A polypeptide compound with anti-inflammatory activity, its preparation method and application

Abstract

This invention discloses a polypeptide compound with anti-inflammatory activity or a pharmaceutical salt thereof. The polypeptide is obtained by screening and identifying key residue sequences of interaction between the binary complex crystal structure of Sting. The polypeptide compound provided by this invention can target STING to produce an anti-inflammatory effect and can be used to treat knee osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a polypeptide compound with anti-inflammatory activity, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a degenerative joint disease with a high incidence and disability rate, imposing a heavy burden on patients, families, and society. According to literature reports, there are currently over 500 million OA patients worldwide, accounting for approximately 15% of the adult population. With the increasing aging of the population, the prevalence of OA is gradually rising. Currently, the first-line treatment for early-stage OA is topical nonsteroidal anti-inflammatory drugs (NSAIDs), while moderate to severe OA patients primarily rely on oral NSAIDs and selective cyclooxygenase inhibitors to control symptoms. These treatments mainly focus on short-term benefits, but long-term use can lead to adverse reactions such as cardiovascular toxicity, gastrointestinal bleeding, and liver and kidney damage. Given the limitations of existing treatments, developing safe and effective drugs that target the pathological mechanisms of OA and improve the joint microenvironment is crucial.

[0003] Apoptosis of articular chondrocytes and activation of innate inflammatory pathways play crucial roles in the development and progression of osteoarthritis (OA). STING, a key receptor for inflammation-induced senescent damage in articular chondrocytes, recognizes cyclic dinucleotides in the cytoplasm, recruits TBK1 kinase and IRF3 transcription factor, leading to IRF3 phosphorylation and dimer formation, inducing the expression of type I interferon and inflammatory factors, and driving the inflammatory response. STING deficiency or mutation can delay the inflammatory response and cartilage degradation in spontaneous osteoarthritis and alleviate joint damage in an adult mouse DMM model, suggesting that STING may be a novel therapeutic target for OA. An ideal drug delivery system can improve drug efficacy while reducing dosage and side effects. Peptide drugs, due to their broad applicability, high safety, and significant efficacy, are currently widely used in the treatment of diseases such as asthma, allergies, and pain. Peptide drugs targeting STING have great potential as an alternative to traditional anti-inflammatory drugs for arthritis, and no reports have yet been published on their use. Summary of the Invention

[0004] The first objective of this invention is to provide a polypeptide compound with anti-inflammatory activity.

[0005] A second objective of this invention is to provide a method for preparing the aforementioned polypeptide compound with anti-inflammatory activity.

[0006] A third objective of this invention is to provide the use of the aforementioned anti-inflammatory polypeptide compound in the preparation of a medicament for treating knee or osteoarthritis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a polypeptide compound with anti-inflammatory activity or a pharmaceutical salt thereof, the polypeptide being identified by screening from the key residue sequences of the interaction between the two binary complex crystal structures of Sting, the structure being selected from one of the following structures:

[0009] LA X1 SYY X2 GYLRL (I)

[0010] X3 LRLILPELQARIRTYNQHYNNLLR (II)

[0011] NFNVAHGLAWSYYI X4 (III)

[0012] In formula (I), X1 is selected from tryptophan or (2R)-2-amino-2-methyl-9-decenoic acid;

[0013] X2 is selected from isoleucine or (2R)-2-amino-2-methyl-9-decenoic acid;

[0014] The paired (2R)-2-amino-2-methyl-6-heptenoic acid in the fragment may or may not undergo cyclization via olefin metathesis.

[0015] In formula (II), X3 is selected from tyrosine or IGY;

[0016] In formula (III), X4 is selected from glycine, GYLRLIL sequence, GYLRLILPELQ sequence, GYLRLILPELQARIRTYN sequence, GYLRLILPELQARIRTYNQHYNNL sequence, GYLRLILPELQARIRTYNQHYNNLL sequence, and GYLRLILPELQARIRTYNQHYNNLLR sequence.

[0017] The N-terminal amino group and C-terminal carboxyl group of the polypeptide of formula (I), (II) or (III) and the amino acid side chain group may not be modified, or may be modified without substantially affecting the activity of the polypeptide of the present invention, such as forming a "pharmaceuticalally acceptable ester". The modification of the N-terminal amino group includes, but is not limited to, de-amino, N-lower alkyl, N-dilower alkyl and N-acyl modification. The modification of the C-terminal carboxyl group includes, but is not limited to, amide, lower alkyl amide, dialkyl amide and lower alkyl ester modification. The N-terminal amino group of the polypeptide of the present invention is acetylated, that is, -Ac, and the C-terminal carboxyl group is amidated, that is, -NH2.

[0018] Preferably, the amino acid sequence of the polypeptide compound with anti-inflammatory activity is selected from one of the following amino acid sequences:

[0019] The amino acid sequence shown in SEQ ID NO.1;

[0020] The amino acid sequence shown in SEQ ID NO.2;

[0021] The amino acid sequence shown in SEQ ID NO.3;

[0022] The amino acid sequence shown in SEQ ID NO.4;

[0023] The amino acid sequence shown in SEQ ID NO.5;

[0024] The amino acid sequence shown in SEQ ID NO.6;

[0025] The amino acid sequence shown in SEQ ID NO.7;

[0026] The amino acid sequence shown in SEQ ID NO. 8;

[0027] The amino acid sequence shown in SEQ ID NO.9;

[0028] The amino acid sequence shown in SEQ ID NO.10;

[0029] The amino acid sequence shown in SEQ ID NO.11.

[0030] A second aspect of the present invention provides a method for preparing the aforementioned polypeptide compound with anti-inflammatory activity, comprising the following steps:

[0031] Step 1, resin swelling: Fmoc-Rinkamid-MBHAResin (degree of substitution = 0.44 mmol / g) was placed in a peptide reaction tube, swollen by DCM, and the DCM was removed by filtration;

[0032] The second step is the removal of Fmoc protection: Add 20% piperidine / DMF solution to the reaction tube from the first step, shake, filter to remove the solution, and rinse the resin with DCM.

[0033] The third step is the amino acid linkage: Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH or Fmoc-Gly-OH and HCTU are dissolved in DMF, then DIPEA is added and shaken. The mixture is then poured into the peptide reaction tube from the second step and shaken at room temperature. After the reaction is complete, the solution is removed by filtration and the resin is rinsed with DCM and DMF.

[0034] Repeat steps two and three to sequentially connect Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Leu-OH until all amino acids are connected.

[0035] Alternatively, repeat steps two and three to sequentially connect Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-S5-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-S5-OH, Fmoc-Ala-OH, and Fmoc-Leu-OH until all amino acids are connected.

[0036] Alternatively, repeat steps two and three to sequentially connect Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Arg(Pbf)-OH. Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, until all amino acids are linked;

[0037] Alternatively, repeat steps two and three to sequentially connect Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Ile-OH. Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, until all amino acids are linked;

[0038] Alternatively, repeat steps two and three to sequentially connect Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, and Fmoc-Asn(Trt)-OH until all amino acids are connected.

[0039] Alternatively, repeat steps two and three to sequentially connect Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, until all amino acids are connected;

[0040] Alternatively, repeat steps two and three to sequentially connect Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and Fm Continue linking Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, until all amino acids are linked;

[0041] Alternatively, repeat steps two and three to sequentially connect Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmo c-Leu-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, until all amino acids are connected;

[0042] Alternatively, repeat steps two and three to sequentially connect Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, and Fmoc-Il. e-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tr p(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, until all amino acids are connected;

[0043] Alternatively, repeat the steps of the second and third steps to sequentially connect Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH until all amino acids are connected;

[0044] Alternatively, repeat steps two and three to sequentially connect Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt) ...Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ile f moc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, until all amino acids are connected;

[0045] Step 4, N-terminal acetylation: Add pyridine and acetic anhydride in a volume ratio of 1:1 to the peptide reaction tube from step 3 above and shake at room temperature. After the reaction is complete, filter to remove the solution and rinse the resin with DCM and DMF.

[0046] Step 5: Resin dissociation and side chain protection removal of peptides: Add a cleavage reagent to the peptide reaction tube from step 4 above. The cleavage reagent is: TFA / TIPs / water = 95:2.5:2.5. Shake at room temperature. After the reaction is complete, collect the filtrate. Add ice-cold ether to the filtrate to precipitate the crude peptide. Filter to obtain the crude peptide.

[0047] Step 6, purification of crude peptide: The crude peptide obtained in step 5 was dissolved in a 1:1 mixture of acetonitrile and water, purified by reversed-phase HPLC, and freeze-dried to obtain a polypeptide compound with anti-inflammatory activity.

[0048] The reversed-phase HPLC purification conditions for step six are as follows: column: Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; rinsing gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths 214nm and 254nm.

[0049] The method for preparing the polypeptide compound with anti-inflammatory activity further includes:

[0050] Alternatively, step four may include a side-chain olefin metathesis reaction: a solution of 1,2-dichloroethane containing a first-generation Grubbs catalyst is added to the peptide reaction tube from step four, and the mixture is shaken overnight at room temperature. After the reaction is complete, the solution is removed by filtration, and the resin is washed with DCM and DMF; then step five is carried out.

[0051] A third aspect of the invention provides the use of the aforementioned anti-inflammatory polypeptide compound or its pharmaceutical salt in the preparation of a medicament for treating knee or osteoarthritis.

[0052] This invention induces an osteoarthritis (OA) model in mice through medial meniscectomy. At weeks 6 and 12 post-treatment, three-dimensional and coronal images of the medial tibial plateau of mice were scanned using Micro-CT. The OARSI score of the mouse knee osteoarthritis was assessed based on HE and Safranin-Fix-Green staining. IHC detection revealed that targeting the synthesized peptide SIP-2 reduced the expression of MMP13 and COL2A1 positive cells in cartilage tissue. The present invention's peptide effectively alleviates articular cartilage damage in the mouse OA model, promotes cartilage matrix synthesis, and inhibits osteophyte formation.

[0053] The medicinal salts refer to salts formed by small-molecule acidic or basic compounds and polypeptides. These salts generally increase the solubility of the polypeptides, and the formed salts do not significantly alter the activity of the polypeptides. For example, acids that can typically form salts with the polypeptides of this invention include hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, succinic acid, maleic acid, and citric acid; bases that can form salts with the polypeptides of this invention include alkali metal or alkaline earth metal hydroxides, ammonium, and carbonates.

[0054] The anti-inflammatory effects of the polypeptide compounds of the present invention can be verified by conventional experimental methods, such as cell experiments. In the specific embodiments of the present invention, cell experiments such as quantitative real-time PCR are preferred. Through this experiment, it was found that the polypeptide compounds of the present invention have in vitro anti-inflammatory effects.

[0055] The present invention also provides a pharmaceutical composition, wherein the aforementioned polypeptide compound with anti-inflammatory activity is used as the active pharmaceutical component, and can be used for anti-inflammatory treatment.

[0056] The pharmaceutical composition may contain one or more pharmaceutically acceptable diluents, excipients or carriers, preferably in unit dose form, such as tablets, films, pills, capsules (including sustained-release or delayed-release forms), powders, granules, syrups or emulsions, sterilized solutions for injection, suspensions or lyophilized powder injections, aerosols or liquid sprays, automated dropper injection devices or suppositories.

[0057] The active pharmaceutical component in the pharmaceutical composition may be combined with a non-toxic, pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water, or a combination thereof. The anti-inflammatory polypeptide compounds of the present invention are preferably prepared using sterilized aqueous solutions for injection.

[0058] The pharmaceutical compositions of the present invention can be administered via methods of administration well known to those skilled in the art, such as oral, rectal, sublingual, pulmonary, transdermal, iontophoresis, vaginal, and intranasal administration. The pharmaceutical compositions of the present invention are preferably administered parenterally, such as subcutaneously, intramuscularly, or intravenously.

[0059] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0060] The polypeptide compounds provided by this invention can target STING to produce an anti-inflammatory effect and can be used to treat knee osteoarthritis.

[0061] This invention utilizes the second messenger 2',3'-cGAMP to stimulate mouse RAW264.7 cells, constructing a STING immune signaling pathway activation model. RT-qPCR analysis showed that SIP-2 reduced cGAMP-induced Ifnβ gene expression in a dose-dependent manner. Further Western blot analysis revealed that SIP-2 effectively reduced the activation of STING-related innate immune signaling pathways through the STING-TBK1-IRF3 signaling axis. Similarly, further validation was conducted in mouse primary chondrocytes, showing that SIP-2 may reduce STING expression in articular cartilage and delay articular cartilage degeneration. In a mouse DMM model, the OARSI scoring system was used to quantitatively score cartilage degeneration, subchondral bone changes, and osteophyte formation. Combined with IHC analysis at weeks 6 and 12, the results indicated that SIP-2 significantly slowed the degenerative process of the knee joint in mice with osteoarthritis (OA). Attached Figure Description

[0062] Figure 1 This is a schematic diagram of the structure of the polypeptide compound SIP-2.

[0063] Figure 2 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-1 of the present invention.

[0064] Figure 3 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-2 of the present invention.

[0065] Figure 4 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-3 of the present invention.

[0066] Figure 5 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-4 of the present invention.

[0067] Figure 6 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-5 of the present invention.

[0068] Figure 7 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-6 of the present invention.

[0069] Figure 8 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-7 of the present invention.

[0070] Figure 9 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-8 of the present invention.

[0071] Figure 10 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-9 of the present invention.

[0072] Figure 11 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-10 of the present invention.

[0073] Figure 12 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-11 of the present invention.

[0074] Figure 13 This diagram illustrates the cytotoxic effects of SIP-2 on cartilage at different concentrations over 24 hours.

[0075] Figure 14 This is a schematic diagram illustrating the cytotoxicity of SIP-2 on cartilage at different concentrations over 48 hours.

[0076] Figure 15 This is a schematic diagram illustrating the protective effect of SIP-2 at different concentrations on a 24-hour cartilage inflammatory injury model.

[0077] Figure 16 This is a schematic diagram illustrating the protective effect of SIP-2 at different concentrations on a cartilage inflammatory injury model for 48 hours.

[0078] Figure 17This is a schematic diagram illustrating the data on the regulation of chondrocyte inflammatory factor Ifnb gene release by SIP-2.

[0079] Figure 18 This is a schematic diagram illustrating the data on the regulation of chondrocyte inflammatory factor Tnf gene release by SIP-2.

[0080] Figure 19 This is a schematic diagram illustrating the data on the regulation of chondrocyte inflammatory factor Il6 gene release by SIP-2.

[0081] Figure 20 This is a schematic diagram of the SIP-2 treatment regimen for arthritis lesions in mice.

[0082] Figure 21 This is a schematic diagram illustrating the effect of SIP-2 on cartilage sclerosis at the joints of DMM model mice.

[0083] Figure 22 This is a schematic diagram illustrating the quantification of cartilage area at the joints of DMM model mice using SIP-2.

[0084] Figure 23 A schematic diagram illustrating the quantification of cartilage thickness at the joints of DMM model mice using SIP-2.

[0085] Figure 24 This is a schematic diagram of a lower limb knee joint CT scan illustrating the effect of SIP-2 on osteophyte formation at the knee joint in a DMM model.

[0086] Figure 25 This is a schematic diagram of the osteophyte grading and scoring in the SIP-2 model of osteophyte formation at the knee joint of the DMM model.

[0087] Figure 26 This is a schematic diagram illustrating the quantification of osteophyte levels in the knee joint generation of the DMM model using SIP-2.

[0088] Figure 27 This is a schematic diagram illustrating the quantification of bone volume during osteophyte formation at the knee joint in the SIP-2 model of the DMM.

[0089] Figure 28 This is a schematic diagram of safranin-fixing staining in the HE pathology and SO staining evaluation of cartilage tissue from the DMM model using SIP-2.

[0090] Figure 29 This is a schematic diagram illustrating the quantification of joint area in the evaluation of HE pathology and SO staining of cartilage tissue in the DMM model using SIP-2.

[0091] Figure 30 This is a schematic diagram illustrating the quantification of joint damage in the evaluation of HE pathology and SO staining of cartilage tissue in the DMM model using SIP-2. Detailed Implementation

[0092] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0093] The methods used in this invention to represent polypeptides, amino acids, and chemical groups are all recognized in the art. The abbreviations for amino acids can be found in Table 1. Specific amino acid structures can be found in Table 2. In this invention, unless otherwise specified, amino acids generally refer to L-type amino acids.

[0094] Table 1. Amino Acid Abbreviations

[0095] amino acids Three-letter abbreviation One-letter abbreviation amino acids Three-letter abbreviation One-letter abbreviation Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C proline Pro P glutamine Gln Q Serine Ser S glutamic acid Glu E threonine Thr T glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V

[0096] Table 2. Special Amino Acid Abbreviations

[0097] amino acids abbreviation (2R)-2-amino-2-methyl-6-heptenic acid <![CDATA[S5]]>

[0098] The names, structural formulas, and mass spectrometry data of the compounds synthesized in this invention are shown in Table 3:

[0099] Table 3. Names, structural formulas, and mass spectrometry data of preferred polypeptide active molecules.

[0100]

[0101]

[0102] Example 1

[0103] The preparation method of linear peptide anti-inflammatory compounds, specifically the solid-phase synthesis of SIP-1, is as follows:

[0104] The first step is the swelling of the resin.

[0105] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0106] The second step is to remove Fmoc protection.

[0107] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0108] The third step is the linking of amino acids.

[0109] Fmoc-Leu-OH (466 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0110] Repeat the steps of removing the Fmoc protecting group in step two and linking the amino acids in step three. First, add 6 ml of 20% piperidine / DMF solution, shake for 10 minutes, filter to remove the solution, and wash the resin with 20 ml of DCM. Then, place Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) in a 10 ml centrifuge tube, add 6 ml of DMF to dissolve them, then add DIPEA (170 mg, 1.32 mmol), shake for 5 minutes, and pour the mixture into the peptide reaction tube from step two. Shake at room temperature for 40 minutes. After the reaction is complete, filter to remove the solution, and wash the resin with 20 ml of DCM and 20 ml of DMF. The following amino acids were sequentially linked: Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Ser(tBu)-OH (505 mg, 1.32 mmol), Fmoc-Trp(Boc)-OH (694 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), and Fmoc-Leu-OH (466 mg, 1.32 mmol) until all amino acids were linked.

[0111] Step 4, N-terminal acetylation

[0112] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0113] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0114] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0115] Step 6: Purification of crude peptides

[0116] The 0.31 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a Labconco freeze dryer (USA) to obtain 0.24 g of white lyophilized SIP-1 powder with a purity ≥97.2%.

[0117] Step 7: Identification of peptides

[0118] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z [M+H] + =1558.8344; [M+2H] 2+ =779.9172; [M+3H] 3+ =520.2781.

[0119] The structural formula for SIP-1 is: LAWSYYIGYLRL.

[0120] Example 2

[0121] Solid-phase synthesis of SIP-2: The preparation method of the polypeptide compound SIP-2 includes the following steps:

[0122] The first step is the swelling of the resin.

[0123] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0124] The second step is to remove Fmoc protection.

[0125] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0126] The third step is the linking of amino acids.

[0127] Fmoc-Leu-OH (466 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0128] Repeat steps two and three to sequentially connect Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-S5-OH (350 mg, 0.88 mmol), and Fmoc-Tyr(tBu)- OH (605 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Ser(tBu)-OH (505 mg, 1.32 mmol), Fmoc-S5-OH (350 mg, 0.88 mmol), Fmoc-Ala-OH (410 mg, 0.88 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), until all amino acids are linked.

[0129] Step 4, N-terminal acetylation

[0130] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0131] Step 5: Metathesis reaction of side-chain olefins

[0132] Add 8 mL of a 1,2-dichloroethane solution containing 120 mg (0.15 mmol) of first-generation Grubbs catalyst to the peptide reaction tube from step four above, and shake overnight at room temperature. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 mL of DCM and 20 mL of DMF.

[0133] Step 6: Resin dissociation and side-chain protection removal of the peptide.

[0134] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step 5 above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0135] Step 7: Purification of crude peptides

[0136] The 0.46 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a Labconco freeze dryer (USA) to obtain 0.22 g of white lyophilized SIP-2 powder with a purity ≥97.2%.

[0137] Step 8: Identification of peptides

[0138] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+H] + =1509.8391, [M+2H] 2+ =755.4195, [M+3H] 3+ =503.9463.

[0139] like Figure 1 As shown, Figure 1 This is a schematic diagram of the structural formula of the polypeptide compound SIP-2. Structural formula of SIP-2: S5 is S-2-(4-pentenyl)Ala-OH, and this strategy uses Grubbs metathesis catalyst to fix olefin-containing amino acids at the i and i+4 positions.

[0140] Example 3

[0141] The preparation method of the polypeptide compound SIP-3 includes the following steps:

[0142] The first step is the swelling of the resin.

[0143] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0144] The second step is to remove Fmoc protection.

[0145] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0146] The third step is the linking of amino acids.

[0147] Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0148] Repeat steps two and three to sequentially connect Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (466 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), and Fmoc-His(Trt)-OH (818 mg). g, 1.32mmol), Fmoc-Gln(Trt)-OH (805mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Tyr(tBu)-OH (6 05mg, 1.32mmol), Fmoc-Thr(tBu)-OH (525mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Ile-OH (466m g, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Ala-OH (410mg, 1.32mmol), Fmoc-Gln(Trt)-OH (805mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Glu(tBu)-OH (563mg, 1.32mmol), Fmoc-Pro-OH (445mg, 1.32mmol) l), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Ile-OH (466mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Tyr(tBu)-OH (605mg, 1.32mmol), until all amino acids are linked.

[0149] Step 4, N-terminal acetylation

[0150] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0151] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0152] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0153] Step 6: Purification of crude peptides

[0154] The 0.38 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a freeze dryer (Labconco, USA). 0.24 g of white lyophilized SIP-3 powder with a purity ≥97.2% was obtained.

[0155] Step 7: Identification of peptides

[0156] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+2H] 2+ =1606.3945; [M+3H] 3+ =1071.1630.

[0157] The structural formula for SIP-3 is: YLRLILPELQARIRTYNQHYNNLLR.

[0158] Example 4

[0159] The preparation method of the polypeptide compound SIP-4 includes the following steps:

[0160] The first step is the swelling of the resin.

[0161] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0162] The second step is to remove Fmoc protection.

[0163] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0164] The third step is the linking of amino acids.

[0165] Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0166] Repeat steps two and three to sequentially connect Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (596 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (596 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), and Fm oc-Gln(Trt)-OH (805mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Tyr(tBu)-OH (605mg, 1.32mmol), Fmoc-T hr(tBu)-OH (525mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Ile-OH (466mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Ala-OH (410mg, 1.32mmol), Fmoc-Gln(Trt)-OH (805mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol) l), Fmoc-Glu(tBu)-OH (563mg, 1.32mmol), Fmoc-Pro-OH (445mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Ile-OH (466mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Tyr(tBu)-OH (605mg, 1.32mmol), Fmoc-Gly-OH (392mg, 1.32mmol), Fmoc-Ile-OH (466mg, 1.32mmol), until all amino acids are linked.

[0167] Step 4, N-terminal acetylation

[0168] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0169] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0170] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0171] Step 6: Purification of crude peptides

[0172] The 0.68 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a Labconco freeze dryer (USA). 0.36 g of white lyophilized SIP-4 powder with a purity ≥97.2% was obtained.

[0173] Step 7: Identification of peptides

[0174] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+H] + =3341.86444; [M+2H] 2+ =1671.5384; [M+3H] 3+ =1114.6431.

[0175] The structural formula for SIP-4 is: IGYLRLILPELQARIRTYNQHYNNLLR.

[0176] Example 5

[0177] The preparation method of the polypeptide compound SIP-5 includes the following steps:

[0178] The first step is the swelling of the resin.

[0179] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0180] The second step is to remove Fmoc protection.

[0181] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0182] The third step is the linking of amino acids.

[0183] Fmoc-Gly-OH (392 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0184] Repeat steps two and three to sequentially connect Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Ser(tBu)-OH (505 mg, 1.32 mmol), Fmoc-Trp(Boc)-OH (694 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), and Fmoc-Le u-OH (466 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Val-OH (447 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (466 mg, 1.32 mmol), Fmoc-Phe-OH (387 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (466 mg, 1.32 mmol), until all amino acids are linked.

[0185] Step 4, N-terminal acetylation

[0186] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0187] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0188] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0189] Step 6: Purification of crude peptides

[0190] The 0.53 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a freeze dryer (Labconco, USA). 0.31 g of white lyophilized SIP-5 powder with a purity ≥97.2% was obtained.

[0191] Step 7: Identification of peptides

[0192] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+H] + =1712.5436; [M+2H] 2+ =857.6453.

[0193] The structural formula for SIP-5 is: NFNVAHGLAWSYYIG.

[0194] Example 6

[0195] The preparation method of the polypeptide compound SIP-6 includes the following steps:

[0196] The first step is the swelling of the resin.

[0197] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0198] The second step is to remove Fmoc protection.

[0199] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0200] The third step is the linking of amino acids.

[0201] Fmoc-Leu-OH (466 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0202] Repeat steps two and three to sequentially connect Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), and Fmoc-Ser(tBu)-OH (505 mg, 1.32 mmol). The following amino acids were linked together: Fmoc-Tyr(tBu)-OH (694 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Val-OH (447 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Phe-OH (387 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), until all amino acids were linked.

[0203] Step 4, N-terminal acetylation

[0204] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0205] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0206] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0207] Step 6: Purification of crude peptides

[0208] The 0.76 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a Labconco freeze dryer (USA). 0.47 g of white lyophilized SIP-6 powder with a purity ≥97.2% was obtained.

[0209] Step 7: Identification of peptides

[0210] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+2H] 2+ =1422.5842; [M+3H] 3+ =813.5840.

[0211] The structural formula for SIP-6 is: NFNVAHGLAWSYYIGYLRLIL.

[0212] Example 7

[0213] The preparation method of the polypeptide compound SIP-7 includes the following steps:

[0214] The first step is the swelling of the resin.

[0215] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0216] The second step is to remove Fmoc protection.

[0217] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0218] The third step is the linking of amino acids.

[0219] Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, and then DIPEA (170 mg, 1.32 mmol) was added. After shaking for 5 min, the mixture was poured into the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was completed, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0220] Repeat steps two and three to sequentially connect Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Glu(tBu)-OH (563 mg, 1.32 mmol), Fmoc-Pro-OH (445 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), and Fmoc-Leu-OH (466 mg, 1.32 mmol). ), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Tyr(tBu)-OH (605mg, 1.32mmol) ), Fmoc-Gly-OH (392mg, 1.32mmol), Fmoc-Ile-OH (466mg, 1.32mmol), Fmoc-Tyr(tBu)-OH (605mg, 1.32mmol), Fmo c-Tyr(tBu)-OH (605mg, 1.32mmol), Fmoc-Ser(tBu)-OH (505mg, 1.32mmol), Fmoc-Trp(Boc)-OH (694mg, 1.32mmo l), Fmoc-Ala-OH (410mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Gly-OH (392mg, 1.32mmol), Fmoc-Hi s(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Val-OH (447 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Phe-OH (387 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), until all amino acids are linked.

[0221] Step 4, N-terminal acetylation

[0222] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0223] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0224] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0225] Step 6: Purification of crude peptides

[0226] The 0.66 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a freeze dryer (Labconco, USA). 0.42 g of white lyophilized SIP-7 powder with a purity ≥97.2% was obtained.

[0227] Step 7: Identification of peptides

[0228] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+2H] 2+ =1476.2105; [M+3H] 3+ =984.6531. SIP-7 structure formula: NFNVAHGLAWSYYIGYLRLILPELQ.

[0229] Example 8

[0230] The preparation method of the polypeptide compound SIP-8 includes the following steps:

[0231] The first step is the swelling of the resin.

[0232] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0233] The second step is to remove Fmoc protection.

[0234] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0235] The third step is the linking of amino acids.

[0236] Fmoc-Asn(Trt)-OH (800 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0237] Repeat steps two and three to sequentially connect Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Thr(tBu)-OH (525 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol), Fmoc- Leu-OH (466mg, 1.32mmol), Fmoc-Glu(tBu)-OH (563mg, 1.32mmol), Fmoc-Pro-OH (445mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Ile- OH (466mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Arg(Pbf)-OH (855mg, 1.32mmol), Fmoc-Leu-OH (466mg, 1.32mmol), Fmoc-Tyr(tBu)- OH (605mg, 1.32mmol), Fmoc-Gly-OH (392mg, 1.32mmol), Fmoc-Ile-OH (466mg, 1.32mmol), Fmoc-Tyr(tBu)-OH (605mg, 1.32mmol), Fmoc-Tyr(tBu) -OH (605mg, 1.32mmol), Fmoc-Ser(tBu)-OH (505mg, 1.32mmol), Fmoc-Trp(Boc)-OH (694mg, 1.32mmol), Fmoc-Ala-OH (410mg, 1.32mmol), Fmoc-Leu -OH (466mg, 1.32mmol), Fmoc-Gly-OH (392mg, 1.32mmol), Fmoc-His(Trt)-OH (818mg, 1.32mmol), Fmoc-Ala-OH (410mg, 1.32mmol), Fmoc-Val-OH (447mg, 1.32mmol), Fmoc-Asn(Trt)-OH (786mg, 1.32mmol), Fmoc-Phe-OH (387mg, 1.32mmol), Fmoc-Asn(Trt)-OH (786mg, 1.32mmol), until all amino acids are linked.

[0238] Step 4, N-terminal acetylation

[0239] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0240] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0241] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0242] Step 6: Purification of crude peptides

[0243] The 0.81 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a freeze dryer (Labconco, USA). 0.66 g of white lyophilized SIP-8 powder with a purity ≥97.2% was obtained.

[0244] Step 7: Identification of peptides

[0245] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+2H] 2+ =1914.2496; [M+3H] 3+ =1276.9350. SIP-8 structure: NFNVAHGLAWSYYIGYLRLILPELQARIRTYN.

[0246] Example 9

[0247] The preparation method of the polypeptide compound SIP-9 includes the following steps:

[0248] The first step is the swelling of the resin.

[0249] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0250] The second step is to remove Fmoc protection.

[0251] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0252] The third step is the linking of amino acids.

[0253] Fmoc-Leu-OH (466 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0254] Repeat the steps of the second and third steps to sequentially couple Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Thr(tBu)-OH (525 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Glu(tBu)-OH (563 mg, 1.32 mmol), Fmoc-Pro-OH (445 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Ser(tBu)-OH (505 mg, 1.32 mmol), Fmoc-Trp(Boc)-OH (694 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Val-OH (447 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Phe-OH (387 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), until all amino acids are linked.

[0255] Step 4, N-terminal acetylation

[0256] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0257] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0258] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0259] Step 6: Purification of crude peptides

[0260] The 0.86 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a Labconco freeze dryer (USA). 0.65 g of white lyophilized SIP-9 powder with a purity ≥97.2% was obtained.

[0261] Step 7: Identification of peptides

[0262] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+3H] 3+ =1533.6832; [M+4H] 4+ =1150.0943. SIP-9 structure: NFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNL.

[0263] Example 10

[0264] The preparation method of the polypeptide compound SIP-10 includes the following steps:

[0265] The first step is the swelling of the resin.

[0266] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0267] The second step is to remove Fmoc protection.

[0268] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0269] The third step is the linking of amino acids.

[0270] Fmoc-Leu-OH (466 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0271] Repeat the steps of the second and third steps to successively couple Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Thr(tBu)-OH (525 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Glu(tBu)-OH (563 mg, 1.32 mmol), Fmoc-Pro-OH (445 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Ser(tBu)-OH (505 mg, 1.32 mmol), Fmoc-Trp(Boc)-OH (694 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.The following amino acids were added: Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Val-OH (447 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Phe-OH (387 mg, 1.32 mmol), and Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), until all amino acids were linked.

[0272] Step 4, N-terminal acetylation

[0273] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0274] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0275] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0276] Step 6: Purification of crude peptides

[0277] The 0.78 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a freeze dryer (Labconco, USA). 0.55 g of white lyophilized SIP-10 powder with a purity ≥97.2% was obtained.

[0278] Step 7: Identification of peptides

[0279] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+3H] 3+ =1570.0223; [M+4H] 4+=1150.8321. SIP-10 structure: NFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLL.

[0280] Example 11

[0281] The preparation method of the polypeptide compound SIP-11 includes the following steps:

[0282] The first step is the swelling of the resin.

[0283] Weigh 1g of Fmoc-Rinkamid-MBHAResin (Shanghai Jier Biochemical) (degree of substitution = 0.44mmol / g) and place it in a polypeptide reaction tube (self-made). Swell with 10ml of DCM for 20min and remove DCM by filtration.

[0284] The second step is to remove Fmoc protection.

[0285] Add 6 ml of 20% piperidine / DMF solution to the reaction tube for the first step, shake for 10 minutes, filter to remove the solution, and rinse the resin with 20 ml of DCM.

[0286] The third step is the linking of amino acids.

[0287] Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol) and HCTU (545 mg, 1.32 mmol) were placed in a 10 ml centrifuge tube, dissolved in 6 ml of DMF, followed by the addition of DIPEA (170 mg, 1.32 mmol). After shaking for 5 min, the solution was transferred to the peptide reaction tube for the second step and shaken at room temperature for 40 min. After the reaction was complete, the solution was removed by filtration, and the resin was rinsed with 20 ml of DCM and 20 ml of DMF.

[0288] Repeat the steps of the second and third steps to successively couple Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Thr(tBu)-OH (525 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Gln(Trt)-OH (805 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Glu(tBu)-OH (563 mg, 1.32 mmol), Fmoc-Pro-OH (445 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Arg(Pbf)-OH (855 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-Ile-OH (466 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Tyr(tBu)-OH (605 mg, 1.32 mmol), Fmoc-Ser(tBu)-OH (505 mg, 1.32 mmol), Fmoc-Trp(Boc)-OH (694 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Leu-OH (466 mg, 1.The following amino acids were added: Fmoc-Gly-OH (392 mg, 1.32 mmol), Fmoc-His(Trt)-OH (818 mg, 1.32 mmol), Fmoc-Ala-OH (410 mg, 1.32 mmol), Fmoc-Val-OH (447 mg, 1.32 mmol), Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), Fmoc-Phe-OH (387 mg, 1.32 mmol), and Fmoc-Asn(Trt)-OH (786 mg, 1.32 mmol), until all amino acids were linked.

[0289] Step 4, N-terminal acetylation

[0290] Add 5 ml of pyridine and 5 ml of acetic anhydride to the peptide reaction tube from step 3 above, and shake at room temperature for 30 min. After the reaction is complete, filter to remove the solution, and rinse the resin with 20 ml of DCM and 20 ml of DMF.

[0291] Step 5: Resin dissociation and removal of side chain protection of the peptide.

[0292] Add 10 ml of cleavage reagent (TFA / TIPs / water = 95:2.5:2.5, volume ratio) to the peptide reaction tube from step four above, and shake at room temperature for 2 hours. After the reaction is complete, collect the filtrate. Add 100 ml of ice-cold ether to the filtrate to precipitate the crude peptide, and filter to obtain the crude peptide.

[0293] Step 6: Purification of crude peptides

[0294] The 0.80 g crude peptide obtained in step 5 was dissolved in 10 mL of a 1:1 mixture of acetonitrile and water, and purified by reversed-phase HPLC. The chromatographic column was a Shim-pack PREP-ODS15UM 20X250MM (Shimadzu, Japan); pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths (214 nm and 254 nm). After purification, the collected liquid was lyophilized to powder using a freeze dryer (Labconco, USA). 0.66 g of white lyophilized SIP-11 powder with a purity ≥97.2% was obtained.

[0295] Step 7: Identification of peptides

[0296] The molecular weight of the purified peptides was determined using high-resolution mass spectrometry (Waters Xevo G2-XSQTOF, Waters Corporation, USA). HRMS m / z: [M+3H] 3+ =1570.0223; [M+4H] 4+=1150.8321.

[0297] The structural formula of SIP-11 is: NFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLR.

[0298] Example 12

[0299] Real-time quantitative PCR was used to detect the inhibitory activity of SIP-1 to SIP11 against IFN-β. The specific steps are as follows:

[0300] Using RAW264.7 (mouse mononuclear macrophage leukemia cells), when the cells reached 80-90% confluence, the supernatant was removed, and the cells were washed twice with PBS. The cells were then digested with trypsin containing 0.25% EDTA until they detached from the cell wall. An equal volume of complete culture medium was added to stop the digestion, and the cells were transferred to 15 mL centrifuge tubes to collect the cells. The supernatant was discarded, and the cells were resuspended in 1-2 mL of culture medium. The cells were counted and seeded at a density of 8000 cells / well in 48-well plates. For initial screening, the cells were cultured in fresh culture medium containing the same concentration (10 μM) of peptide for 6 hours, followed by stimulation with 2'3'-cGAMP (5 μg / mL) for 4 hours. RNA was extracted using a TRIzol kit, quantified, and reverse transcribed into cDNA. Changes in Ifnb mRNA were detected at the mRNA level using real-time quantitative PCR. For efficacy validation of the selected compounds, cells were cultured in fresh medium containing different concentrations of the drug (0, 1, 2.5, 5, 7.5, 10, 20, 40, 80 μM) for 6 h, followed by stimulation with 2'3'-cGAMP (5 μg / mL) for 4 h. RNA was extracted, and the expression level of Ifnb mRNA was detected using quantitative real-time PCR. The IC50 value was then fitted using GraphpadPrism 9.0 software. 50 value.

[0301] Experimental results: such as Figures 2-12 As shown, Figure 2 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-1 of the present invention. Figure 3 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-2 of the present invention. Figure 4 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-3 of the present invention. Figure 5 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-4 of the present invention. Figure 6 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-5 of the present invention. Figure 7 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-6 of the present invention. Figure 8This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-7 of the present invention. Figure 9 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-8 of the present invention. Figure 10 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-9 of the present invention. Figure 11 This is a schematic diagram of the initial activity screening and verification data of the polypeptide compound SIP-10 of the present invention. Figure 12 This is a schematic diagram illustrating the initial screening and validation data of the peptide compound SIP-11 of the present invention. Each peptide compound inhibits IfnbmRNAIC. 50 The values ​​are shown in the figure: SIP-1 inhibits Ifnb mRNAIC 50 The values ​​were 8.6 μM and 8.6 μM, respectively, indicating that SIP-2 inhibited Ifnb mRNA IC. 50 The values ​​were 9.2 μM and 9.2 μM, respectively, for SIP-3 inhibition of Ifnb mRNA IC. 50 The values ​​were 14.3 μM and 14.3 μM, respectively, for SIP-4 inhibition of Ifnb mRNA IC. 50 The values ​​were 22.31 μM and 22.31 μM, respectively, indicating that SIP-5 inhibited Ifnb mRNA IC. 50 The values ​​were 28.52 μM, and SIP-6 inhibited Ifnb mRNA IC. 50 The values ​​were 35.31 μM and 35.31 μM, respectively, indicating that SIP-7 inhibited Ifnb mRNA IC. 50 The values ​​were 18.94 μM and 18.94 μM, respectively, for SIP-8 inhibition of Ifnb mRNA IC50. 50 The values ​​were 32.6 μM and 32.6 μM, respectively, indicating that SIP-9 inhibited Ifnb mRNA IC. 50 The values ​​were 41.87 μM and SIP-10, respectively, which inhibited Ifnb mRNA IC. 50 The values ​​were 66.28 μM and 66.28 μM, respectively, indicating that SIP-11 inhibited Ifnb mRNA IC. 50 The values ​​were 53.17 μM. All 11 polypeptide molecules significantly inhibited the expression of Ifnb mRNA, and showed a good dose-response relationship.

[0302] Example 13

[0303] The specific steps for determining the chondrogenicity of SIP-2 using the CCK-8 assay are as follows:

[0304] Primary chondrocytes were extracted and passaged when the cells reached 80-90% confluence. The culture medium was removed, and the cells were washed twice with PBS. The cells were digested with trypsin containing 0.25% EDTA until they detached from the cell wall. An equal volume of complete culture medium was added to stop the reaction. The cells were then transferred to 15 ml centrifuge tubes to collect the cells. The supernatant was discarded, and 1-2 ml of culture medium was added to prepare a cell suspension. The cells were counted, and the obtained chondrocytes were seeded at a density of 5000 cells per well in 96-well plates. After overnight adhesion, the cells were cultured in complete culture medium containing different concentrations of drugs (6.25, 12.5, 25, 50, 100, and 200 μM) for 24 h and 48 h, respectively. CCK-8 working solution was added, and the cells were incubated in a 37°C incubator in the dark for 1-1.5 h. The OD value at 450 nm was measured using a microplate reader.

[0305] Experimental results: The results are as follows Figure 13 and Figure 14 As shown, Figure 13 This diagram illustrates the cytotoxic effects of SIP-2 on cartilage at different concentrations over 24 hours. Figure 14 This diagram illustrates the cytotoxicity of SIP-2 at different concentrations to cartilage cells over 48 hours. The figure shows that SIP-2 at working concentrations below 50 μM exhibits good safety for primary chondrocytes.

[0306] Example 14

[0307] The CCK-8 assay was used to determine the protective effect of SIP-2 on a chondrocyte inflammatory injury model. The specific steps are as follows:

[0308] Chondrocytes were seeded into 96-well plates at a density of 5000 cells / well. After 24 hours of culture, an inflammatory injury model was induced using IL-1β (10 ng / ml). The cells were co-cultured with different concentrations of the drug (10 and 50 μM) for 24 hours and 48 hours, respectively. CCK-8 working solution was then added, and the cells were incubated in a 37°C incubator in the dark for 1–1.5 hours. The OD value at 450 nm was measured using a microplate reader.

[0309] The results are as follows Figure 15 and 16 As shown, Figure 15 This is a schematic diagram illustrating the protective effect of SIP-2 at different concentrations on a 24-hour cartilage inflammatory injury model. Figure 16 This diagram illustrates the protective effect of SIP-2 at different concentrations on a cartilage inflammatory injury model over 48 hours. The figure shows that, compared to the IL-1β-induced injury model, the SIP-2 treatment group exhibited significantly increased cell viability, indicating that SIP-2 can effectively improve cartilage inflammatory injury.

[0310] Example 15

[0311] The specific steps for detecting the effects of SIP-2 on Ifnβ, Tnf, and Il6 mRNA in chondrocytes using real-time quantitative PCR are as follows:

[0312] Chondrocytes were seeded into 48-well plates at a density of 8000 cells / well and cultured overnight in an incubator. Then, the cells were pretreated with different concentrations of SIP-2 (10, 50 μM) for 6 h. An inflammatory injury model was induced using IL-1β (10 ng / ml). RNA was extracted, and the changes in Ifnb, Tnf, and Il6 genes were detected by real-time PCR.

[0313] Experimental results: The results are as follows Figures 17-19 As shown, Figure 17 This is a schematic diagram illustrating the data on the regulation of chondrocyte inflammatory factor Ifnb gene release by SIP-2. Figure 18 This is a schematic diagram illustrating the data on the regulation of chondrocyte inflammatory factor Tnf gene release by SIP-2. Figure 19 This is a schematic diagram illustrating the regulation of chondrocyte inflammatory factor Il6 gene release by SIP-2. The diagram shows that IL-1β stimulation significantly increased the levels of Ifnb, Tnf, and Il6, while the SIP-2 treatment group significantly reduced the transcriptional levels of these inflammatory cytokines, exhibiting a good dose-response relationship. SIP-2 can improve the IL-1β (10 ng / ml)-induced inflammatory injury model.

[0314] Example 16

[0315] The therapeutic effect of SIP-2 on a mouse model of arthritis induced by medial meniscus instability surgery was studied, and the specific steps are as follows:

[0316] Test animals: 36 male C57BL / 6 mice, aged 8-9 weeks, purchased from Changzhou Kevins Company.

[0317] Experimental drugs: Model group (n=12), low concentration (1 mg / kg, n=12), high concentration (10 mg / kg, n=12).

[0318] The method for constructing a medial meniscus instability (DMM)-induced arthritis model is as follows: Mice were anesthetized with isoflurane and fixed supine on the operating table. The left knee joint was routinely prepared and disinfected. Using sterile surgical instruments, the skin and subcutaneous tissue were sequentially incised through the medial side of the patella, and the patellar ligament was gently separated to expose the knee joint. The anteromedial meniscal ligament and medial collateral ligament were cut, and the medial meniscus was freed to induce instability in the left knee joint. The incision was sutured and disinfected with povidone-iodine.

[0319] The experimental mice were divided into three groups: model group (n=12), low-concentration group (1 mg / kg, n=12), and high-concentration group (10 mg / kg, n=12). The dosing regimen was as follows: Figure 20 ( Figure 20 The diagram shows the dosing regimen for SIP-2 treatment of arthritis lesions in mice. As shown, the drug was administered via the tail vein after DMM surgery, once every 3 days. Joint tissues were collected at 6 and 12 weeks of treatment for histological evaluation.

[0320] Experimental results: Compared with the DMM model group, the SIP-2 treatment group (10 mg / kg) showed wider knee joint space and significantly reduced sclerotic cartilage thickness, such as... Figures 21-23 As shown, Figure 21 This is a schematic diagram illustrating the effect of SIP-2 on cartilage sclerosis at the joints of DMM model mice. Figure 22 This is a schematic diagram illustrating the quantification of cartilage area at the joints of DMM model mice using SIP-2. Figure 23 This is a schematic diagram illustrating the quantitative effect of SIP-2 on cartilage thickness at the joints of DMM model mice. The figure shows that SIP-2 effectively improves the cartilage sclerosis process in the joints of DMM model mice; three-dimensional imaging observation of the effect of SIP-2 on osteophytes at the joints revealed that SIP-2 treatment significantly reduced the area and volume of osteophytes after DMM surgery.

[0321] like Figures 24-27 As shown, Figure 24 This is a schematic diagram of a lower limb knee joint CT scan illustrating the effect of SIP-2 on osteophyte formation at the knee joint in a DMM model. Figure 25 This is a schematic diagram of the osteophyte grading and scoring in the SIP-2 model of osteophyte formation at the knee joint of the DMM model. Figure 26 This is a schematic diagram illustrating the quantification of osteophyte levels in the knee joint generation of the DMM model using SIP-2. Figure 27 This is a schematic diagram illustrating the quantitative effect of SIP-2 on osteophyte formation in the knee joint of a DMM model. The figure shows that SIP-2 effectively improved osteophyte formation in DMM model mice. Morphological observation of cartilage tissue revealed changes in cartilage morphology. HE staining showed that, compared to the model group, SIP-2 treatment resulted in restored smooth cartilage surface, reduced hypertrophic chondrocytes, tidal line displacement, reduced cartilage lesions and defects in the distal femur, intact cartilage tissue, and a significant increase in the OARSI score.

[0322] like Figures 28-30 As shown, Figure 28 This is a schematic diagram of safranin-fixing staining in the HE pathology and SO staining evaluation of cartilage tissue from the DMM model using SIP-2. Figure 29 This is a schematic diagram illustrating the quantification of joint area in the evaluation of HE pathology and SO staining of cartilage tissue in the DMM model using SIP-2. Figure 30This diagram illustrates the quantification of joint damage in the evaluation of cartilage tissue in a DMM model using HE pathology and SO staining with SIP-2. The diagram demonstrates that high-concentration SIP-2 treatment effectively protects the normal morphology and structure of cartilage in the knee joint.

[0323] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A polypeptide compound with anti-inflammatory activity or a pharmaceutical salt thereof, characterized in that, The amino acid sequence of the peptide compound with anti-inflammatory activity is selected from one of the following amino acid sequences: The amino acid sequence shown in SEQ ID NO.1; The structure of the polypeptide compound SIP-2 is shown below: S5 is (2R)-2-amino-2-methyl-6-heptenoic acid.

2. A method for preparing a polypeptide compound with anti-inflammatory activity as described in claim 1, characterized in that, Includes the following steps: Step 1, resin swelling: Fmoc-Rink amid-MBHAResin is placed in a peptide reaction tube, swollen by DCM, and the DCM is removed by filtration; The second step is the removal of Fmoc protection: Add 20% piperidine / DMF solution to the reaction tube from the first step, shake, filter to remove the solution, and rinse the resin with DCM. The third step is the amino acid linkage: Fmoc-Leu-OH and HCTU are dissolved in DMF, then DIPEA is added and shaken. The mixture is then poured into the peptide reaction tube from the second step and shaken at room temperature. After the reaction is complete, the solution is removed by filtration and the resin is rinsed with DCM and DMF. Repeat steps two and three to sequentially connect Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Leu-OH until all amino acids are connected. Alternatively, repeat steps two and three to sequentially connect Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-S5-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-S5-OH, Fmoc-Ala-OH, and Fmoc-Leu-OH until all amino acids are connected. Step 4, N-terminal acetylation: Add pyridine and acetic anhydride in a volume ratio of 1:1 to the peptide reaction tube from step 3 above and shake at room temperature. After the reaction is complete, filter to remove the solution and rinse the resin with DCM and DMF. Step 5: Resin dissociation and side chain protection removal of peptides: Add a cleavage reagent to the peptide reaction tube from step 4 above. The cleavage reagent is: TFA / TIPs / water = 95:2.5:2.

5. Shake at room temperature. After the reaction is complete, collect the filtrate. Add ice-cold ether to the filtrate to precipitate the crude peptide. Filter to obtain the crude peptide. Step 6, purification of crude peptide: The crude peptide obtained in step 5 was dissolved in a 1:1 mixture of acetonitrile and water, purified by reversed-phase HPLC, and freeze-dried to obtain a polypeptide compound with anti-inflammatory activity.

3. The method for preparing the anti-inflammatory polypeptide compound according to claim 2, characterized in that, The reversed-phase HPLC purification conditions for step six are as follows: column: Shim-pack PREP-ODS15UM 20X250MM; pump A: acetonitrile containing 1 / 1000 trifluoroacetic acid; pump B: water containing 1 / 1000 trifluoroacetic acid; wash gradient: from 90% pump B to 0% pump B over 20 minutes; detector: dual wavelengths 214nm and 254nm.

4. The method for preparing the polypeptide compound with anti-inflammatory activity according to claim 2 or 3, characterized in that, The method for preparing the polypeptide compound with anti-inflammatory activity further includes: When the polypeptide compound is SIP-2, the fourth step includes a side-chain olefin metathesis reaction: a 1,2-dichloroethane solution of the first-generation Grubbs catalyst is added to the polypeptide reaction tube of the fourth step above, and the mixture is shaken at room temperature overnight; after the reaction is completed, the solution is removed by filtration, and the resin is washed with DCM and DMF; the fifth step reaction is then carried out.

5. The use of the polypeptide compound with anti-inflammatory activity as described in claim 1 or its pharmaceutical salt in the preparation of a medicament for treating osteoarthritis.

6. The application according to claim 5, characterized in that, The osteoarthritis mentioned is knee osteoarthritis.

7. A pharmaceutical composition comprising, as the active pharmaceutical ingredient, the polypeptide compound having anti-inflammatory activity as described in claim 1.

Images