WO2017133258A1 - 1h-吲唑类衍生物及其作为ido抑制剂的用途 - Google Patents

1h-吲唑类衍生物及其作为ido抑制剂的用途 Download PDF

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WO2017133258A1
WO2017133258A1 PCT/CN2016/099845 CN2016099845W WO2017133258A1 WO 2017133258 A1 WO2017133258 A1 WO 2017133258A1 CN 2016099845 W CN2016099845 W CN 2016099845W WO 2017133258 A1 WO2017133258 A1 WO 2017133258A1
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compound
group
indazole
formula
preparation
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PCT/CN2016/099845
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English (en)
French (fr)
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钱珊
王周玉
杨羚羚
李国菠
赖朋
刘思言
李会周
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西华大学
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Priority claimed from CN201610081017.3A external-priority patent/CN107033087B/zh
Priority claimed from CN201610560610.6A external-priority patent/CN107619392B/zh
Priority claimed from CN201610831550.7A external-priority patent/CN107840826B/zh
Application filed by 西华大学 filed Critical 西华大学
Publication of WO2017133258A1 publication Critical patent/WO2017133258A1/zh

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    • 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
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
    • 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

  • This invention relates to 1H-carbazole derivatives, to processes for their preparation and to their use as IDO inhibitors.
  • Indoleamine 2,3-dioxygenase is a catalyzed cleavage of guanidine urate pathway in guanamine molecules such as tryptophan. Speed enzyme.
  • IDO plays an important role in tumor immune exemption and tumorigenesis. Under normal circumstances, IDO is expressed at a low level in the body, and most tumor cells constitute a high expression of IDO, which converts L-tryptophan to N-formyl kynurenine, which reduces the color in the cell microenvironment. The concentration of tryptophan makes tryptophan-dependent T cell synthesis arrest in G1 phase, and T cell proliferation is inhibited, thereby inhibiting the killing effect of the body's immune system on tumor tissues. At the same time, the metabolite of tryptophan under the action of IDO is cytotoxic and can directly dissolve T cells.
  • IDO inhibitors can also be combined with chemotherapeutic drugs to reduce the resistance of tumor cells, thereby enhancing the anti-tumor activity of conventional cytotoxic therapies. Simultaneous administration of IDO inhibitors can also increase the efficacy of therapeutic vaccines for cancer patients.
  • IDO In addition to its important role in tumor cell resistance, IDO is also closely associated with the pathogenesis of a variety of diseases associated with cellular immune activation. IDO has been identified as a target for major diseases such as infections, malignancies, autoimmune diseases, and AIDS associated with cellular immune activation. At the same time, inhibition of IDO is an important therapeutic strategy for patients with neurological diseases such as depression and Alzheimer's disease. Therefore, IDO inhibitors have broad clinical application prospects.
  • the present invention mainly provides a novel class of IDO inhibitor drugs, which are all 1H-carbazole derivatives.
  • the present invention provides a compound or a pharmaceutically acceptable salt thereof or a solvate thereof, which has a structure as shown in the formula (A):
  • Y means -NH-, Or -O-;
  • R 1 is selected from H, halogen, C 1 -C 6 alkyl or C 1 -C 6 haloalkyl;
  • R 2 is selected from -(CH 2 ) b -R 3 , -CH 2 NHR 3 , -CH 2 CONH-R 3 , -CH(CH 2 OH)R 3 or -CH 2 CH(OH)R 3 ;
  • R 3 is selected from a substituted or unsubstituted aryl group, a heteroaryl group, a cycloalkyl group or a heterocyclic group, and the substituted aryl group, heteroaryl group, cycloalkyl group or heterocyclic group are independently one or more One selected from the group consisting of halogen, -(CH 2 ) c COOH, -(CH 2 ) d COOR 4 , -(CH 2 ) e OH, -(CH 2 ) f NH 2 , nitro, C 1 -C 6 alkyl, Substituted by a C 1 -C 6 haloalkyl group, -(CH) 2 CONH-R 4 or a substituent of -(CH) 2 NH-R 5 ;
  • R 4 is selected from C 1 -C 6 alkyl
  • R 5 is selected from -CH 2 COOH, 5-6 membered cycloalkyl, C 1 -C 6 alkyl substituted or hydroxy substituted cycloalkyl;
  • a, b, c, d, e, and f each independently represent 0 or 1.
  • the C 1 -C 6 alkyl group means a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert. Butyl, sec-butyl, pentyl, hexyl and the like.
  • Y means -NH-
  • R 1a is selected from the group consisting of H, X, -CX 3 , -CHX 2 , -CH 2 X, -(CH 2 ) a CH 3 ;
  • R 2a is selected from -(CH 2 ) b -R 3a , -CH 2 NHR 3a , -CH 2 CONH-R 3a , -CH(CH 2 OH)R 3a or -CH 2 CH(OH)R 3a ;
  • R 3a is selected from a substituted or unsubstituted aryl, cycloalkyl or heterocyclic group, and the substituted aryl, cycloalkyl or heterocyclic group is independently selected from one or more selected from X, -(CH). 2 ) a substituent substituted by c COOH, -(CH 2 ) d COOR 4a , -(CH 2 ) e OH, (CH 2 ) f NH 2 , a nitro group, a C 1 -C 6 alkyl group;
  • R 4a is selected from methyl or ethyl
  • X represents F, Cl, Br or I
  • a, b, c, d, e, and f each independently represent 0 or 1.
  • R 1a is selected from the group consisting of H, Br, —CF 3 , —CH 3 or —CH 2 CH 3 .
  • R 1a is selected from the group consisting of Br.
  • the cycloalkyl group is a cyclohexyl group; the substituted cycloalkyl group is a cycloalkyl group substituted by one hydroxyl group.
  • heterocyclic group is a tetrahydropyranyl group.
  • substituted aryl group is selected from a phenyl group substituted with one -(CH 2 ) c COOH, chlorine, amino, hydroxy, nitro or methyl.
  • R 3 is selected from one of the following groups:
  • the compound is selected from one of the following compounds:
  • the invention also provides a process for the preparation of the compound of formula (II) or a preparation intermediate thereof, comprising the steps of:
  • the compound of the formula (II) or a preparation intermediate thereof is prepared by reacting a compound represented by SM and S2 with a reducing agent.
  • reaction can be carried out in the presence of an acidic catalyst.
  • the reducing agent may be selected from DHP, NaBH 4 or NaBH 3 (CN), most preferably DHP;
  • the acidic catalyst may be TFA, formic acid, acetic acid or binaphthyl phosphate, most preferably TFA;
  • the solvent is CH 2 Cl 2 , MeOH or DMF, most preferably CH 2 Cl 2 .
  • the preparation intermediate refers to a synthetic intermediate of the previous step of the target compound.
  • a certain compound R 3 when a certain compound R 3 is selected from -(CH 2 ) d COOR 4 , it may be used as a drug itself, or as another intermediate for the preparation of a compound in which R 3 is selected from -(CH 2 ) d COOR 4 compound.
  • the compound 1m can be used as a preparation intermediate of the compound 1l
  • the compound 1o can be used as an intermediate for the preparation of the compound 1n.
  • Compounds 11 and 1n can be prepared as intermediates of compounds 1m and 1o, respectively, and further by ester hydrolysis by conventional ester hydrolysis techniques in the art.
  • R 3 when R 3 is selected from a nitro group, it may be used as a drug itself, or as another intermediate for the preparation of R 3 from the -NH 2 compound.
  • compound 1p can be used as a preparation intermediate of compound 1q, and 1q is prepared by a nitro reduction reaction on 1p.
  • the invention also provides a process for the preparation of the compound of formula (III) or a process for the preparation thereof, comprising the steps of:
  • the compound represented by SM and S2 is used as a raw material, and a compound of the formula (III) or a preparation intermediate thereof is prepared by a condensation reaction.
  • the synthetic condensing agent can be EDCI or DCC, preferably EDCI; the base can be HOBT or HATU, and most preferably HOBT.
  • the invention also provides a process for the preparation of the compound of formula (IV) or a process for the preparation thereof, comprising the steps of:
  • the compound represented by S4a and the bis(trichloromethyl) carbonate are used as raw materials to prepare a compound represented by S4b;
  • the compound represented by S4b and SM is used as a raw material to prepare a compound of the formula (IV) or a preparation intermediate thereof.
  • the invention also provides a process for the preparation of the compound of formula (V), formula (VI), or a precursor thereof, comprising the steps of:
  • the compound represented by S5 and SM is used as a raw material to prepare a compound of the formula (V) or the formula (VI), or an intermediate thereof.
  • the pH of the synthesis reaction can be between 7 and 10, with a most preferred pH being 7.
  • the reaction temperature may be from 20 to 80 ° C, most preferably 80 ° C.
  • the solvent may be selected from any one or more of EtOH, H 2 O, DMF, MeOH, THF, CH 2 Cl 2 , EtOAc or dioxane, and most preferably a mixed solution of EtOH and H 2 O.
  • the invention also provides a process for the preparation of a compound of formula (VII) or a process for the preparation thereof, comprising the steps of:
  • the compound represented by SM and ethyl bromoacetate are used as raw materials to prepare a compound represented by S7a;
  • reaction it can be carried out in the presence of a base.
  • the base may be selected from K 2 CO 3 , NaH, NaOH or KOH, most preferably K 2 CO 3 ;
  • the solvent may be selected from CH 2 Cl 2 , MeOH, tOH, THF, CH 3 COCH 3 , EtOAc or DMF, most preferably DMF.
  • the compound represented by the formula (VII) or a preparation intermediate thereof is prepared by a condensation reaction using the compound represented by S7b and S7c as a raw material.
  • the present invention also provides a compound or a pharmaceutically acceptable salt thereof or a solvate thereof, which has a structure as shown in the formula (VIII):
  • R 1b is selected from H, halogen, C 1 -C 6 alkyl or C 1 -C 6 haloalkyl;
  • R 2b is selected from substituted or unsubstituted aryl, heteroaryl, cycloalkyl or heterocyclic groups, each of which is independently one or more One selected from halogen, -NH 2 , -(CH 2 ) a OH, -COOH, nitro, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -CONH-R 3b or -NH-R 4b Substituted by a substituent;
  • R 3b is selected from C 1 -C 6 alkyl
  • R 4b is selected from -CH 2 COOH, a 5-6 membered cycloalkyl group, a C 1 -C 6 alkyl group-substituted or hydroxy-substituted cycloalkyl group;
  • R 1b is selected from H, Cl, -Br, CF 3 , -CHX 2 , -CH 2 X or -CH 3 , and X represents a halogen atom.
  • the C 1 -C 6 alkyl group is selected from a methyl group.
  • the C 1 -C 6 haloalkyl group is a trifluoromethyl group.
  • the aryl group is selected from a phenyl group
  • the heteroaryl group is selected from the group consisting of benzothiophenes
  • the cycloalkyl group is selected from a cyclohexyl group
  • the heterocyclic group is selected from a 4- to 6-membered nitrogen heterocyclic group.
  • R 2b is selected from one of the following groups:
  • the compound is selected from one of the following compounds:
  • the present invention also provides a compound or a pharmaceutically acceptable salt thereof or a solvate thereof, which has a structure as shown in the formula (IX):
  • A represents a substituent at any position on the benzene ring, the substituent is selected from -COOR, -OH or -NH 2 , R is selected from H or a C 1 -C 4 alkyl group; and X is selected from a halogen.
  • X is selected from Cl, Br or I.
  • the compound is selected from one of the following compounds:
  • the invention also provides a process for the preparation of the aforementioned compounds or intermediates for their preparation, comprising the steps of:
  • Pg represents an amino protecting group
  • the compound S92 is condensed with the compound S92 and the compound S96 to obtain the compound S97, and the amino group is deprotected.
  • the temperature of the heating cyclization is 80 to 160 ° C, preferably 95 ° C.
  • the solvent may be a high boiling point solvent such as ethylene glycol, DMF or DMSO, preferably ethylene glycol.
  • the base is selected from the group consisting of potassium carbonate, NaOH, KOH, Na 2 CO 3 , LiOH or triethylamine, preferably potassium carbonate.
  • the hydrolysis is carried out at a temperature of from 25 to 100 ° C, preferably at 90 ° C.
  • the present invention also provides a process for the preparation of the aforementioned compound or a preparation intermediate thereof, comprising the steps of:
  • the compound SM2 is prepared by using the compound SM1, 1,1,3,3-tetramethyldisiloxane and I 2 as a raw material; wherein R is selected from a C 1 -C 4 alkyl group; in a specific embodiment of the present invention , R is selected from methyl; SM2 can be replaced by Br, I 2 of the reaction raw material can be replaced by NaI, LiBr; 1,1,3,3-tetramethyldisiloxane can be replaced by trimethylchlorosilane a silylating agent such as tert-butyldimethylchlorosilane.
  • SM4 is reacted with di-tert-butyl dicarbonate to prepare compound SM5;
  • Boc is a protecting group, and di-tert-butyl dicarbonate can also be replaced with a common protecting group for an amino group such as benzyloxycarbonyl chloride.
  • Compound SM2 is condensed with compound SM2 and compound SM6 to obtain a compound SM7, which is obtained by removing a tert-butoxycarbonyl group in the presence of trifluoroacetic acid.
  • Synthetic SM7 is a conventional ether-forming reaction, and the base can be K2CO3, Et3N, NaOH, solvent. DMF, MeCN, acetone, THF, dioxane can be used. The temperature is from room temperature to 120 degrees.
  • X is selected from Br.
  • the temperature of the heating cyclization is 95 °C.
  • the catalyst is selected from the group consisting of 4-dimethylaminopyridine.
  • the alkaline environment is an aqueous solution of potassium carbonate, and the temperature of the hydrolysis reaction is 90 °C.
  • the preparation intermediate refers to a synthetic intermediate of the previous step of the target compound.
  • a certain compound R when a certain compound R is selected from an alkyl group, it may be used either as a drug itself or as a preparation intermediate in which another R is selected from the H compound.
  • the compound O12 can be used as an intermediate for the preparation of the compound O14
  • the compound O13 can be used as an intermediate for the preparation of the compound O15.
  • the compounds O14 and O15 can be prepared by the ester hydrolysis reaction of the compounds O12 and O14, respectively, and further by ester hydrolysis techniques conventional in the art.
  • the present invention also provides the use of the aforementioned compound, the compound of the formula (X), or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof, for the preparation of an IDO inhibitor-like drug,
  • R 1b is as defined above.
  • the medicament is a medicament for preventing and/or treating Alzheimer's disease, cataract, infection related to cellular immune activation, autoimmune disease, AIDS, cancer, depression or abnormal metabolism of tryptophan.
  • the present invention also provides a pharmaceutical composition which is prepared by using the compound described above or a pharmaceutically acceptable salt thereof as an active ingredient, together with a pharmaceutically acceptable adjuvant.
  • composition is a medicament for treating or preventing and/or treating Alzheimer's disease, cataract, infection related to cellular immune activation, autoimmune disease, AIDS, cancer, depression or abnormal metabolism of tryptophan.
  • the prodrugs are derivatives of the aforementioned compounds which may themselves have weak or no activity, but are converted to corresponding conditions under physiological conditions (for example by metabolism, solvolysis or otherwise) after administration. Biologically active form.
  • the preparation may include an injection or an oral preparation.
  • the key intermediates and compounds of the present invention are isolated and purified in a manner that is commonly used in organic chemistry for separation and purification.
  • One or more compounds of the invention may be used in combination with one another, or the compounds of the invention may be used in combination with any other active agent for the preparation of IDO inhibitors. If a group of compounds is used, the compounds can be administered to the subject simultaneously, separately or sequentially.
  • the pharmaceutically acceptable excipient of the present invention means a substance which is contained in a dosage form in addition to the active ingredient.
  • the 1H-carbazole derivatives provided by the present invention have an excellent inhibitory effect on IDO and can be used for preventing and/or treating various diseases such as Alzheimer's disease, cataract, and cellular immune activation-related infections. , autoimmune diseases, AIDS, cancer, depression or abnormal metabolism of tryptophan.
  • Boc tert-butoxycarbonyl
  • DHP 1,4-Dihydropyridine, dihydropyridyl ester.
  • DCC Dicyclohexylcarbodiimide, dicyclohexylcarbodiimide.
  • DIBAL-H Diisobutylaluminum bydride, diisobutylaluminum hydride
  • DIEA N, N-Diisopropylethylamine, N,N-diisopropylethylamine.
  • EA Ethyl acetate, ethyl acetate.
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.
  • HOBT 1-Hydroxybenzotriazole, 1-hydroxybenzotriazole.
  • HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.
  • PE Petroleum ether, petroleum ether.
  • TFA Tallow Fatty Acid, trifluoroacetic acid.
  • THF Tetrahydrofuran, tetrahydrofuran.
  • the reagents and raw materials were all from commercially available products, and the reagents were purchased from Chengdu Kelon Chemical Reagent Co., Ltd. except for the starting materials specifically labeled with the source.
  • the compound O2 was replaced with the compound O2, and the compound O6 was obtained by the method of the compound O5.
  • the o-hydroxybenzaldehyde O7 (244.0 mg, 2.00 mmol) was dissolved in 10 mL of dichloromethane, and MeOH (0.19 mL, 4.00 mmol) and DIEA (0.30 mL, 6.00 mmol) was added with stirring at 0 ° C. The reaction was stirred at room temperature for 1 h and TLC showed a complete reaction. Water was added, and the mixture was combined with EtOAc EtOAc. The compound O3 was used in place of the compound O3, and the compound O5 was synthesized by the method of the compound O5 to obtain a pale yellow oil, O9, yield 50%.
  • the synthesis of the compound O11 was carried out with reference to the compound O15 in a yield of 90%.
  • the synthesis of the compound O12 was carried out with reference to the synthesis of the compound O5 in a yield of 45%.
  • the starting material 4-nitro-1H-carbazole 5a (CAS: 2942-40-7, 1.4 g, 8.26 mmol, purchased from Anne Chemical Reagent Co., Ltd.) or 6-bromo-4-nitro-1H-oxime Azole 5b (CAS: 885518-46-7, 2.0 g, 8.26 mmol, purchased from Jiangsu Nantong Biotechnology Co., Ltd.) was dissolved in a mixed solvent of ethanol (20 mL) and water (10 mL), and ammonium chloride (221.5 mg, 4.13 mmol), a part of iron powder (1.3 g, 23.46 mmol) was first added thereto, and the mixture was heated to 80 ° C, stirred for 5 minutes, and the remaining iron powder (1.0 g, 17.86 mmol) was added thereto, and the reaction was further stirred for 20 minutes.
  • reaction solution was filtered while hot, and the residue was washed with ethanol (10 mL). Ethanol was evaporated under reduced pressure and the aqueous layer was extracted with ethyl acetate (20 mL). The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate The yield of 6a was 94%, and the yield of 6b was 92%.
  • P-xylene (7) (360.0 mg, 3.40 mmol) was placed in a round bottom flask, cooled to 0 ° C in an ice bath, slowly added dropwise, mixed acid solution of 90% nitric acid solution and concentrated sulfuric acid (2:1) (1 mL) Solid precipitated, stirred at room temperature for 3 h, TLC detection of the starting material (7) reaction was completed, the reaction solution was poured into ice water, filtered, and the filter cake was dried in a vacuum oven for 12 h. The pale yellow solid was 545.8 mg, yield 82%.
  • 2,5-Dimethyl-3-nitroaniline (9) (83.0 mg, 0.50 mmol) was dissolved in glacial acetic acid (3 mL), then NaNO 2 (69.0 mg, 1.00 mmol) was dissolved in water (0.5 mL) The aqueous solution of NaNO 2 was slowly added dropwise to a solution of 2,5-dimethyl-3-nitroaniline (9) in glacial acetic acid under stirring at room temperature. A solid precipitated and the mixture was stirred at room temperature for 13 h. After the TLC test raw material (9) was completely reacted, the reaction liquid was poured into ice water, filtered, and the filter cake was dried in a vacuum oven for 12 hours to obtain a yellow solid 79.6 mg, yield 90%.
  • the starting material 6-methyl-4-nitro-1H-carbazole (10) (50 mg, 0.28 mmol) was dissolved in a mixed solvent of ethanol (2 mL) and water (1 mL), and ammonium chloride (1.6 mg, 0.03) was added. Further, iron powder (79.1 mg, 1.41 mmol) was added thereto, and the mixture was heated to 80 ° C to stir the reaction for 0.5 h. After the reaction of the starting material (10) was completed by TLC, the reaction mixture was filtered while hot, and the residue was washed with ethanol. Ethanol was turned off under reduced pressure and the aqueous layer was extracted three times with ethyl acetate. The organic layer was combined, washed with brine, dried over anhydrous magnesium sulfate
  • the starting material 6-methyl-4-nitro-1H-carbazole (n4a) (50 mg, 0.28 mmol) was dissolved in a mixed solvent of ethanol (2 mL) and water (1 mL), and ammonium chloride (1.6 mg, 0.03) was added. After adding iron powder (79.1 mg, 1.41 mmol), the mixture was heated to 80 ° C and stirred for 0.5 h. The reaction solution was filtered while hot, and the residue was washed with ethanol. The ethanol was rotated under reduced pressure, and the aqueous layer was extracted three times with EA.
  • the compound 4-trifluoromethyl-toluene (n4b) was used as a raw material.
  • the compound n5b was obtained by the method of synthesizing the compound n5a. Yellow solid, yield 95%. The purity was 98% by HPLC; 1 H-NMR (400 MHz, CDCl 3 , ppm): ⁇ 13.0 (br, 1H, NH), 8.10 (s, 1H, indazole-H), 7.21 (s, 1H, indazole -H), 6.58 (s, 1H, indazole-H), 4.37 (br, 2H, NH2).
  • ESI-MS 202.05 [M+H].
  • reaction solution was filtered while hot, and the residue was washed with ethanol (10 mL). Ethanol was decanted under reduced pressure and the aqueous layer was extracted three times with EA (20 mL).
  • 6-Bromo-4-fluoro-1H-indazole-1-carbonyl tert-butyl ester O15 (100.0 mg, 0.32 mmol) was firstly dissolved in 5 mL of DMF, and K 2 CO 3 (77.3 mg, 0.56 mmol) was added under stirring at room temperature.
  • 6-Bromo-4-fluoro-1H-indazole-1-carbonyl tert-butyl ester O15 (100.0 mg, 0.32 mmol) was firstly dissolved in 5 mL of DMF, and NaOH (22.4 mg, 0.56 mmol) and water (28.8) were added with stirring at room temperature.
  • 6-Bromo-1H-indazole-1-carbonyl tert-butyl ester O16 (60.0 mg, 0.13 mmol) was dissolved in 2.5 mL of CH 2 Cl 2 , and then added with 0.5 mL of trifluoroacetic acid under stirring at room temperature, and stirred at room temperature for 2 h. Complete reaction. Spin off the solvent, made basic with saturated NaHCO 3, extracted three times with ethyl acetate, washed with saturated brine, dried and concentrated to give a pale yellow solid compound O17 (46.0mg, yield 98%).
  • 6-Bromo-1H-indazole-4-amino 6b (212.0 mg, 1.00 mmol) was dissolved in DMF (5 mL) and potassium carbonate (345.0 mg, 2.50 mmol) and potassium iodide (14.9 mg, 0.09 mmol).
  • Ethyl bromoacetate (167.0 ⁇ L, 1.50 mmol) was added under argon, and the mixture was reacted at 65 ° C overnight.
  • Ethyl 2-(6-bromo-1H-indazole-4-amino)acetate (11) (80.0 mg, 0.27 mmol) was dissolved in ethanol (1.5 mL) and water (1. 32.2 mg, 0.81 mmol), and the reaction was stirred at room temperature overnight. After the TLC detection reaction of the raw material is completed, the pH is adjusted to 5 with 1 mol/L of dilute hydrochloric acid, and the reaction liquid is spin-dried under reduced pressure, and directly used for the next reaction, the yield is 100%.
  • the acid 12 (50.0 mg, 0.19 mmol) was dissolved in dichloromethane (3 mL), and then placed in ice bath, and tetrahydro-2H-pyran-4-amine 13 (57 uL, 0.56 mmol) was added dropwise to EDCI (42.6 mg, 0.22 mmol), HoBt (32.4 mg, 0.22 mmol), DIEA (61 uL, 0.37 mmol) were added and reacted at 0 ° C for 5 minutes, and then stirred at room temperature (25 ° C) overnight.
  • the amide 3c (40.0 mg, 0.10 mmol) was dissolved in ethanol (1.0 mL) and water (1.0 mL). After the reaction of the starting material was completed by TLC, the pH was adjusted to 5 with 1 mol/L of dilute hydrochloric acid, and a white solid was precipitated, filtered, and dried under reduced pressure to give 15.6 mg of white solid.
  • the synthetic route is as follows:
  • Epoxy 18 (229.2 mg, 1.42 mmol) and aniline 6b (300.0 mg, 1.42 mmol) were dissolved in a mixed 1:1 solution of ethanol and water (20 mL), and the mixture was warmed to 100 ° C and refluxed overnight. After the reaction of the starting material (18) was completed by TLC, the ethanol was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate (15 mL), and the organic phase was combined, washed with brine, dried over anhydrous magnesium sulfate column.
  • the compound 1l (38 mg, 0.10 mmol) was obtained as a brown solid.
  • the nitro compound 1p (2.9 g, 8.26 mmol) was dissolved in a mixed solvent of ethanol (20 mL) and water (10 mL), and ammonium chloride (221 mg, 4.13 mmol) was added, and a portion of iron powder (1.314 g, 23.46 mmol) was first added. After the addition, the mixture was heated to 80 ° C and stirred for 5 minutes, and then another iron powder (1 g, 17.86 mmol) was added thereto, and the reaction was further stirred for 20 minutes. After the TLC was used to detect the completion of the 1p reaction of the starting material, the reaction solution was filtered while hot, and the residue was washed with ethanol (10 mL). Ethanol was evaporated under reduced pressure and the aqueous layer was extracted with ethyl acetate (20 mL). The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate
  • the starting material (6b) (50 mg, 0.185 mmol) was dissolved in dichloromethane (3 mL), placed in an ice bath, and dropped.
  • Compound 21 (97.6 mg, 0.708 mmol), EDCI (62.4 mg, 0.329 mmol), HOBT (48 mg, 0.329 mmol), DIEA (77.5 uL, 0.470 mmol) were added, and reacted at 0 ° C for 5 minutes and then allowed to warm to room temperature. (30 ° C) Stir overnight.
  • the reaction mixture was washed with water (1 mL), and washed with saturated sodium hydrogen carbonate (1.5 mL).
  • the yellow solid was 27.1 mg and the structure was characterized as follows:
  • the compound (25) (0.34 mmol) was dissolved in a round bottom flask with 4 mL of CH 2 Cl 2 and 4 mL of saturated NaHCO 3 , and the compound (23) (0.03 g, 0.10 mmol) was added with stirring at 0 ° C, and stirred at room temperature for 15 min. TLC showed complete reaction. The reaction solution was poured into a sep. funnel and the organic layer was collected, dried and concentrated,
  • the starting material (n5b) (150 mg, 0.746 mmol) and the starting material (n8) (185.4 mg, 0.896 mmol) were dissolved in DCM (7 mL), DHP (264.2 mg, 1.04 mmol) was added, and trifluoroacetic acid (55.8 uL, The reaction mixture was refluxed for 12 hours at 40 ° C. The reaction mixture was filtered,jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj
  • the starting material (N15) (68.3 mg, 0.223 mmol) and the starting material (n12) (30.0 mg, 0.268 mmol) were dissolved in DCM (2mL), DHP (79.1mg, 0.312mmol) was added, and trifluoroacetic acid (16.6uL) was added dropwise. , 0.223 mmol), and refluxed at 40 ° C for 12 hours.
  • the reaction mixture was filtered, dried and evaporated,jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj
  • N18, N19 and N20 were synthesized in a similar manner, except that the starting materials were replaced by n19, n20, n21.
  • the results and characterization are as follows:
  • Example 21 Inhibitory activity of the compound of the present invention on IDO protein
  • the recombinant human IDO protein was expressed by E. coli and purified by nickel affinity chromatography.
  • the compound was tested for IDO inhibition activity using L-tryptophan as a substrate.
  • the test compound was dissolved in 10% DMSO solution to prepare a dilution. 5 uL of the dilution was added to 100 ⁇ L of the reaction system.
  • the 100 ⁇ L reaction system contained 0.5% DMSO, 40 nmol/L IDO, 900 ⁇ mol/L L-tryptophan, and other reaction coexistences (potassium phosphate buffer, ascorbic acid, catalase, methylene blue).
  • the reaction mixture was incubated at 37 °C for 180 minutes and the reaction was stopped by the addition of trichloroacetic acid.
  • the concentration of N-formyl kynurenine produced was measured at 321 nm using a Tecan Infinite M1000 plate reader to evaluate the inhibitory activity of the compound against IDO.
  • the negative control replaced the IDO with 5 ⁇ L of buffer.
  • the clinical phase III IDO inhibitor INCB024360 was used as a positive control to verify whether the IDO activity detection system established in this experiment was effective.
  • the inhibitory activity of some of the compounds of the present invention against IDO was tested by the above experimental methods.
  • the inhibitory activities of the specific compound at a concentration of 1 ⁇ M, 10 ⁇ M, and 100 ⁇ M are shown in Table 1.
  • A indicates an inhibition rate greater than 90%
  • B indicates an inhibition rate of 70-90%
  • C indicates an inhibition rate of 50-69%
  • D indicates an inhibition rate of 10-49%
  • E indicates an inhibition rate of less than 10%
  • a positive control The inhibition rate at a concentration of 0.05 ⁇ M was 46%.
  • the 1H-carbazole derivatives provided by the present invention have an excellent inhibitory effect on IDO and can be used for preventing and/or treating various diseases such as Alzheimer's disease, cataract, and cellular immune activation-related infections. , autoimmune diseases, AIDS, cancer, depression or abnormal metabolism of tryptophan.

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Abstract

本发明公开了式(A)所示的1H-吲唑类衍生物，还公开了所述化合物的制备方法和作为IDO抑制剂的用途。本发明的化合物可以用于预防和/或治疗多种疾病，如阿尔茨海默病、白内障、细胞免疫激活相关的感染、自身免疫性疾病、艾滋病、癌症、抑郁症或色氨酸代谢异常等。

Description

1H-吲唑类衍生物及其作为IDO抑制剂的用途 技术领域
本发明涉及1H-吲唑类衍生物，还涉及其制备方法和作为IDO抑制剂的用途。
背景技术
吲哚胺2,3-双加氧酶(Indoleamine 2,3-dioxygenase，IDO)是催化色氨酸等吲哚胺类分子中吲哚环氧化裂解，使其按犬尿酸途径分解代谢的限速酶。
IDO在肿瘤免疫豁免及肿瘤发生过程中起着重要作用。正常情况下，IDO在体内呈低水平表达，而大多数肿瘤细胞则会组成的高表达IDO，将L-色氨酸转化为N-甲酰犬尿氨酸，降低了细胞微环境中的色氨酸浓度，使得色氨酸依赖的T细胞合成停滞于G1期，T细胞增殖受到抑制，从而抑制了机体免疫系统对肿瘤组织的杀伤作用。同时，IDO作用下色氨酸的代谢产物存在细胞毒性，可对T细胞产生直接溶解作用。
因此，抑制IDO的活性可以有效地阻止肿瘤细胞周围色氨酸的降解，促进T细胞的增殖，从而增强机体对肿瘤细胞的攻击能力。并且，IDO抑制剂还可以与化疗药物合用，降低肿瘤细胞的耐药性，从而增强常规细胞毒疗法的抗肿瘤活性。同时服用IDO抑制剂也可提高癌症病人的治疗性疫苗的疗效。
除了在肿瘤细胞耐药性方面发挥着重要作用，IDO还与多种与细胞免疫激活相关的疾病的发病机制密切相关。IDO已被证实是与细胞免疫激活相关的感染、恶性肿瘤、自身免疫性疾病、艾滋病等重大疾病的靶标。同时，抑制IDO还是对于患有神经系统疾病如抑郁症，阿尔茨海默病的病人的重要治疗策略。因此，IDO抑制剂具有广阔的临床应用前景。
发明内容
为解决上述问题，本发明主要提供了一类新型的IDO抑制剂类药物，它们均为1H-吲唑类衍生物。
本发明提供了一种化合物或其药学上可接受的盐或其溶剂合物，所述化合物的结构如式(A)所示：
Figure PCTCN2016099845-appb-000001
Y表示-NH-、
Figure PCTCN2016099845-appb-000002
或-O-；
R1选自H、卤素、C1～C6烷基或C1～C6卤代烷基；
R2选自-(CH2)b-R3、-CH2NHR3、-CH2CONH-R3、-CH(CH2OH)R3或-CH2CH(OH)R3；
R3选自取代的或非取代的芳基、杂芳基、环烷基或杂环基，所述取代的芳基、杂芳基、环烷基或杂环基分别独立地被一个或多个选自卤素、-(CH2)cCOOH、-(CH2)dCOOR4、-(CH2)eOH、-(CH2)fNH2、硝基、C1～C6烷基、C1～C6卤代烷基、-(CH)2CONH-R4或-(CH)2NH-R5 的取代基所取代；
R4选自C1～C6烷基，R5选自-CH2COOH、5-6元的环烷基、C1～C6烷基取代的或羟基取代的环烷基；
a、b、c、d、e、f分别独立地表示0或1。
所述C1～C6烷基是指具有1～6个碳原子的直链或支链的烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、仲丁基、戊基、己基等等。
进一步地，所述化合物的结构如式(Ⅰ)所示：
Figure PCTCN2016099845-appb-000003
其中，
Y表示-NH-、
Figure PCTCN2016099845-appb-000004
R1a选自H、X、-CX3、-CHX2、-CH2X、-(CH2)aCH3；
R2a选自-(CH2)b-R3a、-CH2NHR3a、-CH2CONH-R3a、-CH(CH2OH)R3a或-CH2CH(OH)R3a；
R3a选自取代的或非取代的芳基、环烷基或杂环基，所述取代的芳基、环烷基或杂环基分别独立地被一个或多个选自X、-(CH2)cCOOH、-(CH2)dCOOR4a、-(CH2)eOH、(CH2)fNH2、硝基、C1～C6烷基的取代基所取代；
R4a选自甲基或乙基；
X表示F、Cl、Br或I，a、b、c、d、e、f分别独立地表示0或1。
进一步地，所述R1a选自H、Br、-CF3、-CH3或-CH2CH3。
更进一步地，所述R1a选自Br。
进一步地，所述化合物的结构如式(II)、式(III)、式(IV)、式(V)、式(Ⅵ)或式(Ⅶ)所示：
Figure PCTCN2016099845-appb-000005
进一步地，所述环烷基为环己基；所述取代的环烷基为被一个羟基所取代的环烷基。
进一步地，所述杂环基为四氢吡喃基。
进一步地，所述取代的芳基选自被一个-(CH2)cCOOH、氯、氨基、羟基、硝基或甲基所取代的苯基。
进一步地，所述R3选自下述基团之一：
Figure PCTCN2016099845-appb-000006
Figure PCTCN2016099845-appb-000007
进一步地，所述化合物选自如下化合物之一：
Figure PCTCN2016099845-appb-000008
本发明还提供了一种制备所述式(Ⅱ)化合物或其制备中间体的方法，包括下述步骤：
Figure PCTCN2016099845-appb-000009
以SM和S2所示化合物为原料，在还原剂的作用下反应制备得到式(Ⅱ)化合物或其制备中间体。
在具体的实施方式中，该反应可在酸性催化剂的存在下进行。
还原剂可选自DHP、NaBH4或NaBH3(CN)，最优选DHP；酸性催化剂可用TFA、甲酸、乙酸或联萘磷酸，最优选TFA；溶剂为CH2Cl2、MeOH或DMF，最优选CH2Cl2。
在本发明提供的制备方法中，所述制备中间体指的是目标化合物前一步的合成中间体。
例如，当某个化合物R3选自-(CH2)dCOOR4时，其可以本身既作为药物使用，也可以作为另一个R3选自-(CH2)dCOOR4化合物的制备中间体。在本发明具体的实施方式中，化合物1m可以作为化合物1l的制备中间体，化合物1o可以作为化合物1n的制备中间体。化合物1l和1n可以分别以化合物1m和1o的制备中间体，再进一步通过本领域常规的酯水解技术手段，酯水解反应制备得到。
又例如，当R3选自硝基时，其可以本身既作为药物使用，也可以作为另一个R3选自-NH2化合物的制备中间体。在本发明具体的实施例中，化合物1p可以作为化合物1q的制备中间体，1q通过对1p上的硝基还原反应制备得到。
本发明还提供了一种制备所述式(Ⅲ)化合物或其制备中间体的方法，包括下述步骤：
Figure PCTCN2016099845-appb-000010
以SM和S2所示化合物为原料，缩合反应制备得到式(Ⅲ)化合物或其制备中间体。合成的缩合剂可用EDCI或DCC，最优为EDCI；碱可用HOBT或HATU，最优为HOBT。
本发明还提供了一种制备所述式(Ⅳ)化合物或其制备中间体的方法，包括下述步骤：
(1)
Figure PCTCN2016099845-appb-000011
以S4a所示化合物和二(三氯甲基)碳酸酯为原料，反应制备得到S4b所示化合物；
(2)
Figure PCTCN2016099845-appb-000012
以S4b和SM所示化合物为原料，反应制备得到式(Ⅳ)化合物或其制备中间体。
本发明还提供了一种制备所述式(Ⅴ)、式(Ⅵ)化合物，或其制备中间体的方法，包括下述步骤：
Figure PCTCN2016099845-appb-000013
以S5和SM所示化合物为原料，反应制备得到式(Ⅴ)或式(Ⅵ)化合物，或其制备中间体。
在具体的实施方式中，合成反应的pH值可在7-10之间，最优选的pH值为7。反应温度可在20-80℃，最优选80℃。溶剂可选自EtOH、H2O、DMF、MeOH、THF、CH2Cl2、EtOAc或二氧六环中的任一种或多种，最优选EtOH和H2O的混合溶液。
本发明还提供了一种制备所述式(Ⅶ)化合物或其制备中间体的方法，包括下述步骤：
(1)
Figure PCTCN2016099845-appb-000014
以SM所示化合物和溴乙酸乙酯为原料，反应制备得到S7a所示化合物；
该反应具体的实施方式中，可在碱的存在下进行。
碱可选自K2CO3、NaH、NaOH或KOH，最优选K2CO3；溶剂可选自CH2Cl2、MeOH,tOH、THF、CH3COCH3、EtOAc或DMF，最优选DMF。
(2)
Figure PCTCN2016099845-appb-000015
将S7a所示化合物水解，反应制备得到S7b所示化合物；
(3)
Figure PCTCN2016099845-appb-000016
以S7b和S7c所示化合物为原料，缩合反应制备得到(Ⅶ)所示化合物或其制备中间体。
本发明还提供了一种化合物或其药学上可接受的盐或其溶剂合物，所述化合物的结构如式(Ⅷ)所示：
Figure PCTCN2016099845-appb-000017
其中，
R1b选自H、卤素、C1～C6烷基或C1～C6卤代烷基；
R2b选自取代的或非取代的芳基、杂芳基、环烷基或杂环基，所述取代的芳基、杂芳基、环烷基或杂环基分别独立地被一个或多个选自卤素、-NH2、-(CH2)aOH、-COOH、硝基、C1～C6烷基、C1～C6卤代烷基、-CONH-R3b或-NH-R4b的取代基所取代；
R3b选自C1～C6烷基；
R4b选-CH2COOH、5-6元的环烷基、C1～C6烷基取代的或羟基取代的环烷基；
a＝0或1。
进一步地，所述R1b选自H、Cl、-Br、CF3、-CHX2、-CH2X或-CH3，X表示卤原子。
进一步地，所述C1～C6烷基选自甲基。
进一步地，所述C1～C6卤代烷基为三氟甲基。
进一步地，所述芳基选自苯基，杂芳基选自苯并噻吩，环烷基选自环己基，杂环基选自4～6元的氮杂环基。
进一步地，所述R2b选自下述基团之一：
Figure PCTCN2016099845-appb-000018
Figure PCTCN2016099845-appb-000019
进一步地，所述化合物选自如下化合物之一：
Figure PCTCN2016099845-appb-000020
Figure PCTCN2016099845-appb-000021
本发明还提供了一种化合物或其药学上可接受的盐或其溶剂合物，所述化合物的结构如式(Ⅸ)所示：
Figure PCTCN2016099845-appb-000022
其中，A表示苯环上任意位置的取代基，所述取代基选自-COOR、-OH或-NH2，R选自H或C1～C4烷基；X选自卤素。
进一步地，X选自Cl、Br或I。
进一步地，所述化合物选自如下化合物之一：
Figure PCTCN2016099845-appb-000023
本发明还提供了一种制备前述化合物或其制备中间体的方法，包括以下步骤：
(1)
Figure PCTCN2016099845-appb-000024
以化合物S91为原料制备得到化合物S92；
(2)
Figure PCTCN2016099845-appb-000025
其中，Pg表示氨基保护基；
A、以化合物S93和水合肼为原料，在惰性气体环境中加热环化制备得到化合物S94；
B、将S94上的氨基保护得到化合物S95；
C、在碱的存在下，S95水解制备得到化合物S96；
(3)
Figure PCTCN2016099845-appb-000026
以化合物S92和化合物S96为原料缩合得到化合物S97，脱去氨基保护基即得。
进一步地，步骤A中，所述加热环化的温度为80-160℃，优选95℃。
步骤A中，溶剂可为乙二醇、DMF、DMSO等高沸点溶剂，优选乙二醇。
进一步地，步骤C中，所述碱选自碳酸钾、NaOH、KOH、Na2CO3、LiOH或三乙胺，优选碳酸钾。
进一步地，步骤C中，所述水解的温度为25-100℃，优选90℃。
更为具体地，本发明还提供了一种制备前述化合物或其制备中间体的方法，包括以下步骤：
(1)
Figure PCTCN2016099845-appb-000027
以化合物SM1、1,1,3,3-四甲基二硅氧烷和I2为原料制备得到化合物SM2；其中，R选自C1～C4烷基；在本发明具体的实施方式中，R选自甲基；SM2中的可换为Br，反应原料的I2可换为NaI、LiBr；1,1,3,3-四甲基二硅氧烷可换为三甲基氯硅烷，叔丁基二甲基氯硅烷等硅烷化试剂。
(2)
Figure PCTCN2016099845-appb-000028
A、以化合物SM3和水合肼为原料，在惰性气体环境中加热环化制备得到化合物SM4；
B、在催化剂的存在下，SM4与二碳酸二叔丁酯反应制备得到化合物SM5；Boc是保护基，二碳酸二叔丁酯也可以换为苄氧羰酰氯等氨基的常用保护基。
C、SM5在碱性环境下水解制备得到化合物SM6；
(3)
Figure PCTCN2016099845-appb-000029
以化合物SM2和化合物SM6为原料缩合得到化合物SM7，在三氟乙酸的存在下脱去叔丁氧羰基即得。合成SM7是常规的成醚反应，碱可用K2CO3、Et3N、NaOH，溶剂 可用DMF、MeCN、丙酮、THF、二氧六环。温度在室温至120度。
对于上述制备方法，在本发明具体的实施方式中，X选自Br。
进一步地，步骤A中，所述加热环化的温度为95℃。
进一步地，步骤B中，所述催化剂选自4-二甲氨基吡啶。
进一步地，步骤C中，所述碱性环境为碳酸钾水溶液，水解反应的温度为90℃。
在本发明提供的制备方法中，所述制备中间体指的是目标化合物前一步的合成中间体。
例如，当某个化合物R选自烷基时，其可以本身既作为药物使用，也可以作为另一个R选自H化合物的制备中间体。在本发明具体的实施方式中，化合物O12可以作为化合物O14的制备中间体，化合物O13可以作为化合物O15的制备中间体。化合物O14和O15可以分别以化合物O12和O14的制备中间体，再进一步通过本领域常规的酯水解技术手段，酯水解反应制备得到。
本发明还提供了前述化合物、式(Ⅹ)所示化合物、或其药学上可接受的盐、或其前药、或其溶剂合物在制备IDO抑制剂类药物上的用途，
Figure PCTCN2016099845-appb-000030
R1b如前述所定义。
进一步地，所述药物是预防和/或治疗阿尔茨海默病、白内障、细胞免疫激活相关的感染、自身免疫性疾病、艾滋病、癌症、抑郁症或色氨酸代谢异常的药物。
本发明还提供了一种药物组合物，它是以前述所述的化合物或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
其中，所述组合物是治疗或预防和/或治疗阿尔茨海默病、白内障、细胞免疫激活相关的感染、自身免疫性疾病、艾滋病、癌症、抑郁症或色氨酸代谢异常的药物。
所述前药是前述化合物的衍生物，它们自身可能具有较弱的活性或甚至没有活性，但是在给药后，在生理条件下(例如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。
所述的制剂可以包括注射剂或口服制剂。
本发明中的关键中间体和化合物进行分离和纯化，所使用的方式是有机化学中常用的分离和纯化方法。
本发明的一种或多种化合物可以彼此联合使用，也可选择将本发明的化合物与任何其它的活性试剂结合使用，用于制备IDO抑制剂。如果使用的是一组化合物，则可将这些化合物同时、分别或有序地对受试对象进行给药。
本发明所述药学上可接受的辅料，是指除活性成分以外包含在剂型中的物质。
经试验证明，本发明提供的1H-吲唑类衍生物对IDO具有优异的抑制作用，可以用于预防和/或治疗多种疾病，如阿尔茨海默病、白内障、细胞免疫激活相关的感染、自身免疫性疾病、艾滋病、癌症、抑郁症或色氨酸代谢异常等。
本发明中，英文缩写的涵义如下所示：
Boc：叔-丁氧羰基。
DHP：1,4-Dihydropyridine，二氢吡啶酯。
DMAP：4-二甲氨基吡啶。
DMF：Dimethylformamide，二甲基甲酰胺。
DCC：Dicyclohexylcarbodiimide，二环己基碳二亚胺。
DIBAL-H：Diisobutylaluminum bydride，二异丁基氢化铝
DIEA：N,N-Diisopropylethylamine，N,N-二异丙基乙胺。
EA：Ethyl acetate，乙酸乙酯。
EDCI：1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐。
Et3N：Triethylamine，三乙胺。
HOBT：1-Hydroxybenzotriazole，1-羟基苯并三氮唑。
HATU：2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯。
PE：Petroleum ether，石油醚。
TFA：Tallow Fatty Acid，三氟乙酸。
THF：Tetrahydrofuran，四氢呋喃。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
具体实施方式
所述试剂和原料均来自市售的商品，除专门标注了来源的起始原料外，其余试剂购于成都科龙化学试剂公司。
Varian INOVA 400超导高分辨核磁共振谱仪(以TMS为内标)；Agilent 1946B质谱仪；薄层层析硅胶GF254(0.2mm)和快速柱层析用硅胶(200～300目)为青岛海洋化工有限公司产品.
4-溴-2,6-二氟苯甲醛(成都欧瑞克科技有限公司)；对羧基苯甲醛(成都欧瑞克科技有限公司)；间羧基苯甲醛(成都欧瑞克科技有限公司)；其他试剂均为市售化学纯。
实施例1 中间体原料的合成
1、间碘甲基苯甲酸甲酯(O5)的合成
Figure PCTCN2016099845-appb-000031
将化合物O1(1.00g，6.67mmol)溶于15mL甲醇中，0℃搅拌下加入SOCl2(1.45mL，20.00mmol)，升至室温搅拌反应12h后用TLC检测反应完全。浓缩反应液，向残余物中加水，用饱和NaHCO3水溶液调至pH＝7～8。用乙酸乙酯提取3次。有机层经水洗，干燥，浓 缩后得到淡黄色油状物化合物O3(0.83g，收率76％)。未经柱层析纯化直接用于下步合成反应。
将化合物O3(1.00g，6.09mmol)和I2(0.93g，3.67mmol)溶于10mLCH2Cl2中，冷却至-5℃搅拌。将1,1,3,3-四甲基二硅氧烷(1.08mL，6.09mmol)溶于10mLCH2Cl2中。-5℃下，将1,1,3,3-四甲基二硅氧烷的CH2Cl2溶液加入到化合物O3和I2的CH2Cl2溶液中。加毕，升至室温搅拌0.5h后TLC监测反应完全。用50mLCH2Cl2反应液稀释，分别用水和饱和NaHCO3水溶液各洗涤3次，干燥，浓缩，得淡黄色固体化合物O5(0.57g，收率56％)。未经柱层析纯化直接用于下步合成反应。
2、对碘甲基苯甲酸甲酯(O6)的合成
Figure PCTCN2016099845-appb-000032
以化合物O2替代化合物O1，参照化合物O5的合成方法制得淡黄色固体化合物O6，收率61％。
3、1-(碘甲基)-2-((2-甲氧乙氧)甲氧)苯(O9)的合成
Figure PCTCN2016099845-appb-000033
将邻羟基苯甲醛O7(244.0mg,2.00mmol)溶于10mL二氯甲烷中，在0℃搅拌下加入MEMCl(0.19mL,4.00mmol)和DIEA(0.30mL,6.00mmol)，将反应液升至室温搅拌反应1h，TLC显示完全反应。加水，乙酸乙酯提取3次，用饱和食盐水洗涤，干燥，浓缩，得淡黄色油状物O8(253.0mg)。以化合物O8替代化合物O3，参照化合物O5的合成方法制得淡黄色油状物O9，收率50％。
4、叔丁基(2-(碘甲基)苯基)碳酸酯(O12)的合成
Figure PCTCN2016099845-appb-000034
化合物O11的合成参照化合物O15进行，收率90％。化合物O12的合成参照化合物O5的合成进行，收率45％。
5、6-溴-4-氟-1H-吲唑-1-羰基叔丁酯(O15)的合成
Figure PCTCN2016099845-appb-000035
先用10mL乙二醇将4-溴-2,6-二氟苯甲醛O13(CAS 537013-51-7,442.0mg,2.00mmol)，在常温搅拌下加入水合肼(0.19mL,4.00mmol)，换气(氮气保护)，将反应液升至95℃搅拌反应2h，TLC显示完全反应。加水，乙酸乙酯提取3次，用饱和食盐水洗涤， 干燥，浓缩，得淡黄色固体化合物O14(430.0mg)，收率100％。
可选方法：
先用10mLDMSO将4-溴-2,6-二氟苯甲醛O13(CAS 537013-51-7,442.0mg,2.00mmol)，在常温搅拌下加入水合肼(0.19mL,4.00mmol)，换气(氮气保护)，将反应液升至160℃搅拌反应1h，TLC显示完全反应。加水，乙酸乙酯提取3次，用饱和食盐水洗涤，干燥，浓缩，得淡黄色固体化合物O14(225.0mg)，收率52％。用10mLCH2Cl2将4-二甲氨基吡啶(DMAP)(12.2mg,0.10mmol)和化合物O14(430.0mg,2.00mmol)溶解，在常温搅拌下加入二碳酸二叔丁酯(872.0mg,4.00mmol)，室温搅拌反应2h，TLC显示完全反应。直接旋干粗品经柱层析(PE:EA＝60：1)纯化得无色透明油状物化合物O15(0.32g，收率50％)
6、6a和6b的合成
Figure PCTCN2016099845-appb-000036
将原料4-硝基-1H-吲唑5a(CAS:2942-40-7,1.4g,8.26mmol，购于安耐吉化学试剂有限公司)或6-溴-4-硝基-1H-吲唑5b(CAS:885518-46-7,2.0g,8.26mmol，购于江苏南通生物科技有限公司)溶于乙醇(20mL)和水(10mL)的混合溶剂中，加入氯化铵(221.5mg,4.13mmol)，先将一部分铁粉(1.3g,23.46mmol)加入其中，升温至80℃搅拌反应5分钟，再将另剩余铁粉(1.0g,17.86mmol)加入，继续搅拌反应20分钟。TLC检测原料反应完全后，将反应液趁热过滤，滤渣用乙醇(10mL)洗。减压旋去乙醇，用乙酸乙酯(20mL)萃取水层三次。合并有机相，用饱和食盐水洗涤，无水硫酸镁干燥，旋干，过柱(PE:EA＝8:1)，得淡黄色固体，收率92-94％。6a的收率为94％，6b的收率为92％。
7、6c的合成
Figure PCTCN2016099845-appb-000037
2,5-二甲基-1,3-二硝基苯(8)的合成
将对二甲苯(7)(360.0mg,3.40mmol)装于圆底烧瓶中，冰浴冷却至0℃，缓慢滴加，90％硝酸溶液和浓硫酸(2:1)的混酸溶液(1mL)，有固体析出，室温搅拌反应3h，TLC检测原料(7)反应完全后，将反应液倒入冰水中，过滤，滤饼真空干燥箱干燥12h，得 淡黄色固体545.8mg，收率82％。
2,5-二甲基-3-硝基苯胺(9)的合成
将2,5-二甲基-1,3-二硝基苯(8)(196.0mg,1.00mmol)溶于MeOH(6mL)和二氧六环(3mL)中，加入浓盐酸(0.60mL)和Fe(168.90mg,3.00mmol)，升至80℃搅拌反应12h。TLC检测原料(8)反应完全后，先过滤，再将反应液旋干，加少量水，饱和NaHCO3溶液将PH调至7～8，乙酸乙酯提取三次，乙酸乙酯层用饱和食盐水洗涤，无水硫酸镁干燥，旋干得淡黄色固体142.8mg，收率86％。
6-甲基-4-硝基-1H-吲唑(10)的合成
将2,5-二甲基-3-硝基苯胺(9)(83.0mg,0.50mmol)溶于冰醋酸(3mL)中，再将NaNO2(69.0mg,1.00mmol)溶于水(0.5mL)中，室温搅拌下将NaNO2的水溶液缓慢滴加到2,5-二甲基-3-硝基苯胺(9)的冰醋酸溶液中，有固体析出，室温搅拌反应13h。TLC检测原料(9)反应完全后，将反应液倒入冰水中，过滤，滤饼真空干燥箱干燥12h，得黄色固体79.6mg，收率90％。
6-甲基-1H-吲哚-4-胺(6c)的合成
将原料6-甲基-4-硝基-1H-吲唑(10)(50mg,0.28mmol)溶于乙醇(2mL)和水(1mL)的混合溶剂中，加入氯化铵(1.6mg,0.03mmol)，再加入铁粉(79.1mg,1.41mmol)加入其中，升温至80℃搅拌反应0.5h，TLC检测原料(10)反应完全后，将反应液趁热过滤，滤渣用乙醇洗。减压旋去乙醇，用乙酸乙酯萃取水层三次。合并有机相，用饱和食盐水洗涤，无水硫酸镁干燥，旋干，过柱(PE:EA＝10:1)，得34.9mg淡黄色固体，收率84％。
8、n5a和n5b的合成
Figure PCTCN2016099845-appb-000038
2,5-二甲基-1，3-二硝基苯(n2a)的合成
取干燥的50mL梨形瓶，以20mL浓硫酸将二甲苯(n1a)(2.00mL，16.23mmol)溶解，室温搅拌下缓慢加入硝酸钾(4.91g，48.68mmol)，加毕，室温搅拌反应2h，将反应液缓慢倒入冰水中，过滤，滤饼真空干燥，再经柱层析(PE:EA＝80：1)纯化得淡黄色固体(n2a)(1.62g，收率51％)。结构鉴定：1H-NMR(400MHz,CDCl3,ppm):δ13.0(br,1H,NH),7.42(s,2H,Ar-H),2.45(s,6H,CH3).ESI-MS:197.05[M+H].
2-甲基-5-三氟甲基-1,3-二硝基苯(n2b)的合成
以4-三氟甲基-甲苯(n1b)(CAS：6140-17-6，购于成都瑞欧克试剂公司)为原料， 按照制备2,5-二甲基-1，3-二硝基苯(n2a)的方法，制备得到2-甲基-5-三氟甲基-1,3-二硝基苯(n2b)，黄色固体，收率75％。结构鉴定：1H-NMR(400MHz,CDCl3,ppm):δ13.0(br,1H,NH),8.29(s,2H,Ar-H),2.69(s,3H,CH3).ESI-MS:251.02[M+H].
2,5-二甲基-3-硝基苯胺(3a)的合成
将2,5-二甲基-1,3-二硝基苯(n2a)(196.0mg,1.00mmol)溶于MeOH(6mL)和二氧六环(3mL)中，加入浓盐酸(0.60mL)和Fe(168.90mg,3.00mmol)，升至80℃搅拌反应12h。先过滤，再将反应液旋干，饱和NaHCO3溶液将pH调至7～8，EA提取三次，有机层用饱和食盐水洗涤，无水硫酸镁干燥，旋干得淡黄色固体142.8mg，收率86％。
2-甲基-5-三氟甲基-3-硝基苯胺(n3b)的合成
按照实施例1中制备5-溴-2-甲基-3-硝基苯胺(n3a)的方法，制备得到2-甲基-5-三氟甲基-3-硝基苯胺(n3b)，黄色固体，收率68％。
6-甲基-4-硝基-1H-吲唑(n4a)的合成
将2,5-二甲基-3-硝基苯胺(n9a)(83.0mg,0.50mmol)溶于冰醋酸(3mL)中，再将NaNO2(69.0mg,1.00mmol)溶于水(0.5mL)中，室温搅拌下将NaNO2的水溶液缓慢滴加到2,5-二甲基-3-硝基苯胺(n3a)的冰醋酸溶液中，有固体析出，室温搅拌反应13h后，将反应液倒入冰水中，过滤，滤饼真空干燥箱干燥12h，得黄色固体79.6mg，收率90％。纯度经HPLC测试为98％；结构鉴定：1H-NMR(400MHz,d6-DMSO,ppm):δ12.46(br,1H),8.20(s,1H),8.10(s,1H),7.87(s,1H),2.35(s,3H).ESI-MS:178.18[M+H].
6-三氟甲基-4硝基-1H-吲唑(n4b)的合成
按照制备6-溴-4硝基-1H-吲唑(n4a)的方法，制备得到6-三氟甲基-4硝基-1H-吲唑(n4b)，收率79％。纯度经HPLC测试为98％。结构鉴定：1H-NMR(400MHz,d6-DMSO,ppm):δ12.46(br,1H),8.20(s,1H),8.12(s,1H),7.80(s,1H).ESI-MS:232.13[M+H].
6-甲基-1H-吲哚-4-胺(n5a)的合成
将原料6-甲基-4-硝基-1H-吲唑(n4a)(50mg,0.28mmol)溶于乙醇(2mL)和水(1mL)的混合溶剂中，加入氯化铵(1.6mg,0.03mmol)，再加入铁粉(79.1mg,1.41mmol)，升温至80℃搅拌反应0.5h后，将反应液趁热过滤，滤渣用乙醇洗。减压旋去乙醇，用EA萃取水层三次。合并有机相，用饱和食盐水洗涤，无水硫酸镁干燥，旋干，过柱(PE:EA＝10:1)，得34.9mg淡黄色固体，收率84％。纯度经HPLC测试为98％；1H-NMR(400MHz,d6-DMSO,ppm):δ12.46(br,1H),7.70(s,1H),7.16(s,1H),6.85(s,1H),4.60(br,2H),2.21(s,3H).ESI-MS:148.10[M+H].
6-三氟甲基-1H-吲唑-4-胺(n5b)的合成
以化合物4-三氟甲基-甲苯(n4b)为原料。参照化合物n5a的合成方法得化合物n5b。黄色固体，收率95％。纯度经HPLC测试为98％；1H-NMR(400MHz,CDCl3,ppm):δ13.0(br,1H,NH),8.10(s,1H,indazole-H),7.21(s,1H,indazole-H),6.58(s,1H,indazole-H),4.37(br,2H,NH2).ESI-MS:202.05[M+H].
9、n 5c和n5d的合成
Figure PCTCN2016099845-appb-000039
将原料6-溴-4-硝基-1H-吲唑(n4c)或6-氯-4-硝基-1H-吲唑(n4d)(8.26mmol，购于江苏南通生物科技有限公司)溶于乙醇(20mL)和水(10mL)的混合溶剂中，加入氯化铵(221.5mg,4.13mmol)，先将一部分铁粉(1.3g,23.46mmol)加入其中，升温至80℃搅拌反应45分钟，再将另剩余铁粉(1.0g,17.86mmol)加入，继续搅拌反应20分钟。TLC检测原料反应完全后，将反应液趁热过滤，滤渣用乙醇(10mL)洗。减压旋去乙醇，用EA(20mL)萃取水层三次。合并有机相，用饱和食盐水洗涤，无水硫酸镁干燥，旋干，过柱(PE:EA＝8:1)，得淡黄色固体，收率为92-94％。纯度经HPLC测试均为98％；n5c的结构表征：1H-NMR(400MHz,d6-DMSO,ppm):δ12.46(br,1H),8.15(s,1H),7.56(s,1H),6.88(s,1H),5.80(br,2H).ESI-MS:213.05[M+H].n5d的结构表征：1H-NMR(400MHz,d6-DMSO,ppm):δ12.46(br,1H),8.14(s,1H),7.54(s,1H),6.89(s,1H),5.78(br,2H).ESI-MS:168.03[M+H].
实施例2 本发明化合物O17和O19的合成
Figure PCTCN2016099845-appb-000040
先用5mLDMF将6-溴-4-氟-1H-吲唑-1-羰基叔丁酯O15(100.0mg,0.32mmol)溶解，在常温搅拌下加入K2CO3(77.3mg,0.56mmol)和水(28.8μL,1.60mmol)，将反应液升至90℃搅拌反应1h，再加入水(5.76μL,0.32mmol)90℃继续搅拌反应1h，将反应液冷却至室温，加入K2CO3(88.3mg,0.64mmol)和3-(碘甲基)苯甲酸甲酯O5(176.7mg,0.64mmol)室温搅拌反应12h，TLC显示完全反应。加水，乙酸乙酯提取3次，用饱和食盐水洗涤，干燥，浓缩，粗品经柱层析(PE:EA＝40：1)纯化得白色固体化合物O16(60.0mg,收率41％)。
可选方法：
先用5mLDMF将6-溴-4-氟-1H-吲唑-1-羰基叔丁酯O15(100.0mg,0.32mmol)溶解，在常温搅拌下加入NaOH(22.4mg,0.56mmol)和水(28.8μL,1.60mmol)，将反应液室温搅拌反应1h，再加入水(5.76μL,0.32mmol)100℃继续搅拌反应1h，将反应液冷却至室温，加入Et3N(2mL,0.64mmol)和3-(碘甲基)苯甲酸甲酯O5(176.7mg,0.64mmol)室温搅拌反应12h，TLC显示完全反应。加水，乙酸乙酯提取3次，用饱和食盐水洗涤，干燥，浓缩，粗品经柱层析(PE:EA＝40：1)纯化得白色固体化合物O16(45.0mg,收率30％)。
用2.5mLCH2Cl2将6-溴-1H-吲唑-1-羰基叔丁酯O16(60.0mg，0.13mmol)溶解，在室温搅拌下加入0.5mL三氟乙酸，室温搅拌2h，TLC显示原料完全反应。旋去溶剂，用饱 和NaHCO3调至碱性，乙酸乙酯提取3次，用饱和食盐水洗涤，干燥，浓缩，得淡黄色固体化合物O17(46.0mg,收率98％)。
1H-NMR(400MHz,d6-DMSO,ppm):δ8.02(s,1H,indazole-H3),7.72(d,1H,J＝7.6Hz,Ar-H6),7.68(s,1H,Ar-H2),7.69(s,1H,indazole-NH),7.60(d,1H,J＝7.6Hz,Ar-H4),7.50(s,1H,indazole-H7),7.49(s,1H,indazole-H5),7.42(t,1H,J＝7.6Hz,Ar-H5),4.31(s,2H,benzyl-CH2),4.10(s,3H,CH3).ESI-MS:383.01[M+Na].
Figure PCTCN2016099845-appb-000041
以化合物O15和O6为原料，参照化合物O16的合成方法，制得白色固体化合物O18，收率55％。
以化合物O18为原料，参照化合物O17的合成方法制得淡黄色油状物化合物O19(0.046g，收率98％)。
1H-NMR(400MHz,d6-DMSO,ppm):δ7.98(s,1H,indazole-NH),7.81(d,2H,J＝8.0Hz,Ar-H2 and Ar-H6),7.62(s,1H,indazole-H3),7.50(d,2H,J＝8.0Hz,Ar-H3 and Ar-H5),7.51(s,1H,indazole-H7),7.49(s,1H,indazole-H5),4.30(s,2H,benzyl-CH2),4.10(s,3H,CH3).ESI-MS:383.01[M+Na].
实施例3 本发明化合物O20-25的制备
Figure PCTCN2016099845-appb-000042
用3mL水和3mL乙醇将化合物O19(50.5mg,0.14mmol)和NaOH(16.8mg,0.42mmol)溶解，反应液升温至100℃搅拌1h，TLC显示原料完全反应。将反应液冷却至室温，旋去溶剂，加水，用稀HCl将pH调至6～7时有白色固体析出，过滤，真空干燥箱干燥得白色固体化合物O20(39.8mg,收率82％)。
1H-NMR(400MHz,d6-DMSO,ppm):δ12.89(br,1H,COOH),7.93(s,1H,indazole-NH),7.87(d,2H,J＝8.0Hz,Ar-H2 and Ar-H6),7.66(s,1H,indazole-H3),7.53(d,2H,J＝8.0Hz,Ar-H3 and Ar-H5),7.51(s,1H,indazole-H7),7.49(s,1H,indazole-H5),4.34(s,2H,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ167.7,162.8,160.4,140.9,134.2,130.1,129.9,129.7,128.2,125.3,121.3,116.0,115.7,114.4.ESI-MS:369.00[M+Na].
Figure PCTCN2016099845-appb-000043
化合物O21(32.1mg,收率84％)以化合物O17(39.7mg,0.11mmol)为原料，按照 化合物O20的制备方法得到，无色固体。
1H-NMR(400MHz,d6-DMSO,ppm):δ12.85(br,1H,COOH),8.04(s,1H,indazole-H3),7.78(d,1H,J＝7.6Hz,Ar-H6),7.70(s,1H,Ar-H2),7.69(s,1H,indazole-NH),7.67(d,1H,J＝7.6Hz,Ar-H4),7.50(s,1H,indazole-H7),7.49(s,1H,indazole-H5),7.44(t,1H,J＝7.6Hz,Ar-H5),4.33(s,2H,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ167.8,162.9,160.4,137.1,135.0,131.8,129.6,129.3,128.5,126.3,121.3,116.0,115.7,114.5.ESI-MS:369.00[M+Na].
Figure PCTCN2016099845-appb-000044
化合物O22的合成与化合物O16类似，收率52％。
将化合物O22(57.0mg,0.14mmol)溶于10mL的4mol/L盐酸中，室温搅拌10h，TLC显示原料完全反应。将反应液冷却至室温，乙酸乙酯提取3次，用饱和食盐水洗涤，干燥，浓缩，得白色固体化合物O23(31.2mg,收率70％)。1H-NMR(400MHz,d6-DMSO,ppm):δ8.05(s,1H,indazole-H3),7.69(s,1H,indazole-NH),7.50(s,1H,indazole-H7),7.49(s,1H,indazole-H5),7.40-6.80(m,4H,Ar-H),4.20(s,2H,benzyl-CH2).ESI-MS:319.00[M+H].
Figure PCTCN2016099845-appb-000045
化合物O24的合成与化合物O16类似，收率50％。
化合物O25的合成与化合物O19类似，收率40％。
1H-NMR(400MHz,d6-DMSO,ppm):δ8.05(s,1H,indazole-H3),7.69(s,1H,indazole-NH),7.50(s,1H,indazole-H7),7.49(s,1H,indazole-H5),7.25-6.68(m,4H,Ar-H),4.18(s,2H,benzyl-CH2),2.5(br,2H,NH2).ESI-MS:318.02[M+H].
实施例4 本发明化合物3a、3b和3c的合成
Figure PCTCN2016099845-appb-000046
2-(6-溴-1H-吲唑-4-氨基)乙酸乙酯(11)的合成
将6-溴-1H-吲唑-4-氨基6b(212.0mg,1.00mmol)溶于DMF(5mL)中，加入碳酸钾(345.0mg,2.50mmol)和碘化钾(14.9mg,0.09mmol)。氩气保护下，加入溴乙酸乙酯(167.0μL,1.50mmol)，65℃反应过夜。TLC检测原料(6b)反应完全后，用油泵抽走大量的DMF，加入乙酸乙酯(25mL)，用水(20mL)洗三次。乙酸乙酯层用饱和食盐水洗涤，无水硫酸镁干燥，旋干过柱(PE:EA＝15:1)。得淡黄色固体206.9mg，收率70％。
2-(6-溴-1H-吲唑-4-氨基)乙酸(12)的合成
将2-(6-溴-1H-吲唑-4-氨基)乙酸乙酯(11)(80.0mg,0.27mmol)溶于乙醇(1.5mL)和水(1.5mL)中，加入氢氧化钠(32.2mg,0.81mmol)，室温搅拌反应过夜。TLC检测原料反应完毕后，用1mol/L的稀盐酸调pH为5，减压旋干反应液，直接用于下一步反应，收率100％
2-(6-溴-1H-吲唑-4-氨基)-N-四氢-2H-吡喃-4-乙酰胺(3a)的合成
将酸12(50.0mg,0.19mmol)溶于二氯甲烷(3mL)中，置于冰浴下，滴入四氢-2H-吡喃-4-胺13(57uL,0.56mmol)，分别将EDCI(42.6mg,0.22mmol)、HoBt(32.4mg,0.22mmol)、DIEA(61uL,0.37mmol)加入，0℃反应5分钟后，升至室温(25℃)搅拌过夜。TLC检测待原料(12)反应完全后，反应液用饱和碳酸氢钠(1.5mL)洗涤，有机相旋干、过柱(PE:EA＝10:1)，得白色粉末42.1mg，收率65％。
1H-NMR(400MHz,d6-DMSO,ppm):δ8.22(d,1H,J＝7.5Hz,amide),8.07(s,1H,indazole-H3),6.87(s,1H,indazole-H7),6.29(d,1H,J＝1.1Hz,indazole-NH),6.14(s,1H,indazole-H5),4.90(s,1H,CH2),3.85-3.76(m,4H,pyran-H2 and pyran-H6),3.52-3.48(m,1H,pyran-H4),3.42-3.34(m,4H,pyran-H3 and pyran-H5).ESI-MS:375.0452[M+Na].
2-(6-溴-1H-吲唑-4-氨基)-N-(4-乙氧羰基苯基)乙酰胺(3c)的合成
以酸12(50.0mg,0.19mmol)和4-氨基苯甲酸乙酯14(91.5mg,0.56mmol)为原料，按照3a的制备方法得到，得到3c 46.4mg，收率60％。1H-NMR(400MHz,d6-DMSO,ppm):δ10.67(s,1H,amide),8.13(s,1H,indazole-H3),7.82(d,2H,J＝8.7Hz,Ar-H2 and Ar-H6),7.74(d,2H,J＝8.7Hz,Ar-H3 and Ar-H5),6.98(s,1H,indazole-H7),6.32(d,1H,J＝1.2Hz,indazole-NH),6.18(s,1H,indazole-H5),5.21(s,2H,benzyl-CH2),4.48(q,2H,J＝6.8Hz,CH 2CH3),1.30(t,3H,J＝6.8Hz,CH2CH 3).ESI-MS:417.03[M+H].
2-(6-溴-1H-吲唑-4-氨基)-N-(4-羧基苯基)乙酰胺(3b)的合成
将酰胺3c(40.0mg,0.10mmol)溶于乙醇(1.0mL)和水(1.0mL)中，加入氢氧化钠(11.5mg,0.29mmol)，室温搅拌过夜。TLC检测待原料反应完全后，用1mol/L的稀盐酸调pH为5，有白色固体析出，过滤，减压干燥，得15.6mg白色固体，收率42％。
1H-NMR(400MHz,d6-DMSO,ppm):δ12.80(br,1H,COOH),10.67(s,1H,amide),8.13(s,1H,indazole-H3),7.91(d,2H,J＝8.7Hz,Ar-H2 and Ar-H6),7.79(d,2H,J＝8.7Hz,Ar-H3 and Ar-H5),6.98(s,1H,indazole-H7),6.32(d,1H,J＝1.2Hz,indazole-NH),6.18(s,1H,indazole-H5),5.21(s,2H,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ207.0,167.3,166.7,143.9,143.1,132.9,131.0,126.0,121.9,118.9,113.2,104.7,99.6,52.2.ESI-MS:389.03[M+H].
实施例5 本发明化合物1a、1c-j和1p的合成
Figure PCTCN2016099845-appb-000047
将胺6b(0.28mmol)和苯甲醛16(0.24mmol)溶于二氯甲烷(DCM，3mL)中，加入二氢吡啶(DHP，83.5mg,0.33mmol)和适量4A分子筛(840.2mg)，滴入三氟醋酸(TFA，17.6μL,0.24mmol)，40℃回流12小时，将反应液过滤，旋干，过柱得化合物1a。
4-((6-溴-1H-吲唑-4-氨基)甲基)苯甲酸(1a).收率64％；棕色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.80(br,2H,COOH and indazole-NH),8.22(s,1H,indazole-H3),7.91(d,2H,J＝8.3Hz,Ar-H2 and Ar-H6),7.49(d,2H,J＝8.3Hz,Ar-H3 and Ar-H5),6.64(s,1H,indazole-H7),6.01(s,1H,indazole-H5),4.53(s,2H,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ167.7,145.3,143.0,142.2,132.5,130.0,129.9,127.5,121.7,112.6,101.2,100.6,46.2.ESI-MS:344.0035[M-H].
选择相应的醛原料，按照类似的方法制备得到化合物1c-j和1p，结果和表征如下：
Figure PCTCN2016099845-appb-000048
N-苄基-6-溴-1H-吲唑-4-胺(1c).收率43％；棕色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.83(s,1H,indazole-NH),8.23(s,1H,indazole-H3),7.40-7.23(m,5H,Ar-H),6.83(s,1H,indazole-H7),6.06(s,1H,indazole-H5),4.44(d,2H,J＝6.0Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ143.2,142.1,139.9,132.7,128.9,127.5,127.3,121.7,112.6,101.2,100.3,46.4.ESI-MS:302.02[M+H].
Figure PCTCN2016099845-appb-000049
3-((6-溴-1H-吲唑-4-氨基)甲基)苯甲酸(1d).收率60％；棕色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.90(br,2H,COOH and indazole-NH),8.22(s,1H,indazole-H3),7.98(s,1H,Ar-H2),7.83(d,1H,J＝7.6Hz,Ar-H4),7.64(d,1H,J＝7.6Hz,Ar-H6),7.48(t,1H,J＝7.6Hz,Ar-H5),7.37(t,1H,J＝6.0Hz,NH),6.84(s,1H,indazole-H7),6.05(s,1H,indazole-H5),4.51(d,2H,J＝6.0Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ167.8,143.0,142.2,140.6,132.5,132.1,131.4,129.1,128.3,128.3,121.7,112.6,101.2,100.5,46.0.ESI-MS:344.00[M-H].
Figure PCTCN2016099845-appb-000050
6-溴-N-(4-甲基苄基)-1H-吲唑-4-胺(1e).收率65％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.82(s,1H,indazole-NH),8.22(s,1H,indazole-H3),7.26(d,2H,J＝7.9Hz,Ar-H2 and Ar-H6),7.14(d,2H,J＝7.9Hz,Ar-H3 and Ar-H5),6.82(s,1H,indazole-H7),6.04(s,1H,indazole-H5),4.38(d,2H,J＝6.0Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ143.2,141.2,136.8,136.3,132.7,129.4,127.5,121.7,112.6,101.2,100.2,46.2,21.1.ESI-MS:316.03[M+H].
Figure PCTCN2016099845-appb-000051
6-溴-N-(4-氯苄基)-1H-吲唑-4-胺(1f).收率71％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.85(s,1H,indazole-NH),8.21(s,1H,indazole-H3),7.40(s,4H,Ar-H),7.32(t,1H,J＝6.0Hz,NH),6.84(s,1H,indazole-H7),6.03(s,1H,indazole-H5),4.43(d,2H,J＝6.0Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ142.9,142.1,139.0,132.6,131.8,129.3,127.5,121.7,112.6,101.3,100.5,45.7.ESI-MS:335.97[M+H].
Figure PCTCN2016099845-appb-000052
4-((6-溴-1H-吲唑-4-氨基)甲基)苯酚(1g).收率59％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.80(s,1H,indazole-NH),9.29(s,1H,indazole-H3),8.21(s,1H,OH),7.17(d,2H,J＝8.4Hz,Ar-H3 and Ar-H5),6.81(s,1H,indazole-H7),6.72(d,2H,J＝8.4Hz,Ar-H2 and Ar-H6),6.06(s,1H,indazole-H5),4.29(d,2H,J＝6.0Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ156.7,143.2,142.8,132.7,129.7,128.8,121.8,115.6,112.6,101.2,100.0,46.1.ESI-MS:318.03[M+H].
Figure PCTCN2016099845-appb-000053
5-(((6-溴-1H-吲唑-4-氨基)甲基)-2-羟基苯甲酸hydroxybenzoic acid(1h).收率82％；棕色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.80(br,2H,COOH and indazole-NH),8.21(s,1H,indazole-H3),7.82(s,1H,Ar-H6),7.46(d,1H,J＝8.4Hz,Ar-H4),7.24(t,1H,J＝6.0Hz,NH),6.87(d,2H,J＝8.4Hz,Ar-H3),6.83(s,1H,indazole-H7),6.06(s,1H,indazole-H5),4.35(d,2H,J＝6.0Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ167.8,143.0,142.2,140.6,132.5,132.1,131.4,129.2,128.3,121.7,117.3,112.6,101.1,100.4,49.1.ESI-MS:360.02[M-H].
Figure PCTCN2016099845-appb-000054
2-(4-((6-溴-1H-吲唑-4-氨基)甲基)苯基)乙酸(1i).收率81％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.80(br,2H,COOH and indazole-NH),8.23(s,1H,indazole-H3),7.33(d,2H,J＝8.0Hz,Ar-H2 and Ar-H6),7.23(d,2H,J＝8.0Hz,Ar-H3 and Ar-H5),6.83(s,1H,indazole-H7),6.05(s,1H,indazole-H5),4.40(s,2H,CH 2NH),3.18(s,2H,CH 2COOH).13C-NMR(100MHz,d6-DMSO,ppm):δ173.2,143.2,142.2,138.1,134.0,132.6,129.9,127.5,121.8,112.6,101.2,100.3,49.1,46.2.ESI-MS:358.03[M-H].
Figure PCTCN2016099845-appb-000055
2-((6-溴-1H-吲唑-4-氨基)甲基)苯酚(1j).收率63％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.81(s,1H,indazole-NH),9.63(s,1H,indazole-H3),8.22(s,1H,indazole-H7),7.19-7.05(m,2H,Ar-H4 and Ar-H6),6.87-6.72(m,2H,Ar-H3 and Ar-H5),6.05(s,1H,indazole-H5),4.34(d,2H,J＝5.8Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ155.4,143.4,142.1,132.7,128.5,128.2,125.3,121.8,119.4,115.4,112.5,100.9,100.0,49.1.ESI-MS:318.02[M+H].
Figure PCTCN2016099845-appb-000056
6-溴-N-(2-硝基苄基)-1H-吲唑-4-胺(1p).收率70％，黄色固体。1H-NMR(400MHz,d6-DMSO,ppm):δ12.20(s,1H,indazole-NH),8.31(s,1H,indazole-H3),7.98-7.55(m,4H,Ar-H),6.69(d,1H,J＝7.9Hz,indazole-H7),6.01(d,1H,J＝7.1Hz,indazole-H5),4.56(s,2H,benzyl-CH2).ESI-MS:346.11[M+H]。
实施例6 本发明化合物1b的合成
Figure PCTCN2016099845-appb-000057
按照实施例3中类似的方法合成化合物1b，区别在于以化合物6a替代化合物6b，得化合物1b。
4-((1H-吲唑-4-氨基)甲基)苯甲酸(1b).收率62％；棕色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.79(br,2H,COOH and indazole-NH),8.22(s,1H,indazole-H3),7.90(d,2H,J＝7.4Hz,Ar-H2 and Ar-H6),7.50(d,2H,J＝7.4Hz,Ar-H3 and Ar-H5),6.99-6.96(m,1H,indazole-H6),6.66(d,1H,J＝7.9Hz,indazole-H7),5.90(d,1H,J＝7.1Hz,indazole-H5),4.52(s,2H,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ167.7,146.1,142.0,141.7,132.1,129.9,129.7,128.0,127.5,113.8,98.2,98.1,46.5.ESI-MS:266.11[M-H].
实施例7 本发明化合物1k的合成
Figure PCTCN2016099845-appb-000058
(1S,2S)-2-羟基环己烷甲醛(17)的合成
将庚二醛(40.0mg,0.31mmol)溶于DCM(2mL)中，加入L-脯氨酸(3.6mg,0.031mmol)，室温搅拌反应两小时。TLC检测待原料庚二醛反应完全后。旋干，过柱(PE:EA＝6:1)，得白色液体34.5mg，收率86％。
(1S,2R)-2-((6-溴-1H-吲唑-4-氨基)甲基)环己醇(1k)的合成
制备化合物1k(56.3mg,0.17mmol，收率62％)：以胺6b(60.0mg,0.28mmol)和醛17(30.7mg,0.24mmol)为原料，按照1a的制备方法得到。
黄色固体。1H-NMR(400MHz,CDCl3,ppm):δ7.94(s,1H,indazole-H3),6.96(s,1H,indazole-H7),6.33(s,1H,indazole-H5),3.56-3.50(m,1H,cyclohexanol-H1),3.35-3.25(m,2H,CH2NH),2.03-2.00(m,1H,cyclohexanol-H3),1.88-1.71(m,4H,cyclohexanol-H3 and H6),1.39-1.25(m,4H,cyclohexanol-H4 and H5).13C-NMR(100MHz,d6-DMSO,ppm):δ142.8,141.8,131.9,123.0,112.7,102.8,101.1,49.3,44.1,36.1,30.9,29.7,25.2,24.6.ESI-MS:346.0498[M+Na].
实施例8 本发明化合物1l、1m、1n和1o的合成
合成路线如下所示：
Figure PCTCN2016099845-appb-000059
(1)4-(1-((6-溴-1H-吲唑-4-氨基)-2-羟乙基)苯甲酸甲酯(1m)和4-(2-((6-溴-1H-吲唑-4-氨基)-1-羟乙基)苯甲酸甲酯(1o)的合成
将环氧18(229.2mg,1.42mmol)和苯胺6b(300.0mg,1.42mmol)溶于乙醇和水1:1的混合溶液(20mL)中，升温至100℃回流反应过夜。TLC检测待原料(18)反应完全后，减压旋走乙醇，用乙酸乙酯(15mL)萃取反应液3次，合并有机相，用饱和食盐水洗涤，无水硫酸镁干燥，旋干，过柱。先用PE:EA＝15:1冲去小极性杂质，再用PE:EA＝6:1洗脱化合物1m，最后用PE:EA＝3:1洗脱化合物1o(总收率43％)。
化合物1m:收率20％；棕色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.87(s,1H,indazole-NH),8.33(s,1H,indazole-H3),7.92(d,2H,J＝8.3Hz,Ar-H2 and Ar-H6),7.57(d,2H,J＝8.3Hz,Ar-H3 and Ar-H5),6.94(d,1H,J＝6.8Hz,NH),6.82(s,1H,indazole-H7),5.88(s,1H,indazole-H5),5.11(t,1H,J＝5.3Hz,OH),4.69-4.64(m,1H,NHCH),3.83(s,3H,OCH3),3.76-3.71(m,2H,OHCH 2).
化合物1o:收率23％；棕色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.81(s,1H,indazole-NH),8.18(s,1H,indazole-H3),7.94(d,2H,J＝8.3Hz,Ar-H2 and Ar-H6),7.56(d,2H,J＝8.3Hz,Ar-H3 and Ar-H5),6.83(s,1H,indazole-H7),6.67(t,1H,J＝5.7Hz,NH),6.19(s,1H,indazole-H5),5.77(d,1H,J＝4.4Hz,OH),4.93-4.89(m,1H,OHCH),3.86(s,3H,OCH3),3.34(m,2H,NHCH 2).
(2)4-(1-((6-溴-1H-吲唑-4-氨基)-2-羟乙基)苯甲酸(1l)和4-(2-(6-溴-1H-吲唑-4-氨基)-1-羟乙基)苯甲酸(1n)的合成
以酯1m(45.0mg,0.11mmol)为原料，按照实施例2中3b的制备方法得到化合物1l(38mg,0.10mmol)，棕色固体。
1H-NMR(400MHz,d6-DMSO,ppm):δ12.87(br,2H,COOH and indazole-NH),8.35(s,1H,indazole-H3),7.91(d,2H,J＝8.2Hz,Ar-H2 and Ar-H6),7.54(d,2H,J＝8.2Hz,Ar-H3and Ar-H5),6.97(d,1H,J＝6.7Hz,NH),6.81(s,1H,indazole-H7),5.89(s,1H,indazole-H5),5.13(br,1H,OH),4.68-4.63(m,1H,NHCH),3.75-3.71(m,2H,OHCH 2).13C-NMR(100MHz,d6-DMSO,ppm):δ167.7,147.0,142.5,142.2,132.7,131.0,129.9,127.5,121.5,112.7,101.9,100.6,65.8,59.8,49.1.ESI-MS:376.0299[M+H].
以酯1o(45.0mg,0.11mmol)为原料，按照实施例2中3b的制备方法得到化合物1n(38mg，收率87％)，棕色固体。
1H-NMR(400MHz,d6-DMSO,ppm):δ12.85(br,2H,COOH and indazole-NH),8.20(s,1H,indazole-H3),7.92(d,2H,J＝8.3Hz,Ar-H2 and Ar-H6),7.53(d,2H,J＝8.3Hz,Ar-H3 and Ar-H5),6.84(s,1H,indazole-H7),6.71(t,1H,J＝5.7Hz,NH),6.19(s,1H,indazole-H5),5.78(br,1H,OH),4.91(m,1H,OHCH),4.13(m,1H,NHCH).13C-NMR(100MHz,d6-DMSO,ppm):δ167.8,149.4,143.2,142.2,132.6,129.6,126.7,121.9,112.6,100.8,100.2,70.6,51.2.ESI-MS:376.0293[M+H].
实施例9 本发明化合物1q的合成
Figure PCTCN2016099845-appb-000060
将硝基化合物1p(2.9g,8.26mmol)溶于乙醇(20mL)和水(10mL)的混合溶剂中，加入氯化铵(221mg,4.13mmol)，先将一部分铁粉(1.314g,23.46mmol)加入其中，升温至80℃搅拌反应5分钟，再将另剩余铁粉(1g,17.86mmol)加入，继续搅拌反应20分钟。TLC检测原料1p反应完全后，将反应液趁热过滤，滤渣用乙醇(10mL)洗。减压旋去乙醇，用乙酸乙酯(20mL)萃取水层三次。合并有机相，用饱和食盐水洗涤，无水硫酸镁干燥，旋干，过柱((PE:EA＝3:1))，得2.4g淡黄色固体，收率92.1％。
1H-NMR(400MHz,d6-DMSO,ppm):δ12.21(s,1H,indazole-NH),8.30(s,1H,indazole-H3),6.69(d,1H,J＝7.9Hz,indazole-H7),6.91-6.01(m,5H,Ar-H,indazole-H5),4.35(s,2H,benzyl-CH2).ESI-MS:316.13[M+H].
实施例10 本发明化合物1r的合成
Figure PCTCN2016099845-appb-000061
按照实施例3中类似的方法合成化合物1r，区别在于以化合物6c替代化合物6b，得化合物1r。
4-((6-甲基-1H-吲唑-4-氨基)甲基)苯甲酸(1r).收率70％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.79(br,2H,COOH and indazole-NH),8.23(s,1H,indazole-H3),7.91(d,2H,J＝7.4Hz,Ar-H2 and Ar-H6),7.43(d,2H,J＝7.4Hz,Ar-H3 and Ar-H5),6.62(d,1H,J＝7.9Hz,indazole-H7),5.84(d,1H,J＝7.1Hz,indazole-H5),4.48(s,2H,benzyl-CH2),2.38(s,3H,CH3).13C-NMR(100MHz,d6-DMSO,ppm):δ167.7,146.1,142.0,141.7,132.1,129.9,129.7,128.0,127.5,113.8,98.2,98.1,46.5,21.5.ESI-MS:281.12[M+H].
实施例11 本发明化合物2a-d的合成
Figure PCTCN2016099845-appb-000062
将原料(6b)(50mg,0.185mmol)溶于二氯甲烷(3mL)中，置于冰浴下，滴入化 合物21(97.6mg,0.708mmol)，分别将EDCI(62.4mg,0.329mmol)、HOBT(48mg,0.329mmol)、DIEA(77.5uL,0.470mmol)加入，0℃反应5分钟后，升至室温(30℃)搅拌过夜。TLC检测待原料(6a)反应完全后，反应液用水(1mL)洗涤，再用饱和碳酸氢钠(1.5mL)洗涤，有机相旋干、过柱，得2a。黄色固体27.1mg，结构表征如下：
3-((6-溴-1H-吲唑-4-氨基甲酰))苯酚(2a)
1H-NMR(400MHz,d6-DMSO,ppm):δ12.83(br,2H,indazole-NH and Ar-OH),8.52(s,1H,indazole-H3),7.62(s,1H,indazole-H7),6.76-8.09(m,4H,Ar-H),7.15(m,1H,Ar-H5),6.76(s,1H,indazole-H5),6.26(s,1H,NHCO).ESI-MS:332.01[M+H].
按照类似的方法，选择相应取代基的苯甲酸，制备得到2b黄色固体35.3mg，收率45.3％。棕黄色固体2c，36.3mg，收率41.5％。结构表征如下：
Figure PCTCN2016099845-appb-000063
3-((6-溴-1H-吲唑-4-氨基甲酰))苯胺(2b)
1H-NMR(400MHz,d6-DMSO,ppm):δ12.75(s,1H,indazole-NH),8.50(s,1H,indazole-H3),7.64(s,1H,indazole-H7),7.18(d,1H,J＝7.9Hz,Ar-H6),7.15(m,1H,Ar-H5),7.13(s,1H,indazole-H5),7.12(s,1H,Ar-H2),7.05(d,1H,J＝7.6,Ar-H4),6.54(s,1H,NHCO).ESI-MS:331.01[M+H].
Figure PCTCN2016099845-appb-000064
4-((6-溴-1H-吲唑-4-氨基甲酰))苯甲酸(2c)
1H-NMR(400MHz,d6-DMSO,ppm):δ13.12(br,2H,COOH and indazole-NH),8.56(s,1H,indazole-H3),8.07(d,2H,J＝8.3Hz,Ar-H2 and Ar-H6),8.03(d,2H,J＝8.5Hz,Ar-H3 and Ar-H5),7.67(s,1H,indazole-H7),6.70(s,1H,indazole-H5),6.62(s,1H,NHCO).ESI-MS:359.98[M+H].
实施例12 本发明化合物4a和4b的合成
Figure PCTCN2016099845-appb-000065
用5mLTHF将化合物(22)(0.63mmol)和三乙胺(0.27ml，1.95mmol)溶解，在0℃ 搅拌下加入化合物(23)(0.06g，0.20mmol)，同温搅拌反应3h后加入化合物(6b)(0.09g，0.42mmol)，加毕，升至室温搅拌反应12h后TLC显示完全反应。直接将反应液旋干得粗品，粗品经柱层析(PE:EA＝2：1)纯化得白色固体粉末0.07g，收率41％。
化合物4a：1H-NMR(400MHz,d6-DMSO,ppm):δ12.40(s,1H,indazole-NH),10.12(br,1H,OH),8.90(s,1H,NHCONH),8.35(s,1H,NHCONH),8.20(s,1H,indazole-H3),7.31-7.68(m,4H,Ar-H),6.87(d,1H,J＝7.9Hz,indazole-H7),5.99(d,1H,J＝7.1Hz,indazole-H5).ESI-MS:347.11[M+H].
按照类似的方法合成化合物4b，区别在于以化合物3-氨基苯酚替代化合物22，得化合物4b，白色固体粉末，收率45％。
4b:1H-NMR(400MHz,d6-DMSO,ppm):δ12.40(s,1H,indazole-NH),10.12(br,1H,OH),8.90(s,1H,NHCONH),8.65(s,1H,NHCONH),8.20(s,1H,indazole-H3),7.38(d,2H,J＝7.4Hz,Ar-H2 and Ar-H6),6.87(d,1H,J＝7.9Hz,indazole-H7),6.73(d,2H,J＝7.4Hz,Ar-H3 and Ar-H5),5.99(d,1H,J＝7.1Hz,indazole-H5).ESI-MS:347.11[M+H].
实施例13 本发明化合物4c和4d的合成
Figure PCTCN2016099845-appb-000066
用4mLCH2Cl2和4mL饱和NaHCO3将化合物(25)(0.34mmol)溶于圆底烧瓶中，在0℃搅拌下加入化合物(23)(0.03g，0.10mmol)，同温搅拌反应15min后TLC显示完全反应。将反应液倒入分液漏斗中，收集有机层，干燥，浓缩，未经纯化直接投下一步。
用5mLCH2Cl2将化合物(26)(0.34mmol)和化合物(6b)(0.06g，0.28mmol)溶解，置于0℃搅拌反应10h后TLC显示完全反应。直接将反应液旋干得粗品，粗品经柱层析(PE:EA＝3：1)纯化得白色固体粉末0.05g，收率51％。
4c:1H-NMR(400MHz,d6-DMSO,ppm):δ12.40(s,1H,indazole-NH),10.12(br,1H,OH),8.90(s,1H,NHCONH),7.62(s,1H,NHCONH),8.20(s,1H,indazole-H3),6.95(d,2H,J＝7.4Hz,Ar-H2 and Ar-H6),6.71(d,1H,J＝7.9Hz,indazole-H7),6.73(d,2H,J＝7.4Hz,Ar-H3 and Ar-H5),5.99(d,1H,J＝7.1Hz,indazole-H5),4.25(s,2H,benzyl-CH2).ESI-MS:361.01[M+H].
按照类似的方法合成化合物4d，区别在于以化合物3-羟基苄胺替代化合物25，得化合物4d，白色固体粉末，收率55％
4d:1H-NMR(400MHz,d6-DMSO,ppm):δ12.40(s,1H,indazole-NH),10.12(br,1H,OH),8.90(s,1H,NHCONH),7.62(s,1H,NHCONH),8.20(s,1H,indazole-H3),6.71(d,1H,J＝7.9Hz,indazole-H7),5.99(d,1H,J＝7.1Hz,indazole-H5),7.10-6.72(m,4H,Ar-H),4.43(s,2H,benzyl-CH2).ESI-MS:361.01[M+H].
实施例14 本发明化合物N1、N2、N3、N4和N5的合成
Figure PCTCN2016099845-appb-000067
6-溴-N-(3-三氟甲基苄基)-1H-吲唑-4-胺(N1).
将胺n5c(0.28mmol)和苯甲醛n6(0.24mmol)溶于二氯甲烷(DCM，3mL)中，加入DHP(83.5mg,0.33mmol)和适量
Figure PCTCN2016099845-appb-000068
(840.2mg)，滴入三氟醋酸(TFA，17.6μL,0.24mmol)，40℃回流12小时，将反应液过滤，旋干，过柱(PE:EA＝10:1)得化合物N1。收率53.9％；红色粉末状固体；1H-NMR(400MHz,d6-CDCl3,ppm):δ8.00(s,1H,indazole-H3),7.69(s,1H,Ar-H),7.63-7.50(m,3H,Ar-H),7.06(s,1H,Ar-H),6.34(s,1H,Ar-H),4.56(d,2H,J＝4.4Hz,benzyl-CH2).13C-NMR(100MHz,d6-CDCl3,ppm):δ141.9,141.7,139.2,131.7,131.4,130.8,129.4,124.6,124.3,122.9,112.5,103.5,102.2,47.6.ESI-MS:370.01[M+H].
选择相应的醛原料，按照类似的方法制备得到化合物N2、N3、N4、N5，结果和表征如下：
Figure PCTCN2016099845-appb-000069
4-((6-溴-1H-吲唑-4-氨基)甲基)苯并噻吩(N2).收率31.8％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ14.09(s,1H,indazole-NH),7.90(d,1H,J＝7.0Hz,Ar-H),7.80(d,1H,J＝7.0Hz,Ar-H),7.48(s,1H,NH),7.44(s,1H,Ar-H),7.22(s,1H,Ar-H),6.70(s,1H,Ar-H),6.59(s,1H,Ar-H),4.76(d,2H,J＝5.0Hz,benzyl-CH2).ESI-MS:356.99[M+H].
Figure PCTCN2016099845-appb-000070
3-((6-溴-1H-吲唑-4-氨基)甲基)苯酚(N3).收率49.6％；淡黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.97(s,1H,indazole-NH),9.63(s,1H,OH),8.23(s,1H,indazole-H),7.12(t,2H,J＝7.7Hz,NH),6.82-6.78(m,3H,Ar-H),6.03(d,1H,J＝0.9Hz,Ar-H),5.77(s,1H,Ar-H),4.36(d,2H,J＝5.8Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ158.0,143.2,141.4,132.6,129.9,121.8,118.0,115.3,114.2,114.0,112.6,101.1,100.1,46.3.ESI-MS:318.02[M+H].
Figure PCTCN2016099845-appb-000071
4-((6-溴-1H-吲唑-4-氨基)甲基)苯甲醇(N4).收率49.6％；淡黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.84(s,1H,indazole-NH),8.22(s,1H,indazole-H3),7.35-7.27(m,5H,Ar-H),6.82(s,1H,NH),6.03(d,1H,J＝1.1Hz,Ar-H),5.15(s,1H,OH),4.47(s,2H,CH 2NH),4.42(s,2H,CH 2COOH).13C-NMR(100MHz,d6-DMSO,ppm):δ143.2,142.2,141.6,138.2,132.6,127.2,127.1,121.7,112.6,101.2,100.3,63.2,48.2.ESI-MS:332.03[M-H].
Figure PCTCN2016099845-appb-000072
6-溴-N-(4-硝基苄基)-1H-吲唑-4-胺(N5).淡棕色固体；收率63.1％；1H-NMR(400MHz,d6-DMSO,ppm):δ12.90(s,1H,indazole-NH),8.23(d,2H,J＝3.8Hz,Ar-H),8.22(s,1H,indazole-H),7.65(d,2H,J＝8.6Hz,Ar-H),7.48(s,1H,-NH),6.86(s,1H,Ar-H),6.01(s,1H,Ar-H),4.62(d,2H,J＝4.8Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ148.5,147.8,147.0,142.7,132.5,128.5,124.1,121.7,112.6,101.3,45.8.ESI-MS:347.01[M+H].
实施例15 本发明化合物N6和N7的合成
Figure PCTCN2016099845-appb-000073
4-((6-甲基-1H-吲唑-4-氨基)甲基)苯甲酸(N6).
按照实施例14中类似的方法合成化合物N6，区别在于以化合物n5a替代化合物n5c，得化合物N6。收率42.0％；淡黄色粉末状固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.72(s,2H,COOH and indazole-NH),8.12(s,2H,indazole-H3 and NH),7.91(d,2H,J＝8.1Hz,Ar-H),7.50(d,2H,J＝8.1Hz,Ar-H),6.47(s,1H,Ar-H),5.77(s,1H,Ar-H),4.50(s,2H,benzyl-CH2),2.21(s,3H,CH3),ESI-MS:282.12[M+H].
4-((6-甲基-1H-吲唑-4-氨基)甲基)苯甲醇(N7).
Figure PCTCN2016099845-appb-000074
选择相应的醛原料，按照类似的方法制备得到化合物N7，收率53.7％；淡黄色粉末状固体；1H-NMR(400MHz,d6-DMSO,ppm):δ9.23(s,2H,indazole-NH and OH),8.12(s,1H,indazole-H),7.19(d,2H,J＝8.4Hz,Ar-H),6.72(d,2H,J＝8.4Hz,Ar-H),6.47(s,1H,Ar-H),5.87(s,1H,Ar-H),4.29(d,2H,J＝8.4Hz,benzyl-CH2),4.29(s,3H,CH3).ESI-MS:254.12[M+H].
实施例16 本发明化合物N8、N9、N10、N11和N12的合成
Figure PCTCN2016099845-appb-000075
6-三氟甲基-N-(3-三氟甲基苄基)-1H-吲唑-4-胺(N8).
按照实施例14中类似的方法合成化合物N8，区别在于以化合物n5b替代化合物n5c，得化合物N8。收率34.5％；黄色固体；1H-NMR(400MHz,d6-CDCl3,ppm):δ10.92(s,1H,indazole-NH),8.11(s,1H,indazole-H3),7.72(s,1H,Ar-H),7.65-7.51(m,2H,Ar-H and NH),7.20(s,1H,Ar-H),6.41(s,1H,Ar-H),4.61(d,2H,J＝5.2Hz,benzyl-CH2).13C-NMR(100MHz,d6-CDCl3,ppm):δ166.0,162.2,141.8,141.0,140.4,139.1,131.7,130.9,129.4,124.7,124.6,124.5,124.4,123.1,114.9,97.1,96.0,61.5.ESI-MS:360.09[M+H].
选择相应的醛原料，按照类似的方法制备得到化合物N9、N10、N11和N12。结果和表征如下:
Figure PCTCN2016099845-appb-000076
4-((6-三氟甲基-1H-吲唑-4-氨基)甲基)苯并噻吩(N9).收率54.7％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.21(s,1H,indazole-NH),8.37(s,1H,indazole-H3),7.88(d,1H,J＝7.8Hz,Ar-H),7.78(d,1H,J＝7.6Hz,Ar-H),7.60(s,1H,NH),7.05(s,1H,Ar-H),6.36(s,1H,Ar-H),4.82(d,2H,J＝5.9Hz,benzyl-CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ145.3,142.7,140.5,140.0,139.2,132.7,128.6,126.7,124.8,124.4,123.7,122.9,121.8,115.3,95.8,94.0,42.6.ESI-MS:348.07[M+H].
Figure PCTCN2016099845-appb-000077
N-甲基-3-(((6-三氟甲基-1H-吲哚-4-)氨基)甲基)苯甲酰胺(N10).收率60.5％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.18(s,1H,indazole-NH),8.43(s,1H,CONH),8.37(s,1H,indazole-H),7.91(s,1H,Ar-H),7.72(d,1H,J＝7.7Hz,Ar-H).7.55(d,1H,J＝7.7Hz,Ar-H).7.46-7.41(m,2H,Ar-H),6.15(s,1H,Ar-H),4.55(d,2H,J＝5.8Hz,benzyl-CH2),2.78(d,3H,J＝4.5Hz,-CH3).13C-NMR(100MHz,d6-DMSO,ppm):δ167.1,143.1,140.5,140.1,135.2,132.7,130.2,128.6,125.9,124.0,115.1,95.4,93.7,46.5,31.1.ESI-MS:349.12[M+H].
Figure PCTCN2016099845-appb-000078
(1S,2S)-2-((6-溴-1H-吲唑-4-氨基)甲基)环己醇N11.收率52.7％；米黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.10(s,1H,indazole-NH),8.34(s,1H,indazole-H),6.95(s,1H,Ar-H),6.27(s,1H,Ar-H)),3.69-3.65(m,1H,cyclohexanol-H),3.24-3.18(m,1H,cyclohexanol-H),2.98-2.92(m,1H,cyclohexanol-H),1.97-1.88(m,2H,CH2NH),1.59-0.98(m,8H,cyclohexanol-H).13C-NMR(100MHz,d6-DMSO,ppm):δ143.8,140.7,132.6,129.1,126.9,124.2,114.9,93.0,71.7,46.3,44.7,36.0,29.7,25.5,24.9.ESI-MS:314.15[M+H].
Figure PCTCN2016099845-appb-000079
3-((6-三氟甲基-1H-吲唑-4-氨基)甲基)苯甲酸(N12).收率81.2％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.11(s,2H,COOH and indazole-NH),8.36(d,1H,J＝0.6Hz,Ar-H),8.01(s,1H,Ar-H),7.84(d,1H,J＝7.7Hz,Ar-H),7.66(d,1H,J＝7.7Hz,Ar-H),7.51-7.46(m,2H,NH and Ar-H),7.00(s,1H,Ar-H),6.15(s,1H,Ar-H),4.58(d,2H,J＝6.0Hz,).ESI-MS:336.09[M+H].
实施例17 本发明化合物N13的合成
Figure PCTCN2016099845-appb-000080
6-氯-N-(3-三氟甲基苄基)-1H-吲唑-4-胺(N13).
按照实施例14中类似的方法合成化合物N13，区别在于以化合物n5d替代化合物n5c，得化合物N13。收率58％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ12.42(s,2H,COOH and indazole-NH),8.14(s,2H,indazole-H3 and NH),8.05(d,2H,J＝8.1Hz,Ar-H),7.76(d,2H,J＝8.1Hz,Ar-H),6.58(s,1H,Ar-H),5.79(s,1H,Ar-H),4.58(s,2H,benzyl-CH2)；ESI-MS:302.02[M+H].
实施例18 本发明化合物N14的合成
Figure PCTCN2016099845-appb-000081
化合物(n9)的合成
将原料(n5b)(150mg,0.746mmol)和原料(n8)(185.4mg,0.896mmol)溶于DCM(7mL)中，加入DHP(264.2mg,1.04mmol)，滴入三氟醋酸(55.8uL,0.746mmol)，40℃回流反应12小时，将反应液过滤，旋干，过柱(DCM:MeOH＝80:1)，得化合物(n9)203.8mg黄色固体，收率72.3％。
(3-((6-(三氟甲基)-1H-吲哚-4-)氨基)苯基)氨基乙酸(N14)的合成
将化合物(n9)(100mg,0.255mmol)溶于乙醇和水(3:3mL)中，加入氢氧化钠(30.6mg,0.765mmol)，室温搅拌反应12小时，减压将反应液中乙醇旋走，用1N盐酸调pH为4左右，EA萃取，无水硫酸镁干燥，旋干过柱(DCM:MeOH＝30:1)得黄色固体化合物N14。收率42.3％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.12(s,1H,indazole-NH),8.36(s,1H,indazole-H3),7.33-6.61(m,5H,Ar-H),6.41(s,1H,Ar-H),6.27(s,1H,NH),4.36(d,2H,J＝5.8Hz,benzyl-CH2),3.75(s,2H,NHCH2).13C-NMR(100MHz,d6-DMSO,ppm):δ173.0,148.8,143.4,140.6,140.4,129.4,128.9,124.5,120.0,115.6,115.1,111.6,111.0,95.2,93.6.ESI-MS:365.12[M+H].
实施例19 本发明化合物N15和N16的合成
Figure PCTCN2016099845-appb-000082
化合物(n11)的合成
将原料(n5b)(200.0mg,1.0mmol)和原料(n10)(181.2mg,1.2mmol)溶于DCM (10mL)中，加入DHP(354.2mg,1.4mmol)，滴入三氟醋酸(74.5uL,1.0mmol)，40℃回流反应12小时。将反应液过滤，旋干，过柱(DCM 60mL,DCM:MeOH＝50：1)，得化合物(n11)278.8mg黄色固体，收率83.0％。
化合物(N15)的合成
将化合物(n11)(200mg,0.595mmol)溶于乙醇和水(1:1)中，加入氯化铵(16.5mg,0.3mmol)，分批加入铁粉(166.7mg,2.98mmol)，于80℃反应30分钟，将反应液趁热过滤，减压旋干溶剂，过柱(DCM:MeOH＝30:1)，得化合物(N15)155.9mg淡黄色固体,收率85.6％。
化合物(13)的合成
将原料(N15)(68.3mg,0.223mmol)和原料(n12)(30.0mg,0.268mmol)溶于DCM(2mL)中，加入DHP(79.1mg,0.312mmol)，滴入三氟醋酸(16.6uL,0.223mmol)，40℃回流反应12小时。将反应液过滤，旋干，过柱(DCM:MeOH＝100:1)，得化合物(n13)57.3mg淡黄色固体，收率66.2％。
3-((3-((6-(三氟甲基)-1H-吲哚-4-)氨基)苯基)氨基)-1-环己醇(N16)的合成
Figure PCTCN2016099845-appb-000083
将化合物(n13)(55mg,0.137mmol)溶于乙醇(1mL)中，置于0℃，加入硼氢化钠(76.6mg,0.684mmol)，升至室温搅拌反应12小时，将反应液降温至0℃，加入丙酮(2mL)淬灭多余的硼氢化钠，将反应液旋干过柱(DCM:MeOH＝20:1)，得淡黄色固体化合物(N16)。收率68.7％；淡黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.13(s,1H,indazole-NH),8.37(s,1H,indazole-H),7.32(t,1H,J＝5.2Hz,NH),7.01-6.42(m,5H,Ar-H),6.18(s,1H,Ar-H),4.59(s,1H,NH),4.42(s,1H,OH),4.35(d,2H,J＝5.6Hz,benzyl-CH2),3.57(t,1H,J＝4.1Hz,CH),2.51(d,1H,J＝1.8Hz,CH),1.66-0.89(m,8H,CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ148.5,143.4,140.5,140.4,132.8,128.7,126.8,124.1,115.1,114.7,111.5,111.4,95.0,93.7,68.3,65.3,50.0,42.8,35.6,32.2,19.6.ESI-MS:405.19[M+H].
实施例20 本发明化合物N17、N18、N19和N20的合成
Figure PCTCN2016099845-appb-000084
化合物n15的合成
将化合物n14(2.47g,24.46mmol)溶解于无水甲醇(30mL)中,冰浴下滴加SOCl2(8.78mL,122.30mmol),加毕，升温至室温搅拌12h后，旋干反应液，得到粗产品为白色固体化合物n15，直接投下步反应，收率99％。
化合物n16的合成
将化合物n15(3.68g,24.46mmol)溶解于乙腈(25mL)中,冰浴下滴加Et3N(3.39mL,24.46mmol)。将(Boc)2O(5.87g,26.91mmol)溶解于乙腈(5mL)中，滴加到反应液中。加毕，升温至室温搅拌4h后，滤去反应液中的固体，旋干滤液，加水溶解残余物。用EA萃取3次，合并有机层，用饱和氯化钠水溶液洗涤一次，用无水硫酸镁干燥有机层，过滤，旋干得粗产品。粗产品经柱层析纯化(PE:EA＝20:1)得淡黄色油状液体化合物n16(3.00g,9.73mmol),收率为65％。
化合物n17的合成
将化合物n16(2.00g,10.64mmol)置于三颈瓶中，氩气保护下加入DCM使之溶解，后置于-78℃下搅拌。10min后，缓慢滴加DIBAL-H(2.3mL,1M)。加毕，保持-78℃搅拌30min后，用2mL无水甲醇淬灭反应。5min后，移至冰浴。向反应液中加入200mL的10％柠檬酸水溶液，有固体析出。冰浴下搅拌30min至固体消失，用EA萃取水层3次，合并有机层，用饱和氯化钠水溶液洗涤一次，无水硫酸镁干燥。过滤，旋干滤液，得粗产品淡黄色透明油状液体化合物n17。直接投下步反应，收率99％。
化合物n18的合成
将化合物n5b(200mg,1.00mmol)、化合物n17(471mg,2.99mmol)和DHP(352mg,1.39mmol)溶解于DCM中，搅拌下滴加TFA(74μl,1.00mmol)。加毕，升温至回流搅拌12h后，旋干反应液，得粗品。粗品经柱层析(DCM:MeOH＝60:1)纯化得橘黄色固体化合物n18(100mg,0.27mg)。收率为27％。
N-(3-甲基-杂氮环丁烷)-6-三氟甲基-1H-吲哚-4-胺(N17)的合成
将化合物n18(50mg,0.14mmol)溶解于DCM(2mL)中，搅拌下滴加TFA(0.30mL,2.63mmol)。加毕，室温下搅拌3h后，旋干反应液得粗品，经柱层析(DCM:MeOH＝20:1)纯化得黄色固体化合物N17。收率63.0％；土黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ8.31(s,1H,indazole-H),6.99(s,1H,Ar-H),6.74(t,1H,J＝5.3Hz,NH),6.19(s,1H,Ar-H),3.59(m,2H,CH2),3.45-3.42(m,2H,CH2),3.28(d,2H,J＝6.5Hz,benzyl-CH2),2.97-2.90(m,2H,CH and NH).13C-NMR(100MHz,d6-DMSO,ppm):δ143.5,140.6,132.6,128.8,126.8,124.1,114.9,95.0,92.9,50.7,47.0,33.9.ESI-MS:271.11[M+H].
以类似的方法合成N18、N19和N20，区别是将初始原料换成n19、n20、n21。结果和表征如下：
Figure PCTCN2016099845-appb-000085
(R)-N-(吡咯-2-甲基-)-6-(三氟甲基)-1H-吲哚-4-胺(N18).收率50.0％；黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ8.33(s,1H,indazole-H),6.98(s,1H,NH),6.95(s,1H,Ar-H),6.65(t,1H,NH),6.22(s,1H,Ar-H),3.37-3.31(m,2H,benzyl-CH2)),3.21-3.09(m,3H, CH2 and CH),2.89-2.76(m,1H,NH)1.89-1.38(m,6H,CH2).13C-NMR(100MHz,d6-DMSO,ppm):δ143.5,410.7,132.5,129.1,128.8,114.9,95.0,93.0,57.1,48.7,46.3,29.9,25.4.ESI-MS:285.13[M+H].
Figure PCTCN2016099845-appb-000086
(R)-N-(哌啶-2-甲基)-6-(三氟甲基)-1H-吲哚-4-胺(N19).收率57.0％；淡黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.23(s,1H,indazole-NH),8.43(s,1H,indazole-H3),7.05(s,2H,NH and Ar-H),6.30(s,1H,Ar-H),3.51-3.18(m,6H,CH2 and NH)1.92-1.43(m,6H,CH2).ESI-MS:399.14[M+H].
Figure PCTCN2016099845-appb-000087
(S)-N-(哌啶-2-甲基)-6-(三氟甲基)-1H-吲哚-4-胺(N20).收率57.0％；淡黄色固体；1H-NMR(400MHz,d6-DMSO,ppm):δ13.26(s,1H,indazole-NH),8.48(s,1H,indazole-H),7.14(t,1H,J＝5.5Hz,NH),7.06(s,1H,Ar-H),6.30(s,1H,Ar-H),3.56-3.40(m,2H,CH),2.90-2.85(m,2H,benzyl-CH2),1.92(m,1H,NH)1.70-1.44(m,5H,CH2),1.12(m,2H,CH2)13C-NMR(100MHz,d6-DMSO,ppm):δ142.9,140.6,132.9,129.0,115.3,100.0,96.0,93.2,55.2,45.9,44.5,27.1,22.7,22.2.ESI-MS:299.15[M+H].
实施例21 本发明化合物对IDO蛋白的抑制活性
重组人IDO蛋白经大肠杆菌表达，镍亲合层析纯化而得。化合物对IDO抑制活性实验采用L-色氨酸作为底物。待测化合物溶解在10％DMSO溶液中配制成稀释液。取5uL稀释液加入到100μL反应体系中。100μL反应体系中含有0.5％DMSO，40nmol/L IDO，900μmol/L L-色氨酸，以及其他反应共存物(磷酸钾缓冲液、抗坏血酸、过氧化氢酶，亚甲基蓝)。反应混合物于37度下培育180分钟，再加入三氯乙酸终止反应。使用Tecan Infinite M1000酶标仪在321nm处测定产生的N-甲酰基犬尿氨酸的浓度，从而评价化合物对IDO的抑制活性。阴性对照物是以5μL的缓冲液代替IDO。临床III期的IDO抑制剂INCB024360作为阳性对照，验证本实验建立的IDO活性检测体系是否有效。
每个浓度设立三复孔。使用软件Graphpad Prism进行数据分析。在不含待测化合物的反应液中，吸光度(At)定义为100％活性。在不含IDO的反应液中，吸光度(Ab)定义为0％活性。对于待测化合物，活性的计算公式为：％activity＝[(A-Ab)/(At-Ab)]×100，其中A为含待测化合物的反应液的吸光度。抑制率的计算公式为：％inhibition＝100-％activity.
通过以上实验方法，测试了本发明中的部分化合物针对IDO的抑制活性。具体部化合物在1μM、10μM、100μM浓度下的抑制活性见表1。
其中A表示抑制率大于90％、B表示抑制率为70-90％，C表示抑制率为50-69％；D表示抑制率为10-49％，E表示抑制率小于10％；阳性对照物在浓度为0.05μM时的抑制率为46％。
表1 本发明化合物对IDO的抑制活性
Figure PCTCN2016099845-appb-000088
经试验证明，本发明提供的1H-吲唑类衍生物对IDO具有优异的抑制作用，可以用于预防和/或治疗多种疾病，如阿尔茨海默病、白内障、细胞免疫激活相关的感染、自身免疫性疾病、艾滋病、癌症、抑郁症或色氨酸代谢异常等。

Claims (35)

  1. 一种化合物或其药学上可接受的盐或其溶剂合物，所述化合物的结构如式(A)所示：
    Figure PCTCN2016099845-appb-100001
    Y表示-NH-、
    Figure PCTCN2016099845-appb-100002
    或-O-；
    R1选自H、卤素、C1～C6烷基或C1～C6卤代烷基；
    R2选自-(CH2)b-R3、-CH2NHR3、-CH2CONH-R3、-CH(CH2OH)R3或-CH2CH(OH)R3；
    R3选自取代的或非取代的芳基、杂芳基、环烷基或杂环基，所述取代的芳基、杂芳基、环烷基或杂环基分别独立地被一个或多个选自卤素、-(CH2)cCOOH、-(CH2)dCOOR4、-(CH2)eOH、-(CH2)fNH2、硝基、C1～C6烷基、C1～C6卤代烷基、-(CH)2CONH-R4或-(CH)2NH-R5的取代基所取代；
    R4选自C1～C6烷基，R5选自-CH2COOH、5-6元的环烷基、C1～C6烷基取代的或羟基取代的环烷基；
    a、b、c、d、e、f分别独立地表示0或1。
  2. 根据权利要求1所述的化合物，其特征在于：所述化合物的结构如式(Ⅰ)所示：
    Figure PCTCN2016099845-appb-100003
    其中，
    Y表示-NH-、
    Figure PCTCN2016099845-appb-100004
    R1a选自H、X、-CX3、-CHX2、-CH2X、-(CH2)aCH3；
    R2a选自-(CH2)b-R3a、-CH2NHR3a、-CH2CONH-R3a、-CH(CH2OH)R3a或-CH2CH(OH)R3a；
    R3a选自取代的或非取代的芳基、环烷基或杂环基，所述取代的芳基、环烷基或杂环基分别独立地被一个或多个选自X、-(CH2)cCOOH、-(CH2)dCOOR4a、-(CH2)eOH、(CH2)fNH2、硝基、C1～C6烷基的取代基所取代；
    R4a选自甲基或乙基；
    X表示F、Cl、Br或I，a、b、c、d、e、f分别独立地表示0或1。
  3. 根据权利要求2所述的化合物，其特征在于：所述R1a选自H、Br、-CF3、-CH3或-CH2CH3。
  4. 根据权利要求3所述的化合物，其特征在于：所述R1a选自Br。
  5. 根据权利要求2-4任一项所述的化合物，其特征在于：所述化合物的结构如式(II)、式(III)、式(IV)、式(V)、式(Ⅵ)或式(Ⅶ)所示：
    Figure PCTCN2016099845-appb-100005
  6. 根据权利要求2-5任一项所述的化合物，其特征在于：所述环烷基为环己基；所述取代的环烷基为被一个羟基所取代的环烷基。
  7. 根据权利要求2-5任一项所述的化合物，其特征在于：所述杂环基为四氢吡喃基。
  8. 根据权利要求2-5任一项所述的化合物，其特征在于：所述取代的芳基选自被一个-(CH2)cCOOH、氯、氨基、羟基、硝基或甲基所取代的苯基。
  9. 根据权利要求2-5任一项所述的化合物，其特征在于：所述R3选自下述基团之一：
    Figure PCTCN2016099845-appb-100006
  10. 根据权利要求9所述的化合物，其特征在于：所述化合物选自如下化合物之一：
    Figure PCTCN2016099845-appb-100007
  11. 一种制备权利要求5中式(Ⅱ)化合物或其制备中间体的方法，包括下述步骤：
    Figure PCTCN2016099845-appb-100008
    以SM和S2所示化合物为原料，在还原剂的作用下反应制备得到式(Ⅱ)化合物或其制备中间体。
  12. 一种制备权利要求5中式(Ⅲ)化合物或其制备中间体的方法，包括下述步骤：
    Figure PCTCN2016099845-appb-100009
    以SM和S2所示化合物为原料，缩合反应制备得到式(Ⅲ)化合物或其制备中间体。
  13. 一种制备权利要求5中式(Ⅳ)化合物或其制备中间体的方法，包括下述步骤：
    (1)
    Figure PCTCN2016099845-appb-100010
    以S4a所示化合物和二(三氯甲基)碳酸酯为原料，反应制备得到S4b所示化合物；
    (2)
    Figure PCTCN2016099845-appb-100011
    以S4b和SM所示化合物为原料，反应制备得到式(Ⅳ)化合物或其制备中间体。
  14. 一种制备权利要求5中式(Ⅴ)、式(Ⅵ)化合物，或其制备中间体的方法，包括下述步骤：
    Figure PCTCN2016099845-appb-100012
    以S5和SM所示化合物为原料，反应制备得到式(Ⅴ)或式(Ⅵ)化合物，或其制备中间体。
  15. 一种制备权利要求5中式(Ⅶ)化合物或其制备中间体的方法，包括下述步骤：
    (1)
    Figure PCTCN2016099845-appb-100013
    以SM所示化合物和溴乙酸乙酯为原料，反应制备得到S7a所示化合物；
    (2)
    Figure PCTCN2016099845-appb-100014
    将S7a所示化合物水解，反应制备得到S7b所示化合物；
    (3)
    Figure PCTCN2016099845-appb-100015
    以S7b和S7c所示化合物为原料，缩合反应制备得到(Ⅶ)所示化合物或其制备中间体。
  16. 根据权利要求1所述的化合物，所述化合物的结构如式(Ⅷ)所示：
    Figure PCTCN2016099845-appb-100016
    其中，
    R1b选自H、卤素、C1～C6烷基或C1～C6卤代烷基；
    R2b选自取代的或非取代的芳基、杂芳基、环烷基或杂环基，所述取代的芳基、杂芳基、环烷基或杂环基分别独立地被一个或多个选自卤素、-NH2、-(CH2)aOH、-COOH、硝基、C1～C6烷基、C1～C6卤代烷基、-CONH-R3b或-NH-R4b的取代基所取代；
    R3b选自C1～C6烷基；
    R4b选自-CH2COOH、5-6元的环烷基、C1～C6烷基取代的环烷基或羟基取代的环烷基；
    a＝0或1。
  17. 根据权利要求16所述的化合物，其特征在于：所述R1b选自H、Cl、-Br、CF3、-CHX2、-CH2X或-CH3，X表示卤原子。
  18. 根据权利要求16所述的化合物，其特征在于：所述C1～C6烷基选自甲基。
  19. 根据权利要求16所述的化合物，其特征在于：所述C1～C6卤代烷基为三氟甲基。
  20. 根据权利要求16所述的化合物，其特征在于：所述芳基选自苯基，杂芳基选自苯并噻吩，环烷基选自环己基，杂环基选自4～6元的氮杂环基。
  21. 根据权利要求16-20任一项所述的化合物，其特征在于：所述R2b选自下述基团之一：
    Figure PCTCN2016099845-appb-100017
    Figure PCTCN2016099845-appb-100018
    Figure PCTCN2016099845-appb-100019
  22. 根据权利要求16所述的化合物，其特征在于：所述化合物选自如下化合物之一：
    Figure PCTCN2016099845-appb-100020
  23. 一种化合物或其药学上可接受的盐或其溶剂合物，所述化合物的结构如式(Ⅸ)所示：
    Figure PCTCN2016099845-appb-100021
    其中，A表示苯环上任意位置的取代基，所述取代基选自-COOR、-OH或-NH2，R选自H或C1～C4烷基；X选自卤素。
  24. 根据权利要求23所述的化合物，其特征在于：X选自Cl、Br或I。
  25. 根据权利要求24所述的化合物，其特征在于：所述化合物选自如下化合物之一：
    Figure PCTCN2016099845-appb-100022
  26. 一种制备权利要求23-25任一项所述化合物或其制备中间体的方法，其特征在于：包括以下步骤：
    (1)
    Figure PCTCN2016099845-appb-100023
    以化合物S91为原料制备得到化合物S92；
    (2)
    Figure PCTCN2016099845-appb-100024
    其中，Pg表示氨基保护基；
    A、以化合物S93和水合肼为原料，在惰性气体环境中加热环化制备得到化合物S94；
    B、将S94上的氨基保护得到化合物S95；
    C、在碱的存在下，S95水解制备得到化合物S96；
    (3)
    Figure PCTCN2016099845-appb-100025
    以化合物S92和化合物S96为原料缩合得到化合物S97，脱去氨基保护基即得。
  27. 根据权利要求26所述的方法，其特征在于：步骤A中，所述加热环化的温度为80-160℃。
  28. 根据权利要求27所述的方法，其特征在于：步骤A中，所述加热环化的温度为95℃。
  29. 根据权利要求26所述的方法，其特征在于：步骤C中，所述碱选自碳酸钾、NaOH、KOH、Na2CO3、LiOH或三乙胺。
  30. 根据权利要求29所述的方法，其特征在于：步骤C中，所述碱选自碳酸钾。
  31. 根据权利要求26、29或30所述的方法，其特征在于：步骤C中，所述水解的温度为25-100℃。
  32. 根据权利要求31所述的方法，其特征在于：步骤C中，所述水解的温度为90℃。
  33. 权利要求1-10、16-25任一项所述化合物、式(Ⅹ)所示化合物、或其药学上可接受的盐、或其前药、或其溶剂合物在制备IDO抑制剂类药物上的用途，
    Figure PCTCN2016099845-appb-100026
    R1b如权利要求16-19任一项所定义。
  34. 根据权利要求33所述的用途，其特征在于：所述药物是预防和/或治疗阿尔茨海默病、白内障、细胞免疫激活相关的感染、自身免疫性疾病、艾滋病、癌症、抑郁症或色氨酸代谢异常的药物。
  35. 一种药物组合物，其特征在于：它是以权利要求求1-10、16-25任一项所述的化合物或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
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CN108467365A (zh) * 2018-03-12 2018-08-31 郑州工程技术学院 一种ido酶抑制剂化合物及其制备方法与应用
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EP4052705A1 (en) 2021-03-05 2022-09-07 Universität Basel Vizerektorat Forschung Compositions for the treatment of ebv associated diseases or conditions
WO2022184930A2 (en) 2021-03-05 2022-09-09 Universität Basel Compositions for the treatment of ebv associated diseases or conditions

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