A group of cyclic polypeptide compounds and their preparation method and application

Abstract

The present invention discloses a group of cyclic polypeptide compounds and their preparation methods and applications. The cyclic polypeptide compounds have a structure as shown in general formula I. The preparation method of the cyclic polypeptide compounds comprises the following steps: (1) reacting the protected free amino group of the side chain of the basic amino acid Fmoc-AA-OP with a halogenated resin to obtain Fmoc-AA-OP-resin; (2) coupling the Fmoc-AA-OP-resins one by one to obtain linear polypeptide-resins; (3) selectively removing the protecting groups of the linear polypeptide-resins and solid-phase cyclization to obtain cyclic polypeptide-resins; (4) acid hydrolyzing the cyclic polypeptide-resins and precipitating them with ether to obtain a crude cyclic polypeptide; (5) purifying and / or converting the crude cyclic polypeptides and freeze-drying them to obtain a pure cyclic polypeptide compound. Experiments have shown that the antibacterial activity and antibacterial spectrum of the cyclic polypeptide compounds of the present invention are significantly improved compared with the natural product octapeptidin. The invention provides a new technical means for the development and application of antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to a series of cyclic polypeptide compounds containing 8-9 amino acid residues and a preparation method thereof, and also relates to the use of the cyclic polypeptide compounds in the preparation of antibacterial drugs, as well as a pharmaceutical composition containing the cyclic polypeptide compounds as active ingredients and the application thereof. The present invention belongs to the field of biomedicine. Background Art

[0002] The rapid emergence of drug-resistant pathogens has become a global health problem. The World Health Organization points out that antibiotic resistance is a global public health crisis that must be managed with the utmost urgency. The overuse and abuse of antibiotics has stimulated the rapid emergence of antibiotic-resistant bacteria. Drug-resistant bacteria have severely restricted the use of existing antibiotics (such as carbapenems, fluoroquinolones, etc.), and clinical treatment options have little effect on infections caused by drug-resistant bacteria. Polymyxin, as the last line of defense for the clinical treatment of Gram-negative bacterial infections, has also developed drug resistance cases. Therefore, people are in urgent need of new antibacterial agents.

[0003] Polypeptide antimicrobial agents, namely antimicrobial peptides (AMPs), have attracted extensive attention due to their broad antimicrobial activity, rapid sterilization, high cell selectivity, and difficulty in developing drug resistance. They are expected to solve the problem of drug-resistant bacterial infections. AMPs are ubiquitous in nature, and some AMPs have been used, such as using polymyxin E as a first-line antimicrobial agent to treat severe infections caused by multidrug-resistant microorganisms.

[0004] Octapeptide was discovered in 1973 and is an antimicrobial peptide produced by Bacillus circulans. Natural octapeptide is divided into four structural types: A, B, C, and D. The main structure is composed of a fatty acyl chain containing a hydroxyl group and a cyclic heptapeptide connected by a D-configuration amino acid. It has a broad-spectrum antibacterial activity, poor activity against Gram-positive bacteria, moderate activity against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and activity against Pseudomonas aeruginosa is comparable to that of polymyxin, but it still has antibacterial activity against polymyxin-resistant Pseudomonas aeruginosa, and has strong antibacterial activity against yeasts and fungi, and its acute toxicity is lower than that of polymyxin B.

[0005] Due to the increasing number of drug-resistant bacteria and the urgent clinical need for new antibiotics, the preparation and application research of new octapeptides and their derivatives with broad antibacterial spectrum, low toxicity and good activity against drug-resistant bacteria is particularly important.

[0006] It was not until 2015 that natural octapeptide was first synthesized by chemical preparation method. In the method reported in the literature (De Zoysa Gayan Heruka, Cameron Alan James, Hegde Veena V et al. Antimicrobial peptides with potential for biofilm eradication: synthesis and structure activity relationship studies of battacin peptides. J Med Chem, 2015, 58: 625-39.), the Fmoc solid phase peptide synthesis method was used to first obtain the corresponding linear peptide on the resin and then cut the linear peptide from the resin and perform cyclization under liquid phase conditions. This method is complicated to operate, has a low yield of only 10%, and is not easy to purify. Then Tony et al. designed and modified the derivatives of natural octapeptin C compounds, optimized the synthesis route, and obtained a derivative with simple operation and a yield of 20% through solid-phase condensation and solid-phase cyclization (Velkov Tony, Gallardo-Godoy Alejandra, Swarbrick James D et al. Structure, Function, and Biosynthetic Origin of Octapeptin Antibiotics Active against Extensively Drug-Resistant Gram-Negative Bacteria. Cell Chem Biol, 2018, 25: 380-391.).

[0007] Based on the above foundation, the present invention studies the design, synthesis and application of a group of cyclic polypeptides containing 8-9 amino acids, and further optimizes the reaction route, simplifies the operation and increases the yield to 30%.

[0008] Regarding the preparation of new derivatives, the present invention prepares the side chain acyl chain by changing the length and volume of the side chain acyl chain to increase or decrease the side chain acyl chain (changing R 0 ) hydrophobic derivatives, basic amino acids or polar amino acids replacing amino acids at positions 1 and / or 3 (changing R 1 , R 2 ), wherein the hydrophobic amino acid or polar amino acid replaces the amino acid at position 4 and / or position 5 (changing R 3 , R 4 ), a new derivative of the present invention, wherein a hydrophobic amino acid, an acidic amino acid, or a polar amino acid replaces the amino acid at position 8 (changing R 7), and the length of z can be changed to expand or reduce the ring. By changing the number of amino groups or hydrophobicity, the antibacterial spectrum can be expanded or the antibacterial activity can be improved or the nephrotoxicity can be reduced.

[0009] The present invention has discovered a group of cyclic polypeptide compounds with new structures containing 8-9 amino acid residues. The antibacterial activity and antibacterial spectrum of the compounds are significantly improved compared with the natural product octapeptide. The present invention provides a new technical means for the development and application of antibacterial drugs. Summary of the invention

[0010] One of the purposes of the present invention is to provide a group of cyclic polypeptide compounds with new structures containing 8-9 amino acid residues, whose antibacterial activity and antibacterial spectrum are significantly improved compared with the natural product octapeptide.

[0011] The second object of the present invention is to provide a method for preparing the cyclic polypeptide compound, in particular a method comprising solid phase condensation and solid phase cyclization.

[0012] The third object of the present invention is to provide the use of the cyclic polypeptide compound in the preparation of antibacterial drugs.

[0013] In order to achieve the above object, the present invention adopts the following technical solutions:

[0014] The present invention provides a group of cyclic polypeptide compounds containing 8-9 amino acid residues or pharmaceutically acceptable salts thereof, the structure of which is shown in general formula Ⅰ':

[0015]

[0016] The numbers 1 to 8 in Formula I' represent the specific positions of the amino acid residues in Formula I', and the amino acids at positions 1 to 8 are separated by square brackets. Each square bracket corresponds to a specific number, for example, the square bracket corresponding to number 1 is the amino acid at position 1, and the square bracket corresponding to number 2 is the amino acid at position 2.

[0017] To simplify the description, the brackets in the general formula I' are removed to form the general formula I:

[0018]

[0019] The present invention provides a group of cyclic polypeptide compounds having a structure shown in general formula I or a pharmaceutically acceptable salt thereof, wherein the cyclic polypeptide compound is composed of three parts: a cyclic heptapeptide, an amino acid linear peptide side chain and a terminal acyl chain;

[0020] Where:

[0021] R 0Selected from the group consisting of: a straight chain alkyl group containing 6-11 carbon atoms, a branched chain alkyl group containing 7-12 carbon atoms, a straight chain alkyl group containing 6-11 carbon atoms substituted with a hydroxyl group, a branched chain alkyl group containing 7-12 carbon atoms substituted with a hydroxyl group, a straight chain alkyl group containing 6-11 carbon atoms and containing an ether bond, a phenyl group substituted with a straight chain alkyl group containing 4-8 carbon atoms or a branched chain alkyl group containing 5-9 carbon atoms, a tetrahydrofuran ring substituted with a straight chain alkyl group containing 2-8 carbon atoms,

[0022]

[0023] R 1 Selected from the group consisting of: -CH 2 OH, -CH(CH 3 )OH、NH 2 -(CH 2 ) x -、CH 3 -NH-(CH 2 ) x -、 NH 2 -CO-(CH 2 ) x -, x is an integer from 1 to 4; the configuration of the amino acid at position 1 is D or L;

[0024] R 2 , R 5 and R 6 Independently selected from the group consisting of: -CH 2 OH, -CH(CH 3 )OH、-(CH 2 ) 2 -S-CH 3 , a straight chain alkyl group containing 1-4 carbon atoms or a branched chain alkyl group containing 3-4 carbon atoms, NH 2 -(CH 2 ) y -、CH 3 -NH-(CH 2 ) y -、 y is an integer from 1 to 4; the configuration of the amino acids at positions 3, 6, and 7 is L-type;

[0025] R 3 and R 4 independently selected from the group consisting of: H, a straight chain alkyl group containing 1 to 8 carbon atoms or a branched chain alkyl group containing 3 to 8 carbon atoms, -CH 2 OH, -CH(CH 3 )OH、-(CH 2 ) 2 -S-CH 3 、-CH2 COOH and -CH 2 -R 8 ; R 8 Selected from the group consisting of phenyl, cyclohexyl, 3-indolyl, The configuration of amino acids at positions 4 and 5 is D or L; R 9 , R 10 and R 11 Independently selected from the group consisting of: -OH, -NH 2 、-F、-Cl、-Br、-CN、-NO 2 , -CF 3 , CH 3 O-、CH 3 CH 2 O-, straight-chain alkyl containing 1-4 carbon atoms or branched-chain alkyl containing 3-4 carbon atoms, phenyl, benzyl, benzoyl;

[0026] R 7 Selected from the group consisting of: -CH 2 OH, -CH(CH 3 )OH、-CH 2 COOH, phenyl, benzyl, and straight-chain alkyl containing 1-4 carbons or branched-chain alkyl containing 3-4 carbons; the configuration of the amino acid at position 8 is L-type;

[0027] z is an integer of 0-3, and the configuration of the amino acid at position 2 is L-type.

[0028] When z in the general formula I is 1, it has a structure as shown in the general formula II:

[0029]

[0030] In a specific embodiment of the present invention, the cyclic polypeptide compound includes a group selected from the following: (HX-1)(R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Thr-Dab-Dab-Thr]

[0031]

[0032] (HX-2)(S)-6-Methyloctanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0033]

[0034] (HX-3)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0035]

[0036] (HX-4)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe-Leu-Dab-Dab-Leu]

[0037]

[0038] (HX-5)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu]

[0039]

[0040] (HX-6)(S)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0041]

[0042] (HX-7)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe-Leu-Dab-Dab-Thr]

[0043]

[0044] (HX-8)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0045]

[0046] (HX-9)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-Leu-D-Phe-Dab-Dab-Leu]

[0047]

[0048] (HX-10)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe-Thr-Dab-Dab-Leu]

[0049]

[0050] (HX-11)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-Leu-D-Phe-Dab-Dab-Thr]

[0051]

[0052] (HX-12)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Thr-Dab-Dab-Leu]

[0053]

[0054] (HX-13)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]

[0055]

[0056] (HX-14) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0057]

[0058] (HX-15) nonanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0059]

[0060] (HX-16) Succinic acid mono(1-methylbutyl) ester-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0061]

[0062] (HX-17)(R)-3-Hydroxydecanoyl-D-Ser-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0063]

[0064] (HX-18)(R)-3-Hydroxydecanoyl-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0065]

[0066] (HX-19)(R)-3-Hydroxydecanoyl-Dab-Dab-Cyclo(3-9)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu]

[0067]

[0068] (HX-20)(R)-3-Hydroxydecanoyl-D-Ser-D-Dab-cyclo(3-9)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu]

[0069]

[0070] (HX-21) Octanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0071]

[0072] (HX-22) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-Leu-Leu-Dab-Dab-Thr]

[0073]

[0074] (HX-23) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-D-Leu-Dab-Dab-Thr]

[0075]

[0076] (HX-24) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Bip-Dab-Dab-Thr]

[0077]

[0078] (HX-25) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Bip-Leu-Dab-Dab-Thr]

[0079]

[0080] (HX-26) n-Decanoyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Chg-Leu-Dab-Dab-Thr]

[0081]

[0082] (HX-27) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-octyl-Gly-Dab-Dab-Thr]

[0083]

[0084] (HX-28) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Asp-Dab-Dab-Thr]

[0085]

[0086] (HX-29) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Asp]

[0087]

[0088] (HX-30) Phenylhexanoyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0089]

[0090] (HX-31)7,7-Dimethyloctanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0091]

[0092] (HX-32) n-Decanyl-D-Dap-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0093]

[0094] (HX-33) n-Decanoyl-D-Orn-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0095]

[0096] (HX-34) n-Decanyl-D-Thr-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0097]

[0098] (HX-35) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Lys-D-Leu-Leu-Dab-Dab-Thr]

[0099]

[0100] (HX-36) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Orn-D-Leu-Leu-Dab-Dab-Thr]

[0101]

[0102] (HX-37) n-Decanyl-D-Dab-Cyclo(2-8)[Dap-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0103]

[0104] (HX-38) n-Decanyl-D-Dab-Cyclo(2-8)[Orn-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0105]

[0106] (HX-39) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-tBu)-Leu-Dab-Dab-Thr]

[0107]

[0108] (HX-40) n-Decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-Cl)-Leu-Dab-Dab-Thr]

[0109]

[0110] (HX-41) n-octanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu]

[0111]

[0112] (HX-42) nonanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu]

[0113]

[0114] (HX-43) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu]

[0115]

[0116] (HX-44) n-Decanoyl-D-Asn-cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu]

[0117]

[0118] (HX-45) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Phe-Bip-Dab-Dab-Thr]

[0119]

[0120] (HX-46) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Trp-Dab-Dab-Leu]

[0121]

[0122] (HX-47) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Phe-Bip-Dab-Dab-Leu]

[0123]

[0124] (HX-48) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Phe-Phe-Dab-Dab-Leu]

[0125]

[0126] (HX-49) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Ser]

[0127]

[0128] (HX-50) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Phe]

[0129]

[0130] (HX-51) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe(4-Me)-Dab-Dab-Leu]

[0131]

[0132] (HX-52) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe(4-OMe)-Dab-Dab-Leu]

[0133]

[0134] (HX-53) n-Decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(3-Cl)-Leu-Dab-Dab-Thr]

[0135]

[0136] (HX-54) n-decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-NH 2 )-Leu-Dab-Dab-Thr]

[0137]

[0138] (HX-55) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(2-Cl)-Leu-Dab-Dab-Thr]

[0139]

[0140] (HX-56) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(3,4-DiCl)-Leu-Dab-Dab-Thr]

[0141]

[0142] (HX-57) n-Decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-Me)-Leu-Dab-Dab-Thr]

[0143]

[0144] (HX-58) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Ala]

[0145]

[0146] (HX-59) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Gly]

[0147]

[0148] (HX-60) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Val]

[0149]

[0150] (HX-61) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Nva]

[0151]

[0152] (HX-62) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Ile]

[0153]

[0154] (HX-63) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Val-Leu-Dab-Dab-Thr]

[0155]

[0156] (HX-64) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Nva-Leu-Dab-Dab-Thr]

[0157]

[0158] (HX-65) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Ile-Leu-Dab-Dab-Thr]

[0159]

[0160] (HX-66) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Val-Dab-Dab-Thr]

[0161]

[0162] (HX-67) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Nva-Dab-Dab-Thr]

[0163]

[0164] (HX-68) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Ile-Dab-Dab-Thr]

[0165]

[0166] (HX-69)(R)-3-Hydroxy-6-methylheptanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0167]

[0168] (HX-70)(R)-2-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0169]

[0170] (HX-71) 9-Hydroxynonanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0171]

[0172] (HX-72)(3R,6S)-3-Hydroxy-6-methyloctanoyl-D-Dab-cyclo(2-8)

[0173] [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0174]

[0175] (HX-73) 4-Pentyloxybutyryl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0176]

[0177] (HX-74) 3-(Tetrahydrofuran-2-yl)propanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0178]

[0179] (HX-75) 6-Methoxyhexanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr]

[0180]

[0181] (HX-76)(R)-3-Hydroxydecanoyl-DN-methyl-Dab-Cyclo(2-8)

[0182] [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0183]

[0184] (HX-77)(R)-3-Hydroxydecanoyl-D-Arg-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0185]

[0186] (HX-78)(R)-3-Hydroxydecanoyl-D-3-guanidinoalanine-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0187]

[0188] (HX-79)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-N-methyl-Dab-D-Leu-Leu-Dab-Dab-Leu]

[0189]

[0190] (HX-80)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-N-methyl-Dab-Dab-Leu]

[0191]

[0192] The term "ring (2-8)" refers to a heptapeptide ring formed by connecting the terminal carboxyl group of the amino acid at position 8 to the side chain amino group of the basic amino acid at position 2 via an amide bond, and the structure is shown in general formula I and general formula II.

[0193] The configuration of D-amino acids is represented by D. When the configuration is not mentioned, it is understood that the amino acid configuration is L. Dab represents α,γ-diaminobutyric acid, Orn represents 2,5-diaminopentanoic acid, Dap represents α,β-diaminopropionic acid, Bip represents 3-(4-biphenyl)alanine, octyl-Gly represents octylglycine, Chg represents cyclohexylglycine, Nva represents 2-aminopentanoic acid, and N-methyl-Dab represents 2-amino-4-methylaminobutyric acid.

[0194] The pharmaceutically acceptable salts of the compounds of general formula I described in the present invention include salts formed by the compounds of general formula I and an acid, wherein the acid is selected from the group consisting of: an inorganic acid or an organic acid, wherein the inorganic acid is, for example, perchloric acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid; the organic acid is, for example, acetic acid, trifluoroacetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, benzoic acid, methanesulfonic acid or p-toluenesulfonic acid.

[0195] The present invention also provides a new solid phase synthesis method for a cyclic polypeptide compound or a pharmaceutically acceptable salt thereof. The method prepares a cyclic polypeptide compound or a pharmaceutically acceptable salt thereof by solid phase condensation and solid phase cyclization, comprising the following steps:

[0196] (1) The protected free amino group of the side chain of the basic amino acid Fmoc-AA-OP is reacted with a halogenated resin to obtain Fmoc-AA-OP-resin; wherein P is a carboxyl protecting group, for example, allyl (Allyl), benzyl (Bn):

[0197] When Fmoc-AA-OP is Fmoc-Dab-OP, its structure is as shown in Formula III:

[0198]

[0199] (2) Fmoc-AA-OP-resin is coupled one by one to obtain linear peptide-resin;

[0200] (3) selectively removing the protecting group from the linear peptide-resin and performing solid phase cyclization to obtain the cyclic peptide-resin;

[0201] (4) Acid hydrolysis of the cyclic polypeptide-resin to obtain a crude cyclic polypeptide compound;

[0202] (5) The crude cyclic polypeptide is purified and / or converted to salt, and then freeze-dried to obtain a pure cyclic polypeptide compound.

[0203] Wherein, preferably, in step (1), the halogenated resin is selected from the group consisting of trityl chloride resin, 4-methyltrityl chloride resin, 4-methoxytrityl chloride resin, 2-chlorotrityl chloride resin, bromo-(4-methylphenyl)-methyl resin or bromo-(4-methoxyphenyl)-methyl resin, for example, the resin is 2-chlorotrityl chloride resin.

[0204] The degree of substitution of the halogenated resin is 0.1-1.6 mmol / g, for example, the degree of substitution is 0.5-1.0 mmol / g.

[0205] The amount of each Fmoc-protected amino acid used is 1.2-6 times, for example, 2.0-3.5 times, the total molar number of the resin fed.

[0206] The base is selected from the group consisting of: at least one of N,N-diisopropylethylamine (DIEA), triethylamine (TEA), and pyridine, for example, DIEA; the molar amount of the base is 1.5-3 times the molar amount of the Fmoc-protected amino acid, for example, 2 times the molar amount of the Fmoc-protected amino acid.

[0207] The substitution reaction time is 1-12 h, for example, 2-3 h.

[0208] Preferably, in step (2), coupling Fmoc-AA-OP-resins one by one to obtain linear polypeptide-resins comprises the following steps:

[0209] Reagents for removing the α-amino Fmoc protecting group include, but are not limited to, a solution of piperidine (PIP) in DMF at a concentration of 10-30%. A solution of PIP in DMF, for example, at a concentration of 20%. The amount of the deprotecting reagent used is 5-15 mL per gram of the resin fed, for example, 10 mL per gram of the resin fed. The deprotection reaction time is 10-60 min, for example, 10-20 min. Reagents for removing the amino ivDde or Dde protecting group of the amino acid side chain at position 2 include, but are not limited to, a solution of hydrazine hydrate in DMF at a concentration of 1-10%. A solution of hydrazine hydrate in DMF, for example, at a concentration of 3%. The amount of the deprotecting reagent used is 5-15 mL per gram of the resin fed, for example, 10 mL per gram of the resin fed. The deprotection reaction time is 30-100 min, for example, 30-60 min.

[0210] In the coupling reaction, the coupling agent is selected from the group consisting of: N,N-diisopropylcarbodiimide (DIC), N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), for example, N,N-diisopropylcarbodiimide (DIC).

[0211] The molar amount of the coupling agent is 1.2-6 times, for example, 2.0-3.5 times, the total molar amount of the feed resin.

[0212] The activator is selected from the group consisting of 1-hydroxybenzotriazole (HOBT), 6-chloro-1-hydroxybenzotriazole (Cl-HOBT), 1-hydroxy-7-azobenzotriazole (HOAT), for example, 1-hydroxybenzotriazole (HOBT).

[0213] The molar amount of the activator is 1.2-6 times, for example, 2.0-3.5 times, of the total molar amount of the feed resin.

[0214] The coupling reaction time is 60-300 min, for example, 60-120 min.

[0215] In the coupling reaction, part of the coupling agent needs to be added with a catalyst, and the catalyst is an organic base selected from the group consisting of: N,N-diisopropylethylamine (DIEA), triethylamine (TEA), N-methylmorpholine (NMM), for example, N,N-diisopropylethylamine (DIEA).

[0216] The solvent is an aprotic polar solvent selected from the group consisting of dimethylformamide (DMF) or N-methylpyrrolidone (NMP) or a mixture thereof, for example, DMF.

[0217] Preferably, in step (3), selectively removing the protecting group from the linear polypeptide-resin and solid phase cyclization to obtain the cyclic polypeptide-resin comprises the following steps:

[0218] The reagent for removing the carboxyl allyl protecting group is a solution of tetrakis(triphenylphosphine)palladium / phenylsilane in DCM and DMF (a mixed solution with a volume ratio of DCM:DMF of 5:5). The molar amount of tetrakis(triphenylphosphine)palladium is 0.1-2 times the total mole number of the feed resin, for example, 0.1-0.3 times. The molar amount of phenylsilane is 2-10 times the total mole number of the feed resin, for example, 3-5 times. The amount of the deprotection reagent used is 10-30 mL per gram of feed resin, for example, 20 mL per gram of feed resin. The deprotection reaction time is 60-300 min, for example, 60-120 min.

[0219] The solid phase cyclization coupling agent is selected from the group consisting of: (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), for example, (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP).

[0220] The molar amount of the coupling agent is 1.2-6 times, for example, 2.0-3.5 times, the total molar amount of the feed resin.

[0221] The activator is selected from the group consisting of 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azobenzotriazole (HOAT), for example, 1-hydroxy-7-azobenzotriazole (HOAT).

[0222] The molar amount of the activator is 1.2-6 times, for example, 2.0-3.5 times, of the total molar amount of the feed resin.

[0223] The cyclization reaction time is 1-20 h, for example, 1-3 h.

[0224] The catalyst is an organic base selected from the group consisting of: N,N-diisopropylethylamine (DIEA), triethylamine (TEA), N-methylmorpholine (NMM), for example, N-methylmorpholine (NMM).

[0225] The solvent is an aprotic polar solvent selected from the group consisting of dimethylformamide (DMF) or N-methylpyrrolidone (NMP) or a mixture thereof, for example, DMF.

[0226] Preferably, in step (4), the acid hydrolysis of the cyclic polypeptide-resin to obtain a crude cyclic polypeptide comprises the following steps:

[0227] The acid hydrolysis uses an acid hydrolysis solution containing hydrofluoric acid (HF) or trifluoroacetic acid (TFA), for example, trifluoroacetic acid.

[0228] The amount of the acidolysis solution is 5-30 mL per gram of the resin, for example, 10 mL per gram of the resin. The acidolysis solution comprises trifluoroacetic acid and a side chain protecting group removing agent.

[0229] The concentration of trifluoroacetic acid is 80%-95%, and the rest is the side chain protecting group removing agent.

[0230] The side chain protecting group removing agent is selected from the group consisting of thioanisole, triisopropylsilane, phenol, water, 1,2-ethanedithiol, for example, water.

[0231] The acid hydrolysis time is 60-300 min, for example, 100-120 min.

[0232] The acid hydrolyzate containing the polypeptide after acid hydrolysis was added to cold ether (the volume ratio of the acid hydrolyzate to cold ether was 1:20) to precipitate the peptide, centrifuge, and dry to obtain the crude peptide.

[0233] Preferably, in step (5), the crude cyclic polypeptide is purified and / or salted, and freeze-dried to obtain a pure cyclic polypeptide, which comprises the following steps:

[0234] The crude peptide is dissolved in water, filtered through a 0.22 μm filter membrane, and purified by preparative high performance liquid chromatography, using mobile phase A 0.08% TFA / water solution, mobile phase B 0.08% TFA / acetonitrile solution, gradient elution, detection wavelength 214 nm, and the product is dried by freeze drying. The final purity that can be achieved by this method is greater than 95%, for example, greater than 95%.

[0235] Wherein, preferably, the method comprises the following steps:

[0236] (1) Preparation of Fmoc-AA-OP resin: Add the halogenated resin to a peptide solid phase synthesis tube, add DCM to swell, wash with DMF three times, wash with DCM three times after swelling, dissolve the protected starting amino acid Fmoc-AA-OP and the base with DCM and add to the peptide synthesis tube, react at room temperature for 2 h, remove the reaction solution under vacuum, wash the resin with DMF three times, and wash with DCM three times to obtain Fmoc-AA-OP resin;

[0237] (2) Coupling synthesis method: The Fmoc-AA-OP-resin obtained in step (1) was treated with 20% piperidine / DMF for 10 min each time, for a total of 2 times to remove the α-amino Fmoc protecting group, and then washed with DMF three times and DCM three times. The amino acid or side chain carboxylic acid, DIEA and HCTU were dissolved in DMF and added to a peptide synthesis tube, reacted at room temperature for 120 min, and the reaction solution was removed by vacuum, and washed with DMF three times and DCM three times; starting from the starting amino acid, i.e., the amino acid at position x, where x is 6 or 7, it was coupled to the amino acid at position 1 one by one, and then the side chain carboxylic acid was coupled to the protecting on a polypeptide-resin; remove the ivDde or Dde protecting group of the amino group of the amino acid side chain at position 2 with a 2% hydrazine hydrate / DMF solution, wash with DMF three times, wash with DCM three times, couple the carboxyl group of the amino acid at position 8 to the amino acid side chain at position 2; couple one by one from the amino acid at position 8 to the amino acid after the starting amino acid, i.e., the amino acid at position x+1, to obtain a linear fully protected polypeptide-resin; the one-by-one coupling sequence includes two parts, the first part is the starting amino acid, i.e., the amino acid at position x, where x is 6 or 7, to the amino acid at position 1, and then to the side chain carboxylic acid, and the second part is from the amino acid at position 8 to the amino acid at position x+1;

[0238] (3) Selective removal of protecting groups and solid phase cyclization: The linear fully protected peptide-resin in step (2) is treated with 20% piperidine / DMF for 10 min each time, for a total of 2 times, to remove the α-amino Fmoc protecting group, and then washed with DMF three times and DCM three times to form a free amino group; the carboxyl allyl protecting group is removed with tetrakis(triphenylphosphine)palladium / phenylsilane in a mixed solution of DCM and DMF to form a free carboxyl group; PyAOP and HOAT are dissolved in DMF and then added with NMM, and then added to the peptide synthesis tube, reacted at room temperature for 3 h, the reaction solution was removed by vacuum, and washed with DMF three times and DCM three times to obtain a cyclic fully protected peptide-resin;

[0239] (4) The crude cyclic basic polypeptide obtained by acid hydrolysis: TFA:H 2 The acid solution with a volume ratio of 95:5 was added to the peptide synthesis tube, and the reaction was carried out at room temperature for 120 min. The acid solution was added to cold ether, and the volume ratio of TFA lysis solution to cold ether was 1:20. The peptide was precipitated, centrifuged, and dried to obtain the crude peptide.

[0240] (5) Purification, salt conversion and freeze-drying of crude product: The crude product was dissolved in water and filtered with a 0.22 μm filter membrane for later use. The crude product was subjected to preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, the mobile phase A was 0.08% TFA / water solution, the mobile phase B was 0.08% TFA / acetonitrile solution, the flow rate of the 22 mm×250 mm chromatographic column was 8 mL / min, the detection wavelength was 214 nm, and the gradient system was used for elution and cyclic injection purification. The crude product solution was loaded on the chromatographic column, and the mobile phase elution was started. The fractions corresponding to the main peak of the chromatogram were collected. The acetonitrile was evaporated from the fractions to obtain a polypeptide aqueous solution, which was freeze-dried to obtain the product.

[0241] Among them, preferably, the halogenated resin is selected from the group consisting of trityl chloride resin, 4-methyltrityl chloride resin, 4-methoxytrityl chloride resin, 2-chlorotrityl chloride resin, bromo-(4-methylphenyl)-methyl resin or bromo-(4-methoxyphenyl)-methyl resin, for example, the resin is 2-chlorotrityl chloride resin.

[0242] Compared with the existing synthesis methods, this method has a wide range of applications, is green and environmentally friendly, has high purity of crude polypeptides, is easy to separate and purify, and can achieve a final purity greater than 95.0%, for example, greater than 95.0%. Calculated on the resin, the yield of most products is greater than 30.0%.

[0243] According to the above method, the present invention prepares a novel cyclic polypeptide compound. The cyclic polypeptide compound of the present invention is easy to prepare according to the chemical synthesis method, is simple to operate, and has a high yield. The structures of some cyclic polypeptide compounds are shown in Table 1:

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254] The present invention also provides the use of the cyclic polypeptide compound or its pharmaceutically acceptable salt in the preparation of antibacterial drugs. Preferably, the drug is a drug that inhibits Gram-negative bacteria and Gram-positive bacteria.

[0255] Pharmaceutically relevant Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii, Salmonella, Moraxella, Helicobacter, Legionella, Haemophilus influenzae, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Morganella morganii, Providentia rettgeri, Proteus vulgaris, Proteus mirabilis, Stenotrophomonas maltophilia maltophilia), Citrobacter freundii, etc. Pharmaceutically relevant Gram-positive bacteria include Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, etc.

[0256] Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumanii. Gram-positive bacteria include Staphylococcus epidermidis and Staphylococcus aureus.

[0257] The present invention also provides a pharmaceutical composition of the cyclic polypeptide compound, which comprises a therapeutically effective amount of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the content of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.1% to 99.5% by weight based on the total weight of the pharmaceutical composition. The compound itself or a mixture of its pharmaceutically acceptable salt and a pharmaceutical excipient, a diluent, etc. can be administered orally in the form of tablets, capsules, granules, powders or syrups, or non-orally in the form of injections, sprays, aerosols, ointments, eye drops.

[0258] The above-mentioned preparations can be prepared by conventional pharmaceutical methods. Examples of usable pharmaceutical excipients and diluents include excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, dextrin and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxymethyl cellulose calcium, hydroxymethyl cellulose sodium; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate, phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.); binders (e.g., gelatin, polyvinyl pyrrolidone and polyethylene glycol); disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose, polyvinyl pyrrolidone); lubricants (e.g., talc, calcium stearate, magnesium stearate, spermaceti, boric acid, sodium benzoate, leucine), stabilizers (methyl parahydroxybenzoate, propyl parahydroxybenzoate, etc.); flavoring agents (e.g., commonly used sweeteners, sour agents and flavors, etc.); diluents and injection solvents (e.g., water, ethanol and glycerol, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0259] Figure 1 The following is a roadmap for synthesizing cyclic polypeptide compounds using the protected amino acid Fmoc-Dab-OP at position 7 as the starting amino acid.

[0260] P stands for: Allyl

[0261] P1 represents: tert-butyloxycarbonyl (Boc)

[0262] P2 represents: 1-(4,4-dimethyl-2,6-dioxacyclohexane-1-ylidene)-ethyl (Dde)

[0263] P3 represents: tert-butyl (tBu)

[0264] Fmoc stands for: 9-fluorenylmethoxycarbonyl DETAILED DESCRIPTION

[0265] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0266] A novel synthetic route for a cyclic polypeptide compound or a pharmaceutically acceptable salt thereof, wherein the synthetic route prepares the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof by solid phase condensation and solid phase cyclization, and comprises the following steps:

[0267] (1) The protected free amino group of the side chain of the basic amino acid Fmoc-AA-OP is reacted with a halogenated resin to obtain Fmoc-AA-OP-resin; wherein P is a carboxyl protecting group, for example, allyl (Allyl), benzyl (Bn):

[0268] The route for synthesizing cyclic peptide compounds using the protected amino acid Fmoc-Dab-OP at position 7 as the starting amino acid is as follows: Figure 1 As shown: the protected amino acid Fmoc-Dab-OP at position 7 is the starting amino acid, and its structure is shown in Formula III:

[0269]

[0270] The halogenated resin is selected from the group consisting of trityl chloride resin, 4-methyltrityl chloride resin, 4-methoxytrityl chloride resin, 2-chlorotrityl chloride resin, bromo-(4-methylphenyl)-methyl resin or bromo-(4-methoxyphenyl)-methyl resin, for example, the resin is 2-chlorotrityl chloride resin.

[0271] The degree of substitution of the halogenated resin is 0.1-1.6 mmol / g, for example, the degree of substitution is 0.5-1.0 mmol / g.

[0272] The amount of each Fmoc-protected amino acid used is 1.2-6 times, for example, 2.0-3.5 times, the total molar number of the resin fed.

[0273] The base is selected from the group consisting of: at least one of N,N-diisopropylethylamine (DIEA), triethylamine (TEA), and pyridine, for example, DIEA; the molar amount of the base is 1.5-3 times the molar amount of the Fmoc-protected amino acid, for example, 2 times the molar amount of the Fmoc-protected amino acid.

[0274] The substitution reaction time is 1-12 h, for example, 2-3 h.

[0275] (2) Fmoc-AA-OP-resin is coupled one by one to obtain linear peptide-resin:

[0276] Reagents for removing the α-amino Fmoc protecting group include, but are not limited to, a solution of piperidine (PIP) in DMF at a concentration of 10-30%. A solution of PIP in DMF, for example, at a concentration of 20%. The amount of the deprotecting reagent used is 5-15 mL per gram of the resin fed, for example, 10 mL per gram of the resin fed. The deprotection reaction time is 10-60 min, for example, 10-20 min. Reagents for removing the amino ivDde or Dde protecting group of the amino acid side chain at position 2 include, but are not limited to, a solution of hydrazine hydrate in DMF at a concentration of 1-10%. A solution of hydrazine hydrate in DMF, for example, at a concentration of 3%. The amount of the deprotecting reagent used is 5-15 mL per gram of the resin fed, for example, 10 mL per gram of the resin fed. The deprotection reaction time is 30-100 min, for example, 30-60 min.

[0277] In the coupling reaction, the coupling agent is selected from the group consisting of: N,N-diisopropylcarbodiimide (DIC), N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), for example, N,N-diisopropylcarbodiimide (DIC).

[0278] The molar amount of the coupling agent is 1.2-6 times, for example, 2.0-3.5 times, the total molar amount of the feed resin.

[0279] The activator is selected from the group consisting of 1-hydroxybenzotriazole (HOBT), 6-chloro-1-hydroxybenzotriazole (Cl-HOBT), 1-hydroxy-7-azobenzotriazole (HOAT), for example, 1-hydroxybenzotriazole (HOBT).

[0280] The molar amount of the activator is 1.2-6 times, for example, 2.0-3.5 times, of the total molar amount of the feed resin.

[0281] The coupling reaction time is 60-300 min, for example, 60-120 min.

[0282] In the coupling reaction, part of the coupling agent needs to be added with a catalyst, and the catalyst is an organic base selected from the group consisting of: N,N-diisopropylethylamine (DIEA), triethylamine (TEA), N-methylmorpholine (NMM), for example, N,N-diisopropylethylamine (DIEA).

[0283] The solvent is an aprotic polar solvent selected from the group consisting of dimethylformamide (DMF) or N-methylpyrrolidone (NMP) or a mixture thereof, for example, DMF.

[0284] (3) Selectively removing the protecting group from the linear peptide-resin and solid phase cyclization to obtain the cyclic peptide-resin:

[0285] The reagent for removing the carboxyl allyl protecting group is a solution of tetrakis(triphenylphosphine)palladium / phenylsilane in DCM and DMF (a mixed solution with a volume ratio of DCM:DMF of 5:5). The molar amount of tetrakis(triphenylphosphine)palladium is 0.1-2 times the total mole number of the feed resin, for example, 0.1-0.3 times. The molar amount of phenylsilane is 2-10 times the total mole number of the feed resin, for example, 3-5 times. The amount of the deprotection reagent used is 10-30 mL per gram of feed resin, for example, 20 mL per gram of feed resin. The deprotection reaction time is 60-300 min, for example, 60-120 min.

[0286] The solid phase cyclization coupling agent is selected from the group consisting of: (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), for example, (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP).

[0287] The molar amount of the coupling agent is 1.2-6 times, for example, 2.0-3.5 times, the total molar amount of the feed resin.

[0288] The activator is selected from the group consisting of 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azobenzotriazole (HOAT), for example, 1-hydroxy-7-azobenzotriazole (HOAT).

[0289] The molar amount of the activator is 1.2-6 times, for example, 2.0-3.5 times, of the total molar amount of the feed resin.

[0290] The cyclization reaction time is 1-20 h, for example, 1-3 h.

[0291] The catalyst is an organic base selected from the group consisting of: N,N-diisopropylethylamine (DIEA), triethylamine (TEA), N-methylmorpholine (NMM), for example, N-methylmorpholine (NMM).

[0292] The solvent is an aprotic polar solvent selected from the group consisting of dimethylformamide (DMF) or N-methylpyrrolidone (NMP) or a mixture thereof, for example, DMF.

[0293] (4) Cyclic polypeptide-resin is subjected to acid hydrolysis to obtain a crude cyclic polypeptide:

[0294] The acid hydrolysis uses an acid hydrolysis solution containing hydrofluoric acid (HF) or trifluoroacetic acid (TFA), for example, trifluoroacetic acid.

[0295] The amount of the acidolysis solution is 5-30 mL per gram of the resin, for example, 10 mL per gram of the resin. The acidolysis solution comprises trifluoroacetic acid and a side chain protecting group removing agent.

[0296] The concentration of trifluoroacetic acid is 80%-95%, and the rest is the side chain protecting group removing agent.

[0297] The side chain protecting group removing agent is selected from the group consisting of thioanisole, triisopropylsilane, phenol, water, 1,2-ethanedithiol, for example, water.

[0298] The acid hydrolysis time is 60-300 min, for example, 100-120 min.

[0299] The acid hydrolyzate containing the polypeptide after acid hydrolysis was added to cold ether (the volume ratio of the acid hydrolyzate to cold ether was 1:20) to precipitate the peptide, centrifuge, and dry to obtain the crude peptide.

[0300] (5) The crude cyclic polypeptide is purified and / or converted to salt, and then freeze-dried to obtain a pure cyclic polypeptide.

[0301] The crude peptide is dissolved in water, filtered through a 0.22 μm filter membrane, and purified by preparative high performance liquid chromatography, using mobile phase A 0.08% TFA / water solution, mobile phase B 0.08% TFA / acetonitrile solution, gradient elution, detection wavelength 214 nm, and the product is dried by freeze drying. The final purity that can be achieved by this method is greater than 95%, for example, greater than 95%.

[0302] In step (1), the preparation of Fmoc-AA-OP-resin is performed, for example, as follows: the halogenated resin is added to a polypeptide solid phase synthesis tube, DCM is added to swell it, and after swelling is completed, it is washed three times with DMF and three times with DCM. The protected starting amino acids Fmoc-AA-OP and DIEA are dissolved in DCM and added to the polypeptide synthesis tube, reacted at room temperature for 2 hours, the reaction solution is removed by vacuum, and the resin is washed three times with DMF and three times with DCM to obtain Fmoc-AA-OP-resin.

[0303] In step (2), the coupling synthesis method comprises: treating the Fmoc-AA-OP-resin obtained in step (1) with 20% piperidine / DMF (twice, 10 minutes each time) to remove the α-amino Fmoc protecting group, washing with DMF three times, washing with DCM three times, and adding the amino acid or side chain carboxylic acid (R 0 -COOH), DIEA and HCTU were dissolved in DMF and added to the peptide synthesis tube, reacted at room temperature for 120 minutes, the reaction solution was removed by vacuum, washed with DMF three times, and washed with DCM three times. From the starting amino acid (i.e., the amino acid at position x, x is 6 or 7) to the amino acid at position 1 one by one, and then the side chain carboxylic acid was coupled to the protected peptide-resin. The ivDde or Dde protecting group of the side chain amino group of the amino acid at position 2 was removed with 2% hydrazine hydrate / DMF solution (30 minutes), washed with DMF three times, washed with DCM three times, and the carboxyl group of the amino acid at position 8 was coupled to the side chain amino group of the amino acid at position 2; from the amino acid at position 8, one by one, it was coupled to the amino acid after the starting amino acid (amino acid at position x+1), and a linear fully protected peptide-resin was obtained. The one-by-one coupling sequence includes two parts, the first part is from the starting amino acid (i.e., the amino acid at position x, x is 6 or 7) to the amino acid at position 1, and then to the side chain carboxylic acid, and the second part is from the amino acid at position 8 to the amino acid at position (x+1). If x is 7, the order of the first part is from amino acid 7 to amino acid 1, and then to side chain carboxylic acid, and the second part is only amino acid 8; if x is 6, the order of the first part is from amino acid 6 to amino acid 1, and then to side chain carboxylic acid, and the order of the second part is from amino acid 8 to amino acid 7.

[0304] In step (3), the specific method of selectively removing the protecting group and solid phase cyclization is, for example, as follows: the linear fully protected peptide-resin in step (2) is treated with 20% piperidine / DMF (twice, 10 minutes each time) to remove the α-amino Fmoc protecting group, washed three times with DMF and three times with DCM to form a free amino group; the carboxyl allyl protecting group is removed with a solution of tetrakis(triphenylphosphine)palladium / phenylsilane in DCM and DMF (a mixed solution of DCM:DMF with a volume ratio of 5:5) (120 minutes) to form a free carboxyl group. PyAOP and HOAT are dissolved in DMF and then added to NMM, added to the peptide synthesis tube, reacted at room temperature for 3 hours, the reaction solution was removed by vacuum, washed three times with DMF and three times with DCM to obtain a cyclic fully protected peptide-resin.

[0305] In step (4), the specific method of obtaining the crude cyclic basic polypeptide by acid hydrolysis is, for example, as follows: the acid hydrolysis solution (TFA:H 2 O volume ratio of 95:5) was added to the peptide synthesis tube, reacted at room temperature for 120 minutes, the acid hydrolyzate was added to cold ether (the ratio of TFA lysis solution to cold ether was 1:20) to precipitate the peptide, centrifuged and dried to obtain the crude peptide.

[0306] In step (5), the specific method of crude product purification, salt conversion and freeze-drying is as follows: the crude product is dissolved in water, filtered with a 0.22 μm filter membrane for later use, and subjected to preparative high performance liquid chromatography, the chromatographic filler is 10 μm reverse phase C18, the mobile phase A is 0.08% TFA / water solution, the mobile phase B is 0.08% TFA / acetonitrile solution, the flow rate of the 22 mm×250 mm chromatographic column is 8 mL / min, the detection wavelength is 214 nm, a gradient system is used for elution, and cyclic injection purification is performed. The crude product solution is loaded on the chromatographic column, the mobile phase elution is started, the fractions corresponding to the main peak of the chromatogram are collected, the acetonitrile is evaporated from the fractions to obtain a polypeptide aqueous solution, and the product is freeze-dried to obtain the product.

[0307] Example 1: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Thr-Dab-Dab-Thr] (Compound HX-1)

[0308] Figure 1 The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Thr(tBu)-OH.

[0309] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Thr-Dab-Dab-Thr] according to the synthetic route.

[0310] 472 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 164 mg, and the yield was 33.2% based on the 0.5 mmol of 2-Cl-Trt resin.

[0311] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=986.63263 ([M+H] + ).

[0312] Example 2: Preparation of (S)-6-methyloctanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-2)

[0313] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (S)-6-methyloctanoic acid, and Fmoc-Leu-OH.

[0314] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (S)-6-methyloctanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0315] 438 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 152 mg, and the yield was 31.0% based on the 0.5 mmol of 2-Cl-Trt resin.

[0316] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=980.69604 ([M+H] + ).

[0317] Example 3: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-3)

[0318] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0319] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0320] 472 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 126 mg, and the yield was 25.1% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0321] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1010.70630 ([M+H] + ).

[0322] Example 4: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Leu-Dab-Dab-Leu] (Compound HX-4)

[0323] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0324] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Leu-Dab-Dab-Leu] according to the synthetic route.

[0325] 433 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 168 mg, and the yield was 32.1% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0326] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1044.69006 ([M+H] + ).

[0327] Example 5: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (Compound HX-5)

[0328] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0329] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0330] 429 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 156 mg, and the yield was 29.8% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0331] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1044.69092 ([M+H] + ).

[0332] Example 6: Preparation of (S)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-6)

[0333] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (S)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0334] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (S)-3-hydroxydecanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0335] 392 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 153 mg, and the yield was 30.3% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0336] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum) > 95.0%; ESI: m / z = 1010.70575 ([M+H] + ).

[0337] Example 7: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Leu-Dab-Dab-Thr] (Compound HX-7)

[0338] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Thr(tBu)-OH.

[0339] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Leu-Dab-Dab-Thr] according to the synthetic route.

[0340] 386 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for standby use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 180 mg, and the yield was 34.8% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0341] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1032.65479 ([M+H] + ).

[0342] Example 8: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-8)

[0343] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Thr(tBu)-OH.

[0344] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0345] 432 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 153 mg, and the yield was 30.6% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0346] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=998.66882 ([M+H] + ).

[0347] Example 9: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-Leu-D-Phe-Dab-Dab-Leu] (Compound HX-9)

[0348] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0349] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-Leu-D-Phe-Dab-Dab-Leu] according to the synthetic route.

[0350] 491 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 164 mg, and the yield was 31.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0351] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1044.69006 ([M+H] + ).

[0352] Example 10: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Thr-Dab-Dab-Leu] (Compound HX-10)

[0353] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0354] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Thr-Dab-Dab-Leu] according to the synthetic route.

[0355] 342 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 192 mg, and the yield was 37.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0356] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1032.65320 ([M+H] + ).

[0357] Example 11: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-Leu-D-Phe-Dab-Dab-Thr] (Compound HX-11)

[0358] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Thr(tBu)-OH.

[0359] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-Leu-D-Phe-Dab-Dab-Thr] according to the synthetic route.

[0360] 431 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 178 mg, and the yield was 34.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0361] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1032.65442 ([M+H] + ).

[0362] Example 12: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Thr-Dab-Dab-Leu] (Compound HX-12)

[0363] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0364] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Thr-Dab-Dab-Leu] according to the synthetic route.

[0365] 368 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 146 mg, and the yield was 29.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0366] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=998.67059 ([M+H] + ).

[0367] Example 13: Preparation of (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] (Compound HX-13)

[0368] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Thr(tBu)-OH.

[0369] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] according to the synthetic route.

[0370] 521 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 156 mg, and the yield was 30.6% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0371] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1020.61707 ([M+H] + ).

[0372] Example 14: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-14)

[0373] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0374] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0375] 356 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 162 mg, and the yield was 32.7% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0376] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=994.71088 ([M+H] + ).

[0377] Example 15: Preparation of n-nonanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-15)

[0378] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, nonanoic acid, and Fmoc-Leu-OH.

[0379] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-nonanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0380] 366 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 176 mg, and the yield was 36.1% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0381] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=980.69611 ([M+H] + ).

[0382] Example 16: Preparation of succinic acid mono(1-methylbutyl) ester-D-Dab-ring (2-8)

[0383] [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr](Compound HX-16)

[0384] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, mono(1-methylbutyl)succinate, and Fmoc-Thr(tBu)-OH.

[0385] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare succinic acid mono(1-methylbutyl) ester-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0386] 431 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 159 mg, and the yield was 31.7% based on the 0.5 mmol of 2-Cl-Trt resin.

[0387] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=998.63854 ([M+H] + ).

[0388] Example 17: Preparation of (R)-3-hydroxydecanoyl-D-Ser-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-17)

[0389] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Ser(tBu)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0390] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Ser-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0391] 472 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 189 mg, and the yield was 37.5% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0392] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=997.67596 ([M+H] + ).

[0393] Example 18: Preparation of (R)-3-hydroxydecanoyl-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-18)

[0394] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0395] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0396] 456 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 156 mg, and the yield was 30.9% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0397] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1010.70593 ([M+H] + ).

[0398] Example 19: Preparation of (R)-3-hydroxydecanoyl-Dab-Dab-cyclo (3-9) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (Compound HX-19)

[0399] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0400] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-Dab-Dab-Dab-cyclo (3-9) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0401] 513 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 195 mg, and the yield was 34.0% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0402] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1144.75281 ([M+H] + ).

[0403] Example 20: Preparation of (R)-3-hydroxydecanoyl-D-Ser-D-Dab-ring (3-9)

[0404] [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu](Compound HX-20)

[0405] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, Fmoc-D-Ser(tBu)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0406] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Ser-D-Dab-cyclo (3-9) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0407] 503 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for standby use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 160 mg, and the yield was 28.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0408] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1131.72131 ([M+H] + ).

[0409] Example 21: Preparation of n-octanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-21)

[0410] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-octanoic acid, and Fmoc-Leu-OH.

[0411] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-octanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0412] 341 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 172 mg, and the yield was 35.6% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0413] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=966.67920 ([M+H] + ).

[0414] Example 22: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-Leu-Leu-Dab-Dab-Thr] (Compound HX-22)

[0415] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0416] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0417] 361 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 159 mg, and the yield was 32.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0418] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=982.67316 ([M+H] + ).

[0419] Example 23: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-D-Leu-Dab-Dab-Thr] (Compound HX-23)

[0420] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0421] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-D-Leu-Dab-Dab-Thr] according to the synthetic route.

[0422] 392 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 204 mg, and the yield was 41.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0423] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=982.67535 ([M+H] + ).

[0424] Example 24: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Bip-Dab-Dab-Thr] (Compound HX-24)

[0425] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Bip-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0426] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Bip-Dab-Dab-Thr] according to the synthetic route.

[0427] 494 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 204 mg, and the yield was 37.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0428] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1092.69324 ([M+H] + ).

[0429] Example 25: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Bip-Leu-Dab-Dab-Thr] (Compound HX-25)

[0430] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Bip-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0431] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Bip-Leu-Dab-Dab-Thr] according to the synthetic route.

[0432] 467 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 186 mg, and the yield was 34.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0433] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1092.69043 ([M+H] + ).

[0434] Example 26: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Chg-Leu-Dab-Dab-Thr] (Compound HX-26)

[0435] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Chg-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0436] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Chg-Leu-Dab-Dab-Thr] according to the synthetic route.

[0437] 381 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 172 mg, and the yield was 33.5% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0438] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum) > 95.0%; ESI: m / z = 1008.69141 ([M+H] + ).

[0439] Example 27: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-octyl-Gly-Dab-Dab-Thr] (Compound HX-27)

[0440] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-octyl-Gly-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0441] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-octyl-Gly-Dab-Dab-Thr] according to the synthetic route.

[0442] 493 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 165 mg, and the yield was 31.8% based on the 0.5 mmol of 2-Cl-Trt resin.

[0443] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1038.74121 ([M+H] + ).

[0444] Example 28: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Asp-Dab-Dab-Thr] (Compound HX-28)

[0445] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0446] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Asp-Dab-Dab-Thr] according to the synthetic route.

[0447] 417 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 167 mg, and the yield was 34.1% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0448] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=984.61798 ([M+H] + ).

[0449] Example 29: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Asp] (Compound HX-29)

[0450] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Asp(tBu)-OH.

[0451] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Asp] according to the synthetic route.

[0452] 476 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 186 mg, and the yield was 37.4% based on the 0.5 mmol of 2-Cl-Trt resin.

[0453] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=996.65570 ([M+H] + ).

[0454] Example 30: Preparation of phenylhexanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-30)

[0455] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, phenylhexanoic acid, and Fmoc-Thr(tBu)-OH.

[0456] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it into the peptide solid phase synthesis tube, and prepare phenylhexanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0457] 384 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 148 mg, and the yield was 29.6% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0458] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1002.64447 ([M+H] + ).

[0459] Example 31: Preparation of 7,7-dimethyloctanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-31)

[0460] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, neodecanoic acid, and Fmoc-Thr(tBu)-OH.

[0461] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare 7,7-dimethyloctanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0462] 466 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 182 mg, and the yield was 37.1% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0463] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=982.67615 ([M+H] + ).

[0464] Example 32: Preparation of n-decanoyl-D-Dap-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-32)

[0465] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dap(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0466] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dap-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0467] 439 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 157 mg, and the yield was 32.4% based on the 0.5 mmol of 2-Cl-Trt resin.

[0468] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=968.65967 ([M+H] + ).

[0469] Example 33: Preparation of n-decanoyl-D-Orn-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-33)

[0470] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Orn(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0471] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Orn-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0472] 407 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 182 mg, and the yield was 36.4% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0473] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=996.69183 ([M+H] + ).

[0474] Example 34: Preparation of n-decanoyl-D-Thr-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-34)

[0475] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Thr(tBu)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0476] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Thr-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0477] 402 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 198 mg, and the yield was 40.2% based on the 0.5 mmol of 2-Cl-Trt resin.

[0478] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=983.65930 ([M+H] + ).

[0479] Example 35: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Lys-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-35)

[0480] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0481] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Lys-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0482] 465 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 202 mg, and the yield was 40.0% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0483] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1010.70697 ([M+H] + ).

[0484] Example 36: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Orn-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-36)

[0485] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0486] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Orn-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0487] 383 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 162 mg, and the yield was 32.4% based on the 0.5 mmol of 2-Cl-Trt resin.

[0488] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=996.69232 ([M+H] + ).

[0489] Example 37: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dap-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-37)

[0490] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dap(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0491] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dap-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0492] 371 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 166 mg, and the yield was 33.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0493] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=968.65985 ([M+H] + ).

[0494] Example 38: Preparation of n-decanoyl-D-Dab-ring (2-8) [Orn-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-38)

[0495] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Orn(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0496] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Orn-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0497] 447 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 138 mg, and the yield was 27.6% based on the 0.5 mmol of 2-Cl-Trt resin.

[0498] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=996.69263 ([M+H] + ).

[0499] Example 39: Preparation of n-decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-tBu)-Leu-Dab-Dab-Thr] (Compound HX-39)

[0500] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe(4-tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0501] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe(4-tBu)-Leu-Dab-Dab-Thr] according to the synthetic route.

[0502] 489 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 157 mg, and the yield was 29.6% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0503] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1072.72351 ([M+H] + ).

[0504] Example 40: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe(4-Cl)-Leu-Dab-Dab-Thr] (Compound HX-40)

[0505] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0506] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Phe(4-Cl)-Leu-Dab-Dab-Thr] according to the synthetic route.

[0507] 490 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 213 mg, and the yield was 40.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0508] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1050.62020 ([M+H] + ).

[0509] Example 41: Preparation of n-octanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (Compound HX-41)

[0510] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-octanoic acid, and Fmoc-Leu-OH.

[0511] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-octanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0512] 407 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 180 mg, and the yield was 36.1% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0513] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1000.66265 ([M+H] + ).

[0514] Example 42: Preparation of n-nonanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (Compound HX-42)

[0515] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, nonanoic acid, and Fmoc-Leu-OH.

[0516] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-nonanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0517] 381 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 169 mg, and the yield was 33.3% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0518] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1014.68030 ([M+H] + ).

[0519] Example 43: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (Compound HX-43)

[0520] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0521] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0522] 389 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 149 mg, and the yield was 29.1% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0523] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1028.69629 ([M+H] + ).

[0524] Example 44: Preparation of n-decanoyl-D-Asn-cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (Compound HX-44)

[0525] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Asn(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0526] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Asn-cyclo (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0527] 342 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for standby use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 191 mg, and the yield was 36.5% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0528] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1042.67573 ([M+H] + ).

[0529] Example 45: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Bip-Dab-Dab-Thr] (Compound HX-45)

[0530] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Bip-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0531] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Phe-Bip-Dab-Dab-Thr] according to the synthetic route.

[0532] 509 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 226 mg, and the yield was 40.3% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0533] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1126.67373 ([M+H] + ).

[0534] Example 46: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Trp-Dab-Dab-Leu] (Compound HX-46)

[0535] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0536] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Trp-Dab-Dab-Leu] according to the synthetic route.

[0537] 423 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 147 mg, and the yield was 27.5% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0538] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1067.70598 ([M+H] + ).

[0539] Example 47: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe-Bip-Dab-Dab-Leu] (Compound HX-47)

[0540] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Bip-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0541] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Phe-Bip-Dab-Dab-Leu] according to the synthetic route.

[0542] 466 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 183 mg, and the yield was 32.1% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0543] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1138.70894 ([M+H] + ).

[0544] Example 48: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Phe-Phe-Dab-Dab-Leu] (Compound HX-48)

[0545] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0546] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Phe-Phe-Dab-Dab-Leu] according to the synthetic route.

[0547] 399 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 146 mg, and the yield was 27.3% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0548] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1062.67710 ([M+H] + ).

[0549] Example 49: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Ser] (Compound HX-49)

[0550] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Ser(tBu)-OH.

[0551] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Ser] according to the synthetic route.

[0552] 369 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for standby use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 141 mg, and the yield was 28.1% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0553] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1002.64622 ([M+H] + ).

[0554] Example 50: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Phe] (Compound HX-50)

[0555] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Phe-OH.

[0556] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] according to the synthetic route.

[0557] 394 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 179 mg, and the yield was 33.8% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0558] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1062.67961 ([M+H] + ).

[0559] Example 51: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Phe (4-Me)-Dab-Dab-Leu] (Compound HX-51)

[0560] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe(4-Me)-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0561] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe(4-Me)-Dab-Dab-Leu] according to the synthetic route.

[0562] 406 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 178 mg, and the yield was 34.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0563] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1042.70524 ([M+H] + ).

[0564] Example 52: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Phe (4-OMe) -Dab-Dab-Leu] (Compound HX-52)

[0565] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Phe(4-OMe)-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Leu-OH.

[0566] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Phe(4-OMe)-Dab-Dab-Leu] according to the synthetic route.

[0567] 427 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 148 mg, and the yield was 28.1% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0568] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1058.70971 ([M+H] + ).

[0569] Example 53: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe(3-Cl)-Leu-Dab-Dab-Thr] (Compound HX-53)

[0570] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe(3-Cl)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0571] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Phe(3-Cl)-Leu-Dab-Dab-Thr] according to the synthetic route.

[0572] 458 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 193 mg, and the yield was 36.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0573] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1050.62169 ([M+H] + ).

[0574] Example 54: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe (4-NH 2 )-Leu-Dab-Dab-Thr](Compound HX-54)

[0575] The order of adding protected amino acids and side chain carboxylic acids to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe(4-NH 2 )-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, Fmoc-Thr(tBu)-OH.

[0576] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it into the peptide solid phase synthesis tube. Prepare n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe (4-NH 2 )-Leu-Dab-Dab-Thr].

[0577] 411 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 198 mg, and the yield was 38.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0578] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1031.66847 ([M+H] + ).

[0579] Example 55: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe(2-Cl)-Leu-Dab-Dab-Thr] (Compound HX-55)

[0580] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe(2-Cl)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0581] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Phe(2-Cl)-Leu-Dab-Dab-Thr] according to the synthetic route.

[0582] 339 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 195 mg, and the yield was 37.2% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0583] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1050.62089 ([M+H] + ).

[0584] Example 56: Preparation of n-decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(3,4-DiCl)-Leu-Dab-Dab-Thr] (Compound HX-56)

[0585] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe(3,4-DiCl)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0586] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe(3,4-DiCl)-Leu-Dab-Dab-Thr] according to the synthetic route.

[0587] 384 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 147 mg, and the yield was 27.1% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0588] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1084.58171 ([M+H] + ).

[0589] Example 57: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe (4-Me) -Leu-Dab-Dab-Thr] (Compound HX-57)

[0590] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe(4-Me)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0591] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Phe(4-Me)-Leu-Dab-Dab-Thr] according to the synthetic route.

[0592] 373 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried to obtain the product. The yield was 172 mg, and the yield was 33.5% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0593] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum) > 95.0%; ESI: m / z = 1030.67965 ([M+H] + ).

[0594] Example 58: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Ala] (Compound HX-58)

[0595] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Ala-OH.

[0596] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Ala] according to the synthetic route.

[0597] 173 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 204 mg, and the yield was 36.4% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0598] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=952.66986 ([M+H] + ).

[0599] Example 59: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Gly] (Compound HX-59)

[0600] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Gly-OH.

[0601] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Gly] according to the synthetic route.

[0602] 387 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 159 mg, and the yield was 33.9% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0603] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=938.65367 ([M+H] + ).

[0604] Example 60: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Val] (Compound HX-60)

[0605] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Val-OH.

[0606] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Val] according to the synthetic route.

[0607] 407 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 167 mg, and the yield was 34.1% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0608] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=980.69541 ([M+H] + ).

[0609] Example 61: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Nva] (Compound HX-61)

[0610] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Nva-OH.

[0611] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Nva] according to the synthetic route.

[0612] 384 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 189 mg, and the yield was 38.6% based on the 0.5 mmol of 2-Cl-Trt resin.

[0613] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=980.69863 ([M+H] + ).

[0614] Example 62: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Ile] (Compound HX-62)

[0615] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Ile-OH.

[0616] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Ile] according to the synthetic route.

[0617] 438 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 216 mg, and the yield was 43.5% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0618] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=994.71671 ([M+H] + ).

[0619] Example 63: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Val-Leu-Dab-Dab-Thr] (Compound HX-63)

[0620] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Val-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0621] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Val-Leu-Dab-Dab-Thr] according to the synthetic route.

[0622] 368 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 179 mg, and the yield was 37.0% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0623] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=968.66435 ([M+H] + ).

[0624] Example 64: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Nva-Leu-Dab-Dab-Thr] (Compound HX-64)

[0625] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Nva-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0626] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Nva-Leu-Dab-Dab-Thr] according to the synthetic route.

[0627] 388 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain a polypeptide compound aqueous solution, and the product was freeze-dried. The yield was 173 mg, and the yield was 35.8% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0628] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=968.66314 ([M+H] + ).

[0629] Example 65: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Ile-Leu-Dab-Dab-Thr] (Compound HX-65)

[0630] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Ile-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0631] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-D-Leu-Dab-Dab-Thr] according to the synthetic route.

[0632] 357 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 170 mg, and the yield was 34.7% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0633] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=982.67916 ([M+H] + ).

[0634] Example 66: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Val-Dab-Dab-Thr] (Compound HX-66)

[0635] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Val-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0636] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Val-Dab-Dab-Thr] according to the synthetic route.

[0637] 374 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 159 mg, and the yield was 32.9% based on the 0.5 mmol of 2-Cl-Trt resin.

[0638] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=968.66427 ([M+H] + ).

[0639] Example 67: Preparation of n-decanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Nva-Dab-Dab-Thr] (Compound HX-67)

[0640] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Nva-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0641] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Nva-Dab-Dab-Thr] according to the synthetic route.

[0642] 383 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 188 mg, and the yield was 38.9% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0643] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=968.66363 ([M+H] + ).

[0644] Example 68: Preparation of n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Ile-Dab-Dab-Thr] (Compound HX-68)

[0645] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Ile-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, n-decanoic acid, and Fmoc-Thr(tBu)-OH.

[0646] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare n-decanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Ile-Dab-Dab-Thr] according to the synthetic route.

[0647] 367 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for standby use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 158 mg, and the yield was 32.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0648] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=982.67549 ([M+H] + ).

[0649] Example 69: Preparation of (R)-3-hydroxy-6-methylheptanoyl-D-Dab-ring (2-8)

[0650] [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-69)

[0651] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxy-6-methylheptanoic acid, and Fmoc-Thr(tBu)-OH.

[0652] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxy-6-methylheptanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0653] 391 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 188 mg, and the yield was 38.8% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0654] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=970.64352 ([M+H] + ).

[0655] Example 70: Preparation of (R)-2-hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-70)

[0656] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-2-hydroxydecanoic acid, and Fmoc-Thr(tBu)-OH.

[0657] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-2-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0658] 416 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 213 mg, and the yield was 42.7% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0659] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=998.67586 ([M+H] + ).

[0660] Example 71: Preparation of 9-hydroxynonanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-71)

[0661] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, 9-hydroxynonanoic acid, and Fmoc-Thr(tBu)-OH.

[0662] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare 9-hydroxynonanoyl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0663] 377 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 199 mg, and the yield was 40.5% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0664] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=984.65981 ([M+H] + ).

[0665] Example 72: Preparation of (3R, 6S)-3-hydroxy-6-methyloctanoyl-D-Dab-cyclo (2-8)

[0666] [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-72)

[0667] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (3R,6S)-3-hydroxy-6-methyloctanoic acid, and Fmoc-Thr(tBu)-OH.

[0668] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (3R, 6S)-3-hydroxy-6-methyloctanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0669] 369 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 168 mg, and the yield was 34.2% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0670] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=984.65752 ([M+H] + ).

[0671] Example 73: Preparation of 4-pentyloxybutyryl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-73)

[0672] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, 4-pentyloxybutyric acid, and Fmoc-Thr(tBu)-OH.

[0673] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare 4-pentyloxybutyryl-D-Dab-ring (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0674] 377 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 159 mg, and the yield was 32.3% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0675] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=984.66013 ([M+H] + ).

[0676] Example 74: Preparation of 3-(tetrahydrofuran-2-yl)propanoyl-D-Dab-ring (2-8)

[0677] [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-74)

[0678] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, 3-(tetrahydrofuran-2-yl)propionic acid, and Fmoc-Thr(tBu)-OH.

[0679] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare 3-(tetrahydrofuran-2-yl)propanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0680] 374 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 154 mg, and the yield was 32.3% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0681] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=954.61148 ([M+H] + ).

[0682] Example 75: Preparation of 6-methoxyhexanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (Compound HX-75)

[0683] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, 6-methoxyhexanoic acid, and Fmoc-Thr(tBu)-OH.

[0684] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare 6-methoxyhexanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] according to the synthetic route.

[0685] 369 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 158 mg, and the yield was 33.0% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0686] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=956.62883 ([M+H] + ).

[0687] Example 76: Preparation of (R)-3-hydroxydecanoyl-DN-methyl-Dab-ring (2-8)

[0688] [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu](Compound HX-76)

[0689] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-DN-methyl-Dab-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0690] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-DN-methyl-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0691] 496 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 147 mg, and the yield was 28.7% calculated based on the 0.5 mmol of 2-Cl-Trt resin.

[0692] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1024.72688 ([M+H] + ).

[0693] Example 77: Preparation of (R)-3-hydroxydecanoyl-D-Arg-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (Compound HX-77)

[0694] The order of adding protected amino acids and side chain carboxylic acids to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Arg(Boc) 2 -OH, (R)-3-hydroxydecanoic acid, Fmoc-Leu-OH.

[0695] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Arg-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0696] 506 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 174 mg, and the yield was 32.6% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0697] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1066.74679 ([M+H] + ).

[0698] Example 78: Preparation of (R)-3-hydroxydecanoyl-D-3-guanidinoalanine-cyclo (2-8)

[0699] [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu](Compound HX-78)

[0700] The order of adding protected amino acids and side chain carboxylic acids to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Alg(Boc) 2 -OH, (R)-3-hydroxydecanoic acid, Fmoc-Leu-OH.

[0701] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-3-guanidinoalanine-cyclo (2-8) [Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0702] 493 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 166 mg, and the yield was 32.0% based on the 0.5 mmol of 2-Cl-Trt resin.

[0703] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1038.71983 ([M+H] + ).

[0704] Example 79: Preparation of (R)-3-hydroxydecanoyl-D-Dab-ring (2-8)

[0705] [Dab-N-methyl-Dab-D-Leu-Leu-Dab-Dab-Leu](Compound HX-79)

[0706] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-N-methyl-Dab-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0707] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-N-methyl-Dab-D-Leu-Leu-Dab-Dab-Leu] according to the synthetic route.

[0708] 481 mg of crude peptide was obtained. The crude peptide was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography. The chromatographic filler was 10 μm reverse phase C18, mobile phase A was 0.08% TFA / water solution, mobile phase B was 0.08% TFA / acetonitrile solution, 22 mm×250 mm chromatographic column, mobile phase flow rate was 8 mL / min, detection wavelength was 214 nm, gradient system elution was adopted, cyclic injection purification was adopted, crude product solution was loaded on the chromatographic column, mobile phase elution was started, fractions corresponding to the main peak of the chromatogram were collected and acetonitrile was evaporated, and the polypeptide compound aqueous solution was obtained, and the product was freeze-dried. The yield was 157 mg, and the yield was 30.7% based on the 0.5 mmol of 2-Cl-Trt resin.

[0709] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1024.72842 ([M+H] + ).

[0710] Example 80: Preparation of (R)-3-hydroxydecanoyl-D-Dab-ring (2-8)

[0711] [Dab-Dab-D-Leu-Leu-N-methyl-Dab-Dab-Leu](Compound HX-80)

[0712] The order in which the protected amino acids and side chain carboxylic acids are added to the reaction in the synthetic route is: Fmoc-Dab-OAllyl, Fmoc-N-methyl-Dab-OH, Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-D-Dab(Boc)-OH, (R)-3-hydroxydecanoic acid, and Fmoc-Leu-OH.

[0713] Take 2-Cl-Trt resin (0.5 mmol, degree of substitution = 0.5 mmol / g) and add it to the peptide solid phase synthesis tube, and prepare (R)-3-hydroxydecanoyl-D-Dab-cyclo (2-8) [Dab-Dab-D-Leu-Leu-N-methyl-Dab-Dab-Leu] according to the synthetic route.

[0714] 500 mg of crude peptide was obtained, which was dissolved in water, filtered with a 0.22 μm filter membrane for later use, and purified by preparative high performance liquid chromatography, with a 10 μm reverse phase C18 as the chromatographic filler, a 0.08% TFA / water solution as the mobile phase A, a 0.08% TFA / acetonitrile solution as the mobile phase B, a 22 mm×250 mm chromatographic column, a mobile phase flow rate of 8 mL / min, a detection wavelength of 214 nm, a gradient system elution, and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the fraction corresponding to the main peak of the chromatogram was collected and the acetonitrile was evaporated to obtain an aqueous solution of the polypeptide compound, and the product was freeze-dried. The yield was 141 mg, and the yield was 27.6% based on the 0.5 mmol of the 2-Cl-Trt resin.

[0715] Characterization of the purified peptide: Purity (by area integration of HPLC spectrum)>95.0%; ESI: m / z=1024.72395 ([M+H] + ).

[0716] Experimental Example 1: Antibacterial Activity Experiment

[0717] The minimum inhibitory concentration (MIC) was determined by the broth microdilution method according to the CLSI recommended method.

[0718] 1) Sample preparation

[0719] Weigh appropriate amounts of the cyclic polypeptide compounds and reference substances prepared in Examples 1-80, and dissolve the samples in sterile pure water according to the solubility of the samples, so that the concentration of the mother solution is 2.56 mg / mL. Take an appropriate amount of the mother solution and dilute it tenfold with sterile MH broth to 0.256 mg / mL, and dispense half of the volume of the drug solution into a 96-well loading tank, and dilute the other half of the volume of the drug solution twice with sterile MH broth and add it to the deep-well loading tank. Repeat the above steps to make the drug concentrations in the loading tank 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06 mg / L, respectively, and prepare and use immediately.

[0720] 2) Preparation of inoculum

[0721] Select several colonies from the agar plate cultured for 18-24 hours and directly prepare a bacterial suspension in sterile saline. Adjust the concentration of the bacterial suspension to 0.5 McFarland units. Dilute the corrected bacterial suspension with MH broth to (4-8)×10 5 CFU / mL, ready for use.

[0722] 3) Sample addition and inoculation

[0723] 100 μL of the above-mentioned compounds of the present invention (e.g., compounds prepared in the examples) and reference solutions of different concentrations were respectively pipetted into the first to twelfth wells of a sterile 96-well polystyrene plate, and 100 μL of the above-mentioned inoculum was added to each well. The final concentrations of the samples in the wells were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg / L (the final concentration range of polymyxin B was 512 to 0.25 mg / L), and the final inoculum concentrations were all (2 to 4)×10 5 CFU / mL. A growth control well was set up, containing 100 μL of inoculum and 100 μL of sterile MH broth. The test substance and inoculum in each well were mixed and sealed.

[0724] 4) Incubation

[0725] The inoculated 96-well plate was placed in a 35-37°C incubator for 16-20 hours.

[0726] 5) MIC endpoint interpretation

[0727] After the culture was completed, the growth of bacteria in each well was observed, and the lowest drug concentration that completely inhibited the growth of bacteria in the well was taken as the minimum inhibitory concentration (MIC).

[0728] The strains used in the antibacterial activity experiments were from the American Type Culture Collection (ATCC) and clinical isolates.

[0729] The strains used in the antibacterial activity experiments included Staphylococcus aureus ATCC33591, ATCC29213, Staphylococcus epidermidis MRSE19-5, MRSE19-6, Enterococcus faecium EFM19-2, VRE EFM19-13, Enterococcus faecalis ATCC29212, Escherichia coli ATCC BAA-2340 (KPC), ATCC BAA-2523, ECO-19-2, Klebsiella pneumoniae ATCC BAA-1705 (KPC), KPN+19-15, KPN+19-17, Pseudomonas aeruginosa ATCC27853, PAE19-1, PAE19-3, PAE17-1, Acinetobacter baumannii NCTC 13304 (OXA-27), ABA19-1, ABA19-8 and polymyxin-resistant Pseudomonas aeruginosa.

[0730] Test product: a cyclic polypeptide compound prepared according to the technical solution of the present invention;

[0731] Reference substances: polymyxin B, meropenem.

[0732] result:

[0733] The activities of the cyclic polypeptide compounds of the present invention against Gram-positive bacteria and Gram-negative bacteria are shown in Table 2.

[0734] Table 2 Gram-positive and Gram-negative bacterial activity of the compounds of the present invention (MIC, unit μg / mL)

[0735]

[0736]

[0737]

[0738]

[0739] ND means not tested. Staphylococcus aureus, 2 strains; Staphylococcus epidermidis, 2 strains; Enterococcus faecium, 2 strains; Enterococcus faecalis, 1 strain; Escherichia coli, 3 strains; Klebsiella pneumoniae, 3 strains; Pseudomonas aeruginosa, 4 strains; Acinetobacter baumannii, 3 strains; Polymyxin-resistant Pseudomonas aeruginosa, 1 strain.

[0740] Experimental Example 2: Nephrotoxicity Experiment

[0741] The CCK-8 method was used to detect HK-2 cell cytotoxicity in vitro.

[0742] 1) Cell culture

[0743] The culture medium of human kidney HK-2 cells was RPMI-1640 complete medium (Invitrogen) containing 10% FBS (Gibco), and the cryopreservation medium was 90% FBS + 10% DMSO. The cells were stored in liquid nitrogen for a long time at 37°C with 5% CO. 2 The cells were cultured in a sterile incubator with a humid environment and digested and passaged when the cell density grew to 70%-80%.

[0744] 2) Dilution of cyclic peptide compounds

[0745] The cyclic polypeptide compounds prepared in Examples 1-80 were weighed and dissolved with sterile water to a 1 mg / mL stock solution, and the drug-containing culture medium was prepared with final concentrations of 500, 250, 200, 100, 50, 25, and 12.5 μg / mL, respectively.

[0746] 3) Detection method

[0747] Total 5×10 4100 μL of cell suspension was placed in a 96-well plate. After 24 hours of stable culture, the drug-containing culture medium was replaced and placed in an incubator for 72 hours. The culture medium was gently aspirated, and 100 μL of blank culture medium containing 10% CCK-8 was added. After incubation in an incubator for 3 hours, the OD 450nm value was measured using a BIOTEK plate reader. The experiment was repeated three times, and each time polymyxin B was set as the positive drug and the blank treatment group without drug was set as the control. Calculate the cell survival rate (%) = (OD 给药组 -OD 调零 ) / (OD 未含药 -OD 调零 )*100%, IC calculated using Log regression 50 value.

[0748] The cells used in the renal cell toxicity experiment were from ATCC (American Type Culture Collection).

[0749] The Cell Counting Kit-8 (CCK-8) kit used for renal cell toxicity experiment was from Beyotime Biotechnology.

[0750] Test product: a polypeptide compound prepared according to the technical scheme of the present invention;

[0751] Reference substance: polymyxin B.

[0752] result:

[0753] The renal cell toxicity results of some cyclic polypeptide compounds of the present invention are shown in Table 3.

[0754] Table 3 Renal cell toxicity (IC 50 , unit μg / mL)

[0755]

[0756]

[0757] In summary, some of the novel cyclic polypeptides prepared in the present invention have low nephrotoxicity, high and broad antibacterial activity, and activity against drug-resistant bacteria, and are entirely likely to become a new class of clinical antibiotics.

Claims

1. A cyclic polypeptide compound or a pharmaceutically acceptable salt thereof, It is characterized in that The cyclic polypeptide compound is selected from the following group: (HX-1)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Thr-Dab-Dab-Thr] (HX-2)(S)-6-Methyloctanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (HX-6)(S)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (HX-8)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-10)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe-Thr-Dab-Dab-Leu] (HX-12)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Thr-Dab-Dab-Leu] (HX-13)(R)-3-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] (HX-15) nonanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (HX-16) Succinic acid mono(1-methylbutyl) ester-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-18)(R)-3-Hydroxydecanoyl-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (HX-19)(R)-3-Hydroxydecanoyl-Dab-Dab-Cyclo(3-9)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (HX-20)(R)-3-Hydroxydecanoyl-D-Ser-D-Dab-cyclo(3-9)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (HX-24) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Bip-Dab-Dab-Thr] (HX-25) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Bip-Leu-Dab-Dab-Thr] (HX-26) n-Decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Chg-Leu-Dab-Dab-Thr] (HX-27) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-octyl-Gly-Dab-Dab-Thr] (HX-30) Phenylhexanoyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-31)7,7-Dimethyloctanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-32) n-Decanyl-D-Dap-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-33) n-Decanoyl-D-Orn-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-38) n-Decanyl-D-Dab-Cyclo(2-8)[Orn-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-39) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-tBu)-Leu-Dab-Dab-Thr] (HX-40) n-Decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-Cl)-Leu-Dab-Dab-Thr] (HX-41) n-octanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (HX-42) nonanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Leu] (HX-45) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Phe-Bip-Dab-Dab-Thr] (HX-46) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Trp-Dab-Dab-Leu] (HX-47) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Phe-Bip-Dab-Dab-Leu] (HX-48) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Phe-Phe-Dab-Dab-Leu] (HX-50) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Phe-Dab-Dab-Phe] (HX-51) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe(4-Me)-Dab-Dab-Leu] (HX-52) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Phe(4-OMe)-Dab-Dab-Leu] (HX-53) n-Decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(3-Cl)-Leu-Dab-Dab-Thr] (HX-55) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(2-Cl)-Leu-Dab-Dab-Thr] (HX-56) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(3,4-DiCl)-Leu-Dab-Dab-Thr] (HX-57) n-Decanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Phe(4-Me)-Leu-Dab-Dab-Thr] (HX-60) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Val] (HX-61) n-Decanyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Nva] (HX-62) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Ile] (HX-63) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Val-Leu-Dab-Dab-Thr] (HX-64) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Nva-Leu-Dab-Dab-Thr] (HX-65) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Ile-Leu-Dab-Dab-Thr] (HX-66) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Val-Dab-Dab-Thr] (HX-67) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Nva-Dab-Dab-Thr] (HX-68) n-Decanyl-D-Dab-Cyclo(2-8)[Dab-Dab-D-Leu-Ile-Dab-Dab-Thr] (HX-69)(R)-3-Hydroxy-6-methylheptanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-70)(R)-2-Hydroxydecanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-71) 9-Hydroxynonanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-72)(3R,6S)-3-Hydroxy-6-methyloctanoyl-D-Dab-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Thr] (HX-77)(R)-3-Hydroxydecanoyl-D-Arg-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] (HX-78)(R)-3-Hydroxydecanoyl-D-3-guanidinoalanine-cyclo(2-8)[Dab-Dab-D-Leu-Leu-Dab-Dab-Leu] 2. The cyclic polypeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, It is characterized in that The pharmaceutically acceptable salts include salts formed by cyclic polypeptide compounds and acids, wherein the acid is selected from the group consisting of: inorganic acids or organic acids, wherein the inorganic acid is perchloric acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid; and the organic acid is acetic acid, trifluoroacetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, benzoic acid, methanesulfonic acid or p-toluenesulfonic acid.

3. A pharmaceutical composition, It is characterized in that The pharmaceutical composition contains the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 and a pharmaceutically acceptable carrier or excipient.

4. The use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Staphylococcus aureus drug, wherein the cyclic polypeptide compound is selected from the group consisting of HX-19, HX-24, HX-25, HX-27, HX-39, HX-40, HX-42, HX-45, HX-46, HX-47, HX-48, HX-50, HX-51, HX-52, HX-53, HX-56, HX-57, HX-60, HX-61, HX-62, HX-63, HX-64, HX-65, HX-66, HX-67, HX-68, and HX-78.

5. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Staphylococcus epidermidis drug, wherein the cyclic polypeptide compound is selected from the group consisting of HX-2, HX-6, HX-8, HX-10, HX-12, HX-15, HX-16, HX-18, HX-19, HX-20, HX-24, HX-25, HX-26, HX-27, HX-30, HX-31, HX-32, HX-33, HX-38, HX-3 9. HX-40, HX-41, HX-42, HX-45, HX-46, HX-47, HX-48, HX-50, HX-51, HX-52, HX-53, HX-55, HX-56, HX-57 , HX-60, HX-61, HX-62, HX-63, HX-64, HX-65, HX-66, HX-67, HX-68, HX-69, HX-70, HX-72, HX-77, HX-78.

6. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Enterococcus faecium drug, wherein the cyclic polypeptide compound is selected from the group consisting of HX-19, HX-20, HX-24, HX-25, HX-26, HX-27, HX-39, HX-40, HX-42, HX-45, HX-46, HX-47, HX-48, HX-50, HX-51, HX-52, HX-53, HX-55, HX-56, HX-57, HX-60, HX-61, HX-62, HX-63, HX-64, HX-65, HX-66, HX-67, HX-68, and HX-78.

7. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Enterococcus faecalis drug, wherein the cyclic polypeptide compound is selected from the group consisting of HX-24, HX-25, HX-27, HX-39, HX-45, HX-46, HX-47, HX-48, HX-50, HX-51, HX-56, HX-60, HX-61, HX-62, HX-63, HX-64, HX-65, HX-66, HX-67, and HX-68.

8. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Escherichia coli drug, wherein the cyclic polypeptide compound is selected from the group consisting of HX-1, HX-2, HX-6, HX-8, HX-10, HX-12, HX-13, HX-15, HX-16, HX-18, HX-19, HX-20, HX-24, HX-25, HX-26, HX-27, HX-30, HX-31, HX-32, HX-33, HX-38, HX -39, HX-40, HX-41, HX-42, HX-45, HX-46, HX-47, HX-48, HX-50, HX-51, HX-52, HX-53, HX-55, HX-56, HX-57, H X-60, HX-61, HX-62, HX-63, HX-64, HX-65, HX-66, HX-67, HX-68, HX-69, HX-70, HX-71, HX-72, HX-77, HX-78.

9. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Klebsiella pneumoniae drug, wherein the cyclic polypeptide compound is selected from HX-1, HX-2, HX-6, HX-8, HX-10, HX-12, HX-13, HX-15, HX-16, HX-18, HX-19, HX-20, HX-24, HX-25, HX-26, HX-27, HX-30, HX-31, HX-32, HX-33, HX-38, HX-39, HX-40, HX-41, HX-42, HX-43, HX-44, HX-45, HX-46, HX-47, HX-48, HX-49, HX-50, HX-51, HX-52, HX-53, HX-54, HX-55, HX-56, HX-57, HX-58, HX-59, HX-60, HX-61, HX-62, HX-63, HX-64, HX-65 X-39, HX-40, HX-41, HX-42, HX-45, HX-46, HX-47, HX-48, HX-50, HX-51, HX-52, HX-53, HX-55, HX-56, HX-57, HX-60, HX-61, HX-62, HX-63, HX-64, HX-65, HX-66, HX-67, HX-68, HX-69, HX-70, HX-71, HX-72, HX-77, HX-78.

10. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Pseudomonas aeruginosa drug, wherein the cyclic polypeptide compound is selected from the group consisting of HX-1, HX-2, HX-6, HX-8, HX-10, HX-12, HX-13, HX-15, HX-16, HX-18, HX-19, HX-20, HX-24, HX-25, HX-26, HX-27, HX-30, HX-31, HX-32, HX-33, HX-38, HX-39, HX-40, HX-41, HX-42, HX-43, HX-44, HX-45, HX-46, HX-47, HX-48, HX-49, HX-50, HX-51, HX-52, HX-53, HX-54, HX-55, HX-56, HX-57, HX-58, HX-59, HX-60, HX-61, HX-62, HX-63, HX-64, HX-65 X-39, HX-40, HX-41, HX-42, HX-45, HX-46, HX-47, HX-48, HX-50, HX-51, HX-52, HX-53, HX-55, HX-56, HX-57, HX-60, HX-61, HX-62, HX-63, HX-64, HX-65, HX-66, HX-67, HX-68, HX-69, HX-70, HX-71, HX-72, HX-77, HX-78.

11. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of an anti-Acinetobacter baumannii drug, wherein the cyclic polypeptide compound is selected from HX-2, HX-6, HX-10, HX-13, HX-15, HX-16, HX-18, HX-19, HX-20, HX-24, HX-25, HX-27, HX-39, HX-40, HX-41, HX-42, HX- 45. HX-46, HX-47, HX-48, HX-50, HX-51, HX-52, HX-53, HX-55, HX-56, HX-57, HX-60, HX-61, H X-62, HX-63, HX-64, HX-65, HX-66, HX-67, HX-68, HX-69, HX-70, HX-71, HX-72, HX-77, HX-78.

12. Use of the cyclic polypeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of a drug against polymyxin-resistant Pseudomonas aeruginosa, wherein the cyclic polypeptide compound is selected from the group consisting of HX-2, HX-6, HX-8, HX-10, HX-12, HX-18, HX-24, HX-25, HX-27, HX-39, HX-40, and HX-42.

13. A method for preparing the cyclic polypeptide compound according to claim 1 or 2, It is characterized in that The following steps are involved: (1) The protected free amino group of the side chain of the basic amino acid Fmoc-AA-OP is reacted with a halogenated resin to obtain Fmoc-AA-OP-resin; wherein P is a carboxyl protecting group; when Fmoc-AA-OP is Fmoc-Dab-OP, its structure is shown in Formula III: (2) Fmoc-AA-OP-resin is coupled one by one to obtain linear peptide-resin; (3) selectively removing the protecting group from the linear peptide-resin and performing solid phase cyclization to obtain the cyclic peptide-resin; (4) Acid hydrolysis of the cyclic polypeptide-resin to obtain a crude cyclic polypeptide compound; (5) The crude cyclic polypeptide compound is purified and / or converted to salt, and then freeze-dried to obtain a pure cyclic polypeptide compound.

14. The method according to claim 13, It is characterized in that P is allyl (Allyl) or benzyl (Bn).

15. The method of claim 13, It is characterized in that The following steps are involved: (1) Preparation of Fmoc-AA-OP resin: Add the halogenated resin to a peptide solid phase synthesis tube, add DCM to swell, wash with DMF three times, wash with DCM three times after swelling, dissolve the protected starting amino acid Fmoc-AA-OP and the base with DCM and add to the peptide synthesis tube, react at room temperature for 2 h, remove the reaction solution under vacuum, wash the resin with DMF three times, and wash with DCM three times to obtain Fmoc-AA-OP resin; (2) Coupling synthesis method: The Fmoc-AA-OP-resin obtained from the reaction in step (1) is treated with 20% piperidine / DMF for 10 min each time, for a total of 2 times, to remove the Fmoc protecting group of the α-amino group. It is washed three times with DMF and three times with DCM. The amino acid or side-chain carboxylic acid, DIEA, and HCTU are dissolved in DMF and added to the polypeptide synthesis tube, and the reaction is carried out at room temperature for 120 min. The reaction solution is evacuated under vacuum, washed three times with DMF and three times with DCM; starting from the starting amino acid, i.e., the x-position amino acid, where x is 6 or 7, it is coupled one by one to the 1-position amino acid, and then the side-chain carboxylic acid is coupled to the protected polypeptide-resin; the ivDde or Dde protecting group of the side-chain amino group of the 2-position amino acid is removed with a 2% hydrazine hydrate / DMF solution, washed three times with DMF and three times with DCM, and the carboxyl group of the 8-position amino acid is coupled to the side-chain amino group of the 2-position amino acid; starting from the 8-position amino acid, it is coupled one by one to the amino acid after the starting amino acid, i.e., the x + 1-position amino acid, to obtain a linearly fully protected polypeptide-resin; the one-by-one coupling sequence includes two parts. The first part is from the starting-position amino acid, i.e., the x-position amino acid, where x is 6 or 7, to the 1-position amino acid, and then to the side-chain carboxylic acid. The second part is from the 8-position amino acid to the x + 1-position amino acid; (3) Selective deprotection and solid-phase cyclization: The linearly fully protected polypeptide-resin in step (2) is treated with 20% piperidine / DMF for 10 min each time, for a total of 2 times, to remove the Fmoc protecting group of the α-amino group, washed three times with DMF and three times with DCM to form a free amino group; tetrakis(triphenylphosphine)palladium / phenylsilane is used to remove the allyl protecting group of the carboxyl group in a mixed solution of DCM and DMF to form a free carboxyl group; PyAOP and HOAT are dissolved in DMF, NMM is added, and it is added to the polypeptide synthesis tube. The reaction is carried out at room temperature for 3 h. The reaction solution is evacuated under vacuum, washed three times with DMF and three times with DCM to obtain a cyclic fully protected polypeptide-resin; (4) The crude cyclic basic polypeptide obtained by acid hydrolysis: TFA:H 2 The acid solution with a volume ratio of 95:5 was added to the peptide synthesis tube, and the reaction was carried out at room temperature for 120 min. The acid solution was added to cold ether, and the volume ratio of TFA lysis solution to cold ether was 1:

20. The peptide was precipitated, centrifuged, and dried to obtain the crude peptide. (5) Crude product purification, salt conversion, and freeze-drying: The crude product is dissolved in water, filtered through a 0.22 μm pore size filter membrane for standby. It is subjected to preparative high-performance liquid chromatography. The chromatographic packing material is 10 μm reversed-phase C18. The mobile phase A is 0.08% TFA / aqueous solution, and the mobile phase B is 0.08% TFA / acetonitrile solution. The flow rate of the 22 mm × 250 mm chromatographic column is 8 mL / min, the detection wavelength is 214 nm, gradient system elution is used, and cyclic injection purification is carried out. The crude product solution is loaded onto the chromatographic column, the mobile phase elution is started, and the fraction corresponding to the chromatographic main peak is collected. The fraction is evaporated to remove acetonitrile to obtain an aqueous polypeptide solution, which is freeze-dried to obtain the product.

16. The method according to claim 15, wherein, the halogenated resin is selected from the group consisting of trityl chloride resin, 4-methyltrityl chloride resin, 4-methoxytrityl chloride resin, 2-chlorotrityl chloride resin, bromo-(4-methylphenyl)-methyl resin, or bromo-(4-methoxyphenyl)-methyl resin.

17. The method according to claim 16, wherein, the halogenated resin is 2-chlorotrityl chloride resin.

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