Cyclic polypeptide compounds and uses thereof

Abstract

The application discloses a cyclic polypeptide compound and application thereof, and the cyclic polypeptide compound is shown as formula (I), wherein the definitions of the groups are shown in the specification; and the compound can be used as a PD-1 / PD-L1 inhibitor.

Description

Technical Field

[0001] This invention relates to a class of cyclic polypeptide compounds or their pharmaceutically acceptable salts, esters, stereoisomers or solvents, and pharmaceutical compositions thereof. This invention relates to, but is not limited to, the field of pharmaceutical chemistry, and particularly to their use in the preparation of 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-based 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] Immune mechanisms 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, Lambruizumab, 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 cyclic polypeptide molecules provided in this application have been shown 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.

[0006] This invention relates to a class of cyclic polypeptide compounds or pharmaceutically acceptable salts, esters, stereoisomers, solvent compounds or prodrugs of the same as PD-1 / PD-L1 inhibitors for the treatment of malignant tumors.

[0007] This application provides cyclic polypeptide compounds of general formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, solvent compounds or prodrugs thereof:

[0008]

[0009] In formula (I), R is hydrogen or methyl;

[0010] R0 is group L3, or any of the following groups substituted by group L3: The group L3 is one of the following groups: amino, hydroxyl;

[0011] X1 and X2 are each independently selected from oxygen, sulfur, and nitrogen;

[0012] R1 and R2 are each independently selected from hydrogen, C1-C6 alkyl groups substituted with group L4, C2-C6 alkenyl groups substituted with group L4, C2-C6 alkynyl groups substituted with group L4, and C3-C8 cycloalkyl groups substituted with group L4; when X1 or X2 is oxygen or sulfur, R1 or R2 is absent accordingly; said group L4 is one or more of the following groups: C1-C6 alkoxy, C1-C8 alkoxycarbonyl, carboxyl, hydroxyl, amino, mono(C1-C6 alkyl)amino, bis(C1-C6 alkyl)amino, -CONH2, phenyl, biphenyl, naphthyl;

[0013] R3 and R4 are each independently selected from: hydrogen, C1-C6 alkoxy, C1-C6 alkyl, nitro, cyano, hydroxyl, carboxyl, amino, halogen and trifluoromethyl;

[0014] At most one of Y1, Y2, Y3 and Y4 is a nitrogen atom, and the rest are carbon atoms;

[0015] At most one of Y5, Y6, Y7 and Y8 is a nitrogen atom, and the rest are carbon atoms;

[0016] R5 is hydrogen, or a substituted or unsubstituted C1-C6 alkyl group; the substituent is selected from: H, C1-C6 alkoxy, C1-C8 alkoxycarbonyl, carboxyl, hydroxyl, amino, mono(C1-C6 alkyl)amino, bis(C1-C6 alkyl)amino, -CONH2, phenyl, biphenyl, naphthyl; in one embodiment, R5 is a substituted or unsubstituted C1-C3 alkyl group; in some specific embodiments, the substituent is selected from carboxyl, hydroxyl, amino, Z-substituted amino, -CONH2;

[0017] R8 is hydrogen, substituted or unsubstituted C1-C6 alkyl; the substituent is selected from: H, C1-C6 alkoxy, C1-C8 alkoxycarbonyl, carboxyl, hydroxyl, amino, mono(C1-C6 alkyl)amino, bis(C1-C6 alkyl)amino, -CONH2, phenyl, biphenyl, naphthyl.

[0018] R9 is hydrogen, a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted C1-C6 aminoalkyl group; the substituent is selected from the group Z, -CONH2 or -CH2CONH2;

[0019] R 10 It can be hydrogen, a C5-C14 aromatic heterocycle, a C1-C6 alkyl group substituted with a C5-C14 aromatic heterocycle, an unsubstituted C1-C6 alkyl group, or a C1-C6 alkyl group substituted with an L4 group;

[0020] R 13 It is hydrogen, substituted or unsubstituted C1-C6 alkyl; or when R 11 and R 12 When R forms a 4-7 membered ring with the atoms connected to it, 13 With R 11 and R 12 The 4-7 membered rings formed together with the atoms attached thereto form a fused ring structure; the substituents are selected from: H, C1-C6 alkoxy, C1-C8 alkoxycarbonyl, carboxyl, hydroxyl, amino, mono(C1-C6 alkyl)amino, bis(C1-C6 alkyl)amino, -CONH2, phenyl, biphenyl, naphthyl; preferably, the substituents are selected from H, carboxyl, hydroxyl, amino, -CONH2, phenyl, biphenyl, naphthyl;

[0021] R6 and R7, R 11 and R 12 and R 14 and R 15 Each of the atoms independently forms a 4-7 membered heterocycle with the atom it is attached to. Optionally, the 4-7 membered ring can be substituted with a substituent or can form a fused ring or a spiro ring with other rings. The substituent is selected from H, carboxyl, hydroxyl, amino, and cyano. Optionally, the 4-7 membered heterocycle is a heterocycle containing 1-3 heteroatoms selected from N, O, or S.

[0022] R 16 The following groups are allowed: hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, mono(C1-C6 alkyl)amino, bis(C1-C6 alkyl)amino, phenyl, halogen;

[0023] Fragment L1 is independently selected from the following structural fragments:

[0024] Fragment L2 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 19 R 22 R 26 R 30 and R 31 Each is independently: hydrogen, or a C1-C6 alkyl group;

[0025] R 17 R 18 R 20 and R 21 Each independently is: hydrogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C2-C6 alkenyl; or, R 17 With R 18 Carbon chains link together to form 8-16 membered rings, with or without unsaturated bonds, or R 18 With R 20 Carbon chains link together to form 8-16 membered rings, with or without unsaturated bonds, or R 20 With R 21 The carbon chains are linked to form 8-16 membered rings with or without unsaturated bonds; when the L2 segment does not contain a ring and R 20 When it is hydrogen, R 18 and R 21 Not simultaneously butyl; the substituent is selected from H, carboxyl, hydroxyl, amino, -CONH2, phenyl, biphenyl, naphthyl;

[0026] R 23 R 24 and R 25 Each independently consists of: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl, or R 24 With R 25 They are linked by carbon chains to form 8-16 membered rings with or without unsaturated bonds;

[0027] R 27 R 28 and R 29 Each is independently hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl, or R 27 With R 28 They are linked by carbon chains to form 8-16 membered rings with or without unsaturated bonds.

[0028] The group Z is one of the following groups, with the dashed lines representing connecting bonds:

[0029]

[0030] In embodiments of this application, compounds of general formula (II) are provided:

[0031]

[0032] The definitions of each group in formula (II) are as in formula (I).

[0033] In some embodiments, group R is hydrogen; in some embodiments, group R is methyl.

[0034] 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 L3-substituted amino acid residue; in some embodiments, R0 is an amino-substituted glycine residue; in some embodiments, R0 is an amino-substituted alanine residue; in one specific embodiment, R0 is... In one specific embodiment, L3 is an amino group.

[0035] 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.

[0036] In some embodiments, both R1 and R2 are H; in some embodiments, R1 is selected from C1-C6 alkyl groups substituted with group L4; said group L4 is one or more of the following groups: C1-C6 alkoxy, C1-C6 alkoxycarbonyl, carboxyl, hydroxyl, amino, mono(C1-C6 alkyl)amino, bis(C1-C6 alkyl)amino, -CONH2, phenyl, biphenyl, naphthyl, and R2 is H; or it is a carboxyl-substituted C1-C6 alkyl group, and R2 is H.

[0037] 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, cyano, hydroxy, carboxyl, amino, halogen, trifluoromethyl.

[0038] In some implementations, one of Y1, Y2, Y3, and Y4 is a nitrogen atom; in other implementations, Y1, Y2, Y3, and Y4 are all carbon atoms.

[0039] In some embodiments, one of Y5, Y6, Y7, and Y8 is a nitrogen atom; in some embodiments, Y5, Y6, Y7, and Y8 are all carbon atoms; in some specific embodiments, Y5, Y6, and Y7 are all carbon atoms.

[0040] In some embodiments, R5 is hydrogen; in some embodiments, R5 is an amino-substituted C1-C6 alkyl group; R5 is an amino-substituted C1-C6 alkyl group substituted with group F; in some more specific embodiments, R5 is an amino-substituted group of the following: methyl, ethyl, propyl; in some more specific embodiments, R5 is an amino-substituted group of the following: methyl, ethyl, propyl.

[0041] In some implementation schemes, R6 and R7, R 11 and R 12 R 14 and R 15 Each atom independently forms a 4-7 membered ring with the atoms it is attached to, wherein the 4-7 membered ring is selected from one or more of the following structures:

[0042] In some embodiments, the substituent is selected from H, C1-C3 alkoxy, C1-C3 alkoxycarbonyl, carboxyl, hydroxyl, amino, mono(C1-C3 alkyl)amino, bis(C1-C3 alkyl)amino, -CONH2, phenyl, biphenyl, naphthyl; in some specific embodiments, R8 is selected from methyl, ethyl, isopropyl, isobutyl, 2-methylpropyl, isopentyl, phenyl, biphenyl; in some embodiments, R8 is selected from hydroxyl, amino, amino, and amino-substituted groups of the following: methyl, ethyl, propyl; in some embodiments, the amino substituent of R8 is methylamino or dimethylamino.

[0043] In some embodiments, R9 is hydrogen, a substituted or unsubstituted C1-C3 alkyl group, or a substituted or unsubstituted C1-C3 aminoalkyl group; the substituent is selected from groups Z, -CONH2, or -CH2CONH2.

[0044] In some embodiments, R9 is hydrogen; in some embodiments, R9 is an amino-substituted group of the following: methyl, ethyl, propyl, butyl; in some embodiments, R9 is an amino-substituted group of the following: methyl, ethyl, propyl, butyl, substituted with -CONH2 or -CH2CONH2; in some embodiments, R9 is an amino-substituted group of the following: methyl, ethyl, propyl, butyl, preferably methyl, substituted with group Z.

[0045] In some implementation schemes, R 10 For hydrogen; in some implementations, R 10For example, benzimidazole and indole; in some embodiments, R 10 It is an ethyl group substituted with L4.

[0046] In some implementation schemes, R 13 For -CH2CONH2; in some embodiments, R13 is adjacent to R 11 and R 12 Together with the atoms connected to it, they form 4-7 membered rings, which together form a fused ring structure.

[0047] In some implementation schemes, R 16 The group is selected from the following groups: hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkoxy, mono(C1-C6 alkyl)amino, bis(C1-C6 alkyl)amino, phenyl, halogen, trifluoromethyl; in some embodiments, R16 is the following group: hydrogen, hydroxyl, amino, C1-C3 alkyl, trifluoromethyl, C1-C3 alkoxy, mono(C1-C3 alkyl)amino, bis(C1-C3 alkyl)amino, phenyl, halogen.

[0048] In some implementations, segment L2 is: In some implementations, segment L2 is: In some implementations, segment L2 is: (in the formula) This indicates that it is attached to a carbonyl group. (Indicates that it is attached to an amino group);

[0049] In some embodiments, R 19 R 22 R 26 R 30 and R 31 Each is independently: hydrogen, or a C1-C3 alkyl group; in some embodiments, R 19 R 22 R 26 R 30 and R 31 Each is independently selected from: hydrogen, methyl.

[0050] In some implementations, segment L2 is At that time, R 17 R 18 R 20 and R 21 Each is independently selected from: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, when R 20 When it is hydrogen, R 18 and R 21 Not simultaneously n-butyl; in some implementations, R 17 With R 18The carbon chains link together to form 8-16 membered rings, with or without unsaturated bonds; in some embodiments, R 18 With R 20 The carbon chains link together to form 8-16 membered rings, with or without unsaturated bonds; in some embodiments, R 20 With R 21 They are linked by carbon chains to form 8-16 membered rings with or without unsaturated bonds.

[0051] In some implementations, in some embodiments, R 23 Selected from: hydrogen, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C1-C6 alkenyl groups; R 24 With R 25 The carbon chains form 8-16 membered rings with or without unsaturated bonds; the substituents are selected from H, carboxyl, hydroxyl, amino, -CONH2, phenyl, biphenyl, and naphthyl; in some embodiments, R 23 R 24 and R 25 Each is independently selected from: C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; in some embodiments, R 24 With R 25 They are linked by carbon chains to form 8-16 membered rings with or without unsaturated bonds.

[0052] In some implementation schemes, R 27 R 28 and R 29 Each is independently selected from: C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; in some embodiments, R 27 With R 28 The carbon chains form 8-16 membered rings, with or without unsaturated bonds; in some embodiments, R 29 Selected from: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl, R 27 With R 28 They are linked by carbon chains to form 8-16 membered rings with or without unsaturated bonds.

[0053] In some embodiments, group L4 is a C1-C6 alkoxycarbonyl group; in some embodiments, group L4 is a carboxyl, hydroxyl, or amino group; in some embodiments, group L4 is a dimethylamino or methylamino group; in some embodiments, group L4 is a phenyl, biphenyl, or naphthyl group.

[0054] In some embodiments, group L3 is a hydroxyl group; in other embodiments, group L3 is an amino group.

[0055] In some implementations, group Z is

[0056]

[0057] In some implementations, group Z is

[0058]

[0059] In some implementations, group Z is

[0060] 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;

[0061] 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;

[0062] In embodiments of this application, the C1-C6 hydrocarbon carbonyl group represents an ester bond formed between a carboxyl group and a C1-C6 hydrocarbon oxygen group, including straight-chain, branched-chain, or cyclic structures;

[0063] 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.

[0064] In the embodiments of this application, the unsaturated aliphatic hydrocarbon group with 1-6 carbon atoms includes straight-chain, branched 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;

[0065] 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.

[0066] 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.

[0067] In the embodiments of this application, the 8-16 membered rings containing or without unsaturated bonds refer to rings containing 8 to 16 atoms, wherein the 8-16 membered rings containing unsaturated bonds refer to rings containing at least one carbon-carbon double bond or carbon-carbon triple bond, or both.

[0068] In the embodiments of this application, R6 and R7, R 11 and R 12 R 14 and R 15 Each atom independently forms a 4-7 membered ring with the atoms it is connected to, wherein the R (n) and R (n+1) Together with the atoms attached to it, they form a 4-7 membered ring, which refers to R (n) and R (n+1) The ring, together with the nitrogen and carbon atoms attached thereto, forms a 4- to 7-membered ring, where n is 6, 11, or 14. This ring may be substituted by other substituents, or the ring and substituents may form a fused ring or a spiro ring. The ring may have one heteroatom (such as O, N, or S) or no heteroatom, in addition to the nitrogen atom. The substituents on the ring include, but are not limited to, the following groups: C1-C6 hydrocarbon groups, C6-C12 aryl groups, halogens, hydroxyl groups, amino groups, and C1-C6 hydrocarbon groups substituted with hydroxyl or amino groups.

[0069] 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.

[0070] 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.

[0071] In embodiments of this application, the aromatic ring structure of the 6-12 carbon atoms of the aryl group in the C6-C12 group includes, but is not limited to, phenyl, naphthyl, and biphenyl.

[0072] On the other hand, compounds of formula (I) or formula (II) or pharmaceutically acceptable salts, esters, stereoisomers, solvent compounds or prodrugs thereof are used to develop peptide inhibitors targeting PD-1 / PD-L1.

[0073] The use of the compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, solvent compound or prodrug compound thereof for the prevention and treatment of tumors.

[0074] The prodrugs described in formula (I) include, but are not limited to: X1 and / or X2 being nitrogen atoms, and one or both of R1 and R2 being hydrogen atoms, wherein the nitrogen atom is associated with -((CH2) n3 -O) n4 Phosphate prodrugs formed by the -P(O)(OH)2 phase, wherein n3 and n4 are independently 1, 2, 3, and 4.

[0075] The tumors include blood cancers, nervous system cancers, gastrointestinal cancers, esophageal cancers, urinary system cancers, lung cancer, liver cancer, and skin cancer; including but not limited to lymphoma, non-small cell lung cancer, small cell lung cancer, head and neck cell carcinoma, glioma, neuroblastoma, squamous cell carcinoma of the lung, adenocarcinoma of the lung, bladder cancer, stomach cancer, colon cancer, colorectal cancer, kidney cancer, bile duct cancer, stomach cancer, squamous cell carcinoma of the esophagus, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, brain cancer, melanoma, multiple myeloma, skin cancer, epithelial cell carcinoma, leukemia, or cervical cancer, as well as metastatic lesions in other tissues or organs far from the primary tumor site.

[0076] The tumor prevention or treatment drugs described in this invention are cancer immunotherapy drugs, cancer chemotherapy drugs, or cancer targeted therapy drugs.

[0077] A pharmaceutical composition comprising a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, solvent compound or prodrug thereof, and a pharmaceutically acceptable carrier.

[0078] The pharmaceutical composition is in the form of tablets, capsules, granules, powders, syrups, oral liquids, or injections.

[0079] The present invention unexpectedly discovers that compounds obtained by modifying the cyclic peptide structure in the prior art can effectively bind to PD-1 / PD-L1, thereby blocking or inhibiting the binding of PD-1 and PD-L1, blocking negative regulatory signals, restoring T cell activity, and thus enhancing the immune response to treat tumors. Attached Figure Description

[0080] Figure 1 This is a trend chart of subcutaneous tumor (lung cancer) in mice. Detailed Implementation

[0081] 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.

[0082] Example 1:

[0083]

[0084] Synthesis of compound 1c:

[0085] 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 1 N 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.

[0086] Synthesis of compound 1d:

[0087] Take 25 ml of 3 mol·L⁻¹ solution at 0 °C. -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, slurried with diethyl ether, and filtered to obtain 3.16 g of yellow solid.

[0088] 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].

[0089] Synthesis of compound 1e:

[0090] 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].

[0091] Synthesis of compound 1f:

[0092] Under nitrogen protection, 1 g of compound 1e and 30 mL of anhydrous acetonitrile were added to a 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 the ESI-MS (+) result was m / z 584.18 [M+H].

[0093] Synthesis of Compound 1:

[0094] 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 mixture 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].

[0095] Synthesis of compound 2:

[0096] 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].

[0097] Example 2:

[0098]

[0099] Synthesis of compound 2d:

[0100] 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 then added, and the mixture was reacted at room temperature for 1 hour. 14.42 g of compound 2b was added, and the mixture was heated to 80°C and refluxed overnight. After the reaction was complete, the mixture 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, and 13.67 g of potassium hydroxide were added sequentially to the mixture. The mixture was refluxed for 2 hours, cooled to room temperature, and the pH was adjusted with acetic acid. Crystallization was allowed to occur for 12 hours. The mixture 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.

[0101] Synthesis of compound 2e:

[0102] 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 the ESI-MS (+) result was m / z 353.17 [M+H].

[0103] Synthesis of compound 3:

[0104] 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].

[0105] Synthesis of compound 4:

[0106] 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].

[0107] Example 3:

[0108]

[0109] Synthesis of compound 3b:

[0110] 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].

[0111] Synthesis of compound 3c:

[0112] 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].

[0113] Synthesis of compound 3f:

[0114] 1.3 g of compound 3c and a mixed solution of 5 mL THF and 5 mL DMF were added to a reaction flask, followed by 477 mg of N-hydroxysuccinimide. The system was cooled to 0 °C. 1.1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was slowly added. The reaction was carried out at 25 °C for 24 h. The mixture was extracted with ethyl acetate and washed successively 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.

[0115] 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].

[0116] Synthesis of compound 5:

[0117] 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].

[0118] Synthesis of compound 6:

[0119] 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].

[0120] Example 4:

[0121]

[0122] Synthesis of compound 4b:

[0123] 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].

[0124] Synthesis of compound 4c:

[0125] 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].

[0126] Synthesis of compound 4d:

[0127] 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].

[0128] Synthesis of compound 7:

[0129] 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. The system was maintained at 0 °C for 1 hour, then heated 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, yield 92%, ESI-MS (+): m / z 394.2 [M+H].

[0130] Example 5:

[0131]

[0132] Synthesis of compound 5b:

[0133] 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].

[0134] Synthesis of compound 5c:

[0135] 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 solution of compound 5b (3 g) in dichloromethane (25 mL) 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].

[0136] Synthesis of compound 5d:

[0137] 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.

[0138] Synthesis of compound 5e:

[0139] 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].

[0140] Synthesis of compound 8:

[0141] 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, the pH of the aqueous phase was adjusted to 1–2, and the mixture was extracted with ethyl acetate. After washing the organic phase, the mixture was concentrated to dryness and purified 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].

[0142] Example 6:

[0143]

[0144] Synthesis of compound 6b:

[0145] 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 reaction was carried out to completion at room temperature. The system was concentrated to dryness, extracted with ethyl acetate and water, and the organic phase was concentrated to dryness. Purification by silica gel column chromatography yielded 4.64 g of compound 6b, 74% yield, ESI-MS (+): m / z 567.3 [M+H]. Synthesis of compounds 6c / 6d:

[0146] 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]).

[0147] Synthesis of compound 9:

[0148] 0.5 g of compound 6c, 10 ml of dichloromethane, and 6 ml of trifluoroacetic acid were added to a reaction flask, followed by 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.

[0149] 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, the pH of the aqueous phase was adjusted to 1-2, and the mixture was extracted with ethyl acetate. After washing the organic phase, the mixture was 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].

[0150] Example 7:

[0151]

[0152] Synthesis of compound 7c:

[0153] 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. The system was stirred at low temperature for 1 hour. 15 mL of n-hexane (2.75 g) in tetrahydrofuran solution 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].

[0154] Synthesis of compound 7e:

[0155] 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].

[0156] Synthesis of compound 7f:

[0157] 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].

[0158] Synthesis of 7g of compound:

[0159] 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 obtain 1.33 g of compound 7f, yield 96%, ESI-MS (+): m / z 188.16 [M+H];

[0160] Synthesis of compound 10:

[0161] 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].

[0162] Example 8:

[0163]

[0164] Synthesis of compound 11:

[0165] 2g of compound 8a, 4ml of 37% formaldehyde aqueous solution, and 30ml of methanol were added to the reaction flask. 0.6g of 10% palladium on carbon was added to the system, and hydrogenation was carried out at room temperature and atmospheric pressure for 24 hours. After the reaction was completed, the system was filtered with diatomaceous earth as an aid.

[0166] The system was concentrated to dryness under reduced pressure, and 15 ml of methanol and 10 ml of trifluoroacetic acid were added. The system was stirred at room temperature for 3 hours. After the reaction was completed, the system was concentrated to dryness and purified by silica gel column chromatography to give 1.06 g of compound 11, with a two-step yield of 81%. ESI-MS (+): m / z 133.05 [M+H];

[0167] Example 9:

[0168]

[0169] Synthesis of compound 9b:

[0170] 2 g of compound 9a, 2.48 g of diethyl acetaminomalonate, 90 mg of sodium hydroxide, and 30 ml of xylene were added to a reaction flask. The system was refluxed for 10 hours. The system was hot-filtered, and the solid was washed with methyl tert-butyl ether. The organic phases were combined, concentrated to dryness, and purified by silica gel column chromatography to give 3.33 g of compound 9b, yield 83%, ESI-MS (+): m / z 348.15 [M+H]; Synthesis of compound 12:

[0171] 3g of compound 9b and 20ml of concentrated hydrochloric acid were added to a reaction flask. The system was heated and stirred for 10 hours, then concentrated to dryness. 10ml of water was added, and the pH of the system was adjusted to approximately 8 with concentrated ammonia. Approximately 15ml of acetone was added to the system, and the system was cooled to 0℃ to crystallize. After filtration, 1.54g of compound 12 was obtained, with a two-step yield of 87%. ESI-MS (+): m / z 206.10 [M+H];

[0172] Example 10:

[0173]

[0174] Synthesis of compound 10b:

[0175] Under nitrogen protection, 690 mg of compound 10a, 295 mg of phenylboronic acid, 76 mg of PdCl2 (dppf), 1.38 g of sodium bicarbonate, and 20 mL of isopropanol aqueous solution (1:20) were added to the reaction flask. The system was reacted at 80 °C for 3 hours. After the reaction, the system was cooled to room temperature, and 5 N hydrochloric acid was added to adjust the pH to 3. The system was extracted four times with dichloromethane. The organic phases were combined, concentrated to dryness, and purified by silica gel column chromatography to give 560 mg of compound 10b, yield 82%, ESI-MS (+): m / z 342.19 [M+H].

[0176] Synthesis of compound 13:

[0177] 550 mg of compound 10b, 10 mL of dichloromethane, and 3 mL of trifluoroacetic acid were added to a reaction flask, and the mixture was stirred at room temperature for 3 hours. The mixture was then concentrated to dryness to give 380 mg of compound 10c, which was used in the next reaction without further purification.

[0178] 380 mg of compound 4d, 6 mL of 10% sodium carbonate, and 6 mL of acetone were added to a reaction flask. The system was cooled to 0 °C, and 490 mg of fluorenemethyloxycarbonyl chloride was added. The system was maintained at 0 °C for 1 hour, then heated 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 657 mg of compound 13, 90% yield, ESI-MS (+): m / z 464.22 [M+H].

[0179] Example 11:

[0180]

[0181] Fmoc-Gly-OH was uploaded to Rink Amide-AM Resin:

[0182] 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.

[0183] Remove Fmoc protection:

[0184] 20% Pip (piperidine) / DMF solution for Fmoc protection removal, followed by DMF washing of the resin;

[0185] Fmoc-N-Me-Cys(Mmt)-OH is coupled to the peptide resin:

[0186] A DMF solution of Fmoc-N-Me-Cys(Mmt)-OH is added and coupled to the resin under the action of condensing agents HBTU / DIEA, DIC / HOBt, PyBOP, or other similar condensing agents, and then the resin is washed.

[0187] Amino acid coupling:

[0188] 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.

[0189] The coupling and deprotection cycles are performed sequentially until the peptide resin is coupled (for coupling, refer to the above "Fmoc-N-Me-Cys(Mmt)-OH coupling to peptide resin", and for deprotection, refer to the above "deprotection of Fmoc").

[0190] Remove the Mmt group from Cys on the peptide chain:

[0191] Add the resin to a 1% TFA (trifluoroacetic acid) DCM solution and wash the resin.

[0192] Cyclic peptides are formed by cyclizing linear peptide chains in a solid-phase resin:

[0193] The resin was swollen in DCM, and the linear peptide was cyclized under the action of tris(2-carboxyethyl)phosphine (TCEP) and diisopropylethylamine (DIEA).

[0194] The resin was cleaved and all protecting groups on the cyclic peptide were removed to obtain the product:

[0195] 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.

[0196] Chromatographic purification:

[0197] 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. DSC2301, ESI-MS(+): m / z 1896.76 [M+H], m / z 949.33 [M+2H], m / z 633.06 [M+3H].

[0198] Example 12:

[0199]

[0200] Fmoc-Gly-OH was uploaded to Rink Amide-AM Resin:

[0201] 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.

[0202] Remove Fmoc protection:

[0203] 20% Pip (piperidine) / DMF solution for Fmoc protection removal, followed by DMF washing of the resin;

[0204] Fmoc-N-Me-Cys(Trt)-OH is coupled to the peptide resin:

[0205] 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.

[0206] Amino acid coupling:

[0207] According to the amino acid sequence of the molecular structure of this invention, Fmoc-2-butyl-3-(methylamino)heptanoic acid, Fmoc-2-aminopentanoic acid, Fmoc-1-(2-(tert-butoxy)-2-oxoethyl)-Trp-OH, Fmoc-Dab(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Dap(Dde)-OH, Fmoc-D-Hyp(tBu)-OH, Fmoc-D-Asn(Trt)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Tyr(tbu)-OH, and chloroacetic acid are added. 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).

[0208] Remove the Dde group from the Dab of the peptide chain:

[0209] The Dde group on Dab was removed using a 3% hydrazine hydrate / N,N-dimethylformamide solution.

[0210] Attach the protected long fatty side chain to the peptide chain:

[0211] The protected long fatty side chain (see formula below) is coupled to the peptide chain under the action of the condensing agent TBTU / DIEA.

[0212]

[0213] Remove the Mmt group from Cys on the peptide chain:

[0214] Add the resin to a 1% TFA (trifluoroacetic acid) DCM solution and wash the resin.

[0215] Cyclic peptides are formed by cyclizing linear peptide chains in a solid-phase resin:

[0216] The resin was swollen in DCM, and the linear peptide was cyclized under the action of tris(2-carboxyethyl)phosphine (TCEP) and diisopropylethylamine (DIEA).

[0217] The resin was cleaved and all protecting groups on the cyclic peptide and side chains were removed to obtain the product:

[0218] 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.

[0219] Chromatographic purification:

[0220] 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.

[0221] 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.

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] Example 13

[0232] ELISA detection of binding to human PD-L1

[0233] 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, then 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.

[0234]

[0235]

[0236] Example 14

[0237] Mouse subcutaneous tumor-bearing (lung cancer) experiment

[0238] Thirty Balb / c mice were subcutaneously inoculated with mouse LLC cells (mouse Lewis lung 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.

[0239] The results are as follows Figure 1 As shown in the trend graph of the subcutaneous tumor-bearing experiment in mice, the compound of this invention can significantly inhibit the growth of Lewis lung cancer xenografts. Simultaneously, during the experiment, the body weight of mice in the invention group remained essentially unchanged compared to the blank control group, demonstrating that the compound administration did not cause any change in body weight.

[0240] Example 15: Animal Model Experiment of Sepsis

[0241] 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 penetration through the entire 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. Group 1 was a blank control group (administered with water for injection twice daily), 3 groups were experimental drug groups (DSC2312 and DSC2336 groups were administered 700 mg / kg twice daily, and DSC2345 group was administered 1 g / kg once every two days), and 1 group was an antibiotic (ceftazidime) 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% (5 / 8) in the DSC2312 group, 42.9% (3 / 7) in the DSC2336 group, 57.1% (4 / 7) in the DSC2345 group, and 75% (6 / 8) in the antibiotic (ceftazidime) 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 is understood that it exerts its unique pharmacological effect through the immune system.

[0242] Example 16 Chronic HBV Mouse Model Experiment

[0243] Several 4-6 week old Balb / c mice were selected. 20 μg of the 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 using real-time quantitative PCR. When a stable plateau of approximately 10⁴-10⁵ copies / ml was reached, grouping and drug administration began. The experimental mice were randomly divided into four groups, with at least six mice in each group: one group was the blank control group (administered with water for injection once daily); two groups were the experimental drug groups (DSC2304 group administered 0.5 mg / kg once daily, DSC2343 group administered 0.75 mg / kg every two days); and one group was 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 blank control group still had a level of 10... 4 ~10 5copies / ml, DSC2304 and DSC2343 groups were 10 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 on chronic HBV, possibly exerting their unique pharmacological effects by regulating the immune system through the inhibition of PD-1 and PD-L1.

Claims

1. Cyclic polypeptide compounds and their pharmaceutically acceptable salts, selected from one of the following compounds: 。 2. A pharmaceutical composition comprising the cyclic polypeptide compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. The use of the cyclic polypeptide compound of claim 1 and its pharmaceutically acceptable salt or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating lung cancer.

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