CN113754736A - Hydrazide structure-containing PD-L1 cyclopeptide inhibitor - Google Patents

Abstract

The invention relates to a hydrazide structure-containing macrocyclic peptide compound, a racemate, a stereoisomer, a tautomer, an isotopic marker, a nitrogen oxide, a solvate, a polymorph, a metabolite, an ester, a prodrug or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same and medical application thereof, wherein the compound has remarkable inhibitory activity on PD-1/PD-L1 protein-protein interaction, and the inhibitory activity of some compounds on PD-1/PD-L1 interaction is remarkably superior to that of a positive control medicament.

Description

Hydrazide structure-containing PD-L1 cyclopeptide inhibitor

Technical Field

The invention relates to a cyclic peptide compound which is used as an immunomodulator in treatment, in particular to a PD-L1 cyclic peptide inhibitor containing a hydrazide structure.

Background

Programmed cell death protein 1 (PD-1 is also called PDCD 1and CD279), is a type I transmembrane protein which is coded by gene PDCD 1and consists of 288 amino acid residues and belongs to a B7-CD28 receptor superfamily member. Its structure includes 4 parts: immunoglobulin variable domains (igvs), transmembrane domains, immunoreceptor tyrosine inhibitory motifs, immunoreceptor tyrosine switch motifs. It is used in bone marrow cell, dendritic cell, natural killer cell, monocyte, CD4^-CD8^-The surface expression of various immune cells such as thymocytes, regulatory T cells, B cells and antigen presenting cells. PD-1 has two ligands, PD-L1(CD274) and PD-L2(CD273), which are type I transmembrane proteins consisting of 290 and 270 amino acids, belonging to the B7 family, and having 37% homologous sequences. PD-L1 is expressed on antigen presenting cells, non-lymphoid organs and a variety of tumor cells. Although PD-L1 and PD-L2 are both ligands of PD-1, PD-L2 has a narrow expression range and is mainly expressed on immune cells such as dendritic cells, monocytes and the like, and the research finds that PD-L1 plays a main role in the immune escape process of tumors.

Under normal physiological conditions, T cells do not express PD-1 in large quantities, and when exposed to antigen stimuli for a long time, the T cells cause the expression of PD-1 to be up-regulated. Meanwhile, the activated T cells further induce other cells to over-express PD-L1 by releasing cytokines such as gamma interferon (TNF-gamma), interleukin and the like. Binding of PD-L1 to PD-1 results in phosphorylation of the Immunoreceptor Tyrosine Inhibitory Motif (ITIM) and Immunoreceptor Tyrosine Switch Motif (ITSM) of the PD-1 endodomain, thereby recruiting the tyrosine phosphatases SHP-1and SHP-2(Chemnitz JM, Parry RV, Nichols KE, et al. SHP-1and SHP-2 association with immunoreceptor type-based switch motif of programmed death 1 uplink human T cell stimulation, but not the receptor expression vectors T cell activation [ J ]. J Immunol,2004,173(2):945 954). These phosphatases can dephosphorylate a plurality of key proteins on a T cell antigen receptor (TCR) signal path, inhibit signal paths downstream of the TCR, such as PI3K/AKT/Mtor, RAS/MEK/ERK, c-Myc and the like, further inhibit transcription of related genes, prevent T cell cycle progression and expression of related proteins. These inhibit the proliferation and differentiation of T cells and the production of cytokines. This regulatory mechanism prevents over-activation of T cells, maintains the immune system in humans immune tolerant to self-antigens, and reduces the damage of the immune response to surrounding normal tissues (Sharpe AH, Pauken KE. the reverse functions of the PD1 inhibition pathway [ J ]. Nat Rev Immunol,2017,18(3): 153-.

Tumor cells cause multiple immunosuppressions by continuously activating the PD-1/PD-L1 signaling pathway through overexpression of PD-L1. These mechanisms are currently roughly classified into the following 3 classes: promoting the apoptosis of tumor specific T cells; ② T cells of peripheral and lymphoid tissues can be transformed into regulatory T cells with loss of function and 'exhaustion' T cells; ③ inhibiting the activation of effector T cells and naive T cells, and the expression of PD-L1 on the surface of immune cells can also affect the anti-tumor CD8⁺T cell response. Through such immune escape mechanisms, tumor cells can easily escape from immune system recognition and attack (Tianjiping, Zhang Jian, etc., Chinese university of pharmacy, 2019, 50 (1): 1-10).

Therefore, blocking the combination of PD-1 and PD-L1 can reverse the immunosuppressive mechanism, which can help to improve the tumor killing capability of the immune system of the organism, and provides a reliable theoretical basis for blocking the tumor immunotherapy mediated by PD-1/PD-L1.

Research shows that polypeptide compounds with molecular weights between those of traditional small molecules and antibody drugs also have the inhibitory activity of a PD-1/PD-L1 channel, and the compounds have lower toxicity and smaller drug nonspecific interaction compared with the traditional small molecules and have better tissue permeability compared with antibodies. Aurigene company researches the compounds, develops a series of polypeptides, and pharmacodynamic data on a CT26 colon cancer model show that the compound a can effectively inhibit tumor growth with an inhibition rate of 46% (P <0.01,2-way, ANOVA). Pharmacodynamic results on a pseudomonas aeruginosa lung infection model show that the survival time of the mice is as high as 15d (patent WO 2013132317). The pharmacodynamic research result of the compound B on a lung metastasis model of a melanoma high-metastasis strain B16F10 shows that the tumor metastasis inhibition rate is 54%, and the inhibition rate of the mouse spleen cell proliferation inhibition by inhibiting the PD1/PD-L1 interaction on an MDA-MB-231 model of PD-L1 overexpression is 60% (patent WO 2013144704).

Macrocyclic polypeptide compounds are also researched by Baishimei-ShiGuibao company, and the compounds can effectively inhibit the combination effect of PD-1 and PD-L1 and represent that the activity of the compounds is as high as nanomolar. The test results show that the compound can increase the release of IFN gamma in the memory T cell population generated by the previous exposure to the persistent antigen by combining with PD-L1. Similar to anti-PD-L1 antibodies, such compounds, when bound to PD-L1, are capable of enhancing the release of IFN γ in a population of T cells that are resistant to persistent chronic viral infection. The earliest patent was published in 2014, and the results of intracellular binding experiments of IC50 and PD-L1 showed that the compounds can bind to PD-L1 on Juekat, a mouse B cell line (LK35.2) and a human lung adenocarcinoma cell line (L2987), and have the same binding site as that of anti-PD-L1 monoclonal antibody. Meanwhile, test results show that the compound can be combined with PD-L1, and the IC50 of the compound c is 1 nmol/L. The compound can selectively interfere the combination of PD-L1 and PD-1 and CD80 so as to combine with PD-L1, but cannot block the interaction of PD-1/PD-L2 or CD80/CTLA 4. In addition, the compounds can also prevent the combination of the recombinant PD-1-Ig and Jurkat-PD-1 cells, and can also prevent the combination of the recombinant PD-1-Ig and a mouse B cell line LK35.2-hPD-L1 which has an adenocarcinoma cell line L2987 or hPD-L1 over-expressed and is expressed by endogenous PD-L1 (WO 2014151634). Macrocyclic peptides capable of inhibiting the PD-L1 signalling pathway are reported in patents by behamei-schurinbao corporation, such as CN107108698A and CN 107223129A.

Hydrazide structural compounds have wide biological activity and are well applied to pesticide science and pharmaceutical chemistry. Many approved drugs on the market contain hydrazide structures, such as isoniazid with excellent anti-tubercular activity; the drug isocarboxazid for the treatment of chronic depression; and the hydrazinopeptide mimetic goserelin, are used in the treatment of hormone-treatable prostate cancer, premenopausal and perimenopausal breast cancer, endometriosis, and the like.

The molecular hybridization technique is a common structure modification strategy in the field of drug design and development, and can splice pharmacophores in different bioactive molecules to possibly generate hybrid molecules with potential high activity, low toxicity and multiple action mechanisms. Based on the principle, hydrazide structural groups are fused into the cyclic peptide with the anti-PD 1/PD-L1 binding activity, so as to obtain the anti-tumor immune cyclic peptide with better activity.

Disclosure of Invention

The invention provides a PD-L1 cyclic peptide inhibitor containing a hydrazide structure, which can inhibit a PD-L1 signal transduction pathway.

The present invention provides compounds of formula (I) and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof:

R_cand R_dIs CH₃,

R_a、R_b、R_e、R_f、R_g、R_h、R_i、R_j、R_k、R_l、R_m、R_nIndependently selected from H, or C₁-C₆An alkyl group;

R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃independently selected from natural amino acid side chains or non-natural amino acid side chains, or may form, together with the respective ortho-R groups and the nitrogen atom to which they are attached, a ring selected from azetidine, pyrrolidine, morpholine, piperidine, piperazine and thiazolidine, wherein each ring is optionally substituted with 1 to 4 groups independently selected from: amino, cyano, methyl, halogen, and hydroxy;

further, R_zTogether with the corresponding ortho-R groups and the nitrogen atom to which they are attached form a pyrrolidine, wherein z ═ 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13.

Further, R₁₂And R_mAnd R_mThe attached nitrogen atoms together form a pyrrolidine;

R₁₀and R_kAnd R_kThe attached nitrogen atoms together form a pyrrolidine;

R₇and R_hAnd R_hThe attached nitrogen atoms together form a pyrrolidine.

Further, R₁₃Is an aromatic benzyl radical, R₄、R₆And R₉Is heterocyclic benzyl.

A compound and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs thereof, or pharmaceutically acceptable salts thereof, wherein the structure of formula (I) is selected from the following structures, as exemplified in table 2: compound I-1, compound I-2, compound I-3, compound I-4, compound I-5, compound I-6, compound I-7, compound I-8, compound I-9, compound I-10, compound I-11, compound I-12, compound I-13, compound I-14, compound I-15, compound I-16, compound I-17, compound I-18, compound I-19, compound I-20, compound I-21, compound I-22, compound I-23, compound I-24, compound I-25, compound I-26, and compound I-27.

Further, acceptable salts of the above compounds include acetate, trifluoroacetate or hydrochloride.

The invention also provides a pharmaceutical composition, which comprises the compound and racemate, stereoisomer, tautomer, isotopic marker, nitrogen oxide, solvate, polymorphic substance, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carrier or excipient.

The invention also provides application of the compound, which is used for preparing a medicament for preventing and/or treating diseases related to a PD-1/PD-L1 signal pathway, preferably, the diseases related to the PD-1/PD-L1 signal pathway are selected from cancers, infectious diseases and autoimmune diseases.

The cancer is selected from: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer); examples of other cancers include bone cancer; pancreatic cancer; skin cancer; head and neck cancer; cutaneous or intraocular malignant melanoma; uterine cancer; ovarian cancer; rectal cancer; cancer of the anal region; gastric cancer; testicular cancer; uterine cancer; endometrial cancer of fallopian tube cancer; cervical cancer; vaginal cancer; vulvar cancer; hodgkin's disease; non-hodgkin lymphoma; esophageal cancer; small bowel cancer; cancer of the endocrine system; thyroid cancer; parathyroid cancer; adrenal cancer; soft tissue sarcoma; cancer of the urethra; penile cancer; chronic or acute leukemias, including acute myeloleukemia, chronic myeloleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; a solid tumor in children; lymphocytic lymphomas; bladder cancer; renal or urethral cancer; renal pelvis cancer; central Nervous System (CNS) neoplasms; primary CNS lymphoma; tumor angiogenesis; spinal axis tumors; brain stem glioma; pituitary adenoma; kaposi's sarcoma; epidermoid carcinoma; squamous cell carcinoma; t cell lymphoma; environmentally induced cancers, including cancers induced by asbestos; and combinations of said cancers; the use also includes the treatment of metastatic cancer, particularly metastatic cancer expressing PD-L1.

The carrier in the pharmaceutical composition is "acceptable" in that it is compatible with (and preferably capable of stabilizing) the active ingredient of the composition and is not deleterious to the subject being treated. One or more solubilizing agents may be used as pharmaceutical excipients for the delivery of the active compound.

The compounds of the invention inhibit the PD-1/PD-L1 protein-protein interaction, thereby causing PD-L1 blockade. Blocking PD-L1 can enhance the immune response to cancer cells and infectious diseases in mammals including humans.

The present invention also provides a method of treatment of cancer comprising administering a compound of formula I of the present invention and pharmaceutically acceptable salts thereof or said pharmaceutical composition.

The methods of the invention may comprise administering a compound of the invention alone, as well as in combination with one or more other chemotherapeutic agents. Administration of multiple drugs can be simultaneous or sequential.

Interpretation of terms:

the compounds of the invention are those of formula (I) of the present patent application.

Hydrazide structure as used herein means a structure of

A natural amino acid side chain refers to the side chain of any naturally occurring amino acid (i.e., L-amino acid) that is normally in the S configuration (i.e., alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

A non-natural amino acid side chain refers to the side chain of any naturally occurring amino acid, usually in the R configuration (i.e., a D-amino acid) or to the side chain of an amino acid other than the naturally occurring amino acid in the R or S configuration (i.e., a D-or L-amino acid, respectively)A group selected from: c₂-C₇Alkenyl radical, C₁-C₃Alkoxy radical C₁-C₃Alkyl radical, C₁-C₆Alkoxycarbonyl radical C₁-C₃Alkyl radical, C₁-C₇Alkyl radical, C₁-C₃Alkylthio radical C₁-C₃Alkyl, acylamino C₁-C₃Alkyl, amino C₁-C₃Alkyl, azaindolyl C₁-C₃Alkyl, benzothiazolyl C₁-C₃Alkyl, benzothienyl C₁-C₃Alkyl, benzyloxy C₁-C₃Alkyl, carboxyl C₁-C₃Alkyl radical, C₃-C₁₄Cycloalkyl radical C₁-C₃Alkyl, biphenylmethyl, furyl C₁-C₃Alkyl, imidazolyl C₁-C₃Alkyl, naphthyl C₁-C₃Alkyl, pyridyl C₁-C₃Alkyl, thiazolyl C₁-C₃Alkyl, thienyl C₁-C₃An alkyl group.

C₁-C₆The alkyl group is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl and hexyl. C₃-C₆The cycloalkyl group is a cyclic alkyl group having 3 to 6 carbon atoms, and specific examples include cyclopropane, cyclobutane and cyclopentane.

C₁-C₆Substituted alkyl refers to C₁-C₆Alkyl being optionally substituted in one or more positions, C₃-C₆Substituted cycloalkyl means C₃-C₆Cycloalkyl groups are optionally substituted at one or more positions, with optional substituents including halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, mercapto, imino, amido, carbonyl, carboxyl, ether, heterocyclyl, aromatic, and the like.

The term "halogen" refers to F, Cl, Br and I.

Aromatic benzyl group: including monocyclic aryl substituted with at least one methylene group, wherein each atom of the ring is carbon, preferably the ring is a 5-7 membered ring, more preferably a 6-membered ring; aromatic benzyl groups also include those in which at least one ring is aromatic, the other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclyl or heteroaryl, and any of the rings is substituted at least at any position with a methylene group, and aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. The aromatic benzyl group may be a phenyl group substituted with at least one methylene group, a phenol group, a biphenyl group, or the like.

The heterocyclic benzyl group includes a heterocyclic group substituted with at least one methylene group, preferably a 3-to 10-membered ring, more preferably a 3-to 7-membered ring, the ring structure of which includes at least one heteroatom; it may also be a polycyclic ring system having two or more rings, at least one of which is a heterocyclic ring, at any one position of at least one of which is substituted with a methylene group, and the heterocyclic group includes, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, and the like. The heterocyclic benzyl group may be a benzopyrrole substituted with at least one methylene group.

Unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present application, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. The definitions of the groups and the structures of the compounds in such combinations and after the combination should fall within the scope of the present specification.

Where a range of numerical values is recited in the specification and claims herein, and where the range of numerical values is defined as an "integer," it is understood that the two endpoints of the range are recited and each integer within the range is recited. For example, "an integer of 0 to 6" should be understood to describe each integer of 0, 1,2, 3,4, 5, and 6.

Depending on their molecular structure, the compounds of the invention may be chiral and may therefore exist in various enantiomeric forms. These compounds may thus be present in racemic or optically active form. The compounds of the invention or intermediates thereof may be separated into enantiomeric compounds by chemical or physical methods well known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are prepared from mixtures by reaction with optically active resolving agents. Examples of suitable resolving agents are optically active acids such as the R and S forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g. N-benzoylproline or N-benzenesulfonylproline) or various optically active camphorsulphonic acids. The chromatographic enantiomeric resolution can also advantageously be carried out with the aid of optically active resolving agents, such as dinitrobenzoylphenylglycine, cellulose triacetate or other carbohydrate derivatives or chirally derivatized methacrylate polymers, which are immobilized on silica gel. Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, for example hexane/isopropanol/acetonitrile.

Pharmaceutically acceptable salts may be acid addition salts of the compounds of the invention having sufficient basicity, for example having a nitrogen atom in the chain or ring, for example with the following inorganic acids: for example hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid, or hydrogen sulfates, or acid addition salts with organic acids such as: such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, and mixtures thereof, Mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid. Hydrochloric acid, trifluoroacetic acid and acetic acid are preferred.

In addition, another suitable pharmaceutically acceptable salt of a compound of the invention which is sufficiently acidic is an alkali metal salt (e.g., sodium or potassium salt), an alkaline earth metal salt (e.g., calcium or magnesium salt), an ammonium salt, or a salt with an organic base which affords a physiologically acceptable cation, such as a salt with: sodium ions, potassium ions, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol. By way of example, the pharmaceutically acceptable salts include salts of the group-COOH with: sodium ion, potassium ion, calcium ion, magnesium ion, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol.

Since the compound of the present invention may exist at a plurality of salt-forming sites, the "pharmaceutically acceptable salt" includes not only the salt formed at 1 salt-forming site among the compounds of the present invention but also the salt formed at 2,3 or all of the salt-forming sites among them. For this purpose, the molar ratio of the "pharmaceutically acceptable salt" of the compound of formula (I) to the cation of the acid (anion) or base required for salt formation may vary within wide limits, and may be, for example, 4:1 to 1:4, such as 3:1, 2:1, 1:2, 1:3, etc.

Depending on the position and nature of the various substituents, the compounds of the present invention may also contain one or more asymmetric centers. Asymmetric carbon atoms may exist in either the (R) or (S) configuration, with only one asymmetric center yielding a racemic mixture and multiple asymmetric centers yielding a diastereomeric mixture. In some cases, asymmetry may also exist due to hindered rotation about a particular bond, for example, the central bond connects two substituted aromatic rings of a particular compound. Also, the substituents may exist in cis or trans isomeric forms.

The compounds of the invention also include all possible stereoisomers of each, either as a single stereoisomer or as any mixture of said stereoisomers (e.g. the R-or S-isomers, or the E-or Z-isomers) in any proportion. Separation of individual stereoisomers (e.g. individual enantiomers or individual diastereomers) of the compounds of the invention may be achieved by any suitable prior art method (e.g. chromatography, particularly, for example, chiral chromatography).

The term "tautomer" refers to an isomer of a functional group resulting from the rapid movement of an atom in two positions in a molecule. The compounds of the invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; whereas in phenol the enol type predominates. The present invention encompasses all tautomeric forms of the compounds.

In the present invention, reference to compounds also includes isotopically-labeled compounds, which are identical to those shown in formula I, but wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of H, C, N, O, S, F and Cl, such as 2H, 3H, 13C, 11C, 14C, 15N, 18O, 17O, 32P, 35S, 18F, and 36Cl, respectively. Compounds of the present invention, prodrugs thereof, or pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example, those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritium (i.e., 3H) and carbon 14 (i.e., 14C) isotopes are particularly preferred for ease of preparation and detectability. Furthermore, substitution with heavier isotopes such as deuterium (i.e. 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g. increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in certain circumstances. The compounds of the invention as claimed may be particularly limited to replacement with deuterium or tritium. Furthermore, the absence of hydrogen in the substituents indicating the term deuterium or tritium alone is not meant to exclude deuterium or tritium, but may equally well comprise deuterium or tritium.

The term "solvate" is those forms of the compounds of the present invention which form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a particular form of solvates in which the coordination is with water. In the present invention, the preferred solvate is a hydrate. Further, pharmaceutically acceptable solvates (hydrates) of the compounds of general formula I according to the invention refer to co-crystals and clathrates of compound I with one or more molecules of water or other solvents in stoichiometric amounts. Solvents that may be used for the solvate include, but are not limited to: water, methanol, ethanol, ethylene glycol and acetic acid.

The term "prodrug", or "prodrug" refers to a compound that is converted in vivo to a compound of the general formula or a particular compound. Such conversion is effected by hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissue. The prodrugs of the present invention may be esters, and in the present invention esters may be used as prodrugs of esters of benzene, aliphatic (C)_1-24) Esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For example, a compound of the present invention comprises a hydroxy/carboxy group, i.e., it may be acylated to provide the compound in prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a hydroxyl group on the parent.

Advantageous effects

1) The invention provides a novel compound with a general formula I, which has obvious inhibitory activity of PD-1/PD-L1 protein-protein interaction, and the inhibitory activity of some compounds on the PD-1/PD-L1 interaction is obviously superior to that of a positive control drug;

2) the compound shows good pharmacokinetic characteristics on rats, has appropriate half-life and excellent bioavailability, is suitable for patent medicine, and has wide medicinal prospect;

3) the compound has obvious inhibition effect on the growth of tumor cells in a mouse model of subcutaneous transplantation tumor, and the treatment effect of the compound I-1 on the tumor is better than that of a positive control drug Durvalumab monoclonal antibody, thereby showing that the compound has good patent drug and market prospect.

Drawings

FIG. 1 is a graph showing the results of pharmacodynamic experiments in a mouse model of subcutaneous transplantation tumor.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

The abbreviations or English letters used in the description and claims of the present invention have the following meanings:

TABLE 1 abbreviation or English full name table

The synthesis of each compound (see table two for details) can be accomplished by reference to the following specific examples.

EXAMPLE 1 preparation of Compound I-1

1) Synthesis of fragment 1-T1 resin

50g of Rink Amino MBHAResin with a degree of substitution of 0.324mmol/g were weighed, 500mL of DCM were added, nitrogen bubbling was carried out, swelling was carried out for 30min, and the solvent was removed by suction filtration. DCM (150mL x 3) was added to wash the cake. Adding 400mL of 20% Pip/DMF (DMF: Pip; 80: 20, volume ratio) solution, mixing, stirring for 5 minutes, and performing suction filtration to obtain a filter cake; 400mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. The filter cake was washed successively with DMF, DCM and drained. To the flask was added 200mL of a DCM solution containing 9.84g of p-nitrophenyl chloroformate. 12.60g DIEA was added dropwise with stirring under nitrogen bubbling, and the reaction was monitored by ninhydrin detection. Filtering and pumping to dry. The filter cake was washed successively with DMF, DCM and dried by suction and used directly in the next step.

2) Synthesis of fragment 1-T2 resin

200mL of DMF was added to the reaction flask containing the filter cake, and the mixture was stirred under nitrogen protection. 25mL of hydrazine hydrate and 20mL of DIEA were added in this order and stirred overnight. Suction filtration, the filter cake washed successively with appropriate amount of DMF, DCM and suction dried, used directly in the next step.

3) Synthesis of fragment 1-T3 resin

28.50g of Fmoc-Cys (Trt) -OH, 7.90g of HOBt and 150mL of DMF are sequentially added into a new reaction bottle, stirred and dissolved, 7.40g of DIC is added at 0-10 ℃, and stirred for 10min to obtain a solution. The freshly prepared solution was transferred to the above reaction flask and stirred until completion of the reaction was monitored by ninhydrin detection. Filtering and pumping to dry. The filter cake was washed successively with DMF, DCM and dried by suction and used directly in the next step.

4) Synthesis of fragment 1-T4 resin

Mixing the filter cake with a mixed solution of 20% Pip/DMF350mL, stirring for 5 minutes, and performing suction filtration to obtain a filter cake; 400mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. The filter cake was washed successively with DMF, DCM and drained. 15.15g of Fmoc-Ala-OH, 7.89g of HOBt and 150mL of DMF are sequentially added into a reaction bottle, stirred and dissolved, 7.36g of DIC is added at 0-10 ℃, and stirred for 10 min. Transferring the newly prepared solution into the reaction bottle, and stirring the solution for reaction until the reaction is completely monitored by a ninhydrin detection method. Filtering and pumping to dry. The filter cake was washed successively with DMF, DCM and dried by suction and used directly in the next step.

5) Synthesis of fragment 1-T16 resin

Deprotecting, condensing and connecting amino acids (Fmoc-N-Me-Nle-OH, Fmoc-N-Me-Nle-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Pro-OH, Fmoc-Gln (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Tyr (tBu) -OH) in sequence according to the method for synthesizing the T4 fragment resin, and filtering to obtain the fragment 1-T16 resin which is directly used in the next step.

6) Synthesis of Compound precursor 1-T17A 20% Pip/DMF350mL mixed solution was added to the resin obtained above, stirred for 5 minutes, and filtered to obtain a filter cake; then adding a proper amount of 20% Pip/DMF solution, stirring for 15 minutes, and filtering by suction. The filter cake was washed successively with DMF, DCM and drained. The filter cake was mixed with 200mL of DCM, stirred well, and 50mL of a dichloromethane solution containing 3.68g of chloroacetyl chloride was added dropwise slowly. 6.30g DIEA was subsequently added dropwise thereto. After the addition was complete, the reaction was monitored by ninhydrin assay. Filtering and pumping to dry. The filter cake was washed sequentially with appropriate amount of DMF, DCM and suction dried to give fragment 1-T17 resin. The resin, 600mL TFA, 30mL EDT and 30mL purified water were added to the reactor in this order, and the reaction was stirred for 3 h. And (4) carrying out suction filtration, washing a filter cake by 120ml of LTFA, carrying out suction filtration, and combining filtrates. The filtrate was added to 8L of ethyl acetate with stirring to precipitate a white solid. Centrifuging, discarding supernatant to obtain precipitate, washing with diethyl ether for 4 times, and drying. To obtain crude fragment 1-T17.

7) Synthesis of Compound 1

5.02g of 1-T17 was weighed into a 500mL single neck flask, 100mL of acetonitrile and 200mL of 0.1M NH were added₄HCO₃The solution is clear, colorless and transparent after being stirred. And (3) adjusting the pH value of the reaction solution to be approximately equal to 9 by using 1M NaOH solution, stirring at room temperature for reaction until HPLC detection, and finishing the reaction of the raw materials. Concentrating under reduced pressure at 37 deg.C to remove most of acetonitrile to obtain crude product of compound 1.

8) Preparation of Compound 1

The crude product is purified by a Hanbang preparative HPLC system (wavelength is 210nm, chromatographic column is C8 reverse phase column, 20M ammonium acetate solution, pH3.0/acetonitrile is mobile phase) at room temperature to obtain refined peptide solution with purity of more than 99.0%. Mixing the fine peptide solutionTransferring salt with preparative HPLC system (C18 reverse phase column, 0.2% acetic acid/acetonitrile as mobile phase), concentrating under reduced pressure, and lyophilizing to obtain white compound 1, HPLC 98.2%, and mass spectrum (M + H)⁺＝1837.9。

TABLE 2 list of compounds

Evaluation test of biological Activity

Example 7 evaluation of inhibitory Activity of the Compound of the present invention against PD-1/PD-L1 protein-protein interaction these effects indicate that the inhibitory effect of the Compound of the present invention against PD1/PD-L1 is significant.

The specific test method is as follows:

purpose and principle of experiment

HTRF (Homogeneous time-Resolved Fluorescence) is a technique for detecting analytes in a pure liquid phase system. The technology is based on two technologies of Fluorescence Resonance Energy Transfer (FRET) and Time-Resolved Fluorescence (TRF), and opens a high-throughput drug screening apparatus.

Experimental materials and instruments

HTRF kits are purchased from Cisbio (CAT #63ADK000 CPACPEG) and comprise Anti-Tag 1-captate, Anti-Tag2-XL665/d2, Tag1-PD-L1, Tag2-PD-1, Dilution Buffer, Detection Buffer and other reagents required by experiments. SpectraMax i3X multifunctional microplate reader was purchased from Molecular Devices. 384 shallow well plates, available from Nunc (CAT # 264706).

Experimental procedure

Detection was performed using HTRF kit. And setting a negative control group, a positive control group and an administration group. PD-1 recombinant protein and PD-L1 recombinant protein were diluted to 250nM and 25nM, respectively, with Dilution Buffer. 100 μ M DMSO-solubilized small molecule compounds were diluted with Dilution Buffer to 1 μ M and 0.1 μ M. To 384 wells, 2ul of diluted test compound, 4ul of diluted PD-1 and 4ul of diluted PDL-1 were added in sequence. Mixing, and standing at room temperature for 15 min. Diluting anti-Tag1-Eu with Detection buffer³⁺(1:25) and anti-Tag2-XL665(1:100), and then mixing the diluted detection reagent in equal volume, and adding 10. mu.l of antibody mixture to each reaction well. And (5) sealing the membrane and incubating for 2h at room temperature. Fluorescence signals (320nm excitation, 665nm, 615nm emission) were detected with an ENVISION (Perkinelmer) instrument.

Data analysis

Inhibition ratio (ER)_positive―ER _sample)/(ER_positive―ER_negative) Data analysis was performed at 100%.

Results of the experiment

The inhibitory activity of compounds I-1 to I-27 was initially screened (100nM &10nM) using the Kit PD1/PD-L1 Binding Assay Kit (Cisbio/Cat #63ADK000 CPACPEG) -HTRF technique, while PD-1/PD-L1inhibitor 1(Selleck, Cat # S7911) was used as a positive reference compound.

TABLE 1 protein level Activity of Compounds to inhibit PD-1/PD-L1 protein-protein interaction

Examples	IC50nM	Examples	IC50nM
				I-1	A	I-14	B
I-2	A	I-15	A
				I-3	A	I-16	B
I-4	A	I-17	B
				I-5	A	I-18	B
I-6	A	I-19	A
				I-7	A	I-20	A
I-8	A	I-21	A
				I-9	A	I-22	B
I-10	B	I-23	A
				I-11	B	I-24	B
I-12	A	I-25	A
				I-13	A	I-26	A
BMS-986189	C	I-27	A

A represents 1 to 10nm

B represents 10 to 100nm

C represents 100-500 nm'

Experimental results show that the compound has obvious inhibition activity of PD-1/PD-L1 protein-protein interaction. The PD-1/PD-L1 inhibitory activity of some compounds is significantly better than that of the positive control drug. This suggests that such compounds of the invention may be useful as immune checkpoint PD-1/PD-L1 inhibitors.

Example 8 pharmacokinetic experiments

Pharmacokinetic study of Compound I-1 in SD rats by Single intravenous or subcutaneous administration of Compound PD-1

SD rats, female and male half, SPF grade.

Preparation of test article

5% DMSO + 10% Solutol + 85% sterile water for injection for intravenous and subcutaneous injection;

experimental groups compound I-1 of the present example was administered in groups according to the administration modes of groups 1-5, respectively, as shown in the table below.

Mode of administration

The weight was weighed before administration, and the amount administered was calculated from the body weight. Administration is by intravenous and subcutaneous injection.

Time point of blood sampling

IV: before administration, 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h and 24h after administration. PO: before administration, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h and 24h after administration.

Sample collection and disposal

Blood is collected via jugular vein or other suitable method, each sample is collected about 0.20mL, heparin sodium is anticoagulated, the blood sample is placed on ice after collection, and plasma is centrifugally separated within 2 hours (centrifugal force: 6800g, 6 minutes, 2-8 ℃). The collected plasma sample is stored in a refrigerator at minus 80 ℃ before analysis, and the residual plasma sample after analysis is continuously stored in the refrigerator at minus 80 ℃ for temporary storage, wherein the storage life is one month.

Biological analysis and data processing

And (3) detecting the blood concentration of the sample, and calculating pharmacokinetic parameters such as AUC (0-T), T1/2, Cmax, Tmax, MRT and the like by using Phoenix WinNonlin through the blood concentration data of different time points. When calculating the drug-induced parameters, the concentration before administration is calculated according to 0; BLQ (including "No peak") before Cmax is calculated as 0; BLQ (including "No peak") appearing after Cmax is not involved in the calculation uniformly, as in the table below.

Dosage form	Route of administration	T1/2	Tmax h	AUC(0～t)	MRT(0～t)h	F％
							10mg/kg	IV	1.85	0.083	120608.54	1.36
10mg/kg	SC	3.17	6.25	23638.28	7.81	19.60
							20mg/kg	SC	3.20	10	39731.75	8.96	20.92
40mg/kg	SC	3.30	8.5	90411.53	8.50	24.63

Results of the experiment

The pharmacokinetics and subcutaneous bioavailability of the compounds of the present invention were studied in rats after a single intravenous and subcutaneous administration. Experiments prove that: the compound of the invention shows good pharmacokinetic characteristics on rats, and has appropriate half-life and excellent bioavailability.

Example 9 pharmacodynamic experiments

Establishing a mouse model of subcutaneous transplanted tumor, wherein the adopted animal is C57BL/6, the adopted tumor cell is hPD-L1-MC38, the tumor growth is observed to a specific size after inoculation, the animals are divided into groups, 10 animals are used in each group, the animals are weighed and dosed, and the groups comprise a blank control group, a positive control group and a compound group; mice were tested twice weekly for tumor growth with a dosing cycle of 21 days, and after tumor volume reached the end of tumor, mice were weighed and euthanized, and tumor tissue, spleen tissue and blood samples were stripped. In vivo pharmacodynamic studies were performed on compound I-1, with the route of administration being subcutaneous injection and the route of administration of Durvalumab being intravenous injection. The results of the experiments are shown in the table below and in FIG. 1.

Pharmacodynamic test data in animal bodies show that the compound I-1 has a much stronger treatment effect on tumors than Durvalumab monoclonal antibody, the compound I-1 belongs to a small molecule inhibitor, and compared with monoclonal antibody medicines, the compound I-1 has many advantages, such as low production cost, good stability, preservation and transportation, and more importantly, the small molecule has no immunogenicity, and certain adverse reactions can be avoided in clinical application; the compounds have good market scenes.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. A compound of formula (I) and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof:

R_cand R_dIs CH₃,

R_a、R_b、R_e、R_f、R_g、R_h、R_i、R_j、R_k、R_l、R_m、R_nIndependently selected from H, or C₁-C₆An alkyl group;

R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃independently selected from natural amino acid side chains or non-natural amino acid side chains, or may form, together with the respective ortho-R groups and the nitrogen atom to which they are attached, a ring selected from azetidine, pyrrolidine, morpholine, piperidine, piperazine and thiazolidine, wherein each ring is optionally substituted with 1 to 4 groups independently selected from: amino, cyano, methyl, halogen and hydroxy.

2. The compound according to claim 1, and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, wherein: r_zTogether with the corresponding ortho-R groups and the nitrogen atom to which they are attached form a pyrrolidine, in which z ═ 1,2, 3,4, 5, 6, 7,8、9、10、11、12、13。

3. the compound according to claim 2, and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, wherein:

R₁₂and R_mAnd R_mThe attached nitrogen atoms together form a pyrrolidine;

R₁₀and R_kAnd R_kThe attached nitrogen atoms together form a pyrrolidine;

R₇and R_hAnd R_hThe attached nitrogen atoms together form a pyrrolidine.

4. The compound according to claim 1, and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, wherein: r₁₃Is an aromatic benzyl radical, R₄、R₆And R₉Is heterocyclic benzyl.

5. The compound according to claim 1, wherein the structure of formula (I) is selected from the following structures, such as those listed in table 2: compound I-1, compound I-2, compound I-3, compound I-4, compound I-5, compound I-6, compound I-7, compound I-8, compound I-9, compound I-10, compound I-11, compound I-12, compound I-13, compound I-14, compound I-15, compound I-16, compound I-17, compound I-18, compound I-19, compound I-20, compound I-21, compound I-22, compound I-23, compound I-24, compound I-25, compound I-26, and compound I-27.

6. The compound according to any one of claims 1 to 5, and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, wherein said acceptable salts comprise acetate, trifluoroacetate or hydrochloride salt.

7. A pharmaceutical composition comprising a compound of any one of claims 1-5 and racemates, stereoisomers, tautomers, isotopic labels, nitric oxides, solvates, polymorphs, metabolites, esters, prodrugs thereof or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or excipient.

8. Use of a compound according to any one of claims 1-5 for the preparation of a medicament for the prevention and/or treatment of a disease associated with the PD-1/PD-L1 signaling pathway, preferably selected from the group consisting of cancer, infectious disease, autoimmune disease.

9. Use of a compound according to claim 8, wherein the cancer is selected from: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer); examples of other cancers include bone cancer; pancreatic cancer; skin cancer; head and neck cancer; cutaneous or intraocular malignant melanoma; uterine cancer; ovarian cancer; rectal cancer; cancer of the anal region; gastric cancer; testicular cancer; uterine cancer; endometrial cancer of fallopian tube cancer; cervical cancer; vaginal cancer; vulvar cancer; hodgkin's disease; non-hodgkin lymphoma; esophageal cancer; small bowel cancer; cancer of the endocrine system; thyroid cancer; parathyroid cancer; adrenal cancer; soft tissue sarcoma; cancer of the urethra; penile cancer; chronic or acute leukemias, including acute myeloleukemia, chronic myeloleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; a solid tumor in children; lymphocytic lymphomas; bladder cancer; renal or urethral cancer; renal pelvis cancer; central Nervous System (CNS) neoplasms; primary CNS lymphoma; tumor angiogenesis; spinal axis tumors; brain stem glioma; pituitary adenoma; kaposi's sarcoma; epidermoid carcinoma; squamous cell carcinoma; t cell lymphoma; environmentally induced cancers, including cancers induced by asbestos; and combinations of said cancers; the use also includes the treatment of metastatic cancer, particularly metastatic cancer expressing PD-L1.

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