Dithiocyclopeptide compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a disulfide cyclic peptide compound of a melanocortin receptor agonist based on polypeptide small molecules, and the disulfide cyclic peptide compound has the following structure: R2-R1-c (Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-R3-R4, wherein R2 and R4 are respectively and independently selected from an amino group, an acetyl group or a palmitoyl group; r1 and R3 are independently selected from Ile, Arg, D-Phe, Pro or deletion, R1 and R3 are not deleted at the same time, and when R1 or R3 is deleted, at least one of R2 and R4 is a palmitoyl group. The disulfide cyclopeptide compound provided by the invention has relatively good selectivity on melanocortin receptors (MCR1, MCR3, MCR4 and MCR5).

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a disulfide cyclic peptide compound and a preparation method and application thereof. Background Art

[0002] The melanocortin system includes melanocortin and its receptor (melanocortin receptor; MCR), neuropeptide Y (NPY), and endogenous melanocortin antagonists agouti and agouti related protein (AgRP); melanocortin is a large class of polypeptides that regulate different functions, including α-, β-, γ-melanocyte stimulating hormones (MSH), adrenocorticotropin (ACTH). They are all derived from the tissue-specific post-translational processing of pro-opiomelanocortin (POMC). The melanocortin receptor system includes five MCRs (MC1R~MC5R) and two receptor auxiliary proteins MRAP (melanocortin receptor associated protein). In addition to MC2R specifically binding to ACTH, MC1R, MC3R, MC4R, and MC5R can bind to all melanocortins, but their affinity varies between different receptors.

[0003] The function of MCR receptors is related to the specific expression of MCR in tissues. MC2R is mainly located in the adrenal cortex and is a key component of the hypothalamus-pituitary-adrenal axis, stimulating the biosynthesis of glucocorticoids; the melanocortin receptors related to energy metabolism regulation are MC3R and MC4R; MC4R is considered to be a key factor in regulating individual energy metabolism because its loss of function shows an obesity-like disease phenotype in both mice and humans. In addition to MC4R, MC3R is also widely distributed in the brain and is mainly expressed in the hypothalamus, mainly the arcuate nucleus and ventromedial hypothalamus, which are involved in regulating energy homeostasis, metabolism and appetite. Many studies have shown that MC3R and MC4R can function independently and play complementary rather than redundant roles in controlling energy balance. MC5R is widely expressed in peripheral organs and tissues. MC5R seems to play a key role in immune and inflammatory responses and is essential for temperature control and exocrine function.

[0004] Currently, a variety of melanocortin peptide analogs have been developed as potential drugs for the treatment of skin diseases, obesity, anorexia and type 2 diabetes. Setmelanotide is a cyclic high-affinity peptide. Compared with natural α-MSH, its G protein signaling spectrum is biased towards Gq / 11 (phospholipase C activation) and its receptor subtype selectivity for MC4R is 20 times. As a targeted therapy, setmelanotide restores the function of the damaged MC4R pathway, re-establishes energy consumption and appetite control in patients with rare genetic diseases of obesity, reduces hunger and reduces weight. However, setmelanotide also has certain side effects, including injection site reactions, skin pigmentation (skin patches that are darker than the surrounding skin), headaches and gastrointestinal side effects (such as nausea, diarrhea and abdominal pain). Adverse reactions of the reproductive system also occur during treatment. Depression and suicidal thoughts have also occurred during the use of setmelanotide.

[0005] Therefore, there is still a need to develop a melanocortin receptor ligand with better selectivity, stronger stability and fewer side effects for melanocortin receptors (MC1R-MC5R). Summary of the invention

[0006] The purpose of the present invention is to provide a disulfide cyclic peptide compound and a preparation method and application thereof.

[0007] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0008] In a first aspect of an embodiment of the present invention, a disulfide cyclic peptide compound is provided, wherein the disulfide cyclic peptide compound has the following structure: R2-R1-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-R3-R4; wherein R2 and R4 are each independently selected from an amino group, an acetyl group or a palmitoyl group; R1 and R3 are each independently selected from Ile, Arg, D-Phe, Pro or are missing, R1 and R3 are not missing at the same time, and when R1 or R3 is missing, at least one of R2 and R4 is a palmitoyl group.

[0009] In one aspect of the present invention, the disulfide cyclic peptide compound has the following structural formula:

[0010]

[0011] Wherein, R1, R2, R3 and R4 are as defined above.

[0012] In one aspect of the invention, R3 is selected from Ile, D-Phe, Pro or deletion, and R1 is Arg.

[0013] In one aspect of the present invention, R2 is selected from acetyl or palmitoyl, and R4 is amino.

[0014] In one aspect of the invention, when R3 is absent, R4 is a palmitoyl group.

[0015] In one aspect of the present invention, the disulfide cyclic peptide compound is selected from at least one of the following compounds:

[0016]

[0017]

[0018] In one aspect of the present invention, the present invention provides a disulfide cyclic peptide compound, wherein the disulfide cyclic peptide compound is compound WP302-1.

[0019] Specifically, compound WP302-1 has the following structure:

[0020]

[0021] Specifically, compound WP302-1 is Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-D-Phe-NH2.

[0022] Specifically, the compound WP302-1 has an amino acid sequence as shown in SEQ ID NO.1.

[0023] Specifically, the molecular formula of compound WP302-1 is C 58 H 77 O 10 N 19 S2.

[0024] In one aspect of the present invention, the present invention provides a disulfide cyclic peptide compound, wherein the disulfide cyclic peptide compound is compound WP302-2.

[0025] Specifically, compound WP302-2 has the following structure:

[0026]

[0027] Specifically, compound WP302-2 is Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-Pro-NH2.

[0028] Specifically, the compound WP302-2 has an amino acid sequence as shown in SEQ ID NO.2.

[0029] Specifically, the molecular formula of compound WP302-2 is C 54 H 75 O 10 N19 S2.

[0030] In one aspect of the present invention, the present invention provides a disulfide cyclic peptide compound, wherein the disulfide cyclic peptide compound is compound WP302-3.

[0031] Specifically, compound WP302-3 has the following structure:

[0032]

[0033] Specifically, compound WP302-3 is Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-Ile-NH2.

[0034] Specifically, the compound WP302-3 has an amino acid sequence as shown in SEQ ID NO.3.

[0035] Specifically, the molecular formula of compound WP302-3 is C 55 H 79 O 10 N 19 S2.

[0036] In one aspect of the present invention, the present invention provides a disulfide cyclic peptide compound, wherein the disulfide cyclic peptide compound is compound WP302-4.

[0037]

[0038] Specifically, compound WP302-4 is Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2.

[0039] Specifically, the compound WP302-4 has an amino acid sequence as shown in SEQ ID NO.4.

[0040] Specifically, the molecular formula of compound WP302-4 is C 63 H 96 N 18 O9S2.

[0041] In one aspect of the present invention, the present invention provides a disulfide cyclic peptide compound, wherein the disulfide cyclic peptide compound is compound WP302-5.

[0042] Specifically, compound WP302-5 has the following structure:

[0043]

[0044] Specifically, compound WP302-5 is Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-D-Phe-NH2.

[0045] Specifically, the compound WP302-5 has an amino acid sequence as shown in SEQ ID NO.5.

[0046] Specifically, the molecular formula of compound WP302-5 is C 72 H 105 N 19 O 10 S2.

[0047] In one aspect of the present invention, the present invention provides a disulfide cyclic peptide compound, wherein the disulfide cyclic peptide compound is compound WP302-6.

[0048] Specifically, compound WP302-6 has the following structure:

[0049]

[0050] Specifically, compound WP302-6 is Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-Pro-NH2.

[0051] Specifically, the compound WP302-6 has an amino acid sequence as shown in SEQ ID NO.6.

[0052] Specifically, the molecular formula of compound WP302-6 is C 68 H 103 N 19 O 10 S2.

[0053] In one aspect of the present invention, the present invention provides a disulfide cyclic peptide compound, wherein the disulfide cyclic peptide compound is compound WP302-7.

[0054] Specifically, compound WP302-7 has the following structure:

[0055]

[0056] Specifically, compound WP302-7 is Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-Ile-NH2.

[0057] Specifically, the compound WP302-7 has an amino acid sequence as shown in SEQ ID NO.7.

[0058] Specifically, the molecular formula of compound WP302-7 is C 69 H 107 N 19 O 10 S2.

[0059] In a second aspect of the embodiments of the present invention, a method for preparing the aforementioned disulfide cyclic peptide compound is provided, the method comprising the following steps:

[0060] Step 1: Select a resin and remove the Fmoc protecting group to obtain a resin from which the Fmoc protecting group has been removed;

[0061] Step 2: Weigh Fmoc-R3-OH or Fmoc-Cys(Trt)-OH and PyBop, and perform coupling reaction on the resin with the Fmoc protecting group removed to obtain a coupling resin;

[0062] Step 3: According to the peptide sequence, for the coupling resin, coupling is performed from the C-terminus to the N-terminus to obtain a linear peptide resin;

[0063] Step 4: reacting the cleavage solution with the linear peptide resin to remove the protecting group and obtain a polypeptide;

[0064] Step 5: dissolving the polypeptide in water, adjusting the pH and adding H2O2 for reaction to obtain the disulfide cyclic peptide compound.

[0065] In a third aspect of the embodiments of the present invention, there is provided a use of the aforementioned disulfide cyclic peptide compound or the disulfide cyclic peptide compound prepared by the aforementioned method in preparing a drug.

[0066] In one aspect of the invention, the medicament includes a medicament for treating or preventing acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection, metabolic diseases associated with weight gain, metabolic diseases associated with weight loss, diabetes, diabetic complications, cancer, cancerous hyperplasia, reproductive system diseases, peripheral or central nervous system diseases, cardiovascular diseases or respiratory system diseases.

[0067] Specifically, the acute or chronic inflammatory diseases include, but are not limited to, systemic inflammation, inflammatory bowel disease, cerebral inflammation, sepsis, and septic shock.

[0068] Specifically, the autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, multiple sclerosis, psoriasis, pemphigus, vitiligo, narcolepsy, neuromyelitis optica, hyperthyroidism, hypothyroidism, autoimmune gastritis, autoimmune hepatitis, primary biliary cholangitis, Crohn's disease, ulcerative colitis, celiac disease, lupus nephritis, Goodpasture's syndrome, autoimmune oophoritis, autoimmune orchitis, polymyositis, vasculitis or diffuse connective tissue diseases such as Sjögren's disease.

[0069] Specifically, the organs causing transplant rejection include, but are not limited to, kidney, heart, liver, pancreas and pancreatic islets, parathyroid glands, heart and lungs, bone marrow, and cornea.

[0070] Specifically, the metabolic diseases associated with weight gain include, but are not limited to, obesity, eating disorders, and Prader-Willi syndrome.

[0071] Specifically, the metabolic disease accompanied by weight loss includes, but is not limited to, anorexia or bulimia.

[0072] Specifically, the diabetic complications include, but are not limited to, diabetic nephropathy, diabetic retinopathy, diabetic cataracts, diabetic foot, cardiovascular complications of diabetes, diabetic cerebrovascular disease, and diabetic neuropathy.

[0073] Specifically, the cancer includes, but is not limited to, medullary tumors, neuroblastoma, rectal cancer, colon cancer, familiar adenomatous polyposis and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urinary cancer , melanoma, brain tumor, lymphoma, head and neck cancer, acute lymphoid leukemia, chronic lymphoid leukemia, acute myeloid leukemia, chronic myeloid leukemia, hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal sarcoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma.

[0074] Specifically, the cancerous hyperplasia includes but is not limited to squamous epithelial dysplasia and glandular epithelial dysplasia.

[0075] Specifically, the reproductive system diseases include, but are not limited to, abnormal urination, pyuria, abnormal urethral discharge, pain, lumps, sexual dysfunction, male infertility, female infertility, polycystic ovary syndrome, menstrual disorders, dysmenorrhea, abnormal pregnancy, and uterine lesions.

[0076] Specifically, the peripheral or central nervous system diseases include, but are not limited to, depression, bipolar depression or manic depression, acute and chronic anxiety states, schizophrenia, Alzheimer's disease, vascular dementia, Parkinson's disease, acute and chronic multiple sclerosis or acute and chronic pain and brain damage caused by stroke, hypoxia or craniocerebral trauma.

[0077] Specifically, the cardiovascular diseases include but are not limited to coronary heart disease, stroke, ischemic stroke, cerebral hemorrhage, subarachnoid hemorrhage, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, abnormal heart rhythm, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolic disease, and venous thrombosis.

[0078] Specifically, the respiratory diseases include but are not limited to upper respiratory tract infection, acute bronchitis, acute pharyngitis, pneumonia, chronic bronchitis, chronic obstructive pulmonary disease, tuberculosis, lung tumors, bronchiectasis, lung abscess, pulmonary interstitial fibrosis, pulmonary embolism, acute respiratory distress syndrome, and cor pulmonale.

[0079] In one aspect of the present invention, the use is achieved by binding the disulfide cyclic peptide compound to a melanocortin receptor.

[0080] In one aspect of the present invention, the melanocortin receptor includes at least one of MC1R, MC3R, MC4R and MC5R.

[0081] In the fourth aspect of the embodiments of the present invention, a pharmaceutical composition is provided, which includes the aforementioned disulfide cyclic peptide compound or its analogs, wherein the analogs include derivatives of the disulfide cyclic peptide compound, pharmaceutically acceptable salts thereof, tautomers thereof or stereoisomers thereof.

[0082] In one aspect of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0083] In one aspect of the present invention, pharmaceutically acceptable carriers or excipients refer to any preparation or carrier medium representative of carriers that can deliver an effective amount of active substance of the present invention, do not interfere with the biological activity of the active substance, and have no toxic side effects on the host or subject, including water, oil, vegetables and minerals, cream base, lotion base, ointment base, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc.

[0084] The beneficial effects of the present invention are:

[0085] The present invention prepares a disulfide cyclic peptide compound for the first time, and the disulfide cyclic peptide compound has the following structure: R2-R1-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-R3-R4. The disulfide cyclic peptide compound provided by the present invention can be well combined with the melanocortin receptor as a ligand of one or more melanocortin receptors to produce a good effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is the MS spectrum of compound WP302-1 of Example 1;

[0087] Figure 2 is the HPLC spectrum of the compound WP302-1 of Example 1;

[0088] Figure 3 is the MS spectrum of compound WP302-2 of Example 2;

[0089] Figure 4 is the HPLC spectrum of the compound WP302-2 of Example 2;

[0090] Figure 5 is the MS spectrum of compound WP302-3 of Example 3;

[0091] Figure 6 is the HPLC spectrum of the compound WP302-3 of Example 3;

[0092] Figure 7 is the MS spectrum of compound WP302-4 of Example 4;

[0093] Figure 8 is the HPLC spectrum of the compound WP302-4 of Example 4;

[0094] Fig. 9 is the MS spectrum of compound WP302-5 of Example 5;

[0095] Fig.10 is the HPLC spectrum of the compound WP302-5 of Example 5;

[0096] Fig.11 is the MS spectrum of compound WP302-6 of Example 6;

[0097] Fig.12 is the HPLC spectrum of the compound WP302-6 of Example 6;

[0098] Fig.13 is the MS spectrum of compound WP302-7 of Example 7;

[0099] Fig.14is the HPLC spectrum of the compound WP302-7 of Example 7;

[0100] Fig.15 HPLC spectrum of compound WP300 of Comparative Example 1;

[0101] Fig.16 This is the MS spectrum of compound WP300 of Comparative Example 1. DETAILED DESCRIPTION

[0102] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present application.

[0103] Terms and Abbreviations:

[0104]

[0105]

[0106]

[0107] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0108] Example 1: Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-D-Phe-NH2 (SEQ ID NO.1)

[0109] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 100 mL reactor.

[0110] Select 1g of Ramage Amide AM resin with a loading of 0.30mmol / g, remove the Fmoc protecting group after swelling; weigh 0.35g of Fmoc-D-Phe-OH, 468mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.34mL of DIEA, dissolve in 25mL of DMF at 0°C, add to the reaction column and react at room temperature for 1 hour after complete dissolution, and use the ninhydrin detection method to detect negative to judge the reaction progress. After the coupling reaction is completed, drain the reaction solution, wash with 30mL of DMF, 40mL of 20% hexahydropyridine DMF solution, react for 5min, wash once with DMF, add 30mL of 20% hexahydropyridine DMF solution again after washing, react for 10min, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF.

[0111] According to the peptide sequence, coupling was performed from C-terminus to N-terminus until Fmoc-Arg(Pbf)-OH was removed and acetic anhydride and pyridine were used to block the amino group to form acetylation, wherein the side chain protecting groups of Arg, Cys, His, and Trp were Pbf, Trt, Trt, and Boc respectively; all amino acids were protected with Fmoc at the α-position amino group;

[0112] By reacting the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin, about 386.2 mg of the polypeptide with all side chain protecting groups removed was obtained;

[0113] The polypeptide was dissolved in 400 mL of pure water, the pH was adjusted to 8.0, 1 mL of H2O2 was added, and the reaction was carried out at room temperature. After 30 minutes of reaction, the polypeptide was completely converted into a cyclic polypeptide by monitoring by analytical HPLC;

[0114] The crude linear peptide aqueous solution was filtered with a 0.45 μm filter membrane, and the improved crude peptide was purified using a high-performance liquid chromatography instrument: through a DAC-HB50 dynamic axial compression column, the mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and B was a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution separation and purification was performed, and the sample was detected by an ultraviolet detector, and the peptide solution of the target peak was collected in sections. After high-performance liquid purification, 80 mL of finished peptide liquid with a purity greater than 95% was obtained. The obtained liquid was balanced with 1 / 1000 acetic acid and acetonitrile, and 30 mL of liquid was obtained after rotary evaporation concentration, and then the liquid was pre-lyophilized and freeze-dried, and finally 45.7 mg of fine peptide was obtained, namely compound WP302-1.

[0115] The structure of compound WP302-1 is shown below:

[0116]

[0117] The MS spectrum and HPLC mass spectrum of compound WP302-1 are shown as follows: Figure 1 and Figure 2 shown.

[0118] Example 2: Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-Pro-NH2 (SEQ ID NO. 2)

[0119] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 100 mL reactor.

[0120] Select 1g of Ramage Amide AM resin with a loading of 0.30mmol / g, remove the Fmoc protecting group after swelling; weigh 0.53g of Fmoc-Cys(Trt)-OH, 468mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.34mL of DIEA, dissolve in 25mL DMF at 0°C, add to the reaction column and react at room temperature for 1 hour after complete dissolution, and use the ninhydrin detection method to detect negative to judge the reaction progress. After the coupling reaction is completed, drain the reaction solution, wash with 30mL DMF, 40mL of 20% hexahydropyridine DMF solution, react for 5min, wash once with DMF, add 30mL of 20% hexahydropyridine DMF solution again after washing, react for 10min, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF.

[0121] According to the peptide sequence, coupling was performed from C-terminus to N-terminus until Fmoc-Arg(Pbf)-OH was removed and acetic anhydride and pyridine were used to block the amino group to form acetylation, wherein the side chain protecting groups of Arg, Cys, His, and Trp were Pbf, Trt, Trt, and Boc respectively; all amino acids were protected with Fmoc at the α-position amino group;

[0122] By reacting the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin, about 368.9 mg of the polypeptide with all side chain protecting groups removed was obtained;

[0123] The polypeptide was dissolved in 400 mL of pure water, the pH was adjusted to 8.0, 1 mL of H2O2 was added, and the reaction was carried out at room temperature. After 30 min of reaction, the polypeptide was completely converted into a cyclic polypeptide by monitoring by analytical HPLC;

[0124] The crude linear peptide aqueous solution was filtered with a 0.45 μm filter membrane, and the improved crude peptide was purified using a high-performance liquid chromatography instrument: through a DAC-HB50 dynamic axial compression column, the mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and B was a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution separation and purification was performed, and the sample was detected by an ultraviolet detector, and the peptide solution of the target peak was collected in sections. After high-performance liquid purification, 80 mL of finished peptide liquid with a purity greater than 95% was obtained. The obtained liquid was balanced with 1 / 1000 acetic acid and acetonitrile, and 30 mL of liquid was obtained after rotary evaporation concentration, and then the liquid was pre-lyophilized and freeze-dried to finally obtain 42.5 mg of fine peptide, namely compound WP302-2.

[0125] The structure of compound WP302-2 is shown below:

[0126]

[0127] The MS spectrum and HPLC mass spectrum of compound WP302-2 are shown as follows: Figure 3 and Figure 4 shown.

[0128] Example 3: Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-Ile-NH2 (SEQ ID NO.3)

[0129] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 100 mL reactor.

[0130] Select 1g of Ramage Amide AM resin with a loading of 0.30mmol / g, remove the Fmoc protecting group after swelling; weigh 0.32g of Fmoc-Ile-OH, 468mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.34mL of DIEA, dissolve in 250mL of DMF at 0°C, add to the reaction column and react at room temperature for 1 hour after complete dissolution, and use the ninhydrin detection method to detect negative to judge the reaction progress. After the coupling reaction is completed, drain the reaction liquid, wash with 30mL of DMF, 40mL of 20% hexahydropyridine DMF solution, react for 5min, wash once with DMF, add 30mL of 20% hexahydropyridine DMF solution again after washing, react for 10min, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF.

[0131] According to the peptide sequence, coupling was performed from C-terminus to N-terminus until Fmoc-Arg(Pbf)-OH was removed and acetic anhydride and pyridine were used to block the amino group to form acetylation, wherein the side chain protecting groups of Arg, Cys, His, and Trp were Pbf, Trt, Trt, and Boc respectively; all amino acids were protected with Fmoc at the α-position amino group;

[0132] By reacting the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin, about 396.3 mg of the polypeptide with all side chain protecting groups removed was obtained;

[0133] The polypeptide was dissolved in 400 mL of pure water, the pH was adjusted to 8.0, 1 mL of H2O2 was added, and the reaction was carried out at room temperature. After 30 min of reaction, the polypeptide was completely converted into a cyclic polypeptide by monitoring by analytical HPLC;

[0134] The crude linear peptide aqueous solution was filtered with a 0.45 μm filter membrane, and the improved crude peptide was purified using a high-performance liquid chromatography instrument: through a DAC-HB50 dynamic axial compression column, the mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and B was a 0.05% trifluoroacetic acid acetonitrile solution, and gradient elution separation and purification were performed. The sample was detected by an ultraviolet detector, and the peptide solution of the target peak was collected in sections. After high-performance liquid purification, 75 mL of a finished peptide liquid with a purity greater than 95% was obtained. The obtained liquid was balanced with 1 / 1000 acetic acid and acetonitrile, and 30 mL of liquid was obtained after rotary evaporation concentration, and then the liquid was pre-lyophilized and freeze-dried to finally obtain 35.6 mg of a fine peptide, namely compound WP302-3.

[0135] The structure of compound WP302-3 is shown below:

[0136]

[0137] The MS spectrum and HPLC mass spectrum of compound WP302-3 are shown as follows: Figure 5 and Figure 6 shown.

[0138] Example 4: Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2 (SEQ ID NO.4)

[0139] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 100 mL reactor.

[0140] Select 1g of Ramage Amide AM resin with a loading of 0.30mmol / g, remove the Fmoc protecting group after swelling; weigh 0.53g of Fmoc-Cys(Trt)-OH, 468mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.34mL of DIEA, dissolve in 25mL DMF at 0°C, add to the reaction column and react at room temperature for 1 hour after complete dissolution, and use the ninhydrin detection method to detect negative to judge the reaction progress. After the coupling reaction is completed, drain the reaction solution, wash with 30mL DMF, 40mL of 20% hexahydropyridine DMF solution, react for 5min, wash once with DMF, add 30mL of 20% hexahydropyridine DMF solution again after washing, react for 10min, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF.

[0141] According to the peptide sequence, coupling was performed from C-terminus to N-terminus until Fmoc-Arg(Pbf)-OH was obtained, and Fmoc was removed and palmitic acid was used to react with the α-amino group of Arg to form an amide bond, wherein the side chain protecting groups of Arg, Cys, His, and Trp were Pbf, Trt, Trt, and Boc, respectively; the α-amino groups of all amino acids were protected with Fmoc;

[0142] By reacting the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin, about 4.5.2 mg of the polypeptide with all side chain protecting groups removed was obtained;

[0143] The polypeptide was dissolved in 450 mL of pure water, the pH was adjusted to 8.0, 1 mL of H2O2 was added, and the reaction was carried out at room temperature. After 30 min of reaction, the polypeptide was completely converted into a cyclic polypeptide by monitoring by analytical HPLC;

[0144] The crude linear peptide aqueous solution was filtered with a 0.45 μm filter membrane, and the improved crude peptide was purified using a high-performance liquid chromatography instrument: through a DAC-HB50 dynamic axial compression column, the mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and B was a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution separation and purification was performed, and the sample was detected by an ultraviolet detector, and the peptide solution of the target peak was collected in sections. After high-performance liquid purification, 120 mL of finished peptide liquid with a purity greater than 95% was obtained. The obtained liquid was balanced with 1 / 1000 acetic acid and acetonitrile, and 50 mL of liquid was obtained after rotary evaporation concentration, and then the liquid was pre-lyophilized and freeze-dried, and finally 32.7 mg of fine peptide was obtained, namely compound WP302-4.

[0145] The structure of compound WP302-4 is shown below:

[0146]

[0147] The MS spectrum and HPLC mass spectrum of compound WP302-4 are shown as follows: Figure 7 and Figure 8 shown.

[0148] Example 5: Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-D-Phe-NH2 (SEQ ID NO.5)

[0149] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 100 mL reactor.

[0150] Select 1 g of Ramage Amide AM resin with a loading of 0.30 mmol / g, and remove the Fmoc protecting group after swelling;

[0151] Weigh 0.34g of Fmoc-D-Phe-OH, 468mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.34mL of DIEA, dissolve in 25mL DMF at 0°C, add to the reaction column and react at room temperature for 1 hour after complete dissolution, and judge the progress of the reaction by negative detection by ninhydrin detection method. After the coupling reaction is completed, drain the reaction solution, wash with 30mL DMF, 40mL of 20% hexahydropyridine DMF solution, react for 5min, wash once with DMF, add 30mL of 20% hexahydropyridine DMF solution again, react for 10min, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF.

[0152] According to the peptide sequence, coupling was performed from C-terminus to N-terminus until Fmoc-Arg(Pbf)-OH was obtained, and Fmoc was removed and palmitic acid was used to react with the α-amino group of Arg to form an amide bond, wherein the side chain protecting groups of Arg, Cys, His, and Trp were Pbf, Trt, Trt, and Boc, respectively; the α-amino groups of all amino acids were protected with Fmoc;

[0153] By reacting the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin, about 488.89 mg of the polypeptide with all side chain protecting groups removed was obtained;

[0154] The polypeptide was dissolved in 500 mL of pure water, the pH was adjusted to 8.0, 1 mL of H2O2 was added, and the reaction was carried out at room temperature. After 30 min of reaction, the polypeptide was completely converted into a cyclic polypeptide by monitoring by analytical HPLC;

[0155] The crude linear peptide aqueous solution was filtered with a 0.45 μm filter membrane, and the improved crude peptide was purified using a high-performance liquid chromatography instrument: through a DAC-HB50 dynamic axial compression column, the mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and B was a 0.05% trifluoroacetic acid acetonitrile solution, and gradient elution separation and purification were performed. The sample was detected by an ultraviolet detector, and the peptide solution of the target peak was collected in sections. After high-performance liquid purification, 150 mL of finished peptide liquid with a purity greater than 95% was obtained. The obtained liquid was balanced with 1 / 1000 acetic acid and acetonitrile, and 65 mL of liquid was obtained after rotary evaporation concentration, and then the liquid was pre-lyophilized and freeze-dried, and finally 35.1 mg of fine peptide was obtained, namely compound WP302-5.

[0156] The structure of compound WP302-5 is shown below:

[0157]

[0158] The MS spectrum and HPLC mass spectrum of compound WP302-5 are shown as follows: Fig. 9 and Fig.10 shown.

[0159] Example 6: Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-Pro-NH2 (SEQ ID NO.6)

[0160] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 100 mL reactor.

[0161] Select 1g of Ramage Amide AM resin with a loading of 0.30mmol / g, remove the Fmoc protecting group after swelling; weigh 0.30g of Fmoc-Pro-OH, 468mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.34mL of DIEA, dissolve in 25mL DMF at 0°C, add to the reaction column and react at room temperature for 1 hour after complete dissolution, and use the ninhydrin detection method to detect negative to judge the reaction progress. After the coupling reaction is completed, drain the reaction liquid, wash with 30mL DMF, 40mL of 20% hexahydropyridine DMF solution, react for 5min, wash once with DMF, add 30mL of 20% hexahydropyridine DMF solution again after washing, react for 10min, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF.

[0162] According to the peptide sequence, coupling was performed from C-terminus to N-terminus until Fmoc-Arg(Pbf)-OH was obtained, and Fmoc was removed and palmitic acid was used to react with the α-amino group of Arg to form an amide bond, wherein the side chain protecting groups of Arg, Cys, His, and Trp were Pbf, Trt, Trt, and Boc, respectively; the α-amino groups of all amino acids were protected with Fmoc;

[0163] By reacting the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin, about 436.12 mg of the polypeptide with all side chain protecting groups removed was obtained;

[0164] The polypeptide was dissolved in 450 mL of pure water, the pH was adjusted to 8.0, 1 mL of H2O2 was added, and the reaction was carried out at room temperature. After 30 min of reaction, the polypeptide was completely converted into a cyclic polypeptide by monitoring by analytical HPLC;

[0165] The crude linear peptide aqueous solution was filtered with a 0.45 μm filter membrane, and the improved crude peptide was purified using a high-performance liquid chromatography instrument: through a DAC-HB50 dynamic axial compression column, the mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and B was a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution separation and purification was performed, and the sample was detected by an ultraviolet detector, and the peptide solution of the target peak was collected in sections. After high-performance liquid purification, 120 mL of finished peptide liquid with a purity greater than 95% was obtained. The obtained liquid was balanced with 1 / 1000 acetic acid and acetonitrile, and 50 mL of liquid was obtained after rotary evaporation concentration, and then the liquid was pre-lyophilized and freeze-dried, and finally 29.6 mg of fine peptide was obtained, namely compound WP302-6.

[0166] The structure of compound WP302-6 is shown below:

[0167]

[0168] The MS spectrum and HPLC mass spectrum of compound WP302-6 are shown as follows: Fig.11 and Fig.12 shown.

[0169] Example 7: Palm-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-D-Phe-NH2 (SEQ ID NO.7)

[0170] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 100 mL reactor.

[0171] Select 1g of Ramage Amide AM resin with a loading of 0.30mmol / g, remove the Fmoc protecting group after swelling; weigh 0.31g of Fmoc-Ile-OH, 468mg of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), and 0.34mL of DIEA, dissolve in 25mL DMF at 0°C, add to the reaction column and react at room temperature for 1 hour after complete dissolution, and use the ninhydrin detection method to detect negative to judge the reaction progress. After the coupling reaction is completed, drain the reaction liquid, wash with 30mL DMF, 40mL of 20% hexahydropyridine DMF solution, react for 5min, wash once with DMF, add 30mL of 20% hexahydropyridine DMF solution again after washing, react for 10min, drain, wash 3 times with DMF, wash 2 times with DCM, and wash once with DMF.

[0172] According to the peptide sequence, coupling was performed from C-terminus to N-terminus until Fmoc-Arg(Pbf)-OH was removed and palmitic acid was used to react with the α-amino group of Arg to form an amide bond, wherein the side chain protecting groups of Arg, Cys, D-His, and Trp were Pbf, Trt, Trt, and Boc, respectively; the α-amino groups of all amino acids were protected with Fmoc;

[0173] By reacting the cleavage solution (TFA: triisopropylsilane: water = 95:2.5:2.5) with the linear peptide resin, about 469.52 mg of the polypeptide with all side chain protecting groups removed was obtained;

[0174] The polypeptide was dissolved in 480 mL of pure water, the pH was adjusted to 8.0, 1 mL of H2O2 was added, and the reaction was carried out at room temperature. After 30 min of reaction, the polypeptide was completely converted into a cyclic polypeptide by monitoring by analytical HPLC;

[0175] The crude linear peptide aqueous solution was filtered with a 0.45 μm filter membrane, and the improved crude peptide was purified using a high-performance liquid chromatography instrument: through a DAC-HB50 dynamic axial compression column, the mobile phase A was a 0.05% trifluoroacetic acid aqueous solution, and B was a 0.05% trifluoroacetic acid acetonitrile solution. Gradient elution separation and purification was performed, and the sample was detected by an ultraviolet detector, and the peptide solution of the target peak was collected in sections. After high-performance liquid purification, 135 mL of finished peptide liquid with a purity greater than 95% was obtained. The obtained liquid was balanced with 1 / 1000 acetic acid and acetonitrile, and 55 mL of liquid was obtained after rotary evaporation concentration, and then the liquid was pre-lyophilized and freeze-dried, and finally 36.8 mg of fine peptide was obtained, namely compound WP302-7.

[0176] The structure of compound WP302-7 is shown below:

[0177]

[0178] The MS spectrum and HPLC mass spectrum of compound WP302-7 are shown as follows: Fig.13 and Fig.14 shown.

[0179] Comparative Example 1: Ac-Arg-c(Cys-DAla-His-DPhe-Arg-Trp-Cys)-NH2 (SEQ ID NO.8)

[0180] The title peptide was synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry in a 5 L reactor. RinkAmide 4-methylbenzhydrylamine (MBHA) resin ( San Diego, CA). The Fmoc amino acids used in the synthesis were Fmoc-Arg(pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-D-Phe-OH, and Fmoc-Trp(Boc)-OH. The synthesis was performed on a 50 mmol scale. The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (DMF) for 30 minutes. In each coupling step, Fmoc amino acid (3 eq, 150 mmol), N,N-diisopropylcarbodiimide (DIC) (3 eq, 150 mmol) and 1-hydroxy-benzotriazole (HOBT) (3 eq, 150 mmol) were coupled in DMF. The resin was subjected to the following cyclic reaction in a reactor: (1) washed with DMF, (2) treated with 20% piperidine in DMF for 30 min to remove the Fmoc protecting group, (3) washed with DMF (4) coupled with Fmoc amino acid in the presence of DIC and HOBT for 1 hour. The resin was successfully coupled according to the sequence of the title peptide. After the peptide chain was assembled and the last Fmoc protecting group was removed, acetylation was performed with a solution of acetic anhydride, N-methylmorpholine (NMM) and DMF (v / v / v: 6 / 10 / 84). The resin was washed thoroughly with dichloromethane (DCM) and methanol and then drained to obtain a dry resin.

[0181] To cleave the title peptide, the resin was treated with a solution of TFA, phenol, EDT, H2O, thioanisole (v / v / v / v / v: 87.5 / 2.5 / 2.5 / 2.5 / 5) at room temperature for 3 hours. The resin was filtered and the filtrate was poured into ether and the precipitate was collected by centrifugation. The centrifugal precipitate was washed 3 times with ether to obtain the final precipitate and dried in vacuo to obtain the crude peptide.

[0182] The crude peptide was dissolved in 0.1% TFA aqueous solution, and then 10 g / L iodine methanol solution was added to the solution for oxidation bridging. When the solution changed from colorless to light yellow, the addition of iodine solution was stopped. After standing for 3 minutes, VC was added to the solution. When the solution changed from light yellow to colorless, the addition was stopped. The oxidation was completed. The crude product after oxidation was purified on a reverse phase preparative HPLC system using a Luna C18100A (650*650*2350mm) (Varian) column. The column was eluted with a linear gradient of 80% A:20% B to 50% A:50% B for about 1 hour, wherein A was a 0.1% TFA aqueous solution, and B was a mixture of 0.1% TFA in 80% acetonitrile and 20% water. The TFA salt product with a concentration of more than 90% was purified on a reverse phase preparative HPLC system using Luna C18

[0183] 100A (650*650*2350mm) (Varian) column was used to exchange Ac salt, and the column was eluted with a linear gradient from 100% A:0% B to 40% A:60% B for about 1 hour, where A was a 0.5% HAc aqueous solution and B was a mixture of 0.5% HAc in 80% acetonitrile and 20% water. Analytical HPLC detection ( Figure 5 ) The qualified product was freeze-dried to obtain 21635 mg, 95% white solid with a yield of 34.7%, namely compound WP300.

[0184] The structure of compound WP300 is shown below:

[0185]

[0186] The MS spectrum and HPLC mass spectrum of compound WP300 are shown in Fig.16 and Fig.15 shown.

[0187] Test 1: Serum stability assay

[0188] 1. Add 594 μL of serum to a 96-well plate and pre-incubate at 37°C for 5 minutes.

[0189] 2. Add 6 μL of the test substance / positive control to the 96-well plate and mix thoroughly by pipetting. Continue to react at 37°C with constant shaking for 48 hours.

[0190] 3. For the test substance, take out 50 μL at 0, 0.5, 1.5, 3, 6, 24 and 48 hours and add to 150 μL 0.1% FA (formic acid) in methanol solution containing internal standard. For the positive control, take out 50 μL at 0, 15, 30, 60 and 120 minutes and add to 150 μL 0.1% FA in methanol solution containing internal standard.

[0191] 4. Mix by oscillation for 10 minutes, then centrifuge at 6000 x g for 10 minutes. Inject the supernatant into the LC-MS / MS system for analysis.

[0192] The measurement results are shown in Table 1:

[0193] Table 1

[0194]

[0195] Test 2: EC50 determination

[0196] Electrochemiluminescence (ECL) assay (Meso Scale Gaithersburg, MD; hereinafter referred to as MSD) measured intracellular cyclic AMP (cAMP) levels.

[0197] 1. Take out the MC1, MC3, MC4 and MC5 stable cell lines from the liquid nitrogen storage system, and quickly thaw them in a 37℃ electric thermostatic water bath. Use a pipette to transfer the cell suspension to a 15mL centrifuge tube and add 10mL complete medium. (MC1 cells: F12K+10% FBS+400μg / mL G418, MC3 cells: F12K+10% FBS+100μg / mL Hygromycin B+200μg / mL Zeocin, MC4 cells: F12K+10% FBS+100μg / mL Hygromycin B+8μg / mL Puromycin, MC5 cells: F12K+10% FBS+100μg / mL Hygromycin B+400μg / mL G418).

[0198] 2. After centrifugation at 1000 rpm for 4 minutes, discard the supernatant, resuspend the cell pellet with 5 mL of complete culture medium, transfer to a T75 culture flask, add 15 mL of culture medium, and culture in a 37°C, 5% carbon dioxide incubator. After the cells are passaged once, they are used for the cell experiment.

[0199] 3. When the cell density reaches 80%-90%, discard the culture medium and wash the cells with 5 mL of phosphate buffer.

[0200] 4. Remove the phosphate buffer, add 3 mL of pancreatin, and place in a 37°C carbon dioxide incubator for 2-5 minutes.

[0201] 5. Add 10 mL of complete culture medium to collect cells, centrifuge at 1000 rpm for 4 min, and discard the supernatant.

[0202] 6. Use Stimulation Buffer to adjust the cell suspension to the appropriate density: 1000 cells / well for MC1 cells, MC3 cells and MC5 cells. 2000 cells / well for MC4 cells.

[0203] Transfer 10 μL of cell solution to the assay plate. Centrifuge at 600 rpm for 3 minutes and incubate at room temperature for 60 minutes. After incubation, add 5 μL Eu-cAMP tracer solution and 5 μL ULight TM -anti-cAMP solution. Centrifuge again at 600 rpm for 3 minutes and incubate at room temperature for 60 minutes.

[0204] The cAMP signal was read on a multifunctional microplate reader, and the data were processed, analyzed, and reported as EC50 values ​​using GraphPad Prism 6. The experimental results are shown in Tables 2 to 5.

[0205] Table 2: MC1 agonism experiments

[0206] Test samples BOTTOM TOP LOGEC50 HILLSLOPE EC50(nM) NDP-α-MSH 2.69 99.96 -1.11 0.87 0.077 WP300 -2.93 93.73 0.13 0.98 1.35 WP302-1 -1.34 97.38 -0.84 0.88 0.14 WP302-2 -2.18 100.70 0.77 1.11 5.85 WP302-3 -1.84 99.13 -0.60 0.98 0.25 WP302-4 -0.24 103.30 -0.08 1.25 0.84 WP302-5 -2.93 93.73 0.13 0.98 1.35 WP302-6 2.58 102.90 -0.05 0.97 0.90 WP302-7 2.68 102.70 0.41 0.97 2.55

[0207] Table 3: MC3 agonist experiments

[0208]

[0209]

[0210] Table 4: MC4 agonist experiments

[0211] Test samples BOTTOM TOP LOGEC50 HILLSLOPE EC50(nM) NDP-α-MSH -0.13 99.10 -1.44 1.30 0.036 WP300 2.16 99.49 -1.96 1.66 0.011 WP302-1 -0.67 100.50 -2.00 1.37 0.010 WP302-2 0.53 100.70 -1.33 1.47 0.047 WP302-3 1.00 99.96 -2.10 1.44 0.0080 WP302-4 -1.68 99.79 -0.86 1.31 0.14 WP302-5 -0.69 99.21 0.56 1.53 3.62 WP302-6 0.53 98.28 -0.80 1.47 0.16 WP302-7 0.04 99.98 -0.16 1.48 0.70

[0212] Table 5: MC5 agonist experiment

[0213]

[0214]

[0215] The cellular effect produced by the binding of the compound WP302 provided by the present invention to the melanocortin receptor is measured by the intracellular cyclic adenosine monophosphate (cAMP) level. EC50 represents the concentration of the agonist compound required to obtain 50% of the maximum response. The measurement results show that the compound provided by the present invention has a good effect on the melanocortin receptor.

[0216] The present invention provides a series of compounds with the general formula R2-R1-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-R3-R4; and from the above experimental data, it can be seen that as the molecular weight of the N-terminal substitution increases, the activity of the MC5 target decreases and the selectivity increases, that is, the target is related to the N-terminal substituent.

[0217] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A disulfide cyclic peptide compound, characterized in that: The disulfide cyclic peptide compound has the following structure: R2-R1-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-R3-R4 wherein R2 and R4 are each independently selected from an amino group, an acetyl group or a palmitoyl group; R1 and R3 are each independently selected from Ile, Arg, D-Phe, Pro or are missing, R1 and R3 are not missing at the same time, and when R1 or R3 is missing, at least one of R2 and R4 is a palmitoyl group.

2. The disulfide cyclic peptide compound according to claim 1, characterized in that The disulfide cyclic peptide compound has the following structural formula: Wherein, R1, R2, R3 and R4 are as defined in claim 1.

3. The disulfide cyclic peptide compound according to claim 1, characterized in that R3 is selected from Ile, D-Phe, Pro or missing, and R1 is Arg.

4. The disulfide cyclic peptide compound according to claim 1, characterized in that R2 is selected from an acetyl group or a palmitoyl group, and R4 is an amino group.

5. The disulfide cyclic peptide compound according to claim 3 or 4, characterized in that: When R3 is absent, R4 is a palmitoyl group.

6. The disulfide cyclic peptide compound according to claim 1 or 2, characterized in that: The disulfide cyclic peptide compound is selected from at least one of the following compounds:

7. A method for preparing the disulfide cyclic peptide compound according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: Step 1: Select a resin and remove the Fmoc protecting group to obtain a resin from which the Fmoc protecting group has been removed; Step 2: Weigh Fmoc-R3-OH or Fmoc-Cys(Trt)-OH and PyBop, and perform coupling reaction on the resin with the Fmoc protecting group removed to obtain a coupling resin; Step 3: According to the peptide sequence, for the coupling resin, coupling is performed from the C-terminus to the N-terminus to obtain a linear peptide resin; Step 4: reacting the cleavage solution with the linear peptide resin to remove the protecting group and obtain a polypeptide; Step 5: dissolving the polypeptide in water, adjusting the pH and adding H2O2 for reaction to obtain the disulfide cyclic peptide compound.

8. Use of the disulfide cyclic peptide compound according to any one of claims 1 to 6 or the disulfide cyclic peptide compound prepared by the method according to claim 7 in the preparation of a drug, characterized in that: The drugs include drugs for treating or preventing acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection, metabolic diseases associated with weight gain, metabolic diseases associated with weight loss, diabetes, diabetic complications, cancer, cancerous hyperplasia, reproductive system diseases, peripheral or central nervous system diseases, cardiovascular diseases or respiratory system diseases.

9. The use according to claim 8, characterized in that: The application is achieved by combining the disulfide cyclic peptide compound with the melanocortin receptor.

10. The use according to claim 9, characterized in that: The melanocortin receptors include at least one of MC1R, MC3R, MC4R and MC5R.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the disulfide cyclic peptide compound or its analogue according to any one of claims 1 to 6, wherein the analogue comprises a derivative of the disulfide cyclic peptide compound, a pharmaceutically acceptable salt thereof, a tautomer thereof or a stereoisomer thereof.

12. The pharmaceutical composition according to claim 11, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

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