Macrocyclic polypeptide compounds and uses thereof

Abstract

The present application discloses a kind of macrocyclic polypeptide compound and its purposes, the macrocyclic polypeptide compound is as shown in formula (I), wherein the definition of each group is detailed in specification;The compound can be used for preparing PD1 / PDL1 inhibitor.

Description

Technical Field

[0001] This invention relates to, but is not limited to, the field of medicinal chemistry, particularly to a class of macrocyclic polypeptide compounds or their pharmaceutically acceptable salts, esters, stereoisomers or solvents and pharmaceutical compositions thereof, and also to their use in PD-1 / PD-L1 inhibitor drugs. Background Technology

[0002] PD-1 (programmed death receptor 1), also known as CD279 (differentiation cluster 279), is an important immunosuppressive molecule, belonging to the type I transmembrane protein family and the immunoglobulin superfamily. PD-1 contains a proximal membrane immunoreceptor tyrosine inhibitory unit and a long-range membrane tyrosine-based switching motif. It modulates the immune system's response to human cells by downregulating the immune system and by inhibiting T-cell inflammatory activity, thereby promoting self-tolerance. PD-1 is mainly expressed on activated CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes, and dendritic cells, promoting T-cell maturation. Two ligands for PD-1 have been identified: PD-L1 and PD-L2. PD-L1, also a type I transmembrane protein, is mainly expressed on antigen-presenting cells, B cells, T cells, epithelial cells, muscle cells, and endothelial cells. PD-L1 ligands are abundant in a variety of human cancers. The interaction between PD-1 and PD-L1 leads to a reduction in tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and suppression of the immune system in cancer, pregnancy, tissue transplantation, and autoimmune diseases.

[0003] The immune mechanism can be reversed by inhibiting the interaction between PD-1 and PD-L1. Evidence suggests that interfering with or blocking this interaction can weaken or eliminate immunosuppression. Currently, the development of PD-1 / PD-L1 inhibitors mainly focuses on monoclonal antibodies. Monoclonal antibodies such as Nivolumab, Lambrolizumab, Atezolizumab, Durvalumab, and Avelumab are already marketed domestically and internationally, showing significant therapeutic effects in treating diseases such as non-small cell lung cancer and melanoma that do not respond well to conventional treatments. Compared to monoclonal antibody research and development, progress in peptide inhibitors in this field has been slow. Therefore, researching and developing inhibitors that inhibit the PD-1 / PD-L1 interaction has significant clinical implications.

[0004] There is still a need in this field for peptide inhibitors with novel structures. Summary of the Invention

[0005] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of protection of this application.

[0006] The macrocyclic polypeptide molecules provided in this application have been demonstrated in biochemical and cell-based experiments to block the interaction between PD-1 and PD-L1, and are a good class of PD-1 / PD-L1 inhibitors.

[0007] This invention relates to a class of macrocyclic polypeptide compounds or pharmaceutically acceptable salts, esters, stereoisomers or solvent compounds thereof as PD-1 / PD-L1 inhibitors for the treatment of malignant tumors.

[0008] This application provides macrocyclic polypeptide compounds of general formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, or solvent compounds thereof:

[0009]

[0010] Its characteristic is that, in formula (I), R' is 1-methylpropyl or isobutyl;

[0011] R is either methyl or hydrogen;

[0012] R0 is a group E or a group substituted by group E.

[0013] X1 and X2 are each independently selected from oxygen, sulfur, and nitrogen;

[0014] R1 and R2 are each independently selected from hydrogen and C1-C6 hydrocarbon groups substituted with a group C. When X1 or X2 is oxygen or sulfur, R1 and R2 are not present.

[0015] R3 and R4 are each independently selected from: hydrogen, C1-C6 alkyloxy groups, nitro groups, nitrile groups, hydroxyl groups, carboxyl groups, amino groups, halogens, and trifluoromethyl groups;

[0016] At most one of Y1, Y2, Y3 and Y4 is a nitrogen atom, and the rest are carbon atoms;

[0017] At most one of Y5, Y6, Y7 and Y8 is a nitrogen atom, and the rest are carbon atoms;

[0018] R5 is selected from hydrogen, C1-C6 hydrocarbon groups substituted with D, and F groups;

[0019] R6 is selected from hydrogen, the group D, or a C1-C6 hydrocarbon group substituted with one or more groups D;

[0020] R7 is selected from hydrogen, -CONH2, -CH2CONH2, and the group F;

[0021] Furthermore, R5 and R7 are not both groups F.

[0022] R8 is selected from hydrogen, C5-C14 aromatic heterocycles, and C1-C6 hydrocarbon groups substituted with C5-C14 aromatic heterocycles;

[0023] Y9 can be C, O, or S;

[0024] n1 is 1 or 2;

[0025] R9 is -CH2CONH2, or R9 together with the adjacent nitrogen-containing heterocycle forms such as The ring structure; where Y 10 For C or O; n2 is 1 or 2;

[0026] R 10 It is a hydrogen or C1-C6 hydrocarbon group;

[0027] Fragment A is independently selected from the following structural fragments:

[0028] Fragment B is independently selected from: (in the formula) This indicates that it is attached to a carbonyl group. (Indicates that it is attached to an amino group); where R 13 R 14 R 19 and R 23 Each is independently selected from: hydrogen, C1-C6 alkyl groups;

[0029] R 11 R 12 R 15 and R 16 Each is independently selected from C1-C6 hydrocarbon groups, or R 11 With R 12 The carbon chains are linked to form 8-16 membered rings with or without alkene bonds, or R 12 With R 15 The carbon chains are linked to form 8-16 membered rings with or without alkene bonds, or R 15 With R 16 They are linked by carbon chains to form 8-16 membered rings, with or without alkene bonds;

[0030] R 17 R 18 and R 20 Each is independently selected from C1-C6 hydrocarbon groups; or R 17 With R 18 They are linked by carbon chains to form 8-16 membered rings, with or without alkene bonds;

[0031] R 21 R 22 and R 24 Each is independently selected from C1-C6 hydrocarbon groups; or R 21 With R 24 They are linked by carbon chains to form 8-16 membered rings, with or without alkene bonds;

[0032] The group C is one or more of the following groups: C1-C6 hydrocarbon carbonyl, carboxyl, hydroxyl, and amino groups;

[0033] The group D is one or more of the following groups: carboxyl, hydroxyl, and amino;

[0034] The group E is one or more of the following groups: amino, hydroxyl, C1-C6 alkoxy;

[0035] The group F is one of the following groups, and the dashed lines represent connecting bonds;

[0036]

[0037] In embodiments of this application, compounds of general formula (II) are provided:

[0038]

[0039] The definitions of each group in formula (II) are as in formula (I).

[0040] In embodiments of this application, compounds of general formula (III) are provided:

[0041]

[0042] The definitions of each group in formula (III) are as in formula (I).

[0043] In the embodiments of this application, the C1-C6 hydrocarbon group represents a saturated or unsaturated aliphatic hydrocarbon group with 1-6 carbon atoms, including straight chain, branched chain or cyclic structure, such as including but not limited to: alkyl with 1-6 carbon atoms, hydrocarbon group with 1-6 carbon atoms containing unsaturated bonds;

[0044] In the embodiments of this application, the C1-C6 hydrocarbon oxygen group represents a saturated or unsaturated aliphatic hydrocarbon oxygen group with 1-6 carbon atoms, including straight-chain, branched-chain or cyclic structures;

[0045] In embodiments of this application, the C1-C6 hydrocarbon carbonyl group represents the carbonyl group forming an ester bond with a C1-C6 hydrocarbon oxygen group, including straight-chain, branched-chain, or cyclic structures;

[0046] In embodiments of this application, the C1-C6 alkyl group represents a saturated aliphatic hydrocarbon group with 1-6 carbon atoms, including straight-chain, branched, or cyclic structures, such as including but not limited to: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, cyclopropylmethyl, 2-cyclopropyl-ethyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0047] In embodiments of this application, the 1-6 carbon atom unsaturated aliphatic hydrocarbon group includes straight-chain, branched-chain, or cyclic structures, such as including but not limited to: vinyl, allyl, 1-buten-4-oxy, 3-hexyn-1-yl, 1-enpentoxy, 2-ethyl-1-en-butyl, (1-cyclopenten)methyl, cyclohexen-4-yl;

[0048] In embodiments of this application, the saturated aliphatic hydrocarbon oxy groups of 1-6 carbon atoms include straight-chain, branched, or cyclic structures, such as including but not limited to: methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, cyclopropylmethoxy, 2-cyclopropyl-ethoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, and 2-ethylbutoxy.

[0049] In embodiments of this application, the unsaturated aliphatic hydrocarbon oxy groups of 1-6 carbon atoms include straight-chain, branched-chain, or cyclic structures, such as including but not limited to: ethyleneoxy, allyloxy, 1-methyl-enoxy, 1-butenoxy, 4-hexyn-1-oxy, 2-methyl-allyloxy, 1-enpentoxy, 2-ethyl-1-en-butoxy, (1-cyclopentene)methoxy, and cyclohexene-4-oxy.

[0050] In the embodiments of this application, the 8-16 membered rings with or without alkene bonds refer to rings containing 8 to 16 atoms, and the 8-16 membered rings with alkene bonds refer to rings with at least one carbon-carbon double bond.

[0051] In the embodiments of this application, the halogen refers to fluorine, chlorine, bromine, and iodine; in some specific embodiments, it is preferably fluorine, chlorine, or bromine.

[0052] In the embodiments of this application, the C5-C14 aromatic heterocycle represents an aromatic ring structure containing 5-14 atoms and at least one heteroatom, which can be a monocyclic, fused, or biphenyl-type aromatic heterocycle, including but not limited to: thiophene, furan, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, triazole, thiadiazole, oxadiazole, tetraazole, thiatriazole, oxtriazole, pyridine, pyrimidine, pyrazine, pyridazine, triazine, tetraazine, purine, benzoxazole, benzofuran, benzothiazole, benzothiadiazole, benzotriazole, benzimidazole, indole, (2-pyridyl)benzene, (3-pyridyl)benzene, (4-pyridyl)benzene, and 5-phenyl-2-pyridine.

[0053] In some embodiments, the group R' is 1-methylpropyl; in some embodiments, the group R' is 1-methylpropyl or isobutyl.

[0054] In some embodiments, group R is methyl; in some embodiments, group R is hydrogen.

[0055] In some embodiments, group R0 is a hydroxyl group; in some embodiments, group R0 is an amino group; in some embodiments, group R0 is an amino acid residue substituted by group E; preferably, R0 is an amino-substituted glycine residue or an alanine residue; more preferably, R0 is an amino-substituted glycine residue.

[0056] In some embodiments, X1 and X2 are both nitrogen; in some embodiments, X1 and X2 are both sulfur; in some embodiments, X1 is sulfur and X2 is nitrogen; in some embodiments, X1 is sulfur and X2 is oxygen; in some embodiments, X1 is nitrogen and X2 is sulfur; in some embodiments, X1 is nitrogen and X2 is oxygen; preferably, X1 and X2 are both nitrogen.

[0057] In some embodiments, both R1 and R2 are H; in some embodiments, R1 is a carboxyl-substituted C1-C6 alkyl group, and R2 is H;

[0058] In some embodiments, R3 and R4 are both hydrogen; in some embodiments, R3 and R4 can be one or both of the following groups: C1-C6 alkoxy, nitro, nitrile, hydroxy, carboxyl, amino, halogen, trifluoromethyl;

[0059] In some embodiments, Y1 or Y4 is a nitrogen atom; in some embodiments, Y1, Y2, Y3, and Y4 are all carbon atoms.

[0060] In some embodiments, Y5 or Y8 is a nitrogen atom; in some embodiments, Y5, Y6, Y7 and Y8 are all carbon atoms.

[0061] In some embodiments, R5 is a C1-C6 hydrocarbon group or F substituted with hydrogen or a D-group; preferably, R5 is a hydrogen or F-group.

[0062] In some embodiments, R6 is selected from hydroxyl, amino, or carboxyl groups; in some embodiments, R6 is selected from hydroxyl, amino, or carboxyl groups that replace the following groups: methyl, ethyl, propyl; preferably, R6 is selected from hydroxyl or hydroxy-substituted C1-C3 hydrocarbon groups;

[0063] R7 is selected from hydrogen, -CONH2, -CH2CONH2, and the group F; preferably, R7 is hydrogen or the group F.

[0064] In some embodiments, R8 is hydrogen; in other embodiments, R8 is benzimidazole or indole.

[0065] In some implementations, Y9 is C; in some implementations, Y9 is O; in some implementations, Y9 is S;

[0066] In some implementations, n1 is 1; in other implementations, n1 is 2.

[0067] In some embodiments, R9 is -CH2CONH2; in some embodiments, R9 together with the adjacent nitrogen-containing heterocycle forms, for example... The ring structure; in some more specific implementations, Y2 is C or O; n2 is 1;

[0068] In some implementations, group R 10 For hydrogen; in some embodiments, the group R 10 It is a C1-C6 alkyl group; in some specific embodiments, the group R 10 It is methyl;

[0069] In some implementations, fragment B is: In some implementations, fragment B is: In some implementations, fragment B is: (in the formula) This indicates that it is attached to a carbonyl group. (Indicates that it is attached to an amino group);

[0070] In some implementation schemes, R 13 R 14 R 19 and R 23 Each is independently selected from: hydrogen, methyl;

[0071] In some implementation schemes, R 11 R 12 R 15 and R 16 Each is independently selected from: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl; in some embodiments, R 11 With R 12 The carbon chains link together to form 8-16 membered rings, with or without alkene bonds; in some embodiments, R 12 With R 15 The carbon chains link together to form 8-16 membered rings, with or without alkene bonds; in some embodiments, R 15 With R 16 They are linked by carbon chains to form 8-16 membered rings, with or without alkene bonds;

[0072] In some implementation schemes, R 17 R 18 and R 20Each is independently selected from: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl; in some embodiments, R 17 With R 18 They are linked by carbon chains to form 8-16 membered rings, with or without alkene bonds;

[0073] In some implementation schemes, R 21 R 22 and R 24 Each is independently selected from: C1-C6 alkyl, C1-C6 alkenyl, and C1-C6 alkynyl; in some embodiments, R 21 With R 24 They are linked by carbon chains to form 8-16 membered rings, with or without alkene bonds;

[0074] In some embodiments, group C is a C1-C6 alkoxycarbonyl group; in some embodiments, group C is a carboxyl group.

[0075] In some embodiments, group D is a carboxyl group; in some embodiments, group D is a hydroxyl group; in some embodiments, group D is an amino group.

[0076] In some embodiments, group E is a carboxyl group; in some embodiments, group E is a hydroxyl group; in some embodiments, group E is an amino group.

[0077] In some implementations, group F is

[0078]

[0079] In some implementations, base F is

[0080]

[0081] In some implementations, group F is

[0082] In some embodiments, the substituted macrocyclic polypeptide compounds provided by the present invention are selected from one of the following compounds:

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] On the other hand, compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts, esters, stereoisomers or solvents are used to develop peptide inhibitors targeting PD-1 / PD-L1.

[0099] The use of the compound of formula (I) or its pharmaceutically acceptable salt, ester, stereoisomer or solvent compound for the prevention and treatment of tumors.

[0100] The tumors include blood cancers, nervous system cancers, gastrointestinal cancers, esophageal cancers, urinary system cancers, lung cancers, liver cancers, and skin cancers.

[0101] The compound in formula (I) or its pharmaceutically acceptable salt, ester, stereoisomer or solvent compound is used for the prevention or treatment of sepsis and hepatitis B virus.

[0102] A pharmaceutical composition comprising a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt or ester or solvate thereof, and a pharmaceutically acceptable carrier.

[0103] The pharmaceutical composition is in the form of tablets, capsules, granules, powders, syrups, oral liquids, or injections.

[0104] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application may be realized and obtained by means of the methods described in the description. Attached Figure Description

[0105] Figure 1 This is a trend chart of subcutaneous tumor-bearing (colon cancer) experiments in mice.

[0106] Figure 2 This is a trend chart of subcutaneous tumor (breast cancer) in mice. Detailed Implementation

[0107] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of the present invention will be described in detail below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other.

[0108] The following examples are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way.

[0109] Example 1:

[0110]

[0111] Synthesis of compound 1c:

[0112] At room temperature, 3.23 g of compound 1a, 2.88 g of EDCI, 2.45 g of DMAP, and 1.97 g of compound 1b were added to a reaction flask. 35 mL of anhydrous dichloromethane was added, and the reaction mixture was stirred at 55 °C for 12 hours. The system was then cooled, and the reaction was quenched with 50 mL of water. Extraction was performed with dichloromethane. The organic phase was washed successively with 1N hydrochloric acid, saturated sodium carbonate solution, and saturated brine. The system was concentrated to dryness to obtain 3.82 g of crude compound 1c, which required no further purification.

[0113] Synthesis of compound 1d:

[0114] At 0°C, 25 mL of 3 mol·L⁻¹ HCl / EtOAc was added to the reaction flask from the previous step, and the system was stirred until the reaction was complete. The system was concentrated to dryness, and the mixture was filtered through ether to obtain 3.16 g of a yellow solid.

[0115] 1 g of the above solid, 0.83 g of potassium carbonate, and 30 ml of acetonitrile were added to a reaction flask. The system was cooled to 0 °C, and 0.35 ml of 2-fluorobenzyl bromide was added. After the addition was complete, the system was heated to 65 °C and reacted for 8 hours. The reaction system was filtered. The filtrate was concentrated to dryness and purified by silica gel column chromatography to give 0.92 g of compound 1d, yield 90%, ESI-MS(+): m / z 403.18 [M+H].

[0116] Synthesis of compound 1e:

[0117] 5 g of compound 1d, 7.23 g of Ni(NO3)2·6H2O, 2.8 g of glycine, and 40 ml of methanol were added to a reaction flask. A methanol (20 ml) solution of 3.48 g of potassium hydroxide was added, and the system was heated to 65 °C and reacted for 1 hour. The system was then cooled to 0 °C, and 4.48 g of acetic acid was added. The system was stirred at room temperature for 1 hour. Water was added for dilution, and the product precipitated. The crude product was crystallized using acetone / Et2O = 1:6, and filtered to obtain 5.9 g of compound 1e, yield 92%, ESI-MS (+): m / z 516.12 [M+H].

[0118] Synthesis of compound 1f:

[0119] Under nitrogen protection, 1 g of compound 1e and 30 mL of anhydrous acetonitrile were added to the reaction flask. The system was cooled to 0 °C, and 0.31 g of sodium hydroxide was added. The mixture was stirred for 10 min, and then 1.14 g of 5-iodopentene was added. The system was stirred at room temperature until the reaction was complete. The system was filtered, the filtrate was concentrated, and the mixture was slurried with n-heptane and filtered to obtain 2.15 g of compound 1f, which did not require further purification. The yield was 88%, and ESI-MS (+): m / z 584.18 [M+H].

[0120] Synthesis of Compound 1:

[0121] 2 g of compound 1f and 20 mL of methanol were added to a reaction flask. The system was heated to 70 °C, and 10 mL of 3 M hydrochloric acid was slowly added dropwise. The system was refluxed until the reaction was complete. After cooling, the methanol was removed by concentration, and the mixture was extracted multiple times with dichloromethane. The chiral auxiliaries were recovered from the organic phase. The aqueous phase was concentrated to dryness, and the pH was adjusted to 9-10 with 6% sodium carbonate solution. 1.15 g of EDTA-2Na was added, and the mixture was stirred for about 10 minutes. The system was cooled to -10 °C, and a solution of fluorenemethyloxycarbonyl succinimide (1.27 g) in acetonitrile (15 mL) was added dropwise. The system was stirred at room temperature for 15 hours. The acetonitrile was removed by concentration, and the pH of the aqueous phase was adjusted to 1-2. The mixture was extracted with ethyl acetate, and the organic phase was washed and concentrated to dryness. The solution was purified by silica gel column chromatography to give 0.813 g of compound 1, with a yield of 65%. ESI-MS (+): m / z 366.17 [M+H].

[0122] Synthesis of compound 2:

[0123] 450 mg of compound 1 and 10 mL of tetrahydrofuran were added to the reaction flask, followed by 100 mg of 10% Pd / C. The system was hydrogenated at 45 °C for 8 h. After the reaction was complete, diatomaceous earth was used as a filter aid, and the filter cake was washed twice with ethyl acetate. The organic phases were combined, concentrated to dryness, and separated by silica gel column chromatography to obtain 429 mg of compound 2, yield 95%, ESI-MS (+): m / z 368.18 [M+H].

[0124] Example 2:

[0125]

[0126] Synthesis of compound 2d:

[0127] 10 g of compound 2a, 8.22 g of triethylamine, and 100 ml of tetrahydrofuran were added to a reaction flask. 12.2 g of isobutyl chloroformate was added to the system, and the reaction was carried out at room temperature for 1 hour. 14.42 g of compound 2b was then added, and the system was heated to 80°C and refluxed overnight. After the reaction was complete, the system was concentrated to dryness, and 200 ml of methanol was added. 30.49 g of glycine, 37.17 g of nickel nitrate hexahydrate, 19.49 g of sodium hydride (DSC), and 13.67 g of potassium hydroxide were added sequentially to the system. The system was refluxed for 2 hours, cooled to room temperature, and the pH was adjusted with acetic acid. Crystallization was carried out for 12 hours. The system was filtered, slurried with methanol and water, and filtered again to obtain product 2d. This product did not require purification and was directly added to the next reaction step.

[0128] Synthesis of compound 2e:

[0129] Under nitrogen protection, 3 g of compound 2d and 50 mL of anhydrous acetonitrile were added to a reaction flask. The system was cooled to 0 °C, and 1.15 g of sodium hydroxide was added. The mixture was stirred for 10 min, and then 3.53 g of 5-iodopentene was added. The system was stirred at room temperature until the reaction was complete. The system was filtered, the filtrate was concentrated, and the mixture was slurried with n-heptane and filtered to obtain 3.5 g of compound 2e, which did not require further purification. The yield was 88%, and ESI-MS (+): m / z 353.17 [M+H].

[0130] Synthesis of compound 3:

[0131] 3g of compound 2e and 30ml of methanol were added to a reaction flask. The system was heated to 70℃, and 15ml of 3M hydrochloric acid was slowly added dropwise. The system was refluxed until the reaction was complete. After cooling, the methanol was removed by concentration, and the mixture was extracted multiple times with dichloromethane. The chiral auxiliaries were recovered from the organic phase. The aqueous phase was concentrated to dryness, and the pH was adjusted to 9-10 with 6% sodium carbonate solution. 1.83g of EDTA-2Na was added, and the mixture was stirred for about 10 minutes. The system was cooled to -10℃, and a solution of fluorenemethyloxycarbonyl succinimide (2.02g) in acetonitrile (20ml) was added dropwise. The system was stirred at room temperature for 15 hours. The acetonitrile was removed by concentration, and the pH of the aqueous phase was adjusted to 1-2. The mixture was extracted with ethyl acetate, and the organic phase was washed and concentrated to dryness. The mixture was purified by silica gel column chromatography to give 1.37g of compound 3, with a yield of 58%. ESI-MS (+): m / z 434.23 [M+H].

[0132] Synthesis of compound 4:

[0133] 1 g of compound 3 was added to the reaction flask, followed by 10 mL of anhydrous dichloromethane and 80 mg of Grubbs' 2nd catalyst. The reaction was carried out under nitrogen protection and refluxed for 2 days. After the reaction was completed, the reaction solution was diluted with dichloromethane, washed successively with water and saturated brine, and the organic phase was concentrated to dryness. Separation by silica gel column chromatography yielded 710 mg of compound 4, with a yield of 76%. ESI-MS (+): m / z 406.21 [M+H].

[0134] Example 3:

[0135]

[0136] Synthesis of compound 3b:

[0137] 2 g of compound 3a was added to a reaction flask and dissolved in toluene (60 mL), along with 760 mg of paraformaldehyde and 88 mg of p-toluenesulfonic acid. The system was connected to a water separator and heated to 130 °C, and reacted for 1 h. After the reaction was complete, the system was concentrated to dryness, extracted with ethyl acetate, and washed successively with saturated sodium bicarbonate, water, and saturated brine. The organic phase was concentrated to dryness, and the crude product was separated by silica gel column chromatography to obtain 1.89 g of compound 3b, yield 92%, ESI-MS(+): m / z 406.21 [M+H].

[0138] Synthesis of compound 3c:

[0139] 1.6 g of compound 3b and 30 mL of chloroform were added to a reaction flask. The system was cooled to 0 °C, and 3 mL of triisopropylsilane was added. Then, 30 mL of trifluoroacetic acid was slowly added dropwise. The system was reacted at 25 °C for 24 h. The system was concentrated to dryness, extracted with ethyl acetate, and washed successively with saturated sodium bicarbonate, water, and saturated brine. The organic phase was concentrated to dryness, and the crude product was separated by silica gel column chromatography to give 1.43 g of compound 3c, yield 89%, ESI-MS(+): m / z 408.22 [M+H].

[0140] Synthesis of compound 3f:

[0141] 1.3 g of compound 3c and a mixed solution of 5 mL THF and 5 mL DMF were added to a reaction flask. 477 mg of N-hydroxysuccinimide was added, and the system was cooled to 0 °C. 1.1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was slowly added. The system was reacted at 25 °C for 24 h. Extraction was performed with ethyl acetate, followed by washing with saturated sodium bicarbonate, water, and saturated brine. The organic phase was concentrated to dryness to obtain crude compound 3d, which could be used directly in the next reaction without further purification.

[0142] The compound 3d obtained in the previous step was dissolved in 15 mL of acetone. The reaction system was cooled to 0 °C, and 573 mg of the hydrochloride salt of compound 3e was slowly added. 15 mL of a 10% sodium carbonate aqueous solution was added dropwise to the system. The system was reacted at 25 °C for 24 h. The system was concentrated to dryness, extracted with ethyl acetate, and washed successively with saturated sodium bicarbonate, water, and saturated brine. The organic phase was concentrated to dryness, and the crude product was separated by silica gel column chromatography to obtain 1.46 g of compound 3f, yield 86%, ESI-MS(+): m / z 533.30 [M+H].

[0143] Synthesis of compound 5:

[0144] 1.3 g of compound 3f was added to the reaction flask, along with 10 mL of anhydrous dichloromethane and 85 mg of Grubbs' 2nd catalyst. The reaction was carried out under nitrogen protection and refluxed for 2 days. After the reaction was completed, the reaction solution was diluted with dichloromethane, washed successively with water and saturated brine, and the organic phase was concentrated to dryness. Separation by silica gel column chromatography yielded 960 mg of compound 5, with a yield of 78%. ESI-MS (+): m / z 505.27 [M+H].

[0145] Synthesis of compound 6:

[0146] 960 mg of compound 5 and 15 mL of tetrahydrofuran were added to the reaction flask, followed by 225.3 mg of 10% Pd / C. The system was hydrogenated at 45 °C for 8 h. After the reaction was complete, diatomaceous earth was used as a filter aid, and the filter cake was washed twice with ethyl acetate. The organic phases were combined, concentrated to dryness, and separated by silica gel column chromatography to obtain 896 mg of compound 6, yield 93%, ESI-MS (+): m / z 507.28 [M+H].

[0147] Example 4:

[0148]

[0149] Synthesis of compound 4b:

[0150] 1.4 ml of compound 4a and 20 ml of anhydrous dichloromethane were added to the reaction flask. The system was cooled to below 10 °C, and 1 ml of chlorosulfonyl isocyanate was slowly added dropwise. The system was refluxed under nitrogen protection. After the reaction was complete, 10 ml of 10% sodium sulfite and 10 ml of 10% potassium hydroxide aqueous solution were added to adjust the pH to 9. The positive phase was separated, washed with water, and concentrated to dryness. The system was purified by silica gel column chromatography to give 1.2 g of compound 4b, yield 77%, ESI-MS (+): m / z 154.12 [M+H].

[0151] Synthesis of compound 4c:

[0152] 1.1 g of compound 4b, 231 mg of tetrabutylammonium bromide, and 15 mL of tetrahydrofuran were added to a reaction flask. 290 mg of sodium hydroxide and 1.12 g of iodomethane were added at 25 °C. The reaction was maintained at this temperature for 8 hours. After the reaction was complete, the pH was adjusted to 4, and the mixture was extracted with dichloromethane. The organic phase was washed with water and concentrated to dryness. The system was purified by silica gel column chromatography to give 1.13 g of compound 4c, 94% yield, ESI-MS (+): m / z 168.13 [M+H].

[0153] Synthesis of compound 4d:

[0154] 1 g of compound 4e was added to a reaction flask, followed by 15 mL of 6M hydrochloric acid. The reaction was carried out at 60 °C for 8 hours. After the reaction was complete, the mixture was concentrated to dryness, extracted multiple times with dichloromethane, and the organic phase was washed with water and concentrated to dryness. The mixture was purified by silica gel column chromatography to give 720 mg of compound 4d, with a yield of 65%. ESI-MS (+): m / z 186.14 [M+H].

[0155] Synthesis of compound 7:

[0156] 0.7 g of compound 4d was added to a mixture of 10 mL of 10% sodium carbonate and 10 mL of acetone in a reaction flask. The system was cooled to 0 °C, and 1.17 g of Fmoc-Cl was added. After reacting at 0 °C for 1 hour, the system was raised to room temperature and reacted for 5 hours. The pH was adjusted to 3, and the mixture was extracted multiple times with ethyl acetate. The organic phase was washed with water and concentrated to dryness. The system was purified by silica gel column chromatography to give 1.37 g of compound 7, with a yield of 92%. ESI-MS (+): m / z 394.2 [M+H].

[0157] Example 5:

[0158]

[0159] Synthesis of compound 5b:

[0160] 3 g of compound 5a, 3.74 g of cesium carbonate, and 30 mL of DMF were added to a reaction flask. The system was cooled to 0 °C, and 3.78 g of tert-butyl bromoacetate was added. The system was slowly heated to room temperature and reacted for 5 hours. Water was added to the system, and the mixture was extracted multiple times with ethyl acetate. The organic phase was washed with water and concentrated to dryness. The system was purified by silica gel column chromatography to give 4.26 g of compound 5b, yield 85%, ESI-MS (+): m / z 285.12 [M+H].

[0161] Synthesis of compound 5c:

[0162] Under nitrogen protection, 4.73 g of 2-(((benzyloxy)carbonyl)amino)-2-(dimethoxyphosphono)benzyl acetate, 1.78 g of DBU, and 25 mL of dichloromethane were added to a reaction flask. The system was stirred at room temperature for 20 minutes. A dichloromethane (25 mL) solution of compound 5b (3 g) was slowly added dropwise. The system was reacted at room temperature for 18 hours. The system was concentrated to dryness, dissolved in ethyl acetate, washed with saturated brine, and the organic phase was concentrated to dryness. The organic phase was purified by silica gel column chromatography to give 4.66 g of compound 5c, yield 78%, ESI-MS (+): m / z 566.22 [M+H].

[0163] Synthesis of compound 5d:

[0164] 3g of compound 5c, 20ml of methanol, and 35mg of (+)-1,2-bis((2S,5S)-2,5-diethylphosphacyclopentan-1-yl)phenyl(cyclooctadiene)tetrafluoroborate (I) were added to a reaction flask. The system was reduced under hydrogen atmosphere at 60psi. After the reaction was completed, the system was filtered through diatomaceous earth, and the filtrate was concentrated to dryness to obtain the crude product, which was directly used in the next reaction.

[0165] Synthesis of compound 5e:

[0166] 3 g of compound 5d and 25 mL of methanol were added to the reaction flask, followed by 0.6 g of 10% Pd / C. The system was hydrogenated at 40 °C for 12 h. After the reaction was complete, diatomaceous earth was used as a filter aid. The organic phase was concentrated to dryness and separated by silica gel column chromatography to obtain 1.6 g of compound 5e, yield 92%, ESI-MS (+): m / z 330.14 [M+H].

[0167] Synthesis of compound 8:

[0168] 1.5 g of compound 5e, 15 ml of tetrahydrofuran, and 10 ml of water were added to a reaction flask. 1 g of sodium bicarbonate and 1.62 g of fluorenemethoxycarbonylsuccinimide were added while stirring. The system was stirred at room temperature for 15 hours. The system was concentrated to remove tetrahydrofuran, and the pH of the aqueous phase was adjusted to dryness 1–2. Extraction was performed with ethyl acetate, followed by washing with the organic phase and concentration to dryness. Purification was achieved by silica gel column chromatography to give 2 g of compound 8, with a yield of 81%. ESI-MS (+): m / z 566.22 [M+H].

[0169] Example 6:

[0170]

[0171] Synthesis of compound 6b:

[0172] 3 g of compound 6a, 3.59 g of tert-butyl N-(diphenylmethylene)glycine ester, 30 mL of anhydrous tetrahydrofuran, and 2.13 g of cesium carbonate were added to a reaction flask. The system was reacted to completion at room temperature. The system was concentrated to dryness, extracted with ethyl acetate and water, concentrated to dryness, and purified by silica gel column chromatography to give 4.64 g of compound 6b, yield 74%, ESI-MS(+): m / z 567.3 [M+H].

[0173] Synthesis of compound 6c / 6d:

[0174] 3 g of compound 6b, 0.6 g of 10% palladium on carbon, and 30 mL of a mixed solution of ethanol and acetic acid (9:1) were added to a reaction flask. The system was hydrogenated at 30 psi for 48 hours. After the reaction, the system was filtered with diatomaceous earth as an aid. The filtrate was concentrated to dryness, extracted with dichloromethane and water, and the organic phase was concentrated to dryness. The organic phase was purified by silica gel column chromatography to give 0.62 g of compound 6c (yield 33%, ESI-MS(+): m / z 355.22 [M+H]) and 0.71 g of compound 6d (yield 38%, ESI-MS(+): m / z 355.22 [M+H]).

[0175] Synthesis of compound 9:

[0176] 0.5 g of compound 6c, 10 ml of dichloromethane, and 6 ml of trifluoroacetic acid were added to a reaction flask, followed by dropwise addition of 0.2 ml of triethylsilane. The reaction was allowed to proceed for 1 hour. The mixture was then concentrated to dryness, extracted with dichloromethane and water, and the organic phase was concentrated to dryness.

[0177] 9 ml of tetrahydrofuran and 3 ml of water were added to the system, followed by the addition of 0.3 g of sodium carbonate and 0.475 g of fluorenemethoxycarbonyl succinimide under stirring. The system was stirred at room temperature for 15 hours. The system was concentrated to remove tetrahydrofuran, and the pH of the aqueous phase was adjusted to 1–2. The mixture was extracted with ethyl acetate, washed with the organic phase, concentrated to dryness, and purified by silica gel column chromatography to give 0.47 g of compound 9, with a yield of 79%. ESI-MS (+): m / z 421.17 [M+H].

[0178] Example 7:

[0179]

[0180] Synthesis of compound 7c:

[0181] Under nitrogen protection, 5 g of compound 7a and 40 mL of anhydrous tetrahydrofuran were added to a reaction flask. The system was cooled to -78 °C, and 19 mL of butyllithium in hexane (1.6 M) was added dropwise using DSC. The system was stirred at low temperature for 1 hour. 15 mL of n-hexane (2.75 g) in tetrahydrofuran was added dropwise, and the system was heated to room temperature and reacted for 8 hours. The reaction was quenched with water, and the solvent was removed under reduced pressure. The mixture was extracted with dichloromethane and water, and the organic phase was concentrated to dryness. The organic phase was purified by silica gel column chromatography to give 3.26 g of compound 7c, yield 76%, ESI-MS (+): m / z 157.12 [M+H].

[0182] Synthesis of compound 7e:

[0183] 3.38 g of compound 7d and 30 mL of anhydrous tetrahydrofuran were added to a reaction flask, and the system was cooled to 0 °C. 1.28 g of sodium hydride was added, and after reacting for 1 hour, 2.5 g of compound 7c was added. The system was heated to 40 °C until the reaction was complete. The system was quenched with water, concentrated to dryness, extracted with ethyl acetate and water, concentrated to dryness, and purified by silica gel column chromatography to give 4.29 g of compound 7e, yield 73%, ESI-MS(+): m / z 368.25 [M+H].

[0184] Synthesis of compound 7f:

[0185] 4 g of compound 7e and 40 mL of anhydrous tetrahydrofuran were added to a reaction flask. The system was cooled to 0 °C, and 1.85 g of iodomethane and 4.87 g of KHMDS were added. The system was stirred at low temperature for 1 hour. The system was then heated to room temperature and reacted for 8 hours. The reaction was quenched with water, and the solvent was removed under reduced pressure. The mixture was extracted with dichloromethane and water, and the organic phase was concentrated to dryness. The organic phase was purified by silica gel column chromatography to give 2.95 g of compound 7f, yield 71%, ESI-MS (+): m / z 382.27 [M+H].

[0186] Synthesis of 7g of compound:

[0187] 2.8 g of compound 7f, 0.4 g of 10% palladium on carbon, and 30 ml of methanol were added to a reaction flask, and the system was hydrogenated at room temperature for 48 hours. After the reaction was completed, the system was filtered with diatomaceous earth as an aid, the filtrate was concentrated to dryness, and purified by silica gel column chromatography to give 1.33 g of compound 7f, yield 96%, ESI-MS (+): m / z 188.16 [M+H];

[0188] Synthesis of compound 10:

[0189] 1.25 g of the crude compound was added to a reaction flask, along with 8 ml of diethyl acetate and 8 ml of 6N hydrochloric acid. The system was heated to 40 °C and reacted for 5 hours. The system was then cooled, extracted with ethyl acetate and water, and the organic phase was concentrated to dryness. The organic phase was purified by silica gel column chromatography to give 1.05 g of compound 10, yield 91%, ESI-MS (+): m / z 174.12 [M+H].

[0190] Example 8:

[0191]

[0192] Fmoc-Gly-OH was uploaded to Rink Amide-AM Resin:

[0193] DCM (dichloromethane) is used to swell Rink Amide-AM Resin resin. Fmoc-Gly-OH is dissolved in DMF solution and coupled to the resin under the action of condensing agents HBTU / DIEA, DIC / HOBt, PyBOP, or other similar condensing agents. The resin is then washed.

[0194] Remove Fmoc protection:

[0195] 20% Pip (piperidine) / DMF solution for Fmoc protection removal, followed by DMF washing of the resin;

[0196] Fmoc-N-Me-Cys(Trt)-OH is coupled to the peptide resin:

[0197] Add a DMF solution of Fmoc-N-Me-Cys(Trt)-OH and couple it to the resin under the action of condensing agents HBTU / DIEA, DIC / HOBt, PyBOP, or other similar condensing agents, then wash the resin.

[0198] Amino acid coupling:

[0199] According to the amino acid sequence of the molecular structure of this invention, Fmoc-Leu-OH, Fmoc-1-methyl-11-(methylamino)-12-oxoazacyclododecane-2-carboxylic acid, Fmoc-(1-(2-(tert-butoxy)-2-oxoethyl)-L-Trp-OH, Fmoc-Dab(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-tras-D-Hmp(tBu)-OH, Fmoc-Ile-OH, Fmoc-L-Dap(Boc)-OH, Fmoc-D-Pro-OH, Fmoc-D-Asn(Trt)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, and chloroacetic acid are added.

[0200] The coupling and deprotection cycles are performed sequentially until the peptide resin is coupled (for coupling, refer to the above "Fmoc-N-Me-Cys(Trt)-OH coupling to peptide resin", and for deprotection, refer to the above "deprotection of Fmoc").

[0201] Remove the Mmt group from Cys on the peptide chain:

[0202] Add the resin to a 1% TFA (trifluoroacetic acid) DCM solution and wash the resin.

[0203] Cyclic peptides are formed by cyclizing linear peptide chains in a solid-phase resin:

[0204] The resin was swollen in DCM, and the linear peptide was cyclized under the action of tris(2-carboxyethyl)phosphine (TCEP) and diisopropylethylamine (DIEA).

[0205] The resin was cleaved and all protecting groups on the cyclic peptide were removed to obtain the product:

[0206] Prepare a lysis buffer according to the volume ratio (TFA:EDT:TIS:H2O = 95:2:2:1). Add the cyclic peptide resin to the lysis buffer and react to remove the side chains. Remove TFA by vacuum rotary evaporation. Add MTBE (methyl tert-butyl ether) to the concentrate to precipitate a white solid. Collect the precipitate, wash repeatedly, and dry.

[0207] Chromatographic purification:

[0208] The mixture was purified by multi-step reversed-phase chromatography using a C8 or C18 column. The final solution was converted to a salt solution or free solution of a specific acid, and then freeze-dried to obtain a powdered mixture. ESI-MS(+): m / z 1870.91 [M+H], m / z 936.21 [M+2H], m / z 624.44 [M+3H]

[0209] Example 9:

[0210]

[0211] Fmoc-Gly-OH was uploaded to Rink Amide-AM Resin:

[0212] DCM (dichloromethane) is used to swell Rink Amide-AM Resin resin. Fmoc-Gly-OH is dissolved in DMF solution and coupled to the resin under the action of condensing agents HBTU / DIEA, DIC / HOBt, PyBOP, or other similar condensing agents. The resin is then washed.

[0213] Remove Fmoc protection:

[0214] 20% Pip (piperidine) / DMF solution for Fmoc protection removal, followed by DMF washing of the resin;

[0215] Fmoc-N-Me-Cys(Trt)-OH is coupled to the peptide resin:

[0216] Add a DMF solution of Fmoc-N-Me-Cys(Trt)-OH and couple it to the resin under the action of condensing agents HBTU / DIEA, DIC / HOBt, PyBOP, or other similar condensing agents, then wash the resin.

[0217] Amino acid coupling:

[0218] According to the amino acid sequence of the molecular structure of this invention, Fmoc-Leu-OH, Fmoc-2-(methylamino)hex-5-enoic acid, Fmoc-2-aminohex-5-enoic acid, Fmoc-(S)-2-amino-3-(1-(2-(tert-butoxy)-2-oxoethyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)propanoic acid, Fmoc-Dab(Dde)-OH, Fmoc-(S)-2-amino-3-(1H-pyrrolo[3,2-b]pyridin-3-yl)propanoic acid, Fmoc-tras-D-Hmp(tBu)-OH, Fmoc-Leu-OH, Fmoc-L-Dap(Boc- OH,Fmoc-D-Pro-OH,Fmoc-D-Asn(Trt)-OH,Fmoc-Ala-OH,Fmoc-Tyr(tBu)-OH, chloroacetic acid.

[0219] The coupling and deprotection cycles are performed sequentially until the peptide resin is coupled (for coupling, refer to the aforementioned "Fmoc-N-Me-Cys(Trt)-OH coupling to peptide resin" step; for deprotection, refer to the aforementioned "deprotection of Fmoc" step).

[0220] Remove the Dde group from the Dab of the peptide chain:

[0221] The Dde group on Dab was removed using a 3% hydrazine hydrate / N,N-dimethylformamide solution.

[0222] Protect the long fatty side chain coupling to the peptide chain

[0223] The protected long fatty side chain (see formula SM-0) is coupled to the peptide chain under the action of the condensing agent HBTU / DIEA.

[0224]

[0225] Remove the Mmt group from Cys on the peptide chain:

[0226] Add the resin to a 1% TFA (trifluoroacetic acid) DCM solution and wash the resin.

[0227] Cyclic peptides are formed by cyclizing linear peptide chains in a solid-phase resin:

[0228] The resin was swollen in DCM, and the linear peptide was cyclized under the action of tris(2-carboxyethyl)phosphine (TCEP) and diisopropylethylamine (DIEA).

[0229] The resin was cleaved and all protecting groups on the cyclic peptide and side chains were removed to obtain the product:

[0230] Prepare a lysis buffer according to the volume ratio (TFA:EDT:TIS:H2O = 95:2:2:1). Add the cyclic peptide resin to the lysis buffer and react to remove the side chains. Remove TFA by vacuum rotary evaporation. Add MTBE (methyl tert-butyl ether) to the concentrate to precipitate a white solid. Collect the precipitate, wash repeatedly, and dry.

[0231] Chromatographic purification:

[0232] The mixture was purified by multi-step reversed-phase chromatography using a C8 or C18 column, and finally converted to a salt solution of a certain acid. After freeze-drying, a powdered mixture was obtained.

[0233] The cyclic peptide compounds of the following examples were synthesized using the same method as in the above examples, using commercially available compounds or amino acids, or amino acids appropriately synthesized from commercially available compounds.

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that there are many more embodiments and implementations within the scope of the embodiments described herein.

[0250] Example 10:

[0251] ELISA detection of binding to human PD-L1

[0252] Plated on 96-well microplates, coated with PD-L1 (Human PD-L1 [28-8] SimpleStep) Kit (catalog number ab214565), incubated at 37°C for 1.5 hours. Discard the solution in the wells, wash three times with wash buffer, and block with PBS solution containing 2% BSA for 1 hour. Wash three more times with wash buffer, add biotinylated human PD-L1 antibody and horseradish peroxidase-labeled avidin to each well, incubate at 37°C for 1.5 hours, wash three times with wash buffer, then add different dilutions of the compound of this invention, incubate at 37°C for 1.5 hours, wash three times with wash buffer, add 100 μl of chromogenic substrate TMB solution, react at room temperature for 30 minutes, stop the reaction with 100 μl of 2M hydrochloric acid solution, and read the absorbance at 450 nm using a microplate reader to calculate the OD value. 8-12 concentrations of each compound were detected, and calculations were performed using Graphpad software.

[0253]

[0254]

[0255] Example 11

[0256] Mouse subcutaneous tumor-bearing (colon cancer) experiment

[0257] Thirty Balb / c mice were subcutaneously inoculated with CT-26 mouse colon cancer cells. When the tumor volume reached 50–100 mm... 3 Mice were randomly divided into 6 groups, with 5 mice in each group. Group 1 was intravenously injected with physiological saline as a blank control; groups 2-5 were intravenously injected with physiological saline solution of the compound of this invention (4 mg / kg). -1 Group 1 received one dose daily as the compound group; Group 3 received intraperitoneal injection of mouse PD-L1 monoclonal antibody (clone number 10F.9G2) at a dose of 10 mg / kg. -1 Every two days, a positive control group was observed. The tumor diameter was measured daily, and its volume was approximately calculated as v = ab. 2 / 2.

[0258] The results are as follows Figure 1 As shown in the trend graph of the mouse subcutaneous tumor (colon cancer) test, the compound of the present invention can significantly inhibit the growth of CT-26 xenograft tumors, and the weight of mice is not affected during the process.

[0259] Example 12 Mouse Subcutaneous Tumor (Breast Cancer) Experiment

[0260] Thirty Nude mice were subcutaneously inoculated with the human breast cancer cell line SKBR-3 in their axillae. When the tumor volume reached 50–100 mm, the cells were inoculated. 3Mice were randomly divided into 6 groups, with 5 mice in each group. Group 1 was intravenously injected with physiological saline as a blank control; groups 2-5 were intravenously injected with physiological saline solution of the compound of this invention (4 mg / kg). -1 Group 1 received the drug once daily as the compound group; Group 6 received intraperitoneal injection of mouse PD-L1 monoclonal antibody (clone number 10F.9G2) at a dose of 10 mg / kg. -1 Every two days, a positive control group was observed. The tumor diameter was measured daily, and its volume was approximately calculated as v = ab. 2 / 2. The result is as follows: Figure 2 The compound shown in the (trend graph of subcutaneous tumor (breast cancer) in mice) can significantly inhibit the growth of transplanted tumors without affecting the weight of mice.

[0261] Example 13: Animal Model Experiment of Sepsis

[0262] Several rats were selected for surgical modeling using the classic cecal ligation and perforation (CLP) method. Preoperatively, the rats were anesthetized with ether. After skin preparation, a longitudinal incision of approximately 2-3 cm was made slightly to the right of the midline of the rat's abdomen under aseptic conditions. The abdominal cavity was opened, and the intestines were dissected and exposed. The cecum was ligated approximately 0.5-1 cm from its free distal end. A perforation was then made at the midpoint of the ligated cecum, ensuring complete penetration of the intestinal wall. Subsequently, 1 mL of sterile saline was injected distal to the ligated cecum to expel a small amount of intestinal contents. All intestinal tracts and other internal organs were returned to the abdominal cavity, and the cavity was closed layer by layer by suture, followed by disinfection of the incision. Postoperatively, the rats were returned to sterilized cages and allowed free access to food. One day later, surviving rats were randomly divided into 5 groups, with at least 6 rats in each group. One group served as a blank control (administered with water for injection twice daily), three groups served as experimental drug groups (DSC1923 and DSC2016 groups were administered 700 mg / kg twice daily, and DSC1939 group was administered 1 g / kg once every two days), and one group served as an antibiotic (cefminox) group (administered 200 mg / kg twice daily). Survival rates were assessed over 6 weeks. The survival rate was 14.2% (1 / 7) in the blank control group, 62.5% (6 / 8) in the DSC1923 group, 57.1% (4 / 7) in the DSC2016 group, 57.1% (4 / 7) in the DSC1939 group, and 87.5% (7 / 8) in the antibiotic (cefminox) group. This experiment demonstrates that the compound of the present invention has a therapeutic effect on sepsis in rats. Furthermore, because its mechanism is significantly different from that of antibiotics and it has the effect of inhibiting PD-1 and PD-L1, it can be understood that it exerts its unique pharmacological effect through the immune system.

[0263] Example 14 Chronic HBV Mouse Model Experiment

[0264] Several 4-6 week old Balb / c mice were selected, and 20 μg of HBV expression plasmid (pCI-neo-attB-CMV-HB1.3) was mixed with physiological saline and rapidly injected into Balb / c mice via the tail vein using a hydrodynamic transfection method. The viral DNA content in mouse serum was detected by real-time quantitative PCR. The viral load was measured after approximately 10 μg of the plasmid had reached a stable plateau. 4 ~10 5 The administration of drugs began when the viral load reached 10 copies / ml. Experimental mice were randomly divided into 5 groups, with at least 6 mice in each group. One group served as the blank control group (administered with water for injection once daily), three groups served as the experimental drug groups (DSC1914 and DSC2027 groups were administered 0.5 mg / kg once daily, and DSC2041 group was administered 0.75 mg / kg every two days), and one group served as the entecavir group (administered 0.07 mg / kg once daily). Viral load was assessed after 20 days. The viral DNA content in mouse serum was detected by PCR; the level in the blank control group remained at 10. 4 ~10 5 copies / ml, DSC1914, DSC2027 and DSC2041 groups were 10 copies / ml, respectively. 3 ~10 4 Level, entecavir group is 10 2 ~10 3 Level. This experiment demonstrates that the compounds of this invention have a therapeutic effect against chronic HBV, possibly exerting their unique pharmacological effects by regulating the immune system through the inhibition of PD-1 and PD-L1.

[0265] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that there are many more embodiments and implementations within the scope of the embodiments described herein.

Claims

1. A macrocyclic polypeptide compound, which is selected from the following compounds: ###0001### ###0002### or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the macrocyclic polypeptide compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of the macrocyclic polypeptide compound of claim 1 and a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 in the manufacture of a medicament for treating colon cancer, breast cancer.

4. Use of the macrocyclic polypeptide compound of claim 1 and a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 in the manufacture of a medicament for preventing or treating sepsis.

5. Use of the macrocyclic polypeptide compound of claim 1 and a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 in the manufacture of a medicament for preventing or treating hepatitis B. ​

Images