Diselenocyclopeptide, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of medicine. Provided in the present invention are a diselenocyclopeptide, and a preparation method therefor and the use thereof. The present invention develops 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 an amino acid sequence as set forth in SEQ ID NO. 1. The diselenocyclopeptide provided in the present invention acts as a ligand of one or more melanocortin receptors and can bind well to the melanocortin receptors to exert a good effect. The diselenocyclopeptide provided in the present invention can effectively reduce body weight and control blood glucose and blood lipid levels, thus showing good application prospects in the treatment of obesity, diabetes or hyperlipidemia.

Description

Diselenide cyclic peptide and preparation method and application thereof

[0001] The present application claims priority to the Chinese patent application No. 202410607198.3, filed on May 15, 2024, and entitled "Diselenide cyclic peptide and preparation method and application thereof". The entire content of the aforementioned application is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the technical field of medicine, and particularly relates to a diselenide cyclic peptide and a preparation method and application thereof. BACKGROUND

[0003] The secretion of melanocortins is regulated by hormones such as ghrelin, leptin and insulin. At present, a variety of melanocortin polypeptide analogs have been developed as potential drugs for treating skin diseases, obesity, anorexia and type 2 diabetes, etc. Melanocortins are a class of endogenous polypeptide hormones composed of adrenocorticotropic hormone (ACTH) and three melanocyte-stimulating hormones (alpha-MSH, beta-MSH and gamma-MSH), which participate in physiological activities such as 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, which belongs to the G-protein coupled receptor family. MC1R is the receptor of MSH and ACTH. The activity of MC1R is mediated by G-protein, which activates adenylate cyclase. MC1R receptors are found in melanocyte and adrenal cortex tissues, and in a variety of other tissues such as adrenal glands, white blood cells, lung lymph nodes, ovaries, testes, pituitary glands, placenta, spleen and uterus. MC2R, also known as adrenocorticotropic hormone receptor (ACTH-R), is a transmembrane protein of 297 amino acids found in melanocyte and adrenal cortex tissues. MC2R mediates the adrenocortical effect of ACTH. In humans, MC3R is a 360 AA protein found in brain tissue, and in mice and rats MC3R is a 323 AA protein. MC4R is a 332 amino acid membrane protein expressed in the brain as well as in placental and intestinal tissues. MC5R is a 325 amino acid transmembrane protein expressed in the adrenal glands, stomach, lungs and spleen and at very low levels in the brain. MC5-R is also expressed in the three-layer adrenal cortex, mainly in the globular band cells that produce aldosterone.

[0005] Melanocortin receptors (MC1R-MC5R) have different roles in the regulation of physiological processes, MC1R is mainly expressed in melanocytes, melanoma cells and immune cells, controls skin and hair color, regulates immune response. MC2R is mainly located in the adrenal cortex, is a key component of the hypothalamic-pituitary-adrenal axis, stimulates the biosynthesis of glucocorticoids. MC4R is called neural MCR due to its high expression in various brain regions including hypothalamus, brainstem and cortex, MC4R is involved in energy homeostasis, feeding and sexual behavior and male erectile function. In addition to MC4R, MC3R is also widely distributed in the brain, mainly expressed in the hypothalamus, mainly in the arcuate nucleus and ventromedial hypothalamus, these structures are involved in the regulation of energy homeostasis, metabolism and appetite. Many studies have shown that MC3R and MC4R can independently play a role, and play a complementary rather than redundant role 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.

[0006] At present, a variety of melanocortin polypeptide analogs have been developed as potential drugs for treating skin diseases, obesity, anorexia and type 2 diabetes and other diseases. 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 times. As a targeted therapy, setmelanotide restores the function of the impaired MC4R pathway, reestablishes energy expenditure and appetite control in patients with rare genetic obesity diseases, reduces hunger and reduces body weight. However, setmelanotide also has certain side effects, including injection site reactions, skin pigmentation (dark patches of skin darker than surrounding skin), headaches and gastrointestinal side effects (such as nausea, diarrhea and abdominal pain), etc. Adverse reactions of the reproductive system also occur during treatment. Depression and suicidal thoughts also occur during the use of setmelanotide.

[0007] Therefore, it is still necessary to develop a melanocortin receptor ligand with better selectivity for melanocortin receptors (MC1R-MC5R), better stability and fewer side effects. SUMMARY

[0008] To solve the above problems, the application provides a diseleno ring peptide as well as a preparation method and application thereof. The application first develops a diseleno ring peptide, i.e., Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&Sec Bridge), namely, compound WP302, which has an amino acid sequence as shown in SEQ ID NO. 1. The diseleno ring peptide provided by the application can be used as a ligand of one or more melanocortin receptors, and can be well combined with the melanocortin receptors to produce a good effect. The diseleno ring peptide provided by the application can effectively reduce body weight, control blood glucose and blood lipid levels, and has a good application prospect in the treatment of obesity or diabetes.

[0009] Terms and abbreviations:

[0010] To achieve the above application purposes, the technical solutions of the application are as follows:

[0011] In one aspect, the application provides a diseleno ring peptide, which is compound WP302. Specifically, the diseleno ring peptide has the following structure:

[0012] Specifically, the diseleno ring peptide is Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&Sec Bridge).

[0013] Specifically, the diseleno ring peptide has an amino acid sequence as shown in SEQ ID NO. 1.

[0014] Specifically, the molecular formula of the diseleno ring peptide is C 49 H 66 N 18 O9Se2.

[0015] In another aspect, the application provides a preparation method of the above diseleno ring peptide, which comprises synthesizing a coupling resin by using an Fmoc chemical synthesis method or a Boc synthesis method.

[0016] Specifically, the preparation method comprises synthesizing a coupling resin by using an Fmoc chemical synthesis method, and sequentially coupling corresponding amino acids from a C-terminal to an N-terminal according to a peptide chain sequence.

[0017] Specifically, the resin used in the Fmoc chemical synthesis method includes but is not limited to Wang-resin, CTC-resin, Rink amide-resin or Sieber-resin.

[0018] Further specifically, the resin used in the Fmoc chemical synthesis method is Rink amide-resin.

[0019] Preferably, the resin used in the Fmoc chemical synthesis method is Rink Amide MDHA resin.

[0020] Further preferably, the initial substitution degree of the Rink Amide MDHA resin is 0.4-0.5 mmol / g.

[0021] In further preferably, the initial substitution degree of the Rink Amide MDHA resin is 0.451 mmol / g.

[0022] Specifically, the Fmoc chemical synthesis method selects Fmoc-protected amino acids for the amino part of the amino acid.

[0023] 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.

[0024] Specifically, the coupling system used in the coupling is HOBt / DIC.

[0025] Specifically, the preparation method further comprises the following steps:

[0026] (1) treating the coupled resin with a cleavage solution, precipitating, washing, and drying to obtain a crude polypeptide;

[0027] (2) oxidizing, purifying, salt-exchanging, and lyophilizing the crude polypeptide to obtain a pure diselenoic cyclic peptide.

[0028] Preferably, the cleavage solution in step (1) comprises one or more of TFA, phenol, EDT, H2O, and thioanisole.

[0029] Further preferably, the cleavage solution is TFA: phenol: EDT: H2O: thioanisole = 85-95: 1-5: 1-5: 1-5: 1-5.

[0030] Still further preferably, the cleavage solution is TFA: phenol: EDT: H2O: thioanisole = 87.5-90: 2.5-3: 2.5-3: 2.5-3: 1-5.

[0031] Still further preferably, the cleavage solution is TFA: phenol: EDT: H2O: thioanisole = 87.5: 2.5: 2.5: 2.5: 5.

[0032] Preferably, the purification in step (2) is performed by reverse phase high performance liquid chromatography using C18 packing as the stationary phase of the chromatographic column, and 0.1% TFA aqueous solution as the mobile phase A phase, and 0.1% TFA in 80% acetonitrile and 20% water as the B phase.

[0033] Further preferably, the purification in step (2) is performed by first using a linear gradient of 78% A phase: 22% B phase, and then using a linear gradient of 48% A phase: 52% B phase to elute the column for 1 hour.

[0034] Preferably, the salt exchange purification in step (2) is performed by reverse phase high performance liquid chromatography using C18 packing as the stationary phase of the chromatographic column, and 0.5% HAc aqueous solution as the mobile phase A phase, and 0.5% HAc in 80% acetonitrile and 20% water as the B phase.

[0035] Further preferably, the salt exchange purification in step (2) is performed by first using a linear gradient of 95% A phase: 5% B phase, and then using a linear gradient of 35% A phase: 65% B phase to elute the column for 1 hour.

[0036] In another aspect, the present application provides the use of the above-mentioned diselenide cyclic peptide in the preparation of a medicament.

[0037] Specifically, the medicament includes but is not limited to 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, hyperlipidemia, cancer, cancerous hyperplasia, reproductive system diseases, peripheral or central nervous system diseases, cardiovascular diseases, or respiratory system diseases.

[0038] Preferably, the acute or chronic inflammatory diseases include but are not limited to systemic inflammation, inflammatory bowel disease, brain inflammation, sepsis, septic shock.

[0039] 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.

[0040] Preferably, the organs of transplant rejection include but are not limited to kidney, heart, liver, pancreas and islets, parathyroid, heart-lung, bone marrow, cornea.

[0041] Preferably, the metabolic disease associated with weight gain includes, but is not limited to, obesity, eating disorders, Prader-Willi syndrome.

[0042] Preferably, the metabolic disease associated with weight loss includes, but is not limited to, anorexia, bulimia, AIDS wasting, cancer cachexia, wasting in the frail elderly.

[0043] Preferably, the diabetic complications include, but are not limited to, diabetic nephropathy, diabetic retinopathy, diabetic cataracts, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease, diabetic neuropathy.

[0044] Preferably, the cancer includes, but is not limited to, dermatofibroma, neuroblastoma, rectal cancer, colon cancer, familiar adenomatous polyposis and hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urological cancer, melanoma, brain tumor, lymphoma, head and neck cancer, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myelocytic 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, myoma, liposarcoma, fibrosarcoma, Ewing's sarcoma and plasmacytoma.

[0045] Preferably, the cancerous proliferation includes, but is not limited to, squamous epithelial dysplasia, glandular epithelial dysplasia.

[0046] Preferably, the reproductive system disease includes, but is not limited to, abnormal urination, pyuria, abnormal urethral discharge, pain, mass, sexual dysfunction, male infertility, female infertility, polycystic ovary syndrome, menstrual disorder, dysmenorrhea, abnormal pregnancy, uterine disease.

[0047] Preferably, the peripheral or central nervous system disease includes, but is not limited to, depression, bipolar depression or manic depression, 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.

[0048] Preferably, the cardiovascular disease includes, but is 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.

[0049] Preferably, the respiratory system disease includes, but is 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, and cor pulmonale.

[0050] Further preferably, the drug is a drug for treating obesity or diabetes or hyperlipidemia.

[0051] Specifically, the application is achieved by binding of the diselenide cyclic peptide to melanocortin receptors.

[0052] Preferably, the melanocortin receptors include any one or more of MC1R, MC2R, MC3R, MC4R, and MC5R.

[0053] In another aspect, the present application provides a pharmaceutical composition comprising the above-mentioned diselenide cyclic peptide or its analog.

[0054] Specifically, the analog includes, but is not limited to, a derivative of the diselenide cyclic peptide, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a stereoisomer thereof.

[0055] Specifically, the analog further 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).

[0056] Preferably, the Ac-Arg-Cys-DAla-His-DPhe-Arg-Trp-Sec-NH2(Cys&Sec Bridge) has an amino acid sequence as shown in SEQ ID NO. 3, and has the following structure:

[0057] Preferably, the Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Cys-NH2(Sec&Cys Bridge) has an amino acid sequence as shown in SEQ ID NO. 4, and has the following structure:

[0058] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0059] The positive and beneficial effects of the present application are:

[0060] The present application first prepares a diseleno ring peptide, the diseleno ring peptide is Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec&Sec Bridge), namely compound WP302, having an amino acid sequence as shown in SEQ ID NO. 1. The diseleno ring peptide provided by the present application is a ligand of one or more melanocortin receptors, and can be well combined with the melanocortin receptor to produce a good effect. The diseleno ring peptide provided by the present application can effectively reduce body weight, control blood glucose and blood lipid levels, and has shown good application prospects in the treatment of obesity or diabetes, hyperlipidemia. BRIEF DESCRIPTION OF DRAWINGS

[0061] Fig. 1 is a HPLC chromatogram of compound WP302.

[0062] Fig. 2 is an electrospray ionization mass spectrum of compound WP302.

[0063] Fig. 3 is a nuclear magnetic resonance spectrum 1 of compound WP302.

[0064] Fig. 4 is a nuclear magnetic resonance spectrum 2 of compound WP302.

[0065] Fig. 5 is a HPLC chromatogram of compound WP300.

[0066] Fig. 6 is an electrospray ionization mass spectrum of compound WP300.

[0067] Fig. 7 is a nuclear magnetic resonance spectrum 1 of compound WP300.

[0068] Fig. 8 is a nuclear magnetic resonance spectrum 2 of compound WP300.

[0069] Fig. 9 is a body weight change result.

[0070] Fig. 10 is a blood glucose determination result.

[0071] Fig. 11 is a blood glucose change rate.

[0072] Fig. 12 is a TC determination result. DETAILED DESCRIPTION

[0073] The present application will be further described in detail below in combination with specific examples, and the following examples are not used to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0074] Example 1 Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec & Sec Bridge) SEQ ID NO.1

[0075] The title peptide was synthesized using a fluorenylmethoxycarbonyl (Fmoc) chemical method in a 100 mL reactor. Rink Amide 4-methyldiphenylmethylamine (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-Sec(Mob)-OH, Fmoc-D-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-D-Phe-OH, and Fmoc-Trp(Boc)-OH. Synthesis was carried out on a 0.2 mmol scale. The Fmoc groups were removed by treatment with 20% piperidine in N,N-dimethylformamide (DMF) for 30 min. In each coupling step, Fmoc amino acids (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 cyclic reaction 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 Fmoc amino acids for 1 h in the presence of DIC and HOBT. 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, it was acetylated 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 to obtain a dry resin.

[0076] 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. The precipitate was collected by centrifugation. The precipitate was washed three times with diethyl ether and centrifuged to obtain the final precipitate, which was then vacuum dried to obtain the crude peptide.

[0077] The crude peptide was dissolved in 20% acetonitrile in water and then 10 g / L iodomethane solution was added dropwise to the solution for oxidative bridging. The addition of iodine solution was stopped when the solution changed from colorless to light yellow. After 3 min, VC was added dropwise to the solution. The addition of VC was stopped when the solution changed from light yellow to colorless. At this point, the oxidation was completed. The crude product after oxidation was purified on a reverse phase preparative HPLC system using a Luna C18 100A (25.4*250 mm) (Hanbon NP7000) column with a linear gradient of 78% A:22% B to 48% A:52% B over 1 h, where A was 0.1% TFA in water and B was 0.1% TFA in 80% acetonitrile and 20% water. The obtained TFA salt product of more than 90% was exchanged to Ac salt on a reverse phase preparative HPLC system using a Luna C18 100A (25.4*250 mm) (Hanbon NP7000) column with a linear gradient of 95% A:5% B to 35% A:65% B over 1 h, where A was 0.5% HAc in water and B was 0.5% HAc in 80% acetonitrile and 20% water. The qualified product was lyophilized to obtain 25 mg of white solid with 95%, and the yield was 23.5%, i.e., compound WP302.

[0078] The structure of compound WP302 is shown below:

[0079] Electrospray ionization mass spectrometry (ESI-MS) analysis gave a molecular weight of 1211.3 (consistent with the theoretical molecular weight of 1211.1), as shown in FIG. 2. The nuclear magnetic resonance spectrum of compound WP302 is shown in FIGS. 3-4.

[0080] Comparative Example 1 Ac-Arg-Cys-DAla-His-DPhe-Arg-Trp-Cys-NH2(Cys & Cys Bridge) SEQ ID NO. 2

[0081] The title peptide was synthesized in a 5 L reactor using fluorenylmethyloxycarbonyl (Fmoc) chemistry. Rink Amide 4-methylbenzhydrylamine (MBHA) resin (0.451 mmol / g substitution) was used as the solid support. Fmoc-D-Phe-OH, and Fmoc-Trp(Boc)-OH. The synthesis was performed on a 50 mmol scale. Fmoc groups were removed by treatment with 20% piperidine in N,N-dimethylformamide (DMF) for 30 min. In each coupling step, Fmoc amino acids (3 eq, 150 mmol), N,N-diisopropylcarbodiimide (DIC) (3 eq, 150 mmol), and 1-hydroxy-benzotriazole (HOBT) (3 eq, 150 mmol) in DMF were used for coupling. The following cycle was performed on the resin in the reactor: (1) washing with DMF, (2) removal of the Fmoc protecting group by treatment with 20% piperidine in DMF for 30 min, (3) washing with DMF (4) coupling with Fmoc amino acid in the presence of DIC and HOBT for 1 h. The resin was successfully coupled according to the sequence of the title peptide. After assembly of the peptide chain and removal of the last Fmoc protecting group, acetylation was performed with a solution of acetic anhydride, N-methylmorpholine (NMM), and DMF (v / v / v: 6 / 10 / 84). The dry resin was obtained by extensive washing of the resin with dichloromethane (DCM) and methanol, followed by suction-drying.

[0082] For cleavage of 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) for 3 h at room temperature. The resin was filtered off and the filtrate was poured into diethyl ether, and the precipitate was collected by centrifugation. The centrifuged precipitate was washed with diethyl ether 3 times, and the final precipitate was obtained and vacuum-dried to give the crude peptide.

[0083] The crude peptide was dissolved in 0.1% TFA in water and then oxidized by the dropwise addition of 10 g / L iodomethanol solution. The addition was stopped when the solution changed from colorless to light yellow. After 3 min, the solution was added with VC dropwise until the solution changed from light yellow to colorless. The oxidation was completed. The crude product was purified on a preparative HPLC system using a Luna C18 100A (650*650*2350mm) (Varian) column with a linear gradient of 80% A:20% B to 50% A:50% B over 1 hour, where A was 0.1% TFA in water and B was 0.1% TFA in 80% acetonitrile and 20% water. The TFA salt product was exchanged to Ac salt on a preparative HPLC system using a Luna C18 100A (650*650*2350mm) (Varian) column with a linear gradient of 100% A:0% B to 40% A:60% B over 1 hour, where A was 0.5% HAc in water and B was 0.5% HAc in 80% acetonitrile and 20% water. The product was lyophilized to give 21635 mg of white solid, 95%, with a yield of 34.7%, i.e., compound WP300.

[0084] The structure of compound WP300 is shown below:

[0085] Electrospray ionization mass spectrometry (ESI-MS) analysis gave a molecular weight of 1117.4 (consistent with the theoretical molecular weight of 1117.3), as shown in Figure 6. The nuclear magnetic resonance spectrum of compound WP302 is shown in Figures 7-8.

[0086] Experimental Example 1 Serum Stability Assay

[0087] 1. Add 594 μL of serum to a 96-well plate and pre-incubate at 37°C for 5 minutes.

[0088] 2. Add 6 μL of test article / positive control to the 96-well plate and mix well by pipetting. Continue to react at 37°C with constant shaking for 48 hours.

[0089] 3. For test articles, at 0, 0.5, 1.5, 3, 6, 24, and 48 hours, remove 50 μL and add to 150 μL of 0.1% FA (formic acid) in methanol containing an internal standard. For positive controls, at 0, 15, 30, 60, and 120 minutes, remove 50 μL and add to 150 μL of 0.1% FA in methanol containing an internal standard.

[0090] 4. Shake mix for 10 minutes, then centrifuge at 6000 x g for 10 minutes. Inject the supernatant into the LC-MS / MS system for analysis.

[0091] The results of the assay are shown in Table 1:

[0092] Table 1. Results of serum stability assay

[0093] The serum stability of the compounds of the application was tested using the above assay. The results of the assay show that the compound WP302 provided by the application has an improved serum stability with an increase of 11.34% in the half-life in rat serum compared to WP300.

[0094] Experimental Example 2 EC 50 Assay

[0095] Intracellular cyclic AMP (cAMP) levels were determined by an electrochemiluminescence (ECL) assay (Meso Scale Diagnostics, Gaithersburg, MD; hereinafter referred to as MSD).

[0096] 1. Take out the cell MCI, MC3, MC4 and MC5 stable cell lines from the liquid nitrogen storage system, quickly thaw in a 37°C electric thermostatic water tank, and then transfer the cell suspension to a 15 mL centrifuge tube with a pipette and add 10 mL of complete medium.

[0097] 2. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant, resuspend the cell pellet with 5 mL of complete medium, and then transfer it to a T75 culture flask, add 15 mL of medium, and place it in a 37°C, 5% carbon dioxide incubator for culture. The cells are passaged once and used for the cell experiment.

[0098] 3. When the cell density reaches 80%-90%, discard the culture medium and wash the cells with 5 mL of phosphate buffer. Remove the phosphate buffer and 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 for 4 min, and discard the supernatant.

[0099] Adjust the cell suspension to the appropriate density with Stimulation Buffer. MCI cells, MC3 cells and MC5 cells are 1000 cells / well. MC4 cells are 2000 cells / well.

[0100] ​Take 10 μL of cell solution to the detection plate. Centrifuge at 600 rpm for 3 minutes on the centrifuge, and incubate at room temperature for 60 minutes. After incubation, add 5 μL of Eu-cAMP tracer and 5 μL of ULight™-anti-cAMP solution in the cAMP detection kit to the experimental plate. Centrifuge again at 600 rpm for 3 minutes, and incubate at room temperature for 60 minutes.

[0101] Read the cAMP signal on the multifunctional enzyme label meter, process and analyze the data with GraphPad Prism 6, and report as EC50 values. The experimental results are shown in Tables 2-5.

[0102] Table 2 MC1 agonist experiment

[0103] Table 3 MC3 agonist experiment

[0104] Table 4 MC4 agonist experiment

[0105] Table 5 MC5 agonist experiment

[0106] The compound WP302 provided by the present application binds to the melanocortin receptor to produce a cellular level of effect, which is measured by the intracellular cyclic adenosine monophosphate (cAMP) level. EC 50 The concentration of the agonist compound required to obtain 50% of the maximum response of the reaction is represented. The determination results show that WP302 has a good effect on the melanocortin receptor.

[0107] Pharmacokinetic study of subcutaneous injection of WP302 in DIO model mice

[0108] This experimental example studies the pharmacokinetic behavior of compound WP302 in male DIO model mice after subcutaneous injection of compound WP302 for 30 consecutive days.

[0109] 1. Experimental animals

[0110] This experiment uses 6 male SPF obese and diabetic model (DIO model) mice, 20-22 weeks old, weighing more than 38g, purchased from Changzhou Kaivens Experimental Animal Co., Ltd., and the facility experimental animal use license number is SYXK(Su)2022-0002. Before starting the experiment, the animals will be adapted to the laboratory for at least 3 days. After the start of the experiment, the animals are free to eat and drink, and the blood glucose is tested after fasting. Fasting starts around 8 am, and fasting lasts about 8 hours. Blood glucose is measured around 4 pm as a baseline.

[0111] 2. Test product information

[0112] The test product information is shown in Table 6:

[0113] Table 6 Test product information

[0114] 3. Preparation of administration solution

[0115] The administration solution will be prepared in advance with stability data support and stored at 2-8°C before administration and used within 3 days after preparation. An appropriate amount of test product is accurately weighed; 1x PBS of calculated volume is added to completely dissolve the test product; and the administration solution is obtained by thoroughly stirring and mixing.

[0116] 4. Grouping and administration dose

[0117] Two days before administration is recorded as Day-1, one day before administration is recorded as Day 0, and the administration day is recorded as Day 1; the animals are evenly grouped according to the body weight on Day-1. The mice are administered according to the administration design described in Table 7.

[0118] Table 7 Animal grouping and administration dose Note: QD in the table represents administration once a day.

[0119] 5. Test index

[0120] 5.1 Body weight test and food intake monitoring

[0121] The weight is recorded before administration each day and used to calculate the administration volume. The body weight of the mice in each group from Day 1 to Day 37 is recorded. The body weight change of the mice in each group is shown in FIG. 9, and the results show that the compound WP302 can effectively reduce the body weight of the DIO model mice.

[0122] 5.2 Blood glucose test

[0123] The blood glucose test is performed before administration. The mice are fasted at about 8:00 am on the day of the blood glucose test, and blood samples are collected for testing at about 4:00 pm. After the test, the mice are fed again. The tail tip blood of the mice is collected for blood glucose test on Day 0, Day 7, Day 14, Day 21, Day 28, and Day 31. The collected blood samples are placed on wet ice for more than 30 minutes, and then centrifuged. After the whole blood is collected, the serum is obtained by centrifugation at 3500g, 4°C for 5 minutes after waiting for more than 30 minutes.

[0124] The blood glucose test results are shown in FIG. 10, and the blood glucose change rate is shown in FIG. 11. The results show that the compound WP302 can effectively reduce the blood glucose level of the DIO model mice and has a hypoglycemic effect.

[0125] 5.3 Total cholesterol (TC) and triglyceride (TG) test

[0126] Blood samples collected prior to fasting were placed on wet ice for 30 minutes or more before centrifugation. After collection of whole blood, serum was obtained for testing after waiting 30 minutes or more using 3500g, 4°C, centrifugation for 5 minutes.

[0127] The results of the TC assay are shown in Figure 12, which shows that compound WP302 effectively reduced total cholesterol levels.

[0128] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application shall be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A diseleno cyclotide, characterized in that, The diselenide cyclic peptide is Ac-Arg-Sec-DAla-His-DPhe-Arg-Trp-Sec-NH2 (Sec & Sec Bridge) having the following structure:

2. The method of claim 1, wherein the diseleno cyclized peptide is prepared by the steps of: The preparation method comprises synthesizing a coupling resin by using an Fmoc chemical synthesis method or a Boc synthesis method.

3. The production method according to claim 2, characterized by, The resin used in the Fmoc chemical synthesis method comprises Wang-resin, CTC-resin, Rink amide-resin or Sieber-resin.

4. The production method according to claim 2, characterized by, The Fmoc chemical synthesis method selects Fmoc-protected amino acids to protect the amino part.

5. Use of the diselenide cyclic peptide according to claim 1 for the preparation of a medicament, characterized in that, The drug comprises a drug for treating or preventing acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection, 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.

6. Use according to claim 5, characterized in that, The drug is a drug for treating obesity or diabetes or hyperlipidemia.

7. Use according to claim 6, characterized in that, The application is achieved by binding of the diselenide cyclic peptide to a melanocortin receptor.

8. Use according to claim 7, characterized in that, The melanocortin receptor comprises any one or more of MC1R, MC2R, MC3R, MC4R and MC5R.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the diselenide cyclic peptide or an analog thereof of claim 1, wherein the analog comprises a derivative of the diselenide cyclic peptide, a pharmaceutically acceptable salt thereof, a tautomer thereof or a stereoisomer thereof.

10. The pharmaceutical composition of claim 9, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

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