Biphenyl compounds as immunomodulators, processes for their preparation and use

Abstract

A kind of biphenyl compound shown in formula (I), its preparation method and application. It is also related to pharmaceutical compositions comprising the compound as active ingredient. The compound is a novel small molecule immunomodulator with excellent oral absorption characteristics, which can be used for treating and / or preventing a variety of diseases related to immunity.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to biphenyl compounds as immunomodulators, their preparation methods, and applications.

[0002] Background Introduction

[0003] Tumor immunotherapy is a novel treatment method that stimulates the body's immune system, enhancing its own anti-tumor immunity to inhibit or kill tumor cells. This method has achieved groundbreaking progress after more than a century of effort. In 2013, *Science* magazine listed tumor immunotherapy as the top scientific breakthrough of the year (Couzin-Frankel J., 2013, *Science*, 342:1432-1433), making it one of the most promising fields of anti-tumor treatment.

[0004] Compared to normal cells, tumor cells have a variety of genetic and epigenetic changes. The immune system can use the surface antigens produced by tumor cells to distinguish between the two, thereby triggering an anti-tumor immune response. In the process of T cell anti-tumor immunity, after T cells are activated by antigen recognition signals mediated by T cell receptors (TCRs), they comprehensively regulate T cell effects through co-stimulatory and co-inhibitory signals. These include inhibitory receptors such as the V-domain immunoglobulinspressor of T-cell activation (VISTA), T-cell immunoglobulin and mucin domain-containing-3 (TIM3), and lymphocyte activation gene 3 (LAG3), as well as inhibitory receptors such as CD28, CD134 (OX40), and glucocorticoid-induced TNFR-related protein. Immune checkpoints are activating receptors for stimulatory signals such as protein GITR, CD137, CD27, and HVEM (Mellman I., Coukos G., Dranoff G., 2011, Nature, 480:480-489). Under normal physiological conditions, immune checkpoints participate in maintaining immune tolerance to self-antigens to avoid autoimmune diseases, and also prevent excessive activation of the immune response leading to tissue damage. However, in tumor cells, they can evade immune killing by inhibiting T cell activation through immune checkpoints. Therefore, it is necessary to reactivate T cells to attack tumor cells by activating co-stimulatory signals (stepping on the "accelerator") and inhibiting co-inhibitory signals (releasing the "brake"), thereby achieving tumor immunotherapy.

[0005] PD-1 is expressed on activated T cells, B cells, and bone marrow cells. Belonging to the CD28 family, it is a type 1 transmembrane glycoprotein on T cells, composed of 288 amino acids. The molecular structure of PD-1 consists of an extracellular region similar to immunoglobulin IgV (amino acids 35-145), a transmembrane region, and a cytoplasmic tail region that connects to a signal peptide. The extracellular region binds to ligands and plays an important role (Cheng X., Veverka V., Radhakrishnan A., et al. 2013, J. Biol. Chem., 288:11771-11785). Programmed death protein ligand 1 (PD-L1) is one of the ligands of PD-1, belonging to the B7 family. It is continuously expressed on various tumor cells, T cells, antigen-presenting cells (APCs), and various non-hematopoietic cells. It is also a type 1 transmembrane glycoprotein, composed of 290 amino acids. The interaction between PD-1 and PD-L1 inhibits T cell activation, which is crucial for maintaining immune tolerance in normal organisms. However, in tumor cells and during viral infection, PD-1 is induced to be highly expressed on T cells, and PD-L1 expression is upregulated, leading to sustained activation of the PD-1 signaling pathway and inhibition of T cell proliferation, resulting in immune escape by tumor cells and pathogens (Fuller MJ, Callendret B., Zhu B., et al. 2013, Proc. Natl. Acad. Sci. USA., 110: 15001-15006; Dolan DE, Gupta S., 2014, Cancer Control, 21: 231-237; Chen L., Han X., 2015, J. Clin. Invest., 125: 3384-3391; Postow MA, Callahan MK, Wolchok). JD, 2015, J.Clin.Oncol., 33:1974-1982). The numerous PD-1 and PD-L1 antibody drugs launched in recent years have fully demonstrated that blocking the PD-1 / PD-L1 interaction is a highly effective treatment approach in immunotherapy for tumors and in many other immune-related diseases.

[0006] Studies have found that PD-L1 can interact with CD80 and inhibit the binding of PD-L1 and PD-1, as well as the ability to suppress T cell activation. Therefore, blocking the immune activation caused by the CD80 / PD-L1 interaction may also promote enhanced T cell activity, thus providing new therapeutic opportunities for immune-related diseases (Sugiura D., Maruhashi T., Okazaki ll-mi, et al. 2019, Science, 364:558-566).

[0007] Significant progress has been made in targeting PD-1 / PD-L1 antibody drugs to date. However, all antibody drugs require injection, have various ADMET issues, and suffer from serious immune system-related side effects. Compared to antibody drugs, small-molecule immunomodulators offer certain advantages, including oral administration, greater tissue penetration, and the ability to minimize side effects through pharmacological modifications. Furthermore, small-molecule inhibitors will have a lower price advantage.

[0008] Clinical studies of PD-1 / PD-L1 antibody drugs have shown that nivolumab T 1 / 2 The treatment duration is 25.2 days, with a dosing frequency of once every two weeks; Pembrolizumab T 1 / 2 The treatment duration is 25 days, with a dosing frequency of once every three weeks; atezolizumab T 1 / 2 The dosing period was 27 days, with a dosing frequency of once every three weeks. The dosing frequency of the above drugs was shorter than their half-life, indicating that sustained exposure in vivo is crucial for achieving ideal clinical efficacy for these target drugs. However, existing small-molecule immunomodulators have low in vivo exposure levels and short durations of sustained exposure, which will affect clinical efficacy. Summary of the Invention

[0009] One aspect of the present invention relates to a small molecule biphenyl compound, or an isomer thereof, a pharmaceutically acceptable salt, a precursor, and a metabolite capable of targeting PD-L1.

[0010] Another aspect of the present invention relates to a method for preparing the compounds described herein.

[0011] Another aspect of the present invention relates to pharmaceutical compositions comprising compounds of the present invention as active ingredients, and to the clinical use of compounds or pharmaceutical compositions of the present invention for the treatment and / or prevention of various other immune-related diseases, including immunotherapy targeting PD-L1 for tumors.

[0012] As small-molecule PD-L1 inhibitors, compared to antibodies, they require optimization in terms of target binding strength and duration of in vivo exposure to narrow the potential gap in efficacy. Therefore, the inventors aim to develop novel small-molecule PD-L1 immunomodulators with higher activity and superior oral absorption characteristics, particularly with sufficient in vivo exposure and duration of exposure, as well as greater targeting to tumor tissue, to meet unmet clinical needs.

[0013] This invention overcomes the drawback of existing PD-1 / PD-L1 antibody drugs, which all require injection administration, and provides a novel small molecule immunomodulator with excellent oral absorption characteristics. In particular, the compound of this invention has ideal in vivo exposure levels and duration of exposure, and is targeted to tumor tissues, which can accumulate in tumor tissues and form higher tumor tissue exposure concentrations, thus helping to better exert anti-tumor activity during treatment and achieve better therapeutic effects.

[0014] This invention relates to compounds of formula (I), or isomers thereof, pharmaceutically acceptable salts, precursors, and metabolites.

[0015]

[0016] in,

[0017] R 1 and R 2 They may be selected from C1-C6 alkyl, cyano, and halogen groups, either the same or different.

[0018] R 3 Selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl C1-C6 alkyl, C2-C6 alkynyl C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, mono-C1-C6 alkylamino C1-C6 alkyl, bis-C1-C6 alkylamino C1-C6 alkyl, halo-C1-C6 alkoxy C1-C6 alkyl, C3-C 14 Cycloalkyl, 3- to 14-membered heterocycloalkyl, C3-C 14 Cycloalkyl-C1-C4 alkyl, 3- to 14-membered heterocycloalkyl-C1-C4 alkyl;

[0019] R 4 Selected from hydrogen and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with one or more hydroxyl, carboxyl, or halogen substituents;

[0020] X is selected from -O-, -S-, -N(R) a )-;

[0021] R a Selected from hydrogen, C1-C6 alkyl, and halo-C1-C6 alkyl;

[0022] m is selected from 1, 2, and 3;

[0023] n is selected from 1, 2, and 3;

[0024] In some implementation schemes, R 1 and R 2 They can be selected from methyl, cyano, and halogen, either the same or different;

[0025] In some implementation schemes, R 1 and R 2 They can be selected from methyl, cyano, fluorine, and chlorine, either the same or different;

[0026] In some implementation schemes, R 1 Selected from C1-C6 alkyl, cyano, and halogen groups;

[0027] In some implementation schemes, R 2 Selected from C1-C6 alkyl groups and halogens;

[0028] In some implementation schemes, R 1 Selected from methyl, cyano, fluorine, and chlorine;

[0029] In some implementation schemes, R 2 Selected from methyl, chlorine;

[0030] In some implementation schemes, R 3 Selected from hydrogen, C1-C6 alkyl, and halo-C1-C6 alkyl;

[0031] In some implementation schemes, R 3 Selected from hydrogen, C1-C6 alkyl, and fluorinated C1-C6 alkyl;

[0032] In some implementation schemes, R 3 Selected from methyl, ethyl, and -CHF2;

[0033] In some implementation schemes, R 4 Selected from hydrogen and C1-C6 alkyl groups;

[0034] In some implementation schemes, R 4 Selected from hydrogen and methyl;

[0035] In some implementations, X is selected from -O-, -N(R) a )-;

[0036] In some implementations, X is selected from -O-;

[0037] In some implementation schemes, R a Selected from hydrogen and C1-C6 alkyl groups;

[0038] In some implementation schemes, R aIt is methyl;

[0039] In some implementation schemes, m is selected from 1 and 2;

[0040] In a preferred aspect, the present invention relates to compounds of formula (I), or isomers thereof, pharmaceutically acceptable salts, precursors, and metabolites thereof, wherein...

[0041] R 1 and R 2 They can be selected from methyl, cyano, fluorine, chlorine, and bromine, either the same or different;

[0042] R 3 Selected from C1-C6 alkyl, C2-C6 alkenyl C1-C6 alkyl, C2-C6 alkynyl C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, mono-C1-C6 alkylamino C1-C6 alkyl, bis-C1-C6 alkylamino C1-C6 alkyl, halo-C1-C6 alkoxy C1-C6 alkyl, C3-C 14 Cycloalkyl, 3- to 14-membered heterocycloalkyl, C3-C 14 Cycloalkyl-C1-C4 alkyl, 3- to 14-membered heterocycloalkyl-C1-C4 alkyl;

[0043] R 4 Selected from hydrogen and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with one or more hydroxyl or halogen substituents;

[0044] X is selected from -O-, -N(R) a )-;

[0045] R a Selected from hydrogen, methyl, ethyl, isopropyl, and halogenated C1-C6 alkyl groups;

[0046] m is selected from 1, 2, and 3;

[0047] n is selected from 1, 2, and 3;

[0048] In some implementation schemes, R 1 and R 2 They can be selected from methyl, cyano, fluorine, and chlorine, either the same or different;

[0049] In some implementation schemes, R 1 Selected from methyl, cyano, fluorine, and chlorine;

[0050] In some implementation schemes, R 2 Selected from methyl, chlorine;

[0051] In some implementation schemes, R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl;

[0052] In some implementation schemes, R 3 Selected from C1-C6 alkyl and fluorinated C1-C6 alkyl;

[0053] In some implementation schemes, R 3 Selected from methyl, ethyl, and -CHF2;

[0054] In some implementation schemes, R 4 Selected from hydrogen and C1-C6 alkyl groups;

[0055] In some implementation schemes, R 4 Selected from hydrogen and methyl;

[0056] In some implementation schemes, R a Selected from hydrogen, methyl, ethyl, and isopropyl;

[0057] In some implementation schemes, R a It is methyl;

[0058] In some implementation schemes, m is selected from 1 and 2;

[0059] In some implementations, X is selected from -O-;

[0060] In another preferred aspect, the present invention relates to compounds of formula (I), or isomers thereof, pharmaceutically acceptable salts, precursors, and metabolites thereof, wherein,

[0061] R 1 and R 2 They can be selected from methyl, cyano, fluorine, and chlorine, either the same or different;

[0062] R 3 Selected from C1-C6 alkyl, C2-C6 alkenyl C1-C6 alkyl, C2-C6 alkynyl C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, mono-C1-C6 alkylamino C1-C6 alkyl, bis-C1-C6 alkylamino C1-C6 alkyl, halo-C1-C6 alkoxy C1-C6 alkyl, C3-C 14 Cycloalkyl, 3- to 14-membered heterocycloalkyl;

[0063] R 4 Selected from hydrogen and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with halogen substituents;

[0064] X is selected from -O-, -N(R) a )-;

[0065] R a Selected from hydrogen, methyl, ethyl, and halogenated C1-C6 alkyl groups;

[0066] m is selected from 1 and 2;

[0067] n is selected from 1, 2, and 3;

[0068] In some implementation schemes, R 1 Selected from methyl, cyano, fluorine, and chlorine;

[0069] In some implementation schemes, R 2 Selected from methyl, chlorine;

[0070] In some implementation schemes, R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl;

[0071] In some implementation schemes, R 3 Selected from C1-C6 alkyl and fluorinated C1-C6 alkyl;

[0072] In some implementation schemes, R 3 Selected from methyl, ethyl, and -CHF2;

[0073] In some implementation schemes, R 4 Selected from hydrogen and C1-C6 alkyl groups;

[0074] In some implementation schemes, R 4 Selected from hydrogen and methyl;

[0075] In some implementation schemes, R a Selected from hydrogen, methyl, and ethyl;

[0076] In some implementation schemes, R a It is methyl;

[0077] In some implementations, X is selected from -O-;

[0078] In another, more preferred aspect, the present invention relates to compounds of formula (I), or isomers thereof, pharmaceutically acceptable salts, precursors, and metabolites thereof, wherein,

[0079] R 1 and R 2 They can be selected from methyl, cyano, fluorine, and chlorine, either the same or different;

[0080] R 3 Selected from C1-C6 alkyl, C2-C6 alkenyl C1-C6 alkyl, C2-C6 alkynyl C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, mono-C1-C6 alkylamino C1-C6 alkyl, bis-C1-C6 alkylamino C1-C6 alkyl, halo-C1-C6 alkoxy C1-C6 alkyl;

[0081] R 4The components are selected from hydrogen and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with fluorine, chlorine, or bromine substituents;

[0082] X is selected from -O-, -N(R) a )-;

[0083] R a Selected from hydrogen, methyl, ethyl, fluorinated C1-C6 alkyl, and chlorolated C1-C6 alkyl;

[0084] m is selected from 1 and 2;

[0085] n is selected from 1, 2, and 3;

[0086] In some implementation schemes, R 1 Selected from methyl, cyano, fluorine, and chlorine;

[0087] In some implementation schemes, R 2 Selected from methyl, chlorine;

[0088] In some implementation schemes, R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl;

[0089] In some implementation schemes, R 3 Selected from C1-C6 alkyl and fluorinated C1-C6 alkyl;

[0090] In some implementation schemes, R 3 Selected from methyl, ethyl, and -CHF2;

[0091] In some implementation schemes, R 4 Selected from hydrogen and C1-C6 alkyl groups;

[0092] In some implementation schemes, R 4 Selected from hydrogen and methyl;

[0093] In some implementation schemes, R a Selected from hydrogen, methyl, and ethyl;

[0094] In some implementation schemes, R a It is methyl;

[0095] In some implementations, X is selected from -O-;

[0096] In a particularly preferred aspect, the present invention relates to compounds of formula (I), or isomers thereof, pharmaceutically acceptable salts, precursors, and metabolites thereof, wherein,

[0097] R 1 and R 2 They can be selected from methyl, cyano, fluorine, and chlorine, either the same or different;

[0098] R3 Selected from C1-C6 alkyl, C2-C6 alkenyl C1-C6 alkyl, C2-C6 alkynyl C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, halo-C1-C6 alkoxy C1-C6 alkyl;

[0099] R 4 Selected from hydrogen and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with fluorine or chlorine substituents;

[0100] X is selected from -O-, -N(R) a )-;

[0101] m is selected from 1 and 2;

[0102] n is selected from 1, 2, and 3;

[0103] R a It is methyl;

[0104] In some implementation schemes, R 1 Selected from methyl, cyano, fluorine, and chlorine;

[0105] In some implementation schemes, R 2 Selected from methyl, chlorine;

[0106] In some implementation schemes, R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl;

[0107] In some implementation schemes, R 3 Selected from C1-C6 alkyl and fluorinated C1-C6 alkyl;

[0108] In some implementation schemes, R 3 Selected from methyl, ethyl, and -CHF2;

[0109] In some implementation schemes, R 4 Selected from hydrogen and C1-C6 alkyl groups;

[0110] In some implementation schemes, R 4 Selected from hydrogen and methyl;

[0111] In some implementations, X is selected from -O-;

[0112] In a particularly preferred aspect, the present invention relates to compounds of formula (I), or isomers thereof, pharmaceutically acceptable salts, precursors, and metabolites thereof, wherein,

[0113] R 1 and R 2 They can be selected from methyl, cyano, fluorine, and chlorine, either the same or different;

[0114] R3 Selected from C1-C6 alkyl, C2-C6 alkenyl C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, and halo-C1-C6 alkoxy-C1-C6 alkyl;

[0115] R 4 Selected from hydrogen and methyl;

[0116] X is selected from -O-, -N(R) a )-;

[0117] m is selected from 1 and 2;

[0118] n is selected from 1, 2, and 3;

[0119] R a It is methyl;

[0120] In some implementation schemes, R 1 Selected from methyl, cyano, fluorine, and chlorine;

[0121] In some implementation schemes, R 2 Selected from methyl, chlorine;

[0122] In some implementation schemes, R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl;

[0123] In some implementation schemes, R 3 Selected from C1-C6 alkyl and fluorinated C1-C6 alkyl;

[0124] In some implementation schemes, R 3 Selected from methyl, ethyl, and -CHF2;

[0125] In some implementations, X is selected from -O-.

[0126] This invention relates to compounds represented by formula (I), including but not limited to:

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] Definitions of each term

[0133] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or scope of the groups. In particular, this invention includes every independent secondary combination of the members of these types and scopes. For example, the term "C1-C6 alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl, or independently disclosed "C1-C4 alkyl", or independently disclosed "C1-C3 alkyl".

[0134] The "halogen" mentioned in this invention is fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

[0135] The term "alkyl" as used in this invention includes straight-chain or branched alkyl groups. C1-C6 alkyl groups as used in this invention refer to alkyl groups with 1-6 carbon atoms, preferably methyl, ethyl, n-propyl or isopropyl, n-butyl, isobutyl or tert-butyl. The alkyl groups in the compounds of this invention can be optionally substituted or unsubstituted, and the substituents can include alkyl, halogen, alkoxy, haloalkyl, cyano, hydroxyl, etc. Examples of alkyl groups in this invention include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc.

[0136] The "alkenyl" in this invention refers to a straight-chain or branched monovalent hydrocarbon group with 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one position is unsaturated, i.e., one CC is sp. 2 The double bond, wherein the alkenyl group may be independently and optionally replaced by one or more substituents described in this invention, including groups having "trans", "cis" or "E", "Z" orientations, wherein the alkenyl group may be C2-C6 alkenyl, specific examples including, but not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), etc.

[0137] The "alkynyl" in this invention refers to a straight-chain or branched monovalent hydrocarbon group with 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one position is unsaturated, i.e., one C C is an sp triple bond, wherein the alkynyl group can be independently and optionally replaced by one or more substituents described in this invention, wherein the alkynyl can be a C2-C6 alkynyl, specific examples include, but are not limited to, ethynyl (-C≡CH), propynyl (-CH2C≡CH), etc.

[0138] The "alkoxy group" mentioned in this invention refers to the group formed by the above-mentioned alkyl group and oxygen atom, wherein the oxygen atom has the ability to form bonds freely, such as "C1-C6 alkoxy group", specifically such as methoxy, ethoxy, n-propoxy, n-butoxy, isopropoxy, tert-butoxy, cyclopropoxy, etc.

[0139] The "alkylamino" mentioned in this invention refers to the group formed by the above-mentioned alkyl group and amino group, such as "C1-C6 alkylamino", specifically methylamino, ethylamino, dimethylamino, methylisopropylamino, etc.

[0140] The "halogenated C1-C6 alkyl" and "halogenated C1-C6 alkoxy" of this invention refer to alkyl and alkoxy groups in which one or more hydrogen atoms are substituted with halogen atoms, particularly fluorine or chlorine atoms. In some embodiments, fluorination is preferred, for example, -CF3, -CHF2, -CH2F, -CH2CH2F, -CH2CHF2, -CH2CF3, -OCF3, -OCHF2, -OCH2F, -OCH2CH2F, -OCH2CHF2, or -OCH2CF3.

[0141] The "cycloalkyl" mentioned in this invention refers to a hydrocarbon monocyclic structure having a specified number of ring carbon atoms, wherein it does not contain unsaturated bonds such as double bonds, and includes C3-C... 14 Cycloalkyl, C3-C6 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The cycloalkyl in the compounds of this invention may be optionally substituted or unsubstituted, and the substituents may include alkyl, halogen, alkoxy, hydrocarbon, hydroxyl, etc.

[0142] The term "heterocycle" as used in this invention refers to unsubstituted or substituted monocyclic or polycyclic non-aromatic ring systems containing one or more heteroatoms, and is either partially unsaturated or fully saturated. Preferred heteroatoms include N, O, and S. Monocyclic heterocycles include, but are not limited to, pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, dihydroimidazoyl, dihydrofuranyl, piperidinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclic groups include spirocyclic, bridged, and fused-ring heterocyclic groups, wherein the heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring.

[0143] From all the above descriptions, it will be apparent to those skilled in the art that the name is any group of a compound name, such as "C2-C6 alkenyl C1-C6 alkyl," and should generally refer to a construction derived therefrom, for example, from a C1-C6 alkyl group substituted with a C2-C6 alkenyl group, wherein the C2-C6 alkenyl group and the C1-C6 alkyl group are as defined above. Other similar compound names include "C2-C6 ynyl C1-C6 alkyl," "C3-C," etc. 14 The term "cycloalkyl-C1-C4 alkyl" can be understood with reference to the aforementioned content.

[0144] Unless otherwise specifically defined, "substituted" as used herein refers to a group in which one or more hydrogen atoms can be independently substituted by a corresponding number of substituents. Those skilled in the art can determine (experimentally or theoretically) possible or impossible substitution positions without much effort. The substituents referred to as "substituted" include, but are not limited to: cyano, carboxyl, halogen, hydroxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, mono-C1-C6 alkylamino-C1-C6 alkyl, bis-C1-C6 alkylamino-C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, halo-C1-C6 alkoxy-C1-C6 alkyl, C3-C6 alkyl, C4-C6 alkyl, C5-C6 alkyl, C6-C6 alkyl, C6-C6 alkyl, C3-C6 alkyl, C4-C6 alkyl, C6 ... 14 Cycloalkyl, 3- to 14-membered heterocycloalkyl, C3-C 14 Cycloalkyl-C1-C4 alkyl, 3- to 14-membered heterocycloalkyl-C1-C4 alkyl.

[0145] When the stereoisomers of the compounds described herein are specifically designated by chemical name as (R)- or (S)- isomers, they should be understood as having a predominant configuration of (R)- or (S)-, respectively. Any asymmetric carbon atom may exist in (R)-, (S)-, or (R, S)- configurations, preferably in the (R)- or (S)- configuration.

[0146] The "pharmaceutically acceptable salt" as described in this invention refers to an acid addition salt obtained by reacting the compounds of this invention with a pharmaceutically acceptable acid, or a salt formed by reacting a compound having an acidic group with a basic compound. Preferably, the acid is selected from inorganic acids (such as hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid) and organic acids (such as oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, lysine, histidine, citric acid, or benzoic acid); the basic compound is preferably selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, or potassium bicarbonate. The above-mentioned pharmaceutically acceptable salts are easily separated and can be purified using conventional separation methods, such as solvent extraction, dilution, recrystallization, column chromatography, and preparative thin-layer chromatography.

[0147] For the compound of formula (I) of the present invention If m is 1, then the structural formula of the compound shown in equation (I) is: Other similar definitions can be understood by referring to the foregoing content.

[0148] Another aspect of the present invention relates to pharmaceutical compositions containing the above-described compound, or isomers thereof, pharmaceutically acceptable salts, precursors and metabolites as active ingredients.

[0149] The compounds described in this invention may optionally be used in combination with one or more other active ingredients, and the dosage and ratio of each ingredient may be adjusted by those skilled in the art according to the specific disease, the patient's specific condition, and clinical needs.

[0150] The compounds of general formula (I) described in this invention, or their isomers, pharmaceutically acceptable salts, precursors and metabolites, can be prepared by those skilled in the art (based on experience or references).

[0151] When the structural formula of a compound of general formula (I) described in this invention does not match its Chinese name, the chemical structural formula shall prevail.

[0152] Therefore, another aspect of the present invention also provides a method for preparing the compound according to the present invention.

[0153] The following synthetic routes describe the preparation of compounds of formula (I) of the present invention. The raw materials, reagents, catalysts, solvents, etc., used in the following synthetic diagrams can be prepared by methods well known to those skilled in the art of organic chemistry or are commercially available. All final derivatives of the present invention can be obtained by the methods described in the diagrams or similar methods, which are well known to those skilled in the art of organic chemistry. All variables used in these diagrams are defined in the context.

[0154] Preparation method

[0155] The definitions of the variables below are as described above, while the definitions of the new variables are as described in this section. Furthermore, the compounds described in general formula (I) and the intermediates involved can all be purified using common separation methods, such as extraction, recrystallization, and silica gel column chromatography. The 200-300 mesh silica gel and thin-layer chromatography silica gel plates used were produced by Qingdao Marine Chemical Plant. The chemical reagents used were commercially available analytical grade or chemically pure reagents, and were used without further purification.

[0156] This invention provides a method for preparing a compound of general formula (I), comprising the following steps:

[0157]

[0158] 1) The compound shown in formula (Ia) loses its protecting group P by first acid, first base or catalytic hydrogenolysis in the first solvent. 1 Without separation and purification, the compound shown in formula (Ib) was further reacted with the compound in a second solvent and in the presence of a first catalyst and a second base to undergo a Suzuki reaction to give the compound shown in formula (Ic).

[0159] 2) In a third solvent, the compounds shown in formula (Ic) and (Id) undergo a reductive amination reaction in the presence of a first reducing agent to give the compound shown in formula (Ie);

[0160] 3) In a fourth solvent, the compound shown in formula (Ie) and the compound shown in formula (If) undergo a reductive amination reaction in the presence of a second reducing agent to give the compound shown in formula (I');

[0161] Optionally, it also includes 4) in the presence of a third base, the compound shown in formula (I') undergoes an ester hydrolysis reaction to give the compound shown in formula (I”).

[0162] The compound shown in formula (I') can be used as the final product formula (I). Alternatively, the compound shown in formula (I') can be further subjected to ester hydrolysis under alkaline conditions to obtain the compound shown in formula (I”), which can also be used as the final product formula (I).

[0163] in:

[0164] X, R 1 R 2 R 3 The definition is as stated above;

[0165] m' is selected from 0, 1, and 2, with a preference for 0 and 1;

[0166] n' is selected from 0, 1, and 2;

[0167] R 4' Except that it is not hydrogen, its definition is the same as R. 4 Same, R 4 The definition is as stated above;

[0168] R 4” It is hydrogen;

[0169] M is selected from borate esters or boric acids, including but not limited to 4,4,5,5-tetramethyl-1,3,2-dioxapentylborane, neopentyl glycol diborate, 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxacyclopentylborane)B(OBu-n)3, B(OPr-i)3; or...

[0170] M is selected from bromine, iodine, chlorine, and CF3SO3-(OTf);

[0171] W is selected from borate esters or boric acids, including but not limited to 4,4,5,5-tetramethyl-1,3,2-dioxapentylborane, neopentyl glycol diborate, 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxacyclopentylborane)B(OBu-n)3, B(OPr-i)3; or...

[0172] W is selected from bromine, iodine, chlorine, and CF3SO3-(OTf);

[0173] P 1 and P 2 It is a protecting group, which may be the same as or different from Boc (tert-butyloxycarbonyl), Fmoc (9-fluorenmethoxycarbonyl), Cbz (N-benzyloxycarbonyl), methanesulfonyl, p-toluenesulfonyl, acetyl, methoxycarbonyl, ethoxycarbonyl, ((2-trimethylsilyl)ethoxy)methyl (SEM), and tetrahydro-2H-pyran-2-yl (THP).

[0174] In some embodiments, the first acid includes, but is not limited to, trifluoroacetic acid (TFA), hydrochloric acid (HCl), acetic acid (HOAc), and hydrobromic acid (HBr);

[0175] The first base includes, but is not limited to, piperidine and diethylamine;

[0176] The first solvent includes, but is not limited to, dichloromethane (DCM), 1,2-dichloroethane, methanol (MeOH), ethanol (EtOH), 1,4-dioxane, tetrahydrofuran (THF), acetonitrile (MeCN), and N,N'-dimethylformamide (DMF).

[0177] In some embodiments, the first catalyst includes, but is not limited to, 1,1'-bis(dicyclohexylphosphino)ferrocene palladium dichloride (PdCl2(dcypf)), palladium acetate (Pd(OAc)2), palladium dichloride (PdCl2), tris(dibenzylacetone)palladium (Pd2(dba)3), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (PdCl2(dppf)), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (PdCl2(dppf)·CH2Cl2), tetra(triphenylphosphine)palladium (Pd(PPh3)4), bis(tricyclohexylphosphine)palladium dichloride (PdCl2(P(Cy)3)2), and 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (SPhos);

[0178] The second base includes organic and inorganic bases, such as triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), n-butyllithium, diisopropylaminolithium, bistrimethylsilylaminolithium, potassium acetate (KOAc), sodium tert-butoxide (NaOBu-t), potassium tert-butoxide (KOBu-t), sodium hydride (NaH), potassium phosphate (K3PO4), sodium carbonate (Na2CO3), potassium carbonate (K2CO3), lithium hydroxide (KOH), and sodium hydroxide (NaOH);

[0179] The second solvent includes, but is not limited to, 1,4-dioxane, tetrahydrofuran (THF), acetonitrile (MeCN), N,N'-dimethylformamide (DMF), and mixed solvents formed by these solvents and water in different proportions.

[0180] In some embodiments, the first reducing agent and the second reducing agent include, but are not limited to, sodium borohydride acetate, sodium borohydride, and sodium cyanoborohydride.

[0181] The third and fourth solvents include, but are not limited to, dichloromethane (DCM), 1,2-dichloroethane, methanol (MeOH), ethanol (EtOH), 1,4-dioxane, tetrahydrofuran (THF), acetonitrile (MeCN), and N,N'-dimethylformamide (DMF).

[0182] In some embodiments, the third base includes, but is not limited to, lithium hydroxide (LiOH), lithium hydroxide (KOH), and sodium hydroxide (NaOH).

[0183] Specifically, the compound shown in formula (Ia) can have its protecting group P removed by appropriate acid, base, or catalytic hydrogenolysis in a suitable solvent. 1Without separation and purification, the compound is further reacted with the compound shown in formula (Ib) under suitable alkaline conditions and solvent, and in the presence of a catalyst via a Suzuki reaction to obtain the compound shown in formula (Ic). Suitable solvents for deprotection include, but are not limited to, dichloromethane (DCM), 1,2-dichloroethane, methanol (MeOH), ethanol (EtOH), 1,4-dioxane, tetrahydrofuran (THF), acetonitrile (MeCN), and N,N'-dimethylformamide (DMF); the acids include, but are not limited to, trifluoroacetic acid (TFA). The bases include, but are not limited to, piperidine and diethylamine; the catalysts include, but are not limited to, 1,1'-bis(dicyclohexylphosphino)ferrocene palladium dichloride (PdCl2(dcypf)), palladium acetate (Pd(OAc)2), palladium dichloride (PdCl2), tris(dibenzylacetone)palladium (Pd2(dba)3), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (PdCl2(dppf)), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (PdCl2(dppf)). The reagents used in the alkaline conditions include palladium chloride dichloromethane complex (PdCl2(dppf)·CH2Cl2), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), bis(tricyclohexylphosphine)palladium dichloride (PdCl2(P(Cy)3)2), and 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (SPhos); the reagents used in the alkaline conditions include organic and inorganic bases, including, but not limited to, triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and potassium acetate (KOAc). Sodium tert-butoxide (NaOBu-t), potassium tert-butoxide (KOBu-t), sodium hydride (NaH), potassium phosphate (K3PO4), sodium carbonate (Na2CO3), potassium carbonate (K2CO3), lithium hydroxide (KOH), and sodium hydroxide (NaOH); the solvents that undergo the Suzuki reaction include, but are not limited to, 1,4-dioxane, tetrahydrofuran (THF), acetonitrile (MeCN), N,N'-dimethylformamide (DMF), and mixed solvents formed by these solvents and water in different proportions;

[0184] The compound shown in formula (Ic) undergoes a reductive amination reaction with the compound shown in formula (Id) in the presence of a suitable solvent and a reducing agent to obtain the compound shown in formula (Ie). The reducing agent includes, but is not limited to, sodium borohydride acetate, sodium borohydride, and sodium cyanoborohydride. The solvent includes, but is not limited to, dichloromethane (DCM), 1,2-dichloroethane, methanol (MeOH), ethanol (EtOH), 1,4-dioxane, tetrahydrofuran (THF), acetonitrile (MeCN), and N,N'-dimethylformamide (DMF).

[0185] The compound shown in formula (Ie) can have its protecting group P removed by appropriate acid, base, or catalytic hydrogenolysis in a suitable solvent. 2 Without separation and purification, the compound is further subjected to a reducing amination reaction with the compound shown in formula (If) in the presence of a suitable solvent and reducing agent to obtain the compound shown in formula (I'). The acid includes, but is not limited to, trifluoroacetic acid (TFA), hydrochloric acid (HCl), acetic acid (HOAc), and hydrobromic acid (HBr); the base includes, but is not limited to, piperidine and diethylamine; the reducing agent includes, but is not limited to, sodium borohydride acetate, sodium borohydride, and sodium cyanoborohydride; the solvent includes, but is not limited to, dichloromethane (DCM), 1,2-dichloroethane, methanol (MeOH), ethanol (EtOH), 1,4-dioxane, tetrahydrofuran (THF), acetonitrile (MeCN), and N,N'-dimethylformamide (DMF).

[0186] The compound shown in formula (I') can be used as the final product formula (I). Furthermore, the compound shown in formula (I') can be further subjected to ester hydrolysis under alkaline conditions to obtain the compound shown in formula (I”), which can also be used as the final product formula (I). The alkaline is, but is not limited to, lithium hydroxide (LiOH), lithium hydroxide (KOH), and sodium hydroxide (NaOH).

[0187] M is selected from borate esters or boric acids, including but not limited to 4,4,5,5-tetramethyl-1,3,2-dioxapentylborane, neopentyl glycol diborate, 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxacyclopentylborane)B(OBu-n)3, B(OPr-i)3; or...

[0188] M is selected from bromine, iodine, chlorine, and CF3SO3-(OTf);

[0189] W is selected from borate esters or boric acids, including but not limited to 4,4,5,5-tetramethyl-1,3,2-dioxapentylborane, neopentyl glycol diborate, 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxacyclopentylborane)B(OBu-n)3, B(OPr-i)3; or...

[0190] W is selected from bromine, iodine, chlorine, and CF3SO3-(OTf);

[0191] P 1 and P 2It is a protecting group, which may be the same as or different from Boc (tert-butyloxycarbonyl), Fmoc (9-fluorenmethoxycarbonyl), Cbz (N-benzyloxycarbonyl), methanesulfonyl, p-toluenesulfonyl, acetyl, methoxycarbonyl, ethoxycarbonyl, ((2-trimethylsilyl)ethoxy)methyl (SEM), and tetrahydro-2H-pyran-2-yl (THP).

[0192] The present invention also provides another method for preparing the compound represented by general formula (I), which includes the following steps:

[0193]

[0194] 1) The compound shown in formula (Ia) loses its protecting group P by first acid, first base or catalytic hydrogenolysis in the first solvent. 1 Without separation and purification, the compound shown in formula (Ib) was further reacted with the compound in a second solvent and in the presence of a first catalyst and a second base to undergo a Suzuki reaction to give the compound shown in formula (Ic).

[0195] 2) In a fourth solvent, the compound shown in formula (Ic) and the compound shown in formula (If) undergo a reductive amination reaction in the presence of a second reducing agent to give the compound shown in formula (Ih);

[0196] 3) In a third solvent, the compound shown in formula (Ih) and the compound shown in formula (Id) undergo a reductive amination reaction in the presence of a first reducing agent to give the compound shown in formula (I');

[0197] Optionally, it also includes 4) in the presence of a third base, the compound shown in formula (I') undergoes an ester hydrolysis reaction to give the compound shown in formula (I”).

[0198] The compound shown in formula (I') can be used as the final product formula (I). Alternatively, the compound shown in formula (I') can be further subjected to ester hydrolysis under alkaline conditions to obtain the compound shown in formula (I”), which can also be used as the final product formula (I).

[0199] in:

[0200] X, R 1 R 2 R 3 The definition is as stated above;

[0201] m'、n'、R 4’ M, W, P 1 and P 2 The definition is as stated above;

[0202] The first acid, the first base, and the first solvent are as described above;

[0203] The first catalyst, the second base, and the second solvent are as described above;

[0204] The first reducing agent, the second reducing agent, the third solvent, and the fourth solvent are as described above;

[0205] The third alkali is as described above.

[0206] Specifically, the compound preparation method of the present invention can also be achieved by removing the protecting group P from the compound shown in formula (Ia). 1 Without separation and purification, the compound is further reacted with the compound shown in formula (Ib) in the Suzuki reaction as described above to obtain the compound shown in formula (Ic); the compound shown in formula (Ic) is first reacted with the compound shown in formula (If) in the reductive amination reaction as described above to obtain the compound shown in formula (Ih), and then the compound shown in (Ih) is deprotected and then reacted with the compound shown in formula (Id) in the reductive amination reaction as described above to obtain the compound shown in formula (I'), or optionally further reacted with the ester hydrolysis reaction as described above to obtain the compound shown in formula (I”). The substituents are defined as described above; the compounds shown in formulas (I') and (I”) can be used as the final product formula (I).

[0207] The present invention further provides another method for preparing a compound of general formula (I), which includes the following steps:

[0208]

[0209] 1) The compound shown in formula (Ia) loses its protecting group P by first acid, first base or catalytic hydrogenolysis in the first solvent. 1 Without separation and purification, the compound shown in formula (Ib) was further reacted with the compound in a second solvent and in the presence of a first catalyst and a second base to undergo a Suzuki reaction to give the compound shown in formula (Ic).

[0210] 2) In a fourth solvent, the compound shown in formula (Ic) and the compound shown in formula (If) undergo a reductive amination reaction in the presence of a second reducing agent to give the compound shown in formula (Ih);

[0211] 3) In the presence of a third base, the compound shown in formula (Ih) undergoes ester hydrolysis to yield the compound shown in formula (Ii);

[0212] 4) In a third solvent, the compounds shown in formula (Ii) and (Id) undergo a reductive amination reaction in the presence of a first reducing agent to give the compound shown in formula (I”’).

[0213] The compound shown in formula (I”') above can be used as the final product formula (I).

[0214] in:

[0215] X, R 1 R 2 R 3 The definition is as stated above;

[0216] m'、n'、R 4’ M, W, P 1 and P 2 The definition is as stated above;

[0217] The first acid, the first base, and the first solvent are as described above;

[0218] The first catalyst, the second base, and the second solvent are as described above;

[0219] The first reducing agent, the second reducing agent, the third solvent, and the fourth solvent are as described above; the third base is as described above.

[0220] Specifically, the compound preparation method of the present invention can also be achieved by removing the protecting group P from the compound shown in formula (Ia). 1 Without separation and purification, the compound is further reacted with the compound shown in formula (Ib) in the Suzuki reaction as described above to obtain the compound shown in formula (Ic); the compound shown in formula (Ic) is first reacted with the compound shown in formula (If) in the reductive amination reaction as described above to obtain the compound shown in formula (Ih), then the compound shown in formula (Ih) undergoes the ester hydrolysis reaction as described above to obtain the compound shown in formula (Ii), and further, the compound shown in formula (Ii) is deprotected and then reacted with the compound shown in formula (Id) in the reductive amination reaction as described above to obtain the compound shown in formula (I”'). The definitions of each substituent are as described above; the compound shown in formula (I”') can be used as the final product formula (I).

[0221] In addition, the present invention provides the use of the aforementioned compounds or their stereoisomers, pharmaceutically acceptable salts, precursors and metabolites, or the aforementioned pharmaceutical compositions in the preparation of medicaments for treating and / or preventing diseases related to the target PD-L1, or

[0222] Use in the preparation of drugs for inhibiting PD-L1 activity, or

[0223] Use in the preparation of drugs as PD-L1 inhibitors, or

[0224] Use in the preparation of drugs as immunomodulators targeting the PD-L1 signaling pathway.

[0225] In some implementations, the diseases associated with targeting PD-L1 include tumors, cancer, or other immune-related diseases.

[0226] In another aspect, the present invention provides the aforementioned compounds or stereoisomers thereof, pharmaceutically acceptable salts, precursors and metabolites, or the aforementioned pharmaceutical compositions for the treatment and / or prevention of diseases related to the target PD-L1, or

[0227] Used to inhibit PD-L1 activity, or

[0228] As a PD-L1 inhibitor, or

[0229] As an immunomodulator targeting the PD-L1 signaling pathway.

[0230] In some implementations, the diseases associated with targeting PD-L1 include tumors, cancer, or other immune-related diseases.

[0231] In another aspect, the present invention provides a method for treating and / or preventing diseases associated with the target PD-L1, comprising administering to a subject in need a therapeutic and / or preventative effective amount of the aforementioned compound or its stereoisomers, pharmaceutically acceptable salts, precursors and metabolites, or the aforementioned pharmaceutical compositions.

[0232] The present invention also provides a method for inhibiting PD-L1 activity, comprising administering to cells (e.g., mammalian cells) an effective amount of the aforementioned compound or its stereoisomer, pharmaceutically acceptable salt, precursor and metabolite, or the aforementioned pharmaceutical composition.

[0233] In some implementations, the diseases associated with targeting PD-L1 include tumors, cancer, or other immune-related diseases.

[0234] In this invention, "subject" refers to a vertebrate. In some embodiments, vertebrate refers to a mammal. The mammal includes bovines, equines, sheep, suidae, canines, felines, rodents, and primates, such as humans, cats, dogs, or pigs. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, mammal refers to a human.

[0235] In this invention, the terms "therapeutic effective amount" or "preventive effective amount" refer to an amount sufficient, within reasonable medical judgment, to treat or prevent a patient's disease while avoiding serious side effects with a sufficiently low risk-reward ratio. The therapeutic effective amount of a compound will vary depending on factors such as the specific compound chosen (e.g., considering the compound's potency, effectiveness, and half-life), the chosen route of administration, the disease being treated, the severity of the disease, the patient's age, size, weight, and physical condition, the patient's medical history, the duration of treatment, the nature of concurrent therapies, and the desired therapeutic effect, but can still be conventionally determined by those skilled in the art.

[0236] It should also be noted that the specific dosage and method of administration of the compound or its stereoisomers, pharmaceutically acceptable salts, precursors, and metabolites for different patients depend on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, drug activity, timing of administration, metabolic rate, severity of illness, and the subjective judgment of the treating physician. The preferred dosage is between 0.001 and 1000 mg / kg body weight / day.

[0237] This invention provides a novel biphenyl-based small molecule immunosuppressant with excellent oral absorption characteristics for the treatment or prevention of immune-related diseases. Furthermore, these compounds, or pharmaceutical compositions containing them as active ingredients, can maximize clinical efficacy against these diseases within a safe therapeutic window. Attached Figure Description

[0238] Figure 1 This indicates the plasma and tumor tissue distribution of compound I-6 from Examples;

[0239] Figure 2 This shows the tumor tissue distribution of compounds I-6 and control molecules in the examples;

[0240] Figure 3 The tumor growth of compounds I-6 and control molecules in the examples is shown. Detailed Implementation

[0241] The embodiments and preparation examples provided in this invention further illustrate and demonstrate the compounds and their preparation methods described herein. It should be understood that the following preparation examples and embodiments do not limit the scope of this invention in any way.

[0242] LC-MS analysis method:

[0243] Mass spectrometry conditions: Instrument: Thermo MSQ Plus; Ion source: ESI (EA+EA-); Cone voltage: 30V; Capillary voltage: 3.00KV; Source temperature: 350℃;

[0244] Chromatographic conditions: Instrument: Thermo U3000; Detector: DAD-3000(RS) (diode array detector); Column: Shimadzu Inertsil ODS-HL HP 3μm 3.0×100mm; Flow rate: 0.4mL / min; Column temperature: 30℃; Mobile phase: CH3OH / H2O / HCOOH (75 / 25 / 0.2).

[0245] HPLC Analysis Method (I):

[0246] Instrument: Thermo U3000; Detector: VWD-3×00(RS) (UV detector); Column: Shimadzu Shim-pack VP-ODS 5μm 4.6×150mm; Flow rate: 1.0mL / min; Column temperature: 30℃; Mobile phase: ACH3OH / H2O / TEA / HOAc (65 / 35 / 0.2 / 0.1).

[0247] HPLC Analysis Method (II):

[0248] Instrument: Thermo U3000; Detector: VWD-3×00(RS) (UV detector); Column: Shimadzu Shim-pack VP-ODS 5μm 4.6×150mm; Flow rate: 1.0mL / min; Column temperature: 30℃; Mobile phase: BCH3OH / H2O / TEA / HOAc (80 / 20 / 0.2 / 0.1).

[0249] 1 H-NMR analysis method:

[0250] 1 H-NMR was performed at room temperature using a BRUKER AVANCE-400MHz nuclear magnetic resonance spectrometer in DMSO-d6 or CDCl3 with TMS as an internal standard. Signal peaks were represented as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and dd (doublet). The coupling constant (J) is expressed in Hertz (Hz).

[0251] Key abbreviations:

[0252] 1,4-dioxane; DCM (dichloromethane)

[0253] MeOH (methanol); EtOAc (ethyl acetate)

[0254] TEA Triethylamine DIPEA N,N'-Diisopropylethylamine

[0255] TFA (trifluoroacetic acid); THF (tetrahydrofuran)

[0256] NaHCO3, sodium bicarbonate, Na2SO4, sodium sulfate

[0257] NaBH(OAc)3 sodium borohydride acetate; LiOH·H2O hydrated lithium hydroxide

[0258] PdCl2(dcypf) 1,1'-bis(dicyclohexylphosphino)ferrocene palladium dichloride

[0259] TLC (Thin Layer Chromatography)

[0260] Following the methods described above, representative compounds such as I-1—I-12 were prepared in this invention (see Table 1).

[0261] Table 1. Representative compounds I-1—I-12 described in this invention

[0262]

[0263] The invention is further illustrated below with specific examples, but the scope of protection of the invention is not limited to these examples. Unless otherwise specified, all percentages mentioned in this invention are weight percentages. The numerical ranges described in the specification, such as units of measurement, reaction conditions, physical states of compounds, or percentages, are provided for clear and unambiguous written reference. Those skilled in the art may still obtain the expected results by using temperatures, concentrations, quantities, carbon numbers, etc., outside these ranges or different from individual values ​​when implementing this invention. Furthermore, unless otherwise specified, the raw materials used in the following examples are commercially available, for example, from Shanghai Bied Pharmaceutical Technology Co., Ltd., Jiangsu Aikon Biomedical R&D Co., Ltd., Nanjing Yaoshi Technology Co., Ltd., Shanghai Shaoyuan Reagent Co., Ltd., and Hechun Biotechnology (Shanghai) Co., Ltd.

[0264] Example 1

[0265] 4-(2-(2-((2'-chloro-3'-(5-(3-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)propyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-1

[0266]

[0267]

[0268] Preparation of intermediate: 2-((2'-chloro-2-methyl-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-carboxylic acid tert-butyl ester I-1c

[0269] 2-((3-bromo-2-chlorophenyl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-1a (1.18 g, 2.52 mmol, 1.0 eq, synthesis reference CN202010997428.3) was dissolved in DCM (10 mL), TFA (10 mL) was added, and the mixture was stirred at ambient temperature for 1 h. The reaction solution was concentrated, and the residue was dissolved in 1,4-dioxane (10 mL). 1-Methyl-2-((2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)carbamoyl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-1b (1.25 g, 2.52 mmol, 1.0 eq, synthesis reference CN202010997428.3), PdCl2 (dcypf) (188.75 mg, 0.25 mmol, 0.1 eq), anhydrous Na2CO3 (801.36 mg, 7.56 mmol, 3.0 eq), and water (5 mL) were added. The resulting mixture was heated to 110 °C in a microwave oven and reacted for 1 h, then cooled to ambient temperature. The concentrated reaction solution and crude product were separated by silica gel column chromatography (DCM / MeOH (v / v) = 15 / 1) to give a pale yellow solid I-1c (1.08 g, yield 65.1%). LC-MS MS-ESI (m / z) 659.2 [M+H) + .

[0270] Preparation of intermediate: 2-((2'-chloro-3'-(5-(3-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)propyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-1e

[0271] Intermediate I-1c (132.00 mg, 0.20 mmol, 1.0 eq) was dissolved in DCM (10 mL), followed by TEA (1 mL) and 2-(4-(methoxymethyl)bicyclo[2.2.1]heptan-1-yl)acetaldehyde I-1d (43.20 mg, 0.22 mmol, 1.1 eq, synthesis reference CN202010997428.3). The resulting mixture was stirred at ambient temperature for 1 h, then NaBH(OAc)3 (212.00 mg, 1.00 mmol, 5.0 eq) was added and stirring continued for 16 h. The reaction solution was quenched with saturated NaHCO3 solution and extracted three times with DCM / MeOH (10 / 1, 100 mL). The organic phases were combined, dried over anhydrous Na2SO4, and concentrated. The crude product was separated by preparative TLC (DCM / MeOH (v / v) = 8 / 1) to give a pale yellow solid I-1e (115.00 mg, yield 68.5%). LC-MS MS-ESI (m / z) 839.5 [M+H) + .

[0272] Preparation of intermediate: methyl 4-(2-(2-((2'-chloro-3'-(5-(3-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)propyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid ester I-1g

[0273] Intermediate I-1e (115.00 mg, 0.13 mmol, 1.0 eq) was dissolved in DCM (10 mL), and TFA (10 mL) was added. The resulting solution was stirred at ambient temperature for 1 h. The reaction solution was concentrated, and the residue was dissolved in DCM (10 mL) and concentrated again. The resulting yellow solid was used directly in the next stage. Trifluoroacetate was dissolved in DCM (10 mL), and TEA (1 mL) and commercially available methyl 4-(2-oxoethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-1f (29.56 mg, 0.15 mmol, 1.1 eq) were added. The resulting mixture was stirred at ambient temperature for 1 h, and NaBH(OAc)3 (145.22 mg, 0.68 mmol, 5.0 eq) was added. Stirring was continued for 16 h. The reaction solution was quenched with saturated NaHCO3 solution and extracted three times with DCM / MeOH (10 / 1, 100 mL). The organic phases were combined, dried over anhydrous Na₂SO₄, and concentrated. The crude product was separated by preparative TLC (DCM / MeOH (v / v) = 6 / 1) to give 1 g (86.00 mg, yield 68.2%). LC-MS MS-ESI (m / z) 919.6 [M+H) + .

[0274] Preparation of compound: 4-(2-(2-((2'-chloro-3'-(5-(3-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)propyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-1

[0275] Intermediate I-1 g (86.00 mg, 0.09 mmol, 1.0 eq) was dissolved in THF (10 mL), and water (10 mL) and LiOH·H₂O (75.60 mg, 1.80 mmol, 20.0 eq) were added. The resulting solution was stirred at ambient temperature for 16 h. THF was removed by concentration, and the pH was adjusted to 5-6 with 1 M hydrochloric acid. The solid was collected by filtration and dried to give a yellow solid I-1 (56.00 mg, yield 66.1%). LC-MS-ESI (m / z) 905.6 [M+H] + . 1H-NMR(400MHz,DMSO-d6)δppm 9.90(s,1H),9.74(s,1H),8.34(d,J=8.3Hz,1H),7.72(d,J=8.1Hz,1H),7.46(t,J=7.9Hz,1H) ,7.30(t,J=7.8Hz,1H),7.07(d,J=7.6Hz,1H),7.01(d,J=7.4Hz,1H),3.90(s,3H),3.87(s,3H) ,3.41-3.38(m,4H),3.33(s,2H),3.23(s,3H),2.81-2.70(m,4H),2.70-2.61(m,4H),2.58-2.4 3(m,4H),1.99(s,3H),1.88-1.79(m,2H),1.74-1.65(m,2H),1.57-1.20(m,20H),1.08(s,2H).

[0276] Example 2

[0277] 4-(2-(2-((2-chloro-3'-(5-(3-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)propyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-2

[0278]

[0279] Preparation of intermediate: 2-((2'-chloro-3'-(5-(2-(4-(methoxycarbonyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-1h

[0280] Intermediate I-1h was prepared from intermediate I-1c (720.00 mg, 1.09 mmol, 1.0 eq), TEA (1 mL), methyl 4-(2-oxoethyl)bicyclo[2.2.1]heptane-1-carboxylate I-1f (320.46 mg, 1.63 mmol, 1.5 eq), and NaBH(OAc)3 (1.39 g, 6.54 mmol, 6.0 eq) following a similar procedure to intermediate I-1e. (563.00 mg, yield 61.5%). LC-MS MS-ESI (m / z) 839.4 [M+H] + .

[0281] Preparation of intermediate: methyl 4-(2-(2-((2-chloro-3'-(5-(3-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)propyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid ester I-2g

[0282] Intermediate I-2g was prepared from intermediate I-1h (95.00 mg, 0.11 mmol, 1.0 eq), TFA (5 mL), TEA (1 mL), 2-(4-(methoxymethyl)bicyclo[2.2.1]heptan-1-yl)acetaldehyde I-1d (24.42 mg, 0.12 mmol, 1.1 eq), and NaBH(OAc)3 (120.00 mg, 0.56 mmol, 5.0 eq) following similar steps to those in intermediate I-1g. (75.00 mg, yield 72.1%). LC-MS MS-ESI (m / z) 919.5 [M+H] + .

[0283] Preparation of compound: 4-(2-(2-((2-chloro-3'-(5-(3-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)propyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-2

[0284] White solid I-2 was prepared from intermediate I-2 g (75.00 mg, 0.08 mmol, 1.0 eq) and LiOH·H₂O (67.20 mg, 1.60 mmol, 20.0 eq) using a similar procedure to that in compound I-1. (27.00 mg, yield 36.5%). LC-MS-ESI (m / z) 905.6 [M+H] + . 1 H-NMR(400MHz,DMSO-d6)δppm 9.91(s,1H),9.73(s,1H),8.34(d,J=8.3Hz,1H),7.72(d,J=8.1Hz,1H),7.46(t,J=7.9Hz,1 H),7.30(t,J=7.8Hz,1H),7.06(d,J=7.6Hz,1H),7.01(d,J=7.4Hz,1H),3.90(s,3H),3.87( s,3H),3.42-3.33(m,6H),3.23(s,3H),2.80-2.71(m,4H),2.70-2.60(m,4H),2.57-2.42(m ,4H),1.98(s,3H),1.88-1.78(m,2H),1.74-1.65(m,2H),1.57-1.20(m,20H),1.08(s,2H).

[0285] Example 3

[0286] 4-(2-(2-((2'-chloro-3'-(5-(2-(4-(ethoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-3

[0287]

[0288] Preparation of intermediate: 2-((2'-chloro-3'-(5-(2-(4-(ethoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-3e

[0289] Intermediate I-3e was prepared from intermediate I-1c (700.00 mg, 1.06 mmol, 1.0 eq), TEA (1 mL), 2-(4-(ethoxymethyl)bicyclo[2.2.1]heptan-1-yl)acetaldehyde I-3d (312.12 mg, 1.59 mmol, 1.5 eq, synthesis reference CN202010997428.3), and NaBH(OAc)3 (1.35 g, 6.36 mmol, 6.0 eq) following similar steps to intermediate I-1e. (595.00 mg, yield 66.9%). LC-MS MS-ESI (m / z) 839.5 [M+H] + .

[0290] Preparation of intermediate: methyl 4-(2-(2-((2'-chloro-3'-(5-(2-(4-(ethoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid ester I-3g

[0291] Intermediate I-3g was prepared from intermediate I-3e (595.00 mg, 0.71 mmol, 1.0 eq), TFA (5 mL), TEA (1 mL), methyl 4-(2-oxoethyl)bicyclo[2.2.1]heptane-1-carboxylate I-1f (209.72 mg, 1.07 mmol, 1.5 eq), and NaBH(OAc)3 (903.12 mg, 4.26 mmol, 6.0 eq) following similar steps to those in intermediate I-1g. (450.00 mg, yield 68.9%). LC-MS MS-ESI (m / z) 919.6 [M+H] + .

[0292] Preparation of compound: 4-(2-(2-((2'-chloro-3'-(5-(2-(4-(ethoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-3

[0293] The pale yellow solid I-3 was prepared from intermediate I-3 g (450.00 mg, 0.49 mmol, 1.0 eq) and LiOH·H₂O (411.60 mg, 9.80 mmol, 20.0 eq) using a similar procedure to that in compound I-1. (51.00 mg, yield 11.5%). LC-MS MS-ESI (m / z) 905.6 [M+H] + . 1 H-NMR(400MHz,DMSO-d6)δppm 9.90(s,1H),9.72(s,1H),8.35(d,J=8.2Hz,1H),7.74(d,J=8.0Hz,1H),7.45(t,J=7.9Hz,1H),7.30(t ,J=7.7Hz,1H),7.06(d,J=7.6Hz,1H),7.00(d,J=7.7Hz,1H),3.89(s,3H),3.87(s,3H),3.43-3.38(m, 4H),3.36(s,2H),3.34-3.30(m,2H),2.79-2.70(m,4H),2.69-2.60(m,4H),2.57-2.51(m,4H),1.99(s ,3H),1.92-1.80(m,2H),1.76-1.65(m,4H),1.58-1.42(m,10H),1.41-1.20(m,6H),1.14-1.06(m,5H).

[0294] Example 4

[0295] 4-(2-(2-((2'-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-4

[0296]

[0297] Preparation of intermediate: 2-((2'-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-4e

[0298] Intermediate I-4e was prepared from intermediate I-1c (600.00 mg, 0.91 mmol, 1.0 eq), TEA (1 mL), 2-(4-(methoxymethyl)bicyclo[2.2.1]heptan-1-yl)acetaldehyde I-4d (248.43 mg, 1.36 mmol, 1.5 eq, synthesis reference CN202010997428.3), and NaBH(OAc)3 (1.16 g, 5.46 mmol, 6.0 eq) following similar steps to intermediate I-1e. (504.00 mg, yield 67.1%). LC-MS MS-ESI (m / z) 825.5 [M+H] + .

[0299] Preparation of intermediate: methyl 4-(2-(2-((2'-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid ester I-4g

[0300] Intermediate I-4g was prepared from intermediate I-4e (504.00 mg, 0.61 mmol, 1.0 eq), TFA (5 mL), TEA (1 mL), methyl 4-(2-oxoethyl)bicyclo[2.2.1]heptane-1-carboxylate I-1f (180.32 mg, 0.92 mmol, 1.5 eq), and NaBH(OAc)3 (775.92 mg, 3.66 mmol, 6.0 eq) following similar steps to those in intermediate I-1g. (400.00 mg, yield 72.4%). LC-MS MS-ESI (m / z) 905.6 [M+H] + .

[0301] Preparation of compound: 4-(2-(2-((2'-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-4

[0302] The pale yellow solid I-4 was prepared from intermediate I-4 g (400.00 mg, 0.44 mmol, 1.0 eq) and LiOH·H₂O (369.60 mg, 8.80 mmol, 20.0 eq) using a similar procedure to that in compound I-1. (102.00 mg, yield 26.0%). LC-MS MS-ESI (m / z) 891.6 [M+H] + . 1 H-NMR(400MHz,DMSO-d6)δppm 9.90(s,1H),9.73(s,1H),8.34(d,J=8.2Hz,1H),7.73(d,J=7.9Hz,1H),7.45(t,J=7.8Hz,1H) ,7.29(t,J=7.7Hz,1H),7.06(d,J=7.3Hz,1H),7.00(d,J=7.6Hz,1H),3.89(s,3H),3.86(s,3H) ,3.42-3.38(m,4H),3.32(s,2H),3.23(s,3H),2.77-2.70(m,4H),2.68-2.60(m,4H),2.51-2.5 6(m,4H),1.98(s,3H),1.90-1.80(m,2H),1.76-1.64(m,4H),1.55-1.20(m,16H),1.11(s,2H).

[0303] Example 5

[0304] 4-(2-(2-((2-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-5

[0305]

[0306] Preparation of intermediate: methyl 4-(2-(2-((2-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid ester I-5g

[0307] Intermediate I-5g was prepared from intermediate I-1h (563.00 mg, 0.67 mmol, 1.0 eq), TFA (5 mL), TEA (1 mL), 2-(4-(methoxymethyl)bicyclo[2.2.1]heptan-1-yl)acetaldehyde I-4d (183.82 mg, 1.01 mmol, 1.5 eq, synthesis reference CN202010997428.3), and NaBH(OAc)3 (852.24 mg, 4.02 mmol, 6.0 eq) following similar steps to those in intermediate I-1g. (525.00 mg, yield 86.5%). LC-MS MS-ESI (m / z) 905.6 [M+H] + .

[0308] Preparation of compound: 4-(2-(2-((2-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-5

[0309] Off-white solid I-5 was prepared from intermediate I-5 g (525.00 mg, 0.58 mmol, 1.0 eq) and LiOH·H₂O (487.20 mg, 11.6 mmol, 20.0 eq) using a similar procedure to that in compound I-1. (116.0 mg, yield 22.4%). LC-MS MS-ESI (m / z) 891.6 [M+H] + . 1H NMR(400MHz,DMSO-d6)δppm 9.90(s,1H),9.72(s,1H),8.35(d,J=8.2Hz,1H),7.74(d,J=8.1Hz,1H),7.46(t,J=7.7Hz,1H) ,7.30(t,J=7.8Hz,1H),7.07(d,J=7.4Hz,1H),7.01(d,J=7.7Hz,1H),3.90(s,3H),3.87(s,3H) ,3.47-3.40(m,4H),3.33(s,2H),3.24(s,3H),2.79-2.70(m,4H),2.69-2.60(m,4H),2.56-2.5 1(m,4H),1.99(s,3H),1.89-1.81(m,2H),1.76-1.69(m,4H),1.58-1.22(m,16H),1.12(s,2H).

[0310] Example 6

[0311] 4-(2-(2-((2,2'-dichloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-6

[0312]

[0313] Preparation of intermediate: 2-((2,2'-dichloro-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-6c

[0314] Intermediate I-6c is composed of 2-((3-bromo-2-chlorophenyl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-1a (530.00 mg, 1.13 mmol, 1.0 eq), TFA (5 mL), 1,4-dioxane (10 mL), 2-((2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)carbamoyl)-1-methyl-1,4, 6,7-Tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-6b (583.08 mg, 1.13 mmol, 1.0 eq, synthesis reference CN202010997428.3), PdCl2 (dcypf) (83.05 mg, 0.11 mmol, 0.1 eq), anhydrous Na2CO3 (359.34 mg, 3.39 mmol, 3.0 eq), and water (5 mL) were prepared according to a similar procedure to intermediate I-1c. (363.00 mg, yield 47.3%). LC-MS MS-ESI (m / z) 679.6 [M+H]+.

[0315] Preparation of intermediate: 2-((2,2'-dichloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-6e

[0316] Intermediate I-6e was prepared from intermediate I-6c (363.00 mg, 0.53 mmol, 1.0 eq), TEA (1 mL), 2-(4-(methoxymethyl)bicyclo[2.2.1]heptan-1-yl)acetaldehyde I-4d (143.78 mg, 0.79 mmol, 1.5 eq), and NaBH(OAc)3 (674.16 mg, 3.18 mmol, 6.0 eq) using a similar procedure to that of intermediate I-1e. (338.00 mg, yield 75.4%). LC-MS MS-ESI (m / z) 845.9 [M+H] + .

[0317] Preparation of intermediate: methyl 4-(2-(2-((2,2'-dichloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido))-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid ester I-6g

[0318] Intermediate I-6g was prepared from intermediate I-6e (338.00 mg, 0.40 mmol, 1.0 eq), TFA (5 mL), TEA (1 mL), methyl 4-(2-oxoethyl)bicyclo[2.2.1]heptane-1-carboxylate I-1f (117.6 mg, 0.60 mmol, 1.5 eq), and NaBH(OAc)3 (508.80 mg, 2.40 mmol, 6.0 eq) following similar steps to those in intermediate I-1g. (307.00 mg, yield 82.9%). LC-MS MS-ESI (m / z) 926.0 [M+H] + .

[0319] Preparation of compound: 4-(2-(2-((2,2'-dichloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-6

[0320] Off-white solid I-6 was prepared from intermediate I-6 g (307.00 mg, 0.33 mmol, 1.0 eq) and LiOH·H₂O (277.20 mg, 6.60 mmol, 20.0 eq) using a similar procedure to that in compound I-1. (84.00 mg, yield 27.9%). LC-MS MS-ESI (m / z) 912.0 [M+H] + . 1H-NMR(400MHz,DMSO-d6)δppm 9.89(s,2H),8.38(d,J=8.4Hz,2H),7.49(t,J=8.0Hz,2H),7.14(d,J=7.4Hz,2H),3.90(s,6H),3.48-3.41(m,4H),3.33(s,2H),3.24(s ,3H),2.78-2.70(m,4H),2.69-2.62(m,4H),2.56-2.51(m,4H),1.89-1.83(m,2H),1.75-1.69(m,4H),1.58-1.22(m,16H),1.12(s,2H).

[0321] Example 7

[0322] 4-(2-(2-((2,2'-dichloro-3'-(5-(2-(4-((difluoromethoxy)methyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-7

[0323]

[0324]

[0325] Preparation of intermediate: 2-(4-((difluoromethoxy)methyl)bicyclo[2.2.1]heptane-1-yl)acetaldehyde I-7d

[0326]

[0327] Commercially available I-7d1 (300.0 g, 1.31 mol, 1.0 eq) was dissolved in THF (2.5 L). Under nitrogen protection, the reaction solution was cooled to 0 °C, and BH3-Me2S (1.57 mol, 157 mL, 1.2 eq) was added dropwise. After the addition was complete, the reaction solution was naturally heated to 20 °C and stirred for 16 h. TLC showed the reaction was complete. The reaction solution was then cooled to 0 °C, and MeOH (500 mL) was added dropwise to quench the reaction. After the addition was complete, the reaction solution was directly concentrated to dryness to obtain a colorless oily substance I-7d2 (280.00 g, yield 99.4%). 1 H-NMR (400MHz, CDCl3) δppm 4.48 (t, J = 5.5Hz, 1H), 3.82 (s, 2H), 3.32 (s, 6H), 1.81 (d, 2H), 1.32-1.58 (m, 10H).

[0328] I-7d2 (21.00 g, 97.99 mmol, 1.0 eq), KHF2 (23.17 g, 391.97 mmol, 4.0 eq), DCM (100 mL), and water (100 mL) were added to a reaction flask. Under nitrogen protection, the mixture was cooled to 10 °C, and difluorobromomethyltrimethylsilane (39.80 g, 195.99 mmol, 2.0 eq) was added dropwise. The system was slowly heated to 12 °C and reacted at 10-15 °C for 1 h. The temperature was then raised to room temperature and reacted for 1 h. TLC analysis showed that some reactants were not completely reacted. The mixture was stirred at room temperature for 16 h. TLC analysis still showed that a small amount of reactants were not completely reacted. The mixture was separated, and the aqueous phase was extracted once with DCM (50 mL). The organic phases were combined, dried over anhydrous magnesium sulfate, and evaporated to dryness to obtain a light yellow oily substance, I-7d3 (14.60 g, yield 56.4%).

[0329] I-7d3 (14.60 g, 55.24 mmol, 1.0 eq) and Acetone (172 mL) were added to a reaction flask. Under nitrogen protection, the mixture was cooled to 10 °C, and 2 M HCl (100 mL, 3.62 eq) was added. The mixture was reacted at this temperature for 30 min, then heated to room temperature and reacted for 1 h. GC analysis confirmed that the reaction was complete. The organic solvent was evaporated under reduced pressure at 45 °C, and the mixture was cooled to room temperature. It was extracted with MTBE (50 mL x 2), dried, and evaporated to dryness to obtain an oil. The crude product was separated by silica gel column chromatography (PE / EA (v / v) = 30 / 1 to 50 / 1) to obtain a colorless oil I-7d (9.21 g, yield 76.4%). 1 H-NMR(400MHz, CDCl3)δppm 9.82(t,1H),6.22(t, 2 J F-H =75.1Hz,1H),3.91(s,2H),2.60(s,2H),1.64(m,6H),1.42(m,4H).

[0330] Preparation of intermediate: 2-((2,2'-dichloro-3'-(5-(2-(4-(methoxycarbonyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-7h

[0331] The yellow solid intermediate I-7h was prepared from intermediate 2-((2,2'-dichloro-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-carboxylic acid tert-butyl ester I-6c (400.00 mg, 0.59 mmol, 1.0 eq), TEA (1 mL), I-1f (172.48 mg, 0.88 mmol, 1.5 eq) and NaBH(OAc)3 (750.48 mg, 3.54 mmol, 6.0 eq) using a similar procedure to that in intermediate I-1e. (457.00 mg, yield 90.2%) LC-MS MS-ESI (m / z) 859.3 [M+H] + .

[0332] Preparation of intermediate: 4-(2-(2-((3'-(5-(tert-butoxycarbonyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-7i

[0333] The off-white intermediate I-7i was prepared from intermediate I-7h (457.00 mg, 0.53 mmol, 1.0 eq) and LiOH·H₂O (445.20 mg, 10.6 mmol, 20.0 eq) according to a similar procedure to that in Example I-1. (376.00 mg, yield 84.0%). LC-MS MS-ESI (m / z) 845.3 [M+H] + .

[0334] Preparation of compound: 4-(2-(2-((2,2'-dichloro-3'-(5-(2-(4-((difluoromethoxy)methyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-7

[0335] Off-white solid I-7 was prepared from intermediate I-7i (376.00 mg, 0.44 mmol, 1.0 eq), TFA (5 mL), TEA (1 mL), I-7d (143.88 mg, 0.66 mmol, 1.5 eq), and NaBH(OAc)3 (559.68 mg, 2.64 mmol, 6.0 eq) following similar steps to intermediate I-1e. (202.00 mg, yield 48.4%). LC-MS MS-ESI (m / z) 947.4 [M+H] + . 1 H-NMR (400MHz, DMSO-d6) δppm9.89(s,2H),8.38(d,J=8.2Hz,2H),7.47(t,J=7.9Hz,2H),7.13(d,J=7.6Hz,2H),6.63(t, 2 J F-H =76.0Hz,1H),3.88(s,6H),3.84(s,2H),3.39(s,4H),2.78-2.70(m,4H),2.67-2.60(m,4H) ,2.56-2.51(m,4H),1.89-1.80(m,2H),1.76-1.67(m,4H),1.58-1.26(m,16H),1.15(s,2H).

[0336] Example 8

[0337] 4-(2-(2-((2,2'-dichloro-3'-(5-((4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)methyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-8

[0338]

[0339] Preparation of intermediate: 4-(methoxymethyl)bicyclo[2.2.1]heptane-1-carboxaldehyde I-8d

[0340]

[0341] Commercially available I-8d1 (60.00 g, 302.70 mmol, 1.0 eq) was dissolved in THF (0.6 L) and placed in a four-necked flask. Under nitrogen protection, the solution was cooled to 0-10 °C, and BH3-Me2S (60.5 mL, 2.0 eq) was added dropwise. During the addition, the temperature change was not significant, but there was obvious gas release. After the addition was complete, the solution was naturally heated to room temperature (20 °C) and stirred overnight for 16 h. TLC showed that the starting material reacted completely. The solution was cooled to 0-10 °C, and saturated NH4Cl solution was added dropwise. After the addition was complete, the solution was stirred for 1 h. The system separated into layers. TLC monitoring of the aqueous phase showed almost no product, which was discarded. The organic phase was dried over magnesium sulfate and evaporated to dryness. The crude product was separated by silica gel column chromatography (PE / EA(v / v) = 20 / 1 to 10 / 1) to obtain a pale yellow liquid I-8d2 (49.70 g, yield 89.12%). 1 H-NMR (400MHz, CDCl3) δppm 3.66(d,5H),2.33(s,1H),1.98(m,2H),1.66(m,4H),1.55(d,2H),1.40(m,2H).

[0342] I-8d2 (25.00 g, 135.70 mmol, 1.0 eq) was dissolved in DMF (100 mL), and MeI (192.61 g, 1.36 mol, 10.0 eq) and Ag2O (94.34 g, 407.10 mmol, 3.0 eq) were added in a single batch. After the addition, no significant exothermic or gas-releasing phenomena were observed in the reaction system. The reaction was stirred overnight at room temperature (20 °C) for 16 h. TLC showed that the reaction was complete. Insoluble matter was removed by filtration, and the filtrate was poured into 1 L of water and extracted with EA (100 mL x 3). The aqueous phase was discarded, and the organic phase was washed once with saturated brine (200 mL). The organic phase was dried over magnesium sulfate, filtered, and evaporated to dryness. The crude product was separated by silica gel column chromatography (PE / EA (v / v) = 30 / 1) to obtain a pale yellow liquid I-8d3 (24.00 g, yield 89.21%). 1 H-NMR (400MHz, CDCl3) δppm 3.66(s,3H),3.42(s,2H),3.35(s,3H),2.04(m,2H),1.94(m,4H),1.56(s,2H),1.38(m,2H).

[0343] I-8d3 (18.00 g, 90.79 mmol, 1.0 eq) was dissolved in THF (200 mL), cooled to -10⁻⁰ °C, and LAH (3.45 g, 90.79 mmol, 1.0 eq) was added in portions. During the addition, vigorous exothermic and gas release occurred, and a large amount of insoluble white solid was produced. After the addition was complete, the mixture was allowed to warm naturally to room temperature (20 °C) and stirred for 2 h. TLC showed that the reaction was complete. The mixture was cooled to -10 °C, and 2 mL of water, 2 mL of 15% NaOH aqueous solution, and finally 6 mL of water were added dropwise. The mixture was quenched for 10 min, anhydrous magnesium sulfate was added, and the mixture was dried for 10 min, filtered, and evaporated to dryness. The crude product was separated by silica gel column chromatography (PE / EA(v / v) = 10 / 1) to obtain a pale yellow liquid I-8d4 (13.00 g, yield 84.10%). 1 H-NMR (400MHz, CDCl3) δppm 3.66(s,2H),3.41(s,2H),3.35(s,3H),1.75(s,1H),1.62(m,4H),1.38(m,4H),1.19(s,2H).

[0344] Dissolve (COCl)₂ (9.69 g, 76.36 mmol, 1.3 eq) in DCM (200 mL) and cool to approximately -70 °C in a liquid nitrogen-ethanol bath under nitrogen protection. Add DMSO (9.18 g, 117.48 mmol, 2.0 eq) dropwise, and incubate for 15 min. Add I-8d₄ (10.00 g, 58.74 mmol, 1.0 eq) in DCM (200 mL) dropwise, and incubate for 15 min. Add TEA (17.83 g, 176.21 mmol, 3.0 eq) dropwise, and allow to warm naturally to room temperature (20 °C) for 4 h with stirring. TLC showed the reaction was complete. Pour the reaction solution directly into 0.5 L of water, adjust the pH to approximately 3-4 with 1 M dilute hydrochloric acid, separate the layers, and discard the aqueous phase. The organic phase was cooled to 15°C, and a 200 mL solution of NaHSO3 (14.81 g, 117.48 mmol, 2.0 eq) in water was added in a single batch. No significant exothermic or gas-releasing phenomena were observed in the reaction. After the addition was complete, the mixture was allowed to return to room temperature (20°C) and stirred overnight for 16 h. TLC monitoring showed the product spot disappeared, indicating the formation of sodium salt. The reaction solution was extracted with DCM (100 x 2 mL), and the organic phase was discarded. The aqueous phase was adjusted to pH ≈ 10 with Na2CO3 (21.79 g, 205.58 mmol, 3.5 eq) solid, stirred for 10 min, extracted with DCM (200 mL), and washed once with saturated brine (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness. The crude product was separated by silica gel column chromatography (PE / EA(v / v) = 30 / 1) to obtain a colorless, transparent liquid I-8d (3.26 g, yield 32.99%). 1H-NMR (400MHz, CDCl3) δppm 9.80(s,1H),3.45(s,2H),3.37(s,3H),2.03(m,2H),1.70(m,2H),1.50(s,3H),1.43(m,3H).

[0345] Preparation of compound: 4-(2-(2-((2,2'-dichloro-3'-(5-((4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)methyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-8

[0346] White solid I-8 was prepared from intermediate I-7i (112.00 mg, 0.13 mmol, 1.0 eq), TFA (5 mL), TEA (1 mL), I-8d (33.60 mg, 0.20 mmol, 1.5 eq), and NaBH(OAc)3 (165.36 mg, 0.78 mmol, 6.0 eq) using a similar procedure to that of intermediate I-1e. (73.00 mg, yield 62.6%). LC-MS MS-ESI (m / z) 897.4 [M+H] + . 1 H-NMR (400MHz, DMSO-d6) δppm 9.88(s,2H),8.36(d,J=8.2Hz,2H),7.47(t,J=7.9Hz,2H),7.12(d,J=7.5Hz, 2H),3.88(s,6H),3.47(s,2H),3.39(s,2H),3.32(s,2H),3.22(s,3H),2.82- 2.70(m,4H),2.69-2.61(m,4H),2.57(s,2H),2.55-2.51(m,2H),1.88-1.78( m,2H),1.73-1.66(m,2H),1.60-1.41(m,8H),1.40-1.20(m,8H),1.15(s,2H).

[0347] Example 9

[0348] 4-(2-(2-((2-chloro-2'-fluoro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-9

[0349]

[0350] Preparation of intermediate: 2-((2-chloro-2'-fluoro-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-carboxylic acid tert-butyl ester I-9c

[0351] Intermediate I-9c was prepared from 2-((3-bromo-2-fluorophenyl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-9a (490.00 mg, 1.08 mmol, 1.0 eq, synthesis reference CN202010997428.3), TFA (3 mL), 1,4-dioxane (10 mL), 2-((2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)carbamoyl) 1-Methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-6b (613.01 mg, 1.13 mmol, 1.1 eq, synthesis reference CN202010997428.3), PdCl2 (dcypf) (83.05 mg, 0.11 mmol, 0.1 eq), anhydrous Na2CO3 (343.44 mg, 3.24 mmol, 3.0 eq) and water (5 mL) were prepared according to a similar procedure to intermediate I-1c. (312.00 mg, yield 43.6%). LC-MS MS-ESI (m / z) 663.3 [M+H]+.

[0352] Preparation of intermediate: 2-((2-chloro-2'-fluoro-3'-(5-(2-(4-(methoxycarbonyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-9h

[0353] The yellow solid intermediate I-9h was prepared from intermediate I-9c (312.00 mg, 0.47 mmol, 1.0 eq), TEA (1 mL), I-1f (138.36 mg, 0.71 mmol, 1.5 eq), and NaBH(OAc)3 (716.28 mg, 2.82 mmol, 6.0 eq) using a similar procedure to that of intermediate I-1e. (335.00 mg, yield 84.6%). LC-MS MS-ESI (m / z) 843.4 [M+H] + .

[0354] Preparation of intermediate: 4-(2-(2-((3'-(5-(tert-butoxycarbonyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-chloro-2-fluoro-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-9i

[0355] The off-white intermediate I-9i was prepared from intermediate I-9h (330.00 mg, 0.39 mmol, 1.0 eq) and LiOH·H₂O (327.60 mg, 7.80 mmol, 20.0 eq) according to similar steps as in Example I-1. (252.00 mg, yield 77.9%). LC-MS MS-ESI (m / z) 829.4 [M+H] + .

[0356] Preparation of compound: 4-(2-(2-((2-chloro-2'-fluoro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-9

[0357] Off-white solid I-9 was prepared from intermediate I-9i (152.00 mg, 0.18 mmol, 1.0 eq), TFA (5 mL), TEA (2 mL), I-8d (49.14 mg, 0.27 mmol, 1.5 eq), and NaBH(OAc)3 (228.96 mg, 1.08 mmol, 6.0 eq) using a similar procedure to that of intermediate I-1e. (78.00 mg, yield 48.4%). LC-MS MS-ESI (m / z) 895.4 [M+H] + . 1 H-NMR (400MHz, DMSO-d6) δppm9.93(s,1H),9.78(s,1H),8.35(d,J=8.1Hz,1H),8.07(t,J=7.4Hz,1H ),7.48(t,J=8.0Hz,1H),7.31(t,J=7.8Hz,1H),7.21(d,J=7.5Hz,1H),7.16(t,J=7.2Hz,1H),3.89(s ,3H),3.87(s,3H),3.41(s,2H),3.39(s,2H),3.32(s,2H),3.23(s,3H),2.79-2.70(m,4H),2.69-2.6 0(m,4H),2.58-2.50(m,4H),1.94-1.78(m,2H),1.72-1.69(m,4H),1.58-1.18(m,16H),1.11(s,2H).

[0358] Example 10

[0359] 4-(2-(2-((2-chloro-3'-(5-(2-(4-((difluoromethoxy)methyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-fluoro-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-10

[0360]

[0361] Preparation of compound: 4-(2-(2-((2-chloro-3'-(5-(2-(4-((difluoromethoxy)methyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-fluoro-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-10

[0362] Off-white solid I-10 was prepared from intermediates I-9i (152.00 mg, 0.18 mmol, 1.0 eq), TFA (5.0 mL), TEA (2.0 mL), I-7d (58.86 mg, 0.27 mmol, 1.5 eq), and NaBH(OAc)3 (228.96 mg, 1.08 mmol, 6.0 eq) using a similar procedure to that of intermediate I-1e. (78.00 mg, yield 46.5%). LC-MS MS-ESI (m / z) 931.4 [M+H] + . 1 H-NMR(400MHz,DMSO-d6)δppm 9.93(s,1H),9.77(s,1H),8.35(d,J=8.0Hz,1H),8.08(t,J=7.3Hz,1H),7.48(t,J=7. 8Hz,1H),7.31(t,J=7.8Hz,1H),7.20(d,J=7.4Hz,1H),7.15(t,J=6.7Hz,1H),6.63(t, 2 J F-H =76.0Hz,1H),3.89(s,3H),3.87(s,3H),3.85(s,2H),3.40(s,4H),2.74(s,4H),2.65(s,4H) ,2.58-2.51(m,4H),1.94-1.79(m,2H),1.77-1.65(m,4H),1.61-1.25(m,16H),1.16(s,2H).

[0363] Example 11

[0364] 4-(2-(2-((2-chloro-2'-cyano-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-11

[0365]

[0366] Preparation of intermediate: 2-((2'-chloro-2-cyano-3'-(1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-carboxylic acid tert-butyl ester I-11c

[0367] The brown solid intermediate I-11c is composed of 2-((3-bromo-2-chlorophenyl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-1a (500.00 mg, 1.06 mmol, 1.0 eq), TFA (5 mL), 1,4-dioxane (10 mL), 2-((2-cyano-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)carbamoyl)-1-methyl-1, 4,6,7-Tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-11b (537.84 mg, 1.06 mmol, 1.0 eq, synthesis reference CN202010997428.3), PdCl2 (dcypf) (83.05 mg, 0.11 mmol, 0.1 eq), anhydrous Na2CO3 (337.08 mg, 3.18 mmol, 3.0 eq), and water (5 mL) were prepared according to a similar procedure to intermediate I-1c. (344.00 mg, yield 48.4%). LC-MS MS-ESI (m / z) 670.3 [M+H] + .

[0368] Preparation of intermediate: 2-((2'-chloro-2-cyano-3'-(5-(2-(4-(methoxycarbonyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-11h

[0369] The yellow solid intermediate I-11h was prepared from intermediate I-11c (344.00 mg, 0.51 mmol, 1.0 eq), TEA (1 mL), I-1f (149.15 mg, 0.76 mmol, 1.5 eq), and NaBH(OAc)3 (648.72 mg, 3.06 mmol, 6.0 eq) using a similar procedure to that of intermediate I-1e. (383.40 mg, yield 88.4%). LC-MS MS-ESI (m / z) 850.4 [M+H] + .

[0370] Preparation of intermediate: 4-(2-(2-((3'-(5-(tert-butoxycarbonyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-chloro-2-cyano-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-11i

[0371] The off-white intermediate I-11i was prepared from intermediate I-11h (383.40 mg, 0.45 mmol, 1.0 eq) and LiOH·H₂O (378.00 mg, 9.0 mmol, 20.0 eq) according to a similar procedure to that in Example I-1. (256.00 mg, yield 68.0%). LC-MS MS-ESI (m / z) 836.4 [M+H] + .

[0372] Preparation of compound: 4-(2-(2-((2-chloro-2'-cyano-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-11

[0373] The pale yellow solid I-11 was prepared from intermediate I-11i (30.0 mg, 0.036 mmol, 1.0 eq), TFA (2.0 mL), TEA (0.2 mL), I-4d (9.83 mg, 0.054 mmol, 1.5 eq), and NaBH(OAc)3 (45.79 mg, 0.22 mmol, 6.0 eq) using a similar procedure to that of intermediate I-1e. (19.90 mg, yield 61.2%). LC-MS MS-ESI (m / z) 902.5 [M+H] + . 1 H-NMR(400MHz,DMSO-d6)δppm 10.38(s,1H),9.94(s,1H),8.42(d,J=7.3Hz,1H),8.02(d,J=8.3Hz,1H),7.80(t,J=8.0Hz, 1H),7.53(t,J=8.0Hz,1H),7.33(d,J=7.6Hz,1H),7.23(d,J=7.6Hz,1H),3.90(s,3H),3.87 (s,3H),3.46-3.42(m,4H),3.32(s,2H),3.23(s,3H),2.78-2.71(m,4H),2.70-2.64(m,4H) ,2.57-2.52(m,4H),1.88-1.84(m,2H),1.72-1.70(m,4H),1.52-1.32(m,16H),1.12(s,2H).

[0374] Example 12

[0375] 4-((2-((2-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-12

[0376]

[0377] Preparation of intermediate: 2-((2'-chloro-3'-(5-((4-(methoxycarbonyl)bicyclo[2.2.1]heptane-1-yl)methyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester I-12h

[0378] The yellow solid intermediate I-12h was prepared from intermediate I-1c (132.0 mg, 0.20 mmol, 1.0 eq), TEA (1.0 mL), and commercially available methyl 4-formylbicyclo[2.2.1]heptane-1-carboxylate I-12f (54.66 mg, 0.30 mmol, 1.5 eq, supplied by Nanjing Yaoshi Technology Co., Ltd.) and NaBH(OAc)3 (254.40 mg, 1.20 mmol, 6.0 eq) using a similar procedure to that in intermediate I-1e. (85.0 mg, yield 51.5%). LC-MS MS-ESI (m / z) 825.4 [M+H] + .

[0379] Preparation of intermediate: 4-((2-((2-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methyl)bicyclo[2.2.1]heptane-1-carboxylic acid methyl ester I'-1

[0380] Intermediate I'-1 was prepared from intermediate I-12h (85.0 mg, 0.10 mol, 1.0 eq), TFA (5 mL), I-4d (27.34 mg, 0.15 mmol, 1.5 eq), TEA (1 mL), and NaBH(OAc)3 (127.20 mg, 0.60 mmol, 6.0 eq) following similar steps to intermediate I-1e. (74.0 mg, yield 80.6%). LC-MS MS-ESI (m / z) 891.5 [M+H] + .

[0381] Preparation of compound: 4-((2-((2-chloro-3'-(5-(2-(4-(methoxymethyl)bicyclo[2.2.1]heptane-1-yl)ethyl)-1-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-carboxamido)-2'-methyl-[1,1'-biphenyl]-3-yl)carbamoyl)-1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methyl)bicyclo[2.2.1]heptane-1-carboxylic acid I-12

[0382] Off-white solid I-12 was prepared from intermediate I'-1 (74.0 mg, 0.08 mmol, 1.0 eq) and LiOH·H₂O (67.20 mg, 1.60 mmol, 20.0 eq) according to similar steps as in Example I-1. (37.0 mg, yield 50.8%). LC-MS MS-ESI (m / z) 877.5 [M+H] + . 1 H-NMR(400MHz,DMSO-d6)δppm 9.90(s,1H),9.73(s,1H),8.33(d,J=8.5Hz,1H),7.72(d,J=7.8Hz,1H),7.45(t,J=8.0Hz,1H),7.29 (t,J=7.8Hz,1H),7.06(d,J=7.5Hz,1H),7.00(d,J=7.5Hz,1H),3.89(s,3H),3.86(s,3H),3.48(s,2 H),3.40(s,2H),3.33(s,2H),3.23(s,3H),2.82-2.70(m,4H),2.69-2.61(m,4H),2.58(s,2H),2.55 -2.51(m,2H),1.98(s,3H),1.87-1.77(m,2H),1.73-1.65(m,2H),1.61-1.20(m,16H),1.12(s,2H).

[0383] For those skilled in the art, other compounds of this application can be prepared by referring to the above method and their chemical structures shall prevail.

[0384] In vitro biological evaluation

[0385] This detection method is used for the in vitro biological activity evaluation of the compounds described in this invention, including in vitro protein-level binding inhibition activity evaluation method and cellular-level biological functional activity evaluation method.

[0386] The purpose of this assay is to comprehensively evaluate the inhibitory activity of different compounds on the binding of PD-1 and PD-L1 and CD80 and PD-L1 in vitro and their blocking effect on the inhibition of T cell activation signals after PD-1 and PD-L1 binding in cell models.

[0387] Example A: Evaluation of in vitro inhibitory activity against PD-1 and PD-L1 binding

[0388] Main principles of the experiment

[0389] Homogeneous time-resolved fluorescence (HTRF): This method utilizes recombinant human PD-L1 protein fused with an hFc tag and recombinant human PD-1 protein fused with a His tag, which act as interacting ligand and receptor. When anti-hFc antibodies containing Eu chelate and anti-His antibodies containing XL665 fluorescein bind to their respective tags and are excited by a 320 nm laser, energy is transferred from Eu to XL665 fluorescein due to ligand-receptor binding, causing the latter to emit light at a wavelength of 665 nm. However, when an inhibitor of the PD-L1-PD-1 interaction is added, the ligand-receptor binding is disrupted, resulting in a greater distance between Eu and XL665, preventing energy transfer and thus preventing XL665 from being excited.

[0390] Experimental materials and equipment

[0391] Recombinant human PD-1 protein with a His tag (His-PD-1 protein, Cat#: 10377-H08H-50) and recombinant human PD-L1-Fc fusion protein (PD-L1-Fc fusion protein, Cat#: 10084-H02H-100) were purchased from SinoBiological Inc., anti-hFc-Eu 3+ Antibodies and anti-His-XL665 antibody were purchased from Cisbio. Other related reagents, such as dilution buffer (Diluent buffer 5, Cat#: 62DL5DDC) and detection buffer (PPI-Europium detection buffer, Cat#: 61DB9RDF), were also purchased from Cisbio. The fluorescence detection instrument, Tecan (Spark10M), was purchased from Tecan GmbH, Switzerland.

[0392] Main experimental procedures

[0393] The experiment was conducted according to the procedure specified in the instructions for use of the testing reagents. The procedure is as follows:

[0394] (1) Experimental preparation: The test compounds were diluted to different concentration gradients using dilution buffer (the highest final concentration in a 20 μL final reaction system was 10 μM). His-PD-1 protein was diluted to 800 nM (final concentration of 100 nM in a 20 μL final reaction system), and PD-L1-Fc fusion protein was diluted to 16 nM (final concentration of 2 nM). The anti-His-XL665 antibody and anti-hFc-Eu were diluted with detection buffer according to the reagent requirements. 3+ Antibodies were diluted 20 times and 100 times.

[0395] (2) First, mix 5 μL of the test compound, 2.5 μL of PD-L1-Fc fusion protein, and 2.5 μL of His-PD-1 protein solution, and incubate at room temperature for 15 min; then add 5 μL of anti-His-XL665 antibody and 5 μL of anti-hFc-Eu to the system. 3+ Antibodies were incubated for another 3 hours before testing.

[0396] (3) Control groups were set up for the detection reaction, including a 0% inhibition positive control without the addition of the test compound and a 100% inhibition negative control without the addition of PD-1 protein. All tests were performed in duplicate.

[0397] (4) The fluorescence signal of each well was detected using a Tecan (Spark 10M) fluorescence detector. The excitation wavelength was 320 nm, and the emission wavelengths detected were 620 nm and 665 nm, respectively. The intensity of the binding between PD-1 and PD-L1 was determined by the fluorescence signal ratio Em665 / Em620.

[0398] (5) Formula for calculating the binding inhibition rate of the test compounds: Inhibition rate (%) = [1 – (fluorescence signal ratio of detection well – fluorescence signal ratio of 100% inhibition of negative control)] / (fluorescence signal ratio of 0% inhibition of positive control – fluorescence signal ratio of 100% inhibition of negative control) × 100%. After calculating the PD-1 / PD-L1 binding inhibition rate of the test compounds at different concentration gradients, the 50% inhibition concentration (IC50) is calculated. 50 The representative compound of this invention inhibits the binding of PD-1 and PD-L1 in vitro. 50 The data is shown in Table 2 below:

[0399] Table 2. IC50 of representative compounds of this invention inhibiting in vitro PD-1 / PD-L1 binding. 50 data

[0400]

[0401] aExample 180 is an example disclosed by Incyte in patent CN110267953A. The inventors synthesized this compound using the synthetic method described therein as a control molecule. The chemical structure of Example 180 was analyzed by LC-MS MS-ESI (m / z) 775.0 [M+H]. + and 1 ¹H-NMR (400MHz, DMSO) δppm 9.90 (s, 2H), 8.38 (dd, J = 7.6, 2.7Hz, 2H), 7.49 (t, J = 8.0Hz, 2H), 7.14 (d, J = 7.7Hz, 2H), 3.90 (s, 6H), 3.57 (t, J = 6.0Hz, 2H), 3.51–3.46 (m, 4H), 2.85–2.77 (m, 4H), 2.70–2.64 (m, 4H), 2.64–2.59 (m, 4H), 1.90–1.76 (m, 2H), 1.68–1.55 (m, 2H), 1.54–1.22 (m, 6H). The structure of this compound was confirmed, and its structural formula is as follows:

[0402]

[0403] As can be seen from the above results, the compounds of this invention exhibit excellent inhibitory activity against PD-1 / PD-L1 in vitro, and the compounds of general formula (I) of this invention also exhibit inhibitory activity against PD-1 / PD-L1. Compared with the control molecule, the compounds of this invention exhibit significantly superior inhibitory activity against PD-1 / PD-L1.

[0404] Example B: Evaluation of in vitro inhibitory activity against CD80 and PD-L1 binding

[0405] Main principles of the experiment

[0406] Besides PD-1, PD-L1 can also exert immunosuppressive activity by binding to CD80. Similarly, in vitro CD80 binding to PD-L1 or binding inhibition assays can be detected using homogeneous time-resolved fluorescence spectrometry (HTRF). When using anti-hFc-Eu... 3+ The antibody and the anti-His-XL665 antibody bind to the hFC tag fused to PD-L1 and the His tag fused to CD80, respectively. Upon excitation with a 320 nm laser, the binding of PD-L1 and CD80 allows energy to be transferred from Eu to the XL665 fluorescein, exciting it to emit light. When an inhibitor of the PD-L1-CD80 interaction is added, the binding is disrupted, resulting in a greater distance between Eu and XL665, preventing energy transfer and thus preventing XL665 excitation.

[0407] Experimental materials and equipment

[0408] Recombinant human CD80 protein with a His tag (His-CD80 protein, Cat#: 10698-H08H-100) and recombinant human PD-L1-Fc fusion protein (PD-L1-Fc fusion protein, Cat#: 10084-H02H-100) were purchased from SinoBiological Inc., anti-hFc-Eu 3+ Antibodies and anti-His-XL665 antibody were purchased from Cisbio. Other related reagents, such as dilution buffer (Diluent buffer 5, Cat#: 62DL5DDC) and detection buffer (PPI-Europium detection buffer, Cat#: 61DB9RDF), were also purchased from Cisbio. The fluorescence detection instrument, Tecan (Spark10M), was purchased from Tecan GmbH, Switzerland.

[0409] Main experimental procedures

[0410] The experiment was conducted according to the procedure specified in the instructions for use of the test reagent (Invitrogen). The procedure is as follows:

[0411] (1) Experimental preparation: The test compounds were diluted to different concentration gradients using dilution buffer (the highest final concentration in a 20 μL final reaction system was 10 μM). His-CD80 protein was diluted to 800 nM (final concentration of 100 nM in a 20 μL final reaction system), and PD-L1-Fc fusion protein was diluted to 16 nM (final concentration of 2 nM). The anti-His-XL665 antibody and anti-hFc-Eu were diluted with detection buffer according to the reagent requirements. 3+ Antibodies were diluted 20 times and 100 times.

[0412] (2) First, mix 5 μL of the test compound, 2.5 μL of His-CD80 protein, and 2.5 μL of PD-1-Fc fusion protein solution, and incubate at room temperature for 15 min; then add 5 μL of anti-His-XL665 antibody and 5 μL of anti-hFc-Eu to the system. 3+ Antibodies were incubated for another 3 hours before testing.

[0413] (3) Control groups were set up for the detection reaction, including a 0% inhibition positive control without the addition of the test compound and a 100% inhibition negative control without the addition of CD80 protein. All tests were performed in duplicate.

[0414] (4) The fluorescence signal of each well was detected using a Tecan (Spark 10M) fluorescence detector. The excitation wavelength was 320 nm, and the emission wavelengths detected were 620 nm and 665 nm, respectively. The intensity of CD80 / PD-L1 binding was determined by the fluorescence signal ratio Em665 / Em620.

[0415] (5) Formula for calculating the binding inhibition rate of the test compound: Inhibition rate (%) = [1 – (fluorescence signal ratio of detection well – fluorescence signal ratio of 100% inhibition of negative control)] / (fluorescence signal ratio of 0% inhibition of positive control – fluorescence signal ratio of 100% inhibition of negative control) × 100%. After calculating the CD80 / PD-L1 binding inhibition rate of the test compounds at different concentration gradients, the 50% inhibition concentration (IC50) is calculated. 50 The representative compound of this invention inhibits the binding of CD80 and PD-L1 in vitro. 50 The data is shown in Table 3:

[0416] Table 3. IC50 of representative compounds of this invention inhibiting in vitro CD80 / PD-L1 binding. 50 data

[0417]

[0418] As can be seen from the above results, the compounds of the present invention exhibit excellent inhibitory activity against CD80 / PD-L1 in vitro, and the compounds of general formula (I) of the present invention also exhibit inhibitory activity against CD80 / PD-L1. Compared with the control molecule (i.e., Example 180 above), the compounds of the present invention exhibit significantly superior inhibitory activity against CD80 / PD-L1.

[0419] Example C: Evaluation of T cell activation-inhibiting signaling mediated by PD-1 and PD-L1 immune checkpoints at the cell level.

[0420] As an immune checkpoint molecule, PD-1 is primarily expressed on the surface of activated T cells, while its ligand PD-L1 is widely expressed. Besides antigen-presenting cells such as dendritic cells, macrophages, and B cells, many tumor cells can also suppress anti-tumor immune responses by upregulating PD-L1 expression. In normal immune responses, antigen-presenting cells, in addition to activating T cells through immune co-stimulatory molecules, also express PD-L1 ligand molecules, which bind to PD-1 molecules on the surface of activated T cells, thereby inhibiting T cell activation and preventing excessive T cell proliferation and activation that could damage surrounding normal tissues.

[0421] Main principles of the experiment

[0422] To investigate the effect of PD-1 and PD-L1 interaction on T cell activation signaling in the immune response, CHO-PD-L1-CD3L cells stably expressing human PD-L1 molecules and an anti-CD3 single-chain antibody (ScFv) and Jurkat-PD-1-NFAT cells stably expressing human PD-1 molecules and the NFAT reporter gene were constructed. When both cell types were co-incubated, the anti-CD3ScFv on the surface of CHO cells bound to the membrane CD3 molecules of Jurkat cells, transmitting an activation signal into the Jurkat cells. However, due to the simultaneous binding of PD-L1 molecules on the surface of CHO cells and PD-1 molecules on the surface of Jurkat cells, an inhibitory activation signal was transmitted inward, preventing luciferase reporter gene expression. Upon the addition of immune checkpoint antibodies or small molecule inhibitors, the binding of PD-1 to PD-L1 was blocked. The NFAT pathway activated by the cross-linking of CD3ScFv antibody with CD3 was no longer affected by the inhibitory signal, and the downstream luciferase reporter gene began to express. A chemiluminescent signal proportional to reporter gene activation was detected by adding a catalytic substrate.

[0423] Experimental materials and equipment

[0424] CHO-PD-L1-CD3L cells expressing human PD-L1 and anti-CD3 single-chain antibody (ScFv), and Jurkat-PD-1-NFAT cells stably expressing human PD-1 and NFAT reporter gene were all constructed and donated by Dr. Bo Chen (Connoya Biopharmaceutical Technology (Chengdu) Co., Ltd.). Puromycin (Cat#540411) and Hygromycin B (Cat#V900372) used for stable cell culture were purchased from Sigma-Aldrich, PMA (Cat#P1585) was purchased from Sigma-Aldrich, and anti-human PD-L1 antibody (Cat#GMP-A066) was purchased from Novoprotein. Luciferase substrate solution (Cat#E6485) and luciferase-specific cell lysis buffer 5× (Cat#E1531) were both purchased from Promega. The Tecan (Spark10M) fluorescence detection instrument was purchased from Tecan GmbH, Switzerland.

[0425] Main experimental procedures

[0426] (1) One day before the experiment, 100 μL of CHO-PD-L1-CD3L cells (approximately 4 × 10⁻⁶ cells) were seeded into 96-well cell culture plates. 4 (cells / well), cultured in DMEM / F12 medium containing 10% FBS, 8 μg / mL Puromycin and 200 μg / mL Hygromycin B, and incubated overnight at 37°C;

[0427] (2) The test compounds were first diluted to different concentration gradients using 0.1% PBST and added to 96-well plates, then pre-incubated for 30 min; the Jurkat-PD-1-NFAT cell count was adjusted to 2 × 10⁻⁶ cells using RPMI 1640 complete medium containing 10% FBS, 8 μg / mL Puromycin, and 200 μg / mL Hygromycin B. 5 The cells were added at 100 ng / mL, and 100 ng / mL of PMA (prepared with DMSO stock solution at a concentration of 10 mg / mL) was added to amplify the T cell activation signal; 100 μL of the above Jurkat-PD-1-NFAT cells were added to each well of a 96-well plate for co-culture.

[0428] (3) A control group was set up for the detection reaction, including a solvent control without the test compound and a positive control with anti-human PD-1 antibody added. All detections were performed in duplicate.

[0429] (4) After incubating at 37°C for 6 hours, add 40 μL of 5× cell lysis buffer, mix well and place at room temperature for 10 minutes to completely lyse the cells; take 50 μL of the lysed cell solution and transfer it into a fluorescence detection plate, add 30 μL of luciferase substrate solution, and immediately select the chemiluminescence detection program on the fluorescence detector for measurement.

[0430] (5) Formula for calculating the inhibition rate of T cell activation signal at the cellular level of the test compound: Inhibition rate of T cell activation signal (%) = (raw chemiluminescence value of the detection well – solvent control) / (highest raw chemiluminescence value measured in the compound detection well – solvent control) × 100%. After calculating the inhibition rate of T cell activation signal for test compounds at different concentration gradients, the 50% inhibition concentration (EC50) was further calculated. 50 The compounds of this invention block PD-1 and PD-L1-mediated T cell activation inhibition signals in ECGs. 50 The data is shown in Table 4:

[0431] Table 4. EC5 values ​​of the compounds of this invention blocking PD-1 / PD-L1-mediated T cell activation inhibition signals 50

[0432]

[0433] As can be seen from the above results, the compounds of the present invention possess the activity of effectively blocking immune checkpoint-mediated T cell activation inhibition signals at the cellular level. Compared with the control molecule (i.e., Example 180 above), most compounds of general formula (I) of the present invention have higher activity in effectively blocking immune checkpoint-mediated T cell activation inhibition signals at the cellular level.

[0434] Example D: Single-dose pharmacokinetic study

[0435] Twenty-four 6-week-old female B-hPD-1 / hPD-L1 mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were used. The test compounds were prepared in a solvent containing 5% DMSO, 60% PEG400, and 35% purified water, including I-6 and three control molecules: Example 17, Compound 14 (INCB086550), and Example 180. The compounds were administered once by gavage at a dose of 100 mg / kg. Blood samples were collected from the fundus venous plexus at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 32 h, and 48 h. Approximately 0.1 mL of blood was collected in centrifuge tubes (anticoagulated with sodium heparin), centrifuged at 5000 rpm for 5 min, and the plasma was separated and stored at -20°C for analysis. After plasma sample processing, the concentration of compounds in the plasma was determined using liquid chromatography-mass spectrometry (LC-MS / MS). Pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0 based on average plasma drug concentration data at different time points. The data summary is shown in Table 5.

[0436] Table 5. Pharmacokinetic parameters of the compound in humanized mice after a single administration of 100 mg / kg.

[0437]

[0438] a Example 17 is a compound disclosed on page 64 of Incyte's patent document WO2019 / 217821. The inventors synthesized this compound using the method described therein as a control molecule. The chemical structure of Example 17 was determined by LC-MS MS-ESI (m / z) 911.4 [M+H]. + Confirmed, and due to the poor solubility of Example 17, a sodium hydroxide solution (molar ratio of Example 17 to sodium hydroxide of 1:2) was added to the NMR sample to aid dissolution, and then MeOD was added to confirm the structure. 1 H-NMR (400MHz, MeOD) δppm 8.45 (dd, J=8.3, 1.2Hz, 2H), 7.43 (t, J=8.0Hz, 2H), 7.12-7.07 (m, 2H), 3.95 (d, J=14.4Hz, 6H), 3.55 (s, 4H), 2.96-2.84 (m, 4H), 2.72-2.82 (m, 4H), 2.70-2.62 (m, 4H), 2.00-1.88 (m, 4H), 1.87-1.78 (m, 4H), 1.66-1.37 (m, 16H). The structure of this compound was confirmed, and its structural formula is as follows:

[0439]

[0440] b Compound 14 (INCB086550) is a compound disclosed in Table 2 of Incyte's patent CN110267953A. The inventors synthesized this compound using the synthetic method described therein as a control molecule. Compound 14 (INCB086550) is one of the most advanced small molecule PD-L1 inhibitors in clinical trials and is currently in Phase 2 research.

[0441] The chemical structure of compound 14 (INCB086550) was determined by LC-MS / MS-ESI (m / z) 694.2 [M+H]. + and 1 H-NMR(400MHz,DMSO)δppm 9.31(s,1H),8.84(s,1H),8.48(d,J=8.0Hz,1H),8.17(s,1H),8.13(d,J=7.7Hz,1H),8.09-8.03(m,2H),7.83(s,1H), 7.54(t,J=7.7Hz,1H),7.43(d,J=7.4Hz,1H),7.34(t,J=7.9Hz,1H),7.18(d,J=5.8Hz,1H),6.91(d,J=7.4Hz,1H),4.76 The structure of the compound was confirmed as follows: (s, 1H), 4.26-4.18(m, 1H), 3.85-3.65(m, 4H), 2.84-2.69(m, 3H), 2.69-2.60(m, 2H), 2.58-2.46(m, 3H), 2.45(s, 3H), 2.38(dd, J=9.6, 3.6Hz, 1H), 2.08(s, 3H), 2.05-1.95(m, 2H), 1.94-1.83(m, 1H), 1.63-1.52(m, 1H).

[0442]

[0443] As can be seen from the above results, the representative compound of this invention, after a single oral administration of 100 mg / kg to mice, showed a plasma exposure (AUC) of... (0-t) Mean time to residence (MRT) and half-life (T) 1 / 2 The levels of these compounds were significantly higher than those of the three control molecules. Compared to the control molecules, the compounds described in this invention exhibit unexpectedly higher levels of exposure and duration of exposure in vivo, which helps to better exert anti-tumor activity and achieve superior therapeutic effects in clinical treatment.

[0444] Example E: Repeated-dose pharmacokinetic study

[0445] Twenty-four 6-week-old humanized female B-hPD-1 / hPD-L1 mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were used. The test compounds were prepared in a solvent containing 5% DMSO, 60% PEG400, and 35% purified water, including I-6, three control molecules (Example 17), compound 14 (INCB086550), and the aforementioned Example 180. The compounds were administered by gavage at a dose of 50 mg / kg once daily for 12 consecutive days. On day 12, blood was collected alternately from the fundus venous plexus at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 32 h, and 48 h. Approximately 0.1 mL of blood was collected in centrifuge tubes (anticoagulated with sodium heparin), centrifuged at 5000 rpm for 5 min, and the plasma was separated and stored at -20°C for analysis. The concentration of the compounds in the plasma was determined using liquid chromatography-mass spectrometry (LC-MS / MS). Pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0 based on average plasma drug concentration data at different time points. Data summary is shown in Table 6.

[0446] Table 6. Pharmacokinetic parameters in humanized mice after repeated administration.

[0447]

[0448] As can be seen from the above results, after repeated administration of the representative compound described in this invention at 50 mg / kg, the plasma exposure (AUC) was [not specified]. (0-t) Mean time to residence (MRT) and half-life (T) 1 / 2 The levels of these compounds were significantly higher than those of the three control molecules. Repeated administration better reflects the pharmacokinetic characteristics under real clinical treatment conditions. Compared with the control molecules, the compounds of this invention showed significantly increased in vivo exposure and duration of exposure under repeated administration conditions, which helps to better exert antitumor activity in clinical treatment.

[0449] Example F: Tumor Tissue Distribution Test

[0450] Eighteen humanized female B-hPD-1 / hPD-L1 mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with MC38-PD-L1 cells after one week of acclimatization, at a dose of 2×10⁶ cells. 6 One tumor per location, until the tumor grows to approximately 200m. 3Groups were then administered the drugs. The test compounds, including compound I-6 and two aforementioned control molecules (Example 17 and compound 14, INCB086550), were prepared in a solvent containing 5% DMSO, 60% PEG400, and 35% purified water. The compounds were administered by gavage at a dose of 100 mg / kg once daily. Blood and tumor tissue samples were collected at 1 hour, 4 hours, and 24 hours after administration on day 16. A certain amount of tissue was weighed, homogenized with phosphate-buffered saline (PBS) solution, and the homogenate was extracted and analyzed by LC-MS / MS to calculate the concentration of the compounds in the tissue. Experimental results are shown below. Figure 1 and Figure 2 .

[0451] As the results above show, after repeated administration, the concentration of the representative compound described in this invention in tumor tissue was significantly higher than that in plasma, and also significantly higher than that of the control molecule, demonstrating excellent tumor tissue targeting. Compared with the control molecule, the compound of this invention exhibits unexpected enrichment and targeting effects on tumor tissue.

[0452] Example G: Drug Efficacy Test in Mouse Xenografts

[0453] Twenty-four humanized female B-hPD-1 / hPD-L1 mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with MC38-PD-L1 cells after one week of acclimatization, at a dose of 2 × 10⁻⁶ cells. 6 One tumor per location, until the tumor reaches approximately 100m in size. 3 Subsequently, patients were randomly assigned to three groups: a solvent control group; compound 14 (INCB086550) at 100 mg / kg; and compound I-6 at 100 mg / kg. The test compounds were prepared in a solvent containing 5% DMSO, 60% PEG400, and 35% purified water, and administered by gavage at a dose of 100 mg / kg once daily for 19 consecutive days. Tumor volume (length × width) was recorded twice weekly. 2 ×0.5). Experimental results are shown in […]. Figure 3 .

[0454] The results above demonstrate that the representative compounds described in this invention can significantly inhibit tumor growth in a humanized mouse MC38-PD-L1 tumor model. Compared to the control molecule, the compounds of this invention exhibit more significant pharmacological efficacy in the humanized mouse tumor model.

[0455] All compounds described in this invention, including the compounds of this invention and control molecules, are based on their chemical structural formulas.

Claims

1. A compound of formula (I), Or its pharmaceutically acceptable salt. in, R 1 and R 2 They may be selected from C1-C6 alkyl, cyano, and halogen groups, either the same or different. R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl; R 4 It is hydrogen; X is -O-; m is selected from 1, 2, and 3; n is selected from 1, 2, and 3.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, in, R 1 and R 2 They can be selected from methyl, cyano, fluorine, chlorine, and bromine, either the same or different; R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl; R 4 It is hydrogen; X is -O-; m is selected from 1, 2, and 3; n is selected from 1, 2, and 3.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, in, R 1 and R 2 They can be selected from methyl, cyano, fluorine, and chlorine, either the same or different; R 3 Selected from C1-C6 alkyl and halo-C1-C6 alkyl; R 4 It is hydrogen; X is -O-; m is selected from 1 and 2; n is selected from 1, 2, and 3.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R 1 Selected from methyl, cyano, fluorine, and chlorine, R 2 Selected from methyl and chlorine.

5. A pharmaceutical composition comprising the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. Use of the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 5, in the preparation of a medicament for treating and / or preventing diseases associated with the target PD-L1, or Use in the preparation of drugs for inhibiting PD-L1 activity, or Use in the preparation of drugs as PD-L1 inhibitors, or Use in the preparation of drugs as immunomodulators targeting the PD-L1 signaling pathway.

7. The use according to claim 6, wherein, The diseases associated with targeting PD-L1 include tumors or other immune-related diseases.

8. The use according to claim 6, wherein, The disease associated with targeting PD-L1 is cancer.

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