CN109180649B

CN109180649B - IDO inhibitor containing indole ring and preparation method thereof

Info

Publication number: CN109180649B
Application number: CN201810945673.2A
Authority: CN
Inventors: 钟刚; 李�杰; 何桂佳; 杨秀娟; 李志勇
Original assignee: Sichuan Baili Pharmaceutical Co Ltd
Current assignee: Sichuan Baili Pharmaceutical Co Ltd
Priority date: 2017-08-18
Filing date: 2018-08-18
Publication date: 2021-03-12
Anticipated expiration: 2038-08-18
Also published as: CN109180649A; WO2019034179A1

Abstract

The invention discloses an IDO inhibitor containing indole ring and a preparation method thereof, and a medicament taking the compound as an active ingredient can be used for treating immune activation, inflammatory diseases, cardiovascular diseases and the like related to indoleamine-2, 3-dioxygenase activity.

Description

IDO inhibitor containing indole ring and preparation method thereof

Technical Field

The invention relates to tetrazole phenyl indole derivatives, and a medicament taking the compounds as active ingredients can be used for treating immune activation, inflammation diseases, cardiovascular diseases and the like related to indoleamine-2, 3-dioxygenase activity.

Background

Tryptophan, an essential amino acid in the human body, has two decomposition pathways: the 5-hydroxytryptamine pathway (about 5%) and the kynurenine pathway (about 95%) (Neuroch em Res,1980,5(3): 223-. Indoleamine-2, 3-dioxygenase (IDO), which is widely present in mammalian tissue cells other than the liver, and tryptophan-2, 3-dioxygenase (TDO), which is mostly expressed in the liver, are the rate-limiting enzymes of the tryptophan/kynurenine pathway.

The indoleamine-2, 3-dioxygenase is the only enzyme except the liver which can catalyze the epoxidation and the cleavage of indole in tryptophan molecules, so that the indole can be decomposed into various metabolites such as L-kynurenine, picolinic acid, quinolinic acid and the like along the kynurenic acid pathway. The expression of this enzyme and its related metabolites is distributed mainly in the medulla of the thymus and the T cell region of the secondary lymphoid organs and is interspersed among several immune-tolerant or immune-privileged tissues, such as the thymus, the gastrointestinal mucosa, the placenta, etc.

Under normal physiological conditions, IDO and TDO protect tissues from an overstimulated immune system through immunosuppression. INF-gamma generated by the activated T cells induces the expression and activation of indoleamine-2, 3-dioxygenase, thereby negatively feeding back and regulating CD4+ T cells, CD8+ T cells and NK cells. However, IDO and TDO have higher expression in various tumor tissues, and the high expression of IDO and TDO is one of the reasons for causing tumor immune tolerance and is closely related to the poorer prognosis of tumor patients. Both IDO and TDO can inactivate tumor surveillance by the immune system and prevent tumor rejection. Tumor cells and specific types of immune cells use this mechanism to limit the anti-tumor immune response.

Studies show that the inhibition of indoleamine-2, 3-dioxygenase can regulate the immunosuppressive function, thereby achieving the effect of inhibiting tumor growth (nat. ReV. Immunol 2004,4, 762-74; nat. ReV. cancer,2006,6, 613-.

Numerous studies have demonstrated that IDO is also associated with inflammation, cardiovascular disease, degenerative diseases of the nervous system, psychiatric diseases, viral infections, autoimmune diseases and other chronic diseases associated with tryptophan degradation.

However, most of the IDO small molecule inhibitors currently entering clinical research stage have only micromolar inhibition on IDO (such as Indoximod of new link Genetics), and poor oral bioavailability (such as Epacadostat of Incyte Corporation), so that drugs with better inhibition and bioavailability are needed to meet the current clinical needs.

Meanwhile, the action mechanisms of the IDO small-molecule inhibitors currently entering clinical research are different, and Epacadostat has affinity to IDO holoenzyme; indoximod can relieve the function inhibition of T cells caused by the activity reduction of mTORC1 due to tryptophan deletion; navoximod has an inhibitory effect on tumors simultaneously expressing IDO 1/TDO; PF-06840003 is a noncompetitive inhibitor of tryptophan and does not bind to the cofactor heme of the IDO enzyme; BMS-986205 was inhibited by binding to cofactor-free apo-IDO 1.

Definition of

The formulae representing chemical structures in the terms used in the present invention may contain a single dash "-", a double dash "-", or a symbol, as is customary in the art

For chemical elements or chemical bond structures between named substituents and their parent moieties: a single dash "-" represents a single bond, a double dash "-" represents a double bond, and the symbols

Represents a bond as the point of attachment of a moiety or substituent to the core or nucleus of a compound structure. If no single or double dash is present in the formula representing the chemical structure, it is understood that a single bond is formed between the substituent and its parent moiety.

Hetero (atom, alkyl, aryl, cyclic) refers to the corresponding chemical structure containing atoms other than carbon, as is standard practice in the art.

Disclosure of Invention

The invention provides a tetrazole phenyl indole derivative which is an indoleamine-2, 3-dioxygenase inhibitor and has the main effect of playing a role in regulating immune system functions and inflammatory factors by inhibiting the activity of indoleamine-2, 3-dioxygenase, and particularly relates to a compound with a structural formula (I):

and/or stereoisomers, tautomers, or pharmaceutically acceptable salts, solvates thereof, wherein:

R¹is optionally substituted straight chain or branched chain alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl with or without heteroatom, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic radical, substituted or unsubstituted alkanoyl, substituted or unsubstituted amido, substituted or unsubstituted amidoalkyl.

R²Selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, and substituted or unsubstituted alkynyl.

A is a carbon chain with or without heteroatoms and has the structure of- (CH)₂)_n-, where n is 0 to 5; or-NH (CH)₂)_n-n is 0-5, wherein the NH terminus is attached to the indole ring.

R³Is substituted or unsubstituted aromatic group, and the aromatic group can be a full carbon skeleton or a skeleton containing one or more heteroatoms such as oxygen, sulfur, nitrogen and the like; wherein the substitution on the aryl group can be mono-or polysubstituted, and the substituents are independently selected from the group consisting of halogen, hydroxy, amino, nitro, cyano, trifluoromethyl, azido, sulfonyl, sulfonamido, substituted or unsubstituted alkylA group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted heteroalkyl group, C₁-C₈Substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkanoyl, substituted or unsubstituted amido, substituted or unsubstituted amidoalkyl.

Wherein said substituents are independently selected from the group consisting of halogen, hydroxy, amino, nitro, cyano, trifluoromethyl, azido, sulfonyl, acyl.

Wherein said alkyl is C₁-C₈Substituted or unsubstituted alkanyl, - (CH)₂)_n-Ar-、-(CH₂)_n-Cy-, wherein Ar is substituted or unsubstituted aryl or heteroaryl and Cy is substituted or unsubstituted cycloalkyl or heterocycloalkyl.

Wherein said alkenyl is C containing one or more- (C ═ C) -, group₂-C₈A chain alkyl group.

Wherein said alkynyl is C containing one or more- (C.ident.C) -₂-C₈A chain alkyl group.

Wherein said heteroalkyl is substituted or unsubstituted C containing one or more atoms other than carbon (oxygen, sulfur or nitrogen atoms)_1-C₈A chain alkyl group.

Wherein said alkoxy is-O-R⁴Wherein R is⁴Is a linear or branched substituted or unsubstituted C₁-C₈Alkyl, alkenyl or alkynyl.

The aromatic group is a skeleton which can be an all-carbon skeleton or a skeleton containing one or more heteroatoms such as oxygen, sulfur, nitrogen and the like, and can be provided with one or more substituents, wherein in some cases, any two adjacent substituents can form C₁-C₆Cyclic structures with or without heteroatoms (oxygen, sulfur or nitrogen).

Wherein said cycloalkyl is C_3-C₇A carbocyclic group.

Wherein said heterocyclic group is a substituted or unsubstituted C containing one or more atoms other than carbon (oxygen, sulfur or nitrogen atoms)_3-C₇A carbocyclic group.

Wherein said alkanoyl is- (C ═ O) -R⁵Wherein R is⁵Is a linear or branched substituted or unsubstituted C_1-C₇An alkyl group.

Wherein said amide group is- (C ═ O) -NH-R⁶Wherein R is⁶Is hydrogen, linear or branched, substituted or unsubstituted C_1-C₇An alkyl group.

Wherein said amidoalkyl is-R⁷-(C＝O)-NH-R⁸Wherein R is⁷Is a linear or branched substituted or unsubstituted C_1-C₇Alkyl radical, R⁸Is hydrogen, linear or branched, substituted or unsubstituted C_1-C₅An alkyl group.

Wherein said sulfonyl is substituted or unsubstituted C_1-C₆An alkylsulfonyl group.

The invention provides a compound (I) characterized in that R³Is a substituted or unsubstituted phenyl group or a six-membered aromatic group containing one or more heteroatoms, wherein the heteroatoms are N, O or S.

In certain embodiments, the present invention provides compounds of structural formula (II):

R²Selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstitutedSubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkynyl.

n＝0～5。

R³Is substituted or unsubstituted aromatic phenyl or six-membered aromatic group containing one or more hetero atoms (nitrogen, oxygen or sulfur), wherein the substitution on the aromatic group can be one or more, and the substituents are independently selected from halogen, hydroxyl, amino, nitro, cyano, trifluoromethyl, azido, sulfonyl, sulfonamide, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, C₁-C₈Substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkanoyl, substituted or unsubstituted amido, substituted or unsubstituted amidoalkyl.

Wherein the aromatic group may be an all-carbon skeleton or contain one or moreA skeleton containing hetero atoms such as oxygen, sulfur, nitrogen and the like, which may have one or more substituents, and in some cases, any two adjacent substituents may form C₁-C₆Cyclic structures with or without heteroatoms (oxygen, sulfur or nitrogen).

Wherein said cycloalkyl is C_3-C₇A carbocyclic group.

In certain embodiments, the present invention provides compounds of structural formula (III):

R¹is optionally substituted straight chain or branched chain alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl with or without hetero atoms, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic radical, substituted or unsubstituted alkanoyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted orSubstituted or unsubstituted amide group, substituted or unsubstituted amide alkyl group, the aforementioned hetero atom being oxygen, sulfur or nitrogen atom.

n＝0～5。

Wherein said cycloalkyl is C_3-C₇A carbocyclic group.

The invention surprisingly discovers that the tetrazole phenyl indole derivatives can regulate the IDO bioactivity through inhibiting IDO enzyme in biological cells, and can generate synergistic action with IDO inhibitors with other action mechanisms due to slight difference of action mechanisms, thereby meeting the current demand on IDO regulators.

Synthesis method

The invention isIn the related compound (II), when n ═ 0, 4-bromo-2-iodoaniline and R may be used²The substituted ethyl acylacetate is subjected to construction of an indole skeleton, and then is subjected to alkyl substitution of hydrogen on N, amidation reaction and suzuki-Miyaura reaction in sequence, and the synthetic route is shown as the following formula:

in the compound (II), when N is 0, the target compound may be prepared by the suzuki reaction and the amidation reaction in this order after the alkyl substitution on indole N:

after cyclohexylamine and ethyl acetoacetate are coupled, an indole ring is constructed by p-benzoquinone, and then a compound (II) (wherein n is 0):

in the compound (II), when n is more than or equal to 1, an indole acid intermediate can be prepared into acyl chloride, carboxylic acid with one carbon chain increased is obtained through an Arndt-Eister reaction, condensation is carried out on the carboxylic acid and corresponding amine, and then tetrazolylphenyl is coupled through Suzuki reaction to obtain a target product; or after an indole ring is constructed, synthesizing aldehyde by reducing and oxidizing ester, obtaining carboxylic acid with 2-5 carbons through ylide reaction, and obtaining a target compound through amidation and Suzuki reaction, wherein the synthetic route is shown as the following formula:

in the compound (III), when n is 0, an indole ring can be constructed by coupling cyclohexylamine and ethyl acetoacetate and p-benzoquinone, and then the indole ring is obtained by Suzuki reaction and Kraus rearrangement in sequence, wherein the synthetic route is shown as the following formula:

in the compound (III), when n is 0, 4-bromo-2-iodoaniline and ethyl acetoacetate can be used for constructing an indole skeleton, and then the indole skeleton is connected with a tetrazolyl phenyl segment through a suzuki reaction, and a urea bond is constructed through a krusei rearrangement, so that the compound (III) is obtained, and the synthetic route is shown as the following formula:

in the compound (III), when n is 1-5, an indole carboxylate intermediate is hydrolyzed and subjected to Kraus rearrangement to generate amine, the amine is connected with bromo-ester containing linear chains of methylene with different lengths, the ester is hydrolyzed and then coupled with corresponding amine, and finally the compound is obtained through Suzuki reaction, wherein the synthetic route is shown as the following formula:

application method

The compounds of the present invention modulate the activity of indoleamine-2, 3-dioxygenase (IDO). "modulation" as referred to above refers to the ability to increase or decrease the activity of an enzyme or receptor. The compounds described herein can be used in methods of modulating IDO by contacting an enzyme with any one or more of the compounds or compositions described herein. In some embodiments, the compounds described herein may act as inhibitors of IDO. In a further embodiment, the compounds described herein may be used to modulate the IDO activity of a cell or subject in need of modulation of the enzyme by administering a modulating (e.g., inhibiting) dose of a compound described herein.

The present invention provides methods for altering (e.g., increasing) extracellular tryptophan levels in a mammal in a system (e.g., tissue, cell, or culture of a living organism) containing IDO expressed by the cells, comprising administering an effective amount of a compound or composition provided herein. Methods for determining tryptophan levels and tryptophan degradation are conventional in the art.

The present invention provides methods of inhibiting immunosuppression (e.g., IDO-mediated immunosuppression) in a subject by administering to the subject an effective amount of a compound or composition described herein. IDO-mediated immunosuppression is associated with cancer, tumor growth, metastasis, infectious diseases (e.g., viral infections), viral replication, and the like.

The present invention treats a disease associated with aberrant IDO activity or expression in an individual (e.g., a patient) by administering to the patient an effective amount of a compound or moiety described herein. Diseases include any disease, disorder or condition associated directly or indirectly with aberrant IDO enzyme expression or activity. Examples of diseases related to IDO include cancer, neurodegenerative disorders (e.g., alzheimer's disease, huntington's chorea, etc.), viral infections (e.g., HIV infection, etc.), depression, trauma, cataracts, autoimmune diseases (e.g., rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, systemic lupus erythematosus, inflammatory bowel disease, etc.), organ transplantation (e.g., organ transplant rejection, etc.).

Examples of tumor-specific immunosuppression associated with cancer that may be treated by the methods of the present invention include immunosuppression associated with colon, pancreatic, breast, prostate, lung, brain, ovarian, cervical, testicular, renal, head and neck cancers, lymphomas, leukemias, melanomas, and the like.

The IDO-mediated immunosuppression associated with viral infections described herein is associated with viral infections selected from the group consisting of: hepatitis C Virus (HCV), Human Papilloma Virus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), poliovirus, varicella zoster virus, Coxsackie virus, Human Immunodeficiency Virus (HIV).

In another embodiment, IDO-mediated immunosuppression associated with infectious disease is associated with tuberculosis or leishmaniasis.

For example, a patient undergoing or having completed a course of chemotherapy and/or radiation therapy for a disease state (e.g., cancer) may benefit from administering to the patient a therapeutically effective amount of a compound or composition of the present invention to inhibit immunosuppression due to the disease state and/or treatment thereof.

Further provided are methods of treating a disease associated with IDO activity or expression (including aberrant activity and/or overexpression) in an individual by administering to the individual (e.g., a patient) in need of such treatment a therapeutically effective amount of a compound of the present invention or a pharmaceutical composition thereof. Examples of diseases may include any disease, disorder or condition that is directly or indirectly associated with the expression or activity of an IDO enzyme, such as an overexpression or an activity abnormality. IDO-related diseases may also include any disease, disorder or condition that can be prevented, alleviated or cured by modulating the activity of an enzyme.

As used herein, "contacting" refers to associating a specified moiety with an in vitro system or an in vivo system. For example, "contacting" an IDO enzyme with a compound or component described herein includes administering a compound of the invention to an individual or patient (e.g., a human) having IDO, or introducing a compound of the invention into a sample of cells or purified preparations containing IDO enzyme.

As used herein, the term "individual" or "patient" refers to any animal or human, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

As used herein, a "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, subject, or human that is being sought by a researcher, physician, or veterinarian, and includes one or more of preventing a disease, ameliorating a disease, inhibiting a disease, and the like.

Combination therapy

The compounds of the present invention may be used in combination with one or more other therapeutic agents, such as antiviral, chemotherapeutic or other anticancer agents, immunopotentiators, immunosuppressive agents, radiation, antitumor and antiviral vaccines, cytokine therapy (e.g., IL2, GM-CSF, etc.), and/or tyrosine kinase inhibitors, to treat IDO-related diseases, disorders or conditions (as described above) or to enhance the efficacy of treatment of disease states or conditions (e.g., cancer). These agents may be used in combination with the compounds of the present invention in one dosage form, or the agents may be administered in different dosage forms, either simultaneously or sequentially.

Suitable antiviral drugs contemplated for use in combination with the compounds of the present invention may include nucleoside and Nucleotide Reverse Transcriptase Inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and other antiviral drugs.

Examples of suitable nucleoside reverse transcriptase inhibitors include, but are not limited to, zidovudine (AZT); didanosine (ddI); zalcitabine (ddC); stavudine (d 4T); lamivudine (3 TC); abacavir (1592U 89); adefovir dipivoxil [ bisacrylamide (POM) -PMEA ]; lobbucavir (BMS-180194); BCH-10652; emtricitabine [ (-) -FTC ]; β -L-FD4 (also known as β -L-D4C and by the name β -L-2 ', 3' -dideoxy-5-fluoro-cytidine); DAPD ((-) - β -D-2, 6-diamino-purine dioxolane); and lodenosine (FddA). Typical suitable non-nucleoside reverse transcriptase inhibitors include, but are not limited to, nevirapine (BI-RG-587); delavirdine (BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442[1- (ethoxy-methyl) -5- (1-methylethyl) -6- (benzyl) - (2,4- (1H,3H) -pyrimidine-dione ]; and (+) -calanolide a (NSC-675451) and b. typical suitable protease inhibitors include, but are not limited to, saquinavir (Ro 31-8959); ritonavir (ABT-538); indinavir (MK-639); nelfinavir (AG-1343); amprenavir (141W 94); lacinavir (BMS-234475); DMP-450; BMS-2322623; ABT-378; and AG-1549 other antiviral drugs include, but are not limited to, hydroxyurea, ribavirin, IL-2, IL-12, pentafusi, and Yissum (item No. 11607).

Suitable chemotherapeutic or other anti-cancer drugs include, but are not limited to, alkylating agents (including without limitation nitrogen mustards, aziridine derivatives, alkyl sulfonates, nitrosoureas, and triazenes), such as uramustine, pipobroman, cyclophosphamide (cytoxan (tm)), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide; antimetabolites (including without limitation, f-folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors), such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine; some natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, daunorubicin, cytarabine, paclitaxel (taxol), mithramycin, desoxymesendomycin, mitomycin-C, levo asparaginase, interferons, etoposide, and teniposide.

Suitable other cytotoxic drugs include, but are not limited to, noviban, CPT-11, anastrozole, letrozole, capecitabine, raloxifene, cyclophosphamide, ifosfamide and droloxifene.

The following cytotoxic drugs (including the same mechanism) are also suitable: such as epiphyllotoxin; a tumor enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum complexes such as cisplatin and carboplatin; a biological response modifier; a growth inhibitor; an anti-hormone therapeutic agent; calcium folinate; tegafur; and hematopoietic growth factors.

Suitable additional anti-cancer drugs include antibody therapies such as trastuzumab (herceptin), costimulatory molecule antibodies such as CTLA-4, 4-1BB and PD-1 or cytokine antibodies (IL-10, TGF- β, etc.); drugs that block migration of immune cells, such as chemokine receptor antagonists (CCR2, CCR4, and CCR6), and the like; drugs that enhance the immune system, such as helper or adaptive T cell transfer.

Suitable anti-cancer vaccines include dendritic cells, synthetic peptides, DNA vaccines and recombinant viruses.

Those skilled in the art are aware of methods for safely and effectively administering most of these chemotherapeutic agents. In addition, their administration is generally described in the standard literature. For example, many methods of administration of chemotherapeutic agents are described in the "Physicians' Desk Reference" (PDR, e.g., 1996 edition, Medical Economics Company, Montevir, N.J.), disclosing that this document is incorporated by Reference herein as if listed in its entirety.

Pharmaceutical formulations and dosage forms

When the compound of the invention is used as a medicament, the compound can be administered in the form of a pharmaceutical composition which is a combination of the compound of the invention and a pharmaceutically acceptable carrier. These compositions can be prepared in a manner well known in the pharmaceutical art and administered by various routes depending on whether the composition is to be used for local or systemic treatment and the area of treatment. Administration may be topical (including ophthalmic and intranasal, vaginal and rectal mucocutaneous administration), pulmonary (e.g., by inhalation or insufflation of powders or aerosols via nebulizer, intratracheal, intranasal, epidermal and transdermal routes), ocular, oral or parenteral.

Methods of ocular administration may include topical administration (eye drops), subconjunctival, periocular or intravitreal injection, introduction of ocular inserts placed in the conjunctival sac by balloon catheter or surgery, and the like.

Methods of parenteral administration include intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Parenteral administration may be in the form of a single bolus dose or may be by means of a continuous infusion pump or the like.

Methods of topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, creams, powders, liquids and powders. It may be necessary or desirable to use conventional pharmaceutical carriers, water, powder or oil bases, thickeners, and the like.

The pharmaceutical compositions of the present invention may contain one or more of the compounds of the present invention described above as active ingredients in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the present invention, the active ingredient is typically mixed with the adjuvant, diluted by the adjuvant or contained within a carrier in the form of a capsule, sachet, paper or other container. When the adjuvant acts as a diluent, the adjuvant may be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. The compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or liquid medium), ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In making the formulations, the active compounds of the present invention may be ground to a powder to provide the appropriate particle size prior to combination with the other ingredients. If the active compound is substantially insoluble, it may be milled to a particle size of less than 200 mesh. If the active compound is soluble in water, the particle size may be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.

Some examples of excipients for use in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Formulation formulations may also include, but are not limited to: lubricants (e.g., talc, magnesium stearate, mineral oil, etc.), wetting agents, emulsifying and suspending agents, preservatives (e.g., methyl and propyl parabens, etc.), sweetening agents, and flavoring agents. The compositions of the present invention may be formulated using procedures known in the art such that the active ingredient is released rapidly, slowly or with a delay after administration to the patient.

The compositions of the present invention may also be formulated in unit dosage forms containing from about 5 to about 200mg, more typically from about 10 to about 100mg, of the active ingredient per dose. The "unit dosage form" described above refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect.

The active compounds of the present invention may be effective in a variety of dosage forms and are generally administered in a pharmaceutically effective amount. It is to be expressly understood that the actual amount of the compound administered will usually be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

In preparing solid compositions, such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a preformulation composition containing a homogeneous mixture of the compounds of the present invention. By "homogeneous" is meant that the active ingredient is generally uniformly dispersed throughout the composition so that the composition is readily subdivided into equivalent unit dosage forms, such as tablets, pills, capsules, and the like. This solid pre-formulation is then subdivided into unit dosage forms of the type described above containing, for example, from 0.1 to about 500mg of the active compound of the present invention.

The tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, a tablet or pill may contain an inner dose and an outer dose component, the latter being in the form of a capsule of the former. The two components may be separated by an enteric layer which serves to prevent disintegration in the stomach and allow the inner component to pass intact through the duodenum or to be delayed in release. A variety of materials may be used for the enteric layer or coating, including but not limited to various polymeric acids and mixtures of polymeric acids with shellac, cetyl alcohol and cellulose acetate.

Liquid forms that may be added to the compounds and compositions of the present invention for oral or injectable administration include: aqueous solutions, suitably flavored syrups, aqueous or oily suspensions and flavored emulsions with edible oils (e.g., cottonseed oil, sesame oil, coconut oil or peanut oil), elixirs and similar pharmaceutical vehicles and the like.

Compositions for inhalation or insufflation include dissolving a compound of the present invention in a pharmaceutically acceptable solution, suspension, water or organic solvent, or mixtures and powders thereof. Suitable pharmaceutically acceptable excipients may be included in the liquid or solid compositions. In some embodiments, the composition is administered by the oral or nasal respiratory route to produce a local or systemic effect. The composition may be atomized using an inert gas. Nebulized solutions can be inhaled directly by nebulizing devices, or by connecting the nebulizing device to a face mask or intermittent positive pressure ventilator. The solution, suspension or powder compositions may be administered orally or nasally using equipment for delivering the formulation in a suitable manner.

The amount of the compound or composition administered to a patient varies depending on the administration component, the purpose of administration (e.g., prevention or treatment), the state of the patient, the mode of administration, and the like. In therapeutic applications, the compositions may be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. The effective dosage will depend on the condition being treated and on the judgment of the attending physician in light of such factors as the severity of the condition, the age, weight and general condition of the patient.

The composition administered to the patient may be in the form of a pharmaceutical composition as described above. These compositions may be sterilized or sterile filtered by conventional sterilization techniques. The aqueous solutions can be packaged for use as is or as a lyophilized formulation for use after mixing with a sterile aqueous carrier prior to administration. The pH of the compound formulation is generally between 3 and 11, more preferably 5 to 9 and most preferably 7 to 8. It will be appreciated that the use of some of the excipients, carriers or stabilizers described above will result in the drug being present in the form of a salt.

The therapeutic dosage of the compounds of the present invention may vary depending upon the particular use being treated, the mode of administration of the compound, the health and condition of the patient, the judgment of the prescribing physician, and the like. The proportion or concentration of the compounds of the invention in the pharmaceutical composition may vary depending on various factors, including dosage, chemical characteristics (e.g., hydrophobicity), route of administration, and the like. For example, the compounds of the present invention may be provided in a physiologically buffered aqueous solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dosage ranges are between about 1. mu.g/kg to about 1g/kg body weight per day. In some embodiments, the dosage range is between about 0.01mg/kg to about 100mg/kg body weight per day. The dosage may depend on such factors as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the adjuvant, and the route of administration. Effective doses can be extrapolated from dose-response curves from in vitro or animal model test systems.

The compounds of the present invention may also be formulated in combination with one or more other active ingredients, which may include any drug, such as antiviral drugs, vaccines, antibodies, immunopotentiators, immunosuppressive agents, anti-inflammatory agents, and the like.

Labeled compound and measurement method

Another aspect of the invention relates to fluorescent dyes, spin labels, heavy metals or radiolabeled derivatives of said compounds, which are useful not only in imaging but also in vitro and in vivo assays to locate and quantify IDO enzymes in tissue samples including humans and to identify IDO enzyme ligands by inhibiting binding to labeled compounds. The invention further provides for IDO enzyme assays comprising such labeled compounds.

The invention further provides isotopically-labelled compounds of said compounds. An "isotopically labeled" or "radio-labeled" compound is a compound of the present invention in which one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (i.e., present in nature). Suitable radionuclides include, but are not limited to²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、¹⁸F、³⁵S、³⁶Cl、⁸²Br、⁷⁵Br、⁷⁶Br、⁷⁷Br、¹²³I、¹²⁴I、¹²⁵I and¹³¹I. the radionuclide that is included in the radiolabeled compound will depend on the particular application of the radiolabeled compound. For example, in vitro IDO enzyme labeling and competitive detection are generally used with³H、¹⁴C、⁸²Br、¹²⁵I、¹³¹I or³⁵A compound of S. For radiographic applications, the general use includes¹¹C、¹⁸F、¹²⁵I、¹²³I、¹²⁴I、¹³¹I、⁷⁵Br、⁷⁶Br or⁷⁷A compound of Br.

The above-mentioned "radiolabel" or "labelled compound" is a compound comprising at least one radionuclide. In some embodiments, theRadionuclide is selected from the group consisting of:³H、¹⁴C、¹²⁵I、³⁵s or⁸²Br。

Methods of incorporating radioisotopes into organic compounds are suitable for use with the compounds of the present invention and are well known in the art.

The radiolabeled compounds of the invention may be used in screening assays to identify or evaluate compounds. In general, a newly synthesized or discovered compound (i.e., test compound) can be evaluated for its ability to reduce binding of the radiolabeled compound of the invention to the IDO enzyme. The ability of the test compound to compete with the radiolabeled compound for binding to the IDO enzyme is directly related to its binding affinity.

Medicament box

The invention also includes kits for treating diseases, such as treating or preventing diseases or disorders associated with IDO, obesity, diabetes, and other diseases to which the invention relates. Such a kit is a container containing one or more pharmaceutical compositions of the present invention in a therapeutically effective amount. Such kits may also include one or more of a variety of conventional kit components as appropriate, e.g., containers containing one or more pharmaceutically acceptable carriers, other containers readily apparent to those skilled in the art, and the like. The kit may also include instructions as an insert or label indicating the amount of compound to be administered, directions for administration, and/or directions for mixing the compounds.

General abbreviations and symbols

g: keke (Chinese character of 'Keke')

mg: milligrams of

mL: milliliter (ml)

mol: mole of

DEG C: degree centigrade

BINOL: 1,1' -binaphthol

BOP: benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphates

DCM: methylene dichloride

DMAP: 4-dimethylaminopyridine

DMF: dimethyl formamide

DMSO, DMSO: dimethyl sulfoxide

DPBS: du's phosphate buffer

EA: ethyl acetate

EDTA: ethylenediaminetetraacetic acid

H₂O: water (W)

LDA: lithium diisopropylamide

NBS: n-bromosuccinimide

min: minute (min)

PCC: pyridinium chlorochromate

SOCl₂: thionyl chloride

TEA: triethylamine

THF: tetrahydrofuran (THF)

TLC: thin layer chromatography

Trypan Blue: trypan blue

Trypsin: trypsin

Drawings

FIG. 1 is a graph showing the antitumor effect of a compound.

Detailed Description

Reagents and solvents were purchased from commercial sources, the reaction was monitored on a 0.25mm HSGF254 thin layer silica gel plate and flash column chromatography was performed on 200-300 mesh particle size thin layer silica gel. NMR spectroscopy was performed using a Bruker Avance III NMR spectrometer. And carrying out mass spectrum detection by using Agilent liquid chromatography devices 1260, 6110 and 1260, 6120. All reactions were magnetically stirred at ambient temperature unless otherwise indicated.

The following specific examples are provided only for illustrating the present invention in detail and are not to be construed as limiting the present invention in any way. Those skilled in the art will readily recognize that various noncritical parameters may be altered or adjusted to produce approximately the same results. The compounds of the following examples were found to be IDO inhibitors according to one or more of the assays described herein.

Example 1: 5- (2- (1H-tetrazol-5-yl) phenyl) -N- (4-methylphenyl) -1-isobutyl-2-methyl-1H-indole-3-carboxamide

1A, 5-bromo-2-methyl-1H-indole-3-carboxylic acid ethyl ester

4-bromo-2-iodoaniline (10g, 33.57mmol), ethyl acetoacetate (6.55g, 50.35mmol), cuprous iodide (640mg, 3.36mmol), BINOL (1.922g, 6.71mmol), cesium carbonate (10.94g, 33.57mmol) and DMSO (60mL) were added to the reaction flask. The reaction was stirred with heating to 55 ℃ and monitored by TLC. After the reaction, the reaction mixture was diluted with a large amount of water, extracted with EA (200mL × 4), the organic phases were combined, washed with a large amount of clear water to remove residual DMSO, washed with saturated brine, dried over anhydrous sodium sulfate, and the reaction mixture was concentrated to give a crude brown oil, which was purified by silica gel column chromatography using a solvent mixture of EA and hexane as an eluent to give 3.12g, yield 21%.¹H NMR(400MHz，CDCl₃)δppm：8.41(s,1H)；8.10(d,1H)； 7.14(d,1H)；4.40(q,2H)；2.38(s,3H)；1.39(t,3H)。LCMS(ES-API)：m/z＝282[M+H]⁺

1B, 5-bromo-1-isobutyl-2-methyl-1H-indole-3-carboxylic acid ethyl ester

Compound 2A (3g, 11.5mmol) and DMF (50mL) were added to the reaction flask, the temperature of the reaction solution was allowed to decrease with an ice-water bath, sodium hydride (1.1g, 46mmol), 1-bromo-2-methylpropane (6.3g, 46mmol) and potassium iodide (100mg) were added, the ice-water bath was removed, the temperature was raised to 50 ℃ for reaction, and TLC was used for monitoring. After the reaction, adding water to the reaction solution to quench the reaction, adding a large amount of water to dilute the reaction solution, extracting the reaction solution by EA (150 mL multiplied by 4), combining organic phases, washing DMF (dimethyl formamide) by a large amount of clear water, washing the organic phases by saturated saline, drying the organic phases by anhydrous sodium sulfate, concentrating the reaction mixture to obtain yellow oily matter, and dissolving the yellow oily matter by using EA and hexane as solventsThe mixture was purified by silica gel column chromatography using eluent to give 2.85g of product in 79.3% yield.¹H NMR(400MHz，CDCl₃)δppm：7.86(d,1H)；7.63(s,1H)；7.34(m,2H)；7.18 (d,1H)；4.40(q,2H)；4.13(d,2H)；2.71(s,3H)；2.15(m,1H)；1.39(t,3H)；1.01(d,6H)。LCMS(ES-API)： m/z＝338[M+H]⁺

1C, 5-bromo-1-isobutyl-2-methyl-1H-indole-3-carboxylic acid

Compound 2B (800mg, 2.37mmol) and 50% potassium hydroxide solution (15mL, methanol: water ═ 4:1v/v) were added to the reaction flask, warmed to reflux and monitored by TLC. After the reaction, the reaction solution was cooled to room temperature, the pH was adjusted to 5-6 with 10N hydrochloric acid, and a large amount of white solid was precipitated, extracted with EA (100 mL. times.3), the organic phases were combined, washed with water, saturated brine, dried over anhydrous sodium sulfate, and the reaction mixture was concentrated to give 760mg of white solid.¹H NMR(400MHz，CDCl₃)δppm：11.2(s, 1H)；7.97(d,1H)；7.49(m,2H)；4.23(d,2H)；2.81(s,3H)；2.25(m,1H)；1.11(d,6H)。LCMS(ES-API)： m/z＝308[M-H]^-

1D, 5-bromo-N- (4-methylphenyl) -1-isobutyl-2-methyl-1H-indole-3-carboxamide

Compound 1C (400mg, 1.29mmol) was added to the reaction flask, cooled in an ice-water bath and then thionyl chloride (6mL) was added, stirred for reaction and monitored by TLC. After the reaction, the reaction mixture was concentrated. The residue was dissolved in dry THF (10mL), cooled in an ice-water bath, TEA (1mL), p-toluidine (346mg, 3.23mmol) added, warmed to room temperature for reaction, and monitored by TLC. After the reaction is finished, spin-drying the reaction liquid, adding sufficient water to dissolve the reaction liquid, extracting the reaction liquid by EA (50mLx3), combining organic phases, washing the organic phases by clean water and saturated salt water in sequence, drying the organic phases by anhydrous sodium sulfate, and mixing the reaction liquid and the organic phasesThe mixture was concentrated, and purified by silica gel column chromatography using a solvent mixture of EA and hexane as an eluent, to give 479mg of a product in 80% yield.¹H NMR(400MHz，CDCl₃)δppm：9.25(s,1H)；7.87(d,1H)；7.66 (d,2H)；7.40(d,1H)；7.31(d,2H)；4.13(d,2H)；2.71(s,3H)；2.44(s,3H)；2.15(m,1H)；1.01(m,6H)。 LCMS(ES-API)：m/z＝399[M+H]⁺

1. 5- (2- (1H-tetrazol-5-yl) phenyl) -N- (4-methylphenyl) -1-isobutyl-2-methyl-1H-indole-3-carboxamide

Compound 1D (200mg, 0.548mmol), 2- (5-tetrapyrazole) phenylboronic acid (200mg, 1.096mmol), potassium phosphate heptahydrate (920mg, 2.74mmol) and 10mL of degassed solvent (DMF: H)₂O ═ 10:1) was charged into a sealed tube, the gas in the sealed tube was replaced once with nitrogen, then tetrakistriphenylphosphine palladium (60mg, 0.05mmol) was rapidly added, the gas in the sealed tube was replaced 3 times or more with nitrogen, and the sealed tube was heated to 90 ℃ for reaction for 7 hours. The reaction was cooled to room temperature, diluted with 10g/L KOH and a small amount of solid precipitated and washed with EA (100mL) to give a dark yellow organic phase and a lighter aqueous phase. The plate had less product in the organic phase and retained the aqueous phase. The aqueous phase was adjusted to pH 4-5 with 1N HCl, a large amount of white solid precipitated, extracted with EA (60mLx3), the organic phases combined, washed successively with clear water and saturated brine, dried over anhydrous sodium sulfate, the reaction mixture concentrated, and purified by silica gel column chromatography using a solvent mixture of EA and hexane as the eluent to give 142mg of product, 49% yield.¹H NMR(400MHz，CDCl₃)δppm：7.97 (dd,1H)；7.72(s,1H)；7.53(m,2H)；7.41(dd,1H)；7.18(t,3H)；6.89(d,2H)；6.77(dd,1H)；3.81(d,2H)； 2.43(s,3H)；2.24(s,3H)；2.12(m,1H)；0.90(m,6H)。LCMS(ES-API)：m/z＝465[M+H]⁺

Examples 2 to 5: the following compounds were obtained by reacting intermediate compound 1C with the corresponding amine and then suzuki reaction using a synthetic method similar to compound 1.

TABLE 1 list of compounds

Example 6: 5- (2- (1H-tetrazol-5-yl) phenyl) -N- (4-methylphenyl) -1-cyclohexyl-2-methyl-1H-indole-3-carboxamide

6A, (3-cyclohexylamino) ethyl crotonate

Ethylacetoacetate (4.85mL,38.4mmol) was added to a 50mL single-neck flask, cyclohexylamine (5.50mL, 48.0mmol) was added dropwise to the reaction mixture at room temperature, and the mixture was stirred at the same temperature for 5 hours after the addition. TLC shows that the raw materials completely react, 100mL of petroleum ether is added into the reaction solution, an organic layer is separated, and then the mixture is washed by water and saturated saline solution in sequence once, dried by anhydrous sodium sulfate, filtered and concentrated under reduced pressure at 50 ℃ to obtain 8.25g of light yellow oily matter, and the crude product is directly used for the next reaction without purification.¹H NMR(400MHz，CDCl₃) δppm：4.82(s,1H)；4.40(q,2H)；2.77(m,1H)；2.46(s,3H)；1.94(m,2H)；1.69(m,4H)；1.56(t,3H)；1.37 (m,4H)。LCMS(ES-API)：m/z＝212[M+H]⁺

6B, 1-cyclohexyl-5-hydroxy-2-methyl-1H-indole-3-carboxylic acid ethyl ester

In a 500mL single-neck flask were added p-benzoquinone (14.52g,134.3mmol) and 200mL of absolute ethanol, and after stirring and dissolution, intermediate 6A (8.25g,39.0mmol) and absolute ethanol were added dropwise under cooling in an ice-water bath(25mL) and then moved to room temperature for 18h, and TLC showed the starting material was completely reacted. The reaction solution was transferred to an ice bath and stirred for 30min to precipitate a large amount of coffee solid, filtered, the filter cake was washed with a small amount of cold ethanol, and the solid was dried under vacuum at 50 ℃ to obtain 5.42g of coffee solid, which was used directly in the next reaction without purification of the crude product.¹H NMR(400MHz，CDCl₃)δppm：7.17(s,1H)；7.29(d,1H)；7.12(d,2H)；4.05(q,2H)；3.38(m, 1H)；2.33(s,3H)；1.82(m,2H)；1.55(m,2H)；1.31(m,6H)；1.02(t,3H)。LCMS(ES-API)：m/z＝ 302[M+H]⁺

6C, 1-cyclohexyl-2-methyl-5-trifluoromethanesulfonyl-1H-indole-3-carboxylic acid ethyl ester

A250 mL single vial was charged with intermediate 6B (5.4g,17.9mmol) and 60mL of dichloromethane, 9mL of pyridine was added with stirring, a mixture of trifluoromethanesulfonic anhydride (6.0mL,35.7mmol) and dichloromethane (30mL) was added dropwise with cooling in an ice-water bath, and the reaction was allowed to warm to room temperature for 18h, TLC showed the starting material was completely reacted. The organic layer was separated, washed with 10% HCl, water, and saturated brine in this order once, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 50 ℃ to give 11.5g of a brown oil, which was purified by silica gel column chromatography using a solvent mixture of EA and hexane as an eluent to give 6.2g of a product with a yield of 72.0%.¹H NMR(400MHz，CDCl₃) δppm：7.84(s,1H)；7.29(d,1H)；7.12(d,2H)；4.05(q,2H)；3.38(m,1H)；2.33(s,3H)；1.82(m,2H)；1.55 (m,2H)；1.31(m,6H)；1.02(t,3H)。LCMS(ES-API)：m/z＝434[M+H]⁺

6D, 5- (2- (1H-tetrazol-5-yl) phenyl) -1-cyclohexyl-2-methyl-1H-indole-3-carboxylic acid ethyl ester

Add intermediate 6C (in order) to a 50mL Schlenk flask2.0g,4.62mmol), 2- (tetrazol-5-yl) phenylboronic acid (1.05g, 5.6mmol), cesium carbonate (4.52g,13.9mmol), palladium tetrakistriphenylphosphine (0.53g,0.46mmol), 20mL DMF and 2mL water, then under N₂Raising the temperature to 90 ℃ under protection and reacting for 3 h. The reaction solution was poured into 80mL of water, the pH was adjusted to 1 with 10% HCl, extraction was performed with 100mL of ethyl acetate, and the organic layer was washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 50 ℃ to obtain 3.44g of a yellow oily substance, which was purified by silica gel column chromatography using a solvent mixture of EA and hexane as an eluent to obtain 1.39g of a product with a yield of 70.2%.¹H NMR(400MHz，CDCl₃)δppm：8.39(d,1H)；7.55(m,2H)；7.28(s,1H)；7.17(d,2H)； 6.95(d,1H)；4.01(q,2H)；3.37(m,1H)；2.35(s,3H)；1.78(m,2H)；1.51(m,2H)；1.29(m,6H)；0.99(t, 3H)。LCMS(ES-API)：m/z＝430[M+H]⁺

6E, 5- (2- (1H-tetrazol-5-yl) phenyl) -1-cyclohexyl-2-methyl-1H-indole-3-carboxylic acid

Intermediate 6D (1.3g,3.03mmol) and 15mL of methanol were added to a 100mL single-neck flask, stirred to dissolve, 5mL of 50% KOH solution was added, then the temperature was raised to 65 ℃ for reaction for 2h, and TLC showed complete reaction of the starting materials. Methanol was removed under reduced pressure at 45 ℃ to precipitate a solid, 20mL of water was added to dissolve the solid, the pH was adjusted to 5-6 with 10% HCl to precipitate a large amount of white solid, the mixture was filtered, the filter cake was washed with 30mL of water, and the solid was dried under vacuum at 45 ℃ to give 0.95g of white solid in 78.0% yield.¹H NMR(400MHz，CDCl₃)δppm：12.89 (s,1H)；7.97(d,1H)；7.55(d,2H)；7.17(m,3H)；7.05(s,1H)；3.34(m,1H)；2.31(s,3H)；1.70(m,2H)；1.45 (m,2H)；1.48(m,6H)。LCMS(ES-API)：m/z＝400[M-H]^-

6. 5- (2- (1H-tetrazol-5-yl) phenyl) -N- (4-methylphenyl) -1-cyclohexyl-2-methyl-1H-indole-3-carboxamide

A25 mL single neck flask was charged with intermediate 6E (0.15g,0.37mmol) and 5mL of anhydrous dichloromethane, and p-methylbenzene was added sequentially with stirringAmine (0.05g,0.45mmol), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP reagent, 0.25g, 0.56mmol) and diisopropylethylamine (0.15ml,0.89mmol) were then reacted at room temperature for 4h and TLC showed complete reaction of starting material. 15ml of methylene chloride was added thereto, the organic layer was separated, washed with 10% HCl, water and saturated brine in this order, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 40 ℃ to obtain a crude product, which was purified by silica gel column chromatography using a solvent mixture of EA and hexane as an eluent, as a yellow solid 0.16g with a yield of 87.4%.¹H NMR(400MHz，CDCl₃)δppm：9.30(s,1H)；8.42(d,1H)；8.01(m,2H)；7.71 (d,2H)；7.63(m,2H)；7.60(d,1H)；7.50(s,1H)；7.36(d,2H)；3.79(m,1H)；2.76(s,3H)；2.49(s,3H)；2.02 (m,4H)；1.63(m,5H)。LCMS(ES-API)：m/z＝491[M+H]⁺

Example 7: reacting 4-bromo-2-iodoaniline with the corresponding

After cyclization, the intermediate compound 6E was coupled with the corresponding amine using a synthetic method similar to compound 6.

Example 8: 1- (5- (2- (1H-tetrazol-5-yl) phenyl) -1-cyclohexyl-2-methyl-1H-indol-3-yl) -3-toluidineurea

According to the synthesis method of the compound 6, the intermediate compound 6E and DPPA are reacted to generate isocyanate, and then the isocyanate and p-methylaniline are reacted to generate a compound 8, and the yield is 23.8%.¹H NMR(400MHz，CDCl₃)δppm：7.87(d,1H)；7.39(m,4H)；7.07(d,2H)；6.97 (m,3H)；6.72(d,1H)；4.07(m,1H)；2.28(s,3H)；2.23(s,3H)；2.10(m,2H)；1.93(m,2H)；1.80(m,2H)； 1.42(m,2H)；1.27(m,2H)。LCMS(ES-API)：m/z＝506[M+H]⁺

Evaluation of biological Activity of Compounds

Experimental materials and instruments

The positive drug INCB024360 is purchased from MedChem Express company, Hela cells are from cell bank of Chinese academy of sciences, complete medium is prepared by laboratory of the company, DPBS, 0.25% Trypsin-EDTA and Trypan Blue are Gibco of Saimer Feishell science and technology (China) Limited^TMThe microplate reader is SpectraMax 190 from Molecular Device, the cytometer is Countstar from Shanghai Rui Yu Biotech, and the cytospin is TDL-40B from Shanghai' an Tingning scientific instruments.

Reagent

INF-gamma (R & D,28-IF-100) was dissolved with sterile purified water to a concentration of 0.2 mg/mL;

4-dimethylaminobenzaldehyde solution (Sigma, CAT #100107) was prepared at a concentration of 2% (m/v) with glacial acetic acid (Colon, CAT # 64197);

samples were dissolved in DMSO (Amresco, CAT # N182) to a concentration of 10 mM;

6.1N trichloroacetic acid (Sigma, CAT # 76039).

Cell lines and culture conditions

Hela cells were cultured in MEM medium (Gibco), 90%;

Sodium Pyruvate(Gibco)，1mM；

fetal bovine serum (Hyclone), 10%;

penicillin streptomycin solution (100 ×, Hyclone), 1%;

gas phase: air, 95%; 5% of carbon dioxide;

the temperature was 37 ℃.

Determination of the inhibition Rate of the IDO enzyme

Hela cells were plated in 96-well plates (2500/well, 200 ul/well) and after overnight adherence, INF- γ was diluted with medium to a final concentration of 50ng/mL, and then the drug was diluted to 1uM and 0.1nM in medium containing 50ng/mL INF- γ, while controls were set: negative and blank controls of 50ng/mL INF-. gamma.in culture medium + cells with 0.1% DMSO: 50ng/mL INF- γ medium with 0.1% DMSO only; the original medium was aspirated from the 96-well plate, 200ul of drug and control groups were added per well, 3 replicate wells per concentration were set, and cells were incubated for 48h under normal culture conditions. Sucking 140 ul/well of cell culture supernatant after 48h, adding 10ul of 6.1N trichloroacetic acid into a 96-well round bottom plate, mixing uniformly, and standing at 50 ℃ for 30 min; then centrifuging at 2500rpm for 10 min; pipette 100ul of supernatant into a new 96-well flat bottom plate; finally, 100ul of 2% 4-dimethylaminobenzaldehyde solution is added, mixed evenly, placed for 10min at room temperature in a dark place, and detected at 480nm of an enzyme labeling instrument.

IDO inhibition rate is [1- (OD drug group-OD blank group)/(OD negative control group-OD blank group) ] × 100%,

wherein the OD drug group is a drug adding hole; OD negative control group is cells cultured in 50ng/mL INF-gamma medium with 0.1% DMSO; OD blank was 50ng/mL INF-gamma medium without 0.1% DMSO of cells.

TABLE 2 percentage inhibition of IDO by some compounds and positive drugs at different concentrations

IDO kynurenine assay with human IDO cells

Hela cells were plated in 96-well plates (2500/well, 200 ul/well) and after overnight adherence, INF- γ was diluted with medium to a final concentration of 50ng/mL, and then drug was diluted to 1uM (10-fold down to 5 concentrations to 0.1nM) in medium containing 50ng/mL INF- γ, while controls were set: negative and blank controls of 50ng/mL INF-. gamma.in culture medium + cells with 0.1% DMSO: 50ng/mL INF- γ medium with 0.1% DMSO only; the original medium was aspirated from the 96-well plate, 200ul of drug and control groups were added per well, 3 replicate wells per concentration were set, and cells were incubated for 48h under normal culture conditions. Sucking 140 ul/well of cell culture supernatant after 48h, adding 10ul of 6.1N trichloroacetic acid into a 96-well round bottom plate, mixing uniformly, and standing at 50 ℃ for 30 min; then with 2Centrifuging at 500rpm for 10 min; pipette 100ul of supernatant into a new 96-well flat bottom plate; finally, 100ul of 2% 4-dimethylaminobenzaldehyde solution is added, the mixture is mixed evenly, the mixture is placed for 10min at room temperature in a dark place, the detection is carried out at 480nm of an enzyme labeling instrument, and GraphPad prism 5 software is used for calculating IC₅₀。

TABLE 3 IDO inhibition assay results for Compounds 2, 8

Determination of the Combined inhibition Rate of IDO enzyme inhibitors

Hela cells are paved in a 96-well plate (2500/well and 200 ul/well), after overnight adherence, INF-gamma is diluted by a culture medium to a final concentration of 50ng/mL, then the medicine is diluted by the culture medium containing 50ng/mL INF-gamma, BL0019 and BL0030 are diluted by 5 concentrations from 120nM to 2 times, and INCB024360 is diluted by 5 concentrations from 60nM to 2 times; the combined administration is that BL0019: BL0030: 1, BL0019: INCB 024360: 2:1, BL0030: INCB 024360: 2:1, the initial concentration is started according to the single drug concentration, 2 times diluted, and total 6 concentrations; and setting comparison: negative and blank controls of 50ng/mL INF-. gamma.in culture medium + cells with 0.1% DMSO: 50ng/mL INF- γ medium with 0.1% DMSO only; the original medium was aspirated from the 96-well plate, 200ul of drug and control groups were added per well, 3 replicate wells per concentration were set, and cells were incubated for 48h under normal culture conditions. Sucking 140 ul/well of cell culture supernatant after 48h, adding 10ul of 6.1N trichloroacetic acid into a 96-well round bottom plate, mixing uniformly, and standing at 50 ℃ for 30 min; then centrifuging at 2500rpm for 10 min; pipette 100ul of supernatant into a new 96-well flat bottom plate; and finally, adding 100ul of 2% 4-dimethylaminobenzaldehyde solution, uniformly mixing, standing at room temperature in a dark place for 10min, detecting at 480nm of an enzyme labeling instrument, and calculating the CI value of the combined medicine by using Calcusyn software.

TABLE 4 measurement of the combination

Example numbering	Fa-50% corresponding CI
		2:8	0.92
2:INCB 024360	0.89
		8:INCB 024360	0.80

Detection of inhibition of T cell proliferation mediated by expressed IDO

Collecting human monocytes from peripheral monocytes by leukocyte isolation at 1 × 10⁶Cell/well Density human monocytes were cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2mM L-glutamine in 96-well culture plates. Adherent cells were retained and cultured with 250ng/ml IL-4, 100ng/ml GM-CSF for 7 days. Cells were matured for 2 days with a combination of LPS cytokines containing 50ng/mL INF- γ at 50 μ g/mL to induce dendritic cell maturation. The medium was replaced with 100-200U/mL IL-2, 100ng/mL anti-CD 3 antibody and complete RPMI 1640 of human allogeneic T cells from normal donors and different dilutions of IDO compound to give final concentrations of IDO inhibitor between 500 and 1. mu.M, and incubated for two additional days. BrdU was added to the overnight shock and T cell proliferation was measured using a colorimetric cell proliferation ELISA kit. Continuously culturing cells in 10 μ M BrdU labeling solution for 18 hours, removing the labeling medium, adding 200 μ L FixDenat/well, incubating at room temperature for 30 minutes, removing the FixDenat solution, incubating with 100 μ L/well anti-BrdU-POD antibody conjugate solution for 90 minutes, removing the antibody conjugate, washing the cells with 200 μ L/well washing solution for 3 times, adding 100200 μ L/well substrate solution, reading the plate data with microplate reader, and reading the plate data with microplate readerMultiple readings are recorded at intermediate points to ensure that the data is within a linear range. Compounds of the invention having an IC50 of less than about 100 μ M are considered active compounds.

In vivo detection of IDO inhibition

A mouse colorectal cancer CT26 transplanted tumor model is established, a compound 5, a compound 161 and INCB024360 (positive drugs) with certain concentrations are respectively given, and the curative effect of each compound on the transplanted tumor of a CT26 mouse is evaluated. In vitro cultured CT26 cells are inoculated under the back subcutaneous skin of a nude mouse, after tumors grow out, the nude mouse is randomly divided into 6 groups, 7-8 tumors in each group are given, different medicines (except special instructions, the medicines are given for 2 times a day), the body weight is weighed regularly, the tumor volume is measured, and the drug effect on the CT26 model is evaluated by observing the indexes of tumor inhibition curative effect and the like of each compound. The results show that: the compound 8 is superior to the compound 2 and the positive medicine, and has a certain tumor inhibition effect.

Claims

1. A compound or pharmaceutically acceptable salt represented by structural formula (I):

wherein:

R¹is selected from C₁-C₈Alkyl or C₃-C₇A cycloalkyl group;

R²is selected from C₁-C₈Alkyl or C₃-C₇A cycloalkyl group;

R³is selected from substituted or unsubstituted phenyl, and the substituent is independently selected from halogen or C₁-C₈An alkyl group;

a is a carbon chain with or without heteroatoms and has the structure of- (CH)₂) n-, wherein n ═ 0; or-NH (CH)₂) n-, n-0, wherein the NH terminus is attached to the indole ring.

2. A compound or pharmaceutically acceptable salt as claimed in claim 1, characterized in that: has a structural formula shown as formula (II):

wherein:

R¹is selected from C₁-C₈Alkyl or C₃-C₇A cycloalkyl group;

R²is selected from C₁-C₈Alkyl or C₃-C₇A cycloalkyl group;

n＝0。

3. the compound or pharmaceutically acceptable salt of claim 1, having a formula as shown in formula (III):

wherein:

R¹is selected from C₁-C₈Alkyl or C₃-C₇A cycloalkyl group;

R²is selected from C₁-C₈Alkyl or C₃-C₇A cycloalkyl group;

n＝0。

4. use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer, inflammation, cardiovascular disease, neurodegenerative disease, psychiatric disease, viral infection, autoimmune disease or other chronic disease associated with tryptophan metabolism disorders.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.