Selenium cyclic peptide as well as preparation method and application thereof

Abstract

The invention provides diselenide cyclic peptide as well as a preparation method and application thereof, and belongs to the technical field of medicines. According to the present invention, the diselenide cyclic peptide is developed for the first time, and the diselenide cyclic peptide is an Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Secamp; the compound (Sec Bridge) is a compound WP302, and has an amino acid sequence as shown in SEQ ID NO.1. The invention also discloses a preparation method of the compound WP302. The diselenide cyclopeptide provided by the invention is used as a ligand of one or more melanocortin receptors, and can be well combined with the melanocortin receptors to generate a good effect. The diselenide cyclic peptide provided by the invention can effectively reduce body weight and control blood sugar and blood fat levels, and shows a good application prospect in the aspect of treating obesity or diabetes and hyperlipidemia.

Description

[0001] This invention claims the priority of a Chinese patent application titled "A Diselenocyclopeptide and Its Preparation Method and Application", with the application number 202410607198.3 and filed with the Chinese Patent Office on May 15, 2024. The entire content is incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, and particularly relates to a diselenocyclopeptide and its preparation method and application. Background Art

[0003] The secretion of melanocortin is regulated by hormones such as ghrelin, leptin, and insulin. Currently, a variety of melanocortin polypeptide analogs have been developed as potential drugs for treating diseases such as skin diseases, obesity, anorexia, and type 2 diabetes. Melanocortin is a class of endogenous polypeptide hormones composed of adrenocorticotropic hormone (ACTH) and three melanocyte-stimulating hormones (α-MSH, β-MSH, and γ-MSH). They participate in physiological activities such as the body's stress response, energy balance, immune regulation, and pigmentation by acting on melanocortin receptors (MC1R-MC5R).

[0004] MC1R is a transmembrane protein of 315 amino acids and belongs to the G-protein coupled receptor family. MC1R is the receptor for MSH and ACTH. The activity of MC1R is mediated by G-protein, which activates adenylate cyclase. The MC1R receptor is found in melanocytes and adrenal cortical tissues, as well as in a variety of other tissues such as the adrenal gland, white blood cells, pulmonary lymph nodes, ovary, testis, pituitary gland, placenta, spleen, and uterus. MC2R, also known as the adrenocorticotropic hormone receptor (ACTH-R), is a transmembrane protein of 297 amino acids found in melanocytes and adrenal cortical tissues. MC2R mediates the adrenocorticotropic effect of ACTH. In humans, MC3R is a 360AA protein found in the brain, and in mice and rats, MC3R is a 323AA protein. MC4R is a membrane protein of 332 amino acids, which is expressed in the brain, as well as in placenta and intestinal tissues. MC5R is a transmembrane protein of 325 amino acids expressed in the adrenal gland, stomach, lung, and spleen and is expressed at a very low level in the brain. MC5-R is also present in the three layers of the adrenal cortex, mainly in the zona glomerulosa cells that produce aldosterone.

[0005] Melanocortin receptors (MC1R - MC5R) play different roles in the regulation of physiological processes. MC1R is mainly expressed in melanocytes, melanoma cells and immune cells, controlling skin and hair color and regulating immune responses. MC2R is mainly located in the adrenal cortex and is a key component of the hypothalamic - pituitary - adrenal axis, stimulating the biosynthesis of glucocorticoids. MC4R is known as the neuronal MCR due to its high expression in various brain regions including the hypothalamus, brainstem and cortex. MC4R is involved in energy homeostasis, feeding and sexual behavior as well as male erectile function. In addition to MC4R, MC3R is also widely distributed in the brain, mainly expressed in the hypothalamus, mainly the arcuate nucleus and the ventromedial hypothalamus, and these structures are involved in regulating energy homeostasis, metabolism and appetite. Many studies have shown that MC3R and MC4R can act 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 crucial for temperature control and exocrine function.

[0006] Currently, a variety of melanocortin peptide analogues have been developed as potential drugs for treating diseases such as skin diseases, obesity, anorexia and type 2 diabetes. Setmelanotide is a cyclic high - affinity peptide. Compared with natural α - MSH, its G - protein signaling profile is biased towards Gq / 11 (phospholipase C activation), and its receptor subtype selectivity for MC4R is 20 - fold. As a targeted therapy, Setmelanotide restores the function of the damaged MC4R pathway, re - establishes energy expenditure and appetite control in patients with rare genetic obesity diseases, reduces hunger and decreases body weight. However, Setmelanotide also has certain side effects, including injection - site reactions, skin pigmentation (skin patches darker than the surrounding skin), headache and gastrointestinal side effects (such as nausea, diarrhea and abdominal pain). There are also adverse reproductive system reactions during treatment. There are also cases of depression and suicidal thoughts during the use of Setmelanotide.

[0007] Therefore, there is still a need to develop a ligand for melanocortin receptors with better selectivity, stronger stability and fewer side effects for melanocortin receptors (MC1R - MC5R). Summary of the Invention

[0008] In view of the above problems, the present invention provides a diselenocyclic peptide, a preparation method thereof, and an application thereof. The present invention develops a diselenocyclic peptide for the first time. The diselenocyclic peptide is Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&Sec Bridge), that is, compound WP302, and has an amino acid sequence shown in SEQ ID NO.1. The diselenocyclic peptide provided by the present invention, as a ligand of one or more melanocortin receptors, can bind well to the melanocortin receptors and produce good effects. The diselenocyclic peptide provided by the present invention can effectively reduce body weight and control blood glucose and blood lipid levels, and shows good application prospects in the treatment of obesity or diabetes.

[0009] Terms and abbreviations:

[0010]

[0011]

[0012] In order to achieve the above-mentioned invention object, the technical solution of the present invention is as follows:

[0013] On the one hand, the present invention provides a diselenocyclic peptide, and the diselenocyclic peptide is compound WP302.

[0014] Specifically, the diselenocyclic peptide has the following structure:

[0015]

[0016] Specifically, the diselenocyclic peptide is Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&SecBridge).

[0017] Specifically, the diselenocyclic peptide has an amino acid sequence shown in SEQ ID NO.1.

[0018] Specifically, the molecular formula of the diselenocyclic peptide is C 49 H 66 N 18 O9Se2.

[0019] On the other hand, the present invention provides a preparation method of the above-mentioned diselenocyclic peptide. The preparation method includes using the Fmoc chemical synthesis method or the Boc synthesis method to synthesize a coupled resin.

[0020] Specifically, the preparation method includes using the Fmoc chemical synthesis method to sequentially couple corresponding amino acids from the C-terminus to the N-terminus according to the peptide chain sequence to synthesize a coupled resin.

[0021] Specifically, the resins used in the Fmoc chemical synthesis method include, but are not limited to, Wang-resin, CTC-resin, Rink amide-resin, or Sieber-resin.

[0022] More specifically, the resin used in the Fmoc chemical synthesis method is Rink amide-resin.

[0023] Preferably, the resin used in the Fmoc chemical synthesis method is Rink Amide MDHA resin.

[0024] More preferably, the initial substitution degree of Rink Amide MDHA resin is 0.4 - 0.5 mmol / g.

[0025] Even more preferably, the initial substitution degree of the Rink Amide MDHA resin is 0.451 mmol / g.

[0026] Specifically, the Fmoc chemical synthesis method selects Fmoc to protect the amino part of the amino acid.

[0027] Preferably, the Fmoc amino acid is selected from any one or more of Fmoc-Arg(pbf)-OH, Fmoc-Sec(Mob)-OH, Fmoc-D-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH.

[0028] Specifically, the coupling system used during coupling is HOBt / DIC.

[0029] Specifically, the preparation method further includes the following steps:

[0030] (1) Treat the coupled resin with a cleavage solution, precipitate, wash, and dry to obtain a crude polypeptide.

[0031] (2) Oxidize, purify, desalt and purify, and lyophilize the crude polypeptide to obtain a pure diselenocyclic peptide.

[0032] Preferably, the cleavage solution in step (1) includes one or more of TFA, phenol, EDT, H2O, and anisole.

[0033] More preferably, the cleavage solution is TFA:phenol:EDT:H2O:anisole = 85 - 95:1 - 5:1 - 5:1 - 5:1 - 5.

[0034] Still further preferably, the cutting fluid is TFA:phenol:EDT:H2O:thioanisole = 87.5-90:2.5-3:2.5-3:2.5-3:1-5.

[0035] Even further preferably, the cutting fluid is TFA:phenol:EDT:H2O:thioanisole = 87.5:2.5:2.5:2.5:5.

[0036] Preferably, in step (2), the purification is carried out by reverse-phase high-performance liquid chromatography, using C18 packing as the stationary phase of the chromatographic column, 0.1% TFA aqueous solution as mobile phase A, and a mixture of 80% acetonitrile and 20% water with 0.1% TFA as mobile phase B.

[0037] Even further preferably, in step (2), the purification first uses 78% A phase:22% B phase, and then uses a linear gradient of 48% A phase:52% B phase to elute the column for 1 hour.

[0038] Preferably, in step (2), the salt exchange purification is carried out by reverse-phase high-performance liquid chromatography, using C18 packing as the stationary phase of the chromatographic column, 0.5% HAc aqueous solution as mobile phase A, and a mixture of 80% acetonitrile and 20% water with 0.5% HAc as mobile phase B.

[0039] Even further preferably, in step (2), the salt exchange purification first uses 95% A phase:5% B phase, and then uses a linear gradient of 35% A phase:65% B phase to elute the column for 1 hour.

[0040] On the other hand, the present invention provides the use of the above-mentioned diselenocyclopeptide in the preparation of drugs.

[0041] Specifically, the drugs include but are not limited to drugs for treating or preventing acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection reactions, metabolic diseases accompanied by weight gain, metabolic diseases accompanied by weight loss, diabetes, diabetic complications, hyperlipidemia, cancer, cancerous hyperplasia, reproductive system diseases, peripheral or central nervous system diseases, cardiovascular diseases or respiratory system diseases.

[0042] Preferably, the acute or chronic inflammatory diseases include but are not limited to systemic inflammation, inflammatory bowel disease, brain inflammation, sepsis, septic shock.

[0043] Preferably, 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 Sjogren's syndrome.

[0044] Preferably, the organs for transplant rejection include, but are not limited to, kidney, heart, liver, pancreas and islets, parathyroid gland, heart-lung, bone marrow, and cornea.

[0045] Preferably, the metabolic diseases accompanied by weight gain include, but are not limited to, obesity, eating disorders, and Prader-Willi syndrome.

[0046] Preferably, the metabolic diseases accompanied by weight loss include, but are not limited to, anorexia nervosa, bulimia nervosa, AIDS wasting, cancer cachexia, and cachexia of the elderly with lean body mass.

[0047] Preferably, the diabetic complications include, but are not limited to, diabetic nephropathy, diabetic retinopathy, diabetic cataract, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular diseases, and diabetic neuropathy.

[0048] Preferably, the cancers include, but are not limited to, dermatofibrosarcoma protuberans, neuroblastoma, rectal cancer, colon cancer, familial 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 lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, hepatocellular carcinoma, gallbladder cancer, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal sarcoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, leiomyoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, and plasmacytoma.

[0049] Preferably, the cancerous hyperplasia includes, but is not limited to, squamous epithelial dysplasia and glandular epithelial dysplasia.

[0050] Preferably, the reproductive system diseases include, but are not limited to, abnormal urination, pyuria, abnormal urethral discharge, pain, mass, sexual dysfunction, male infertility, female infertility, polycystic ovary syndrome, menstrual disorder, dysmenorrhea, pregnancy abnormality, uterine lesion.

[0051] Preferably, the peripheral or central nervous system diseases include, but are not limited to, depression, bipolar depression or manic-depressive disorder, acute and chronic anxiety states, schizophrenia, Alzheimer's disease, Parkinson's disease, acute and chronic multiple sclerosis or acute and chronic pain and brain injury caused by stroke, hypoxia or craniocerebral trauma.

[0052] Preferably, the cardiovascular diseases include, but are not limited to, coronary heart disease, stroke, 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, venous thrombosis.

[0053] Preferably, the respiratory system diseases include, but are not limited to, upper respiratory tract infection, acute bronchitis, acute pharyngitis, pneumonia, chronic bronchitis, chronic obstructive pulmonary disease, pulmonary tuberculosis, lung tumor, bronchiectasis, lung abscess, pulmonary interstitial fibrosis, pulmonary embolism, acute respiratory distress syndrome, cor pulmonale.

[0054] More preferably, the drug is a drug for treating obesity, diabetes or hyperlipidemia.

[0055] Specifically, the application is achieved by the binding of diselenocyclopeptide to melanocortin receptor.

[0056] Preferably, the melanocortin receptor includes any one or more of MC1R, MC2R, MC3R, MC4R, MC5R.

[0057] In another aspect, the present invention provides a pharmaceutical composition, which comprises the above-mentioned diselenocyclopeptide or its analog.

[0058] Specifically, the analog includes, but is not limited to, derivatives of diselenocyclopeptide, pharmaceutically acceptable salts thereof, tautomers thereof or stereoisomers thereof.

[0059] Specifically, the analog also includes Ac-Arg-Cys-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Cys&Sec Bridge) or Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Cys-NH2 (Sec&Cys Bridge).

[0060] Preferably, the Ac-Arg-Cys-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Cys&Sec Bridge) has the amino acid sequence shown in SEQ ID NO.3 and has the following structure:

[0061]

[0062] Preferably, the Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Cys-NH2 (Sec&Cys Bridge) has the amino acid sequence shown in SEQ ID NO.4 and has the following structure:

[0063]

[0064] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0065] The positive and beneficial effects of the present invention are as follows:

[0066] The present invention has prepared a diselenocyclopeptide for the first time. The diselenocyclopeptide is Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&Sec Bridge), namely compound WP302, which has the amino acid sequence shown in SEQ ID NO.1. The diselenocyclopeptide provided by the present invention, as a ligand of one or more melanocortin receptors, can bind well to melanocortin receptors and produce good effects. The diselenocyclopeptide provided by the present invention can effectively reduce body weight, control blood glucose and blood lipid levels, and shows good application prospects in the treatment of obesity, diabetes or hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is the HPLC chromatogram of compound WP302.

[0068] Figure 2 It is the electrospray ionization mass spectrum of compound WP302.

[0069] Figure 3 It is the nuclear magnetic resonance spectrum 1 of compound WP302.

[0070] Figure 4 It is the nuclear magnetic resonance spectrum 2 of compound WP302.

[0071] Figure 5 It is the HPLC chromatogram of compound WP300.

[0072] Figure 6 It is the electrospray ionization mass spectrum of compound WP300.

[0073] Figure 7 It is the nuclear magnetic resonance spectrum 1 of compound WP300.

[0074] Figure 8 It is the nuclear magnetic resonance spectrum 2 of compound WP300.

[0075] Figure 9 It is the result of body weight change.

[0076] Figure 10 It is the result of blood glucose measurement.

[0077] Figure 11 It is the blood glucose change rate.

[0078] Figure 12 It is the result of TC measurement. Specific Embodiments

[0079] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are conventional conditions unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0080] Example 1 Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&Sec Bridge) SEQ ID NO.1

[0081] The title peptide was synthesized using fluorenylmethyloxycarbonyl (Fmoc) chemistry in a 100 mL reactor. Using Rink Amide 4-methylbenzhydrylamine (MBHA) resin with 0.451 mmol / g substitution (San Diego, CA). The Fmoc amino acids used in the synthesis were Fmoc-Arg(pbf)-OH, Fmoc-Sec(Mob)-OH, Fmoc-D-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-D-Phe-OH, and Fmoc-Trp(Boc)-OH. The synthesis was carried out on a 0.2 mmol scale. The Fmoc group was removed by treating with 20% piperidine in N,N-dimethylformamide (DMF) for 30 minutes. In each coupling step, the Fmoc amino acid (3 eq, 0.6 mmol), N,N-diisopropylcarbodiimide (DIC) (3 eq, 0.6 mmol), and 1-hydroxy-benzotriazole (HOBT) (3 eq, 0.6 mmol) were coupled in DMF. The resin was subjected to the following cycle of reactions in the reactor: (1) washing with DMF, (2) removing the Fmoc protecting group by treating with 20% piperidine in DMF for 30 min, (3) washing with DMF, and (4) coupling with the 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 final Fmoc protecting group was removed, acetylation was carried out with a solution of acetic anhydride, N-methylmorpholine (NMM), and DMF (v / v / v: 6 / 10 / 84). The resin was thoroughly washed with dichloromethane (DCM) and methanol and then dried by suction to obtain the dry resin.

[0082] To cleave the title peptide, the resin was treated with a solution of TFA, phenol, EDT, H2O, and anisole (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 diethyl ether, and the precipitate was collected by centrifugation. The centrifuged precipitate was washed 3 times with diethyl ether, and the final precipitate was obtained and dried in vacuo to obtain the crude peptide.

[0083] The crude peptide was dissolved in 20% acetonitrile and water, and then a 10 g / L iodine methanol solution was added dropwise to the solution for oxidative bridging. When the solution changed from colorless to light yellow, the addition of iodine solution was stopped. After standing for 3 min, VC was added dropwise to the solution. When the solution changed from light yellow to colorless, the addition was stopped, and the oxidation was completed. The oxidized crude product was purified on a reverse-phase preparative HPLC system using a Luna C18 100A (25.4*250 mm) (Hanbang NP7000) column. The column was eluted with a linear gradient of 78% A: 22% B to 48% A: 52% B for about 1 hour, where A was a 0.1% TFA aqueous solution and B was a mixture of 80% acetonitrile and 20% water with 0.1% TFA. More than 90% of the TFA salt product obtained was converted to the Ac salt on a reverse-phase preparative HPLC system using a Luna C18 100A (25.4*250 mm) (Hanbang NP7000) column. The column was eluted with a linear gradient of 95% A: 5% B to 35% A: 65% B for about 1 hour, where A was a 0.5% HAc aqueous solution and B was a mixture of 80% acetonitrile and 20% water with 0.5% HAc. The qualified product was detected by analytical HPLC ( Figure 1 ) After lyophilization, 25 mg of a 95% white solid was obtained, and the yield was 23.5%, which was the compound WP302.

[0084] The structure of compound WP302 is shown below:

[0085]

[0086] Electrospray ionization mass spectrometry (ESI-MS) analysis gave a molecular weight of 1211.3 (consistent with the theoretical molecular weight of 1211.1), see Figure 2 . The nuclear magnetic resonance spectrum of compound WP302 is shown in Figure 3-4 .

[0087] Comparative Example 1 Ac-Arg-Cys-DAla-His-DPhe-Arg-Trp-Cys-NH2 (Cys&Cys Bridge) SEQ IDNO.2

[0088] The title peptide was synthesized using fluorenylmethyloxycarbonyl (Fmoc) chemistry in a 5 L reactor. Rink Amide 4-methylbenzhydrylamine (MBHA) resin with 0.451 mmol / g substitution was used ( (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 carried out 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, the 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 cycle of reactions in the reactor: (1) washing with DMF, (2) removing the Fmoc protecting group by treatment with 20% piperidine in DMF for 30 min, (3) washing with DMF, and (4) coupling with the 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 final Fmoc protecting group was removed, acetylation was carried out with a solution of acetic anhydride, N-methylmorpholine (NMM), and DMF (v / v / v: 6 / 10 / 84). The resin was thoroughly washed with dichloromethane (DCM) and methanol and then dried by suction to obtain the dry resin.

[0089] To cleave the title peptide, the resin was treated with a solution of TFA, phenol, EDT, H2O, and anisole (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 diethyl ether, and the precipitate was collected by centrifugation. The centrifugal precipitate was washed 3 times with diethyl ether, and the final precipitate was obtained and dried in vacuo to obtain the crude peptide.

[0090] The crude peptide was dissolved in 0.1% TFA aqueous solution, and then a 10 g / L iodine methanol solution was added dropwise to the solution for oxidative bridging. When the solution changed from colorless to light yellow, the addition of iodine solution was stopped. After standing for 3 min, VC was added dropwise to the solution. When the solution changed from light yellow to colorless, the addition was stopped, and the oxidation was completed. The oxidized crude product was purified on a reverse-phase preparative HPLC system using a Luna C18 100A (650*650*2350 mm) (Varian) column. The column was eluted with a linear gradient of 80% A:20% B to 50% A:50% B for about 1 hour, where A was 0.1% TFA aqueous solution and B was a mixture of 80% acetonitrile and 20% water with 0.1% TFA. More than 90% of the TFA salt product obtained was converted to the Ac salt on a reverse-phase preparative HPLC system using a Luna C18 100A (650*650*2350 mm) (Varian) column. The column was eluted with a linear gradient of 100% A:0% B to 40% A:60% B for about 1 hour, where A was 0.5% HAc aqueous solution and B was a mixture of 80% acetonitrile and 20% water with 0.5% HAc. The qualified product was detected by analytical HPLC ( Figure 5 ) After lyophilization, 21635 mg of a 95% white solid was obtained, with a yield of 34.7%, which was compound WP300.

[0091] The structure of compound WP300 is shown below:

[0092]

[0093] Electrospray ionization mass spectrometry (ESI-MS) analysis gave a molecular weight of 1117.4 (consistent with the theoretical molecular weight of 1117.3), see Figure 6 . The nuclear magnetic resonance spectrum of compound WP302 is shown in Figure 7-8 .

[0094] Experimental Example 1 Determination of Serum Stability

[0095] 1. Add 594 μL of serum to a 96-well plate and pre-incubate at 37 °C for 5 minutes.

[0096] 2. Add 6 μL of the test substance / positive control to the 96-well plate and mix well by pipette. Continue to react at 37 °C with continuous shaking for 48 hours.

[0097] 3. For the test substance, at 0, 0.5, 1.5, 3, 6, 24, and 48 hours, 50 μL was taken out each time and added to 150 μL of a solution of 0.1% FA (formic acid) in methanol containing an internal standard. In the positive control, at 0, 15, 30, 60, and 120 minutes, 50 μL was taken out and added to 150 μL of a solution of 0.1% FA in methanol containing an internal standard.

[0098] 4. Vortex and mix for 10 minutes, then centrifuge at 6000 x g for 10 minutes. Inject the supernatant into the LC-MS / MS system for analysis.

[0099] The measurement results are shown in Table 1:

[0100] Table 1 Serum stability measurement results

[0101]

[0102] The rat serum stability of the compounds of the present invention was tested using the above assay method. The measurement results showed that for the compound WP302 provided by the present invention in rat serum, the half-life was increased by 11.34%, and it had better serum stability than WP300.

[0103] Experimental Example 2 EC 50 Measurement

[0104] The intracellular cyclic AMP (cAMP) level was measured by an electrochemiluminescence (ECL) assay (Meso Scale Gaithersburg, MD; hereinafter referred to as MSD).

[0105] 1. Respectively take out the cell lines MC1, MC3, MC4, and MC5 stably transfected cells from the liquid nitrogen storage system. After quickly thawing in a 37 °C electrothermal constant temperature water bath, transfer the cell suspension to a 15 mL centrifuge tube with a pipette, and supplement with 10 mL of complete medium.

[0106] 2. After centrifuging at 1000 rpm for 4 minutes, discard the supernatant. Resuspend the cell pellet with 5 mL of complete medium, then transfer it to a T75 culture flask, supplement with 15 mL of medium, and place it in an incubator at 37 °C and 5% carbon dioxide for culture. After the cells are passaged once, they are used for this cell experiment.

[0107] 3. When the cell density reaches 80% - 90%, discard the medium and wash the cells with 5 mL of phosphate buffer. Remove the phosphate buffer, add 3 mL of trypsin, and place it in a 37 °C carbon dioxide incubator for 2 - 5 min. Add 10 mL of complete medium to collect the cells, centrifuge at 1000 rpm x 4 min, and discard the supernatant.

[0108] Adjust the cell suspension to an appropriate density with Stimulation Buffer. For MC1 cells, MC3 cells, and MC5 cells, it is 1000 cells / well. For MC4 cells, it is 2000 cells / well.

[0109] Transfer 10 μL of the cell solution to the detection plate. Centrifuge at 600 rpm for 3 minutes on a centrifuge and incubate at room temperature for 60 minutes. After incubation, add 5 μL of Eu-cAMP tracer solution and 5 μL of ULight from the cAMP detection kit to the experimental plate. TM -anti-cAMP solution. Centrifuge again at 600 rpm for 3 minutes and incubate at room temperature for 60 minutes.

[0110] Read the cAMP signal on a multi-functional microplate reader, process and analyze the data using GraphPad Prism 6 and report as the EC50 value. The experimental results are shown in Tables 2 - 5.

[0111] Table 2 MC1 Agonist Experiment

[0112] No. ID BOTTOM TOP <![CDATA[LOGEC 50 > HILLSLOPE <![CDATA[EC 50 (nM) <!-- 10 -->]]> 1 NDP-α-MSH -1.94 101.50 -0.86 0.81 0.14 2 WP300 -2.93 93.73 0.13 0.98 1.35 3 WP302 -2.53 99.43 -0.09 0.86 0.81

[0113] Table 3 MC3 Agonist Experiment

[0114] No. ID BOTTOM TOP <![CDATA LOGEC50 > HILLSLOPE <![CDATA[EC 50 (nM)]]> 1 NDP-α-MSH 0.86 100.80 -0.49 1.46 0.32 2 WP300 0.22 93.17 0.04 0.88 1.09 3 WP302 -1.14 90.86 0.15 0.84 1.40

[0115] Table 4 MC4 Agonist Experiment

[0116] No. ID BOTTOM TOP <![CDATA[LOGEC 50 > HILLSLOPE <![CDATA[EC 50 (nM)]]> 1 NDP-α-MSH -1.19 98.43 -1.34 1.52 0.046 2 WP300 2.16 99.49 -1.96 1.66 0.011 3 WP302 0.39 99.86 -2.03 1.51 0.0094

[0117] Table 5 MC5 Agonist Experiment

[0118]

[0119]

[0120] The effect at the cellular level 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. EC 50 represents the concentration of the agonist compound required to obtain 50% of the maximum response. The measurement results show that WP302 has a good effect on the melanocortin receptor.

[0121] Pharmacodynamic Study of Subcutaneous Injection of WP302 in DIO Model Mice in Experimental Example 3

[0122] In this experimental example, the pharmacodynamic behavior of the compound WP302 in male DIO model mice was studied after subcutaneous injection of the compound WP302 for 30 consecutive days.

[0123] 1. Experimental Animals

[0124] Six male SPF-grade obese and diabetic model (DIO model) mice, 20 - 22 weeks old and weighing over 38 g, were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The facility's experimental animal use license number is SYXK (Jiangsu) 2022 - 0002. Before the experiment began, the animals were acclimated in the laboratory for at least 3 days. After the experiment started, the animals had free access to food and water, and were fasted before blood glucose testing. Fasting began at around 8 am and lasted for about 8 hours, and blood glucose was measured at around 4 pm as a baseline.

[0125] 2. Test article information

[0126] The test article information for this experiment is shown in Table 6 as follows:

[0127] Table 6 Test article information

[0128]

[0129] 3. Preparation of administration solution

[0130] The administration solution will be prepared in advance with the support of stability data, stored at 2 - 8 °C before administration, and used within 3 days after preparation. Weigh an appropriate amount of the test article accurately; add a calculated volume of 1×PBS to dissolve the test article completely; stir well to mix evenly to obtain the administration solution.

[0131] 4. Grouping and administration dose

[0132] Two days before administration is denoted as Day - 1, the day before administration is denoted as Day0, and the day of administration is denoted as Day1; the animals are evenly grouped according to their body weights on Day - 1. The mice are administered according to the administration design described in Table 7.

[0133] Table 7 Animal grouping and administration dose

[0134]

[0135] Note: QD in the table represents once - daily administration.

[0136] 5. Detection indexes

[0137] 5.1 Body weight detection and monitoring of feeding situation

[0138] Weighing records are taken before administration every day and used to calculate the administration volume. The body weights of each group of mice from Day1 to Day37 are recorded. The changes in the body weights of each group of mice are as Figure 9 shown, and the results show that compound WP302 can effectively reduce the body weight of DIO model mice.

[0139] 5.2 Blood glucose test

[0140] Blood glucose tests were performed before drug administration. Fasting was carried out around 8:00 am on the day of the blood glucose test. Blood samples were collected for testing at around 4:00 pm, and feeding was resumed after the test. Mouse tail tip blood was collected on Day0, Day7, Day14, Day21, Day28, and Day31 for blood glucose tests. The collected blood samples were placed on wet ice and waited for more than 30 minutes, and then centrifuged. After collecting whole blood, after waiting for more than 30 minutes, centrifugation was performed at 3500g and 4°C for 5 minutes to obtain serum for detection.

[0141] The results of blood glucose measurement are as Figure 10 shown, and the blood glucose change rate is as Figure 11 shown. The results show that compound WP302 can effectively reduce the blood glucose level of DIO model mice and can play a hypoglycemic role.

[0142] 5.3 Total cholesterol (TC) and triglyceride (TG) tests

[0143] Mouse medial canthus blood was collected on Day31 for TC and TG tests. Fasting was not required before blood collection. The collected blood samples were placed on wet ice and waited for more than 30 minutes, and then centrifuged. After collecting whole blood, after waiting for more than 30 minutes, centrifugation was performed at 3500g and 4°C for 5 minutes to obtain serum for detection.

[0144] The results of TC measurement are as Figure 12 shown, and the results show that compound WP302 can effectively reduce the total cholesterol level.

[0145] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent should be subject to the appended claims.

Claims

1. A diselenocyclic peptide, characterized in that, The diselenocyclopeptide is Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&Sec Bridge), and has the following structure:

2. The preparation method of the diselenocyclopeptide according to claim 1, characterized in that, The preparation method includes using the Fmoc chemical synthesis method or the Boc synthesis method to synthesize a coupled resin.

3. The preparation method according to claim 2, wherein The resins used in the Fmoc chemical synthesis method include Wang-resin, CTC-resin, Rink amide-resin or Sieber-resin.

4. The preparation method according to claim 2, wherein, The Fmoc chemical synthesis method selects Fmoc to protect the amino part of the amino acid.

5. Use of the diselenocyclic peptide according to claim 1 in the preparation of a drug, characterized in that, The drug includes drugs for treating or preventing acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection reactions, metabolic diseases accompanied by weight gain, metabolic diseases accompanied by weight loss, diabetes, diabetic complications, hyperlipidemia, cancer, cancerous hyperplasia, reproductive system diseases, peripheral or central nervous system diseases, cardiovascular diseases or respiratory diseases.

6. The application according to claim 5, wherein The drug is a drug for treating obesity, diabetes or hyperlipidemia.

7. The application according to claim 6, wherein The application is achieved by the binding of the diselenocyclopeptide to the melanocortin receptor.

8. The application according to claim 7, wherein The melanocortin receptor includes any one or more of MC1R, MC2R, MC3R, MC4R, MC5R.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the diselenocyclopeptide or its analogs described in claim 1, and the analogs include derivatives of the diselenocyclopeptide, pharmaceutically acceptable salts thereof, tautomers thereof or stereoisomers thereof.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

Images