CN111153850B - Indole compound, preparation method thereof, pharmaceutical composition and application - Google Patents

Abstract

The invention discloses an indole compound with the structural characteristics of a formula (I), a metabolite, a metabolic precursor, a prodrug, a solvate, a crystal or a pharmaceutically acceptable salt thereof, a preparation method thereof and application thereof as an indoleamine 2,3-dioxygenase 1(IDO1) inhibitor. The experimental result shows that the compound has a remarkable inhibiting effect on the activity of IDO1, can effectively promote the proliferation of T lymphocytes, inhibit the differentiation of initial T lymphocytes into regulatory T cells and reverse the immunosuppressive action mediated by IDO1, and can be used for treating related diseases with the pathological characteristics of IDO 1-mediated kynurenine metabolic pathway, such as cancers, virus infection, neurodegenerative diseases, cataracts, organ transplant rejection, depression, autoimmune diseases and the like.

Description

Indole compound, preparation method thereof, pharmaceutical composition and application

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to indole compounds serving as indoleamine 2,3-dioxygenase 1(IDO1) inhibitors, metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof, a preparation method thereof, pharmaceutical compositions containing the compounds, and application of the compounds or the compositions in treating diseases related to IDO1 mediated immunosuppression, such as cancer, viral infection, neurodegenerative diseases, cataract, organ transplant rejection, depression or autoimmune diseases.

Background

Tryptophan is an amino acid essential for cell proliferation and survival in humans and is useful in the biosynthesis of proteins, nicotinic acid and 5-hydroxytryptamine. Tryptophan is normally taken from food, and more than 95% of tryptophan is metabolized by the kynurenine pathway, and the remainder is converted to 5-hydroxytryptophan and 5-hydroxytryptamine in the nervous system and intestinal tract, or melatonin is synthesized in the pineal gland. Indoleamine 2,3-dioxygenase 1(IDO 2,3-dioxygenase, IDO1) is the rate-limiting enzyme in the kynurenine metabolic pathway that catalyzes tryptophan outside the human liver. IDO1 is expressed in a variety of tissues (e.g., lung, kidney, brain, placenta, thymus) as well as in a variety of cells (e.g., macrophages and dendritic cells). IDO1 decomposes tryptophan by oxidation to reduce the concentration of tryptophan in the body's microenvironment and produces a series of metabolites such as kynurenine, 3-hydroxykynurenine, 2-amino-3-hydroxybenzoic acid, quinolinic acid, and the like. Cytokines such as IFN-. gamma.TNF-. alpha.IL-1. beta.and IL-6 induce up-regulation of the expression of IDO1 (Bernhardt R.chem Rev,1996,96(1): 2841-2888).

Munn DH et al reported that IDO1 not only catalyzes tryptophan oxidative metabolism, but also has important regulatory effects on innate and adaptive immunity of the body (Munn DH, et al. trends Immonol,2013,34(3): 137-. IDO1 primarily catalyzes tryptophan depletion and its metabolite accumulation to regulate the immune system by catalyzing tryptophan: in one aspect, tryptophan depletion can inhibit T cell proliferation by activating the GCN2 pathway to induce T cell division cycle arrest at G1, while also inhibiting primary CD4⁺T cells differentiate into helper T cells 17(Th17) and thus produce immunosuppression (Munn DH, et al. immunity,2005,22(5): 633-. On the other hand, tryptophan metabolites such as kynurenine are cytotoxic and can kill T cells and Natural Killer (NK) cells (Frumento G, et al. J Exp Med,2002,196(4):459-⁺Differentiation of T cells into regulatory T cells (Tregs) and promotion of Dendritic Cell (DC) conversion to tolerogenic DC (Mezrich JD, et al. J Immunol,2010,185(6): 3190-; in addition, tryptophan metabolites can inhibit NK cell function by down-regulating NK cell receptor expression, which can further inhibit the immune response of the body (Della Chiesa M, et al. blood,2006,108(13):4118 and 4125).

IDO1 is involved in many pathophysiological processes. Research shows that during the stress of IDO1 in the physiological processes of host immune defense, maternal and fetal immune tolerance and the like, the secretion of cytokines such as IFN-gamma is obviously increased, so that IDO1 expression is induced to be up-regulated, tryptophan depletion, kynurenine and other metabolites are accumulated, the T cell reaction of a mother body is inhibited, maternal immune tolerance is induced, and the fetus is ensured not to be rejected by the immune system of the mother body; depletion of tryptophan in the host microenvironment renders it unable to provide the tryptophan necessary for replication of pathogenic microorganisms, thereby leading to death of pathogenic microorganisms, while IDO 1-mediated immunosuppression may prevent over-activation of the body' S immune system (Mellor AL, et AL nat Rev Immunol,2008,8(1): 74-80; terns P, et AL am J Reprod Immunol,2007,58(3): 238-. When an inhibitor of IDO1 was administered to pregnant mice, it caused T cell-mediated embryo rejection, resulting in abortion in the mice, indicating that IDO1 could protect the fetus from maternal rejection (Munn DH, et al science,1998,281(5380): 1191-. IDO1 also exerts an immunosuppressive effect on the survival of transplanted tissue in new hosts (Radu CA, et al. plant Reconstr Surg,2007,119(7): 2023-. These findings indicate that IDO is an immunomodulatory enzyme involved in immune tolerance in the body.

Numerous studies have shown that IDO 1-mediated immune tolerance is closely related to diseases such as tumor immune escape, viral infection, neurodegenerative diseases, organ transplant rejection, autoimmune diseases, neuropsychiatric diseases and cataracts (Munn DH, et al trends Immunol, 2013,34(3): 137-. In these diseases, tryptophan depletion mediated by over-expressed IDO1 and accumulation of its metabolites may inhibit T cell activation, resulting in immune tolerance in the body.

In a mouse model of viral infection, administration of an IDO1 inhibitor significantly promoted CD8⁺Proliferation of T cells restores the immune response of T cells and inhibits the virus from infecting host monocytes macrophages. In influenza virus infection, over-expressed IDO 1-mediated immunosuppression is likely to lead to secondary lung infection (Van Der Sluijs KF, et al. J infection Dis,2006,193(2): 214-222). During HIV infection, IDO1 is up-regulated and expressed to promote the proliferation of Treg cells and inhibit the proliferation of Th17 cells, resulting in the imbalance of Tregs/Th17 cell ratio and immunosuppression of patients (Favre D, et al. Sci Transl Med,2010,2(32): 32-36). Furthermore, IDO 1-mediated tryptophan depletion and the increase in the concentration of its metabolites are also associated with parasitic infections (Knubel CP, et al. FASEB J,2010,24(8): 2689-2701).

Studies have shown that tryptophan metabolites catalyzed by IDO1, such as kynurenine and quinolinic acid, are neurotoxic and that these metabolites are closely related to the development of neurodegenerative diseases such as memory impairment, Alzheimer's Disease (AD), cognitive impairment, senile dementia, parkinson's disease and dyskinetic diseases (Malpass k. nat Rev Neurol,2011,7(8): 417; Maddison DC, et al. semin Cell Dev Biol,2015,40: 134-. Both IDO1 expression and quinolinic acid concentration were higher in brains of AD patients than in normal individuals, with the highest levels in microglial nuclear astrocytes surrounding senile plaques. In addition, the concentration of tryptophan in the blood of AD patients is lower than that of normal people, the concentration of kynurenine is higher than that of normal people, and the ratio of the tryptophan to the kynurenine is closely related to the cognitive deficiency degree of the AD patients (Guillemin GJ, et al. neuropathol Appl Neurobiol,2005,31(4): 395-404; Widner B, et al. adv Exp Med Biol,1999,467: 133-138). Neuropsychiatric disorders such as depression, schizophrenia, anxiety are also associated with an overexpression of IDO1 and an elevated level of metabolic products such as kynurenine. Overexpression of IDO1 results in depletion of tryptophan, thereby reducing the amount of tryptophan used to synthesize the neurotransmitter 5-hydroxytryptamine, resulting in 5-hydroxytryptamine deficiency, coupled with accumulation of neurotoxic metabolites such as kynurenine and quinolinic acid, which together contribute to the development of neuropsychiatric diseases and are a factor in a variety of mood disorders (tint am. febs J,2012,279(8): 1375-. Therefore, inhibition of IDO1 is an important therapeutic strategy for patients with neurodegenerative and neuropsychiatric diseases.

Tryptophan overexpression mediated by high expression of IDO1 is also present in various autoimmune diseases (Nguyen NT, et al Front Immunol,2014,5: 551). IDO1 was highly expressed in DCs in synovial joint tissues of patients with rheumatoid arthritis, and the serum tryptophan concentration was decreased, while the kynurenine concentration and the kynurenine/tryptophan ratio were both significantly increased (Widner B, et al. immunology, 2000,201(5): 621-630). Therefore, inhibition of IDO1 is also an important therapeutic strategy for autoimmune patients.

Numerous studies have shown that IDO 1-induced immunosuppression plays an important role in tumor immune escape. IDO1 is overexpressed in various tumors and their cells in the microenvironment, such as DC cells and stromal cells, leading to local tryptophan depletion and accumulation of tryptophan metabolites in the tumor, thereby inducing tumor immune escape and helping tumor cells evade attack by the body's immune system (Munn DH, et al. trends Immonol,2016,37(3): 193-207). The Uyttenhove group detected the expression of IDO1 in 24 human tumors, including melanoma, lung, breast, stomach, colon, bladder, pancreas, lymph, prostate, kidney, brain, head and neck, ovary, cervix, endometrium, mesothelial, thyroid, liver, and esophagus, using immunohistochemical labeling (Uyttenhove C, et al. nat. Med., 2003,9(10): 1269-. It was subsequently confirmed in tumor tissues such as ovarian Cancer, melanoma, lung Cancer, leukemia, etc., and found that the expression level of IDO1 in tumor tissues is closely related to the malignancy of tumors and affects the prognosis of tumor patients (Th ate I, et al Cancer Immunol Res,2015,3(2): 161-172; Curti A, et al blood,2007,109(7): 2871-2877; De Jong RA, et al int J Gynecol cer,2011,21(7): 1320-1327; Okamoto A, et al Clin Cancer Res,2005,11(16): 6030-6039; Ino K, et al Br J Cancer 2012, 2006,95(11 1555-1561; Speecka R, Eustill. J. Cancer, 48 (2011 13). Inhibitors of IDO1 can activate T cells, overcome tumor immune escape mediated by IDO1, and can enhance therapy with other tumor therapeutics (Koblish HK, et al. mol Cancer Ther,2010,9(2): 489-.

Various preclinical and clinical studies show that the IDO1 inhibitor can reduce accumulation of metabolites such as tryptophan metabolism and kynurenine, thereby reversing IDO 1-mediated immunosuppression, restoring proliferation and functions of T cells and NK cells, and inhibiting proliferation of Treg cells, thereby enhancing immune response of the body, so that the IDO1 inhibitor can be used for treating the related diseases caused by IDO 1-mediated immunosuppression, including cancer, viral infection, neurodegenerative diseases, cataract, organ transplantation rejection, depression and autoimmune diseases. In addition, the IDO1 inhibitor can also be combined with other chemotherapeutic agents, targeted antineoplastic agents, immune checkpoint inhibitors, immune checkpoint agonists, anti-tumor vaccines, antiviral agents, antiviral vaccines, cytokine therapy, adoptive cellular immunotherapy and radiotherapy to act as a synergistic or therapeutic enhancement (Vacchelli E, et al. Oncoimmunology,2014,3(10): E957994; Jochems C, et al. Oncoimagent, 2016,7(25): 37762) 37772; Liu X, et al. blood,2010,115(17): 3520) 3530; Zamararin D, et al. Pharmacol Ther,2015,150: 23-32).

Based on the fact that IDO1 is closely related to pathogenesis of various diseases such as cancer, virus infection, neurodegenerative diseases, cataract, organ transplant rejection, depression and autoimmune diseases, an IDO1 inhibitor can be adopted to inhibit activity of IDO1, so that tryptophan metabolism and accumulation of metabolic products such as kynurenine are reduced, immune function of an organism is restored, and the purpose of treating the diseases is achieved. The compound can obviously inhibit the activity of IDO1, and can be used for treating related diseases caused by IDO1 mediated immunosuppression, including cancer, virus infection, neurodegenerative diseases, cataract, organ transplant rejection, depression and autoimmune diseases.

Furthermore, it is worth noting that since the 4-month old default month old Shadong company of 2018 declared the failure of phase III clinical trial (ECHO-301) of the IDO1 inhibitor epacadostat in combination with PD-1 monoclonal antibody pembrolizumab for metastatic melanoma, clinical emphasis was placed on developing a new generation of apo-IDO1 inhibitors that bind to heme-free IDO 1. It is expected that a breakthrough in clinical trials will be obtained by developing a first generation IDO1 inhibitor (acting on heme-containing IDO1) that is distinct from those found earlier. Among them, apo-IDO1 inhibitors represented by BMS-986205 developed by Bethes-Messajou-Guibao, Inc. have restarted several phase I-III clinical trials. Therefore, apo-IDO1 inhibitors may be the mainstream direction for the development of future IDO1 inhibitors. The compound is an apo-IDO1 inhibitor and has good development value and application prospect.

Disclosure of Invention

The purpose of the invention is as follows:

the invention aims to provide a compound with structural characteristics of a formula (I), a metabolite, a metabolic precursor, a prodrug, a solvate, a crystal or a pharmaceutically acceptable salt thereof, a preparation method, a pharmaceutical composition and application thereof. The compound of the invention has good IDO1 inhibitory activity, and can be used for treating and/or preventing various related diseases caused by IDO1 mediated immunosuppression.

The technical scheme is as follows:

the present invention provides compounds, metabolites, metabolic precursors, prodrugs, solvates, crystals, or pharmaceutically acceptable salts thereof, structurally characterized by the general formula (I):

wherein:

R¹represents hydrogen, halogen, C₁-C₈Alkyl radical, C₁-C₈Alkoxy or-NR⁶R⁷；

R²Represents cyano, -CO₂R⁸or-CONR⁶R⁷；

R³Represents hydrogen, halogen, cyano, hydroxy or nitro;

R⁴represents-NR⁶R⁷or-CONHR⁸；

R⁵Represents nitro, -NR⁸CONR⁸R⁹、-NR⁸CO(CH₂)_nR⁹or-NR⁸SO₂R⁹；

n represents an integer of 0 to 6;

R⁶and R⁷Each independently represents hydrogen, C₁-C₈Alkyl radical, C₃-C₈Cycloalkyl radical, C₂-C₈Alkenyl radical, C₂-C₈Alkynyl, C₁-C₈Alkylamino or R⁶And R⁷Together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; wherein said heterocyclic ring may optionally comprise one or more heteroatoms selected from O, S or N; wherein said heterocyclic ring may be optionally substituted with one or more of the following groups: halogen, nitro, cyano, hydroxy, amino, C₁-C₈Alkyl radical, C₁-C₈Alkoxy or C₃-C₆A cycloalkyl group;

R⁸represents hydrogen, C₁-C₈Alkyl radical, C₃-C₈Cycloalkyl radical, C₂-C₈Alkenyl or C₂-C₈An alkynyl group;

R⁹represents aryl or aromatic heterocycle, wherein said aryl or aromatic heterocycle may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen, cyano, hydroxy, mercapto, C₁-C₈Alkyl radical, C₁-C₈Alkoxy radical, C₁-C₈Alkylamino, haloalkyl or-NR⁶R⁷；

Wherein said-NR⁸CONR⁸R⁹Two R in the formula⁸May be selected from the same or different groups;

wherein said alkyl represents a straight chain alkyl group, a branched chain alkyl group or a cyclic alkyl group;

wherein said alkoxy represents a linear alkoxy group, a branched alkoxy group or a cyclic alkoxy group;

wherein said alkylamino represents a linear, branched or cyclic alkylamino group;

wherein said alkenyl represents a linear, branched or cyclic alkenyl;

wherein said alkynyl represents a linear alkynyl group or a branched alkynyl group;

wherein said aryl group represents phenyl, naphthyl, acenaphthenyl or tetrahydronaphthyl;

wherein said heteroaromatic ring represents a monocyclic heterocycle of pyrrolyl, pyrazolyl, imidazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl; or a bicyclic heterocycle of quinolinyl, quinoxalinyl, indolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzofuranyl, benzothienyl, 2, 3-dihydrobenzo [1,4] dioxinyl or benzo [1,3] dioxolyl;

wherein the haloalkyl is a straight or branched chain saturated hydrocarbon group having 1 to 8 carbon atoms, or a cyclic saturated hydrocarbon group having 3 to 8 carbon atoms to which a straight or branched chain saturated hydrocarbon group having 1 to 8 carbon atoms is attached; wherein one or more hydrogen atoms are replaced by one or more halogen atoms.

Further, indole compounds represented by general formula (I), metabolites, metabolic precursors, prodrugs, solvates, crystals, or pharmaceutically acceptable salts thereof, wherein:

R¹represents hydrogen, halogen, C₁-C₈An alkyl group;

R²represents cyano, -CO₂R⁸or-CONR⁶R⁷；

R³Represents hydrogen or halogen;

R⁴represents-NR⁶R⁷or-CONHR⁸；

R⁵Represents nitro, -NR⁸CONR⁸R⁹、-NR⁸CO(CH₂)_nR⁹or-NR⁸SO₂R⁹；

n represents an integer of 0 to 6;

R⁶and R⁷Each independently represents hydrogen, C₁-C₈Alkyl radical, C₃-C₈Cycloalkyl radical, C₁-C₈Alkylamino or R⁶And R⁷Together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; wherein said heterocyclic ring may optionally comprise one or more heteroatoms selected from O, S or N; wherein said heterocyclic ring may be optionally substituted with one or more of the following groups: c₁-C₈Alkyl or C₃-C₆A cycloalkyl group;

R⁸represents hydrogen, C₁-C₈Alkyl or C₃-C₈A cycloalkyl group;

R⁹represents aryl or aromatic heterocycle, wherein said aryl or aromatic heterocycle may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen, cyano, hydroxy, mercapto, C₁-C₈Alkyl radical, C₁-C₈Alkoxy radical, C₁-C₈Alkylamino, haloalkyl or-NR⁶R⁷。

Further, the indole compound has a general formula (I), a metabolite, a metabolic precursor, a prodrug, a solvate, a crystal, or a pharmaceutically acceptable salt thereof, wherein:

R¹represents hydrogen or halogen;

R²represents cyano, carboxyl, formamido, methoxycarbonyl or ethoxycarbonyl;

R³represents hydrogen or halogen;

R⁴represents-NR⁶R⁷or-CONHR⁸；

R⁵Represents nitro, -NR⁸CONR⁸R⁹、-NR⁸CO(CH₂)_nR⁹or-NR⁸SO₂R⁹；

n represents an integer of 0 to 3;

R⁶and R⁷Each independently represents hydrogen, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl radical, C₁-C₆Alkylamino or R⁶And R⁷Together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; wherein said heterocyclic ring may optionally comprise one or more heteroatoms selected from O, S or N; wherein said heterocyclic ring is optionally substituted with one or more methyl groups;

R⁸represents hydrogen, C₁-C₃Alkyl or C₃-C₆A cycloalkyl group;

R⁹represents a benzene ring or an isoxazolyl group, wherein said aryl group may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen, cyano, C₁-C₅Alkyl radical, C₁-C₅Alkoxy, trifluoromethyl or N-methylpiperazinyl.

More preferably, the indole compound has a general formula (I), a metabolite, a metabolic precursor, a prodrug, a solvate, a crystal, or a pharmaceutically acceptable salt thereof, wherein:

R¹represents hydrogen;

R²represents COOH;

R³represents hydrogen or halogen;

R⁴represents-NR⁶R⁷；

R⁵represents-NR⁸CONR⁸R⁹；

R⁶And R⁷Each independently represents C₁-C₄Alkyl or C₃-C₆A cycloalkyl group;

R⁸represents hydrogen;

R⁹represents a phenyl ring, wherein said phenyl ring may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen or C₁-C₅An alkyl group.

Specifically, the indole compound shown in the general formula (I), metabolite, metabolic precursor, prodrug, solvate, crystal or pharmaceutically acceptable salt thereof is preferably selected from:

ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (1),

1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (2),

1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxamide (3),

1- (3-nitro-4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (4),

1- (3- (3- (4-chlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (5),

1- (3- (3- (4- (4-methylpiperazin-1-yl) phenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (6),

1- (3- (3- (3-trifluoromethylphenyl) -4- ((ethyl) cyclohexylamino) ureido) phenyl) -1H-indole-2-carboxylic acid (7),

1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (8),

1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (9),

1- (3- ((4-methylphenyl) sulfonamide) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (10),

1- (3- (2- (4-methylphenyl) acetamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (11),

1- (3- ((4-methylphenyl) carboxamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (12),

1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -6-chloro-1H-indole-2-carboxylic acid (13),

1- (3- (3- (4-methylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (14),

1- (3- (3- (4-fluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (15),

1- (3- (3- (4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (16),

1- (3- (3- (2, 4-difluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (17),

1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (18),

1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (19),

1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (20),

1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (21),

1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (22),

1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (23),

1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (24),

1- (3- (3- (4-methylphenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (25),

1- (3- (3- (4-fluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (26),

1- (3- (3- (4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (27),

1- (3- (3- (2, 4-difluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (28),

1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (29),

1- (3-chloro-4- (diisobutylamino) -5- (3- (4-methylphenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (30),

1- (3-chloro-4- (diisobutylamino) -5- (3- (4-fluorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (31),

1- (3-chloro-4- (diisobutylamino) -5- (3- (4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (32),

1- (3-chloro-4- (diisobutylamino) -5- (3- ((2, 4-difluorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (33),

1- (3-chloro-4- (diisobutylamino) -5- (3- (2-fluoro-4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (34),

1- (3- (3- (4-methylphenyl) ureido) -4- (methyl- (1-methylpiperidin-4-yl) amino) phenyl) -1H-indole-2-carboxylic acid (35),

1- (3- (3- (4-methylphenyl) ureido) -4- (4-methylpiperazin-1-yl) phenyl) -1H-indole-2-carboxylic acid (36),

1- (3- (3- (4-methylphenyl) ureido) -4- (cyclohexylcarbamoyl) phenyl) -1H-indole-2-carboxylic acid (37),

another object of the present invention is to provide a process for preparing a compound represented by the general formula (I), which comprises the steps of:

a) when R is⁴is-NR⁶R⁷The preparation method of the compound shown in the general formula (I) comprises the following steps:

1) substituted nitrobenzene is taken as raw material and reacts with amine compound HNR under the action of alkali⁶R⁷Reacting to obtain an intermediate i;

2) i and indole-2-carboxylic acid ethyl ester are reacted by Ullmann to prepare an intermediate ii;

3) ii, reducing by a reducing agent to obtain an intermediate iii;

4) iii with substituted phenylisocyanates R⁹NCO is condensed to prepare a compound iv, or iii is firstly reacted with 4-nitrophenyl chloroformate to generate an active intermediate, and then the active intermediate is reacted with an amine compound R⁹NH₂Reacting to obtain a target compound iv;

iv hydrolyzing to obtain a target compound v;

v reacting with oxalyl chloride or thionyl chloride to prepare acyl chloride, and then reacting with amine compound HNR⁷R⁸Reacting to obtain a target compound vi;

intermediate iii with substituted phenylsulfonyl chlorides R⁹SO₂Cl to prepare a target compound vii;

vii, hydrolyzing to obtain a target compound viii;

intermediate iii and acid compound R⁹(CH₂)_nCO₂H is reacted under the action of a condensing agent to prepareObtaining a target compound ix;

ix, hydrolyzing with sodium hydroxide to obtain a target compound x;

the synthetic route is as follows:

wherein R is¹、R³、R⁶、R⁷、R⁸And R⁹As defined in claim 1;

wherein the base is selected from triethylamine, DIPEA and Na₂CO₃、K₂CO₃、Cs₂CO₃NaOH or KOH;

wherein, the reducing agent in the step 3) is selected from zinc powder and ammonium chloride or iron powder and ammonium chloride.

b) When R is⁴is-CONHR⁸The preparation method of the compound shown in the general formula (I) comprises the following steps:

1) substituted benzoic acid is taken as a raw material and reacts with oxalyl chloride or thionyl chloride to prepare acyl chloride xi;

2) xi and amine compound R⁸NH₂Condensing to obtain amide xii;

3) xii and indole-2-carboxylic acid ethyl ester are reacted by Ullmann to prepare an intermediate xiii;

4) xiii is reduced by a reducing agent to prepare an intermediate xiv;

5) xiv and substituted phenyl isocyanates R⁹NCO is condensed to prepare a target compound xv;

xv is hydrolyzed to prepare a target compound xvi;

the synthetic route is as follows:

wherein R is¹、R³、R⁸And R⁹As defined in claim 1;

wherein the base used in step 2) is selected from triethylamine, DIPEA and Na₂CO₃、K₂CO₃、Cs₂CO₃NaOH or KOH;

wherein, the reducing agent in the step 4) is selected from zinc powder and ammonium chloride or iron powder and ammonium chloride.

c) When R is³Is chlorine, R⁴is-NR⁶R⁷The preparation method of the compound shown in the general formula (I) comprises the following steps:

1) taking 2-fluoro-5-bromonitrobenzene as a raw material, and reacting the raw material with an amine compound HNR under the action of alkali⁶R⁷Reacting to obtain an intermediate i;

2) i, carrying out NCS chlorination reaction to obtain an intermediate xvii;

3) reacting xvii with indole-2-carboxylic acid ethyl ester through Ullmann to obtain an intermediate xviii;

4) xviii is reduced by a reducing agent to prepare an intermediate xix;

5) xix and substituted phenyl isocyanate R⁹NCO is condensed to prepare a target compound xx;

xx is hydrolyzed to prepare a target compound xxi;

wherein R is¹、R⁶、R⁷、R⁸And R⁹Is as defined in claim 1.

Wherein, the reducing agent in the step 4) is selected from zinc powder and ammonium chloride or iron powder and ammonium chloride.

The pharmaceutically acceptable salts of the compounds of general formula (I) can be synthesized by general chemical methods.

In general, salts can be prepared by reacting the free base or acid with a stoichiometric equivalent or excess of an acid (inorganic or organic) or base (inorganic or organic) in a suitable solvent or solvent composition.

The invention also provides a pharmaceutical composition which mainly comprises active components with effective dose in treatment and pharmaceutically acceptable auxiliary materials; the active component comprises one or more compounds of general formula (I) or pharmaceutically acceptable salts thereof. In the pharmaceutical composition, the auxiliary materials comprise pharmaceutically acceptable carriers, diluents and/or excipients.

The pharmaceutical composition may be formulated into various types of administration unit dosage forms according to the therapeutic purpose, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, injections (solutions and suspensions), and the like, preferably tablets, capsules, liquids, suspensions, and injections (solutions and suspensions).

For shaping the pharmaceutical composition in the form of tablets, pills or suppositories, any excipient known and widely used in the art can be used.

For preparing the pharmaceutical composition in the form of injection, the solution or suspension may be sterilized (preferably by adding appropriate amount of sodium chloride, glucose or glycerol) and made into injection with blood isotonic pressure. In the preparation of injection, any carrier commonly used in the art may also be used. For example: water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyethoxylated isostearyl alcohol, and fatty acid esters of polyethylene sorbitan, and the like. In addition, usual dissolving agents, buffers and the like may be added.

The content of the composition of the present invention in the pharmaceutical composition is particularly limited, and can be selected from a wide range, usually 5 to 95% by mass, preferably 30 to 85% by mass.

The method of administration of the pharmaceutical composition of the present invention is not particularly limited. The formulation of various dosage forms can be selected for administration according to the age, sex and other conditions and symptoms of the patient.

The invention also provides application of the compound with the general formula (I), the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing an indoleamine 2,3-dioxygenase 1(IDO1) inhibitor. The IDO1 inhibitor is used for treating patients with diseases related to IDO1 mediated immunosuppression, such as cancer, viral infection, neurodegenerative diseases, cataract, organ transplant rejection, depression or autoimmune diseases.

The invention also provides the use of the compound of general formula (I), a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a medicament for the treatment of cancer, viral infection, neurodegenerative disease, cataract, organ transplant rejection, depression or autoimmune disease in a patient.

Such cancers include, but are not limited to: malignant melanoma, lung cancer, breast cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, lymphatic cancer, leukemia, prostate cancer, testicular cancer, renal cancer, brain cancer, head and neck cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, thyroid tumor, liver cancer, and esophageal cancer.

Such viral infections include, but are not limited to: infections caused by one or more of human immunodeficiency virus, hepatitis b virus, hepatitis c virus, influenza virus, poliovirus, cytomegalovirus, coxsackievirus, human papilloma virus, epstein-barr virus and varicella-zoster virus.

Such neurodegenerative diseases include, but are not limited to: one or more of memory disorders, alzheimer's disease, cognitive disorders, senile dementia, parkinson's disease and dyskinetic disorders.

The autoimmune diseases include but are not limited to: one or more of rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, nephropathy, myasthenia gravis, mixed connective tissue disease, psoriasis, liver disease, endocrine-related diseases, and autoimmune reactions due to infection.

Further, the present invention provides that said compounds of general formula (i), pharmaceutically acceptable salts thereof or said pharmaceutical compositions may be used in combination with one or more other kinds of therapeutic agents and/or methods of treatment for the treatment of related diseases mediated by IDO 1.

Such other classes of therapeutic agents and/or methods of treatment include, but are not limited to: chemotherapeutic agents, targeted antineoplastic agents, immune checkpoint inhibitors, immune checkpoint agonists, anti-tumor vaccines, antiviral agents, antiviral vaccines, cytokine therapy, adoptive cellular immunotherapy, or radiation therapy.

Such chemotherapeutic agents include, but are not limited to: alkylating agents, tubulin inhibitors, topoisomerase inhibitors, platinum drugs, antimetabolites or hormone antineoplastic drugs.

The targeted antitumor drugs include but are not limited to: protein kinase inhibitors, proteasome inhibitors, isocitrate dehydrogenase inhibitors, epigenetic-based antineoplastic agents or cell cycle signaling pathway inhibitors.

Such immune checkpoint inhibitors include, but are not limited to: a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, a LAG3 inhibitor, a TIGIT inhibitor, an A2AR inhibitor, or a VTCN1 inhibitor.

Such immune checkpoint agonists include, but are not limited to: a STING agonist, a 4-1BB agonist, an OX40 agonist, a rory agonist, or an ICOS agonist.

Has the advantages that:

the compound of the present invention has high IDO1 inhibiting activity. Pharmacological experiment results show that the indole compounds can effectively reverse the IDO 1-mediated immunosuppression effect and promote CD8⁺Proliferation of T lymphocytes, increased secretion of granzyme B and interferon-gamma, and decreased CD4⁺CD25⁺Foxp3⁺Production of regulatory T cells, reducing expression of PCNA protein. The results of in vivo pharmacodynamic evaluation show that the compounds of the invention can obviously inhibit the growth of mouse transplantable tumors of various tumor types, but have no influence on the growth of nude mouse transplantable tumors with defects of immune system, which indicates that the compounds play an anti-tumor role by activating host immune response.

Drawings

Figure 1 is a graph of the effect of compounds of the invention on IDO1 protein expression wherein: a is the effect of compounds 2, 18, 28, 29 and 34 on IDO1 protein expression and B is the grayscale scan statistics of panel a. Hela cells at 2X 10⁵The density of each well was cultured in 6-well plates at 37 ℃ with 5% CO₂Culturing under the condition for 12 h. Blank control (medium only), test group (100ng/Ml IFN-. gamma., compound), at 37 ℃,5％CO₂culturing for 24h under the condition, collecting cells, and detecting IDO1 expression by Western blot.

FIG. 2 is a graph of the effect of compounds of the invention on T lymphocyte proliferation and IFN- γ release, wherein: a is the effect of compounds 2, 18, 28, 29 and 34 on T lymphocyte proliferation and B is the effect of compounds 2, 18, 28, 29 and 34 on IFN- γ release. The treated tumor cell supernatant and spleen cells (density 1X 10)⁶One/well) were mixed at 50. mu.L each, and added to a 96-well plate, 100. mu.L of RPMI 1640 medium containing 10% FBS was added to each well, so that the final system was 200. mu.L/well. In all wells except the blank, ConA (final concentration 5. mu.g/mL) was added and CO was added₂The culture is carried out in an incubator for 48h, and the absorbance value is detected at 570nm by an MTT method.

FIG. 3 is a graph showing the effect of compounds of the present invention on the growth of B16F1 melanoma mouse graft tumors. Will be 1 × 10⁶B16F1 cells were transplanted subcutaneously into the axilla of C57BL/6 mice. 3 days after transplantation, mice were randomly divided into a model group, a control group and a treatment group (n ═ 6). Administered on days 1-11. P<0.05，**P<0.01vs. model set.

FIG. 4 is a graph showing the effect of compounds of the present invention on the growth of LLC lung cancer mouse transplantable tumors, wherein: a is the influence of the compound 2 on the volume of the LLC lung cancer transplantable tumor, and B is the influence of the compound 2 on the weight of the LLC lung cancer transplantable tumor. Will be 1 × 10⁶LLC lung cancer cells were transplanted subcutaneously into the axilla of C57BL/6 mice. Mice were randomized 3 days after transplantation into model, control and treatment groups (n ═ 6). Administration was on days 1-15. P<0.01vs. model set.

FIG. 5 is a graph showing the effect of compounds of the present invention on the growth of BALB/c mouse transplants from CT26 rectal cancer. Will be 1 × 10⁶CT26 cells were transplanted subcutaneously into the axilla of BALB/c mice. Mice were randomly divided into model, control and treatment groups 3 days after transplantation (n-8).

FIG. 6 is a graph of the effect of compounds of the invention on the growth of immune system deficient nude mouse transplants, wherein: a is the effect of Compound 2 on the volume of immune system deficient nude mouse transplants, and B is the effect of Compound 2 on the weight of immune system deficient nude mouse transplants. Will be 1 × 10⁷CT26 cells were transplanted subcutaneously into axilla of BALB/c nude mice. 5 days after transplantationMice were randomly divided into model, control and treatment groups (n-8). Administered on days 1-9. P<0.001vs. model set.

FIG. 7 is a graph showing the effect of compounds of the present invention on the growth of transplanted PAN02 pancreatic cancer mice. Will be 1 × 10⁶PAN02 cells were transplanted subcutaneously into the axilla of C57BL/6 mice. 3 days after transplantation, mice were randomly divided into a model group, a control group and a treatment group (n ═ 8).

FIG. 8 shows that the compound of the present invention binds to IDO1 protein and improves its thermostability. (A) Compound 2 in combination with IDO1 improved the stability of IDO1 at different temperatures. (B) Compound 2 binds to IDO1 in a dose-dependent manner and inhibits degradation of IDO1 at 58 ℃.

Fig. 9 shows that the compounds of the present invention have a very strong selective binding force to IDO1, where: a is the binding affinity of compound 2 to IDO1 protein at various concentrations, and B is the equilibrium dissociation constant of compound 2 to IDO1 protein. Mixing IDO1 protein and compound, and detecting K by Biacore T200 system_D。

Detailed Description

To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.

Example 1

Synthesis of ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (1)

Synthesis of 2-nitro-4-bromo-N, N- (ethyl) cyclohexylaniline (1A)

2-fluoro-5-bromonitrobenzene (3g,13.6mmol) and N-ethylcyclohexylamine (2.61g,20.5mmol) were dissolved in N, N-dimethyldiamide (50mL), N-diisopropylethylamine (3.53g,27.3mmol) was added, and the mixture was stirred at 80 ℃ for 4 hours. After cooling, 200mL of ethyl acetate and 200mL of water were added, the organic layer was separated, the aqueous layer was extracted with ethyl acetate (100mL), the organic layers were combined, washed with saturated brine, and dried over anhydrous magnesium sulfateDrying, suction filtering, decompression concentrating, column chromatography purifying to obtain red solid 4.03g with yield 90.4%.¹H NMR(300MHz,Chloroform-d)δ(ppm)7.75(t,J＝2.1Hz,1H),7.49(dt,J＝8.8,2.1Hz,1H),7.08(dd,J＝8.8,1.7Hz,1H),3.13(m,2H),3.00-2.85(m,1H),1.75(d,J＝15.0Hz,3H),1.63-1.55(m,1H),1.47-1.01(m,6H),0.95(td,J＝7.1,1.7Hz,3H)；MS(EI)m/z 325.1[M-H]^-.

Synthesis of ethyl 1- (3-nitro-4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (1B)

Compound 1A (3g,9.2mmol) was dissolved in DMF (50mL), ethyl indole-2-carboxylate (1.74g,9.2mmol), cuprous iodide (3.49g,18.4mmol), cesium carbonate (5.97g,18.4mmol) and L-proline (2.12g,18.4mmol) were added, and the mixture was stirred at 80 ℃ for 12 hours under nitrogen. Cooling, adding 200mL ethyl acetate and 200mL water, suction-filtering with diatomaceous earth, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, drying over anhydrous magnesium sulfate, suction-filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain 1.68g of yellow solid with a yield of 42.1%.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.18(s,1H),7.94(d,J＝7.9Hz,1H),7.89(d,J＝8.4Hz,1H),7.73(d,J＝7.8Hz,1H),7.65(s,1H),7.23(t,J＝7.6Hz,1H),7.13(t,J＝7.6Hz,1H),7.05(d,J＝8.4Hz,1H),3.47-3.38(m,3H),1.80-1.70(m,2H),1.57-1.46(m,4H),1.43-1.31(m,4H),1.21(t,J＝7.2Hz,3H)；MS(EI)m/z 434.5[M-H]^-.

Synthesis of ethyl 1- (3-amino-4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (1C)

Compound 1B (1.68g,3.85mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (1.25g,19.25mmol) and ammonium chloride (1.23g,19.25mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, after adding 100mL ethyl acetate and 100mL water, separated organic layer, water layer using ethyl acetate (50mL) extraction twice, combined organic layer, saturated salt water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get light yellow solid 1.54 g.

Compound 1C (1.54g,3.79mmol) was dissolved in anhydrous tetrahydrofuran (20mL), p-tolylene isocyanate (0.51g,3.79mmol) was added, and the reaction was carried out at room temperature for 4 hours. ColumnPurification by chromatography gave 1.84g of a white solid in 89.8% yield.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.52(d,J＝12.8Hz,2H),8.34(s,1H),7.97(d,J＝7.8Hz,1H),7.71-7.65(m,2H),7.49(t,J＝7.6Hz,1H),7.43(t,J＝7.6Hz,1H),7.26(d,J＝8.1Hz,2H),7.05-6.98(m,3H),6.85(d,J＝8.4Hz,1H),4.35-4.27(q,J＝7.0Hz,2H),3.48-3.40(q,J＝7.3Hz,2H),3.33(s,1H),2.33(s,3H),1.79-1.66(m,4H),1.53-1.42(m,2H),1.42-1.32(m,7H),1.19(t,J＝7.2Hz,3H)；MS(EI)m/z 537.2[M-H]^-.

Example 2

Synthesis of 1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (2)

Compound 1(1.84g,3.44mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.69g,17.22mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.96g of white solid with the yield of 55.0%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.68(s,1H),8.65(s,1H),8.17(d,J＝2.4Hz,1H),7.64(d,J＝7.7Hz,1H),7.36(d,J＝8.1Hz,2H),7.27(d,J＝8.4Hz,1H),7.2-6.96(m,7H),6.87(dd,J＝8.3,2.5Hz,1H),3.06(q,J＝7.0Hz,2H),2.78(s,1H),2.22(s,3H),1.97(d,J＝11.3Hz,3H),1.80-1.42(m,4H),1.40-0.95(m,8H)；MS(EI)m/z 509.2[M-H]^-.

Example 3

Synthesis of 1- (3- (3- (p-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxamide (3)

Compound 2(1g,1.96mmol) was dissolved in 15mL of anhydrous dichloromethane, cooled in ice bath for 10 minutes, added with 2mL of oxalyl chloride, and added dropwiseTwo drops of DMF initiate the reaction, the system generates bubbles, the reaction is carried out at room temperature overnight, the reaction solution is slowly poured into ice ammonia water to generate a large amount of solid, the solid is filtered and extracted by dichloromethane, anhydrous magnesium sulfate is dried, and the white solid is obtained by column chromatography purification, wherein the yield is 50.1%.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.46(s,1H),8.28(d,J＝2.5Hz,1H),7.69(d,J＝7.8Hz,1H),7.42(s,1H),7.33-7.09(m,8H),6.92(dd,J＝8.4,2.5Hz,1H),6.48(s,1H),2.91(q,J＝7.0Hz,2H),2.61(m,1H),2.35(s,3H),1.25(s,5H),1.17-1.03(m,4H),0.98(d,J＝12.0Hz,3H),0.85(d,J＝6.7Hz,3H)；MS(EI)m/z 508.2[M-H]⁺.

Example 4

Synthesis of 1- (3-nitro-4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (4)

Compound 1B (1g,2.3mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.46g,11.49mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 200mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.51g of yellow solid with the yield of 54.5%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)7.74-7.58(m,2H),7.40(d,J＝2.7Hz,2H),7.28(s,1H),7.19(t,J＝7.5Hz,1H),7.07(t,J＝7.4Hz,1H),6.97(d,J＝8.4Hz,1H),3.05(d,J＝7.1Hz,2H),1.63(t,J＝12.7Hz,4H),1.46-1.20(m,5H),1.18-0.88(m,7H)；MS(EI)m/z 406.2[M-H]^-.

Example 5

Synthesis of 1- (3- (3- (4-chlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (5)

Synthesis of ethyl 1- (3- (3- (4-chlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (5D)

Compound 1C (1.54g,3.79mmol) was dissolved in anhydrous tetrahydrofuran (20mL), p-chlorobenzene isocyanate (0.58g,3.79mmol) was added, and the reaction was carried out at normal temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 1.92g of a white solid.

Compound 5D (1.92g,3.44mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.68g,17.21mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 1.01g of white solid with the yield of 54.8%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.75(s,1H),9.81(s,1H),8.75(s,1H),8.17(s,1H),7.75(s,1H),7.49(s,2H),7.38(d,J＝4.4Hz,2H),7.31(d,J＝3.3Hz,3H),7.12(d,J＝7.7Hz,1H),6.96(s,1H),3.10(s,2H),2.83(s,1H),1.99(s,2H),1.73(s,2H),1.22(s,5H),0.91(s,4H)；MS(EI)m/z 529.2[M-H]^-.

Example 6

Synthesis of 1- (3- (3- (4- (4-methylpiperazin-1-yl) phenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (6)

Synthesis of ethyl 1- (3- (3- (4- (4-methylpiperazin-1-yl) phenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (6D)

Compound 1C (0.19g,0.56mmol) was dissolved in anhydrous dichloromethane (10mL), p-nitrophenyl chloroformate (0.11g,0.67mmol) was added, the reaction was allowed to react at 60 ℃ for 1 hour, the reaction was observed on a dot plate, the reaction mixture was concentrated under reduced pressure, the concentrated solution was dissolved in anhydrous dichloromethane (10mL), triethylamine (0.05g,0.46mmol) and 4-N-methylpiperazine aniline (0.08g,0.46mmol) were added, and the mixture was stirred at room temperature for 8 hours. Purifying by column chromatography to obtain white solid 0.08 g.

Compound 6D (0.08g,0.13mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.03g,0.64mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting pH to 3E with dilute hydrochloric acid (1M)4, adding 100mL of ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain a white solid of 0.03g with the yield of 39.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.91(s,1H),8.76(s,1H),8.16(d,J＝2.5Hz,1H),7.75(d,J＝7.9Hz,1H),7.40-7.26(m,5H),7.17(t,J＝7.4Hz,1H),7.10(d,J＝8.4Hz,1H),6.95-6.86(m,3H),3.22(s,4H),3.07(d,J＝7.1Hz,2H),2.81(s,1H),2.72(s,3H),1.97(d,J＝9.9Hz,2H),1.72(s,2H),1.56(d,J＝11.6Hz,1H),1.21(s,5H),1.07(s,1H),0.90(t,J＝6.9Hz,3H)；MS(EI)m/z 593.3[M-H]^-.

Example 7

Synthesis of 1- (3- (3- (3-trifluoromethylphenyl) -4- ((ethyl) cyclohexylamino) ureido) phenyl) -1H-indole-2-carboxylic acid (7)

Synthesis of ethyl 1- (3- (3- (3-trifluoromethylphenyl) -4- (cyclohexyl (ethyl) amino) ureido) phenyl) -1H-indole-2-carboxylate (7D)

Compound 1C (0.21g,0.52mmol) was dissolved in anhydrous tetrahydrofuran (20mL), 3-trifluoromethylphenylisocyanate (0.1g,0.52mmol) was added, and the reaction was carried out at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.23g of a white solid.

Compound 7D (0.23g,0.39mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.08g,1.94mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.11g of white solid with the yield of 50.1%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)10.03(s,1H),8.80(s,1H),8.17(d,J＝2.5Hz,1H),7.97(d,J＝2.3Hz,1H),7.77(d,J＝7.9Hz,1H),7.62(d,J＝8.5Hz,1H),7.50(t,J＝7.9Hz,1H),7.43-7.37(m,2H),7.35-7.27(m,2H),7.19(t,J＝7.4Hz,1H),7.10(d,J＝8.4Hz,1H),6.98(dd,J＝8.3,2.5Hz,1H),3.11(q,J＝6.9Hz,2H),2.84(s,1H),1.74(s,2H),1.58(d,J＝12.0Hz,1H),1.30-1.17(m,4H),0.92(t,J＝6.9Hz,3H)；MS(EI)m/z 563.2[M-H]^-.

Example 8

Synthesis of 1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (8)

Synthesis of ethyl 1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (8D)

Compound 1C (0.19g,0.47mmol) was dissolved in anhydrous tetrahydrofuran (20mL), 4-chloro-3-trifluoromethylphenylisocyanate (0.1g,0.47mmol) was added, and the reaction was carried out at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.24g of a white solid.

Compound 8D (0.24g,0.38mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.08g,1.92mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.13g of white solid with the yield of 56.7%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)8.82(s,1H),7.76(d,J＝7.9Hz,1H),7.67(dd,J＝8.8,2.5Hz,1H),7.59(d,J＝8.8Hz,1H),7.39(d,J＝6.9Hz,2H),7.30(t,J＝7.7Hz,1H),7.18(t,J＝7.4Hz,1H),7.09(d,J＝8.3Hz,1H),6.98(dd,J＝8.3,2.5Hz,1H),3.10(q,J＝7.0Hz,2H),2.82(s,1H),2.05-1.91(m,2H),1.72(s,2H),1.57(d,J＝12.1Hz,1H),1.20(dd,J＝11.3,7.9Hz,3H),0.99(m,5H)；MS(EI)m/z 597.2[M-H]^-.

Example 9

Synthesis of 1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (9)

Synthesis of ethyl 1- (3- (3, 4-dichlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (9D)

Compound 1C (0.21g,0.52mmol) was dissolved in anhydrous tetrahydrofuran (20mL), 3, 4-dichlorophenylisocyanate (0.1g,0.52mmol) was added, the reaction was carried out at room temperature for 4 hours, concentration under reduced pressure was carried out, and purification by column chromatography gave 0.2g of a white solid.

Compound 9D (0.2g,0.34mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.07g,1.68mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.12g of white solid with the yield of 62.1%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.99(s,1H),8.79(s,1H),8.16(d,J＝2.5Hz,1H),7.89(d,J＝2.5Hz,1H),7.76(d,J＝7.9Hz,1H),7.51(d,J＝8.8Hz,1H),7.39(t,J＝4.2Hz,2H),7.35-7.26(m,2H),7.19(t,J＝7.4Hz,1H),7.10(d,J＝8.4Hz,1H),6.98(dd,J＝8.4,2.6Hz,1H),3.10(d,J＝7.3Hz,2H),2.83(s,1H),1.99(s,2H),1.73(s,2H),1.57(d,J＝11.8Hz,1H),1.22(s,5H),0.90(t,J＝6.9Hz,3H)；MS(EI)m/z 563.1[M-H]^-.

Example 10

Synthesis of 1- (3- ((4-methylphenyl) sulfonamide) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid

Synthesis of ethyl 1- (3- ((4-methylphenyl) sulfonylamino) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (10D)

Compound 1C (0.5g,1.23mmol) was dissolved in dichloromethane (20mL), p-toluenesulfonyl chloride (2.28g,1.48mmol) was added, and triethylamine (0.25g,2.47mmol) was added dropwise under ice bath, and the reaction was carried out at room temperature for 3 hours. 50mL of methylene chloride and 100mL of water were added to separate an organic layer, which was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered under suction, and the filtrate was distilled under reduced pressure to give 0.5g of a pale yellow solid which was used in the next step.

Compound 10D (0.5g, 1.28)mmol) was dissolved in anhydrous ethanol (20mL), sodium hydroxide (0.26g,6.39mmol) was added, and the mixture was stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.3g of white solid with the yield of 61.6%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)7.80(dd,J＝7.5,1.5Hz,1H),7.71-7.65(m,2H),7.34(dd,J＝7.4,1.3Hz,2H),7.19(m,2H),6.73-6.63(m,3H),3.29(q,J＝8.0Hz,2H),2.42(d,J＝1.3Hz,3H),2.29(m,1H),1.90(m,2H),1.76-1.65(m,2H),1.60(m,2H),1.43-1.33(m,2H),1.17(m,2H),1.06(t,J＝8.0Hz,3H)；MS(EI)m/z 530.2[M-H]^-.

Example 11

Synthesis of 1- (3- (2- (4-methylphenyl) acetamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (11)

Synthesis of ethyl 1- (3- (2- (4-methylphenyl) acetamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (11D)

Compound 1C (0.81g,1.99mmol) was dissolved in methylene chloride (20mL), p-methylphenylacetic acid (0.45g,2.99mmol), dicyclohexylcarbodiimide (0.82g,3.98mmol) and 4-dimethylaminopyridine (0.02g,0.19mmol) were added, and the reaction was carried out at room temperature for 24 hours. And (5) purifying by column chromatography to obtain 0.5g of white solid.

Compound 11D (0.5g,0.93mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.19g,4.64mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), and performing suction filtration to obtain 0.24g of white solid with the yield of 50.6%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)7.66(d,J＝7.7Hz,1H),7.33-7.26(m,3H),7.24(d,J＝8.0Hz,2H),7.18-7.14(m,1H),7.12(d,J＝2.6Hz,1H),7.10(s,1H),7.07(s,1H),6.99(d,J＝7.8Hz,1H),6.97-6.93(m,1H),3.71(s,2H),2.81(q,J＝6.9Hz,2H),2.34(s,3H),2.27(d,J＝4.0Hz,1H),1.25(d,J＝11.7Hz,4H),1.18(s,2H),1.10(d,J＝3.0Hz,2H),1.06(s,3H),1.02(s,2H)；MS(EI)m/z 508.2[M-H]^-.

Example 12

Synthesis of 1- (3- ((4-methylphenyl) carboxamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (12)

Synthesis of ethyl 1- (3- ((4-methylphenyl) carboxamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (12D)

Compound 1C (0.71g,1.75mmol) was dissolved in anhydrous tetrahydrofuran (20mL), and p-toluic acid (0.39g,2.63mmol), dicyclohexylcarbodiimide (0.72g,3.5mmol) and 4-dimethylaminopyridine (0.02g,0.17mmol) were added to react at room temperature for 24 hours. And (5) purifying by column chromatography to obtain 0.34g of white solid.

Compound 12D (0.34g,0.65mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.13g,3.25mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), and performing suction filtration to obtain 0.11g of white solid with the yield of 34.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.94(s,1H),8.38(d,J＝2.5Hz,1H),7.78(d,J＝8.0Hz,2H),7.65(d,J＝7.7Hz,1H),7.42(dd,J＝8.2,5.2Hz,3H),7.21-7.01(m,5H),3.16-3.09(m,2H),2.83(s,1H),2.40(s,3H),1.69(d,J＝10.5Hz,2H),1.54(d,J＝11.5Hz,1H),1.19(m,6H),0.92(t,J＝6.9Hz,3H)；MS(EI)m/z494.2[M-H]^-.

Example 13

Synthesis of 1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -6-chloro-1H-indole-2-carboxylic acid (13)

Synthesis of ethyl 1- (3-nitro-4- ((ethyl) cyclohexylamino) phenyl) -6-chloro-1H-indole-2-carboxylate (13B)

Compound 1A (1g,3.1mmol) was dissolved in DMF (50mL), 6-chloro-indole-2-carboxylic acid ethyl ester (0.68g,3.1mmol), cuprous iodide (1.16g,6.2mmol), copper acetate (1.11g,6.2mmol), potassium carbonate (0.84g,6.2mmol) and potassium hydroxide (0.34g,6.2mmol) were added, and stirred under nitrogen at 120 ℃ for 12 hours. Cooling, adding 200mL ethyl acetate and 200mL water, filtering with diatomaceous earth, separating organic layer, extracting water layer with ethyl acetate (100mL), mixing organic layers, washing with saturated saline solution, drying with anhydrous magnesium sulfate, filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain yellow solid 0.8 g.

Synthesis of ethyl 1- (3-amino-4- ((ethyl) cyclohexylamino) phenyl) -6-chloro-1H-indole-2-carboxylate (13C)

Compound 13B (0.8g,1.7mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (0.55g,8.5mmol) and ammonium chloride (0.54g,8.5mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, adding 100mL ethyl acetate and 100mL water, separating the organic layer, water layer with ethyl acetate (50mL) extraction, organic layer, combined, saturated saline water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get light yellow solid 0.64 g.

Synthesis of ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -6-chloro-1H-indole-2-carboxylate (13D)

Compound 13C (0.64g,1.45mmol) was dissolved in anhydrous tetrahydrofuran (20mL), p-tolylene isocyanate (0.20g,1.45mmol) was added, and the reaction was carried out at room temperature for 4 hours. And (5) purifying by column chromatography to obtain 0.52g of white solid.

Compound 13D (0.52g,0.89mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.18g,4.54mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.24g of white solid with the yield of 48.5%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.81(s,1H),9.56(s,1H),8.69(s,1H),8.17(dd,J＝2.5,1.2Hz,1H),7.80(d,J＝8.4Hz,1H),7.37(dd,J＝16.4,7.2Hz,4H),7.22(dt,J＝8.6,1.6Hz,1H),7.07(d,J＝7.7Hz,3H),6.95(dd,J＝8.6,2.4Hz,1H),3.09(d,J＝7.3Hz,2H),2.81(s,1H),2.23(s,3H),1.97(s,2H),1.72(s,2H),1.57(d,J＝11.8Hz,1H),1.20(s,4H),1.06(s,1H),0.90(t,J＝6.9Hz,3H)；MS(EI)m/z 543.2[M-H]^-.

Example 14

Synthesis of 1- (3- (3- (4-methylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid

Synthesis of 2-nitro-4-bromo-N, N-diisobutylaniline (14A)

2-fluoro-5-bromonitrobenzene (4g,18.2mmol) and diisobutylamine (4.7g,36.4mmol) were dissolved in DMF (50mL), cesium carbonate (11.85g,36.4mmol) was added, and stirring was carried out at 30 ℃ for 36 hours. Cooling, adding 200mL of ethyl acetate and 200mL of water, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining, washing with saturated brine, and drying over anhydrous magnesium sulfate; filtering, decompressing, concentrating, purifying by column chromatography to obtain 5.83g of red solid with 97.3 percent of yield.¹H NMR(300MHz,DMSO-d₆)δ(ppm)8.05(td,J＝2.4,1.3Hz,1H),8.01(m,1H),3.00-2.65(m,4H),1.70(dt,J＝13.4,6.7Hz,2H),0.81(s,12H)；MS(EI)m/z 327.1[M-H]^-Synthesis of ethyl 1- (3-nitro-4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (14B)

Compound 14A (4g,12.15mmol) was dissolved in DMF (50mL), ethyl indole-2-carboxylate (1.92g,10.13mmol), cuprous iodide (3.86g,20.25mmol), cesium carbonate (6.6g,20.25mmol) and sarcosine (2.33g,20.25mmol) were added, and the mixture was stirred at 80 ℃ for 24 hours under nitrogen. Cooling, adding 200mL ethyl acetate and 200mL water, filtering with celite, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, drying over anhydrous magnesium sulfate, filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain a yellow solid 1.82g, with a yield of 41.9%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)7.80(d,J＝2.2Hz,1H),7.68-7.63(m,2H),7.44(q,J＝1.0Hz,1H),7.37-7.30(m,1H),7.29-7.20(m,3H),4.34(q,J＝7.1Hz,4H),4.15(q,J＝7.1Hz,2H),1.93(m,2H),1.14(q,J＝7.4Hz,3H),0.85(d,J＝6.5Hz,12H)；MS(EI)m/z 438.2[M-H]⁺.

Synthesis of ethyl 1- (3-amino-4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (14C)

Compound 14B (1.82g,4.15mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (1.35g,20.8mmol) and ammonium chloride (1.33g,20.8mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, adding 100mL ethyl acetate and 100mL water, separating the organic layer, water layer with ethyl acetate (50mL) extraction, organic layer, combined, saturated saline water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get light yellow solid 1.58 g.

Synthesis of ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (14D)

Compound 14C (1.58g,3.88mmol) was dissolved in tetrahydrofuran (40mL), p-tolylene isocyanate (0.52g,3.88mmol) was added, and the reaction was carried out at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 1.68g of a white solid.

Compound 14D (1.68g,3.09mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.63g,15.27mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.66g of a white solid with the yield of 41.4%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.49(s,1H),8.12-7.97(m,2H),7.75(d,J＝7.9Hz,1H),7.42-7.25(m,5H),7.18(t,J＝7.4Hz,1H),7.08(t,J＝7.7Hz,3H),6.94(dd,J＝8.4,2.5Hz,1H),2.78(d,J＝6.8Hz,4H),2.23(s,3H),1.76(dt,J＝13.1,6.6Hz,2H),0.90(d,J＝6.5Hz,12H)；MS(EI)m/z 511.3[M-H]^-.

Example 15

Synthesis of 1- (3- (3- (4-fluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (15)

Synthesis of ethyl 1- (3- (3- (4-fluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (15D)

Compound 14C (0.17g,0.42mmol) was dissolved in tetrahydrofuran (20mL), 4-fluorobenzeneisocyanate (0.06g,0.42mmol) was added, reacted at normal temperature for 4 hours, concentrated under reduced pressure, and purified by column chromatography to give 0.13g of a white solid.

Compound 15D (0.13g,0.24mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.05g,1.19mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.07g of white solid with the yield of 56.8%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.62(s,1H),8.08(s,1H),8.00(d,J＝2.5Hz,1H),7.73(d,J＝7.9Hz,1H),7.49-7.43(m,2H),7.36(d,J＝8.4Hz,1H),7.31(d,J＝5.3Hz,1H),7.29-7.24(m,1H),7.17-7.06(m,4H),6.94(dd,J＝8.4,2.5Hz,1H),2.78(d,J＝6.8Hz,4H),1.75(p,J＝6.6Hz,2H),0.90(d,J＝6.6Hz,12H)；MS(EI)m/z 515.2[M-H]^-.

Example 16

Synthesis of 1- (3- (3- (4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (16)

Synthesis of ethyl 1- (3- (3- (4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (16D)

Compound 14C (0.5g,1.23mmol) was dissolved in tetrahydrofuran (20mL), 4-chlorobenzene isocyanate (0.19g,1.23mmol) was added, the reaction was carried out at normal temperature for 4 hours, concentration under reduced pressure and purification by column chromatography gave 0.48g of a white solid.

Compound 16D (0.48g,0.86mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.17g,4.29mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.27g of white solid with the yield of 59.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.72(s,1H),8.11(s,1H),7.99(d,J＝2.5Hz,1H),7.75(d,J＝7.9Hz,1H),7.49(d,J＝2.2Hz,1H),7.47(d,J＝2.2Hz,1H),7.39-7.35(m,2H),7.34-7.26(m,3H),7.21-7.15(m,1H),7.12-7.07(m,1H),6.96(dd,J＝8.4,2.5Hz,1H),2.79(d,J＝6.8Hz,4H),1.76(m,2H),0.90(d,J＝6.6Hz,12H)；MS(EI)m/z 531.2[M-H]^-.

Example 17

Synthesis of 1- (3- (3- (2, 4-difluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (17)

Synthesis of ethyl 1- (3- (3- (2, 4-difluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (17D)

Compound 14C (0.4g,0.98mmol) was dissolved in tetrahydrofuran (20mL), and 2, 4-difluoropolyisocyanate (0.15g,0.98mmol) was added thereto, reacted at room temperature for 4 hours, concentrated under reduced pressure, and purified by column chromatography to obtain 0.44g of a white solid.

Compound 17D (0.44g,0.78mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.16g,3.91mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.22g of white solid with the yield of 52.6%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.95(s,1H),9.46(s,1H),8.31(s,1H),7.94(s,1H),7.75(d,J＝7.9Hz,1H),7.64(d,J＝8.0Hz,3H),7.44(d,J＝7.5Hz,3H),7.37(s,1H),7.26(d,J＝1.2Hz,1H),7.24(t,J＝1.3Hz,1H),2.79(d,J＝7.3Hz,4H),1.78(d,J＝8.6Hz,2H),0.97-0.82(m,12H)；MS(EI)m/z 533.2[M-H]^-.

Example 18

Synthesis of 1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (18)

Synthesis of ethyl 1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (18D)

Compound 14C (0.19g,0.47mmol) was dissolved in tetrahydrofuran (20mL), and 2-fluoro-4-chlorobenzene isocyanate (0.08g,0.47mmol) was added to react at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.23g of a white solid.

Compound 18D (0.23g,0.4mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.08g,1.99mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.11g of white solid with the yield of 50.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.70(s,1H),9.57(s,1H),8.35(s,1H),8.08-7.96(m,1H),7.91(d,J＝2.5Hz,1H),7.73(d,J＝8.0Hz,1H),7.44(dd,J＝11.1,2.4Hz,1H),7.34(s,1H),7.33-7.23(m,1H),7.16(t,J＝8.0Hz,2H),7.08(d,J＝8.3Hz,1H),6.95(dd,J＝8.4,2.5Hz,1H),2.79(d,J＝6.8Hz,4H),1.76(dt,J＝13.2,6.7Hz,2H),0.88(d,J＝6.6Hz,10H)；MS(EI)m/z 549.2[M-H]^-.

Example 19

Synthesis of 1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (19)

Synthesis of ethyl 1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (19D)

Compound 14C (0.19g,0.47mmol) was dissolved in tetrahydrofuran (40mL), and 3-trifluoromethylphenylisocyanate (0.09g,0.47mmol) was added thereto, followed by reaction at room temperature for 4 hours, concentration under reduced pressure, and purification by column chromatography to give 0.24g of a white solid.

Compound 19D (0.24g,0.4mmol) was dissolved in absolute ethanol (10mL), sodium hydroxide (0.08g,2.02mmol) was added, and the mixture was stirred at 65 ℃For 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.1g of white solid with the yield of 43.7%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.96(s,1H),8.18(s,1H),7.97(d,J＝13.1Hz,2H),7.75(d,J＝8.0Hz,1H),7.63(d,J＝8.2Hz,1H),7.50(t,J＝8.1Hz,1H),7.38(d,J＝10.0Hz,2H),7.29(t,J＝7.9Hz,2H),7.17(t,J＝7.6Hz,1H),7.08(d,J＝8.3Hz,1H),6.98(d,J＝8.3Hz,1H),2.79(d,J＝6.7Hz,4H),1.76(s,2H),1.09(t,J＝7.1Hz,4H),0.91(d,J＝6.5Hz,8H)；MS(EI)m/z 565.3[M-H]^-.

Example 20

Synthesis of 1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (20)

Synthesis of ethyl 1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (20D)

Compound 14C (0.19g,0.47mmol) was dissolved in tetrahydrofuran (40mL), and 4-chloro-3-trifluoromethylphenylisocyanate (0.1g,0.47mmol) was added thereto, followed by reaction at room temperature for 4 hours, concentration under reduced pressure, and purification by column chromatography to give 0.25g of a white solid.

Compound 20D (0.24g,0.38mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.08g,1.91mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.12g of white solid with the yield of 52.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)8.20(s,1H),8.03(s,1H),7.99(d,J＝2.5Hz,1H),7.78-7.70(m,2H),7.60(d,J＝8.8Hz,1H),7.42-7.35(m,2H),7.29(t,J＝7.6Hz,1H),7.17(t,J＝7.4Hz,1H),7.07(d,J＝8.5Hz,1H),6.99(d,J＝8.7Hz,1H),2.79(d,J＝6.7Hz,4H),1.80-1.72(m,2H),1.09(t,J＝7.0Hz,2H),0.90(d,J＝6.5Hz,10H)；MS(EI)m/z 599.2[M-H]^-.

Example 21

Synthesis of 1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid (21)

Synthesis of ethyl 1- (3- (3, 4-dichlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (21D)

Compound 14C (0.21g,0.51mmol) was dissolved in tetrahydrofuran (40mL), and 3, 4-dichlorophenylisocyanate (0.1g,0.51mmol) was added to react at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.24g of a white solid.

Compound 21D (0.24g,0.4mmol) was dissolved in absolute ethanol (10mL), and sodium hydroxide (0.08g,2.01mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.11g of white solid with the yield of 52.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.91(s,1H),8.16(s,1H),7.87(d,J＝2.4Hz,1H),7.75(d,J＝8.0Hz,1H),7.39(d,J＝8.9Hz,2H),7.36-7.26(m,2H),7.18(t,J＝7.5Hz,1H),7.08(d,J＝8.4Hz,1H),3.17(d,J＝4.9Hz,4H),2.78(d,J＝6.7Hz,4H),1.81-1.69(m,2H),0.90(d,J＝6.5Hz,8H)；MS(EI)m/z565.2[M-H]^-.

Example 22

Synthesis of 1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- (cyclohexyl (methyl) amino) phenyl) -1H-indole-2-carboxylic acid (22)

Synthesis of 2-nitro-4-bromo-N, N- (methyl) cyclohexylaniline (22A)

2-fluoro-5-bromonitrobenzene (3g,13.6mmol) and N-methylcyclohexylamine (2.34g,20.5mmol) were dissolved in DMF (50mL), N-diisopropylethylamine (8.88g,27.4mmol) was added, and stirring was continued at 80 ℃ for 4 hours. Cooling, adding 200mL of ethyl acetate and 200mL of water, separating an organic layer, extracting a water layer with ethyl acetate (100mL), combining the organic layers, washing with saturated saline, drying with anhydrous magnesium sulfate, carrying out suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 3.99g of a red solid with the yield of 93.1%.

Synthesis of ethyl 1- (3-nitro-4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (22B)

Compound 22A (3g,9.5mmol) was dissolved in DMF (50mL), ethyl indole-2-carboxylate (1.79g,9.5mmol), cuprous iodide (3.60g,19.1mmol), cesium carbonate (6.16g,19.1mmol) and L-proline (2.19g,19.1mmol) were added, and the mixture was stirred at 80 ℃ for 12 hours under nitrogen. Cooling, adding 200mL ethyl acetate and 200mL water, suction filtering with diatomaceous earth, separating organic layer, extracting water layer with ethyl acetate (100mL), combining organic layers, washing with saturated saline solution, drying with anhydrous magnesium sulfate, suction filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain white solid 1.57 g.

Synthesis of ethyl 1- (3-amino-4- ((methyl) cyclohexylamino) -phenyl) -1H-indole-2-carboxylate (22C)

Compound 22B (1.57g,3.64mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (1.18g,18.21mmol) and ammonium chloride (1.16g,18.21mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, adding 100mL ethyl acetate and 100mL water, separating the organic layer, water layer with ethyl acetate (50mL) extraction, organic layer, combined, saturated saline water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get light yellow solid 1.44 g.

Synthesis of ethyl 1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (22D)

Compound 22C (1.44g,3.59mmol) was dissolved in tetrahydrofuran (20mL), and 3-trifluoromethylphenylisocyanate (0.67g,3.59mmol) was added thereto, followed by reaction at room temperature for 4 hours, concentration under reduced pressure and purification by column chromatography to give 1.38g of a white solid.

Compound 22D (1.38g,2.38mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.48g,11.94mmol) was added at 65 deg.CStirred for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.68g of white solid with the yield of 51.7%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.92(s,1H),8.51(s,1H),7.99(d,J＝2.4Hz,1H),7.84(s,1H),7.60(d,J＝7.9Hz,1H),7.46(d,J＝8.1Hz,1H),7.36(t,J＝8.0Hz,1H),7.28-7.09(m,4H),7.03(t,J＝7.4Hz,1H),6.96(d,J＝8.4Hz,1H),6.83(dd,J＝8.6,2.5Hz,1H),2.55(s,4H),1.86-1.76(m,2H),1.60(d,J＝10.8Hz,2H),1.44(d,J＝11.5Hz,1H),1.20-0.98(m,5H)；MS(EI)m/z 549.2[M-H]^-.

Example 23

Synthesis of 1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (23)

Synthesis of ethyl 1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (22D)

Compound 22C (0.3g,0.77mmol) was dissolved in tetrahydrofuran (20mL), and 4-chloro-3-trifluoromethylphenyl isocyanate (0.17g,0.77mmol) was added thereto, followed by reaction at room temperature for 4 hours, concentration under reduced pressure, and purification by column chromatography to give 0.31g of a white solid.

Compound 23D (0.31g,0.51mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.1g,2.53mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.17g of white solid with the yield of 57.5%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)8.53(s,1H),7.99-7.91(m,2H),7.55(d,J＝8.6Hz,2H),7.44(d,J＝8.8Hz,1H),7.19(d,J＝8.3Hz,1H),7.00(m,5H),6.79(d,J＝8.2Hz,1H),2.55(m,4H),1.77(s,2H),1.57(s,2H),1.42(d,J＝10.8Hz,1H),1.12(d,J＝18.2Hz,5H)；MS(EI)m/z 583.1[M-H]^-.

Example 24

Synthesis of 1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (24)

Synthesis of ethyl 1- (3- (3, 4-dichlorophenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (24D)

Compound 22C (0.3g,0.77mmol) was dissolved in tetrahydrofuran (20mL), and 3, 4-dichlorophenylisocyanate (0.14g,0.77mmol) was added thereto, reacted at room temperature for 4 hours, concentrated under reduced pressure, and purified by column chromatography to give 0.3g of a white solid.

Compound 24D (0.3g,0.51mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.1g,2.59mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.15g of white solid with the yield of 52.5%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.61(s,1H),9.87(s,1H),8.50(s,1H),7.98(s,1H),7.75(s,1H),7.62(d,J＝8.0Hz,1H),7.37(d,J＝8.8Hz,1H),7.25(d,J＝8.0Hz,2H),7.15(t,J＝7.7Hz,2H),7.04(t,J＝7.5Hz,1H),6.96(d,J＝8.4Hz,1H),6.84(d,J＝8.3Hz,1H),2.57(d,J＝16.1Hz,4H),1.79(s,2H),1.66-1.54(m,2H),1.44(d,J＝11.0Hz,1H),1.23-0.96(m,5H)；MS(EI)m/z 549.2[M-H]^-.

Example 25

Synthesis of 1- (3- (3- (4-methylphenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (25)

Synthesis of 2-nitro-4-bromo-N- (isobutyl) cyclohexylaniline (25A)

2-fluoro-5-bromonitrobenzene (4.88g,22.2mmol) andn-isobutyl cyclohexylamine (4.15g,26.6mmol) was dissolved in N-methylpyrrolidone (50mL), N-diisopropylethylamine (5.76g,44.4mmol) was added, and the mixture was stirred at 120 ℃ for 12 hours under nitrogen. Cooling, adding 200mL of ethyl acetate and 200mL of water, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, and drying over anhydrous magnesium sulfate; filtering, decompressing, concentrating, purifying by column chromatography to obtain red solid 6.99g with 88.9% yield.¹H NMR(300MHz,DMSO-d₆)δ(ppm)7.95(d,J＝2.4Hz,1H),7.65(dd,J＝8.9,2.5Hz,1H),7.35(d,J＝8.9Hz,1H),2.86(d,J＝7.2Hz,2H),2.76(d,J＝11.8Hz,1H),1.68(t,J＝13.0Hz,4H),1.56-1.32(m,4H),1.08(s,3H),0.81(d,J＝6.6Hz,6H)；MS(EI)m/z 353.1[M-H]^-.

Synthesis of ethyl 1- (3-nitro-4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (25B)

Compound 25A (3g,8.5mmol) was dissolved in DMF (50mL), ethyl indole-2-carboxylate (1.6g,8.5mmol), cuprous iodide (3.22g,17.1mmol), cesium carbonate (5.51g,17.1mmol) and sarcosine (1.95g,17.1mmol) were added, and the mixture was stirred at 80 ℃ for 12 hours under nitrogen. Cooling, adding 200mL ethyl acetate and 200mL water, filtering with diatomaceous earth, separating organic layer, extracting water layer with ethyl acetate (100mL), combining organic layers, washing with saturated saline solution, drying with anhydrous magnesium sulfate, filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain yellow solid 1.45g, with yield 39.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)7.85-7.74(m,2H),7.55-7.46(m,3H),7.37-7.30(m,1H),7.21(t,J＝7.4Hz,1H),7.12(d,J＝8.4Hz,1H),4.14(q,J＝7.1Hz,2H),2.93(dd,J＝16.4,9.3Hz,3H),1.76(d,J＝11.2Hz,4H),1.53(s,5H),1.10(t,J＝7.1Hz,5H),0.88(d,J＝6.5Hz,6H)；MS(EI)m/z 462.2[M-H]^-.

Synthesis of ethyl 1- (3-amino-4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (25C)

Compound 25B (1.45g,3.12mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (1.02g,15.61mmol) and ammonium chloride (1g,15.61mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, adding 100mL ethyl acetate and 100mL water, separating the organic layer, water layer with ethyl acetate (50mL) extraction, organic layer, combined, saturated saline water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get light yellow solid 1.31 g.

Synthesis of ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (25D)

Compound 25C (1.31g,3.02mmol) was dissolved in tetrahydrofuran (20mL), p-tolylene isocyanate (0.41g,3.02mmol) was added, and the reaction was carried out at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 1.12g of a white solid.

Compound 25D (1.12g,1.96mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.38g,9.88mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.56g of white solid with the yield of 52.6%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.50(s,1H),8.20(s,1H),8.09(d,J＝2.5Hz,1H),7.73(d,J＝8.0Hz,1H),7.38-7.22(m,5H),7.20-7.03(m,4H),6.91(dd,J＝8.4,2.5Hz,1H),2.87(s,2H),2.65(s,1H),2.23(s,3H),1.97(d,J＝11.0Hz,3H),1.73(d,J＝11.2Hz,2H),1.56(d,J＝11.3Hz,1H),1.44(m,1H),1.33-0.96(m,6H),0.88(d,J＝6.6Hz,6H)；MS(EI)m/z 537.3[M-H]^-.

Example 26

Synthesis of 1- (3- (3- (4-fluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (26)

Synthesis of ethyl 1- (3- (3- (4-fluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (26D)

Compound 25C (0.5g,1.15mmol) was dissolved in tetrahydrofuran (20mL), 4-fluorobenzeneisocyanate (0.16g,1.15mmol) was added, reacted at normal temperature for 4 hours, concentrated under reduced pressure, and purified by column chromatography to give 0.46g of a white solid.

Compound 26D (0.46g,0.81mmol) was dissolved in chloroformTo aqueous ethanol (20mL) was added sodium hydroxide (0.18g,4.03mmol), and the mixture was stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.27g of white solid with the yield of 61.7%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.69(s,1H),8.22(s,1H),8.07(d,J＝2.5Hz,1H),7.70(d,J＝7.8Hz,1H),7.53-7.40(m,2H),7.32(d,J＝8.5Hz,1H),7.27-7.18(m,2H),7.16-7.03(m,4H),6.91(dd,J＝8.4,2.5Hz,1H),2.87(s,2H),2.66(s,1H),1.99(d,J＝11.2Hz,2H),1.74(d,J＝11.3Hz,2H),1.56(d,J＝11.1Hz,1H),1.16(m,6H),0.88(d,J＝6.6Hz,6H)；MS(EI)m/z 541.3[M-H]^-.

Example 27

Synthesis of 1- (3- (3- (4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (27)

Synthesis of ethyl 1- (3- (3- (4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (27D)

Compound 25C (0.5g,1.15mmol) was dissolved in tetrahydrofuran (20mL), 4-chlorobenzene isocyanate (0.18g,1.15mmol) was added, and the reaction was carried out at normal temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to obtain 0.49g of a white solid.

Compound 27D (0.49g,0.84mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.17g,4.18mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.28g of white solid with the yield of 60.0%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.79(s,1H),8.29(s,1H),8.08(d,J＝2.5Hz,1H),7.75(d,J＝8.0Hz,1H),7.55-7.44(m,2H),7.40-7.23(m,5H),7.22-7.07(m,2H),6.95(dd,J＝8.4,2.5Hz,1H),2.88(d,J＝6.5Hz,2H),2.00(d,J＝11.1Hz,2H),1.74(d,J＝11.3Hz,2H),1.56(d,J＝11.4Hz,1H),1.44(dd,J＝13.1,6.6Hz,1H),1.36-0.99(m,6H),0.89(d,J＝6.6Hz,6H)；MS(EI)m/z 557.2[M-H]^-.

Example 28

Synthesis of 1- (3- (3- (2, 4-difluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (28)

Synthesis of ethyl 1- (3- (3- (2, 4-difluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (28D)

Compound 25C (0.5g,1.15mmol) was dissolved in anhydrous tetrahydrofuran (20mL), and 2, 4-difluoropolyisocyanate (0.18g,1.15mmol) was added thereto, reacted at room temperature for 4 hours, concentrated under reduced pressure, and purified by column chromatography to give 0.48g of a white solid.

Compound 28D (0.48g,0.82mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.16g,4.11mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.27g of white solid with the yield of 59.1%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)8.40(s,1H),8.00(d,J＝2.5Hz,1H),7.85(td,J＝9.2,6.2Hz,1H),7.76-7.67(m,1H),7.39-7.22(m,4H),7.22-7.08(m,2H),7.07-6.98(m,1H),6.94(dd,J＝8.4,2.5Hz,1H),2.86(d,J＝6.8Hz,2H),2.67(t,J＝11.0Hz,1H),1.96(d,J＝10.8Hz,2H),1.74(d,J＝11.2Hz,2H),1.56(d,J＝11.3Hz,1H),1.51-1.39(m,1H),1.16(m,6H),0.88(d,J＝6.6Hz,7H)；MS(EI)m/z 559.2[M-H]^-.

Example 29

Synthesis of 1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid (29)

Synthesis of ethyl 1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylate (29D)

Compound 25C (0.5g,1.15mmol) was dissolved in anhydrous tetrahydrofuran (20mL), and 2-fluoro-4-chlorophenylisocyanate (0.19g,1.15mmol) was added thereto, followed by reaction at room temperature for 4 hours, concentration under reduced pressure, and purification by column chromatography to give 0.48g of a white solid.

Compound 29D (0.48g,0.79mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.16g,3.97mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.25g of white solid with the yield of 54.6%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.64(s,1H),8.49(s,1H),7.98(s,2H),7.73(d,J＝7.8Hz,1H),7.45(d,J＝11.2Hz,1H),7.35-7.23(m,3H),7.14(m,3H),6.95(d,J＝8.3Hz,1H),2.86(s,2H),2.69(s,2H),1.96(s,2H),1.72(s,2H),1.53(s,3H),1.35-1.00(m,6H),0.87(d,J＝6.6Hz,5H)；MS(EI)m/z575.2[M-H]^-.

Example 30

Synthesis of 1- (3-chloro-4- (diisobutylamino) -5- (3- (4-methylphenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (30)

Synthesis of 2-chloro-4-bromo-6-nitro-N-diisobutylaniline (30B)

Compound 14A (15g,45.56mmol) was dissolved in DMF (100mL), and N-chlorosuccinimide (11.1g,83.12mmol) was slowly added under ice-cooling, followed by reaction at room temperature for 8 hours. Adding 200mL of ethyl acetate and 200mL of water, separating an organic layer, extracting an aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, and drying over anhydrous magnesium sulfate; suction filtration, reduced pressure concentration and column chromatography purification are carried out to obtain 7g of orange solid with the yield of 42.8 percent.¹H NMR(300MHz,DMSO-d₆)δ(ppm)7.91-7.81(m,2H),7.38(t,J＝2.3Hz,1H),7.07(dd,J＝3.9,1.7Hz,1H),6.40-6.32(m,1H),3.06-2.71(m,4H),1.75(m,2H),0.87(d,J＝7.4Hz,12H)；MS(EI)m/z 361.0[M-H]^-.

Synthesis of ethyl 1- (3-chloro-5-nitro-4- (diisobutylamino) phenyl) -1H-indole-2-carboxylate (30C)

Compound 30B (7g,19.25mmol) was dissolved in DMF (50mL), ethyl indole-2-carboxylate (3.64g,19.25mmol), cuprous iodide (7.33g,38.5mmol), cesium carbonate (12.54g,38.5mmol) and sarcosine (3.64g,38.5mmol) were added, and the mixture was stirred at 100 ℃ for 12 hours under nitrogen protection. Cooling, adding 200mL ethyl acetate and 200mL water, suction-filtering with diatomaceous earth, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, drying over anhydrous magnesium sulfate, suction-filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain 3.81g of yellow solid.

Synthesis of ethyl 1- (3-amino-4- (diisobutylamino) -5-chlorophenyl) -1H-indole-2-carboxylate (30D)

Compound 30C (3.81g,8.09mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (2.63g,40.42mmol) and ammonium chloride (2.59g,40.42mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, adding 100mL ethyl acetate and 100mL water, separating the organic layer, aqueous layer with ethyl acetate (50mL) extraction, combined organic layer, saturated saline water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get pale yellow solid 3.48 g.

Synthesis of ethyl 1- (3-chloro-4- (diisobutylamino) -5- (3- (4-methylphenyl) ureido) phenyl) -1H-indole-2-carboxylate (30E)

Compound 30D (3.48g,7.89mmol) was dissolved in anhydrous tetrahydrofuran (50mL), p-tolylene isocyanate (1.05g,7.89mmol) was added, and the reaction was carried out at normal temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 3.12g of a white solid.

Compound 30E (3.12g,5.44mmol) was dissolved in absolute ethanol (30mL), and sodium hydroxide (1.08g,27.16mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 1.38g of a white solid with the yield of 46.5%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.84(s,1H),9.54(s,1H),8.47(s,1H),8.11(d,J＝2.5Hz,1H),7.40(d,J＝0.8Hz,1H),7.35(d,J＝2.6Hz,1H),7.32(d,J＝2.9Hz,1H),7.20(m,1H),7.13(dd,J＝8.3,1.1Hz,1H),7.09(s,1H),7.08-7.03(m,2H),3.07(dd,J＝12.6,6.6Hz,2H),2.93(dd,J＝12.6,6.4Hz,2H),2.23(s,3H),1.75(m,2H),0.89(dd,J＝13.8,6.6Hz,12H)；MS(EI)m/z 545.2[M-H]^-.

Example 31

Synthesis of 1- (3-chloro-4- (diisobutylamino) -5- (3- (4-fluorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (31)

Synthesis of ethyl 1- (3-chloro-4- (diisobutylamino) -5- (3- (4-fluorophenyl) ureido) phenyl) -1H-indole-2-carboxylate (31E)

Compound 30D (0.4g,0.9mmol) was dissolved in anhydrous tetrahydrofuran (20mL), 4-fluorobenzeneisocyanate (0.12g,0.9mmol) was added, reacted at normal temperature for 4 hours, concentrated under reduced pressure, and purified by column chromatography to give 0.38g of a white solid.

Compound 31E (0.38g,0.66mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.13g,3.29mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.19g of a white solid with the yield of 52.5%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.84(s,1H),9.67(s,1H),8.49(s,1H),8.10(d,J＝2.5Hz,1H),7.76(d,J＝8.0Hz,1H),7.47(d,J＝4.9Hz,1H),7.46-7.43(m,1H),7.40(s,1H),7.36-7.29(m,1H),7.20(t,J＝7.3Hz,1H),7.14(s,1H),7.12(d,J＝1.7Hz,1H),7.09-7.05(m,1H),3.07(dd,J＝12.5,6.6Hz,2H),2.94(dd,J＝12.6,6.4Hz,2H),1.76(dt,J＝13.1,6.6Hz,2H),0.90(dd,J＝13.7,6.6Hz,12H)；MS(EI)m/z 549.2[M-H]^-.

Example 32

Synthesis of 1- (3-chloro-4- (diisobutylamino) -5- (3- (4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (32)

Synthesis of ethyl 1- (3-chloro-4- (diisobutylamino) -5- (3- (4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylate (32E)

Compound 30D (0.4g,0.9mmol) was dissolved in anhydrous tetrahydrofuran (20mL), 4-chlorobenzene isocyanate (0.14g,0.9mmol) was added, and the reaction was carried out at normal temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.39g of a white solid.

Compound 32E (0.39g,0.66mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.13g,3.28mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.21g of a white solid with the yield of 56.5%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.85(s,1H),9.79(s,1H),8.53(s,1H),8.10(d,J＝2.5Hz,1H),7.77(dt,J＝8.0,1.0Hz,1H),7.52-7.45(m,2H),7.40(d,J＝0.8Hz,1H),7.33(d,J＝2.2Hz,1H),7.31(d,J＝2.2Hz,1H),7.20(m,1H),7.16-7.11(m,1H),7.08(d,J＝2.5Hz,1H),3.08(dd,J＝12.6,6.6Hz,2H),2.94(dd,J＝12.6,6.5Hz,2H),1.77(dt,J＝13.1,6.6Hz,2H),0.90(dd,J＝13.6,6.6Hz,12H)；MS(EI)m/z 565.2[M-H]^-.

Example 33

Synthesis of 1- (3-chloro-4- (diisobutylamino) -5- (3- ((2, 4-difluorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (33)

Synthesis of ethyl 1- (3-chloro-4- (diisobutylamino) -5- (3- ((2, 4-difluorophenyl) ureido) phenyl) -1H-indole-2-carboxylate (33E)

Compound 30D (0.4g,0.9mmol) was dissolved in anhydrous tetrahydrofuran (20mL), and 2, 4-difluoropolyisocyanate (0.14g,0.9mmol) was added thereto, followed by reaction at room temperature for 4 hours, concentration under reduced pressure, and purification by column chromatography to give 0.38g of a white solid.

Compound 33E (0.39g,0.64mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.13g,3.19mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.2g of white solid with the yield of 55.2%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.53(s,1H),8.67(s,1H),8.04(d,J＝2.5Hz,1H),7.83(td,J＝9.2,6.2Hz,1H),7.74(d,J＝8.0Hz,1H),7.32(s,1H),7.30(d,J＝1.3Hz,1H),7.18(m,1H),7.12(dd,J＝8.2,1.0Hz,1H),7.06(d,J＝2.5Hz,1H),7.04-6.98(m,1H),3.07(dd,J＝12.6,6.6Hz,2H),2.92(dd,J＝12.6,6.5Hz,2H),1.76(m,2H),0.89(dd,J＝13.0,6.6Hz,12H)；MS(EI)m/z 567.2[M-H]^-.

Example 34

Synthesis of 1- (3-chloro-4- (diisobutylamino) -5- (3- (2-fluoro-4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid (34)

Synthesis of ethyl 1- (3-chloro-4- (diisobutylamino) -5- (3- (2-fluoro-4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylate (34E)

Compound 30D (0.4g,0.9mmol) was dissolved in anhydrous tetrahydrofuran (20mL), and 2-fluoro-4-chlorophenylisocyanate (0.15g,0.9mmol) was added thereto, followed by reaction at room temperature for 4 hours, concentration under reduced pressure, and purification by column chromatography to give 0.37g of a white solid.

Compound 34E (0.37g,0.6mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.12g,3.02mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.19g of a white solid with the yield of 53.8%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.83(s,1H),9.68(s,1H),8.76(s,1H),8.04(d,J＝2.5Hz,1H),7.95(t,J＝8.8Hz,1H),7.76(d,J＝8.0Hz,1H),7.47(dd,J＝11.1,2.4Hz,1H),7.39(s,1H),7.32(t,J＝7.6Hz,1H),7.24-7.16(m,2H),7.13(d,J＝8.2Hz,1H),7.10(d,J＝2.6Hz,1H),3.10(dd,J＝12.6,6.5Hz,2H),2.92(dd,J＝12.6,6.5Hz,2H),1.77(m,2H),0.89(dd,J＝13.2,6.6Hz,12H)；MS(EI)m/z 583.1[M-H]^-.

Example 35

Synthesis of 1- (3- (3- (4-methylphenyl) ureido) -4- (methyl- (1-methylpiperidin-4-yl) amino) phenyl) -1H-indole-2-carboxylic acid (35)

Synthesis of N- (2-nitro-4-bromophenyl) -N, 1-dimethylpiperidin-4-amine (35A)

2-fluoro-5-bromonitrobenzene (3.09g,14.05mmol) and 1-methyl-4- (methylamino) piperidine (3.6g,28.09mmol) were dissolved in DMF (50mL), N-diisopropylethylamine (2.79g,21.07mmol) was added, and the mixture was stirred at 80 ℃ for 5 hours. Cooling, adding 200mL of ethyl acetate and 200mL of water, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, and drying over anhydrous magnesium sulfate; suction filtration, reduced pressure concentration and column chromatography purification are carried out to obtain 3.81g of red solid.

Synthesis of ethyl 1- (3-nitro-4- (methyl- (1-methylpiperidin-4-yl) amino) phenyl) -1H-indole-2-carboxylate (35B)

Compound 35A (1.5g,4.57mmol) was dissolved in DMF (50mL), indole-2-carboxylic acid ethyl ester (1.3g,6.86mmol), cuprous iodide (1.74g,9.14mmol), cesium carbonate (2.98g,9.14mmol) and L-proline (1.05g,9.14mmol) were added, and the mixture was stirred under nitrogen at 100 ℃ for 12 hours. Cooling, adding 200mL ethyl acetate and 200mL water, filtering with diatomaceous earth, separating organic layer, extracting water layer with ethyl acetate (100mL), combining organic layers, washing with saturated saline solution, drying with anhydrous magnesium sulfate, filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain yellow solid 0.55g, with yield 27.6%.¹H NMR(300MHz,DMSO-d₆)δ(qqm)8.57(d,J＝7.9Hz,2H),8.27(d,J＝1.9Hz,2H),8.00(dd,J＝8.2,1.9Hz,2H),7.53(d,J＝8.1Hz,2H),3.67(q,J＝9.0Hz,2H),1.83(d,J＝11.0Hz,4H),1.71(d,J＝12.5Hz,4H),1.58(d,J＝12.7Hz,2H),1.30(d,J＝14.6Hz,4H),1.26-1.14(m,6H)；MS(EI)m/z 435.2[M-H]^-.

Synthesis of ethyl 1- (3-amino-4- (methyl- (1-methylpiperidin-4-yl) amino) -phenyl) -1H-indole-2-carboxylate (35C) compound 35B (0.55g,1.26mmol) was dissolved in a mixed solution (20mL) of ethanol and water ═ 5:1, and zinc powder (0.41g,6.3mmol) and ammonium chloride (0.4g,6.3mmol) were added and reacted at room temperature for 4 hours under nitrogen atmosphere. Suction filtration, filtrate reduced pressure concentration, adding 100mL ethyl acetate and 100mL water, separating the organic layer, aqueous layer using ethyl acetate (50mL) extraction twice, the organic layer, combined with saturated salt solution washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get light yellow solid 0.49 g.

Synthesis of ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- (methyl- (1-methylpiperidin-4-yl) amino) phenyl) -1H-indole-2-carboxylate (35D)

Compound 35C (0.49g,1.21mmol) was dissolved in anhydrous tetrahydrofuran (20mL), p-tolylene isocyanate (0.16g,1.21mmol) was added, and the reaction was carried out at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.48g of a white solid.

Compound 35D (0.48g,0.89mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.18g,4.45mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.28g of white solid with the yield of 46.2%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.64(s,1H),8.59(s,1H),8.23(s,1H),7.69(d,J＝7.9Hz,2H),7.32(d,J＝8.1Hz,3H),7.17(dd,J＝17.6,8.5Hz,4H),7.04(d,J＝8.0Hz,2H),2.89(s,3H),2.60(s,4H),2.32(s,3H),2.21(s,3H),1.80(s,2H),1.58(s,2H)；MS(EI)m/z 510.3[M-H]^-.

Example 36

Synthesis of 1- (3- (3- (4-methylphenyl) ureido) -4- (4-methylpiperazin-1-yl) phenyl) -1H-indole-2-carboxylic acid

Synthesis of 1- (2-nitro-4-bromophenyl) -4-methylpiperazine (36A)

2-fluoro-5-bromonitrobenzene (1g,4.55mmol) and 1-methylpiperazine (0.68g,6.82mmol) were dissolved in DMF (50mL), N-diisopropylethylamine (1.17g,9.1mmol) was added, and stirring was carried out at 80 ℃ for 5 hours. Cooling, adding 200mL of ethyl acetate and 200mL of water, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, and drying over anhydrous magnesium sulfate; filtering, decompressing, concentrating, purifying by column chromatography to obtain red solid 1.17g, the yield is 86.3%.

Synthesis of ethyl 1- (3-nitro-4- (4-methylpiperazin-1-yl) phenyl) -1H-indole-2-carboxylate (36B)

Compound 36A (1.17g,3.91mmol) was dissolved in DMF (50mL), indole-2-carboxylic acid ethyl ester (1.11g,5.87mmol), cuprous iodide (1.49g,7.83mmol), cesium carbonate (2.55g,7.83mmol) and sarcosine (0.7g,7.83mmol) were added, and the mixture was stirred at 100 ℃ under nitrogen for 12 hours. Cooling, adding 200mL ethyl acetate and 200mL water, suction-filtering with diatomaceous earth, separating organic layer, extracting water layer with ethyl acetate (100mL), combining organic layers, washing with saturated saline solution, drying with anhydrous magnesium sulfate, suction-filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain yellow solid 0.35 g.

Synthesis of ethyl 1- (3-amino-4- (4-methylpiperazin-1-yl) -phenyl) -1H-indole-2-carboxylate (35C)

Compound 36B (0.35g,0.86mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (0.28g,4.29mmol) and ammonium chloride (0.27g,4.29mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, after adding 100mL ethyl acetate and 100mL water, separated organic layer, water layer with ethyl acetate (50mL) extraction, combined organic layer, saturated saline water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, light yellow solid 0.32g, yield 98.7%.

Synthesis of ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- (4-methylpiperazin-1-yl) phenyl) -1H-indole-2-carboxylate (36D)

Compound 36C (0.32g,0.85mmol) was dissolved in anhydrous tetrahydrofuran (20mL), p-tolylene isocyanate (0.11g,0.85mmol) was added, and the reaction was carried out at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.36g of a white solid.

Compound 36D (0.36g,0.72mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.16g,3.52mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.16g of white solid with the yield of 47.1%. MS (EI) M/z 482.2[ M-H ]]^-.

Example 37

Synthesis of 1- (3- (3- (4-methylphenyl) ureido) -4- (cyclohexylcarbamoyl) phenyl) -1H-indole-2-carboxylic acid (37)

Synthesis of 2-nitro-4-bromobenzoyl chloride (37A)

4-bromo-2-nitrobenzoic acid (2g,8.17mmol) was dissolved in anhydrous dichloromethane (15mL), cooled in ice bath for 10min, added with 2mL of sulfonyl chloride, added dropwise with a small amount of triethylamine, reacted at 50 ℃ for 4h, and spin-dried to give 2.15g of oil. Synthesis of 2-nitro-4-bromobenzoyl-N-cyclohexylamine (37B)

Compound 37A (2.15g,8.13mmol) was dissolved in anhydrous tetrahydrofuran (25mL), added dropwise to a solution of 25mL of anhydrous tetrahydrofuran-dissolved cyclohexylamine (1.61g,16.26mmol) while cooling on ice for 10 minutes, and then triethylamine (2.47g,24.39mmol) was added slowly and stirred at room temperature for 4 hours. Concentrating under reduced pressure, adding 200mL ethyl acetate and 200mL water, separating organic layer, extracting water layer with ethyl acetate (100mL), combining organic layers, washing with saturated saline, and drying with anhydrous magnesium sulfate; suction filtration is carried out, and the filtrate is decompressed and concentrated to obtain 1.8g of light yellow solid.¹H NMR(300MHz,DMSO-d₆)δ(ppm)8.57(d,J＝7.9Hz,1H),8.27(d,J＝1.8Hz,1H),8.00(dd,J＝8.2,1.9Hz,1H),7.53(d,J＝8.2Hz,1H),3.67(d,J＝9.6Hz,1H),1.83(d,J＝11.1Hz,2H),1.71(d,J＝12.1Hz,2H),1.58(d,J＝12.7Hz,1H),1.24(m,5H)；MS(EI)m/z 325.1[M-H]^-.

Synthesis of ethyl 1- (3-nitro-4- (cyclohexylcarbamoyl) phenyl) -1H-indole-2-carboxylate (37C)

Compound 37B (1.8g,5.52mmol) was dissolved in DMF (50mL), indole-2-carboxylic acid ethyl ester (1.25g,6.63mmol), cuprous iodide (2.1g,1.1mmol), cesium carbonate (3.6g,1.1mmol) and L-proline (1.27g,1.1mmol) were added, and the mixture was stirred under nitrogen at 100 ℃ for 12 hours. Cooling, adding 200mL ethyl acetate and 200mL water, suction-filtering with diatomaceous earth, separating the organic layer, extracting the aqueous layer with ethyl acetate (100mL), combining the organic layers, washing with saturated brine, drying over anhydrous magnesium sulfate, suction-filtering, concentrating under reduced pressure, and purifying by column chromatography to obtain 1.08g of a yellow solid.

Synthesis of ethyl 1- (3-amino-4- (cyclohexylcarbamoyl) phenyl) -1H-indole-2-carboxylate (37D)

Compound 37C (1.08g,2.48mmol) was dissolved in a mixed solution (20mL) of ethanol and water at a ratio of 5:1, and zinc powder (0.81g,12.41mmol) and ammonium chloride (0.79g,12.41mmol) were added, followed by reaction under nitrogen atmosphere at room temperature for 4 hours. Suction filtration, filtrate reduced pressure concentration, adding 100mL ethyl acetate and 100mL water, separating the organic layer, water layer with ethyl acetate (50mL) extraction, organic layer, combined, saturated saline water washing, anhydrous magnesium sulfate drying, suction filtration, reduced pressure concentration, get light yellow solid 0.88 g.

Synthesis of ethyl 1- (3- (3- (4-methylphenyl) ureido) -4- (cyclohexylcarbamoyl) phenyl) -1H-indole-2-carboxylate (37E)

Compound 37D (0.88g,2.17mmol) was dissolved in anhydrous tetrahydrofuran (20mL), p-tolylene isocyanate (0.29g,2.17mmol) was added, and the reaction was carried out at room temperature for 4 hours, followed by concentration under reduced pressure and purification by column chromatography to give 0.78g of a white solid.

Compound 37E (0.78g,1.44mmol) was dissolved in absolute ethanol (20mL), and sodium hydroxide (0.28g,7.24mmol) was added and stirred at 65 ℃ for 8 hours. Cooling, concentrating under reduced pressure, adjusting the pH value to 3-4 with dilute hydrochloric acid (1M), adding 100mL ethyl acetate, separating an organic layer, washing with saturated saline solution, drying with anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by column chromatography to obtain 0.32g of white solid with the yield of 43.3%.¹H NMR(300MHz,DMSO-d₆)δ(ppm)12.92(s,1H),10.49(s,1H),9.70(s,1H),8.65(d,J＝7.8Hz,1H),8.33(d,J＝2.1Hz,1H),7.80(t,J＝7.6Hz,2H),7.44(s,1H),7.39-7.29(m,3H),7.24-7.17(m,2H),7.05(dd,J＝8.3,2.3Hz,3H),3.84(s,1H),2.22(s,3H),1.90(d,J＝9.4Hz,2H),1.78(d,J＝9.5Hz,2H),1.64(d,J＝12.3Hz,1H),1.37(m,4H)；MS(EI)m/z 509.2[M-H]^-.

Evaluation of pharmacological Activity

1. HeLa cell-based IDO1 inhibitory Activity test

1.1 Experimental materials and Main instruments

1.2 Experimental methods

HeLa cells purchased from ATCC were stored in minimal basal medium (2mM L-glutamine and Earle's BSS adjusted to contain 1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids, 1mM sodium cuprate and 10% fetal bovine serum). Hela cells were stored at 37 ℃ to provide 5% CO₂In a humidity-controlled incubator.

By 5X 10³Density per well HeLa cells were seeded in 96-well culture plates and cultured overnight. The next day, IFN-. gamma.was added to the cells (final concentration 100ng/mL) and serial dilutions of the compound (total volume 200. mu.L of medium). After 24 hours of incubation, 140. mu.L of the supernatant/well was transferred to a new 96-well plate, 10. mu.L of 6.1mol/L trichloroacetic acid was added, and incubated at 50 ℃ for 30min in a constant temperature oven to hydrolyze the produced N-formyl kynurenine to kynurenine. The reaction mixture was then centrifuged at 4000rpm for 10min to remove the precipitate. 100 μ L of supernatant/well was transferred to another 96-well plate and mixed with an equal volume of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. Detecting light absorption value at 480nm by using enzyme-labeling instrument, and using IC to obtain the result₅₀And calculating by a calculator. The experiment was performed in 3 duplicate wells.

In addition, the survival rate of each group of HeLa cells was examined by the MTT method to examine whether the test substance had an effect on the proliferation of HeLa cells. The method comprises the following specific operations: adding 20 mu L of 4mg/mL MTT solution into each well of a 96-well plate, putting the 96-well plate into a cell incubator for incubation for 4 hours, centrifuging the 96-well plate, carefully sucking liquid in each well, adding 200 mu L of dimethyl sulfoxide into each well, placing the 96-well plate on a shaking table for 300r and shaking for 10min to fully dissolve the purple crystalline substance. Finally, the absorbance was measured at 570nm of the microplate reader.

1.3 results of the experiment

The experimental results are shown in table 1, and the results show that the compound of the present invention has a significant inhibitory effect on IDO1 activity. Among them, compounds 2, 5, 17, 18, 25, 26, 27, 28, 29, 32, 33 and 34 had significantly better activity than positive control BMS-52. In addition, MTT test results show that the survival rate of HeLa cells in each group is kept above 90%, and the compounds do not inhibit the activity of IDO1 by inhibiting the proliferation of HeLa cells.

TABLE 1 inhibitory Activity of the Compounds of the present invention against IDO1

Positive control: BMS-52 is compound No. 52 of WO2015031295A 1.

2. Effect of Compounds of the invention on IDO1 protein expression

The purpose of this experiment was to investigate whether the compounds of the present invention inhibit IDO1 activity by down-regulating IDO1 protein expression. The effect of the compounds on IDO1 protein expression was examined using immunoblotting methods.

2.1 Experimental methods

Hela cells were cultured at 2X 10⁵The density of each well was cultured in 6-well plates at 37 ℃ with 5% CO₂Culturing under the condition for 12 h. Blank control (medium only), model group (IFN-. gamma.added, corresponding positive drug), drug treatment group (IFN-. gamma.added, corresponding compound), 5% CO at 37 deg.C₂Culturing for 24h under the condition, collecting cells, and detecting IDO1 expression by Western blot.

2.2 results of the experiment

The experimental results (figure 1) show that the compounds 2, 18, 28, 29 and 34 of the invention do not affect the expression of IDO1 protein, and the gray-scale scanning results also show that the ratio of IDO1 protein/Actin protein of the drug-adding group is not changed compared with the control group. In addition, we also validated compound 2 at different high dose concentrations and found that it had an IC higher than that₅₀At 100-fold concentration, compound 2 still did not affect IDO1 protein expression. Demonstrating that the compounds of the present invention have no effect on IDO1 protein expression, it is further demonstrated that these compounds do not inhibit IDO1 activity by down-regulating IDO1 protein expression.

3. Effect of Compounds of the invention on T lymphocyte proliferation and IFN- γ Release

T lymphocytes are the core performers of the immune system in humans and play a central role in the tumor immune response. Local tryptophan depletion and kynurenine accumulation caused by excessive expression of IDO1 can inhibit proliferation and induce apoptosis of T lymphocyte, promote differentiation of initial T lymphocyte to regulatory T lymphocyte, and inhibit secretion of cytokines such as IFN-gamma, IL-2 and TNF-alpha. The purpose of this experiment was to test the ability of the compounds of the present invention to reverse IDO 1-mediated immunosuppression.

Local tryptophan depletion and kynurenine accumulation caused by over-expression of IDO1 inhibit the proliferation and induce apoptosis of effector T lymphocytes, and also promote differentiation of naive T lymphocytes into regulatory T lymphocytes, thereby inhibiting the secretion of cytokines such as IFN- γ, IL-2, TNF- α, etc. The purpose of this experiment was to test the ability of the compounds of the present invention to reverse IDO 1-mediated immunosuppression.

3.1 Experimental methods

B16F1 cell treatment: the medium was aspirated (high-glucose DMEM, 10% FBS) and washed 1-2 times with PBS. Adding 0.25% pancreatin for digestion. Pancreatin was aspirated, media was added, cells were blown down, transferred to a 1.5mL centrifuge tube, centrifuged, supernatant aspirated, and cells resuspended in 1mL DMEM media. Adding mitomycin C (final concentration 25 mug/mL), blowing, beating, mixing, washing with water bath at 37 deg.C for 30min, RP1640 washing for 3 times, and counting cells.

Preparation of spleen cells: C57/BL6 mice were harvested, sacrificed by exsanguination, and spleens were aseptically removed into 35mm petri dishes containing 2mL of sterile, pre-cooled RPMI 1640 medium, and splenocytes gently squeezed out with a 5mL syringe needle. An additional 2mL of medium was added and the suspension was repeatedly pipetted using a 5mL pipette until uniform. The cell suspension was filtered through a 70 μm filter and centrifuged at 300g for 5min (4 ℃). 10mL Tris-NH was added to splenocytes after discarding supernatant₄Cl, blowing uniformly, standing for 2-3min, centrifuging for 5min (4 ℃) at 300g, and removing red blood cells. After discarding the supernatant, it was washed twice with PRMI 1640 and used.

1) 2X 10 treated B16F1 cells⁴Cell/well (stimulated cells), spleen lymphocytes 1X 10⁶Per well (reaction cells) was added to a 96-well plate, RP1640 (10% FBS) was added, and the volume was made up to 200. mu.L.

2) Grouping: administration group (stimulator cells + reaction cells + corresponding compound), blank control (reaction cells only), model group (stimulator cells + reaction cells), and other groups except blank control were added with ConA (final concentration 5. mu.g/mL) and placed at 37 ℃ with 95% humidity and 5% CO₂Culturing in the incubator for 48 h.

3) Adding into 20 μ L MTT (final concentration 4mg/mL) incubator, culturing for 4h, and measuring absorbance value at 570nm wavelength with microplate reader; calculating the proliferation rate of the T lymphocytes:

3.2 results of the experiment

The experimental result (figure 2) shows that in the mixed lymphocyte system, the B16F1 cell highly expresses IDO1 and can generate an inhibiting effect on the proliferation of T lymphocytes. When example compounds 2, 18, 28, 29, 34(3 times IC) were added₅₀Concentration) for 48h, they can effectively promote the proliferation of T lymphocytes and can also obviously improve the release of cell factor IFN-gamma. These experiments show that the compound of the present invention can reverse IDO1 mediated immune suppression effectively, so as to strengthen the proliferation capacity of T lymphocyte, promote IFN-gamma secretion and raise T cell qualityThe immune function of (1).

4. Effect of Compounds of the invention on regulatory T lymphocytes

CD4⁺CD25⁺Foxp3⁺T lymphocytes are an important class of regulatory T lymphocytes that play an important negative regulatory role in the human immune system. Studies have shown that IDO1 mediates the transformation of naive T cells into regulatory T lymphocytes, leading to an increased proportion of regulatory T lymphocytes in the tumor microenvironment, thereby inducing the formation of an immunosuppressive tumor microenvironment. Therefore, a mouse melanoma B16F1 cell line with high IDO1 expression is selected, treated by adding a compound, and then taken out of the cell line to be co-cultured with mouse spleen cells to simulate a tumor microenvironment. After cell collection, the effect of the compounds on the differentiation of naive T cells into regulatory T cells in the co-culture system was examined using flow cytometry.

4.1 Experimental methods

Treated B16F1 cells (8X 10)⁴One/well), spleen lymphocytes (10)⁶One per well, using ConA stimulation of 5. mu.g/mL) into 24-well plates, adding the corresponding concentration of compound, and standing at 37 ℃ with 95% humidity, 5% CO₂Culturing for 48h in an incubator; supernatants were collected for testing ELISA, and T cell differentiation was detected in a flow cytometer using anti-CD 4, anti-CD 25, anti-Foxp 3 antibody staining.

4.2 results of the experiment

The experimental results showed that when primary T lymphocytes were co-cultured with the melanoma B16F1 cell line, the number of regulatory T lymphocytes increased 4-fold (12.7%) compared to the experimental group containing only primary T lymphocytes (3.2%). When compounds 2, 18, 28, 29, 34 of the examples (3 times IC)₅₀Concentration) can significantly reverse this effect by down-regulating the regulatory T lymphocyte fraction to 7.1%, 9.5%, 6.7%, 8.3% and 5.87%, respectively, indicating that the compounds of the present invention can reverse the conversion of naive T lymphocytes to regulatory T lymphocytes by inhibiting the activity of IDO 1.

5. In vivo pharmacodynamic evaluation of Compounds of the invention

Granzyme B is a serine protease commonly found in Cytotoxic T Lymphocytes (CTL) and Natural Killer (NK) cell granules, and is a major effector of CTL and NK cells to exert cytotoxicity. The Proliferating Cell Nuclear Antigen (PCNA) is a nuclear protein necessary for DNA synthesis of eukaryotic cells, and the proliferation state of tumor cells can be objectively evaluated by detecting the PCNA. To this end, granzyme B, IFN- γ and PCNA levels were detected in tumor tissues during in vivo pharmacodynamic evaluation using immunohistochemistry and TUNEL assays.

5.1 Experimental methods

Culturing the mice: female mice of 7-8 weeks are selected and bred in an SPF animal breeding room for one week, and the weight of each mouse is about 18-20 g.

Treatment of tumor cells: collecting CT26, B16F1, PAN02 and LLC cells in logarithmic growth phase, centrifuging at 180g for 5min (4 deg.C), washing with precooled PBS for 2 times, and blowing uniformly to obtain final cell concentration of 1 × 10⁷mL, ice bath for standby.

Transplantation of tumor cells: CT26, B16F1, PAN02 and LLC cell suspensions are inoculated to the right armpit of the mouse to inoculate tumor cells of 1 × 10⁶A/only. The tumor size of the mice was measured every two days using a vernier caliper, and the body weight of the mice was weighed once. The tumor volume was calculated according to the following formula: v (volume) ═ A × B²And/2, wherein A is the length of the long side of the tumor and B is the length of the short side of the tumor. When the mean value of the tumor volume reaches 40mm³On the left and right sides, administration was started.

When the tumor volume reaches a certain size, the animal experiment is ended. The mice were weighed, subjected to eyeball bleeding, euthanized, tumor tissue stripped, weighed and photographed. Meanwhile, a part of the tissue was placed in 10% neutral fixative, and sent to paraffin-embedded tissue, paraffin tissue sections were made, and H & E staining, TUNEL and immunohistochemical analysis were performed. The experimental procedures refer to the instructions of the test kit.

5.1.1B 16F1 melanoma mouse graft tumor model

The C57BL/6 female mice transplanted with B16F1 melanoma were divided into 5 groups of 6 mice each. Model group (PBS + 2% Tween 20+ 2% DMSO, i.p., qd.), control group (cisplatin, dose: 1mg/kg, i.p., qod.), administration groups 1-3 (example Compound 2, dose: 1.25mg/kg, 2.5mg/kg, 5mg/kg, i.p., qd.).

5.1.2 LLC Lung cancer mouse transplantation tumor model

BALB/c female mice transplanted with LLC lung cancer were divided into 5 groups of 6 mice each. Model group (PBS + 2% Tween 20+ 2% DMSO, i.g., qd.), control group 1 (cisplatin, dose: 1mg/kg, i.p., qod.), administration groups 1-3 (example Compound 2, dose: 3.25mg/kg, 7.5mg/kg, 15mg/kg, i.g., qd.).

5.1.3 CT26 colorectal cancer BALB/c mouse transplantation tumor model

BALB/c females transplanted with CT26 colorectal tumors were divided into 3 groups of 8 mice each. Model group (PBS + 2% Tween 20+ 2% DMSO, i.p., qd.), control group (5-FU, dose: 25mg/kg, i.p., qod.), administration group (example Compound 2, dose: 5mg/kg, i.p., qd.).

5.1.4 CT26 colorectal cancer nude mouse transplantation tumor model

To verify whether the compounds of the present invention exert anti-tumor effects via the immune system, a model of CT26 colon cancer transplantation tumor was constructed in mice deficient in the immune system.

BALB/c (nu/nu) nude mice transplanted with CT26 colorectal tumor were divided into 3 groups of 8 mice each. Model group (PBS + 2% Tween 20+ 2% DMSO, i.p., qd.), control group (5-FU, dose: 25mg/kg, i.p., qod.), administration group (example Compound 2, dose: 5mg/kg, i.p., qd.).

5.1.5 PAN02 pancreatic cancer mouse graft tumor model

The C57BL/6 female mice transplanted with PAN02 pancreatic cancer were divided into 4 groups of 8 mice each. Model group (PBS + 2% Tween 20+ 2% DMSO, i.g., qd.), control group 1 (gemcitabine, dose: 30mg/kg, i.p., qod.), control group 2(Epacadostat, dose: 50mg/kg, i.g., bid.), administration group (Compound 2, dose: 15mg/kg, i.g., qd.).

5.2 results of the experiment

5.2.1 dose-dependent inhibition of the growth of B16F1 melanoma mouse graft tumors by Compounds of the invention

The results of the experiment (fig. 3) show that the inhibitory effect of example compound 2 on B16F1 melanoma mouse graft tumor increases with the increase of the administered dose, showing dose dependence. Compared with the model group, the compound 2 has 43.5 percent of tumor inhibition rate at the dose of 5mg/kg, and the positive control cisplatin group has 39.1 percent of tumor inhibition rate. In addition, the body weight of the mice in each group of compound 2 was not significantly changed from that in the Vehicle group, whereas the body weight of the mice in the cisplatin group tended to decrease after day 9.

The H & E staining result of the tumor tissue shows that the number of the tumor cells of the compound 2 in the middle and high dose groups is obviously reduced, the arrangement of the tumor cells is disordered, and the shrinkage of the nucleoplasm is obvious. The TUNEL experiment results show that the tumor cell apoptosis of the groups administered with 2.5mg/kg and 5mg/kg is obviously increased. Shows that the compound 2 has the effect of anti-tumor immunity by promoting the apoptosis of tumor cells.

5.2.2 the compounds of the present invention inhibit the growth of LLC lung cancer mouse transplantable tumor dose-dependently

The experimental results (figure 4) show that the inhibition effect of the compound 2 of the example on the transplanted tumor of the LLC lung cancer mouse is improved along with the increase of the dosage compared with the model group, and particularly under the administration dosage of 15mg/kg, the tumor inhibition rate of the compound is equivalent to that of the positive control cisplatin group in terms of tumor volume and tumor mass. However, the body weight of the mice in the cisplatin group showed a significant decrease after the administration of the cisplatin from day 9, indicating that the cisplatin group was more toxic, while the body weight of the mice in each group administered with compound 2 remained relatively stable.

5.2.3 the compound of the invention can obviously inhibit the growth of the transplantation tumor of a BALB/c mouse with the rectal cancer CT26

The experimental results (fig. 5) show that compound 2 of example is able to significantly inhibit the growth of BALB/c mouse graft tumor of CT26 rectal cancer compared to the model group, and does not affect the body weight of the mouse. The TUNEL results showed that the tumor cells in the Vehicle group were less apoptotic, while the numbers of apoptosis in the 5-FU group and the compound 2 group were significantly increased.

5.2.4 the Compounds of the invention do not have an effect on immune system deficient nude mouse transplantable tumors

The experimental results (fig. 6) show that the tumor volume and tumor mass of the compound 2 administration group of the example are unchanged compared with the model group, which indicates that the compound 2 does not produce inhibitory effect on the transplanted tumor of the immune system-deficient nude mouse. In contrast, the cytotoxic drug 5-FU can remarkably inhibit the growth of the transplanted tumor of the nude mouse with the colorectal cancer of CT 26. Indicating that the compound 2 plays an anti-tumor role through an immune system.

5.2.5 the compound of the invention can obviously inhibit the growth of PAN02 pancreatic cancer mouse transplantation tumor

The experimental results (fig. 7) show that example compound 2 was able to significantly inhibit the growth of tumor cells in PAN02 pancreatic cancer mouse transplantable tumors, and did not affect the body weight of the mice, compared to the model group.

It is noted that other compounds of the present invention show significant anti-tumor effects in mouse graft tumor models of various tumor types, such as CT26, EMT6, B16F1, PAN02, LLC, and the like. For example, compounds 5, 18, 28, 29, 32, 33, and the like of the examples significantly inhibited the growth of mouse transplantable tumors at low doses (2.5mg/kg to 15 mg/kg). Immunohistochemistry and TUNEL experiments showed that these compounds are capable of increasing CD8⁺T cell infiltration, promoting the secretion of granzyme B, improving the expression of IFN-gamma in tumor tissues and simultaneously reducing Foxp3⁺The number of Treg cell populations, reduces expression of PCNA protein. These experiments indicate that the compounds of the present invention are effective in reversing IDO 1-mediated immunosuppression.

6. Interaction of Compounds of the invention with IDO1 protein

6.1 Experimental methods

1) Cell thermomigration assay (CETSA): the principle is that the tertiary structure of the protein is affected and then gradually degraded along with the rise of the temperature, but when the small molecules are combined with the protein, the thermal stability of the protein can be increased, and the degradation trend is slowed down.

The method comprises the following specific operations: B16F1 cells were evenly divided into 2 groups 24h after stimulation with IFN- γ (final concentration 100 ng/mL): control and compound treated groups. Example compound 2 was added to ensure a final concentration of 1 μ M, an equal volume of DMSO was added to the control, cells were harvested after 3h, washed twice with PBS, 500 μ L of PBS resuspended cells, and placed in inlet PCR tubes in 10 aliquots. The PCR instrument was set to 10 temperatures (43, 46, 49, 52, 55, 58, 61, 64, 67, 70 ℃), and each sample of the control group and the experimental group was heated at the corresponding temperature: each group was heated for 3min, then left at room temperature for 3min, and then placed on ice. Finally, the samples were placed at-80 ℃ overnight, taken out the next day, thawed at room temperature, and then repeatedly frozen and thawed 3 times with liquid nitrogen. The treated sample was transferred to a 1.5mL EP tube, 12000g, centrifuged for 20min, and 40. mu.L of the supernatant was mixed with 6 × loading and subjected to immunoblotting.

2) Surface plasmon resonance experiments (SPR): the technology detects the interaction between biological molecules by measuring the refractive index change near the thin metal layer, including protein-small molecule interaction, and can detect the existence of the combination between the small molecules and the protein and the combination selectivity in real time with high sensitivity.

The method comprises the following specific operations: firstly, preparing a solution A: 210mL of 10 XPBS buffer, 1780mL of ddH₂And mixing O with 10mL of P20 surfactant, stirring uniformly, and metering to 2L. Preparing a solution B: 50mL of DMSO solution was added to 950mL of solution A to a volume of 1L. Finally, solution C is prepared, the prepared solution C and 5.8% DMSO solution are used for preparing a solvent correction solution, and the concentration of the compound 2 solution in the embodiment is prepared according to the instruction of the instrument operation instruction. After preparing each solution, each buffer solution was placed at the corresponding position of the instrument, the Biacore T200 system was turned on, the program was set according to the operation, and the sample was loaded for detection and data analysis.

6.2 results of the experiment

The results of cell thermo migration experiments show that the IDO1 protein of the control group (containing only DMSO) is completely degraded at 58 ℃, while compared with the control group, the compound 2 of the example can still maintain the stability of the IDO1 protein at 61 ℃ so that the IDO1 protein is not easily degraded (fig. 8A). Based on this, we chose to set up different concentration gradients of compound 2 at 58 ℃ and showed that compound was able to increase the stability of IDO1 protein with increasing concentration (fig. 8B). Indicating that compound 2 is able to enter B16F1 cells and bind to IDO1 protein.

The surface plasma resonance experiment result shows that the equilibrium dissociation constant K of the compound 2 of the example and the IDO1 protein_D＝1.9×10^-10M, indicating that dissociation rarely occurs and the affinity between the two is strong when the two are in equilibrium (fig. 9). Compound 2 was also shown to be very selective for IDO 1.

Claims (10)

1. An indole compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R¹represents hydrogen or halogen;

R²represents-CO₂R⁸or-CONR⁶R⁷；

R³Represents hydrogen or halogen;

R⁴represents-NR⁶R⁷or-CONHR⁸；

R⁵Represents nitro, -NR⁸CONR⁸R⁹、-NR⁸CO(CH₂)_nR⁹or-NR⁸SO₂R⁹；

n represents an integer of 0 to 6;

R⁶and R⁷Each independently represents hydrogen, C₁-C₈Alkyl radical, C₃-C₈Cycloalkyl radical, C₁-C₈Alkylamino or R⁶And R⁷Together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; wherein said heterocyclic ring may optionally contain one or more N heteroatoms; wherein said heterocyclic ring may be optionally substituted with one or more of the following groups: c₁-C₈An alkyl group;

R⁸represents hydrogen, C₁-C₈Alkyl or C₃-C₈A cycloalkyl group;

R⁹represents aryl, wherein said aryl may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen, C₁-C₈Alkyl, haloalkyl or-NR⁶R⁷。

2. The indole compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein:

R¹represents hydrogen or halogen;

R²represents-CO₂R⁸or-CONR⁶R⁷；

R³Represents hydrogen or halogen;

R⁴represents-NR⁶R⁷or-CONHR⁸；

R⁵Represents nitro, -NR⁸CONR⁸R⁹、-NR⁸CO(CH₂)_nR⁹or-NR⁸SO₂R⁹；

n represents an integer of 0 to 6;

R⁶and R⁷Each independently represents hydrogen, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl radical, C₁-C₆Alkylamino or R⁶And R⁷Together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; wherein said heterocyclic ring may optionally contain one or more N heteroatoms; wherein said heterocyclic ring is optionally substituted with one or more methyl groups;

R⁸represents hydrogen, C₁-C₈Alkyl or C₃-C₈A cycloalkyl group;

R⁹represents aryl, wherein said aryl may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen, C₁-C₈Alkyl, haloalkyl or-NR⁶R⁷。

3. The indole compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein:

R¹represents hydrogen or halogen;

R²represents carboxy, -CONH₂Or an ethoxycarbonyl group;

R³represents hydrogen or halogen;

R⁴represents-NR⁶R⁷or-CONHR⁸；

R⁵Represents nitro, -NR⁸CONR⁸R⁹、-NR⁸CO(CH₂)_nR⁹or-NR⁸SO₂R⁹；

n represents an integer of 0 to 3;

R⁶and R⁷Each independently represents hydrogen, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl radical, C₁-C₆Alkylamino or R⁶And R⁷Together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; wherein said heterocyclic ring may optionally contain one or more N heteroatoms; wherein said heterocyclic ring is optionally substituted with one or more methyl groups;

R⁸represents hydrogen, C₁-C₃Alkyl or C₃-C₆A cycloalkyl group;

R⁹represents a phenyl ring, wherein said phenyl ring may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen, C₁-C₅Alkyl, trifluoromethyl or N-methylpiperazinyl.

4. The indole compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein:

R¹represents hydrogen;

R²represents COOH;

R³represents hydrogen or halogen;

R⁴represents-NR⁶R⁷；

R⁵represents-NR⁸CONR⁸R⁹；

R⁶And R⁷Each independently represents C₁-C₄Alkyl or C₃-C₆A cycloalkyl group;

R⁸represents hydrogen;

R⁹represents a phenyl ring, wherein said phenyl ring may optionally be substituted by one or more R¹⁰Substitution;

R¹⁰represents hydrogen, halogen or C₁-C₅An alkyl group.

5. The indole compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of:

1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid ethyl ester,

1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxamide,

1- (3-nitro-4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-chlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4- (4-methylpiperazin-1-yl) phenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (3-trifluoromethylphenyl) -4- ((ethyl) cyclohexylamino) ureido) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- ((4-methylphenyl) sulfonylamino) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (2- (4-methylphenyl) acetamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- ((4-methylphenyl) carboxamido) -4- ((ethyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-methylphenyl) ureido) -4- ((ethyl) cyclohexylamino) phenyl) -6-chloro-1H-indole-2-carboxylic acid,

1- (3- (3- (4-methylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-fluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (2, 4-difluorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- (diisobutylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (3-trifluoromethylphenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-chloro-3-trifluoromethylphenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (3, 4-dichlorophenyl) ureido) -4- ((methyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-methylphenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-fluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (2, 4-difluorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (2-fluoro-4-chlorophenyl) ureido) -4- ((isobutyl) cyclohexylamino) phenyl) -1H-indole-2-carboxylic acid,

1- (3-chloro-4- (diisobutylamino) -5- (3- (4-methylphenyl) ureido) phenyl) -1H-indole-2-carboxylic acid,

1- (3-chloro-4- (diisobutylamino) -5- (3- (4-fluorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid,

1- (3-chloro-4- (diisobutylamino) -5- (3- (4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid,

1- (3-chloro-4- (diisobutylamino) -5- (3- ((2, 4-difluorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid,

1- (3-chloro-4- (diisobutylamino) -5- (3- (2-fluoro-4-chlorophenyl) ureido) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-methylphenyl) ureido) -4- (methyl- (1-methylpiperidin-4-yl) amino) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-methylphenyl) ureido) -4- (4-methylpiperazin-1-yl) phenyl) -1H-indole-2-carboxylic acid,

1- (3- (3- (4-methylphenyl) ureido) -4- (cyclohexylcarbamoyl) phenyl) -1H-indole-2-carboxylic acid,

6. a pharmaceutical composition, it is mainly made up of active ingredient and acceptable supplementary product pharmaceutically in effective quantity on the treatment; the active ingredient comprising a compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof.

7. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the manufacture of an indoleamine 2,3-dioxygenase 1 inhibitor.

8. Use of a pharmaceutical composition according to claim 6 for the manufacture of a medicament for the treatment of an indoleamine 2,3-dioxygenase 1 inhibitor.

9. Use of a compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 6, in the manufacture of a medicament for treating cancer, a viral infection, a neurodegenerative disease, a cataract, organ transplant rejection, depression, or an autoimmune disease in a patient.

10. The use of claim 9, wherein the cancer is one or more of malignant melanoma, lung cancer, breast cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, lymphatic cancer, leukemia, prostate cancer, testicular cancer, kidney cancer, brain cancer, head and neck cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, thyroid tumor, liver cancer, or esophageal cancer; the virus infection is infection caused by one or more of human immunodeficiency virus, hepatitis B virus, hepatitis C virus, influenza virus, poliovirus, cytomegalovirus, coxsackievirus, human papilloma virus, Epstein-Barr virus or varicella-zoster virus; the neurodegenerative disease is one or more of dysmnesia, Alzheimer disease, cognitive impairment, senile dementia, Parkinson disease or dyskinesia; the autoimmune disease is one or more of rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, myasthenia gravis, mixed connective tissue disease, psoriasis, endocrine-related diseases or autoimmune reaction caused by infection.

Images