WO2021143937A1 - Pyrrole compounds, preparation method therefor, and pharmaceutical composition and use thereof - Google Patents

Abstract

Disclosed are pyrrole compounds with structural characteristics of formula (I), metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof, a preparation method therefor, and the use thereof in the preparation of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor drugs. Experimental results indicate that the pyrrole compounds of the present invention have a significant inhibitory effect on the activity of IDO1, and can effectively promote the proliferation of T lymphocytes, inhibit the differentiation of naive T lymphocytes into regulatory T cells, and reverse IDO1-mediated immunosuppression, and can thus be used in the treatment of related diseases having a pathological feature of IDO1-mediated kynurenine metabolic pathway, such diseases comprising cancer, viral infections, neurodegenerative diseases, cataracts, organ transplant rejection, depression, autoimmune diseases, etc.

Description

Pyrrole compound, its preparation method, pharmaceutical composition and use Technical field

The present invention belongs to the field of new compounds, and specifically relates to a class of pyrrole compounds as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, their metabolites, metabolic precursors, prodrugs, solvates, crystals or The pharmaceutically acceptable salts thereof, their preparation methods, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions in the treatment of diseases related to IDO1-mediated immunosuppression.

Background technique

Indoleamine 2,3-dioxygenase 1 (IDO1) is the rate-limiting enzyme in the kynurenine metabolism pathway that catalyzes tryptophan outside the human liver. IDO1 is expressed in a variety of tissues (such as lung, kidney, brain, placenta, thymus) and a variety of cells (such as macrophages and dendritic cells), cytokines such as IFN-γ, TNF-α, IL-1β and IL-6 can induce IDO1 expression.

IDO1 can participate in the regulation of the body's innate immunity and adaptive immunity by catalyzing the oxidative metabolism of tryptophan. IDO1 achieves its regulatory effect on the immune system by catalyzing the local depletion of tryptophan and the accumulation of its metabolites by catalyzing tryptophan. On the one hand, the depletion of tryptophan can induce the T cell division cycle to stop at G1 by activating the GCN2 pathway. At the same time, it inhibits the proliferation of T cells, and at the same time inhibits the ^{differentiation of initial CD4 +} T cells into helper T cell 17 (Th17), which leads to immunosuppression; on the other hand, tryptophan metabolites such as kynurenine are cytotoxic It can kill T cells and natural killer (NK) cells, and these metabolites can also induce CD4 ⁺ T cells to differentiate into regulatory T cells (Treg) by activating aryl hydrocarbon receptors (AhR), and promote dendrites Cells (DC) are transformed into tolerogenic DCs; in addition, tryptophan metabolites can inhibit the function of NK cells by down-regulating the expression of NK cell receptors, which can further inhibit the body's immune response.

IDO1 is related to many physiological and pathological processes. Studies have shown that IDO1 plays an important role in host immune defense and maternal-fetal immune tolerance and other physiological stress processes. During this period, the secretion of cytokines such as IFN-γ significantly increases, thereby inducing IDO1 expression, leading to tryptophan depletion and kynurenine, etc. The accumulation of metabolites inhibits the mother's T cell response, induces the mother's immune tolerance, and ensures that the fetus is not rejected by the mother's immune system. The depletion of tryptophan in the host microenvironment makes it unable to provide the necessary tryptophan for the replication of pathogenic microorganisms, leading to the death of pathogenic microorganisms. At the same time, IDO1-mediated immunosuppression can avoid excessive activation of the body's immune system. IDO1 also exerts an immunosuppressive effect on the survival of transplanted tissue in the new host. These findings indicate that IDO1 is an immunomodulatory enzyme that participates in the body's immune tolerance.

Numerous studies have shown that IDO1-mediated immune tolerance is closely related to tumor immune escape, viral infection, neurodegenerative diseases, organ transplant rejection, autoimmune diseases, neuropsychiatric diseases and cataracts. In these diseases, the local depletion of tryptophan and the accumulation of its metabolites mediated by over-expression of IDO1 can inhibit the activation of T cells, leading to immune tolerance of the body.

In addition, IDO1 catalyzed tryptophan metabolites such as kynurenine and quinolinic acid are neurotoxic, and these metabolites are related to neurodegenerative diseases such as memory disorders, Alzheimer's disease (AD), and dementia. , Alzheimer's disease, Parkinson's disease, Parkinson's syndrome and movement disorders are closely related to the occurrence. Neuropsychiatric diseases such as depression, schizophrenia, and anxiety are also related to the overexpression of IDO1 and increased levels of metabolites such as kynurenine. The overexpression of IDO1 causes tryptophan depletion, thereby reducing the amount of tryptophan used to synthesize the neurotransmitter serotonin, leading to serotonin deficiency, plus neurotoxic kynurenine and quinolinic acid, etc. The accumulation of metabolites together promote the occurrence of neuropsychiatric diseases, and it is a factor of a variety of mood disorders.

Tryptophan depletion mediated by IDO1 overexpression also exists in various autoimmune diseases. In rheumatoid arthritis patients, the DCs of synovial joint tissues highly express IDO1. The serum tryptophan concentration of the patients decreases, while the kynurenine concentration and the kynurenine/tryptophan ratio are significantly increased.

Immune suppression induced by IDO1 plays an important role in tumor immune escape. IDO1 is overexpressed in various tumors and cells in their microenvironment such as DC cells and stromal cells, leading to local tumor depletion of tryptophan and accumulation of tryptophan metabolites, thereby inducing tumor immune escape and helping tumor cells to escape the body's immune system. attack.

IDO1 inhibitors can reduce the accumulation of tryptophan metabolism and kynurenine and other metabolites, thereby reversing the immunosuppressive effect mediated by IDO1, restoring the proliferation and function of T cells and NK cells, and inhibiting the proliferation of Treg cells, thereby enhancing The immune response of the body, therefore IDO1 inhibitors can be used to treat or prevent the above-mentioned related diseases caused by IDO1 mediated immunosuppression, including cancer, viral infections, neurodegenerative diseases, cataracts, organ transplant rejection, depression and autoimmunity Diseases etc. In addition, IDO1 inhibitors can also be combined with other chemotherapeutics, targeted antitumor drugs, immune checkpoint inhibitors, immune checkpoint agonists, antitumor vaccines, antiviral agents, antiviral vaccines, cytokine therapy, and adoptive cellular immunity Treatment and radiotherapy are used in combination to achieve the purpose of synergistic or enhanced therapy.

Summary of the invention

Objective of the invention: The technical problem to be solved by the present invention is to provide a compound with the structural characteristics of general formula (I), its metabolites, metabolic precursors, prodrugs, solvates, crystals and pharmaceutically acceptable salts thereof, Preparation method, pharmaceutical composition and use. The compound of the present invention has excellent IDO1 inhibitory activity and can be used to treat and/or prevent various related diseases caused by IDO1 mediated immunosuppression.

Technical Solution: The present invention provides pyrrole compounds, their metabolites, metabolic precursors, prodrugs, solvates, crystals or their pharmaceutically acceptable salts with the structural characteristics of general formula (I):

in:

R ¹ represents cyano, -CO ₂ R ⁶ or -CONR ⁷ R ⁸ ;

R ² represents hydrogen, halogen, cyano, hydroxyl or nitro;

R ³ and R ⁴ each independently represent hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ cycloalkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₁ -C ₈ alkane The amino group or R ³ and R ⁴ together with the nitrogen atom attached to them form a 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein The heterocyclic ring may be optionally substituted by one or more of the following groups: halogen, nitro, cyano, hydroxyl, amino, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy or C ₃ -C ₆ cycloalkyl;

R ⁵ represents an aryl group or an aromatic heterocyclic ring, wherein the aryl group or an aromatic heterocyclic ring may be optionally substituted ^{by one or more R 9;}

R ⁶ , R ⁷ and R ⁸ each independently represent hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ cycloalkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C _1- C ₈ alkylamino;

R ⁹ represents hydrogen, halogen, cyano, hydroxyl, mercapto, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylamino or haloalkyl;

The alkyl group represents a straight chain alkyl group, a branched chain alkyl group or a cyclic alkyl group; the alkoxy group represents a straight chain alkoxy group, a branched chain alkoxy group or a cyclic alkoxy group; Amino represents straight-chain alkylamino, branched alkylamino or cyclic alkylamino; said alkenyl represents straight-chain alkenyl, branched alkenyl or cyclic alkenyl; said alkynyl represents straight-chain alkynyl or branched Alkynyl

The aryl group represents phenyl, naphthyl, acenaphthyl or tetrahydronaphthyl; the aromatic heterocycle represents pyrrolyl, pyrazolyl, imidazolyl, furyl, thienyl, oxazolyl, isoxazole Monocyclic heterocycles of pyridyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl; or quinolinyl, quinoxalinyl, indolyl, benzimidazolyl, benzoxanyl Azolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzofuranyl, benzothienyl, 2,3-dihydrobenzo[1,4]dioxanyl Group, or a bicyclic heterocycle of benzo[1,3]dioxolyl;

The halogenated alkyl group is a linear or branched saturated hydrocarbon group with 1-8 carbon atoms, or a cyclic saturated hydrocarbon group with 3-8 carbon atoms, or a straight chain with 1-8 carbon atoms. Or a cyclic saturated hydrocarbon group with 3-8 carbon atoms of a branched saturated hydrocarbon group; wherein one or more hydrogen atoms are replaced by one or more halogen atoms.

Preferably, in the structure of the pyrrole compound and its derivative:

R ¹ represents cyano, -CO ₂ R ⁶ or -CONR ⁷ R ⁸ ;

R ² represents hydrogen or halogen;

R ³ and R ⁴ each independently represent hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ cycloalkyl, C ₁ -C ₈ alkylamino or R ³ and R ⁴ are formed together with the nitrogen atom to which they are attached 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein the heterocyclic ring may optionally be substituted by one or more of the following groups Group substitution: C ₁ -C ₈ alkyl or C ₃ -C ₆ cycloalkyl;

R ⁵ represents an aryl group or an aromatic heterocyclic ring, wherein the aryl group or an aromatic heterocyclic ring may be optionally substituted ^{by one or more R 9;}

R ⁶ , R ⁷ and R ⁸ each independently represent hydrogen, C ₁ -C ₈ alkyl or C ₃ -C ₈ cycloalkyl;

R ⁹ represents hydrogen, halogen, cyano, hydroxyl, mercapto, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylamino or haloalkyl.

Preferably, in the structure of the pyrrole compound and its derivative:

R ¹ represents cyano, -CO ₂ R ⁶ or -CONR ⁷ R ⁸ ;

R ² represents hydrogen or halogen;

R ³ and R ⁴ each independently represent hydrogen, C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylamino or R ³ and R ⁴ are formed together with the nitrogen atom to which they are attached 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein the heterocyclic ring may optionally be substituted by one or more methyl groups replace;

R ⁵ represents a benzene ring or an isoxazolyl group, wherein the benzene ring may be optionally substituted ^{by one or more R 9;}

R ⁶ , R ⁷ and R ⁸ each independently represent hydrogen, C ₁ -C ₃ alkyl or C ₃ -C ₆ cycloalkyl;

R ⁹ represents hydrogen, halogen, cyano, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy or trifluoromethyl.

Preferably, in the structure of the pyrrole compound and its derivative:

R ¹ stands for COOH;

R ² represents hydrogen or halogen;

R ³ and R ⁴ each independently represent a C ₁ -C ₄ alkyl group or a C ₃ -C ₆ cycloalkyl group;

R ⁵ represents a benzene ring, and the benzene ring may be optionally substituted ^{by one or more R 9;}

R ⁹ represents hydrogen, halogen, cyano, C ₁ -C ₅ alkyl or C ₁ -C ₅ alkoxy.

More specifically, the pyrrole compound is any one of the following compounds:

Another object of the present invention is to provide a preparation method of the compound represented by general formula (I), the preparation method being any one of the following methods:

Method 1: Using substituted nitrobenzene as raw material, reacting with amine compound HNR ³ R ⁴ under the action of alkali to prepare intermediate i, i and pyrrole-2-carboxylate are reacted with Ullmann to prepare intermediate ii, ii Intermediate iii is obtained by reduction, and ^{compound iv is obtained by condensation of iii with substituted benzene isocyanate R 5} NCO, or iii firstly forms active intermediate with 4-nitrophenyl chloroformate, and then reacts with amine compound R ⁵ NH ₂ The target compound iv is prepared; iv is hydrolyzed to obtain the target compound v; v is reacted with oxalyl chloride or thionyl chloride to form acid chloride and then reacted with the amine compound HNR ⁷ R ⁸ to obtain the target compound vi; vi is dehydrated at high temperature Obtain the target compound vii;

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are as defined above;

The base is selected from triethylamine, DIPEA, Na ₂ CO ₃ , K ₂ CO ₃ or Cs ₂ CO ₃ ; the reducing agent is selected from zinc powder and ammonium chloride or iron powder and ammonium chloride; vi Phosphorus oxychloride undergoes high-temperature dehydration to obtain the target compound vii.

Method 2: Using 2-fluoro-5-bromonitrobenzene as the raw material, reacting with the amine compound HNR ³ R ⁴ under the action of a base to obtain intermediate i, and obtaining intermediates viii, viii and pyrrole via NCS chlorination reaction. -2-carboxylic acid ester is reacted by Ullmann to obtain intermediate ix, ix is reduced by reducing agent to obtain intermediate x, x is ^{condensed with substituted phenyl isocyanate R 5} NCO to obtain intermediate xi, and xi is hydrolyzed to obtain target compound xii;

Wherein, R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above.

Wherein, the base described in step 1) is selected from triethylamine, DIPEA, Na ₂ CO ₃ , K ₂ CO ₃ or Cs ₂ CO ₃ .

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

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

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

The present invention also provides a pharmaceutical composition, which is mainly composed of a therapeutically effective amount of active components and pharmaceutically acceptable excipients; the active components include a compound of general formula (I) or its pharmaceutically acceptable One or more of the accepted salt. In the pharmaceutical composition, the adjuvant includes a pharmaceutically acceptable carrier, diluent and/or excipient.

According to the purpose of treatment, the pharmaceutical composition can be made into various types of dosage unit dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories and injections (solutions and suspensions), etc. Preference is given to tablets, capsules, liquids, suspensions and injections (solutions and suspensions).

In order to shape the pharmaceutical composition in the form of tablets, pills or suppositories, any excipient known and widely used in the art can be used.

In order to prepare a pharmaceutical composition in the form of an injection, the solution or suspension can be sterilized (preferably by adding an appropriate amount of sodium chloride, glucose or glycerin) to prepare an injection that is isotonic with blood. When preparing injections, any carriers commonly used in the art can also be used. For example: water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyethoxylated isostearyl alcohol and fatty acid esters of polyethylene sorbitan, etc. In addition, common dissolving agents and buffering agents can also be added.

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

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

The present invention also provides the use of the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor drugs use. The IDO1 inhibitor drugs are used to treat IDO1 mediated immune suppression related diseases, and the related diseases include cancer, viral infections, neurodegenerative diseases, cataracts, organ transplant rejection, depression or autoimmune diseases.

The present invention also provides the use of the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of medicines for the treatment of cancer, viral infections, neurodegenerative diseases, and cataracts , Organ transplant rejection, depression or autoimmune disease.

The cancers include but are not limited to: malignant melanoma, lung cancer, breast cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, lymphoma, leukemia, prostate cancer, testicular cancer, kidney cancer, brain cancer, head and neck cancer, ovarian cancer One or more of cancer, cervical cancer, endometrial cancer, mesothelial cancer, thyroid tumor, liver cancer, and esophageal cancer.

The viral infections include but are not limited to: human immunodeficiency virus, hepatitis B virus, hepatitis C virus, influenza virus, polio virus, cytomegalovirus, coxsackie virus, human papilloma virus, Infection caused by one or more of Stan-Barr virus and varicella-zoster virus.

The neurodegenerative diseases include, but are not limited to: one or more of memory disorder, Alzheimer's disease, dementia, Alzheimer's disease, Parkinson's disease, and movement disorders.

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

Furthermore, the present invention also provides that the compound of general formula (I), its pharmaceutically acceptable salt or the pharmaceutical composition can be used in combination with one or more other types of therapeutic agents and/or therapeutic methods for treatment Related diseases mediated by IDO1.

The other types of therapeutic agents and/or treatment methods include, but are not limited to: chemotherapeutics, targeted antitumor drugs, immune checkpoint inhibitors, immune checkpoint agonists, antitumor vaccines, antiviral agents, antiviral vaccines, Cytokine therapy, adoptive cellular immunotherapy or radiation therapy.

Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

The compound of the present invention has high inhibitory activity on IDO1. The results of pharmacological experiments show that these azole compounds can effectively reverse the immunosuppressive effect mediated by IDO1, promote ^{the proliferation of CD8 +} T lymphocytes, increase the secretion of granzyme B and interferon-γ, and reduce CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulation The generation of T cells reduces the expression of PCNA protein. The results of in vivo pharmacodynamic evaluation showed that the compounds of the present invention can significantly inhibit the growth of transplanted tumors in mice with various tumor types, but have no effect on the growth of transplanted tumors in nude mice with immune system defects, indicating that these compounds are activated by the host Immune response and play an anti-tumor effect.

Description of the drawings

Figure 1 shows the effect of the compounds of the present invention on IDO1 protein expression, in which: Figure 1A shows the effects of compounds 2, 14, 26, 27 and 39 on IDO1 protein expression, and Figure 1B shows the gray-scale scanning statistical results of Figure 1A;

Figure 2 shows the effect of the compound of the present invention on the growth of B16F1 melanoma transplanted tumors in mice. Figure 2A shows the effect of compound 2 on the volume of B16F1 melanoma transplanted tumors, and Figure 2B shows the effect of compound 2 on the weight of B16F1 melanoma transplanted tumors;

Figure 3 shows the effect of the compound of the present invention on the growth of CT26 colorectal cancer BALB/c mice transplanted tumor;

Figure 4 shows that the compound of the present invention does not inhibit transplanted tumors in nude mice with immune system deficiency;

Figure 5 shows the effect of the compound of the invention on the growth of PAN02 pancreatic cancer transplanted tumor in mice;

Figure 6 shows the effect of the compound of the present invention on IDO1 activity at different temperatures. Figure 6A shows the effect of Epacadostat on IDO1 activity at different temperatures. Figure 6B shows the effect of compound 26 on IDO1 activity at different temperatures. Figure 6C shows the effect of compound 26 on IDO1 activity at different temperatures. The effect of compound 39 on IDO1 activity at different temperatures.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with embodiments.

Reagents and materials: The reagents required for the experiment are all commercially available chemical or analytical products without special instructions.

Apparatus: ¹ HNMR is measured with Bruker AV-300 or 400MHz nuclear magnetic resonance instrument, the coupling constant (J) value is in Hz, and TMS is the internal standard. The mass spectrometer is determined by Shimadzu LCMS-2020 mass spectrometer. Thin layer chromatography uses HG/T2354-92 GF254 thin layer chromatography silica gel produced by Qingdao Ocean Chemical Co., Ltd. ZF7 type three-purpose ultraviolet analyzer 254nm color development.

Example 1: 1-(3-(3-(4-Methylphenyl)ureido)-4-((ethyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid methyl ester (1 )Synthesis

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

Combine 2-fluoro-5-bromonitrobenzene (3g, 13.6mmol), N-ethylcyclohexylamine (2.61g, 20.5mmol) and N,N-diisopropylethylamine (3.53g, 27.3mmol) Add N,N-dimethyl diamide (50 mL), stir at 80°C for 4 hours, extract with ethyl acetate (100 mL×3), and purify by column chromatography to obtain 4.03 g of a red solid with a yield of 90.4%. ¹ H 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[MH] ^- .

(2) Synthesis of 1-(3-nitro-4-((ethyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid methyl ester (1B)

Combine 1A (3g, 9.2mmol), pyrrole-2-methyl formate (1.15g, 9.2mmol), cuprous iodide (3.49g, 18.4mmol), cesium carbonate (5.97g, 18.4mmol) and L-proline Acid (2.12g, 18.4mmol) was added to DMF (50mL), stirred at 80°C for 12 hours under nitrogen protection, extracted with ethyl acetate (100mL×3), and purified by column chromatography to obtain 1.52g of yellow solid with a yield of 44.6%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm) 7.76 (d, J = 2.5 Hz, 1H), 7.57-7.37 (m, 2H), 7.30 (t, J = 2.2 Hz, 1H), 7.05 ( dd, J = 3.9, 1.88 Hz, 1H), 6.33 (dd, J = 3.9, 2.7 Hz, 1H), 3.65 (s, 3H), 3.15 (q, J = 7.0 Hz, 2H), 2.95 (tt, J = 11.7, 2.7Hz, 1H), 1.84-1.63 (m, 4H), 1.55 (d, J = 11.3Hz, 1H), 1.37 (td, J = 14.6, 13.7, 6.8 Hz, 2H), 1.28-1.01 ( m,3H),0.91(t,J=7.0Hz,3H); MS(EI)m/z 370.5[MH] ^- .

(3) Synthesis of 1-(3-amino-4-((ethyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid methyl ester (1C)

Add 1B (1.52g, 4.09mmol), zinc powder (1.33g, 20.46mmol) and ammonium chloride (1.31g, 20.46mmol) to 20mL of ethanol, react at room temperature for 4 hours under nitrogen protection, extract with ethyl acetate and reduce pressure Concentrate to obtain 1.34 g of light yellow solid.

(4) 1-(3-(3-(4-Methylphenyl)ureido)-4-((ethyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid methyl ester (1) Synthesis

Dissolve 1C (1.54g, 3.92mmol) in anhydrous tetrahydrofuran (20mL), add p-toluene isocyanate (0.53g, 3.92mmol), react at room temperature for 4 hours, concentrate under reduced pressure, and purify by column chromatography to obtain 1.32g of white solid , The yield is 63.7%. ¹ H NMR(300MHz,DMSO-d ₆ )δ(ppm)9.50(s,1H),8.6(s,1H), 8.17(d,J=2.4Hz,1H), 7.36(d,J=8.1Hz, 2H), 7.30-7.17 (m, 2H), 7.14-7.00 (m, 3H), 6.88 (dd, J = 8.6, 2.6 Hz, 1H), 6.30 (t, J = 3.2 Hz, 1H), 3.61 (s ,3H),3.05(q,J=7.0Hz,2H),2.77(s,1H),2.24(s,3H),1.96(d,J=14.6Hz,2H),1.71(s,2H),1.55 (d,J=11.9Hz,1H),1.18(s,5H),0.87(t,J=6.8Hz,3H); MS(EI)m/z 473.2[MH] ^- .

Example 2: 1-(3-(3-(4-Methylphenyl)ureido)-4-(cyclohexyl(ethyl)amino)phenyl)-1H-pyrrole-2-carboxylic acid (2) synthesis

Add 1 (1.32g, 2.78mmol) and sodium hydroxide (0.56g, 13.92mmol) to 20mL of ethanol, stir at 65°C for 8 hours, concentrate under reduced pressure, adjust the pH to 3～4 with dilute hydrochloric acid (1M), ethyl acetate Ester extraction and column chromatography purification yielded 0.68 g of white solid with a yield of 53.1%. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 8.51 (s, 1H), 8.26 (d, J = 2.4 Hz, 1H), 7.28 (s, 1H), 7.21 (d, J = 2.0 Hz, 3H ), 7.16(dt,J=3.9,2.0Hz,1H),7.12-6.99(m,3H),6.91(dd,J=8.4,2.4Hz,1H), 6.27(dd,J=3.9,2.6Hz, 1H), 2.91-2.8 (m, 2H), 2.55 (s, 1H), 2.37 (s, 3H), 1.59 (d, J = 9.7 Hz, 5H), 1.07 (t, J = 12.9 Hz, 5H), 0.77(t,J=6.9Hz,3H); MS(EI)m/z 459.3[MH] ^- .

Example 3: 1-(3-(3-(4-methylphenyl)ureido)-4-((ethyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxamide (3) Synthesis

Dissolve 2 (1g, 1.96mmol) in 15mL dry dichloromethane, cool in an ice bath, add 2mL oxalyl chloride, react overnight at room temperature, remove excess oxalyl chloride under reduced pressure, add 15mL dry dichloromethane to dissolve, ice bath After cooling, 3 mL of ammonia was added dropwise, reacted at room temperature for 5 hours, extracted with dichloromethane, and purified by column chromatography to obtain 0.5 g of white solid with a yield of 50.1%. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 8.58 (d, J = 2.1Hz, 1H), 7.80 (dd, J = 7.4, 1.5 Hz), 7.80 (s, 1H), 7.28 (s, 1H) ), 7.22 (dd, J = 7.6, 2.3 Hz, 3H), 7.09 (dd, J = 7.5, 1.5 Hz, 1H), 6.96 (dd, J = 7.5, 1.3 Hz, 2H), 6.81 (d, J = 7.5Hz, 1H), 6.76 (s, 2H), 6.64 (t, J = 7.5 Hz, 1H), 3.29 (q, J = 8.0 Hz, 2H), 2.33 (d, J = 1.2 Hz, 3H), 1.79 -1.67(m,4H),1.60(t,J=5.6Hz,2H),1.45-1.29(m,4H),1.18(t,J=8.0Hz,3H); MS(EI)m/z 458.3[ MH] ^- .

Example 4: 1-(2-((ethyl)cyclohexylamino)-5-(2-cyano-1H-pyrrolyl-1)phenyl)-3-(4-methylphenyl)urea ( 4) Synthesis

Dissolve 3 (0.4g, 0.87mmol) in 10mL of dichloromethane, add 3mL of phosphorus oxychloride, and react at 65°C for 8 hours. After cooling, the reaction solution was poured into ice water, sodium hydroxide aqueous solution (4M) was added to adjust the pH value to 9, extracted with dichloromethane, and purified by column chromatography to obtain 0.2 g of white solid with a yield of 52.0%. ¹ H NMR (300MHz, Chloroform-d) δ (ppm) 8.53 (s, 1H), 8.45 (d, J = 2.6 Hz, 1H), 7.28 (s, 3H), 7.23-7.12 (m, 3H), 7.10 (dd,J=8.4,2.6Hz,1H), 6.99(dd,J=4.0,1.6Hz,1H), 6.33(t,J=3.3Hz,2H), 2.88(q,J=7.1Hz,2H) ,2.56(t,J=10.8Hz,1H),2.39(s,3H),1.27(d,J=2.9Hz,4H),1.16-0.99(m,3H),0.90(t,J=8.8Hz, 3H),0.80(t,J=7.0Hz,3H); MS(EI)m/z 440.3[MH] ^- .

Using operations similar to those in Example 1 and Example 2, the following compounds were prepared:

Example 5: Evaluation of Pharmacological Activity

1. IDO1 inhibitory activity test based on HeLa cells

1.1 Experimental materials and main instruments

HeLa cell line: ATCC, centrifuge: Eppendorf (CHINA), electrothermal constant temperature blast drying oven (DHG-924385-Ⅲ): Shanghai Xinmiao Medical Equipment Manufacturing Co., Ltd. Acetic acid (glacial acetic acid): Nanjing Chemical Reagent Co., Ltd., Trifluoroacetic acid: Shanghai Lingfeng Chemical Reagent Co., Ltd., electronic balance: Sartorius, p-dimethylaminobenzaldehyde (CAS: 100-10-7): Aladdin, Recombinant Human IFN-γ (Catalog#AF-300-02): PEPROTECH.

1.2 Experimental method

HeLa cells purchased from ATCC are stored in the minimum basal medium (2mM L-glutamine and Earle adjusted to contain 1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids, 1mM sodium propionate and 10% fetal bovine serum BSS). The HeLa cells were stored in a humidity-controlled incubator _{with 5% CO 2 at 37°C.}

HeLa cells were seeded in a 96-well culture plate at a density of 5×10 ^{3 /well and cultured overnight.} On the next day, IFN-γ (final concentration 100 ng/mL) and serial dilutions of the compound (total volume 200 μL of medium) were added to the cells. After 24 hours of incubation, transfer 140 μL of supernatant/well to a new 96-well plate, add 10 μL of 6.1 mol/L trichloroacetic acid, and incubate in a constant temperature oven at 50°C for 30 minutes to produce N-formyl dogs. Urine is hydrolyzed to kynurenine. The reaction mixture was then centrifuged at 4000 rpm for 10 min to remove the precipitate. Transfer 100 μL of supernatant/well to another 96-well plate and mix with an equal volume of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. Use a microplate reader to detect the absorbance at 480nm, and calculate the _{result using an IC 50 calculator.} The experiment has 3 multiple holes.

In addition, the MTT method was used to detect the survival rate of HeLa cells in each group in order to investigate whether the compound inhibits the activity of IDO1 by inhibiting the proliferation of HeLa cells.

Specific operation: Add 20μL 4mg/mL MTT solution to each well of a 96-well plate, place it in the cell culture incubator and incubate for 4 hours, centrifuge the 96-well plate, carefully aspirate the liquid in the well, and add 200μL dimethyl sulfoxide to each well , Placed on a shaker at 300r and shaken for 10 minutes to fully dissolve the purple crystalline substance. Finally, the absorbance value was detected at 570nm in the microplate reader.

1.3 Experimental results

The experimental results (Table 1) show that the compound of the present invention has a significant inhibitory effect on the activity of IDO1. Among them, compound 27 has the strongest activity (IC ₅₀ :0.010 nM). In addition, the results of MTT test showed that the survival rate of HeLa cells in each group remained above 90%, indicating that these compounds did not inhibit the activity of IDO1 by inhibiting the proliferation of HeLa cells.

Table 1. Inhibitory activity of the compounds of the present invention on IDO1

*Positive control: BMS-52 is compound No. 52 in WO2015031295A1.

2. The effect of the compound of the present invention on the expression of IDO1 protein

The purpose of this experiment is to investigate whether the compound of the present invention inhibits the activity of IDO1 by down-regulating the expression of IDO1 protein. The immunoblotting method was used to detect the effect of the compound on the expression of IDO1 protein.

2.1 Experimental method

HeLa cells were ^{seeded in a 6-well plate at a density of 2×10 5} per well, and cultured at 37°C and 5% CO ₂ for 12 hours. Blank control (only add culture medium), model group (add IFN-γ, corresponding positive drug), drug treatment group (add IFN-γ, corresponding compound), _{culture at 37℃, 5% CO 2} for 24h, collect cells , Western blot detection of IDO1 expression.

2.2 Experimental results

The experimental results (Figure 1) show that the compound of the present invention does not affect the expression of IDO1 protein, and the gray-scale scanning results also show that the IDO1 protein/Actin protein ratio of the drug-added group has no change compared with the control group. It shows that the compound of the present invention does not inhibit the activity of IDO1 by down-regulating the expression of IDO1 protein.

3. The effect of the compound of the present invention on T lymphocyte proliferation and IFN-γ release

T lymphocytes are the core executors of the immune system in the human body and play a central role in tumor immune response. The local tryptophan depletion and kynurenine accumulation caused by IDO1 overexpression can inhibit the proliferation of T lymphocytes and induce their apoptosis. At the same time, it can also promote the differentiation of initial T lymphocytes into regulatory T lymphocytes and inhibit cytokines such as IFN. -Secretion of γ, IL-2 and TNF-α. The purpose of this experiment is to test the ability of the compounds of the present invention to reverse IDO1-mediated immunosuppression.

3.1 Experimental method

B16F1 cell treatment: aspirate the medium (high sugar DMEM, 10% FBS), wash 1-2 times with PBS. Add 0.25% trypsin for digestion. Aspirate the trypsin, add medium, pipette down the cells, transfer to a 1.5mL centrifuge tube, centrifuge, aspirate the supernatant, add 1mL DMEM medium to resuspend the cells. Add mitomycin C (final concentration 25μg/mL), mix by pipetting, 37°C, water bath for 30min, wash 3 times with RP1640, count the cells, and set aside.

Preparation of spleen cells: Take C57/BL6 mice, remove the eyeballs and bleed to death, remove the spleen aseptically and place it in a 35mm petri dish containing 2mL of sterile pre-cooled RPMI 1640 medium, and gently push the spleen with a 5mL syringe needle. Cell extrusion. Then add 2 mL of medium and pipette repeatedly with a 5 mL pipette until the suspension is uniform. The cell suspension was filtered with a 70 μm filter, and centrifuged at 300 g for 5 min (4° C.). After discarding the supernatant, add 10 mL of Tris-NH ₄ Cl to the spleen cells, blow them evenly, let stand for 2-3 min, and centrifuge at 300 g for 5 min (4° C.) to remove red blood cells. After discarding the supernatant, wash twice with PRMI 1640 and set aside.

1) Add the treated B16F1 cells 2×10 ⁴ cells/well (stimulator cells) and spleen lymphocytes 1×10 ⁶ cells/well (reactive cells) into a 96-well plate, add RP1640 (10% FBS), supplement Qi to 200μL.

2) Grouping: administration group (stimulator cell + reactive cell + corresponding compound), blank control (only reactive cell), model group (stimulator cell + reactive cell), except blank control, all other groups are added with ConA( The final concentration is 5μg/mL), placed in an incubator at 37°C, 95% humidity, and 5% CO ₂ for 48 hours.

3) Add 20μL MTT (final concentration 4mg/mL) in the incubator and continue to incubate for 4 hours, measure the absorbance value at 570nm wavelength with a microplate reader; calculate the proliferation rate of T lymphocytes:

3.2 Experimental results

The experimental results show that in the mixed lymphocyte system, B16F1 cells highly express IDO1, which can inhibit the proliferation of T lymphocytes. After adding compound 2, 14, 26, 27, 39 (3 times IC ₅₀ concentration) and culturing for 48 hours, using MTT to detect the proliferation of T lymphocytes, these compounds can significantly increase the proliferation of T lymphocytes, and the proliferation rate reaches 139.8%~ 173.0%, and these compounds can also increase the release of cytokine IFN-γ (increasing rate: 125.8% to 134.5%). These experiments show that the compound of the present invention can effectively reverse IDO1-mediated immune suppression, thereby enhancing the proliferation ability of T lymphocytes, promoting the secretion of IFN-γ, and improving the immune function of T cells.

4. The effect of the compound of the present invention on regulatory T lymphocytes

CD4 ⁺ CD25 ⁺ Foxp3 ⁺ T lymphocytes are an important type of regulatory T lymphocytes, which play an important negative regulatory role in the human immune system. Studies have shown that IDO1 can mediate the transformation of initial T cells into regulatory T lymphocytes, leading to an increase in the proportion of regulatory T lymphocytes in the tumor microenvironment, thereby inducing the formation of an immunosuppressed tumor microenvironment. To this end, we selected the mouse melanoma B16F1 cell line with high IDO1 expression, treated it with compounds, and took its supernatant to co-culture with mouse spleen cells to simulate the tumor microenvironment. After the cells were collected, flow cytometry was used to detect the effect of the compound on the differentiation of naive T cells into regulatory T cells in the co-culture system.

4.1 Experimental method

Add the treated B16F1 cells (8×10 ⁴ cells/well), splenic lymphocytes (10 ⁶ cells/well, stimulated with 5μg/mL ConA) into a 24-well plate, add the corresponding concentration of compound and place at 37℃ Incubate in an incubator with 95% humidity and 5% CO ₂ for 48 hours; collect the supernatant to test ELISA, stain with anti-CD4, anti-CD25, and anti-Foxp3 antibodies, and detect T cell differentiation in a flow cytometer.

4.2 Experimental results

The experimental results showed that when the initial T lymphocytes were co-cultured with the melanoma B16F1 cell line, the number of regulatory T lymphocytes increased by 4 times (12.7%) compared with the experimental group containing only the initial T lymphocytes (3.2%) . When compounds 2, 14, 26, 27, 39 (3 times IC ₅₀ concentration) are added to the system, this effect can be significantly reversed, which reduces the proportion of regulatory T lymphocytes to 7.8%, 5.3%, 6.2%, and 4.7, respectively. % And 4.5%, indicating that the compound of the present invention can reverse the differentiation of initial T lymphocytes into regulatory T lymphocytes by inhibiting the activity of IDO1.

5. In vivo pharmacodynamic evaluation of the compound of the present invention

Granzyme B is a serine protease commonly found in cytotoxic T lymphocytes (CTL) and natural killer (NK) cell granules, and is the main effector of CTL and NK cells to exert cytotoxicity. Proliferating cell nuclear antigen (PCNA) is a nuclear protein necessary for DNA synthesis in eukaryotic cells. The detection of PCNA can objectively evaluate the proliferation status of tumor cells. To this end, in the process of in vivo pharmacodynamic evaluation, immunohistochemistry and TUNEL analysis were used to detect the levels of granzyme B, IFN-γ and PCNA in tumor tissues.

5.1 Experimental method

Cultivation of mice: select female mice aged 7-8 weeks and raise them in an SPF-class animal breeding room for one week. Each mouse weighs about 18-20g.

Treatment of tumor cells: Collect CT26, B16F1, PAN02 cells in logarithmic growth phase, centrifuge at 180g for 5min (4℃), wash twice with pre-cooled PBS, pipette evenly, and the final cell concentration is 1×10 ⁷ /mL , Ice bath for spare.

Tumor cell transplantation: CT26, B16F1, and PAN02 cell suspension were respectively inoculated into the right axillary of the mouse subcutaneously, and the number of tumor cells inoculated was 1×10 ⁶ /mouse. The tumor size of the mouse was measured with a vernier caliper every two days, and the mouse weight was weighed once. The volume of the tumor is calculated according to the following formula: V (volume)=A×B ² /2, where 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 average tumor volume reaches about 40mm ³ , the drug is started.

When the tumor volume reached a certain size, the animal experiment was ended. Weigh the weight of the mice, take blood from their eyeballs, and euthanize the mice, strip the tumor tissues, weigh the tumor tissues and take pictures. At the same time, some tissues were placed in 10% neutral fixative, samples were sent for paraffin-embedded tissues, paraffin tissue sections were made, and H&E staining, TUNEL and immunohistochemical analysis were performed. Refer to the test kit instructions for experimental operations.

5.1.1 B16F1 melanoma transplanted tumor model in mice

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

5.1.2 CT26 colorectal cancer BALB/c mouse xenograft model

The BALB/c female mice transplanted with CT26 colorectal tumors were divided into 5 groups with 8 mice in each group. Model group (PBS+2% Tween 20+2% DMSO, ip, qd.), positive control group (5-FU, dose: 25 mg/kg, ip, qod.), administration group (Example Compound 2 , Dose: 5mg/kg, ip, qd.).

5.1.3 CT26 colorectal cancer transplanted tumor model in nude mice

In order to verify whether the compound of the present invention exerts an anti-tumor effect through the immune system, a CT26 colon cancer xenograft model was constructed on mice with immune system deficiency.

BALB/c(nu/nu) nude mice transplanted with CT26 colorectal tumors were divided into 3 groups, each with 8 mice. Model group (PBS+2% Tween 20+2% DMSO, ip, qd.), positive control group (5-FU, dose: 25 mg/kg, ip, qod.), administration group (Example Compound 2 , Dose: 10mg/kg, ip, qd.).

5.1.4 PAN02 Pancreatic Cancer Mouse Xenograft Model

C57BL/6 female mice transplanted with PAN02 pancreatic cancer were divided into 4 groups with 6 mice in each group. Model group (PBS+2% Tween 20+2% DMSO, ig, qd.), positive control group 1 (gemcitabine, dose: 30 mg/kg, ip, qod.), positive control group 2 (Epacadostat, dose: 50 mg/kg, ig, bid.), administration group (Example Compound 9, dose: 15 mg/kg, ig, qd.).

5.2 Experimental results

5.2.1 The compound of the present invention inhibits the growth of transplanted tumors of B16F1 melanoma mice in a dose-dependent manner

The experimental results (Figure 2) showed that with the increase of the administered dose, the inhibitory effect of compound 2 on the transplanted tumor of B16F1 melanoma mice also increased, showing a dose-dependence. The body weight of mice in all administration groups remained between 20-23 g, indicating that compound 2 did not affect the body weight of mice. Immunohistochemistry and TUNEL experiments showed that compound 2 (5mg/kg) can increase CD8 ⁺ T cell infiltration, reduce ^{the number of Foxp3 +} Treg cells, promote the secretion of granzyme B and IFN-γ, and increase tumor cell apoptosis.

5.2.2 The compound of the present invention can significantly inhibit the growth of CT26 colorectal cancer BALB/c mice transplanted tumor

The experimental results (Figure 3) show that Example Compound 2 can significantly inhibit the growth of CT26 colorectal cancer BALB/c mice transplanted tumors, and there is no obvious fibrosis and inflammation in the liver of the mice, and other organs such as the heart, The morphology and structure of the kidney, spleen and lung did not change significantly, indicating that these compounds did not have significant drug-induced damage to the various organs of mice. In addition, compared with the model group, the body weight of the mice in the 5-FU group began to decrease after the 9th day, while the body weight of the mice in the compound 2 administration group remained stable, indicating that the compound 2 did not affect the body weight of the mice.

The results of immunohistochemistry and TUNEL experiments show that compound 2 can increase the ^{infiltration of CD8 +} T cells, increase the expression of IFN-γ in tumor tissues, reduce ^{the number of Foxp3 +} Treg cell populations, and can also significantly promote the secretion of granzyme B , Reduce the expression of PCNA protein, and further increase tumor cell apoptosis.

5.2.3 The compound of the present invention does not inhibit the transplanted tumor of nude mice with defective immune system

The experimental results (Figure 4) show that the cytotoxic drug 5-FU can significantly inhibit the growth of CT26 colorectal cancer transplanted tumors in nude mice. However, Example Compound 2 has no inhibitory effect on transplanted tumors in nude mice with immune system deficiency. It shows that compound 2 exerts anti-tumor effect through the immune system.

5.2.4 The compound of the present invention can significantly inhibit the growth of PAN02 pancreatic cancer transplanted tumor in mice

The experimental results (Figure 5) show that, compared with the model group, compound 9 can significantly inhibit tumor growth in the PAN02 pancreatic cancer mouse xenograft model. Immunohistochemistry and TUNEL experiments show that compound 9 can effectively reverse IDO1-mediated immunosuppression.

It should be pointed out that the other compounds in the present invention also show significant anti-tumor effects in various tumor types such as CT26, EMT6, B16F1, PAN02, LLC and other mouse xenograft tumor models. For example, compounds 20, 23, 24, 26, 27, 28, 30, 31, 33, 34, 35, 36, 38, 39, 40, and 41 will defecate at low doses (2.5 mg/kg to 15 mg/kg). Can significantly inhibit the growth of transplanted tumors in mice. In addition, the results of immunohistochemistry and TUNEL experiments on tumor tissues show that these compounds can also increase CD8 ⁺ T cell infiltration, promote the secretion of granzyme B, increase the expression of IFN-γ in tumor tissues, and reduce Foxp3 ⁺ Treg. The number of cell populations reduces the expression of PCNA protein. These experiments show that the compound of the present invention can effectively reverse the immunosuppressive effect mediated by IDO1.

6. Interaction between the compound of the present invention and IDO1 protein

6.1 Experimental method

1) Cell Thermal Migration Test (CETSA): The principle is that as the temperature rises, the tertiary structure of the protein will be affected and gradually degraded, but when small molecules are combined with the protein, the thermal stability of the protein can be increased. Slow down the degradation trend.

Specific operation: B16F1 cells were stimulated with IFN-γ (final concentration 100ng/mL) for 24 hours and then divided into 2 groups evenly: control group and compound treatment group. Add compounds 2 and 27 to ensure that the final concentration is 1μM. Add an equal volume of DMSO to the control group. After 3 hours, collect the cells, wash twice with PBS, resuspend the cells in 500μL PBS, and divide them into 10 parts and place them in imported PCR tubes. . Set the PCR instrument to 10 temperatures (43, 46, 49, 52, 55, 58, 61, 64, 67, 70°C), and heat each sample of the control group and the experimental group according to the corresponding temperature: each group is heated for 3 minutes Leave it at room temperature for 3 minutes, and then place it on ice. Finally, the sample was placed at -80°C overnight, and then taken out the next day. After being thawed at room temperature, the sample was repeatedly frozen and thawed 3 times with liquid nitrogen. Transfer the processed sample to a 1.5mL EP tube, centrifuge at 12000g for 20min, take 40μL of the supernatant, add 6×loading and mix, and then perform Western blot analysis.

2) Micro thermophoresis (MST): This technology is based on the directional movement of molecules in a temperature gradient to cause changes in molecular properties, such as molecular size, charge, hydration layer, and conformation. MST is a new type of technology for measuring the interaction between molecules, which can measure different binding modes, including dimerization, synergy and competition. The technology is highly adaptable, can be used in different environments, different biomolecules and different solutions, and can be completed in complex biological solutions or even cell lysates without sample purification.

Specific operation: Fluorescently label the IDO protein, and measure the fluorescence value of the protein at a certain dilution before performing the formal operation to observe whether there are problems such as adsorption. Subsequently, 16 different concentration gradients were set for 27 of compound 2 to be tested, and the protein buffer was prepared in an imported PCR tube, each tube having a volume of 10 μL. Add the diluted protein to the PCR tube, the total volume is 20μL. Use a capillary tube to draw 10μL on the machine to detect and analyze the data.

6.2 Experimental results

CETSA experimental results show that the IDO1 protein of the control group (only DMSO) has been completely degraded at 58°C. Compared with the control group, the compounds 2 and 27 of the present invention can still maintain the stability of IDO1 protein at a high temperature of 61°C. Make it difficult to be degraded. It shows that compounds 2 and 27 can enter B16F1 cells and bind to IDO1 protein.

The results of the MST experiment show that the dissociation constants K _{D of the} compounds 2 and 27 of the present invention and IDO1 protein are 3.9×10 ^-8 M and 1.3×10 ^-10 M, respectively, indicating that the affinity between compounds 2 and 27 and IDO1 protein is very strong. , And can have a strong selective binding force to IDO1.

7. The binding mode of the compound of the present invention and IDO1 protein

The IDO1 inhibitors developed in the early stage mainly inhibit the activity of IDO1 by combining with heme-containing IDO1 (holo-IDO1). Holo-IDO1 inhibitors represented by Epacadostat mainly occupy the catalytic pocket of IDO1 by complexing with heme iron ions, thereby producing inhibitory activity. The purpose of this experiment is to verify the binding mode of the compound of the present invention and IDO1 protein.

7.1 Experimental method

1) Enzymology experiments at different temperatures: Studies have shown that the stability of IDO1 protein changes with temperature. At 25°C, the binding of heme to IDO1 protein is relatively strong. At this time, when it is incubated with the apo-IDO1 inhibitor, the inhibitor cannot squeeze the heme out of the IDO1 protein binding pocket. When the temperature rises to 37°C, the stability of the binding of heme to IDO1 protein will decrease. At this time, heme is easily squeezed out of the IDO1 binding pocket by the apo-IDO1 inhibitor. However, holo-IDO1 inhibitors such as Epacadostat can stabilize the binding of heme to IDO1 protein by complexing with iron ions at any temperature. Therefore, enzyme experiments at different temperatures can be used to detect the binding of inhibitors to IDO1 (Ortizmeoz RF, et al. bioRxiv, 2018).

Specific operation: Divide the test substance and IDO1 protein of different concentrations into two groups, mix them and add them to the EP tube, pre-incubate at 25°C and 37°C for 2h, add catalase solution, methylene blue solution, and TritonX-100 solution , VCNa solution, incubate at 37°C for 15 min. Add D-Trp solution, the total reaction system is 250μL, continue to incubate for 60min, and perform detection according to 1.2 detection method.

2) X-ray diffraction experiment: In order to cultivate the compound crystals of the compound of the present invention and IDO1 protein binding, under the condition of 50mM Tris buffer, pH 7.4, incubate compound 39 and hIDO1 in a water bath at 42°C for 2h. After centrifugation at 15000 rpm for 5 minutes, the supernatant was placed in 96-well sitting-drop trays, and crystal growth was performed after adding the crystallization solution to obtain 39/hIDO1 complex crystals and then X-ray diffraction data were collected at Shanghai Light Source BL19U line station .

7.2 Experimental results

The results of enzymatic experiments showed that Epacadostat can bind to IDO1 containing heme, no matter at 25℃ or 37℃, so it has strong inhibitory activity on IDO1 at two different temperatures (Figure 6A). However, at 25°C, the inhibitory effects of Example Compounds 26 and 39 on IDO1 were very weak, and only showed strong inhibitory activity at 37°C (Figure 6B and Figure 6C). It shows that the compound of the present invention mainly inhibits the activity of IDO1 protein by competitively binding with heme.

Furthermore, crystals of compound 39 of Example compound 39 and IDO1 protein were obtained by crystal culture. X-ray diffraction results showed that compound 39 of Example compound could bind to apo-IDO1 protein, which further confirmed that the compound of the present invention is a type of apo-IDO1 protein. IDO1 inhibitor.

Claims (10)

1. A pyrrole compound represented by general formula (I), its metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof:

   

   in:

   R ¹ represents cyano, -CO ₂ R ⁶ or -CONR ⁷ R ⁸ ;

   R ² represents hydrogen, halogen, cyano, hydroxyl or nitro;

   R ³ and R ⁴ each independently represent hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ cycloalkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₁ -C ₈ alkane The amino group or R ³ and R ⁴ together with the nitrogen atom attached to them form a 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein The heterocyclic ring may be optionally substituted by one or more of the following groups: halogen, nitro, cyano, hydroxyl, amino, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy or C ₃ -C ₆ cycloalkyl;

   R ⁵ represents an aryl group or an aromatic heterocyclic ring, wherein the aryl group or an aromatic heterocyclic ring may be optionally substituted ^{by one or more R 9;}

   R ⁶ , R ⁷ and R ⁸ each independently represent hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ cycloalkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C _1- C ₈ alkylamino;

   R ⁹ represents hydrogen, halogen, cyano, hydroxyl, mercapto, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylamino or haloalkyl.
2. The pyrrole compound, its metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to claim 1, characterized in that:

   R ¹ represents cyano, -CO ₂ R ⁶ or -CONR ⁷ R ⁸ ;

   R ² represents hydrogen or halogen;

   R ³ and R ⁴ each independently represent hydrogen, C ₁ -C ₈ alkyl, C ₃ -C ₈ cycloalkyl, C ₁ -C ₈ alkylamino or R ³ and R ⁴ are formed together with the nitrogen atom to which they are attached 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein the heterocyclic ring may optionally be substituted by one or more of the following groups Group substitution: C ₁ -C ₈ alkyl or C ₃ -C ₆ cycloalkyl;

   R ⁵ represents an aryl group or an aromatic heterocyclic ring, wherein the aryl group or an aromatic heterocyclic ring may be optionally substituted ^{by one or more R 9;}

   R ⁶ , R ⁷ and R ⁸ each independently represent hydrogen, C ₁ -C ₈ alkyl or C ₃ -C ₈ cycloalkyl;

   R ⁹ represents hydrogen, halogen, cyano, hydroxyl, mercapto, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylamino or haloalkyl.
3. The pyrrole compound, its metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to claim 1, characterized in that:

   R ¹ represents cyano, -CO ₂ R ⁶ or -CONR ⁷ R ⁸ ;

   R ² represents hydrogen or halogen;

   R ³ and R ⁴ each independently represent hydrogen, C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylamino or R ³ and R ⁴ are formed together with the nitrogen atom to which they are attached 5-7 membered heterocyclic ring; wherein the heterocyclic ring may optionally contain one or more heteroatoms selected from O, S or N; wherein the heterocyclic ring may optionally be substituted by one or more methyl groups replace;

   R ⁵ represents a benzene ring or an isoxazolyl group, wherein the benzene ring may be optionally substituted ^{by one or more R 9;}

   R ⁶ , R ⁷ and R ⁸ each independently represent hydrogen, C ₁ -C ₃ alkyl or C ₃ -C ₆ cycloalkyl;

   R ⁹ represents hydrogen, halogen, cyano, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy or trifluoromethyl.
4. The pyrrole compound, its metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to claim 1, characterized in that:

   R ¹ stands for COOH;

   R ² represents hydrogen or halogen;

   R ³ and R ⁴ each independently represent a C ₁ -C ₄ alkyl group or a C ₃ -C ₆ cycloalkyl group;

   R ⁵ represents a benzene ring, and the benzene ring may be optionally substituted ^{by one or more R 9;}

   R ⁹ represents hydrogen, halogen, cyano, C ₁ -C ₅ alkyl or C ₁ -C ₅ alkoxy.
5. The pyrrole compound, its metabolite, metabolic precursor, prodrug, solvate, crystal, or pharmaceutically acceptable salt thereof according to claim 1, wherein the pyrrole compound is selected from any of the following compounds :

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

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

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

   1-(2-((ethyl)cyclohexylamino)-5-(2-cyano-1H-pyrrolyl-1)phenyl)-3-(4-methylphenyl)urea (4),

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

   1-(3-(3-(3-trifluoromethylphenyl)ureido)-4-((ethyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (6),

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

   1-(3-(3-(3,4-Dichlorophenyl)ureido)-4-((ethyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (8),

   1-(3-(3-(4-methylphenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (9),

   1-(3-(3-(4-fluorophenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (10),

   1-(3-(3-(4-chlorophenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (11),

   1-(3-(3-(4-Trifluoromethylphenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (12),

   1-(3-(3-(2,4-Difluorophenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (13),

   1-(3-(3-(2-Fluoro-4-chlorophenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (14),

   1-(3-(3-(3-Trifluoromethylphenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (15),

   1-(3-(3-(4-chloro-3-trifluoromethylphenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (16),

   1-(3-(3-(3,4-Dichlorophenyl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (17),

   1-(3-(3-(3-Methylisoxazol-5-yl)ureido)-4-(diisobutylamino)phenyl)-1H-pyrrole-2-carboxylic acid (18),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(4-methylphenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (19),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(4-methoxyphenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (20),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(4-fluorophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (21),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(4-chlorophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (22),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(4-bromophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (23),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(4-cyanophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (24),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(2-chlorophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (25),

   1-(3-Chloro-4-(diisobutylamino)-5-(3-(2,4-difluorophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (26),

   1-(3-chloro-4-(diisobutylamino)-5-(3-(2-fluoro-4-chlorophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (27),

   1-(3-chloro-4-(diisobutylamino)-5-(3-(2-fluoro-4-cyanophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (28) ,

   1-(3-chloro-4-(diisobutylamino)-5-(3-(3-fluoro-4-chlorophenyl)ureido)phenyl)-1H-pyrrole-2-carboxylic acid (29),

   1-(3-(3-(4-methylphenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (30),

   1-(3-(3-(4-methoxyphenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (31),

   1-(3-(3-(4-Fluorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (32),

   1-(3-(3-(4-chlorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (33),

   1-(3-(3-(4-Bromophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (34),

   1-(3-(3-(4-cyanophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (35),

   1-(3-(3-(2-Fluorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (36),

   1-(3-(3-(2-chlorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (37),

   1-(3-(3-(2,4-Difluorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (38),

   1-(3-(3-(2-Fluoro-4-chlorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (39),

   1-(3-(3-(2-Fluoro-4-bromophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (40),

   1-(3-(3-(2-Fluoro-4-cyanophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (41) ,

   1-(3-(3-(2,4-Dichlorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (42),

   1-(3-(3-(3-Fluoro-4-chlorophenyl)ureido)-4-((isobutyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (43),

   1-(3-(3-(3-Trifluoromethylphenyl)ureido)-4-((methyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (44),

   1-(3-(3-(4-Chloro-3-trifluoromethylphenyl)ureido)-4-((methyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (45 ),

   1-(3-(3-(3,4-Dichlorophenyl)ureido)-4-((methyl)cyclohexylamino)phenyl)-1H-pyrrole-2-carboxylic acid (46).
6. A preparation method of pyrrole compounds according to claim 1, wherein the preparation method is any one of the following methods:

   Method 1: Using substituted nitrobenzene as raw material, reacting with amine compound HNR ³ R ⁴ under the action of alkali to prepare intermediate i, i and pyrrole-2-carboxylate are reacted with Ullmann to prepare intermediate ii, ii Intermediate iii is obtained by reduction, and ^{compound iv is obtained by condensation of iii with substituted benzene isocyanate R 5} NCO, or iii firstly forms active intermediate with 4-nitrophenyl chloroformate, and then reacts with amine compound R ⁵ NH ₂ The target compound iv is prepared; iv is hydrolyzed to obtain the target compound v; v is reacted with oxalyl chloride or thionyl chloride to form acid chloride and then reacted with the amine compound HNR ⁷ R ⁸ to obtain the target compound vi; vi is dehydrated at high temperature Obtain the target compound vii;

   

   Wherein, R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are as defined in claim 1;

   Method 2: Using 2-fluoro-5-bromonitrobenzene as the raw material, reacting with the amine compound HNR ³ R ⁴ under the action of a base to obtain intermediate i, and obtaining intermediates viii, viii and pyrrole via NCS chlorination reaction. -2-carboxylic acid ester is reacted by Ullmann to obtain intermediate ix, ix is reduced by reducing agent to obtain intermediate x, x is ^{condensed with substituted phenyl isocyanate R 5} NCO to obtain intermediate xi, and xi is hydrolyzed to obtain target compound xii;

   

   Wherein, R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in claim 1.
7. A pharmaceutical composition, characterized in that, the pharmaceutical composition includes a therapeutically effective amount of active components and pharmaceutically acceptable auxiliary materials; the active components include any one of claims 1-5 The pyrrole compound, its metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof.
8. A pyrrole compound, its metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to any one of claims 1-5 are used in the preparation of indoleamine 2,3- Application of dioxygenase 1 inhibitor drugs.
9. A pyrrole compound, its metabolites, metabolic precursors, prodrugs, solvates, crystals or pharmaceutically acceptable salts thereof according to any one of claims 1-5 are used in the preparation of indoleamine 2,3 -Use of dioxygenase 1-mediated immunosuppressive drugs for related diseases.
10. The use according to claim 8 or 9, wherein the medicament is used to treat cancer, viral infection, neurodegenerative disease, cataract, organ transplant rejection, depression or autoimmune disease.

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