Xipuya pharmaceutical science and technology Co., Ltd.)

11. Results of the experiment

The pharmacokinetic data in mice are shown in table 7, and the results indicate that compound 55 can be orally absorbed and its oral bioavailability in mice is 19.54%. Other compounds of the invention may also be orally absorbed. This shows that the benzothiazole compound of the invention has better druggability.

TABLE 7 pharmacokinetic parameters of Compound 55 of the invention in Balb/C mice

Example 104

Anti-inflammatory Effect of Compound 55 on Raw264.7 cells

1. The experimental method comprises the following steps:

raw264.7 cells (purchased from the cell bank of the Chinese academy of sciences) were cultured in DMEM medium containing 10% inactivated fetal bovine serum. Raw264.7 was inoculated into 12-well plates at 30 ten thousand per well and cultured adherently for 12 hours. Compound 55 was dispensed as 10mM stock solution in DMSO and diluted sequentially in DMEM medium containing 10% inactivated fetal calf serum to obtain a corresponding working concentration of drug solution. The culture medium in the 12-well plate is discarded, and the culture medium containing the liquid medicine is added for pretreatment for 1 h. Lipopolysaccharide (LPS) stock solution with a concentration of 1mg/mL was diluted with medium to a medium solution of 100ng/mL and the compound was diluted with the lipopolysaccharide-containing medium solution to the corresponding working concentration (0-20. mu.M). The pretreated medium was discarded, the medium solution was added to the control well, the lipopolysaccharide medium solution at 100ng/mL was added to the positive well, and the lipopolysaccharide medium solution containing the compound was added to the administration well. After 1h of stimulation, the medium was discarded and the cells were frozen in a-80 ℃ freezer for subsequent testing.

Add 500. mu.L of precooled Trizol reagent per well, lyse on ice for 15 minutes, add 100. mu.L of chloroform and shake vigorously for 15s, leave on ice for 10 minutes, centrifuge at 12000rpm for 15 minutes. The upper aqueous phase was transferred to a clean 1.5mL EP tube, 200. mu.L of isopropanol was added to precipitate RNA, and after standing on ice for 10 minutes, the mixture was centrifuged at 12000rpm for 10 minutes at 4 ℃. The supernatant was discarded, and the pellet was washed once with 75% ethanol, centrifuged to remove the supernatant, and the RNA pellet was dissolved in 15. mu.L of DEPC-treated water. RNA concentration was quantified using Nano, and a reverse transcription reagent from Takara was added as described, and mRNA was reverse-transcribed into cDNA using a conventional PCR instrument. Finally, the upstream and downstream primers (Primer sequences from Primer Bank), q-PCR reagent (SYBR Green) and cDNA of the target gene (Gapdh, IL6 or IL1b) were added to a q-PCR-dedicated 96-well plate, and amplification and quantification were performed using a q-PCR apparatus. The delta Ct value is selected to represent the difference of gene expression, and corresponding software is adopted to carry out data processing and statistical test.

2. Results of the experiment

As shown in FIG. 5, compound 55 significantly inhibited LPS-induced IL-6 and IL-1b and was dose-dependent, and other compounds of the present invention also had similar effects, indicating that the compounds of the present invention have anti-inflammatory activity.

Example 105

Tablet formulation

Compound 60(50g) obtained in example 60, hydroxypropylmethylcellulose E (150g), starch (200g), povidone K30, and magnesium stearate (1g) were mixed, granulated, and tabletted.

In addition, the compounds prepared in examples 1 to 94 can be formulated into capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, patches, and the like, with various pharmaceutical excipients according to the conventional formulation method of pharmacopoeia 2015 edition.

Claims

1. A benzothiazole compound as shown in formula I or a pharmaceutically acceptable salt or ester or solvate thereof:

X is methylene, carbonyl or sulfonyl;

Y is hydrogen or XR ¹ ;

R ¹ , R ² are each independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted substituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

^R3 is _hydrogen , hydroxy, heterocyclyl, alkyl, _NH2 , _NO2 , COOH, CN, SH, _CF3 , _SO3H , _SO2CH3 , or halogen.

2. The benzothiazole compound according to claim 1 or a pharmaceutically acceptable salt or ester or solvate thereof, wherein the benzothiazole compound shown in formula I or a pharmaceutically acceptable compound thereof In the accepted salt or ester or solvate,

X is methylene, carbonyl or sulfonyl;

Y is hydrogen or XR ¹ ;

R ¹ is substituted alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroarylmethyl, substituted or unsubstituted aryl or heteroaryl;

R ² is substituted and unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl;

R ³ is hydrogen, hydroxy or heterocyclyl.

3. The benzothiazole compound according to claim 1 or a pharmaceutically acceptable salt or ester or solvate thereof, wherein the benzothiazole compound is a compound represented by the following formula II or formula III or a pharmaceutically acceptable salt or ester or solvate thereof:

Y is hydrogen or SO ₂ R ¹ ;

R ¹ , R ² are each independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Substituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

4. The benzothiazole compound or a pharmaceutically acceptable salt or ester solvate thereof according to any one of claims 1 to 3, wherein the compound or a pharmaceutically acceptable salt or ester or The solvate is selected from any one of the following compounds:

5. The use of the benzothiazole compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, ester or solvate thereof in the preparation of a USP7 regulator.

6. The benzothiazole compound as claimed in any one of claims 1 to 3 or a pharmaceutically acceptable salt or ester or solvate thereof is prepared for preventing or treating inflammation, autoimmune disease, myelodysplastic syndrome or Use in medicines for malignant tumors.

7. A pharmaceutical composition for preventing or treating inflammation, autoimmune disease, myelodysplastic syndrome or malignant tumor, comprising a therapeutically effective amount of any one of the benzothiazoles according to any one of claims 1 to 3 Class compounds or their pharmaceutically acceptable salts or esters or solvates and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is preferably an ordinary tablet or capsule, a sustained-release tablet or capsule, a controlled-release tablet or capsule, granule, powder, syrup medicine, oral liquid or injection.