Polypeptide inhibitor as well as preparation method and application thereof

Abstract

The invention relates to a polypeptide inhibitor as well as a preparation method and application thereof. In particular, the present invention relates to a compound represented by general formula (I), a preparation method thereof, a pharmaceutical composition containing the compound, and a use thereof as an inhibitor in the treatment of a variety of inflammatory, autoimmune diseases and cancers, each substituent in the general formula (I) being as defined in the specification. # imgabs0 #

Description

A polypeptide inhibitor, its preparation method and application

[0001] This application claims priority to Chinese patent application No. 2022115304381, filed on November 30, 2022. This application incorporates the entire text of the aforementioned Chinese patent application.

[0002] This application claims the priority of Chinese Patent Application No. 2023102062995, filed on March 3, 2023. This application incorporates the entire text of the aforementioned Chinese Patent Application.

[0003] This application claims the priority of Chinese Patent Application No. 2023102618188, filed on March 15, 2023. This application incorporates the entire text of the aforementioned Chinese Patent Application.

[0004] This application claims the priority of Chinese patent application No. 202310349519X, filed on March 31, 2023. This application incorporates the entire text of the aforementioned Chinese patent application.

[0005] This application claims the benefit of Chinese patent application No. 2023111444895, filed on September 5, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0006] The present invention belongs to the field of biomedicine, and specifically relates to a polypeptide inhibitor and a preparation method and application thereof. Background Art

[0007] The cytokine IL23 plays a crucial role in both innate and adaptive immunity. IL-23 induces the expression of inflammatory cytokines by lymphocytes, most notably helper T cells (TH17), innate lymphoid cells (ILCs), and γδT cells. The IL-23 heterodimeric receptor is composed of two subunits: IL-23R and IL-12Rβ1. IL-23R is a subunit unique to the IL-23 pathway. IL-12Rβ1 is shared with the IL-12 receptor. Similarly, the IL-23 cytokine is composed of two subunits: p19 and p40. The p19 subunit is unique to IL-23, while p40 is shared with IL-12.

[0008] IL-23 provides essential conditions for the generation and survival of Th17 cells. At the same time, a large amount of evidence from preclinical models and clinical practice shows that Th17 cells play a vital role in the pathology of many autoimmune diseases, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE), and multiple sclerosis (MS). Evidence from preclinical models and clinical practice shows that blocking IL23 signal transduction is effective in the treatment of autoimmune diseases, but the nature of the IL-23 and IL-12 pathways sharing ligands and receptor subunits means a more complex biological process, and data on tumorigenesis, susceptibility to infection, and autoimmune disorders indicate that IL-23 blockade appears to have therapeutic advantages over IL-12 blockade in terms of efficacy and safety.

[0009] The limitations of antibody therapy include high production costs, poorer stability, higher requirements for transportation, storage, use and production conditions, and usually require infusion or injection during treatment, which is a challenge for patient compliance. Antibody immunosuppressive therapy is usually systemic, and tuberculosis patients receiving treatment are at risk of relapse and other serious infections. Therefore, in many developing countries, a high proportion of patients with latent tuberculosis or hepatitis B (HBV) need to be excluded when conducting anti-TNF or anti-IL23 therapy. The half-life of systemic antibody therapy is very long, so that anti-drug antibodies (ADA) are produced to neutralize the antibody drug and lead to a decrease in efficacy. The intermittent dosing of anti-TNF antibodies greatly increases the possibility of ADA development, which increases the treatment risk for patients.

[0010] Therefore, the present invention aims to provide molecules that specifically block the IL-23 heterodimeric receptor by blocking IL-23R, compositions comprising these molecules, methods for screening these molecules, and methods of using these molecules to treat various inflammatory and autoimmune diseases and cancers. These molecules should have good affinity and specificity and be localized to inflammatory disease tissues without significantly affecting systemic immunity.

[0011] Summary of the Invention

[0012] The object of the present invention is to provide a compound represented by general formula (IA), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound represented by general formula (IA) has the following structure:

[0013] in:

[0014] R A Selected from -(CH2) mA -R AA ;

[0015] R AA Selected from -NH2, -N+ H3, -N + (CH3)3 or -NH-C(O)-CH3;

[0016] m A An integer selected from 1 to 6;

[0017] Preferably, R A Selected from

[0018] R B Selected from -(CH2) mB -R BB ;

[0019] R BB Selected from-NR 1 R 2 , -N + (R 1 )2R 2 or -NH-C(O)-R 1 ;

[0020] R 1 Selected from H, alkyl, or amino acids, preferably, R 1 selected from H, alkyl;

[0021] R 2 Selected from H, alkyl, or amino acids;

[0022] R a Selected from H, alkyl, or amino acids;

[0023] R aa Selected from alkyl groups, or amino acids;

[0024] X1 is selected from amino acids;

[0025] X'1 is selected from amino acids;

[0026] Preferably, R BB Selected from -NH2, -N + H3, -N + (CH3)3 or -NH-C(O)-CH3;

[0027] m B An integer selected from 1 to 6;

[0028] Preferably, R B Selected from

[0029] R 3Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids;

[0030] R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0031] R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0032] R c Selected from H, alkyl, or amino acids;

[0033] R cc Selected from alkyl groups, or amino acids;

[0034] X5 is selected from amino acids;

[0035] X'5 is selected from amino acids;

[0036] X6 is selected from amino acids;

[0037] X'6 is selected from amino acids;

[0038] X7 is selected from amino acids;

[0039] X'7 is selected from amino acids;

[0040] R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0041] R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; preferably, selected from

[0042] R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group;

[0043] R bb Selected from alkyl groups, or amino acids;

[0044] X2 is selected from amino acids;

[0045] X'2 is selected from amino acids;

[0046] X3 is selected from amino acids;

[0047] X'3 is selected from amino acids;

[0048] X4 is selected from amino acids;

[0049] X'4 is selected from amino acids;

[0050] n1 to n2 are each independently selected from an integer of 0 to 12;

[0051] t2 to t9 are each independently selected from an integer of 0 to 12;

[0052] m4 to m22 are each independently selected from integers of 0-24.

[0053] The object of the present invention is to provide a compound represented by general formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the compound represented by general formula (I) has the following structure:

[0054] in:

[0055] R 1 and R 2 Each independently selected from H, alkyl, or amino acids;

[0056] R a Selected from H, alkyl, or amino acids;

[0057] R aa Selected from alkyl groups, or amino acids;

[0058] X1 is selected from amino acids;

[0059] Preferably, R aa Selected from alkyl groups, or amino acids;

[0060] X1 is selected from amino acids;

[0061] X'1 is selected from amino acids;

[0062] R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids;

[0063] R6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0064] R c Selected from H, alkyl, or amino acids;

[0065] R cc Selected from alkyl groups, or amino acids;

[0066] X5 is selected from amino acids;

[0067] X6 is selected from amino acids;

[0068] X7 is selected from amino acids;

[0069] Preferably, R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids;

[0070] R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0071] R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0072] R c Selected from H, alkyl, or amino acids;

[0073] R cc Selected from alkyl groups, or amino acids;

[0074] X5 is selected from amino acids;

[0075] X'5 is selected from amino acids;

[0076] X6 is selected from amino acids;

[0077] X'6 is selected from amino acids;

[0078] X7 is selected from amino acids;

[0079] X'7 is selected from amino acids;

[0080] R4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0081] R b Selected from H, alkyl, or amino acids;

[0082] R bb Selected from alkyl groups, or amino acids;

[0083] X2 is selected from amino acids;

[0084] X3 is selected from amino acids;

[0085] X4 is selected from amino acids;

[0086] Preferably, R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0087] R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; preferably, selected from

[0088] R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group;

[0089] R bb Selected from alkyl groups, or amino acids;

[0090] X2 is selected from amino acids;

[0091] X'2 is selected from amino acids;

[0092] X3 is selected from amino acids;

[0093] X'3 is selected from amino acids;

[0094] X4 is selected from amino acids;

[0095] X'4 is selected from amino acids;

[0096] n to n2 are each independently selected from an integer of 0 to 12;

[0097] t to t9 are each independently selected from an integer of 0 to 12;

[0098] m to m21 are each independently selected from an integer of 0 to 24;

[0099] m22 is an integer selected from 0 to 24;

[0100] The compound is not

[0101] Another object of the present invention is to provide a compound represented by general formula (IV), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound represented by general formula (IV) has the following structure:

[0102] in:

[0103] X 1 selected from amino acids;

[0104] X 2 selected from amino acids;

[0105] R 1 and R 2 Each independently selected from H, alkyl, or amino acids;

[0106] R a Selected from H, alkyl, or amino acids;

[0107] R aa Selected from alkyl groups, or amino acids;

[0108] X1 is selected from amino acids;

[0109] X'1 is selected from amino acids;

[0110] R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids;

[0111] R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0112] R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0113] Rc Selected from H, alkyl, or amino acids;

[0114] R cc Selected from alkyl groups, or amino acids;

[0115] X5 is selected from amino acids;

[0116] X'5 is selected from amino acids;

[0117] X6 is selected from amino acids;

[0118] X'6 is selected from amino acids;

[0119] X7 is selected from amino acids;

[0120] X'7 is selected from amino acids;

[0121] R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0122] R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0123] R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group;

[0124] R bb Selected from alkyl groups, or amino acids;

[0125] X2 is selected from amino acids;

[0126] X'2 is selected from amino acids;

[0127] X3 is selected from amino acids;

[0128] X'3 is selected from amino acids;

[0129] X4 is selected from amino acids;

[0130] X'4 is selected from amino acids;

[0131] n to n2 are each independently selected from an integer of 0 to 12;

[0132] t to t9 are each independently selected from an integer of 0 to 12;

[0133] m to m21 are each independently selected from an integer of 0 to 24;

[0134] m22 is an integer selected from 0 to 24;

[0135] The compound is not

[0136] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 1 and R 2 Each independently selected from H, alkyl, or amino acids;

[0137] R a Selected from H, alkyl, or amino acids;

[0138] R aa Selected from alkyl groups, or amino acids;

[0139] X1 is selected from amino acids;

[0140] Preferably, R aa Selected from alkyl groups, or amino acids;

[0141] X1 is selected from amino acids;

[0142] X'1 is selected from amino acids;

[0143] R 3 Selected from hydroxyl groups;

[0144] R 4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2;

[0145] n is an integer selected from 0 to 12;

[0146] t to t1 are each independently selected from an integer of 0 to 12;

[0147] m to m3 are each independently selected from an integer of 0-24.

[0148] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 1 and R 2 Selected from H;

[0149] R 3 Selected from hydroxy, alkyl, -NR 6 R 7or amino acids;

[0150] R 6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0151] R c Selected from H, alkyl, or amino acids;

[0152] R cc Selected from alkyl groups, or amino acids;

[0153] X5 is selected from amino acids;

[0154] X6 is selected from amino acids;

[0155] X7 is selected from amino acids;

[0156] Preferably, R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids;

[0157] R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0158] R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0159] R c Selected from H, alkyl, or amino acids;

[0160] R cc Selected from alkyl groups, or amino acids;

[0161] X5 is selected from amino acids;

[0162] X'5 is selected from amino acids;

[0163] X6 is selected from amino acids;

[0164] X'6 is selected from amino acids;

[0165] X7 is selected from amino acids;

[0166] X'7 is selected from amino acids;

[0167] R 4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2;

[0168] n2 is an integer selected from 0 to 12;

[0169] t6 to t9 are each independently selected from an integer of 0 to 12;

[0170] m13 to m21 are each independently selected from integers of 0-24.

[0171] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 1 and R 2 Selected from H;

[0172] R 3 Selected from hydroxyl group;

[0173] R 4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0174] R b Selected from H, alkyl, or amino acids;

[0175] R bb Selected from alkyl groups, or amino acids;

[0176] X2 is selected from amino acids;

[0177] X3 is selected from amino acids;

[0178] X4 is selected from amino acids;

[0179] Preferably, R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0180] R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; preferably, selected from

[0181] R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group;

[0182] R bb Selected from alkyl groups, or amino acids;

[0183] X2 is selected from amino acids;

[0184] X'2 is selected from amino acids;

[0185] X3 is selected from amino acids;

[0186] X'3 is selected from amino acids;

[0187] X4 is selected from amino acids;

[0188] X'4 is selected from amino acids;

[0189] n1 is an integer selected from 0 to 12;

[0190] t2 to t5 are each independently selected from an integer of 0 to 12;

[0191] m4 to m12 are each independently selected from an integer of 0 to 24;

[0192] m22 is selected from integers of 0-24.

[0193] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that the compound is further represented by general formula (II):

[0194] in:

[0195] R 1 and R 2 Each independently selected from H, alkyl, or amino acids;

[0196] R a Selected from H, alkyl, or amino acids;

[0197] R aa Selected from alkyl groups, or amino acids;

[0198] X1 is selected from amino acids;

[0199] Preferably, R aa Selected from alkyl groups, or amino acids;

[0200] X1 is selected from amino acids;

[0201] X'1 is selected from amino acids;

[0202] R 4 Selected from H or alkyl;

[0203] n is an integer selected from 0 to 12;

[0204] t to t1 are each independently selected from an integer of 0 to 12;

[0205] m to m3 are each independently selected from an integer of 0-24.

[0206] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that the compound is further represented by general formula (III):

[0207] in:

[0208] R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids;

[0209] R 6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0210] R c Selected from H, alkyl, or amino acids;

[0211] R cc Selected from alkyl groups, or amino acids;

[0212] X5 is selected from amino acids;

[0213] X6 is selected from amino acids;

[0214] X7 is selected from amino acids;

[0215] Preferably, R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids;

[0216] R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0217] R 7Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0218] R c Selected from H, alkyl, or amino acids;

[0219] R cc Selected from alkyl groups, or amino acids;

[0220] X5 is selected from amino acids;

[0221] X'5 is selected from amino acids;

[0222] X6 is selected from amino acids;

[0223] X'6 is selected from amino acids;

[0224] X7 is selected from amino acids;

[0225] X'7 is selected from amino acids;

[0226] R 4 Selected from H or alkyl;

[0227] n2 is an integer selected from 0 to 12;

[0228] t6 to t9 are each independently selected from an integer of 0 to 12;

[0229] m13 to m21 are each independently selected from integers of 0-24.

[0230] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that the compound further has a compound structure as shown in general formula (V) as follows:

[0231] in:

[0232] X 1 selected from amino acids;

[0233] X 2 selected from amino acids;

[0234] R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0235] R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0236] R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group;

[0237] R bb Selected from alkyl groups, or amino acids;

[0238] X'2 is selected from amino acids;

[0239] X'3 is selected from amino acids;

[0240] X4 is selected from amino acids;

[0241] n1 is an integer selected from 0 to 12;

[0242] t2 to t5 are each independently selected from an integer of 0 to 12;

[0243] m4 to m12 are each independently selected from an integer of 0 to 24;

[0244] m22 is an integer selected from 0 to 12;

[0245] The compound is not

[0246] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that X 1 Selected from Thr, Preferably

[0247] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that X 2 Selected from Preferably

[0248] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that the compound is further represented by general formula (IV):

[0249] in:

[0250] R 4 Selected from H or alkyl;

[0251] R 5 Selected from alkyl, -CH2C(O)NH2, or amino acids;

[0252] R b Selected from H, alkyl, or amino acids;

[0253] R bb Selected from alkyl groups, or amino acids;

[0254] X2 is selected from amino acids;

[0255] X3 is selected from amino acids;

[0256] X4 is selected from amino acids;

[0257] Preferably, R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0258] R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; preferably, selected from

[0259] R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group;

[0260] R bb Selected from alkyl groups, or amino acids;

[0261] X2 is selected from amino acids;

[0262] X'2 is selected from amino acids;

[0263] X3 is selected from amino acids;

[0264] X'3 is selected from amino acids;

[0265] X4 is selected from amino acids;

[0266] X'4 is selected from amino acids;

[0267] n1 is an integer selected from 0 to 12;

[0268] t2 to t5 are each independently selected from an integer of 0 to 12;

[0269] m4 to m12 are each independently selected from an integer of 0 to 24;

[0270] m22 is selected from integers of 0-24.

[0271] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 1 Selected from H, C 1-6 alkyl, or amino acids;

[0272] R a Selected from H, C 1-6 alkyl, or amino acids;

[0273] R aa Selected from C 1-6 alkyl, or amino acids;

[0274] X1 is selected from amino acids;

[0275] Preferably, R aa Selected from C 1-6 alkyl, or amino acids;

[0276] X1 is selected from amino acids;

[0277] X'1 is selected from amino acids;

[0278] n is an integer selected from 0 to 12;

[0279] t to t1 are each independently selected from an integer of 0 to 12;

[0280] m to m3 are each independently selected from integers of 0-24.

[0281] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 2 Selected from H, C 1-6 alkyl, or amino acids;

[0282] R a Selected from H, C 1-6 alkyl, or amino acids;

[0283] R aa Selected from C 1-6 alkyl, or amino acids;

[0284] X1 is selected from amino acids;

[0285] Preferably, R aa Selected from C 1-6alkyl, or amino acids;

[0286] X1 is selected from amino acids;

[0287] X'1 is selected from amino acids;

[0288] n is an integer selected from 0 to 12;

[0289] t to t1 are each independently selected from an integer of 0 to 12;

[0290] m to m3 are each independently selected from an integer of 0-24.

[0291] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 3 Selected from hydroxyl, C 1-6 Alkyl, -NR 6 R 7 or amino acids;

[0292] R 6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids;

[0293] R c Selected from H, alkyl, or amino acids;

[0294] R cc Selected from alkyl groups, or amino acids;

[0295] X5 is selected from amino acids;

[0296] X6 is selected from amino acids;

[0297] X7 is selected from amino acids;

[0298] Preferably, R 3 Selected from hydroxyl, C 1-6 Alkyl, -NR 6 R 7 or amino acids;

[0299] R 6 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids;

[0300] R 7 Selected from H, C 1-6Alkyl, -CH2C(O)NH2, or amino acids;

[0301] R c Selected from H, C 1-6 alkyl, or amino acids;

[0302] R cc Selected from C 1-6 alkyl, or amino acids;

[0303] X5 is selected from amino acids;

[0304] X'5 is selected from amino acids;

[0305] X6 is selected from amino acids;

[0306] X'6 is selected from amino acids;

[0307] X7 is selected from amino acids;

[0308] X'7 is selected from amino acids;

[0309] n2 is an integer selected from 0 to 12;

[0310] t6 to t9 are each independently selected from an integer of 0 to 12;

[0311] m13 to m21 are each independently selected from integers of 0-24.

[0312] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 4 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids;

[0313] R b Selected from H, C 1-6 alkyl, or amino acids;

[0314] R bb Selected from C 1-6 alkyl, or amino acids;

[0315] X2 is selected from amino acids;

[0316] X3 is selected from amino acids;

[0317] X4 is selected from amino acids;

[0318] Preferably, R 4 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids;

[0319] R b Selected from H, C 1-6 alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group;

[0320] R bb Selected from C 1-6 alkyl, or amino acids;

[0321] X2 is selected from amino acids;

[0322] X'2 is selected from amino acids;

[0323] X3 is selected from amino acids;

[0324] X'3 is selected from amino acids;

[0325] X4 is selected from amino acids;

[0326] X'4 is selected from amino acids;

[0327] n1 is an integer selected from 0 to 12;

[0328] t2 to t5 are each independently selected from an integer of 0 to 12;

[0329] m4 to m12 are each independently selected from an integer of 0 to 24;

[0330] m22 is selected from integers of 0-24.

[0331] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that R 5 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids;

[0332] R b Selected from H, C 1-6 alkyl, or amino acids;

[0333] R bb Selected from C 1-6 alkyl, or amino acids;

[0334] X2 is selected from amino acids;

[0335] X3 is selected from amino acids;

[0336] X4 is selected from amino acids;

[0337] Preferably, R 5 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids, further preferably, selected from

[0338] R b Selected from H, C 1-6 alkyl, or amino acid, wherein the alkyl group may be further substituted by a hydroxyl group or an alkoxy group; R bb Selected from C 1-6 alkyl, or amino acids;

[0339] X2 is selected from amino acids;

[0340] X'2 is selected from amino acids;

[0341] X3 is selected from amino acids;

[0342] X'3 is selected from amino acids;

[0343] X4 is selected from amino acids;

[0344] X'4 is selected from amino acids;

[0345] n1 is an integer selected from 0 to 12;

[0346] t2 to t5 are each independently selected from an integer of 0 to 12;

[0347] m4 to m12 are each independently selected from an integer of 0 to 24;

[0348] m22 is selected from integers of 0-24.

[0349] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from

[0350] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from

[0351] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from

[0352] or, Selected from

[0353] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from

[0354] or, Selected from

[0355] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from Selected from

[0356] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from Selected from

[0357] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from

[0358] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from

[0359] or, Selected from

[0360] In a preferred embodiment of the present invention, the compound, its stereoisomer or pharmaceutically acceptable salt thereof is characterized in that: Selected from

[0361] or Selected from

[0362] In a preferred embodiment of the present invention, the amino acid is Gly, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Asp, His, Asn, Glu, Lys, Gln, Met, Arg, Ser, Thr, Cys or Pro;

[0363] Preferably, the amino acid is Gln;

[0364] More preferably, the amino acid is Gln or Glu.

[0365] In a further embodiment of the present invention, said n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0366] n1 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0367] n2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0368] t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0369] t1 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0370] t2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0371] t3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0372] t4 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0373] t5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0374] t6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0375] t7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0376] t8 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0377] t9 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0378] m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0379] m1 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0380] m2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0381] m3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0382] m4 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0383] m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0384] m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0385] m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0386] m8 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0387] m9 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0388] m10 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0389] m11 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0390] m12 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0391] m13 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0392] m14 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0393] m15 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0394] m16 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0395] m17 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0396] m18 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0397] m19 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0398] m20 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0399] m21 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0400] m22 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0401] In a preferred embodiment of the present invention, the compound, its stereoisomer or a pharmaceutically acceptable salt thereof is characterized in that the amino acid is Gly, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Asp, His, Asn, Glu, Lys, Gln, Met, Arg, Ser, Thr, Cys, Pro,

[0402] Preferably, the amino acids are Glu, Thr,

[0403] The present invention also provides a method for preparing the above-mentioned compound, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that it is based on solid phase or liquid phase synthesis;

[0404] Preferably, the synthesis method comprises:

[0405] 1) Synthesize resin peptides based on solid phase synthesis method;

[0406] 2) cleaving the resin peptide obtained in step 1) to obtain a polypeptide intermediate;

[0407] 3) The polypeptide intermediate undergoes condensation reaction with the side chain;

[0408] 4) removing the protecting group of the peptide obtained in step 3) and cyclizing the peptide to obtain the final product;

[0409] Further preferably, the synthesis method comprises:

[0410] 1) Solid-phase synthesis method based on the Fmoc method, synthesizing resin peptides and acetic anhydride end-capping;

[0411] 2) cleaving the resin peptide obtained in step 1) to obtain a polypeptide intermediate;

[0412] 3) The polypeptide intermediate and the side chain undergo condensation reaction using a coupling agent;

[0413] 4) After removing the protecting group of the peptide segment obtained in step 3), the peptide segment is oxidized into a ring through a disulfide bond to obtain the final product.

[0414] The present invention further relates to a pharmaceutical composition comprising a therapeutically effective dose of the compound, its stereoisomers or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0415] In a preferred embodiment of the present invention, the pharmaceutical composition is selected from tablets, capsules, liquid preparations or injections.

[0416] In a preferred embodiment of the present invention, the pharmaceutical composition is a rapid-release preparation or a sustained-release preparation.

[0417] In certain embodiments of the present invention, the compound, its stereoisomer or a pharmaceutically acceptable salt thereof, can be administered by any convenient method, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, intrathecal or transdermal administration, and the pharmaceutical composition adjusted accordingly.

[0418] In certain embodiments of the present invention, the compound, its stereoisomers or pharmaceutically acceptable salts thereof can be formulated into liquid or solid preparations, such as syrups, suspensions, emulsions, tablets, capsules, powders, granules, or lozenges.

[0419] The present invention further relates to the use of the compound, its stereoisomer or pharmaceutically acceptable salt, or its pharmaceutical composition in the preparation of IL-23R inhibitor drugs. The present invention further relates to the use of the compound, its stereoisomer or pharmaceutically acceptable salt, or its pharmaceutical composition in the preparation of inflammatory, autoimmune diseases and cancers, such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, colitis associated with radiotherapy or chemotherapy, colitis associated with innate immune disorders such as leukocyte adhesion retardation-1, chronic granulomatous disease, type 1b glycogen storage disease, Hermansky- Prak's syndrome, Shedgkin-Donghal syndrome, Weed-Olland syndrome, pouchitis following proctocolectomy and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, psoriasis, psoriatic arthritis, irritable bowel syndrome (IBS), multiple sclerosis (MS), psoriasis, psoriatic arthritis, rheumatoid arthritis, pemphigus vulgaris, organ transplant rejection, Crohn's disease, systemic lupus erythematosus (SLE), or use of medications for diabetes.

[0420] The present invention further relates to the use of the compound, its stereoisomers or pharmaceutically acceptable salts, or its pharmaceutical compositions in the preparation of a pharmaceutical composition for treating inflammatory, autoimmune diseases and cancers, such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, colitis associated with radiotherapy or chemotherapy, colitis associated with innate immune disorders such as leukocyte adhesion retardation-1, chronic granulomatous disease, type 1b glycogen storage disease, Hermansky's disease, and inflammatory bowel disease. - Prak's syndrome, Shedgkin's-Eastern syndrome, Weeds-Oberweiss syndrome, pouchitis following proctocolectomy and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, psoriasis, psoriatic arthritis, irritable bowel syndrome (IBS), multiple sclerosis (MS), psoriasis, psoriatic arthritis, rheumatoid arthritis, pemphigus vulgaris, organ transplant rejection, Crohn's disease, systemic lupus erythematosus (SLE), or diabetic disease.

[0421] The present invention further relates to a therapeutic, preventive and / or therapeutic preparative treatment of inflammatory, autoimmune diseases and cancers, such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, colitis associated with radiotherapy or chemotherapy, colitis associated with innate immune disorders such as leukocyte adhesion retardation-1, chronic granulomatous disease, glycogen storage disease type 1b, Hermansky-Prague syndrome, Shedgkin-Eastern syndrome and Weed-Oedipus syndrome, in colorectal cancer. A method for treating pouchitis following resection and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, psoriasis, psoriatic arthritis, irritable bowel syndrome (IBS), multiple sclerosis (MS), psoriasis, psoriatic arthritis, rheumatoid arthritis, pemphigus vulgaris, organ transplant rejection, Crohn's disease, systemic lupus erythematosus (SLE) or diabetes mellitus, comprising administering to the patient a therapeutically effective dose of a stereoisomer of the compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0422] The present invention also relates to the treatment of inflammatory, autoimmune diseases and cancer in mammals, such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, colitis associated with radiation therapy or chemotherapy, colitis associated with innate immune disorders such as leukocyte adhesion deficiency-1, chronic granulomatous disease, type 1b glycogen storage disease, Hermansky-Prague syndrome, Shedgkin-Eastern syndrome and Weed-Oller syndrome, and in the setting of proctocolectomy and ileoanal anastomosis. A method for treating a condition such as pouchitis caused by surgery, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, psoriasis, psoriatic arthritis, irritable bowel syndrome (IBS), multiple sclerosis (MS), psoriasis, psoriatic arthritis, rheumatoid arthritis, pemphigus vulgaris, organ transplant rejection, Crohn's disease, systemic lupus erythematosus (SLE) or diabetes, which comprises administering to the mammal a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate or derivative thereof.

[0423] In some embodiments, the present methods relate to inflammatory, autoimmune diseases and cancers, such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, colitis associated with radiation therapy or chemotherapy, colitis associated with leukocyte adhesion molecule deficiency-1, colitis associated with an innate immune disorder, chronic granulomatous disease, glycogen storage disease type 1b, Hermansky-Präkurg syndrome, Shedgkin-Eastern syndrome, Weed-Olland syndrome, pouchitis following proctocolectomy and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, psoriasis, psoriatic arthritis, irritable bowel syndrome (IBS), multiple sclerosis (MS), psoriasis, psoriatic arthritis, rheumatoid arthritis, pemphigus vulgaris, organ transplant rejection, Crohn's disease, systemic lupus erythematosus (SLE), or diabetes mellitus;

[0424] Inflammatory bowel disease, arthritis or psoriasis are preferred.

[0425] Detailed Description of the Invention

[0426] Unless otherwise defined herein, the scientific and technical terms used in this patent application shall have the meanings commonly understood by those skilled in the art. Generally, the nomenclature used in connection with chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry and the techniques of such disciplines described herein are the nomenclature and techniques well known and commonly used in the art.

[0427] The term "peptide" broadly refers to a sequence of two or more amino acids joined together by peptide bonds. It should be understood that this term does not imply a specific length of the amino acid polymer, nor is it intended to suggest or distinguish between polypeptides produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring polypeptides. The term peptide includes cyclic peptides.

[0428] The term "amino acid" refers to any and all amino acids and their residues, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, synthetic amino acids, or rare amino acids, including both D- and L-amino acids. Natural amino acids include amino acids found in nature, such as the 23 amino acids that combine into peptide chains to form the building blocks of a large number of proteins. These stereoisomers are primarily L stereoisomers, but some D-amino acids are present in bacterial envelopes and some antibiotics. The 20 "standard" natural amino acids are alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). The "non-standard" natural amino acids are pyrrolysine (found in methanogens and other eukaryotic organisms), selenocysteine ​​(present in many non-eukaryotic organisms as well as most eukaryotic organisms), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Non-natural" or "unnatural amino acids" are naturally occurring or chemically synthesized non-proteinogenic amino acids (i.e., amino acids that are not naturally encoded or found in the genetic code). More than 140 unnatural amino acids are known and thousands of possible combinations. Examples of "unnatural" amino acids include β-amino acids (β 3 and β 2 ), homoamino acids, proline derivatives and pyruvate derivatives, tri-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear nuclear amino acids, diamino acids, D-diamino acids, α-methyl amino acids and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. "Modified" amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include groups or chemical moieties that are not naturally present on amino acids. Preferably, the non-natural amino acids of the present invention include, but are not limited to,

[0429] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like.

[0430] The hydrogen atoms described in the present invention can all be replaced by their isotope deuterium, and any hydrogen atom in the example compounds of the present invention can also be replaced by a deuterium atom.

[0431] "Hydroxy" refers to an -OH group.

[0432] "THF" refers to tetrahydrofuran.

[0433] "MeOH" refers to methanol.

[0434] "DMF" refers to N,N-dimethylformamide.

[0435] "TFA" refers to trifluoroacetic acid.

[0436] "TEA" refers to triethylamine.

[0437] "MeI" refers to methyl iodide.

[0438] "DMA" refers to N,N-dimethylacetamide.

[0439] "Et2O" refers to diethyl ether.

[0440] "DCM" refers to dichloromethane.

[0441] "DMAP" refers to 4-dimethylaminopyridine.

[0442] "DCC" refers to dicyclohexylcarbodiimide.

[0443] "DCE" refers to 1,2-dichloroethane.

[0444] "DIEA" refers to N,N-diisopropylethylamine.

[0445] "NBS" refers to N-bromosuccinimide.

[0446] "NIS" refers to N-iodosuccinimide.

[0447] "Cbz-Cl" refers to benzyl chloroformate.

[0448] "Pd2(dba)3" refers to tris(dibenzylideneacetone)dipalladium.

[0449] "Dppf" refers to 1,1'-bis(diphenylphosphino)ferrocene.

[0450] "HATU" refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0451] "KHMDS" refers to potassium hexamethyldisilazide.

[0452] "LiHMDS" refers to lithium bis(trimethylsilylamide).

[0453] "MeLi" refers to methyllithium.

[0454] "n-BuLi" refers to n-butyllithium.

[0455] "NaBH(OAc)3" refers to sodium triacetoxyborohydride.

[0456] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0457] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that are safe and effective when used in mammals and have the desired biological activity. DETAILED DESCRIPTION

[0458] The present invention is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present invention.

[0459] Example

[0460] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements are performed using a Bruker AVANCE-400 nuclear magnetic spectrometer, using deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or deuterated water (D2O) as the internal standard, with tetramethylsilane (TMS).

[0461] Liquid chromatography-mass spectrometry (LC-MS) was performed using an electrospray ion chromatography (EI) system: Thermo Scientific-LTQ-XL. HPLC was performed using an Agilent 1260 high-pressure liquid chromatograph (Phenomenex Gemini C18, 4.6×150 mm, 5 μm column). Thin-layer chromatography (TLC) silica gel plates were Yantai Huanghai HSGF254 or Qingdao GF254. The plate size used for TLC was 0.15 mm to 0.20 mm, and the plate size used for TLC separation and purification was 0.4 mm to 0.5 mm. Column chromatography generally used Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0462] The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

[0463] Unless otherwise specified, all reactions of the present invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, with dry solvents and reaction temperatures in degrees Celsius.

[0464] The eluent systems for silica gel column chromatography and the developing solvent systems for thin-layer chromatography used for the intermediates and purified compounds in the examples include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, and C: dichloromethane and acetone system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

[0465] Intermediate A

[0466] first step

[0467] To a solution of compound A1 (50 g, 0.19 mol) in DMF (350 mL) was added NaHCO₃ (79.8 g, 0.95 mol) and MeI (80.9 g, 0.57 mol) at room temperature. The mixture was stirred at 30°C for 16 hours, then diluted with water and extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over Na₂SO₄, and filtered. The filtrate was concentrated to give compound A2 (55 g), which was used directly in the next step.

[0468] LC / MS: [M+H] + -100=178.0.

[0469] Step 2

[0470] Compound A2 (10 g, 36.06 mmol) was dissolved in TFA / DCM (v:v = 1:2, 100 mL) and stirred at room temperature for 2 hours. The reaction solution was concentrated to obtain compound A3 (10 g), which was used directly in the next step.

[0471] LC / MS: [M+H] + =178.1.

[0472] Step 3

[0473] To a solution of compound A1 (9.5 g, 36 mmol) in CHCN (250 mL) at 0°C were added compound A3 (6.4 g, 36 mmol), DIEA (23.3 g, 180 mmol), and HATU (13.6 g, 36 mmol). The reaction mixture was stirred at room temperature for 1 hour, diluted with water, and extracted three times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over NaSO, and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (0%-5%, MeOH / DCM) to afford compound A4 (15 g) in a 98.7% yield.

[0474] LC / MS: [M+H] +-100=323.2.

[0475] Step 4

[0476] Compound A4 (9.1 g, 21.54 mmol) was dissolved in HCl / dioxane (4N, 100 mL) and stirred at room temperature for 1 hour. The reaction solution was concentrated to obtain compound A5 (7 g), which was used directly in the next step.

[0477] LC / MS: [M+H] + =323.4.

[0478] Step 5

[0479] To a solution of (S)-4-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (7.2 g, 21.34 mmol) in CHCN (80 mL) was added compound A5 (6.88 g, 21.34 mmol), DIEA (13.79 g, 106.71 mmol), and HATU (8.05 g, 21.34 mmol) at 0°C. The reaction was stirred at room temperature for 16 hours. The mixture was diluted with brine and extracted three times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over NaSO, and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (0%-5%, MeOH / DCM) to afford compound A6 (7 g) in a 51.1% yield.

[0480] LC / MS: [M+H] + =642.6.

[0481] Step 6

[0482] To a solution of compound A6 (7 g, 10.91 mmol) in THF (150 mL) was added 10% Pd / C (700 mg) at room temperature. The mixture was stirred under a hydrogen atmosphere (1.5 atm) at room temperature for 16 hours. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to provide compound A7 (5.07 g) in a 91.6% yield.

[0483] LC / MS: [M+H] + =508.4.

[0484] Step 7

[0485] To a solution of mono-tert-butyl hexadecanedioate (4.45 g, 12.99 mmol) in CHCN (80 mL) at 0°C was added compound A7 (5.07 g, 10 mmol), DIEA (6.45 g, 50 mmol), and HATU (4.45 g, 13 mmol). The reaction mixture was stirred at room temperature for 16 hours, diluted with brine, and extracted three times with EtOAc. The combined organic layers were washed with saturated brine, dried over NaSO, and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (0%-5%, MeOH / DCM) to afford compound A8 (7.7 g) in a 92.6% yield.

[0486] LC / MS: [M+H] + =832.7;

[0487] 1 H NMR(400MHz,CD3OD)δ4.24(dd,J=9.1,5.1Hz,1H),4.16(s,2H),4.00(s,2H),3.73(s,3H),3.69(d t,J=5.3,2.7Hz,4H),3.66–3.62(m,4H),3.56(dt,J=8.4,5.5Hz,4H),3.44(t,J=5.4Hz,2H),3.37 (t,J=5.5Hz,2H),2.29(t,J=7.7Hz,2H),2.25–2.16(m,4H),2.10(qd,J=7.6,5.3Hz,1H) ,1.89(dt,J=16.5,7.5Hz,1H),1.64–1.52(m,4H),1.45(d,J=9.1Hz,18H),1.29(s,20H).

[0488] Step 8

[0489] To a solution of compound A8 (4 g, 4.81 mmol) in THF (40 mL) was added a solution of LiOH (0.23 g, 9.61 mmol) in water (10 mL) at 0°C and allowed to react for 1 hour. 1N HCl was added to the reaction solution to adjust the pH to 4, and the aqueous phase was extracted three times with EtOAc. The combined organic layers were washed with saturated brine, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to afford compound A9 (4 g), which was used directly in the next step.

[0490] LC / MS: [M+H] + =818.7.

[0491] Step 9

[0492] To a solution of compound A9 (3.5 g, 4.28 mmol) in CH3CN (40 mL) were added DIEA (3.32 g, 25.67 mmol), HATU (1.94 g, 5.13 mmol), and 2-amino-N-(2-(2-(3-methoxy-3-oxopropoxy)ethoxy)ethyl)-N,N-dimethylethane-1-aminium hydrochloride (1.92 g, 6.42 mmol) in sequence at 0°C, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound A10 (as a TFA salt, 2.16 g) in a yield of 43.7%.

[0493] LC / MS: [M] + =1062.8.

[0494] Step 10

[0495] To a solution of compound A10 (2 g, 1.88 mmol) in THF (20 mL) was added aqueous LiOH (135 mg, 5.64 mmol, 10 mL) at 0°C, and the mixture was allowed to react for 1 hour. The reaction solution was concentrated to remove THF, and 1N HCl was added dropwise to the residue to adjust the pH to 7. The mixture was purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to afford compound A11 (as the TFA salt, 0.7 g) in a yield of 35.5%.

[0496] LC / MS: [M] + =1048.8.

[0497] Step 11

[0498] To a solution of compound A11 (0.7 g, 0.67 mmol) in CH3CN (10 mL) were added DIEA (517 mg, 4.0 mmol), HATU (302 mg, 0.8 mmol), and 2-amino-N-(2-(2-(3-methoxy-3-oxopropoxy)ethoxy)ethyl)-N,N-dimethylethane-1-aminium hydrochloride (208 mg, 1.0 mmol) in sequence at 0°C, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound A12 (as a TFA salt, 820 mg) in a yield of 99.2%.

[0499] LC / MS: [(M+H) / 2] + =620.4.

[0500] Step 12

[0501] To a solution of compound A12 (700 mg, 0.56 mmol) in THF (20 mL) was added 10% Pd / C (70 mg) at room temperature. The reaction was stirred under a hydrogen atmosphere (1.5 atm) at room temperature for 16 hours. The reaction solution was filtered through celite, the filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to provide compound intermediate A (as the TFA salt, 600 mg) in a 95.1% yield.

[0502] LC / MS: [M] + =1104.7.

[0503] 1 H NMR (400MHz, CD3OD) δ4.24 (dd, J=9.2, 5.2Hz, 1H), 4.02 (d, J=14.0Hz, 4H), 3.94 (s, 2H), 3.74 (q, J=6.1Hz, 4H) ,3.71–3.63(m,14H),3.61–3.54(m,6H),3.48(dt,J=10.6,5.6Hz,4H),3.40–3.35(m,2H),3.22(s,6H),3.13( t,J=5.8Hz,2H),2.73(s,3H),2.51(t,J=6.2Hz,2H),2.29(t,J=7.6Hz,2H),2.26–2.17(m,4H),2.11(ddd,J=1 5.6,10.3,6.6Hz,1H),1.95–1.82(m,1H),1.59(dt,J=22.3,7.1Hz,4H),1.45(d,J=9.0Hz,18H),1.29(s,20H).

[0504] Intermediate A1

[0505] first step

[0506] L-phenylglycine (3.35 g, 40.26 mmol) was dissolved in methanol (150 mL) and cooled to -78°C. A methanol solution (20 mL) of 6,6-dimethylbicyclo[3.1.0]hexan-3-one A1a (CAS: 13855-29-3, 5.0 g, 40.26 mmol) and a methanol solution (20 mL) of tert-butyl isocyanate were added dropwise. The mixture was gradually warmed to room temperature and allowed to react for 24 hours. The reaction solution was concentrated, dissolved in 200 mL of diethyl ether, and the insoluble material was filtered off. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (EA / PE gradient elution) to obtain compound (2S)-methyl 2-((3-(tert-butylcarbamoyl)-6,6-dimethylbicyclo[3.1.0]hexan-3-yl)amino)-2-phenylacetate A1b (10.5 g) in a 70.0% yield.

[0507] LC / MS: [M+H] + =373.3.

[0508] Step 2

[0509] Methyl (2S)-2-((3-(tert-butylcarbamoyl)-6,6-dimethylbicyclo[3.1.0]hexan-3-yl)amino)-2-phenylacetate A1b (10.0 g, 26.84 mmol) and palladium hydroxide (1.13 g, 8.05 mmol) were dissolved in methanol (200 mL). The atmosphere was replaced with hydrogen three times, and the reaction was allowed to proceed at room temperature for 16 hours. The reaction solution was filtered through celite, the filtrate was concentrated, dissolved in diethyl ether (150 mL), and extracted twice with 2N dilute hydrochloric acid (50 mL). The combined aqueous phases were adjusted to pH 8 with solid potassium carbonate, extracted twice with diethyl ether (100 mL), and concentrated under reduced pressure to afford 3-amino-N-(tert-butyl)-6,6-dimethylbicyclo[3.1.0]hexane-3-carboxamide A1c (4.9 g) in an 81.4% yield.

[0510] LC / MS: [M+H] + =225.2.

[0511] Step 3

[0512] 3-Amino-N-(tert-butyl)-6,6-dimethylbicyclo[3.1.0]hexane-3-carboxamide Alc (4.9 g, 21.84 mmol) was dissolved in 6N hydrochloric acid (50 mL) and refluxed for 16 hours. The reaction mixture was cooled to room temperature, extracted and washed with diethyl ether, and the aqueous phase was collected and concentrated under reduced pressure to obtain 3-amino-6,6-dimethylbicyclo[3.1.0]hexane-3-carboxylic acid Ald (hydrochloride, 4.4 g) in a 98% yield.

[0513] LC / MS: [M+H] + =170.1.

[0514] Step 4

[0515] 3-Amino-6,6-dimethylbicyclo[3.1.0]hexane-3-carboxylic acid A1d (hydrochloride, 4.4 g, 21.39 mmol) was dissolved in sodium bicarbonate solution (100 mL). A solution of 9-fluorenylmethyl-N-succinimidyl carbonate (10.79 g, 32.09 mmol) in dioxane (80 mL) was added and allowed to react at room temperature for 1 hour. The reaction solution was extracted twice with ethyl acetate (100 mL). The combined organic phases were washed with water. The aqueous phase was adjusted to pH 2 with 2N dilute hydrochloric acid and extracted again twice with ethyl acetate (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (MeOH / DCM gradient elution) to give intermediate A1 3-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6,6-dimethylbicyclo[3.1.0]hexane-3-carboxylic acid (5.6 g) in a yield of 66.9%.

[0516] LC / MS: [MH] - =390.2.

[0517] Intermediate A2

[0518] Referring to the synthesis method of intermediate A1, intermediate A2 1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(difluoromethylene)cyclohexane-1-carboxylic acid was obtained by using 4-(difluoromethylene)cyclohexanone (CAS: 137780-61-1) and subsequent similar experimental operations.

[0519] LC / MS: [MH] - =412.1.

[0520] Intermediate A3

[0521] first step

[0522] To a solution of tetrahydrothiopyran-4-one A3a (50 g, 431.0 mmol) in methanol (30 mL) was added a 7 M methanolic ammonia solution (300 mL) at 0°C and stirred at 0°C for 3 h. Trimethylsilyl cyanide (44.9 g, 452.6 mmol) was then added dropwise. After completion, the mixture was gradually warmed to room temperature and stirred for 1.5 h. The reaction solution was concentrated to yield a light brown oil. This was then slurried with petroleum ether (300 mL) at 0°C, resulting in the precipitation of solids. The product was filtered and dried to yield 4-aminotetrahydrothiopyran-4-carbonitrile A3b (61.5 g), which was used directly in the next step without further purification.

[0523] LC / MS: [M+H] + =143.1.

[0524] Step 2

[0525] A3b (61.5 g, 433.0 mmol) was dissolved in a mixture of dioxane (300 mL) and water (300 mL), and sodium carbonate (55.1 g, 519.7 mmol) was added. A solution of 9-fluorenylmethyl chloroformate (117.6 g, 454.7 mmol) in dioxane (100 mL) was added dropwise to the reaction mixture at 0°C, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was slurried with water (400 mL) and filtered to obtain a white solid. The resulting solid was slurried with petroleum ether (300 mL), filtered, and dried to obtain (9H-fluoren-9-yl)methyl (4-cyanotetrahydro-2H-thiopyran-4-yl)carbamate A3c (124 g) in a yield of 79%.

[0526] LC / MS: [M+H] + =365.2.

[0527] Step 3

[0528] (9H-fluoren-9-yl)methyl (4-cyanotetrahydro-2H-thiopyran-4-yl)carbamate A3c (50.0 g, 137.4 mmol) was dissolved in dichloromethane (150 mL). 85% m-chloroperbenzoic acid (83.7 g, 412.1 mmol) was added portionwise at 0°C. The mixture was gradually warmed to room temperature and stirred overnight. The reaction mixture was filtered, and the filter cake was dissolved in dichloromethane / methanol (10:1) (800 mL) and washed with saturated sodium bicarbonate solution (300 mL x 4). The organic phase was dried and concentrated to afford (9H-fluoren-9-yl)methyl (4-cyanotetrahydro-1,1-dioxotetrahydro-2H-thiopyran-4-yl)carbamate A3d (38.1 g) in a 28.2% yield.

[0529] LC / MS: [M+H] + =396.2.

[0530] Step 4

[0531] A3d (38.1 g, 96.2 mmol) was dissolved in 4N hydrochloric acid in dioxane (300 mL), followed by the addition of concentrated hydrochloric acid (400 mL) and stirring at 90°C for 30 h. The reaction mixture was concentrated to 200 mL and slurried with water (400 mL). Solid precipitated, and the resulting solid was filtered and slurried with ethyl acetate (200 mL). The resulting solid was filtered and dried to obtain intermediate A3 (12.77 g, purity: 97.80%).

[0532] LC / MS: [M+H] + =416.2;

[0533] 1H NMR (400MHz, DMSO-d6) δ7.91(d,3H),7.72(d,2H),7.43(t,2H),7.35(td,2H),4.35(d,2H),4.25(t,1H),3.09(s,4H),2.46(s,2H),2.29(s,2H).

[0534] Intermediate B

[0535] first step

[0536] To a solution of compound B1 (2.1 g, 16.13 mmol) in DCM (21 mL) at room temperature were added compound B2 (3.5 g, 16.25 mmol) and TEA (4.93 g, 48.72 mmol). T3P (50%, 20.69 g, 32.51 mmol) was then added under ice-cooling. The mixture was stirred at room temperature for 16 hours, then concentrated, ethyl acetate and water were added, and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were washed with 1M HCl, then with saturated NaHCO3 solution, dried over Na2SO4, and filtered. The filtrate was concentrated to give compound B3 (5 g) in a 94.6% yield. The crude product was used directly in the next reaction.

[0537] LC / MS: [M+H] + -56=272.2.

[0538] Step 2

[0539] Compound B3 (5 g, 15.27 mmol) was dissolved in TFA / DCM (v:v = 1:1, 50 mL) and stirred at room temperature for 2 hours. The reaction solution was concentrated, dissolved in an appropriate amount of DCM, and then concentrated three times. After drying and concentrating the DCM, compound B4 (2.7 g) was obtained in a 65.2% yield. The crude product was used directly in the next reaction.

[0540] LC / MS: [M+H] + =272.2.

[0541] Step 3

[0542] To a solution of compound B4 (2.7 g, 9.95 mmol) in DMF (30 mL) was added compound B5 (4.47 g, 14.92 mmol) at room temperature. After cooling in an ice bath, HATU (4.54 g, 11.94 mmol) and DIEA (7.71 g, 59.69 mmol) were added to the solution. The mixture was stirred at 0°C for 2 hours and then concentrated. An appropriate amount of CH3CN was added to the residue for dissolution. The residue was purified by reverse phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound B6 (1.5 g) in a yield of 29.4%.

[0543] LC / MS: [M] + =516.4.

[0544] Step 4

[0545] To a solution of compound B6 (1.5 g, 2.9 mmol) in THF (15 mL) was added LiOH (244 mg, 5.8 mmol) under ice-cooling, and the mixture was stirred at 0°C for 2 hours. The reaction solution was adjusted to neutral pH with 1 M HCl and concentrated. The residue was dissolved in an appropriate amount of CH3CN and purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound B7 (1.3 g) in a yield of 89.6%.

[0546] LC / MS: [M] + =502.4.

[0547] Step 5

[0548] To a solution of compound B7 (1.1 g, 2.2 mmol) in CH3CN (10 mL) was added compound B8 (763 mg, 4.4 mmol) at room temperature. After cooling in an ice bath, HATU (1.0 g, 2.6 mmol) and DIEA (1.41 g, 10.9 mmol) were added. The mixture was stirred at 0°C for 2 hours and then concentrated. An appropriate amount of CH3CN was added to the residue for dissolution. The residue was purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to afford compound B9 (1.3 g) in an 89.8% yield.

[0549] LC / MS: [M] + =658.6.

[0550] Step 6

[0551] Compound B9 (1.3 g, 1.97 mmol) was dissolved in HCl / dioxane (4N, 100 mL) and stirred at room temperature for 2 hours. The reaction solution was concentrated to obtain compound intermediate B (hydrochloride salt, 1.08 g) with a yield of 98.2%.

[0552] LC / MS: [M] + =558.6;

[0553] 1 H NMR (400MHz, MeOD) δ3.94(s,2H),3.76(t,J=6.2Hz,2H),3.66(t,J=4.5Hz,8H),3.52(dd,J=12.9,6.7Hz,4H),3.27–3.07(m,10H),2.73(s, 3H), 2.52(t,J=6.1Hz,2H),2.23(td,J=7.6,3.8Hz,4H),1.61(s,4H),1.50(d,J=6.6Hz,2H),1.33(d,J=9.9Hz,12H),0.91(t,J=6.7Hz,3H).

[0554] Intermediate C

[0555] Intermediate C was synthesized according to the preparation method of WO 2021 / 127460.

[0556] Intermediate D

[0557] Intermediate D was synthesized with reference to the preparation method of WO 2021 / 041770.

[0558] Intermediate L1

[0559] first step

[0560] To a solution of compound L1-1 (0.9 g, 3.52 mmol) in CH3CN (25 mL) were added DIEA (2.73 g, 21.1 mmol), HATU (1.61 g, 4.22 mmol), and N-Cbz-N-methylethylenediamine (hydrochloride, 1.29 g, 5.28 mmol) in sequence at 0°C, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (CH3CN / 0.03% TFA / water, 0-100% gradient elution) to give compound L1-2 (as a TFA salt, 1.35 g) in a yield of 73%.

[0561] LC / MS: [M] + =410.3.

[0562] Step 2

[0563] To a solution of compound L1-2 (1.2 g, 2.29 mmol) in THF (50 mL) was added 10% Pd / C (120 mg) at room temperature. The reaction was stirred under a hydrogen atmosphere (1.5 atm) at room temperature for 16 hours. The reaction solution was filtered through celite, the filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (CH3CN / 0.03% TFA / water, 0-100% gradient elution) to provide compound intermediate L1 (as the TFA salt, 760 mg) in an 85% yield.

[0564] LC / MS: [M] + =276.2.

[0565] 1 H NMR(400MHz,MeOD)δ3.99–3.91(m,2H),3.77(t,J=6.2Hz,2H),3.70–3.62(m,4H),3.62 –3.52(m,4H),3.23(s,9H),3.10(t,J=5.6Hz,2H),2.70(s,3H),2.57(t,J=6.2Hz,2H).

[0566] Intermediate L2

[0567] first step

[0568] To a solution of compound L2-1 (0.6 g, 2.86 mmol) in CH3CN (20 mL) at 0°C were added DIEA (2.22 g, 17.2 mmol), HATU (1.31 g, 3.43 mmol), and N-Cbz-N-methylethylenediamine (hydrochloride, 1.05 g, 4.29 mmol) in sequence, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (CH3CN / 0.03% TFA / water, 0-100% gradient elution) to give compound L2-2 (as a TFA salt, 1.17 g) in an 86% yield.

[0569] LC / MS: [M] + =364.3.

[0570] Step 2

[0571] To a solution of compound L2-2 (1.0 g, 2.29 mmol) in THF (50 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction was stirred under a hydrogen atmosphere (1.5 atm) at room temperature for 16 hours. The reaction solution was filtered through celite, the filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (CH3CN / 0.03% TFA / water, 0-100% gradient elution) to provide compound intermediate L2 (as the TFA salt, 0.66 mg) in a 92% yield.

[0572] LC / MS: [M] + =230.

[0573] 1 H NMR (400MHz, DMSO-d6) δ8.47-8.37(m,2H),3.35–3.21(m,4H),3.05(s,9H),2.79(t,J=6.0Hz,2H) ,2.53–2.45(m,3H),2.16-2.09(m,2H),1.72–1.62(m,2H),1.60-1.51(m,2H),1.29-1.21(m,2H).

[0574] Intermediate L3

[0575] first step

[0576] To a solution of compound L3-1 (CAS: 1118767-16-0, 4.0 g, 4.73 mmol) in CH3CN (45 mL) were added DIEA (3.67 g, 28.37 mmol), HATU (2.16 g, 5.67 mmol) and 2-amino-N-(2-(2-(3-methoxy-3-oxopropoxy)ethoxy)ethyl)-N,N-dimethylethane-1-ammonium hydrochloride (2.13 g, 7.09 mmol) in sequence at 0°C, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound L3-2 (as a TFA salt, 3.05 g) in a yield of 53.6%.

[0577] LC / MS: [M] + =1090.8.

[0578] Step 2

[0579] To a solution of compound L3-2 (2.4 g, 1.99 mmol) in THF (24 mL) was added aqueous LiOH (143 mg, 5.97 mmol, 12 mL) at 0°C, and the mixture was allowed to react for 1 hour. The reaction solution was concentrated to remove THF, and 1N HCl was added dropwise to the residue to adjust the pH to 7. The mixture was purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to afford compound L3-3 (as a TFA salt, 1.1 g) in a yield of 46.4%.

[0580] LC / MS: [M] + =1076.7.

[0581] Step 3

[0582] To a solution of compound L3-3 (0.8 g, 0.67 mmol) in CH3CN (12 mL) were added DIEA (521 mg, 4.03 mmol), HATU (306 mg, 0.81 mmol), and N-Cbz-N-methylethylenediamine (hydrochloride, 245 mg, 1.01 mmol) in sequence at 0°C, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound L3-4 (as a TFA salt, 870 mg) in a yield of 93.8%.

[0583] LC / MS: [(M+H) / 2] + =632.9.

[0584] Step 4

[0585] To a solution of compound L3-4 (650 mg, 0.56 mmol) in THF (20 mL) was added 10% Pd / C (65 mg) at room temperature. The reaction was stirred under a hydrogen atmosphere (1.5 atm) at room temperature for 16 hours. The reaction solution was filtered through celite, the filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to afford compound intermediate L3 (as a TFA salt, 570 mg) in a 97.1% yield.

[0586] LC / MS: [M] + =1132.8.

[0587] Intermediate L4

[0588] first step

[0589] To a solution of compound A9 (3.0 g, 3.67 mmol) in CH3CN (40 mL) were added DIEA (2.84 g, 22.0 mmol), HATU (1.67 g, 4.40 mmol), and N-(2-aminoethyl)-6-methoxy-N,N-dimethyl-6-oxohexane-1-aminium hydrochloride (1.4 g, 5.50 mmol) in sequence at 0°C, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound L4-1 (as a TFA salt, 1.75 g) in a yield of 42.2%.

[0590] LC / MS: [M] + =1016.7.

[0591] Step 2

[0592] To a solution of compound L4-1 (1.4 g, 1.24 mmol) in THF (15 mL) was added aqueous LiOH (89 mg, 3.71 mmol, 5 mL) at 0°C, and the mixture was allowed to react for 1 hour. The reaction solution was concentrated to remove THF, and 1N HCl was added dropwise to the residue to adjust the pH to 7. The mixture was purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to afford compound L4-2 (as a TFA salt, 740 mg) in a yield of 53.6%.

[0593] LC / MS: [M] + =1002.7.

[0594] Step 3

[0595] To a solution of compound L3-3 (0.7 g, 0.63 mmol) in CH3CN (10 mL) were added DIEA (486 mg, 3.76 mmol), HATU (286 mg, 0.75 mmol), and N-Cbz-N-methylethylenediamine (hydrochloride, 229 mg, 0.94 mmol) in sequence at 0°C, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to give compound L4-3 (as a TFA salt, 770 mg) in a yield of 94.0%.

[0596] LC / MS: [(M+H) / 2] + =596.9.

[0597] Step 4

[0598] To a solution of compound L4-3 (0.7 g, 0.56 mmol) in THF (20 mL) was added 10% Pd / C (70 mg) at room temperature. The reaction was stirred under a hydrogen atmosphere (1.5 atm) at room temperature for 16 hours. The reaction solution was filtered through celite, the filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (5%-40%, 40%-70%, CH3CN / 0.03% TFA / water) to obtain compound intermediate L4 (as the TFA salt, 590 mg) in a yield of 93.9%.

[0599] LC / MS: [M] + =1044.8.

[0600] 1 H NMR(400MHz,MeOD)δ4.31-4.23(m,1H),4.04(d,J=10.8Hz,4H),3.77-3.67(m ,10H),3.64-3.56(m,4H),3.53-3.45(m,6H),3.43-3.36(m,4H),3.20-3.12( m,8H),2.75(s,3H),2.37-2.20(m,8H),2.18-2.09(m,1H),1.98-1.81(m,4H) ,1.79-1.70(m,2H),1.69-1.53(m,5H),1.47(d,J=8.0Hz,18H),1.31(s,20H).

[0601] Intermediate L5

[0602] first step

[0603] To a solution of compound L3-1 (2 g, 2.36 mmol) in CH3CN (20 mL) at 0°C were added DIEA (1.83 g, 14.2 mmol), HATU (1.08 g, 2.84 mmol), and N-Cbz-N-methylethylenediamine (hydrochloride, 864 mg, 3.55 mmol) in sequence, and the solution was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (CH3CN / 0.03% TFA / water, 0-100% gradient elution) to give compound L5-1 (1.9 g) in a yield of 77.6%.

[0604] LC / MS: [M+H] + =1036.7.

[0605] Step 2

[0606] To a solution of compound L1-2 (1.2 g, 1.16 mmol) in THF (30 mL) was added 10% Pd / C (120 mg) at room temperature. The reaction was stirred under a hydrogen atmosphere (1.5 atm) at room temperature for 16 hours. The reaction solution was filtered through celite, the filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (CH3CN / 0.03% TFA / water, 0-100% gradient elution) to provide compound intermediate L5 (940 mg) in a 90% yield.

[0607] LC / MS: [M+H] + =902.6.

[0608] 1 H NMR(400MHz,MeOD)δ4.27(dd,J=9.1,5.2Hz,1H),4.04(d,J=5.9Hz,4H),3.70(s,6H),3.68(d,J=5.6Hz,3H),3 .62(dd,J=12.1,6.8Hz,3H),3.56(dd,J=9.5,5.7Hz,4H),3.51–3.44(m,3H),3.41(d,J=5.9Hz,2H),3.08(t,J =5.8Hz,2H),2.67(s,3H),2.31(t,J=7.6Hz,2H),2.28–2.19(m,5H),2.13(dd,J=13.7,5.9Hz,1H),2.05(d,J= 5.5Hz, 1H), 1.91 (dd, J=13.9, 9.1Hz, 1H), 1.61 (dd, J=16.0, 8.1Hz, 4H), 1.48 (d, J=9.0Hz, 18H), 1.31 (s, 24H).

[0609] Example 1

[0610] Step 1: Synthesis of fully protected linear peptides

[0611] 1.1 Hanging resin

[0612] 1.1.1 Weigh 0.6 g of 2-CTC Resin (degree of substitution S = 1.08 mmol / g) and 78 mg of N-FMOC-L-3-pyridylalanine (0.2 mmol, degree of substitution 0.33) and add them to a reaction column. Then add DCM (10 mL) and then add 0.6 mL of DIEA to the reaction column and bubble nitrogen into the column for 2 hr. Then, add 0.6 mL of MeOH to the reaction column and continue bubbling nitrogen into the column for 30 min. Drain the column until no more liquid flows out. Add DMF (30 mL) and wash the column five times for 1 min each. Drain the column until no more liquid flows out.

[0613] 1.1.2 Add 20% piperidine / DMF (15 mL) to the reaction column, blow nitrogen for 20 min, and drain until no more liquid flows out. Add DMF (30 mL) and wash five times for 1 min each, draining until no more liquid flows out. Ninhydrin test: the resin should turn blue.

[0614] 1.2. Amino acid coupling

[0615] 1.2.1 Coupling of Fmoc-Asn(Trt)-OH

[0616] 1. Weigh Fmoc-Asn(Trt)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0617] 2. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0618] 3. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0619] 1.2.2 Coupling of Fmoc-Glu(OtBu)-OH

[0620] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0621] 2. Weigh Fmoc-Glu(OtBu)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0622] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0623] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0624] 1.2.3 Coupling of Fmoc-Thp(Gly)-OH

[0625] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0626] 2. Weigh Fmoc-Thp(Gly)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HATU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0627] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0628] 4. Draw off the reaction liquid, wash with DMF 5 times, 1 min each time, and drain until no liquid flows out.

[0629] 1.2.4 Coupling of Fmoc-2Nal-OH

[0630] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Chloranil detection should indicate a green resin color.

[0631] 2. Weigh Fmoc-2-Nal-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HATU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0632] 3. React at 25℃ for 0.5h and test with chloranil. The resin is colorless and transparent.

[0633] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0634] 1.2.5 Coupling of Fmoc-4-[2-(Boc-amino)ethoxy]-L-Phenylalanine

[0635] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0636] 2. Weigh Fmoc-4-[2-(Boc-amino)ethoxy]-L-Phenylalanine (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0637] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0638] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0639] 1.2.6 Coupling of Fmoc-Pen(Trt)-OH

[0640] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0641] 2. Weigh Fmoc-Pen(Trt)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0642] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0643] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0644] 1.2.7 Coupling of Fmoc-Lys(Ac)-OH

[0645] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0646] 2. Weigh Fmoc-Lys(Ac)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0647] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0648] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0649] 1.2.8 Coupling of Fmoc-(7Me)Trp-OH

[0650] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0651] 2. Weigh Fmoc-(7Me)Trp-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0652] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0653] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0654] 1.2.9 Coupling of Fmoc-Thr(Tbu)-OH

[0655] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0656] 2. Weigh Fmoc-Thr(Tbu)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0657] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0658] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0659] 1.2.10 Coupling of Fmoc-Asn(Trt)-OH

[0660] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0661] 2. Weigh Fmoc-Asn(Trt)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0662] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0663] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0664] 1.2.11 Coupling of Fmoc-Pen(Trt)-OH

[0665] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0666] 2. Weigh Fmoc-Pen(Trt)-OH (3.0 eq) and add it to the above resin. Add DIEA (6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0667] 3. React at 25℃ for 0.5h and test with ninhydrin. The resin is colorless and transparent.

[0668] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0669] 1.2.12 Ac2O Capping

[0670] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times, 1 minute each time, draining until no more liquid flows out. Ninhydrin detection: the resin turns blue.

[0671] 2. Add DMF (10 mL), DIEA (6.00 eq), and Ac2O (5.0 eq) to the above resin, and bubbling nitrogen. Adjust the nitrogen so that the resin is evenly bulged.

[0672] 3. React at 25°C for 10 minutes and test with ninhydrin. The resin is colorless and transparent.

[0673] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out.

[0674] Step 2: Cleavage of fully protected linear peptides

[0675] 2.1 Wash with DMF 5 times (20 mL each time), 1 min each time, and drain until no liquid flows out

[0676] 2.2 Shrink the resin with MeOH (20 mL) for 3 min each time, drain until no liquid flows out, then wash the resin three times with 20 mL of tertiary methyl ether, blow dry the resin with nitrogen, and set aside.

[0677] 2.3 Pour 50 mL of the prepared 20% HFIP / DCM solution into the reactor, purge with nitrogen, react for 30 min once, cut three times, collect all the filtrates, concentrate under reduced pressure, add 50 mL of DCM and continue to concentrate under reduced pressure.

[0678] 2.4 Add 100 mL of acetonitrile and 100 mL of water and sonicate until the product is evenly dispersed. Then, freeze-dry using a freeze dryer to obtain 500 mg of intermediate 1c with a purity of 75% and a yield of 62%.

[0679] LC / MS: [M+H] + =3012.7; [M+H-243] + =2769.7

[0680] Step 3: Condensation of intermediate 1c with intermediate A

[0681] To 200 mg (0.06 mmol) of the intermediate 1c, 1 mL of DMF and DIEA (3 eq, 0.18 mmol) were added, followed by the addition of intermediate A (77 mg, 0.06 mmol, 1 eq). HATU (23 mg, 0.06 mmol, 1 eq) dissolved in 0.2 mL of DMF was then slowly added dropwise. Tof-LCMS confirmed complete consumption of the starting material, indicating a very low polarity product. The gradient was 80-100 over 2 min. LC / MS: [M] + =4099.20,[M-243] + =3856.2.

[0682] After the reaction is complete, use an oil pump to spin dry the DMF and set aside.

[0683] Step 4: Cleavage of linear peptides

[0684] 4.1. Prepare cutting fluid according to the following volume

[0685] The crude peptide obtained in step 3 was added to the prepared cutting solution and shaken on a shaker for 2.5 hours. Filtered, the filtrate was added to 10 volumes of icy isopropyl ether, centrifuged, and washed three times with isopropyl ether. Dry under vacuum for 2 hours to obtain the crude peptide. LC / MS: [(M+H) / 2] + =1402.8; [(M+2H) / 3] + =936.1.

[0686] Step 5: Oxidation of disulfide bonds

[0687] 5.1 160 mg of the crude peptide obtained in Step 4 was added to 100 mL of water and 100 mL of acetonitrile. After dissolution and clarification, 0.1 M I2 / MeOH was slowly added dropwise until the solution turned bright yellow. Stirring continued for 5 minutes. If the yellow color did not disappear, 0.1 M sodium thiosulfate was then added dropwise until the yellow color disappeared. After stirring for 2 minutes without any change, the sample was lyophilized and purified to obtain 43 mg of Example 1 with a purity of 97.41% and a yield of 18.7%.

[0688] LC / MS: [(M+H) / 2] + =1401.8; [(M+2H) / 3] + =935.1.

[0689] Purification conditions:

[0690] Example 2

[0691] Referring to the synthesis method of Example 1, 26.4 mg of the product Example 2 was obtained through the reaction of intermediate B with polypeptide intermediate 1c and subsequent similar experimental operations with a purity of 92.94% and a yield of 13.6%.

[0692] LC / MS: [(M+H) / 2] + =1184.8; [(M+2H) / 3] + =790.2.

[0693] Example 3

[0694] Referring to the synthesis method of Example 1, 42.0 mg of the product Example 3 was obtained with a purity of 96.4% and a yield of 20.2% by reacting the intermediate L1 with the polypeptide intermediate 1c and subsequent similar experimental operations.

[0695] LC / MS: [(M+H) / 2] + =1043.5.

[0696] Example 4

[0697] Referring to the synthesis method of Example 1, 28.1 mg of the product Example 4 was obtained by reacting the intermediate L3 with the polypeptide intermediate 1c and subsequent similar experimental operations with a purity of 96.2% and a yield of 14.9%.

[0698] LC / MS: [(M+H) / 2] + =1415.7; [(M+2H) / 3] + =944.2.

[0699] Example 6

[0700] Referring to the synthesis method of Example 1, 31.2 mg of the product Example 6 was obtained by reacting the intermediate L5 with the polypeptide intermediate 1c and subsequent similar experimental operations with a purity of 97.8% and a yield of 18.1%.

[0701] LC / MS: [(M+H) / 2] + =1300.1; [(M+2H) / 3] + =867.1.

[0702] Example 12

[0703] Referring to the synthesis method of Example 1, intermediate L2 was reacted with polypeptide intermediate 1c and subsequent similar experimental operations to obtain 23.8 mg of product Example 12 with a purity of 95.7% and a yield of 17.5%.

[0704] LC / MS: [(M+H) / 2] + =1020.5.

[0705] Example 12 can also be prepared by the following method:

[0706] Step 1: Condensation of intermediate 1c with intermediate L2

[0707] To the above intermediate 1c (200 mg, 0.06 mmol), DMF (1 mL) and DIEA (10 eq, 0.6 mmol) were added, followed by the addition of intermediate L2 (28 mg, 0.12 mmol, 2 eq). HATU (69 mg, 0.18 mmol, 3 eq) dissolved in DMF (0.2 mL) was then slowly added dropwise. Tof-LCMS confirmed complete consumption of the starting material. After the reaction was complete, the DMF was dried to afford intermediate 12d, which was then set aside.

[0708] Step 2: Deprotection

[0709] Prepare cutting fluid in the following volumes

[0710] The intermediate 12d obtained in the previous step was added to the prepared cutting solution, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 volumes of ice-cold isopropyl ether, centrifuged, washed three times with isopropyl ether, and vacuum dried for 2 hours to obtain intermediate 12e.

[0711] Step 3: Oxidation and purification of disulfide bonds and salt conversion

[0712] Intermediate 12e (190 mg) obtained above was added to water (160 mL) and acetonitrile (40 mL). After dissolution and clarification, 0.1 M I / MeOH was slowly added dropwise until the solution turned bright yellow. Stirring continued for 5 minutes, if the yellow color did not disappear. 0.1 M sodium thiosulfate was then added dropwise until the yellow color disappeared. After stirring for 2 minutes without any change, the reaction solution of Example 12 was obtained.

[0713] 3.1 Purification

[0714] The reaction solution was purified under the following purification conditions.

[0715] Purification conditions:

[0716] After the purified solution was tested by RP-HPLC, the qualified parts were combined and diluted to half with purified water for later use.

[0717] 3.2 Salt conversion and freeze-drying

[0718] Salt conversion conditions:

[0719] The same preparative RP-HPLC column was equilibrated at 15 ml / min for 15 minutes with MPA containing 5% MPB (MPA = 0.5% AcOH / water, MPB = 0.5% AcOH / ACN, MPC = 0.15 M NH4OAc / water). The diluted purified solution was loaded onto the column at 15 ml / min. The column was washed with 5% MPB / MPC for 20 minutes, followed by 5% MPB / MPA for 20 minutes, and finally eluted with 60% MPB / MPA. The organic solvent was removed by concentration under reduced pressure and lyophilization to yield the final product, Example 12, 35 mg, with a purity of 96.1% and a yield of 18.4%.

[0720] LC / MS: [(M+H) / 2] + =1020.5.

[0721] Example 20

[0722] Referring to the synthesis method of Example 1, 35.1 mg of the product Example 20 was obtained by reacting the intermediate L4 with the polypeptide intermediate 1c and subsequent similar experimental operations with a purity of 97.1% and a yield of 19.1%.

[0723] LC / MS: [(M+H) / 2] + =1378.7; [(M+2H) / 3] + =919.5.

[0724] Other examples were prepared with reference to the above preparation method:

[0725] Examples 1-14

[0726] Step 1: Solid-phase synthesis of fully protected linear peptides

[0727] 1.1 Hanging resin

[0728] 1.1.1 Swelling

[0729] Rink Amide-AM Resin (1-14a, 0.4 g, degree of substitution 0.5-0.7 mmol / g) was weighed and added to the reaction column. DMF (10 mL) was added and nitrogen was bubbled for 20 minutes and then discharged until no liquid flowed out.

[0730] 1.1.2 Resin hanging

[0731] 1) Add 20% piperidine / DMF (10 mL), bubble nitrogen for 30 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times for 1 minute each time, and drain until no liquid flows out.

[0732] Sampling was performed and the resin turned blue after ninhydrin testing.

[0733] 2) Fmocylsarcosine (186.6 mg, 0.6 mmol), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0734] 3) DMF (10 mL) was added to dissolve the mixture, and nitrogen was bubbled for 4 hours.

[0735] 4) Drain the solvent. Wash the resin sequentially with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL). Stir the mixture with nitrogen for 2-3 minutes after each wash, then drain the solvent.

[0736] Sampling was performed and tested with ninhydrin, and the resin was colorless.

[0737] 1.2. Amino acid coupling

[0738] 1.2.1 Coupling of Fmoc-3-(4-pyridyl)-L-alanine

[0739] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0740] Sampling, chloranil test, resin blue.

[0741] 2) Fmoc-3-(4-pyridyl)-L-alanine (234 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0742] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0743] Sampling and chloranil testing showed that the resin was colorless and transparent.

[0744] 4) Drain the solvent and wash the resin with 10 mL of DMF (10 mL), 10 mL of isopropanol (10 mL), 10 mL of DMF (10 mL), 10 mL of DMF (10 mL), and 10 mL of DMF (10 mL). Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0745] 1.2.2 Coupling of Fmoc-Asn(Trt)-OH

[0746] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0747] Sampling, ninhydrin test, resin blue.

[0748] 2) Fmoc-Asn(Trt)-OH (358 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0749] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled through the mixture, and the mixture was reacted at room temperature for 3 hours.

[0750] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0751] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0752] 1.2.3 Coupling of Fmoc-Glu(OtBu)-OH

[0753] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0754] Sampling, ninhydrin test, resin blue.

[0755] 2) Weigh Fmoc-Glu(OtBu)-OH (255 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) and add them to the reaction column.

[0756] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled through the mixture, and the mixture was reacted at room temperature for 3 hours.

[0757] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0758] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0759] 1.2.4 Coupling of 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid

[0760] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0761] Sampling, ninhydrin test, resin blue.

[0762] 2) Weigh 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid (241 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) and add them to the reaction column.

[0763] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled through the mixture, and the mixture was reacted at room temperature for 3 hours.

[0764] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0765] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0766] 1.2.5 Coupling of Fmoc-2Nal-OH

[0767] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0768] Sampling was done and chloranil was tested, the resin was reddish brown.

[0769] 2) Weigh Fmoc-2Nal-OH (262 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) and add them to the reaction column.

[0770] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0771] Sampling and chloranil testing showed that the resin was colorless and transparent.

[0772] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0773] 1.2.6 Coupling of Fmoc-4-[2-(Boc-amino)ethoxy]-L-Phenylalanine

[0774] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0775] Sampling, ninhydrin test, resin blue.

[0776] 2) Weigh Fmoc-4-[2-(Boc-amino)ethoxy]-L-Phenylalanine (328 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) and add them to the reaction column.

[0777] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0778] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0779] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0780] 1.2.7 Coupling of Fmoc-Pen(Trt)-OH

[0781] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0782] Sampling, ninhydrin test, resin blue.

[0783] 2) Fmoc-Pen(Trt)-OH (368 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0784] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0785] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0786] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0787] 1.2.8 Coupling of Fmoc-Lys(Ac)-OH

[0788] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0789] Sampling, ninhydrin test, resin blue.

[0790] 2) Fmoc-Lys(Ac)-OH (246 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0791] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0792] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0793] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0794] 1.2.9 Coupling of Fmoc-(7Me)Trp-OH

[0795] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0796] Sampling, ninhydrin test, resin blue.

[0797] 2) Fmoc-(7Me)Trp-OH (264 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0798] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0799] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0800] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0801] 1.2.10 Coupling of Fmoc-Thr(Tbu)-OH

[0802] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0803] Sampling, ninhydrin test, resin blue.

[0804] 2) Weigh Fmoc-Thr(Tbu)-OH (238 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) and add them to the reaction column.

[0805] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0806] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0807] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0808] 1.2.11 Coupling of Fmoc-Asn(Trt)-OH

[0809] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0810] Sampling, ninhydrin test, resin blue.

[0811] 2) Fmoc-Asn(Trt)-OH (358 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0812] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0813] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0814] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0815] 1.2.12 Coupling of Fmoc-Pen(Trt)-OH

[0816] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0817] Sampling, ninhydrin test, resin blue.

[0818] 2) Fmoc-Pen(Trt)-OH (368 mg), N,N'-diisopropylcarbodiimide (76 mg) and 1-hydroxybenzotriazole (81 mg) were weighed and added to the reaction column.

[0819] 3) DMF (10 mL) was added to dissolve the mixture, nitrogen was bubbled, and the mixture was reacted at room temperature for 3 hours.

[0820] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0821] 4) Drain the solvent and wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent.

[0822] 1.2.13 Ac2O Capping

[0823] 1) Add 20% piperidine / DMF (10 mL) to the reaction column, bubble nitrogen for 20 minutes, and drain until no liquid flows out. Add DMF (10 mL) and wash 5 times, 1 minute each time, and drain until no liquid flows out.

[0824] Sampling, ninhydrin test, resin blue.

[0825] 2) DCM (10 mL), DIEA (6.0 eq), and Ac2O (5.0 eq) were added to the above resin and nitrogen was added. The nitrogen was adjusted to ensure uniform bubbling of the resin.

[0826] 3) React at room temperature for 30 minutes.

[0827] Sampling was performed and ninhydrin was tested, and the resin was colorless and transparent.

[0828] 4) Wash the resin with DMF (10 mL), isopropanol (10 mL), DMF (10 mL), DMF (10 mL), and DMF (10 mL) in sequence. Bubble nitrogen and stir for 2-3 minutes before draining the solvent after each wash.

[0829] 5) Sample washing: The resin was washed with isopropyl ether (10 mL) bubbling nitrogen with stirring for 2-3 minutes, then drained. This operation was repeated three times to obtain resin peptide 1-14b.

[0830] Step 2: Cleavage and deprotection of linear peptides

[0831] Prepare cutting fluid in the following volumes

[0832] Add 10 mL of the prepared cleavage solution to the resulting resin peptide 1-14b and shake in a 25°C water bath for 2.5 hours. Filter and add the filtrate to 10 volumes of icy isopropyl ether. Centrifuge and discard the supernatant. Wash three times with isopropyl ether and vacuum dry for 2 hours to obtain crude peptide 1-14c.

[0833] Step 3: Oxidation of disulfide bonds

[0834] The crude peptide 1-14c (190 mg) obtained above was added to water (100 mL) and acetonitrile (100 mL). After dissolution and clarification, 0.1 M I2 / MeOH was slowly added dropwise until the solution turned bright yellow. If the yellow color persisted after stirring for 5 minutes, 0.1 M sodium thiosulfate was then added dropwise until the yellow color disappeared. After stirring for 2 minutes without any change, the sample was purified using the following purification conditions.

[0835] Purification conditions:

[0836] The purified solution was concentrated under reduced pressure to remove the organic solvent and lyophilized to give 45 mg of the final product Example 1-14 with a purity of 97.5% and a yield of 11.7%.

[0837] MS: m / z = 1931.8, [M+H] + ; m / z=966.4,[M+2H] 2+ .

[0838] Example 1-16-1

[0839] Step 1: Synthesis of fully protected linear peptides

[0840] 1.1 Hanging resin

[0841] 1.1.1 Weigh 0.6 g of 2-CTC Resin (1-16-1a, degree of substitution S = 1.08 mmol / g) and 78 mg of N-Fmoc-L-3-pyridylalanine (0.2 mmol, degree of substitution 0.33) and add them to a reaction column. Then add DCM (10 mL) and then add 0.6 mL of DIEA to the reaction column. Sparge with nitrogen for 2 hours. Then add 0.6 mL of MeOH to the reaction column and continue bubbling with nitrogen for 30 minutes. Drain the column until no more liquid flows out. Add DMF (30 mL) and wash the column five times for 1 minute each. Drain the column until no more liquid flows out.

[0842] 1.1.2 Add 20% piperidine / DMF (15 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (30 mL) and wash five times for 1 minute each, draining until no more liquid flows out. Ninhydrin test: The resin should turn blue.

[0843] 1.2. Amino acid coupling

[0844] 1.2.1 Coupling of Fmoc-Asn(Trt)-OH

[0845] 1. Weigh Fmoc-Asn(Trt)-OH (358 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0846] 2. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0847] 3. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0848] 1.2.2 Coupling of Fmoc-Glu(OtBu)-OH

[0849] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0850] 2. Weigh Fmoc-Glu(OtBu)-OH (255 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0851] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0852] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0853] 1.2.3 Coupling of 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid

[0854] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0855] 2. Weigh 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid (CAS: 1986905-26-3, 241 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HATU (217 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0856] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0857] 4. Draw off the reaction liquid, wash with DMF 5 times, 1 minute each time, and drain until no liquid flows out.

[0858] 1.2.4 Coupling of Fmoc-2Nal-OH

[0859] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, draining until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, draining until no more liquid flows out. Test with chloranil; the resin will turn green.

[0860] 2. Weigh Fmoc-2-Nal-OH (262 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HATU (217 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0861] 3. React in an environment of 25℃ for 0.5 hours and test with tetrachlorobenzoquinone. The resin is colorless and transparent.

[0862] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0863] 1.2.5 Coupling of Fmoc-4-[2-(Boc-amino)ethoxy]-L-Phenylalanine

[0864] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0865] 2. Weigh Fmoc-4-[2-(Boc-amino)ethoxy]-L-Phenylalanine (328 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0866] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0867] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0868] 1.2.6 Coupling of Fmoc-Pen(Trt)-OH

[0869] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0870] 2. Weigh Fmoc-Pen(Trt)-OH (368 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0871] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0872] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0873] 1.2.7 Coupling of Fmoc-Lys(Ac)-OH

[0874] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0875] 2. Weigh Fmoc-Lys(Ac)-OH (246 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0876] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0877] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0878] 1.2.8 Coupling of Fmoc-(7Me)Trp-OH

[0879] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0880] 2. Weigh Fmoc-(7Me)Trp-OH (264 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HATU (217 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0881] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0882] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0883] 1.2.9 Coupling of Fmoc-Thr(Tbu)-OH

[0884] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0885] 2. Weigh Fmoc-Thr(Tbu)-OH (238 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0886] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0887] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0888] 1.2.10 Coupling of Fmoc-Asn(Trt)-OH

[0889] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0890] 2. Weigh Fmoc-Asn(Trt)-OH (358 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0891] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0892] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0893] 1.2.11 Coupling of Fmoc-Pen(Trt)-OH

[0894] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0895] 2. Weigh Fmoc-Pen(Trt)-OH (368 mg, 3.0 eq) and add it to the above resin. Add DIEA (155 mg, 6.00 eq) and 5 mL of DMF to the reaction column. Purge nitrogen. After the amino acid dissolves, add HBTU (216 mg, 2.85 eq). Adjust the nitrogen flow to ensure that the resin is evenly inflated.

[0896] 3. React at 25°C for 0.5 hours and test with ninhydrin. The resin is colorless and transparent.

[0897] 4. Draw off the reaction liquid, wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out.

[0898] 1.2.12 Ac2O Capping

[0899] 1. Add 20% piperidine / DMF (20 mL) to the reaction column and purge with nitrogen for 20 minutes, then drain until no more liquid flows out. Add DMF (20 mL) and wash five times for 1 minute each, then drain until no more liquid flows out. Ninhydrin detection: the resin will turn blue.

[0900] 2. Add DMF (10 mL), DIEA (155 mg, 6.00 eq), and Ac2O (102 mg, 5.0 eq) to the above resin and bubbling with nitrogen. Adjust the nitrogen so that the resin is evenly inflated.

[0901] 3. React in an environment of 25℃ for 10 minutes and test with ninhydrin. The resin is colorless and transparent.

[0902] 4. The reaction solution was removed and washed with DMF 5 times (20 mL each time), each time for 1 minute, and then discharged until no liquid flowed out to obtain linear peptide 1-16-1b.

[0903] Step 2: Cleavage of fully protected linear peptides

[0904] 2.1 Wash with DMF 5 times (20 mL each time), 1 minute each time, and drain until no liquid flows out

[0905] 2.2 Shrink the resin with MeOH (20 mL) for 3 minutes each time, drain until no liquid flows out, then wash the resin three times with 20 mL of tertiary methyl ether, blow dry the resin with nitrogen, and set aside.

[0906] 2.3 Pour 50 mL of the prepared 20% HFIP / DCM solution into the reactor, purge with nitrogen, and react once for 30 minutes. After cutting three times, all the filtrates were collected and concentrated to dryness under reduced pressure. Then, 50 mL of DCM was added and further concentrated to dryness under reduced pressure.

[0907] 2.4 Add 100 mL of acetonitrile and 100 mL of water and sonicate until the product is evenly dispersed. Then, freeze-dry using a freeze dryer to obtain 480 mg of intermediate 1-16-1c with a purity of 76% and a yield of 60%.

[0908] LC / MS: [M+H] + =3044.4; [M+H-243] + =2801.4

[0909] Step 3: Condensation of intermediate 1-16-1c with intermediate A

[0910] Take the above intermediate 1-16-1c (200 mg, 0.06 mmol), add DMF 1 mL, DIEA (3 eq, 0.18 mmol), then add intermediate A (77 mg, 0.06 mmol, 1 eq), and then slowly add HATU (23 mg, 0.06 mmol, 1 eq, dissolved in DMF 0.2 mL) dropwise. Use Tof-LCMS to detect complete consumption of the starting material. Use an oil pump to spin dry the DMF to obtain the crude intermediate 1-16-1d for later use.

[0911] LC / MS: [M] + =4130.2,[M-243] + =3887.2.

[0912] Step 4: Cleavage of linear peptides

[0913] 4.1. Prepare cutting fluid according to the following volume

[0914] The crude peptide obtained in step 3 was added to the prepared cutting solution, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 volumes of ice-cold isopropyl ether, centrifuged, and washed three times with isopropyl ether. The crude peptide 1-16-1e was obtained by vacuum drying for 2 hours.

[0915] LC / MS: [(M+H) / 2] + =1419.2; [(M+2H) / 3] + =946.5.

[0916] Step 5: Oxidation of disulfide bonds

[0917] 5.1 160 mg of the crude peptide 1-16-1e obtained in Step 4 was dissolved in 100 mL of water and 100 mL of acetonitrile. After dissolution and clarification, 0.1 M I2 / MeOH was slowly added dropwise until the solution turned bright yellow. Stirring continued for 5 minutes, if the yellow color did not disappear. 0.1 M sodium thiosulfate was then added dropwise until the yellow color disappeared. After stirring for two minutes without any change, the sample was lyophilized and purified to obtain 42 mg of Example 1-16-1 with a purity of 97.1% and a yield of 18.0%.

[0918] LC / MS: [(M+H) / 2] + =1418.2; [(M+2H) / 3] + =945.8.

[0919] Purification conditions:

[0920] Example 3-16-1

[0921] Example 3-16-1 can be prepared by referring to the following method:

[0922] Refer to the preparation method of Example 1-16-1, wherein 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid in the first step of the linear peptide solid phase synthesis is replaced by (9H-fluoren-9-yl)methyl(4-cyanotetrahydro-2H-thiopyran-4-yl)carbamate A3c to obtain 120 mg of product Example 3-16-1 with a purity of 98.5% and a yield of 23.0%.

[0923] LC / MS: [(M+H) / 2] + =1409.6; [(M+2H) / 3] + =940.8.

[0924] Example 4-16-1

[0925] Example 4-16-1 can be prepared by referring to the following method:

[0926] Refer to the preparation method of Example 1-16-1, wherein 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid in the first step of the linear peptide solid phase synthesis is replaced by intermediate A3 to obtain 110 mg of product Example 4-16-1 with a purity of 99.4% and a yield of 21.0%.

[0927] LC / MS: [(M+H) / 2] + =1425.7; [(M+2H) / 3] + =951.0.

[0928] Purification conditions:

[0929] Examples 1-19

[0930] Examples 1-19 can be prepared by referring to the following method:

[0931] Refer to the preparation method of Example 1-16-1, wherein the intermediate A in the third step of coupling the linear peptide with the side chain is replaced by intermediate L2 to obtain 110 mg of product Example 1-19 with a purity of 95.1% and a yield of 15.4%.

[0932] LC / MS: [(M+H) / 2] + =1037.5; [(M+2H) / 3] + =692.4.

[0933] Example 3-19

[0934] Examples 3-19 can be prepared by referring to the following method:

[0935] Refer to the preparation method of Example 1-16-1, wherein 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid in the first step of the solid-phase synthesis of the linear peptide is replaced by (9H-fluoren-9-yl)methyl(4-cyanotetrahydro-2H-thiopyran-4-yl)carbamate A3c, and the side chain intermediate A in the third step of the coupling of the linear peptide and the side chain is replaced by the intermediate L2 to obtain 110 mg of the product Example 3-19 with a purity of 99.0% and a yield of 15.4%.

[0936] LC / MS: [(M+H) / 2] + =1028.5; [(M+2H) / 3] + =686.4.

[0937] Example 4-19

[0938] Examples 4-19 can be prepared by referring to the following method:

[0939] Refer to the preparation method of Example 1-16-1, wherein 1-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-difluoro-cyclohexanecarboxylic acid in the first step of solid-phase synthesis of the linear peptide is replaced by intermediate A3, and the side chain intermediate A in the third step of coupling the linear peptide with the side chain is replaced by intermediate L2 to obtain 102 mg of product Example 4-19 with a purity of 99.8% and a yield of 14.2%.

[0940] LC / MS: [(M+H) / 2] + =1044.5; [(M+2H) / 3] + =697.0.

[0941] The following examples can be prepared with reference to the above preparation method:

[0942] Among them, R 1 and R 2 Select from the following structures

[0943] Table 1

[0944] Biological test evaluation

[0945] The present invention is further described and explained below in conjunction with test examples, but these examples are not intended to limit the scope of the present invention.

[0946] Test Example 1: Experiment on the competition between the compound of the present invention and IL23 for binding to IL23R

[0947] 1.1 Experimental purpose: to determine the efficiency of the compounds of the present invention competing with IL23 for binding to IL23R

[0948] 1.2 Experimental instruments and reagents:

[0949] 1.2.1 Instruments

[0950] 1.2.2 Reagents

[0951] 1.3 Experimental methods:

[0952] The compounds of the present invention were screened in IL23 / IL23R BINDING ASSAY KITS (PerkinElmer Cat#64BDPIL23PEH). The screening of the compounds of the present invention was carried out in the form of a multi-well plate (suitable for HTRF determination). Usually, 2 μl of several compounds of the present invention and STANDARD SOLUTIONS in the KIT were added to the wells of the multi-well plate. All wells needed to be added with 4 μl Tag1-IL23, 4 μl Tag2-IL23R and 10 μL of pre-mixed Anti-Tag1 Eu Cryptate Antibody and Anti-Tag2d2reagent. The multi-well plate was protected from light using an opaque film. After incubation at room temperature for 2 hours, the opaque film was removed and the signal value was read using an HTRF-compatible detection instrument to detect IC50 value.

[0953] 1.4 Experimental data processing method

[0954] The inhibition rate was calculated as follows:

[0955] %inhibition=(Signal cmpd-Signal Ave_PC) / (Signal Ave_VC-Signal Ave_PC)×100. H=Ave(DMSO); L=Ave(Guselkumab)

[0956] Calculate the IC of the compound 50 value:

[0957] The HTRF experimental data were fitted with nonlinear regression using the log(inhibitor) vs. response--Variable slope (four parameters) function in Graphpad. The curve was fitted and the IC was obtained. 50 value.

[0958] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0959] X:log of inhibitor concentration;Y:%Inhibition

[0960] 1.5 Experimental results:

[0961] 1.6 Experimental Conclusion: The preferred embodiment compound of the present invention showed excellent biological activity in the IL23R inhibition test by competing with IL23.

[0962] Test Example 2: Experiment on the competition between the compound of the present invention and IL12 for binding to IL12Rβ1

[0963] 2.1 Experimental purpose: Determine the efficiency of the compounds of the present invention in competing with IL12 for binding to IL12Rβ1

[0964] 2.2 Experimental instruments and reagents

[0965] 2.2.1 Instruments

[0966] 2.2.2 Reagents

[0967] 2.3 Experimental methods:

[0968] The compounds of the present invention were screened in IL12 / IL12RB1 BINDING ASSAY KITS (PerkinElmer Cat#64BDIL12PEG). The screening of the compounds of the present invention was carried out in the form of a multi-well plate (suitable for HTRF determination). Usually, 2 μl of several compounds of the present invention and STANDARD SOLUTIONS in the KIT were added to the wells of the multi-well plate. All wells needed to be added with 4 μl Tag1-IL12, 4 μl Tag2-IL12Rb1 and 10 μL of pre-mixed Anti-Tag1 Eu Cryptate Antibody and Anti-Tag2XL665reagent. The multi-well plate was protected from light using an opaque film. After incubation at room temperature for 2 hours, the opaque film was removed and the signal value was read using an HTRF-compatible detection instrument to detect IC 50 value.

[0969] 2.4 Experimental data processing method

[0970] The inhibition rate was calculated as follows:

[0971] %inhibition=(Signal cmpd-Signal Ave_PC) / (Signal Ave_VC-Signal Ave_PC)×100. H=Ave(DMSO); L=Ave(Guselkumab)

[0972] Calculate the IC of the compound 50 value:

[0973] The HTRF experimental data were fitted with nonlinear regression using the log(inhibitor) vs. response--Variable slope (four parameters) function in Graphpad. The curve was fitted and the IC was obtained. 50 value.

[0974] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0975] X:log of inhibitor concentration;Y:%Inhibition

[0976] 2.5 Experimental Conclusions:

[0977] The above scheme shows that the compound of the present invention shows an inhibition of more than 1000nM (IC50 )'s biological activity.

[0978] Test Example 3: Experiment on the inhibition of hIL23-activated STAT3 phosphorylation in DB cells by the compounds of the present invention

[0979] 3.1 Experimental purpose: Determine the inhibitory activity of the compounds of the present invention on hIL23-activated STAT3 phosphorylation in DB cells

[0980] 3.2 Experimental instruments and reagents

[0981] 3.2.1 Instruments

[0982] 3.2.2 Reagents

[0983] 3.3 Experimental Method: The compounds generated above were added to DB (ATCC Cat#CRL-2289) cells to inhibit rhIL-23 cytokine signal transduction. Compound screening was performed using a multi-well plate format (suitable for ELISA assays). Typically, rhIL-23 (0.5 nM, R&D SYSTEMS) was used to screen 6.25×10 6 DB cells were stimulated at 50 μL / mL in RPMI medium (Invitrogen) supplemented with 10% FBS. Half an hour later, the cells were lysed on ice using 50 μL of 2× lysis buffer (CST). The lysates were assayed for p-STAT3 levels using the PathScan Phospho-Stat3 (Tyr705) Sandwich ELISA Kit (CST Cat#7300C). This was used to calculate the inhibitory activity of the compounds of the present invention on rhIL23 signaling in DB cells.

[0984] 3.4 Experimental data processing method

[0985] The inhibition rate was calculated as follows:

[0986] Inhibition%=(Ave_H-Sample) / (Ave_H-Ave_L)×100

[0987] H=Ave(DMSO); L=Ave(Guselkumab)

[0988] Calculate the IC of the compound 50 value:

[0989] Use the log(inhibitor) vs.response--Variable slope (four parameters) in Graphpad to perform nonlinear regression fitting on the ELISA experimental results data, fit the curve and obtain the IC 50 value.

[0990] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0991] X:log of inhibitor concentration;Y:%Inhibition

[0992] 3.5 Experimental results:

[0993] 3.6 Experimental conclusion: The preferred example compounds of the present invention showed excellent inhibitory activity in the inhibition test on hIL23-activated STAT3 phosphorylation.

[0994] Test Example 4: Pharmacokinetics in rats

[0995] 4.1. Study purpose: SD rats were used as test animals to study the pharmacokinetic behavior of the example compounds in rats after oral or IV administration (plasma).

[0996] Experimental plan

[0997] 4.2.1 Investigational Drugs:

[0998] The compound implemented by the present invention is self-made.

[0999] 4.2.2 Experimental Animals:

[1000] Three male SD rats were used in each group. Shanghai JXJ Laboratory Animal Co., Ltd., Animal Production License No. (SCXK(Shanghai)2013-0006N0.311620400001794).

[1001] 4.2.3 Drug preparation:

[1002] Oral drug preparation: 20% Labrasol

[1003] 20% Labrasol: Weigh 40 ml of Labrasol into a 500 ml glass bottle, add PBS to 200 ml, and stir magnetically to completely dissolve to prepare 20% Labrasol.

[1004] The example compound was weighed and dissolved in the solution, shaken and sonicated for 5 minutes to obtain a colorless clear solution with a concentration of 0.5 mg / mL.

[1005] Intravenous drug preparation: PBS

[1006] The example compound was weighed and dissolved in the solution, vortexed and sonicated for 5 minutes, and filtered through a 0.22 μm filter to obtain a colorless clear solution with a concentration of 0.2 mg / mL.

[1007] 4.2.4 Administration:

[1008] Three male SD rats were administered PO after overnight fasting at a dose of 5 mg / kg in a dosing volume of 10 mL / kg.

[1009] Three male SD rats were administered IV after overnight fasting at a dose of 1 mg / kg in a volume of 5 mL / kg.

[1010] 4.2.5 Sample collection:

[1011] Before administration and at 0.083 h (IV), 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h, and 24.0 h after administration, 0.2 mL of blood was collected from the jugular vein, placed in an EDTA-2K tube, and centrifuged at 6000 rpm at 4°C for 6 min to separate the plasma, which was then stored at -20°C. Food was consumed 4 h after administration.

[1012] 4.3 Sample processing:

[1013] 1) 50 μL of plasma sample was added to 200 μL of acetonitrile for precipitation, mixed and centrifuged at 4500 rpm for 15 minutes.

[1014] 2) The supernatant solution after treatment was subjected to LC / MS / MS analysis to determine the concentration of the test compound. The LC / MS / MS analysis instrument was AB Sciex Triple Quad 6500+.

[1015] 4.4 Liquid phase analysis:

[1016] Liquid phase conditions: Shimadzu LC-30AD pump

[1017] Column: HALO 90A PFP 2.7μm 2.1×30mm Mobile phase: Liquid A: 5% acetonitrile in water (0.1% formic acid), Liquid B: 95% acetonitrile in water (0.1% formic acid)

[1018] Flow rate: 0.6 mL / min

[1019] Elution time: 0-2.0 minutes, eluent is as follows:

[1020] 4.5. Experimental results and analysis

[1021] The main pharmacokinetic parameters were calculated using WinNonlin 8.1.

[1022] 4.6 Experimental Conclusions:

[1023] The data showed that in the rat pharmacokinetic evaluation experiment, the preferred embodiment compound of the present invention had a stronger blood concentration and a higher exposure than PN-235.

[1024] Test Example 5: Pharmacokinetic Determination in Beagle Dogs

[1025] 5.1. Study Objective: Beagle dogs were used as test animals to study the pharmacokinetic behavior of Example Compound IV in rats (plasma).

[1026] Experimental plan

[1027] 5.2.1 Investigational Drugs:

[1028] The compound implemented by the present invention is self-made.

[1029] 5.2.2 Experimental Animals:

[1030] Each group included 3 male beagles, purchased from Yizheng Anlimao Biotechnology Co., Ltd.

[1031] 5.2.3 Drug preparation:

[1032] Intravenous drug preparation: PBS

[1033] The example compound was weighed and dissolved in the solution, vortexed and sonicated for 5 minutes, and filtered through a 0.22 μm filter to obtain a colorless clear solution with a concentration of 0.1 mg / mL.

[1034] 5.2.4 Administration:

[1035] Three male beagle dogs were administered IV after overnight fasting at a dose of 0.2 mg / kg in a dosing volume of 2 mL / kg.

[1036] 5.2.5 Sample collection:

[1037] Before administration and at 0.083 h (IV), 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after administration, 0.5 mL of blood was collected from the forelimb vein, placed in an EDTA-2K tube, and centrifuged at 6000 rpm at 4°C for 6 min to separate the plasma, which was then stored at -20°C. Food was consumed 4 h after administration.

[1038] 5.3 Sample processing:

[1039] 1) 50 μL of plasma sample was added to 200 μL of acetonitrile for precipitation, mixed and centrifuged at 4500 rpm for 15 minutes.

[1040] 2) The supernatant solution after treatment was subjected to LC / MS / MS analysis to determine the concentration of the test compound. The LC / MS / MS analysis instrument was AB Sciex Triple Quad 6500+.

[1041] 5.4 Liquid phase analysis:

[1042] Liquid phase conditions: ExionLC

[1043] Column: ACQUITY™ Premier Peptide CSH C18 130A 1.7μm 2.1*100mm Column Mobile Phase: Liquid A: 5% acetonitrile in water (0.1% formic acid), Liquid B: 95% acetonitrile in water (0.1% formic acid)

[1044] Flow rate: 0.6 mL / min

[1045] Elution time: 0-2.0 minutes, eluent is as follows:

[1046] 5.5. Test results and analysis

[1047] The main pharmacokinetic parameters were calculated using WinNonlin 8.1.

[1048] 5.6 Experimental Conclusions:

[1049] The data showed that in the canine pharmacokinetic evaluation experiment, the preferred embodiment compound of the present invention had a stronger blood concentration and a higher exposure than PN-235.

[1050] Test Example 6: Solubility determination of the compound of the present invention

[1051] 6.1 Study objective: To study the solubility of the compound in PBS.

[1052] 6.2 Experimental instruments and reagents:

[1053] 6.3 Experimental methods:

[1054] Weigh approximately 2 mg of compound, add an appropriate amount of PBS solution, and sonicate for 10 seconds to dissolve. No precipitation is observed after leaving the solution at room temperature overnight.

[1055] 6.4 Experimental results and conclusions:

[1056] At room temperature, the preferred embodiment compounds of the present invention exhibit excellent solubility advantages. For example, the solubility of Example 1-16-1 is 39.58 to 59.38 mg / mL; the solubility of Example 4-16-1 is greater than 124.75 mg / mL.

[1057] Test Example 7: Stability of the compound of the present invention in gastrointestinal tract simulation fluid

[1058] 7.1 Study objective: To study the stability of the example compounds in simulated gastrointestinal fluid.

[1059] 7.2 Experimental instruments and reagents:

[1060] 7.2.1 Reagents

[1061] Acetonitrile (F22M4L201, Fisher), trifluoroacetic acid (17110655, TEDIA), sodium hydroxide (20170616, Sinopharm), pancreatin (20220811, Shanghai trial), pepsin (20220909, Shanghai trial), potassium dihydrogen phosphate (20210528, Wokai), sodium chloride (20200105, Shanghai trial), hydrochloric acid (20181203, Sinopharm), anhydrous sodium dihydrogen phosphate (20200602, Shanghai trial), 3F Powder (FFF-0723-B, Biorelevant), glacial acetic acid (20181112, Sinopharm).

[1062] 7.2.2 Instruments

[1063] High performance liquid chromatograph (1260, Agilent), electronic balance (MCE-C, Sartorius), pH meter (Five Easy Plus, METTLER TOLEDO), pipettes (500-5000 μL, 100-1000 μL, 10-100 μL, Eppendorf), and ultrasonic cleaner (SK5200LHC, Shanghai Kedao Ultrasonic Instrument Co., Ltd.) were used.

[1064] 7.3 Experimental methods:

[1065] 7.3.1 Preparation of mobile phase and gastrointestinal tract simulation fluid

[1066] 0.05% trifluoroacetic acid aqueous solution: Measure 2 L of purified water, add 1 mL of trifluoroacetic acid, mix well and then sonicate.

[1067] 0.05% trifluoroacetic acid acetonitrile solution: Measure 2 L of acetonitrile, add 1 mL of trifluoroacetic acid, mix well and then sonicate.

[1068] SIF (E): Weigh 69.48 mg of potassium dihydrogen phosphate and 108.49 mg of pancreatin, add 10 mL of purified water, mix well, and adjust the pH to 6.76 with 1N sodium hydroxide aqueous solution (about 210 μL).

[1069] SGF(E): Weigh 21.64 mg of sodium chloride and 33.70 mg of pepsin, add 10 mL of purified water, add 20 μL of hydrochloric acid, and adjust the pH to 1.98 with 1N sodium hydroxide aqueous solution (about 120 μL).

[1070] FaSSIF: Weigh 0.42 g of sodium hydroxide, 3.43 g of anhydrous sodium dihydrogen phosphate, and 6.19 g of sodium chloride, add 1 L of purified water, and sonicate to dissolve. Adjust the pH to 6.50 with 1N sodium hydroxide or 1N hydrochloric acid. Add 2.24 g of 3F powder and stir until dissolved.

[1071] FeSSIF: Weigh 4.04 g of sodium hydroxide, 8.65 g of glacial acetic acid, and 11.87 g of sodium chloride, add 1 L of purified water, sonicate to dissolve, adjust the pH to 5.0 with 1N sodium hydroxide or 1N hydrochloric acid, add 11.2 g of 3F powder, and stir until dissolved.

[1072] 7.3.2 Stability determination

[1073] Weigh approximately 2 mg of compound, add an appropriate amount of purified water, and sonicate to dissolve. This serves as the compound stock solution. Add 50 μL of the stock solution to 950 μL of SGF, FaSSIF, FeSSIF, SGF(E), and SIF(E), respectively. Mix thoroughly and examine the stability of the samples at different time points.

[1074] 7.4 Liquid phase analysis:

[1075] Liquid chromatography conditions are as follows:

[1076] 7.5 Test results and analysis

[1077] The preferred embodiment compounds of the present invention have excellent stability in simulated gastrointestinal fluid.

[1078] Test Example 8: Investigation of in vitro metabolic stability of the compounds of the present invention in liver microsomes

[1079] 8.1 Study Objective: To investigate the metabolic stability of the compound of this example in phase I and partially phase II in mouse, rat, dog and human liver microsomes.

[1080] 8.2 Experimental Reagents

[1081] 8.2.1 Reagents

[1082] Compounds of the present invention (self-made), liver microsomes (H0610 / M1000 / D1000, Xenotech), phosphate buffer (Lot#SLBS7904 and Lot#SLBR3106V, pH 7.4, Gibco), NADPH (reduced nicotinamide adenine dinucleotide phosphate, Shanghai Bid Pharmaceutical Technology Co., Ltd.), UDPGA (Sigma), Alamethicin (Lot#GR3226732-1, J&K), methanol (Merck), acetonitrile (Merck), DMSO (Sigma), 7-Hydroxycoumarin (J&K).

[1083] 8.2.2 Drug configuration

[1084] The test compounds were prepared into 10 mM stock solutions with DMSO and stored in a refrigerator at -20°C until use.

[1085] 8.3 Experimental Procedure

[1086] 1) Prepare buffer solution

[1087] Prepare phosphate buffer with a final concentration of 100 mM by dissolving 4.01 mL of 1 M K2HPO4·PO2O (AR grade) and 0.99 mL of 1 M KH2PO4 (AR grade) in ultrapure water and diluting to 50 mL.

[1088] 2) Prepare compound working solution

[1089] Prepare compound working solution: Add 1 μL of compound stock solution to 999 μL of phosphate buffer for a final concentration of 10 μM. Adjust the ratio and final concentration appropriately based on the properties of the compound.

[1090] 3) Preparation of liver microsome working solution

[1091] 156.3 μL of 20 mg / mL microsomes were diluted to 5 mL with 100 mM phosphate buffer and mixed to a final concentration of 0.625 mg / mL.

[1092] 4) Prepare NADPH and UDPGA

[1093] Weigh 33.3 mg of NADPH and 25.8 mg of UDPGA, add 2 mL of 100 mM phosphate buffer, and the final concentration of both is 20 mM.

[1094] 5) Prepare the punching agent (Alamethicin)

[1095] Weigh 1 mg of Alamethicin and add it to 200 μL of methanol to prepare a 5 mg / mL solution. Then, remove 10 μL of this solution and add it to 990 μL of phosphate buffer (pH 7.4) for a final concentration of 50 μg / mL.

[1096] 6) Prepare reaction termination solution

[1097] Dilute the internal standard with acetonitrile to make the stop solution and store it in a 2-8 dilution refrigerator.

[1098] 7) Incubation process

[1099] 400 μL of prepared liver microsomes, 25 μL of compound working solution (10 μM), and 25 μL of alamethicin (50 μg / mL) were added sequentially to a 96-well plate and pre-incubated at 37°C for 10 min. The reaction was then initiated by adding 50 μL of prepared NADPH / UDPGA and incubated at 37°C for a total volume of 500 μL. The final concentrations of the components were: compound of this example (0.5 μM), liver microsomes (0.5 mg / mL), NADPH (1 mM), UDPGA (1 mM), and alamethicin (2.5 μg / mL).

[1100] 50 μL was taken out at the time points of 0, 5, 15, 30, 60 and 120 min, and 200 μL of cold stop solution containing internal standard was added to terminate the sample reaction. The sample was centrifuged at 3500 rpm for 10 min, and the supernatant was taken for LC-MS / MS analysis.

[1101] 8.4 Bioanalysis

[1102] 1) Chromatographic conditions

[1103] Instrument: Shimadzu LC-20AD

[1104] Column: Phenomenex C18 (50*4.6mm, 5μm particle size)

[1105] Mobile phase: A: acetonitrile, B: 0.1% formic acid solution

[1106] Wash gradient: 0.2-1.6 min 5% A to 95% A, 3.0-3.1 min 95% A to 5% A

[1107] Flow rate: 1.0ml / min

[1108] Run time: 4.0 minutes

[1109] Injection volume: 5 μL

[1110] 2) Mass spectrometry conditions

[1111] Instrument: API4000 liquid chromatography-mass spectrometer, AB Sciex

[1112] Ion source: electrospray ionization (ESI)

[1113] Drying gas: N2, temperature 500°C

[1114] Electrospray voltage: 5000 V

[1115] Detection method: positive ion detection

[1116] Scanning mode: reaction monitoring (MRM) mode

[1117] Scan time: 0.8401s

[1118] 8.5 Data Processing

[1119] The original data is calculated according to the following formula:

[1120] Residual rate % = peak area ratio of compound to internal standard at any time point / peak area ratio of compound to internal standard at 0 minutes × clock time

[1121] T 1 / 2 =0.693 / K e , where K e represents the elimination rate constant.

[1122] By K e Calculation of in vitro liver microsomal intrinsic clearance (CL int ) and hepatic intrinsic clearance (CL int,liver )

[1123] CL int =0.693 / T 1 / 2 / Microsomal protein content (microsomal concentration during incubation mg / mL)

[1124] CL int,liver =CL int nt microsomal protein content in liver (mg / g) × liver weight to body weight ratio

[1125] Based on the well-stirred model, the in vivo hepatic clearance (CL) was estimated.

[1126] CL=(CL int,liver nt,liver rate) / (CL int,liver nt, liver rate), where fu represents the free fraction in the blood and the default value is 1.

[1127] The parameters in the formula are shown in the table below.

[1128] 8.6 Experimental Conclusions:

[1129] The results showed that the preferred embodiment compounds of the present invention exhibited stable metabolic effects in various liver microsomes.

[1130] Test Example 9: Metabolic Stability of the Compounds of the Invention in Liver / Kidney Tissue Homogenates

[1131] 9.1 Experimental purpose: To detect the metabolic stability of the compound of this example in the liver / kidney tissues of rats, dogs and monkeys.

[1132] 9.2 Experimental Reagents

[1133] Rat liver tissue homogenate (WuXi AppTec), monkey liver tissue homogenate (WuXi AppTec), dog liver tissue homogenate (WuXi AppTec), human liver tissue homogenate (Bioreclamation IVT, S06585), rat kidney tissue homogenate (WuXi AppTec), monkey kidney tissue homogenate (WuXi AppTec), dog kidney tissue homogenate (WuXi AppTec), human kidney tissue homogenate (Bioreclamation IVT, S06585).

[1134] 9.3 Experimental Procedure

[1135] 1) Solution preparation

[1136] Test compound working solution: dilute 5 μL of compound stock solution (10 mM dimethyl sulfoxide solution) with 995 μL of dimethyl sulfoxide (working solution concentration: 50 μM, 100% DMSO);

[1137] Control Deslorelin working solution: dilute 5 μL of Deslorelin stock solution (10 mM in dimethyl sulfoxide (DMSO)) with 495 μL of dimethyl sulfoxide (working solution concentration: 100 μM, 100% DMSO);

[1138] Control Semagulide working solution: dilute 50 μL of Semaguliide stock solution (1 mM dimethyl sulfoxide solution) with 450 μL of dimethyl sulfoxide (working solution concentration: 100 μM, 100% DMSO).

[1139] 2) Incubation process

[1140] Before the experiment, the pooled frozen liver / kidney homogenate was thawed in a 37°C water bath.

[1141] Aliquot 98 μL / well of blank liver / kidney homogenate into all 96-well reaction plates (blank, T0, T10, T30, T60, and T120).

[1142] Aliquot 2 μL / well of the working solution (50 or 100 μM) into all reaction plates except the blank (T0, T10, T30, T60, and T120).

[1143] All reaction plates containing compound and liver / kidney homogenate mixtures were incubated in a 37°C water bath.

[1144] The reaction plate was incubated at 37°C and a timer was started.

[1145] At the end of the incubation, 100 μL of 4% H 3 PO 4 was mixed with 100 μL of sample, and then 800 μL of stop solution (methanol with tolbutamide and labetalol as internal standards) was added to precipitate the protein. Mix thoroughly.

[1146] Seal each plate and shake for 20 minutes.

[1147] After shaking, each plate was centrifuged at 4000 rpm and 4 °C for 20 min.

[1148] After centrifugation, transfer 150 μL of supernatant from each reaction plate to its corresponding biotinylation plate.

[1149] Prior to LC-MS / MS analysis, each bioassay plate was sealed and shaken for 10 minutes.

[1150] 9.4 Data Processing

[1151] The percentage of test compound remaining in liver / kidney homogenates after incubation was calculated using the following equation:

[1152] % Residual = 100x (PAR incubation time point / PAR at T0 time), where PAR is the peak area ratio of analyte to internal standard (is), and the incubation time points are T0 (0 min), Tn (n = 0, 10, 30, 60, 120 min).

[1153] 9.5 Experimental Conclusion

[1154] The results showed that the preferred example compound of the present invention had excellent stability in liver and kidney tissue homogenates.

[1155] Test Example 10: Stability Study of the Compound of the Invention in Plasma

[1156] 10.1 Study Objective: To study the stability of the compound of this example in the plasma of mice, rats, dogs and humans.

[1157] 10.2 Experimental Instruments

[1158] Centrifuge (Eppendorf 5804R / 5424R), vortexer (IKA VORTEX GENIUS 3), pipettes (Eppendorf 10-100 μL, Eppendorf 100-1000 μL, RAININ 0.5-10 μL), and water bath (Shanghai Hengke).

[1159] 10.3 Experimental Procedure

[1160] 1) Solution preparation

[1161] Plasma preparation: After collecting whole blood from animals or humans, place it into a test tube containing anticoagulant, centrifuge it at 3500 rpm for 10 minutes, and collect the upper pale yellow plasma;

[1162] 10 μM test compound working solution: Prepare the stock solution in DMSO and the working solution in 100 mM phosphate buffer;

[1163] 10 μM positive control working solution:

[1164] Procaine: Weigh 2.36 mg of procaine and dilute to a 10 mM stock solution with 1 mL of DMSO. Pipette 10 μL of the 10 mM stock solution into 1 mL of 100 mM phosphate buffer for a final concentration of 100 μM.

[1165] Enalapril: Weigh 4.93 mg of enalapril and dilute it with 1 mL of DMSO to a 10 mM stock solution. Pipette 10 μL of the 10 mM stock solution into 1 mL of 100 mM phosphate buffer for a final concentration of 100 μM.

[1166] 2) Incubation process

[1167] In a 96-well plate, 285 μL of plasma and 15 μL of 10 μM compound (test compound, positive control) were added sequentially and incubated at 37°C.

[1168] Take out 40 μL at 0, 15, 30, 60, 90, and 120 min (the sampling point can be fine-tuned) and add 160 μL of acetonitrile stop solution containing internal standard.

[1169] After centrifugation (3500 rpm, 10 min), 50 μL of supernatant was collected, diluted with 50 μL of DD H2O, and then injected into LC-MS / MS.

[1170] 10.4 Bioanalysis

[1171] 1) Chromatographic conditions

[1172] Instrument: Shimadzu LC-20AD

[1173] Chromatographic column: Phenomenex Gemiu C 18 (50*4.6mm, 5μm particle size)

[1174] Mobile phase: A: acetonitrile, B: 0.1% formic acid solution

[1175] Washing gradient: 0-8 min: 5% A → 95% A, 2.0-2.1 min: 90% A → 5% A

[1176] Flow rate: 0.8 mL / min

[1177] Run time: 5.0 min

[1178] Injection volume: 5 μL

[1179] 2) Mass spectrometry conditions

[1180] Instrument: API4000 liquid chromatography-mass spectrometer, AB, USA

[1181] Ion source: electrospray ionization (ESI)

[1182] Drying gas: N2, temperature 500℃

[1183] Electrospray voltage: 5500 V

[1184] Detection method: positive ion detection

[1185] Scanning mode: reaction monitoring (MRM) mode

[1186] Scan time: 0.1s

[1187] 10.5 Experimental Results and Data Processing

[1188] All calculations were performed using Microsoft Excel. Peak area ratios were determined from extracted ion chromatograms.

[1189] The percentage of compound remaining at each time point was calculated using the following equation:

[1190] Remaining percentage t hour (%) = peak area ratio t hour / peak area ratio 0 hour ' hour ' ratio

[1191] Wherein, the peak area ratio thr is the peak area ratio of the control compound and the test compound at tmin;

[1192] The slope value, k, was determined by linear regression of the percent parent drug remaining versus the natural logarithm of the incubation time curve.

[1193] In vitro half-life (in vitro t 1 / 2 ) is determined by the slope value:

[1194] In vitro 1 / 2 =-(0.693 / k)

[1195] 10.6 Experimental Conclusions:

[1196] The preferred embodiment compounds of the present invention exhibit excellent stability in mouse, rat, dog and human plasma.

[1197] Test Example 11: In vivo pharmacodynamic study of the compound of the present invention in IL23-induced rat otitis model

[1198] 11.1 Experimental Objective: To evaluate the in vivo efficacy of the compound in the IL23-induced rat otitis model.

[1199] 11.2 Experimental Instruments and Reagents

[1200] 11.2.1 Experimental Instruments

[1201] Refrigerator (BCD-268TN, Haier), biological safety cabinet (BSC-1300II A2, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), clean bench (CJ-2F, Suzhou Fengshi Experimental Animal Equipment Co., Ltd.), electric pipette assistant (Easypet 3, Eppendorf), constant temperature water bath (HWS-12, Shanghai Yiheng Science), ultrasonic cleaner (115F0032, Shanghai Kedao), water purifier (Pacific TII, Thermo), magnetic stirrer (08-2G, Chijiu), electronic balance (CPA2202S, Sartorius), electronic balance (BSA2202S-CW, Sartorius), ultrasonic cell disruptor (JY92-IIN, Ningbo Xinzhi), micrometer (MDC-25PX, Sanfeng).

[1202] 11.2.2 Experimental Reagents

[1203] IL-23 Protein, Rat, Recombinant (CT045-R08H, Sino Biological), PBS (10010-049, Gibco), permeation enhancer, isoflurane (R510-22-10, Reward), 4% tissue cell fixative (AR-0211, Dingguo).

[1204] 11.3 Experimental Procedure

[1205] 11.3.1 Animal Procurement

[1206] SD rats, 6-8 weeks old, male, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[1207] 11.3.2 Establishment of Otitis Model

[1208] a, After one week of acclimatization, the animals were marked with tail numbers using a marker, weighed, and randomly grouped according to their weight.

[1209] b, One day before modeling (i.e., D-1), animals were gavaged and administered drugs according to the experimental design and grouping;

[1210] c, rrIL-23 was diluted to 50 μg / ml with PBS, aliquoted into 1 ml centrifuge tubes, and stored at −80°C until use;

[1211] d, On the first day of modeling (i.e., D0), animals were gavage-administered according to the experimental design and grouping. Half an hour later, the rats were anesthetized with isoflurane. After measuring the initial thickness of the right ear, IL-23 (1 μg, 20 μL) or 20 μL PBS was injected intradermally into the right ear.

[1212] e, On the second day of modeling (i.e., D1), animals were gavage-administered according to the experimental design and grouping. Half an hour later, rats were anesthetized with isoflurane. After measuring the initial thickness of the right ear, IL-23 (1 μg, 20 μL) or 20 μL PBS was injected intradermally into the right ear.

[1213] f, On the third day of modeling (i.e., D2), animals were gavage-administered according to the experimental design and grouping. Half an hour later, the rats were anesthetized with isoflurane. After measuring the initial thickness of the right ear, IL-23 (1 μg, 20 μL) or 20 μL PBS was injected intradermally into the right ear.

[1214] g, On the fourth day of modeling (i.e., D3), animals were gavage-administered according to the experimental design and grouping. Half an hour later, the rats were anesthetized with isoflurane. After measuring the initial thickness of the right ear, IL-23 (1 μg, 20 μL) or 20 μL PBS was injected intradermally into the right ear.

[1215] On day 5 (D4), the final right ear thickness was measured. Animals were gavage-administered according to the experimental design and grouping. One hour later, the animals were euthanized, and blood and ear tissue samples were collected. The ears were divided into two aliquots: one was quick-frozen for inflammatory factor mRNA analysis; the other was fixed with 4% histological fixative for histological processing and analysis (HE staining). Plasma was used for PK analysis.

[1216] 11.4 Data Processing:

[1217] a, The daily ear thickness of each group of animals minus the initial ear thickness (ear thickening) was compared with that of the Model group (Vehicle).

[1218] b. Ear thickness results from Day 4 to Day 0, the final day of the experiment, were plotted and analyzed using Graphpad Prism 9. Inter-group differences in ear thickness were analyzed using a one-way ANOVA Dunnett test; p < 0.05 was considered significant. Ear thickness inhibition rate (%) = [1 - (mean value of the treatment group / mean value of the model group)] × 100%.

[1219] 11.5 Experimental results:

[1220] 11.6 Experimental Conclusion: In the IL23-induced rat otitis model, the preferred embodiment compound of the present invention can effectively inhibit ear thickening. Its inhibitory effect on ear thickening when administered once daily is more significant than that of PN-235 administered twice daily, thus achieving the purpose of extending the dosing cycle.

Claims

1. A compound represented by general formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: in: R 1 and R 2 Each independently selected from H, alkyl, or amino acids; R a Selected from H, alkyl, or amino acids; R aa Selected from alkyl groups, or amino acids; X1 is selected from amino acids; Preferably, R aa Selected from alkyl groups, or amino acids; X1 is selected from amino acids; X'1 is selected from amino acids; R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids; R 6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X6 is selected from amino acids; X7 is selected from amino acids; Preferably, R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids; R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X'5 is selected from amino acids; X6 is selected from amino acids; X'6 is selected from amino acids; X7 is selected from amino acids; X'7 is selected from amino acids; R 4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, alkyl, or amino acids; R bb Selected from alkyl groups, or amino acids; X2 is selected from amino acids; X3 is selected from amino acids; X4 is selected from amino acids; Preferably, R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; preferably, selected from R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group; R bb Selected from alkyl groups, or amino acids; X2 is selected from amino acids; X'2 is selected from amino acids; X3 is selected from amino acids; X'3 is selected from amino acids; X4 is selected from amino acids; X'4 is selected from amino acids; n to n2 are each independently selected from an integer of 0 to 12; t to t9 are each independently selected from an integer of 0 to 12; m to m21 are each independently selected from an integer of 0 to 24; m22 is an integer selected from 0 to 24; The compound is not Preferably, R 1 and R 2 Each independently selected from H, alkyl, or amino acids; R a Selected from H, alkyl, or amino acids; R aa Selected from alkyl groups, or amino acids; X1 is selected from amino acids; Preferably, R aa Selected from alkyl groups, or amino acids; X1 is selected from amino acids; X'1 is selected from amino acids; R 3 Selected from hydroxyl group; R 4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2; n is an integer selected from 0 to 12; t to t1 are each independently selected from an integer of 0 to 12; m to m3 are each independently selected from an integer of 0 to 24; or, R 1 and R 2 Selected from H; R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids; R 6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X6 is selected from amino acids; X7 is selected from amino acids; Preferably, R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids; R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X'5 is selected from amino acids; X6 is selected from amino acids; X'6 is selected from amino acids; X7 is selected from amino acids; X'7 is selected from amino acids; R 4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2; n2 is an integer selected from 0 to 12; t6 to t9 are each independently selected from an integer of 0 to 12; m13 to m21 are each independently selected from an integer of 0 to 24; or, R 1 and R 2 Selected from H; R 3 Selected from hydroxyl group; R 4 and R 5 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, alkyl, or amino acids; R bb Selected from alkyl groups, or amino acids; X2 is selected from amino acids; X3 is selected from amino acids; X4 is selected from amino acids; Preferably, R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; preferably, selected from R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group; R bb Selected from alkyl groups, or amino acids; X2 is selected from amino acids; X'2 is selected from amino acids; X3 is selected from amino acids; X'3 is selected from amino acids; X4 is selected from amino acids; X'4 is selected from amino acids; n1 is an integer selected from 0 to 12; t2 to t5 are each independently selected from an integer of 0 to 12; m4 to m12 are each independently selected from an integer of 0 to 24; m22 is selected from integers of 0-24.

2. A compound represented by general formula (IV), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: in: X 1 selected from amino acids; X 2 selected from amino acids; R 1 and R 2 Each independently selected from H, alkyl, or amino acids; R a Selected from H, alkyl, or amino acids; R aa Selected from alkyl groups, or amino acids; X1 is selected from amino acids; X'1 is selected from amino acids; R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids; R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X'5 is selected from amino acids; X6 is selected from amino acids; X'6 is selected from amino acids; X7 is selected from amino acids; X'7 is selected from amino acids; R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group; R bb Selected from alkyl groups, or amino acids; X2 is selected from amino acids; X'2 is selected from amino acids; X3 is selected from amino acids; X'3 is selected from amino acids; X4 is selected from amino acids; X'4 is selected from amino acids; n to n2 are each independently selected from an integer of 0 to 12; t to t9 are each independently selected from an integer of 0 to 12; m to m21 are each independently selected from an integer of 0 to 24; m22 is an integer selected from 0 to 24; The compound is not 3. The compound according to any one of claims 1-2, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound is further represented by general formula (II) or (III): in: R 1 and R 2 Each independently selected from H, alkyl, or amino acids; R a Selected from H, alkyl, or amino acids; R aa Selected from alkyl groups, or amino acids; X1 is selected from amino acids; Preferably, R aa Selected from alkyl groups, or amino acids; X1 is selected from amino acids; X'1 is selected from amino acids; R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids; R 6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X6 is selected from amino acids; X7 is selected from amino acids; Preferably, R 3 Selected from hydroxy, alkyl, -NR 6 R 7 or amino acids; R 6 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 7 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X'5 is selected from amino acids; X6 is selected from amino acids; X'6 is selected from amino acids; X7 is selected from amino acids; X'7 is selected from amino acids; R 4 Selected from H or alkyl; n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t1 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m1 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; n2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t8 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t9 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; m13 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m14 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m15 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m16 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m17 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m18 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m19 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m20 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m21 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

4. The compound according to any one of claims 1 to 3, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: R 1 Selected from H, C 1-6 alkyl, or amino acids; R a Selected from H, C 1-6 alkyl, or amino acids; R aa Selected from C 1-6 alkyl, or amino acids; X1 is selected from amino acids; Preferably, R aa Selected from C 1-6 alkyl, or amino acids; X1 is selected from amino acids; X'1 is selected from amino acids; n is an integer selected from 0 to 12; t to t1 are each independently selected from an integer of 0 to 12; m to m3 are each independently selected from an integer of 0 to 24; and / or, R 2 Selected from H, C 1-6 alkyl, or amino acids; R a Selected from H, C 1-6 alkyl, or amino acids; R aa Selected from C 1-6 alkyl, or amino acids; X1 is selected from amino acids; Preferably, R aa Selected from C 1-6 alkyl, or amino acids; X1 is selected from amino acids; X'1 is selected from amino acids; n is an integer selected from 0 to 12; t to t1 are each independently selected from an integer of 0 to 12; m to m3 are each independently selected from an integer of 0 to 24; and / or, R 3 Selected from hydroxyl, C 1-6 Alkyl, -NR 6 R 7 or amino acids; R 6 and R 7 Each is independently selected from H, alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, alkyl, or amino acids; R cc Selected from alkyl groups, or amino acids; X5 is selected from amino acids; X6 is selected from amino acids; X7 is selected from amino acids; Preferably, R 3 Selected from hydroxyl, C 1-6 Alkyl, -NR 6 R 7 or amino acids; R 6 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids; R 7 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids; R c Selected from H, C 1-6 alkyl, or amino acids; R cc Selected from C 1-6 alkyl, or amino acids; X5 is selected from amino acids; X'5 is selected from amino acids; X6 is selected from amino acids; X'6 is selected from amino acids; X7 is selected from amino acids; X'7 is selected from amino acids; n2 is an integer selected from 0 to 12; t6 to t9 are each independently selected from an integer of 0 to 12; m13 to m21 are each independently selected from integers of 0-24.

5. The compound according to claim 2, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound is further represented by the general formula (V): in: X 1 selected from amino acids; X 2 selected from amino acids; R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group; R bb Selected from alkyl groups, or amino acids; X'2 is selected from amino acids; X'3 is selected from amino acids; X4 is selected from amino acids; n1 is an integer selected from 0 to 12; t2 to t5 are each independently selected from an integer of 0 to 12; m4 to m12 are each independently selected from an integer of 0 to 24; m22 is an integer selected from 0 to 12; The compound is not 6. The compound according to any one of claims 2 or 5, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: X 1 Selected from Thr, Preferably and / or, X 2 Selected from Preferably 7. The compound according to any one of claims 1 to 2 or 5, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound is further represented by the general formula (IV): in: R 4 Selected from H or alkyl; R 5 Selected from alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, alkyl, or amino acids; R bb Selected from alkyl groups, or amino acids; X2 is selected from amino acids; X3 is selected from amino acids; X4 is selected from amino acids; Preferably, R 4 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; R 5 Selected from H, alkyl, -CH2C(O)NH2, or amino acids; preferably, selected from R b Selected from H, alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group; R bb Selected from alkyl groups, or amino acids; X2 is selected from amino acids; X'2 is selected from amino acids; X3 is selected from amino acids; X'3 is selected from amino acids; X4 is selected from amino acids; X'4 is selected from amino acids; n1 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t4 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; t5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; m4 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m8 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m9 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m10 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m11 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m12 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; m22 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

8. The compound according to any one of claims 1-2 or 4-7, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: R 4 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, C 1-6 alkyl, or amino acids; R bb Selected from C 1-6 alkyl, or amino acids; X2 is selected from amino acids; X3 is selected from amino acids; X4 is selected from amino acids; Preferably, R 4 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, C 1-6 alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group; R bb Selected from C 1-6 alkyl, or amino acids; X2 is selected from amino acids; X'2 is selected from amino acids; X3 is selected from amino acids; X'3 is selected from amino acids; X4 is selected from amino acids; X'4 is selected from amino acids; n1 is an integer selected from 0 to 12; t2 to t5 are each independently selected from an integer of 0 to 12; m4 to m12 are each independently selected from an integer of 0 to 24; m22 is an integer selected from 0 to 24; and / or, R 5 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids; R b Selected from H, C 1-6 alkyl, or amino acids; R bb Selected from C 1-6 alkyl, or amino acids; X2 is selected from amino acids; X3 is selected from amino acids; X4 is selected from amino acids; Preferably, R 5 Selected from H, C 1-6 Alkyl, -CH2C(O)NH2, or amino acids, further preferably, selected from R b Selected from H, C 1-6 alkyl, or amino acids, wherein the alkyl group may be further substituted with a hydroxyl group or an alkoxy group; R bb Selected from C 1-6 alkyl, or amino acids; X2 is selected from amino acids; X'2 is selected from amino acids; X3 is selected from amino acids; X'3 is selected from amino acids; X4 is selected from amino acids; X'4 is selected from amino acids; n1 is an integer selected from 0 to 12; t2 to t5 are each independently selected from an integer of 0 to 12; m4 to m12 are each independently selected from an integer of 0 to 24; m22 is an integer selected from 0 to 24; Preferably, Selected from Selected from Selected from Selected from 9. The compound, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: Selected from the following compounds:

10. A method for preparing the compound according to any one of claims 1 to 9, its stereoisomers or pharmaceutically acceptable salts thereof, characterized in that: Based on solid-phase or liquid-phase synthesis; Preferably, the synthesis method comprises: 1) Synthesize resin peptides based on solid phase synthesis method; 2) cleaving the resin peptide obtained in step 1) to obtain a polypeptide intermediate; 3) The polypeptide intermediate undergoes condensation reaction with the side chain; 4) removing the protecting group of the peptide obtained in step 3) and cyclizing the peptide to obtain the final product; Further preferably, the synthesis method comprises: 1) Solid-phase synthesis method based on the Fmoc method, synthesizing resin peptides and acetic anhydride end-capping; 2) cleaving the resin peptide obtained in step 1) to obtain a polypeptide intermediate; 3) The polypeptide intermediate and the side chain undergo condensation reaction using a coupling agent; 4) After removing the protecting group of the peptide segment obtained in step 3), the peptide segment is oxidized into a ring through a disulfide bond to obtain the final product.

11. A pharmaceutical composition comprising a therapeutically effective dose of the compound according to any one of claims 1 to 9, its stereoisomers or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers, diluents or excipients.

12. Use of the compound according to any one of claims 1 to 9, its stereoisomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 11 in the preparation of an IL-23R inhibitor.

13. The compound according to any one of claims 1 to 9, its stereoisomer or pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 11 for use in the preparation of a pharmaceutical composition for treating inflammatory, autoimmune diseases and cancers, such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, colitis associated with radiotherapy or chemotherapy, colitis associated with innate immune disorders such as leukocyte adhesion retardation-1, chronic granulomatous disease, glycogen storage disease type 1b, Hermansky-Prague syndrome The invention also relates to the use of the present invention in the treatment of inflammatory bowel disease (IBS), inflammatory bowel disease (IBD), rheumatoid arthritis, pemphigus vulgaris, organ transplant rejection, Crohn's disease, systemic lupus erythematosus (SLE), or diabetes mellitus; preferably, inflammatory bowel disease (IBD), rheumatoid arthritis, or psoriasis.

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