WO2023213211A1 - 2,4-二取代-5-氟嘧啶衍生物及其制备方法和应用 - Google Patents

2,4-二取代-5-氟嘧啶衍生物及其制备方法和应用 Download PDF

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WO2023213211A1
WO2023213211A1 PCT/CN2023/090579 CN2023090579W WO2023213211A1 WO 2023213211 A1 WO2023213211 A1 WO 2023213211A1 CN 2023090579 W CN2023090579 W CN 2023090579W WO 2023213211 A1 WO2023213211 A1 WO 2023213211A1
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amino
phenyl
benzo
compound
fluoro
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WO2023213211A9 (zh
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李乾斌
胡高云
陈卓
陶立坚
彭张哲
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长沙晶易医药科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • Renal interstitial fibrosis is a chronic and progressive process that affects the normal function of the kidney in aging and chronic kidney disease, thus endangering human health.
  • NF- ⁇ B, TGF- ⁇ /Smad3, Wnt/ ⁇ -catenin, Notch and p53 signaling pathways play an important role in the occurrence and development of renal interstitial fibrosis. It can be seen from the existing drug research results that the strategy of drug discovery targeting one or two of the above channels has not been successful in the field of drug development for renal interstitial fibrosis treatment (Nat Rev Drug Discov, 2016, 15(8): 568; Am J Kidney Dis. 2022, doi:10.1053/j.ajkd.2021.11.010).
  • homologous domain interacting protein kinase 2 is highly expressed in the lesions of renal interstitial tubules and participates in fibrosis and through its regulation. inflammatory process. Therefore, inhibiting HIPK2 can simultaneously regulate fibrosis and inflammation to achieve the purpose of treating the disease.
  • HIPK2 inhibitors reported in literature studies are classified into three categories ( Figure 1): HIPK2 kinase inhibitors, HIPK2/SMAD interaction inhibitors and HIPK2 expression inhibitors.
  • TBID Pan one.2014,9(2):e89176
  • TAE-226 Nat Med. 2012, 18 (4): 580
  • CTx-0294857 J. Proteome Res.
  • BT173 J Am Soc Nephrol. 2017, 28:2133 is a HIPK2/SMAD interaction inhibitor that can exert anti-fibrotic effects by blocking the protein-protein interaction between HIPK2 and Smad3, but does not inhibit HIPK2 of kinase activity.
  • the HIPK2 expression inhibitor phosphate niclosamide (Kidney International.2017,92:612) can inhibit the transcription of HIPK2 by interfering with the binding of Smad3 to the promoter sequence of the HIPK2 gene, thus inhibiting multiple signaling pathways downstream of HIPK2. .
  • Invention patent 201911152583.9 and PCT/CN2020/129308 disclose a ureido-containing benzimidazole derivative and its preparation method and application.
  • Invention patent CN106565673 discloses a 5-fluoropyrimidine heterocyclic compound with Wnt signaling pathway inhibitory activity, and is used to treat or prevent diseases caused by Wnt signaling pathway disorders.
  • Invention patent CN105017159 discloses a 5-fluoro-2,4-disubstituted aminopyrimidine derivative, its preparation method and anti-tumor application, but its inhibitory HIPK2 kinase and anti-renal interstitial fibrosis activities are not reported.
  • the present invention relates to the field of pharmaceutical chemical synthesis, specifically to a class of 2,4-disubstituted-5-fluoropyrimidine derivatives, their preparation methods and applications.
  • R 1 and R 2 can be independently selected from:
  • R 4 is selected from:
  • X, Y are independently selected from nitrogen atoms or carbon atoms;
  • Z is selected from hydrogen atom, tert-butoxycarbonyl group, C1-C5 alkyl group, C1-C5 alkyl acyl group, C1-C3 alkyl hydroxyl group, C1-C3 alkyl sulfonate Acyl group, C1-C3 substituted phenyl group, C1-C5 alkylamido group.
  • R 4 is Z is selected from hydrogen atom, C1-C5 alkyl group, C1-C5 alkyl acyl group, C1-C3 alkyl hydroxyl group, C1-C3 alkylsulfonyl group, C1-C3 substituted phenyl group, C1-C5 alkyl amide group.
  • R 1 or R 2 is:
  • R 4 is Z is butyl, pentyl, isobutyl, isopentyl, and the other of R 1 and R 2 is:
  • X and Y are not carbon atoms at the same time.
  • R 4 is Z is selected from C4-C5 alkyl, R 1 and R 2 The other is not:
  • R 1 or R 2 is:
  • R 4 is Z is hydrogen, R 1 and R 2 are not:
  • R 1 or R 2 is: R 1 and R 2 and the other is: Z is selected from hydrogen atom, C4-C5 alkyl group, C1-C3 alkyl acyl group, C1-C3 substituted phenyl group, C1-C5 alkyl amido group.
  • the present invention also provides another 2,4-disubstituted-5-fluoropyrimidine derivative, the general structural formula of which is shown in formula II or formula III:
  • R 3 is:
  • R 4 is selected from:
  • X, Y are independently selected from nitrogen atoms or carbon atoms;
  • Z is selected from hydrogen atom, tert-butoxycarbonyl group, C1-C5 alkyl group, C1-C5 alkyl acyl group, C1-C3 alkyl hydroxyl group, C1-C3 alkyl sulfonate Acyl group, C1-C3 substituted phenyl group, C1-C5 alkylamido group.
  • C1-C5 alkyl includes methyl, ethyl, propyl, butyl, pentyl, isobutyl, isopentyl and isopropyl.
  • R 3 is:
  • R 4 is Z is selected from hydrogen atom, C1-C3 alkyl group, C1-C5 alkyl acyl group, C1-C3 alkyl hydroxyl group, C1-C3 alkylsulfonyl group, C1-C3 substituted phenyl group, C1-C5 alkyl amido group.
  • R 4 is When, Z is not tert-butoxycarbonyl.
  • R 4 in formula III is When, Z is not tert-butoxycarbonyl, C4-C5 alkyl, C4-C5 alkylacyl, C4-C5 alkylamide.
  • C1-C5 alkyl acyl includes formyl, acetyl, propionyl, butyryl, valeryl, 3,3-dimethyl-1-butyryl, and isovaleryl.
  • the C1-C5 alkylamido group includes formamide group, acetamide group, propionamide group, butylamido group, valeramide group, N-tert-butylaminoformyl group, and isovaleramide group.
  • C1-C3 alkyl hydroxyl groups include hydroxymethyl, 1-hydroxyethyl, and 1-hydroxypropyl.
  • C1-C3 alkylsulfonyl group includes sulfonylmethyl, sulfonylethyl, and sulfonylpropyl.
  • the C1-C3 substituted phenyl group includes benzyl, phenethyl, and phenylpropyl.
  • the pharmaceutically acceptable salts of the above compounds are hydrochloride, sulfate, phosphate, perchlorate, methanesulfonate, triflate, formate, acetate, and propionate. , butyrate, maleate, succinate, trifluoroacetate, succinate, salicylate, DL-aspartate, D-aspartate, L-aspartate Acid salt, DL-glutamate, D-glutamate, L-glutamate, glycerate, stearate, DL-tartrate, D-tartrate, L-tartrate, ( ⁇ )mandelate, (R)-(-)mandelate, (S)-(+)mandelate, citrate, mucate, malonate, benzoate, DL-malic acid Salt, ( ⁇ )lactate, L-(+)-lactate, D-(+)-lactate, paramitate, D- ⁇ -galacturonate, glycerate,
  • the invention also discloses a method for preparing 2,4-disubstituted-5-fluoropyrimidine derivatives, which is characterized by comprising the following steps:
  • 2,4-dichloro-5-fluoro-pyrimidine reacts with different amine compounds successively to obtain compound I or II, and the intermediate generated by the reaction of 2,4-dichloro-5-fluoro-pyrimidine and borate ester continues to react with 2-
  • the reaction of aminobenzimidazole gives compound III.
  • the present invention includes the use of the above-mentioned 2,4-disubstituted-5-fluoropyrimidine derivatives in preparing drugs for treating fibrosis.
  • the fibrosis is renal interstitial fibrosis.
  • the present invention includes 2,4-disubstituted-5-fluoropyrimidine derivatives or pharmaceutically acceptable salts thereof as anti-fibrosis drugs.
  • the invention provides a pharmaceutical composition, comprising an effective amount of a compound, stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof, and Its pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipients include solvents, diluents, other liquid excipients, dispersants or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders or lubricants. Agents, etc.
  • the pharmaceutical composition may be in liquid, solid, semi-solid, gel or spray form.
  • the compounds or pharmaceutically acceptable compositions of the present invention can be administered in any suitable manner, and can be administered orally, rectum, parenterally, intracisternally, intravaginally, intraperitoneally, topically, etc. to humans or according to the severity of the disease. Other animals are administered the compounds and pharmaceutically acceptable compositions described above.
  • pharmaceutically acceptable salts refers to organic and inorganic salts of the compounds of the invention.
  • pharmaceutically acceptable means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients of the formulation and/or the mammal being treated therewith.
  • the compounds described in the present invention include compounds as defined in the present invention labeled with various isotopes, for example, those compounds in which radioactive isotopes are present, such as 3H, 14C and 18F, or in which non-radioactive isotopes are present, such as 2H and 18F. 13C compound.
  • Figure 1 shows compounds reported in the literature to have HIPK 2 inhibitory activity.
  • Figure 2 shows the effect of compound 7a on the expression of ⁇ -SMA and Fn 1 in NRK-49F cells in Example 16.
  • Figure 3 shows the effect of compound 7a in Example 17 on the expression of P65 in the inflammatory signaling pathway.
  • Figure 4 shows HE staining (1 ⁇ ) of mouse kidney tissue in each group in Example 18.
  • the left side is the Sham group, the middle is the UUO group, and the right side is the UUO+Compound 7a group.
  • Figure 5 shows HE staining (200 ⁇ ) of mouse kidney tissue in each group in Example 18.
  • the left side is the Sham group, the middle is the UUO group, and the right side is the UUO+Compound 7a group.
  • FIG. 6 shows the renal tissue damage scores of mice in each group in Example 18.
  • the left side is the Sham group
  • the middle is the UUO group
  • the right side is the UUO+Compound 7a group.
  • Figure 7 shows masson staining ( ⁇ 1) of mouse kidney tissue in each group of mice in Example 18.
  • the left side is the Sham group, the middle is the UUO group, and the right side is the UUO+Compound 7a group.
  • Figure 8 shows masson staining (200 ⁇ ) of mouse kidney tissue in each group in Example 18.
  • the left side is the Sham group, the middle is the UUO group, and the right side is the UUO+Compound 7a group.
  • Figure 9 shows masson staining scores of kidney tissue of mice in each group of Example 18.
  • the left side is the Sham group
  • the middle is the UUO group
  • the right side is the UUO+Compound 7a group.
  • Figure 10 shows the renal tissue staining and damage score of the compound 25f group in Example 18.
  • Conditions and reagents aK 2 CO 3 , DMSO, 90°C, 5h; b.Fe, NH 4 Cl, EtOH, H 2 O, reflux, 5h; cH 2 O, MeOH, 60°C, 12h; d.
  • Conditions and reagents aK 2 CO 3 , DMSO, 90°C, 5-18h; b.NH 4 Cl, Fe, H 2 O, EtOH, reflux, 5h; cH 2 O, MeOH, 60°C, 12h; d.Cs 2 CO 3 ,Xantphos,Pd 2 (dba) 3 ,1,4-dioxane,N 2 protection,sealed tube,120°C,12h.
  • Compound 5f-g and 2-aminobenzimidazole use Cs 2 CO 3 as base, Xantphos as carrier, Pd 2 (dba) 3 as catalyst, and 1,4-dioxane as solvent to generate Buchwald–Hartwig coupling. A combined reaction was carried out to obtain target compound 6f-g and compound 6g-1.
  • Compound 10 and 2-aminobenzimidazole underwent Buchwald–Hartwig coupling reaction to obtain the target compound 11h.
  • Compound 11h was removed with Boc using trifluoroacetic acid at room temperature to obtain the target compound 12h.
  • Compound 12h undergoes Bauch reduction reaction with isovaleraldehyde to obtain target compound 13h.
  • Conditions and reagents a. Corresponding amines, DMSO, K 2 CO 3 , 90°C, 5h or diethylamine, DMSO, K 2 CO 3 , 50°C, 18h; b. Fe, NH 4 Cl, H 2 O, EtOH, reflux ,5h;c.DCM,Et 3 N,reflux,4h;dH 2 ,Pd/C;MeOH,18h;e.MeOH,H 2 O,60°C,12h;f.17,Pd 2 (dba) 3 , Xanphos,Cs 2 CO 3 ,1,4-dioxane,N 2 protection,sealed tube,120°C,12h.
  • the benzimidazole fragment was synthesized.
  • Compounds 14 and 15 were used as raw materials for cyclization to obtain the nitro-containing benzimidazole fragment 16.
  • the nitro group was reduced to the amino group through hydrogen reduction to synthesize the amino-containing benzimidazole fragment. 17.
  • the nucleophilic substitution reaction connects the corresponding aliphatic amino group to reach an aromatic cyclic aliphatic with a nitro group in the para position. Heterocyclic fragments.
  • the nitro group was reduced using the iron powder reduction method to obtain the aromatic ring-linked aliphatic heterocyclic fragments 20a-h with the amino position in the para position.
  • 20i was not synthesized by this method and was directly purchased from Anaiji Company. 20a-i and raw material 21 undergo nucleophilic substitution reaction in methanol as solvent without any alkaline environment to obtain compound 22a-i. 22a-i is then combined with compound 17 using Pd 2 (dba) 3 as catalyst and Xanphos As the carrier, Cs 2 CO 3 was used to provide an alkaline environment, and the Buchwald–Hartwig coupling reaction occurred with 1,4-dioxane as the solvent to obtain the target compound 23a-i. Among them, 23f-h was removed from Boc to obtain target compounds 24a-c.
  • Compound 25a was obtained by reacting 24a with methyl iodide at minus 10°C using acetonitrile as the solvent and triethylamine as the catalyst.
  • Compound 25b was obtained by reacting 24a with acetic anhydride at room temperature using acetonitrile as the solvent and triethylamine as the catalyst.
  • Compound 25c was obtained by reacting 24a with 2-iodopropane at 65°C using acetonitrile as the solvent.
  • Compound 25d was obtained by reacting 24a with 2-bromoethanol for 16 h using acetonitrile as the solvent and triethylamine as the catalyst at room temperature.
  • Compound 25e was obtained by reacting 24a with ethanesulfonyl chloride for 1 hour at room temperature using acetonitrile as the solvent and triethylamine as the catalyst.
  • Compound 25f was obtained by reacting 24a with trimethylacetaldehyde, using dichloroethane as the solvent, glacial acetic acid, and sodium trimethoxyborohydride as the catalyst at 60°C for 16 hours. 24a reacted with benzyl bromide, using acetonitrile as the solvent and triethylamine as the catalyst, and reacted at minus 10°C for 10 minutes to obtain 25g of compound.
  • 24a reacted with 3,3-dimethylbutyryl chloride, using acetonitrile as the reaction solvent and triethylamine as the catalyst, and reacted at minus 10°C for 10 minutes to obtain compound 25h.
  • 24a and tert-butyl cyanate were reacted with acetonitrile as the reaction solvent and triethylamine as the catalyst at minus 10°C for 10 minutes to obtain compound 25i.
  • compound 17 and raw material 12 undergo a nucleophilic substitution reaction in methanol as a solvent without any alkaline reagent to obtain compound 26, and then react with di-tert-butyl dicarbonate, DMAP and triethylamine as catalysts, and dichloromethane.
  • the reaction solvent As the reaction solvent, the reaction was carried out at room temperature for 24 hours to obtain compound 27.
  • Compound 27 reacts with the corresponding amine or compound 17 respectively, using Pd 2 (dba) 3 as the catalyst, Xanphos as the carrier, Cs 2 CO 3 to provide an alkaline environment, and 1,4-dioxane as the solvent. –Hartwig coupling reaction afforded target compounds 28a-e and 28f.
  • the sodium sulfate was removed by suction filtration, and the organic solvent was spin-dried to obtain a gray oily substance. Total 4.5g, yield 84%. Because this product is easily oxidized in air, it is directly added to the next step.
  • 20b-20h were prepared using the same method as 20a, and were also directly added to the next step without structural confirmation.
  • N 6 -(5-fluoro-4-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-2-yl)-N 2 , N 2 -dimethyl-1H-benzo[ d] Imidazole-2,6-diamine (0.45g, 1mmol) and acetic acid (0.06g, 1mmol) were added to an eggplant-shaped flask containing 10mL DCE, and stirred at 60°C for 20min. Then trimethylacetaldehyde (0.26g, 3mmol) and ethyl triacetate sodium borohydride (0.64g, 3mmol) were added to the reaction solution, and stirred at 60°C for 16h.
  • reaction solution was extracted with dichloromethane and water, and the aqueous layer was washed three times with dichloromethane until the water layer had no fluorescence at 254 nm.
  • N6-(2-chloro-5-fluoropyrimidin-4-yl)-N 2,N 2-dimethyl-1H-benzo[d]imidazole-2,6-diamine (3.06g, 10mmol) was added To 100 mL of DCM in a 250 mL eggplant-shaped flask, add (Boc) 2 O (21.81 g, 100 mmol), triethylamine (5.05 g, 50 mmol) and DMAP (0.49 g, 4 mmol) and react at room temperature for 24 h. , spin the reaction liquid to dryness, and obtain an orange-red oil.
  • Extract with dichloromethane and water wash the organic layer three times with saturated aqueous citric acid solution, once with saturated aqueous sodium bicarbonate solution, and finally wash once with saturated brine.
  • the organic layer was dried over anhydrous Na2SO4 and left for 4h. Filter with suction, spin dry the organic solvent, and obtain an orange-red oil. Place it in an oil pump and drain it for 2 hours to obtain 4.72 g of an orange-red solid with a yield of 93%.
  • Compound 29a was prepared as a gray solid with a yield of 50% using compound 28a as raw material using the method of Example 11 above.
  • Example 15 The synthesized compound was subjected to a HIPK2 inhibitory activity experiment, and the synthesized compound was subjected to a rat kidney fibroblast (NRK-49F) proliferation inhibition experiment.
  • HIPK2 kinase (Carna); MBP, ATP (Promega), ADP-Glo TM detection kit, Cytation 5.
  • the synthesized compound was used to inhibit the proliferation of rat kidney fibroblasts (NRK-49F).
  • Reagents and instruments 96-well plate (Corning); fetal calf serum (Gibco), F12 medium, DMEM medium (BI); biological safety cabinet, carbon dioxide incubator (ESCO); Cytation 5 multi-function imager ( Bio-Tek).
  • Cell culture NRK-49F cells were cultured conventionally. Cells in the logarithmic growth phase with a viable cell ratio higher than 90% were used in the experiments.
  • Target compounds have inhibitory activity against HIPK2 kinase and NRK-49F cell proliferation.
  • the maximum concentration of a compound is 10 ⁇ M.
  • the data in brackets are the inhibition rate at the maximum concentration. For example, >10 (2.7%) means that the concentration is 10 ⁇ M.
  • the maximum concentration of the compound is 100 ⁇ M, and the data in brackets are the inhibition rate at the maximum concentration;
  • the active Wnt inhibitor disclosed in patent CN106565673 d The 5-fluoro-2,4-disubstituted pyrimidine derivative disclosed in patent CN105017159 .
  • Example 16 Effect of target compound on fibrosis
  • ultra-sensitive ECL chemiluminescence reagents were purchased from Abbkine; goat anti-rabbit secondary antibodies, developer, fixer, RIPA lysis buffer, BSA, BCA protein concentration detection kits, and pre-stained protein markers were all purchased. From Nanjing Enjing Biotechnology Co., Ltd.
  • 6-well cell culture plates were purchased from Corning Company; ultra-clean workbench (ESCO); carbon dioxide incubator (ESCO); fluorescence inverted biological microscope (optec); desktop high-speed centrifuge (SCILOGEX); micro vertical Electrophoresis tank (Bio-RAD); transfer electrophoresis tank (Bio-RAD); electrophoresis instrument (Junyi Co., Ltd.); destaining shaker (Hualida Co., Ltd.); Cytation 5 microplate reader (Bio-tek Co., Ltd.).
  • NRK-49F cells in the logarithmic growth phase were used for experiments.
  • the cells were digested, counted, and diluted into a cell suspension of 2 ⁇ 105 cells/mL, and inoculated into a 6-well plate at 2 mL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours; replace with fresh culture medium.
  • a blank control group and an experimental group were set up. Different concentrations of compound 7a were added.
  • the culture medium was poured out, and the cells in each well were washed three times with 2 mL of pre-cooled PBS at 4°C. The dish was then placed on ice.
  • ECL luminescent solution A and B in a ratio of 1:1 and let stand for 1 minute at room temperature. Place the PVDF membrane protein side up on the plate. After 1 minute, apply ECL luminescent liquid for development and fixation.
  • GAPDH GAPDH as the internal reference, use Adobe Photoshop CS5 software to analyze the net optical density value of the target band.
  • the scanner scanned the developed strip, and the obtained results were scanned with ImageJ software to obtain their grayscale values, and graphed with Graphpad software.
  • Example 17 Effect of compounds on inflammation-related pathways
  • Antibodies P65, p-P65, and GAPDH were purchased from CST. Others are the same as in Embodiment 16.
  • Example 18 Effect of compounds on unilateral ureteral ligation mouse renal interstitial fibrosis model
  • the unilateral ureteral ligation model (UUO model) is a classic renal interstitial fibrosis model, which simulates the entire dynamic process from acute renal injury to gradual progression to renal interstitial fibrosis. It is generally believed that 3 days after ureteral ligation is the acute phase of injury, which is mainly characterized by cell edema, death and inflammatory cell infiltration; 3-7 days is the subacute phase, which is mainly characterized by renal interstitial edema, renal tubular dilation, inflammatory Cell infiltration is dominant, and extracellular matrix deposition begins to occur; after 7 days, renal interstitial edema and renal tubular dilation gradually disappear, and are replaced by renal interstitial atrophy, interstitial extracellular matrix deposition significantly increases, and fibrosis worsens, entering the chronic phase. .
  • the purpose of this study to observe the efficacy of the target compound (HIPK2-inhibitor) of the present invention on the UUO 14-day model, so as to clarify the therapeutic effect of
  • Target compound HIPK2-inhibitor
  • mice C57-BL6 mice, male, weighing 22-25g, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., experimental animal production license number: SCXK (Hunan) 2015-0017.
  • mice SPF grade male mice were divided into sham operation group (Sham), unilateral ureteral ligation group (UUO), and UUO + target compound (75 mg/kg) group. After anesthesia, the back hair was shaved and disinfected with iodine, except for the sham operation group. In all mice, a small incision was made in the skin on the upper left side of the midline of the back to expose the posterior peritoneum. The peritoneum was then cut, the left kidney and ureter were squeezed out, and 3-0 sutures were ligated near the renal hilus and below the ureter. Cut the ureter in the middle of the two ligation points, push the kidney back into the abdominal cavity, and suture the peritoneum and back skin.
  • Sham sham operation group
  • UUO unilateral ureteral ligation group
  • UUO + target compound 75 mg/kg
  • mice in the sham operation group were only subjected to abdominal opening and closing.
  • the UUO + target compound (75 mg/kg) group was administered the target compound by gavage at 75 mg/kg/6 mL, once a day for 14 consecutive days.
  • the mice were sacrificed on the 14th day after surgery.
  • HE staining Classify renal tissue lesions into renal interstitial fibrosis, renal interstitial inflammation, renal tubular vacuolar degeneration, renal tubular atrophy, renal tubular dilation, renal interstitial edema, red blood cell casts, and protein casts. Score the above-mentioned various types of lesions respectively, including normal: score "0”; mild: score “1”; moderate: score “2”; severe: score “3”, and assign each animal The scores are added up to get the total score. Calculate the above-mentioned types of scores and the total score respectively.
  • Masson staining Calculated based on the area of extracellular matrix deposition, 0-25%, 25-50%, 50-75%, and greater than 75% are scored as 1, 2, 3, and 4 points respectively.
  • the software used for statistics is SPSS20.0. If the variances are homogeneous, use One-Way ANOVA for statistical analysis. If the variances are not equal, use the Kruskal-Wallis test. If the Kruskal-Wallis test is statistically significant (P ⁇ 0.05), Dunnett’s Test (non-parametric method) is used for comparative analysis. Statistical results are expressed as statistically significant with P ⁇ 0.05, and P ⁇ 0.01 indicates that the tested difference is very significant.
  • mice in the UUO group had obvious renal interstitial fibrosis deposition at 14 days, and the masson staining pathological scores were significantly increased (P ⁇ 0.01); Compared with the UUO group, the kidney interstitial fibrosis deposition in the compound group (only data for 7a and 25f are listed) was significantly reduced, and the masson staining pathological score was significantly reduced (P ⁇ 0.05).
  • Other target compound groups also showed a significant reduction in renal interstitial fibrosis deposition and a significant reduction in Masson staining pathology scores.
  • the 2,4-disubstituted-5-fluoropyrimidine derivative of the present invention can reduce renal damage and renal interstitial fibrosis deposition.

Abstract

本发明公开了2，4-二取代-5-氟嘧啶类衍生物，其结构通式如式I所示： < 其中，R1和R2分别独立选自： R4选自：本发明还公开了所述化合物的制备方法和它们在制备抗肾间质纤维化药物中的用途。

Description

2,4-二取代-5-氟嘧啶衍生物及其制备方法和应用 技术领域
肾间质纤维化是一种慢性和进行性的过程，在衰老和慢性肾病中影响肾脏的正常功能，从而危害人类健康。在目前已知的病理机制中，NF-κB、TGF-β/Smad3、Wnt/β-catenin、Notch和p53信号通路在肾间质纤维化的发生发展中具有重要作用。从现有的药物研究结果可知，针对上述一条或两条通道进行药物发现的策略在肾间质纤维化治疗药物的研发领域没有成功的药物上市(Nat Rev Drug Discov,2016,15(8):568；Am J Kidney Dis.2022,doi:10.1053/j.ajkd.2021.11.010)。研究结果表明，尽管上述各通路已被证明是疾病过程中的重要环节，但由于各信号通路之间存在复杂的相互作用，单独抑制其中一条或两条通路会打开与炎症、纤维化相关的其他通路(Front Cell Dev Biol.2021,9:696542)。这种复杂的情况是当前肾间质纤维化治疗药物研发一直没有取得突破的重要原因。因此，单独针对这些下游靶标寻找高效的肾间质纤维化治疗药物并不是理想的策略。
在肾间质纤维化发病过程中，氧化应激、炎症与纤维化是关键性环节，同时针对上述关键环节寻找多通道药物有望避免各通路相互作用，提高药物疗效。然而，从药物设计学的角度来看，设计同时抑制上述五条通路的单一结构抑制剂难度非常大，十分有必要从另一种角度进行探索，以求开辟新的研究领域，为肾间质纤维化治疗药物的研发提供创新性的思路。
John He等人发表文章指出(Nat Med.2012,18(4):580)，同源结构域相互作用蛋白激酶2在肾间质小管病变部位高表达，并通过对的调节，参与纤维化和炎症过程。因此，抑制HIPK2可同时调控纤维化和炎症，达到治疗疾病的目的。
文献研究报道的HIPK2抑制剂分类三类(图1)：HIPK2激酶抑制剂、HIPK2/SMAD相互作用抑制剂和HIPK2表达抑制剂。TBID(Plos one.2014,9(2):e89176)是HIPK2激酶抑制剂，它通过占据ATP结合口袋抑制HIPK2的激酶活性。除TBID外，其他具有HIPK2激酶抑制活性的化合物均是通过脱靶效应研究发现的，如TAE-226(Nat Med.2012,18(4):580)、CTx-0294857(J.Proteome Res.2013,12:3104)、Sorafenib(Plos one.2015,10(2):e0117757)、GW-5074、Compound C(Biochem.J.2007,408:297)。BT173(J Am Soc Nephrol.2017,28:2133)是一种HIPK2/SMAD相互作用抑制剂，它可以通过阻断HIPK2和Smad3之间的蛋白-蛋白相互作用发挥抗纤维化作用，但无抑制HIPK2的激酶活性。HIPK2表达抑制剂磷酸氯硝柳胺(phosphate niclosamide)(Kidney International.2017,92:612)可以通过干扰Smad3与HIPK2基因的启动子序列结合来抑制HIPK2的转录，从而抑制HIPK2下游内多条信号通路。
发明专利201911152583.9和PCT/CN2020/129308公开了一种含脲基苯并咪唑类衍生物及其制备方法和应用。
发明专利CN106565673公开了一种具有Wnt信号通路抑制活性的5-氟嘧啶杂环化合物，并将其用于治疗或预防因Wnt信号通路失常引起的病症。
发明专利CN105017159公开了一种5-氟-2,4-二取代氨基嘧啶衍生物及其制备方法和抗肿瘤应用，但并未报道其抑制HIPK2激酶及抗肾间质纤维化活性。
因此，到目前为止，尚没有HIPK2抑制剂成功用于临床。鉴于肾间质纤维化疾病的重大临床需求和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
本发明涉及药物化学合成领域，具体涉一类2,4-二取代-5-氟嘧啶衍生物，及其制备方法和应用。
一种2，4-二取代-5-氟嘧啶类衍生物，其结构通式如式I所示：
其中，R1和R2可分别独立选自：
R4选自：
X,Y分别独立选自氮原子或碳原子；Z选自氢原子，叔丁氧羰基，C1-C5烷基，C1-C5烷基酰基，C1-C3烷基羟基，C1-C3烷基磺酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
优选的，R1和R2任一为：R4为Z选自氢原子，C1-C5烷基，C1-C5烷基酰基，C1-C3烷基羟基，C1-C3烷基磺酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
优选的，R1和R2任一为：R4为Z为丁基，戊基，异丁基，异戊基，R1和R2另一为：时，X,Y不同时为碳原子。
优选的，R1和R2任一为：R4为Z选自C4-C5烷基，R1和R2 另一不为：
优选的，R1和R2任一为：R4为Z为氢，R1和R2另一不为：
优选的，R1和R2任一为：R1和R2另一为：为Z选自氢原子，C4-C5烷基，C1-C3烷基酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
本发明还提供另外一种2，4-二取代-5-氟嘧啶类衍生物，其结构通式如式II或式III所示：
其中，R3为：
R4选自：
X,Y分别独立选自氮原子或碳原子；Z选自氢原子，叔丁氧羰基，C1-C5烷基，C1-C5烷基酰基，C1-C3烷基羟基，C1-C3烷基磺酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
优选的，C1-C5烷基包括甲基，乙基，丙基，丁基，戊基，异丁基，异戊基，异丙基。
优选的，R3为：R4为Z选自氢原子，C1-C3烷基，C1-C5烷基酰基，C1-C3烷基羟基，C1-C3烷基磺酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
优选的，在R3中，R4为时，Z不为叔丁氧羰基。
优选的，式III中的R4为时，Z不为叔丁氧羰基、C4-C5烷基、C4-C5烷基酰基，C4-C5烷基酰胺基。
优选的，C1-C5烷基酰基包括甲酰基，乙酰基，丙酰基，丁酰基，戊酰基，3，3-二甲基-1-丁酰基，异戊酰基。
优选的，C1-C5烷基酰胺基包括甲酰胺基，乙酰胺基，丙酰胺基，丁酰胺基，戊酰胺基，N-叔丁基胺基甲酰基，异戊酰胺基。
优选的，C1-C3烷基羟基包括羟甲基，1-羟基乙基，1-羟基丙基。
优选的，C1-C3烷基磺酰基包括磺酰基甲基，磺酰基乙基，磺酰基丙基。
优选的，C1-C3取代苯基包括苄基，苯乙基，苯丙基。
优选的，上述化合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，具体为以下化合物：
叔丁基-4-(4-((2-(2-氨基-1H-苯并[d]咪唑-1-基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯(化合物6a)；
叔丁基-4-(4-((2-((1H-苯并[d]咪唑-2-基)氨基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯(化合物6h)；
叔丁基-4-(4-((5-氟-2-(异喹啉-3-基氨基)嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯(化合物6b)；
叔丁基-4-(4-((5-氟-2-(((四氢呋喃-2-基)甲基)氨基)嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯(化合物6c)；
叔丁基-4-(4-((5-氟-2-((吡啶-2-基甲基)氨基)嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯(化合物6d)；
叔丁基-4-(4-((5-氟-2-((4-(甲氧甲酰基)苯基)氨基)吡啶-4-基)氨基)苯基)哌嗪-1-羧酸酯(化合物6e)；
1-(5-氟-4-((4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺(化合物6f)；
1-(4-((4-(4-乙基哌嗪-1-基)苯基)氨基)-5-氟嘧啶-2-基)-1H-苯并[d]咪唑-2-胺(化合物6g)；
N2-(1H-苯并[d]咪唑-2-基)-N4-(4-(4-乙基哌嗪-1-基)苯基)-5-氟嘧啶-2,4-二胺(化合物6g-1)；
1-(5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺(化合物7a)；
5-氟-N2-(异喹啉-3-基)-N4-(4-(哌嗪-1-基)苯基)嘧啶-2,4-二胺(化合物7b)；
5-氟-N4-(4-哌嗪-1-基)苯基)-N2-((四氢呋喃-2-基)甲基)嘧啶-2,4-二胺(化合物7c)；
5-氟-N4-(4-(哌嗪-1-基)苯基-N2-(吡啶-2-基甲基)嘧啶-2,4-二胺(化合物7d)；
甲基-4-((5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)氨基苯甲酸酯(化合物7e)；
1-(5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2- 胺(化合物8a)；
5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-异喹啉-3-基)嘧啶-2,4-二胺(化合物8b)；
5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-((四氢呋喃-2-基)甲基)嘧啶-2，4-二胺(化合物8c)；
5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-(吡啶-2-基甲基)嘧啶-2,4-二胺(化合物8d)；
甲基-4-((5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)氨基)嘧啶-2-基)氨基)苯甲酸酯(化合物8e)；
叔丁基-4-(5-(2-(2-氨基-1H-苯并[d]咪唑-1-基)-5-氟嘧啶-4-基)吡啶-2-基)哌嗪-1-羧酸酯(化合物11h)；
1-(5-氟-4-(6-(哌嗪-1-基)吡啶-3-基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺(化合物12h)；
1-(5-氟-4-(6-(4-异戊基哌嗪-1-基)吡啶-3-基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺(化合物13h)；
N6-(5-氟-4-((4-吗啉苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23a)；
N6-(5-氟-4-((4-(吡咯-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23b)；
N6-(4-((4-(二乙基胺基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23c)；
N6-(4-((4-(二丙基胺基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23d)；
N6-(5-氟-4-((4-(哌啶-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23e)；
叔丁基-4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-羧酸酯(化合物23f)；
N6-(4-((4-(二甲基胺基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23i)；
叔丁基-4-(5-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)吡啶-2-基)哌嗪-1-羧酸酯(化合物23g)；
叔丁基-4-(5-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)吡啶-3-基)哌嗪-1-羧酸酯(化合物23h)；
N6-(5-氟-4-((4-(哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物24a)；
N6-(5-氟-4-((6-(哌嗪-1-基)吡啶-3-基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物24b)；
N6-(5-氟-4-((5-(哌嗪-1-基)吡啶-2-基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物24c)；
N6-(5-氟-4-((4-(4-甲基哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25a)；
1-(4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-基)乙酮(化合物25b)；
N6-(5-氟-4-((4-(4-异丙基哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25c)；
2-(4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-基)乙醇(化合物25d)；
N6-(4-((4-(4-(乙基磺酰基)哌嗪-1-基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2- 二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25e)；
N6-(5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25f)；
N6-(4-((4-(4-苄基哌嗪-1-基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25g)；
1-(4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-基)-3,3-二甲基丁-1-酮(化合物25h)；
N-(叔丁基)-4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-甲酰胺(化合物25i)；
N6-(5-氟-2-((4-(吡咯-1-基)苯基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29a)；
N6-(5-氟-2-((4-(哌嗪-1-基)苯基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29b)；
N6-(5-氟-2-((6-(哌嗪-1-基)吡啶-3-基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29c)；
N6-(2-((4-(二甲基胺基)苯基)胺基)-5-氟嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29d)；
N6-(5-氟-2-((5-(哌嗪-1-基)吡啶-2-基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29e)；
1-(4-(4-((4-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-2-基)胺基)苯基)哌嗪-1-基)乙酮(化合物29f)；
N6-(5-氟-2-((4-(4-异戊基哌嗪-1-基)苯基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29g)；
N6，N6-(5-氟嘧啶-2,4-二基)双(N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺)(化合物29h)。
本发明还公开了一种2，4-二取代-5-氟嘧啶类衍生物的制备方法，其特征在于包括以下步骤：
2，4-二氯-5-氟-嘧啶先后与不同胺类化合物反应得到化合物I或II，2，4-二氯-5-氟-嘧啶与硼酸酯反应生成的中间体继续与2-氨基苯并咪唑反应得到化合物III。
本发明包含上述2，4-二取代-5-氟嘧啶类衍生物在制备治疗纤维化的药物中的应用。
优选的，所述纤维化为肾间质纤维化。
本发明包含2，4-二取代-5-氟嘧啶类衍生物或其药学上可用的盐，作为抗纤维化的药物。
本发明提供一种药物组合物，包含有效量的化合物、化合物的立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐或它的前药及其药学上可接受的辅料。
所述药学上可接受的辅料包括溶剂、稀释剂、其他液体赋形剂、分散剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、固体粘合剂或润滑剂等。
所述药物组合物可以是液体，固体，半固体，凝胶或喷雾剂型。
本发明的化合物或药学上可接受的组合物可以通过任何合适方式给药，可根据疾病的严重程度经口、直肠、肠胃外、脑池内、阴道内、腹膜内、局部给药等向人或其它动物施用以上所述化合物和药学上可接受的组合物。
术语“药学上可接受的盐”是指本发明的化合物的有机盐和无机盐。术语“药学上可接受的”是指物质或组合物必须与包含制剂的其它成分和/或用其治疗的哺乳动物化学上和/或毒理学上相容。
另一方面，本发明所述化合物包括使用各种同位素标记的本发明所定义的化合物，例如，其中存在放射性同位素，如3H，14C和18F的那些化合物，或者其中存在非放射性同位素，如2H和13C的化合物。
附图说明
图1为文献报道具有HIPK2抑制活性的化合物。
图2为实施例16中化合物7a对NRK-49F细胞α-SMA、Fn 1表达的影响。
图3为实施例17中化合物7a对炎症信号通路中P65表达的影响
图4为实施例18中各组小鼠肾组织HE染色(1×)，左侧为Sham组，中间为UUO组，右侧为UUO+化合物7a组。
图5为实施例18中各组小鼠肾组织HE染色(200×)，左侧为Sham组，中间为UUO组，右侧为UUO+化合物7a组。
图6为实施例18中各组小鼠肾组织损伤评分，左侧为Sham组，中间为UUO组，右侧为UUO+化合物7a组。
图7为实施例18中各组小鼠肾组织masson染色(×1)，左侧为Sham组，中间为UUO组，右侧为UUO+化合物7a组。
图8为实施例18中各组小鼠肾组织masson染色(200×)，左侧为Sham组，中间为UUO组，右侧为UUO+化合物7a组。
图9各实施例18中组小鼠肾组织masson染色评分，左侧为Sham组，中间为UUO组，右侧为UUO+化合物7a组。
图10为实施例18中化合物25f组的肾组织染色和损伤评分。
具体实施方式
条件和试剂:a.K2CO3,DMSO,90℃,5h；b.Fe,NH4Cl,EtOH,H2O,reflux,5h；c.H2O,MeOH,60℃,12h；d.Corresponding amines,Cs2CO3,Xantphos,Pd2(dba)3,1,4-dioxane,N2protection,sealed tube,120℃,12h；e.TFA,DCM,r.t.,4h；f.3-Methyl butanal,DCE,AcOH,NaHB(OCOCH3)3,60℃,16h.
以对氟硝基苯、1-Boc-哌嗪作为起始原料，用二甲亚砜作为反应溶剂，在回流5h的条件下发生亲核取代反应，得到中间体3。然后用铁粉和氯化铵将硝基还原为氨基，得到中间体4。中间体4和2，4-二氯-5-氟嘧啶在甲醇中发生亲核取代反应，得到化合物5。化合物5和各种不同的胺以Cs2CO3为碱,以Xantphos做载体,以Pd2(dba)3做催化剂,以1,4-dioxane做溶剂发生Buchwald–Hartwig偶联反应，得到目标化合物6a-e,6h。化合物6a-e在室温下用三氟乙酸脱Boc，得到目标化合物7a-e。化合物7a-e与异戊醛发生鲍奇还原反应，得到目标化合物8a-e。
条件和试剂:a.K2CO3,DMSO,90℃,5-18h；b.NH4Cl,Fe,H2O,EtOH,reflux,5h；c.H2O,MeOH,60℃,12h；d.Cs2CO3,Xantphos,Pd2(dba)3,1,4-dioxane,N2protection,sealed tube,120℃,12h.
以对氟硝基苯，1-甲基哌嗪或者1-乙基哌嗪作为起始原料，以二甲基亚砜作为反应溶剂，90度的条件下反应得到中间体3f-g。以铁粉为还原剂，乙醇和水为反应溶剂，加入适量的氯化铵，回流条件下反应5小时得到还原产物4f-g，然后与2，4-二氯-5-氟嘧啶在甲醇中发生亲核取代反应，得到化合物5f-g。化合物5f-g和2-氨基苯并咪唑以Cs2CO3为碱，以Xantphos做载体,以Pd2(dba)3做催化剂,以1,4-dioxane做溶剂发生Buchwald–Hartwig偶 联反应，得到目标化合物6f-g和化合物6g-1。
条件和试剂:a.K2CO3,Pd(dppf)Cl2,H2O:THF＝1:5,reflux,5h；b.Xantphos,Pd2(dba)3,Cs2CO3,1,4-dioxane,N2protection,sealed tube,120℃,12h；c.TFA,DCM,r.t.,4h；d.Trimethyl acetaldehyde,DCE,AcOH,NaHB(OCOCH3)3,60℃,16h.
2-(4-BOC-哌嗪基)吡啶-5-硼酸频哪醇酯在SUZUKI反应条件下(H2O：THF＝1：5为溶剂，醋酸钯为催化剂，碳酸钾为碱)与2，4-二氯-5-氟嘧啶反应5h，得到化合物10。化合物10和2-氨基苯并咪唑发生Buchwald–Hartwig偶联反应，得到目标化合物11h。化合物11h在室温下用三氟乙酸脱Boc，得到目标化合物12h。化合物12h与异戊醛发生鲍奇还原反应，得到目标化合物13h。
条件和试剂:a.Corresponding amines,DMSO,K2CO3,90℃,5h or diethylamine,DMSO,K2CO3,50℃,18h；b.Fe,NH4Cl,H2O,EtOH,reflux,5h；c.DCM,Et3N,reflux,4h；d.H2,Pd/C；MeOH,18h；e.MeOH,H2O,60℃,12h；f.17,Pd2(dba)3,Xanphos,Cs2CO3,1,4-dioxane,N2protection,sealed tube,120℃,12h.
首先对苯并咪唑片段进行合成，以化合物14和15为原料环合得到含硝基的苯并咪唑片段16，在通过氢气还原的方法将硝基还原成氨基合成得到含氨基的苯并咪唑片段17。然后以18a-c作为起始原料，亲核取代反应接入对应脂肪氨基达到对位为硝基的芳香环联脂肪 杂环片段。然后对采用铁粉还原的方法对硝基进行还原得到对位为氨基的芳香环联脂肪杂环片段20a-h，20i没有经过此方法合成，直接外购于安耐吉公司。20a-i和原料21在甲醇做溶剂不需要任何碱性环境的条件下发生亲核取代反应得到化合物22a-i，22a-i再和化合物17在以Pd2(dba)3做催化剂,以Xanphos做载体,以Cs2CO3提供碱性环境,以1,4-dioxane做溶剂的条件下发生Buchwald–Hartwig偶联反应得到目标化合物23a-i。其中23f-h脱Boc得到目标化合物24a-c。
条件和试剂:g.DCM,TAF,r.t.,4h；h.for 25a,methyl iodide,ACN,Et3N,-10℃,10min；for 25b,Acetic anhydride,ACN,Et3N,r.t.,2h；for 25c,2-iodopropane,ACN,K2CO3,65℃,16h；for 25d,2-bromoethan-1-ol,ACN,Et3N,r.t.,16h；for 25e,ethanesulfonyl chloride,ACN,Et3N,r.t.,1h；for 25f,Trimethyl acetaldehyde,AcOH,DCE,NaHB(OCOCH3)3,60℃,16h；for 25g,(bromomethyl)benzene,ACN,Et3N,-10℃,10min；for 25h,3,3-dimethylbutanoyl chloride,ACN,Et3N,-10℃,10min；for 25i,2-isocyanato-2-methylpropane,ACN,Et3N,-10℃,10min.
24a与碘甲烷，在负10℃条件下，以乙腈为溶剂，三乙胺为催化剂反应得到化合物25a。24a与醋酸酐，以乙腈为溶剂，三乙胺为催化剂，在室温下反应得到化合物25b。24a与2-碘丙烷，在65℃条件下，以乙腈为溶剂，反应得到化合物25c。24a与2-溴乙醇，以乙腈为溶剂，三乙胺为催化剂，室温条件下反应16h得到化合物25d。24a与乙烷磺酰氯，以乙腈为溶剂，三乙胺为催化剂，室温条件下反应1h得到化合物25e。24a与三甲基乙醛，以二氯乙烷为溶剂，以冰醋酸，三甲氧基硼氢化钠为催化剂，在60℃条件下，反应16h得到化合物25f。24a与溴苄反应，以乙腈为溶剂，三乙胺为催化剂，在负10℃条件下反应10分钟得到化合物25g。24a与3，3-二甲基丁酰氯反应，以乙腈为反应溶剂，三乙胺为催化剂，在负10℃条件下反应10分钟得到化合物25h。24a与叔丁基氰酸酯，以乙腈为反应溶剂，三乙胺为催化剂，在负10℃条件下反应10分钟得到化合物25i。
条件和试剂:a.17,MeOH,H2O,60℃,12h；b.(Boc)2O,DMAP,Et3N,DCM,r.t.,24h；c1.Corresponding amines,Pd2(dba)3,Xanphos,Cs2CO3,1,4-dioxane,N2protection,sealed tube,120℃,12h；c2.17,Pd2(dba)3,Xanphos,Cs2CO3,1,4-dioxane,N2protection,sealed tube,120℃,12h；d.DCM,TAF,r.t.,4h；e.for 29f,Acetic anhydride,ACN,Et3N,r.t.,2h；for 29g Trimethylacetaldehyde,AcOH,DCE,NaBH(OCOCH3)3,60℃,16h.
首先化合物17和原料12在甲醇做溶剂不需要任何碱性试剂的条件下发生亲核取代反应得到化合物26，然后与二碳酸二叔丁酯，以DMAP和三乙胺为催化剂，以二氯甲烷为反应溶剂，室温条件下反应24小时得到化合物27。化合物27分别与相应的胺或者化合物17，在以Pd2(dba)3做催化剂,以Xanphos做载体,以Cs2CO3提供碱性环境,以1,4-dioxane做溶剂的条件下发生Buchwald–Hartwig偶联反应得到目标化合物28a-e和28f。对28a-e和28f脱去Boc保护得到目标化合物29a-e和29h。29b和乙酸酐，以乙腈为溶剂，三乙胺为催化剂，室温条件下反应2小时得到化合物29f。29b和三甲基乙醛，以二氯乙烷为反应溶剂，以冰醋酸，三甲氧基硼氢化钠为催化剂，60℃反应16小时得到化合物29g。
实施例1：叔丁基-4-(4-((2-(2-氨基-1H-苯并[d]咪唑-1-基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯的制备(化合物6a)
化合物5由实验室前期工作合成，经氢谱、质谱确证结构。1H NMR(500MHz,DMSO-d6)δ9.83(s,1H),8.23(d,J＝3.5Hz,1H),7.51(d,J＝9.1Hz,2H),6.97(d,J＝9.1Hz,2H),3.52-3.41(m,4H),3.14-3.03(m,4H),1.42(s,9H).HRMS(ESI)m/z calcd for[C19H23ClFN5O2+H]+:408.1597；found:408.1601[M+H]+。
将化合物5(2.03g，5mmol)，2-氨基苯并咪唑(20mmol)，Xantphos(0.046g，0.08mmol)， Pd2(dba)3(0.018g，0.02mmol)，碳酸铯(4.864g，15mmol)，加入到含搅拌子的100mL封管中，加入30mL二氧六环(4A分子筛干燥)，用氮气置换瓶中空气，拧上盖子，置于超声溶解。放入120℃油浴中反应12h。置于室温冷却后，旋干反应液，用二氯甲烷/水进行萃取，再用二氯甲烷对水层洗涤3次，用无水Na2SO4干燥有机层4h。
抽滤，旋干有机溶剂，得黄绿色固体，用200-300目硅胶柱层析对其分离纯化(DCM:MeOH＝40:1)，得到白色固体粉末1.169g，收率47％。1H NMR(500MHz,DMSO-d6)δ9.87(s,1H),8.39(d,J＝3.4Hz,1H),8.00(d,J＝8.0Hz,1H),7.60(s,2H),7.44-7.38(m,2H),7.17-7.12(m,1H),7.07-7.00(m,3H),6.82-6.75(m,1H),3.50(t,J＝5.1Hz,4H),3.14(t,J＝5.1Hz,4H),1.44(s,9H).13C NMR(125MHz,DMSO-d6)δ154.51,154.35,149.20,143.37,139.86,139.70,131.92,129.53,125.63,123.19,119.23,116.73,115.26,115.18,79.46,49.09,28.55.HRMS(ESI)m/z calcd for[C26H29FN8O2+H]+:505.2476；found:505.2491。
叔丁基-4-(4-((2-((1H-苯并[d]咪唑-2-基)氨基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯的制备(化合物6h)
使用上述实施例1的方法，以2-氨基苯并咪唑为原料，制备得到化合物6h，为白色固体粉末，收率16％。1H NMR(500MHz,DMSO-d6)δ11.60(s,1H),10.95(s,1H),9.54(s,1H),8.16(d,J＝3.7Hz,1H),7.64(d,J＝8.6Hz,2H),7.43-7.15(m,2H),7.02(dd,J＝9.3,3.4Hz,4H),3.51(t,J＝5.1Hz,4H),3.14(t,J＝5.1Hz,4H),1.43(s,9H).13C NMR(125MHz,DMSO-d6)δ154.37,154.20,150.96,150.87,149.47,148.25,142.63,140.66,140.13,139.97,130.78,124.32,116.68,79.49,49.06,28.55.HRMS(ESI)m/z calcd for[C26H29FN8O2+H]+:505.2476；found:505.2515.
叔丁基-4-(4-((5-氟-2-(异喹啉-3-基氨基)嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯的制备(化合物6b)
使用上述实施例1的方法，以2-氨基异喹啉为原料，制备得到化合物6b,为黄色固体粉末，收率86％。1H NMR(500MHz,DMSO-d6)δ9.44(s,1H),9.34(s,1H),9.05(s,1H),8.34(s,1H),8.15(d,J＝3.6Hz,1H),7.97(d,J＝8.2Hz,1H),7.62(s,1H),7.60-7.54(m,2H),7.49(d,J＝8.3Hz,1H),7.41(t,J＝7.5Hz,1H),7.01(d,J＝8.5Hz,2H),3.53(t,J＝4.9Hz,4H),3.14(t,J＝5.1Hz,4H),1.45(s,9H).13C NMR(125MHz,DMSO-d6)δ154.96,154.36,151.45,149.20,148.07,140.71,140.55,137.94,131.10,130.69,128.08,126.25,125.07,124.77,124.56,116.71,104.44,79.48,49.25,28.55.HRMS(ESI)m/z calcd for[C28H30FN7O2+H]+:516.2523；found:516.2554.
叔丁基-4-(4-((5-氟-2-(((四氢呋喃-2-基)甲基)氨基)嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯的制备(化合物6c)
使用上述实施例1的方法，以2-甲胺基四氢呋喃为原料，制备得到化合物6c,为白色固体粉末，收率35％。1H NMR(500MHz,DMSO-d6)δ8.95(s,1H),7.85(d,J＝3.9Hz,1H),7.70-7.63(m,2H),6.93-6.87(m,2H),6.61(s,1H),3.97(p,J＝6.4Hz,1H),3.79-3.71(m,1H),3.60(q,J＝7.4Hz,1H),3.46(t,J＝5.0Hz,4H),3.30-3.15(m,2H),3.03(t,J＝5.1Hz,4H),1.92-1.72(m,3H),1.56(ddt,J＝12.3,7.9,6.4Hz,1H),1.42(s,9H).13C NMR(125MHz,DMSO-d6)δ158.71,154.31,147.12,132.35,122.14,116.70,79.40,77.57,67.46,49.51,45.93,29.12,28.52,25.54.HRMS(ESI)m/z calcd for[C24H33FN6O3+H]+:473.2676；found:473.2723.
叔丁基-4-(4-((5-氟-2-((吡啶-2-基甲基)氨基)嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯的制备(化合物6d)
使用上述实施例1的方法，以2-甲胺基吡啶为原料，制备得到化合物6d,为白色固体粉末，收率13％。1H NMR(500MHz,DMSO-d6)δ8.97(s,1H),8.54-8.50(m,1H),7.87(d,J＝3.8Hz,1H),7.71(t,J＝7.8Hz,1H),7.51(s,2H),7.28(t,J＝8.4Hz,2H),7.22(t,J＝6.2Hz,1H),6.88-6.73(m,2H),4.48(d,J＝6.1Hz,2H),3.46(t,J＝4.9Hz,4H),3.01(t,J＝5.1Hz,4H),1.42(d,J＝2.0Hz,9H).13C NMR(125MHz,DMSO-d6)δ160.54,158.61,154.31,149.17,147.03,136.92,132.18,122.17,120.86,116.62,79.41,49.47,47.22,28.53.HRMS(ESI)m/z calcd for [C25H30FN7O2+H]+:480.2523；found:480.2578.
叔丁基-4-(4-((5-氟-2-((4-(甲氧甲酰基)苯基)氨基)吡啶-4-基)氨基)苯基)哌嗪-1-羧酸酯的制备(化合物6e)
使用上述实施例1的方法，以对氨基苯甲酸甲酯为原料，制备得到化合物6e,为白色固体粉末，收率55％。1H NMR(500MHz,DMSO-d6)δ9.65(s,1H),9.33(s,1H),8.10(d,J＝3.6Hz,1H),7.82-7.74(m,4H),7.54(d,J＝8.6Hz,2H),6.98(d,J＝8.6Hz,2H),3.79(s,3H),3.49(t,J＝5.1Hz,4H),3.09(t,J＝5.1Hz,4H),1.43(s,9H).13C NMR(125MHz,DMSO-d6)δ166.49,155.37,155.35,154.30,150.74,148.12,146.12,140.49,140.38,140.23,131.18,130.42,124.18,121.34,117.59,116.70,79.44,52.06,49.41,28.52.HRMS(ESI)m/z calcd for[C27H31FN6O4+H]+：523.2469；found:523.2527.
叔丁基-4-(5-(2-(2-氨基-1H-苯并[d]咪唑-1-基)-5-氟嘧啶-4-基)吡啶-2-基)哌嗪-1-羧酸酯的制备(化合物11h)
2-(4-BOC-哌嗪基)吡啶-5-硼酸频哪醇酯在SUZUKI反应条件下(H2O：THF＝1：5为溶剂，醋酸钯为催化剂，碳酸钾为碱)与2，4-二氯-5-氟嘧啶反应5h，旋干溶剂，用水打浆，抽滤得到化合物10直接投入下一步。
使用上述实施例1的方法，以2-氨基苯并咪唑为原料，制备得到化合物11h，为白色固体粉末，收率53％。1H NMR(500MHz,DMSO-d6)δ8.83-8.77(m,2H),8.13(q,J＝4.9,4.0Hz,2H),7.63(s,2H),7.21(d,J＝7.6Hz,1H),7.10(t,J＝7.6Hz,1H),6.98(t,J＝7.8Hz,1H),6.94(dd,J＝9.3,3.1Hz,1H),3.67-3.62(m,4H),3.46-3.40(m,4H),1.44(s,9H).13C NMR(125MHz,DMSO-d6)δ159.70,154.34,153.29,152.92,151.37,151.26,149.88,149.79,147.12,146.86,143.53,137.90,137.83,131.92,123.63,119.79,117.23,117.17,115.70,114.22,107.10,79.61,44.32,28.53.HRMS(ESI)m/z calcd for[C25H27FN8O2+H]+:491.2319；found:491.2361.
实施例2：1-(5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺的制备(化合物7a)
将化合物6a(1.2g，15mmol)加入60mL的DCM/TFA＝2/1中于150mL的茄形瓶中，室温搅拌4h，用NaOH调pH至碱性，用水洗3次，旋干有机层，得白色固体粉末0.8g，收率83％。1H NMR(500MHz,DMSO-d6)δ9.86(s,1H),8.37(d,J＝3.4Hz,1H),7.99(d,J＝8.0Hz,1H),7.65(s,2H),7.38(d,J＝8.8Hz,2H),7.15(d,J＝7.7Hz,1H),7.04(t,J＝7.6Hz,1H),6.98(d,J＝8.9Hz,2H),6.78(t,J＝7.7Hz,1H),3.07(dd,J＝6.3,3.7Hz,4H),2.86(dd,J＝6.4,3.7Hz,4H).13C NMR(125MHz,DMSO-d6)δ154.54,152.42,150.04,143.93,143.37,141.91,139.74,139.57,131.93,128.82,125.67,123.19,119.20,115.97,115.26,115.23,50.04,46.06.HRMS(ESI)m/z calcd for[C21H21FN8+H]+:405.1951；found:405.1975.
5-氟-N2-(异喹啉-3-基)-N4-(4-(哌嗪-1-基)苯基)嘧啶-2,4-二胺的制备(化合物7b)
使用上述实施例2的方法，以化合物6b为原料，制备得到化合物7b，白色固体粉末，收率83％。1H NMR(500MHz,DMSO-d6)δ9.56(s,1H),9.35-9.31(m,1H),9.07(s,1H),8.34(s,1H),8.17(d,J＝3.8Hz,1H),7.96(d,J＝8.1Hz,1H),7.60(t,J＝7.6Hz,1H),7.55(d,J＝8.6Hz,2H),7.47(d,J＝8.3Hz,1H),7.41(t,J＝7.5Hz,1H),7.01-6.95(m,2H),3.29(s,1H),3.11(t,J＝4.9Hz,4H),2.92(dd,J＝6.0,3.6Hz,4H).13C NMR(125MHz,DMSO-d6)δ154.98,151.45,150.81,149.18,142.20,140.73,140.57,140.23,137.95,130.73,128.05,126.28,125.05,124.82,124.77,116.08,104.43,49.06,45.86.HRMS(ESI)m/z calcd for[C23H22FN7+H]+:416.1999；found:416.2017.
5-氟-N4-(4-哌嗪-1-基)苯基)-N2-((四氢呋喃-2-基)甲基)嘧啶-2,4-二胺的制备(化合物7c)
使用上述实施例2的方法，以化合物6c为原料，制备得到化合物7c，为白色固体粉末，收率为75％。1H NMR(500MHz,Chloroform-d)δ7.76(d,J＝3.2Hz,1H),7.47(d,J＝8.4Hz, 2H),6.91(s,1H),6.87(d,J＝8.5Hz,2H),5.33(t,J＝6.0Hz,1H),4.04(dt,J＝11.3,5.1Hz,1H),3.85(q,J＝7.3Hz,1H),3.73(q,J＝7.3Hz,1H),3.53(dt,J＝14.2,5.0Hz,1H),3.31(dt,J＝13.2,6.2Hz,1H),3.10–3.05(m,4H),3.00(t,J＝4.8Hz,4H),2.16(s,1H),1.94(dt,J＝16.6,6.4Hz,1H),1.86(hept,J＝6.0Hz,2H),1.58(dq,J＝15.2,7.8Hz,1H).13C NMR(125MHz,Chloroform-d)δ158.56,158.53,150.34,150.26,148.35,139.84,139.68,130.76,121.92,116.55,77.92,67.97,50.89,46.10,45.89,28.74,25.78.HRMS(ESI)m/z calcd for[C19H25FN6O+H]+:373.2152；found:373.2188.
5-氟-N4-(4-(哌嗪-1-基)苯基-N2-(吡啶-2-基甲基)嘧啶-2,4-二胺的制备(化合物7d)
使用上述实施例2的方法，以化合物6d为原料，制备得到化合物7d，为白色固体粉末，收率为75％。1H NMR(500MHz,DMSO-d6)δ9.01(s,1H),8.52(d,J＝4.8Hz,1H),7.88(d,J＝3.9Hz,1H),7.69(t,J＝7.8Hz,1H),7.65-7.40(m,2H),7.40-7.26(m,2H),7.20(t,J＝6.2Hz,1H),6.79(s,2H),4.52(d,J＝6.1Hz,2H),3.03(t,J＝4.8Hz,4H),2.91(t,J＝4.8Hz,4H).13C NMR(125MHz,DMSO-d6)δ160.52,158.65,149.13,147.58,141.47,139.54,136.92,131.73,122.17,120.94,116.04,49.62,47.25,45.48.HRMS(ESI)m/z calcd for[C20H22FN7+H]+:380.1999；found:380.2026.
甲基-4-((5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)氨基苯甲酸酯的制备(化合物7e)
使用上述实施例2的方法，以化合物6e为原料，制备得到化合物7e，为白色固体粉末，收率为72％。1H NMR(400MHz,DMSO-d6)δ9.63(s,1H),9.30(d,J＝1.7Hz,1H),8.10(d,J＝3.7Hz,1H),7.83-7.72(m,4H),7.55-7.47(m,2H),6.99-6.90(m,2H),3.80(s,3H),3.10-3.03(m,4H),2.92-2.85(m,4H).13C NMR(101MHz,DMSO-d6)δ166.51,155.40,155.37,150.78,150.67,148.88,146.14,140.34,140.15,130.49,130.42,124.24,121.34,117.59,115.96,52.10,50.11,45.93.HRMS(ESI)m/z calcd for[C22H23FN6O2+H]+:423.1945；found:423.1968.
1-(5-氟-4-(6-(哌嗪-1-基)吡啶-3-基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺的制备(化合物12h)
使用上述实施例2的方法，以化合物11h为原料，制备得到化合物12h，为白色固体粉末，收率为92％。1H NMR(400MHz,DMSO-d6)δ8.80(dd,J＝15.8,3.0Hz,2H),8.19-8.07(m,2H),7.65(s,2H),7.22(d,J＝7.7Hz,1H),7.10(t,J＝7.5Hz,1H),6.98(t,J＝7.7Hz,1H),6.92(d,J＝9.2Hz,1H),3.58(t,J＝5.0Hz,4H),2.78(d,J＝10.0Hz,4H).13C NMR(101MHz,DMSO-d6)δ159.91,154.35,153.14,152.89,151.30,150.57,149.96,149.86,146.84,146.57,143.50,137.58,131.89,123.56,119.73,116.60,115.65,114.25,106.72,45.76,45.68.HRMS(ESI)m/z calcd for[C20H19FN8+H]+:391.1795；found:391.1826.
实施例3：1-(5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺的制备(化合物8a)
将化合物7a(0.2g，0.5mmol)和乙酸(0.03g，0.5mmol)加入装有10mLDCE的茄形瓶，60℃搅拌20min。再将异戊醛(0.22g，2.5mmol)和三乙酰氧基硼氢化钠(0.3g，1.5mmol)加入反应液中，60℃搅拌13h。用二氯甲烷和水萃取反应液，水层用再用二氯甲烷洗涤3次直至水层在254nm无荧光，合并有机层，旋干，用200-300目硅胶柱层析进一步分离纯化(DCM:MeOH:Et3N＝20：1：1)得白色固体粉末0.1g，收率50％。1H NMR(500MHz,DMSO-d6)δ9.87(s,1H),8.38(d,J＝3.4Hz,1H),7.99(d,J＝8.0Hz,1H),7.64(s,2H),7.38(d,J＝8.5Hz,2H),7.14(d,J＝7.7Hz,1H),7.03(t,J＝7.5Hz,1H),7.01-6.96(m,2H),6.81-6.74(m,1H),3.15(t,J＝5.0Hz,4H),2.53-2.47(m,5H),2.32(t,J＝7.6Hz,2H),1.66-1.54(m,J＝6.8Hz,1H),1.35(q,J＝7.2Hz,2H),0.89(d,J＝6.7Hz,6H).13C NMR(125MHz,DMSO-d6)δ154.51,149.39,143.36,139.77,139.60,131.91,128.89,125.63,123.19,119.20,115.98,115.23,56.55,53.30,48.90,35.85,26.32,23.11.HRMS(ESI)m/z calcd for[C26H31FN8+H]+:475.2734； found:475.2762.
5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-异喹啉-3-基)嘧啶-2,4-二胺的制备(化合物8b)
使用上述实施例3的方法，以化合物7b为原料，制备得到化合物8b，为白色固体粉末，收率为72％。1H NMR(500MHz,DMSO-d6)δ9.47(s,1H),9.32(s,1H),9.06(s,1H),8.33(s,1H),8.15(d,J＝3.7Hz,1H),7.96(d,J＝8.1Hz,1H),7.56(dd,J＝7.9,5.6Hz,3H),7.48(d,J＝8.3Hz,1H),7.41(t,J＝7.5Hz,1H),6.98(d,J＝8.5Hz,2H),3.17(t,J＝4.9Hz,4H),2.55(t,J＝4.9Hz,4H),2.36(t,J＝7.6Hz,2H),1.63(dp,J＝13.3,6.7Hz,1H),1.39(q,J＝7.4Hz,2H),0.91(d,J＝6.6Hz,6H).13C NMR(125MHz,DMSO-d6)δ151.40,149.19,137.94,130.67,128.04,126.30,125.07,124.74,115.96,104.43,56.56,53.45,49.05,39.54,35.87,26.36,23.13.HRMS(ESI)m/z calcd for[C28H32FN7+H]+:486.2781；found:486.2826.
5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-((四氢呋喃-2-基)甲基)嘧啶-2，4-二胺的制备(化合物8c)
使用上述实施例3的方法，以化合物7c为原料，制备得到化合物8c，为白色固体粉末，收率为36％。1H NMR(500MHz,Chloroform-d)δ7.81(d,J＝3.4Hz,1H),7.51(d,J＝8.4Hz,2H),6.93(d,J＝8.4Hz,2H),6.60(s,1H),5.17(d,J＝6.2Hz,1H),4.08(p,J＝6.4Hz,1H),3.89(q,J＝7.3Hz,1H),3.77(q,J＝7.3Hz,1H),3.57(dt,J＝10.4,4.9Hz,1H),3.35(dt,J＝13.2,6.1Hz,1H),3.20(t,J＝4.9Hz,4H),2.63(t,J＝4.9Hz,4H),2.42(t,J＝7.9Hz,2H),1.98(dt,J＝12.1,6.6Hz,1H),1.92(d,J＝6.7Hz,1H),1.89(d,J＝7.1Hz,1H),1.64(dt,J＝13.9,7.0Hz,2H),1.45(q,J＝7.5Hz,2H),0.94(d,J＝6.6Hz,6H).13C NMR(125MHz,Chloroform-d)δ158.50,150.27,147.91,130.56,121.75,116.54,68.06,57.05,53.37,49.64,45.87,35.91,28.73,26.73,25.84,22.76.HRMS(ESI)m/z calcd for[C24H35FN6O+H]+:443.2935；found:443.2966.5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-(吡啶-2-基甲基)嘧啶-2,4-二胺的制备(化合物8d)
使用上述实施例3的方法，以化合物7d为原料，制备得到化合物8d，为白色固体粉末，收率为39％。1H NMR(400MHz,Chloroform-d)δ8.59(dd,J＝5.3,2.9Hz,1H),7.88-7.81(m,1H),7.63(ddq,J＝9.6,5.4,1.8Hz,1H),7.46(dd,J＝9.1,3.0Hz,2H),7.32(d,J＝7.9Hz,1H),7.22–7.14(m,1H),6.94-6.85(m,2H),6.62(dd,J＝11.6,4.0Hz,1H),5.91-5.78(m,1H),3.20(dt,J＝6.2,3.2Hz,4H),2.68–2.61(m,4H),2.44(td,J＝7.7,3.2Hz,2H),1.63(dp,J＝13.3,6.7Hz,1H),1.45(dd,J＝9.5,6.1Hz,2H),1.27(d,J＝4.2Hz,2H),0.94(dd,J＝6.6,1.3Hz,6H).13C NMR(101MHz,Chloroform-d)δ154.96,149.07,147.77,139.84,139.68,136.54,130.43,126.07,121.59,121.46,120.27,116.57,116.08,56.94,53.19,49.44,35.62,29.69,29.43,26.72,23.35,22.73.HRMS(ESI)m/z calcd for[C25H32FN7+H]+:450.2781；found:450.2808.
甲基-4-((5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)氨基)嘧啶-2-基)氨基)苯甲酸酯的制备(化合物8e)
使用上述实施例3的方法，以化合物7e为原料，制备得到化合物8e，为白色固体粉末，收率为57％。1H NMR(500MHz,Chloroform-d)δ7.98–7.91(m,3H),7.63-7.57(m,2H),7.48-7.43(m,2H),7.42(s,1H),6.99-6.94(m,2H),6.80(d,J＝2.9Hz,1H),3.90(s,3H),3.24(t,J＝5.0Hz,4H),2.65(t,J＝4.9Hz,4H),2.44(dd,J＝9.3,6.6Hz,2H),1.70-1.58(m,J＝6.8Hz,1H),1.46(q,J＝7.4Hz,2H),0.95(d,J＝6.6Hz,6H).13C NMR(125MHz,Chloroform-d)δ166.96,148.81,144.36,139.73,139.58,130.76,129.25,123.30,122.84,117.34,116.37,57.04,53.32,51.81,49.41,35.91,26.72,22.77.HRMS(ESI)m/z calcd for[C27H33FN6O2+H]+:493.2727；found:493.2754.
1-(5-氟-4-(6-(4-异戊基哌嗪-1-基)吡啶-3-基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺的制备(化合物13h)
使用上述实施例3的方法，以化合物12h为原料，制备得到化合物13h，为黄色固体粉末，收率为54％。1H NMR(400MHz,Chloroform-d)δ9.08(d,J＝2.5Hz,1H),8.52(d,J＝3.4Hz,1H),8.36(d,J＝8.0Hz,1H),8.30(dd,J＝9.1,2.5Hz,1H),7.44(d,J＝7.8Hz,1H),7.25(t,J＝7.6Hz,1H),7.14(t,J＝7.7Hz,1H),6.86(s,2H),6.78(d,J＝9.2Hz,1H),3.82-3.75(m,4H),2.60(t,J＝5.1Hz,4H),2.48-2.39(m,2H),1.72-1.58(m,J＝6.6Hz,1H),1.46(q,J＝7.1Hz,2H),0.95(d,J＝6.6Hz,6H).13C NMR(101MHz,Chloroform-d)δ159.84,153.77,153.07,150.49,150.35,150.25,145.50,145.23,142.52,137.64,131.93,123.73,120.49,116.14,114.49,106.10,57.01,53.05,44.68,35.85,26.67,22.76.HRMS(ESI)m/z calcd for[C25H29FN8+H]+:461.2577；found:461.2616.
实施例4：1-(5-氟-4-((4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺的制备(化合物6f)
以对氟硝基苯、2f-g作为起始原料，在二甲亚砜为溶剂，回流的条件下反应5h发生亲核取代反应得到中间体3f-g。然后采用铁粉还原的方法对硝基进行还原得到对位为氨基的中间体4f-g。中间体4g-f和2，4-二氯-5-氟嘧啶在甲醇中发生亲核取代反应得到化合物5f-g。前三步合成比较简单，且反应完成后直接析出固体，因此直接抽滤得到产物未经打谱确证直接投于下一步。将化合物5f(2g，6mmol)，2-氨基苯并咪唑(1.655g，12mmol)，Pd2(dba)3(0.285g，0.28mmol)，碳酸铯(3.038g，9.3mmol)，Xantphos(0.180g，0.31mmol)加入120mL耐压瓶中，加入50mL经过4A分子筛干燥过的二氧六环，加入到含搅拌子的100mL封管中，加入30mL二氧六环(4A分子筛干燥)，用氮气置换瓶中空气，拧上盖子，置于超声溶解。放入120℃油浴中反应12h。置于室温冷却后，旋干反应液，用二氯甲烷/水进行萃取，萃取3次，用无水Na2SO4干燥有机层4h。抽滤，旋干有机溶剂，得黄绿色固体，用200-300目硅胶柱层析对其分离纯化(DCM:MeOH＝40:1),白色固体粉末，收率46％。1H NMR(500MHz,DMSO-d6)δ9.86(s,1H),8.39(dd,J＝3.5,1.6Hz,1H),7.97(d,J＝8.0Hz,1H),7.60(s,2H),7.41-7.36(m,2H),7.14(d,J＝7.8Hz,1H),7.07-6.98(m,3H),6.77(t,J＝7.7Hz,1H),3.21-3.15(m,4H),2.49(s,4H),2.25(d,J＝1.6Hz,3H).13C NMR(125MHz,DMSO-d6)δ154.49,149.34,143.36,141.66,139.81,139.61,131.91,128.95,125.66,123.19,119.20,116.07,115.25,115.21,55.02,48.72,46.20.HRMS(ESI)m/z calcd for[C22H23FN8+H]+:419.2108；found:419.2134.
1-(4-((4-(4-乙基哌嗪-1-基)苯基)氨基)-5-氟嘧啶-2-基)-1H-苯并[d]咪唑-2-胺的制备(化合物6g)
使用上述实施例4的方法，以化合物5g为原料，制备得到化合物6g，为白色固体粉末，收率54％。1H NMR(400MHz,DMSO-d6)δ9.79(s,1H),8.36(d,J＝3.5Hz,1H),7.99(d,J＝8.0Hz,1H),7.55(s,2H),7.39(d,J＝8.5Hz,2H),7.15(d,J＝7.7Hz,1H),7.04(t,J＝7.6Hz,1H),6.99(d,J＝8.6Hz,2H),6.78(t,J＝7.7Hz,1H),3.20-3.15(m,4H),2.56(t,J＝5.0Hz,4H),2.42(q,J＝7.2Hz,2H),1.06(t,J＝7.2Hz,3H).13C NMR(101MHz,DMSO-d6)δ154.50,149.35,143.36,139.80,139.60,131.90,128.95,125.66,123.20,119.20,116.03,115.23,52.73,52.09,48.79,12.39.HRMS(ESI)m/z calcd for[C23H25FN8+H]+:433.2264；found:433.2289.
N2-(1H-苯并[d]咪唑-2-基)-N4-(4-(4-乙基哌嗪-1-基)苯基)-5-氟嘧啶-2,4-二胺(化合物6g-1)
使用上述实施例4的方法，以化合物5g为原料，制备得到化合物6g-1，为白色固体粉末，收率30％。1H NMR(500MHz,DMSO-d6)δ11.44(s,1H),9.57(s,1H),8.17(s,1H),7.52(d,J＝8.3Hz,3H),7.38-7.34(m,1H),7.02(s,2H),6.97(d,J＝8.5Hz,3H),3.40(s,4H),2.38(q,J＝7.3Hz,4H),1.04(t,J＝7.3Hz,5H).13C NMR(125MHz,DMSO-d6)δ154.19,151.19,151.10,149.60,148.62,142.59,140.62,129.79,115.89,52.93,52.14,48.66,12.46.HRMS(ESI)m/z calcd for[C23H25FN8+H]+:433.2264；found:433.2289.
实施例5:N,N-二甲基-6-硝基-1H-苯并[d]咪唑-2-胺(化合物16)
将二氯亚甲基二甲基氯化铵(19.44g，120mmol)溶于300mL的DCM中于500mL茄型瓶中，加入4-硝基邻苯二胺(15.30g，100mmol)，将茄形瓶放于室温搅拌，茄形瓶上放置回流冷凝管。将三乙胺(30.30g，300mmol)加到恒压滴液漏斗中，用气球封住恒压滴液漏斗的瓶口，将其插入回流冷凝管上，各个衔接口都用封口膜封好。缓慢向茄形瓶中滴加三乙胺，20min滴加完毕。升温至回流继续反应4h。旋干反应液，得黄白色固体。用甲醇重结晶得黄白色固体18.1g。收率88％。1H NMR(500MHz,DMSO-d6)δ13.91(s,1H),8.16(dd,J＝8.7,2.0Hz,1H),8.11(d,J＝1.9Hz,1H),7.55(d,J＝8.7Hz,1H),3.28(s,6H).HRMS(ESI)m/z calcd for[C9H10N4O2+H]+:207.0877；found:207.0877[M+H]+.
实施例6：N2，N2-二甲基-1H-苯并[d]咪唑-2，6-二胺(化合物17)
将N，N-二甲基-5-硝基-1H-苯并[d]咪唑-2-胺(2.06g，10mmol)溶于40mL的MeOH中于100mL的氢化仪专用反应容器中，加入含量10％的钯碳(含水约55％)0.20g。在0.4MPa氢气环境下室温搅拌反应18h。抽滤除去钯碳，旋干滤液，得灰固体。用甲醇重结晶得灰色固体1.45g。收率82％。1H NMR(500MHz,DMSO-d6)δ7.05(d,J＝8.4Hz,1H),6.69(s,1H),6.50(d,J＝8.4Hz,1H),3.18(s,6H).HRMS(ESI)m/z calcd for[C9H12N4+H]+:177.1135；found:117.1107[M+H]+.
实施例7：4-(4-硝基苯基)吗啉(化合物19a)
将对氟硝基苯(7.05g，50mmol)，吗啉(5.22g，60mmol)加入250mL茄型瓶中，加入碳酸钾(10.35g，75mmol)于90℃下反应5h。之后放于室温冷却，冷却到室温后，用乙酸乙酯萃取，有机层再水洗2次，再用饱和NaCl溶液洗涤有机层1次，无水Na2SO4干燥有机层并放置4h。抽滤，旋干有机溶剂，得黄色固体。用乙酸乙酯重结晶得黄色结晶19a共9.5g，收率91％。1H NMR(500MHz,DMSO-d6)δ8.08(d,J＝9.5Hz,2H),7.04(d,J＝9.5Hz,2H),3.76–3.70(m,4H),3.44–3.38(m,4H).HRMS(ESI)m/z calcd for[C10H12N2O3+H]+:209.0921；found:209.0921[M+H]+.
1-(4-硝基苯基)吡咯(化合物19b)
使用上述实施例7的方法，以化合物18b为原料，制备得到化合物19b，收率90％，黄色固体。1H NMR(500MHz,DMSO-d6)δ8.04(d,J＝9.4Hz,2H),6.60(d,J＝9.4Hz,2H),3.37(t,J＝6.7Hz,4H),2.04–1.94(m,4H).HRMS(ESI)m/z calcd for[C10H12N2O2+H]+:193.0972；found:193.0972[M+H]+.
N，N-二乙基-4-硝基苯胺(化合物19c)
使用上述实施例7的方法，以化合物18c为原料，制备得到化合物19c，收率88％，黄色固体。1H NMR(500MHz,DMSO-d6)δ8.03(d,J＝9.5Hz,2H),6.75(d,J＝9.5Hz,2H),3.48(q,J＝7.1Hz,4H),1.14(t,J＝7.1Hz,6H).HRMS(ESI)m/z calcd for[C10H14N2O2+H]+:195.1128；found:195.1131[M+H]+.
4-硝基-N，N-二丙基苯胺(化合物19d)
使用上述实施例7的方法，以化合物18d为原料，制备得到化合物19d，收率87％，黄色固体。1H NMR(500MHz,DMSO-d6)δ8.01(d,J＝9.4Hz,2H),6.75(d,J＝9.5Hz,2H),3.47–3.34(m,4H),1.66–1.46(m,4H),0.91(t,J＝7.3Hz,6H).HRMS(ESI)m/z calcd for[C12H18N2O2+H]+:223.1441；found:223.1445[M+H]+.
1-(4-硝基苯基)哌啶(化合物19e)
使用上述实施例7的方法，以化合物18e为原料，制备得到化合物19e，收率90％，黄色固体。1H NMR(500MHz,DMSO-d6)δ8.02(d,J＝9.5Hz,2H),6.98(d,J＝9.6Hz,2H),3.54–3.44(m,4H),1.69–1.60(m,2H),1.57(m,4H).HRMS(ESI)m/z calcd for[C11H14N2O2+H]+:207.1128；found:207.1133[M+H]+.
叔丁基-4-(4-硝基苯基)哌嗪-1-羧酸酯(化合物19f)
使用上述实施例7的方法，以化合物18f为原料，制备得到化合物19f，收率92％，黄色固体。1H NMR(500MHz,DMSO-d6)δ8.07(d,J＝9.4Hz,2H),7.01(d,J＝9.5Hz,2H),3.48(m,8H),1.43(s,9H).HRMS(ESI)m/z calcd for[C15H21N3O4+Na]+:330.1426；found:330.1424[M+Na]+.
叔丁基-4-(5-硝基吡啶-2-基)哌嗪-1-羧酸酯(化合物19g)
使用上述实施例7的方法，以化合物18g为原料，制备得到化合物19g，收率90％，黄色固体。1H NMR(500MHz,DMSO-d6)δ8.97(d,J＝2.7Hz,1H),8.25(dd,J＝9.6,2.8Hz,1H),6.95(t,J＝11.9Hz,1H),3.91–3.66(m,4H),3.61–3.38(m,4H),1.43(s,9H).HRMS(ESI)m/z calcd for[C14H20N4O4+Na]+:331.1377；found:331.1382[M+Na]+.
叔丁基-4-(5-硝基吡啶-2-基)哌嗪-1-羧酸酯(化合物19h)
使用上述实施例7的方法，以化合物18h为原料，制备得到化合物19h，收率80％，黄色固体。1H NMR(500MHz,DMSO-d6)δ8.24(d,J＝2.9Hz,1H),8.16(d,J＝9.2Hz,1H),7.46(dd,J＝9.2,3.0Hz,1H),3.51(dd,J＝14.0,5.6Hz,8H),1.43(s,9H).HRMS(ESI)m/z calcd for[C14H20N4O4+Na]+:309.1557；found:309.1571[M+H]+.
实施例8：4-吗啉苯胺(化合物20a)
将4-吗啉硝基苯(6.24g，30mmol)，铁粉(8.40g，150mmol)，氯化铵(7.95g，150mmol)加入250mL茄型瓶中，加入乙醇/水＝5/1的溶剂120mL回流反应5h。之后放于室温冷却，冷却到室温后，用装有硅藻土的漏斗抽滤，滤液中加入少量亚硫酸钠以防产物被空气中氧气氧化。低温旋蒸出溶剂中的乙醇，用二氯甲烷/甲醇＝10/1的溶剂和水萃取，再用二氯甲烷/甲醇＝10/1的溶剂洗涤水溶液3次，合并有机层，无水Na2SO4干燥有机层并放置4h。抽滤出去硫酸钠，旋干有机溶剂，得灰色油状物。共4.5g，收率84％。因为此产物在空气中易氧化，故直接投下一步。20b-20h采用和20a相同的方法制备，同样未经结构确证直接投下一步。
实施例9：2-氯-5-氟-N-(4-吗啉苯基)嘧啶-4-胺(化合物22a)
将4-吗啉苯胺(3.56g，20mmol)，2,4-二氯-5-氟嘧啶(4.65g，28mmol)，加入250mL茄型瓶中，加入甲醇/水＝1/1的溶剂120mL，60℃反应12h。之后放于室温过夜，瓶中有白色固体析出，抽滤得灰白色固体，用甲醇重结晶得灰白色固体5.0g，收率81％。1H NMR(500MHz,DMSO-d6)δ9.83(s,1H),8.24(d,J＝3.5Hz,1H),7.50(d,J＝9.0Hz,2H),6.97(d,J＝9.1Hz,2H),3.89–3.53(m,4H),3.22–2.92(m,4H).HRMS(ESI)m/z calcd for[C14H14ClFN4O+H]+:309.0913；found:309.0923[M+H]+.
2-氯-5-氟-N-(4-(吡咯-1-基)苯基)嘧啶-4-胺(化合物22b)
使用上述实施例9的方法，以化合物20b为原料，制备得到化合物22b，收率77％，白色固体。1H NMR(500MHz,DMSO-d6)δ9.71(s,1H),8.17(d,J＝3.6Hz,1H),7.40(d,J＝8.9Hz,2H),6.53(d,J＝9.0Hz,2H),3.21(t,J＝6.5Hz,4H),2.06–1.78(m,4H).HRMS(ESI)m/z calcd for[C14H14ClFN4+H]+:293.0964；found:293.0976[M+H]+.
N1-(2-氯-5-氟嘧啶-4-基)-N4，N4-二乙基苯-1，4-二胺(化合物22c)
使用上述实施例9的方法，以化合物20c为原料，制备得到化合物22c，收率81％，白色固体。1H NMR(500MHz,DMSO-d6)δ9.70(s,1H),8.19(d,J＝3.6Hz,1H),7.38(d,J＝9.1Hz,2H),6.67(d,J＝9.1Hz,2H),3.34(t,J＝7.0Hz,4H),1.09(t,J＝7.0Hz,6H).HRMS(ESI)m/z calcd for[C14H16ClFN4+H]+:295.1120；found:295.1131[M+H]+.
N1-(2-氯-5-氟嘧啶-4-基)-N4，N4-二丙基苯-1，4-二胺(化合物22d)
使用上述实施例9的方法，以化合物20d为原料，制备得到化合物22d，收率75％，黄色油状物。1H NMR(500MHz,DMSO-d6)δ9.70(s,1H),8.18(d,J＝3.6Hz,1H),7.37(d,J＝9.0Hz,2H),6.64(d,J＝9.1Hz,2H),3.29–3.16(m,4H),1.61–1.46(m,4H),0.89(t,J＝7.4Hz,6H).HRMS(ESI)m/z calcd for[C16H20ClFN4+H]+:323.1433；found:323.1454[M+H]+.
2-氯-5-氟-N-(4-哌啶-1-基)苯基)嘧啶-4-胺(化合物22e)
使用上述实施例9的方法，以化合物20e为原料，制备得到化合物22e，收率83％，白色固体。1H NMR(500MHz,DMSO-d6)δ9.80(s,1H),8.22(d,J＝3.5Hz,1H),7.46(d,J＝8.9Hz,2H),6.93(d,J＝9.0Hz,2H),3.18–3.05(m,4H),1.66–1.56(m,4H),1.56–1.47(m,2H).HRMS(ESI)m/z calcd for[C15H16ClFN4+H]+:307.1120；found:307.1135[M+H]+.
叔丁基-1-(4-((2-氯-5-氟嘧啶-4-基)氨基)苯基)哌嗪-4-羧酸酯(化合物22f)
使用上述实施例9的方法，以化合物20f为原料，制备得到化合物22f，收率85％，白色固体。1H NMR(500MHz,DMSO-d6)δ9.83(s,1H),8.23(d,J＝3.5Hz,1H),7.51(d,J＝9.1Hz,2H),6.97(d,J＝9.1Hz,2H),3.52–3.41(m,4H),3.14–3.03(m,4H),1.42(s,9H).HRMS(ESI)m/z calcd for[C19H23ClFN5O2+H]+:408.1597；found:408.1601[M+H]+.
叔丁基-4-(5-((2-氯-5-氟嘧啶-4-基)氨基)吡啶-2-基)哌嗪-1-羧酸酯(化合物22g)
使用上述实施例9的方法，以化合物20g为原料，制备得到化合物22g，收率75％，白色固体。1H NMR(500MHz,DMSO-d6)δ9.90(s,1H),8.34(d,J＝2.7Hz,1H),8.26(d,J＝3.4Hz,1H),7.78(dd,J＝9.1,2.7Hz,1H),6.91(d,J＝9.1Hz,1H),3.46(m,8H),1.43(s,9H).HRMS(ESI)m/z calcd for[C18H22ClFN6O2+H]+:409.1550；found:409.1557[M+H]+.
叔丁基-4-(6-((2-氯-5-氟嘧啶-4-基)氨基)吡啶-3-基)哌嗪-1-羧酸酯(化合物22h)
使用上述实施例9的方法，以化合物20h为原料，制备得到化合物22h，收率72％，白色固体。1H NMR(400MHz,DMSO-d6)δ10.25(s,1H),8.31(d,J＝3.3Hz,1H),8.11(d,J＝2.9Hz,1H),7.78(d,J＝9.0Hz,1H),7.49(dd,J＝9.1,3.0Hz,1H),3.57–3.41(m,4H),3.28–3.01(m,4H),1.42(s,9H).HRMS(ESI)m/z calcd for[C18H22ClFN6O2+H]+:409.1550；found:409.1581[M+H]+.
N1-(2-氯-5-氟嘧啶-4-基)-N4，N4-二甲基苯-1，4-二胺(化合物22i)
使用上述实施例9的方法,以化合物20i为原料，制备得到化合物22i，收率79％，白色固体。1H NMR(500MHz,DMSO-d6)δ9.75(s,1H),8.20(d,J＝3.6Hz,1H),7.42(d,J＝9.0Hz,2H),6.75(d,J＝9.1Hz,2H),2.89(s,6H).HRMS(ESI)m/z calcd for[C12H12ClFN4+H]+:267.0807；found:267.0817[M+H]+.
实施例10：N6-(5-氟-4-((4-吗啉苯基)氨基)嘧啶-2-基)-N2,N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23a)
将化合物22a(2.53g，8mmol)，化合物9(1,4g，8mmol)，Pd2(dba)3(0.18g，0.02mmol)，Xanphos(0.46g，0.08mmol)，碳酸铯(4.89g，15mmol)加入120mL耐压瓶中，加入50mL二氧六环，加入搅拌子，用氮气置换瓶中空气，封好口后超声使原料尽可能溶解于溶剂中。放入120℃油浴中反应12h。之后放于室温冷却，冷却到室温后，旋干反应液，用二氯甲烷/水进行萃取，再用二氯甲烷对水层洗涤2次，无水Na2SO4干燥有机层并放置4h。抽滤，旋干有机溶剂，得黑色油状物，用硅胶柱层析对其分离纯化(DCM:MeOH:Et3N＝200:1:1),得到白色固体2.34g，收率65％。1H NMR(500MHz,DMSO-d6)δ11.14(s,1H),9.07(s,1H),8.83(s,1H),7.98(d,J＝3.7Hz,1H),7.67(d,J＝8.9Hz,2H),7.57(s,1H),7.13(d,J＝8.1Hz,1H),7.01(d,J＝8.4Hz,1H),6.89(d,J＝8.9Hz,2H),3.83–3.67(m,4H),3.11–3.04(m,4H),3.03(s,6H).13C NMR(125MHz,DMSO-d6)δ157.17,156.76,156.74,150.10,150.01,147.44,141.70,140.36,140.21,139.75,134.03,131.69,122.63,115.80,113.18,66.62,49.43,38.56.HRMS(ESI)m/z calcd for[C23H25FN8O+H]+:449.2208；found:449.2211[M+H]+.
N6-(5-氟-4-((4-(吡咯-1-基)苯基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23b)
使用上述实施例10的方法，以化合物22b为原料，制备得到化合物23b，收率62％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ11.05(s,1H),8.92(s,1H),8.74(s,1H),7.94(d,J＝3.4Hz,1H),7.59(s,1H),7.57(d,J＝8.4Hz,2H),7.16(d,J＝8.4Hz,1H),7.00(d,J＝8.3Hz,1H),6.51(d,J＝8.4Hz,2H),3.21(m,4H),3.03(s,6H),1.94(m 4H).13C NMR(125MHz, DMSO-d6)δ170.77,157.31,156.76,150.29,150.20,144.83,141.76,139.89,139.81,139.75,133.96,127.96,123.37,112.83,111.87,47.53,38.02,24.80.HRMS(ESI)m/z calcd for[C23H25FN8+H]+:433.2259；found:443.2261[M+H]+.
N6-(4-((4-(二乙基氨基)苯基)氨基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23c)
使用上述实施例10的方法，以化合物22c为原料，制备得到化合物23c，收率65％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ13.10(s,1H),9.16(s,1H),9.05(s,1H),8.00(s,1H),7.83(s,1H),7.51(m,3H),7.20(d,J＝8.6Hz,1H),6.66(s,2H),3.32(d,J＝5.3Hz,4H),3.22(s,6H),1.08(t,J＝6.9Hz,6H).13C NMR(125MHz,DMSO-d6)δ155.99,151.44,150.54,150.44,142.19,140.25,139.46,137.34,131.32,125.66,123.88,114.96,112.27,111.05,102.42,44.30,39.29,12.91.HRMS(ESI)m/z calcd for[C23H27FN8+H]+:435.2415；found:435.2420[M+H]+.
N6-(4-((4-(二丙基氨基)苯基)氨基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23d)
使用上述实施例10的方法，以化合物22d为原料，制备得到化合物23d，收率61％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ11.22(d,J＝29.3Hz,1H),8.93(s,1H),8.73(d,J＝32.5Hz,1H),7.92(s,1H),7.55(m,3H),7.12(m,1H),6.98(m,1H),6.58(d,J＝6.9Hz,2H),3.28–3.14(m,4H),3.02(s,6H),1.63–1.42(m,4H),0.88(t,J＝7.3Hz,6H).(互变异构)13C NMR(125MHz,DMSO-d6)δ157.70,157.11,156.80,150.25,150.17,144.81,144.40,141.72,141.70,139.84,139.74,139.57,139.41,134.78,133.25,130.23,127.78,123.41,114.35,113.79,112.06,111.79,108.15,107.60,101.83,52.72,38.64,20.54,11.73.HRMS(ESI)m/z calcd for[C25H31FN8+H]+:463.2728；found:463.2732[M+H]+.
N6-(5-氟-4-((4-(哌啶-1-基)苯基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23e)
使用上述实施例10的方法，以化合物22e为原料，制备得到化合物23e，收率70％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ9.03(s,1H),8.80(s,1H),7.98(d,J＝3.0Hz,1H),7.64(d,J＝8.4Hz,2H),7.58(s,1H),7.12(d,J＝8.3Hz,1H),7.02(d,J＝8.3Hz,1H),6.87(d,J＝8.4Hz,2H),3.12–3.05(m,4H),3.04(s,6H),1.63(s,4H),1.52(d,J＝5.0Hz,2H).13C NMR(125MHz,DMSO-d6)δ157.35,156.77,150.10,150.02,148.19,141.69,140.32,140.17,139.74,133.88,131.12,122.62,116.51,113.15,113.09,50.62,38.55,25.78,24.25.HRMS(ESI)m/z calcd for[C24H27FN8+H]+:447.2415；found:447.2417[M+H]+.
叔丁基-4-(4-((2-((2-(二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-羧酸酯(化合物23f)
使用上述实施例10的方法，以化合物22f为原料，制备得到化合物23f，收率69％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ9.08(s,1H),8.80(s,1H),7.99(d,J＝3.2Hz,1H),7.68(d,J＝8.5Hz,2H),7.60(s,1H),7.16(d,J＝8.4Hz,1H),7.04(d,J＝8.4Hz,1H),6.90(d,J＝8.5Hz,2H),3.48(m,4H),3.05(m,10H),1.44(s,9H).13C NMR(125MHz,DMSO-d6)δ156.79,156.74,150.10,150.01,147.29,141.73,140.29,139.78,134.16,132.04,122.60,116.79,113.19,111.39,104.43,79.36,49.55,38.61,28.55.HRMS(ESI)m/z calcd for[C28H34FN9O2+H]+:548.2892；found:548.2898[M+H]+.
叔丁基-4-(5-((2-((2-(二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-4-基)氨基)吡啶-2-基)哌嗪-1-羧酸酯(化合物23g)
使用上述实施例10的方法，以化合物22g为原料，制备得到化合物23g，收率49％，浅黄色固体。1H NMR(500MHz,DMSO-d6)δ11.19(s,1H),9.17(s,1H),8.90(s,1H),8.53(s,1H),7.99(d,J＝10.7Hz,2H),7.58(s,1H),7.14(d,J＝8.3Hz,1H),7.00(d,J＝8.3Hz,1H),6.84(d,J＝9.0Hz,1H),3.02(s,6H),1.43(s,9H).13C NMR(125MHz,DMSO-d6)δ157.07, 156.60,156.58,155.74,154.41,150.27,150.18,141.81,141.16,140.43,140.28,139.85,136.89,135.94,132.38,127.51,112.95,107.44,79.47,55.39,45.51,38.57,28.55.HRMS(ESI)m/z calcd for[C27H33FN10O2+H]+:549.2845；found:549.2851[M+H]+.
叔丁基-4-(6-((2-((2-(二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-4-基)氨基)吡啶-2-基)哌嗪-1-羧酸酯(化合物23h)
使用上述实施例10的方法，以化合物22h为原料，制备得到化合物23h，收率45％，灰白色固体。1H NMR(400MHz,DMSO-d6)δ9.30(s,1H),9.03(s,1H),8.26–7.99(m,3H),7.64(s,1H),7.35(dd,J＝9.0,2.5Hz,1H),7.15(d,J＝8.3Hz,1H),7.07(d,J＝8.4Hz,1H),3.49(t,4H),3.10(t,4H),3.06(s,6H),1.43(s,9H).13C NMR(101MHz,DMSO-d6)δ156.76,156.52,149.34,149.23,145.16,143.69,141.64,141.42,141.23,139.19,136.46,134.10,125.96,116.05,113.38,111.56,104.59,79.51,52.45,49.08,38.60,28.52.HRMS(ESI)m/z calcd for[C27H33FN10O2+H]+:549.2845；found:549.2849[M+H]+.
N6-(4-((4-(二甲基氨基)苯基)氨基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物23i)
使用上述实施例10的方法，以化合物22i为原料，制备得到化合物23i，收率55％，白色固体。1H NMR(500MHz,DMSO-d6)δ9.02(s,1H),8.83(s,1H),7.98(d,J＝3.2Hz,1H),7.64(s,1H),7.62(d,J＝3.5Hz,2H),7.19(d,J＝8.4Hz,1H),7.04(d,J＝8.3Hz,1H),6.71(d,J＝8.5Hz,2H),3.04(s,6H),2.86(s,6H).13C NMR(125MHz,DMSO-d6)δ157.20,156.79,150.28,150.20,147.40,141.78,140.05,139.90,139.83,134.12,129.14,123.14,113.07,111.32,104.43,40.99,38.56.HRMS(ESI)m/z calcd for[C21H23FN8+H]+:407.2102；found:407.2113[M+H]+.
实施例11：N6-(5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物24a)
将23f(8.2g，15mmol)加入45mL的DCM/TAF＝2/1中于100mL的茄形瓶中，室温搅拌4h。，旋干反应液，加三乙胺和甲醇反复旋干几次，得黑灰色油状物。用二氯甲烷和水萃取，水层用再用二氯甲烷洗涤5次直至水层在254nm无荧光，合并有机层，旋干，得黑灰色油状物，用硅胶柱层析进一步分离纯化(DCM:MeOH:Et3N＝40:1:1)得白色固体3.50g，收率52％。1H NMR(500MHz,DMSO-d6)δ10.95(s,1H),9.04(s,1H),8.80(s,1H),7.97(d,J＝3.3Hz,1H),7.64(d,J＝8.5Hz,2H),7.56(s,1H),7.11(d,J＝8.0Hz,1H),7.00(d,J＝8.2Hz,1H),6.87(d,J＝8.5Hz,2H),4.34(s,1H),3.03(s,6H),3.02–2.97(m,4H),2.86(m,4H).13C NMR(125MHz,DMSO-d6)δ157.40,156.82,156.80,150.10,150.01,148.05,141.68,140.35,140.19,139.73,133.88,131.43,122.63,115.94,113.41,50.25,45.98,38.55.HRMS(ESI)m/z calcd for[C23H26FN9+H]+:448.2368found:448.2372[M+H]+。
N6-(5-氟-4-((6-(哌嗪-1-基)吡啶-3-基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物24b)
使用上述实施例11的方法，以化合物23g为原料，制备得到化合物24b，收率47％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ11.03(d,J＝44.5Hz,1H),9.10(s,1H),8.82(d,J＝30.5Hz,1H),8.47(d,J＝16.6Hz,1H),7.97(t,J＝11.2Hz,2H),7.55(d,J＝34.2Hz,1H),7.12(s,1H),7.01(s,1H),6.77(s,1H),3.38–3.31(m,4H),3.02(s,6H),2.89–2.64(m,4H).(互变异构).13C NMR(125MHz,DMSO-d6)δ162.74,156.59,150.28,141.81,141.34,140.31,134.65,132.47,126.83,114.46,113.54,108.16,107.59,106.86,101.55,46.85,45.93,38.55.HRMS(ESI)m/z calcd for[C22H25FN10+H]+:449.2320；found:449.2323[M+H]+.
N6-(5-氟-4-((5-(哌嗪-1-基)吡啶-2-基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物24c)
使用上述实施例11的方法，以化合物23h为原料，制备得到化合物24c，收率49％，灰白色固体。1H NMR(400MHz,DMSO-d6)δ9.27(s,1H),8.99(s,1H),8.06(dd,J＝13.3,6.3 Hz,3H),7.60(s,1H),7.30(d,J＝8.3Hz,1H),7.10(d,J＝8.2Hz,1H),7.05(d,J＝8.3Hz,1H),3.59(m,4H),3.36(s,1H),3.04(m,10H).13C NMR(101MHz,DMSO-d6)δ157.50,156.79,149.33,149.23,144.56,144.37,141.58,141.40,141.32,141.22,139.13,135.66,133.67,125.17,116.16,113.23,49.78,45.74,38.54.HRMS(ESI)m/z calcd for[C22H25FN10+H]+:449.2320；found:449.2321[M+H]+.
实施例12：N6-(5-氟-4-((4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25a)
将装有10mL的ACN的25mL的茄形瓶放于-10℃冷肼中，将24a(0.45g，1mmol)和三乙胺(0.15g，1.5mmol)加入茄形瓶中，将碘甲烷(0.17g，1.2mmol)溶于2mL ACN降温到-10℃，缓慢将其滴加到茄形瓶中，-10℃搅拌10min。用二氯甲烷和水萃取反应液，水层用再用二氯甲烷洗涤3次直至水层在254nm无荧光，合并有机层，旋干，得无色油状物，用硅胶柱层析进一步分离纯化(DCM:MeOH:Et3N＝80:1:1)得白色固体0.20g，收率44％。1H NMR(500MHz,DMSO-d6)δ9.21(s,1H),9.18(s,1H),8.04(d,J＝3.5Hz,1H),7.76(s,1H),7.68(d,J＝8.7Hz,2H),7.44(d,J＝8.4Hz,1H),7.19(d,J＝8.6Hz,1H),6.97(d,J＝8.8Hz,2H),3.41(s,4H),3.25(s,4H),3.19(s,6H),2.75(s,3H).13C NMR(125MHz,DMSO-d6)δ152.45,150.29,146.31,142.05,140.15,136.73,132.48,132.15,123.07,116.67,114.80,111.20,102.78,52.82,46.67,42.75,39.15.HRMS(ESI)m/z calcd for[C24H28FN9+H]+:462.2524found:462.2535[M+H]+.
实施例13：1-(4-(4-((2-((2-二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-基)乙酮(化合物25b)
将24a(0.45g，1mmol)，三乙胺(0.15g，1.5mmol)和乙酸酐(0.18g，1.5mmol)加入装有10mL ACN的茄形瓶，室温搅拌2h。用二氯甲烷和水萃取反应液，水层用再用二氯甲烷洗涤3次直至水层在254nm无荧光，合并有机层，旋干，得无色油状物，用硅胶柱层析进一步分离纯化(DCM:MeOH:Et3N＝80:1:1)得白色固体0.35g，收率71％。1H NMR(500MHz,DMSO-d6)δ11.33(s,1H),9.09(s,1H),8.87(s,1H),7.99(d,J＝3.5Hz,1H),7.68(d,J＝8.7Hz,2H),7.60(s,1H),7.16(d,J＝8.2Hz,1H),7.03(d,J＝8.4Hz,1H),6.92(d,J＝8.8Hz,2H),3.58(m,4H),3.11–3.00(m,10H),2.05(s,3H).13C NMR(125MHz,DMSO-d6)δ168.69,156.66,150.08,150.00,147.20,141.72,140.38,140.25,139.77,134.32,131.99,122.61,116.71,113.26,49.44,46.01,38.61,21.67.HRMS(ESI)m/z calcd for[C25H28FN9O+H]+:490.2474found:490.2470[M+H]+.
实施例14：N6-(5-氟-4-((4-(4-异丙基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25c)
将24a(0.45g，1mmol)，碳酸钾(0.41g，3mmol)和2-碘丙烷(0.51g，3mmol)加入装有10mL ACN的茄形瓶，65℃搅拌16h。用二氯甲烷和水萃取反应液，水层用再用二氯甲烷洗涤3次直至水层在254nm无荧光，合并有机层，旋干，得无色油状物，用硅胶柱层析进一步分离纯化(DCM:MeOH:Et3N＝80:1:1)得白色固体0.26g，收率53％。1H NMR(500MHz,DMSO-d6)δ9.05(s,1H),8.83(s,1H),7.98(s,1H),7.65(d,J＝7.4Hz,2H),7.58(s,1H),7.14(d,J＝7.2Hz,1H),7.02(d,J＝7.6Hz,1H),6.88(d,J＝7.3Hz,2H),3.10(s,4H),3.04(s,6H),2.76(s,1H),2.65(s,4H),1.04(s,6H).13C NMR(125MHz,DMSO-d6)δ157.08,156.73,150.10,150.02,147.41,141.71,140.35,140.31,140.18,139.75,134.11,131.47,122.66,116.05,113.10,54.39,49.34,48.54,38.57,18.53.HRMS(ESI)m/z calcd for[C26H32FN9+H]+:490.2837found:490.2841[M+H]+.
2-(4-(4-((2-((2-(二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-基)乙-1-醇(化合物25d)
使用上述实施例13的方法，以化合物24a，2-溴乙醇为原料，制备得到化合物25d，收率70％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ9.07(s,1H),8.84(s,1H),7.98(d,J＝ 3.7Hz,1H),7.65(d,J＝8.9Hz,2H),7.58(s,1H),7.13(d,J＝9.9Hz,1H),7.02(d,J＝8.4Hz,1H),6.88(d,J＝9.0Hz,2H),4.51(s,1H),3.56(t,J＝6.2Hz,2H),3.14–3.05(m,4H),3.03(s,6H),2.59(s,4H),2.47(t,J＝6.1Hz,2H).13C NMR(125MHz,DMSO-d6)δ156.98,156.72,156.57,150.10,147.38,141.71,140.33,140.17,139.79,134.26,131.48,122.71,116.05,113.28,111.42,104.52,60.48,58.70,53.47,48.95,38.62.HRMS(ESI)m/z calcd for[C25H30FN9O+H]+:492.2630found:492.2636[M+H]+.
N6-(4-((4-(4-(乙基磺酰基)哌嗪-1-基)苯基)氨基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25e)
使用上述实施例13的方法，以化合物24a，乙烷磺酰氯为原料，制备得到化合物25e，收率68％，灰白色固体。1H NMR(500MHz,DMSO-d6)δ11.13(s,1H),9.08(s,1H),8.82(s,1H),7.99(d,J＝3.7Hz,1H),7.69(d,J＝8.9Hz,2H),7.57(s,1H),7.13(d,J＝7.7Hz,1H),7.02(d,J＝8.4Hz,1H),6.92(d,J＝8.9Hz,2H),3.35–3.31(m,4H),3.22–3.14(m,4H),3.14–3.08(m,2H),3.04(s,6H),1.25(t,J＝7.3Hz,3H).13C NMR(125MHz,DMSO-d6)δ157.10,156.74,156.69,150.02,149.63,146.87,146.54,141.78,140.37,140.17,139.41,133.82,132.44,132.32,132.25,122.58,116.93,112.77,49.62,45.67,42.90,38.56,7.95.HRMS(ESI)m/z calcd for[C25H30FN9O2S+H]+:540.2300found:540.2310[M+H]+.
N6-(5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25f)
将N6-(5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)-N 2，N 2-二甲基-1H-苯并[d]咪唑-2,6-二胺(0.45g，1mmol)和乙酸(0.06g，1mmol)加入装有10mL DCE的茄形瓶，60℃搅拌20min。再将三甲基乙醛(0.26g，3mmol)和三乙酸乙酯硼氢化钠(0.64g，3mmol)加入反应液中，60℃搅拌16h。用二氯甲烷和水萃取反应液，水层用再用二氯甲烷洗涤3次直至水层在254nm无荧光，合并有机层，旋干，得无色油状物，用硅胶柱层析进一步分离纯化(DCM:MeOH:Et3N＝80:1:1)得白色固体0.316g，收率61％。1H NMR(500MHz,DMSO-d6)δ11.01(s,1H),9.04(s,1H),8.81(s,1H),7.97(d,J＝3.3Hz,1H),7.65(d,J＝8.4Hz,2H),7.57(s,1H),7.12(d,J＝8.3Hz,1H),7.01(d,J＝8.3Hz,1H),6.87(d,J＝8.5Hz,2H),3.08(m,4H),3.03(s,6H),2.51(m,4H),2.34(t,J＝7.4Hz,2H),1.61(m,1H),1.36(m,2H),0.90(d,J＝6.6Hz,6H).13C NMR(125MHz,DMSO-d6)δ157.23,156.78,156.76,150.10,150.01,147.46,141.69,140.33,140.19,139.74,134.05,134.01,131.42,122.63,116.00,113.26,56.47,53.31,49.22,38.56,35.76,26.31,23.09.HRMS(ESI)m/z calcd for[C28H36FN9+H]+:518.3150found:518.3155[M+H]+.
N6-(4-((4-(4-苄基哌嗪-1-基)苯基)氨基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物25g)
使用上述实施例12的方法，以化合物24a，溴苄为原料，制备得到化合物25g，收率70％，白色固体。1H NMR(500MHz,DMSO-d6)δ11.12(s,1H),9.07(s,1H),8.85(s,1H),7.98(d,J＝3.4Hz,1H),7.65(d,J＝8.6Hz,2H),7.58(s,1H),7.30(m,5H),7.13(d,J＝8.2Hz,1H),7.01(d,J＝8.3Hz,1H),6.87(d,J＝8.7Hz,2H),3.08(m,4H),3.52(s,2H),3.01(s,6H),2.51(m,4H).13C NMR(125MHz,DMSO-d6)δ157.14,156.82,150.08,149.99,147.45,141.68,140.31,140.18,139.73,138.54,133.97,131.45,129.39,128.67,127.45,122.62,116.07,113.13,113.07,62.53,53.05,49.28,38.54.HRMS(ESI)m/z calcd for[C30H32FN9+H]+:538.2837found:538.2842[M+H]+.
1-(4-(4-((2-((2-(二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-基)-3,3-二甲基丙-1酮(化合物25h)
使用上述实施例12的方法，以化合物24a，3,3-二甲基丁酰氯为原料，制备得到化合物25h，收率73％，白色固体。1H NMR(400MHz,DMSO-d6)δ9.12(s,1H),8.93(s,1H),8.00(d,J＝3.7Hz,1H),7.69(t,J＝8.8Hz,2H),7.64(s,1H),7.21(dd,J＝8.5,1.4Hz,1H),7.07(d,J ＝8.4Hz,1H),6.92(d,J＝9.0Hz,2H),3.67(t,J＝16.6Hz,4H),3.05(d,J＝10.6Hz,10H),2.28(s,2H),1.01(s,9H).13C NMR(101MHz,DMSO-d6)δ169.86,156.60,156.11,150.10,149.98,147.21,141.99,140.35,139.54,134.66,131.96,122.62,116.66,113.47,111.31,104.18,49.58,44.09,38.66,31.49,30.25.HRMS(ESI)m/z calcd for[C29H36FN9O+H]+:546.3100 found:546.3106[M+H]+.
N-(叔丁基)-4-(4-((2-((2-(二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-4-基)氨基)苯基)哌嗪-1-甲酰胺(化合物25i)
使用上述实施例12的方法，以化合物24a，叔丁基异氰酸酯为原料，制备得到化合物25i，收率68％，白色固体。1H NMR(500MHz,DMSO-d6)δ11.09(s,1H),9.08(s,1H),8.84(s,1H),7.99(s,1H),7.68(d,J＝8.1Hz,2H),7.58(s,1H),7.13(d,J＝6.7Hz,1H),7.02(d,J＝7.6Hz,1H),6.91(d,J＝8.1Hz,2H),5.92(s,1H),3.42(m,4H),3.04(m,10H),1.28(s,9H).13C NMR(125MHz,DMSO-d6)δ157.54,157.40,156.81,150.13,150.05,147.44,141.73,140.39,140.23,139.78,133.89,131.87,122.65,116.54,113.08,50.44,49.52,44.14,38.57,29.70.HRMS(ESI)m/z calcd for[C28H35FN10O+H]+:547.3052found:547.3060[M+H]+.
N6-(2-氯-5-氟嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物26)
使用上述实施例9的方法，以化合物12为原料，制备得到化合物26，收率88％，白色固体。1H NMR(500MHz,DMSO-d6)δ13.27(s,1H),10.13(s,1H),8.31(s,1H),7.85(s,1H),7.50(d,J＝8.5Hz,1H),7.37(d,J＝8.5Hz,1H),3.24(s,6H).HRMS(ESI)m/z calcd for[C13H12ClFN6+H]+:307.0869；found:307.0875[M+H]+.
叔丁基-6-((叔丁氧基碳酸酯)(2-氯-5-氟嘧啶-4-基)氨基)-2-(二甲基氨基)-1H-苯并[d]咪唑-1-羧酸酯(化合物27)
将N6-(2-氯-5-氟嘧啶-4-基)-N 2，N 2-二甲基-1H-苯并[d]咪唑-2,6-二胺(3.06g，10mmol)加入100mL的DCM中于250mL的茄形瓶中，加入(Boc)2O(21.81g，100mmol)、三乙胺(5.05g，50mmol)和DMAP(0.49g，4mmol)室温反应24h。，旋干反应液，得橙红色油状物。用二氯甲烷和水萃取，有机层用饱和柠檬酸的水溶液洗涤3次，再用饱和碳酸氢钠水溶液洗涤1次，最后用饱和食盐水洗涤1次。无水Na2SO4干燥有机层并放置4h。抽滤，旋干有机溶剂，得橙红色油状物，放置于油泵中抽干2h，得橙红色固体4.72g，收率93％。1H NMR(500MHz,DMSO-d6)δ8.96(s,1H),7.53(s,1H),7.33(d,J＝8.3Hz,1H),7.09(d,J＝8.3Hz,1H),2.99(s,6H),1.58(s,9H),1.42(s,9H)。HRMS(ESI)m/z calcd for[C23H28ClFN6O4+H]+:507.1917；found:507.1923[M+H]+.
叔丁基-6-((叔丁氧基碳酸酯)(5-氟-2-((4-(吡咯-1-基)苯基)氨基)嘧啶-4-基)氨基)-2-(二甲基氨基)-1H-苯并[d]咪唑-1-羧酸酯(化合物28a)
将6-((叔丁氧基羰基)(2-氯-5-氟嘧啶-4-基)氨基)-2-(二甲基氨基)-1H-苯并[d]咪唑-1-羧酸丁酯(2.53g，5mmol)，4-(吡咯烷-1-基)苯胺(0.81g，5mmol)，Pd2(dba)3(0.18g，0.02mmol)，Xanphos(0.46g，0.08mmol)，碳酸铯(4.89g，15mmol)加入120mL耐压瓶中，加入50mL二氧六环，加入搅拌子，用氮气置换瓶中空气，封好口后超声使原料尽可能溶解于溶剂中。放入120℃油浴中反应12h。之后放于室温冷却，冷却到室温后，旋干反应液，用二氯甲烷/水进行萃取，再用二氯甲烷对水层洗涤2次，无水Na2SO4干燥有机层并放置4h。抽滤，旋干有机溶剂，得黑色油状物，用硅胶柱层析对其分离纯化(DCM:MeOH:Et3N＝200:1:1),得到黑色油状物2.34g，收率74％。结构未确证直接投下一步。28b-28f采用和28a相同的方法制备，同样未经结构确证直接投下一步。
N6-(5-氟-2-((4-(吡咯-1-基)苯基)氨基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29a)
使用上述实施例11的方法，以化合物28a为原料，制备得到化合物29a，收率50％，灰白固体。1H NMR(500MHz,DMSO-d6)δ11.19(d,1H),9.01(d,,1H),8.67(s,1H),7.93(d,J＝3.1Hz,1H),7.47(m 3H),7.25(m 1H),7.09(m 1H),6.40(m,2H),3.15(m,4H),3.05(s,6H), 1.93(m,4H).13C NMR(125MHz,DMSO-d6)δ157.56,156.61,150.63,150.53,144.43,143.62,141.60,141.10,140.22,139.66,134.69,132.46,131.50,130.57,130.49,121.26,121.16,114.38,113.97,111.95,109.99,108.20,104.60,48.08,38.52,25.32.HRMS(ESI)m/z calcd for[C23H25FN8+H]+:433.2259；found:443.2258[M+H]+.
N6-(5-氟-2-((4-(哌嗪-1-基)苯基)氨基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29b)
使用上述实施例11的方法，以化合物28b为原料，制备得到化合物29b，收率45％，灰白固体。1H NMR(500MHz,DMSO-d6)δ11.19(s,1H),9.08(s,1H),8.84(s,1H),7.97(d,J＝3.8Hz,1H),7.50(m,3H),7.21(s,1H),7.12(s,1H),6.74(s,2H),3.33(s,1H),3.06(s,6H),2.96–2.86(m,4H),2.85–2.71(m,4H).13C NMR(125MHz,DMSO-d6)δ157.76,156.37,150.76,150.68,146.73,141.75,140.35,140.34,140.21,140.19,139.80,133.81,120.17,116.31,114.35,50.92,46.19,38.51.HRMS(ESI)m/z calcd for[C23H26FN9+H]+:448.2368found:448.2370[M+H]+.
N6-(5-氟-2-((6-(哌嗪-1-基)吡啶-3-基)氨基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29c)
使用上述实施例11的方法，以化合物28c为原料，制备得到化合物29c，收率65％，浅紫色固体。1H NMR(500MHz,DMSO-d6)δ11.19(s,1H),9.07(s,1H),8.82(s,1H),8.27(d,J＝2.0Hz,1H),7.96(d,J＝3.7Hz,1H),7.94–7.85(m,1H),7.47(s,1H),7.23(d,J＝7.8Hz,1H),7.10(d,J＝8.3Hz,1H),6.65(d,J＝9.1Hz,1H),3.34–3.22(m,4H),3.05(s,6H),2.95–2.64(m,4H).13C NMR(125MHz,DMSO-d6)δ157.77,156.53,155.47,150.90,150.81,142.05,140.18,140.09,140.03,139.34,131.39,130.37,129.43,115.27,106.99,46.82,45.68,38.53.HRMS(ESI)m/z calcd for[C22H25FN10+H]+:449.2320found:449.2321[M+H]+.
N6-(2-((4-(二甲基氨基)苯基)氨基)-5-氟嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29d)
使用上述实施例11的方法，以化合物28d为原料，制备得到化合物29d，收率65％，浅紫色固体。1H NMR(500MHz,DMSO-d6)δ11.37(s,1H),9.04(s,1H),8.73(s,1H),7.95(d,1H),7.51(s,1H),7.46(d,J＝8.0Hz,2H),7.26(d,J＝8.2Hz,1H),7.11(d,J＝8.3Hz,1H),6.61(d,J＝8.1Hz,2H),3.06(s,6H),2.80(s,6H).13C NMR(125MHz,DMSO-d6)δ157.37,156.53,150.67,150.59,146.15,141.69,140.35,140.19,139.74,131.81,131.67,120.76,115.36,113.48,111.16,107.34,41.28,38.58.HRMS(ESI)m/z calcd for[C20H22FN9+H]+:449.2320found:449.2319[M+H]+.
N6-(5-氟-2-((5-(哌嗪-1-基)吡啶-2-基)氨基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29e)
使用上述实施例11的方法，以化合物28e为原料，制备得到化合物29e，收率68％，浅紫色固体。1H NMR(500MHz,DMSO-d6)δ11.37(s,1H),9.04(s,1H),8.73(s,1H),7.95(d,1H),7.51(s,1H),7.46(d,J＝8.0Hz,2H),7.26(d,J＝8.2Hz,1H),7.11(d,J＝8.3Hz,1H),6.61(d,J＝8.1Hz,2H),3.06(s,6H),2.80(s,6H).13C NMR(125MHz,DMSO-d6)δ157.37,156.53,150.67,150.59,146.15,141.69,140.35,140.19,139.74,131.81,131.67,120.76,115.36,113.48,111.16,107.34,41.28,38.58.HRMS(ESI)m/z calcd for[C20H22FN9+H]+:449.2320found:449.2319[M+H]+.
N6，N6-(5-氟嘧啶-2,4-二基)双(N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺)(化合物29h)
使用上述实施例11的方法，以化合物28f为原料，制备得到化合物29h，收率50％，白色固体。1H NMR(500MHz,DMSO-d6)δ8.11(d,J＝4.1Hz,1H),7.69(s,1H),7.66(s,1H),7.58(d,J＝8.2Hz,1H),7.34(m,2H),7.27(d,J＝8.6Hz,1H),3.18(s,6H),3.16(s,6H).13C NMR(125MHz,DMSO-d6)δ152.64,152.55,151.81,151.53,151.42,140.72,138.74,133.43, 132.38,130.94,130.77,128.38,127.52,119.24,117.67,111.44,111.20,107.04,105.32,39.50,39.48.HRMS(ESI)m/z calcd for[C22H23FN10+H]+:447.2164found:447.2169[M+H]+.
1-(4-(4-((4-((2-(二甲基氨基)-1H-苯并[d]咪唑-6-基)氨基)-5-氟嘧啶-2-基)氨基)苯基)哌嗪-1-基)丙-1-酮(化合物29f)
使用上述实施例13的方法，以化合物29b为原料，制备得到化合物29f,收率60％，白色固体。1H NMR(400MHz,DMSO-d6)δ9.19(s,1H),8.92(s,1H),8.00(d,J＝3.7Hz,1H),7.58(s,1H),7.55(d,J＝8.8Hz,2H),7.35(d,J＝8.3Hz,1H),7.18(d,J＝8.4Hz,1H),6.80(d,J＝8.9Hz,2H),3.77–3.33(m,4H),3.10(s,6H),3.06–2.83(m,4H),2.04(s,3H).13C NMR(101MHz,DMSO-d6)δ168.66,156.28,150.64,150.53,145.77,142.05,140.50,140.32,139.61,134.48,132.17,120.17,117.08,115.88,111.24,107.13,50.35,49.92,38.69,21.66.HRMS(ESI)m/z calcd for[C25H28FN9O+H]+:490.2474found:490.2475[M+H]+.
N6-(5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺(化合物29g)
使用上述实施例13的方法，以化合物29b为原料，制备得到化合物29g，收率65％，白色固体。1H NMR(500MHz,DMSO-d6)δ11.51(s,1H),9.19(s,1H),8.92(s,1H),8.00(d,J＝3.0Hz,1H),7.58(s,1H),7.55(d,J＝8.3Hz,2H),7.37(d,J＝8.3Hz,1H),7.18(d,J＝8.4Hz,1H),6.82(d,J＝8.4Hz,2H),3.18(d,J＝68.6Hz,14H),2.89(s,2H),1.66–1.47(m,3H),0.91(d,J＝5.8Hz,6H).13C NMR(125MHz,DMSO-d6)δ156.28,155.93,150.61,150.52,144.91,141.83,140.49,140.34,139.88,134.61,132.41,120.26,116.81,116.05,111.24,106.91,55.09,51.77,47.54,38.79,33.00,26.26,22.77.HRMS(ESI)m/z calcd for[C28H36FN9+H]+:518.3150found:518.3152[M+H]+.
实施例15:对所合成的化合物进行HIPK2的抑制活性实验、对所合成的化合物进行大鼠肾成纤维细胞(NRK-49F)的增殖抑制实验
一、对所合成的化合物进行HIPK2的抑制活性实验
1.实验材料
HIPK2激酶(Carna)；MBP、ATP(Promega)、ADP-GloTM检测试剂盒、Cytation 5。
2.实验方法
(1)在384孔板中加入1μL化合物溶液(或5％DMSO)，2μL HIPK2溶液，2μL ATP和底物的混合溶液，室温培育60min；
(2)加入5μL ADP-GloTM试剂，室温培育40min；
(3)加入10μL激酶消除试剂，室温培育30min；
(4)记录荧光强度。
二、对所合成的化合物进行大鼠肾成纤维细胞(NRK-49F)的增殖抑制实验
1.实验材料
(1)细胞株：大鼠肾成纤维细胞(NRK-49F)
(2)试剂与仪器：96孔板(Corning)；胎牛血清(Gibco),F12培养基，DMEM培养基(BI)；生物安全柜，二氧化碳培养箱(ESCO)；Cytation 5多功能成像仪(Bio-Tek)。
2.实验方法
(1)细胞培养：NRK-49F细胞采用常规培养。实验时均用处于对数生长期且活细胞比例高于90％的细胞进行实验。
(2)细胞生长检测：消化细胞，将细胞稀释成浓度为3-5×104个/mL的细胞悬液，向96孔板中间60孔中每孔加入100μL细胞悬液(每孔3-5×103个细胞)，四周36孔中每孔加入100μL PBS；将96孔板置于37℃，5％CO2的培养箱中培养至贴壁(进入对数生长期)；吸取中间60孔中培养基后，向每个孔中加入100μL含不同浓度化合物的培养基，每组3复孔；将96孔板置于培养箱中培养48h；弃去含化合物的培养基以及PBS，96孔板每孔加入100μL含10％CCK-8的基础培基，将96孔板在培养箱内孵育1-4h后，用酶标仪测定在450nm处的OD值。
3.实验结果
表1目标化合物对HIPK2激酶抑制活性、NRK-49F细胞增殖抑制活性






说明：a化合物最大浓度为10μM，括号内数据为最大浓度下的抑制率，例如>10(2.7％)即在浓度为10μM
下的抑制率；b化合物最大浓度为100μM，括号内数据为最大浓度下的抑制率；c专利CN106565673披露的活性Wnt抑制剂d专利CN105017159披露的5-氟-2,4-二取代嘧啶衍生物。
以上数据结果显示，多数目标化合物化合物的HIPK2的抑制活性实验结果与肾成纤维细胞(NRK-49F)的增殖抑制实验结果趋势一致，且活性强于已上市抗纤维化药物吡非尼酮，并优于对比文献的化合物，具有明显的活性优势。
实施例16：目标化合物对纤维化的影响
1.实验材料
(1)细胞株：大鼠肾成纤维细胞(NRK-49F)
(2)抗体：P65、p-P65、α-SMA、Fn1、Collagen1、GAPDH均购自CST公司。
(3)药品及试剂：超敏ECL化学发光试剂购自Abbkine公司；羊抗兔二抗，显影液、定影液，RIPA裂解液，BSA，BCA蛋白浓度检测试剂盒，预染蛋白marker，均购自南京恩晶生物科技有限公司。
(4)仪器及耗材：6孔细胞培养板购自corning公司；超净工作台(ESCO)；二氧化碳培养箱(ESCO)；荧光倒置生物显微镜(optec)；台式高速离心机(SCILOGEX)；微型垂直电泳槽(Bio-RAD)；转移电泳槽(Bio-RAD)；电泳仪(君意有限公司)；脱色摇床(华利达有限公司)；Cytation 5酶标仪(Bio-tek公司)。
2.实验方法
(1)蛋白样本制备
取对数生长期NRK-49F的细胞进行实验。细胞经消化、计数、稀释成2×105个/mL的细胞悬液，以每孔2mL接种于6孔板中，置于37℃，5％CO2培养箱中培养24小时；更换新鲜培养基，同时设立空白对照组和实验组(加TGF-β刺激)，加入不同浓度的化合物7a，作用24h后，倒掉培养液，每个孔细胞加2mL 4℃预冷的PBS洗涤三次。之后培养皿置于冰上。加入100μL的RIPA裂解液(含磷酸酶抑制剂和PMSF)裂解30min，需要经常来回摇动以使细胞充分裂解。裂解完后，用刮棒将细胞快速刮到孔的一侧，然后用移液枪将孔中液体移至1.5mL离心管中(整个操作均需在冰上进行)。最后在4℃温度下12000rpm离心5min，将离心后的上清液转移到0.5mL的离心管中，用蛋白定量测定试剂盒测定上清液中蛋白的浓度，将上清液放于-20℃中保存，电泳上样前要将样品蛋白变性。
(2)SDS-PAGE电泳
将干净的玻璃板对齐后放入夹中，垂直卡在架子上准备灌胶。吸取5mL的分离胶沿玻璃缓慢灌入，胶面升到绿带中间线的高度时即可停止，然后胶上加一层无水乙醇进行液封。胶凝后就可倒去胶上层水并用吸水纸将水吸干。用浓缩胶将剩余空间灌满后将梳子插入浓缩胶中。浓缩胶凝固后，将梳子竖直向上轻轻将拔出。用水冲洗浓缩胶后，将其放入电泳槽中。上样30-50μg蛋白，在加入足够的电泳缓冲液后上样。先用70V电泳20min,等到蛋白聚集到分离胶界限的时候将电压换成130V，电泳至溴酚兰刚跑出时终止电泳，进行转膜。
(3)转膜
用撬胶板小心地将玻璃板撬开，小心地将胶平铺在三张滤纸上，去除气泡。将浸泡好了的PVDF膜贴在所需目标条带的地方，小心去除气泡后不能再移动。再叠放三张滤纸在PVDF膜上，注意胶两侧上下方的滤纸不能直接接触，以免形成短路损坏转膜设备。确认无误后，盖上海绵垫合上转膜板。按照PVDF膜在正极胶在负极的方式放置转模板，在转膜槽中加入转膜缓冲液，并将转膜槽置于冰水混合物中，80V条件下转膜45min。转膜完成后取出PVDF膜，用TBS浸润后放入封闭液中，4℃过夜封闭。封闭完后，将膜从封闭液中取出，在用滤纸吸除残留的封闭液后，将膜蛋白面朝下放于已经稀释到特定浓度的一抗溶液的液面上，掀动膜的四角以赶出残留气泡后；室温下孵育2h。一抗孵育完毕后将PVDF膜用TBST在水平摇床上10min/次清洗，重复3次。用和孵育一抗相同的方法给PVDF膜孵育二抗(1:5000稀释)，室温条件下孵育1h。随后再次将PVDF膜用TBST在水平摇床上10min/次清洗，重复3次，之后进行化学发光显影。
(4)化学发光显影
将ECL发光液的A液和B液按1：1得比例混合，室温条件下静置1min。将PVDF膜蛋白质面向上贴在平板上。1min后涂上ECL发光液进行显影及定影。以GAPDH为内参，用Adobe Photoshop CS5软件分析目标条带的净光密度值。
(5)统计学处理
扫描仪扫描显影条带，所得结果用ImageJ软件扫描得其灰度值，用Graphpad软件作图。
3.实验结果
得到的结果如图2所示。其中(A)、(C)用Western blot检测经不同浓度的化合物7a处理后NRK-49F细胞α-SMA和Fn 1的表达；(B)、(D)对Western blot数据的定量分析。####表示与阴性对照组(0/-)相比p<0.0001,###表示与阴性对照组(0/-)相比p<0.001,***表示与阳性对照组(0/+)相比p<0.001,****表示与阳性对照组(0/+)相比p<0.0001。
4.实验结论
由图2可知，加入10ng/mL的TGF-β后，细胞内α-SMA、Fn1的水平明显上升；通过7a处理后，细胞内α-SMA、Fn1的水平明显下降。因此7a可以通过抑制HIPK2而抑制其下游纤维化信号通路相关分子的表达。
实施例17：化合物对炎症相关通路的影响
1.实验材料
抗体：P65、p-P65、GAPDH均购自CST公司。其他同实施例16。
2.实验方法(同实施例16)
3.实验结果
得到的结果如图3所示。其中(A)用Western blot检测经不同浓度的化合物7a处理后NRK-49F细胞P-P65和P65的表达；(B)、(C)对左中Western blot数据的定量分析。***表示与阳性对照组(0/+)相比p<0.001,****表示与阳性对照组(0/+)相比p<0.0001。
如图所示，给予不同浓度的7a治疗，低浓度时，P65有所上升；高浓度时，P65相较于低浓度时显著下降，与对照组相差不大。但P65的磷酸化水平有明显的下降。
4.实验结论
7a可以通过抑制HIPK2而抑制其下游炎症信号通路相关分子的表达。
实施例18：化合物对单侧输尿管结扎小鼠肾间质纤维化模型的影响
单侧输尿管结扎模型(UUO模型)是经典的肾间质纤维化模型，其模拟了从肾急性损伤，逐渐进展到肾间质纤维化整个动态过程。一般认为输尿管结扎后3天为损伤的急性期，主要以细胞水肿、死亡及炎性细胞浸润为主；第3-7天为亚急性期，主要以肾间质水肿、肾小管扩张、炎性细胞浸润为主，开始出现细胞外基质沉积；7天以后肾间质水肿、肾小管扩张逐渐消失，取而代之的是肾间质萎缩、间质细胞外基质沉积明显增加、纤维化恶化，进入慢性期。本研究目的：观察本发明的目标化合物(HIPK2-inhibitor)对UUO14天模型的疗效，以明确目标化合物对肾间质纤维化的治疗效果。
1.实验材料
1.1药品：目标化合物(HIPK2-inhibitor)；性状：黄色固体。提供单位：中南大学药学院。双蒸水作为溶剂溶解目标化合物(调节pH＝4.5左右)。
1.2实验动物：C57-BL6小鼠，雄性，体重22-25g，购于湖南斯莱克景达实验动物有限公司，实验动物生产许可证号：SCXK(湘)2015-0017。
2.实验方法
2.1试验设计
SPF级雄性小鼠分为假手术组(Sham)、单侧输尿管结扎组(UUO)、UUO+目标化合物(75mg/kg)组，麻醉后剃去背部毛发，络合碘消毒，除假手术组外所有小鼠取背部正中线左上方剪开皮肤小口，暴露出后侧腹膜，随后剪开腹膜，挤出左侧肾脏及输尿管，在输尿管近肾门处及输尿管下方予以3-0缝线结扎，于两处结扎处中间剪断输尿管后将肾脏推回腹腔，并缝合腹膜及背部皮肤。假手术组小鼠仅予以开腹和关腹处理。模型构建后第2天开始UUO+目标化合物(75mg/kg)组予以目标化合物以75mg/kg/6mL灌胃给药，1次/日，连续14天，术后第14天处死小鼠。
2.2检测指标
2.2.1样品配制及供试品分析：根据小鼠体重称取足够量的化合物7a，调节PH后使用双蒸水作为溶剂配制成相应浓度的药液。
2.2.2对肾脏组织病理学的影响：给药14天后将所有小鼠予以CO2麻醉安乐死，立即放血，取左侧肾脏一半置于福尔马林固定并进行脱水，石蜡包埋，切片，分别进行HE染色、masson染色，其中HE染色的组织切片采用肾脏损伤评分标准进行病理学评分，masson染色的组织切片采用肾脏纤维化评分标准进行病理学评分。具体评分标准和面积计算方法如下：
HE染色：将肾脏组织病变分类，分别为肾间质纤维化、肾间质炎症、肾小管空泡变性、肾小管萎缩、肾小管扩张、肾间质水肿、红细胞管型、蛋白管型。分别对上述各种类型病变进行评分，其中正常：记“0”分；轻度：记“1”分；中度：记“2”分；重度：记“3”分，并将每只动物得分相加，得总分。分别对上述各类型得分及总得分进行统计。
masson染色：按细胞外基质沉积的面积计算，0-25％、25-50％、50-75％、大于75％分别记1、2、3、4分。
2.3统计方法
统计所用软件为SPSS20.0。如果方差齐性，用单因素方差分析(One-Way ANOVA)进行统计分析。如果方差不齐，则用Kruskal-Wallis检验。如果Kruskal-Wallis检验有统计学意义(P＜0.05)，则用Dunnett’s Test(非参数方法)进行比较分析。统计结果以P＜0.05表示有统计学意义，P＜0.01表示所检验的差别有非常显著性意义。
3.实验结果
3.1.对肾脏HE染色病理评分的影响
如图4-6和图10所示，与假手术组(Sham)比较，UUO组小鼠14天的肾脏中炎性细胞浸润、肾小管萎缩、肾间质纤维化明显，HE染色病理评分均显著升高(P＜0.05)；与UUO组比较，化合物组(仅列出7a和25f的数据)肾脏炎性细胞浸润、肾小管萎缩、肾间 质纤维化明显减少，而肾小管扩张病变明显，化合物组(仅列出7a和25f的数据)HE染色病理评分降低(P＜0.05)。其他目标化合物组的也表现出明显的肾脏炎性细胞浸润、肾小管萎缩、肾间质纤维化明显减少。
3.2对肾脏masson染色病理评分的影响
如图7-9和图10所示，与假手术组(Sham)比较，UUO组小鼠14天的肾脏肾间质纤维化沉积明显，masson染色病理评分均显著升高(P＜0.01)；与UUO组比较，化合物组(仅列出7a和25f的数据)肾脏肾间质纤维化沉积明显减少，masson染色病理评分显著降低(P＜0.05)。其他目标化合物组的也表现出明显的肾脏肾间质纤维化沉积明显减少，masson染色病理评分显著降低。
4.结论：
本发明的2,4-二取代-5-氟嘧啶衍生物可以减轻肾损伤和肾间质纤维化沉积。

Claims (14)

  1. 一种2，4-二取代-5-氟嘧啶类衍生物，其特征在于，其结构通式如式I所示：
    其中，R1和R2分别独立选自：
    R4选自：
    X,Y分别独立选自氮原子或碳原子；Z选自氢原子，叔丁氧羰基，C1-C5烷基，C1-C5烷基酰基，C1-C3烷基羟基，C1-C3烷基磺酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
  2. 根据权利要求1所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，R1和R2任一为：R4为Z选自氢原子，C1-C5烷基，C1-C5烷基酰基，C1-C3烷基羟基，C1-C3烷基磺酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
  3. 根据权利要求1所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，R1和R2任一为：R4为Z为丁基，戊基，异丁基，异戊基，R1和R2另一为：时，X,Y不同时为碳原子。
  4. 一种2，4-二取代-5-氟嘧啶类衍生物，其特征在于，其结构通式如式II或式III所示：
    其中，R3选自：
    R4选自：
    X,Y分别独立选自氮原子或碳原子；Z选自氢原子，叔丁氧羰基，C1-C5烷基，C1-C5烷基酰基，C1-C3烷基羟基，C1-C3烷基磺酰基，C1-C3取代苯基，C1-C5烷基酰胺基。
  5. 根据权利要求4所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，在R3中，R4为时，Z不为叔丁氧羰基。
  6. 根据权利要求4所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，式III中的R4为时，Z不为叔丁氧羰基、C4-C5烷基、C4-C5烷基酰基，C4-C5烷基酰胺基。
  7. 根据权利要求1-6任一项所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，C1-C5烷基包括甲基，乙基，丙基，丁基，戊基，异丁基，异戊基，异丙基。
  8. 根据权利要求1-6任一项所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，C1-C5烷基酰基包括甲酰基，乙酰基，丙酰基，丁酰基，戊酰基，3，3-二甲基-1-丁酰基，异戊酰基。
  9. 根据权利要求1-6任一项所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，C1-C5烷基酰胺基包括甲酰胺基，乙酰胺基，丙酰胺基，丁酰胺基，戊酰胺基，N-叔丁基胺基甲酰基，异戊酰胺基。
  10. 根据权利要求1或4所述的2，4-二取代-5-氟嘧啶类衍生物，其特征在于，具体为以下化合物：
    叔丁基-4-(4-((5-氟-2-((4-(甲氧甲酰基)苯基)氨基)吡啶-4-基)氨基)苯基)哌嗪-1-羧酸酯；
    1-(5-氟-4-((4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺；
    1-(4-((4-(4-乙基哌嗪-1-基)苯基)氨基)-5-氟嘧啶-2-基)-1H-苯并[d]咪唑-2-胺；
    N2-(1H-苯并[d]咪唑-2-基)-N4-(4-(4-乙基哌嗪-1-基)苯基)-5-氟嘧啶-2,4-二胺；
    1-(5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺；
    5-氟-N2-(异喹啉-3-基)-N4-(4-(哌嗪-1-基)苯基)嘧啶-2,4-二胺；
    5-氟-N4-(4-哌嗪-1-基)苯基-N2-((四氢呋喃-2-基)甲基)嘧啶-2,4-二胺；
    5-氟-N4-(4-(哌嗪-1-基)苯基-N2-(吡啶-2-基甲基)嘧啶-2,4-二胺；
    甲基-4-(5-氟-4-((4-(哌嗪-1-基)苯基)氨基)嘧啶-2-基)氨基苯甲酸酯；
    1-(5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)氨基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺；
    5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-((四氢呋喃-2-基)甲基)嘧啶-2，4-二胺；
    5-氟-N4-(4-(4-异戊基哌嗪-1-基)苯基)-N2-(吡啶-2-基甲基)嘧啶-2,4-二胺；
    1-(5-氟-4-(6-(哌嗪-1-基)吡啶-3-基)嘧啶-2-基)-1H-苯并[d]咪唑-2-胺；
    N6-(5-氟-4-((4-吗啉苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-4-((4-(吡咯-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(4-((4-(二乙基胺基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(4-((4-(二丙基胺基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-4-((4-(哌啶-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d] 咪唑-2,6-二胺；
    N6-(4-((4-(二甲基胺基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    叔丁基-4-(5-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)吡啶-2-基)哌嗪-1-羧酸酯；
    叔丁基-4-(5-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)吡啶-3-基)哌嗪-1-羧酸酯；
    N6-(5-氟-4-((4-(哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-4-((6-(哌嗪-1-基)吡啶-3-基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-4-((5-(哌嗪-1-基)吡啶-2-基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-4-((4-(4-甲基哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    1-(4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-基)乙酮；
    N6-(5-氟-4-((4-(4-异丙基哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    2-(4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-基)乙醇；
    N6-(4-((4-(4-(乙基磺酰基)哌嗪-1-基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-4-((4-(4-异戊基哌嗪-1-基)苯基)胺基)嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(4-((4-(4-苄基哌嗪-1-基)苯基)胺基)-5-氟嘧啶-2-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    1-(4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-基)-3,3-二甲基丁-1-酮；
    N-(叔丁基)-4-(4-((2-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-4-基)胺基)苯基)哌嗪-1-甲酰胺；
    N6-(5-氟-2-((4-(吡咯-1-基)苯基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-2-((4-(哌嗪-1-基)苯基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-2-((6-(哌嗪-1-基)吡啶-3-基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(2-((4-(二甲基胺基)苯基)胺基)-5-氟嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    N6-(5-氟-2-((5-(哌嗪-1-基)吡啶-2-基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺；
    1-(4-(4-((4-((2-(二甲基胺基)-1H-苯并[d]咪唑-6-基)胺基)-5-氟嘧啶-2-基)胺基)苯基)哌嗪-1-基)乙酮；
    N6-(5-氟-2-((4-(4-异戊基哌嗪-1-基)苯基)胺基)嘧啶-4-基)-N2，N2-二甲基-1H- 苯并[d]咪唑-2,6-二胺；
    N6，N6-(5-氟嘧啶-2,4-二基)双(N2，N2-二甲基-1H-苯并[d]咪唑-2,6-二胺)。
  11. 一种制备权利要求1-10任一项所述的2，4-二取代-5-氟嘧啶类衍生物的方法，其特征在于，包括以下步骤：
    2，4-二氯-5-氟-嘧啶与胺类化合物反应得到化合物I或II，2，4-二氯-5-氟-嘧啶与硼酸酯反应生成的中间体继续与2-氨基苯并咪唑反应得到化合物III。
  12. 一种药物组合物，其特征在于，包含权利要求1-10任一项所述的2，4-二取代-5-氟嘧啶类衍生物或其药学上可用的盐以及药学上可接受的载体和辅料。
  13. 权利要求1-10任一项所述的2，4-二取代-5-氟嘧啶类衍生物及权利要求12所述的组合物在制备治疗纤维化的药物中的应用。
  14. 根据权利要求13所述应用，其特征在于，所述纤维化是指肾间质纤维化。
PCT/CN2023/090579 2022-05-06 2023-04-25 2,4-二取代-5-氟嘧啶衍生物及其制备方法和应用 WO2023213211A1 (zh)

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