WO2021098671A1 - 含脲基苯并咪唑类衍生物及其制备方法和应用 - Google Patents

含脲基苯并咪唑类衍生物及其制备方法和应用 Download PDF

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WO2021098671A1
WO2021098671A1 PCT/CN2020/129308 CN2020129308W WO2021098671A1 WO 2021098671 A1 WO2021098671 A1 WO 2021098671A1 CN 2020129308 W CN2020129308 W CN 2020129308W WO 2021098671 A1 WO2021098671 A1 WO 2021098671A1
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phenyl
urea
benzimidazol
methyl
trifluoromethyl
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French (fr)
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李乾斌
胡高云
王泽瑜
陈卓
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石家庄以岭药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/10—Radicals substituted by halogen atoms or nitro radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/18—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of a saturated carbon skeleton containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/20—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton

Definitions

  • the invention relates to the field of pharmaceutical chemical synthesis, in particular to ureido-containing benzimidazole derivatives, and a preparation method and application thereof.
  • Chronic kidney disease is a general term for a class of diseases, including diabetic nephropathy, hypertensive nephropathy, glomerulonephritis, IgA nephropathy, pyelonephritis, allergic purpura nephritis, lupus erythematosus nephritis, gouty kidney, nephrotic syndrome, polycystic kidney disease Wait.
  • the incidence and (especially) the prevalence of CKD have a trend of increasing year by year.
  • the incidence of CKD is positively correlated with the age of patients, and there has been a trend of younger age.
  • the awareness rate of CKD in the general population is low, and clinically effective prevention and treatment methods are lacking.
  • CKD CKD caused by different causes has the same pathological changes, that is, renal fibrosis accompanied by inflammation.
  • angiotensin converting enzyme inhibitors ACEI
  • ARB angiotensin receptor inhibitors
  • ACEI and ARB drugs in the early stage of diabetic nephropathy can regulate glomerular hemodynamics, inhibit renal fibrosis, and reduce renal protection such as trace proteinuria.
  • ACEI and ARB drugs have no advantages in the late-stage efficacy of diabetic nephropathy, and there may be varying degrees of elevated blood creatinine, which may cause renal insufficiency. Therefore, unlike other diseases, CKD will continue to progress even if the cause is well controlled. The treatment of pathological processes is an important and necessary means to control CKD.
  • drug treatment strategies for pathological processes mainly include anti-inflammatory or anti-fibrosis single strategies and multi-target strategies with dual effects.
  • the multi-target drug pirfenidone has the dual effects of anti-inflammatory and anti-fibrosis, and has a good application prospect in the treatment of diabetic nephropathy, but its half-life is short, the activity is low, the dosage and side effects are large, and it is suitable for the treatment of chronic kidney disease that requires long-term medication. In terms of its application is restricted. Therefore, new ideas and strategies are needed for the development of therapeutic drugs for chronic kidney disease.
  • the homeodomain-interacting protein kinase (HIPK) family consists of 4 members: HIPK1, HIPK2, HIPK3 and HIPK4.
  • the expression level is low at normal levels.
  • HIPK1 and HIPK3 are mainly distributed in tissues such as heart and smooth muscle.
  • HIPK2 is mainly in Distributed in the lesions of the kidney, HIPK4 is less studied.
  • HIPK2 can regulate inflammation and fibrosis in chronic kidney disease models (Nat Med, 2012, 18, 580). Research by Jin Y et al.
  • HIPK2 was expressed at a high level in a variety of patients with kidney disease (HIV infection, diabetic nephropathy, IgA nephropathy, focal segmental glomerulosclerosis).
  • kidney disease HIV infection, diabetic nephropathy, IgA nephropathy, focal segmental glomerulosclerosis.
  • HIPK2 can directly interact with Smad3 to activate the Smad3 pathway and promote the production of fibrosis; regulate epithelial-mesenchymal transition by activating Wnt- ⁇ -catenin and Notch pathways; and inhibit I ⁇ B - ⁇ activates the NF- ⁇ B pathway to activate the occurrence of inflammation; regulates the process of p53-mediated autophagy.
  • HIPK2 can simultaneously regulate fibrosis and inflammatory processes.
  • HIPK2 inhibitors are a promising field for chronic kidney disease treatment drugs.
  • HIPK2 inhibitors can improve unilateral ureteral ligation and Tg26 mice by inhibiting the interaction of HIPK2 and SMAD3 Model kidney fibrosis, but these compounds do not directly inhibit the kinase activity of HIPK2.
  • Z is an oxadiazole ring
  • X is selected from C or NH
  • R 1 and R 2 are independently selected from C1-C8 hydrocarbon groups, -OH, O(C1-C8) hydrocarbon groups, halogens, nitro groups, C1-C3 alkylamino groups , C1-C3 dialkylamino, C1-C3 amido, C1-C3 alkanesulfonyl, C1-C3 alkylthio, C1-C3 haloalkyl, C1-C3 haloalkoxy, C1-C3 haloalkylthio, -COOH , C1-C3 alkoxycarbonyl group, B(OR3)2, BF3K; R 3 is selected from H, C1-C8 hydrocarbon group; or (OR3) 2 is selected from dioxopentaborane or dioxohexaborane as a whole.
  • the technical problem to be solved by the present invention is to solve the above-mentioned problems in the prior art, and provide a ureido-containing benzimidazole derivative and its preparation method and application, which can effectively inhibit HIPK2, and can be effective against IPK1-3 subtypes. Selectively, while regulating fibrosis and inflammatory processes.
  • R 1 is selected from one of hydrogen, C1-C6 aliphatic chain, C3-C6 aliphatic ring, substituted C3-C6 aliphatic ring, aromatic ring, substituted aromatic ring, aromatic heterocyclic ring and substituted aromatic heterocyclic ring;
  • R 2 is selected from one of hydrogen, methyl, trifluoromethyl, dimethylamino, aromatic ring, aromatic heterocyclic ring, substituted aromatic ring and substituted aromatic heterocyclic ring;
  • Part A is selected from one of aromatic ring, aromatic heterocyclic ring, substituted aromatic ring and substituted aromatic heterocyclic ring;
  • Part B is selected from aliphatic heterocycles, aromatic heterocycles, C1-C4 open-chain amine compounds, C1-C6 linear or branched alkoxy groups, C1-C6 linear or branched alkanes, C3-C6 aliphatic rings and substituted One of C3-C6 aliphatic rings;
  • n 1 ⁇ 6.
  • the aromatic ring is selected from one of benzene, naphthalene and anthracene;
  • the aromatic heterocyclic ring is selected from pyrrole, furan, thiophene, imidazole, thiazole, oxazole, pyrazole, isoxazole, thiadiazole, oxadiazole, tetrazolium, pyridine, pyrimidine, pyrazine, and pyridine.
  • azine purine, quinoline, isoquinoline, indole, acridine and carbazole;
  • the aliphatic heterocyclic ring is selected from one of pyrrolidine, piperazine, piperidine, dioxane and tetrahydrofuran;
  • the substituent in the substituted aromatic ring or substituted aromatic heterocyclic ring is selected from one of hydroxy, methyl, methoxy, amino, trifluoromethyl and halogen.
  • the above-mentioned ureido-containing benzimidazole derivatives whose pharmaceutically usable salts are hydrochloride, sulfate, phosphate, perchlorate, methanesulfonate, trifluoromethanesulfonate, and formic acid.
  • part B is selected from aliphatic heterocycles, C1-C4 open-chain amine compounds, C1-C6 linear or branched alkoxy, C1-C6 linear or branched alkanes, C3-C6 aliphatic rings and substituted C3 -One of C6 aliphatic rings.
  • part B is selected from one of aliphatic heterocycles, C3-C6 aliphatic rings and substituted C3-C6 aliphatic rings.
  • part B is an aliphatic heterocyclic ring
  • the aliphatic heterocyclic ring is selected from one of pyrrolidine, piperazine, piperidine, dioxane and tetrahydrofuran.
  • the A part is an aromatic ring or a substituted aromatic ring.
  • R 1 is an aromatic ring or a substituted aromatic ring.
  • R 2 is methyl or trifluoromethyl.
  • the above-mentioned ureido group-containing benzimidazole derivatives are specifically the following compounds:
  • the invention also discloses a preparation method of ureido-containing benzimidazole derivatives, which comprises the following steps:
  • compound II is coupled with a boric acid substitute under the action of palladium acetate catalyst to obtain compound III.
  • the substituent on the boric acid substitute is selected from the group consisting of aminobenzene, aminonaphthalene, aminoanthracene, aminophenanthrene, substituted aminobenzene, and substituted Amino naphthalene, substituted amino anthracene and substituted amino phenanthrene; the structure of the boric acid substitute is shown in formula IV:
  • R1 is selected from hydrogen, C1-C6 aliphatic chain, C3-C6 aliphatic ring, substituted C3-C6 aliphatic ring, aromatic ring, substituted aromatic ring, aromatic heterocyclic ring and substituted aromatic heterocyclic ring;
  • R2 is selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylamino, aromatic ring, aromatic heterocyclic ring, substituted aromatic ring and substituted aromatic heterocyclic ring;
  • A is selected from one of aromatic ring, aromatic heterocyclic ring, substituted aromatic ring and substituted aromatic heterocyclic ring;
  • B is selected from aliphatic heterocycles, aromatic heterocycles, C1-C4 open-chain amino groups, C1-C6 linear or branched alkoxy groups, C1-C6 linear or branched alkanes, C3-C6 aliphatic rings and substituted C3- One of C6 aliphatic rings;
  • n 1 ⁇ 6.
  • the invention also discloses the application of the above-mentioned ureido-containing benzimidazole derivatives in the preparation of medicines for treating chronic kidney disease. Preferably, it is used as an active ingredient to prepare anti-diabetic nephropathy drugs.
  • the present invention can simultaneously down-regulate the protein level of HIPK2 and play a dual role. It has a dual effect on the level of HIPK2 kinase and protein, fibrosis signal molecule SMAD3, and inflammation signal molecule I ⁇ B. - ⁇ phosphorylation level has an inhibitory effect, can simultaneously regulate fibrosis and inflammatory processes, and has a good market application prospect in the treatment of chronic kidney disease.
  • Figure 1 shows the effect of compound 6d1 prepared in Example 7 on HIPK2
  • Figure 2 shows the effect of compound 6d1 prepared in Example 7 on fibrotic pathways
  • Figure 3 shows the effect of compound 6d1 prepared in Example 7 on inflammatory pathways.
  • step 1 In a 50ml three-necked flask, add iron powder (0.4g, 7.2mmol), 20ml of trifluoroacetic acid, add 2-3 drops of concentrated hydrochloric acid to catalyze, and then activate it under ultrasound for 3min.
  • the 4-bromo-2-nitro-N-aniline (0.58 g, 2 mmol) obtained in step 1 was added to the reaction system, and the reaction was carried out under reflux for 6 hours. After the reaction was stopped, it was allowed to stand to room temperature. Use 20 g of diatomaceous earth to remove iron filings with a Buchner funnel, and rinse with ethyl acetate until the diatomaceous earth has almost no product remaining.
  • the preparation method of compound 6e 1 refers to 6a 2 , a light yellow solid, the yield is 60.3%, mp: 210.5-212.3°C.
  • the preparation method of compound 6i 1 refers to 6a 2 , pale yellow solid, yield: 65.0%, mp: 220.3-221.5°C.
  • the preparation method of compound 6i 2 refers to 6a 2 , pale yellow solid, yield: 58.8%, mp: 217.4-219.0°C.
  • the preparation method of compound 6m 1 refers to 6a 2 , a pale yellow oily substance, with a yield of 43.9%.
  • this experiment used the CCK-8 method to determine the inhibitory rate of the synthesized compound on the rat renal fibroblast NRK-49F cell line.
  • Compound C and Sorafenib were used as positive controls. , Calculate the IC 50 value of each compound, and conduct a preliminary structure-activity relationship study.
  • the proliferation inhibitory activity of the compound on the HK-2 cell line of human proximal renal tubular epithelial cells was determined.
  • Compound C and Sorafenib were used as positive controls to calculate the IC 50 value of each compound.
  • IC 50 (HK -2) The ratio of IC 50 (NRK-49F) to investigate the selectivity of the compound to cells to preliminarily explore the nephrotoxicity of the compound.
  • Cell lines rat kidney fibroblast NRK-49F cell line and human proximal tubular epithelial cell HK-2 cell line.
  • 96-well cell culture plate was purchased from Costar; RPMI 1640 medium was purchased from Gibco; DMEM medium was purchased from Gibco; Fetal bovine serum was purchased from Gibco; DMSO was purchased from Sigma; EnoGeneCell TM Counting Kit-8 (CCK-8) cell viability detection kit (E1CK-000208-10) was purchased from Nanjing Enjing Biotechnology Co., Ltd.
  • ChemBase CBS-CJ-1FD ultra-clean workbench MCO-15AC carbon dioxide incubator (SANYO); XD-202 fluorescent inverted biological microscope microscope (Nanjing Jiangnan Yongxin Optics Co., Ltd.); Thermo MK3 microplate reader (American Thermoelectric Company).
  • NRK-49F and HK-2 cells are cultured routinely. In the experiment, cells in the logarithmic growth phase were used, and cells with a ratio of viable cells of more than 90% were used for the experiment.
  • Cell growth detection Cells are digested, counted, and made into a cell suspension with a concentration of 1 ⁇ 105 cells/mL. Add 100 ⁇ L of cell suspension to each well of a 96-well plate (1 ⁇ 104 cells per well); 96-well plate Place in a 37°C, 5% CO2 incubator for 24 hours; add 100 ⁇ L of the corresponding drug-containing medium to each well to make the final concentration of the compound in the well 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125 ⁇ M/mL, and at the same time set up a negative control group, a vehicle control group, and a positive control group, each with 5 multiple wells; the 96-well plate is placed in a 37°C, 5% CO2 incubator after 72 hours of incubation; 10 ⁇ L is added to each well With CCK-8 solution, incubate the culture plate in the incubator for 4 hours, and measure the OD value at 450nm with a microplate reader.
  • Graphpad Prism 5.0 software was used to fit the curve with the concentration as the abscissa and the inhibition rate as the ordinate to calculate the IC 50 value of the compound. Measure the absorbance OD value of each well on the microplate reader at 450nm, and the cell growth inhibition rate is calculated as follows:
  • Ratio of IC 50 IC 50 (HK-2)/IC 50 (NRK-49F); "-" means that the value cannot be determined.
  • the HTRF experiment was used to determine the inhibitory effect of the target compound on HIPK2 enzyme activity.
  • ATP, DTT, MgCl2, CaCl2, and cGMP were purchased from Sigma; Calnexin was purchased from Upstate, and HIPK2 was purchased from Carna; other reagents were purchased from Cisbio without instructions.
  • HTRF KinEASE-STK kit was purchased from Cisbio.
  • Enzyme preparation stage pre-configured buffer (5mM MgCl2, 1mM DTT, 50 ⁇ M AMP); compound solution (100,000nM, 20,000nM, 4,000nM, 800nM, 160nM, 32nM, 6.4nM, 1.3nM, 0.26nM, 0,051nM ); Add 4 ⁇ L of compound solution (or buffer) to each well of a 384-well plate, add 2 ⁇ L of HIPK2 solution, add 2 ⁇ L of ATP, 2 ⁇ LSTK-S2, and incubate for 1h at rt. Select Compound C and Staurosporine as the positive control drugs.
  • Detection stage pre-configure fluorescent substance solution (0.5 ⁇ M SA-XL665, 0.5 ⁇ M STK Ab); add 5 ⁇ L SA-XL665 solution, 5 ⁇ L STK Ab solution to each well; incubate at rt for 1h; use HTRF microplate reader Read the OD values at 620nm and 665nm.
  • Graphpad Prism 5.0 software was used to fit the curve with the concentration as the abscissa and the inhibition rate as the ordinate to calculate the IC 50 value of the compound. Measure the absorbance OD value of each well on the microplate reader at 620nm and 665nm, and the cell growth inhibition rate is calculated as follows:
  • HIPK2 is degraded and expressed by ubiquitin ligase at normal levels. In the disease state, ubiquitin ligase is inactivated. HIPK2 is highly stable. MG132 can inhibit the activity of ubiquitin ligase. The IC 50 in the cell-free test is 100 nM. In this experiment, the compound 6d 1 with the best HIPK2 enzyme inhibitory activity was selected for Western Blot experiment. During the experiment, MG132 was used to increase the concentration of HIPK2 in cells at a concentration of 200ng/ml. The experimental components were: control group, MG132 treatment group, MG132 Treatment/drug group, MG132/control group.
  • Cell lines NRK-49F, HK-2 cell lines.
  • Antibodies smad2/3, p-smad3, I ⁇ B- ⁇ , p-I ⁇ B- ⁇ , ⁇ -SMA, HIPK2, GAPDH were all purchased from EnoGene.
  • ECL chemiluminescence reagents were purchased from Biyuntian; Maxisignal Western Soltion was purchased from SUDGEN; RIPA lysate, BSA, pre-stained protein marker, goat anti-rabbit secondary antibody, developer, fixer, BCA protein concentration
  • the detection kits were all purchased from Nanjing Enjing Biotechnology Co., Ltd.
  • a. Cell sample preparation Take logarithmic growth phase NRK-49F, HK-2 cells for experiment. The cells are digested and counted to prepare a cell suspension of 1 ⁇ 10 5 cells/mL, seeded in a 6-well plate (2ml/well), and placed in a 37°C, 5% CO 2 incubator for 24 hours; each well Add 200nM MG132, and set up a blank control group at the same time; place the plate in the incubator for 72 hours, change the fresh medium, and add different concentrations of compound at the same time, NRK-49F adds 2uM, 10uM compound 6d 1 and Compound C, HK-2 Add 10uM, 50uM compound 6d 1 and BAY 11-7082, and set up a MG132 single-action group at the same time, collect cell samples after 5 hours of action, and detect each target protein by WB.
  • Protein determination trypsin digestion to collect cell samples, add 100 ⁇ l RIPA lysis buffer (including PMSF and phosphatase inhibitor), pipette repeatedly in an ice bath to fully lyse the sample, obtain the lysate and centrifuge (4°C, 12000rpm, 15min) ), collect the supernatant and determine the protein content. Add 5 ⁇ Loading Buffer in a 4:1 ratio according to the volume of the supernatant, and denature the protein in a water bath at 100°C for 10 minutes. After cooling to room temperature, it is centrifuged briefly and stored at -20°C for testing.
  • RIPA lysis buffer including PMSF and phosphatase inhibitor
  • the membrane was blocked in 5% BSA for 1 hour, and then fully washed with TBST (5 min/time, 3 times), and combined with the corresponding target protein antibody, overnight at 4°C. After TBST was fully washed (5min/time, 3 times), combined with the corresponding secondary antibody at room temperature for 1h, TBST was fully rinsed (10min/time, 3 times), and developed and fixed with film and ECL luminescent solution in a dark room.
  • Use Adobe Photoshop CS5 software to analyze the net optical density value of the target band, and use GAPDH as the internal reference.
  • the data are expressed in mean ⁇ SD, using GraphPad Prism 5.0 statistical software, the comparison between the two groups is by t-test test, and the comparison between multiple groups is by One-way Anova (Dunnett), P ⁇ 0.05 is considered statistically significant.
  • the HIPK2 levels in the control cells in Figure 1 are all at a low level, and the HIPK2 levels in the cells are greatly increased after the addition of 200ng/ml MG132 for 72 hours.
  • the phosphorylation level of HIPK2 decreased, and the reduction level was dose-dependent on the compound concentration.
  • Compound 6d1 inhibited HIPK2 phosphorylation level at 10 ⁇ M and 50 ⁇ M stronger than the control drug BAY11- 7082.
  • the phosphorylation level of HIPK2 decreased, and the phosphorylation decreased level was dose-dependent on the compound concentration.
  • the compound has an inhibitory effect on the level of HIPK2 protein.
  • HIPK2 can regulate fibrosis and inflammation pathways.
  • the present invention measured the changes in p-smad3 and ⁇ -SMA phosphorylation levels after compounds stimulated NRK-49F cells. As shown in Figure 2, after adding compound 6d 1 , the phosphorylation level of p-smad3 decreased after Compound C, and the reduction level was dose-dependent on the concentration of compound 6d 1 and Compound C, which proved that the compound has a significant effect on the key signal molecule smad3 of fibrosis. Regulation effect. The compound basically has no effect on the phosphorylation level of ⁇ -SMA.
  • this experiment measured the effect of the compound on the level of p-I ⁇ B- ⁇ . As shown in Figure 3, when the cells were treated with MG132, the p-I ⁇ B- ⁇ level increased. Compound 6d 1 was added. After BAY11-7082, p-I ⁇ B- ⁇ decreased, and the concentration of the compound was dose-dependent, so the compound It has a regulatory effect on the inflammatory signaling molecule I ⁇ B- ⁇ .
  • compound 6d 1 has an inhibitory effect on HIPK2 kinase and protein levels, fibrosis signal molecule smad3, and inflammation signal molecule I ⁇ B- ⁇ phosphorylation level.
  • the compound prepared by the invention directly targets HIPK2, is selective for HIPK1-3 subtypes, has dual effects of anti-inflammatory and anti-fibrosis, and has a better application prospect for the treatment of chronic kidney disease.

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Abstract

本发明公开了含脲基苯并咪唑类衍生物，结构通式如式(I)所示。本发明还公开了所述化合物的制备方法和它们在制备抗慢性肾病药物中的用途。本发明为选择性同源结构域相互作用蛋白激酶-2(HIPK2)的抑制剂，能够同时下调HIPK2的蛋白水平，发挥双重作用，对HIPK2激酶及蛋白水平、纤维化信号分子smad3、炎症信号分子IκB-α磷酸化水平均具有抑制作用，可同时调节纤维化与炎症过程，在慢性肾病治疗药物上有良好的市场应用前景。

Description

含脲基苯并咪唑类衍生物及其制备方法和应用
交叉参考相关引用
本申请要求2019年11月22日递交的申请号为201911152583.9、发明名称为“含脲基苯并咪唑类衍生物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及药物化学合成领域，具体涉及含脲基苯并咪唑类衍生物及其制备方法和应用。
背景技术
慢性肾病(CKD)是一类疾病的总称，包括糖尿病肾病、高血压肾病、肾小球肾炎、IgA肾病、肾盂肾炎、过敏性紫癜肾炎、红斑狼疮肾炎、痛风肾、肾病综合征、多囊肾等。CKD的发病率和(尤其是)患病率有逐年增长的趋势。CKD的发病率与患者年龄呈正相关，且已经出现低龄化趋势。然而，CKD在普通人群中的知晓率低，临床上缺乏有效的防治手段。
导致CKD的诱因较多，有糖尿病、高血压、肥胖、感染等。不同诱因导致的CKD有相同的病理改变，即伴有炎症的肾脏纤维化。临床上除了控制高血糖、高血压、高血脂以及限制蛋白的摄入等基础治疗外，血管紧张素转化酶抑制剂(ACEI)和血管紧张素受体抑制剂(ARB)已成为国际公认的防治糖尿病肾病的一线药物。ACEI和ARB类药物在糖尿病肾病的早期能调节肾小球血流动力学，抑制肾纤维化，降低微量的蛋白尿等肾脏保护作用。然而，ACEI和ARB类药物在糖尿病肾病晚期疗效方面并无优势，且可能出现程度不等的血肌酐升高现象，有引起肾功能不全的风险。因而，与其他疾病不同的是，即使较好地控制了病因，CKD仍会持续进展，针对病理过程的治疗是控制CKD重要且必须的手段。
目前，针对病理过程的药物治疗策略主要包括抗炎或抗纤维化单一策略和兼有双重作用的多靶点策略等。研究表明，纤维化与炎症之间存在复杂的相互作用，单纯抑制纤维化并不能阻断炎症，由于炎症引发的细胞通透性增加会使肾脏产生蛋白尿恶化；同样单纯阻断炎症过程，肾脏的纤维化程度由于TGF-β等促纤维细胞因子的持续刺激，细胞外基质过度沉积，加重肾脏的负担和功能丧失。而且，临床试验结果显示现有的药物多存在潜在风险(如作为抗炎药物的三萜类化合物Bardoxolone methyl通过诱导对NK-κB 信号通路具有抑制作用的核因子Nrf2以减少炎症和氧化应激，发挥对肾脏的保护作用，但由于提升患者死于心血管功能异常的风险，被迫终止III期临床试验)。因此，单纯阻断炎症或纤维化的治疗策略目前没有取得成功。多靶点药物吡非尼酮兼有抗炎和抗纤维化双重作用，具有较好的糖尿病肾病治疗应用前景，但其半衰期短、活性低、用量及副作用大，对于需要长期用药的慢性肾病治疗而言其应用受到限制。因此，慢性肾病治疗药物研发需要新的思路与策略。
同源结构域相互作用蛋白激酶(HIPK)家族包含4个成员：HIPK1，HIPK2，HIPK3及HIPK4，在正常水平下表达水平均较低，HIPK1和HIPK3主要分布在心脏、平滑肌等组织，HIPK2主要在分布在肾脏病变部位，HIPK4研究较少。HIPK2可以调控慢性肾病模型中的炎症及纤维化过程(Nat Med,2012,18,580)。Jin Y等人研究发现，与对照组相比，多种患者肾病组织(HIV感染、糖尿病肾病、IgA肾病、局灶性节段性肾小球硬化)中HIPK2处于高表达水平。在人肾小管上皮细胞HK-2细胞模型中，HIPK2可直接与Smad3相互作用，激活Smad3通路，促进纤维化的产生；通过激活Wnt-β-catenin及Notch通路调控上皮间质转化；通过抑制IκB-α激活NF-κB通路激活炎症的发生；调节p53介导的自噬过程。综上所述，HIPK2可同时调节纤维化与炎症过程，HIPK2抑制剂用作慢性肾病治疗药物是一个很有前景的领域。
近年来，有研究者们为考察其他激酶抑制剂的脱靶效应测定了化合物对HIPK不同亚型的抑制作用，这些抑制剂多数被用于抗肿瘤药物研究，有些已获批上市，如索拉菲尼、舒尼替尼等。
目前，直接以HIPK2为靶点的慢性肾病治疗药物研究报道极少。文献(PLoS ONE,2014,9,e891762)报道了一类2-芳基-4,5,6,7-四溴异吲哚-1,3-二酮衍生物(通式VI)，该类化合物可以抑制肝癌细胞HIPK2对P53蛋白Ser46的磷酸化。但化合物对HIPK1-3亚型无选择性，且并无对纤维化或慢性肾病相关的数据报道。
Figure PCTCN2020129308-appb-000001
文献(J Am Soc Nephrol,2017,28,2133)和专利WO2018129274公开了一类噁二唑类(通式VII)HIPK2抑制剂可通过抑制HIPK2与SMAD3的相互作用改善单侧输尿管结扎和Tg26小鼠模型的肾纤维化，但该类化合物并不直接抑制HIPK2的激酶活性。
Figure PCTCN2020129308-appb-000002
其中Z为噁二唑环；X选自C或NH；R 1和R 2分别独立选自C1-C8烃基，-OH,O(C1-C8)烃基，卤素，硝基，C1-C3烷氨基，C1-C3二烷氨基，C1-C3酰胺基，C1-C3烷磺酰基，C1-C3烷硫基，C1-C3卤代烷基，C1-C3卤代烷氧基，C1-C3卤代烷硫基，-COOH，C1-C3烷氧羰基，B(OR3)2,BF3K；R 3选自H，C1-C8烃基；或(OR3)2整体选自二杂氧戊硼烷或二杂氧己硼烷。
鉴于慢性肾病重大的临床需求和HIPK2抑制剂的良好前景，本领域迫切需要开发有效的HIPK2抑制剂。
发明内容
本发明所要解决的技术问题是：解决上述现有技术存在的问题，提供一种含脲基苯并咪唑类衍生物及其制备方法和应用，有效抑制HIPK2，并能对IPK1-3亚型有选择性，同时调节纤维化与炎症过程。
本发明采用的技术方案是：
一种含脲基苯并咪唑类衍生物，结构通式如式(I)所示：
Figure PCTCN2020129308-appb-000003
其中，
R 1选自氢、C1-C6脂肪链、C3-C6脂肪环、取代C3-C6脂肪环、芳环、取代芳环、芳杂环和取代芳杂环其中一种；
R 2选自氢、甲基、三氟甲基、二甲胺基、芳环、芳杂环、取代芳环和取代芳杂环其中一种；
A部分选自芳环、芳杂环、取代芳环和取代芳杂环其中一种；
B部分选自脂肪杂环、芳杂环、C1-C4开链胺类化合物、C1-C6直链或支链烷氧基、C1-C6直链或支链烷烃、C3-C6脂肪环和取代C3-C6脂肪环其中一种；
n＝1～6。
优选的，所述芳环选自苯、萘和蒽其中一种；
优选的，所述芳杂环选自吡咯、呋喃、噻吩、咪唑、噻唑、噁唑、吡唑、异噁唑、噻二唑、噁二唑、四氮唑、吡啶、嘧啶、吡嗪、哒嗪、嘌呤、喹啉、异喹啉、吲哚、吖啶和咔唑其中一种；
优选的，所述脂肪杂环选自吡咯烷、哌嗪、哌啶、二氧六环和四氢呋喃其中一种；
优选的，所述取代芳环或取代芳杂环中的取代基选自羟基、甲基、甲氧基、氨基、三氟甲基和卤素其中一种。
优选的，上述含脲基苯并咪唑类衍生物，其药学上可用的盐是盐酸盐、硫酸盐、磷酸盐、高氯酸盐、甲磺酸盐、三氟甲磺酸盐、甲酸盐、乙酸盐、丙酸盐、丁酸盐、马来酸盐、丁二酸盐、三氟乙酸盐、琥珀酸盐、水杨酸盐、DL-天冬氨酸盐、D-天冬氨酸盐、L-天冬氨酸盐、DL-谷氨酸盐、D-谷氨酸盐、L-谷氨酸盐、甘油酸盐、硬脂酸盐、DL-酒石酸盐、D-酒石酸盐、L-酒石酸盐、(±)扁桃酸盐、(R)-(-)扁桃酸盐、(S)-(+)扁桃酸盐、柠檬酸盐、粘酸盐、丙二酸盐、苯甲酸盐、DL-苹果酸盐、(±)乳酸盐、L-(+)-乳酸盐、D-(+)-乳酸盐、扑酸盐、D-α-半乳糖醛酸盐、甘油酸盐、DL-半胱氨酸盐、D-半胱氨酸盐、L-半胱氨酸盐、(4S)-羟基-L-脯氨酸盐、环丙烷-1,1-二羧酸盐、2,2-甲基丙二酸盐、酪氨酸盐、脯氨酸盐、富马酸盐、1-羟基-2-萘甲酸盐、膦酰基乙酸盐、碳酸盐、碳酸氢盐、3-膦酰基丙酸盐、DL-焦谷氨酸盐、D-焦谷氨酸盐、L-焦谷氨酸盐、对甲苯磺酸盐、苯磺酸盐、乙磺酸盐、(±)樟脑磺酸盐、萘磺酸盐、1R-(-)-樟脑磺酸盐、1S-(+)-樟脑磺酸盐、1,5-萘二磺酸盐、1,2-乙烷二磺酸盐、1,3-丙烷二磺酸盐、3-(N-吗啉代)丙烷磺酸盐、联苯基磺酸盐，羟乙基磺酸盐、1-羟基-2-萘磺酸盐、磷酸二氢盐、磷酸氢钾盐、磷酸二钾盐、磷酸钾盐、磷酸氢钠盐、磷酸二钠盐、磷酸钠盐、磷酸二氢钠盐、磷酸钙盐、三代磷酸钙盐、六氟代磷酸盐、乙烯基磷酸盐、2-羟基乙基磷酸盐和苯基磷酸盐。
优选的，B部分选自脂肪杂环、C1-C4开链胺类化合物、C1-C6直链或支链烷氧基、C1-C6直链或支链烷烃、C3-C6脂肪环和取代C3-C6脂肪环其中一种。
优选的，B部分选自脂肪杂环、C3-C6脂肪环和取代C3-C6脂肪环其中一种。
优选的，B部分为脂肪杂环，所述脂肪杂环选自吡咯烷、哌嗪、哌啶、二氧六环和四氢呋喃其中一种。
优选的，A部分为芳环或取代芳环。
优选的，R 1为芳环或取代芳环。
优选的，R 2为甲基或三氟甲基。
优选的，上述含脲基苯并咪唑类衍生物具体为以下化合物：
1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-吗啉乙基)脲；
1-(2-吗啉乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(3-吗啉丙基)脲；
1-(3-吗啉丙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-(哌啶-1-基)乙基)脲；
1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(2-(哌啶-1-基)乙基)脲；
1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-(吡咯烷-1-基)乙基)脲；
1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(2-(吡咯烷-1-基)乙基)脲；
1-异戊基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-异戊基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(3-(吡咯烷-1-基)丙基)脲；
1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(3-(吡咯烷-1-基)丙基)脲；
1-(2-环己基乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-(2-环己基乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-戊基脲；
1-戊基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-丁基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-丁基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-(环己基甲基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-(环己基甲基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-异丁基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-异丁基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
1-(2-甲氧基乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-(3-甲氧基乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-yl)苯基)脲；
1-(3-甲氧基丙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-(3-甲氧基丙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-yl)苯基)脲；
1-(3-(二乙胺)乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
1-(3-(二乙胺)丙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
本发明还公开了一种含脲基苯并咪唑类衍生物的制备方法，包括以下步骤：
Figure PCTCN2020129308-appb-000004
S1、化合物II在催化剂醋酸钯作用下与硼酸取代物发生耦联反应得到化合物III，所述硼酸取代物上的取代基选自氨基苯、氨基萘、氨基蒽、氨基菲、取代氨基苯、取代氨基萘、取代氨基蒽和取代氨基菲；所述硼酸取代物结构如式IV所示：
Figure PCTCN2020129308-appb-000005
S2、化合物III与伯胺在缩合剂的作用下形成所述含脲基苯并咪唑类衍生物，所述缩合剂选自三光气、N,N'-羰基二咪唑和氯甲酸对硝基酚酯其中一种；所述伯胺结构如式V所示：
Figure PCTCN2020129308-appb-000006
其中：
R1选自氢、C1-C6脂肪链、C3-C6脂肪环、取代C3-C6脂肪环、芳环、取代芳环、芳杂环和取代芳杂环；
R2选自氢、C1-C6的烷基、C1-C6的卤代烷基、C1-C6的烷基胺基、芳环、芳杂环、取代芳环和取代芳杂环；
A选自芳环、芳杂环、取代芳环和取代芳杂环其中一种；
B选自脂肪杂环、芳杂环、C1-C4开链胺基、C1-C6直链或支链烷氧基、C1-C6直链或支链烷烃、C3-C6脂肪环和取代C3-C6脂肪环其中一种；
n＝1～6。
本发明还公开了上述含脲基苯并咪唑类衍生物在制备治疗慢性肾病药物制备中的应用。优选的，其作为活性成分用于制备抗糖尿病肾病药物。
本发明作为选择性同源结构域相互作用蛋白激酶-2(HIPK2)抑制剂，能够同时下调HIPK2的蛋白水平，发挥双重作用，对HIPK2激酶及蛋白水平、纤维化信号分子 SMAD3、炎症信号分子IκB-α磷酸化水平均具有抑制作用，可同时调节纤维化与炎症过程，在慢性肾病治疗上具有良好的市场应用前景。
附图说明
图1为实施例7制备的化合物6d1对HIPK2的影响；
图2为实施例7制备的化合物6d1对纤维化通路的影响；
图3为实施例7制备的化合物6d1对炎症通路的影响。
具体实施方式
以下实施例以A部分选取苯环进行说明，具体反应方程式如下：
Figure PCTCN2020129308-appb-000007
实施例1 1-(2-吗啉乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6a 2)
1、制取4-溴-2-硝基-N-苯胺(3)
Figure PCTCN2020129308-appb-000008
将根据文献方法由1和2制备或购置2-氟-5-溴硝基苯(4.4g，20mmol)和苯胺(2.2ml，24mmol)加入装有15ml水的微波管中，于120℃下反应4h。冷却后，用乙酸乙酯萃取3次，乙酸乙酯层用饱和NaCl溶液洗涤，无水Na 2SO 4干燥并放置4h。旋干有机溶剂，用石油醚进行硅胶柱层析分离，得到橙红色结晶4.92g，产物3与原料1极性非常相近，即使是用极性最小的石油醚展开，二者位移值依旧非常相近，较难得到纯品，直接投入下一步反应。
2、制取5-溴-1-苯基-2-(三氟甲基)-1H-苯并咪唑(4b)
Figure PCTCN2020129308-appb-000009
在50ml三颈瓶中，加入铁粉(0.4g，7.2mmol)，20ml三氟乙酸，滴加2-3滴浓盐酸催化，随后在超声中活化3min。在反应体系中加入步骤一中得到的4-溴-2-硝基-N-苯胺(0.58g，2mmol)，于回流下反应6h，停止反应后静置至室温。以20g硅藻土用布氏漏斗除去铁屑，用乙酸乙酯冲洗至硅藻土基本无产物残留。减压旋蒸除去大部分溶剂，以饱和KOH溶液调PH至7，乙酸乙酯萃取3次，饱和NaCl溶液洗涤3次，无水Na 2SO 4干燥并放置4h，旋干有机溶剂。粗产物经过硅胶柱层析分离(以PE：EA＝3:1分离反应原料，极性增至1：1分离出产物)，得到4a共0.37g。
白色晶体，收率:80％，mp:70.8-71.9℃。 1H NMR(500MHz,DMSO-d 6):δ8.20(d,J＝1.7Hz,2H),7.67(s,2H),7.60(d,J＝1.9Hz,1H),7.58(d,J＝1.9Hz,1H),7.17(s,1H),7.15(s,1H). 13C NMR(125MHz,DMSO-d 6):δ141.81,136.57,133.98,130.81,130.41,129.43,127.94,123.82,122.18,119.98,117.78,116.67,115.73,113.78.HRMS(ESI)m/z calcd for[C 14H 8BrF 3N 2+H] +:340.9823；found:340.9901[M+H] +.
3、制取叔丁基(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)羰基酰胺(5b)
Figure PCTCN2020129308-appb-000010
在50ml茄形瓶中加入步骤2中制得的5-溴-1-苯基-2-(三氟甲基)-1H-苯并咪唑(0.5g，1.74mmol)，Boc保护的对氨基苯硼酸(0.5g，2.12mmol),Pd(OAc) 2(0.008g，0.04mmol)，K 2CO 3(0.8g，5.78mmol)，加入20mlDMF溶解。Ar保护，于80℃下反应5h。所得溶液在室温下冷却4h。将冷却后的反应液加入盛有80ml水的200ml烧杯中，静置2h，过滤，得灰色黏稠物。将黏稠物烘干，进行硅胶柱柱层析(石油醚：乙酸乙酯＝1:1)，得到5a共0.28g。
白色晶体，收率72％，mp:176.9-177.3℃。 1H NMR(400MHz,DMSO-d 6):δ9.47(s,1H),8.15(d,J＝1.2Hz,1H),7.73(dd,J＝8.7,1.6Hz,1H),7.71–7.61(m,7H),7.58(d,J＝8.7Hz,2H),7.23(d,J＝8.7Hz,1H),1.50(d,J＝4.5Hz,9H).
4、制取1-(2-吗啉乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6a 2)
Figure PCTCN2020129308-appb-000011
在50ml茄形瓶中加入20ml甲苯，三光气(0.187g，0.4mmol)，在-20℃，Ar保护的条件下搅拌10min。将反应温度升温至0℃，将中间体叔丁基(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)羰基酰胺(0.35g，1mmol)溶于5ml甲苯，缓慢滴加约5min，搅拌10min，此时反应体系中有大量白色沉淀生成。将反应瓶移出冷阱，擦干瓶底，升温至回流，反应4.5h至反应液澄清。反应冷却至室温，滴加溶解于5ml甲苯的氨乙基吗啉溶液(0.197ml，1.5mmol)，搅拌30min后停止反应。用二氯甲烷：甲醇＝15:1进行硅胶柱层析，旋干有机溶剂，接着用甲醇作洗脱剂进行凝胶柱层析，得化合物6a 2共0.3g。
黄色油状物，收率:60％。 1H NMR(400MHz,CDCl 3):δ8.10(d,J＝1.1Hz,1H),7.65–7.58(m,6H),7.47(d,J＝8.4Hz,4H),7.29(s,1H),7.23(d,J＝8.6Hz,1H),5.63(s,1H),3.78–3.73(m,4H),3.44(dd,J＝11.1,5.3Hz,2H),2.62(dd,J＝13.0,7.5Hz,4H),2.51(s,2H). 13C NMR(100MHz,CDCl 3):δ155.98,141.01,138.30,137.37,136.44,135.96,134.35,130.02,129.86,128.08,127.39,125.46,120.91,119.06,111.43,66.70,57.45,53.36,36.74.HRMS(ESI)m/z calcd for[C 27H 26F 3N 5O 2+H] +:510.2039；found:510.2101[M+H] +.
实施例2 1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-吗啉乙基)脲(6a 1)
Figure PCTCN2020129308-appb-000012
1、制备4-溴-2-硝基-N-苯胺(3)
Figure PCTCN2020129308-appb-000013
将2-氟-5-溴硝基苯(4.4g，20mmol)，苯胺(2.2ml，24mmol)加入装有15ml水的微波管中，于120℃下反应4h。冷却后，用乙酸乙酯萃取3次，乙酸乙酯层用饱和NaCl溶液洗涤，无水Na 2SO 4干燥并放置4h。旋干有机溶剂，用石油醚进行硅胶柱层 析分离，得到橙红色结晶4.92g，产物3与原料1极性非常相近，即使是用极性最小的石油醚展开，二者位移值依旧非常相近，较难得到纯品，直接投入下一步反应。
2、5-溴-2-甲基-1-苯基-1H-苯并咪唑(4a)
Figure PCTCN2020129308-appb-000014
在50ml三颈瓶中，加入铁粉(0.4g，7.2mmol)，20ml冰醋酸，滴加2-3滴浓盐酸催化，随后在超声中活化3min。在反应体系中加入4-溴-2-硝基-N-苯基苯胺(1)(0.58g，2mmol)，于回流下反应6h，停止反应后静置至室温。以20g硅藻土用布氏漏斗除去铁屑，用乙酸乙酯冲洗至硅藻土基本无产物残留。减压旋蒸除去大部分溶剂，以饱和KOH溶液调PH至7，乙酸乙酯萃取3次，饱和NaCl溶液洗涤3次，无水Na 2SO 4干燥并放置4h，旋干有机溶剂。粗产物经过硅胶柱层析分离(以PE：EA＝3:1分离反应原料，极性增至1：1分离出产物)，得到4a共0.37g。
白色粉末，收率:64％，mp:122.2-123.8℃。 1H NMR(400MHz,DMSO-d 6):δ7.83(d,J＝1.6Hz,1H),7.64(d,J＝7.7Hz,2H),7.59(d,J＝7.3Hz,1H),7.57–7.54(m,2H),7.32(dd,J＝8.5,1.8Hz,1H),7.07(d,J＝8.5Hz,1H),2.44(s,3H). 13C NMR(100MHz,DMSO-d 6):δ153.44,144.18,135.43,130.42,129.40,127.21,125.57,121.30,114.50,112.06,14.37.HRMS(ESI)m/z calcd for[C 14H 11BrN 2+H] +:287.0106；found:287.0184[M+H] +.
3、制备叔丁基(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)羰酰胺(5a)
Figure PCTCN2020129308-appb-000015
在50ml茄形瓶中加入5-溴-2-甲基-1-苯基苯并咪唑(0.5g，1.74mmol)，Boc保护的对氨基苯硼酸(0.5g，2.12mmol),Pd(OAc) 2(0.008g，0.04mmol)，K 2CO 3(0.8g，5.78mmol)，加入20mlDMF溶解。Ar保护，于80℃下反应5h。所得溶液在室温下冷却4h。将冷却后的反应液加入盛有80ml水的200ml烧杯中，静置2h，过滤，得灰色黏稠物。将黏稠物烘干，进行硅胶柱柱层析(石油醚：乙酸乙酯＝1:1)，得到5a共0.28g。
白色固体，收率:40％，mp:125.8-127.1℃。 1H NMR(500MHz,DMSO-d 6):δ9.42(s,1H),7.84(d,J＝1.2Hz,1H),7.66(t,J＝7.6Hz,2H),7.62–7.57(m,4H),7.57–7.53(m,3H),7.45(dd,J＝8.4,1.6Hz,1H),7.16(d,J＝8.4Hz,1H),2.46(s,3H),1.50(s,9H). 13C NMR(100MHz,DMSO-d 6):δ153.18,152.46,143.46,138.84,136.05,135.70,135.08, 134.89,130.49,129.19,127.44,127.31,121.65,118.95,116.41,110.54,79.51,28.61,14.57.
4、1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-吗啉乙基)脲(6a 1)
在50ml茄形瓶中加入20ml甲苯，三光气(0.187g，0.4mmol)，在-20℃，Ar保护的条件下搅拌10min。将反应温度升温至0℃，将中间体5a(0.35g，1mmol)溶于5ml甲苯，缓慢滴加约5min，搅拌10min，此时反应体系中有大量白色沉淀生成。将反应瓶移出冷阱，擦干瓶底，升温至回流，反应4.5h至反应液澄清。反应冷却至室温，滴加溶解于5ml甲苯的氨乙基吗啉溶液(0.197ml，1.5mmol)，搅拌30min后停止反应。用二氯甲烷：甲醇＝15:1进行硅胶柱层析，旋干有机溶剂，接着用甲醇作洗脱剂进行凝胶柱层析，得化合物6a 1共0.3g。
黄色油状物，收率:65.1％。 1H NMR(500MHz,DMSO-d 6):δ8.79(s,1H),8.12(s,1H),7.80–7.69(m,2H),7.64(dd,J＝18.2,9.3Hz,6H),7.51(d,J＝8.1Hz,2H),7.23(d,J＝8.5Hz,1H),6.09(s,1H),3.21(d,J＝5.5Hz,2H),2.36(s,7H),1.40(s,2H). 13C NMR(125MHz,DMSO-d 6):δ155.60,152.38,143.54,140.04,136.06,135.59,135.06,134.04,130.49,129.18,127.48,127.31,121.63,118.42,116.20,110.51,66.64,58.32,53.70,36.43,14.67.HRMS(ESI)m/z calcd for[C 27H 29N 5O 2+H] +:456.2321；found:456.2373[M+H] +.
实施例3 1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(3-吗啉丙基)脲(6b 1)
Figure PCTCN2020129308-appb-000016
化合物6b 1的制备方法参考6a 2，黄色油状物，收率:63.0％。 1H NMR(500MHz,DMSO-d 6):δ8.59(s,1H),7.82(d,J＝1.4Hz,1H),7.73–7.64(m,2H),7.63–7.52(m,5H),7.53–7.46(m,2H),7.45(dd,J＝8.4,1.6Hz,1H),7.16(d,J＝8.4Hz,1H),6.23(s,1H),3.61(s,4H),3.14(dd,J＝12.7,6.6Hz,2H),2.46(s,3H),2.42–2.33(m,2H),1.64(s,2H). 13C NMR(125MHz,DMSO-d 6):δ155.60,152.38,143.54,140.04,136.06,135.59,135.06,134.04,130.49,129.18,127.48,127.31,121.63,118.42,116.20,110.51,66.64,58.32,53.70,36.43,14.67.HRMS(ESI)m/z calcd for[C 28H 31N 5O 2+H] +:470.2478；found:470.2964[M+H] +.
实施例4 1-(3-吗啉丙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6b 2)
Figure PCTCN2020129308-appb-000017
化合物6b 2的制备方法参考6a 2，黄色油状物，收率:58.9％。 1H NMR(400MHz,DMSO-d 6)δ8.56(s,1H),8.12(d,J＝1.2Hz,1H),7.75–7.69(m,2H),7.69–7.65(m,4H),7.63(d,J＝4.3Hz,1H),7.61(s,1H),7.51(d,J＝8.7Hz,2H),7.23(d,J＝8.6Hz,1H),7.23(d,J＝8.6Hz,1H),6.18(t,J＝5.7Hz,1H),3.62–3.56(m,4H),3.14(dd,J＝12.7,6.7Hz,2H),2.34(d,J＝7.9Hz,4H),2.30(d,J＝7.3Hz,2H),1.61(p,J＝7.0Hz,2H). 13C NMR(125MHz,DMSO-d 6)δ155.65,141.35,140.60,140.43,137.21,136.60,134.45,133.07,130.67,130.43,128.02,127.73,125.64,122.50,120.34,118.55,118.25,118.18,116.02,112.15,66.68,56.36,53.87,37.98,27.13.HRMS(ESI)m/z calcd for[C 28H 28F 3N 5O 2+H] +,524.2195；found,524.2243[M+H] +.
实施例5 1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-(哌啶-1-基)乙基)脲(6c 1)
Figure PCTCN2020129308-appb-000018
化合物6c 1的制备方法参考6a 2，黄色油状物，收率:55.3％。 1H NMR(500MHz,DMSO-d 6):δ8.75(s,1H),7.82(s,1H),7.66(t,J＝7.7Hz,2H),7.62–7.53(m,5H),7.52–7.46(m,2H),7.45(dd,J＝8.4,1.3Hz,1H),7.16(d,J＝8.4Hz,1H),6.09(t,J＝5.2Hz,1H),3.26–3.16(m,2H),2.46(s,3H),2.43–2.23(m,6H),1.58–1.47(m,4H),1.40(s,2H). 13C NMR(125MHz,DMSO-d 6):δ155.61,152.38,143.52,140.07,136.06,135.58,135.10,134.01,130.50,129.19,127.48,127.31,121.64,118.41,116.21,110.47,58.56,54.48,36.86,25.92,24.57,14.61.HRMS(ESI)m/z calcd for[C 28H 31N 5O+H] +:454.2529；found:454.2581[M+H] +.
实施例6 1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(2-(哌啶-1-基)乙基)脲(6c 2)
Figure PCTCN2020129308-appb-000019
化合物6c 2的制备方法参考6a 2，黄色油状物，收率:53.1％。 1H NMR(500MHz,DMSO-d 6):δ8.74(s,1H),7.82(d,J＝10.3Hz,1H),7.63(d,J＝21.0Hz,2H),7.53(m,5H),7.50–7.31(m,3H),7.15(s,1H),6.11(s,1H),3.54(m,4H),3.23(s,2H),2.47–2.30(m,8H). 13C NMR(125MHz,DMSO-d 6):δ155.60,141.31,140.66,137.19,134.42,132.99,130.68,130.43,128.03,127.75,127.56,125.66,118.41,118.24,112.16,99.99,58.56,54.49,36.87,26.03,24.56.HRMS(ESI)m/z calcd for[C 28H 28F 3N 5O+H] +:508.2246；found:508.2314[M+H] +.
实施例7 1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-(吡咯烷-1-基)乙基)脲(6d 1)
Figure PCTCN2020129308-appb-000020
化合物6d 1的制备方法参考6a 2，黄色油状物，收率：54.5％。 1H NMR(500MHz,DMSO-d 6):δ9.19(s,1H),7.83(d,J＝1.2Hz,1H),7.69–7.64(m,2H),7.58(m,5H),7.52(d,J＝8.8Hz,2H),7.45(dd,J＝8.4,1.6Hz,1H),7.16(d,J＝8.4Hz,1H),6.77(t,J＝5.8Hz,1H),3.57(d,J＝19.5Hz,2H),3.49(dd,J＝12.5,6.3Hz,2H),3.24(t,J＝6.2Hz,2H),3.03(s,2H),2.46(s,3H),1.92(d,J＝8.9Hz,4H). 13C NMR(125MHz,DMSO-d 6):δ155.97,152.40,143.50,139.80,136.04,135.60,135.03,134.26,130.50,129.20,127.45,127.31,121.65,118.63,116.21,110.49,54.50,53.83,36.18,23.07,14.74.HRMS(ESI)m/z calcd for[C 27H 29N 5O+H] +:440.2372；found:440.2418[M+H] +.
实施例8 1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(2-(吡咯烷-1-基)乙基)脲(6d 2)
Figure PCTCN2020129308-appb-000021
化合物6d 2的制备方法参考6a 2，黄色油状物，收率:47.8％。 1H NMR(500MHz,DMSO-d 6):δ8.78(s,1H),8.13(t,J＝5.7Hz,1H),7.72(dd,J＝8.7,1.5Hz,1H),7.71–7.67(m,3H),7.67–7.63(m,2H),7.61(d,J＝8.7Hz,2H),7.50(dd,J＝17.1,7.2Hz,2H),7.22(d,J＝8.6Hz,1H),6.22–6.10(m,1H),3.23(dd,J＝11.8,6.0Hz,2H),2.57–2.51(m,2H),2.48(s,4H),1.71(s,4H). 13C NMR(125MHz,DMSO-d 6):δ155.65,141.35,140.60,140.43,137.21,136.60,134.45,133.07,130.67,130.43,128.02,127.73,125.64,122.50,120.34,118.55,118.25,118.18,116.02,112.15,66.68,56.36,53.87,37.98,27.13.HRMS(ESI)m/z calcd for[C 27H 26F 3N 5O+H] +:494.2089；found:494.2169[M+H] +.
实施例9 1-异戊基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6e 1)
Figure PCTCN2020129308-appb-000022
化合物6e 1的制备方法参考6a 2，浅黄色固体，收率60.3％，mp:210.5-212.3℃。
1H NMR(500MHz,DMSO-d 6):δ8.49(s,1H),7.83(s,1H),7.65(t,J＝7.6Hz,2H),7.57(dd,J＝9.3,8.2Hz,5H),7.49(d,J＝8.7Hz,2H),7.44(dd,J＝8.4,1.5Hz,1H),7.15(d,J＝8.4Hz,1H),6.12(t,J＝5.6Hz,1H),3.13(dd,J＝13.4,6.5Hz,2H),2.45(s,3H),1.62(m,1H),1.34(dd,J＝14.3,7.0Hz,2H),0.90(d,J＝6.6Hz,6H). 13C NMR(125MHz,DMSO-d 6):δ155.65,152.37,143.52,140.06,136.05,135.54,135.11,134.01,130.48,129.17,127.46,127.29,121.64,118.45,116.22,110.46,39.25,37.69,25.60,22.89,14.63.HRMS(ESI)m/z calcd for[C 26H 28N 4O+H] +:413.2263；found:413.2321[M+H] +.
实施例10 1-异戊基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6e 2)
Figure PCTCN2020129308-appb-000023
化合物6e 2的制备方法参考6a 2，浅黄色固体，收率:65.5％，mp:223.0-225.5℃。 1H NMR(500MHz,DMSO-d 6):δ8.53(s,1H),8.12(s,1H),7.73(d,J＝8.7Hz,1H),7.67(d,J＝8.2Hz,3H),7.62–7.56(m,4H),7.54–7.48(m,2H),7.22(d,J＝8.5Hz,1H),6.13(s,1H),3.13(dd,J＝13.0,6.5Hz,2H),1.62(m,1H),1.35(q,J＝6.9Hz,2H),0.91(d,J＝6.4Hz,6H). 13C NMR(125MHz,DMSO-d 6):δ155.61,141.32,140.61,137.21,136.57,134.43,133.02,130.66,130.43,128.01,127.73,125.63,120.33,118.47,118.24,112.08,39.24,37.75,25.62,22.87.HRMS(ESI)m/z calcd for[C 26H 25F 3N 4O+H] +:467.1980；found:467.2039[M+H] +.
实施例11 1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(3-(吡咯烷-1-基)丙基)脲(6f 1)
Figure PCTCN2020129308-appb-000024
化合物6f 1的制备方法参考6a 2，浅黄色油状物，收率:47.2％。 1H NMR(500MHz,DMSO-d 6):δ8.91(s,1H),7.85(s,1H),7.67(t,J＝7.6Hz,2H),7.64–7.53(m,5H),7.50(d,J＝8.4Hz,2H),7.45(d,J＝8.5Hz,1H),7.16(d,J＝8.4Hz,1H),6.55(s,1H),3.21–3.17(m,2H),3.12(d,J＝7.0Hz,2H),2.46(s,3H),2.01–1.89(m,4H),1.89–1.78(m,2H),1.32–1.16(m,4H). 13C NMR(125MHz,DMSO-d 6):δ155.99,139.95,135.99,135.59,135.10,134.11,130.52,129.24,127.45,127.32,121.68,118.55,116.21,110.50,53.54,52.35,36.83,27.07,23.11,14.58.HRMS(ESI)m/z calcd for[C 28H 31N 5O+H] +:454.2529；found:454.2617[M+H] +.
实施例12 1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(3-(吡咯烷-1-基)丙基)脲(6f 2)
Figure PCTCN2020129308-appb-000025
化合物6f 2的制备方法参考6a 2，浅黄色油状物，收率:45.8％。 1H NMR(500MHz,DMSO-d 6):δ8.62(s,1H),8.12(d,J＝1.2Hz,1H),7.72(dd,J＝8.7,1.6Hz,1H),7.70–7.66(m,3H),7.64(dd,J＝7.1,2.7Hz,2H),7.61(d,J＝8.7Hz,2H),7.52(d,J＝8.7Hz,2H),7.22(d,J＝8.6Hz,1H),6.21(m,1H),3.15(dd,J＝12.7,6.6Hz,2H),2.43(t,J＝6.8Hz,6H),1.71–1.66(m,4H),1.64–1.57(m,2H). 13C NMR(125MHz,DMSO-d 6):δ155.68,141.33,141.00,140.70,140.63,140.40,140.09,137.20,136.55,134.42,133.02,130.64,130.41,127.99,127.71,125.61,120.32,118.50,118.24,112.10,54.08,53.73,38.04,29.49,23.55.HRMS(ESI)m/z calcd for[C 28H 28F 3N 5O+H] +:508.2246；found:508.2325[M+H] +.
实施例13 1-(2-环己基乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6g 1)
Figure PCTCN2020129308-appb-000026
化合物6g 1的制备方法参考6a 2，透明油状物，收率:55.9％。 1H NMR(500MHz,DMSO-d 6):δ8.48(s,1H),7.86(s,1H),7.66(d,J＝7.5Hz,2H),7.64–7.50(m,5H),7.52–7.38(m,3H),7.16(d,J＝8.3Hz,1H),6.11(s,1H),3.12(d,J＝5.5Hz,2H),2.47(s,3H),1.68(m,4H),1.62(d,J＝10.1Hz,1H),1.34(d,J＝6.0Hz,2H),1.25–1.07(m,4H),0.96–0.84(m,2H). 13C NMR(125MHz,DMSO-d 6):δ155.63,152.55,143.30,140.09,135.97,135.46,135.21,133.93,130.51,129.23,127.46,127.34,121.75,118.44,116.16,110.54,37.75,37.20,35.05,33.17,26.60,26.27,14.63.HRMS(ESI)m/z calcd for[C 29H 32N 4O+H] +:453.2576；found:453.2665[M+H] +.
实施例14 1-(2-环己基乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6g 2)
Figure PCTCN2020129308-appb-000027
化合物6g 2的制备方法参考6a 2，透明油状物，收率:53.3％。 1H NMR(500MHz,DMSO-d 6):δ8.53(s,1H),8.12(s,1H),7.73(dd,J＝8.7,1.5Hz,1H),7.71–7.67(m,3H),7.66–7.63(m,2H),7.61(d,J＝8.6Hz,2H),7.50(d,J＝8.6Hz,2H),7.23(dd,J＝11.6,5.6Hz,1H),6.13(t,J＝5.6Hz,1H),3.12(dd,J＝12.8,6.5Hz,2H),1.69(m,4H),1.62(d,J＝11.8Hz,1H),1.34–1.24(m,6H),0.90(m,2H). 13C NMR(125MHz,DMSO-d 6):δ155.65,147.66,141.32,141.00,140.72,140.60,140.42,140.12,137.21,136.58,134.42,133.03,130.68,130.44,128.00,127.73,125.65,118.49,118.19,112.15,37.23,35.07,33.17,30.30,26.59,26.26.HRMS(ESI)m/z calcd for[C 29H 29F 3N 4O+H] +:507.2293；found:507.2377[M+H] +.
实施例15 1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-戊基脲(6h 1)
Figure PCTCN2020129308-appb-000028
化合物6h 1的制备方法参考6a 2，浅黄色油状物，收率:62.4％。 1H NMR(500MHz,DMSO-d 6):δ8.50(s,1H),7.84(s,1H),7.66(t,J＝6.8Hz,2H),7.58(dd,J＝15.7,7.5Hz,5H),7.51–7.43(m,3H),7.16(d,J＝8.4Hz,1H),6.18–6.11(m,1H),3.09(dd,J＝12.8,6.8Hz,2H),2.46(d,J＝4.8Hz,3H),1.48–1.41(m,2H),1.30(m,4H),0.89(t,J＝6.9Hz,3H). 13C NMR(125MHz,DMSO-d 6):δ155.68,141.33,141.00,140.70,140.63,140.40,140.09,137.20,136.55,134.42,133.02,130.64,130.41,127.99,127.71,125.61,120.32,118.50,118.24,112.10,54.08,53.73,38.04,29.49,23.55.HRMS(ESI)m/z calcd for[C 26H 28N 4O+H] +:413.2263；found:413.2349[M+H] +.
实施例16 1-戊基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6h 2)
Figure PCTCN2020129308-appb-000029
化合物6h 2的制备方法参考6a 2，浅黄色油状物，收率:53.5％。 1H NMR(500MHz,DMSO-d 6):δ8.53(s,1H),8.12(s,1H),7.77–7.64(m,6H),7.61(d,J＝8.6Hz,2H),7.51(d,J＝8.6Hz,2H),7.22(d,J＝8.6Hz,1H),6.16(t,J＝5.6Hz,1H),3.09(dd,J＝12.9,6.6Hz,2H),1.49–1.40(m,2H),1.30(dd,J＝13.6,8.3Hz,4H),0.89(t,J＝6.9Hz,3H). 13C NMR(125MHz,DMSO-d 6):δ155.60,141.32,141.00,140.70,140.62,140.4 0,140.17,137.19,136.57,134.43,132.99,130.67,130.43,128.02,127.73,125.62,120.36,118.44,118.24,112.15,31.61,29.91,29.08,22.36,14.44.HRMS(ESI)m/z calcd for[C 26H 25F 3N 4O+H] +:467.1980；found:467.2069[M+H] +.
实施例17 1-丁基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6i 1)
Figure PCTCN2020129308-appb-000030
化合物6i 1的制备方法参考6a 2，浅黄色固体，收率:65.0％，mp:220.3-221.5℃。
1H NMR(500MHz,DMSO-d 6):δ8.48(s,1H),7.83(s,1H),7.69–7.65(m,2H),7.58(m,5H),7.46(dd,J＝15.2,8.6Hz,3H),7.16(d,J＝8.4Hz,1H),6.14(t,J＝5.6Hz,1H),3.10(dd,J＝12.8,6.6Hz,2H),2.46(s,3H),1.47–1.39(m,2H),1.34(dd,J＝14.9,7.4Hz,2H),0.91(t,J＝7.3Hz,3H). 13C NMR(125MHz,DMSO-d 6):δ155.65,152.36,143.49,140.08,136.06,135.57,135.11,133.98,130.49,129.19,127.46,127.31,121.63,118.43,116.17,110.36,39.19,32.37,20.01,14.71,14.18.HRMS(ESI)m/z calcd for[C 25H 26N 4O+H] +:399.2107；found:399.2246[M+H] +.
实施例18 1-丁基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6i 2)
Figure PCTCN2020129308-appb-000031
化合物6i 2的制备方法参考6a 2，浅黄色固体，收率:58.8％，mp:217.4-219.0℃。
1H NMR(500MHz,DMSO-d 6):δ8.53(s,1H),8.12(s,1H),7.73(d,J＝8.6Hz,1H),7.71–7.64(m,5H),7.61(d,J＝8.6Hz,2H),7.51(d,J＝8.6Hz,2H),7.23(d,J＝8.6Hz,1H),6.16(t,J＝5.6Hz,1H),3.10(dd,J＝12.8,6.6Hz,2H),1.47–1.39(m,2H),1.32(dd,J＝15.1,7.6Hz,2H),0.91(t,J＝7.3Hz,3H) 13C NMR(125MHz,DMSO-d 6):δ155.61,141.32,140.99,140.70,140.62,140.40,140.08,137.19,136.57,134.42,132.99,130.66,130.42,128.02,127.73,125.62,120.33,118.44,118.24,112.14,39.19,32.35,20.06,14.17.HRMS(ESI)m/z calcd for[C 25H 23F 3N 4O+H] +:453.1824；found:453.1901[M+H] +.
实施例19 1-(环己基甲基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6j 1)
Figure PCTCN2020129308-appb-000032
化合物6j 1的制备方法参考6a 2，浅黄色油状物，收率:57.7％。 1H NMR(500MHz,DMSO-d 6):δ8.46(s,1H),7.82(d,J＝1.3Hz,1H),7.66(t,J＝7.6Hz,2H),7.62–7.53(m,5H),7.49(t,J＝11.3Hz,2H),7.44(dd,J＝8.4,1.6Hz,1H),7.16(d,J＝8.4Hz,1H),6.23–6.14(m,1H),2.96(t,J＝6.2Hz,2H),2.47(d,J＝14.0Hz,3H),1.68(t,J＝13.4Hz,4H),1.63(d,J＝11.2Hz,1H),1.26–1.16(m,4H),0.98–0.87(m,2H). 13C NMR(100MHz,DMSO-d 6):δ155.71,152.38,143.51,140.06,136.05,135.57,135.10,133.98,130.49,129.18,127.47,127.30,121.63,118.38,116.15,110.47,45.76,38.47,30.81,26.56,25.90,14.67.HRMS(ESI)m/z calcd for[C 28H 30N 4O+H] +:439.2420；found:439.2535[M+H] +.
实施例20 1-(环己基甲基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6j 2)
Figure PCTCN2020129308-appb-000033
化合物6j 2的制备方法参考6a 2，浅黄色油状物，收率:49.8％。 1H NMR(400MHz,DMSO-d 6):δ8.53(s,1H),8.11(s,1H),7.72(dd,J＝8.7,1.3Hz,1H),7.70–7.65(m,3H),7.65–7.57(m,4H),7.50(d,J＝8.5Hz,2H),7.21(d,J＝8.6Hz,1H),6.21(t,J＝5.7Hz,1H),2.96(t,J＝6.2Hz,2H),1.67(t,J＝11.3Hz,4H),1.62(d,J＝9.4Hz,1H),1.27–1.09(m,4H),0.95–0.84(m,2H). 13C NMR(100MHz,DMSO-d 6):δ155.71,152.38,143.51,140.06,136.05,135.57,135.10,133.98,130.49,129.18,127.47,127.30,121.63,118.38,116.15,110.47,45.76,38.47,30.81,26.56,25.90,14.67.HRMS(ESI)m/z calcd for[C 28H 27F 3N 4O+H] +:493.2137；found:493.2217[M+H] +.
实施例21 1-异丁基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6k 1)
Figure PCTCN2020129308-appb-000034
化合物6k 1的制备方法参考6a 2，浅黄色固体，收率:48.8％，mp:226.6-227.9℃。 1H NMR(500MHz,DMSO-d 6):δ8.49(s,1H),7.83(d,J＝1.4Hz,1H),7.69–7.64(m,2H),7.61–7.55(m,5H),7.48(d,J＝8.7Hz,2H),7.45(dd,J＝8.4,1.6Hz,1H),7.16(d,J＝8.4Hz,1H),6.21(t,J＝5.8Hz,1H),2.94(t,J＝6.3Hz,2H),2.46(s,3H),1.76–1.65(m,1H),0.89(d,J＝6.7Hz,6H). 13C NMR(100MHz,DMSO-d 6):δ155.75,152.41,143.41,140.03,136.00,135.54,135.13,134.01,130.50,129.21,127.48,127.29,121.68,118.44,116.15,110.50,47.00,28.96,20.47,14.63.HRMS(ESI)m/z calcd for[C 25H 26N 4O+H] +:399.2107；found:399.2198[M+H] +.
实施例22 1-异丁基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲(6k 2)
Figure PCTCN2020129308-appb-000035
化合物6k 2的制备方法参考6a 2，浅黄色固体，收率:43.9％，mp:219.3-221.6℃。 1H NMR(400MHz,DMSO-d 6):δ8.56(s,1H),8.12(d,J＝1.2Hz,1H),7.73(dd,J＝8.7,1.6Hz,1H),7.70–7.67(m,3H),7.65(dd,J＝7.2,3.0Hz,2H),7.62(d,J＝8.7Hz,2H),7.52(d,J＝8.7Hz,2H),7.22(d,J＝8.7Hz,1H),6.25(t,J＝5.8Hz,1H),2.95(t,J＝6.3Hz,2H),1.79–1.63(m,1H),0.89(d,J＝6.7Hz,6H). 13C NMR(100MHz,DMSO-d 6):δ155.76,141.26,140.53,137.19,136.54,134.36,133.05,130.69,130.45,127.97,127.75,125.67,120.46,118.50,118.20,117.83,112.15,47.00,28.93,20.45.HRMS(ESI)m/z calcd for[C 25H 23F 3N 4O+H] +:453.1824；found:453.1907[M+H] +.
实施例23 1-(2-甲氧基乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6l 1)
Figure PCTCN2020129308-appb-000036
化合物6l 1的制备方法参考6a 2，浅黄色油状物，收率:65.6％。 1H NMR(500MHz,CDCl 3):δ8.04(s,1H),7.90(s,1H),7.60(dd,J＝10.2,4.7Hz,2H),7.56–7.50(m,3H),7.43(d,J＝8.6Hz,2H),7.39(dd,J＝9.7,4.3Hz,3H),7.14(d,J＝8.4Hz,1H),6.00(s,1H),3.52–3.49(m,2H),3.48(s,2H),3.33(s,3H),2.54(s,3H). 13C NMR(125MHz,CDCl 3):δ156.55,152.19,142.69,138.36,136.15,136.01,135.85,135.55,130.00,128.95,127.88,126.98,122.27,120.19,116.70,110.15,72.15,58.78,40.05,14.13.HRMS(ESI)m/z calcd for[C 24H 24N 4O 2+H] +:401.1899；found:401.1996[M+H] +.
实施例24 1-(3-甲氧基乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-yl)苯基)脲(6l 2)
Figure PCTCN2020129308-appb-000037
化合物6l 2的制备方法参考6a 2，浅黄色油状物，收率55.3％。 1H NMR(400MHz,DMSO-d 6):δ8.67(s,1H),8.13(d,J＝1.1Hz,1H),7.73(dd,J＝8.7,1.5Hz,1H),7.71–7.58(m,7H),7.51(d,J＝8.7Hz,2H),7.23(d,J＝8.6Hz,1H),6.25(t,J＝5.5Hz,1H),3.40(t,J＝5.2Hz,2H),3.29(s,3H),3.29–3.24(m,2H). 13C NMR(125MHz,DMSO-d 6):δ155.55,141.32,140.72,140.48,137.17,136.60,134.38,133.12,130.68,130.43,128.03,127.77,125.64,118.44,118.26,112.16,71.74,58.40,39.30.HRMS(ESI)m/z calcd for[C 24H 21F 3N 4O 2+H] +:455.1617；found:455.1708[M+H] +.
实施例25 1-(3-甲氧基丙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6m 1)
Figure PCTCN2020129308-appb-000038
化合物6m 1的制备方法参考6a 2，浅黄色油状物，收率43.9％。 1H NMR(500MHz,CDCl 3):δ7.90(s,1H),7.61(t,J＝7.5Hz,2H),7.58–7.52(m,3H),7.50–7.36(m,6H),7.15(dd,J＝12.1,5.4Hz,1H),5.82(s,1H),3.51–3.45(m,2H),3.40(d,J＝4.7Hz,2H),3.28(s,3H),2.55(s,3H),1.81(m,2H). 13C NMR(100MHz,DMSO-d 6):δ155.76,141.26,140.53,137.19,136.54,134.36,133.05,130.69,130.45,127.97,127.75,125.67,120.46,118.50,118.20,117.83,112.15,47.00,28.93,20.45.HRMS(ESI)m/z calcd for[C 25H 26N 4O 2+H] +:415.2056；found:415.2144[M+H] +.
实施例26 1-(3-甲氧基丙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-yl)苯基)脲(6m 2)
Figure PCTCN2020129308-appb-000039
化合物6m 2的制备方法参考6a 2，浅黄色油状物，收率:47.3％。 1H NMR(400MHz,DMSO-d 6):δ8.59(s,1H),8.13(d,J＝1.2Hz,1H),7.73(dd,J＝8.7,1.6Hz,1H),7.71–7.66(m,4H),7.66–7.59(m,3H),7.55–7.49(m,2H),7.23(d,J＝8.6Hz,1H),6.19(t,J＝5.7Hz,1H),3.38(t,J＝5.6Hz,2H),3.26(s,3H),3.16(dd,J＝12.6,6.7Hz,2H),1.68(m,2H). 13C NMR(100MHz,DMSO-d 6):δ155.61,141.33,140.53,137.21,136.57,134.44,132.99,130.67,130.43,128.03,127.73,125.63,118.49,118.24,112.12,70.37,58.37,37.00,30.14. HRMS(ESI)m/z calcd for[C 25H 23F 3N 4O 2+H] +:469.1773；found:469.1849[M+H] +.
实施例27 1-(3-(二乙胺)乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6n)
Figure PCTCN2020129308-appb-000040
化合物6n的制备方法参考6a 2，浅黄色油状物，收率:37.5％。 1H NMR(500MHz,CDCl 3):δ8.68(s,1H),7.89(s,1H),7.59(t,J＝7.8Hz,2H),7.56–7.49(m,5H),7.43–7.35(m,3H),7.25(d,J＝21.0Hz,1H),7.14(d,J＝8.4Hz,1H),3.65(d,J＝5.1Hz,2H),3.16(dd,J＝12.0,6.9Hz,2H),3.11(m,4H),2.53(s,3H),1.34(t,J＝7.2Hz,6H). 13C NMR(100MHz,DMSO-d 6):δ155.61,141.33,140.53,137.21,136.57,134.44,132.99,130.67,130.43,128.03,127.73,125.63,118.49,118.24,112.12,70.37,58.37,37.00,30.14.HRMS(ESI)m/z calcd for[C 27H 31N 5O+H] +:442.2529；found:442.2604[M+H] +.
实施例28 1-(3-(二乙胺)丙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲(6o)
Figure PCTCN2020129308-appb-000041
化合物6o的制备方法参考6a 2，黄色油状物，收率:28.8％。 1H NMR(500MHz,CDCl 3):δ8.91(s,1H),7.87(s,1H),7.63–7.55(m,4H),7.51(m,3H),7.36(d,J＝5.5Hz,3H),7.12(d,J＝8.3Hz,1H),7.07(s,1H),3.36(s,2H),3.11–3.03(m,6H),2.51(s,3H),1.99(s,2H),1.29(d,J＝6.8Hz,6H). 13C NMR(125MHz,CDCl 3):δ157.10,152.11,142.98,138.79,135.93,135.75,135.57,135.46,129.95,128.84,127.58,126.95,121.97,119.12,116.63,110.10,49.73,46.70,36.67,29.66,14.42,8.61.HRMS(ESI)m/z calcd for[C 28H 33N 5O+H] +:456.2685；found:456.2770[M+H] +.
实施例1-28中所合成的化合物的结构式所对应的取代基如表1所示：
表1化合物的结构式与所对应的取代基
Figure PCTCN2020129308-appb-000042
Figure PCTCN2020129308-appb-000043
Figure PCTCN2020129308-appb-000044
Figure PCTCN2020129308-appb-000045
Figure PCTCN2020129308-appb-000046
Figure PCTCN2020129308-appb-000047
对实施例1-28所合成的化合物分别进行细胞增殖抑制实验、酶活性抑制实验和蛋白免疫印迹实验，具体实验如下所示：
1、细胞增殖抑制实验
为了考察目标化合物对肾成纤维细胞的增殖抑制活性，本实验采用CCK-8法测定所合成的化合物对大鼠肾成纤维细胞NRK-49F细胞株的抑制率，以Compound C、Sorafenib为阳性对照，计算各化合物的IC 50值，进行初步的构效关系研究。进一步为了探讨化合物的肾毒性，测定化合物对人近端肾小管上皮细胞HK-2细胞株的增殖抑制活性，以Compound C、Sorafenib为阳性对照，计算各化合物的IC 50值，利用IC 50(HK-2)、IC 50(NRK-49F)的比值，考察化合物对细胞的选择性，以初步探讨化合物的肾毒性。
(1)实验材料
a.细胞株：大鼠肾成纤维细胞NRK-49F细胞株和人近端肾小管上皮细胞HK-2细胞 株。
b.试剂和仪器：96孔细胞培养板购自Costar公司；RPMI 1640培养基购自Gibco公司；DMEM培养基购自Gibco公司；胎牛血清购自Gibco公司；DMSO购自Sigma公司；EnoGeneCell TM Counting Kit-8(CCK-8)细胞活力检测试剂盒(E1CK-000208-10)购自南京恩晶生物科技有限公司。ChemBase CBS-CJ-1FD超净工作台；MCO-15AC二氧化碳培养箱(SANYO)；XD-202荧光倒置生物显微镜显微镜(南京江南永新光学有限公司)；Thermo MK3酶标仪(美国热电公司)。
(2)实验方法
a.细胞培养：NRK-49F、HK-2细胞采用常规培养。实验时均用对数生长期细胞，且取活细胞比例达90％以上的细胞进行实验。
b.细胞生长检测：细胞消化、计数、制成浓度为1×105个/mL的细胞悬液，96孔板中每孔加入100μL细胞悬液(每孔1×104个细胞)；96孔板置于37℃，5％CO2培养箱中培养24小时；每孔加入100μL相应的含药物的培养基，使孔中化合物终浓度为100、50、25、12.5、6.25、3.125、1.5625、0.78125、0.390625、0.1953125μM/mL，同时设立阴性对照组，溶媒对照组，阳性对照组，每组5复孔；96孔板置于37℃，5％CO2培养箱中培养72小时后；每孔加入10μL CCK-8溶液,将培养板在培养箱内孵育4小时，用酶标仪测定在450nm处的OD值。
(3)数据处理
采用Graphpad Prism 5.0软件，以浓度为横坐标，抑制率为纵坐标拟合曲线，计算化合物的IC 50值。酶标仪上于450nm处测定每孔的吸光度OD值，细胞生长抑制率按下式计算：
Figure PCTCN2020129308-appb-000048
表2：化合物对细胞增殖抑制的抑制作用
Figure PCTCN2020129308-appb-000049
注：本实验细胞增殖抑制实验IC 50值为5次独立实验求平均值所得；
Ratio of IC 50(ROI)＝IC 50(HK-2)/IC 50(NRK-49F)；“-”表示数值无法确定。
2、酶活性抑制实验
本实验通过HTRF实验测定目标化合物对HIPK2酶活性的抑制效果。
(1)实验材料
ATP，DTT，MgCl2，CaCl2，cGMP购自Sigma公司；钙连蛋白购自Upstate公司，HIPK2购自Carna公司；其他试剂未经说明均来自Cisbio公司。HTRF KinEASE–STK试剂盒购自Cisbio公司。
(2)实验方法
a.酶准备阶段：预先配置缓冲液(5mM MgCl2,1mM DTT,50μM AMP)；化合物溶液(100,000nM,20,000nM,4,000nM,800nM,160nM,32nM,6.4nM,1.3nM,0.26nM,0,051nM)；在384孔板中每孔加入4μL化合物溶液(或缓冲液)，加入2μLHIPK2溶液，加入2μLATP，2μLSTK-S2，在rt下孵育1h。选取Compound C和Staurosporine为阳性对照药物。
b.检测阶段：预先配置荧光物溶液(0.5μM SA-XL665，0.5μM STK Ab)；在每孔中加入5μL SA-XL665溶液，5μL STK Ab溶液；在rt下孵育1h；用HTRF酶标仪读取620nm及665nm处的OD值。
(3)数据处理
采用Graphpad Prism 5.0软件，以浓度为横坐标，抑制率为纵坐标拟合曲线，计算化合物的IC 50值。酶标仪上于620nm及665nm处测定每孔的吸光度OD值，细胞生长抑制率按下式计算：
Figure PCTCN2020129308-appb-000050
表3：化合物对激酶的抑制作用
Figure PCTCN2020129308-appb-000051
从表2中可以看出，化合物对HIPK2亚型的选择性远大于对HIPK1和HIPK3亚型的选择性。
3、蛋白免疫印迹实验
HIPK2在正常水平由于受到泛素连接酶的降解表达较低，在疾病状态下泛素连接酶失活，HIPK2高度稳定，MG132可抑制泛素连接酶的活性，无细胞试验中IC 50为100nM。本实验选取HIPK2酶抑制活性最佳的化合物6d 1进行Western Blot实验，在实验过程中采用MG132提高细胞中HIPK2的浓度，浓度为200ng/ml，实验组分为：对照组，MG132处理组，MG132处理/药物组，MG132/对照药组。本论文分别在NRK-49F和HK-2细胞中测定四组细胞HIPK2的水平；在NRK-49F细胞中，以Compound C为对照药，分别测定细胞中总Smad3和磷酸化Smad3水平，α-SMA水平，以探讨化合物对纤维化相关信号分子的影响；在HK-2细胞中，以BAY11-7082为对照药，分别测定细胞中总IκB-α和磷酸化IκB-α的水平，以探讨化合物对炎症相关信号分子的影响。
(1)实验材料
a.细胞株：NRK-49F、HK-2细胞株。
b.抗体：smad2/3、p-smad3、IκB-α、p-IκB-α、α-SMA、HIPK2、GAPDH均购自EnoGene公司。
c.药品及试剂：ECL化学发光试剂购自碧云天公司；Maxisignal Western Soltion购自SUDGEN公司；RIPA裂解液，BSA，预染蛋白marker，羊抗兔二抗，显影液、定影液，BCA蛋白浓度检测试剂盒，均购自南京恩晶生物科技有限公司。
d.仪器及耗材：6孔板；二氧化碳培养箱(SANYO)；荧光倒置生物显微镜(南京江南永新光学有限公司)；台式高速离心机(SCILOGEX)；微型垂直电泳槽(上海天能科技有限公司)；转移电泳槽(上海天能科技有限公司)；电泳仪(上海天能科技有限公司)；脱色摇床(江苏省金坛市荣华仪器制造有限公司)；静音混合器(江苏海门市其林贝尔仪器制造有限公司)；酶标仪(Thermo scientific)。
(2)实验方法：
a.细胞样本制备：取对数生长期NRK-49F、HK-2细胞进行实验。细胞经消化、计数、制成1×10 5个/mL的细胞悬液，接种于6孔板中(2ml/孔)，置于37℃，5％CO 2培养箱中培养24小时；每孔加入200nM MG132，同时设立空白对照组；将板置于培养箱中培养72h后，更换新鲜培养基，同时加入不同浓度化合物，NRK-49F加入2uM、10uM的化合物6d 1及Compound C，HK-2加入10uM、50uM的化合物6d 1及BAY 11-7082，同时设立MG132单独作用组，作用5小时后，收集细胞样本，WB检测各目的蛋白。
b.蛋白测定：胰酶消化收集细胞样本，加入100μl RIPA裂解液(含PMSF及磷酸酶抑制剂)，在冰浴中反复吹打，充分裂解样本，获得裂解液后离心(4℃，12000rpm，15min)，收集上清并进行蛋白含量测定。根据上清体积按4:1比例加入5×Loading Buffer，100℃水浴10min使蛋白变性，冷却至室温后短暂离心后保存于-20℃待测。
根据目的蛋白分子量分别配置8％及10％分离胶，按40μg蛋白上样量进行上样，同 时加入5μl蛋白marker，于恒压80v-100v条件下进行电泳，当marker各分量条带充分分开时可终止电泳。PVDF膜经甲醇活化15s-5min后，于低温环境下转膜，转膜条件：恒压80V，1h-2h。
转膜完成后，膜用5％BSA中封闭1h，经TBST充分洗涤后(5min/次，3次)，与相应目的蛋白抗体结合，4℃过夜。TBST充分洗涤后(5min/次，3次)，与相应二抗室温结合1h，TBST充分漂洗(10min/次，3次)，于暗室中利用胶片及ECL发光液分别进行显影及定影。利用Adobe Photoshop CS5软件分析目的条带的净光密度值，以GAPDH为内参。
(3)数据处理
数据以mean±SD表示，应用GraphPad Prism 5.0统计软件，两组组间比较采用t-test检验，多组间比较采用One-way Anova(Dunnett)，P<0.05为有统计学意义。
(4)实验结果
在正常情况下图1中的对照组(Control)细胞中HIPK2均处于较低的水平，加入200ng/ml的MG132处理72h后细胞中的HIPK2水平大大增加。在HK-2细胞中加入化合物6d1处理5h后HIPK2磷酸化水平降低，且降低水平对化合物浓度具有剂量依赖性，化合物6d1在10μM，50μM下对HIPK2磷酸化水平的抑制均强于对照药BAY11-7082。在NRK-49F细胞中加入化合物6d1处理5h后HIPK2磷酸化水平降低，其磷酸化降低水平对化合物浓度具有剂量依赖性。综上所述，化合物对HIPK2蛋白水平具有抑制作用。
HIPK2可调节纤维化及炎症通路，为了验证化合物对纤维化信号通路的影响，本发明测定了化合物刺激NRK-49F细胞后p-smad3,α-SMA磷酸化水平的改变。如图2所示，加入化合物6d 1，Compound C后p-smad3磷酸化水平降低，且降低水平对化合物6d 1，Compound C浓度均具有剂量依赖性，这证明化合物对纤维化关键信号分子smad3有调节作用。化合物对α-SMA的磷酸化水平基本不影响。
为了验证化合物对炎症通路的影响，本实验测定了化合物对p-IκB-α水平的影响。如图3所示，当细胞中加入MG132处理后p-IκB-α水平增高，加入化合物6d 1，BAY11-7082后p-IκB-α降低，且对化合物的浓度均具有剂量依赖性，故化合物对炎症信号分子IκB-α具有调节作用。
综上所述，化合物6d 1对HIPK2激酶及蛋白水平、纤维化信号分子smad3、炎症信号分子IκB-α磷酸化水平均具有抑制作用。本发明所制得的化合物直接以HIPK2为靶点，同时对HIPK1-3亚型有选择性，兼有抗炎和抗纤维化双重作用，具有较好的慢性肾病治疗应用前景。

Claims (13)

  1. 一种含脲基苯并咪唑类衍生物，结构通式如式(I)所示：
    Figure PCTCN2020129308-appb-100001
    其中，
    R 1选自氢、C1-C6脂肪链、C3-C6脂肪环、取代C3-C6脂肪环、芳环、取代芳环、芳杂环和取代芳杂环；
    R 2选自氢、C1-C6的烷基、C1-C6的卤代烷基、C1-C6的烷基胺基、芳环、芳杂环、取代芳环和取代芳杂环；
    A选自芳环、芳杂环、取代芳环和取代芳杂环其中一种；
    B选自脂肪杂环、芳杂环、C1-C4开链胺基、C1-C6直链或支链烷氧基、C1-C6直链或支链烷烃、C3-C6脂肪环和取代C3-C6脂肪环其中一种；
    n＝1～6。
  2. 根据权利要求1所述的含脲基苯并咪唑类衍生物，其特征在于：
    所述芳环选自苯、萘和蒽；
    所述芳杂环选自吡咯、呋喃、噻吩、咪唑、噻唑、噁唑、吡唑、异噁唑、噻二唑、噁二唑、四氮唑、吡啶、嘧啶、吡嗪、哒嗪、嘌呤、喹啉、异喹啉、吲哚、吖啶和咔唑；
    所述脂肪杂环选自吡咯烷、哌嗪、哌啶、二氧六环和四氢呋喃；
    所述取代芳环或取代芳杂环中的取代基选自羟基、甲基、甲氧基、氨基、三氟甲基和卤素。
  3. 根据权利要求1所述的含脲基苯并咪唑类衍生物，其特征在于：其药学上可用的盐是盐酸盐、硫酸盐、磷酸盐、高氯酸盐、甲磺酸盐、三氟甲磺酸盐、甲酸盐、乙酸盐、丙酸盐、丁酸盐、马来酸盐、丁二酸盐、三氟乙酸盐、琥珀酸盐、水杨酸盐、DL-天冬氨酸盐、D-天冬氨酸盐、L-天冬氨酸盐、DL-谷氨酸盐、D-谷氨酸盐、L-谷氨酸盐、甘油酸盐、硬脂酸盐、DL-酒石酸盐、D-酒石酸盐、L-酒石酸盐、(±)扁桃酸盐、(R)-(-)扁桃酸盐、(S)-(+)扁桃酸盐、柠檬酸盐、粘酸盐、丙二酸盐、苯甲酸盐、DL-苹果酸盐、(±)乳酸盐、L-(+)-乳酸盐、D-(+)-乳酸盐、扑酸盐、D-α-半乳糖 醛酸盐、甘油酸盐、DL-半胱氨酸盐、D-半胱氨酸盐、L-半胱氨酸盐、(4S)-羟基-L-脯氨酸盐、环丙烷-1,1-二羧酸盐、2,2-甲基丙二酸盐、酪氨酸盐、脯氨酸盐、富马酸盐、1-羟基-2-萘甲酸盐、膦酰基乙酸盐、碳酸盐、碳酸氢盐、3-膦酰基丙酸盐、DL-焦谷氨酸盐、D-焦谷氨酸盐、L-焦谷氨酸盐、对甲苯磺酸盐、苯磺酸盐、乙磺酸盐、(±)樟脑磺酸盐、萘磺酸盐、1R-(-)-樟脑磺酸盐、1S-(+)-樟脑磺酸盐、1,5-萘二磺酸盐、1,2-乙烷二磺酸盐、1,3-丙烷二磺酸盐、3-(N-吗啉代)丙烷磺酸盐、联苯基磺酸盐，羟乙基磺酸盐、1-羟基-2-萘磺酸盐、磷酸二氢盐、磷酸氢钾盐、磷酸二钾盐、磷酸钾盐、磷酸氢钠盐、磷酸二钠盐、磷酸钠盐、磷酸二氢钠盐、磷酸钙盐、三代磷酸钙盐、六氟代磷酸盐、乙烯基磷酸盐、2-羟基乙基磷酸盐和苯基磷酸盐。
  4. 根据权利要求1所述的含脲基苯并咪唑类衍生物，其特征在于：B部分选自五元或六元脂肪杂环、C1-C4开链胺类化合物、C1-C6直链或支链烷氧基、C1-C6直链或支链烷烃、C3-C6脂肪环和取代C3-C6脂肪环。
  5. 根据权利要求4所述的含脲基苯并咪唑类衍生物，其特征在于：B部分选自含氮或/和氧的脂肪杂环、C3-C6脂肪环和取代C3-C6脂肪环。
  6. 根据权利要求4所述的含脲基苯并咪唑类衍生物，其特征在于：B部分所述脂肪杂环选自吡咯烷、哌嗪、哌啶、二氧六环和四氢呋喃其中一种。
  7. 根据权利要求1-6任意一项所述的含脲基苯并咪唑类衍生物，其特征在于：A部分为芳环或取代芳环。
  8. 根据权利要求1-6任意一项所述的含脲基苯并咪唑类衍生物，其特征在于：R1为芳环或取代芳环。
  9. 根据权利要求1-6任意一项所述的含脲基苯并咪唑类衍生物，其特征在于：R2为甲基或三氟甲基。
  10. 根据权利要求1-6任意一项所述的含脲基苯并咪唑类衍生物，其特征在于具体为以下化合物：
    1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-吗啉乙基)脲；
    1-(2-吗啉乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(3-吗啉丙基)脲；
    1-(3-吗啉丙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-(哌啶-1-基)乙基)脲；
    1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(2-(哌啶-1-基)乙基)脲；
    1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(2-(吡咯烷-1-基)乙基)脲；
    1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(2-(吡咯烷-1-基)乙基)脲；
    1-异戊基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-异戊基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-(3-(吡咯烷-1-基)丙基)脲；
    1-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)-3-(3-(吡咯烷-1-基)丙基)脲；
    1-(2-环己基乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-(2-环己基乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)-3-戊基脲；
    1-戊基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-丁基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-丁基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-(环己基甲基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-(环己基甲基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-异丁基-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-异丁基-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-基)苯基)脲；
    1-(2-甲氧基乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-(3-甲氧基乙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-yl)苯基)脲；
    1-(3-甲氧基丙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-(3-甲氧基丙基)-3-(4-(1-苯基-2-(三氟甲基)-1H-苯并咪唑-5-yl)苯基)脲；
    1-(3-(二乙胺)乙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
    1-(3-(二乙胺)丙基)-3-(4-(2-甲基-1-苯基-1H-苯并咪唑-5-基)苯基)脲；
  11. 一种含脲基苯并咪唑类衍生物的制备方法，其特征在于包括以下步骤：
    Figure PCTCN2020129308-appb-100002
    S1、化合物II在催化剂醋酸钯作用下与硼酸取代物发生耦联反应得到化合物III，所述硼酸取代物上的取代基选自氨基苯、氨基萘、氨基蒽、氨基菲、取代氨基苯、取代氨基萘、取代氨基蒽和取代氨基菲；所述硼酸取代物结构如式IV所示：
    Figure PCTCN2020129308-appb-100003
    S2、化合物III与伯胺在缩合剂的作用下形成所述含脲基苯并咪唑类衍生物，所述缩合剂选自三光气、N,N'-羰基二咪唑和氯甲酸对硝基酚酯其中一种；所述伯胺结构如式V所示：
    Figure PCTCN2020129308-appb-100004
    其中：
    R1选自氢、C1-C6脂肪链、C3-C6脂肪环、取代C3-C6脂肪环、芳环、取代芳环、芳杂环和取代芳杂环；
    R2选自氢、C1-C6的烷基、C1-C6的卤代烷基、C1-C6的烷基胺基、芳环、芳杂环、取代芳环和取代芳杂环；
    A选自芳环、芳杂环、取代芳环和取代芳杂环其中一种；
    B选自脂肪杂环、芳杂环、C1-C4开链胺基、C1-C6直链或支链烷氧基、C1-C6直链或支链烷烃、C3-C6脂肪环和取代C3-C6脂肪环其中一种；
    n＝1～6。
  12. 权利要求1-10任一项所述含脲基苯并咪唑类衍生物在制备治疗慢性肾病的药物中的应用。
  13. 一种抗糖尿病肾病药物，含权利要求1-10任一项所述脲基苯并咪唑类衍生物或其药学上可用的盐。
PCT/CN2020/129308 2019-11-22 2020-11-17 含脲基苯并咪唑类衍生物及其制备方法和应用 WO2021098671A1 (zh)

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CN1617869A (zh) * 2001-12-04 2005-05-18 埃科特莱茵药品有限公司 作为尿压素ⅱ受体拮抗剂的4－(哌啶基－吡咯烷基－烷基－脲基)－喹啉类化合物
WO2006039718A2 (en) * 2004-10-01 2006-04-13 Amgen Inc Aryl nitrogen-containing bicyclic compounds and their use as kinase inhibitors

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EP2508184A1 (en) * 2011-04-06 2012-10-10 Æterna Zentaris GmbH Pyridopyrazine derivatives and their use

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1527831A (zh) * 2001-01-31 2004-09-08 ̩ Mcp-1功能的拮抗物及其使用方法
CN1617869A (zh) * 2001-12-04 2005-05-18 埃科特莱茵药品有限公司 作为尿压素ⅱ受体拮抗剂的4－(哌啶基－吡咯烷基－烷基－脲基)－喹啉类化合物
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