WO2021175234A1 - Benzothiazole compound and medical use - Google Patents

Benzothiazole compound and medical use Download PDF

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WO2021175234A1
WO2021175234A1 PCT/CN2021/078798 CN2021078798W WO2021175234A1 WO 2021175234 A1 WO2021175234 A1 WO 2021175234A1 CN 2021078798 W CN2021078798 W CN 2021078798W WO 2021175234 A1 WO2021175234 A1 WO 2021175234A1
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compound
substituted
unsubstituted
dmso
nmr
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PCT/CN2021/078798
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French (fr)
Chinese (zh)
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孙宏斌
陈彩萍
周鑫煜
刘胜杰
袁浩亮
温小安
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中国药科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to the field of medicinal chemistry, in particular to benzothiazole compounds and their use in pharmacy, in particular to benzothiazole compounds as USP7 C-terminal domain regulators and their use in the prevention and treatment of myelodysplastic syndromes and malignancies. Use in tumors, inflammations or autoimmune diseases.
  • Myelodysplastic syndrome is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells, characterized by abnormal differentiation and development of myeloid cells, manifested by ineffective hematopoiesis, refractory hematopoietic reduction, and hyperplasia. The risk is transformed into acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the main causes of death of patients are complications caused by the disease itself and death caused by conversion to AML (Cancer 2010, 16: 2174-2179). According to statistics, in the United States, the prevalence rate is about 0.004-0.005%, and the susceptible population is elderly men or those who have previously received chemotherapy (Blood 2008, 112: 45-52).
  • MDS is divided into two types: high-risk and low-risk.
  • the classification is based on the proportion of immature cells in the bone marrow and the analysis of mutant genes.
  • the International Prognostic Scoring System (IPSS) is used to score the test results, based on the proportion of bone marrow blasts, the number of blood cells, and the type of gene mutation (Am.J.Hematol.2014,89:98-108; Blood 1997,89 :2079-2088).
  • IIPSS International Prognostic Scoring System
  • Different types of MDS have different treatment goals.
  • the goal of treatment for low-risk MDS is to reduce the need for blood transfusion, delay the process of transforming AML, and increase survival, while the goal of treatment for high-risk MDS is to prolong survival.
  • DNMT1 DNA methyltransferase 1
  • DNMT1 DNA methyltransferase 1
  • the C-terminus mainly exerts its catalytic function of methylation, while the N-terminus mainly regulates the activity of the catalytic region through allosteric action, thereby controlling the interaction between DNMT1 and other proteins (Prog.Mol.Biol.Transl.Sci.2011, 101:221-254; Epigenetics 2012, 7:994-1007).
  • DNMT1 The basic function of DNMT1 is to methylate newly synthesized DNA in the S phase of the cell cycle (Nature 2007,447:396-398). In mammals, the time point of methylation is precise and fixed, while the human body controls the time point of methylation through regulation of the level of DNMT1 protein: regulation of a series of transcription and post-transcriptional modifications Down, the protein level of DNMT1 changes with changes in the cell cycle, reaching a peak in the early S phase, then decreasing and reaching the lowest point in the G1 phase (Sci.Signal. 2010, 3:ra80).
  • the protein level of DNMT1 is controlled by a variety of post-transcriptional modification methods: ubiquitination, acetylation (Sci.Signal.2011,4:pe3; Mol.Cell Biol.2011,31:4720-4734), methylation (Proc. Natl.Acad.Sci.USA 2009,106:5076-5081; Nat.Genet.2009,41:125-129; Nat.Struct.Mol.Biol.2011,18:42-48) and protein-protein interactions (such as And ⁇ -catenin) (Nucleus 2011, 2:392-402) can affect the level of DNMT1 protein.
  • DNMT1 DNMT1
  • ubiquitination directly mediates the degradation of DNMT1 protein and plays a vital role in its stability.
  • USP7 is a deubiquitinating enzyme, which is a kind of ubiquitin-specific proteases (USPs), which can efficiently hydrolyze the ubiquitin chain on the substrate protein and deubiquitinate the target substrate to stabilize it.
  • USP7 is a nuclear protein, which is mainly distributed in nuclear dots in the nucleus to perform its physiological functions.
  • USP7 structure is highly conserved (human and mouse USP7 structure homology is as high as 98.6%), composed of 1102 amino acids, and its relative molecular mass is about 135kDa (Cell 2009,138:389-403; Nat.Cell Biol. 2002, 4:106-110).
  • USP7 can be divided into N-terminal TRAF domain (residue 53-206), catalytic domain (residue 208-560) and C-terminal UBL domain (residue 564-1084) according to its amino acid sequence and function.
  • USP7 plays an important role in the protein stability, enzyme activity and target DNA recognition of DNMT1.
  • USP7 is the deubiquitinating enzyme of DNMT1. Its C-terminal UBL1-2 region has an acid pocket consisting of four amino acid residues Glu736, Asp758, Glu759 and Asp764 and the KG connecting region of DNMT1 (residue 1109-1119) Interaction (Nat.Commun.2015, 6:7023-7034), thereby deubiquitinating and stabilizing the DNMT1 protein.
  • USP7 plays an important role in the stability of DNMT1.
  • USP7 also It can regulate the enzymatic activity of DNMT1 through protein-protein interaction.
  • USP7 mediates the binding of DNMT1 to the target DNA.
  • DNMT1 itself does not recognize the target DNA sequence of hemimethylation. It needs to form a trimer complex with USP7 and UHRF1 before it can perform demethylation.
  • the N-terminal of USP7 is bound to UHRF1
  • the C-terminal of USP7 is bound to the N-terminal TS region of DNMT1
  • UHRF1 is bound to the N-terminal RFTS region of DNMT1.
  • the SDR region of UHRF1 can specifically recognize DNA hemimethylated CpG islands, and the DNMT1-UHRF1-USP7 complex is then pulled to the DNA target region to play a role (Nucleic Acids Res. 2011, 39: 8355-8365). Therefore, inhibiting DNMT1 by interfering with the interaction between the C-terminal domain of USP7 and DNMT1 is a potential treatment approach for diseases such as myelodysplastic syndrome and malignant tumors.
  • USP7 C-terminal regulator has potentially important medical value.
  • small molecule modulators of USP7 C-terminal protein It is of great significance to develop small molecule modulators of USP7 C-terminal domain with high activity and low toxic and side effects.
  • the present invention provides a class of benzothiazole compounds, which can be used as USP7 C-terminal regulators, or pharmaceutically acceptable salts or esters or solvates thereof. It has a strong binding force to the C-terminal protein of USP7, and can reduce the protein level of DNMT1 in tumor cells. It also has a significant anti-tumor cell proliferation effect. In addition, it can also inhibit the deubiquitination of NF ⁇ B by USP7.
  • the invention also provides preparation methods, pharmaceutical combinations and medical uses of the benzothiazole compounds and intermediates thereof.
  • X is methylene, carbonyl or sulfonyl
  • Y is hydrogen or XR 1 ;
  • R 1 and 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 heterocyclic aryl.
  • R 3 is hydrogen, hydroxyl, heterocyclyl, alkyl, NH 2 , NO 2 , COOH, CN, SH, CF 3 , SO 3 H, SO 2 CH 3 or halogen.
  • X is a methylene group, a carbonyl group or a sulfonyl group
  • Y is hydrogen or XR 1 ;
  • R 1 is a substituted alkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted heteroarylmethyl group, a substituted or unsubstituted aryl group or a heteroaryl group;
  • R 2 is substituted and unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl
  • R 3 is hydrogen, a hydroxyl group or a heterocyclic group.
  • X is a sulfonyl group
  • Y is hydrogen or XR 1 ;
  • R 1 is a substituted alkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted heteroarylmethyl group, a substituted or unsubstituted aryl group or a heteroaryl group;
  • R 2 is substituted or unsubstituted heterocycloalkyl
  • R 3 is hydrogen
  • the benzothiazole compound of the present invention is a compound represented by the following formula II or formula III or a pharmaceutically acceptable salt or solvate thereof:
  • Y is hydrogen or SO 2 R 1 ;
  • R 1 and 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 heterocyclic aryl.
  • the benzothiazole compound of the present invention is any one of the compounds in Table 1 or a pharmaceutically acceptable salt or ester or solvation thereof:
  • the present invention provides the use of benzothiazole compounds represented by formulas I to III or pharmaceutically acceptable salts or esters or solvates thereof in the preparation of USP7 C-terminal regulators.
  • the inventors discovered that the benzothiazole compounds represented by formulas I to III can bind to the C-terminal of USP and cause conformational changes at the C-terminal of USP. This is the first type of USP7 C-terminal small molecule regulators disclosed so far.
  • the present invention also provides the use of the benzothiazole compounds represented by formulas I to III or pharmaceutically acceptable salts or esters or solvates thereof in the preparation of prevention or treatment of inflammation, autoimmune diseases, myelodysplastic syndromes and malignant tumors. Use in medicine.
  • the inflammation and autoimmune diseases include but are not limited to: ulcerative colitis, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, multiple sclerosis or Behçet's disease.
  • the tumor includes, but is not limited to: bone cancer, hematology cancer, nervous system cancer, gastrointestinal cancer, urinary system cancer, lung cancer, liver cancer or skin cancer.
  • the bone cancer includes, but is not limited to: bone-derived sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, Malignant giant cell tumor chordoma, osteochondroma (gu tube exogenous bone wart), benign chondroma, chondroblastoma, cartilage and tumor-like fibroma, osteoid osteoma, and giant cell tumor.
  • bone-derived sarcoma osteosarcoma
  • fibrosarcoma malignant fibrous histiocytoma
  • chondrosarcoma chondrosarcoma
  • Ewing's sarcoma malignant lymphoma
  • multiple myeloma Malignant giant cell tumor chordoma
  • osteochondroma gu tube exogenous bone
  • the hematological cancers include but are not limited to: acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic disease, multiple myeloma and myelodysplastic syndrome, Hodge King's lymphoma (malignant lymphoma) and Waldenstrom's macroglobulinemia.
  • the nervous system cancers include, but are not limited to: meningeal cancer, such as meningiomas, meningiosarcoma, and glioma; brain cancers, such as astrocytoma, medulloblastoma, glioma, ependymoma, Germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; spinal cord tumors, such as fibroneuronoma, meningioma , Glioma and Sarcoma.
  • meningeal cancer such as meningiomas, meningiosarcoma, and glioma
  • brain cancers such as astrocytoma, medulloblastoma, glioma, ependymoma, Germ cell tumor (pineal tumor), glioblastoma multi
  • the 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 cancer, such as ductal adenocarcinoma, insulinoma, Glucagonoma, gastrinoma, carcinoid tumor and vasoactive intestinal peptide tumor; small bowel cancer, such as adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma , Fibroneuronoma and fibroids; colorectal cancer, such as adenocarcinoma, tubular adenocarcinoma, villous adenoma, hamartoma, and leiomyoma.
  • the urinary system cancers include, but are not limited to: kidney cancer, such as adenocarcinoma, Wilms tumor (wilms tumor), lymphoma, and leukemia; bladder and urethral cancer, such as squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma ; Prostate cancer, such as adenocarcinoma and sarcoma; Testicular cancer, such as seminoma, teratoma, embryonic carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, gland Tumor-like tumors and lipomas.
  • kidney cancer such as adenocarcinoma, Wilms tumor (wilms tumor), lymphoma, and leukemia
  • bladder and urethral cancer such as squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma
  • Prostate cancer such as adenocar
  • the lung cancer includes, but is not limited to: bronchial carcinoma, such as squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, and adenocarcinoma; bronchioloalveolar carcinoma; bronchial adenoma; sarcoma; lymphoma; pulmonary chondroma Hamartoma and mesothelioma.
  • bronchial carcinoma such as squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, and adenocarcinoma
  • bronchioloalveolar carcinoma bronchial adenoma
  • sarcoma sarcoma
  • lymphoma pulmonary chondroma Hamartoma
  • mesothelioma mesothelioma.
  • the liver cancer includes but is not limited to: hepatocellular carcinoma, such as hepatocellular carcinoma; cholangiocarcinoma; hepato
  • the skin cancer includes but is not limited to: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis .
  • the compound of the present invention can be used in combination with one or more other types of drugs for the prevention or treatment of the above-mentioned diseases, including but not limited to the following combination drugs:
  • preventive or therapeutic drugs can be one or more anti-cancer drugs, including alkylating agents (such as cisplatin, cyclophosphamide, ifosfamide, melphalan, Chlorambucil, Bendamustine, Estramustine, Cetepa, Iminoquinone, Busulfan, Dibromomannitol, Cyclohexylnitrosene, Carmustine, Pyrimidine Nitrosourea, Methalin Nitrosourea, methazine, procarbazine, etc.), antimetabolites (such as fluorouracil, cytarabine, furfurouracil, difurfurouracil, mercaptopurine, sulfathioprine, azathioprine, thioguanine) , Methotrexate, methotrexate, etc.), anti-tumor antibiotics (such as mitomycin C, bleomycin
  • alkylating agents such as cisplatin,
  • the present invention also provides a pharmaceutical composition for preventing or treating inflammation, autoimmune diseases, myelodysplastic syndromes and tumors, which contains a therapeutically effective amount of any benzothiazole compound represented by formula I to III or its Pharmaceutically acceptable salts or solvates are used as active ingredients and pharmaceutically acceptable excipients.
  • the adjuvants that can be arbitrarily mixed can be changed according to the dosage form, administration form, etc.
  • the carrier includes but is not limited to excipients, binders, disintegrating agents, lubricants, flavoring agents, flavoring agents, coloring agents and sweetening agents.
  • the pharmaceutical composition can be in the form of conventional pharmaceutics such as ordinary tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, granules, powders, syrups, oral liquids or injections.
  • the present invention has the following advantages:
  • the benzothiazole compound of the present invention or its pharmaceutically acceptable salt or ester or solvate has a strong binding force with the C-terminal protein of USP7, and is the first USP7 C-terminal small molecule regulator published so far It can reduce the protein level of DNMT1 in tumor cells and has a significant anti-tumor cell proliferation effect, so it can be used to prepare drugs for the prevention or treatment of myelodysplastic syndromes and malignant tumors.
  • the USP7 C-terminal small molecule regulator of the present invention can also block the binding of USP7 C-terminal to NF ⁇ B, inhibit the deubiquitination of NF ⁇ B, and has significant anti-inflammatory activity, so it can also be used for the preparation of anti-inflammatory drugs and treatments.
  • Drugs for autoimmune diseases can also block the binding of USP7 C-terminal to NF ⁇ B, inhibit the deubiquitination of NF ⁇ B, and has significant anti-inflammatory activity, so it can also be used for the preparation of anti-inflammatory drugs and treatments.
  • Drugs for autoimmune diseases Drugs for autoimmune diseases.
  • the benzothiazole compound of the present invention or its pharmaceutically acceptable salt or ester or solvate has good drug-forming properties in human liver microsomes, and can be absorbed orally with good drug-forming properties.
  • the benzothiazole compound of the present invention has simple structure, ingenious synthesis route design, cheap and readily available raw materials, safe and environmentally friendly synthesis process, and is easy to scale production.
  • Figure 1 is an X-ray co-crystal structure diagram of compound 25 and USP7 C-terminal protein
  • Figure 2 is a graph of the GST Pull-down experiment result of the compound's influence on the interaction between USP7 C-terminal protein and DNMT1;
  • Figure 3 is a Western Blot experimental result of compound 55 concentration-dependently reducing the level of DNMT1 in NB4 cells;
  • Figure 4 is a diagram showing the results of a Western Blot experiment in which compound 60 reduces the level of DNMT1 in NB4 cells in a concentration-dependent manner;
  • Figure 5 shows the effect of compound 55 on LPS-induced IL-1b and IL-6 expression on Raw264.7 cells.
  • Compound 1c (100mg, 0.39mmol) was added to dichloromethane (3mL) (suspension), followed by isonicotinic acid (52mg, 0.47mmol), 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide hydrochloride (96mg, 0.47mmol) and 4-dimethylaminopyridine (47mg, 0.47mmol) were stirred overnight at room temperature. A large amount of gray solids were observed to precipitate.
  • Ethyl p-fluorobenzoate (13a, 100mg, 0.59mmol) was dissolved in dry dimethyl sulfoxide (1mL), and 1-Boc-piperazine (13b, 121mg, 0.65mmol) and potassium carbonate (246mg , 1.78mmol), stirred at 120°C for 5h, TLC monitored the completion of the reaction and added saturated brine (5mL), extracted with ethyl acetate (5mL ⁇ 3).
  • Ethyl 5-chloromethyl-2-furancarboxylate (33f, 200mg, 1.06mmol) was dissolved in acetonitrile (10mL), followed by morpholine (55mg, 1.27mmol), potassium iodide (176mg, 1.27mmol) and potassium carbonate (212mg, 1.27mmol), stirred at room temperature for 7 hours.
  • the mother liquor after recrystallization was evaporated to remove the solvent under reduced pressure, ethyl acetate (30 mL) was added to the obtained solid to make a slurry, and a yellow solid (37b, 2.06 g) was obtained by suction filtration, which was combined with the solid obtained by recrystallization and put into the next step.
  • Ethyl acetate (100 mL) was added to the obtained solid to dissolve it, 15% sodium hydroxide solution was added dropwise to adjust the pH to 6-7, and then saturated sodium bicarbonate solution was used to adjust the pH to 7-8.
  • the ethyl acetate was evaporated under reduced pressure and then filtered with suction, the filter cake was collected and dried.
  • the filtrate was extracted with ethyl acetate (80mL ⁇ 3).
  • the ethyl acetate layer was washed with saturated brine and dried with anhydrous sodium sulfate for 0.5 hours.
  • the anhydrous sodium sulfate was removed by filtration and the solvent was evaporated under reduced pressure.
  • dichloromethane (3mL) to the reaction solution to dilute, transfer the reaction solution to a 15mL centrifuge tube, centrifuge at 2500rpm for 5 minutes, discard the supernatant, add dichloromethane (3mL), and continue centrifugation at 2500rpm for 5 minutes.
  • Dissolve compound 39c (1.78g, 7.10mmol) in dioxane (80mL), add solid sodium hydroxide (1.70g, 42.60mmol) under ice bath, add water (40mL) to the system, and stir under ice bath 30 minutes. After that, 5% sodium hypochlorite solution (29 mL, 42.60 mmol) was added dropwise to the system, and the mixture was stirred at room temperature overnight. After the reaction was monitored by TLC, the stirring was stopped, the solvent was evaporated under reduced pressure and water (10mL) was added, and the compound 39d (white solid, 1.44g, yield 91%) was obtained by suction filtration: 1 H NMR (500MHz, DMSO-d 6 ) ⁇ 7.
  • reaction solution was diluted with anhydrous dichloromethane (30mL), washed with 1N dilute hydrochloric acid (50mL), water (50mL ⁇ 2) and saturated brine (50mL) successively, dried with anhydrous sodium sulfate, filtered, and the filtrate Concentrate and recrystallize with ethyl acetate (10 mL) to obtain compound 1b (light yellow solid, 1.7 g, yield 73%).
  • the furoic acid was replaced with 1-Boc-4-piperidinecarboxylic acid to prepare the N-Boc precursor compound of compound 41 (dark green solid, 140 mg).
  • reaction solution was concentrated, ethyl acetate (30 mL) was added, neutralized with saturated sodium bicarbonate aqueous solution (30 mL), filtered under reduced pressure, and the filter cake was washed with water (30 mL) and ethyl acetate (10 mL) to obtain compound 55c (Brown solid, 1.05g, yield 94%).
  • the monomethyl isophthalate (56a, 500mg, 2.77mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1023mg, 3.32mmol) and triethylamine (561mg, 5.54mmol) were sequentially added to N,N-dimethylformamide (6mL), stirred at room temperature and reacted for 1 hour, then 3-amino-1-propanesulfonic acid was added (579mg, 4.16mmol), continue to stir the reaction at room temperature.
  • Dissolve compound 57b (166mg, 0.54mmol) in a mixed solution of acetic acid (7.5mL) and water (2.5mL), add zinc powder (176mg, 2.7mmol), stir at room temperature until TLC detects the reaction is complete, add saturated carbonic acid first Sodium bicarbonate solution (15ml), then add solid sodium bicarbonate until no bubbles are generated.
  • Example 65 Referring to the synthesis procedure of Example 65, the raw material 65a was replaced with 1-tert-butoxycarbonyl-4-piperidineacetic acid to obtain intermediate 66a (white solid, 497 mg).
  • Example 65 Referring to the synthesis step of Example 65, the raw material 65a was replaced with Boc-glycine, and the 2-amino-6nitrobenzothiazole was replaced with 71e to obtain intermediate 71f (white solid, 240 mg, 85%).
  • Example 65 Referring to the synthetic route of Example 65, the raw material 65a was replaced with Boc-glycine, and 2-thiophenesulfonyl chloride was replaced with 1-methyl-1H-pyrazole-3-sulfonyl chloride to obtain intermediate 72a (yellow solid, 150 mg).
  • Example 80 Refer to the synthesis procedure of Example 80, replace 1-tert-butoxycarbonyl-4-piperidineacetic acid with tetrahydropyran-4-carboxylic acid, and replace 2-thiophenesulfonyl chloride with 3-(chlorosulfonyl)propionate methyl Ester to obtain compound 85 (white solid, 160 mg).
  • Example 85 tetrahydropyran-4-carboxylic acid was replaced with 1-methylpiperidine-4-carboxylic acid to obtain the methyl ester of compound 87.
  • compound 85 was replaced with the methyl ester of compound 87 to obtain compound 87 (white solid, 127 mg).
  • Example 2 Referring to the synthetic experimental procedure of Example 1, the furoyl chloride was replaced with acetyl chloride to prepare N-(6-aminobenzo[d]thiazol-2-yl)acetamide. Again referring to the synthetic experimental procedure in 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 obtain Compound 92 (white solid).
  • MST Micro thermophoresis
  • the MST method was used to determine the binding force of the compound to the C-terminal protein of USP7, and the respective equilibrium dissociation constants (K D ) were calculated.
  • the compound of the present invention (dissolved with dimethyl sulfoxide (DMSO) to prepare a mother liquor, dilute with Tris-HCl buffer to an appropriate concentration before use); USP7 C-terminal protein (10 ⁇ M); Microthermophoresis (NT.115) ; MST standard capillary; MST protein fluorescent labeling kit (NT-647); Tris-HCl buffer.
  • DMSO dimethyl sulfoxide
  • Table 1 shows the binding force (K D ) test results of some compounds with the C-terminal protein of USP7. Experimental results show that the compound of the present invention can bind to the C-terminal protein of USP7, and some compounds have strong affinity, and other compounds also have good affinity.
  • SPR Surface Plasmon Resonance
  • the SPR method was used to determine the binding force of the compound to the C-terminal protein of USP7, and the respective equilibrium dissociation constants (K D ) were calculated.
  • the compound of the present invention (dissolved in DMSO to prepare a mother liquor, diluted with PBST (pH 7.4, Tween 20 content 0.05%) buffer to an appropriate concentration before use); USP7 C-terminal protein (1mg/mL); Biacore T200(GE Healthcare); CM5 chip (GE Healthcare); Amino coupling kit (GE Healthcare); PBST buffer.
  • Table 2 shows the binding force (K D ) test results of some compounds with the C-terminal protein of USP7.
  • the experimental results show that the compound of the present invention can bind to the C-terminal protein of USP7, and some compounds have stronger affinity, and other compounds also have better affinity.
  • SPR Surface Plasmon Resonance
  • the surface plasmon resonance (SPR) method was used to determine the binding ability of the compound to the full-length USP7 protein and different domains to investigate whether the compound is specific to the C-terminal domain protein of USP7.
  • Example 96 Refer to the test method of Example 96 to replace the USP7 C-terminal protein with the USP7 full-length protein or proteins with different domains to perform the surface plasmon resonance (SPR) method to determine the binding force of the protein to the compound.
  • SPR surface plasmon resonance
  • Table 3 shows the binding ability test results of compounds 25, 55 and 60 with the full-length USP7 protein and different domains.
  • the experimental results show that compounds 25, 55 and 60 have strong specificity to the C-terminal domain protein of USP7. They do not bind to the N-terminal domain protein of USP7. Although they also bind to the catalytic domain protein of USP7, their affinity is not as good as With the C-terminal domain protein.
  • the binding of compound 55 to the C-terminal domain protein of USP7 is highly dependent on the C-terminal UB12 domain and amino acid sequence 666-723. When the UB12 domain or amino acid sequence 666-723 is missing, the binding force drops sharply; other compounds have a strong effect on the C-terminal structure of USP7 Domain proteins are also specific.
  • CTD ⁇ UBl2 is the USP7 C-terminal protein with the UBL2 domain deleted
  • CTD ⁇ 666-723 is the USP7 C-terminal protein with the amino acid sequence 666-723 deleted
  • ND means not tested.
  • Compound 25 binds to the UBL2 domain of the C-terminal protein of USP7, and the binding pocket is composed of key amino acid residues such as Asp666, Tyr706 and Arg723.
  • the sulfonyl group of compound 25 forms a hydrogen bond with the Asp666 residue of UBL2 of the USP7 C-terminal protein, and the amide group and thiazole ring form a hydrogen bond with Tyr706 residue.
  • the CellTiter-Glo method was used to evaluate the compound's inhibitory activity against tumor cell proliferation in vitro
  • the compound of the present invention (dissolved in DMSO to prepare a mother liquor, and dilute to an appropriate concentration with complete medium before use); CellTiter-Glo Luminescent cell viability detection kit (purchased from Promega); medium and fetal calf serum (purchased from Biological Industries) Company); 96-well cell culture plate (purchased from Thermo Fisher Scientific Company), EnSpire microplate reader (purchased from PerkinElmer Company).
  • Select cells with more than 90% viable cells for cell plating that is, add 100 ⁇ L of cell suspension to each well of a 96-well plate, including 2500 LNCaP cells per well, RS 4; 5000 cells per well for 11 cells, and MM.1S cells 16,500 cells per well, 2000 cells per well for NB4, MCF7, Huh7 and HCT-116 cells. Place the 96-well plate in a 37°C, 5% CO 2 incubator for 24 hours.
  • the 96-well plate was placed in a 37°C, 5% CO 2 incubator for continuous culture, in which LNCaP cells were cultured for 6 days, MM.1S cells were cultured for 5 days, RS 4;11, NB4, MCF7, Huh7 and HCT-116 cells Continue to cultivate for 3 days.
  • the compound 60 of the present invention has significant in vitro proliferation inhibitory activity against the above-mentioned various tumor cells, especially LNCaP, RS 4; 11, NB4. Some other compounds of the present invention also have an inhibitory effect on the growth of tumor cells. This result suggests that the compounds of the present invention can be used to prepare anti-tumor drugs.
  • the compound of the present invention (dissolved in DMSO to prepare a mother liquor, diluted with a complete medium to a concentration of 1 ⁇ M, 5 ⁇ M, 10 ⁇ M and 20 ⁇ M before use); modified RMPI-1640 medium and fetal calf serum (purchased from Biological Industries); 6 Well cell culture plate (purchased from NEST company).
  • compounds 55 and 60 decreased the DNMT1 level of NB4 cells in a concentration-dependent manner.
  • Other compounds of the present invention such as 25, 44, 56 have the same effect, indicating that the compound of the present invention inhibits the deubiquitination of DNMT1 by USP7 by interfering with the binding of USP7 C-terminal to DNMT1, thereby degrading DNMT1 ubiquitination Increase, and thus decrease the DNMT1 level.
  • the incubation system is incubated at 37°C for one hour. After adding NADPH solution, the timing starts. The reaction was terminated at each time point by adding the stop solution, and the sampling interval was 0, 5, 15, 30, and 60 minutes, a total of 5 points. NADPH was not added to the negative control, and the sampling time point was 0 to 60 minutes.
  • Ultra-high performance liquid chromatography system Waters, ACQUITY UPLC, including binary solvent manager (ACQUITY UPLC Binary Solvent Manager), sample manager (ACQUITY UPLC Sample Manager), high-throughput sample organizer (ACQUTIY UPLC Sample Organizer) ), high temperature column heater (ACQUITY UPLC Column Heater HT).
  • Binary solvent manager ACQUITY UPLC Binary Solvent Manager
  • sample manager ACQUITY UPLC Sample Manager
  • high-throughput sample organizer ACQUTIY UPLC Sample Organizer
  • ACQUITY UPLC Column Heater HT High temperature column heater
  • Mass spectrometer TQ 6500+, American Applied Biosystems
  • electrospray ion source (ESI) tandem quadrupole mass analyzer.
  • Micro analytical balance (XP26, METTLER TOLEDO Instruments (Shanghai) Co., Ltd.); vortex oscillator (SI-A256, Scientific Industries, Inc.; MULTI-TUBE VORTEXER, Fisher Scientific); small desktop high-speed refrigerated centrifuge (5417R, Eppendorf); ultrapure water machine (Millipore); pipette (Eppendorf).
  • the data processing system is Analyst software (American Applied Biosystems, software version number 1.6.3).
  • Methanol (Burdick & Jackson, HPLC), acetonitrile (Burdick & Jackson, HPLC), formic acid (J&K), water is ultrapure water.
  • Solvent 5% DMSO + 10% solutol + 85% saline.
  • mice Balb/C male mice, SPF grade.
  • mice were grouped according to Table 6 for experiment.
  • Blood was collected from the orbit. About 0.03 mL of each sample was collected. Heparin sodium was anticoagulated and placed on ice after collection.
  • Plasma samples were collected on ice and centrifuged to separate plasma within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8°C). Plasma samples are stored in a -80°C refrigerator before analysis, and the plasma samples are analyzed by the analysis department of the laboratory using LC-MS/MS.
  • the blood sampling time points are as follows:
  • Oral group 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h after administration.
  • Intravenous group 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 24h after administration.
  • A 0.1% formic acid aqueous solution
  • B 0.1% formic acid acetonitrile solution
  • column temperature 40°C
  • autosampler temperature 4°C
  • flow rate 0.6 ml/min
  • injection volume 2 ⁇ l.
  • Scan mode negative ion multi-reaction detection mode
  • ion source electrospray ion source
  • atomization mode electrospray
  • Q1 resolution Unit
  • Q3 resolution Unit
  • atomizing gas (Gas1) 50psi
  • auxiliary heater (Gas2 ) 50psi
  • Curtain Air (CUR) 40psi
  • Ion Source Voltage (IS) -450v
  • TEM Ion Source Temperature
  • Preparation of 400,000ng/mL working solution Take compound 55 stock solution and dilute it with methanol to a working solution with a concentration of 400,000ng/mL.
  • Preparation of standard curve and quality control samples Take a certain amount of 400,000ng/mL working solution and add it to a certain amount of blank plasma at a ratio of 1:39 to prepare a plasma sample with a concentration of 10,000ng/mL. Take 10000ng/mL plasma samples and dilute them to 5000, 1000, 500, 100, 50, 10, 5ng/mL standard curve samples and 4000, 800, 15ng/mL quality control samples with blank plasma.
  • Preparation of the internal standard working solution pipet a certain amount of tolbutamide internal standard stock solution with a concentration of 1018,000ng/mL into a certain volume of volumetric flask, dilute to the mark with methanol and mix well to obtain a concentration of 200ng /mL of internal standard working solution.
  • the non-compartmental model calculates the pharmacokinetic parameters AUC 0-t , AUC 0- ⁇ , MRT 0- ⁇ , C max , T max and T 1/2 of the test product respectively.
  • the bioavailability (F) will be calculated by the following formula.
  • mice pharmacokinetic data is shown in Table 7.
  • Table 7 The results show that compound 55 can be absorbed orally, and its oral bioavailability in mice is 19.54%.
  • the other compounds of the present invention can also be absorbed orally. This indicates that the benzothiazole compounds of the present invention have better drug-forming properties.
  • 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 a 12-well plate at 300,000 per well, and cultured adherently for 12 hours.
  • Compound 55 was prepared into a 10 mM stock solution with DMSO, and diluted sequentially with DMEM medium containing 10% inactivated fetal bovine serum to obtain a drug solution with a corresponding working concentration. The medium in the 12-well plate was discarded, and the medium containing the medicinal solution was added for pretreatment for 1 h.
  • the lipopolysaccharide (LPS) stock solution with a concentration of 1 mg/mL was diluted with culture medium to a 100 ng/mL culture medium solution, and the compound was diluted with the lipopolysaccharide-containing medium solution to the corresponding working concentration (0-20 ⁇ M).
  • the pretreated medium was discarded, the medium solution was added to the control wells, the 100ng/mL lipopolysaccharide medium solution was added to the positive wells, and the compound-containing lipopolysaccharide medium solution was added to the dosing wells. After stimulation for 1 hour, the culture medium was discarded, and the cells were frozen in a refrigerator at -80°C for subsequent testing.
  • compound 55 significantly inhibited LPS-induced IL-6 and IL-1b in a dose-dependent manner.
  • Other compounds of the present invention also have similar effects, indicating that the compound of the present invention has anti-inflammatory activity.
  • Example 60 The compound 60 (50g), hydroxypropylmethylcellulose E (150g), starch (200g), povidone K30 and magnesium stearate (1g) prepared in Example 60 were mixed, granulated, and compressed. .
  • the compounds prepared in Examples 1-94 can be given different pharmaceutical excipients into capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments, according to the conventional preparation method of the pharmacopoeia 2015 edition. Agent, suppository or patch, etc.

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Abstract

Disclosed are a benzothiazole compound represented by formula I and a medical use thereof, and specifically refers to a benzothiazole USP7 C-terminal domain regulating agent compound or a pharmaceutically acceptable salt, ester or solvate thereof, and a preparation method therefor and a use thereof. The compound or a pharmaceutically acceptable salt, ester or solvate thereof of the present invention has a strong binding force with a USP7 C-terminal protein, a regulatory effect on a USP7 C-terminal domain, a significant anti-tumor cell proliferation effect and anti-inflammatory activity, and can be used to prepare a drug for preventing or treating myelodysplastic syndrome, malignant tumors, inflammation, or autoimmune diseases.

Description

苯并噻唑类化合物及医药用途Benzothiazole compounds and medical use 技术领域Technical field
本发明涉及药物化学领域，具体涉及苯并噻唑类化合物及其在制药中的用途，尤其涉及苯并噻唑类化合物作为USP7 C端结构域调控剂及其在预防和治疗骨髓增生异常综合征、恶性肿瘤、炎症或自身免疫性疾病中的用途。The present invention relates to the field of medicinal chemistry, in particular to benzothiazole compounds and their use in pharmacy, in particular to benzothiazole compounds as USP7 C-terminal domain regulators and their use in the prevention and treatment of myelodysplastic syndromes and malignancies. Use in tumors, inflammations or autoimmune diseases.
背景技术Background technique
骨髓增生异常综合症(myelodysplastic syndromes,MDS)是起源于造血干细胞的一组异质性髓系克隆性疾病，特点是髓系细胞分化及发育异常，表现为无效造血、难治性血细胞减少、高风险向急性髓系白血病(AML)转化。患者的死亡原因主要为疾病本身引起的并发症以及转化为AML后引发的死亡(Cancer 2010,16:2174-2179)。据统计在美国，其患病比例大约为0.004-0.005％，而其易患人群为年长男性或之前接受过化疗的人群(Blood 2008,112:45-52)。Myelodysplastic syndrome (myelodysplastic syndromes, MDS) is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells, characterized by abnormal differentiation and development of myeloid cells, manifested by ineffective hematopoiesis, refractory hematopoietic reduction, and hyperplasia. The risk is transformed into acute myeloid leukemia (AML). The main causes of death of patients are complications caused by the disease itself and death caused by conversion to AML (Cancer 2010, 16: 2174-2179). According to statistics, in the United States, the prevalence rate is about 0.004-0.005%, and the susceptible population is elderly men or those who have previously received chemotherapy (Blood 2008, 112: 45-52).
MDS分为高危及低危两种，分类依据为骨髓中未成熟细胞的比例以及突变基因分析。临床上使用国际预后评分系统(IPSS)对检测结果进行打分，其打分依据为骨髓原始细胞比例、血细胞数量以及基因突变类型(Am.J.Hematol.2014,89:98-108；Blood 1997,89:2079-2088)。不同类型的MDS，其治疗目标也有所不同。低危MDS的治疗目标为减少输血需求、延缓转化AML的过程以及增加生存率，而高危MDS的治疗目标为延长生存率。MDS is divided into two types: high-risk and low-risk. The classification is based on the proportion of immature cells in the bone marrow and the analysis of mutant genes. Clinically, the International Prognostic Scoring System (IPSS) is used to score the test results, based on the proportion of bone marrow blasts, the number of blood cells, and the type of gene mutation (Am.J.Hematol.2014,89:98-108; Blood 1997,89 :2079-2088). Different types of MDS have different treatment goals. The goal of treatment for low-risk MDS is to reduce the need for blood transfusion, delay the process of transforming AML, and increase survival, while the goal of treatment for high-risk MDS is to prolong survival.
临床上针对不同类型的MDS有着不同的治疗方案。对于低危MDS患者，临床上首选来那度胺(N.Engl.J.Med.2006,355:1456-1465)或血细胞生长因子注射(J.Natl.Cancer Inst.2008,100:1542-1551)，若无效再使用DNMT1抑制剂进行治疗。而对于高危MDS患者，给予DNMT1抑制剂进行治疗则是首选的标准疗法(Leukemia 2014,28:1-14)。Clinically, there are different treatment plans for different types of MDS. For patients with low-risk MDS, lenalidomide (N.Engl.J.Med.2006,355:1456-1465) or blood cell growth factor injection (J.Natl.Cancer Inst.2008,100:1542-1551) is the first choice in clinical practice. ), if ineffective, use DNMT1 inhibitor for treatment. For patients with high-risk MDS, treatment with DNMT1 inhibitors is the preferred standard therapy (Leukemia 2014, 28:1-14).
DNMT1(DNA甲基转移酶1)是一种将甲基转移到基因组DNA的胞嘧啶核苷酸上的酶，由1616个氨基酸组成，结构上可分为C端催化区、N端调节区和中间的KG连接区。C端主要发挥其甲基化的催化功能，而N端主要是通过变构作用来调节催化区的活性，进而控制DNMT1与其他蛋白的相互作用(Prog.Mol.Biol.Transl.Sci.2011,101:221-254；Epigenetics 2012,7:994-1007)。DNMT1的基本功能是在细胞周期中的S期对新合成的DNA进行甲基化修饰(Nature 2007,447:396-398)。在哺乳动物体内，甲基化的时间点是精准且固定的，而人体对甲基化时间点的控制则是通过对DNMT1蛋白水平的调控而实现的：在一系列转录以及转录后修饰的调控下，DNMT1的蛋白水平随细胞周期的变化而变化，在S期早期达到顶峰，之后降低并在G1期达到最低点(Sci.Signal. 2010,3:ra80)。DNMT1 (DNA methyltransferase 1) is an enzyme that transfers methyl groups to the cytosine nucleotides of genomic DNA. It consists of 1616 amino acids and can be divided into C-terminal catalytic region, N-terminal regulatory region and The KG connection area in the middle. The C-terminus mainly exerts its catalytic function of methylation, while the N-terminus mainly regulates the activity of the catalytic region through allosteric action, thereby controlling the interaction between DNMT1 and other proteins (Prog.Mol.Biol.Transl.Sci.2011, 101:221-254; Epigenetics 2012, 7:994-1007). The basic function of DNMT1 is to methylate newly synthesized DNA in the S phase of the cell cycle (Nature 2007,447:396-398). In mammals, the time point of methylation is precise and fixed, while the human body controls the time point of methylation through regulation of the level of DNMT1 protein: regulation of a series of transcription and post-transcriptional modifications Down, the protein level of DNMT1 changes with changes in the cell cycle, reaching a peak in the early S phase, then decreasing and reaching the lowest point in the G1 phase (Sci.Signal. 2010, 3:ra80).
DNMT1的蛋白水平由多种转录后修饰方式控制：泛素化、乙酰化(Sci.Signal.2011,4:pe3；Mol.Cell Biol.2011,31:4720-4734)、甲基化(Proc.Natl.Acad.Sci.USA 2009,106:5076-5081；Nat.Genet.2009,41:125-129；Nat.Struct.Mol.Biol.2011,18:42-48)以及蛋白蛋白相互作用(如与β-catenin)(Nucleus 2011,2:392-402)均可以对DNMT1蛋白水平产生影响。这些机制使得DNMT1可以在细胞周期中的正确时间点被激活，同时接受正确的指令发挥其DNA甲基化作用。在众多转录后调节机制中，泛素化由于直接介导DNMT1蛋白的降解，对其稳定性起着至关重要的作用。The protein level of DNMT1 is controlled by a variety of post-transcriptional modification methods: ubiquitination, acetylation (Sci.Signal.2011,4:pe3; Mol.Cell Biol.2011,31:4720-4734), methylation (Proc. Natl.Acad.Sci.USA 2009,106:5076-5081; Nat.Genet.2009,41:125-129; Nat.Struct.Mol.Biol.2011,18:42-48) and protein-protein interactions (such as And β-catenin) (Nucleus 2011, 2:392-402) can affect the level of DNMT1 protein. These mechanisms allow DNMT1 to be activated at the correct time in the cell cycle and at the same time receive the correct instructions to exert its DNA methylation effect. Among many post-transcriptional regulatory mechanisms, ubiquitination directly mediates the degradation of DNMT1 protein and plays a vital role in its stability.
USP7是一种去泛素化酶，属于泛素特异性蛋白酶(USPs)的一种，能够高效地水解底物蛋白上的泛素链，将目标底物去泛素化从而使之稳定。从分布上讲，USP7是一种核蛋白，其主要分布在细胞核中的核点(nuclear dots)来发挥其生理功能。在哺乳动物中，USP7结构高度保守(人与鼠的USP7结构同源性高达98.6％)，由1102个氨基酸构成，其相对分子质量约为135kDa(Cell 2009,138:389-403；Nat.Cell Biol.2002,4:106-110)。USP7根据其氨基酸顺序以及功能的不同可分为N端类TRAF结构域(residue 53-206)、催化域(residue 208-560)以及C端UBL结构域(residue 564-1084)。USP7 is a deubiquitinating enzyme, which is a kind of ubiquitin-specific proteases (USPs), which can efficiently hydrolyze the ubiquitin chain on the substrate protein and deubiquitinate the target substrate to stabilize it. In terms of distribution, USP7 is a nuclear protein, which is mainly distributed in nuclear dots in the nucleus to perform its physiological functions. In mammals, USP7 structure is highly conserved (human and mouse USP7 structure homology is as high as 98.6%), composed of 1102 amino acids, and its relative molecular mass is about 135kDa (Cell 2009,138:389-403; Nat.Cell Biol. 2002, 4:106-110). USP7 can be divided into N-terminal TRAF domain (residue 53-206), catalytic domain (residue 208-560) and C-terminal UBL domain (residue 564-1084) according to its amino acid sequence and function.
USP7对DNMT1的蛋白稳定性、酶活性以及目标DNA识别均起着重要的作用。一方面，USP7是DNMT1的去泛素化酶，其C端UBL1-2区一个由Glu736、Asp758、Glu759以及Asp764四个氨基酸残基构成的酸性口袋与DNMT1的KG连接区(residue 1109-1119)相互作用(Nat.Commun.2015,6:7023-7034)，从而将DNMT1蛋白去泛素化使之稳定。USP7对DNMT1的稳定性起着重要的作用，在工具细胞HEK293上敲除USP7导致DNMT1水平显著下降(Sci.Signal.2010,3:ra80)，而在人类结肠癌组织中，DNMT1水平与USP7水平呈现正相关(J.Cell Biochem.2011,112:439-444)。另一方面，USP7还能对DNMT1的酶活性产生影响。体外实验表明，当有USP7存在时，DNMT1的酶活性提高两倍，且这种效应并不依赖USP7的去泛素化酶活性(Nucleic Acids Res.2011,39:8355-8365)，说明USP7还能通过蛋白蛋白相互作用对DNMT1的酶活性进行调节。此外，USP7介导DNMT1与目标DNA的结合。DNMT1本身并不能识别半甲基化的目标DNA序列，需要首先与USP7以及UHRF1形成一个三聚体复合物才能发挥去甲基化作用。在该三聚体复合物中，USP7的N端与UHRF1结合，USP7的C端与DNMT1的N端TS区相结合，UHRF1与DNMT1的N端的RFTS区相结合。三聚体复合物形成后，UHRF1的SDR区可特异性识别DNA半甲基化的CpG岛，DNMT1-UHRF1-USP7复合体进而被牵引至DNA目标区域发挥作用(Nucleic Acids Res.2011,39:8355-8365)。因此，通过干扰USP7 C端结构域与DNMT1的相互作用来抑制DNMT1，是骨髓增生异常综合征及恶性肿瘤等疾病的潜在治疗途径。USP7 plays an important role in the protein stability, enzyme activity and target DNA recognition of DNMT1. On the one hand, USP7 is the deubiquitinating enzyme of DNMT1. Its C-terminal UBL1-2 region has an acid pocket consisting of four amino acid residues Glu736, Asp758, Glu759 and Asp764 and the KG connecting region of DNMT1 (residue 1109-1119) Interaction (Nat.Commun.2015, 6:7023-7034), thereby deubiquitinating and stabilizing the DNMT1 protein. USP7 plays an important role in the stability of DNMT1. Knockout of USP7 on the tool cell HEK293 resulted in a significant decrease in DNMT1 levels (Sci.Signal.2010, 3:ra80), while in human colon cancer tissues, DNMT1 levels and USP7 levels Shows a positive correlation (J. Cell Biochem. 2011, 112: 439-444). On the other hand, USP7 can also affect the enzymatic activity of DNMT1. In vitro experiments have shown that when USP7 is present, the enzyme activity of DNMT1 increases twice, and this effect does not depend on the deubiquitination enzyme activity of USP7 (Nucleic Acids Res. 2011, 39:8355-8365), indicating that USP7 also It can regulate the enzymatic activity of DNMT1 through protein-protein interaction. In addition, USP7 mediates the binding of DNMT1 to the target DNA. DNMT1 itself does not recognize the target DNA sequence of hemimethylation. It needs to form a trimer complex with USP7 and UHRF1 before it can perform demethylation. In this trimeric complex, the N-terminal of USP7 is bound to UHRF1, the C-terminal of USP7 is bound to the N-terminal TS region of DNMT1, and UHRF1 is bound to the N-terminal RFTS region of DNMT1. After the trimeric complex is formed, the SDR region of UHRF1 can specifically recognize DNA hemimethylated CpG islands, and the DNMT1-UHRF1-USP7 complex is then pulled to the DNA target region to play a role (Nucleic Acids Res. 2011, 39: 8355-8365). Therefore, inhibiting DNMT1 by interfering with the interaction between the C-terminal domain of USP7 and DNMT1 is a potential treatment approach for diseases such as myelodysplastic syndrome and malignant tumors.
此外，研究表明，USP7还可以通过去泛素化作用而稳定NFκB，进而促进NFκB的 转录调控。有意思的是，USP7 C端的上述“酸性口袋”(尤其是氨基酸757-760)对USP7识别并结合NFκB也至关重要(J.Biol.Chem.295(33):11754-11763)。因此，对USP7 C端结构域进行药理性干预，也可能成为炎症、自身免疫性疾病的新治疗途径。In addition, studies have shown that USP7 can also stabilize NFκB through deubiquitination, thereby promoting the transcriptional regulation of NFκB. Interestingly, the above-mentioned “acid pocket” (especially amino acids 757-760) at the C-terminus of USP7 is also crucial for USP7 to recognize and bind NFκB (J. Biol. Chem. 295(33): 11754-11763). Therefore, pharmacological intervention on the C-terminal domain of USP7 may also become a new treatment approach for inflammation and autoimmune diseases.
综上所述，USP7 C端调控剂具有潜在重要医药价值。然而，目前为止，USP7 C端蛋白的小分子调控剂类化合物尚未见报道，开发活性高、毒副作用小的USP7 C端结构域小分子调控剂具有重要意义。In summary, USP7 C-terminal regulator has potentially important medical value. However, so far, there have been no reports on the small molecule modulators of USP7 C-terminal protein. It is of great significance to develop small molecule modulators of USP7 C-terminal domain with high activity and low toxic and side effects.
发明内容Summary of the invention
发明目的：针对现有技术存在的问题，本发明提供一类苯并噻唑类化合物，该类苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物可以作为USP7 C端调控剂，其与USP7 C端蛋白具有很强的结合力，并可以降低肿瘤细胞中DNMT1的蛋白水平，同时具有显著的抗肿瘤细胞增殖作用，此外还可抑制USP7对NFκB的去泛素化。Objective of the invention: In view of the problems in the prior art, the present invention provides a class of benzothiazole compounds, which can be used as USP7 C-terminal regulators, or pharmaceutically acceptable salts or esters or solvates thereof. It has a strong binding force to the C-terminal protein of USP7, and can reduce the protein level of DNMT1 in tumor cells. It also has a significant anti-tumor cell proliferation effect. In addition, it can also inhibit the deubiquitination of NFκB by USP7.
本发明还提供所述苯并噻唑类化合物及其中间体的制备方法、药物组合及其医药用途。The invention also provides preparation methods, pharmaceutical combinations and medical uses of the benzothiazole compounds and intermediates thereof.
技术方案：为了实现上述目的，本发明所述如式I所示的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物：Technical solution: In order to achieve the above object, the benzothiazole compound represented by formula I or a pharmaceutically acceptable salt or ester or solvate thereof according to the present invention:
Figure PCTCN2021078798-appb-000001
Figure PCTCN2021078798-appb-000001
X为亚甲基、羰基或磺酰基；X is methylene, carbonyl or sulfonyl;
Y为氢或XR 1； Y is hydrogen or XR 1 ;
R 1、R 2各自独立地选自H、D、取代或非取代的烷基、取代或非取代的烯基、取代或非取代的炔基、取代或非取代的环烷基、取代或非取代的杂环烷基、取代或非取代的杂环烯基、取代或非取代的芳基或取代或非取代的杂环芳基。 R 1 and 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 heterocyclic aryl.
R 3为氢、羟基、杂环基、烷基、NH 2、NO 2、COOH、CN、SH、CF 3、SO 3H、SO 2CH 3或卤素。 R 3 is hydrogen, hydroxyl, heterocyclyl, alkyl, NH 2 , NO 2 , COOH, CN, SH, CF 3 , SO 3 H, SO 2 CH 3 or halogen.
作为优选，所述所述如式I所示的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物中，X为亚甲基、羰基或磺酰基；Preferably, in the benzothiazole compound represented by formula I or a pharmaceutically acceptable salt or ester or solvate thereof, X is a methylene group, a carbonyl group or a sulfonyl group;
Y为氢或XR 1； Y is hydrogen or XR 1 ;
R 1为取代的烷基、取代或未取代的杂环基、取代或未取代的苄基、取代或未取代的杂芳基甲基、取代或未取代的芳基或杂芳基； R 1 is a substituted alkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted heteroarylmethyl group, a substituted or unsubstituted aryl group or a heteroaryl group;
R 2为取代及未取代的杂芳基、取代或未取代的杂环烷基； R 2 is substituted and unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl;
R 3为氢、羟基或杂环基。 R 3 is hydrogen, a hydroxyl group or a heterocyclic group.
进一步地，所述如式I所示的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物中所述X为磺酰基；Further, in the benzothiazole compound represented by formula I or a pharmaceutically acceptable salt or ester or solvate thereof, X is a sulfonyl group;
Y为氢或XR 1； Y is hydrogen or XR 1 ;
R 1为取代的烷基、取代或未取代的杂环基、取代或未取代的苄基、取代或未取代的杂芳基甲基、取代或未取代的芳基或杂芳基； R 1 is a substituted alkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted heteroarylmethyl group, a substituted or unsubstituted aryl group or a heteroaryl group;
R 2为取代或未取代的杂环烷基； R 2 is substituted or unsubstituted heterocycloalkyl;
R 3为氢。 R 3 is hydrogen.
在某些优选的实施方案中，本发明的苯并噻唑类化合物是如下式II或式III所示的化合物或其药学上可接受的盐或溶剂化物：In certain preferred embodiments, the benzothiazole compound of the present invention is a compound represented by the following formula II or formula III or a pharmaceutically acceptable salt or solvate thereof:
Figure PCTCN2021078798-appb-000002
Figure PCTCN2021078798-appb-000002
Y为氢或SO 2R 1； Y is hydrogen or SO 2 R 1 ;
R 1、R 2各自独立地选自H、D、取代或非取代的烷基、取代或非取代的烯基、取代或非取代的炔基、取代或非取代的环烷基、取代或非取代的杂环烷基、取代或非取代的杂环烯基、取代或非取代的芳基或取代或非取代的杂环芳基。 R 1 and 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 heterocyclic aryl.
在某些更优选的实施方案中，本发明的苯并噻唑类化合物是如下表1中的任一化合物或其药学上可接受的盐或酯或溶剂化：In some more preferred embodiments, the benzothiazole compound of the present invention is any one of the compounds in Table 1 or a pharmaceutically acceptable salt or ester or solvation thereof:
表1 本发明的部分化合物Table 1 Some compounds of the present invention
Figure PCTCN2021078798-appb-000003
Figure PCTCN2021078798-appb-000003
Figure PCTCN2021078798-appb-000004
Figure PCTCN2021078798-appb-000004
Figure PCTCN2021078798-appb-000005
Figure PCTCN2021078798-appb-000005
Figure PCTCN2021078798-appb-000006
Figure PCTCN2021078798-appb-000006
Figure PCTCN2021078798-appb-000007
Figure PCTCN2021078798-appb-000007
Figure PCTCN2021078798-appb-000008
Figure PCTCN2021078798-appb-000008
Figure PCTCN2021078798-appb-000009
Figure PCTCN2021078798-appb-000009
Figure PCTCN2021078798-appb-000010
Figure PCTCN2021078798-appb-000010
Figure PCTCN2021078798-appb-000011
Figure PCTCN2021078798-appb-000011
Figure PCTCN2021078798-appb-000012
Figure PCTCN2021078798-appb-000012
Figure PCTCN2021078798-appb-000013
Figure PCTCN2021078798-appb-000013
Figure PCTCN2021078798-appb-000014
Figure PCTCN2021078798-appb-000014
本发明提供式I～III所示的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物在制备USP7 C端调控剂中的用途。本发明人发现，式I～III所示的苯并噻唑类化合物可结合于USP C端并引起USP C端构象变化，这是迄今为止公开的首类USP7 C端小分子调控剂。The present invention provides the use of benzothiazole compounds represented by formulas I to III or pharmaceutically acceptable salts or esters or solvates thereof in the preparation of USP7 C-terminal regulators. The inventors discovered that the benzothiazole compounds represented by formulas I to III can bind to the C-terminal of USP and cause conformational changes at the C-terminal of USP. This is the first type of USP7 C-terminal small molecule regulators disclosed so far.
本发明还提供式I～III所示的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物在制备预防或治疗炎症、自身免疫性疾病、骨髓增生异常综合征及恶性肿瘤的药物中的用途。The present invention also provides the use of the benzothiazole compounds represented by formulas I to III or pharmaceutically acceptable salts or esters or solvates thereof in the preparation of prevention or treatment of inflammation, autoimmune diseases, myelodysplastic syndromes and malignant tumors. Use in medicine.
所述炎症、自身免疫性疾病包括但不限于：溃疡性结肠炎、克罗恩病、系统性红斑狼疮、类风湿关节炎、银屑病、多发性硬化症或白塞氏病。The inflammation and autoimmune diseases include but are not limited to: ulcerative colitis, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, multiple sclerosis or Behçet's disease.
所述肿瘤包括但不限于：骨癌、血液科癌症、神经系统癌症、胃肠瘤、泌尿系统癌症、肺癌、肝癌或皮肤癌。The tumor includes, but is not limited to: bone cancer, hematology cancer, nervous system cancer, gastrointestinal cancer, urinary system cancer, lung cancer, liver cancer or skin cancer.
所述骨癌包括但不限于：骨源性肉瘤(骨肉瘤)、纤维肉瘤、恶性纤维组织细胞瘤、软骨肉瘤、尤因氏肉瘤、恶性淋巴瘤(网状细胞肉瘤)、多发性骨髓瘤、恶性巨细胞瘤脊索瘤、骨软骨瘤(顾软管型外生骨疣)、良性软骨瘤、成软骨细胞瘤、软骨及瘤样纤维瘤、骨样骨瘤和巨细胞瘤。所述血液科癌症包括但不限于：急性骨髓性白血病、慢性骨髓性白血病、急性淋巴细胞系白血病、慢性淋巴细胞系白血病、骨髓增生性疾病、多发性骨髓瘤和骨髓增生异常综合征、霍奇金氏淋巴瘤(恶性淋巴瘤)和瓦尔登斯特伦巨球蛋白血症。所述神经系统癌症包括但不限于：脑膜癌，如脑膜瘤、脑膜肉瘤和神经胶质瘤；脑癌，如星形细胞瘤、成神经管细胞瘤、神经胶质瘤、室管膜瘤、生殖细胞瘤(松 果体瘤)、多形性成胶质细胞瘤、少突神经胶质细胞瘤、神经鞘瘤、成视网膜细胞瘤和先天性肿瘤；脊髓瘤，如纤维神经瘤、脑膜瘤、神经胶质瘤和肉瘤。所述胃肠瘤包括但不限于：食道癌症，如鳞状细胞癌、腺癌、平滑肌肉瘤和淋巴瘤；胃癌，如肿瘤、淋巴瘤和平滑肌肉瘤；胰腺癌，如导管腺癌、胰岛瘤、胰高血糖素瘤、胃泌素瘤、类癌瘤和血管活性肠肽瘤；小肠癌，如腺癌、淋巴瘤、类癌瘤、卡波济氏肉瘤、平滑肌瘤、血管瘤、脂肪瘤、纤维神经瘤和纤维瘤；大肠癌，如腺癌、小管腺癌、绒毛状腺瘤、错构瘤和平滑肌瘤。所述泌尿系统癌症包括但不限于：肾癌，如腺癌、维尔姆斯瘤(肾母细胞瘤)、淋巴瘤和白血病；膀胱和尿道癌，如鳞状细胞癌、移行细胞癌和腺癌；前列腺癌，如腺癌和肉瘤；睾丸癌，如精原细胞瘤、畸胎瘤、胚胎性癌、畸胎瘤、绒毛膜癌、肉瘤、间质细胞癌、纤维瘤、纤维腺瘤、腺瘤样瘤和脂肪瘤。所述肺癌包括但不限于：支气管癌，如鳞状细胞癌、未分化小细胞癌、未分化大细胞癌和腺癌；细支气管肺泡癌；支气管腺瘤；肉瘤；淋巴瘤；肺软骨瘤性错构瘤和间皮瘤。所述肝癌包括但不限于：肝细胞癌，如肝细胞癌；胆管癌；肝胚细胞瘤；血管肉瘤；肝细胞腺瘤和血管瘤。所述皮肤癌包括但不限于：恶性黑素瘤、基底细胞癌、鳞状细胞癌、卡波济氏肉瘤、发育异常性痣、脂肪瘤、血管瘤、皮肤纤维瘤、瘢痕瘤、银屑病。The bone cancer includes, but is not limited to: bone-derived sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, Malignant giant cell tumor chordoma, osteochondroma (gu tube exogenous bone wart), benign chondroma, chondroblastoma, cartilage and tumor-like fibroma, osteoid osteoma, and giant cell tumor. The hematological cancers include but are not limited to: acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic disease, multiple myeloma and myelodysplastic syndrome, Hodge King's lymphoma (malignant lymphoma) and Waldenstrom's macroglobulinemia. The nervous system cancers include, but are not limited to: meningeal cancer, such as meningiomas, meningiosarcoma, and glioma; brain cancers, such as astrocytoma, medulloblastoma, glioma, ependymoma, Germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; spinal cord tumors, such as fibroneuronoma, meningioma , Glioma and Sarcoma. The 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 cancer, such as ductal adenocarcinoma, insulinoma, Glucagonoma, gastrinoma, carcinoid tumor and vasoactive intestinal peptide tumor; small bowel cancer, such as adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma , Fibroneuronoma and fibroids; colorectal cancer, such as adenocarcinoma, tubular adenocarcinoma, villous adenoma, hamartoma, and leiomyoma. The urinary system cancers include, but are not limited to: kidney cancer, such as adenocarcinoma, Wilms tumor (wilms tumor), lymphoma, and leukemia; bladder and urethral cancer, such as squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma ; Prostate cancer, such as adenocarcinoma and sarcoma; Testicular cancer, such as seminoma, teratoma, embryonic carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, gland Tumor-like tumors and lipomas. The lung cancer includes, but is not limited to: bronchial carcinoma, such as squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, and adenocarcinoma; bronchioloalveolar carcinoma; bronchial adenoma; sarcoma; lymphoma; pulmonary chondroma Hamartoma and mesothelioma. The liver cancer includes but is not limited to: hepatocellular carcinoma, such as hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hepatocellular adenoma and hemangioma. The skin cancer includes but is not limited to: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis .
在某些实施方案中，本发明的化合物可与一种或多种其他类型的预防或治疗上述疾病的药物联合使用，包括但不限于以下几种联合用药的情形：In certain embodiments, the compound of the present invention can be used in combination with one or more other types of drugs for the prevention or treatment of the above-mentioned diseases, including but not limited to the following combination drugs:
可选择与本发明的化合物联合使用的其他类型的预防或治疗药物可以是一种或多种抗癌药物，包括烷化剂(如顺铂、环磷酰胺、异环磷酰胺、美法仑、苯丁酸氮芥、苯达莫司汀、雌莫司汀、塞替哌、亚胺醌、白消安、二溴甘露醇、环己亚硝、卡氮芥、嘧啶亚硝脲、甲环亚硝脲、甲氮咪胺、甲基苄肼等)、抗代谢药(如氟尿嘧啶、阿糖胞苷、呋氟尿嘧啶、双呋氟尿嘧啶、巯嘌呤、磺巯嘌呤钠、硫唑嘌呤、硫鸟嘌呤、甲氨蝶呤、氨蝶呤扥等)、抗肿瘤抗生素(如丝裂霉素C、博来霉素、放线菌素D、光神霉素、柔红霉素、阿霉素、色霉素A3、恩霉素、新制癌素、抗癌霉素、素道霉素等)、天然抗癌药(如长春新碱、秋水仙碱、喜树碱、羟基喜树碱、斑蝥素、靛玉红等)、激素药物(如泼尼松、氢化泼尼松、氢化可的松、地塞米松、己烯雌酚、溴乙酰己烷雌酚、丙酸睾丸酮、甲睾酮、苯丙酸诺龙、萘氧啶、三苯氧胺等)、免疫治疗剂(如PD-1抑制剂nivolumab和pembrolizumab等；PD-L1抑制剂atezolizumab、durvalumab和avelumab等；CTLA-4抑制剂Ipilimumab等；其他免疫检查点抑制剂；细胞治疗剂等免疫治疗药物)、抗体药物缀合物(如Kadcyla等)、激酶抑制剂(如SHP-2抑制剂、B-RAF抑制剂、MEK抑制剂和Btk抑制剂等)、IDO抑制剂(如Epacadostat)等。Other types of preventive or therapeutic drugs that can be used in combination with the compounds of the present invention can be one or more anti-cancer drugs, including alkylating agents (such as cisplatin, cyclophosphamide, ifosfamide, melphalan, Chlorambucil, Bendamustine, Estramustine, Cetepa, Iminoquinone, Busulfan, Dibromomannitol, Cyclohexylnitrosene, Carmustine, Pyrimidine Nitrosourea, Methalin Nitrosourea, methazine, procarbazine, etc.), antimetabolites (such as fluorouracil, cytarabine, furfurouracil, difurfurouracil, mercaptopurine, sulfathioprine, azathioprine, thioguanine) , Methotrexate, methotrexate, etc.), anti-tumor antibiotics (such as mitomycin C, bleomycin, actinomycin D, mithomycin, daunorubicin, doxorubicin, color A3, Enmycin, Neocarcinogen, Anticancerin, Sudoxin, etc.), natural anticancer drugs (such as vincristine, colchicine, camptothecin, hydroxycamptothecin, cantharidin, etc.) Indirubin, etc.), hormone drugs (such as prednisone, prednisone, hydrocortisone, dexamethasone, diethylstilbestrol, bromoacetestrol, testosterone propionate, methyltestosterone, nandrolone phenylpropionate, Naphthoxidine, tamoxifen, etc.), immunotherapeutics (such as PD-1 inhibitors nivolumab and pembrolizumab, etc.; PD-L1 inhibitors atezolizumab, durvalumab and avelumab, etc.; CTLA-4 inhibitor Ipilimumab, etc.; other immune checkpoint inhibitors; Cell therapy agents and other immunotherapeutic drugs), antibody drug conjugates (such as Kadcyla, etc.), kinase inhibitors (such as SHP-2 inhibitors, B-RAF inhibitors, MEK inhibitors and Btk inhibitors, etc.), IDO inhibitors (Such as Epacadostat) and so on.
本发明还提供一种预防或治疗炎症、自身免疫性疾病、骨髓增生异常综合征及肿瘤的药物组合物，其中含有治疗有效量的式I～III所示的任一苯并噻唑类化合物或其药学上可接受的盐或溶剂化物作为活性成份和药学上可接受的辅料。可任意混合的辅料根据 剂型、给药形式等可以改变，载体包括但不限于赋形剂、粘合剂、崩解剂、润滑剂、矫味剂、香味剂、着色剂和甜味剂等。所述药物组合物可以是普通片剂或胶囊、缓释片剂或胶囊、控释片剂或胶囊、颗粒剂、散剂、糖浆剂、口服液或注射剂等制剂学上常规的制剂形式。The present invention also provides a pharmaceutical composition for preventing or treating inflammation, autoimmune diseases, myelodysplastic syndromes and tumors, which contains a therapeutically effective amount of any benzothiazole compound represented by formula I to III or its Pharmaceutically acceptable salts or solvates are used as active ingredients and pharmaceutically acceptable excipients. The adjuvants that can be arbitrarily mixed can be changed according to the dosage form, administration form, etc. The carrier includes but is not limited to excipients, binders, disintegrating agents, lubricants, flavoring agents, flavoring agents, coloring agents and sweetening agents. The pharmaceutical composition can be in the form of conventional pharmaceutics such as ordinary tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, granules, powders, syrups, oral liquids or injections.
有益效果：与现有技术相比，本发明具有如下优点：Beneficial effects: Compared with the prior art, the present invention has the following advantages:
(1)本发明的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物与USP7 C端蛋白具有较强的结合力，是迄今为止公布的首类USP7 C端小分子调控剂，其可以降低肿瘤细胞中DNMT1的蛋白水平，具有显著的抗肿瘤细胞增殖作用，因而可用于制备预防或治疗骨髓增生异常综合征及恶性肿瘤的药物。此外，本发明的USP7 C端小分子调控剂，也可以阻断USP7 C端与NFκB的结合，抑制NFκB的去泛素化，具有显著的抗炎活性，因而也可用于制备抗炎药物以及治疗自身免疫性疾病的药物。(1) The benzothiazole compound of the present invention or its pharmaceutically acceptable salt or ester or solvate has a strong binding force with the C-terminal protein of USP7, and is the first USP7 C-terminal small molecule regulator published so far It can reduce the protein level of DNMT1 in tumor cells and has a significant anti-tumor cell proliferation effect, so it can be used to prepare drugs for the prevention or treatment of myelodysplastic syndromes and malignant tumors. In addition, the USP7 C-terminal small molecule regulator of the present invention can also block the binding of USP7 C-terminal to NFκB, inhibit the deubiquitination of NFκB, and has significant anti-inflammatory activity, so it can also be used for the preparation of anti-inflammatory drugs and treatments. Drugs for autoimmune diseases.
(2)本发明的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物成药性在人肝微粒体中代谢稳定性较好，并可口服吸收，成药性好。(2) The benzothiazole compound of the present invention or its pharmaceutically acceptable salt or ester or solvate has good drug-forming properties in human liver microsomes, and can be absorbed orally with good drug-forming properties.
(3)本发明的苯并噻唑类化合物结构简单，合成路线设计巧妙，原料便宜易得，合成工艺安全、环保，易于规模化生产。(3) The benzothiazole compound of the present invention has simple structure, ingenious synthesis route design, cheap and readily available raw materials, safe and environmentally friendly synthesis process, and is easy to scale production.
附图说明Description of the drawings
图1为化合物25与USP7 C端蛋白的X-射线共晶结构图；Figure 1 is an X-ray co-crystal structure diagram of compound 25 and USP7 C-terminal protein;
图2为化合物影响USP7 C端蛋白与DNMT1相互作用的GST Pull-down实验结果图；Figure 2 is a graph of the GST Pull-down experiment result of the compound's influence on the interaction between USP7 C-terminal protein and DNMT1;
图3为化合物55浓度依赖性地降低NB4细胞内DNMT1水平的Western Blot实验结果图；Figure 3 is a Western Blot experimental result of compound 55 concentration-dependently reducing the level of DNMT1 in NB4 cells;
图4为化合物60浓度依赖性地降低NB4细胞内DNMT1水平的Western Blot实验结果图；Figure 4 is a diagram showing the results of a Western Blot experiment in which compound 60 reduces the level of DNMT1 in NB4 cells in a concentration-dependent manner;
图5为化合物55在Raw264.7细胞上对LPS诱导的IL-1b和IL-6表达的影响。Figure 5 shows the effect of compound 55 on LPS-induced IL-1b and IL-6 expression on Raw264.7 cells.
具体实施方式Detailed ways
下面通过实施例具体说明本发明的内容。在本发明中，以下所述的实施例是为了更好的阐述本发明，并不是用来限制本发明的范围。在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。The content of the present invention will be described in detail through the following examples. In the present invention, the following embodiments are used to better illustrate the present invention, and are not used to limit the scope of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.
实施例1Example 1
N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)异烟酰胺(化合物1)N-(2-(furan-2-carboxamide)benzo[d]thiazol-6-yl)isonicotinamide (Compound 1)
Figure PCTCN2021078798-appb-000015
Figure PCTCN2021078798-appb-000015
将2-氨基-6-硝基苯并噻唑(1a,1.0g,5.13mmol)溶于吡啶(20mL)中，滴加新制的糠酰氯(758μL，7.70mmol)，加毕后升温至40℃搅拌8小时。TLC监测反应完全后加入1N盐酸(30mL)中和，大量固体析出。抽滤后固体用少量乙酸乙酯(2mL×3)洗涤，得中间体1b的粗品，无需纯化直接投下一步。Dissolve 2-amino-6-nitrobenzothiazole (1a, 1.0g, 5.13mmol) in pyridine (20mL), add fresh furoyl chloride (758μL, 7.70mmol) dropwise, after the addition, heat to 40℃ and stir. 8 hours. After TLC monitoring the reaction was completed, 1N hydrochloric acid (30 mL) was added for neutralization, and a large amount of solids precipitated. After suction filtration, the solid was washed with a small amount of ethyl acetate (2 mL×3) to obtain the crude product of Intermediate 1b, which was directly used in the next step without purification.
将所得全部粗品中间体1b加入甲醇(25mL)中，加入10％钯/碳(100mg)，通入氢气后50℃下搅拌7小时。TLC监测反应完全后停止加热，冷却至室温后溶液用硅藻土抽滤，滤液减压蒸除溶剂即得到化合物1c(棕色固体,520mg,两步收率39％)： 1H NMR(300MHz,DMSO-d 6)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] -。 All the obtained crude intermediate 1b was added to methanol (25 mL), 10% palladium/carbon (100 mg) was added, hydrogen gas was introduced, and the mixture was stirred at 50° C. for 7 hours. After the reaction was monitored by TLC, the heating was stopped. After cooling to room temperature, the solution was suction filtered with diatomaceous earth, and the filtrate was evaporated under reduced pressure to remove the solvent to obtain compound 1c (brown solid, 520mg, two-step yield 39%): 1 H NMR (300MHz, DMSO-d 6 )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[MH] - .
将化合物1c(100mg,0.39mmol)加入到二氯甲烷(3mL)中(混悬液)，依次加入异烟酸(52mg,0.47mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(96mg,0.47mmol)、4-二甲氨基吡啶(47mg,0.47mmol)，室温下搅拌过夜。观察到大量灰色固体析出，TLC监测反应完全后停止搅拌，抽滤，滤饼用少量二氯甲烷(1mL×2)洗涤即得化合物1(灰白色固体,91mg,产率65％)： 1H NMR(500MHz,DMSO-d 6)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] -。 Compound 1c (100mg, 0.39mmol) was added to dichloromethane (3mL) (suspension), followed by isonicotinic acid (52mg, 0.47mmol), 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide hydrochloride (96mg, 0.47mmol) and 4-dimethylaminopyridine (47mg, 0.47mmol) were stirred overnight at room temperature. A large amount of gray solids were observed to precipitate. After the reaction was monitored by TLC, the stirring was stopped, filtered with suction, and the filter cake was washed with a small amount of dichloromethane (1mL×2) to obtain compound 1 (off-white solid, 91mg, yield 65%): 1 H NMR (500MHz,DMSO-d 6 )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[MH] - .
实施例2Example 2
N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)吡啶酰胺(化合物2)N-(2-(furan-2-carboxamide)benzo[d]thiazol-6-yl)pyridine amide (compound 2)
Figure PCTCN2021078798-appb-000016
Figure PCTCN2021078798-appb-000016
参照实施例1的方法，将异烟酸替换成吡啶甲酸，制得化合物2(灰白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with picolinic acid to obtain compound 2 (off-white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.80 (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[MH] - .
实施例3Example 3
N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)吡嗪-2-甲酰胺(化合物3)N-(2-(furan-2-carboxamide)benzo[d]thiazol-6-yl)pyrazine-2-carboxamide (compound 3)
Figure PCTCN2021078798-appb-000017
Figure PCTCN2021078798-appb-000017
参照实施例1的方法，将异烟酸替换成吡嗪-2-羧酸，制得化合物3(灰白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with pyrazine-2-carboxylic acid to obtain compound 3 (off-white solid): 1 H NMR (300MHz, DMSO-d 6 )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 [MH] - .
实施例4Example 4
(±)-N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)哌啶-3-甲酰胺(化合物4)(±)-N-(2-(furan-2-carboxamide)benzo(d)thiazol-6-yl)piperidine-3-carboxamide (Compound 4)
Figure PCTCN2021078798-appb-000018
Figure PCTCN2021078798-appb-000018
Figure PCTCN2021078798-appb-000019
Figure PCTCN2021078798-appb-000019
将化合物1c(20mg,0.08mmol)溶于四氢呋喃(2mL)中，依次加入N-叔丁氧羰基哌啶-3-甲酸4a(21mg,0.09mmol)、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,35mg,0.09mmol)以及N,N-二异丙基乙胺(20μL,0.12mmol)，室温下搅拌过夜。TLC监测反应完全后停止搅拌，减压蒸除溶剂后加二氯甲烷(10mL)溶解，有机相经水洗(5mL)后加入无水硫酸钠干燥30分钟，过滤，制砂。硅胶柱层析(二氯甲烷:甲醇＝300:1)得化合物4b(白色固体,20mg,产率54％)。Compound 1c (20mg, 0.08mmol) was dissolved in tetrahydrofuran (2mL), and N-tert-butoxycarbonylpiperidine-3-carboxylic acid 4a (21mg, 0.09mmol) and 2-(7-azabenzotriazide) were added sequentially. Azole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 35mg, 0.09mmol) and N,N-diisopropylethylamine (20μL, 0.12mmol), stir at room temperature overnight. After the reaction was monitored by TLC, the stirring was stopped, the solvent was evaporated under reduced pressure, and dichloromethane (10 mL) was added to dissolve. The organic phase was washed with water (5 mL) and dried with anhydrous sodium sulfate for 30 minutes, filtered, and sand was made. Silica gel column chromatography (dichloromethane:methanol=300:1) to obtain compound 4b (white solid, 20mg, yield 54%).
将化合物4b置于茄形瓶中，加入三氟醋酸的二氯甲烷溶液(三氟醋酸:二氯甲烷＝1:2，3mL)，室温搅拌0.5小时。TLC监测反应完全后停止搅拌，减压蒸除溶剂后，加入二氯甲烷(2mL)，滴加饱和碳氢酸钠溶液至中性后有固体析出，抽滤，干燥后得化合物4(白色固体,10mg,产率54％)； 1H NMR(300MHz,Methanol-d 4)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] -。 Put compound 4b in an eggplant-shaped flask, add a dichloromethane solution of trifluoroacetic acid (trifluoroacetic acid:dichloromethane=1:2, 3mL), and stir at room temperature for 0.5 hours. After the reaction was monitored by TLC, the stirring was stopped. After the solvent was distilled off under reduced pressure, dichloromethane (2mL) was added, and saturated sodium hydrogen carbonate solution was added dropwise to neutrality. A solid precipitated out, filtered off with suction, and dried to obtain compound 4 (white solid) , 10mg, yield 54%); 1 H NMR (300MHz, Methanol-d 4 ) δ 8.28 (d, J = 2.0 Hz, 1H), 7.84 (dd, J = 1.8, 0.8 Hz, 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[MH] - .
实施例5Example 5
(±)-N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)哌啶-2-甲酰胺(化合物5)(±)-N-(2-(furan-2-carboxamide)benzo(d)thiazol-6-yl)piperidine-2-carboxamide (compound 5)
Figure PCTCN2021078798-appb-000020
Figure PCTCN2021078798-appb-000020
参照实施例4的方法，将N-叔丁氧羰基哌啶-3-甲酸替换成N-叔丁氧羰基哌啶-2-甲酸，制得化合物5(白色固体)： 1H NMR(300MHz,Methanol-d 4)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＝13.1 Hz,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] -。 Referring to the method of Example 4, N-tert-butoxycarbonylpiperidine-3-carboxylic acid was replaced with N-tert-butoxycarbonylpiperidine-2-carboxylic acid to obtain compound 5 (white solid): 1 H NMR (300MHz, Methanol-d 4 )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 = 3.6 Hz, 1H), 6.72 (dd, J = 3.6, 1.8 Hz, 1H), 4.04 (dd, J = 11.6, 3.1 Hz, 1H), 3.47 (d ,J＝13.1 Hz,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[MH] - .
实施例6Example 6
N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)哌啶-4-甲酰胺(化合物6)N-(2-(furan-2-carboxamide)benzo[d]thiazol-6-yl)piperidine-4-carboxamide (Compound 6)
Figure PCTCN2021078798-appb-000021
Figure PCTCN2021078798-appb-000021
参照实施例4的方法，将N-叔丁氧羰基哌啶-3-甲酸替换成N-叔丁氧羰基哌啶-4-甲酸，制得化合物6(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Referring to the method of Example 4, N-tert-butoxycarbonylpiperidine-3-carboxylic acid was replaced with N-tert-butoxycarbonylpiperidine-4-carboxylic acid to obtain compound 6 (white solid): 1 H NMR (300MHz, DMSO-d 6 )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 [MH] - .
实施例7Example 7
N-(6-(4-氨基苯甲酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物7)N-(6-(4-Aminobenzoyl)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 7)
Figure PCTCN2021078798-appb-000022
Figure PCTCN2021078798-appb-000022
将对氨基苯甲酸(7a，493mg，3.60mmol)溶于二氧六环与水的混合溶液(二氧六环:水＝2:1，10mL)中，室温滴加三乙胺(1mL，7.30mmol)，搅拌5分钟后加入Boc酸酐(1.6g,7.30mmol)，室温搅拌36小时。TLC监测反应完全后加入2N盐 酸调节溶液至pH＝5-6，大量固体析出。抽滤，固体烘干即得到化合物7b(白色固体，708mg，产率83％)。P-aminobenzoic acid (7a, 493 mg, 3.60 mmol) was dissolved in a mixed solution of dioxane and water (dioxane: water = 2:1, 10 mL), and triethylamine (1 mL, 7.30) was added dropwise at room temperature. mmol), add Boc anhydride (1.6 g, 7.30 mmol) after stirring for 5 minutes, and stir at room temperature for 36 hours. After the 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 solids precipitated. Suction filtration, drying the solid to obtain compound 7b (white solid, 708 mg, yield 83%).
将化合物1c(100mg,0.39mmol)加入到二氯甲烷(5mL)中(为混悬液)，依次加入中间体7b(103mg,0.47mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(96mg,0.47mmol)、4-二甲氨基吡啶(47mg,0.47mmol)，室温下搅拌过夜。观察到大量白色固体析出，TLC监测反应完全后停止搅拌，抽滤，滤饼用少量二氯甲烷/甲醇混合溶液(1mL×2)洗涤即得化合物7c(灰白色固体)。Compound 1c (100mg, 0.39mmol) was added to dichloromethane (5mL) (for suspension), and Intermediate 7b (103mg, 0.47mmol), 1-(3-dimethylaminopropyl)-3 were added in turn -Ethylcarbodiimide hydrochloride (96mg, 0.47mmol), 4-dimethylaminopyridine (47mg, 0.47mmol), stirred overnight at room temperature. A large amount of white solid was observed to precipitate. After TLC monitoring the reaction was completed, the stirring was stopped, filtered with suction, and the filter cake was washed with a small amount of dichloromethane/methanol mixed solution (1 mL×2) to obtain compound 7c (off-white solid).
将全部化合物7c置于茄形瓶中，加入三氟醋酸的二氯甲烷溶液(三氟醋酸:二氯甲烷＝1:2，6mL)，室温搅拌1小时。TLC监测反应完全后，向体系内加入饱和碳酸氢钠溶液至中性，有大量固体析出，抽滤，滤饼用乙酸乙酯(0.5mL×3)洗涤，干燥后可得化合物7(白色固体,78mg,两步产率53％)： 1H NMR(300MHz,DMSO-d 6)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] -。 Put all the compound 7c in an eggplant-shaped flask, add a dichloromethane solution of trifluoroacetic acid (trifluoroacetic acid:dichloromethane=1:2, 6mL), and stir at room temperature for 1 hour. After the reaction was monitored by TLC, saturated sodium bicarbonate solution was added to the system until it became neutral. A large amount of solids precipitated out. The filter cake was washed with ethyl acetate (0.5 mL×3). After drying, compound 7 (white solid) was obtained. , 78mg, two-step yield 53%): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.75 (s, 1H), 9.88 (s, 1H), 8.39 (d, J = 2.0 Hz, 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[MH] - .
实施例8Example 8
N-(6-(3-氨基苯甲酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物8)N-(6-(3-Aminobenzoyl)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 8)
Figure PCTCN2021078798-appb-000023
Figure PCTCN2021078798-appb-000023
参照实施例7的方法，将对氨基苯甲酸替换成间氨基苯甲酸，制得化合物8(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 7, p-aminobenzoic acid was replaced with meta-aminobenzoic acid to obtain compound 8 (white solid): 1 H NMR (300MHz, DMSO-d 6 )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[MH] - .
实施例9Example 9
N-(6-(4-腈基苯甲酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物9)N-(6-(4-cyanobenzoyl)benzo[d]thiazol-2-yl)furan-2-carboxamide (compound 9)
Figure PCTCN2021078798-appb-000024
Figure PCTCN2021078798-appb-000024
参照实施例1的方法，将异烟酸替换成4-氰基苯甲酸，制得化合物9(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Referring to the method of Example 1, the isonicotinic acid was replaced with 4-cyanobenzoic acid to obtain compound 9 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.81 (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[MH] - .
实施例10Example 10
N-(6-(4-三氟甲基苯甲酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物10)N-(6-(4-Trifluoromethylbenzoyl)benzo(d)thiazol-2-yl)furan-2-carboxamide (compound 10)
Figure PCTCN2021078798-appb-000025
Figure PCTCN2021078798-appb-000025
参照实施例1的方法，将异烟酸替换成4-三氟甲基苯甲酸，制得化合物10(灰色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with 4-trifluoromethyl benzoic acid to obtain compound 10 (gray solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.81 (s, 1H) , 10.63 (s, 1H), 8.48 (s, 1H), 8.19 (d, J = 8.1 Hz, 2H), 8.05 (d, J = 1.7 Hz, 1H), 7.94 (d, J = 8.1 Hz, 2H) , 7.85-7.65 (m, 3H), 6.77 (dd, J=3.7, 1.7 Hz, 1H). ESI-MS m/z 430.1 [MH] - .
实施例11Example 11
N-(6-(2,4-二氟苯甲酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物11)N-(6-(2,4-Difluorobenzoyl)benzo(d)thiazol-2-yl)furan-2-carboxamide (Compound 11)
Figure PCTCN2021078798-appb-000026
Figure PCTCN2021078798-appb-000026
参照实施例1的方法，将异烟酸替换成2,4-二氟苯甲酸，制得化合物11(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with 2,4-difluorobenzoic acid to obtain compound 11 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) 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=9.9,2.4 Hz, 1H), 7.25 (td, J=8.5, 2.4 Hz, 1H), 6.77 (dd, J=3.6, 1.7 Hz, 1H). ESI-MS m/z 398.0 [MH] - .
实施例12Example 12
N-(6-(3-氟-4-氯苯甲酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物12)N-(6-(3-Fluoro-4-chlorobenzoyl)benzo(d)thiazol-2-yl)furan-2-carboxamide (Compound 12)
Figure PCTCN2021078798-appb-000027
Figure PCTCN2021078798-appb-000027
参照实施例1的方法，将异烟酸替换成3-氟-4-氯苯甲酸，制得化合物12(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Refer to the method of Example 1, replacing isonicotinic acid with 3-fluoro-4-chlorobenzoic acid to obtain compound 12 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.72 (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[MH] - .
实施例13Example 13
N-(6-(4-哌嗪-1-基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物13)N-(6-(4-piperazin-1-yl)benzo[d]thiazol-2-yl)furan-2-carboxamide (compound 13)
Figure PCTCN2021078798-appb-000028
Figure PCTCN2021078798-appb-000028
将对氟苯甲酸乙酯(13a,100mg,0.59mmol)溶于干燥的二甲基亚砜(1mL)中，依次加入1-Boc-哌嗪(13b,121mg,0.65mmol)和碳酸钾(246mg,1.78mmol)，120℃下搅拌5h，TLC监测反应完全后加入饱和食盐水(5mL)，乙酸乙酯萃取(5mL×3)。合并有机相，加入无水硫酸钠干燥30分钟后制砂干法上柱，经硅胶柱层析(乙酸乙酯:石油醚＝8:1)得到化合物13c(灰白色固体,141mg,产率78％)。Ethyl p-fluorobenzoate (13a, 100mg, 0.59mmol) was dissolved in dry dimethyl sulfoxide (1mL), and 1-Boc-piperazine (13b, 121mg, 0.65mmol) and potassium carbonate (246mg , 1.78mmol), stirred at 120°C for 5h, TLC monitored the completion of the reaction and added saturated brine (5mL), extracted with ethyl acetate (5mL×3). The organic phases were combined, dried by adding anhydrous sodium sulfate for 30 minutes, and then applied to the column by a dry method of sand preparation, and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 8:1) to obtain compound 13c (off-white solid, 141 mg, yield 78%) ).
将化合物13c(100mg,0.30mmol)加入到2N的氢氧化钠水溶液(5mL)中，80℃下搅拌24小时，溶液澄清，TLC监测反应完全后加入2N盐酸调pH至5-6，大量固体析出。抽滤，滤饼收集烘干；滤液用乙酸乙酯萃取(10mL×3)，有机相用无水硫酸钠干燥后旋干，与烘干的滤饼合并得化合物13d(白色固体,73mg,收率79％)。Compound 13c (100mg, 0.30mmol) was added to 2N sodium hydroxide aqueous solution (5mL) and stirred at 80°C for 24 hours. The solution was clear. After the reaction was monitored by TLC, 2N hydrochloric acid was added to adjust the pH to 5-6. A large amount of solids precipitated . Suction filtration, the filter cake was collected and dried; the filtrate was extracted with ethyl acetate (10mL×3), the organic phase was dried over anhydrous sodium sulfate and spin-dried, and combined with the dried filter cake to obtain compound 13d (white solid, 73mg, yield) Rate 79%).
参照实施例7的方法，将化合物7b替换成化合物13d，制得化合物13(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 7, compound 7b was replaced with compound 13d to obtain compound 13 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) 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] + .
实施例14Example 14
N-(6-(4-甲磺酰基苯磺酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物14)N-(6-(4-Methanesulfonylbenzenesulfonyl)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 14)
Figure PCTCN2021078798-appb-000029
Figure PCTCN2021078798-appb-000029
参照实施例1的方法，将异烟酸替换成4-甲砜基苯甲酸，制得化合物14(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with 4-methylsulfonyl benzoic acid to obtain compound 14 (white solid): 1 H NMR (300MHz, DMSO-d 6 )δ12.82(s, 1H), 10.66 (s, 1H), 8.49 (s, 1H), 8.22 (d, J = 8.2 Hz, 2H), 8.11 (d, J = 8.2 Hz, 2H), 8.06 (d, J = 1.7 Hz, 1H), 7.85-7.65 (m, 3H), 6.77 (dd, J=3.6, 1.7 Hz, 1H), 3.31 (s, 3H). ESI-MS m/z 440.1[MH] - .
实施例15Example 15
N-(6-(4-甲磺酰基苯磺酰)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物15)N-(6-(4-Methanesulfonylbenzenesulfonyl)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 15)
Figure PCTCN2021078798-appb-000030
Figure PCTCN2021078798-appb-000030
将化合物2(50mg,0.14mmol)溶于在N,N-二甲基甲酰胺(3mL)中，室温滴加碘甲烷(86μL,1.37mmol)，加毕后室温搅拌1小时。TLC监测反应完毕后停止搅拌，减压蒸除溶剂后所得固体用二氯甲烷(2mL×2)洗涤，得到化合物15(白色固体,61mg,产率88％)： 1H NMR(300MHz,DMSO-d 6)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] -。 Compound 2 (50 mg, 0.14 mmol) was dissolved in N,N-dimethylformamide (3 mL), methyl iodide (86 μL, 1.37 mmol) was added dropwise at room temperature, and after addition, stirred at room temperature for 1 hour. After the reaction was monitored by TLC, the stirring was stopped. After the solvent was evaporated under reduced pressure, the solid obtained was washed with dichloromethane (2mL×2) to obtain compound 15 (white solid, 61mg, yield 88%): 1 H NMR (300MHz, DMSO- d 6 )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[MH] - .
实施例16Example 16
N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)苯并[1,2,5]二噁唑-5-甲酰胺(化合物16)N-(2-(furan-2-carboxamide)benzo[d]thiazol-6-yl)benzo[1,2,5]dioxazole-5-carboxamide (compound 16)
Figure PCTCN2021078798-appb-000031
Figure PCTCN2021078798-appb-000031
参照实施例1的方法，将异烟酸替换成2,1,3-苯并恶二唑-5-羧酸，制得化合物16(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with 2,1,3-benzoxadiazole-5-carboxylic acid to obtain compound 16 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) 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[MH] - .
实施例17Example 17
N-(6-(呋喃-3-甲酰胺)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物17)N-(6-(furan-3-carboxamide)benzo[d]thiazol-2-yl)furan-2-carboxamide (compound 17)
Figure PCTCN2021078798-appb-000032
Figure PCTCN2021078798-appb-000032
参照实施例1的方法，将异烟酸替换成3-糠酸，制得化合物17(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 1, the isonicotinic acid was replaced with 3-furoic acid to obtain compound 17 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.82 (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, 1.7 Hz, 1H). MS (ESI) m/z 376.1 [M+Na] + .
实施例18Example 18
N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)异喹啉-3-甲酰胺(化合物18)N-(2-(furan-2-carboxamide)benzo[d]thiazol-6-yl)isoquinoline-3-carboxamide (Compound 18)
Figure PCTCN2021078798-appb-000033
Figure PCTCN2021078798-appb-000033
参照实施例1的方法，将异烟酸替换成异喹啉-3-甲酸，制得化合物18(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with isoquinoline-3-carboxylic acid to obtain compound 18 (white solid): 1 H NMR (300MHz, DMSO-d 6 )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[MH] - .
实施例19Example 19
N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)喹啉-3-甲酰胺(化合物19)N-(2-(furan-2-carboxamide)benzo(d)thiazol-6-yl)quinoline-3-carboxamide (Compound 19)
Figure PCTCN2021078798-appb-000034
Figure PCTCN2021078798-appb-000034
参照实施例1的方法，将异烟酸替换成喹啉-3-甲酸，制得化合物19(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 1, the isonicotinic acid was replaced with quinoline-3-carboxylic acid to obtain compound 19 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.83(s, 1H), 11.00 (s, 1H), 9.43 (d, J = 2.3 Hz, 1H), 9.12 (d, J = 2.2 Hz, 1H), 8.56 (d, J = 2.2 Hz, 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[MH] - .
实施例20Example 20
N-(6-(4-硝基苯乙酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物20)N-(6-(4-nitrophenylacetamido)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 20)
Figure PCTCN2021078798-appb-000035
Figure PCTCN2021078798-appb-000035
参照实施例1的方法，将异烟酸替换成对硝基苯乙酸，制得化合物20(黄色固体)： 1H NMR(300MHz,DMSO-d 6)δ12.83(s,1H),10.52(s,1H),8.32(s,1H),8.22(d,J＝8.2 Hz,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] -。 Referring to the method of Example 1, the isonicotinic acid was replaced with p-nitrophenylacetic acid to obtain compound 20 (yellow solid): 1 H NMR (300MHz, DMSO-d 6 )δ12.83(s, 1H), 10.52( s, 1H), 8.32 (s, 1H), 8.22 (d, J = 8.2 Hz, 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[MH] - .
实施例21Example 21
N-(6-(4-甲基苯乙酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物21)N-(6-(4-Methylphenylacetamido)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 21)
Figure PCTCN2021078798-appb-000036
Figure PCTCN2021078798-appb-000036
参照实施例1的方法，将异烟酸替换成对甲基苯乙酸，制得化合物21(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] +。 Referring to the method of Example 1, the isonicotinic acid was replaced with p-toluene acetic acid to obtain compound 21 (white solid): 1 H NMR (300MHz, DMSO-d 6 )δ12.79(s, 1H), 10.48( s, 1H), 8.34 (d, J = 1.9 Hz, 1H), 8.05 (d, J = 1.6 Hz, 1H), 7.83-7.52 (m, 3H), 7.39-7.01 (m, 4H), 6.76 (dd , J=3.6, 1.7 Hz, 1H), 3.63 (s, 2H), 2.28 (s, 3H). ESI-MS m/z 414.1 [M+Na] + .
实施例22Example 22
N-(6-((4-硝基苯乙基)氨基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物22)N-(6-((4-nitrophenethyl)amino)benzo(d)thiazol-2-yl)furan-2-carboxamide (Compound 22)
Figure PCTCN2021078798-appb-000037
Figure PCTCN2021078798-appb-000037
将化合物1c(50mg,0.19mmol)溶于N,N-二甲基甲酰胺(2mL)中，依次加入对硝基溴苄(22a，46mg,0.21mmol)和碳酸钾(50mg,0.39mmol)，室温搅拌过夜。TLC监测反应完毕后，减压蒸除溶剂。向体系内加入乙酸乙酯(10mL)将固体溶解，有机层用饱和食盐水(5mL)洗涤，经无水硫酸钠干燥0.5小时后过滤，滤液制砂后硅胶柱层析(二氯甲烷:甲醇＝400:1)得化合物22(金黄色固体,14mg,产率19％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Compound 1c (50mg, 0.19mmol) was dissolved in N,N-dimethylformamide (2mL), and p-nitrobenzyl bromide (22a, 46mg, 0.21mmol) and potassium carbonate (50mg, 0.39mmol) were added successively, Stir at room temperature overnight. After the reaction was monitored by TLC, the solvent was evaporated under reduced pressure. Ethyl acetate (10mL) was added to the system to dissolve the solids. The organic layer was washed with saturated brine (5mL), dried over anhydrous sodium sulfate for 0.5 hours and filtered. =400:1) to obtain compound 22 (golden yellow solid, 14mg, yield 19%): 1 H NMR (300MHz, DMSO-d 6 )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] + .
实施例23Example 23
N-(6-((4-硝基苯基)磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物23)N-(6-((4-nitrophenyl)sulfonamido)benzo(d)thiazol-2-yl)furan-2-carboxamide (compound 23)
Figure PCTCN2021078798-appb-000038
Figure PCTCN2021078798-appb-000038
将化合物1c(50mg,0.19mmol)置于茄形瓶中，加入二氯甲烷(3mL)使之成混悬液后加入对硝基苯甲酰氯(23a,43mg,0.21mmol)，在搅拌状态下向反应体系中滴加三乙胺(32μL,0.21mmol)，加毕后室温反应过夜，大量固体析出。TLC监测反应完全后停止搅拌，抽滤，滤饼用少量二氯甲烷/甲醇洗，得化合物23(黄色固体,39mg,产率48％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Put compound 1c (50mg, 0.19mmol) in an eggplant-shaped flask, add dichloromethane (3mL) to make a suspension, then add p-nitrobenzoyl chloride (23a, 43mg, 0.21mmol), under stirring Triethylamine (32 μL, 0.21 mmol) was added dropwise to the reaction system, and after the addition, the reaction was carried out at room temperature overnight, and a large amount of solids precipitated. After the reaction was monitored by TLC, the stirring was stopped, filtered with suction, and the filter cake was washed with a small amount of dichloromethane/methanol to obtain compound 23 (yellow solid, 39mg, yield 48%): 1 H NMR (300MHz, DMSO-d 6 )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, 2.2 Hz, 1H), 6.76 (dd, J=3.7, 1.7 Hz, 1H). ESI-MS m/z 467.1 [M+Na] + .
实施例24Example 24
4-(N-(2-(呋喃-2-甲酰胺)苯并[d]噻唑-6-基)磺酰胺基)苯甲酸(化合物24)4-(N-(2-(furan-2-carboxamide)benzo(d)thiazol-6-yl)sulfonamido)benzoic acid (Compound 24)
Figure PCTCN2021078798-appb-000039
Figure PCTCN2021078798-appb-000039
将化合物1c(100mg,0.38mmol)置于茄形瓶中，加入四氢呋喃(4mL)溶液成混悬液后加入4-(氯磺酰基)苯甲酸乙酯(24a,105mg,0.42mmol)，在搅拌状态下向反应体系中滴加N,N-二异丙基乙胺(200μL,0.42mmol)，加毕后室温反应过夜，大量固 体析出。TLC监测反应完全后停止搅拌，抽滤，滤饼用二氯甲烷/甲醇混合溶液(1mL二氯甲烷中加入5滴甲醇)洗，得到24b的粗品。Place compound 1c (100mg, 0.38mmol) in an eggplant-shaped flask, add tetrahydrofuran (4mL) solution to make a suspension, add ethyl 4-(chlorosulfonyl)benzoate (24a, 105mg, 0.42mmol), stir N,N-diisopropylethylamine (200μL, 0.42mmol) was added dropwise to the reaction system under the state, and reacted overnight at room temperature after the addition, and a large amount of solids precipitated. After the reaction was monitored by TLC, the stirring was stopped, filtered with suction, and the filter cake was washed with a dichloromethane/methanol mixed solution (5 drops of methanol added to 1 mL of dichloromethane) to obtain a crude product of 24b.
将所得全部粗品24b置于10mL茄形瓶中，加入质量分数8％的氢氧化钠水溶液(3mL)，40℃下搅拌2小时，溶液澄清。TLC监测反应完全后停止加热，向反应体系中滴加1N盐酸调至中性，大量固体析出，抽滤，得化合物24(白色固体,43mg,两步收率28％)： 1H NMR(300MHz,DMSO-d 6)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] -。 Put all the obtained crude product 24b in a 10 mL eggplant-shaped bottle, add 8% sodium hydroxide aqueous solution (3 mL), and stir at 40° C. for 2 hours. The solution is clear. After the reaction was monitored by TLC, the heating was stopped, 1N hydrochloric acid was added dropwise to the reaction system to adjust to neutrality, a large amount of solid precipitated, and suction filtration, to obtain compound 24 (white solid, 43mg, two-step yield 28%): 1 H NMR (300MHz ,DMSO-d 6 )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=8.7 Hz, 1H), 6.76 (s, 1H). ESI-MS m/z 442.0 [MH] - .
实施例25Example 25
N-(6-(噻吩磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物25)N-(6-(thiophenesulfonamido)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 25)
Figure PCTCN2021078798-appb-000040
Figure PCTCN2021078798-appb-000040
参照实施例23的方法，将对硝基苯甲酰氯替换成2-噻吩磺酰氯，制得化合物25(淡黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 23, p-nitrobenzoyl chloride was replaced with 2-thiophenesulfonyl chloride to obtain compound 25 (light yellow solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.81(s, 1H) ), 10.46 (s, 1H), 8.04 (d, J = 1.6 Hz, 1H), 7.87 (dd, J = 5.0, 1.4 Hz, 1H), 7.79-7.59 (m, 3H), 7.53 (dd, J = 3.8, 1.4 Hz, 1H), 7.20 (dd, J = 8.7, 2.2 Hz, 1H), 7.10 (dd, J = 5.0, 3.8 Hz, 1H), 6.75 (dd, J = 3.6, 1.7 Hz, 1H). ESI-MS m/z 404.0[MH] - .
实施例26Example 26
N-(6-((4-甲磺酰基苯基)磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物26)N-(6-((4-Methanesulfonylphenyl)sulfonamido)benzo(d)thiazol-2-yl)furan-2-carboxamide (Compound 26)
Figure PCTCN2021078798-appb-000041
Figure PCTCN2021078798-appb-000041
参照实施例23的方法，将对硝基苯甲酰氯替换成对甲磺酰基苯磺酰氯，制得化合物26(淡黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 23, p-nitrobenzoyl chloride was replaced with p-methanesulfonylbenzenesulfonyl chloride to obtain compound 26 (light yellow solid): 1 H NMR (300MHz, DMSO-d 6 )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] - .
实施例27Example 27
(±)-N-(2-(四氢呋喃-2-甲酰胺)苯并[d]噻唑-6-基)烟酰胺(化合物27)(±)-N-(2-(Tetrahydrofuran-2-carboxamide)benzo(d)thiazol-6-yl)nicotinamide (Compound 27)
Figure PCTCN2021078798-appb-000042
Figure PCTCN2021078798-appb-000042
将化合物1a(100mg,0.51mmol)溶于四氢呋喃(3mL)溶液中，依次加入2-四氢呋喃甲酸27a(71mg,0.61mmol)、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,232mg,0.61mmol)以及N,N-二异丙基乙胺(134μL,0.77mmol)，40℃下搅拌6小时。TLC监测反应完全后停止搅拌，减压蒸除溶剂后加乙酸乙酯(3mL)打浆，抽滤得化合物27b(白色固体,154mg,产率97％)： 1H NMR(300MHz,DMSO-d 6)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 1a (100mg, 0.51mmol) was dissolved in tetrahydrofuran (3mL) solution, and 2-tetrahydrofurancarboxylic acid 27a (71mg, 0.61mmol), 2-(7-azabenzotriazole)-N,N, N',N'-tetramethylurea hexafluorophosphate (HATU, 232mg, 0.61mmol) and N,N-diisopropylethylamine (134μL, 0.77mmol) were stirred at 40°C for 6 hours. After the reaction was monitored by TLC, the stirring was stopped. After the solvent was evaporated under reduced pressure, ethyl acetate (3mL) was added to make a slurry. The compound 27b (white solid, 154mg, yield 97%) was obtained by suction filtration: 1 H NMR (300MHz, DMSO-d 6 )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 = 7.2 Hz, 1H), 3.85 (d, J = 7.2 Hz, 1H), 2.35-2.20 (m, 1H), 2.17-1.77 (m, 3H).
将化合物27b(250mg,0.85mmol)溶解在甲醇(25mL)中，加入10％钯/碳(25mg)，通入氢气后50℃下搅拌9小时。TLC监测反应完全后停止加热，冷却至室温后溶液用硅藻土抽滤，滤液减压蒸除溶剂后制砂，硅胶柱层析(二氯甲烷：甲醇＝200：1)即得到化合物27c(灰色固体,100mg,产率43％)： 1H NMR(300MHz,DMSO-d 6)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 27b (250 mg, 0.85 mmol) was dissolved in methanol (25 mL), 10% palladium/carbon (25 mg) was added, hydrogen gas was introduced, and the mixture was stirred at 50° C. for 9 hours. After the reaction was monitored by TLC, the heating was stopped. After cooling to room temperature, the solution was filtered with diatomaceous earth. The filtrate was evaporated to remove the solvent under reduced pressure, and then sand was prepared. Gray solid, 100mg, yield 43%): 1 H NMR (300MHz, DMSO-d 6 ) δ 11.67 (s, 1H), 7.33 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 2.2 Hz, 1H), 6.62 (dd, J = 8.6, 2.3 Hz, 1H), 5.09 (s, 2H), 4.44 (dd, J = 8.2, 5.1 Hz, 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).
将化合物27c(25mg,0.10mmol)加入到二氯甲烷(2mL)中，依次加入烟酸(14mg,0.11mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(24mg,0.11mmol)、4-二甲氨基吡啶(14mg,0.11mmol)，室温下搅拌过夜。TLC监测反应完全后停止搅拌，加入二氯甲烷(5mL)稀释，饱和食盐水(3mL)洗一次，有机相用无水硫酸钠干燥后，过滤，制砂，硅胶柱层析(二氯甲烷:甲醇＝200:1)得化合物27(白色固体，31mg，产率84％)： 1H NMR(300MHz,DMSO-d 6)δ11.89(s,1H),10.51(s,1H),9.05(d,J＝2.2 Hz,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] -。 Compound 27c (25mg, 0.10mmol) was added to dichloromethane (2mL), niacin (14mg, 0.11mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added in sequence Hydrochloride (24 mg, 0.11 mmol) and 4-dimethylaminopyridine (14 mg, 0.11 mmol) were stirred overnight at room temperature. After the reaction was monitored by TLC, the stirring was stopped, diluted with dichloromethane (5mL), washed with saturated brine (3mL), the organic phase was dried with anhydrous sodium sulfate, filtered, sand-making, silica gel column chromatography (dichloromethane: Methanol=200:1) to obtain compound 27 (white solid, 31mg, yield 84%): 1 H NMR (300MHz, DMSO-d 6 ) δ 11.89 (s, 1H), 10.51 (s, 1H), 9.05 ( d, J = 2.2 Hz, 1H), 8.69 (dd, J = 4.9, 1.6 Hz, 1H), 8.38 (s, 1H), 8.23 (dt, J = 8.1, 1.9 Hz, 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[MH] - .
实施例28Example 28
(R)-N-(2-(四氢呋喃-2-甲酰胺)苯并[d]噻唑-6-基)烟酰胺(化合物28)(R)-N-(2-(Tetrahydrofuran-2-carboxamide)benzo(d)thiazol-6-yl)nicotinamide (Compound 28)
Figure PCTCN2021078798-appb-000043
Figure PCTCN2021078798-appb-000043
参照实施例27的方法，将2-四氢呋喃甲酸替换成(R)-2-四氢呋喃甲酸，制得化合物28(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 27, 2-tetrahydrofurancarboxylic acid was replaced with (R)-2-tetrahydrofurancarboxylic acid to obtain compound 28 (white solid): 1 H NMR (300MHz, DMSO-d 6 )δ12.04(s, 1H) ), 10.49 (s, 1H), 9.04 (d, J = 2.2 Hz, 1H), 8.68 (dd, J = 5.0, 1.6 Hz, 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[MH] - .
实施例29Example 29
(S)-N-(2-(四氢呋喃-2-甲酰胺)苯并[d]噻唑-6-基)烟酰胺(化合物29)(S)-N-(2-(Tetrahydrofuran-2-carboxamide)benzo(d)thiazol-6-yl)nicotinamide (Compound 29)
Figure PCTCN2021078798-appb-000044
Figure PCTCN2021078798-appb-000044
参照实施例27的方法，将2-四氢呋喃甲酸替换成(S)-2-四氢呋喃甲酸，制得化合物29(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 27, 2-tetrahydrofurancarboxylic acid was replaced with (S)-2-tetrahydrofurancarboxylic acid to obtain compound 29 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.05(s, 1H) ), 10.50 (s, 1H), 9.05 (d, J = 2.3 Hz, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 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 [MH] - .
实施例30Example 30
(±)-N-(6-(4-硝基苯甲酰)苯并[d]噻唑-2-基)四氢呋喃-2-甲酰胺(化合物30)(±)-N-(6-(4-Nitrobenzoyl)benzo(d)thiazol-2-yl)tetrahydrofuran-2-carboxamide (Compound 30)
Figure PCTCN2021078798-appb-000045
Figure PCTCN2021078798-appb-000045
参照实施例24的方法，将24a替换成对硝基苯磺酰氯，将1c替换成27c，制得化合物30(黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Refer to the method of Example 24, replacing 24a with p-nitrobenzenesulfonyl chloride and 1c with 27c to obtain compound 30 (yellow solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.16(s, 1H), 10.63 (s, 1H), 8.35 (d, J = 8.9 Hz, 2H), 7.97 (d, J = 9.0 Hz, 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 [MH] - .
实施例31Example 31
(±)-N-(6-(4-甲基苯甲酰)苯并[d]噻唑-2-基)四氢呋喃-2-甲酰胺(化合物31)(±)-N-(6-(4-Methylbenzoyl)benzo(d)thiazol-2-yl)tetrahydrofuran-2-carboxamide (Compound 31)
Figure PCTCN2021078798-appb-000046
Figure PCTCN2021078798-appb-000046
参照实施例24的方法，将24a替换成对甲基苯磺酰氯，将1c替换成27c，制得化合物31(黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Refer to the method of Example 24, replacing 24a with p-toluenesulfonyl chloride and 1c with 27c to obtain compound 31 (yellow solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.09(s, 1H), 10.22 (s, 1H), 7.84–7.50 (m, 4H), 7.29 (d, J = 8.0 Hz, 2H), 7.11 (dd, J = 8.6, 2.2 Hz, 1H), 4.59–4.44 (m ESI-MS m/z 416.1[MH] - .
实施例32Example 32
(±)-N-(6-噻吩-2-磺酰基苯并[d]噻唑-2-基)四氢呋喃-2-甲酰胺(化合物32)(±)-N-(6-thiophene-2-sulfonylbenzo(d)thiazol-2-yl)tetrahydrofuran-2-carboxamide (Compound 32)
Figure PCTCN2021078798-appb-000047
Figure PCTCN2021078798-appb-000047
参照实施例24的方法，将24a替换成2-噻吩磺酰氯，将1c替换成27c，制得化合物32(黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 24, 24a was replaced with 2-thiophenesulfonyl chloride, and 1c was replaced with 27c, to obtain compound 32 (yellow solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.15(s, 1H) ), 10.45 (s, 1H), 7.88 (dd, J = 5.0, 1.4 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H), 7.65 (d, J = 8.6 Hz, 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 ] + .
实施例33Example 33
N-(6-(4-甲基苯磺酰胺基)苯并[d]噻唑-2基)-5-(吗啉基甲基)呋喃-2-甲酰胺(化合物33)N-(6-(4-Methylbenzenesulfonamido)benzo[d]thiazol-2yl)-5-(morpholinylmethyl)furan-2-carboxamide (Compound 33)
Figure PCTCN2021078798-appb-000048
Figure PCTCN2021078798-appb-000048
Figure PCTCN2021078798-appb-000049
Figure PCTCN2021078798-appb-000049
将2-氨基-6-硝基苯并噻唑(33a,200mg,1.03mmol)加入到二氯甲烷(20mL)中(混悬液)，依次加入Boc酸酐(305mg,1.23mmol)和4-二甲氨基吡啶(150mg,1.23mmol)，室温搅拌6小时。TLC监测反应完全后停止搅拌，抽滤，滤饼用二氯甲烷(2mL)和甲醇(1mL)洗，烘干得化合物33b(白色固体,245mg,产率8％)。Add 2-amino-6-nitrobenzothiazole (33a, 200mg, 1.03mmol) to dichloromethane (20mL) (suspension), then add Boc anhydride (305mg, 1.23mmol) and 4-dimethyl Aminopyridine (150 mg, 1.23 mmol) was stirred at room temperature for 6 hours. After the reaction was monitored by TLC, the stirring was stopped, filtered with suction, the filter cake was washed with dichloromethane (2 mL) and methanol (1 mL), and dried to obtain compound 33b (white solid, 245 mg, yield 8%).
将化合物33b(245mg,0.83mmol)置于50mL茄形瓶中，加入甲醇(15mL)后加入10％钯/碳(25mg)，通入氢气后室温搅拌3小时。TLC监测反应完全后停止搅拌，反应混合物经硅藻土抽滤，滤液减压蒸除溶剂，得化合物33c(白色固体,201mg,产率91％)。Compound 33b (245 mg, 0.83 mmol) was placed in a 50 mL eggplant-shaped flask, methanol (15 mL) was added, and then 10% palladium/carbon (25 mg) was added, and hydrogen was bubbled in and stirred at room temperature for 3 hours. After the reaction was monitored by TLC, the stirring was stopped, the reaction mixture was filtered through diatomaceous earth, and the filtrate was evaporated under reduced pressure to remove the solvent to obtain compound 33c (white solid, 201 mg, yield 91%).
将化合物33c(100mg,0.38mmol)溶于吡啶(5mL)中，冰浴下分批加入对甲基苯磺酰氯(80mg,0.42mmol)，自然升温至室温，搅拌过夜，大量固体析出。TLC监测反应完全后向反应体系内滴加1N盐酸至中性，抽滤，滤饼用少量乙酸乙酯洗，烘干得到33d的粗品。Compound 33c (100 mg, 0.38 mmol) was dissolved in pyridine (5 mL), p-toluenesulfonyl chloride (80 mg, 0.42 mmol) was added in batches under ice bath, the temperature was naturally raised to room temperature, and the mixture was stirred overnight. A large amount of solids precipitated. After the reaction was monitored by TLC, 1N hydrochloric acid was added dropwise to the reaction system until it was neutral, filtered with suction, the filter cake was washed with a small amount of ethyl acetate, and dried to obtain a 33d crude product.
将所得全部粗品33d溶解在二氯甲烷(4mL)中，滴加三氟醋酸(2mL)，室温搅拌0.5小时，TLC监测反应完全后向反应体系中滴加饱和碳酸氢钠溶液至中性，抽滤，滤饼用水洗和少量乙酸乙酯(1mL)洗后得化合物33e(白色固体,77mg,两步产率64％)： 1H NMR(300MHz,DMSO-d 6)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] -。 Dissolve all the crude product 33d obtained in dichloromethane (4mL), add trifluoroacetic acid (2mL) dropwise, stir at room temperature for 0.5 hours, TLC monitor the reaction is complete, add saturated sodium bicarbonate solution to the reaction system until neutral, pump After filtration, the filter cake was washed with water and a small amount of ethyl acetate (1 mL) to obtain compound 33e (white solid, 77 mg, two-step yield 64%): 1 H NMR (300MHz, DMSO-d 6 ) 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[MH] - .
将5-氯甲基-2-呋喃甲酸乙酯(33f,200mg,1.06mmol)溶解在乙腈(10mL)中，依次加入吗啉(55mg,1.27mmol)、碘化钾(176mg,1.27mmol)和碳酸钾(212mg,1.27mmol)，室温下搅拌7小时。TLC监测反应完全后减压蒸除溶剂，制砂，硅胶柱层析(用石油醚：乙酸乙酯＝10：1洗脱小极性杂质后更换洗脱剂为二氯甲烷：甲醇＝40：1)得到化合物33g(白色固体,260mg,产率99％)。Ethyl 5-chloromethyl-2-furancarboxylate (33f, 200mg, 1.06mmol) was dissolved in acetonitrile (10mL), followed by morpholine (55mg, 1.27mmol), potassium iodide (176mg, 1.27mmol) and potassium carbonate (212mg, 1.27mmol), stirred at room temperature for 7 hours. After the reaction was monitored by TLC, the solvent was evaporated under reduced pressure, sand was prepared, and silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 was used to elute small polar impurities, and the eluent was changed to dichloromethane: methanol = 40: 1) Obtain compound 33g (white solid, 260mg, yield 99%).
将所得化合物33g(260mg,1.06mmol)溶解在甲醇(10mL)中，加入氢氧化钠固体(87mg,2.17mmol)，滴加2滴水，40℃下搅拌10小时，TLC监测反应完全后，滴加浓盐酸至溶液pH＝4-6，蒸除溶剂，所得固体用二氯甲烷/甲醇的混合溶液(二氯甲烷:甲醇＝3:1)溶解，抽滤，滤液蒸除溶剂后得化合物33h(浅棕色固体,220mg,产率94％)。The obtained compound 33g (260mg, 1.06mmol) was dissolved in methanol (10mL), sodium hydroxide solid (87mg, 2.17mmol) was added, 2 drops of water were added dropwise, and the mixture was stirred at 40°C for 10 hours. After the reaction was monitored by TLC, the reaction was completed dropwise. Concentrated hydrochloric acid to the solution pH = 4-6, evaporate the solvent, the obtained solid was dissolved in a dichloromethane/methanol mixed solution (dichloromethane: methanol = 3:1), filtered with suction, and the filtrate was evaporated to remove the solvent to obtain compound 33h ( Light brown solid, 220mg, yield 94%).
将化合物33e(40mg,0.13mmol)溶于四氢呋喃(2mL)中，依次加入化合物33h(37mg,0.15mmol)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,57mg,0.15mmol)以及N,N-二异丙基乙胺(48μL,0.15mmol)，40℃下加热48小时。TLC监测反应完全后停止搅拌，减压蒸去溶剂后用甲醇溶解，制砂，硅胶柱层析(二氯甲烷:甲醇＝100:1)得化合物33(白色固体,25mg,产率38％)： 1H NMR(300MHz,DMSO-d 6)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] -。 Compound 33e (40mg, 0.13mmol) was dissolved in tetrahydrofuran (2mL), and compound 33h (37mg, 0.15mmol), 2-(7-azabenzotriazole)-N,N,N',N was added in sequence '-Tetramethylurea hexafluorophosphate (HATU, 57mg, 0.15mmol) and N,N-diisopropylethylamine (48μL, 0.15mmol), heated at 40°C for 48 hours. After the reaction was monitored by TLC, the stirring was stopped, the solvent was evaporated under reduced pressure and dissolved in methanol, sand was prepared, and silica gel column chromatography (dichloromethane:methanol=100:1) was used to obtain compound 33 (white solid, 25mg, yield 38%) : 1 H NMR (300MHz, DMSO-d 6 ) δ 12.73 (s, 1H), 10.24 (s, 1H), 7.75-7.55 (m, 5H), 7.32 (d, J = 7.8 Hz, 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 [MH] - .
实施例34Example 34
5-(吗啉基甲基)-N-(6-噻吩-2-磺酰胺基苯并[d]噻唑-2-基)-呋喃-2-甲酰胺(化合物34)5-(Morpholinylmethyl)-N-(6-thiophen-2-sulfonamidobenzo[d]thiazol-2-yl)-furan-2-carboxamide (Compound 34)
Figure PCTCN2021078798-appb-000050
Figure PCTCN2021078798-appb-000050
参照实施例33的方法，将对甲基苯磺酰氯替换成2-噻吩磺酰氯，制得化合物34(淡黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 33, p-toluenesulfonyl chloride was replaced with 2-thiophenesulfonyl chloride to obtain compound 34 (light yellow solid): 1 H NMR (300MHz, DMSO-d 6 ) 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] + .
实施例35Example 35
5-羟甲基-N-(6-(4-甲基苯磺酰基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物35)5-hydroxymethyl-N-(6-(4-methylbenzenesulfonyl)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 35)
Figure PCTCN2021078798-appb-000051
Figure PCTCN2021078798-appb-000051
参照实施例33的方法，将33f替换成5-羟甲基-2-呋喃甲酸，制得化合物35(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Refer to the method of Example 33, and replace 33f with 5-hydroxymethyl-2-furancarboxylic acid to obtain compound 35 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.67 (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=5.9 Hz, 1H), 4.50 (d, J=5.8 Hz, 2H), 2.32 (s, 3H). ESI-MS m/z 442.1[MH] - .
实施例36Example 36
5-羟甲基-N-(6-噻吩-2-磺酰基苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物36)5-hydroxymethyl-N-(6-thiophene-2-sulfonylbenzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 36)
Figure PCTCN2021078798-appb-000052
Figure PCTCN2021078798-appb-000052
参照实施例33的方法，将对甲基苯磺酰氯替换成2-噻吩磺酰氯，将33f替换成5-羟甲基-2-呋喃甲酸，制得化合物36(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 33, p-toluenesulfonyl chloride was replaced with 2-thiophenesulfonyl chloride, and 33f was replaced with 5-hydroxymethyl-2-furancarboxylic acid to obtain compound 36 (white solid): 1 H NMR( 300MHz, DMSO-d 6 )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 = 3.5 Hz, 1H), 5.47 (s, 1H), 4.49 (d, J = 3.8 Hz, 2H). ESI-MS m/z 434.0 [MH] - .
实施例37Example 37
N-(4-羟基-6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物37)N-(4-hydroxy-6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 37)
Figure PCTCN2021078798-appb-000053
Figure PCTCN2021078798-appb-000053
将2-氨基-5-硝基苯酚(37a,5.00g,32.40mmol)溶于N,N-二甲基甲酰胺(50mL)中，冰浴下滴加溴苄(3.65mL,30.78mmol)，加毕后室温搅拌过夜。TLC监测反应完全后，向反应体系中加入水(400mL)稀释，观察到大量固体析出，抽滤，烘干。所得固体用石油醚/乙酸乙酯混合溶液(石油醚:乙酸乙酯＝1:1,160mL)重结晶，得到橙 黄色结晶(37b,2.08g)。将重结晶后的母液减压蒸除溶剂，向所得固体中加入乙酸乙酯(30mL)打浆，抽滤得黄色固体(37b,2.06g)，与重结晶得到的固体合并投入下一步。2-Amino-5-nitrophenol (37a, 5.00g, 32.40mmol) was dissolved in N,N-dimethylformamide (50mL), and benzyl bromide (3.65mL, 30.78mmol) was added dropwise under ice bath, After the addition, stir at room temperature overnight. After the completion of the reaction monitored by TLC, water (400 mL) was added to the reaction system for dilution, a large amount of solids were observed to precipitate, filtered off with suction, and dried. The obtained solid was recrystallized from a petroleum ether/ethyl acetate mixed solution (petroleum ether: ethyl acetate=1:1, 160 mL) to obtain orange-yellow crystals (37b, 2.08 g). The mother liquor after recrystallization was evaporated to remove the solvent under reduced pressure, ethyl acetate (30 mL) was added to the obtained solid to make a slurry, and a yellow solid (37b, 2.06 g) was obtained by suction filtration, which was combined with the solid obtained by recrystallization and put into the next step.
将所得部分粗品37b(2.08g,8.52mmol)溶于冰醋酸(20mL)中，冰盐浴下加入硫氰酸钾(3.31g,34.06mmol)，搅拌10分钟后，缓慢滴加液溴(873μL,17.03mmol)，冰盐浴下搅拌2小时后缓慢升至室温，继续搅拌7天，有固体析出。TLC监测反应完毕后，加入乙酸乙酯(150mL)稀释，抽滤，收集滤液，蒸除溶剂。向所得固体中加入乙酸乙酯(100mL)使之溶解，向其中滴加15％的氢氧化钠溶液调节pH至6-7，再用饱和碳酸氢钠溶液调节pH至7-8。减压蒸除乙酸乙酯后抽滤，收集滤饼，烘干。滤液用乙酸乙酯萃取(80mL×3)，乙酸乙酯层经饱和食盐水洗后加入无水硫酸钠干燥0.5小时，过滤除去无水硫酸钠后减压蒸除溶剂，所得固体与烘干的滤饼合并得到化合物37c(黄色固体,1.23g,两步收率27％)： 1H NMR(300MHz,DMSO-d 6)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)。 The obtained part of crude product 37b (2.08g, 8.52mmol) was dissolved in glacial acetic acid (20mL), potassium thiocyanate (3.31g, 34.06mmol) was added under an ice-salt bath, and after stirring for 10 minutes, liquid bromine (873μL) was slowly added dropwise. , 17.03mmol), stirred for 2 hours in an ice-salt bath, slowly warmed to room temperature, continued stirring for 7 days, and solids precipitated. After the reaction was monitored by TLC, ethyl acetate (150 mL) was added for dilution, filtered with suction, the filtrate was collected, and the solvent was evaporated. Ethyl acetate (100 mL) was added to the obtained solid to dissolve it, 15% sodium hydroxide solution was added dropwise to adjust the pH to 6-7, and then saturated sodium bicarbonate solution was used to adjust the pH to 7-8. The ethyl acetate was evaporated under reduced pressure and then filtered with suction, the filter cake was collected and dried. The filtrate was extracted with ethyl acetate (80mL×3). The ethyl acetate layer was washed with saturated brine and dried with anhydrous sodium sulfate for 0.5 hours. The anhydrous sodium sulfate was removed by filtration and the solvent was evaporated under reduced pressure. Combine the cakes to obtain compound 37c (yellow solid, 1.23g, two-step yield 27%): 1 H NMR (300MHz, DMSO-d 6 ) δ 8.39 (d, J = 2.3 Hz, 1H), 8.21 (s, 2H) ), 7.75 (d, J = 2.3 Hz, 1H), 7.50 (d, J = 7.5 Hz, 2H), 7.31-7.47 (m, 3H), 5.30 (s, 2H).
将化合物37c(660mg,2.19mmol)溶解在吡啶(15mL)中，室温滴加新制的呋喃-2-甲酰氯(282μL,2.85mmol)，60℃下搅拌5小时后室温反应过夜，TLC监测反应完成后，加入2N盐酸溶液调节pH至5-6，观察到大量灰色固体析出。抽滤，滤饼烘干后制砂，硅胶柱层析(二氯甲烷:甲醇＝1000:1)得化合物37d(淡黄色固体，737mg,产率85％)： 1H NMR(300MHz,DMSO-d 6)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 37c (660mg, 2.19mmol) was dissolved in pyridine (15mL), freshly prepared furan-2-formyl chloride (282μL, 2.85mmol) was added dropwise at room temperature, stirred at 60°C for 5 hours, and reacted at room temperature overnight. TLC monitored the completion of the reaction Afterwards, 2N hydrochloric acid solution was added to adjust the pH to 5-6, and a large amount of gray solid was observed to precipitate. Suction filtration, drying the filter cake and making sand, silica gel column chromatography (dichloromethane:methanol=1000:1) to obtain compound 37d (light yellow solid, 737mg, yield 85%): 1 H NMR (300MHz, DMSO- d 6 )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, 1.8 Hz, 1H), 5.44 (s, 2H).
将化合物37d(512mg,1.29mmol)溶解在95％乙醇(30mL)中，室温下加入二水合氯化亚锡(877mg,3.88mmol)，加毕后升温至80℃搅拌14小时后室温反应过夜，之后再升温至80℃搅拌10小时。TLC监测反应完全后停止加热，冷却至室温后蒸除溶剂，向所得残留物中加入饱和碳酸氢钠溶液(20mL)，大量白色固体析出。抽滤后将滤饼置于四氢呋喃中(混悬液)，氮气保护下搅拌0.5小时，抽滤，收集滤液后减压蒸除溶剂得化合物37e(土黄色固体,300mg,产率59％)： 1H NMR(300MHz,DMSO-d 6)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)。 Compound 37d (512mg, 1.29mmol) was dissolved in 95% ethanol (30mL), and stannous chloride dihydrate (877mg, 3.88mmol) was added at room temperature. After the addition, the temperature was raised to 80°C and stirred for 14 hours and then reacted at room temperature overnight. After that, the temperature was raised to 80°C and stirred for 10 hours. The heating was stopped after the completion of the reaction monitored by TLC, the solvent was evaporated after cooling to room temperature, saturated sodium bicarbonate solution (20 mL) was added to the residue obtained, and a large amount of white solid was precipitated. After suction filtration, the filter cake was placed in tetrahydrofuran (suspension), stirred for 0.5 hours under nitrogen protection, and filtered with suction. After collecting the filtrate, the solvent was evaporated under reduced pressure to obtain compound 37e (yellow solid, 300 mg, yield 59%): 1 H NMR(300MHz,DMSO-d 6 )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=2.0 Hz, 1H), 5.31 (s, 2H).
将2-噻吩磺酰氯(28mg,0.15mmol)溶于吡啶(1mL)中，冰浴氮气保护下滴入化合物37e(50mg,0.28mmol)的吡啶(1mL)溶液中，冰浴下搅拌2小时。TLC监测反应完全后加入乙酸乙酯(20mL)，1N盐酸洗(20mL×3)，有机层加入无水硫酸钠干燥，过滤，蒸除溶剂后得化合物37f(淡紫色固体,72mg,产率97％)： 1H NMR(300MHz,DMSO-d 6)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)。 Dissolve 2-thiophenesulfonyl chloride (28 mg, 0.15 mmol) in pyridine (1 mL), drop it into the pyridine (1 mL) solution of compound 37e (50 mg, 0.28 mmol) under the protection of nitrogen in an ice bath, and stir for 2 hours in an ice bath. After the reaction was monitored by TLC, ethyl acetate (20mL) was added, washed with 1N hydrochloric acid (20mL×3), the organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain compound 37f (lavender solid, 72mg, yield 97 %): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.89 (s, 1H), 10.47 (s, 1H), 8.03 (s, 1H), 7.89 (d, J = 5.0 Hz, 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).
将化合物37f(72mg,0.14mmol)以及五甲基苯(146mg,1.40mmol)置于茄形瓶中，加入二氯甲烷(5mL)，搅拌使之成混悬液，降温至-78℃后缓慢滴加1M的三氯化硼二氯甲烷溶液(369μL,0.36mmol)，保持-78℃搅拌1小时。TLC监测反应完全后滴加甲醇(5滴)淬灭反应，自然升温至室温后搅拌1小时。向反应溶液中补加二氯甲烷(3mL)稀释后将反应液转移至15mL离心管中，2500rpm离心5分钟，弃去上清后补加二氯甲烷(3mL)，继续2500rpm离心5分钟。重复4次后得化合物37(白色固体，56mg，产率93％)： 1H NMR(300MHz,DMSO-d 6)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] -。 Put compound 37f (72mg, 0.14mmol) and pentamethylbenzene (146mg, 1.40mmol) in an eggplant-shaped flask, add dichloromethane (5mL), stir to make a suspension, cool to -78℃ and then slowly 1M boron trichloride dichloromethane solution (369 μL, 0.36 mmol) was added dropwise, and the mixture was stirred at -78°C for 1 hour. After the completion of the reaction was monitored by TLC, methanol (5 drops) was added dropwise to quench the reaction, the temperature was naturally raised to room temperature and then stirred for 1 hour. Add dichloromethane (3mL) to the reaction solution to dilute, transfer the reaction solution to a 15mL centrifuge tube, centrifuge at 2500rpm for 5 minutes, discard the supernatant, add dichloromethane (3mL), and continue centrifugation at 2500rpm for 5 minutes. After repeating 4 times, compound 37 (white solid, 56 mg, yield 93%) was obtained: 1 H NMR (300MHz, DMSO-d 6 ) δ 12.82 (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 = 9.0 Hz, 2H), 6.76 (d, J = 12.8 Hz,2H).ESI-MS m/z 420.0[MH] - .
实施例38Example 38
N-(4-羟基-6-(4-甲基苯磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物38)N-(4-hydroxy-6-(4-methylbenzenesulfonamido)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 38)
Figure PCTCN2021078798-appb-000054
Figure PCTCN2021078798-appb-000054
参照实施例37的方法，将2-噻吩磺酰氯替换成对甲基苯磺酰氯，制得化合物38(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 37, 2-thiophenesulfonyl chloride was replaced with p-toluenesulfonyl chloride to obtain compound 38 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.72(s, 1H) , 10.44 (s, 1H), 7.86 (dd, J = 4.9, 1.4 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H), 7.64 (d, J = 10.4 Hz, 2H), 7.52 (dd, J = 3.8, 1.4 Hz, 1H), 7.18 (dd, J = 8.7, 2.2 Hz, 1H), 7.09 (dd, J = 5.0, 3.7 Hz, 1H), 6.55 (d, J = 3.5 Hz, 1H), 5.47 (s, 1H), 4.49 (d, J=3.8 Hz, 2H). ESI-MS m/z 434.0 [MH] - .
实施例39Example 39
N-(2-(呋喃-2-甲酰胺)-4-吗啉基苯并[d]噻唑-6-基)烟酰胺(化合物39)N-(2-(furan-2-carboxamide)-4-morpholinobenzo[d]thiazol-6-yl)nicotinamide (compound 39)
Figure PCTCN2021078798-appb-000055
Figure PCTCN2021078798-appb-000055
Figure PCTCN2021078798-appb-000056
Figure PCTCN2021078798-appb-000056
将2-氟-4-硝基苯甲酸(39a,10.00g,54.00mmol)溶于二氯甲烷(40mL)中，冰浴下滴加草酰氯(40mL,65.00mmol)，滴加3滴N,N-二甲基甲酰胺作催化剂后室温搅拌3小时。TLC监测反应完全后减压蒸除溶剂，得到酰氯中间体。将该中间体溶于乙腈(20mL)溶液中，冰浴下加入氨水溶液(30mL)，自然升温至室温，室温下搅拌2小时。TLC监测反应完全后停止搅拌，减压蒸除溶剂，所得固体用少量乙酸乙酯(5mL)洗，抽滤，烘干后得到化合物39b(黄色固体,9.60g,产率96％)。Dissolve 2-fluoro-4-nitrobenzoic acid (39a, 10.00g, 54.00mmol) in dichloromethane (40mL), add oxalyl chloride (40mL, 65.00mmol) dropwise under ice bath, and add 3 drops of N, After N-dimethylformamide was used as a catalyst, the mixture was stirred at room temperature for 3 hours. After the completion of the reaction was monitored by TLC, the solvent was evaporated under reduced pressure to obtain the acid chloride intermediate. The intermediate was dissolved in an acetonitrile (20 mL) solution, an aqueous ammonia solution (30 mL) was added under an ice bath, the temperature was naturally raised to room temperature, and the mixture was stirred at room temperature for 2 hours. After the reaction was monitored by TLC, the stirring was stopped, and the solvent was evaporated under reduced pressure. The obtained solid was washed with a small amount of ethyl acetate (5 mL), filtered with suction, and dried to obtain compound 39b (yellow solid, 9.60 g, yield 96%).
将化合物39b(3.20g,25.42mmol)溶于DMSO溶液(25mL)中，依次加入吗啉(4.42mL,50.84mmol)以及碳酸钾(5.27g,38.18mmol)，加毕后升温至90℃搅拌4小时。TLC监测反应完全后停止加热。冷却至室温后将反应液倒入冰水(50mL)中，加入1N盐酸调节pH至中性，抽滤，收集滤饼烘干，滤液用乙酸乙酯萃取(20mL×3)，有机相加入无水硫酸钠干燥，抽滤，蒸除滤液溶剂后所得固体与之前所烘滤饼合并，得到化合物39c(黄色固体,3.98g,产率63％)： 1H NMR(300MHz,DMSO-d 6)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 39b (3.20g, 25.42mmol) was dissolved in DMSO solution (25mL), and morpholine (4.42mL, 50.84mmol) and potassium carbonate (5.27g, 38.18mmol) were added in sequence. After the addition, the temperature was raised to 90°C and stirred. 4 Hour. The heating was stopped after the completion of the reaction as monitored by TLC. 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, filtered with suction, the filter cake was collected and dried, the filtrate was extracted with ethyl acetate (20mL×3), and the organic phase was added without After drying with sodium sulfate and suction filtration, the solid obtained after evaporating the solvent of the filtrate was combined with the previously baked filter cake to obtain compound 39c (yellow solid, 3.98 g, yield 63%): 1 H NMR (300MHz, DMSO-d 6 ) δ 7.98 (s, 1H), 7.77 (dd, J = 8.3, 2.3 Hz, 1H), 7.75-7.47 (m, 3H), 3.94-3.47 (m, 4H), 3.15-2.75 (m, 4H).
将化合物39c(1.78g,7.10mmol)溶于二氧六环(80mL)中，冰浴下加入氢氧化钠固体(1.70g,42.60mmol)，向体系内加入水(40mL)，冰浴下搅拌30分钟。之后，向体系内滴加5％次氯酸钠溶液(29mL,42.60mmol)，室温搅拌过夜。TLC监测反应完毕后停止搅拌，减压蒸除溶剂后加水(10mL)，抽滤得化合物39d(白色固体,1.44g,产率91％)： 1H NMR(500MHz,DMSO-d 6)δ7.84(dd,J＝8.9,2.6Hz,1H),7.75(d,J＝2.6 Hz,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)。 Dissolve compound 39c (1.78g, 7.10mmol) in dioxane (80mL), add solid sodium hydroxide (1.70g, 42.60mmol) under ice bath, add water (40mL) to the system, and stir under ice bath 30 minutes. After that, 5% sodium hypochlorite solution (29 mL, 42.60 mmol) was added dropwise to the system, and the mixture was stirred at room temperature overnight. After the reaction was monitored by TLC, the stirring was stopped, the solvent was evaporated under reduced pressure and water (10mL) was added, and the compound 39d (white solid, 1.44g, yield 91%) was obtained by suction filtration: 1 H NMR (500MHz, DMSO-d 6 )δ7. 84 (dd, J = 8.9, 2.6 Hz, 1H), 7.75 (d, J = 2.6 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H), 6.39 (s, 2H), 3.81 (t, J = 4.5 Hz, 4H), 2.85 (t, J = 4.4 Hz, 4H).
参照实施例37的方法，将37b替换成39d，制得化合物39e(棕色固体)： 1H NMR(300MHz,DMSO-d 6)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)。 According to the method of Example 37, 37b was replaced with 39d to obtain compound 39e (brown solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.30 (s, 1H), 8.01 (d, J = 1.7 Hz) ,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).
参照实施例1的方法，将1c替换成39e，将异烟酸替换成烟酸，制得化合物39(淡黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Refer to the method of Example 1, replacing 1c with 39e and isonicotinic acid with niacin to obtain compound 39 (light yellow solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.66(s, 1H) ), 10.49 (s, 1H), 9.15 (s, 1H), 8.78 (d, J = 5.2 Hz, 1H), 8.33 (d, J = 8.0 Hz, 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[MH] - .
实施例40Example 40
N-(6-(2-(噻吩-2-基)乙酰氨基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物40)N-(6-(2-(Thien-2-yl)acetamido)benzo(d)thiazol-2-yl)furan-2-carboxamide (Compound 40)
Figure PCTCN2021078798-appb-000057
Figure PCTCN2021078798-appb-000057
取糠酸(40a,1.0g,8.9mmol)溶于无水二氯甲烷(15mL)中，冰浴下缓慢加入草酰氯(5mL,44.6mmol)，滴加完毕后，加入N,N-二甲基甲酰胺(1滴)，室温反应1小时。TLC检测反应完全后，减压蒸除溶剂，用无水二氯甲烷(6mL)复溶，缓慢地滴加到溶有2-氨基-6硝基苯并噻唑(1a,1.58g,8.1mmol)的无水二氯甲烷(10mL)中，室温搅拌反应13小时。反应完后反应液加入无水二氯甲烷(30mL)稀释，依次用1N的稀盐酸(50mL)、水(50mL×2)和饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，用乙酸乙酯(10mL)重结晶得到化合物1b(淡黄色固体,1.7g,产率 73％)。Dissolve furoic acid (40a, 1.0g, 8.9mmol) in anhydrous dichloromethane (15mL), slowly add oxalyl chloride (5mL, 44.6mmol) under ice bath, after the addition is complete, add N,N-dimethyl Methyl formamide (1 drop), react at room temperature for 1 hour. After the completion of the reaction was detected by TLC, the solvent was evaporated under reduced pressure, reconstituted with anhydrous dichloromethane (6mL), and slowly added dropwise to dissolve 2-amino-6nitrobenzothiazole (1a, 1.58g, 8.1mmol) In anhydrous dichloromethane (10 mL), the reaction was stirred at room temperature for 13 hours. After the reaction, the reaction solution was diluted with anhydrous dichloromethane (30mL), washed with 1N dilute hydrochloric acid (50mL), water (50mL×2) and saturated brine (50mL) successively, dried with anhydrous sodium sulfate, filtered, and the filtrate Concentrate and recrystallize with ethyl acetate (10 mL) to obtain compound 1b (light yellow solid, 1.7 g, yield 73%).
取化合物1b(600mg,2.07mmol)，悬浮于乙酸(8mL)和水(2mL)的混合溶液中，室温搅拌下缓慢加入锌粉(678mg,10.4mmol)，室温搅拌，TLC检测反应完全后，加入乙酸乙酯(40mL)稀释，加入碳酸氢钠调pH至中性，再以饱和碳酸氢钠水溶液(40mL×2)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，得化合物1c的粗品(黄色固体,420mg,产率78％)，无需纯化直接投下一步。Take compound 1b (600mg, 2.07mmol), suspend it in a mixed solution of acetic acid (8mL) and water (2mL), slowly add zinc powder (678mg, 10.4mmol) under stirring at room temperature, and stir at room temperature. After TLC detects that the reaction is complete, add Dilute with ethyl acetate (40mL), add sodium bicarbonate to adjust the pH to neutral, then wash with saturated sodium bicarbonate aqueous solution (40mL×2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain the crude product of compound 1c (yellow) Solid, 420mg, yield 78%), directly cast to the next step without purification.
取化合物1c(179mg,0.69mmol)溶于无水二氯甲烷(5mL)中，缓慢加入三乙胺(419mg,4.14mmol)和溶有2-噻吩乙酰氯(133mg,0.83mmol)的无水二氯甲烷(10mL)溶液。室温搅拌至TLC检测反应完全。加入无水二氯甲烷(20mL)稀释，以1N的稀盐酸(30mL)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，经硅胶柱层析(二氯甲烷:甲醇＝200:1)纯化，得化合物40的粗品，再以乙酸乙酯(5mL)打浆得到化合物40的纯品(灰白色固体,47mg,产率15％)： 1H NMR(300MHz,DMSO-d 6)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] -。 Take compound 1c (179mg, 0.69mmol) dissolved in dry dichloromethane (5mL), slowly add triethylamine (419mg, 4.14mmol) and 2-thiophene acetyl chloride (133mg, 0.83mmol) dissolved in anhydrous dichloromethane A solution of methyl chloride (10 mL). Stir at room temperature until TLC detects that the reaction is complete. Dilute with anhydrous dichloromethane (20mL), wash with 1N dilute hydrochloric acid (30mL) and saturated brine (30mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate, and chromatograph on silica gel column (dichloromethane: methanol =200:1) to obtain the crude product of compound 40, and then beat with ethyl acetate (5mL) to obtain the pure product of compound 40 (off-white solid, 47mg, yield 15%): 1 H NMR (300MHz, DMSO-d 6 )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[MH ] - .
实施例41Example 41
N-(6-(2-(噻吩-2-基)乙酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺盐酸盐(化合物41)N-(6-(2-(Thien-2-yl)acetamido)benzo(d)thiazol-2-yl)piperidine-4-carboxamide hydrochloride (Compound 41)
Figure PCTCN2021078798-appb-000058
Figure PCTCN2021078798-appb-000058
参照实施例40的方法，将糠酸替换成1-Boc-4-哌啶甲酸，制得化合物41的N-Boc前体化合物(墨绿色固体,140mg)。According to the method of Example 40, the furoic acid was replaced with 1-Boc-4-piperidinecarboxylic acid to prepare the N-Boc precursor compound of compound 41 (dark green solid, 140 mg).
取N-Boc前体化合物(140mg,0.28mmol)溶于四氢呋喃(5mL)，加入盐酸乙醇溶液(10mL)，室温搅拌过夜，抽滤，烘干得到化合物41(灰白色固体，100mg，产率82％)： 1H NMR(300MHz,DMSO-d 6)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] -。 The N-Boc precursor compound (140mg, 0.28mmol) was dissolved in tetrahydrofuran (5mL), added with hydrochloric acid ethanol solution (10mL), stirred overnight at room temperature, filtered with suction, and dried to obtain compound 41 (off-white solid, 100mg, yield 82%) ): 1 H NMR(300MHz,DMSO-d 6 )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 ] - .
实施例42Example 42
N-(2-(呋喃-2-羧酰胺基)苯并[d]噻唑-6-基)四氢-2H-吡喃-4-羧酰胺(化合物42)N-(2-(furan-2-carboxamide)benzo[d]thiazol-6-yl)tetrahydro-2H-pyran-4-carboxamide (Compound 42)
Figure PCTCN2021078798-appb-000059
Figure PCTCN2021078798-appb-000059
参照实施例40的方法，将糠酸替换成四氢吡喃-4-甲酸，制得化合物42(白色固体,103mg)： 1H NMR(300MHz,CDCl 3)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] +。 According to the method of Example 40, the furoic acid was replaced with tetrahydropyran-4-carboxylic acid to obtain compound 42 (white solid, 103mg): 1 H NMR (300MHz, CDCl 3 )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] + .
实施例43Example 43
N-(6-((5-氯噻吩)-2-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺盐酸盐(化合物43)N-(6-((5-Chlorothiophene)-2-sulfonamido)benzo(d)thiazol-2-yl)piperidine-4-carboxamide hydrochloride (Compound 43)
Figure PCTCN2021078798-appb-000060
Figure PCTCN2021078798-appb-000060
取1-Boc-4-哌啶甲酸(43a,2.18g,9.5mmol)溶于N,N-二甲基甲酰胺(20mL)中，依次加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,3.65g,11.85mmol)和三乙胺(1.92g,19.0mmol)，室温反应1小时。TLC检测反应完全后，加入2-氨基-6硝基苯并噻唑(1.0g,5.1mmol)，室温搅拌反应至TLC检测反应完全后，加乙酸乙酯(50mL)稀释反应液，依次用水(50mL×3)和饱和食盐水(50mL)洗涤， 无水硫酸钠干燥，过滤，滤液浓缩，经硅胶柱层析(二氯甲烷:甲醇＝100:1)纯化，得化合物43b(淡黄色固体,1.93g,产率93％)。Dissolve 1-Boc-4-piperidinecarboxylic acid (43a, 2.18g, 9.5mmol) in N,N-dimethylformamide (20mL), and add 2-(7-azabenzotriazole) successively -N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 3.65g, 11.85mmol) and triethylamine (1.92g, 19.0mmol), react at room temperature for 1 hour. After the reaction was completed by TLC, 2-amino-6nitrobenzothiazole (1.0g, 5.1mmol) was added, and the reaction was stirred at room temperature until the reaction was completed by TLC. The reaction solution was diluted with ethyl acetate (50mL), and the reaction solution was diluted with water (50mL). ×3) was washed with saturated brine (50 mL), dried with anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol = 100:1) to obtain compound 43b (light yellow solid, 1.93) g, yield 93%).
取化合物43b(150mg,0.37mmol)，悬浮于乙酸(12mL)和水(3mL)的混合溶液中，室温搅拌下缓慢加入锌粉(121mg,1.85mmol)，室温搅拌至TLC检测反应完全后，加入乙酸乙酯(30mL)稀释，加入碳酸氢钠调pH至中性，再以饱和碳酸氢钠水溶液(30mL×2)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，得化合物43c粗品(黄褐色固体,197mg,产率71％)，无需纯化直接投下一步。Take compound 43b (150mg, 0.37mmol), suspend it in a mixed solution of acetic acid (12mL) and water (3mL), slowly add zinc powder (121mg, 1.85mmol) with stirring at room temperature, and stir at room temperature until TLC detection is complete. After the reaction is complete, add Dilute with ethyl acetate (30mL), add sodium bicarbonate to adjust the pH to neutral, then wash with saturated sodium bicarbonate aqueous solution (30mL×2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain crude compound 43c (yellow brown) Solid, 197mg, yield 71%), directly cast to the next step without purification.
取化合物43c(70mg,0.19mmol)溶于二氯甲烷(4mL)和吡啶(1mL)的混合溶剂中，缓慢加入5-氯噻吩-2-磺酰氯(45mg,0.21mmol)。室温搅拌反应至TLC检测反应完全。加入乙酸乙酯(30mL)稀释，依次用1N的稀盐酸(30mL×2)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，经硅胶柱层析(二氯甲烷:甲醇＝100:1)纯化，得化合物43d(淡黄色固体,85mg,产率83％)。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 TLC detected that the reaction was complete. Add ethyl acetate (30mL) to dilute, wash with 1N dilute hydrochloric acid (30mL×2) and saturated brine (30mL) successively, dry with anhydrous sodium sulfate, filter, concentrate the filtrate, and chromatograph on silica gel column (dichloromethane: Methanol=100:1) was purified to obtain compound 43d (light yellow solid, 85mg, yield 83%).
取化合物43d(85mg,0.15mmol)溶于盐酸乙醇溶液(10mL)，室温搅拌过夜，抽滤，烘干得到化合物43(黄色固体,52mg,，产率69％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Dissolve compound 43d (85mg, 0.15mmol) in hydrochloric acid ethanol solution (10mL), stir overnight at room temperature, filter with suction, and dry to obtain compound 43 (yellow solid, 52mg, yield 69%): 1 H NMR (300MHz, DMSO) -d 6 )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] + .
实施例44Example 44
N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺盐酸盐(化合物44)N-(6-(thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)piperidine-4-carboxamide hydrochloride (compound 44)
Figure PCTCN2021078798-appb-000061
Figure PCTCN2021078798-appb-000061
参照实施例43的方法，将5-氯噻吩-2-磺酰氯替换成2-噻吩磺酰氯，制得化合物44(白色固体,42mg)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 43, 5-chlorothiophene-2-sulfonyl chloride was replaced with 2-thiophenesulfonyl chloride to obtain compound 44 (white solid, 42mg): 1 H NMR (300MHz, DMSO-d 6 ) 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] + .
实施例45Example 45
N-(6-(吗啉-4-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺盐酸盐(化合物45)N-(6-(morpholine-4-sulfonamido)benzo[d]thiazol-2-yl)piperidine-4-carboxamide hydrochloride (compound 45)
Figure PCTCN2021078798-appb-000062
Figure PCTCN2021078798-appb-000062
参照实施例43的方法，将5-氯噻吩-2-磺酰氯替换成4-吗啉磺酰氯，制得化合物45(黄色固体,48mg)： 1H NMR(300MHz,DMSO-d 6)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] +。 Referring to the method of Example 43, 5-chlorothiophene-2-sulfonyl chloride was replaced with 4-morpholinesulfonyl chloride to obtain compound 45 (yellow solid, 48mg): 1 H NMR (300MHz, DMSO-d 6 )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] + .
实施例46Example 46
N-(6-(吗啉-4-磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-羧酰胺盐酸盐(化合物46)N-(6-(morpholine-4-sulfonamido)benzo[d]thiazol-2-yl)furan-2-carboxamide hydrochloride (Compound 46)
Figure PCTCN2021078798-appb-000063
Figure PCTCN2021078798-appb-000063
参照实施例45的方法，将1-Boc-4-哌啶甲酸替换成糠酸，制得化合物46(淡黄色固体,60mg)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method in Example 45, 1-Boc-4-piperidinecarboxylic acid was replaced with furoic acid to obtain compound 46 (light yellow solid, 60mg): 1 H NMR (300MHz, DMSO-d 6 ) 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] + .
实施例47Example 47
N-(6-((5-氯噻吩)-2-磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-羧酰胺(化合物47)N-(6-((5-Chlorothiophene)-2-sulfonamido)benzo(d)thiazol-2-yl)furan-2-carboxamide (Compound 47)
Figure PCTCN2021078798-appb-000064
Figure PCTCN2021078798-appb-000064
参照实施例43的方法，将1-Boc-4-哌啶甲酸替换成糠酸，制得化合物47(淡黄色固体,62mg)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 43, 1-Boc-4-piperidinecarboxylic acid was replaced with furoic acid to obtain compound 47 (light yellow solid, 62mg): 1 H NMR (300MHz, DMSO-d 6 ) 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 = 1.8 Hz, 1H). ESI-MS m/z 462.0 [M+H] + .
实施例48Example 48
N-(6-(吡啶-3-磺酰胺基)苯并[d]噻唑-2-基)呋喃-2-甲酰胺(化合物48)N-(6-(pyridine-3-sulfonamido)benzo[d]thiazol-2-yl)furan-2-carboxamide (Compound 48)
Figure PCTCN2021078798-appb-000065
Figure PCTCN2021078798-appb-000065
参照实施例47的方法，将5-氯噻吩-2-磺酰氯替换成吡啶-3-磺酰氯，制得化合物48(白色固体,109mg)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 47, 5-chlorothiophene-2-sulfonyl chloride was replaced with pyridine-3-sulfonyl chloride to obtain compound 48 (white solid, 109mg): 1 H NMR (300MHz, DMSO-d 6 )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[MH] - .
实施例49Example 49
N-(6-((4-硝基苯基)磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺盐酸盐(化合物49)N-(6-((4-nitrophenyl)sulfonamido)benzo(d)thiazol-2-yl)piperidine-4-carboxamide hydrochloride (Compound 49)
Figure PCTCN2021078798-appb-000066
Figure PCTCN2021078798-appb-000066
参照实施例43的方法，将5-氯噻吩-2-磺酰氯替换成对硝基苯磺酰氯，制得化合物49(淡黄色固体,69mg)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 43, 5-chlorothiophene-2-sulfonyl chloride was replaced with p-nitrobenzenesulfonyl chloride to obtain compound 49 (light yellow solid, 69mg): 1 H NMR (300MHz, DMSO-d 6 )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] + .
实施例50Example 50
N-(6-(吡啶-3-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺盐酸盐(化合物50)N-(6-(pyridine-3-sulfonamido)benzo[d]thiazol-2-yl)piperidine-4-carboxamide hydrochloride (compound 50)
Figure PCTCN2021078798-appb-000067
Figure PCTCN2021078798-appb-000067
参照实施例43的方法，将5-氯噻吩-2-磺酰氯替换成吡啶-3-磺酰氯，制得化合物50(淡黄色固体,54mg)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 43, 5-chlorothiophene-2-sulfonyl chloride was replaced with pyridine-3-sulfonyl chloride to obtain compound 50 (light yellow solid, 54mg): 1 H NMR (300MHz, DMSO-d 6 )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] + .
实施例51Example 51
5-(N-(2-(哌啶-4-甲酰胺基)苯并[d]噻唑-6-基)氨磺酰基)噻吩-3-羧酸盐酸盐(化合物51)5-(N-(2-(piperidine-4-carboxamido)benzo[d]thiazol-6-yl)sulfamoyl)thiophene-3-carboxylic acid hydrochloride (Compound 51)
Figure PCTCN2021078798-appb-000068
Figure PCTCN2021078798-appb-000068
Figure PCTCN2021078798-appb-000069
Figure PCTCN2021078798-appb-000069
取3-噻吩甲酸(51a,1.0g,7.80mmol)溶于乙醇(15mL)中，滴加浓硫酸(2滴)，78℃加热回流至TLC检测反应完全后，冷却至室温，减压蒸除溶剂，加入乙酸乙酯(30mL)复溶，依次用饱和碳酸氢钠溶液(30mL)、水(30mL)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，浓缩，得化合物51b(淡黄色油状液体,1.07g,产率88％)： 1H NMR(300MHz,CDCl 3)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)。 Dissolve 3-thiophenecarboxylic acid (51a, 1.0g, 7.80mmol) in ethanol (15mL), add concentrated sulfuric acid (2 drops) dropwise, heat to reflux at 78°C until TLC detects the completion of the reaction, cool to room temperature, and evaporate under reduced pressure The solvent was reconstituted by adding ethyl acetate (30 mL), washed with saturated sodium bicarbonate solution (30 mL), water (30 mL) and saturated brine (30 mL) in turn, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 51b ( Pale yellow oily liquid, 1.07g, yield 88%): 1 H NMR (300MHz, CDCl 3 ) δ 8.14-8.04 (m, 1H), 7.53 (d, J = 4.8 Hz, 1H), 7.34-7.27 ( m, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H).
取化合物51b(500mg,3.20mmol)溶于无水二氯甲烷(4mL)，冰浴下，氩气保护，缓慢地滴加溶有氯磺酸(746mg,6.40mmol)的无水二氯甲烷(4mL)溶液。自然升至室温反应至TLC检测反应完全。将反应液缓慢地滴加入冰水中(20mL)，用二氯甲烷(20mL×2)萃取，有机相用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(乙酸乙酯：石油醚＝30：1)得到化合物51c(淡黄色液体,404mg,产率50％)： 1H NMR(300MHz,CDCl 3)δ8.47(s,1H),8.26(s,1H),4.38(q,J＝7.1Hz,2H),1.39(t,J＝7.1Hz,3H)。 Take compound 51b (500mg, 3.20mmol) and dissolve it in dry dichloromethane (4mL), under ice bath, under argon protection, slowly add dropwise dry dichloromethane (746mg, 6.40mmol) dissolved in chlorosulfonic acid (746mg, 6.40mmol) 4mL) solution. Spontaneously warm to room temperature and react until TLC detects that the reaction is complete. The reaction solution was slowly added dropwise to ice water (20mL), extracted with dichloromethane (20mL×2), 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%): 1 H NMR (300MHz, CDCl 3 ) δ8.47(s, 1H), 8.26(s, 1H), 4.38 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).
取化合物43c(220mg,0.58mmol)溶于二氯甲烷(4mL)和吡啶(1mL)的混合溶剂中，缓慢地加入化合物51c(178mg,0.70mmol)，室温搅拌至TLC检测反应完全。浓缩，加入乙酸乙酯(30mL)稀释，依次用1N的稀盐酸(30mL×3)和饱和食盐水(30mL)洗，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(二氯甲烷:甲醇＝80:1)得到化合物51d(黄色固体,316mg,产率92％)。Dissolve compound 43c (220 mg, 0.58 mmol) in a mixed solvent of dichloromethane (4 mL) and pyridine (1 mL), slowly add compound 51c (178 mg, 0.70 mmol), and stir at room temperature until TLC detects that the reaction is complete. Concentrate, add ethyl acetate (30mL) to dilute, wash with 1N dilute hydrochloric acid (30mL×3) and saturated brine (30mL) successively, dry with anhydrous sodium sulfate, filter, concentrate, and chromatograph on silica gel column (dichloromethane) : Methanol = 80:1) to obtain compound 51d (yellow solid, 316 mg, yield 92%).
取化合物51d(200mg,0.336mmol)溶于甲醇(6mL)和水(2mL)的混合溶剂中，加入氢氧化锂一水合物(28mg,0.673mmol)，室温反应过夜。TLC检测反应完全后，减压蒸除溶剂，加入水(5mL)复溶，二氯甲烷(5mL×2)洗涤，水相蒸除残留的二氯甲烷，室温搅拌下，用1N的稀盐酸调pH至4-5，抽滤，滤饼烘干，得到化合物51e(白色固体142mg,产率74％)。Compound 51d (200 mg, 0.336 mmol) was dissolved in a mixed solvent of methanol (6 mL) and water (2 mL), lithium hydroxide monohydrate (28 mg, 0.673 mmol) was added, and the reaction was carried out at room temperature overnight. After the completion of the reaction was detected by TLC, the solvent was evaporated under reduced pressure, water (5mL) was added for reconstitution, and the dichloromethane (5mL×2) was added to wash. The aqueous phase was evaporated to remove the residual dichloromethane. The pH was set to 4-5, filtered with suction, and the filter cake was dried to obtain compound 51e (white solid 142 mg, yield 74%).
取化合物51e(142mg,0.25mmol)溶于盐酸乙酸乙酯溶液(10mL)，室温搅拌过夜，TLC检测反应完全后，抽滤，滤饼烘干，得到化合物51(白色固体,77mg,产率62％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Take compound 51e (142mg, 0.25mmol) dissolved in hydrochloric acid ethyl acetate solution (10mL), stir overnight at room temperature, TLC check the reaction is complete, suction filtration, filter cake drying, to obtain compound 51 (white solid, 77mg, yield 62 %): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.49 (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] + .
实施例52Example 52
5-(N-(2-(哌啶-4-羧酰胺基)苯并[d]噻唑-6-基)氨磺酰基)噻吩-3-羧酸乙酯盐酸盐(化合物52)5-(N-(2-(piperidine-4-carboxamide)benzo(d)thiazol-6-yl)sulfamoyl)thiophene-3-carboxylic acid ethyl ester hydrochloride (compound 52)
Figure PCTCN2021078798-appb-000070
Figure PCTCN2021078798-appb-000070
取化合物51d(96mg,0.16mmol)溶于盐酸乙酸乙酯溶液(10mL)，室温搅拌过夜，TLC检测反应完全后，抽滤，滤饼烘干，得到化合物52(白色固体,80mg,产率93％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Take compound 51d (96mg, 0.16mmol) and dissolve it in hydrochloric acid ethyl acetate solution (10mL), stir overnight at room temperature, TLC detects the completion of the reaction, suction filtration, filter cake drying, to obtain compound 52 (white solid, 80mg, yield 93 %): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.52 (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 = 8.6 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H), 4.24 (q, J = 7.1 Hz, 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] + .
实施例53Example 53
3-((5-(N-(2-(哌啶-4-甲酰胺基)苯并[d]噻唑-6-基)氨磺酰基)噻吩-3-甲酰胺基)甲基)苯甲酸盐酸盐(化合物53)3-((5-(N-(2-(Piperidine-4-carboxamido)benzo(d)thiazol-6-yl)sulfamoyl)thiophen-3-carboxamido)methyl)benzyl Hydrochloride (Compound 53)
Figure PCTCN2021078798-appb-000071
Figure PCTCN2021078798-appb-000071
Figure PCTCN2021078798-appb-000072
Figure PCTCN2021078798-appb-000072
取51e(200mg,0.35mmol)溶于N,N-二甲基甲酰胺(4mL)中，依次加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,128mg,0.42mmol)和三乙胺(65mg,0.64mmol)，室温搅拌反应1小时。同时将3-(氨甲基)苯甲酸甲酯盐酸盐(65mg,0.32mmol)、三乙胺(65mg,0.64mmol)依次加入二氯甲烷(4mL)中搅拌1小时，使游离出3-(氨甲基)苯甲酸甲酯。TLC检测反应完全后，将游离后的3-(氨甲基)苯甲酸甲酯二氯甲烷溶液滴加入上述N,N-二甲基甲酰胺反应液中，加入碳酸钾(147mg,1.06mmol)。室温搅拌至TLC检测反应完全后，加入二氯甲烷(30mL)稀释反应液，反应液依次用1N稀盐酸(30mL)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，经硅胶柱层析(乙酸乙酯:石油醚＝1:2)纯化，得化合物53a(白色固体,116mg,产率47％)。Dissolve 51e (200mg, 0.35mmol) in N,N-dimethylformamide (4mL) and add 2-(7-azabenzotriazole)-N,N,N',N'- Tetramethylurea hexafluorophosphate (HATU, 128 mg, 0.42 mmol) and triethylamine (65 mg, 0.64 mmol) were reacted with 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 dichloromethane (4mL) and stirred for 1 hour to free 3- (Aminomethyl) methyl benzoate. After TLC detects that the reaction is complete, the free methyl 3-(aminomethyl)benzoate in dichloromethane solution is added dropwise to the above-mentioned N,N-dimethylformamide reaction solution, and potassium carbonate (147mg, 1.06mmol) is added. . After stirring at room temperature until TLC detects that the reaction is complete, dichloromethane (30mL) is added to dilute the reaction solution. The reaction solution is washed with 1N dilute hydrochloric acid (30mL) and saturated brine (30mL) successively, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated. Purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:2), compound 53a (white solid, 116 mg, yield 47%) was obtained.
取化合物53a(80mg,0.112mmol)溶于甲醇(8mL)和水(2mL)的混合溶剂中，加入氢氧化锂一水合物(19mg,0.449mmol)，室温反应过夜。TLC检测反应完全后，减压蒸除溶剂，加入水(5mL)复溶，二氯甲烷(5mL×2)洗涤，水相蒸除残留的二氯甲烷，室温搅拌下，用1N的稀盐酸调pH至4-5，抽滤，滤饼烘干，得到化合物53b(白色固体57mg,产率73％)。Take compound 53a (80 mg, 0.112 mmol) and dissolve it in a mixed solvent of methanol (8 mL) and water (2 mL), add lithium hydroxide monohydrate (19 mg, 0.449 mmol), and react at room temperature overnight. After the completion of the reaction was detected by TLC, the solvent was evaporated under reduced pressure, water (5mL) was added for reconstitution, and the dichloromethane (5mL×2) was added to wash. The aqueous phase was evaporated to remove the residual dichloromethane. The pH was set to 4-5, filtered with suction, and the filter cake was dried to obtain compound 53b (white solid 57mg, yield 73%).
取化合物53b(57mg,0.083mmol)溶于盐酸乙酸乙酯溶液(10mL)室温搅拌过夜，TLC检测反应完全后，抽滤，滤饼烘干，得到化合物53(白色固体,30mg,产率58％)： 1H NMR(300MHz,DMSO-d 6)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] +。 The compound 53b (57mg, 0.083mmol) was dissolved in a hydrochloric acid ethyl acetate solution (10mL) and stirred overnight at room temperature. After the completion of the reaction was detected by TLC, it was filtered off with suction and the filter cake was dried to obtain compound 53 (white solid, 30mg, yield 58%) ): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.91 (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] + .
实施例54Example 54
3-((5-(N-(2-(哌啶-4-甲酰胺基)苯并[d]噻唑-6-基)氨磺酰基)噻吩-3-甲酰胺基)甲基)苯甲酸甲酯盐酸盐(化合物54)3-((5-(N-(2-(Piperidine-4-carboxamido)benzo(d)thiazol-6-yl)sulfamoyl)thiophen-3-carboxamido)methyl)benzoic acid Methyl ester hydrochloride (Compound 54)
Figure PCTCN2021078798-appb-000073
Figure PCTCN2021078798-appb-000073
参照实施例52的方法，将化合物51d替换成化合物53a，制得化合物54(白色固体,10mg)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 52, compound 51d was replaced with compound 53a to obtain compound 54 (white solid, 10 mg): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.48 (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] + .
实施例55Example 55
1-甲基-N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺(化合物55)1-Methyl-N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)piperidine-4-carboxamide (Compound 55)
Figure PCTCN2021078798-appb-000074
Figure PCTCN2021078798-appb-000074
取55a(500g,3.49mmol)溶于无水二氯甲烷(15mL)中，冰浴下缓慢加入草酰氯(0.59mL,8.98mmol)，滴加完毕后，加入N,N-二甲基甲酰胺(1滴)，室温反应1小时。TLC监测反应完全后，减压蒸除溶剂得到化合物55b，用无水二氯甲烷(10mL)复溶，缓慢地滴加到溶有2-氨基-6硝基苯并噻唑(681mg,3.49mmol)和三乙胺(968μL, 6.98mmol)的无水二氯甲烷(10mL)中，室温搅拌反应13小时。反应完后将反应液浓缩，加入乙酸乙酯(30mL)用饱和碳酸氢钠水溶液(30mL)中和、减压抽滤，滤饼依次用水(30mL)和乙酸乙酯(10mL)洗涤得到化合物55c(褐色固体,1.05g,产率94％)。Take 55a (500g, 3.49mmol) and dissolve it in anhydrous dichloromethane (15mL), slowly add oxalyl chloride (0.59mL, 8.98mmol) under ice bath, after the addition is complete, add N,N-dimethylformamide (1 drop), react at room temperature for 1 hour. After the completion of the reaction was monitored by TLC, the solvent was evaporated under reduced pressure to obtain compound 55b, which was reconstituted with anhydrous dichloromethane (10mL), and slowly added dropwise to dissolve 2-amino-6nitrobenzothiazole (681mg, 3.49mmol) And triethylamine (968μL, 6.98mmol) in anhydrous dichloromethane (10mL), the reaction was stirred at room temperature for 13 hours. After the reaction, the reaction solution was concentrated, ethyl acetate (30 mL) was added, neutralized with saturated sodium bicarbonate aqueous solution (30 mL), filtered under reduced pressure, and the filter cake was washed with water (30 mL) and ethyl acetate (10 mL) to obtain compound 55c (Brown solid, 1.05g, yield 94%).
取化合物55c(1.05g,3.26mmol)，悬浮于乙酸(12mL)和水(3mL)的混合溶液中，室温搅拌下缓慢加入锌粉(1.07g,16.3mmol)，室温搅拌，TLC监测反应完全后，减压蒸除溶剂，加入二氯甲烷(20mL)，用饱和碳酸氢钠水溶液将pH调至8-9，加入浓氨水(10mL)，室温搅拌1小时。加入水(10mL)分液，水相用二氯甲烷(20mL)萃取，合并有机相，用无水硫酸钠干燥，过滤，滤液浓缩，得化合物55d粗品(黄褐色固体,197mg,产率71％)，无需纯化直接投下一步。Take compound 55c (1.05g, 3.26mmol), suspend it in a mixed solution of acetic acid (12mL) and water (3mL), slowly add zinc powder (1.07g, 16.3mmol) with stirring at room temperature, stir at room temperature, TLC monitors after the reaction is complete , The solvent was evaporated under reduced pressure, dichloromethane (20 mL) was added, the pH was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution, concentrated ammonia (10 mL) was added, and the mixture was stirred at room temperature for 1 hour. Water (10 mL) was added for separation, the aqueous phase was extracted with dichloromethane (20 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound 55d (yellow-brown solid, 197 mg, yield 71%) ), no need to be purified directly to the next step.
参照实施例43的方法，将43c替换成55d，将5-氯噻吩-2-磺酰氯替换成2-噻吩磺酰氯得到化合物55(白色固体,47mg)： 1H NMR(300MHz,DMSO-d 6)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] -。 According to the method of Example 43, 43c was replaced with 55d, 5-chlorothiophene-2-sulfonyl chloride was replaced with 2-thiophenesulfonyl chloride to obtain compound 55 (white solid, 47mg): 1 H NMR (300MHz, DMSO-d 6 )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=8.7 Hz,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[MH] - .
实施例56Example 56
3-((3-(N-(2-(1-甲基哌啶-4-甲酰胺基)苯并[d]噻唑-6-基)氨磺酰基)丙基)氨基甲酰基)苯甲酸(化合物56)3-((3-(N-(2-(1-methylpiperidine-4-carboxamido)benzo(d)thiazol-6-yl)sulfamoyl)propyl)carbamoyl)benzoic acid (Compound 56)
Figure PCTCN2021078798-appb-000075
Figure PCTCN2021078798-appb-000075
Figure PCTCN2021078798-appb-000076
Figure PCTCN2021078798-appb-000076
将间苯二甲酸单甲酯(56a,500mg,2.77mmol)、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,1023mg,3.32mmol)和三乙胺(561mg,5.54mmol)依次加入N,N-二甲基甲酰胺(6mL)中，室温搅拌反应1小时后，加入3-氨基-1-丙磺酸(579mg,4.16mmol)，继续室温搅拌反应。TLC检测反应完全后，加入乙酸乙酯(20mL)稀释反应液，反应液用水(20mL)萃取，水相用乙酸乙酯(20mL)洗涤。水相减压蒸除溶剂后，经硅胶柱层析(二氯甲烷:甲醇＝15:1)纯化，得化合物56b(白色固体，420mg，产率50％)。The monomethyl isophthalate (56a, 500mg, 2.77mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate ( HATU, 1023mg, 3.32mmol) and triethylamine (561mg, 5.54mmol) were sequentially added to N,N-dimethylformamide (6mL), stirred at room temperature and reacted for 1 hour, then 3-amino-1-propanesulfonic acid was added (579mg, 4.16mmol), continue to stir the reaction at room temperature. After the completion of the reaction was detected by TLC, ethyl acetate (20 mL) was added to dilute the reaction solution, the reaction solution was extracted with water (20 mL), and the aqueous phase was washed with ethyl acetate (20 mL). After the solvent was evaporated from the aqueous phase under reduced pressure, it was purified by silica gel column chromatography (dichloromethane:methanol=15:1) to obtain compound 56b (white solid, 420 mg, yield 50%).
取化合物56b(400mg,1.33mmol)悬浮于无水二氯甲烷(15mL)中，冰浴下缓慢地加入五氯化磷(553mg,2.66mmol)，加毕后，自然升至室温反应至TLC检测反应完全后，加入水(15mL)淬灭，加入二氯甲烷(10mL)分液，有机相用饱和食盐水(25mL)洗涤，干燥，过滤，浓缩，经硅胶柱层析(乙酸乙酯:石油醚＝3:1)纯化，得到化合物56c(白色固体,140mg,产率33％)。Suspend compound 56b (400mg, 1.33mmol) in anhydrous dichloromethane (15mL), slowly add phosphorus pentachloride (553mg, 2.66mmol) under ice bath, after the addition, warm to room temperature and react until TLC detection After the reaction was completed, it was quenched by adding water (15 mL), and dichloromethane (10 mL) was added for separation. The organic phase was washed with saturated brine (25 mL), dried, filtered, concentrated, and subjected to silica gel column chromatography (ethyl acetate: petroleum Ether=3:1) was purified to obtain compound 56c (white solid, 140 mg, yield 33%).
取化合物55d(76mg,0.26mmol)溶于二氯甲烷(4mL)和吡啶(1mL)的混合溶剂中，缓慢地加入化合物56c(100mg,0.31mmol)，室温搅拌至TLC检测反应完全。浓缩，加入乙酸乙酯(30mL)稀释，依次用水(30mL×3)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(二氯甲烷:甲醇＝25:1)得到化合物56e(淡黄色固体,65mg,产率44％)。Take compound 55d (76 mg, 0.26 mmol) and dissolve it in a mixed solvent of dichloromethane (4 mL) and pyridine (1 mL), slowly add compound 56c (100 mg, 0.31 mmol), and stir at room temperature until TLC detects that the reaction is complete. Concentrate, add ethyl acetate (30mL) to dilute, wash sequentially with water (30mL×3) and saturated brine (30mL), dry with anhydrous sodium sulfate, filter, concentrate, and chromatograph on silica gel column (dichloromethane: methanol = 25 1) Compound 56e (light yellow solid, 65 mg, yield 44%) was obtained.
取化合物56e(65mg,0.11mmol)溶于甲醇(4mL)和水(1mL)的混合溶剂中，加入氢氧化锂一水合物(24mg,0.57mmol)，室温反应过夜。TLC检测反应完全后，减压蒸除溶剂，加入水(5mL)复溶，二氯甲烷(5mL×2)洗涤，水相蒸除残留的二氯甲烷，室温搅拌下，用1N的稀盐酸调pH至6-7，抽滤，滤饼烘干，得到化合物56 (白色固体34mg,产率56％)： 1H NMR(300MHz,DMSO-d 6)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 C 25H 30N 5O 6S 2[M+H] +560.1632,found 560.1632。 Take compound 56e (65 mg, 0.11 mmol) and dissolve it in a mixed solvent of methanol (4 mL) and water (1 mL), add lithium hydroxide monohydrate (24 mg, 0.57 mmol), and react at room temperature overnight. After the completion of the reaction was detected by TLC, the solvent was evaporated under reduced pressure, water (5mL) was added for reconstitution, and the dichloromethane (5mL×2) was added to wash. The aqueous phase was evaporated to remove the residual dichloromethane. pH to 6-7, suction filtration, and drying the filter cake to obtain compound 56 (white solid 34mg, yield 56%): 1 H NMR (300MHz, DMSO-d 6 )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 C 25 H 30 N 5 O 6 S 2 [M+H] + 560.1632, found 560.1632.
实施例57Example 57
N-(6-(2-(噻吩-2-基)乙酰氨基)苯并[d]噻唑-2-基)吗啉-4-羧酰胺(化合物57)N-(6-(2-(Thien-2-yl)acetamido)benzo(d)thiazol-2-yl)morpholine-4-carboxamide (Compound 57)
Figure PCTCN2021078798-appb-000077
Figure PCTCN2021078798-appb-000077
取化合物1a(976mg,5mmol)、三乙胺(2mL,15mmol)溶于二氯甲烷(40ml)中，向其中加入氯甲酸苯酯(1.25mL,10mmol)的二氯甲烷溶液(10mL)，室温搅拌至TLC检测反应完全后，减压蒸除溶剂，用乙酸乙酯(20mL)打浆，抽滤，滤饼用1N的稀盐酸(20mL)洗，抽滤，烘干得中间体57a的粗品(黄色固体，1.168g)。Dissolve compound 1a (976mg, 5mmol) and triethylamine (2mL, 15mmol) in dichloromethane (40ml), add phenyl chloroformate (1.25mL, 10mmol) in dichloromethane (10mL), room temperature After stirring until the reaction is completed by TLC detection, the solvent was evaporated under reduced pressure, slurried with ethyl acetate (20mL), filtered with suction, the filter cake was washed with 1N dilute hydrochloric acid (20mL), filtered with suction, and dried to obtain the crude intermediate 57a ( Yellow solid, 1.168g).
取中间体57a的粗品(315mg,1mmol)溶于二甲基亚砜(5mL)中，加入吗啉(435μL,5mmol)，室温搅拌至TLC检测反应完全后，向反应液中加入水(50mL)和乙酸乙酯(20mL)，分液，水相用乙酸乙酯(20mL×3)萃取，合并有机相，有机相用饱和食盐水(25mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(二氯甲烷:甲醇＝50:1)纯化，得到化合物57b(黄色固体,173mg,两步产率56％)。Dissolve the crude product of Intermediate 57a (315 mg, 1 mmol) in dimethyl sulfoxide (5 mL), add morpholine (435 μL, 5 mmol), stir at room temperature until TLC detects the reaction is complete, add water (50 mL) to the reaction solution And ethyl acetate (20mL), the layers were 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, Purification by silica gel column chromatography (dichloromethane:methanol=50:1) gave compound 57b (yellow solid, 173 mg, yield 56% in two steps).
取化合物57b(166mg,0.54mmol)溶于乙酸(7.5mL)和水(2.5mL)的混合溶液中，加入锌粉(176mg,2.7mmol)，室温搅拌至TLC检测反应完全后，先加入饱和碳酸氢钠溶液(15ml)，再加入碳酸氢钠固体至无气泡产生。向混合物中加入乙酸乙酯(30ml)，分液，水相用乙酸乙酯(30mL×3)萃取，合并有机相，有机相用饱和食盐水(25mL)洗涤，无水硫酸钠干燥，过滤，浓缩，得到化合物57c(棕色固体， 137mg，产率91％)。Dissolve compound 57b (166mg, 0.54mmol) in a mixed solution of acetic acid (7.5mL) and water (2.5mL), add zinc powder (176mg, 2.7mmol), stir at room temperature until TLC detects the reaction is complete, add saturated carbonic acid first Sodium bicarbonate solution (15ml), then add solid sodium bicarbonate until no bubbles are generated. Ethyl acetate (30ml) was added to the mixture, 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 to obtain compound 57c (brown solid, 137 mg, yield 91%).
取2-噻吩乙酰氯(30μL,0.24mmol)溶于干燥二氯甲烷(2mL)中，缓慢滴入三乙胺(166μL,1.2mmol)、57c(56mg,0.2mmol)的干燥二氯甲烷(5mL)溶液中，室温搅拌至TLC检测反应完全后，加入1N的稀盐酸(5mL)和二氯甲烷(5mL)，分液，水相用二氯甲烷(5mL×3)萃取，合并有机相，有机相用饱和食盐水洗涤(10mL×3)，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(二氯甲烷:甲醇＝50:1)纯化，得到化合物57(黄色固体,41mg,产率51％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Take 2-thiophene acetyl chloride (30μL, 0.24mmol) and dissolve it in dry dichloromethane (2mL), slowly drop in triethylamine (166μL, 1.2mmol), 57c (56mg, 0.2mmol) in dry dichloromethane (5mL) ) In the solution, stir at room temperature until the TLC detection reaction is complete, add 1N dilute hydrochloric acid (5mL) and dichloromethane (5mL), separate the layers, extract the aqueous phase with dichloromethane (5mL×3), combine the organic phases, and organic The phase was washed with saturated brine (10mL×3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane:methanol=50:1) to obtain compound 57 (yellow solid, 41mg, product Rate 51%): 1 H NMR (300MHz, DMSO-d 6 ) δ 11.46 (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] + .
实施例58Example 58
N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)吗啉-4-羧酰胺(化合物58)N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)morpholine-4-carboxamide (compound 58)
Figure PCTCN2021078798-appb-000078
Figure PCTCN2021078798-appb-000078
参照实施例37的方法，将37e替换57c，制得化合物58(黄色固体)： 1H NMR(300MHz,DMSO-d 6)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] -。 Refer to the method of Example 37, and replace 37e with 57c to obtain compound 58 (yellow solid): 1 H NMR (300MHz, DMSO-d 6 ) δ 11.21 (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 [MH] - .
实施例59Example 59
N-(6-(四氢-2H-吡喃-4-甲酰胺基)苯并[d]噻唑-2-基)哌嗪-1-甲酰胺盐酸盐(化合物59)N-(6-(Tetrahydro-2H-pyran-4-carboxamido)benzo[d]thiazol-2-yl)piperazine-1-carboxamide hydrochloride (Compound 59)
Figure PCTCN2021078798-appb-000079
Figure PCTCN2021078798-appb-000079
取中间体粗品57a(315mg,1mmol)溶于二甲基亚砜(5mL)中，加入N-Boc-哌嗪(931mg,5mmol)，室温搅拌至TLC检测反应完全后，向反应液中依次加入水(50mL)、乙酸乙酯(20mL)，分液，水相用乙酸乙酯(20mL×3)萃取，合并有机相，有机相用饱和食盐水(25mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(二氯甲烷:甲醇＝50:1)纯化，得到化合物59a(淡黄色固体,213mg,两步产率52％)。Dissolve the crude intermediate 57a (315mg, 1mmol) in dimethyl sulfoxide (5mL), add N-Boc-piperazine (931mg, 5mmol), stir at room temperature until TLC detects the reaction is complete, add to the reaction solution in sequence Water (50 mL) and ethyl acetate (20 mL) were separated, the aqueous phase was extracted with ethyl acetate (20 mL×3), the organic phases were combined, and the organic phase was washed with saturated brine (25 mL), dried with anhydrous sodium sulfate, and filtered , Concentrated, and purified by silica gel column chromatography (dichloromethane:methanol=50:1) to obtain compound 59a (light yellow solid, 213 mg, two-step yield 52%).
取化合物59a(213mg,0.52mmol)溶于乙酸(7.5mL)和水(2.5mL)的混合溶液中，加入锌粉(171mg,2.6mmol)，室温搅拌至TLC检测反应完全后，先加入饱和碳酸氢钠(15mL)溶液，再加入碳酸氢钠固体至无气泡产生。向混合物中加入乙酸乙酯(30mL)，分液，水相用乙酸乙酯(30mL×3)萃取，合并有机相，有机相用饱和食盐水(25mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(二氯甲烷：甲醇＝50：1)纯化，得到化合物59b(黄色固体,180mg,产率92％)。Take compound 59a (213mg, 0.52mmol) and dissolve it in a mixed solution of acetic acid (7.5mL) and water (2.5mL), add zinc powder (171mg, 2.6mmol), stir at room temperature until TLC detects the reaction is complete, add saturated carbonic acid first Sodium bicarbonate (15mL) solution, and then sodium bicarbonate solid was added until no bubbles were generated. Ethyl acetate (30 mL) was added to the mixture, the layers were separated, the aqueous phase was extracted with ethyl acetate (30 mL×3), the organic phases were combined, and the organic phase was washed with saturated brine (25 mL), dried with anhydrous sodium sulfate, and filtered. Concentrated and purified by silica gel column chromatography (dichloromethane: methanol = 50:1) to obtain compound 59b (yellow solid, 180 mg, yield 92%).
取四氢吡喃‐4‐甲酸(20mg,0.15mmol)溶于N,N-二甲基甲酰胺(1.5mL)中，加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,69mg,0.23mmol)和三乙胺(43μL,0.3mmol)，室温搅拌1小时，加入化合物59b(57mg,0.15mmol)，继续室温搅拌至TLC检测反应完全后，反应液直接制砂，经硅胶柱层析(二氯甲烷:甲醇＝20:1)纯化，得到化合物59c(淡黄色固体,64mg,产率87％)。Dissolve tetrahydropyran-4-carboxylic acid (20mg, 0.15mmol) in N,N-dimethylformamide (1.5mL), add 2-(7-azabenzotriazole)-N,N ,N',N'-Tetramethylurea hexafluorophosphate (HATU, 69mg, 0.23mmol) and triethylamine (43μL, 0.3mmol), stir at room temperature for 1 hour, add compound 59b (57mg, 0.15mmol), continue After stirring at room temperature until TLC detects that the reaction is complete, the reaction solution is directly made into sand and purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain compound 59c (light yellow solid, 64 mg, yield 87%).
取化合物59c(48.9mg,0.1mmol)溶于四氢呋喃(1mL)中，加入盐酸乙醇溶液(5mL)，室温搅拌至TLC检测反应完全后，抽滤，滤饼烘干，得到化合物59(棕色固体,32mg,产率75％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Dissolve compound 59c (48.9 mg, 0.1 mmol) in tetrahydrofuran (1 mL), add hydrochloric acid ethanol solution (5 mL), stir at room temperature until TLC detection reaction is complete, filter with suction, and dry the filter cake to obtain compound 59 (brown solid, 32mg, yield 75%): 1 H NMR (300MHz, DMSO-d 6 ) δ 10.09 (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] + .
实施例60Example 60
N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺盐酸盐(化合物60)N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamide)benzo(d)thiazol-2-yl)piperidine-4-carboxamide hydrochloride (Compound 60)
Figure PCTCN2021078798-appb-000080
Figure PCTCN2021078798-appb-000080
取化合物43c(180mg,0.48mmol)溶于二氯甲烷(15mL)中，依次缓慢地加入三乙胺(291mg,2.88mmol)及2-噻吩磺酰氯(105mg,0.57mmol)，室温搅拌至TLC检测反应完全后，加入二氯甲烷(40mL)稀释，依次用1N的稀盐酸水(40mL×3)和饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经硅胶柱层析(二氯甲烷:甲醇＝200:1)得到化合物60a(白色固体,80mg,产率25％)。Dissolve compound 43c (180mg, 0.48mmol) in dichloromethane (15mL), slowly add triethylamine (291mg, 2.88mmol) and 2-thiophenesulfonyl chloride (105mg, 0.57mmol) in sequence, and stir at room temperature until TLC detection After the reaction was completed, dichloromethane (40mL) was added to dilute, washed with 1N dilute hydrochloric acid water (40mL×3) and saturated brine (40mL) successively, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography ( Dichloromethane: methanol = 200:1) to obtain compound 60a (white solid, 80 mg, yield 25%).
取化合物60a(80mg,0.12mmol)溶于盐酸乙酸乙酯溶液(10mL)，室温搅拌过夜，TLC检测反应完全后，抽滤，滤饼烘干，得到化合物60(白色固体,40mg,产率56％)： 1H NMR(300MHz,DMSO-d 6)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] +。 Take compound 60a (80mg, 0.12mmol) and dissolve it in hydrochloric acid ethyl acetate solution (10mL), stir overnight at room temperature, TLC check the reaction is complete, suction filtration, filter cake drying, to obtain compound 60 (white solid, 40mg, yield 56 %): 1 H NMR (300MHz, DMSO-d 6 ) δ 12.71 (s, 1H), 9.14-8.87 (m, 1H), 8.87-8.58 (m, 1H), 8.23 (d, J = 4.7 Hz, 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] + .
实施例61Example 61
1-甲基-N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺(化合物61)1-Methyl-N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)piperidine-4-carboxamide (compound 61)
Figure PCTCN2021078798-appb-000081
Figure PCTCN2021078798-appb-000081
参照实施例60的方法，将1-Boc-4-哌啶甲酸替换成1-甲基哌啶-4-甲酸，制得化合物61(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 60, 1-Boc-4-piperidine carboxylic acid was replaced with 1-methylpiperidine-4-carboxylic acid to obtain compound 61 (white solid): 1 H NMR (300MHz, DMSO-d 6 ) δ12.43 (s, 1H), 8.23 (d, J = 5.0 Hz, 2H), 7.81 (d, J = 2.0 Hz, 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] + .
实施例62Example 62
N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)哌嗪-1-甲酰胺盐酸盐(化合物62)N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)piperazine-1-carboxamide hydrochloride (Compound 62)
Figure PCTCN2021078798-appb-000082
Figure PCTCN2021078798-appb-000082
参照实施例60的方法，将化合物43c替换成化合物59b，制得化合物62(白色固体)： 1H NMR(300MHz,DMSO-d 6)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] +。 According to the method of Example 60, compound 43c was replaced with compound 59b to obtain compound 62 (white solid): 1 H NMR (300MHz, DMSO-d 6 )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] + .
实施例63Example 63
N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环己烷甲酰胺(化合物63)N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)cyclohexanecarboxamide (Compound 63)
Figure PCTCN2021078798-appb-000083
Figure PCTCN2021078798-appb-000083
将2-氨基-6硝基苯并噻唑(500mg,2.56mmol)悬浮于无水二氯甲烷(10mL)中，加入三乙胺(0.71mL,5.12mmol)，缓慢地滴加63a(563mg,3.84mmol)，室温搅拌至TLC监测反应完全。反应液浓缩，加入乙酸乙酯(20mL)稀释，依次用1N的稀盐酸(20mL)、水(20mL)、饱和碳酸氢钠(20mL)和饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，用经硅胶柱层析(二氯甲烷:甲醇＝100:1)纯化，得到化合物63b(淡黄色固体，513mg，产率66％)。Suspend 2-amino-6nitrobenzothiazole (500mg, 2.56mmol) in dry dichloromethane (10mL), add triethylamine (0.71mL, 5.12mmol), slowly add 63a (563mg, 3.84 mmol), stirring at room temperature until TLC monitors the reaction to be complete. The reaction solution was concentrated, diluted with ethyl acetate (20mL), washed with 1N dilute hydrochloric acid (20mL), water (20mL), saturated sodium bicarbonate (20mL) and saturated brine (20mL), and dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane:methanol=100:1) to obtain compound 63b (light yellow solid, 513 mg, yield 66%).
取化合物63b(500mg,1.64mmol)，悬浮于乙酸(8mL)和水(2mL)的混合溶液中，室温搅拌下缓慢加入锌粉(535mg,8.18mmol)，室温搅拌，TLC监测反应完全后，加入水(10mL)和乙酸乙酯(20mL)稀释，加入碳酸氢钠调pH至中性，以饱和碳酸氢钠水溶液(20mL×2)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，得化合物63c的粗品(黄褐色固体,197mg,产率71％)，无需纯化直接投下一步。Take compound 63b (500mg, 1.64mmol), suspend it in a mixed solution of acetic acid (8mL) and water (2mL), slowly add zinc powder (535mg, 8.18mmol) under stirring at room temperature, and stir at room temperature. After TLC monitoring the reaction is complete, add Dilute with water (10mL) and ethyl acetate (20mL), add sodium bicarbonate to adjust the pH to neutral, wash with saturated sodium bicarbonate aqueous solution (20mL×2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain compound 63c The crude product (yellow-brown solid, 197 mg, yield 71%), directly cast to the next step without purification.
将化合物63c(165mg,0.60mmol)悬浮于二氯甲烷(15mL)中，依次加入三乙胺(250μL,1.80mmol)、4-二甲氨基吡啶(DMAP,7mg,0.06mmol)和2-噻吩磺酰氯(274mg,1.50mmol)，室温搅拌至TLC监测反应完全后，加入二氯甲烷(15mL)稀释，依次用1N的稀盐酸(20mL)、水(30mL)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，用经硅胶柱层析(二氯甲烷:甲醇＝350:1)纯化得到化合物63(白色固体,69mg,产率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 C 22H 22N 3O 5S 5[M+H] +568.0158,found 568.0161。 Compound 63c (165mg, 0.60mmol) was suspended in dichloromethane (15mL), and triethylamine (250μL, 1.80mmol), 4-dimethylaminopyridine (DMAP, 7mg, 0.06mmol) and 2-thiophenesulfon were added in sequence Acid chloride (274mg, 1.50mmol), stir at room temperature until TLC monitors the reaction to complete, add dichloromethane (15mL) to dilute, and wash with 1N dilute hydrochloric acid (20mL), water (30mL) and saturated brine (30mL) successively. It was dried over sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane:methanol=350:1) to obtain compound 63 (white solid, 69 mg, 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 C 22 H 22 N 3 O 5 S 5 [M+H] + 568.0158,found 568.0161.
实施例64Example 64
N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环戊烷甲酰胺(化合物64)N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamide)benzo(d)thiazol-2-yl)cyclopentanecarboxamide (Compound 64)
Figure PCTCN2021078798-appb-000084
Figure PCTCN2021078798-appb-000084
参考实施例63的合成步骤，将环己酰氯替换成环戊酰氯，得到化合物64(白色固体,136mg)。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 C 21H 20N 3O 5S 5[M+H] +554.0001,found 544.0010。 Referring to the synthesis procedure of Example 63, cyclohexanoyl chloride was replaced with cyclopentanoyl chloride to obtain compound 64 (white solid, 136 mg). 1H NMR (300MHz, DMSO-d 6 ) δ 12.52 (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 C 21 H 20 N 3 O 5 S 5 [M+H] + 554.0001,found 544.0010.
实施例65Example 65
4-氧代-N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环己烷-1-羧酰胺(化合物65)4-oxo-N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamide)benzo[d]thiazol-2-yl)cyclohexane-1-carboxamide( Compound 65)
Figure PCTCN2021078798-appb-000085
Figure PCTCN2021078798-appb-000085
将2-氨基-6硝基苯并噻唑(1g,5.12mmol)悬浮于二氯甲烷(15mL)中，依次加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI,2.93g,0.71mL,1.53mmol)、4-二甲氨基吡啶(DMAP,187mg,0.153mmol)和65a(2.17g，1.53mmol)，室温搅拌至TLC监测反应完全后，加入二氯甲烷(15mL)稀释，反应液依次用饱和碳酸氢钠水溶液(35mL)、1N的稀盐酸(35mL)和饱和食盐水(35mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩后经二氯甲烷(4mL)和正己烷(4mL)打浆，抽滤得到化合物65b(黄白色固体,775mg,产率48％)。Suspend 2-amino-6nitrobenzothiazole (1g, 5.12mmol) in dichloromethane (15mL), and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloric acid successively Salt (EDCI, 2.93g, 0.71mL, 1.53mmol), 4-dimethylaminopyridine (DMAP, 187mg, 0.153mmol) and 65a (2.17g, 1.53mmol), stir at room temperature until TLC monitoring the reaction is complete, add dichloride Diluted with methane (15mL), the reaction solution was washed with saturated sodium bicarbonate aqueous solution (35mL), 1N dilute hydrochloric acid (35mL) and saturated brine (35mL) successively, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and passed through dichloromethane (4 mL) and n-hexane (4 mL) to be slurried, and filtered with suction to obtain compound 65b (yellow-white solid, 775 mg, yield 48%).
参考实施例63的合成步骤，将中间体63b替换成65b，得到化合物65(白色固体,136mg)。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 C 22H 20N 3O 6S 5[M+H] +581.9950,found 581.9949。 Referring to the synthesis procedure of Example 63, the intermediate 63b was replaced with 65b to obtain compound 65 (white solid, 136 mg). 1H NMR (300MHz, DMSO-d 6 ) δ12.66 (s, 1H), 8.22 (d, J = 4.9 Hz, 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 C 22 H 20 N 3 O 6 S 5 [M+H] + 581.9950, found 581.9949.
实施例66Example 66
2-(哌啶-4-基)-N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)乙酰胺(化合物66)2-(piperidin-4-yl)-N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamide)benzo(d)thiazol-2-yl)acetamide( Compound 66)
Figure PCTCN2021078798-appb-000086
Figure PCTCN2021078798-appb-000086
参考实施例65的合成步骤，将原料65a替换成1-叔丁氧羰基-4-哌啶乙酸得到中间体66a(白色固体,497mg)。Referring to the synthesis procedure of Example 65, the raw material 65a was replaced with 1-tert-butoxycarbonyl-4-piperidineacetic acid to obtain intermediate 66a (white solid, 497 mg).
将化合物66a(497mg,0.73mmol)溶于三氟乙酸(4mL)和二氯甲烷(4mL)中，室温搅拌至TLC监测反应完全后，将反应液浓缩，以饱和碳酸氢钠溶液调pH至8-9，抽滤，红外下干燥得到化合物66(白色固体,420mg,99％)： 1H NMR(300MHz,DMSO-d 6)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 C 22H 23N 4O 5S 5[M+H] +583.0266,found 583.0280。 Compound 66a (497mg, 0.73mmol) was dissolved in trifluoroacetic acid (4mL) and dichloromethane (4mL), stirred at room temperature until TLC monitoring the reaction was complete, the reaction solution was concentrated, and the pH was adjusted to 8 with saturated sodium bicarbonate solution -9, suction filtration, and drying under infrared to obtain compound 66 (white solid, 420mg, 99%): 1 H NMR (300MHz, DMSO-d 6 )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 C 22 H 23 N 4 O 5 S 5 (M +H] + 583.0266,found 583.0280.
实施例67Example 67
1-甲基-N-(6-(N-(甲基磺酰基)甲基磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺(化合物67)1-Methyl-N-(6-(N-(methylsulfonyl)methylsulfonamide)benzo(d)thiazol-2-yl)piperidine-4-carboxamide (Compound 67)
Figure PCTCN2021078798-appb-000087
Figure PCTCN2021078798-appb-000087
参考实施例61的合成步骤，将2-噻吩磺酰氯替换成甲基磺酰氯，得到化合物67(白色固体,85mg)。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 C 16H 23N 4O 5S 3[M+H] +447.0825,found 447.0827。 Referring to the synthesis procedure of Example 61, 2-thiophenesulfonyl chloride was replaced with methylsulfonyl chloride to obtain compound 67 (white solid, 85 mg). 1H NMR (300MHz, DMSO-d 6 ) δ 12.46 (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 C 16 H 23 N 4 O 5 S 3 [M+H] + 447.0825, found 447.0827.
实施例68Example 68
1-甲基-N-(6-(N-(苯磺酰基)苯基磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺(化合物68)1-Methyl-N-(6-(N-(phenylsulfonyl)phenylsulfonamido)benzo[d]thiazol-2-yl)piperidine-4-carboxamide (Compound 68)
Figure PCTCN2021078798-appb-000088
Figure PCTCN2021078798-appb-000088
参考实施例61的合成步骤，将2-噻吩磺酰氯替换成苯磺酰氯，得到化合物68(白色固体,195mg)。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 C 26H 27N 4O 5S 3[M+H] +571.1138,found 571.1148。 Referring to the synthesis procedure of Example 61, 2-thiophenesulfonyl chloride was replaced with benzenesulfonyl chloride to obtain compound 68 (white solid, 195 mg). 1H NMR (300MHz, DMSO-d 6 ) δ 12.40 (s, 1H), 7.91–7.76 (m, 7H), 7.75–7.62 (m, 5H), 6.95 (d, J = 8.6 Hz, 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 C 26 H 27 N 4 O 5 S 3 [M+H] + 571.1138, found 571.1148.
实施例69Example 69
1-甲基-N-(6-(((1-甲基-N-((1-甲基-1H-吡唑-4-基)磺酰基)-1H-吡唑)-4-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺(化合物69)1-methyl-N-(6-(((1-methyl-N-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-1H-pyrazole)-4-sulfonamide Yl)benzo(d)thiazol-2-yl)piperidine-4-carboxamide (Compound 69)
Figure PCTCN2021078798-appb-000089
Figure PCTCN2021078798-appb-000089
参考实施例61的合成步骤，将2-噻吩磺酰氯替换成1-甲基-1H-吡唑-3-磺酰氯，得到化合物69(白色固体,137mg)。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 C 22H 27N 8O 5S 3[M+H] +579.1261,found 579.1267。 Referring to the synthesis procedure of Example 61, 2-thiophenesulfonyl chloride was replaced with 1-methyl-1H-pyrazole-3-sulfonyl chloride to obtain compound 69 (white solid, 137 mg). 1H NMR (300MHz, DMSO-d 6 ) δ 12.46 (s, 1H), 8.40 (s, 2H), 7.85 (d, J = 2.1 Hz, 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 C 22 H 27 N 8 O 5 S 3 [M+H] + 579.1261,found 579.1267.
实施例70Example 70
1-甲基-N-(6-(N-(吡啶-3-基磺酰基)吡啶-3-磺酰胺基)苯并[d]噻唑-2-基)哌啶-4-羧酰胺(化合物70)1-Methyl-N-(6-(N-(pyridin-3-ylsulfonyl)pyridine-3-sulfonamide)benzo(d)thiazol-2-yl)piperidine-4-carboxamide (compound 70)
Figure PCTCN2021078798-appb-000090
Figure PCTCN2021078798-appb-000090
参考实施例61的合成步骤，将2-噻吩磺酰氯替换成3-吡啶磺酰氯，得到化合物70(白色固体,130mg)。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 C 24H 25N 6O 5S 3[M+H] +573.1043,found 573.10396。 Referring to the synthesis procedure of Example 61, 2-thiophenesulfonyl chloride was replaced with 3-pyridinesulfonyl chloride to obtain compound 70 (white solid, 130 mg). 1H NMR (300MHz, DMSO-d 6 ) δ 12.43 (s, 1H), 9.02 (d, J = 4.0 Hz, 2H), 8.97 (d, J = 1.9 Hz, 2H), 8.28 (d, J = 8.2 Hz, 2H), 7.98 (d, J = 1.9 Hz, 1H), 7.87–7.70 (m, 3H), 7.06 (dd, J = 8.6, 2.0 Hz, 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 C 24 H 25 N 6 O 5 S 3 [M+H] + 573.1043,found 573.10396.
实施例71Example 71
2-氧代-2-((6-(N-(噻吩-2-基磺酰基)噻吩-2-磺胺基)苯并[d]噻唑-2-基)氨基)乙烷-1-氯化铵(化合物71)2-oxo-2-((6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonyl)benzo(d)thiazol-2-yl)amino)ethane-1-chloride Ammonium (Compound 71)
Figure PCTCN2021078798-appb-000091
Figure PCTCN2021078798-appb-000091
将2-氨基-6硝基苯并噻唑(1g,5.12mmol)悬浮于二氯甲烷(40mL)中，依次加入Boc 2O(1.34g,6.15mmol)和4-二甲氨基吡啶(DMAP,751mg，6.15mmol)，室温搅拌至TLC监测反应完全。过滤收集固体，以二氯甲烷(4mL)洗涤滤饼，得到化合物71b(白色固体,1.26g,产率84％)。 Suspend 2-amino-6nitrobenzothiazole (1g, 5.12mmol) in dichloromethane (40mL), add Boc 2 O (1.34g, 6.15mmol) and 4-dimethylaminopyridine (DMAP, 751mg , 6.15 mmol), and stir at room temperature until TLC monitors that the reaction is complete. The solid was collected by filtration, and the filter cake was washed with dichloromethane (4 mL) to obtain compound 71b (white solid, 1.26 g, yield 84%).
将化合物71b(1.6mg,5.42mmol)悬浮于甲醇(50mL)中，加入10％钯碳(160mg,10％)，在氢气氛围下室温搅拌至TLC监测反应完全。反应完全后经硅藻土抽滤，滤液减压蒸除溶剂后即得到中间体71c(粉色固体,1.3g,产率90％)。 1H NMR(300MHz,DMSO-d 6)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)。 Compound 71b (1.6 mg, 5.42 mmol) was suspended in methanol (50 mL), 10% palladium on carbon (160 mg, 10%) was added, and stirred at room temperature under a hydrogen atmosphere until the reaction was completed as monitored by TLC. After the completion of the reaction, it was suction filtered through diatomaceous earth, and the filtrate was evaporated under reduced pressure to remove the solvent to obtain Intermediate 71c (pink solid, 1.3 g, yield 90%). 1 H NMR(300MHz,DMSO-d 6 )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).
参考实施例65的合成步骤，将中间体65c替换成71c，得到中间体71d(白色固体,1.2g,产率72％)。 1H NMR(300MHz,DMSO-d 6)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)。 Referring to the synthesis procedure of Example 65, intermediate 65c was replaced with 71c to obtain intermediate 71d (white solid, 1.2 g, yield 72%). 1 H NMR (300MHz, DMSO-d 6 ) δ 11.96 (s, 1H), 8.23 (d, J = 4.9 Hz, 2H), 7.79-7.64 (m, 4H), 7.29 (t, J = 4.3 Hz, 2H), 7.00–6.91 (m, 1H), 1.52 (s, 9H).
参考化实施例66的合成步骤，将中间体66a替换成71d，得到中间体71e(白色固体,806mg,82％)。 1H NMR(300MHz,DMSO-d 6)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)。 Referring to the synthesis procedure of Example 66, the intermediate 66a was replaced with 71d to obtain the intermediate 71e (white solid, 806 mg, 82%). 1 H NMR (300MHz, DMSO-d 6 ) δ 8.21 (d, J = 4.0 Hz, 2H), 7.78 (s, 2H), 7.72 (d, J = 2.8 Hz, 2H), 7.45 (d, J = 2.0Hz, 1H), 7.34–7.22 (m, 3H), 6.75 (dd, J=8.5, 2.0Hz, 1H).
参考实施例65合成步骤，将原料65a替换成Boc-甘氨酸，将2-氨基-6硝基苯并噻唑替换成71e，得到中间体71f(白色固体,240mg,85％)。Referring to the synthesis step of Example 65, the raw material 65a was replaced with Boc-glycine, and the 2-amino-6nitrobenzothiazole was replaced with 71e to obtain intermediate 71f (white solid, 240 mg, 85%).
取中间体71f(240mg,0.39mmol)溶于盐酸乙酸乙酯溶液(6mL)，室温搅拌过夜，TLC监测反应完全后，抽滤，滤饼烘干，得到化合物71(白色固体,101mg,产率47％)。 1H NMR(300MHz,DMSO-d 6)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 C 17H 15N 4O 5S 5[M-Cl] +514.9640,found 514.9645。 Intermediate 71f (240mg, 0.39mmol) was dissolved in a hydrochloric acid ethyl acetate solution (6mL), stirred overnight at room temperature, TLC monitored the completion of the reaction, filtered with suction, and the filter cake was dried to obtain compound 71 (white solid, 101mg, yield 47%). 1 H NMR (300MHz, DMSO-d 6 ) δ 13.07 (s, 1H), 8.44 (s, 3H), 8.24 (d, J = 4.9 Hz, 2H), 7.94 (d, J = 2.1 Hz, 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 C 17 H 15 N 4 O 5 S 5 [M-Cl] + 514.9640, found 514.9645.
实施例72Example 72
2-((6-((1-甲基-N-((1-甲基-1H-吡唑-4-基)磺酰基)-1H-吡唑)-4-磺酰胺基)苯并[d]噻唑-2-基)氨基)-2-氧代乙烷-1-氯化铵(化合物72)2-((6-((1-methyl-N-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-1H-pyrazole)-4-sulfonamido)benzo[ d)thiazol-2-yl)amino)-2-oxoethane-1-ammonium chloride (compound 72)
Figure PCTCN2021078798-appb-000092
Figure PCTCN2021078798-appb-000092
Figure PCTCN2021078798-appb-000093
Figure PCTCN2021078798-appb-000093
参考实施例65的合成路线，将原料65a替换成Boc-甘氨酸，2-噻吩磺酰氯替换成1-甲基-1H-吡唑-3-磺酰氯，得到中间体72a(黄色固体,150mg)。Referring to the synthetic route of Example 65, the raw material 65a was replaced with Boc-glycine, and 2-thiophenesulfonyl chloride was replaced with 1-methyl-1H-pyrazole-3-sulfonyl chloride to obtain intermediate 72a (yellow solid, 150 mg).
参考实施例71的合成路线，将71f替换成72a，得到化合物72(淡黄色固体,81mg,90％)。 1H NMR(300MHz,DMSO-d 6)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 C 17H 19N 8O 5S 5[M-Cl] +511.0635,found 511.0646。 Referring to the synthetic route of Example 71, 71f was replaced with 72a to obtain compound 72 (pale yellow solid, 81 mg, 90%). 1 H NMR (300MHz, DMSO-d 6 ) δ 13.03 (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 C 17 H 19 N 8 O 5 S 5 (M- Cl] + 511.0635, found 511.0646.
实施例73Example 73
2-((6-(N-(环丙基磺酰基)环丙烷磺酰胺基)苯并[d]噻唑-2-基)氨基)-2-氧杂-1-氯化铵(化合物73)2-((6-(N-(Cyclopropylsulfonyl)cyclopropanesulfonamido)benzo(d)thiazol-2-yl)amino)-2-oxa-1-ammonium chloride (Compound 73)
Figure PCTCN2021078798-appb-000094
Figure PCTCN2021078798-appb-000094
参考实施例72的合成路线，将1-甲基-1H-吡唑-3-磺酰氯替换成环丙磺酰氯，得到化合物73(白色固体,80mg)。 1H NMR(300MHz,DMSO-d 6)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 C 15H 19N 4O 5S 3[M-Cl] +431.0512,found 431.0518。 Referring to the synthetic route of Example 72, 1-methyl-1H-pyrazole-3-sulfonyl chloride was replaced with cyclopropanesulfonyl chloride to obtain compound 73 (white solid, 80 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ13.01 (s, 1H), 8.36 (s, 3H), 8.22 (d, J = 2.1 Hz, 1H), 7.84 (d, J = 8.6 Hz, 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 C 15 H 19 N 4 O 5 S 3 [M-Cl] + 431.0512, found 431.0518.
实施例74Example 74
反式-4-氨基-N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环己烷-1-羧酰胺(化合物74)Trans-4-amino-N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)cyclohexane-1-carboxy Amide (Compound 74)
Figure PCTCN2021078798-appb-000095
Figure PCTCN2021078798-appb-000095
参考实施例66的合成步骤，将1-叔丁氧羰基-4-哌啶乙酸替换成反式-4-(Boc-氨基)环己烷羧酸，得到化合物74(白色固体,130mg)。 1H NMR(300MHz,DMSO-d 6)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 C 22H 23N 4O 5S 5[M+H] +583.0266,found 583.0279。 Referring to the synthesis procedure of Example 66, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with trans-4-(Boc-amino)cyclohexanecarboxylic acid to obtain compound 74 (white solid, 130 mg). 1 H NMR(300MHz,DMSO-d 6 )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 C 22 H 23 N 4 O 5 S 5 [M+H] + 583.0266,found 583.0279.
实施例75Example 75
3-((6-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)氨基甲酰基)氮杂环丁烷盐酸盐(化合物75)3-((6-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)carbamoyl)azetidine salt Acid salt (compound 75)
Figure PCTCN2021078798-appb-000096
Figure PCTCN2021078798-appb-000096
参考实施例71的合成步骤，将Boc-甘氨酸替换成1-Boc-氮杂环丁烷-3-羧酸，得到化合物75(白色固体,120mg)。 1H NMR(300MHz,DMSO-d 6)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 C 19H 17N 4O 5S 5[M-Cl] +550.9797,found 550.9805。 Referring to the synthesis procedure of Example 71, Boc-glycine was replaced with 1-Boc-azetidine-3-carboxylic acid to obtain compound 75 (white solid, 120 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.79 (s, 1H), 9.47 (s, 1H), 9.06 (s, 1H), 8.24 (d, J = 4.8 Hz, 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 C 19 H 17 N 4 O 5 S 5 [M-Cl] + 550.9797,found 550.9805.
实施例76Example 76
2-((6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)氨基甲酰基)吡咯盐酸盐(化合物76)2-((6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)carbamoyl)pyrrole hydrochloride (Compound 76)
Figure PCTCN2021078798-appb-000097
Figure PCTCN2021078798-appb-000097
参考实施例71的合成步骤，将Boc-甘氨酸替换成N-Boc-DL-脯氨酸，得到化合物76(白色固体,140mg)。 1H NMR(300MHz,DMSO-d 6)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 C 20H 19N 4O 5S 5[M-Cl] +554.9953,found 554.9963。 Referring to the synthesis procedure of Example 71, Boc-glycine was replaced with N-Boc-DL-proline to obtain compound 76 (white solid, 140 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 13.23 (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 C 20 H 19 N 4 O 5 S 5 [M-Cl] + 554.9953,found 554.9963.
实施例77Example 77
1-甲基-N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)-1H-吡咯-2-羧酰胺(化合物77)1-Methyl-N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamide)benzo[d]thiazol-2-yl)-1H-pyrrole-2-carboxamide (Compound 77)
Figure PCTCN2021078798-appb-000098
Figure PCTCN2021078798-appb-000098
参考实施例71的合成步骤，将Boc-甘氨酸替换成N-甲基-2-吡咯羧酸，得到化合物77(白色固体,129mg)。 1H NMR(300MHz,CDCl 3)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 C 21H 17N 4O 5S 5[M+H] +564.9797,found 564.97935。 Referring to the synthesis procedure of Example 71, Boc-glycine was replaced with N-methyl-2-pyrrole carboxylic acid to obtain compound 77 (white solid, 129 mg). 1 H NMR(300MHz, CDCl 3 )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 C 21 H 17 N 4 O 5 S 5 [M+H] + 564.9797, found 564.97935.
实施例78Example 78
2-(二甲基氨基)-N-(6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)乙酰胺(化合物78)2-(Dimethylamino)-N-(6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)acetamide (Compound 78 )
Figure PCTCN2021078798-appb-000099
Figure PCTCN2021078798-appb-000099
参考实施例71的合成步骤，将Boc-甘氨酸替换成N,N-二甲基甘氨酸得到化合物78(白色固体,75mg)。 1H NMR(300MHz,CDCl 3)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 C 19H 19N 4O 5S 5[M+H] +542.99535,found 542.99463。 Referring to the synthesis procedure of Example 71, Boc-glycine was replaced with N,N-dimethylglycine to obtain compound 78 (white solid, 75 mg). 1 H NMR(300MHz,CDCl 3 )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 C 19 H 19 N 4 O 5 S 5 [M+H] + 542.99535,found 542.99463.
实施例79Example 79
4-氧代-4-((6-(N-(噻吩-2-基磺酰基)噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)氨基)丁酸(化合物79)4-oxo-4-((6-(N-(thiophen-2-ylsulfonyl)thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)amino)butanoic acid (Compound 79)
Figure PCTCN2021078798-appb-000100
Figure PCTCN2021078798-appb-000100
参考实施例71的合成步骤，将Boc-甘氨酸替换成丁二酸酐得到化合物79(白色固体,64mg)。 1H NMR(300MHz,DMSO-d 6)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 C 19H 16N 3O 7S 5[M+H] +557.9586,found 557.9595。 Referring to the synthesis procedure of Example 71, Boc-glycine was replaced with succinic anhydride to obtain compound 79 (white solid, 64 mg). 1 H NMR(300MHz,DMSO-d 6 )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 C 19 H 16 N 3 O 7 S 5 [M+H] + 557.9586,found 557.9595.
实施例80Example 80
2-(哌啶-4-基)-N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)乙酰胺(化合物80)2-(piperidin-4-yl)-N-(6-(thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)acetamide (Compound 80)
Figure PCTCN2021078798-appb-000101
Figure PCTCN2021078798-appb-000101
参考实施例65的合成步骤，将65a替换成1-叔丁氧羰基-4-哌啶乙酸，得到中间体80a(淡红色固体,628mg)。Referring to the synthesis procedure of Example 65, 65a was replaced with 1-tert-butoxycarbonyl-4-piperidineacetic acid to obtain intermediate 80a (pale red solid, 628 mg).
将中间体80a(350mg,0.90mmol)溶于二氯甲烷(8mL)和吡啶(1mL)中，缓慢加入2-噻吩磺酰氯(196mg,1.08mmol)，室温搅拌至TLC监测反应完全，加入乙酸乙酯(20mL)稀释，依次用1N的稀盐酸(20mL×2)和饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤，滤液浓缩，经硅胶柱层析(二氯甲烷:甲醇＝50:1)纯化得到化合物80b(白色固体,454mg,产率94％)。 1H NMR(300MHz,DMSO-d 6)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)。 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 TLC monitoring the reaction was complete, and ethyl acetate was added Ester (20mL) was diluted, washed with 1N dilute hydrochloric acid (20mL×2) and saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and subjected to silica gel column chromatography (dichloromethane: methanol = 50 1) Purification to obtain compound 80b (white solid, 454 mg, yield 94%). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.29 (s, 1H), 10.41 (s, 1H), 7.87 (dd, J = 5.0, 1.3 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.51 (dd, J = 3.7, 1.3 Hz, 1H), 7.16 (dd, J = 8.7, 2.1 Hz, 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).
参考实施例66的合成步骤，将中间体66a替换成80b，得到化合物80(淡黄色固体,304mg,83％)。 1H NMR(300MHz,DMSO-d 6)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 C 18H 21N 4O 3S 3[M+H] +437.0770,found 437.0780。 Referring to the synthesis procedure of Example 66, the intermediate 66a was replaced with 80b to obtain compound 80 (pale yellow solid, 304 mg, 83%). 1 H NMR (300MHz, DMSO-d 6 ) δ 7.65 (d, J = 4.9 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.47 (d, J = 8.7 Hz, 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 C 18 H 21 N 4 O 3 S 3 [M+H] + 437.0770, found 437.0780.
实施例81Example 81
4-氧代-N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环己烷-1-羧酰胺(化合物81)4-oxo-N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)cyclohexane-1-carboxamide (Compound 81)
Figure PCTCN2021078798-appb-000102
Figure PCTCN2021078798-appb-000102
参考实施例80的合成步骤，将1-叔丁氧羰基-4-哌啶乙酸替换成4-羰基环己羧酸，得到化合物81(白色固体,187mg)。 1H NMR(300MHz,DMSO-d 6)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 C 18H 18N 3O 4S 3[M+H] +436.0454,found 436.0456。 Referring to the synthesis procedure of Example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with 4-carbonylcyclohexancarboxylic acid to obtain compound 81 (white solid, 187 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.44 (s, 1H), 10.42 (s, 1H), 7.87 (d, J = 4.9 Hz, 1H), 7.70 (d, J = 2.0 Hz, 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 C 18 H 18 N 3 O 4 S 3 [M+H] + 436.0454, found 436.0456.
实施例82Example 82
N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)氮杂环丁烷-3-羧酰胺盐酸盐(化合物82)N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)azetidine-3-carboxamide hydrochloride (Compound 82)
Figure PCTCN2021078798-appb-000103
Figure PCTCN2021078798-appb-000103
参考实施例80的合成路线，将1-叔丁氧羰基-4-哌啶乙酸替换成1-Boc-氮杂环丁烷-3-羧酸，得到中间体82a(白色固体,102mg)。 1H NMR(300MHz,DMSO-d 6)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 route of Example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with 1-Boc-azetidine-3-carboxylic acid to obtain intermediate 82a (white solid, 102 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.42 (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).
参考实施例72的合成步骤，将中间体72a替换成82a，得到化合物82(白色固体,37mg,产率42％)。 1H NMR(300MHz,DMSO-d 6)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 C 15H 15N 4O 3S 3[M-Cl] +395.0301,found 395.0310。 Referring to the synthesis procedure of Example 72, the intermediate 72a was replaced with 82a to obtain compound 82 (white solid, 37 mg, yield 42%). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.55 (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 C 15 H 15 N 4 O 3 S 3 [M-Cl] + 395.0301, found 395.0310.
实施例83Example 83
反式-4-氨基-N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环己烷-1-羧酰胺(化合物83)Trans-4-amino-N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)cyclohexane-1-carboxamide (Compound 83)
Figure PCTCN2021078798-appb-000104
Figure PCTCN2021078798-appb-000104
参考实施例80的合成步骤，将1-叔丁氧羰基-4-哌啶乙酸替换成反式-4-(Boc-氨基)环己烷羧酸，得到化合物83(白色固体,70mg)。 1H NMR(300MHz,DMSO-d 6)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 C 18H 21N 4O 3S 3[M+H] +437.0770,found 437.0769。 Referring to the synthesis procedure of Example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with trans-4-(Boc-amino)cyclohexanecarboxylic acid to obtain compound 83 (white solid, 70 mg). 1 H NMR(300MHz,DMSO-d 6 )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 C 18 H 21 N 4 O 3 S 3 [M+H] + 437.0770, found 437.0769.
实施例84Example 84
4-(二甲氨基)-N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)苯甲酰胺(化合物84)4-(Dimethylamino)-N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)benzamide (Compound 84)
Figure PCTCN2021078798-appb-000105
Figure PCTCN2021078798-appb-000105
参考实施例80的合成步骤，将1-叔丁氧羰基-4-哌啶乙酸替换成4-二甲氨基苯甲酸，得到化合物84(白色固体,55mg)。 1H NMR(300MHz,DMSO-d 6)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 C 20H 19N 4O 3S 3[M+H] +459.0614,found 459.0620。 Referring to the synthesis procedure of Example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with 4-dimethylaminobenzoic acid to obtain compound 84 (white solid, 55 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.40 (s, 1H), 10.42 (s, 1H), 8.03 (d, J = 8.9 Hz, 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 C 20 H 19 N 4 O 3 S 3 [M+H] + 459.0614, found 459.0620.
实施例85Example 85
3-(N-(2-(四氢-2H-吡喃-4-甲酰胺基)苯并[d]噻唑-6-基)氨磺酰基)丙酸甲酯(化合物85)Methyl 3-(N-(2-(tetrahydro-2H-pyran-4-carboxamido)benzo(d)thiazol-6-yl)sulfamoyl)propionate (Compound 85)
Figure PCTCN2021078798-appb-000106
Figure PCTCN2021078798-appb-000106
参考实施例80的合成步骤，将1-叔丁氧羰基-4-哌啶乙酸替换成四氢吡喃-4-甲酸，将2-噻吩磺酰氯替换成3-(氯磺酰基)丙酸甲酯，得到化合物85(白色固体,160mg)。 1H NMR(300MHz,DMSO-d 6)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 C 17H 22N 3O 6S 2[M+H] +428.0945,found 428.0950。 Refer to the synthesis procedure of Example 80, replace 1-tert-butoxycarbonyl-4-piperidineacetic acid with tetrahydropyran-4-carboxylic acid, and replace 2-thiophenesulfonyl chloride with 3-(chlorosulfonyl)propionate methyl Ester to obtain compound 85 (white solid, 160 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.32 (s, 1H), 9.96 (s, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 8.7 Hz, 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 C 17 H 22 N 3 O 6 S 2 [M+H] + 428.0945, found 428.0950.
实施例86Example 86
3-(N-(2-(四氢-2H-吡喃-4-甲酰胺基)苯并[d]噻唑-6-基)氨磺酰基)丙酸(化合物86)3-(N-(2-(tetrahydro-2H-pyran-4-carboxamido)benzo[d]thiazol-6-yl)sulfamoyl)propionic acid (Compound 86)
Figure PCTCN2021078798-appb-000107
Figure PCTCN2021078798-appb-000107
参考实施例53的合成步骤，将53a替换成85，得到化合物86(白色固体,50mg)。 1H NMR(300MHz,DMSO-d 6)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 C 16H 20N 3O 6S 2[M+H] +414.0788,found 414.0791。 Referring to the synthesis procedure of Example 53, 53a was replaced with 85 to obtain compound 86 (white solid, 50 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.51 (s, 1H), 12.33 (s, 1H), 9.93 (s, 1H), 7.79 (d, J = 2.0 Hz, 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 C 16 H 20 N 3 O 6 S 2 [M+H] + 414.0788, found 414.0791.
实施例87Example 87
3-(N-(2-(1-甲基哌啶-4-甲酰胺基)苯并[d]噻唑-6-基)氨磺酰基)丙酸(化合物87)3-(N-(2-(1-methylpiperidine-4-carboxamido)benzo(d)thiazol-6-yl)sulfamoyl)propionic acid (Compound 87)
Figure PCTCN2021078798-appb-000108
Figure PCTCN2021078798-appb-000108
参考实施例85的合成步骤，将四氢吡喃-4-甲酸替换成1-甲基哌啶-4-甲酸，得到化合物87的甲酯。再参考实施例86的合成步骤，将化合物85替换成化合物87的甲酯，得到化合物87(白色固体,127mg)。 1H NMR(300MHz,DMSO-d 6)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  C 17H 23N 4O 5S 2[M+H] +427.1104,found 427.1111。 Referring to the synthesis procedure of Example 85, tetrahydropyran-4-carboxylic acid was replaced with 1-methylpiperidine-4-carboxylic acid to obtain the methyl ester of compound 87. Again referring to the synthesis procedure of Example 86, compound 85 was replaced with the methyl ester of compound 87 to obtain compound 87 (white solid, 127 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.49 (s, 1H), 10.43 (s, 1H), 9.96 (s, 1H), 7.81 (d, J = 2.0 Hz, 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 C 17 H 23 N 4 O 5 S 2 [M+H] + 427.1104, found 427.1111.
实施例88Example 88
4-甲基-N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)哌嗪-1-甲酰胺(化合物88)4-Methyl-N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)piperazine-1-carboxamide (Compound 88)
Figure PCTCN2021078798-appb-000109
Figure PCTCN2021078798-appb-000109
参考实施例58的合成步骤，将吗啉替换甲基哌嗪得到化合物88(淡黄色固体,105mg)。 1H NMR(300MHz,DMSO-d 6)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 C 17H 20N 5O 3S 3[M+H] +438.0723,found 438.0725。 Referring to the synthesis procedure of Example 58, morpholine was substituted for methylpiperazine to obtain compound 88 (pale yellow solid, 105 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 11.39 (s, 1H), 10.45 (s, 1H), 7.87 (d, J = 4.8 Hz, 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 C 17 H 20 N 5 O 3 S 3 [M+H] + 438.0723, found 438.0725.
实施例89Example 89
2-(二甲基氨基)-N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)乙酰胺(化合物89)2-(Dimethylamino)-N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)acetamide (Compound 89)
Figure PCTCN2021078798-appb-000110
Figure PCTCN2021078798-appb-000110
参考实施例80的合成步骤，将1-叔丁氧羰基-4-哌啶乙酸替换成N,N-二甲基甘氨酸，得到化合物89(白色固体,65mg)。 1H NMR(300MHz,DMSO-d 6)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 C 15H 17N 4O 3S 3[M+H] +397.0457,found 397.0461。 Referring to the synthesis procedure of Example 80, 1-tert-butoxycarbonyl-4-piperidineacetic acid was replaced with N,N-dimethylglycine to obtain compound 89 (white solid, 65 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 11.71 (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 C 15 H 17 N 4 O 3 S 3 [M+H] + 397.0457, found 397.0461.
实施例90Example 90
1-甲基-N-(6-(苯基磺胺基)苯并噻唑-2-基)哌啶-4-甲酰胺(化合物90)1-Methyl-N-(6-(phenylsulfonyl)benzothiazol-2-yl)piperidine-4-carboxamide (Compound 90)
Figure PCTCN2021078798-appb-000111
Figure PCTCN2021078798-appb-000111
参考实施例55的合成步骤，将2-噻吩磺酰氯替换成苯磺酰氯，得到化合物90(白色固体,132mg)。 1H NMR(300MHz,DMSO-d 6)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 C 22H 23N 4O 3S 2[M+H] +431.1206,found 431.1207。 Referring to the synthesis procedure of Example 55, 2-thiophenesulfonyl chloride was replaced with benzenesulfonyl chloride to obtain compound 90 (white solid, 132 mg). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.05 (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 C 22 H 23 N 4 O 3 S 2 [M+H] + 431.1206, found 431.1207.
实施例91Example 91
N-(6-((5-氯噻吩)-2-磺胺基)苯并噻唑-2-基)-1-甲基哌啶-4-甲酰胺(91)N-(6-((5-Chlorothiophene)-2-sulfo)benzothiazol-2-yl)-1-methylpiperidine-4-carboxamide (91)
Figure PCTCN2021078798-appb-000112
Figure PCTCN2021078798-appb-000112
参考实施例85的合成步骤，将2-噻吩磺酰氯替换成5-氯-2-噻吩磺酰氯，得到化合物91(粉色固体,84mg)。 1H NMR(300MHz,DMSO-d 6)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 C 18H 19ClN 4O 3S 3[M+H] +471.0381,found 471.0391。 Referring to the synthesis procedure of Example 85, 2-thiophenesulfonyl chloride was replaced with 5-chloro-2-thiophenesulfonyl chloride to obtain compound 91 (pink solid, 84 mg). 1 H NMR(300MHz,DMSO-d 6 )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 C 18 H 19 ClN 4 O 3 S 3 [M+H] + 471.0381, found 471.0391.
实施例92Example 92
N-(6-(噻吩-2-磺胺基)苯并[d]噻唑-2-基)乙酰胺(化合物92)N-(6-(thiophen-2-sulfonyl)benzo(d)thiazol-2-yl)acetamide (Compound 92)
Figure PCTCN2021078798-appb-000113
Figure PCTCN2021078798-appb-000113
参考实施例1的合成实验步骤，将糠酰氯替换成乙酰氯，制得N-(6-氨基苯并[d]噻唑-2-基)乙酰胺。再参考实施例23的合成实验步骤，将对硝基苯甲酰氯替换成2-噻吩磺酰氯，将1c替换成N-(6-氨基苯并[d]噻唑-2-基)乙酰胺，得到化合物92(白色固体)。 1H NMR(300MHz,DMSO-d 6)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] -。 Referring to the synthetic experimental procedure of Example 1, the furoyl chloride was replaced with acetyl chloride to prepare N-(6-aminobenzo[d]thiazol-2-yl)acetamide. Again referring to the synthetic experimental procedure in 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 obtain Compound 92 (white solid). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.30 (s, 1H), 10.42 (s, 1H), 7.87 (d, J = 4.8 Hz, 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[MH] - .
实施例93Example 93
N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环戊烷甲酰胺(化合物93)N-(6-(thiophen-2-sulfonamido)benzo[d]thiazol-2-yl)cyclopentanecarboxamide (compound 93)
Figure PCTCN2021078798-appb-000114
Figure PCTCN2021078798-appb-000114
参考实施例92的合成实验步骤，将乙酰氯替换成环戊酰氯，得到化合物93(黄色固体)。 1H NMR(300MHz,DMSO-d 6)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] +。 Referring to the synthetic experimental procedure of Example 92, acetyl chloride was replaced with cyclopentanoyl chloride to obtain compound 93 (yellow solid). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.31 (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] + .
实施例94Example 94
N-(6-(噻吩-2-磺酰胺基)苯并[d]噻唑-2-基)环己烷甲酰胺(94)N-(6-(thiophen-2-sulfonamido)benzo(d)thiazol-2-yl)cyclohexanecarboxamide (94)
Figure PCTCN2021078798-appb-000115
Figure PCTCN2021078798-appb-000115
参考实施例92的合成实验步骤，将乙酰氯替换成环己甲酰氯，得到化合物94(白色固体)。 1H NMR(300MHz,DMSO-d 6)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] -。 Referring to the synthetic experimental procedure of Example 92, acetyl chloride was replaced with cyclohexanoyl chloride to obtain compound 94 (white solid). 1 H NMR (300MHz, DMSO-d 6 ) δ 12.22 (s, 1H), 10.40 (s, 1H), 7.93-7.81 (m, 1H), 7.68 (d, J = 2.0 Hz, 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[MH] - .
实施例95Example 95
微量热泳动(MST)法测定化合物与USP7 C端蛋白结合力Micro thermophoresis (MST) method to determine the binding ability of compounds with USP7 C-terminal protein
1.实验目的1. The purpose of the experiment
采用MST法测定化合物与USP7 C端蛋白的结合力，并计算各自的平衡解离常数(K D)。 The MST method was used to determine the binding force of the compound to the C-terminal protein of USP7, and the respective equilibrium dissociation constants (K D ) were calculated.
2.实验材料2. Experimental materials
本发明化合物(用二甲基亚砜(DMSO)溶解配制成母液，使用前采用Tris-HCl缓冲液稀释成适当浓度)；USP7 C端蛋白(10μM)；微量热泳动仪(NT.115)；MST标准毛细管；MST蛋白荧光标记试剂盒(NT-647)；Tris-HCl缓冲液。The compound of the present invention (dissolved with dimethyl sulfoxide (DMSO) to prepare a mother liquor, dilute with Tris-HCl buffer to an appropriate concentration before use); USP7 C-terminal protein (10μM); Microthermophoresis (NT.115) ; MST standard capillary; MST protein fluorescent labeling kit (NT-647); Tris-HCl buffer.
3.实验方法3. Experimental method
(1)用PBS(pH7.4)将USP7 C端蛋白稀释为10μM的蛋白溶液，使用MST蛋白荧光标记试剂盒中的脱盐柱将PBS溶液置换为标记效率较高的标记缓冲液(100μL)，然后加入100μL含有荧光染料(NT-647)的标记缓冲液溶液，使染料终浓度为20μM，在室温下进行避光孵育；(2)孵育1小时后使用蛋白荧光标记试剂盒中的分离柱将标记后的蛋白与多余的荧光染料分离，得到500μL标记后的USP7 C端蛋白溶液；(3)使用MST标准毛细管吸取标记后的蛋白溶液，对其荧光强度进行检测，确定LED power 100％；(4)在0.2mL EP管中使用Tris-HCl缓冲液配制本发明小分子化合物的梯度稀释液，每管中小分子溶液体积为10μL，每管中DMSO含量保持一致(1％)；(5)向装有本发明化合物溶液的EP管中加入等体积(10μL)的蛋白溶液，混合均匀后室温孵育0.5小时；(6)使用MST标准毛细管吸取小分子-蛋白混合溶液(每个浓度一根毛细 管)，上机测试(LED power＝100％，MST power＝40％)，测试结果使用Themophoresis+T-Jump模式进行拟合。(1) Dilute the USP7 C-terminal protein into a 10μM protein solution with PBS (pH7.4), use the desalting column in the MST protein fluorescent labeling kit to replace the PBS solution with a labeling buffer (100μL) with higher labeling efficiency. Then add 100μL of labeling buffer solution containing fluorescent dye (NT-647) to make the final concentration of the dye 20μM, and incubate in the dark at room temperature; (2) After incubating for 1 hour, use the separation column in the protein fluorescent labeling kit to separate The labeled protein is separated from the excess fluorescent dye to obtain 500μL of labeled USP7 C-terminal protein solution; (3) Use MST standard capillary to suck up the labeled protein solution, detect its fluorescence intensity, and determine LED power 100%; ( 4) Prepare a gradient dilution of the small molecule compound of the present invention with Tris-HCl buffer in a 0.2mL EP tube, the volume of the small molecule solution in each tube is 10 μL, and the DMSO content in each tube remains the same (1%); (5) Add an equal volume (10μL) of protein solution to the EP tube containing the compound solution of the present invention, mix well and incubate at room temperature for 0.5 hours; (6) Use MST standard capillary to suck the small molecule-protein mixed solution (one capillary for each concentration) , Computer test (LED power=100%, MST power=40%), the test results are fitted using Thermophoresis+T-Jump mode.
4.实验结果4. Experimental results
部分化合物与USP7 C端蛋白的结合力(K D)测试结果如表1所示。实验结果表明，本发明化合物可与USP7 C端蛋白结合，且部分化合物亲和力较强，其他化合物也具有较好的亲和力。 Table 1 shows the binding force (K D ) test results of some compounds with the C-terminal protein of USP7. Experimental results show that the compound of the present invention can bind to the C-terminal protein of USP7, and some compounds have strong affinity, and other compounds also have good affinity.
表1、化合物1～39与USP7 C端蛋白的结合力Table 1. The binding ability of compounds 1～39 to the C-terminal protein of USP7
化合物Compound K D(μM) K D (μM) 化合物Compound K D(μM) K D (μM) 化合物Compound K D(μM) K D (μM) 化合物Compound K D(μM) K D (μM)
11 85.985.9 1212 68.068.0 23twenty three 0.40.4 3434 >250>250
22 >250>250 1313 45.345.3 24twenty four >250>250 3535 >250>250
33 25.625.6 1414 25.025.0 2525 2.22.2 3636 49.249.2
44 >250>250 1515 127.0127.0 2626 2.52.5 3737 2.62.6
55 105.0105.0 1616 108.0108.0 2727 76.476.4 3838 2.92.9
66 >250>250 1717 36.236.2 2828 85.585.5 3939 >250>250
77 47.047.0 1818 >250>250 2929 84.584.5  To  To
88 56.756.7 1919 >250>250 3030 63.463.4  To  To
99 8.48.4 2020 5.45.4 3131 >250>250  To  To
1010 >250>250 21twenty one >250>250 3232 52.152.1  To  To
1111 >250>250 22twenty two 3.33.3 3333 >250>250  To  To
实施例96Example 96
表面等离子体共振(SPR)法测定化合物与USP7 C端蛋白结合力Surface Plasmon Resonance (SPR) method to determine the binding ability of compounds with USP7 C-terminal protein
1.实验目的1. The purpose of the experiment
采用SPR法测定化合物与USP7 C端蛋白的结合力，并计算各自的平衡解离常数(K D)。 The SPR method was used to determine the binding force of the compound to the C-terminal protein of USP7, and the respective equilibrium dissociation constants (K D ) were calculated.
2.实验材料2. Experimental materials
本发明化合物(用DMSO溶解配制成母液，使用前采用PBST(pH为7.4，吐温20含量为0.05％)缓冲液稀释成适当浓度)；USP7 C端蛋白(1mg/mL)；Biacore T200(GE Healthcare)；CM5芯片(GE Healthcare)；氨基偶联试剂盒(GE Healthcare)；PBST缓冲液。The compound of the present invention (dissolved in DMSO to prepare a mother liquor, diluted with PBST (pH 7.4, Tween 20 content 0.05%) buffer to an appropriate concentration before use); USP7 C-terminal protein (1mg/mL); Biacore T200(GE Healthcare); CM5 chip (GE Healthcare); Amino coupling kit (GE Healthcare); PBST buffer.
3.实验方法3. Experimental method
(1)分别用pH 4.0、4.5、5.0和5.5的10mM醋酸钠溶液将USP7 C端蛋白稀释为20μg/mL的蛋白溶液(稀释50倍)，依次流经一块CM5芯片的第二个通道(第一个通道为参比通道)，对比发现，pH 4.5时，预富集信号较高，且蛋白活性较好，遂采用pH 4.5的10mM醋酸钠溶液稀释的USP7 C端蛋白进行偶联；(2)设置程序，先将1-乙基-3-(3- 二甲基氨丙基)-碳化二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)同时流经第一个和第二个通道(即为活化)，再将稀释好的蛋白流经第二个通道(即为偶联)，最后将乙醇胺同时流经两个通道(即为封闭)，观察到偶联信号为16000RU，偶联效果较好；(3)用含5％DMSO的PBST稀释本发明的化合物，将化合物溶液同时流经两个通道，结合90秒，解离90秒，得到化合物与蛋白的结合和解离曲线，并通过Biacore T200 Evaluation Software拟合得K D。 (1) Dilute the C-terminal protein of USP7 to 20μg/mL protein solution (diluted 50 times) with 10mM sodium acetate solution of pH 4.0, 4.5, 5.0 and 5.5 respectively, and then flow through the second channel of a CM5 chip (No. One channel is the reference channel). The comparison found that at pH 4.5, the pre-enrichment signal was higher and the protein activity was better, so the USP7 C-terminal protein diluted with a 10mM sodium acetate solution at pH 4.5 was used for coupling; (2 ) Set up the program, first flow 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) through the first and second Two channels (i.e. activation), and then the diluted protein flows through the second channel (i.e. coupling), and finally ethanolamine flows through both channels (i.e. closed), the coupling signal is observed to be 16000RU , The coupling effect is better; (3) Dilute the compound of the present invention with PBST containing 5% DMSO, flow the compound solution through two channels at the same time, bind for 90 seconds, and dissociate for 90 seconds to obtain the binding and dissociation of the compound and the protein Curve, and K D is fitted by Biacore T200 Evaluation Software.
4.实验结果4. Experimental results
部分化合物与USP7 C端蛋白的结合力(K D)测试结果如表2所示。实验结果表明，本发明化合物可与USP7 C端蛋白结合，且一些化合物亲和力较强，其他化合物也具有较好的亲和力。 Table 2 shows the binding force (K D ) test results of some compounds with the C-terminal protein of USP7. The experimental results show that the compound of the present invention can bind to the C-terminal protein of USP7, and some compounds have stronger affinity, and other compounds also have better affinity.
表2、部分化合物与USP7 C端蛋白的结合力Table 2. The binding ability of some compounds with USP7 C-terminal protein
化合物Compound K D(μM) K D (μM) 化合物Compound K D(μM) K D (μM) 化合物Compound K D(μM) K D (μM) 化合物Compound K D(μM) K D (μM)
2525 4.264.26 4545 61.261.2 5151 9.79.7 5757 27.727.7
4040 17.817.8 4646 39.139.1 5252 187.3187.3 5858 17.817.8
4141 51.451.4 4747 153.0153.0 5353 37.537.5 5959 17.917.9
4242 13.613.6 4848 49.749.7 5454 22.722.7 6060 2.802.80
4343 68.868.8 4949 31.131.1 5555 0.3600.360 6161 20.220.2
4444 3.73.7 5050 66.666.6 5656 3.23.2 6262 8.98.9
6363 9.269.26 6464 1.991.99 6565 1.481.48 6666 37.437.4
6767 19.319.3 6868 9.019.01 6969 >100>100 7070 35.735.7
7171 21.021.0 7272 3.343.34 7373 7.067.06 7474 1.701.70
7575 15.715.7 7676 4.704.70 7777 >100>100 7878 12.412.4
7979 13.213.2 8080 23.923.9 8181 >100>100 8282 3.933.93
8383 3.573.57 8484 >100>100 8585 >100>100 8686 11.411.4
8787 >100>100 8888 14.414.4 8989 2.982.98 9090 1.991.99
9191 22.222.2 9292 85.7985.79 9393 86.7086.70 9494 42.9942.99
实施例97Example 97
表面等离子体共振(SPR)法测定化合物与USP7全长蛋白和不同结构域的结合力Surface Plasmon Resonance (SPR) Method to Determine the Binding Ability of Compounds to USP7 Full-length Protein and Different Domains
1.实验目的1. The purpose of the experiment
采用表面等离子体共振(SPR)法测定化合物与USP7全长蛋白及不同结构域的结合力，以考察化合物是否对USP7的C端结构域蛋白具有特异性。The surface plasmon resonance (SPR) method was used to determine the binding ability of the compound to the full-length USP7 protein and different domains to investigate whether the compound is specific to the C-terminal domain protein of USP7.
2.实验方法2. Experimental method
参考文献(Nat.Commun.2015,6:7023-7033)表达纯化CTD ΔUBl2和CTD Δ666-723蛋白。 References (Nat.Commun.2015, 6:7023-7033) express and purify CTD ΔUB12 and CTD Δ666-723 proteins.
参考实施例96的测试方法将USP7 C端蛋白替换成USP7全长蛋白或不同结构域蛋 白进行表面等离子体共振(SPR)法测定蛋白与化合物的结合力。Refer to the test method of Example 96 to replace the USP7 C-terminal protein with the USP7 full-length protein or proteins with different domains to perform the surface plasmon resonance (SPR) method to determine the binding force of the protein to the compound.
3.实验结果3. Experimental results
化合物25、55和60与USP7全长蛋白和不同结构域的结合力测试结果如表3所示。实验结果表明，化合物25、55和60对USP7的C端结构域蛋白具有很强的特异性，其不与USP7的N端结构域蛋白结合，尽管也与USP7催化域蛋白有结合，但亲和力不如与C端结构域蛋白的。化合物55与USP7 C端结构域蛋白的结合，高度依赖C端的UBl2域和氨基酸序列666-723，当UBl2域或氨基酸666-723序列缺失时，结合力急剧下降；其他化合物对USP7的C端结构域蛋白也具有特异性。Table 3 shows the binding ability test results of compounds 25, 55 and 60 with the full-length USP7 protein and different domains. The experimental results show that compounds 25, 55 and 60 have strong specificity to the C-terminal domain protein of USP7. They do not bind to the N-terminal domain protein of USP7. Although they also bind to the catalytic domain protein of USP7, their affinity is not as good as With the C-terminal domain protein. The binding of compound 55 to the C-terminal domain protein of USP7 is highly dependent on the C-terminal UB12 domain and amino acid sequence 666-723. When the UB12 domain or amino acid sequence 666-723 is missing, the binding force drops sharply; other compounds have a strong effect on the C-terminal structure of USP7 Domain proteins are also specific.
表3 部分化合物与USP7全长蛋白和不同结构域的结合力Table 3 The binding ability of some compounds with the full-length USP7 protein and different domains
Figure PCTCN2021078798-appb-000116
Figure PCTCN2021078798-appb-000116
注：CTD ΔUBl2为UBL2域缺失的USP7 C端蛋白；CTD Δ666-723为氨基酸666-723序列缺失的USP7 C端蛋白；N.D.表示未测试。 Note: CTD ΔUBl2 is the USP7 C-terminal protein with the UBL2 domain deleted; CTD Δ666-723 is the USP7 C-terminal protein with the amino acid sequence 666-723 deleted; ND means not tested.
实施例98Example 98
USP7 C端蛋白与化合物25复合物共晶的培养、X-射线衍射及结构解析Cultivation, X-ray diffraction and structure analysis of the co-crystal of USP7 C-terminal protein and compound 25 complex
参照文献(Nat.Commun.2015,6:7023-7033)方法制备USP7 C端蛋白。将纯化好的USP7 C端蛋白寄送至清华大学结构生物学高精尖创新中心X-射线晶体学平台，委托其进行USP7 C端蛋白与化合物25复合物共晶的培养及结构解析。结果显示，蛋白浓度为9mg/ml，蛋白:化合物＝1:1.2，结晶缓冲液为200mM碘化铵，20％w/v聚乙二醇3350时，晶体在培养两天之后出现，形态较好。将晶体捞出，液氮速冻后，带至上海同步辐射光源进行X-射线衍射，收集数据。通过分子置换(PDB ID:4YOC)解析出化合物25与USP7 C端蛋白的共晶结构(分辨率
Figure PCTCN2021078798-appb-000117
)。 Refer to the literature (Nat. Commun. 2015, 6:7023-7033) method to prepare USP7 C-terminal protein. The purified C-terminal protein of USP7 was sent to the X-ray crystallography platform of the Advanced Innovation Center for Structural Biology of Tsinghua University, and it was entrusted to cultivate and analyze the structure of the co-crystal of the USP7 C-terminal protein and compound 25 complex. The results showed that when the protein concentration was 9mg/ml, protein: compound=1:1.2, the crystallization buffer was 200mM ammonium iodide, 20%w/v polyethylene glycol 3350, the crystals appeared after two days of culture, and the morphology was better. . The crystals were taken out, liquid nitrogen was quick-frozen, and then taken to the Shanghai synchrotron radiation source for X-ray diffraction to collect data. The co-crystal structure of compound 25 and USP7 C-terminal protein was resolved by molecular replacement (PDB ID: 4YOC) (resolution
Figure PCTCN2021078798-appb-000117
).
晶体结构解析结果如图1所示，化合物25结合于USP7 C端蛋白的UBL2结构域，结合口袋由Asp666、Tyr706及Arg723等关键氨基酸残基构成。化合物25的磺酰基与USP7 C端蛋白的UBL2的Asp666残基形成氢键作用，酰胺基、噻唑环与Tyr706残基形成氢键作用，苯并噻唑骨架与Arg723残基存在π-阳离子相互作用，其苯环还与Tyr706残基形成π-π相互作用。The crystal structure analysis result is shown in Figure 1. Compound 25 binds to the UBL2 domain of the C-terminal protein of USP7, and the binding pocket is composed of key amino acid residues such as Asp666, Tyr706 and Arg723. The sulfonyl group of compound 25 forms a hydrogen bond with the Asp666 residue of UBL2 of the USP7 C-terminal protein, and the amide group and thiazole ring form a hydrogen bond with Tyr706 residue. There is a π-cation interaction between the benzothiazole skeleton and the Arg723 residue. Its benzene ring also forms a π-π interaction with Tyr706 residues.
实施例99Example 99
GST Pull-down实验考察化合物对DNMT1与USP7 C端蛋白相互作用的影响GST Pull-down experiment to investigate the effect of compounds on the interaction between DNMT1 and USP7 C-terminal protein
1.实验方法1. Experimental method
取GST亲和树脂于1.5mL EP管中，3000rpm离心2分钟，小心吸去上清后加入PBS，3000rpm离心2分钟，重复三次。Take the GST affinity resin in a 1.5mL EP tube, centrifuge at 3000 rpm for 2 minutes, carefully aspirate the supernatant, add PBS, centrifuge at 3000 rpm for 2 minutes, and repeat three times.
向弃去上清的树脂中加入PBS，用移液枪吹打混合均匀后将含有树脂的PBS溶液平行分装至数个1.5mL EP管中(每管30μL树脂)，然后以GST蛋白做对照，加入4μg GST-USP7 C端蛋白，再对应加入不同的化合物溶液(以PBS补齐至每管470μL)。将EP管置于4℃冰箱中用360 °旋转仪旋转(转速10rpm)孵育30分钟，使GST-USP7 C端蛋白、GST蛋白、化合物与GST亲和树脂充分接触。 Add PBS to the resin from which the supernatant is discarded, pipette to mix evenly, and distribute the PBS solution containing the resin into several 1.5 mL EP tubes (30 μL resin per tube) in parallel, and then use GST protein as a control. Add 4μg of GST-USP7 C-terminal protein, and then add different compound solutions (fill up to 470μL per tube with PBS). Place the EP tube in a 4°C refrigerator and incubate for 30 minutes with a 360° rotator (rotating speed 10rpm) to make the GST-USP7 C-terminal protein, GST protein, and compound fully contact the GST affinity resin.
取培养好的flag-DNMT1高表达的293T细胞，PBS洗两遍后加入细胞温和裂解液和蛋白酶抑制剂(v/v 100:1)，冰上裂解1小时，将裂解好的细胞从培养皿上刮下，加入1.5mL EP管中，12000rpm和4℃下离心20分钟，取上清，BCA定量，等体积(每管30μL，至最终体积为每管500μL，DMSO含量为0.1％，化合物终浓度为20μM)加入到孵育有蛋白、化合物和树脂的EP管中，4℃继续旋转孵育过夜(转速10rpm)。Take the cultured 293T cells with high expression of flag-DNMT1, wash twice with PBS, add mild cell lysate and protease inhibitor (v/v 100:1), lyse on ice for 1 hour, remove the lysed cells from the culture dish Scrape the top, add it to a 1.5mL EP tube, centrifuge at 12000rpm and 4°C for 20 minutes, take the supernatant, and quantify the BCA in an equal volume (30μL per tube, to a final volume of 500μL per tube, with a DMSO content of 0.1%. Concentration of 20μM) was added to the EP tube incubated with protein, compound and resin, and incubated overnight at 4°C (rotating speed 10rpm).
将树脂放置自然沉降，小心吸去上清，用50mM Tris-HCl(pH7.5)洗五次，每次500μL，前三次自然沉降，后两次离心(3000rpm离心2分钟)。每管加入100μL上样缓冲液，进行Western Blot。Place the resin for natural sedimentation, carefully aspirate the supernatant, wash five times with 500 μL of 50mM Tris-HCl (pH 7.5), the first three times of natural sedimentation, and the last two centrifugation (3000rpm for 2 minutes). Add 100μL of loading buffer to each tube for Western Blot.
2.实验结果2. Experimental results
如图2所示，与USP7 C端蛋白亲和力较强的化合物均能干扰DNMT1与USP7 C端蛋白的相互作用，如其中化合物55和60的影响极显著，而无结合或亲和力较弱的化合物84(K D>100μM)、92(K D＝86.0μM)对DNMT1与USP7 C端蛋白的相互作用无影响。 As shown in Figure 2, compounds with strong affinity for the C-terminal protein of USP7 can interfere with the interaction between DNMT1 and C-terminal protein of USP7. For example, compounds 55 and 60 have extremely significant effects, while compound 84 has no binding or weak affinity. (K D >100 μM) and 92 (K D =86.0 μM) have no effect on the interaction between DNMT1 and the C-terminal protein of USP7.
实施例100Example 100
采用CellTiter-Glo法评价化合物对体外肿瘤细胞的增殖抑制活性The CellTiter-Glo method was used to evaluate the compound's inhibitory activity against tumor cell proliferation in vitro
1.实验目的1. The purpose of the experiment
采用CellTiter-Glo法评价化合物对多种肿瘤细胞体外增殖活性的影响，并计算各自的半数抑制浓度IC 50。 Compounds were evaluated using the CellTiter-Glo method proliferative activity of various tumor cells in vitro, and to calculate the respective half maximal inhibitory concentration IC 50.
2.实验材料2. Experimental materials
本发明化合物(用DMSO溶解配制成母液，使用前采用完全培养基稀释成适当浓度)；CellTiter-Glo Luminescent细胞活力检测试剂盒(购自Promega公司)；培养基和胎牛血清(购自Biological Industries公司)；96孔细胞培养板(购自Thermo Fisher Scientific公司)，EnSpire酶标仪(购自PerkinElmer公司)。The compound of the present invention (dissolved in DMSO to prepare a mother liquor, and dilute to an appropriate concentration with complete medium before use); CellTiter-Glo Luminescent cell viability detection kit (purchased from Promega); medium and fetal calf serum (purchased from Biological Industries) Company); 96-well cell culture plate (purchased from Thermo Fisher Scientific Company), EnSpire microplate reader (purchased from PerkinElmer Company).
3.实验方法3. Experimental method
选择活细胞比例达90％以上的细胞进行细胞铺板，即在96孔板中每孔加入100μL 细胞悬液，其中LNCaP细胞每孔2500个，RS 4；11细胞每孔5000个，MM.1S细胞每孔16500个，NB4、MCF7、Huh7及HCT-116细胞每孔2000个。将96孔板置于37℃，5％CO 2培养箱中培养24小时。用完全培养基稀释药物至所需浓度(最高浓度为200μM，2倍梯度稀释，得9个浓度，即200、100、50、25、12.5、6.25、3.125、1.5625、0.78125μM)，每孔加入100μL含药物的培养基，每化合物设3个复孔，同时设立阴性对照组，溶媒对照组。96孔板置于37℃、5％CO 2培养箱中继续培养，其中LNCaP细胞继续培养6天，MM.1S细胞继续培养5天，RS 4；11、NB4、MCF7、Huh7及HCT-116细胞继续培养3天。将96孔板从培养箱中取出，室温放置30分钟，以使培养板平衡至室温。从-20℃冰箱取出CellTiter-Glo试剂于室温下融化，此过程约需10分钟。向96孔板每孔加入50μL CellTiter-Glo试剂，振荡混匀2-5分钟使细胞充分裂解，室温放置10分钟，用EnSpire酶标仪Luminescence 96程序读板，计算不同浓度的化合物对肿瘤细胞的抑制率，并通过Graphpad prism 5.0拟合出IC 50值。 Select cells with more than 90% viable cells for cell plating, that is, add 100μL of cell suspension to each well of a 96-well plate, including 2500 LNCaP cells per well, RS 4; 5000 cells per well for 11 cells, and MM.1S cells 16,500 cells per well, 2000 cells per well for NB4, MCF7, Huh7 and HCT-116 cells. Place the 96-well plate in a 37°C, 5% CO 2 incubator for 24 hours. Dilute the drug with complete medium to the required concentration (the highest concentration is 200μM, and the 2-fold gradient dilution yields 9 concentrations, namely 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125μM), and add to each well 100 μL of drug-containing medium, 3 multiple wells for each compound, and a negative control group and a vehicle control group. The 96-well plate was placed in a 37°C, 5% CO 2 incubator for continuous culture, in which LNCaP cells were cultured for 6 days, MM.1S cells were cultured for 5 days, RS 4;11, NB4, MCF7, Huh7 and HCT-116 cells Continue to cultivate for 3 days. Take the 96-well plate out of the incubator and place it at room temperature for 30 minutes to equilibrate the plate to room temperature. Take out CellTiter-Glo reagent from the refrigerator at -20℃ and melt it at room temperature. This process takes about 10 minutes. Add 50μL CellTiter-Glo reagent to each well of a 96-well plate, shake and mix for 2-5 minutes to fully lyse the cells, leave it at room temperature for 10 minutes, read the plate with the EnSpire plate reader Luminescence 96 program, and calculate the effects of different concentrations of compounds on tumor cells Inhibition rate, and the IC 50 value was fitted by Graphpad prism 5.0.
4.实验结果4. Experimental results
如表4所示，本发明化合物60对上述各种肿瘤细胞具有显著的体外增殖抑制活性，尤其对LNCaP、RS 4；11、NB4。本发明中的其他一些化合物也对肿瘤细胞的生长具有抑制作用，该结果提示本发明化合物可以用于制备抗肿瘤药物。As shown in Table 4, the compound 60 of the present invention has significant in vitro proliferation inhibitory activity against the above-mentioned various tumor cells, especially LNCaP, RS 4; 11, NB4. Some other compounds of the present invention also have an inhibitory effect on the growth of tumor cells. This result suggests that the compounds of the present invention can be used to prepare anti-tumor drugs.
表4 化合物60对不同肿瘤细胞体外增殖的抑制活性Table 4 Inhibitory activity of compound 60 on the proliferation of different tumor cells in vitro
细胞系Cell line LNCaPLNCaP MCF7MCF7 HCT-116HCT-116 Huh7Huh7 MM.1SMM.1S RS 4；11RS 4; 11 NB4NB4
IC 50(μM) IC 50 (μM) 9.9±2.59.9±2.5 25.6±3.225.6±3.2 22.8±3.722.8±3.7 15.5±1.715.5±1.7 25.5±6.625.5±6.6 8.6±1.88.6±1.8 5.6±1.05.6±1.0
实施例101Example 101
Western blot评价化合物对肿瘤细胞中DNMT1水平的影响Western blot evaluation of the effect of compounds on the level of DNMT1 in tumor cells
1.实验目的1. The purpose of the experiment
采用Western blot方法测定化合物对人早幼粒白血病细胞NB4中DNMT1水平的影响。Western blot method was used to determine the effect of the compound on the level of DNMT1 in human promyelocytic leukemia cells NB4.
2.实验材料2. Experimental materials
本发明化合物(用DMSO溶解配制成母液，使用前采用完全培养基稀释成1μM、5μM、10μM和20μM的浓度)；改良型RMPI-1640培养基和胎牛血清(购自Biological Industries公司)；6孔细胞培养板(购自NEST公司)。The compound of the present invention (dissolved in DMSO to prepare a mother liquor, diluted with a complete medium to a concentration of 1μM, 5μM, 10μM and 20μM before use); modified RMPI-1640 medium and fetal calf serum (purchased from Biological Industries); 6 Well cell culture plate (purchased from NEST company).
3.实验方法3. Experimental method
取活细胞比例达90％以上的细胞进行铺板，即在6孔板中每孔加入1000μL细胞悬液，每孔100万个NB4细胞。用完全培养基稀释药物至所需浓度(0μM、1μM、5μM、10μM和20μM)，每孔加入1000μL含药物的培养基，同时设立阴性对照组。将6孔 板置于37℃、5％CO 2培养箱中培养24小时。收集细胞，离心，用PBS清洗细胞后使用细胞裂解液裂解细胞，提取蛋白，用8％分离胶电泳。 Take cells with a ratio of more than 90% living cells to plate, that is, add 1000 μL of cell suspension to each well of a 6-well plate, and 1 million NB4 cells per well. Dilute the drug with complete medium to the required concentration (0μM, 1μM, 5μM, 10μM, and 20μM), add 1000μL of medium containing the drug to each well, and set up a negative control group at the same time. Place the 6-well plate in a 37°C, 5% CO 2 incubator for 24 hours. Collect the cells, centrifuge, wash the cells with PBS, lyse the cells with cell lysate, extract protein, and use 8% separating gel for electrophoresis.
4.实验结果4. Experimental results
如图3和图4所示，化合物55和60浓度依赖性地降低NB4细胞的DNMT1水平。本发明中的其他化合物如25、44、56等也具有同样的效果，说明本发明的化合物通过干扰USP7 C端与DNMT1的结合而抑制USP7对DNMT1的去泛素化，使DNMT1泛素化降解增加，DNMT1水平因而降低。As shown in Figures 3 and 4, compounds 55 and 60 decreased the DNMT1 level of NB4 cells in a concentration-dependent manner. Other compounds of the present invention such as 25, 44, 56 have the same effect, indicating that the compound of the present invention inhibits the deubiquitination of DNMT1 by USP7 by interfering with the binding of USP7 C-terminal to DNMT1, thereby degrading DNMT1 ubiquitination Increase, and thus decrease the DNMT1 level.
实施例102Example 102
化合物对人肝微粒体代谢稳定性研究Study on the Metabolic Stability of Compounds on Human Liver Microsomes
人肝微粒体代谢稳定性评价是药物研发中临床前评价候选化合物药代动力学性质的重要手段。该部分实验由美迪西普亚医药科技有限公司完成。The evaluation of the metabolic stability of human liver microsomes is an important method for preclinical evaluation of the pharmacokinetic properties of candidate compounds in drug development. This part of the experiment was completed by Medicipuya Pharmaceutical Technology Co., Ltd.
实验温孵体系(体积为250μL，n＝3)由肝微粒体、受试物工作溶液和磷酸盐缓冲液组成，将温孵体系于37℃共孵育一个小时，加入NADPH溶液后计时开始，每个时间点以加入终止液终止反应，取样间点为0，5，15，30，60分钟，共5个点。阴性对照不加NADPH，取样时间点为0、60分钟。用LC-MS/MS进行分析，通过受试物剩余量的百分率的自然对数与时间作图测得斜率的绝对值k，按以下公式进行计算：T 1/2(半衰期)＝ln2/k＝0.693/k，Clint＝(0.693/T1/2)×(1/微粒体蛋白浓度)×换算系数，微粒体蛋白浓度为0.5mg/mL，换算系数1254.2)。实验结果如表5所示。 The experimental incubation system (volume 250μL, n=3) consists of liver microsomes, test substance working solution and phosphate buffer. The incubation system is incubated at 37°C for one hour. After adding NADPH solution, the timing starts. The reaction was terminated at each time point by adding the stop solution, and the sampling interval was 0, 5, 15, 30, and 60 minutes, a total of 5 points. NADPH was not added to the negative control, and the sampling time point was 0 to 60 minutes. Analyze by LC-MS/MS, obtain the absolute value of the slope k by plotting the natural logarithm of the percentage of the remaining amount of the test substance against time, and calculate it according to the following formula: T 1/2 (half-life) = ln2/k =0.693/k, Clint=(0.693/T1/2)×(1/microsomal protein concentration)×conversion coefficient, the microsomal protein concentration is 0.5 mg/mL, the conversion coefficient is 1254.2). The experimental results are shown in Table 5.
表5 部分化合物在人肝微粒体中的代谢稳定性Table 5 Metabolic stability of some compounds in human liver microsomes
化合物编号Compound number T 1/2(min) T 1/2 (min) Cl int(mL/min/kg) Cl int (mL/min/kg)
5555 75.2675.26 72.5272.52
6060 12.7212.72 428.88428.88
6161 7.787.78 701.70701.70
实验结果表明，化合物55在人肝微粒体中的代谢稳定性较好，其T 1/2达75.26分钟。本发明中其他化合物也具有较好的肝微粒体代谢稳定性。 The experimental results show that compound 55 has better metabolic stability in human liver microsomes, with a T 1/2 of 75.26 minutes. Other compounds in the present invention also have better metabolic stability of liver microsomes.
实施例103Example 103
化合物在Balb/C小鼠体内的药代动力学研究Study on the pharmacokinetics of the compound in Balb/C mice
该部分实验由美迪西普亚医药科技有限公司完成。This part of the experiment was completed by Medicipuya Pharmaceutical Technology Co., Ltd.
1.实验仪器1. Experimental instrument
超高效液相色谱系统(Waters公司，ACQUITY UPLC)，包括二元溶剂管理器(ACQUITY UPLC Binary Solvent Manager)、样品管理器(ACQUITY UPLC Sample  Manager)、高通量样品组织管理器(ACQUTIY UPLC Sample Organizer)、高温柱温箱(ACQUITY UPLC Column Heater HT)。质谱仪(TQ 6500+，美国应用生物系统公司)，电喷雾离子源(ESI)，串联四极杆质量分析器。微量分析天平(XP26，梅特勒-托利多仪器(上海)有限公司)；涡旋振荡器(SI-A256，Scientific Industries，Inc.；MULTI-TUBE VORTEXER，Fisher Scientific)；小型台式高速冷冻离心机(5417R，Eppendorf)；超纯水机(Millipore)；移液器(Eppendorf)。数据处理系统为Analyst软件(美国应用生物系统公司，软件版本号1.6.3)。Ultra-high performance liquid chromatography system (Waters, ACQUITY UPLC), including binary solvent manager (ACQUITY UPLC Binary Solvent Manager), sample manager (ACQUITY UPLC Sample Manager), high-throughput sample organizer (ACQUTIY UPLC Sample Organizer) ), high temperature column heater (ACQUITY UPLC Column Heater HT). Mass spectrometer (TQ 6500+, American Applied Biosystems), electrospray ion source (ESI), tandem quadrupole mass analyzer. Micro analytical balance (XP26, METTLER TOLEDO Instruments (Shanghai) Co., Ltd.); vortex oscillator (SI-A256, Scientific Industries, Inc.; MULTI-TUBE VORTEXER, Fisher Scientific); small desktop high-speed refrigerated centrifuge (5417R, Eppendorf); ultrapure water machine (Millipore); pipette (Eppendorf). The data processing system is Analyst software (American Applied Biosystems, software version number 1.6.3).
2.实验试剂2. Experimental reagents
甲醇(Burdick&Jackson，HPLC)，乙腈(Burdick&Jackson，HPLC)，甲酸(J&K)，水为超纯水。Methanol (Burdick & Jackson, HPLC), acetonitrile (Burdick & Jackson, HPLC), formic acid (J&K), water is ultrapure water.
3.溶媒3. Solvent
溶媒：5％DMSO+10％solutol+85％saline。Solvent: 5% DMSO + 10% solutol + 85% saline.
4.实验动物4. Experimental animals
种属：Balb/C雄性小鼠，SPF级。Species: Balb/C male mice, SPF grade.
来源：浙江维通利华实验动物技术有限公司。Source: Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.
5.动物给药5. Animal administration
Balb/C小鼠，按表6进行实验分组。Balb/C mice were grouped according to Table 6 for experiment.
表6 IV和PO给药动物分组Table 6 Animal groups for IV and PO administration
Figure PCTCN2021078798-appb-000118
Figure PCTCN2021078798-appb-000118
注： *在口服给药前，所有动物禁食过夜(10-14小时)，给药4小时后给食。 Note: * All animals are fasted overnight (10-14 hours) before oral administration, and 4 hours after administration.
6.样品采集与处理6. Sample collection and processing
经眼眶采血，每个样品采集约0.03mL，肝素钠抗凝，采集后放置冰上。Blood was collected from the orbit. About 0.03 mL of each sample was collected. Heparin sodium was anticoagulated and placed on ice after collection.
血液样本采集后置于冰上，并于1小时之内离心分离血浆(离心条件：6800g，6分钟，2-8℃)。血浆样本在分析前存放时则放于-80℃冰箱内，血浆样品由实验机构分析部门采用LC-MS/MS进行分析。The blood samples were collected on ice and centrifuged to separate plasma within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8°C). Plasma samples are stored in a -80℃ refrigerator before analysis, and the plasma samples are analyzed by the analysis department of the laboratory using LC-MS/MS.
采血时间点如下：The blood sampling time points are as follows:
口服组：给药后0.25h,0.5h,1h,2h,4h,6h,8h,24h。Oral group: 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h after administration.
静脉组：给药后0.083h,0.25h,0.5h,1h,2h,4h,8h,24h。Intravenous group: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 24h after administration.
7.LC-MS/MS条件7. LC-MS/MS conditions
(1)液相条件(1) Liquid phase conditions
A：0.1％甲酸水溶液，B：0.1％甲酸乙腈溶液；柱温：40℃；自动进样器温度：4℃；流速：0.6ml/min；进样量：2μl。A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution; column temperature: 40°C; autosampler temperature: 4°C; flow rate: 0.6 ml/min; injection volume: 2 μl.
色谱柱:ACQUITY UPLC BEH C18 1.7μm(2.1×50mm)Column: ACQUITY UPLC BEH C18 1.7μm (2.1×50mm)
(2)质谱条件(2) Mass spectrometry conditions
扫描模式：负离子多反应检测模式；离子源：电喷雾离子源；雾化模式：电喷雾；Q1分辨率：Unit；Q3分辨率：Unit；雾化气(Gas1)：50psi；辅助加热器(Gas2)：50psi；气帘气(CUR)：40psi；碰撞气(CAD)：10；离子源电压(IS)：-4500v；离子源温度(TEM)：500℃。Scan mode: negative ion multi-reaction detection mode; ion source: electrospray ion source; atomization mode: electrospray; Q1 resolution: Unit; Q3 resolution: Unit; atomizing gas (Gas1): 50psi; auxiliary heater (Gas2 ): 50psi; Curtain Air (CUR): 40psi; Collision Gas (CAD): 10; Ion Source Voltage (IS): -450v; Ion Source Temperature (TEM): 500°C.
8.标准曲线和质控样品的配制8. Preparation of standard curve and quality control samples
400,000ng/mL的工作溶液的制备：取化合物55储备液，用甲醇稀释成浓度为400,000ng/mL的工作溶液。Preparation of 400,000ng/mL working solution: Take compound 55 stock solution and dilute it with methanol to a working solution with a concentration of 400,000ng/mL.
标准曲线和质控样品的制备：取一定量的400000ng/mL工作溶液，按照1：39的比例，加入到一定量的空白血浆中，制得浓度为10000ng/mL的血浆样品。取10000ng/mL的血浆样品，用空白血浆依次稀释到5000、1000、500、100、50、10、5ng/mL的标准曲线样品和4000、800、15ng/mL的质控样品。Preparation of standard curve and quality control samples: Take a certain amount of 400,000ng/mL working solution and add it to a certain amount of blank plasma at a ratio of 1:39 to prepare a plasma sample with a concentration of 10,000ng/mL. Take 10000ng/mL plasma samples and dilute them to 5000, 1000, 500, 100, 50, 10, 5ng/mL standard curve samples and 4000, 800, 15ng/mL quality control samples with blank plasma.
内标工作液的制备：吸取一定量的浓度为1018,000ng/mL的甲苯磺丁脲内标储备液至一定体积的容量瓶中，用甲醇定容至刻度后混匀，制得浓度为200ng/mL的内标工作液。Preparation of the internal standard working solution: pipet a certain amount of tolbutamide internal standard stock solution with a concentration of 1018,000ng/mL into a certain volume of volumetric flask, dilute to the mark with methanol and mix well to obtain a concentration of 200ng /mL of internal standard working solution.
9.样品前处理9. Sample pretreatment
取10μl血浆样品至1.5ml离心管中，加入400μl内标溶液(含100ng/mL内标物，空白不加内标补加相同体积的甲醇)，涡旋混匀，18000转/分钟，离心7分钟，取200μl上清液加入到96孔进样板中，LC-MS/MS进样分析。Take 10μl of plasma sample into a 1.5ml centrifuge tube, add 400μl of internal standard solution (containing 100ng/mL internal standard, blank without internal standard plus the same volume of methanol), vortex and mix, 18,000 rpm, centrifuge 7 Minutes, 200μl of supernatant was added to the 96-well injection plate, and the LC-MS/MS injection analysis.
10.药物代谢动力学分析10. Pharmacokinetic analysis
根据药物各组各时间点的平均血药浓度数据，使用药代动力学计算软件Phoenix
Figure PCTCN2021078798-appb-000119
非房室模型分别计算供试品的药代动力学参数AUC 0-t、AUC 0-∞、MRT 0-∞、C max、T max和T 1/2等参数。此外，生物利用度(F)将通过下面的公式进行计算。 According to the average blood concentration data of each drug group at each time point, use the pharmacokinetic calculation software Phoenix
Figure PCTCN2021078798-appb-000119
The non-compartmental model calculates the pharmacokinetic parameters AUC 0-t , AUC 0-∞ , MRT 0-∞ , C max , T max and T 1/2 of the test product respectively. In addition, the bioavailability (F) will be calculated by the following formula.
Figure PCTCN2021078798-appb-000120
Figure PCTCN2021078798-appb-000120
对于浓度低于定量下限的样品，在进行药代动力学参数计算时，在达到C max以前取样的样品以零值计算，在达到C max以后取样点样品以无法定量(标注为“BLQ”)计算。 For samples with a concentration lower than the lower limit of quantification, when calculating the pharmacokinetic parameters, the samples sampled before reaching C max are calculated as zero. After reaching C max , the sample at the sampling point cannot be quantified (marked as "BLQ") calculate.
(注：该部分实验由美迪西普亚医药科技有限公司完成)(Note: This part of the experiment was completed by Medicipuya Pharmaceutical Technology Co., Ltd.)
11.实验结果11. Experimental results
小鼠药代动力学数据如表7所示，结果表明化合物55可口服吸收，其小鼠口服生 物利用度为19.54％。本发明的其他化合物也都可口服吸收。这表明本发明的苯并噻唑类化合物具有较好的成药性。The mouse pharmacokinetic data is shown in Table 7. The results show that compound 55 can be absorbed orally, and its oral bioavailability in mice is 19.54%. The other compounds of the present invention can also be absorbed orally. This indicates that the benzothiazole compounds of the present invention have better drug-forming properties.
表7 本发明化合物55在Balb/C小鼠体内的药代动力学参数Table 7 Pharmacokinetic parameters of compound 55 of the present invention in Balb/C mice
Figure PCTCN2021078798-appb-000121
Figure PCTCN2021078798-appb-000121
实施例104Example 104
化合物55在Raw264.7细胞上的抗炎效应Anti-inflammatory effect of compound 55 on Raw264.7 cells
1.实验方法：1. Experimental method:
Raw264.7细胞(购自中科院细胞库)培养在含10％灭活胎牛血清的DMEM培养基中。将Raw264.7按每孔30万个接种至12孔板中，贴壁培养12h。将化合物55用DMSO配置成10mM的储存液，并用含10％灭活胎牛血清的DMEM培养基依次稀释，得到相应工作浓度的药液。将12孔板中的培养基弃去，加入含药液的培养基，预处理1h。将浓度为1mg/mL的脂多糖(LPS)储存液用培养基稀释成100ng/mL的培养基溶液，并用含脂多糖的培养基溶液稀释化合物至相应工作浓度(0-20μM)。将预处理的培养基弃去，对照孔中加入培养基溶液，阳性孔中加入100ng/mL的脂多糖培养基溶液，给药孔中加入含化合物的脂多糖培养基溶液。刺激1h后，将培养基弃去，细胞冻存于-80℃冰箱以便后续检测。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 a 12-well plate at 300,000 per well, and cultured adherently for 12 hours. Compound 55 was prepared into a 10 mM stock solution with DMSO, and diluted sequentially with DMEM medium containing 10% inactivated fetal bovine serum to obtain a drug solution with a corresponding working concentration. The medium in the 12-well plate was discarded, and the medium containing the medicinal solution was added for pretreatment for 1 h. The lipopolysaccharide (LPS) stock solution with a concentration of 1 mg/mL was diluted with culture medium to a 100 ng/mL culture medium solution, and the compound was diluted with the lipopolysaccharide-containing medium solution to the corresponding working concentration (0-20 μM). The pretreated medium was discarded, the medium solution was added to the control wells, the 100ng/mL lipopolysaccharide medium solution was added to the positive wells, and the compound-containing lipopolysaccharide medium solution was added to the dosing wells. After stimulation for 1 hour, the culture medium was discarded, and the cells were frozen in a refrigerator at -80°C for subsequent testing.
每孔加入预冷的Trizol试剂500μL，冰上裂解15分钟，加入100μL氯仿后剧烈震摇15s，在冰上放置10分钟后4℃，12000rpm离心15分钟。转移上层水相至干净的1.5mL EP管中，加入200μL异丙醇沉淀RNA，冰上放置10分钟后，4℃，12000rpm离心10分钟。弃去上清液，将沉淀用75％乙醇洗涤一遍，离心后去除上清，用15μL DEPC处理水溶解RNA沉淀。用Nano进行RNA浓度定量，并按说明书加入Takara公司的逆转录试剂，使用普通PCR仪将mRNA逆转录成cDNA。最后，在q-PCR专用96孔板中分别加入目的基因(Gapdh、IL6或IL1b)的上下游引物(引物序列查自Primer Bank)、q-PCR试剂(SYBR Green)以及cDNA，使用q-PCR仪进行扩增和定量。选用ΔΔCt值来表征基因表达的差异，并采用相应软件进行数据处理以及统计学检验。Add 500 μL of pre-cooled Trizol reagent to each well, lyse on ice for 15 minutes, add 100 μL of chloroform, shake vigorously for 15 seconds, place on ice for 10 minutes, and centrifuge at 4°C at 12000 rpm for 15 minutes. Transfer the upper aqueous phase to a clean 1.5mL EP tube, add 200μL of isopropanol to precipitate RNA, place it on ice for 10 minutes, and centrifuge at 4°C and 12000rpm for 10 minutes. Discard the supernatant, wash the pellet with 75% ethanol, centrifuge to remove the supernatant, and dissolve the RNA pellet with 15 μL DEPC-treated water. Use Nano to quantify the RNA concentration, add Takara's reverse transcription reagent according to the instructions, and use an ordinary PCR machine to reverse transcribe mRNA into cDNA. Finally, add the upstream and downstream primers of the target gene (Gapdh, IL6 or IL1b) (check the primer bank), q-PCR reagent (SYBR Green) and cDNA into the 96-well plate dedicated for q-PCR. Use q-PCR The instrument performs amplification and quantification. Choose the ΔΔCt value to characterize the difference in gene expression, and use the corresponding software for data processing and statistical testing.
2.实验结果2. Experimental results
如图5所示，化合物55显著抑制LPS诱导的IL-6和IL-1b，并具有剂量依赖性，本发明中其他化合物也具有类似效果，说明本发明的化合物具有抗炎活性。As shown in Figure 5, compound 55 significantly inhibited LPS-induced IL-6 and IL-1b in a dose-dependent manner. Other compounds of the present invention also have similar effects, indicating that the compound of the present invention has anti-inflammatory activity.
实施例105Example 105
片剂tablet
将实施例60中制得的化合物60(50g)、羟丙甲基纤维素E(150g)、淀粉(200g)、聚维酮K30适量和硬脂酸镁(1g)混合，制粒，压片。The compound 60 (50g), hydroxypropylmethylcellulose E (150g), starch (200g), povidone K30 and magnesium stearate (1g) prepared in Example 60 were mixed, granulated, and compressed. .
此外，可以根据药典2015版常规制剂法，将实施例1-94制得的化合物赋予不同的药物辅料制成胶囊剂、散剂、颗粒剂、丸剂、注射剂、糖浆剂、口服液、吸入剂、软膏剂、栓剂或贴剂等。In addition, the compounds prepared in Examples 1-94 can be given different pharmaceutical excipients into capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments, according to the conventional preparation method of the pharmacopoeia 2015 edition. Agent, suppository or patch, etc.

Claims (8)

  1. 如式I所示的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物：The benzothiazole compound shown in formula I or a pharmaceutically acceptable salt or ester or solvate thereof:
    Figure PCTCN2021078798-appb-100001
    Figure PCTCN2021078798-appb-100001
    X为亚甲基、羰基或磺酰基；X is methylene, carbonyl or sulfonyl;
    Y为氢或XR 1； Y is hydrogen or XR 1 ;
    R 1、R 2各自独立地选自H、D、取代或非取代的烷基、取代或非取代的烯基、取代或非取代的炔基、取代或非取代的环烷基、取代或非取代的杂环烷基、取代或非取代的杂环烯基、取代或非取代的芳基或取代或非取代的杂环芳基； R 1 and 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 heterocyclic aryl;
    R 3为氢、羟基、杂环基、烷基、NH 2、NO 2、COOH、CN、SH、CF 3、SO 3H、SO 2CH 3或卤素。 R 3 is hydrogen, hydroxyl, heterocyclyl, alkyl, NH 2 , NO 2 , COOH, CN, SH, CF 3 , SO 3 H, SO 2 CH 3 or halogen.
  2. 根据权利要求1所述的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物，其特征在于，所述如式I所示的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物中，The benzothiazole compound or a pharmaceutically acceptable salt or ester or solvate thereof according to claim 1, wherein the benzothiazole compound as shown in formula I or a pharmaceutically acceptable Salt or ester or solvate,
    X为亚甲基、羰基或磺酰基；X is methylene, carbonyl or sulfonyl;
    Y为氢或XR 1； Y is hydrogen or XR 1 ;
    R 1为取代的烷基、取代或未取代的杂环基、取代或未取代的苄基、取代或未取代的杂芳基甲基、取代或未取代的芳基或杂芳基； R 1 is a substituted alkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted heteroarylmethyl group, a substituted or unsubstituted aryl group or a heteroaryl group;
    R 2为取代及未取代的杂芳基、取代或未取代的杂环烷基； R 2 is substituted and unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl;
    R 3为氢、羟基或杂环基。 R 3 is hydrogen, a hydroxyl group or a heterocyclic group.
  3. 根据权利要求1所述的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物，其特征在于，所述苯并噻唑类化合物是如下式II或式III所示的化合物或其药学上可接受的盐或酯或溶剂化物：The benzothiazole compound or a pharmaceutically acceptable salt or ester or solvate thereof according to claim 1, wherein the benzothiazole compound is a compound represented by the following formula II or formula III or Pharmaceutically acceptable salt or ester or solvate:
    Figure PCTCN2021078798-appb-100002
    Figure PCTCN2021078798-appb-100002
    Y为氢或SO 2R 1； Y is hydrogen or SO 2 R 1 ;
    R 1、R 2各自独立地选自H、D、取代或非取代的烷基、取代或非取代的烯基、取代 或非取代的炔基、取代或非取代的环烷基、取代或非取代的杂环烷基、取代或非取代的杂环烯基、取代或非取代的芳基或取代或非取代的杂环芳基。 R 1 and 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 heterocyclic aryl.
  4. 根据权利要求1～3任一所示的苯并噻唑类化合物或其药学上可接受的盐或酯可溶剂化物，其特征在于，所述化合物或其药学上可接受的盐或酯或溶剂化物选自如下任意一种化合物：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 solvate thereof Any one of the following compounds:
    Figure PCTCN2021078798-appb-100003
    Figure PCTCN2021078798-appb-100003
    Figure PCTCN2021078798-appb-100004
    Figure PCTCN2021078798-appb-100004
    Figure PCTCN2021078798-appb-100005
    Figure PCTCN2021078798-appb-100005
    Figure PCTCN2021078798-appb-100006
    Figure PCTCN2021078798-appb-100006
    Figure PCTCN2021078798-appb-100007
    Figure PCTCN2021078798-appb-100007
    Figure PCTCN2021078798-appb-100008
    Figure PCTCN2021078798-appb-100008
    Figure PCTCN2021078798-appb-100009
    Figure PCTCN2021078798-appb-100009
    Figure PCTCN2021078798-appb-100010
    Figure PCTCN2021078798-appb-100010
    Figure PCTCN2021078798-appb-100011
    Figure PCTCN2021078798-appb-100011
    Figure PCTCN2021078798-appb-100012
    Figure PCTCN2021078798-appb-100012
    Figure PCTCN2021078798-appb-100013
    Figure PCTCN2021078798-appb-100013
  5. 如权利要求1～3任一项所述的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物在制备在制备USP7调控剂中的用途。Use of the benzothiazole compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt or ester or solvate thereof in the preparation of a USP7 regulator.
  6. 如权利要求1～3任一项所述的苯并噻唑类化合物或其药学上可接受的盐或酯或溶剂化物在制备预防或治疗炎症、自身免疫性疾病、骨髓增生异常综合征或恶性肿瘤的药物中的用途。The benzothiazole compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt or ester or solvate thereof is used in the preparation of prevention or treatment of inflammation, autoimmune disease, myelodysplastic syndrome or malignant tumor Use in medicines.
  7. 一种预防或治疗炎症、自身免疫性疾病、骨髓增生异常综合征或恶性肿瘤的药物组合物，其中含有治疗有效量的如权利要求1～3任一项所述的任一苯并噻唑类化合物 或其药学上可接受的盐或酯或溶剂化物和药学上可接受的辅料。A pharmaceutical composition for preventing or treating inflammation, autoimmune diseases, myelodysplastic syndromes or malignant tumors, which contains a therapeutically effective amount of any benzothiazole compound according to any one of claims 1 to 3 Or a pharmaceutically acceptable salt or ester or solvate thereof and a pharmaceutically acceptable excipient.
  8. 根据权利要求7所述的药物组合物，其特征在于，所述药物组合物是普通片剂或胶囊、缓释片剂或胶囊、控释片剂或胶囊、颗粒剂、散剂、糖浆剂、口服液或注射剂。The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is an ordinary tablet or capsule, sustained-release tablet or capsule, controlled-release tablet or capsule, granule, powder, syrup, oral Liquid or injection.
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