WO2020113816A1 - 4位芳杂环取代的吲唑类化合物及其作为ido/tdo双重抑制剂的用途 - Google Patents

4位芳杂环取代的吲唑类化合物及其作为ido/tdo双重抑制剂的用途 Download PDF

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WO2020113816A1
WO2020113816A1 PCT/CN2019/074333 CN2019074333W WO2020113816A1 WO 2020113816 A1 WO2020113816 A1 WO 2020113816A1 CN 2019074333 W CN2019074333 W CN 2019074333W WO 2020113816 A1 WO2020113816 A1 WO 2020113816A1
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indazole
compound
tdo
dmso
nmr
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王周玉
钱珊
曹治兴
李玉芝
曹仁旭
王桂
袁欣
马兴梅
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西华大学
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Definitions

  • the invention relates to a class of indazole compounds substituted with 4-position aromatic heterocycle, and also to the use of the indazole compounds as IDO/TDO dual inhibitors.
  • Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (tryptophan 2,3-dioxygenase, TDO) are catalytic indole such as tryptophan
  • IDO and TDO play an important role in tumor immunity and tumorigenesis. Under normal circumstances, IDO and TDO are expressed at low levels in the body, and most tumor cells will be composed of high expression IDO/TDO, which converts L-tryptophan to N-formylkynurenine, which reduces the cell
  • IDO/TDO converts L-tryptophan to N-formylkynurenine
  • the concentration of tryptophan in the environment makes the synthesis of tryptophan-dependent T cells stalled in the G1 phase, and the proliferation of T cells is inhibited, thereby suppressing the killing effect of the body's immune system on tumor tissues.
  • the metabolites of tryptophan under the action of IDO/TDO are cytotoxic and can directly lyse T cells.
  • IDO/TDO inhibitors can also be used in combination with chemotherapeutic drugs to reduce the drug resistance of tumor cells, thereby enhancing the anti-tumor activity of conventional cytotoxic therapy. At the same time taking IDO/TDO inhibitors can also improve the efficacy of therapeutic vaccines for cancer patients.
  • IDO/TDO In addition to playing an important role in drug resistance of tumor cells, IDO/TDO is also closely related to the pathogenesis of various diseases related to cellular immune activation. IDO/TDO has been proved to be the target of major diseases such as infections, malignant tumors, autoimmune diseases and other related to activation of cellular immunity. At the same time, inhibiting IDO/TDO is also an important treatment strategy for patients with neurological diseases such as depression and Alzheimer's disease. Therefore, IDO/TDO inhibitors have broad clinical application prospects.
  • the present invention mainly provides the use of a class of 4-position aromatic heterocyclic substituted indazole compounds as dual inhibitors of IDO/TDO.
  • the present invention provides the use of the structural compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof in the preparation of TDO inhibitor drugs:
  • X is selected from H or C1-C6 alkyl
  • A is selected from substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, benzyl, C1-C6 alkyl, the substituted aryl, heteroaryl, benzyl, cycloalkane
  • the radical or heterocycloalkyl is independently selected by one or more selected from -(CH 2 ) a OH, -(CH 2 ) a NHR 1 , haloalkyl, -(CH 2 ) a CN, -SO 2 NH 2 , -(CH 2 ) a NO 2 , C1 ⁇ C6 alkyl, halogen or Substituted by
  • R 1 is selected from H, —OH, —COR 4 , —(CH 2 ) b COOR 5 , cyclohexanone, cyclohexyl or hydroxy substituted cyclohexyl;
  • R 4 is selected from C1-C6 alkyl, -NHR 6 and -CH 2 R 7 ;
  • R 6 is selected from C3-C6 cycloalkyl or heterocycloalkyl, the heterocycloalkyl contains 1 to 2 heteroatoms selected from O, N, S;
  • R 7 is selected from substituted or unsubstituted aryl or heteroaryl, the substituted aryl or heteroaryl is independently selected by one or more selected from amino, hydroxy, halogen, nitro or C1-C6 alkyl Substituent substitution;
  • R 5 is selected from H or C1-C6 alkyl
  • R 2 and R 3 constitute a 5- to 6-membered heterocycle containing two or more heteroatoms, the heteroatoms are selected from O, N, and S;
  • X is selected from H or methyl; and/or, A is selected from aryl, heteroaryl or methyl.
  • aryl group is selected from phenyl or naphthyl.
  • heteroaryl group is selected from furyl, pyridyl, thienyl, benzothienyl, thiazolyl, or imidazolyl.
  • the compound has the following structure:
  • the present invention provides the use of the above compound, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof for preparing TDO inhibitor drugs.
  • the present invention also provides the use of the above compound, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof in the preparation of IDO/TDO dual inhibitor drugs.
  • the drug is a drug for preventing and/or treating tumors, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, depression, infections related to cellular immune activation, or abnormal metabolism of tryptophan, etc. .
  • the invention also provides compounds having the following structure:
  • substitution refers to the replacement of hydrogen atoms in a molecule with other different atoms or molecules.
  • the minimum and maximum carbon atom content in the hydrocarbon group is represented by a prefix.
  • C ab alkyl indicates any alkyl group containing "a" to "b" carbon atoms, including straight-chain alkanes Radical and branched alkyl.
  • C 1-6 alkyl refers to linear and branched alkyl groups containing 1 to 6 carbon atoms.
  • halogen refers to a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • cycloalkyl refers to a saturated ring formed by all carbon atoms or a non-aromatic unsaturated ring.
  • heterocycloalkyl group in the present invention refers to a saturated ring or a non-aromatic unsaturated ring containing at least one hetero atom; wherein the hetero atom refers to a nitrogen atom, an oxygen atom, and a sulfur atom.
  • aryl refers to an unsaturated ring formed of all carbon atoms and having aromaticity.
  • arylhetero group in the present invention refers to an unsaturated ring having aromaticity containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, and a sulfur atom.
  • pharmaceutically acceptable means that a certain carrier, carrier, diluent, adjuvant, and/or salt formed is usually chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form, and It is physiologically compatible with the receptor.
  • the pharmaceutically acceptable auxiliary materials in the present invention refer to substances contained in the dosage form in addition to the active ingredients.
  • prodrug refers to derivatives of the aforementioned compounds, which may themselves have weak activity or even no activity, but after administration, under physiological conditions (for example, by metabolism, solvolysis, or other means) ) Is converted into the corresponding biologically active form.
  • One or more compounds of the present invention can be used in combination with each other, or the compound of the present invention can be used in combination with any other active agent for preparing IDO/TDO inhibitors. If a group of compounds is used, these compounds can be administered to the subject simultaneously, separately or sequentially.
  • the 4-position aromatic heterocyclic substituted indazole compounds provided by the present invention have excellent inhibitory effects on IDO/TDO enzymes and IDO/TDO cells, and have significant antitumor activity in vivo, which can be used for prevention and/or Treat a variety of diseases, such as tumors, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, depression, infections related to cellular immune activation, or abnormal metabolism of tryptophan.
  • diseases such as tumors, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, depression, infections related to cellular immune activation, or abnormal metabolism of tryptophan.
  • 1 is a graph of the inhibitory effect of compound 35 on the activity of IDO1 enzyme and TDO enzyme; (A) shows the inhibitory effect of compound 35 on the IDO1 enzyme activity, (B) shows the inhibitory effect of compound 35 on the activity of TDO enzyme.
  • Fig. 2 is a graph of Compound 35 of the present invention inhibiting IDO1 and TDO activity in tumor cells; (A) shows that Compound 35 inhibits IDO1 activity in HeLa cells induced by INF ⁇ in a concentration-dependent manner, (B) shows Compound 35 in a concentration-dependent manner Ways to inhibit TDO activity in A172 cells.
  • Figure 3 is a Western blot evaluation of the effect of compound 35 and LWQ-84 on the expression of IDO1 protein; (A) shows the effect of compound 35 on the expression of IDO1 protein, (B) shows the effect of compound LWQ-84 on the expression of IDO1 protein.
  • Figure 4 shows the in vivo effect of compound 35 of the present invention on CT26 tumor xenografts.
  • Figure 5 is the in vivo effect of compound 35 of the present invention on CT26 tumor xenografts.
  • the reagents and raw materials are all commercially available products. Except for the starting materials specifically marked with the source, the remaining reagents were purchased from Chengdu Kelong Chemical Reagent Company.
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • trichlorosilane (20 ⁇ L, 0.19 mmol) was added to a solution of C (30 mg, 0.10 mmol) and DMF (2 ⁇ L, 0.02 mmol) in DCM (8 mL). The reaction was stirred for 1 hour under the protection of argon at 0°C. MeOH (0.1 mL) was added to quench the reaction. The solution was warmed to room temperature. The mixture was dissolved with EA, and the organic matter was washed with an aqueous solution. It was washed with NaHCO 3 and brine, dried over anhydrous Na 2 SO 4 and concentrated.
  • 3-(1,3-dioxolane-2-yl)aniline was obtained by the synthesis method of intermediate A.
  • Ethyl bromoacetate was added to a solution of amine and K 2 CO 3 (1.25 g, 9.09 mmol) dissolved in DMF (5 mL). The mixture was stirred at room temperature. Lasts 8 hours. The reaction mixture was diluted with EA.
  • the EA layer was then dried over anhydrous Na 2 SO 4 and concentrated to obtain a residue, which was purified by silica gel column chromatography using petroleum ether and EA (3:1) as eluents to obtain ethyl (3-( 1,3)-dioxolane-2-yl)phenyl)glycine salt as yellow liquid.
  • Tetrahydro-2H-pyran-4-amine 50 mg, 0.4926 mmol
  • DIEA 205.17 ⁇ L, 1.477 mmol
  • a solution of trichloromethyl carbonate 48.44 mg, 0.1630 mmol
  • DCM 2 mL
  • compound 12 100 mg, 0.2463 mmol
  • the mixture was stirred at room temperature. The mixture was washed with brine for 3 hours, and then dried over anhydrous Na 2 SO 4 .
  • HATU (0.11 g, 0.29 mmol, DIEA (0.08 ⁇ L, 0.29 mmol, EDCI (56 mg, 0.49 mmol) and 2-(2-nitrophenyl)acetic acid (0.08 g, 0.49 mmol) were added to the compound at 0°C 12 in DCM (4 mL) solution. The solution was stirred at room temperature for 4 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was dissolved with EA. The combined organic layers were washed with aqueous solution. Washed with NaHCO 3 then Dry with anhydrous Na 2 SO 4.
  • Human N-terminal IDO1 and TDO are expressed in E. coli and purified by nickel affinity chromatography. The determination was carried out by ultraviolet absorption using recombinant hIDO1, hTDO and L-tryptophan as substrates. In order to detect the inhibitory activity of compounds on TDO and IDO1 enzymes, recombinant hTDO (100nM) and hIDO (100nM) and a certain concentration of compounds at room temperature contain 400mM tryptophan, 40mM ascorbic acid, 200 ⁇ g/ml catalase, 20 ⁇ M methylene blue, Ca2+, Mg2+-potassium phosphate-free buffer cultivation system.
  • the synthesized compound was diluted in 10% DMSO, and 5 L was diluted to 100 L, so that the final concentration of DMSO was 0.5% in all reactions.
  • the compounds of the present invention were tested for their inhibitory activity against IDO and TDO.
  • the inhibitory activity of specific compounds at a concentration of 10 ⁇ M is shown in Table 1.
  • A means that the inhibition rate is greater than 80%
  • B means that the inhibition rate is 60-79%
  • C means that the inhibition rate is 40-59%
  • D means that the inhibition rate is 20-30%
  • E means that the inhibition rate is less than 20%.
  • HeLa cells were seeded in 24-well plates for 24 hours, then INF ⁇ (50 ng/mL) was added, and treated with inhibitors for 24 hours.
  • DMSO 0.5%) and Epacadostat (25nM) were used as negative and positive controls, respectively.
  • IDO1 activity was determined by measuring the concentration of L-kynurenine in the cell culture medium. 400 ⁇ L of medium was mixed with 180 ⁇ L of 30% trichloroacetic acid and centrifuged at 13.000 rpm for 10 minutes. The supernatant (100 ⁇ L) was transferred to a new 96-well plate, and an equal volume of freshly prepared 2% w/v p-dimethylaminobenzaldehyde in acetic acid was added.
  • the optical density was measured at 480 nm using Multiscan spectroscopy Mk3 (Thermo Fisher).
  • the kynurenine concentration was determined from the L-kynurenine standard curve. Each determination was performed in triplicate, and the data was expressed as mean ⁇ standard deviation.
  • the hTDO activity was tested in a cellular environment with the TDO high expression cell line A172. After seeding overnight in 96-well plates (2 ⁇ 10 4 cells/well), A172 cells were treated with a certain concentration of compound and L-Trp (20 ⁇ g/ml) for 24 hours. Then, the cell culture medium (300 ⁇ L/well) was transferred to a culture tube and mixed with 90 ⁇ LTCA (30%, w/v). Next, the culture tube was transferred to a 65°C water bath for 30 minutes to convert N-formylkynurenine to kynurenine.
  • HeLa cells were seeded in 96-well plates and treated with IDO1 inhibitors for 2 hours, and then INF ⁇ (50ng/mL) was added for 24 hours.
  • HeLa cells were seeded in 6-well plates and treated with IDO1 inhibitors (10, 20 and 50 ⁇ M) and JAK inhibitors (1 ⁇ M) for 2 hours, then 50 ng/mL of INF ⁇ was added for 24 hours.
  • a DMSO concentration of 0.5% was used as a negative control.
  • the treated cells were lysed in RIPA lysis buffer, and the same amount of protein samples were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis.
  • the protein was transferred to a nitrocellulose membrane and then blocked with 5% bovine serum albumin in Tris buffered saline containing Tween 20 (TBST).
  • TST Tris buffered saline containing Tween 20
  • the membrane was incubated with anti-IDO1 (rabbit polyclonal antibody, 1:1000, #13268-1-AP, Proteintech) and anti-GAPDH (rabbit polyclonal antibody, 1:4000, #10494-A-AP) at 4°C overnight. .
  • HRP horseradish peroxidase
  • each Balb/c mouse was injected subcutaneously with 100 ⁇ L of 1 ⁇ 107 cells/mL CT26 single cell suspension. After incubation for 4 days, the mice were randomly divided into 5 groups (6 mice per group). Three groups were given orally every day, and ISL was given at doses of 20, 40 and 80 mg/kg/day, respectively. Oral intake of 5% DMSO, 20% PEG400 and 75% deionized water as a carrier group.
  • the Epacadostat (INBC024360, 40 mg/kg) treatment group was used as a positive control.
  • volume (mm 3 ) a ⁇ b 2 /2 (a: longest diameter (length); b: shortest diameter (width)) .
  • the results show that compound 35 has a significant antitumor effect in vivo.
  • the present invention discloses the use of a 4-position aromatic heterocyclic substituted indazole compound as an IDO/TDO dual inhibitor. Tests have shown that the compound has excellent inhibitory effects on IDO/TDO enzymes and IDO/TDO cells, and has significant anti-tumor activity in vivo, which can be used to prevent and/or treat various diseases, such as tumors, neurodegenerative diseases, Al Alzheimer's disease, Parkinson's disease, depression, infection related to cellular immune activation, or abnormal metabolism of tryptophan, etc.
  • various diseases such as tumors, neurodegenerative diseases, Al Alzheimer's disease, Parkinson's disease, depression, infection related to cellular immune activation, or abnormal metabolism of tryptophan, etc.

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Abstract

本发明公开了一种式(I)所示的4位芳杂环取代的吲唑类化合物作为IDO/TDO双重抑制剂的用途。本发明化合物对IDO/TDO酶和IDO/TDO细胞均具有优异的抑制作用，而且体内具有显著抗肿瘤活性，可以用于预防和/或治疗多种疾病，如肿瘤、神经退行性疾病、阿尔茨海默病、帕金森病、抑郁症、细胞免疫激活相关的感染、或色氨酸代谢异常等。

Description

4位芳杂环取代的吲唑类化合物及其作为IDO/TDO双重抑制剂的用途 技术领域
本发明涉及一类4位芳杂环取代的吲唑类化合物，还涉及其作为IDO/TDO双重抑制剂的用途。
背景技术
吲哚胺2,3-双加氧酶(Indoleamine 2,3-dioxygenase，IDO)和色氨酸2,3-双加氧酶(tryptophan 2,3-dioxygenase，TDO)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和TDO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和TDO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/TDO，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/TDO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/TDO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/TDO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/TDO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/TDO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/TDO已被证实是与细胞免疫激活相关的感染、恶性肿瘤、自身免疫性疾病等重大疾病的靶标。同时，抑制IDO/TDO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/TDO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4位芳杂环取代的吲唑类化合物在作为IDO/TDO双重抑制剂中的用途。
本发明提供了式(I)所示结构化合物、或其药学上可接受的盐、或其溶剂合物在制备TDO抑制剂类药物中的用途：
Figure PCTCN2019074333-appb-000001
其中，
X选自H或C1～C6烷基；
A选自取代的或非取代的芳基、杂芳基、环烷基、杂环烷基、苄基、C1～C6烷基，所述取代的芳基、杂芳基、苄基、环烷基或杂环烷基分别独立地被一个或多个选自-(CH 2) aOH、-(CH 2) aNHR 1、卤代烷基、-(CH 2) aCN、-SO 2NH 2、-(CH 2) aNO 2、C1～C6烷基、卤素或
Figure PCTCN2019074333-appb-000002
的取代基取代；
R 1选自H、-OH、-COR 4、-(CH 2) bCOOR 5、环己酮基、环己基或羟基取代的环己基；
R 4选自C1～C6烷基、-NHR 6、-CH 2R 7；
R 6选自C3～C6环烷基或杂环烷基，所述杂环烷基含有1～2个选自O、N、S的杂原子；
R 7选自取代或非取代的芳基或杂芳基，所述取代芳基或杂芳基分别独立地被一个或多个选自氨基、羟基、卤素、硝基或C1～C6烷基的取代基取代；
R 5选自H或C1～C6烷基；
R 2与R 3构成含两个或以上杂原子的5～6元杂环，所述杂原子选自O、N、S；
a＝0或1，b＝1。
进一步地，X选自H或甲基；和/或，A选自芳基、杂芳基或甲基。
进一步地，所述芳基选自苯基或萘基。
进一步地，所述杂芳基选自呋喃基、吡啶基、噻吩基、苯并噻吩基、噻唑基、或咪唑基。
进一步地，所述化合物(I)的结构如式(II)或式(III)所示：
Figure PCTCN2019074333-appb-000003
更进一步地，所述化合物具有如下结构：
Figure PCTCN2019074333-appb-000004
Figure PCTCN2019074333-appb-000005
本发明提供了上述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物在制备TDO抑制剂类药物上的用途。
本发明还提供了上述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物在制备IDO/TDO双重抑制剂类药物上的用途。
更进一步地，所述药物是预防和/或治疗肿瘤、神经退行性疾病、阿尔茨海默病、帕金森病、抑郁症、细胞免疫激活相关的感染、或色氨酸代谢异常等疾病的药物。
本发明还提供了具有如下结构的化合物：
Figure PCTCN2019074333-appb-000006
本发明中，“取代”是指分子中的氢原子被其它不同的原子或分子所替换。
本发明中，碳氢基团中碳原子含量的最小值和最大值通过前缀表示，例如，前缀C a-b烷基表明任何含“a”至“b”个碳原子的烷基，包括直链烷基和支链烷基。因此，例如，C 1-6烷基是指包含1～6个碳原子的直链烷基和支链烷基。
本发明中，卤素指氟原子、氯原子、溴原子、碘原子。
本发明中“环烷基”指全碳原子形成的饱和环或非芳香性的不饱和环。
本发明中的“杂环烷基”指包含至少一个杂原子的饱和环或非芳香性的不饱和环；其中杂原子指氮原子、氧原子、硫原子。
本发明中“芳基”指全碳原子形成的具有芳香性的不饱和环。
本发明中的“芳杂基”指包含至少一个杂原子的具有芳香性的不饱和环；其中杂原子指氮原子、氧原子、硫原子。
本发明中，“药学上可接受的”是指某载体、运载物、稀释剂、辅料，和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容，并在生理上与受体相兼容。
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。
本发明中，“前药”是指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。
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/TDO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。
经试验证明，本发明提供的4位芳杂环取代的吲唑类化合物对IDO/TDO酶和IDO/TDO细胞具有优异的抑制作用，而且体内具有显著抗肿瘤活性，可以用于预防和/或治疗多种疾病，如肿瘤、神经退行性疾病、阿尔茨海默病、帕金森病、抑郁症、细胞免疫激活相关的感染、或色氨酸代谢异常等。
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。
附图说明：
图1为本发明化合物35对IDO1酶和TDO酶的活性抑制效果图；(A)表示化合物35对IDO1酶的活性抑制，(B)表示化合物35对TDO酶的活性抑制。
图2为本发明化合物35抑制肿瘤细胞中的IDO1和TDO活性图；(A)表示化合物35以浓度依赖性方式抑制INFγ诱导的HeLa细胞中的IDO1活性，(B)表示化合物35以浓度依赖性方式抑制A172细胞中的TDO活性。
图3为Western印迹评估化合物35和LWQ-84对IDO1蛋白表达的影响；(A)表示化合物35对IDO1蛋白表达的影响，(B)表示化合物LWQ-84对IDO1蛋白表达的影响。
图4为本发明化合物35对CT26肿瘤异种移植物的体内作用。
图5为本发明化合物35对CT26肿瘤异种移植物的体内作用。
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。
实验例1中间体A的合成
合成路线如下：
Figure PCTCN2019074333-appb-000007
(1)化合物1b的合成：
向2-甲基-1,3-二硝基苯(1a)(10.0g，54.91mmol)的浓H 2SO 4(150mL)溶液中缓慢加入1,3-二溴-5,5-二甲基海因(DBDMH,9.4g,32.94mmol)，并将所得混合物在室温下搅拌反应，反应时间为8h，反应完成后将混合物倒入冰水中，然后搅拌10分钟。过滤混合物，滤渣用水洗涤、干燥。将粗产物过硅胶柱色谱，以石油醚和EA(10:1)作为洗脱剂，得到白色固体化合物1b(14g，99％)。
(2)化合物1c的合成：
将化合物1b(38.31mmol)用MeOH/1,4二氧六环(2:1,18mL)混合溶剂溶解，加入铁粉(3.58g，114.93mmol)和盐酸(14mL)，80℃搅拌4h。反应完成后过滤混合物，利用NaHCO 3将滤液调节至pH＝7-8，并在减压下浓缩。将浓缩产物用EA溶解，并用盐水洗涤，经无水Na 2SO 4干燥。将粗产物过硅胶柱色谱柱，以石油醚和EA(3:1)作为洗脱液，得到化合物1c(92％)。
(3)化合物1d的合成：
在0℃下，将亚硝酸钠(1.45g，0.29mol)的水(50mL)溶液一次全部加入到化合物1c(0.13mol)的冰醋酸溶液中。搅拌下剧烈反应45分钟，然后获得沉淀物，过滤，减压浓缩滤液，将棕色固体在水中重结晶，得到黄色固体化合物1d(62％)，99.2％HPLC纯度；熔点：238-239℃。 1H NMR(400MHz，CDCl3，ppm)：δ14.55(s，1H，吲唑-NH)，8.62(s，1H，吲唑-H3)，8.30(s，1H，吲唑-H7)，8.16(d，J＝3.2Hz，1H，吲唑-H5)。 13C NMR(300MHz，CDCl 3，ppm)：δ140.9,139.4,130.2,119.8,114.4,113.5,113.2。
(4)中间体A的合成：
将化合物1d(2.0g,8.26mmol)、铁粉(2.3g,41.32mmol)和氯化铵(221.5mg,4.13mmol)加入到乙醇/水(3：1)溶液中，80℃反应1小时。将反应液利用硅藻土过滤。用EA萃取滤液。将合并的有机层用盐水洗涤，用无水Na 2SO 4干燥，蒸发溶剂，将粗产物过硅胶柱色谱柱，得到棕色固体的化合物A，收率95％，HPLC纯度99.1％；熔点179-181℃； 1H NMR(400MHz，d6-DMSO，ppm)：δ12.75(s，1H，吲唑-NH)，8.10(s，1H，吲唑-H3)，6.77(s，1H，吲唑-H7)，6.27(d)，J＝1.3Hz，1H，吲唑-H5)，6.12(s，2H，NH 2)。 13C NMR(300MHz，d6-DMSO，ppm)：δ143.8,142.4,132.7,121.4,112.6,104.2,99.4。
实验例2化合物5-8、15、16、21、23-32、34、35的合成
Figure PCTCN2019074333-appb-000008
5-8、15、16、21、23-32、34、35的合成通过方法1还原胺化一锅法获得，在室温下将TFA(0.1当量)加入到用DCM/MeOH(3:1)溶解化合物5(1.0当量)，不同的芳族醛(1.2当量)和酯(1.2当量)的溶液中，和将反应升温至45℃并反应约4小时。完成后(通过TLC监测)，通过加入NaHCO 3将溶液调节至pH 7-8，并通过真空浓缩获得粗残余物。最后，通过柱色谱法纯化粗残余物，得到高产率的目标化合物。
化合物5产率:95％,99.1％HPLC纯度；熔点:179-181℃； 1H NMR(400MHz,d 6-DMSO,ppm):δ12.75(s,1H,indazole-NH),8.10(s,1H,indazole-H3),6.77(s,1H,indazole-H7),6.27(d,J＝1.3Hz,1H,indazole-H5),6.12(s,2H,NH 2). 13C NMR(300MHz, d 6-DMSO,ppm):δ143.8,142.4,132.7,121.4,112.6,104.2,99.4.
化合物6产率：80％,99.3％HPLC纯度.熔点:182-183℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.97(br,1H,indazole-NH),9.35(br,1H,OH),8.23(s,1H,indazole-H3),7.13(t,J＝7.7Hz,1H,N HCH 2),6.88-6.72(m,3H,Ar-H4,H5 and H6),6.68-6.59(m,1H,Ar-H2),6.03(s,1H,indazole-H7),5.77(s,1H,indazole-H5),4.36(d,J＝9.4Hz,2H,NHC H 2). 13C NMR(300MHz,d 6-DMSO,ppm)δ158.0,143.2,142.2,141.4,132.6,129.9,121.8,118.0,114.2,114.0,112.6,101.1,100.2,46.3.HRMS(AP-ESI)Calcd.for C 14H 12BrN 3O:318.0237(M+H)+.Found:318.0233.
化合物7产率：82％,98.5％HPLC纯度.熔点:188-189℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.84(s,1H,indazole-NH),8.22(s,1H,indazole-H3),7.34-7.27(m,5H,N HCH 2,Ar-H2,H3,H5 and H6),6.82(s,1H,indazole-H7),6.03(s,1H,indazole-H5),5.15(s,1H,OH),4.47(s,2H,C H 2OH),4.42(d,J＝3.1Hz,2H,NHC H 2). 13C NMR(300MHz,d 6-DMSO)δ143.2,142.2,141.6,138.2,132.6,127.2,127.1,121.7,112.6,101.2,100.3,63.2,46.2.HRMS(AP-ESI)Calcd.for C 15H 14BrN 3O:332.0393(M+H)+.Found:332.0392.
化合物8产率：73％,98.7％HPLC纯度.熔点:205-206℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.84(s,1H,indazole-NH),9.93(s,1H,N HCOCH 3),8.22(s,1H,indazole-H3),7.54(d,J＝8.5Hz,2H,Ar-H2 and H6),7.30(d,J＝8.5Hz,2H,Ar-H3 and H5),7.25(t,J＝5.9Hz,1H,N HCH 2),6.82(s,1H,indazole-H7),6.05(s,1H,indazole-H5),4.37(d,J＝5.8Hz,2H,C H 2NH),2.03(s,3H,C H 3CO).HRMS(AP-ESI)Calcd.for C 16H 15BrN 4O:359.0502(M+H)+.Found:359.0499.
化合物15产率：87％,98.1％HPLC纯度.熔点:124-125℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.87(s,1H,indazole-NH),8.22(s,1H,indazole-H3),7.76(s,1H,Ar-H2),7.70(d,J＝7.3Hz,1H,Ar-H4),7.65–7.54(m,2H,Ar-H5 and H6),7.36(t,J＝6.1Hz,1H,N HCH 2),6.85(s,1H,indazole-H7),6.08(d,J＝1.1Hz,1H,indazole-H5),4.55(d,J＝6.1Hz,2HNHC H 2). 13C NMR(100MHz,d 6-DMSO,ppm)δ142.9,142.2 141.6,132.6,131.6,129.9129.8 129.5 126.1,123.4,121.8 112.7,101.3,100.7,45.9.HRMS(AP-ESI)Calcd.for C 15H 11BrF 3N 3:370.0161(M+H) +.Found:370.0156.
化合物16产率：85％,95.3％HPLC纯度.熔点:201-202℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.89(s,1H,indazole-NH),8.23(s,1H,indazole-H3),7.82(d,J＝8.3Hz,2H,Ar-H2 and H6),7.58(d,J＝8.3Hz,2H,Ar-H3 and H5),7.41(t,J＝6.1Hz,1H,N HCH 2), 6.87(s,1H,indazole-H7),6.02(s,1H,indazole-H5),4.57(s,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 15H 11BrN 4:327.0240(M+H) +.Found:327.0236
化合物21产率：49.5％,91.2％HPLC纯度.熔点:222-223℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.66(s,1H,indazole-NH),9.47(s,1H,Ar-OH),8.95(s,1H,Ar-OH),8.10(s,1H,indazole-H3),6.37(s,1H,indazole-H7),6.32(d,J＝2.2Hz,1H,Ar-H6),6.19(d,J＝8.3Hz,1H,Ar-H5),5.99(dd,J＝8.3,2.2Hz,1H,Ar-H3),5.91(s,1H,indazole-H5),3.97(s,2H,NHC H 2). 13C NMR(300MHz,d 6-DMSO,ppm)δ156.7,155.9,141.6,133.1,128.8,128.6,123.7,116.7,113.3,108.8,106.2,105.6,102.7,49.1.HRMS(AP-ESI)Calcd.for C 14H 12BrN 3O 2:334.0186(M+H) +.Found:334.0180.
化合物23产率：91％；96.7％HPLC纯度.熔点:182-183℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.80(s,1H,indazole-NH),8.90(s,1H,Ar-OH),8.23(s,1H,indazole-H3),7.13(t,J＝5.5Hz,1H,N HCH 2),6.80(s,1H,indazole-H7),6.67-6.54(m,2H,Ar-H2 and H5),6.43(dd,J＝7.9,1.7Hz,1H,Ar-H6),6.04(s,1H,indazole-H5),4.53(s,1H,Ar-NH 2),4.21(d,J＝5.7Hz,1H,NHC H 2). 13C NMR(300MHz,d 6-DMSO,ppm)δ143.4,142.1,137.0,132.8,130.4,121.8,115.6,114.6,113.5,112.5,101.1,99.8,46.6.HRMS(AP-ESI)Calcd.for C 14H 13BrN 4O:333.0351(M+H)+.Found:333.0348.
化合物24产率：96.3％,96.6％HPLC纯度.熔点:186-187℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.84(s,1H,indazole-NH),10.06(s,1H,OH),8.22(s,1H,indazole-H3),7.35(d,J＝2.0Hz,1H,Ar-H6),7.23-7.13(m,2H,N HCH 2 and Ar-H4),6.95(d,J＝8.3Hz,1H,Ar-H3),6.85(s,1H,indazole-H7),6.09(d,J＝0.8Hz,1H,indazole-H5),4.33(d,J＝5.9Hz,2H,NHC H 2). 13C NMR(300MHz,d 6-DMSO,ppm)δ152.4,143.0,142.1,132.7,131.6,128.9,127.4,121.8,120.0,117.0,112.6,101.3,100.4,45.5.HRMS(AP-ESI)Calcd.for C 14H 11BrClN 3O:351.9847(M+H) +.Found:351.9844.
化合物25产率：92.5％,95.4％HPLC纯度.熔点:182-183℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.83(s,1H,indazole-NH),9.82(s,1H,OH),8.22(s,1H,indazole-H3),7.21(t,J＝8.6Hz,1H,N HCH 2),7.08(t,J＝5.6Hz,1H,Ar-H5),6.85(s,1H,indazole-H7),6.67-6.55(m,2H,Ar-H2 and H6),6.12(d,J＝0.9Hz,1H,indazole-H5),4.33(d,J＝5.6Hz,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 14H 11BrFN 3O:336.0142(M+H) +.Found:336.0132.
化合物26产率：69.1％,95.5％HPLC纯度.熔点:196-197℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.85(s,1H,indazole-NH),9.74(s,1H,Ar-OH),8.22(s,1H,indazole-H3), 7.20(t,J＝5.8Hz,1H,N HCH 2),7.14(dd,J＝12.2,1.7Hz,1H,Ar-H3),7.02(dd,J＝8.3,1.3Hz,1H,Ar-H6),6.92(t,J＝8.6Hz,1H,Ar-H4),6.85(s,1H,indazole-H7),6.08(s,1H,indazole-H5),4.33(d,J＝5.9Hz,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 14H 11BrFN 3O:336.0142(M+H) +.Found:336.0132.
化合物27产率：69.2％,97.8％HPLC纯度.熔点:192-193℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.84(s,1H,indazole-NH),9.23(s,1H,Ar-OH),8.23(s,1H,indazole-H3),7.13(t,J＝5.2Hz,1H,N HCH 2),7.09(s,1H,Ar-H3),7.01(d,J＝8.0Hz,1H,Ar-H5),6.83(s,1H,indazole-H7),6.75(d,J＝8.1Hz,1H,Ar-H6),6.08(s,1H,indazole-H5),4.27(d,J＝5.4Hz,2H,NHC H 2),2.11(s,3H,Ar-CH 3).HRMS(AP-ESI)Calcd.for C 15H 14BrN 3O:332.0393(M+H) +.Found:332.0391.
化合物28产率：64.6％,95.2％HPLC纯度.熔点:193-194℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ8.30(s,1H,indazole-H3),7.08(d,J＝8.2Hz,1H,Ar-H5),6.86(s,1H,indazole-H7),6.65(d,J＝2.4Hz,1H,Ar-H2),6.56(dd,J＝8.2,2.4Hz,1H,Ar-H6),6.11(s,1H,indazole-H5),4.26(d,J＝7.2Hz,1H,NHC H 2),2.26(s,3H,Ar-CH 3).HRMS(AP-ESI)Calcd.for C 15H 14BrN 3O:332.0393(M+H) +.Found:3320390.
化合物29产率：63.7％％,92.8％HPLC纯度.熔点:195-196℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.84(s,1H,indazole-NH),8.20(s,1H,indazole-H3),7.61(dd,J＝1.8,0.8Hz,1H,furyl-H5),7.14(t,J＝5.8Hz,1H,N HCH 2),6.87(s,1H,indazole-H7),6.43-6.40(m,1H,furyl-H4),6.36(dd,J＝3.1,0.5Hz,1H,furyl-H3),6.25(s,1H,indazole-H5),4.42(d,J＝5.8Hz,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 12H 10BrN 3O:292.0080(M+H) +.Found:292.0075.
化合物30产率：69.4％,97.6％HPLC纯度.熔点:183-184℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.85(s,1H,indazole-NH),8.21(s,1H,indazole-H3),7.39(dd,J＝5.1,1.1Hz,1H,Thienyl-H5),7.31(t,J＝5.9Hz,1H,N HCH 2),7.10(d,J＝2.6Hz,1H,thienyl-H3),7.03-6.97(m,1H,thienyl-H4),6.88(s,1H,indazole-H7),6.23(s,1H,indazole-H5),4.63(d,J＝5.9Hz,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 12H 10BrN 3S:307.9852(M+H) +.Found:307.9850.
化合物31产率:66.7％,98.1％HPLC纯度.熔点:194-195℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.82(s,1H,indazole-NH),11.95(s,1H,imidazole-NH),8.22(s,1H,indazole-H3),7.62(d,J＝0.6Hz,1H,imidazole-H2),6.99(s,1H,imidazole-H4),6.94(t,J＝ 5.4Hz,1H,N HCH 2),6.84(s,1H,indazole-H7),6.24(s,1H,indazole-H5),4.31(d,J＝5.4Hz,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 11H 10BrN 5:292.0198(M+H)+.Found:292.0189.
化合物32产率：63.2％,97.3％HPLC纯度.熔点:135-136℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.89(s,1H,indazole-NH),8.52(d,J＝5.9Hz,2H,pyridyl-H2 and H6),8.23(s,1H,indazole-H3),7.42-7.34(m,3H,N HCH 2,pyridyl-H3 and H5),6.87(s,1H,indazole-H7),6.01(s,1H,indazole-H5),4.51(d,J＝6.1Hz,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 13H 11BrN 4:303.0240(M+H) +.Found:303.0238.
化合物34产率：82％,96.5％HPLC纯度.熔点:208-209℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.80(s,1H,indazole-NH),δ8.23(s,1H,indazole-H3),7.21(t,J＝5.8Hz,1H,N HCH 2),6.96(s,1H,benzodioxole-H2),6.88(d,J＝0.8Hz,2H,benzodioxole-H6and H7),6.85(s,1H,indazole-H7),6.09(s,1H,indazole-H5),5.99(s,2H,benzodioxole-H4),4.35(d,J＝6.0Hz,2H,NHC H 2). 13C NMR(300MHz,d 6-DMSO,ppm)δ147.8,146.7,143.1,142.1,133.7,132.7,121.8,120.6,112.6,108.6,108.0,101.3,100.3,46.2.HRMS(AP-ESI)Calcd.for C 15H 12BrN 3O 2:332.0035(M+H)+.Found:332.0032.
化合物35产率：92％,100％HPLC纯度.熔点:195-196℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.90(s,1H,indazole-NH),8.25(s,1H,indazole-H3),7.95(d,J＝8.7Hz,1H,benzofurazan-H7),7.59(dd,J＝9.0,6.6Hz,1H,benzofurazan-H8),7.52–7.36(m,2H,N HCH 2 and benzofurazan-H9),6.89(s,1H,indazole-H7),6.14(d,J＝0.8Hz,1H,indazole-H5),4.85(d,J＝5.8Hz,2H,NHC H 2). 13C NMR(100MHz,d 6-DMSO,ppm)δ149.7,148.7,142.8,142.1,133.3,132.6,128.9,128.6,121.7,114.9,112.6,101.2,100.9,43.0.HRMS(AP-ESI)Calcd.for C 14H 10BrN 5O:329.9990(M+H)+.Found:329.9995.
实验例3化合物9的合成
向化合物8(100mg，0.28mmol)的MeOH(4mL)溶液中加入SOCl 2(70μL，1mmol)。将混合物在65℃下搅拌2小时，然后减压浓缩。将得到的混合物用MeOH稀释。通过加入NaHCO 3将溶液调节至pH 7-8，然后浓缩。所得固体通过硅胶柱色谱纯化，得到化合物9(40mg，45.7％)，为黄色固体；HPLC纯度为97.8％。熔点：178-179℃； 1H NMR(400MHz，d6-DMSO，ppm)δ12.87(s，1H，吲唑-NH)，8.19(s，1H，吲唑-H3)，6.89-6.77(m，3H，吲唑-H7，Ar-H2)和H6)，6.47-6.39(m，3H，吲唑-H5，Ar-H3和H5)，4.11(s，2H，NH2)，4.00(s，2H，NHCH2).HRMS(AP-ESI)Calcd.for C 14H 13BrN 4： 317.0396(M+H)+。Found：317.0397。
实验例4化合物10、11、13、14的合成
Figure PCTCN2019074333-appb-000009
参照合成方法1还原胺化一锅法利用邻间位取代的硝基苯甲醛和化合物反应得到化合物B。
参照中间体A的合成方法利用铁粉和NH 4Cl还原硝基，以B为原料得到化合物10、11。
化合物10：产率71％,97.9％HPLC纯度.熔点:165-166℃； 1H NMR(400MHz,d6-DMSO,ppm)δ12.83(s,1H,indazole-NH),8.22(s,1H,indazole-H3),7.11-7.05(m,2H,Ar-H3 and NHCH2),6.98(t,J＝7.8Hz,1H,Ar-H5),6.85(s,1H,indazole-H7),6.69(d,J＝7.7Hz,1H,Ar-H6),6.54(t,J＝7.4Hz,1H,Ar-H4),6.08(s,1H,indazole-H5),5.03(s,2H,Ar-NH2),4.26(d,J＝5.6Hz,2H,CH2NH). 13C NMR(300MHz,d6-DMSO,ppm)δ146.7,143.4,142.1,132.7,128.0,128.0,122.1,121.8,116.5,115.4,112.6,101.3,100.2,43.58HRMS(AP-ESI)Calcd.for C 14H 13BrN 4:317.0396(M+H)+.Found:317.0398.
化合物11：产率72％,98.6％HPLC纯度.熔点:172-173℃； 1H NMR(400MHz,d6-DMSO,ppm)δ12.81(s,1H,indazole-NH),8.23(s,1H,indazole-H3),7.22(t,J＝4.6Hz,1H,NHCH2),6.98(t,J＝7.6Hz,1H,Ar-H5),6.82(s,1H,indazole-H7),6.58(s,1H,Ar-H2),6.53(d,J＝7.2Hz,1H,Ar-H6),6.44(d,J＝7.7Hz,1H,Ar-H4),6.03(s,1H,indazole-H5),5.04(s,2H,Ar-NH2),4.29(d,J＝5.3Hz,2H,CH2NH). 13C NMR(300MHz,d6-DMSO,ppm)δ149.3,143.4,142.1,140.5,132.7,129.4,121.8,114.9,113.0,112.6,101.1,100.0,46.8.HRMS(AP-ESI)Calcd.for C 14H 13BrN 4:317.0396(M+H)+.Found:317.0393.
化合物13的合成
向化合物9(100mg，0.28mmol)的二恶烷/H 2O(1:1,6mL)溶液中加入NH 4Cl(75mg，1.15mmol)和锌粉(30mg，0.57mmol)。将混合物在室温下搅拌。12小时过滤反应混合 物，滤液用EA萃取。然后将EA层用无水Na 2SO 4干燥，并浓缩，得到残余物，将其通过硅胶柱色谱纯化，得到化合物13为黄色固体(45mg，产率：46.7％)。97.3％HPLC纯度.熔点:205-206℃； 1H NMR(400MHz,d6-DMSO,ppm)δ12.80(s,1H,indazole-NH),8.22(s,1H,indazole-H3),7.11-6.99(m,3H,NHCH2,Ar-H2 and H6),6.80(s,1H,indazole-H7),6.54(d,J＝8.3Hz,2H,Ar-H3 and H5),6.08(s,1H,indazole-H5),4.23(d,J＝5.7Hz,2H,NHCH2). 13C NMR(300MHz,d6-DMSO,ppm)δ148.1,143.3,142.1,132.8,128.5,126.3,121.8,114.3,112.6,101.2,99.9,46.4.HRMS(AP-ESI)Calcd.for C 14H 13BrN 4O:333.0346(M+H)+.Found:333.0349.
化合物14的合成
参照化合13的合成，合成化合物14产率48.9％，96.5％HPLC纯度。熔点：182-183℃； 1H NMR(400MHz,d6-DMSO,ppm)δ12.87(d,J＝6.8Hz,1H,indazole-NH),8.37-7.90(m,3H,indazole-H3,Ar-H4 and NHCH2),7.76-7.33(m,3H,Ar-H2,H5 and H6),6.86(d,J＝5.2Hz,1H,indazole-H7),6.10(d,J＝10.7Hz,1H,indazole-H5),4.57(dd,J＝26.3,6.0Hz,2H,NHCH2).HRMS(AP-ESI)Calcd.for C 14H 13BrN 4O:333.0346(M+H)+.Found:333.0346.
实验例5化合物12的合成
Figure PCTCN2019074333-appb-000010
将化合物5(300mg，1.41mmol)和CsCO 3(1.38g，4.24mmol)的DMF(5mL)溶液在65℃下搅拌4小时，然后加入1-(2-溴乙基)-4-硝基苯(390mg，1.70mmol)。将混合物搅拌2小时。用EA稀释反应混合物。将EA层用盐水洗涤，然后用无水Na 2SO 4干燥，并浓缩，得到残余物，将其通过硅胶柱色谱纯化，得到硝基化合物12a，为黄色固体。使用硝基化合物12a，通过化合物A的和合成方法得到化合物12两步反应产率：62.5％，98.8％HPLC纯度.熔点:203-204℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ8.08(s,1H,indazole-H3),6.89(m,1H,NHCH 2),6.86(d,J＝8.3Hz,2H,Ar-H3 and H5),6.46(d,J＝8.3Hz,2H,Ar-H2 and H6),6.27(d,J＝1.2Hz,1H,indazole-H7),6.12(s,1H,indazole-H5),4.87(s,2H,Ar-NH 2),4.36(t,J＝7.5Hz,2H,NHC H 2),2.90(t,J＝7.5Hz,2H,Ar-CH 2). 13C NMR(300MHz,d 6-DMSO,ppm)δ147.4,143.8,141.7,131.9,129.6,125.6,121.6,114.4, 113.0,104.4,99.3,50.4,35.1.HRMS(AP-ESI)Calcd.for C 15H 15BrN 4:331.0553(M+H) +.Found:331.0547.
实验例6化合物17的合成
合成路线如下：
Figure PCTCN2019074333-appb-000011
在0℃下，向4-氨磺酰基苯甲酸(3a；200mg；0.53mmol)的甲醇(8mL)溶液中加入SOCl 2(1348μL，1.84mmol)，将混合物在40℃下搅拌2小时，然后减压浓缩，得到化合物3b，将其直接用于下一步反应。
将化合物3b(214mg，1.00mmol)和(Boc) 2O(238.6mg，1.09mmol)溶解在DCM(8mL)中，加入三乙胺(138μL，1mmol)和DMAP(12.2mg，0.1mmol)，搅拌，室温下反应1.5小时。反应完成后对反应液进行浓缩，通过硅胶柱色谱纯化产生的粗产物，得到化合物3c。
将化合物3c(300mg，1mmol)溶解在DCM(8mL)中。在-78℃下缓慢加入DIBAL-H(2mL，2mmol)，并将混合物在-78℃氩气氛围下搅拌2小时。在-78℃下利用甲醇(2mL)淬灭反应。将溶液温热至0℃并在搅拌下加入10％柠檬酸。用DCM萃取混合物，用盐水洗涤有机相，利用无水Na 2SO 4干燥并浓缩，通过硅胶柱色谱纯化产生的粗产物，得到化合物3d。
将化合物3d和A采用通用合成方法1，得到化合物3e，在二氯甲烷作溶剂三氟醋酸的作用下3e脱一个Boc，得到化合物17，5步反应总产率：54％,94.0％HPLC纯度.熔点210-212℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.88(s,1H,indazole-NH),8.23(s,1H,indazole-H3),7.85(d,J＝8.4Hz,2H,Ar-H2 and H6),7.62(d,J＝8.4Hz,2H,Ar-H3 and H5),7.44(t,J＝6.0Hz,1H,N HCH 2),6.85(s,1H,indazole-H7),5.97(s,1H,indazole-H5),4.58(d,J＝6.0Hz,2H,NHC H 2).
实验例7化合物18的合成
Figure PCTCN2019074333-appb-000012
将5(50mg，0.24mmol)，丙酮(87μL，1.18mmol)和二氯乙烷(1.5mL)在微波650W并使用4A分子筛除去产物水的条件下缩合反应9小时。在反应完成(TLC监测)后，过滤反应混合物。减压蒸发有机溶剂。获得的粗产物通过柱色谱法纯化，用PE和EA(3：1)作为洗脱剂，得到亚胺中间体C，为白色固体(40mg，0.16mmol)，收率65％。
在0℃下，将三氯硅烷(20μL，0.19mmol)加入到C(30mg，0.10mmol)和DMF(2μL，0.02mmol)的DCM(8mL)溶液中。将反应在0℃氩气保护下搅拌1小时。加入MeOH(0.1mL)淬灭反应。将溶液温热至室温。将混合物用EA溶解，并将有机物用水溶液洗涤。用NaHCO 3和盐水洗涤，用无水Na 2SO 4干燥并浓缩。产生的固体通过硅胶柱色谱纯化，用PE和EA(1：1)作为洗脱剂，得到18，为白色固体(22mg，产率：60％)96.9％HPLC纯度.熔点:180-181℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.78(s,1H,indazole-NH),8.21(s,1H,indazole-H3),6.80(s,1H,indazole-H7),6.30(d,J＝7.8Hz,1H,NH),6.12(s,1H,indazole-H5),3.72(m,1H,NHC H),1.23(d,J＝6.3Hz,6H,CH(C H 3) 2). 13C NMR(300MHz,d 6-DMSO,ppm)δ142.6,142.2,132.8,122.0,112.5,100.8,99.6,43.7,22.7.HRMS(AP-ESI)Calcd.for C 10H 12BrN 3:254.0287(M+H) +.Found:254.0289.
实验例8化合物20的合成
参照化合物18的合成，合成化合物20产率：30％,96.9％HPLC纯度.熔点:192-193℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.85(s,1H,indazole-NH),8.35(s,1H,indazole-H3),8.27–8.12(m,2H,Ar-H3 and H5),7.78–7.59(m,2H,Ar-H2 and H6),7.14(d,J＝7.1Hz,1H,N HCH),6.82(s,1H,indazole-H7),5.89(s,1H,indazole-H5),4.96–4.80(m,1H,NHC H),1.55(d,J＝6.8Hz,3H,NHC H 3). 13C NMR(100MHz,d 6-DMSO,ppm)δ154.1,146.9,142.1,141.9,132.9,127.6,124.3,121.5,112.6,102.2,100.8,52.0,24.3.
HRMS(AP-ESI)Calcd.for C 15H 13BrN 4O 2:361.0295(M+H) +.Found:361.0293.
实验例9化合物33的合成
Figure PCTCN2019074333-appb-000013
在-78℃下，向4-溴苯并[B]噻吩(1.0g，4.69mmol)的THF(10mL)溶液中加入2.5N n-BuLi(3.75μL，9.38mmol)，搅拌混合物50分钟。将反应温热至室温。并用NH 4Cl(30mL)淬灭。用EA萃取混合物，用盐水洗涤有机物，用无水Na 2 SO 4干燥并浓缩。产生的固体通过硅胶柱色谱纯化，得到苯并[B]噻吩-4-甲醛，为黄色液体。使用苯并[b]噻吩-4-甲醛，通过方法1从5得化合物33。产率：72％,90.5％HPLC纯度.熔点:204-205℃； 1H NMR(400MHz,d 6-DMSO,ppm)δ12.70(s,1H,indazole-NH),7.90(d,J＝7.8Hz,2H,indazole-H3),7.84-7.75(m,1H,Ar-H4),7.54-7.39(m,2H,benzothienyl-H4 and H5),7.39-7.11(m,4H,indazole-H7,benzothienyl-H1,benzothienyl-H2 and H6),6.64(d,J＝46.7Hz,1H,indazole-H5),4.76(d,J＝4.9Hz,2H,NHC H 2).HRMS(AP-ESI)Calcd.for C 16H 12BrN 3S:358.0008(M+H) +.Found:358.0000.
实验例10化合物36的合成
使用2-氨基噻唑-4-羧酸乙酯，按照化合物5合成17的一般方法，合成化合物36产率：63％，95％HPLC纯度.熔点:165-166℃； 1H NMR(300MHz,DMSO,ppm)δ12.80(s,1H),8.20(s,1H),7.04(t,J＝5.8Hz,1H),6.90(s,2H),6.82(s,1H),6.31(s,1H),6.13(d,J＝0.9Hz,1H),4.20(d,J＝5.7Hz,2H). 13C NMR(101MHz,DMSO,ppm)δ169.11(s),150.08(s),143.24(s),142.13(s),132.72(s),121.83(s),112.57(s),102.18(s),101.10(s),100.11(s),56.53(s),55.37(s),43.71(s),19.04(s).HRMS(AP-ESI)Calcd.for C 11H 10N 5S:323.9919(M+H)+.Found:323.9916.
实验例11化合物37的合成
Figure PCTCN2019074333-appb-000014
向溶于90％EtOH(10mL)溶液中的1-萘甲醛(50mg，0.32mmol)和甲氧基胺盐酸盐(88.24mg，1.056mmol)加入NaOH(115.25mg，2.88mmol)，并将所得混合物在室温下搅拌30分钟，随后将反应在80℃下回流4小时。减压浓缩混合物，得到甲氧基胺，为浅绿色液体。
将(Z)-1-萘甲醛-O-甲基肟(360mg，1.945mmol)，Pd(OAc) 2(44mg，0.1945mmol)，AgNO 2(600mg，3.89mmol)，K 2S 2O 8(1.05g，3.89mmol)的溶液溶于DCE(10mL)于密封管中于110℃搅拌48小时。浓缩混合物，得到硝基化合物，为棕色固体。
向硝基化合物(450mg，1.948mmol)的THF(10mL)溶液中加入TsOH(670mg，3.896mmol)和7％福尔马林(1.5mL)，将得到的混合物在100℃下回流4小时。浓缩混合物，得到2-硝基-1-萘甲醛，为浅黄色固体。
使用2-硝基-1-萘甲醛，按照通用方法1和中间体A的合成方法依次反应获得化合物37，白色固体，5步反应总产率：20.6％，97.6％HPLC纯度.熔点:171-172℃； 1H NMR(400MHz,d6-DMSO,ppm)δ12.93(s,1H,indazole-NH),8.28(s,1H,indazole-H3),7.74(dd,J＝8.0,1.5Hz,1H,naphthyl-H8),7.43-7.31(m,2H,naphthyl-H4 and Naphthyl-H5),7.28-7.17(m,3H,NHCH2,Naphthyl-H3 and H6),7.00(s,1H,indazole-H7),6.82(dd,J＝5.6,3.2Hz,1H,Naphthyl-H7),6.37(s,1H,indazole-H5),5.52(s,2H,Naphthyl-NH2),4.86(d,J＝4.7Hz,2H,NHCH2).13C NMR(300MHz,d6-DMSO,ppm)δ146.5,142.5,142.1,136.7,134.1,133.0,129.3,128.1,126.8,125.4,124.3,121.8,118.4,112.9,112.4,102.3,101.7,48.7.HRMS(AP-ESI)Calcd.for C 18H 16BrN 4:367.0553(M+H)+.Found:367.0541.
实验例12化合物38、39的合成
Figure PCTCN2019074333-appb-000015
将乙二醇(22.1mL，364mL)和p-TsOH加入到3-硝基苯甲醛(5g，33.1mmol)的无水甲苯(50mL)溶液中，并将反应混合物用分水器在110℃下回流12小时。完成后(通过TLC监测)，将溶液冷却至室温。并用水洗涤。用无水Na 2 SO 4干燥NaHCO 3和盐水，浓缩，得到粗产物2-(3-硝基苯基)-1,3-二氧戊环。
使用硝基化合物，通过中间体A的合成方法获得3-(1,3-二氧戊环-2-基)苯胺。将溴乙酸乙酯加入到胺和K 2CO 3(1.25g，9.09mmol)溶于DMF(5mL)的溶液中。将混合物在室温下搅拌。持续8小时。用EA稀释反应混合物。然后将EA层用无水Na 2 SO 4干燥，并浓缩，得到残余物，将其通过硅胶柱色谱纯化，用石油醚和EA(3:1)作为洗脱液，得到乙基(3-(1,3)-二氧戊环-2-基)苯基)甘氨酸盐，为黄色液体。
向酯(500mg，1.98mmol)的THF(6mL)溶液中加入3N HCl(2mL)。将溶液在室温下搅拌。持续6小时将溶液浓缩并通过硅胶柱色谱纯化，用石油醚和EA(3:1)作为洗脱剂，得到化合物(3-甲酰基苯基)甘氨酸乙酯(1mg，1％)，为黄色液体。
通过通用方法1得到化合物38.对于五个步骤总产率：67％，97.5％HPLC纯度.熔点:181-182℃； 1H NMR(400MHz,d6-DMSO,ppm)δ12.82(s,1H,indazole-NH),8.23(s,1H,indazole-H3),7.23(t,J＝5.8Hz,1H,NHCH2),7.05(t,J＝7.7Hz,1H,Ar-H5),6.82(s,1H,indazole-H7),6.61(d,J＝9.0Hz,2H,Ar-H4 and H6),6.41(d,J＝8.0Hz,1H,indazole-H5),6.10-5.98(m,2H,Ar-H2 and NH),4.32(d,J＝5.8Hz,2H,NHCH2),4.06(q,J＝7.1Hz,2H,CH2CH3),3.85(d,J＝6.4Hz,2H,Ar-NHCH2),1.15(t,J＝7.1Hz,3H,CH2CH3).13C NMR(300MHz,d6-DMSO,ppm)δ171.7,148.7,143.4,142.1,140.6,132.7,129.4,121.8,115.6,112.6,111.2,110.9,101.1,100.1,60.7,46.8,45.2,14.6.HRMS(AP-ESI)Calcd.for C 18H 19BrN 4O 2:403.0770(M+H)+.Found:403.0765.
化合物39的合成
向38的H 2O/EtOH溶液中加入NaOH。回流反应30分钟，用EA稀释溶液。将EA层用饱和食盐水洗涤，然后用无水Na 2SO 4干燥，并浓缩，得到粗品，将其通过硅胶 柱色谱纯化，用DCM和MeOH(20:1)作为洗脱液，得到化合物39(产率：58％)为黄色固体；92.5％HPLC纯度.熔点:202-203℃； 1H NMR(400MHz,d6-DMSO,ppm)δ12.74(br,2H,indazole-NH and COOH),8.24(s,1H,indazole-H3),7.21(t,J＝5.6Hz,1H,NHCH2),7.05(t,J＝7.7Hz,1H,Ar-H5),6.82(s,1H,indazole-H7),6.69-6.54(m,2H,Ar-H2and H6),6.42(dd,J＝8.0,1.5Hz,1H,Ar-H4),6.05(s,1H,indazole-H5),4.31(d,J＝5.2Hz,2H,NHCH2),3.78(s,2H,Ar-NHCH2). 13C NMR(300MHz,d6-DMSO,ppm)δ173.1,148.8,143.4,142.2,140.5,132.6,129.4,121.8,115.5,112.6,111.5,110.8,101.1,100.1,46.9,45.1.HRMS(AP-ESI)Calcd.for C 16H 15BrN 4O 2:375.0451(M+H)+.Found:375.0450.
实验例13化合物40、41的合成
Figure PCTCN2019074333-appb-000016
通过通用合成方法1，以化合物11为原料得到化合物40产率：77.3％.96.9％HPLC纯度.熔点:205-206℃；1H NMR(400MHz,d6-DMSO,ppm)δ12.86(s,1H,indazole-NH),8.84(s,1H,indazole-H3),8.23(s,1H,indazole-H7),7.36-7.07(m,5H,Ar-H2,Ar-H4,Ar-H5Ar-H6and NHCH),6.84(s,1H,NHCH2),6.04(s,1H,indazole-H5),5.35(s,1H,cyclohexyl-H1),4.46(d,J＝5.9Hz,2H,NHCH2),2.48(t,J＝6.0Hz,2H,cyclohexyl-H4),2.15(t,J＝6.4Hz,2H,cyclohexyl-H2),1.96-1.78(m,2H,cyclohexyl-H6),1.00-0.96(m,1H,cyclohexyl-H5). 13C NMR(300MHz,d6-DMSO,ppm)δ196.3,162.4,143.1,141.4,139.8,129.7,123.5,121.9,121.8,121.6,112.6,101.2,98.6,46.3,46.2,36.9,29.0,22.0.HRMS(AP-ESI)Calcd.for C 20H 21BrN 4O:413.0977(M+H)+.Found:413.0979.
化合物41的合成
在0℃下，将NaBH 4(86.2mg，0.771mmol)加入到化合物40(63.6mg，0.154mmol)的EtOH(1.5mL)溶液中，并将反应升温至室温。并反应12小时。用丙酮(3mL)淬灭反应，然后浓缩。最后，将粗残余物通过柱色谱纯化，用DCM和MeOH(20:1)作为洗脱剂，得到化合物41，产率：85.2％；96.7％HPLC purity.mp:211-212℃；1H NMR(400MHz,d6-DMSO,ppm)δ12.86(s,1H,indazole-NH),8.84(s,1H,indazole-H3),8.22(s,1H,indazole-H7),7.36-7.08(m,5H,Ar-H4,Ar-H5,Ar-H6,NHCH and),6.83(s,1H,NHCH2),6.03(d,J＝1.2Hz,1H,indazole-H5),5.77(s,1H,cyclohexyl-H3),5.34(s,1H,cyclohexyl-H1), 4.45(d,J＝5.9Hz,2H,NHCH2),2.48(t,J＝6.1Hz,2H,cyclohexyl-H2),2.15(t,J＝6.4Hz,2H,cyclohexyl-H4 and H6),1.92-1.81(m,2H,cyclohexyl-H4 and H6),1.00(t,J＝7.1Hz,2H,cyclohexyl-H5).13C NMR(300MHz,d6-DMSO,ppm)δ196.2,162.3,143.1,141.4,139.8,129.7,123.5,121.9,121.7,121.6,112.6,101.2,98.7,55.4,46.3,46.2,36.9,29.0,22.0.HRMS(AP-ESI)Calcd.for C 20H 21BrN 4O:415.1128(M+H)+.Found:415.1141.
实验例14化合物42的合成
Figure PCTCN2019074333-appb-000017
将四氢-2H-吡喃-4-胺(50mg，0.4926mmol)和DIEA(205.17μL，1.477mmol)溶解在DCM(5mL)中。在0℃下加入三氯甲基碳酸酯(48.44mg，0.1630mmol)的DCM(2mL)溶液，并将混合物搅拌30分钟。完成后(通过TLC监测)，将化合物12(100mg，0.2463mmol)加入到该溶液中，并将该混合物在室温下搅拌。3小时将混合物用盐水洗涤，然后经无水Na 2SO 4干燥。蒸发溶剂并将粗产物加入硅胶柱色谱上，用PE和EA(2:1)作为洗脱剂，得到白色固体42，产率：51.4％；98.8％HPLC纯度；熔点:215-216℃； 1H NMR(400MHz,d6-DMSO,ppm)δ8.25(s,1H,indazole-H3),8.07(s,1H,Ar-NHCO),7.25(d,J＝8.5Hz,2H,Ar-H3 and Ar-H5),7.05(d,J＝8.4Hz,2H,Ar-H2 and Ar-H6),6.90(s,1H,indazole-H7),6.26(d,J＝1.3Hz,1H,indazole-H5),6.20-5.95(m,2H,NHCH2 and NHCONH),4.42(t,J＝7.3Hz,2H,NHCH2),3.92-3.54(m,5H,furyl-H2,H4 and H6),3.49-3.27(m,4H,furyl-H3 and H5),2.99(t,J＝7.3Hz,2H,Ar-CH2). 13C NMR(300MHz,d6-DMSO,ppm)δ154.9,143.8,141.8,139.2,132.0,131.3,129.5,121.6,118.0,113.0,104.5,99.2,66.3,50.0,45.7,35.1,33.7.HRMS(AP-ESI)Calcd.for C 21H 24BrN 5O 2:458.1192(M+H)+.Found:458.1188.
实验例15化合物43的合成
Figure PCTCN2019074333-appb-000018
在0℃下将HATU(0.11g，0.29mmol，DIEA(0.08μL，0.29mmol，EDCI(56mg，0.49mmol)和2-(2-硝基苯基)乙酸(0.08g，0.49mmol)加入到化合物12的DCM(4mL)溶液中。将溶液在室温下搅拌，持续4小时。过滤混合物，减压浓缩滤液。将残余物用EA溶解。将合并的有机层用水溶液洗涤。用NaHCO 3洗涤，然后用无水Na 2SO 4干燥。蒸发溶剂，将粗产物加到硅胶柱色谱上，用PE和EA(1:1)作为洗脱剂，得到白色固体。再利用中间体A的合成方法得到化合物43，两步产率为52.3％，98.5％HPLC纯度.熔点:176-177℃； 1H NMR(400MHz,CDCl 3,ppm)δ7.91(s,1H,N HCO),7.86(s,1H,indazole-H3),7.32(d,J＝8.4Hz,2H,Ar-H3 and Ar-H5),7.18-7.08(m,2H,Ar′-H3 and Ar′-H5),6.99(d,J＝8.4Hz,2H,Ar-H2 and Ar-H6),6.82-6.71(m,3H,indazole-H7,Ar′-H4and Ar′-H6),6.41(d,J＝1.2Hz,1H,indazole-H5),4.41(t,J＝7.4Hz,2H,NHC H 2),4.27(s,2H,Ar-NH 2),3.61(s,2H,C H 2CONH),3.10(t,J＝7.4Hz,2H,Ar-CH 2).HRMS(AP-ESI)Calcd.for C 23H 22BrN 5O:464.1080(M+H) +.Found:464.1082.
实验例16酶抑制活性测试实验
人N末端IDO1和TDO在大肠杆菌中表达，镍亲合层析纯化而得。使用重组hIDO1，hTDO和L-色氨酸作为底物通过紫外吸收进行测定。为了检测化合物对TDO和IDO1酶的抑制活性，将重组hTDO(100nM)和hIDO(100nM)与一定浓度的化合物在室温下在含有400mM色氨酸，40mM抗坏血酸，200μg/ml的过氧化氢酶，20μM亚甲基蓝，Ca2+，Mg2+-无磷酸钾缓冲液培育系统中培养。将合成的化合物在10％DMSO中稀释，并将5L稀释到100L，使得在所有反应中DMSO的最终浓度为0.5％。对于阴性对照(空白)，加入5μL测定缓冲液代替酶，加入10％DMSO代替抑制剂。对于阳性对照，加入Epacadostat代替抑制剂。温育1小时后，向每个系统中加入30％三氯乙酸，并在65℃温育15分钟以终止酶反应并将N-甲酰基犬尿氨酸转化为犬尿氨酸。然后，将来自各系统的100μL上清液与等体积的含有DMAB(二甲基氨基苯甲醛，3％，w/v)的乙酸混合，并使用Multiscan光谱Mk3(Thermo Fisher)在480nm波长下检测光密度。从L-犬尿氨酸标准曲线确定犬尿氨酸浓度。最后，使用GraphPad Prism 5.0软件处理数据。IC50值由引起半数最大百分比活性的浓度确定。
通过以上实验方法，测试了本发明中的化合物针对IDO和TDO的抑制活性。具体化合物在10μM浓度下的抑制活性见表1。
其中A表示抑制率大于80％、B表示抑制率为60-79％，C表示抑制率为40-59％；D表示抑制率20-30％；E表示抑制率小于20％。
表1本发明化合物对IDO和TDO的抑制活性
Figure PCTCN2019074333-appb-000019
Figure PCTCN2019074333-appb-000020
Figure PCTCN2019074333-appb-000021
经试验证明，本发明提供的吲唑类化合物对IDO/TDO酶均具有优异的抑制作用，特别是化合物35对IDO/TDO都具有较高的抑制效率，对IDO和TDO的IC50值为0.74μM和2.94μM。
实验例17 IDO1细胞的抑制测定
将HeLa细胞接种在24孔板中24小时，然后加入INFγ(50ng/mL)，并用抑制剂处理24小时。DMSO(0.5％)和Epacadostat(25nM)分别用作阴性和阳性对照。通过测量细胞培养基中L-犬尿氨酸的浓度来确定IDO1活性。将400μL培养基与180μL 30％三氯乙酸混合，并以13.000rpm离心10分钟。将上清液(100μL)转移到新的96孔板中，并加入等体积的新鲜制备的2％w/v对二甲基氨基苯甲醛的乙酸溶液。使用Multiscan光谱Mk3(Thermo Fisher)在480nm下测量光密度。从L-犬尿氨酸标准曲线确定犬尿氨酸浓度。每个测定一式三份进行，数据表示为平均值±标准偏差。
经试验证明，本发明提供的吲唑类化合物在细胞水平对IDO具有显著的抑制作用，IC 50为1.37μM。
实验例18 TDO细胞抑制的测定
用TDO高表达细胞系A172在细胞环境中测试hTDO活性。在96孔板(2×10 4个细胞/孔)中接种过夜后，用一定浓度的化合物和L-Trp(20μg/ml)处理A172细胞24小时。然后，将细胞培养基(300μL/孔)转移到培养管中并与90μLTCA(30％，w/v)混合。接下来，将培养管转移到65℃水浴中30分钟将N-甲酰基犬尿氨酸转化为犬尿氨酸。在13.000rpm离心10分钟后，将上清液(100μL)转移到新的96孔微孔板中，并加入等体积的新鲜制备的2％w/v对二甲基氨基苯甲醛的乙酸溶液。使用Multiscan光谱Mk3(Thermo Fisher)在480nm下测量光密度。从L-犬尿氨酸标准曲线确定犬尿氨酸浓度。最后，使用GraphPad Prism 5.0软件处理数据。
经试验证明，本发明提供的吲唑类化合物在细胞水平对TDO具有显著的抑制作用，IC 50为7.54μM。
实验例19蛋白质印迹分析
将HeLa细胞接种在96孔板中并用IDO1抑制剂处理2小时，然后再加入INFγ(50ng/mL)24小时。将HeLa细胞接种在6孔板中，并用IDO1抑制剂(10,20和50μM)和JAK抑制剂(1μM)处理2小时，然后加入50ng/mL的INFγ24小时。DMSO浓度为0.5％用作阴性对照。将处理过的细胞在RIPA裂解缓冲液中裂解，并将相同量的蛋白质样品上样到10％十二烷基硫酸钠-聚丙烯酰胺凝胶上进行电泳。此后，将蛋白质转移到硝酸纤维素膜上，然后用含有Tween 20(TBST)的Tris缓冲盐水中的5％牛血清白蛋白封闭。将膜在4℃下与抗IDO1(兔多克隆抗体，1:1000，#13268-1-AP，Proteintech)和抗GAPDH(兔多克隆抗体，1:4000，#10494-A-AP)培养过夜。然后，在室温下与辣根过氧化物酶(HRP)山羊抗兔抗体IgG(1:3000，#511203，Zen BioScience)培养育90分钟。通过使用Image Quant LAS 500(GE Healthcare Bio Sciences AB，USA)上的增强化学发光试剂(EasySee western印迹试剂盒，TransGen Biotech)使条带可视化。
通过Western blot方法研究了化合物35和LWQ-84对IDO1蛋白表达的影响，定量WB分析表明，化合物35以浓度依赖性方式显著降低INFγ诱导的IDO1表达。
实验例20体内抗肿瘤研究
将7-8周龄雌性Balb/c小鼠并将其圈养在无菌屏障设施中并随意喂食标准饮食。饲养动物与美国国立卫生研究院实验动物护理和使用指南一致。为了建立CT26异种移植模 型，每只Balb/c小鼠皮下注射100μL1×107个细胞/mL CT26单细胞悬浮液。孵育4天后，将小鼠随机分成5组(每组6只小鼠)。每天口服给予三组，分别以20,40和80mg/kg/天的剂量给予ISL。口服摄入5％DMSO，20％PEG400和75％去离子水作为载体组。将Epacadostat(INBC024360,40mg/kg)处理组用作阳性对照。在治疗期间用游标卡尺每3天测量肿瘤生长，并且使用以下公式计算肿瘤体积：体积(mm 3)＝a×b 2/2(a：最长直径(长度)；b：最短直径(宽度))。结果显示，化合物35在体内具有显著的抗肿瘤作用。
综上所述，本发明公开了一种4位芳杂环取代的吲唑类化合物作为IDO/TDO双重抑制剂的用途。经试验证明化合物对IDO/TDO酶和IDO/TDO细胞均具有优异的抑制作用，而且体内具有显著抗肿瘤活性，可以用于预防和/或治疗多种疾病，如肿瘤、神经退行性疾病、阿尔茨海默病、帕金森病、抑郁症、细胞免疫激活相关的感染、或色氨酸代谢异常等。

Claims (10)

  1. 式(I)所示结构化合物、或其药学上可接受的盐、或其溶剂合物在制备TDO抑制剂类药物中的用途：
    Figure PCTCN2019074333-appb-100001
    其中，
    X选自H或C1～C6烷基；
    A选自取代的或非取代的芳基、杂芳基、环烷基、杂环烷基、苄基、C1～C6烷基，所述取代的芳基、杂芳基、苄基、环烷基或杂环烷基分别独立地被一个或多个选自-(CH 2) aOH、-(CH 2) aNHR 1、卤代烷基、-(CH 2) aCN、-SO 2NH 2、-(CH 2) aNO 2、C1～C6烷基、卤素或
    Figure PCTCN2019074333-appb-100002
    的取代基取代；
    R 1选自H、-OH、-COR 4、-(CH 2) bCOOR 5、环己酮基、环己基或羟基取代的环己基；
    R 4选自C1～C6烷基、-NHR 6、-CH 2R 7；
    R 6选自C3～C6环烷基或杂环烷基，所述杂环烷基含有1～2个选自O、N、S的杂原子；
    R 7选自取代或非取代的芳基或杂芳基，所述取代芳基或杂芳基分别独立地被一个或多个选自氨基、羟基、卤素、硝基或C1～C6烷基的取代基取代；
    R 5选自H或C1～C6烷基；
    R 2与R 3构成含两个或以上杂原子的5～6元杂环，所述杂原子选自O、N、S；
    a＝0或1，b＝1。
  2. 根据权利要求1所述用途，其特征在于：所述化合物(I)中X选自H或甲基；和/或，A选自芳基、杂芳基或甲基。
  3. 根据权利要求2所述的用途，其特征在于：所述芳基选自苯基或萘基。
  4. 根据权利要求2所述的用途，其特征在于：所述杂芳基选自呋喃基、吡啶基、噻吩基、苯并噻吩基、噻唑基、或咪唑基。
  5. 根据权利要求1-4任一项所述用途，其特征在于：所述化合物(I)的结构如式(II) 或式(III)所示：
    Figure PCTCN2019074333-appb-100003
  6. 根据权利要求1-5任一项所述化合物，其特征在于：所述化合物具有如下结构：
    Figure PCTCN2019074333-appb-100004
    Figure PCTCN2019074333-appb-100005
  7. 权利要求1-6任一项所述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物在制备TDO抑制剂类药物上的用途。
  8. 权利要求1-6任一项所述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物在制备IDO/TDO双重抑制剂类药物上的用途。
  9. 根据权利要求8所述的用途，其特征在于：所述药物是预防和/或治疗肿瘤、神经退行性疾病、阿尔茨海默病、帕金森病、抑郁症、细胞免疫激活相关的感染、或色氨酸代谢异常等疾病的药物。
  10. 具有如下结构的化合物：
    Figure PCTCN2019074333-appb-100006
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