Preparation and application of an el a-11 cyclic peptide analogue

Abstract

The application belongs to the technical field of biochemistry, and particularly relates to preparation and application of an ELA-11 cyclic peptide analogue. The structure of the ELA-11 cyclic peptide analogue is R1-R2-R3-Pro-Leu-His-Ser-Arg-Val-Hyp-dAla-Cys, wherein R1 is a fatty acid, R2 is a connecting arm containing gammaGlu and / or AEEA, R3 is Cys-Nle or Ala-Cys, and the Cys of R3 and the C-terminal Cys are covalently bonded to form a ring through a side chain mercapto group. The application studies the biological activity of the ELA-11 cyclic peptide analogue, including constructing an in-vitro cell model and an in-vivo animal model of kidney fibrosis to evaluate the in-vitro anti-fibrosis activity and inhibitory effect and in-vivo therapeutic effect of the ELA-11 cyclic peptide analogue, and studying the cytotoxicity, serum stability and pharmacokinetics of the ELA-11 cyclic peptide analogue. The results show that, compared with the ELA-11 linear peptide analogue E28, the ELA-11 cyclic peptide analogue obtained in the application is better in all aspects of activity index and stability, and can be used for preparing a drug for preventing kidney fibrosis diseases.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to the preparation and application of an ELA-11 cyclic peptide analog. Background Technology

[0002] Renal fibrosis is a common terminal pathway in the progression of various chronic kidney diseases to end-stage renal failure, characterized by myofibroblast activation, excessive extracellular matrix deposition, and tubulointerstitial atrophy. However, there are currently no approved drugs for treating renal fibrosis, and clinical management primarily focuses on etiological control and indirect delay of disease progression, lacking effective treatment options. Therefore, the development of novel and effective drugs for treating renal fibrosis is of great significance in clinical practice.

[0003] ELA-11 (CMPLHSRVPFP) is a short active fragment of the APJ receptor agonist peptide ELABELA. It exerts an anti-renal fibrosis effect in a renal ischemia-reperfusion injury model by inhibiting the expression of fibrosis-related genes, collagen deposition, and inflammatory responses. However, the rapid metabolism of ELA-11 in vivo severely limits its clinical application. Existing technologies have designed and modified ELA-11 analogs, such as Pal-E11, which improve in vitro mouse serum stability, but still suffer from rapid in vivo metabolism, high dosing frequency, low bioavailability, and the need for further improvement in efficacy. Summary of the Invention

[0004] To address the above problems, the present invention aims to provide an ELA-11 cyclic peptide analog, its preparation method, and its application in the preparation of medicaments for the prevention and / or treatment of renal fibrosis. Specifically, the ELA-11 cyclic peptide analog provided by the present invention exhibits significantly superior renal protective effects compared to unmodified ELA-11 in a mouse renal ulcerative fibrosis model, and the dosing frequency is reduced from twice daily to twice weekly, with significantly improved drug-likeness.

[0005] According to a specific embodiment of the present invention, an ELA-11 cyclic peptide analog has the following general structural formula:

[0006] R1-R2-R3-Pro-Leu-His-Ser-Arg-Val-Hyp-dAla-Cys, where... R1 is a fatty acid, R2 is a linker arm containing γGlu and / or AEEA, and R3 is Cys-Nle or Ala-Cys. The C-terminal Cys and the R3 Cys side chain thiol groups are covalently cyclized.

[0007] The ELA-11 cyclic peptide analog of the present invention is formed by coupling a fatty acid to the N-terminus of ELA-11 via a linker arm containing γGlu and / or AEEA, and cyclizing the Cys at the C-terminus and Cys of R3. Preferably, the fatty acid contained in R1 is a C6 to C30 fatty acid, more preferably R1 is a C10 to C20 straight-chain saturated fatty acid diacid.

[0008] Preferably, R2 is a connecting arm containing γGlu and / or AEEA, more preferably γGlu-AEEA-AEEA.

[0009] Preferably, R3 is Cys-Nle or Ala-Cys, more preferably Cys-Nle.

[0010] Preferably, the C-terminal Cys and the Cys of R3 are cyclized by a disulfide bond or a C4-C10 alkyl chain, more preferably the C-terminal Cys and the Cys of R3 are cyclized by a disulfide bond.

[0011] The "alkyl chain" in this invention is preferably composed of the following groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, cyclopentyl, n-hexyl, neohexyl, n-heptyl, n-neoyl, n-nonyl, n-decyl, etc.

[0012] In one specific implementation, R1 is a C20 straight-chain saturated fatty diacid, R2 is γGlu-AEEA-AEEA, and R3 is Cys-Nle, with the side chain thiol groups of the two Cys forming a disulfide bond (i.e., E48).

[0013] The ELA-11 cyclic peptide analog according to a specific embodiment of the present invention has the following structure: .

[0014] This invention links cysteine ​​residues in the peptide chain via disulfide bonds, thereby increasing peptide rigidity and enhancing enzymatic resistance, while maintaining the active conformation in the ELA-11-APJ molecular docking result, improving stability while preserving anti-fibrotic activity as much as possible; after cyclization, this invention couples the original N-terminal free amino group to a long-chain fatty acid via a hydrophilic flexible linker, improving the in vivo metabolic stability of the cyclic peptide.

[0015] The ELA-11 cyclic peptide analogs screened through chemical modification in this invention have more stable in vitro and in vivo stability and superior biological activity against renal fibrosis. Compared with the polypeptide ELA-11, the dosing frequency is significantly reduced while retaining the anti-renal fibrosis activity, and the drug potential is enhanced. Compared with small molecule compounds, the analogs are less toxic and have a larger safety window. Compared with large molecule proteins, the preparation cost is more economical and the scalability is higher.

[0016] Another aspect of the present invention provides the use of the above-mentioned ELA-11 cyclic peptide analog in the preparation of a medicament for treating renal fibrosis. The ELA-11 cyclic peptide analog of the present invention has an ameliorative effect on renal fibrosis and can be used to directly or indirectly treat conditions characterized by renal fibrosis. Renal fibrosis includes obstructive nephropathy, diabetic nephropathy, nephrosclerosis, nephritis, renal tumors, and renal fibrosis caused by various factors such as unhealthy lifestyle habits, adverse drug reactions, and pathogenic microbial infections.

[0017] In another aspect, this invention provides the ameliorative effect of the key intermediate E28 of the cyclic peptide on renal fibrosis. E28 is a key intermediate for further obtaining the long-acting cyclic peptide E48, and it also has independent application value (it can be used directly as a therapeutic drug or as a parent compound for further modification). Compared with natural ELA-11, E28, through the introduction of non-natural amino acids such as Nle replacing Met and D-Ala replacing Pro, has an approximately 16-fold increase in serum half-life (see Example 6, Table 5), and its anti-fibrotic activity is maintained or even enhanced (see Example 7). Figure 8 ).

[0018] Another aspect of the present invention is to provide a pharmaceutical composition comprising, as a therapeutically effective amount of any of the above-mentioned chemically modified ELA-11 cyclic peptide analogs and / or E28 as the main components, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0019] Preferably, the pharmaceutical composition is used for the prevention and / or treatment of renal fibrosis.

[0020] The amount of the polypeptide analog in the pharmaceutical composition of the present invention can vary within a wide range, and those skilled in the art can easily determine it based on objective factors such as the type of disease, the severity of the disease, the patient's weight, the dosage form, and the route of administration.

[0021] This invention provides a method for preparing the above-mentioned ELA-11 cyclic peptide analog, comprising the following steps: Step 1: Using the Fmoc solid-phase synthesis method, the linear polypeptide resin corresponding to the ELA-11 cyclic peptide analog is synthesized from the carboxyl terminus to the amino terminus. The resin is then cleaved, and the cleavage solution containing the ELA-11 linear analog is collected. Step 2: Purify the cutting fluid obtained in Step 1 and freeze-dry it to obtain the ELA-11 linear analog solid; Step 3: Dissolve the linear analogue of ELA-11 obtained in Step 2 in a solvent prepared according to AcOH:TFE:DCM (1:2:7), and add it dropwise to an iodine solution dissolved in the same solvent; mix well, and stir at room temperature in the dark for 10 min. Step 4: Purify the reaction solution obtained in step 3 to obtain the ELA-11 cyclic peptide analog.

[0022] Preferably, in step 1, the resin used in the Fmoc solid-phase synthesis method is 2-chlorotrimethylbenzene resin.

[0023] Preferably, in step 1, the cutting fluid is composed of TFA:Tis:H2O in a volume ratio of 95:2.5:2.5.

[0024] Preferably, the amount of iodine used in step 3 is 4-10 times the molar amount of the ELA-11 linear analog.

[0025] The beneficial effects of this invention are: This invention provides an ELA-11 cyclic peptide analog that, compared to ELA-11, exhibits significantly improved in vitro serum stability, a slower in vivo clearance rate, and enhanced in vitro and in vivo anti-fibrotic effects. This demonstrates that the ELA-11 cyclic peptide analog of this invention can be used to prepare medicaments for the prevention and / or treatment of renal fibrosis. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The survival rate of mouse embryonic fibroblasts (NIH-3T3) with the mother peptide ELA-11.

[0028] Figure 2 Survival rate of E16-E29 against NIH-3T3.

[0029] Figure 3 Survival rates of E30-E34, E39-E42, and E46-E48 against NIH-3T3.

[0030] Figure 4 The results of Western blot analysis of the effects of the precursor peptide ELA-11 on the expression of α-SMA, Vimentin, Collagen I and Fibronectin in the NIH-3T3 cell model and the corresponding statistical graphs are shown.

[0031] Figure 5 The survival rate of human renal tubular epithelial cells (HK-2) with the precursor peptide ELA-11.

[0032] Figure 6The survival rate of HK-2 cells for lipotropic cyclic peptides E46, E47, and E48.

[0033] Figure 7 The results of Western blot analysis of the effects of the parent peptide ELA-11 on the expression of α-SMA, Vimentin, Collagen I and Fibronectin in the HK-2 cell model and the corresponding statistical graphs are shown.

[0034] Figure 8 The effects of ELA-11 linear analogues and peptide ELA-11 on the expression of α-SMA and Collagen I in the NIH-3T3 cell model, as detected by Western blot, and the corresponding statistical graphs.

[0035] Figure 9 The effects of linear peptide E28 and polypeptide ELA-11 on the expression of α-SMA, Vimentin, Collagen I and Fibronectin in mouse kidneys, as detected by Western blot, and their corresponding statistical graphs are shown.

[0036] Figure 10 The effects of linear peptide E28 and polypeptide ELA-11 detected by qPCR on the expression of α-SMA, Vimentin, Collagen I and Fibronectin in mouse kidneys and their corresponding statistical graphs are shown.

[0037] Figure 11 The results of H&E and immunohistochemical staining of kidney tissue sections from mice in each group 7 days after administration of linear peptide E28.

[0038] Figure 12 The results of Western blot analysis of the effects of ELA-11 cyclic peptide analogs on the expression of α-SMA and Collagen I in the NIH-3T3 cell model and their corresponding statistical graphs are shown.

[0039] Figure 13 The effects of lipotropic cyclic peptides E46, E47, and E48 on the expression of α-SMA and Collagen I in a mouse NIH-3T3 cell model, as detected by Western blot, and the corresponding statistical graphs are shown.

[0040] Figure 14 The effects of lipotropic cyclic peptides E46, E47, and E48 on the HK-2 cell model as detected by Western blot. Figure 15The effects of lipotropic cyclic peptide E48 and linear peptide E28 on the expression of α-SMA, Vimentin, Collagen I and Fibronectin in mouse kidneys, as detected by Western blot, and their corresponding statistical graphs are shown.

[0041] Figure 16 The results show the effects of lipotropic cyclic peptide E48 and linear peptide E28 on the expression of α-SMA, Vimentin, Collagen I and Fibronectin in mouse kidneys, as detected by qPCR.

[0042] Figure 17 The results of H&E and immunohistochemical staining of kidney tissue sections from mice in each group 7 days after administration of lipotropic cyclic peptide E48.

[0043] Figure 18 The in vivo half-life results for lipotropic cyclic peptide E48 and linear peptide E28 are presented. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0045] The specific meanings of the abbreviations used in this invention are as follows: dAla (abbreviated a) is D-alanine, dPhe (abbreviated f) is D-phenylalanine, Nle is ortholeucine, Hyp is hydroxyproline, HoLeu is homoleucine, Tle is tertiary leucine, Cys (abbreviated C) is cysteine, Met (abbreviated M) is methionine, Pro (abbreviated P) is proline, Leu L (abbreviated) is leucine, Ile (abbreviated I) is isoleucine, His (abbreviated H) is histidine, Ser (abbreviated S) is serine, Arg (abbreviated R) is arginine, Val (abbreviated V) is valine, Phe (abbreviated F) is phenylalanine, γGlu is γ-glutamic acid, AEEA is hydroxyethyl ethylenediamine, AcOH is glacial acetic acid, TFE is trifluoroethanol, DCM is dichloromethane, TFA is trifluoroacetic acid, Tis is triisopropylsilane, H2O is water, MeOH is methanol, and DIPEA is N,N-diisopropylethylamine.

[0046] Materials and Methods: Unless otherwise specified, the experimental conditions and methods described in the following examples are conventional conditions and methods, and the reagents or instruments mentioned are all commercially available.

[0047] Western blot results were analyzed for protein expression using EvolutionCapt software (grayscale analysis), and statistical analysis was performed using GraphPadPrism 8.0 software. Data are expressed as mean ± standard deviation (Mean ± SD), and significant differences were analyzed using one-way ANOVA and Tukey's test. Compared with the control group, ## P <0.01, # P <0.05; compared with the model group, P <0.01, P <0.05.

[0048] Example 1: Design and Synthesis of Cyclic Peptides Step 1: The linear polypeptide was synthesized using the Fmoc solid-phase synthesis method, proceeding from the carboxyl terminus to the amino terminus. The amino acid sequence is shown below: C20 diacid-γGlu-AEEA-AEEA-Cys-Nle-Pro-Leu-His-Ser-Arg-Val-Hyp-dAla-Cys (two Cys cyclize through disulfide bond formation) and Ac-Cys-Nle-Pro-Leu-His-Ser-Arg-Val-Hyp- dAla-Cys (two Cys bonds cyclize through a thioether bond at C4).

[0049] The specific synthesis steps are as follows: Step 1: Resin swelling and amino acid coupling: (1) According to the synthesis requirements, weigh 3.0 eq of dichloro resin and swell it in DCM for 30 min. After drying, wash it with DCM 3 times, 3 min each time. (2) Weigh 3.0 eq of the first amino acid, dissolve it in DCM, add 6 eq of DIPEA, mix well, pour into the resin, and stir at room temperature for 8 h. (3) After the reaction solution is dried, pour in the blocking solution (DCM:MeOH:DIPEA = 8.5:1:0.5) and block for 0.5 h; then wash with DCM and DMF 3 times each, 3 min each time. (4) The N-terminal Fmoc protecting group was removed using a deprotection solution (piperidine:DMF = 1:4), followed by thorough washing with DMF; (5) The amino acids, AEEA, and C20 diacid were then added to the resin after being activated in DMF by Fmoc-protected amino acids (3.0 eq), HBTU (3.0 eq), HOBt (3.0 eq) and DIPEA (6.0 eq). The mixture was stirred at room temperature for 1 hour to perform coupling, and the free amino groups were detected by Kaiser test.

[0050] Step 2: Cutting the peptide chain: The resin was cut for 3 hours under the conditions of cutting solution (TFA:Tis:H2O =95:2.5:2.5), the resin was filtered off, the filtrate was concentrated, precipitated with cold ether, dissolved in the aqueous phase and placed in a -80℃ refrigerator overnight, and then freeze-dried to obtain crude peptide.

[0051] Step 3: Preparation and purification of linear peptides: (1) Weigh about 50 mg of crude peptide and dissolve it in deionized water containing an appropriate amount of acetonitrile. Remove insoluble substances using a 0.22 μm oil filter before loading the sample. (2) High performance liquid chromatography uses a C18 reverse preparative column, with acetonitrile containing 0.1% TFA and deionized water as the elution solvent. The initial concentration of acetonitrile is 15%, and the proportion of acetonitrile in the elution solution is gradually increased over time to separate the sample components. (3) After injection, the absorption peak at 220 nm was detected, the main absorption peak was collected and labeled, and the target compound was identified by mass spectrometry. The centrifuge tube containing the corresponding liquid was sealed with plastic wrap, the vent hole was punched, and the tube was frozen overnight at -80℃ and then freeze-dried to obtain the linear polypeptide compound; (4) After lyophilization, a small amount of the compound was dissolved and eluted with 5%-95% acetonitrile / deionized water for 30 min on a C18 reverse analytical column. The purity was calculated by integrating the peak area of ​​the 220 nm chromatogram. The purity was >95%. The isolated product was identified by mass spectrometry and the synthesized and purified product was confirmed to be the target product.

[0052] Step 4: Preparation and purification of cyclic peptides: (1) The linear polypeptide obtained in step 3 was dissolved in a solvent prepared according to the volume ratio of AcOH:TFE:DCM of 1:2:7, and added dropwise to an iodine solution dissolved in the same solvent. 0.01 mmol of polypeptide was dissolved in 5 mL of the prepared solvent, and 0.04-0.10 mmol of iodine was dissolved in 0.5 mL of the prepared solvent. The reaction was stirred at room temperature in the dark for 10 min to obtain a disulfide bond cyclized polypeptide. The linear peptide obtained in step 3 was dissolved in 250 μL of 1M Tris-HCl (pH=9.0), and the same molar amount of the C5-C10 corresponding thiaanthraium salt was dissolved in 750 μL of DMSO. The mixture was stirred at room temperature for 1 h to obtain the C5-C10 chain alkyl cyclized peptide. Replacing Tris-HCl with Tris-H3PO4 and adding the C4 corresponding thiaanthraium salt, the reaction was carried out under the same conditions to obtain the C4 chain alkyl cyclized peptide.

[0053] The structure of thiaanthraium salts.

[0054] (2) The above reaction solution was purified by high performance liquid chromatography to obtain the ELA-11 cyclic peptide analog, E48, with the following structural formula:

[0055] The E40 structural formula is as follows: .

[0056] (3) After lyophilization, a small amount of the compound was dissolved and eluted with 5%-95% acetonitrile / deionized water on a C18 reverse analytical column for 30 min. The purity was statistically determined by integrating the peak area of ​​the chromatogram at 220 nm. The purity of the compound was >95%, and the isolated product was identified by mass spectrometry. The synthesized and purified product was confirmed to be the target product.

[0057] Table 1. Structure of the cyclic peptide E48 synthesized in Example 1, and its theoretical [M+2H] structure. + Measure [M+2H] + and purity

[0058] Table 2. Structure and theoretical [M+H] of the cyclic peptide E40 synthesized in Example 1. + Measurement [M+H] + and purity

[0059] Following a similar synthetic method to the examples described above, the compounds listed in Tables 3 and 4 were prepared: Table 3 shows the structures and theoretical [M+H] of the compounds synthesized according to Example 1. + Measurement [M+H] + and purity

[0060] Table 4 shows the structure and theoretical composition of the compound synthesized according to Example 1 [M+2H]. + Measure [M+2H] + and purity

[0061] The specific synthesis conditions (such as resin type, cleavage fluid ratio, cyclization reagent and time) described in Example 1 above are only representative embodiments of the present invention. Those skilled in the art can reasonably adjust the parameters to obtain other cyclic peptide analogs of the present invention based on the general preparation method of the present invention.

[0062] Example 2: Evaluation of ELA-11 as the test substance against NIH-3T3 cytotoxicity. The NIH-3T3 cell line was selected to study and observe the cytotoxicity of ELA-11 against NIH-3T3 cells.

[0063] NIH-3T3 cells were seeded at 4000 cells / well in 96-well plates and cultured in DMEM medium containing 10% FBS and 1% penicillin antibody at 37°C and 5% CO2 for 24 h. The old medium was discarded, and DMEM medium with ELA-11 concentrations of 0, 20, 40, 80, 160, and 320 μM was added. After incubation for 24 h, 10 μL of CCK-8 solution was added and incubated for 1.5 h. The absorbance at 450 nm was measured using a microplate reader.

[0064] Figure 1 The study showed that the survival rate of NIH-3T3 cells was not significantly different among the groups within the concentration range of 0-320 μM, indicating that ELA-11 is non-toxic to NIH-3T3 cells.

[0065] Following a similar detection method to the above embodiments, the cytotoxicity of the compounds shown in Tables 1, 3, and 4 to NIH-3T3 cells was tested. The results showed that there was no significant difference in cell survival rate among the groups within the concentration range of 0-320 μM, indicating that the above compounds (except E46-E48) were not toxic to NIH-3T3 cells. Figure 2 , 3 The results for E16-E29 and E30-E48 are shown separately. Within the concentration range of 0-80 μM, there was no significant difference in cell viability among the groups of E46-E48. However, when the drug concentration reached 160 μM, all three peptides produced significant killing effects on NIH-3T3 cells, with E48 showing the most significant killing effect.

[0066] Example 3: Detection of the activity of ELA-11 against NIH-3T3 cell in vitro fibrosis The NIH-3T3 cell line was used in the experiment to study and observe the effects of ELA-11 on the expression of α-smooth muscle actin (α-SMA), vimentin, type I collagen, and fibronectin induced by transforming growth factor TGF-β1 in NIH-3T3 cells.

[0067] NIH-3T3 cells were seeded at a rate of 200,000 cells / well in 6-well plates using DMEM medium containing 10% FBS and 1% penicillin antibiotics. The cells were cultured at 37°C and 5% CO2 for 24 h. After being replaced with serum-free DMEM medium and starved for 12 h, 5 ng / mL TGF-β1 and 1, 2.5, 5, and 10 μM ELA-11 were added and the cells were treated for 24 h. Total cell protein was extracted, and the protein expression levels of GAPDH, α-SMA, Vimentin, Collagen I, and Fibronectin were detected by Western blot.

[0068] Figure 4 After treating NIH-3T3 cells with TGF-β1 and different concentrations of ELA-11, Western blot was used to detect the protein expression of GAPDH, α-SMA, Vimentin, Collagen I and Fibronectin, and the relative expression levels of α-SMA, Vimentin, Collagen I and Fibronectin were analyzed.

[0069] Depend on Figure 4 It was found that 10 μM ELA-11 could significantly inhibit the expression of α-SMA, Vimentin, Collagen I and Fibronectin proteins.

[0070] Example 4: Evaluation of ELA-11 as the test substance against HK-2 cytotoxicity. The HK-2 cell line was selected to study and observe the cytotoxicity of ELA-11 on HK-2 cells.

[0071] HK-2 cells were seeded at a rate of 6000 cells / well in 96-well plates and cultured in DMEM / F12 medium containing 10% FBS and 1% penicillin antibody at 37°C and 5% CO2 for 24 h. DMEM medium with ELA-11 concentrations of 0, 20, 40, 80, 160, and 320 μM was added and incubated for 24 h. After incubation, 10 μL of CCK-8 solution was added and the cells were incubated for 1.5 h. The absorbance at 450 nm was measured using a microplate reader.

[0072] Figure 5The results showed that there was no significant difference in cell survival rate among the groups within the concentration range of 0-320 μM, indicating that ELA-11 is non-toxic to HK-2 cells.

[0073] Using a detection method similar to that described in the above embodiments, the cytotoxicity of compounds named E46-E48 (shown in Tables 1, 3, and 4) to HK-2 cells was evaluated. Figure 6 As shown, the results indicate that there was no significant difference in cell survival rate among the groups of E46-E48 within the concentration range of 0-160 μM, suggesting that it is non-toxic to HK-2 cells.

[0074] Example 5: Detection of the activity of ELA-11 against HK-2 cell in vitro fibrosis The HK-2 cell line was used to study and observe the effect of ELA-11 on the expression of α-SMA, Vimentin, Collagen I, and Fibronectin induced by TGF-β1 in HK-2 cells. HK-2 cells were seeded in 6-well plates with DMEM / F12 medium containing 10% FBS and 1% penicillin antibiotics and cultured at 37℃ and 5% CO2 for 24 h. After 12 h of culture in serum-free medium, 5 ng / mL TGF-β1 and 1, 2.5, 5, and 10 μM ELA-11 were added and treated for 24 h. Total cellular protein was extracted, and the protein expression levels of α-SMA, Vimentin, Collagen I, and Fibronectin were detected by Western blot.

[0075] Figure 7 After treating HK-2 cells with TGF-β1 and different concentrations of ELA-11, Western blot was used to detect the protein expression of GAPDH, α-SMA, Vimentin, Collagen I, and Fibronectin, and the relative expression levels of these proteins were analyzed. The results showed that 5 μM ELA-11 significantly inhibited the expression of α-SMA, Vimentin, Collagen I, and Fibronectin, with 10 μM showing even better inhibitory effects.

[0076] Example 6 compares the serum stability of ELA-11 linear peptide analogs and ELA-11. Blood was collected from male C57BL / 6J mice after anesthesia. After standing at room temperature for 1 h, the samples were centrifuged at 5000 rpm for 30 min, and the supernatant was collected for later use. An appropriate amount of the test sample was weighed and prepared into a 10 mM stock solution with PBS buffer. An appropriate volume of the stock solution was mixed thoroughly with 19 times the volume of mouse serum and incubated at 37°C. 75 μL samples were taken at different time points (0 h, 0.08 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 10 h) and immediately added to a pre-chilled centrifuge tube containing 75 μL of ice-cold acetonitrile. After vortexing, the samples were incubated at low temperature for 10 min and centrifuged at 15000 rpm for 15 min. The supernatant was collected for sample preparation.

[0077] Using a C18 reverse analytical column, with 5% acetonitrile / deionized water as the initial mobile phase, the acetonitrile content was increased to 95% over time for gradient elution. The target absorption peak at 220 nm was detected. The degradation rate of the test sample at different time points was determined by integrating the peak area of ​​the test sample and comparing it with the corresponding peak area at 0 min.

[0078] Table 5 shows the serum half-lives of ELA-11 and its linear peptide analogs. All linear peptide analogs had longer serum half-lives than ELA-11. The serum stability of ELA-11 was significantly improved when Met at position 2 was replaced by Nle, and Pro at position 9 was replaced by D-type Ala or D-type Phe. Among the obtained compounds, E25 and E28 showed the most significant increase in serum half-life, approximately 16 times that of ELA-11.

[0079] Table 5 shows the serum half-life of the compounds as determined in Example 6.

[0080] Following a similar detection method to the above embodiments, the activity of ELA-11 cyclic peptide analogs was evaluated, with sampling time points changed to 0 h, 12 h, 24 h, 36 h, 48 h, and 72 h. The results are shown in Table 6. The experimental results indicate that the shorter the cyclization linker, the longer the half-life, and the stability improvement of disulfide-bonded cyclized peptides is more significant than that of alkyl-bonded peptides. Disulfide-bonded cyclized peptides E32 and E39 exhibited the best serum stability, with half-lives reaching 96.40 and 70.62 hours, respectively.

[0081] Table 6 shows the serum half-life of the compounds measured with reference to Example 6.

[0082] Example 7: Detection of the activity of ELA-11 linear peptide analog against NIH-3T3 cell in vitro fibrosis. The experiment used the NIH-3T3 cell line to study and observe the effects of ELA-11 linear peptide analogs on TGF-β1-induced expression of α-SMA and Collagen I proteins in NIH-3T3 cells.

[0083] NIH-3T3 cells were seeded at a rate of 200,000 cells / well in 6-well plates using DMEM medium containing 10% FBS and 1% penicillin antibiotics. The cells were cultured at 37°C and 5% CO2 for 24 h. After being replaced with serum-free medium and cultured for 12 h, 5 ng / mL TGF-β1 and 10 μM ELA-11 or a linear peptide analog were added and the cells were treated for 24 h. Total cell protein was then extracted, and the protein expression levels of α-SMA and Collagen I were detected by Western blot.

[0084] Control group (labeled as Control): No TGF-β1 and ELA-11 linear peptide analogs were added to the culture medium; Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; ELA-11 linear peptide analogue group (labeled as peptide analogue names shown in Table 3): 5 ng / m LTF-β1 and 10 μM ELA-11 linear peptide analogue were added to the culture medium.

[0085] Figure 8 After treating NIH-3T3 cells with TGF-β1 and ELA-11 linear peptide analogues, Western blot was used to detect the protein expression of α-SMA, Collagen I, and GAPDH, and the relative expression levels of α-SMA and Collagen I proteins were analyzed.

[0086] Depend on Figure 8 It was found that linear peptides E16, E17, E18, E19, E20, E22, E25, E26, E27, E28, and E29 significantly inhibited the expression of α-SMA and Collagen I proteins. Among these, mutations at position 9 (Pro) of ELA-11 to Hyp and position 10 (Phe) to D-type Phe or D-type Ala improved antifibrotic activity. Table 5 shows that mutation at position 2 (Met) to Nle prolonged the in vitro half-life while maintaining the original antifibrotic activity. Some linear peptide analogs obtained through amino acid mutation strategies also exhibited superior in vitro stability and antifibrotic activity compared to ELA-11, demonstrating higher drug-likeness, such as E27, E28, and E29. The in vivo antifibrotic effect of the linear peptide analog E28 in a mouse model of unilateral ureteral obstruction will be further investigated using this analog as an example.

[0087] Example 8 investigated the therapeutic effects of ELA-11 linear peptide analogues and ELA-11 on improving renal fibrosis in mice. (1) Test substances: ELA-11 and linear peptide analog E28.

[0088] (2) Forty male C57BL / 6J mice (6 weeks old, weighing about 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into 5 groups of 8 mice each.

[0089] Groups: Sham surgery group, model group (UUO), ELA-11 twice a day group (UUO+ELA-11-twice), E28 twice a day group (UUO+E28-twice), E28 once a day group (UUO+E28-once).

[0090] (3) Establishment of renal fibrosis model: UUO (unilateral ureteric obstruction, abbreviated as UUO) induces renal fibrosis in mice.

[0091] After anesthesia with 1% sodium pentobarbital solution via intraperitoneal injection, the hair on the left back of the mice was shaved, and the mice were fixed in a prone position on a surgical board. The left ureter was isolated. In the model group and the drug treatment group, the ureter near the renal pelvis was ligated with 4-0 sutures, and the ligated ureter was cut. In the sham surgery group, only the left ureter was isolated without ligation. The UUO-induced renal fibrosis model was characterized by hydronephrosis, thinning of the renal cortex, dilation of renal tubules, and inflammatory cell infiltration. After treatment with the test drug, the kidneys were subjected to pathological analysis.

[0092] (4) UUO+ELA-11-twice group: The model mice were injected intraperitoneally with 1.2 μmol / kg ELA-11 at 9:00 and 21:00 daily; UUO+E28-twice group: The model mice were intraperitoneally injected with 1.2 μmol / kg E28 at 9:00 and 21:00 daily; UUO+E28-once group: The model mice were injected intraperitoneally with 1.2 μmol / kg E28 at 9:00 every day; Mice in the UUO group and the Sham group were injected intraperitoneally with an equal volume of sterile PBS solution. Samples were collected 7 days later.

[0093] Figure 9The graph shows the expression of GAPDH, α-SMA, Vimentin, Collagen I, and Fibronectin proteins in the kidney tissues of mice in each group 7 days after drug administration, along with the statistical analysis of their relative expression levels. It can be seen that in UUO model mice, the levels of fibrosis-related proteins in the kidneys were significantly reduced after treatment with the test substance, indicating that E28 has the effect of improving renal fibrosis in the UUO model.

[0094] Figure 10 The gene expression levels of α-SMA, Vimentin, Collagen I, and Fibronectin in the kidney tissues of mice in each group were analyzed 7 days after drug administration. The results showed that in UUO model mice, treatment with the test substance significantly reduced the gene levels of fibrosis-related proteins in the kidneys, indicating that the test substance has a good effect in improving kidney fibrosis. Furthermore, at the same dosing frequency, E28 showed significantly better therapeutic effects than ELA-11.

[0095] Figure 11 The images show the H&E and immunohistochemical staining results of the kidney tissue sections from each group of mice after tissue collection.

[0096] H&E staining: Paraffin sections were sent to Wuhan Saiweier Biotechnology Co., Ltd. for staining.

[0097] Immunohistochemical staining: Paraffin sections were baked in a 60℃ oven for 1 h, followed by dewaxing and hydration by sequentially soaking in xylene, anhydrous ethanol, 95% ethanol, 80% ethanol, and ultrapure water. The resulting sections were then microwaved twice in sodium citrate buffer (0.01 M) for 10 minutes each time, and cooled to room temperature to complete antigen retrieval. The resulting sections were then incubated sequentially with endogenous peroxidase inhibitor for 10 min; washed three times with PBS for 3 min each time; incubated with primary antibody at 4℃ for 10 h; incubated with reaction enhancement solution at 37℃ for 20 min; washed three times with PBS for 3 min each time; incubated with enzyme-labeled goat anti-rabbit IgG polymer at 37℃ for 20 min; washed three times with PBS for 3 min each time; developed with freshly prepared DAB; counterstained with hematoxylin; and thoroughly washed with tap water. Finally, the sections were sequentially dehydrated and cleared by soaking in 80% ethanol, 95% ethanol, 100% ethanol, and xylene, and then mounted with neutral resin after air drying.

[0098] Figure 11 H&E staining results showed that, compared with the Sham group, the UUO group had significant kidney damage, with extensive cystic dilatation of renal tubules, disappearance, flattening, or necrosis and sloughing of epithelial cells brush borders, hyaline casts and cellular debris visible in the lumen, and significant widening and edema of the renal interstitium, accompanied by pathological changes such as extensive infiltration of lymphocytes and mononuclear cells. After 7 days of treatment with ELA-11 or the linear peptide analog E28, the dilatation of the renal tubule lumen was reduced, the renal interstitial edema was reduced, and the infiltration of inflammatory cells was decreased, showing a significant therapeutic and ameliorative effect. Figure 11 Immunohistochemical staining results showed that α-SMA, Vimentin, Collagen I and Fibronectin proteins were highly expressed in the kidneys of the UUO group. Treatment with ELA-11 or the linear peptide analog E28 reduced the expression levels, indicating that renal fibrosis in mice was improved. The UUO+E28-twice group showed the best improvement, suggesting that there is still a need to improve the stability of E28.

[0099] Example 9: Comparison of the activity of ELA-11 cyclic peptide analog and linear peptide analog E28 against NIH-3T3 cell fibrosis in vitro. The experiment used the NIH-3T3 cell line to study and observe the effects of ELA-11 cyclic peptide analogues on TGF-β1-induced expression of α-SMA and Collagen I proteins in NIH-3T3 cells.

[0100] NIH-3T3 cells were seeded at a rate of 200,000 per well in 6-well plates using DMEM medium containing 10% FBS and 1% penicillin antibiotics. The cells were cultured at 37°C and 5% CO2 for 24 h. After being replaced with serum-free medium and starved for 12 h, the cells were treated with 5 ng / mL TGF-β1 and 10 μM ELA-11 cyclic peptide analogue for 24 h. Total cell protein was then extracted, and the protein expression levels of α-SMA and Collagen I were detected by Western blot.

[0101] Control group (labeled as Control): No TGF-β1 and ELA-11 cyclic peptide analogs were added to the culture medium; Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; ELA-11 cyclic peptide analogue group (labeled as peptide analogue names shown in Tables 1 and 3): 5 ng / m LGF-β1 and 10 μM ELA-11 cyclic peptide analogue were added to the culture medium.

[0102] Figure 12 After treating NIH-3T3 cells with TGF-β1 and ELA-11 cyclic peptide analogues, Western blot was used to detect the protein expression of α-SMA, Collagen I, and GAPDH, and the relative expression levels of α-SMA and Collagen I proteins were analyzed.

[0103] Depend on Figure 12It was found that precyclized peptides E30 and E31 showed poor antifibrotic effects in the TGF-β1-induced NIH-3T3 cell model. Cyclation improved antifibrotic activity, and the antifibrotic activity was better when the cyclized linker was shorter, such as E32, E33, E34, E39, E40, E41, and E42. Cyclation is a common strategy to improve peptide stability. Based on the serum stability results in Table 6, E32, which had the longest serum stability and showed good inhibitory effect on fibrotic protein expression in TGF-β1-induced NIH-3T3 cells, was selected as the research object for lipid modification to delay in vivo metabolism.

[0104] Based on the structure of E32, using the method described in Example 1, AEEA, γGlu, and C20 diacid were sequentially coupled to its N-terminus and purified to obtain the lipotropic cyclic peptide E48. Similarly, by adjusting the fatty acid chain length, E46 (C16 diacid) and E47 (C18 diacid) were obtained.

[0105] Example 10: Detection of the activity of ELA-11 lipotropic cyclic peptide analog against NIH-3T3 cell in vitro fibrosis. The experiment used the NIH-3T3 cell line to study and observe the effects of ELA-11 lipotropic cyclic peptide analogue on TGF-β1-induced expression of α-SMA and Collagen I proteins in NIH-3T3 cells.

[0106] NIH-3T3 cells were seeded at a rate of 200,000 per well in 6-well plates using DMEM medium containing 10% FBS and 1% penicillin antibiotics. The cells were cultured at 37°C and 5% CO2 for 24 h. After being replaced with serum-free medium and starved for 12 h, the cells were treated with 5 ng / mL TGF-β1 and 10 μM ELA-11 lipocyclic peptide analogue for 24 h. Total cell protein was extracted, and the protein expression levels of α-SMA and Collagen I were detected by Western blot.

[0107] Control group (labeled as Control): No TGF-β1 and ELA-11 lipotropic peptide analogs were added to the culture medium; Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; ELA-11 cyclic peptide analogues (labeled as peptide analogue names shown in Tables 1 and 4): 5 ng / m LTF-β1 and 10 μM ELA-11 esterified cyclic peptide analogues were added to the culture medium.

[0108] Figure 13After treating NIH-3T3 cells with TGF-β1 and ELA-11 esterified cyclic peptide analogues, Western blot was used to detect the protein expression of GAPDH, α-SMA, and Collagen I, and the relative expression levels of α-SMA and Collagen I proteins were analyzed.

[0109] Depend on Figure 13 It was found that the relative expression levels of α-SMA and Collagen I in NIH-3T3 cells treated with E47 and E48 were significantly lower than those in the TGF-β1-induced group, indicating that E47 and E48 could significantly inhibit the expression of TGF-β1-induced fibrotic proteins in NIH-3T3 cells. E48 was more effective than E47 and comparable to E28. The effective concentration of E48 (10 μM) was far lower than the initial toxic concentration (160 μM), indicating a large safety window.

[0110] Example 11: Detection of the activity of ELA-11 lipotropic cyclic peptide analog against HK-2 cell in vitro fibrosis. The HK-2 cell line was used in the experiment to study and observe the effects of ELA-11 lipotropic cyclic peptide analogues on the expression of α-SMA, Vimentin and Collagen I proteins induced by TGF-β1 in HK-2 cells.

[0111] HK-2 cells were seeded at a rate of 200,000 cells / well in 6-well plates using DMEM medium containing 10% FBS and 1% penicillin antibiotics. The cells were cultured at 37°C and 5% CO2 for 24 h. After being cultured in serum-free medium for 12 h, 5 ng / mL TGF-β1 and 10 μM ELA-11 lipocyclic peptide analogs were added and the cells were treated for 24 h. Total cell protein was then extracted, and the protein expression levels of α-SMA, Vimentin, and Collagen I were detected by Western blot.

[0112] Control group (labeled as Control): No TGF-β1 and ELA-11 lipotropic peptide analogs were added to the culture medium; Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; ELA-11 cyclic peptide analogues (labeled as peptide analogue names shown in Tables 1 and 4): 5 ng / m LTF-β1 and 10 μM ELA-11 esterified cyclic peptide analogues were added to the culture medium.

[0113] Figure 14After treating HK-2 cells with TGF-β1 and ELA-11 cyclic peptide analogues, Western blot was used to detect the protein expression of GAPDH, α-SMA, Vimentin and Collagen I, and the relative expression levels of α-SMA, Vimentin and Collagen I were analyzed.

[0114] Depend on Figure 14 It was found that after treatment of HK-2 cells with E46, E47, and E48, the relative expression levels of α-SMA, Vimentin, and Collagen I were significantly lower than those in the TGF-β1-induced group, indicating that they could significantly inhibit TGF-β1-induced renal fibrosis in HK-2 cells. Based on the results of Example 10, the in vivo anti-fibrotic effect of the lipotropic cyclic peptide E48 in a mouse model of unilateral ureteral obstruction will be further investigated.

[0115] Example 12: Detection of the therapeutic effect of ELA-11 lipotropic cyclic peptide analog on renal fibrosis in mice. (1) Test substances: ELA-11 linear peptide analog E28 and lipotropic cyclic peptide analog E48.

[0116] (2) Forty male C57BL / 6J mice (6 weeks old, weighing about 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into 5 groups of 8 mice each.

[0117] Groups: Sham surgery group, model group (UUO), E28 twice a day group (UUO+E28 group), E48 once a week group (UUO+E48-once), E48 twice a week group (UUO+E48-twice).

[0118] (3) Establishment of renal fibrosis model: The operation is the same as in Example 8.

[0119] (4) UUO+E28 group: The model mice were injected intraperitoneally with 1.2 μmol / kg E28 at 9:00 and 21:00 daily; UUO+E48-once group: Model mice were injected intraperitoneally with 1.2 μmol / kg E48 0 h after modeling; UUO+E48-twice group: Model mice were intraperitoneally injected with 1.2 μmol / kg and 0.6 μmol / kg E48 at 0 h and 84 h after modeling, respectively; Mice in the UUO group and the Sham group were injected intraperitoneally with an equal volume of sterile PBS solution. Samples were collected 7 days later.

[0120] Figure 15The graph shows the expression levels of GAPDH, α-SMA, Vimentin, Collagen I, and Fibronectin proteins in the kidney tissues of mice in each group 7 days after administration, along with the statistical analysis of their relative expression levels. It can be seen that in UUO model mice, the levels of fibrosis-related proteins in the kidneys were significantly reduced after treatment with the test substance, indicating that E48 has the effect of improving renal fibrosis in the UUO model, and the improvement effect is comparable to that of E28.

[0121] Figure 16 The gene expression of α-SMA, Vimentin, Collagen I, and Fibronectin in the kidney tissue of mice in each group 7 days after administration was shown. It can be seen that in UUO model mice, the gene level of fibrosis-related proteins in the kidney was significantly reduced after treatment with the test substance, indicating that the test substance can improve kidney fibrosis and the effect is comparable to that of E28.

[0122] Figure 17 The images show the H&E and immunohistochemical staining results of the kidney tissue sections from each group of mice after tissue collection. The procedure was the same as in Example 8. H&E staining results showed that, compared with the sham-operated group, the UUO group exhibited significant kidney damage, with widespread cystic dilatation of the renal tubules, loss, flattening, or necrosis and sloughing of the brush border of epithelial cells, hyaline casts and cellular debris visible within the tubules, and significant widening and edema of the renal interstitium, accompanied by extensive infiltration of lymphocytes and mononuclear cells. After 7 days of treatment with E48 or E28, the dilatation of the renal tubules was significantly reduced, the edema of the renal interstitium was alleviated, and the infiltration of inflammatory cells decreased, demonstrating a significant therapeutic and ameliorative effect. Figure 17 Immunohistochemical staining results showed that α-SMA, Vimentin, Collagen I and Fibronectin proteins were highly expressed in the kidneys of the UUO group, and the expression levels were reduced by E48 or E28 treatment, indicating that renal fibrosis in mice was improved.

[0123] In summary, E48 and E28 showed similar effects on improving renal fibrosis in mice, but E48 required a much lower dosing frequency than E28 and had higher drug-like properties. Further investigation of its in vivo metabolic kinetics will be conducted using E48 as an example.

[0124] Example 13: Comparison of pharmacokinetics of linear ELA-11 peptide analogs and esterified cyclic peptides Male C57BL / 6J mice were injected intravenously or intraperitoneally with 3 μmol / kg E28 and E48. Blood samples of 800 μL were collected from the eyeballs at different time points. The blood samples were mixed with 200 μL of 2 mg / kg heparin sodium and centrifuged at 15,000 rpm for 10 min at 4 °C. 190 μL of the supernatant plasma and 10 μL of internal standard solution were added to 800 μL of ice-cold acetonitrile and vortexed. The mixture was centrifuged at 15,000 rpm for 10 min at 4 °C. 700 μL of the supernatant was collected and concentrated under vacuum at 14,000 rpm at 37 °C to remove the liquid. The precipitate was resuspended in 300 μL of the initial mobile phase, filtered, and detected by LC-MS / MS.

[0125] Figure 18 The in vivo pharmacokinetic curves for E28 and E48 show that the concentration of the lipotropic cyclic peptide E48 is significantly higher than that of the linear peptide E28. After intravenous injection, the half-life of E48 is 1241.17 min, while that of E28 is 15.23 min. After intraperitoneal injection, the half-life of E48 is 1552.74 min, while that of E28 is 17.98 min. The half-life of E48 is nearly 100 times longer than that of E28. Furthermore, its clearance after distribution in vivo is very slow, requiring nearly 10 days for complete metabolism in mice, making it a stable polypeptide in vivo.

[0126] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An ELA-11 cyclic peptide analog, characterized in that, Its structure is as follows: R1-R2-R3-Pro-Leu-His-Ser-Arg-Val-Hyp-dAla-Cys, where... R1 is a fatty acid, R2 is a linker containing γGlu and / or AEEA, and R3 is Cys-Nle or Ala-Cys. The C-terminal Cys and the R3 Cys form a covalent cyclization through the side chain thiol group.

2. A linear peptide, characterized in that, The sequence of the linear peptide is Ac-Cys-Nle-Pro-Leu-His-Ser-Arg-Val-Hyp-dAla-Pro.

3. The ELA-11 cyclic peptide analog according to claim 1, characterized in that, R1 is a C10-C20 straight-chain saturated fatty acid diacid.

4. The ELA-11 cyclic peptide analog according to claim 1, characterized in that, The C-terminal Cys group cyclizes with the side chain of the R3 Cys group via a disulfide bond, or cyclizes with the two Cys groups via a C4-C10 alkyl chain to form a thioether bond.

5. The ELA-11 cyclic peptide analog as described in claim 1, having the structure: C20 diacid-γGlu-AEEA-AEEA-Cys-Nle-Pro-Leu-His-Ser-Arg-Val-Hyp-dAla-Cys, wherein the C-terminal Cys group and the Cys group of R3 form a disulfide bond cyclization via a side chain thiol group, as shown below: 。 6. The use of the ELA-11 cyclic peptide analog according to any one of claims 1 to 5 in the preparation of a medicament for treating renal fibrosis.

7. A drug for treating renal fibrosis, characterized in that, The drug has the ELA-11 cyclic peptide analogue as described in any one of claims 1 to 5 as its main component.

8. The medicament for treating renal fibrosis according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

9. A method for preparing the ELA-11 cyclic peptide analog according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) The linear polypeptide resin corresponding to the ELA-11 cyclic peptide analog was synthesized by the Fmoc solid-phase synthesis method from the carboxyl terminus to the amino terminus. Then the resin was cleaved and the cleavage solution containing the linear polypeptide was collected. (2) Purify the cutting solution obtained in step (1) and freeze-dry it to obtain a linear polypeptide solid; (3) Dissolve the linear polypeptide obtained in step (2) in a suitable solvent, add an oxidizing agent or a dialkylating agent to carry out a cyclization reaction to obtain a cyclic peptide; preferably, the cyclization reaction is a disulfide bond cyclization or a thioether bond cyclization. (4) Purify the reaction solution from step (3) to obtain ELA-11 cyclic peptide analog.

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