Polypeptide and modifier thereof promoting bone formation

Abstract

A polypeptide or a modifier thereof. The polypeptide or the modifier thereof has the effect of promoting bone formation, and can be used for preventing and / or treating bone diseases.

Description

A polypeptide promoting bone formation and its modified substance

[0001] This application claims priority to Chinese Patent Application No. 2023118261965 filed on December 27, 2023, and Chinese Patent Application No. 2024112494006 filed on September 6, 2024. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention belongs to the field of biomedicine and relates to a polypeptide for promoting bone formation and a modified product thereof. Background Art

[0003] Throughout life, bones undergo continuous renewal and remodeling. Osteoclasts first remove a certain amount of bone tissue, and then new bone tissue forms in the resorption zone. Generally speaking, bone formation by osteoblasts and bone resorption by osteoclasts are in balance.

[0004] Bone resorption inhibitors include bisphosphonates, anti-RANKL antibodies, calcitonin, selective estrogen receptor modulators, etc. Bone formation promoters include parathyroid hormone analogs, etc.

[0005] Mingxiang Cai et al. (Generation of functional oligopeptides that promote osteogenesis based on unsupervised deep learning of protein IDRs, Bone Res 10, 23 (2022).) disclosed AIB5P (a pentapeptide with the sequence ESSES); AIB5P showed bone-forming promoting effects in multiple mouse models (e.g., osteoporosis, fractures, and implant bone integration). Summary of the Invention

[0006] The first aspect of the present invention provides a polypeptide as follows (a) or (b),

[0007] n = 0, 1, or 2;

[0008] (a) a cyclic polypeptide represented by formula I;

[0009] (b) A linear polypeptide formed by breaking one peptide bond of the cyclic polypeptide represented by formula I.

[0010] In one embodiment of the present invention, the polypeptide is a cyclic polypeptide. In one embodiment of the present invention, the polypeptide is a linear polypeptide. In one embodiment of the present invention, n=0. In one embodiment of the present invention, n=1. In one embodiment of the present invention, n=2.

[0011] In one embodiment of the present invention, the polypeptide is a cyclic polypeptide, and n = 0. In one embodiment of the present invention, the polypeptide is a cyclic polypeptide, and n = 1. In one embodiment of the present invention, the polypeptide is a cyclic polypeptide, and n = 2.

[0012] In one embodiment of the present invention, the polypeptide is as shown in Formula II or Formula III;

[0013] In the present invention, Formula II and Formula IV represent the same polypeptide,

[0014] In one embodiment of the present invention, the polypeptide is a linear polypeptide, n = 0. In one embodiment of the present invention, the polypeptide is a linear polypeptide, n = 1. In one embodiment of the present invention, the polypeptide is a linear polypeptide, n = 2.

[0015] In one embodiment of the present invention, the polypeptide is a linear polypeptide, and when n=0, the linear polypeptide is selected from:

[0016] (b001)Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu;

[0017] (b002)Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser;

[0018] (b003)Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu;

[0019] (b004)Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser;

[0020] (b005)Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser;

[0021] (b006)Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu;

[0022] (b007)Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys;

[0023] (b008) Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser;

[0024] (b009) Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu;

[0025] (b010) Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser; or

[0026] (b011) Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser.

[0027] In one embodiment of the present invention, the polypeptide is a linear polypeptide. When n = 1, the linear polypeptide is selected from:

[0028] (b101) Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu;

[0029] (b102) Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser;

[0030] (b103) Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu;

[0031] (b104) Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser;

[0032] (b105) Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser;

[0033] (b106)Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu;

[0034] (b107)Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys;

[0035] (b108)Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser;

[0036] (b109)Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu;

[0037] (b110)Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser; or

[0038] (b111)Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser.

[0039] The second aspect of the present invention provides a modified form of the polypeptide according to the first aspect of the present invention.

[0040] In one embodiment of the present invention, the modification is selected from a methylation modification, an acetylation modification, an amidation modification, a PEGylation modification, a biotin modification, a fluorescent labeling modification, or a fatty acid modification.

[0041] In one embodiment of the present invention, the modified compound is as shown in Formula II-A,

[0042] In one embodiment of the present invention, the modified compound is as shown in Formula II-B,

[0043] In one embodiment of the present invention, the modified compound represented by formula II-A is preferably the modified compound represented by formula V.

[0044] In one embodiment of the present invention, the modified substance is a modified substance of a linear polypeptide (b001), wherein the N-terminus of the linear polypeptide (b001) is modified by acetylation (i.e., the -NH2 at the N-terminus is replaced by -NHC(O)-CH3), and / or the C-terminus is modified by amidation (i.e., the -COOH at the C-terminus is replaced by -C(O)NH2).

[0045] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b002), wherein the N-terminus of the linear polypeptide (b002) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0046] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b003), wherein the N-terminus of the linear polypeptide (b003) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0047] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b004), wherein the N-terminus of the linear polypeptide (b004) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0048] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b005), wherein the N-terminus of the linear polypeptide (b005) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0049] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b006), wherein the N-terminus of the linear polypeptide (b006) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0050] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b007), wherein the N-terminus of the linear polypeptide (b007) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0051] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b008), wherein the N-terminus of the linear polypeptide (b008) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0052] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b009), wherein the N-terminus of the linear polypeptide (b009) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0053] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b010), wherein the N-terminus of the linear polypeptide (b010) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0054] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b011), wherein the N-terminus of the linear polypeptide (b011) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0055] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b101), wherein the N-terminus of the linear polypeptide (b101) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0056] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b102), wherein the N-terminus of the linear polypeptide (b102) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0057] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b103), wherein the N-terminus of the linear polypeptide (b103) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0058] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b104), wherein the N-terminus of the linear polypeptide (b104) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0059] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b105), wherein the N-terminus of the linear polypeptide (b105) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0060] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b106), wherein the N-terminus of the linear polypeptide (b106) is acetylated and / or the C-terminus is amidated.

[0061] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b107), wherein the N-terminus of the linear polypeptide (b007) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0062] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b108), wherein the N-terminus of the linear polypeptide (b108) is acetylated and / or the C-terminus is amidated.

[0063] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b109), wherein the N-terminus of the linear polypeptide (b109) is acetylated and / or the C-terminus is amidated.

[0064] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b110), wherein the N-terminus of the linear polypeptide (b110) is acetylated and / or the C-terminus is amidated.

[0065] In one embodiment of the present invention, the modified product is a modified product of a linear polypeptide (b111), wherein the N-terminus of the linear polypeptide (b111) is modified by acetylation, and / or the C-terminus is modified by amidation.

[0066] The third aspect of the present invention provides a pharmaceutical composition, which comprises the polypeptide described in the first aspect of the present invention and / or the modified substance described in the second aspect of the present invention, and a pharmaceutically acceptable carrier.

[0067] The fourth aspect of the present invention provides use of the polypeptide according to the first aspect of the present invention and / or the modified product according to the second aspect of the present invention in the preparation of a medicament for promoting bone formation.

[0068] The present invention also provides a method for promoting bone formation, comprising administering the polypeptide according to the first aspect of the present invention and / or the modified substance according to the second aspect of the present invention.

[0069] The present invention also provides use of the polypeptide according to the first aspect of the present invention and / or the modified substance according to the second aspect of the present invention for promoting bone formation.

[0070] In one embodiment of the present invention, the bone formation promotion can prevent and / or treat bone diseases. "Bone disease" refers to any of those diseases that cause various abnormalities or deformities of one or more bones and / or bone cells. For example, the bone disease is bone defect.

[0071] In the present invention, the term "polypeptide" refers to a compound formed by multiple amino acids connected by peptide bonds (-C(O)-NH-). In the present invention, "polypeptide" can be a linear polypeptide or a cyclic polypeptide. The means and methods for preparing such polypeptides are well known in the art; for example, they can be prepared by solid-phase synthesis. Unless otherwise specified, the solid-phase synthesis sequence is generally from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus). For ease of presentation, two peptide bonds are drawn in the form of (-C(O)-NH-) in Formula III, and the remaining peptide bonds in Formula I, Formula II, Formula II-A, Formula II-B, and Formula III are drawn in the form of lines.

[0072] In the present invention, the term "modification" of a peptide refers to any change made to the peptide. A modified polypeptide is also referred to as a modified polypeptide.

[0073] In the present invention, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0074] In the present invention, the term "room temperature" refers to 15-30°C.

[0075] In the present invention, the term "comprising" or "including" may be open, semi-closed or closed. In other words, the term also includes "essentially consisting of" or "consisting of."

[0076] In the present invention, "mass volume percentage" or "% (w / v)" or "% w / v" is a way of expressing mass volume concentration, which means the number of grams of solute contained in every 100 mL of solution. For example, 20% (w / v) means that 20 g of solute is contained in every 100 mL of solution.

[0077] The partial polypeptide sequences used in the present invention are shown in Table 1 below.

[0078] Table 1 Peptide sequence list

[0079] The present invention provides a polypeptide or a modified product thereof, which has the effect of promoting bone formation and can be used for preventing and / or treating bone diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 shows the ALP staining results in Example 6. a is the uninduced group, b is the induced group without drug addition, and c and d are drug addition groups; wherein c is the cyclic peptide compound of formula II, and d is the cyclic peptide compound of formula III.

[0081] FIG2 is a diagram showing the ARS staining results in Example 7. DETAILED DESCRIPTION

[0082] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0083] In the present invention, the abbreviations of chemical reagents have the meanings shown in Table 2 below:

[0084] Table 2 Abbreviations of chemical reagents

[0085] Example 1: Preparation of cyclic peptide compound of formula II

[0086] (1) Preparation of Fmoc-1-1 peptide resin

[0087] 2-CTC resin with a substitution degree between 0.25 mmol / g and 1.0 mmol / g is selected as the starting material.

[0088] Resin swelling and first amino acid loading: Weigh 10 g of 2-CTC resin (degree of substitution 0.59 mmol / g), add 80 mL of DCM and swell for 0.5 h, then filter; weigh Fmoc-Glu-OAll (17.7 mmol, 7.24 g) and DIEA (35.4 mmol, 4.6 g) and dissolve them in 60 mL of DCM, add them to the reactor, react for 3.0 h, and filter.

[0089] Head sealing and washing: Use DCM and DMF to wash alternately, and filter; prepare head sealing liquid DCM / methanol / DIEA (volume ratio: 16 / 3 / 1) and add it to the reactor, seal the head for 0.5h; use DCM and DMF to wash alternately and filter.

[0090] (2) Preparation of Fmoc-1-2 peptide resin

[0091] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0092] Preparation of activation solution and condensation: Fmoc-Ser-OH (17.7 mmol, 5.8 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0093] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0094] (3) Preparation of Fmoc-1-3 peptide resin

[0095] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0096] Preparation of activation solution and condensation: Fmoc-Ser-OH (17.7 mmol, 5.8 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0097] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0098] (4) Preparation of Fmoc-1-4 peptide resin

[0099] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0100] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (17.7 mmol, 7.53 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0101] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0102] (5) Preparation of Fmoc-1-5 peptide resin

[0103] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0104] Preparation of activation solution and condensation: Fmoc-Ser-OH (17.7 mmol, 5.8 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0105] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0106] (6) Preparation of Fmoc-1-6 peptide resin

[0107] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0108] Preparation of activation solution and condensation: Fmoc-Lys(Boc)-OH (17.7 mmol, 8.3 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor, reacted for 1.5 h, and filtered.

[0109] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0110] (7) Preparation of Fmoc-1-7 peptide resin

[0111] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0112] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (17.7 mmol, 7.53 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0113] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0114] (8) Preparation of Fmoc-1-8 peptide resin

[0115] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0116] Preparation of activation solution and condensation: Fmoc-Ser-OH (17.7 mmol, 5.8 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0117] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0118] (9) Preparation of Fmoc-1-9 peptide resin

[0119] Removal of Fmoc and washing: Add 20% Pip / DMF to remove Fmoc three times, wash with DCM and DMF alternately, and filter.

[0120] Preparation of activation solution and condensation: Fmoc-Ser-OH (17.7 mmol, 5.8 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0121] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0122] (10) Preparation of Fmoc-1-10 peptide resin

[0123] Removal of Fmoc and washing: Add 20% Pip / DMF to remove Fmoc three times, wash with DCM and DMF alternately, and filter.

[0124] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (17.7 mmol, 7.53 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0125] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0126] (11) Preparation of Fmoc-1-11 peptide resin

[0127] Removal of Fmoc and washing: Add 20% Pip / DMF to remove Fmoc three times, wash with DCM and DMF alternately, and filter.

[0128] Preparation of activation solution and condensation: Fmoc-Ser-OH (17.7 mmol, 5.8 g), HOBT (19.5 mmol, 2.63 g), and DIC (19.5 mmol, 2.45 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.5 h, and filtered.

[0129] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0130] (12) Preparation of 1-11 peptide resin

[0131] Removal of Fmoc and washing: Add 20% Pip / DMF to remove Fmoc three times, wash with DCM and DMF alternately, and filter.

[0132] Deallylation: Weigh Pd(PPh3)4 (1.77 mmol, 2.1 g) and dissolve it in a THF / DMSO / 0.5 M HCl mixture (2:2:1). Weigh morpholine (295 mmol, 25.67 g) and stir until well combined. Add to a reactor and react at 45°C for 3.0 h. Filter with suction. Wash the mixture three times with DCM and DMF, then filter. Wash the mixture three times with 0.5% palladium wash agent (sodium diethyldithiocarbamate trihydrate), then filter. Wash the mixture alternately with DCM and DMF, then filter.

[0133] (13) Preparation of 1-11 peptide resin cyclization

[0134] PyAOP (17.7 mmol, 9.22 g), HOAT (17.7 mmol, 2.41 g), and DIEA (35.4 mmol, 4.6 g) were weighed and dissolved in 60 mL of DMF and added to the reactor. The mixture was reacted for 3.0 h and filtered.

[0135] Washing after condensation: Use DCM and DMF to wash alternately, filter with suction; add 80 mL of methanol to wash, filter with suction; repeat the methanol washing 3 times, filter with suction.

[0136] Drying: After washing with methanol, the peptide resin was placed in a vacuum dryer at 35°C for 8 hours; the peptide resin after 1-11 cyclization was weighed to obtain 16.5 g.

[0137] (14) Preparation of crude cyclic peptide

[0138] Prepare 140 mL of lysis solution (trifluoroacetic acid: triisopropylsilane: water = 95:2.5:2.5) and precool to -5°C in advance; slowly add the peptide resin under stirring conditions and stir for 10 minutes; heat to 30±5°C, stir for 2 hours, and filter; wash the filter cake three times with 20 mL of trifluoroacetic acid each time and collect the filtrate.

[0139] Beating and drying: The filtrate was vacuum concentrated to 80 mL at 35 ° C, and the concentrate was slowly added dropwise to 400 mL of methyl tert-butyl ether precooled to 0 ° C with stirring for slurrying; filtration was performed to collect the filter cake; the filter cake was slurried in 50 mL of methyl tert-butyl ether solution and filtered; the above operation was repeated twice, and the filter cake was vacuum dried at 30 ± 5 ° C for 12 h to obtain 6.0 g of crude cyclic peptide.

[0140] (15) Purification and freeze-drying of crude cyclic peptides

[0141] Purification equipment: Chuangxin Tongheng DAC-100 (filler: particle size 10 μm, C18-BP, 100A, Dacao)

[0142] Wavelength: 210nm Flow rate: 200mL / min Column temperature: room temperature

[0143] Mobile phase: A-0.1% trifluoroacetic acid aqueous solution; B-acetonitrile; methanol for later use

[0144] Gradient program: 0 min, 100% A + 0% B; 10 min, 100% A + 0% B; 100 min, 80% A + 20% B

[0145] The preparative column was equilibrated with 100% A + 0% B mobile phase for 8 min before injection.

[0146] Dissolve the crude product: Crude product / trifluoroacetic acid (approximately 1g / 10mL). Once dissolved, dilute to 50mL with water. Refer to this ratio for specific amounts. The single injection volume should not exceed 10g.

[0147] The solution was filtered through medium-speed filter paper and injected directly into the sample, running a gradient. After the target fraction was collected, the column was washed with 80% methanol + 20% water, and eluted until the baseline was stable, completing the single-pass purification. Liquid phase monitoring was performed on the collected fractions, and qualified fractions were combined to obtain a preparative purified solution of the target product, which was then concentrated under reduced pressure to obtain an aqueous solution.

[0148] Freeze drying: The aqueous solution of the target compound obtained by preparative liquid phase purification is pre-frozen into a solid, and then freeze-dried in a freeze dryer to obtain a white solid, which is the target product.

[0149] (16) Structural confirmation

[0150] The molecular formula of the cyclic peptide compound of formula II is C 44 H 70 N 12 O 25 The molecular weight is 1167.10 (M) and the theoretical mass-to-charge ratio is 1166.46. Mass spectrometry detection shows that the mass-to-charge ratio of the target product is consistent with the theoretical mass-to-charge ratio.

[0151] The amino acid sequence of the target product was analyzed using electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS / MS). The target product sequence was confirmed to be correct by matching the theoretical sequence with the measured MS data.

[0152] By hydrolyzing and derivatizing the peptide chain of the target product, data on the composition and configuration of different amino acids can be obtained; the results show that the amino acid composition of the target product is all L-type, which is consistent with the theoretical configuration, indicating that its amino acid composition and configuration are correct.

[0153] In summary, the molecular weight was determined by mass spectrometry, the polypeptide sequence was analyzed by secondary mass spectrometry, and the composition and configuration of the amino acids were determined by derivatization methods; the structure of the target product was confirmed, and the target product was the cyclic peptide compound of formula II.

[0154] Example 2: Preparation of cyclic peptide compound of formula III

[0155] In the "1-11 peptide resin cyclization preparation" of Example 1, the NH2 of the peptide chain and the COOH of the peptide chain itself are activated and cyclized, ultimately obtaining a cyclic peptide compound of Formula II. At this time, the NH2 of some peptide chains will also form peptide bonds with the COOH of adjacent peptide chains, ultimately forming a cyclic peptide compound of Formula III (a cyclic dimer compound composed of 22 amino acids). The crude cyclic peptide in step (14) of Example 1 can also be separated by liquid preparative chromatography to obtain a cyclic peptide compound of Formula III.

[0156] At the same time, the crude cyclic peptide was tested by liquid chromatography-mass spectrometry, and substances with molecular weights consistent with those of cyclic trimer compounds were also detected.

[0157] Example 3: Preparation of compound of formula V

[0158] (1) Preparation of Fmoc-1-1 peptide resin

[0159] Wang resin with a degree of substitution between 0.25 mmol / g and 1.0 mmol / g is selected as the starting material.

[0160] Resin swelling and first amino acid loading: 10 g of Wang resin (degree of substitution 0.56 mmol / g) was added to 80 mL of DCM and swelled for 0.5 h, then filtered. Fmoc-Glu-OAll (11.2 mmol, 4.59 g), HOBT (12.32 mmol, 1.67 g), and DIC (12.32 mmol, 1.56 g) were dissolved in 55 mL of DCM and added to the reactor. DMAP (2.8 mmol, 0.34 g) was dissolved in 5 mL of DCM and added to the reactor for 8.0 h, then filtered.

[0161] Seal the reactor and wash it: Use DCM and DMF to wash it alternately, and filter it with suction. Weigh acetic anhydride (16.8 mmol, 1.71 g) and pyridine (16.8 mmol, 1.33 g) and dissolve them in 70 mL of DCM. Add them to the reactor and seal it for 1 hour. Use DCM and DMF to wash it alternately and filter it with suction.

[0162] (2) Preparation of Fmoc-1-2 peptide resin

[0163] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0164] Preparation of activation solution and condensation: Fmoc-Ser-OH (7.5 mmol, 2.46 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0165] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0166] (3) Preparation of Fmoc-1-3 peptide resin

[0167] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0168] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (7.5 mmol, 3.19 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0169] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0170] (4) Preparation of Fmoc-1-4 peptide resin

[0171] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0172] Preparation of activation solution and condensation: Fmoc-Ser-OH (7.5 mmol, 2.46 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0173] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0174] (5) Preparation of Fmoc-1-5 peptide resin

[0175] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter for 20 min.

[0176] Preparation of activation solution and condensation: Fmoc-Ser-OH (7.5 mmol, 2.46 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0177] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0178] (6) Preparation of Fmoc-1-6 peptide resin

[0179] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0180] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (7.5 mmol, 3.19 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0181] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0182] (7) Preparation of Fmoc-1-7 peptide resin

[0183] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter for 20 min.

[0184] Preparation of activation solution and condensation: Fmoc-Ser-OH (7.5 mmol, 2.46 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0185] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0186] (8) Preparation of Fmoc-1-8 peptide resin

[0187] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0188] Preparation of activation solution and condensation: Fmoc-Lys(Dde)-OH (7.5 mmol, 4.0 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor, reacted for 1.0 h, and filtered.

[0189] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0190] (9) Preparation of Fmoc-1-9 peptide resin

[0191] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0192] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (7.5 mmol, 3.19 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0193] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0194] (10) Preparation of Fmoc-1-10 peptide resin

[0195] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0196] Preparation of activation solution and condensation: Fmoc-Ser-OH (7.5 mmol, 2.46 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0197] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0198] (11) Preparation of Fmoc-1-11 peptide resin

[0199] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0200] Preparation of activation solution and condensation: Fmoc-Ser-OH (7.5 mmol, 2.46 g), HOBT (8.25 mmol, 1.11 g), and DIC (8.25 mmol, 1.04 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor and reacted for 1.0 h, and filtered.

[0201] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0202] (12) Preparation of peptide resin

[0203] Removal of Fmoc and washing: Add 20% Pip / DMF to remove Fmoc three times, wash with DCM and DMF alternately, and filter.

[0204] Deallylation: Weigh Pd(PPh3)4 (0.75 mmol, 0.87 g) and dissolve it in a DCM / DMF mixture (1:1). Weigh morpholine (25 mmol, 2.2 g) and add it to the mixture, stirring thoroughly. Add to the reactor, protect from light and react for 3 h under nitrogen atmosphere. Filter with suction. Wash the mixture three times with DCM and DMF, then filter. Wash the mixture three times with 0.5% palladium wash agent (sodium diethyldithiocarbamate trihydrate), filter, and then wash the mixture with DCM and DMF, then filter.

[0205] (13) Preparation of peptide resin cyclization

[0206] Preparation of activation solution and condensation: PyAOP (10 mmol, 5.21 g), HOAT (10 mmol, 1.37 g), and DIEA (20 mmol, 2.6 g) were weighed and dissolved in 60 mL of NMP and added to the reactor. The mixture was reacted for 2.0 h and filtered.

[0207] Washing after condensation: Use DCM and DMF to wash alternately and filter;

[0208] De-Dde and washing: Add 80 mL of 2% hydrazine hydrate / DMF, under nitrogen, stir for 30 min, wash with DCM and DMF alternately, and filter.

[0209] (14) Containing C 18 Preparation of cyclic peptide resin

[0210] Preparation of activation solution and condensation: Stearic acid (10 mmol, 2.85 g), HOAT (10 mmol, 1.37 g), and DIC (10 mmol, 1.26 g) were weighed and dissolved in 60 mL of DMF and added to the reactor. The mixture was reacted for 2.0 h and filtered.

[0211] Washing after condensation: Use DCM and DMF to wash alternately and filter;

[0212] Drying: After washing with methanol, the peptide resin was placed in a vacuum dryer at 35°C for 8 h;

[0213] Weighing C 18 15.5 g of cyclic peptide resin was obtained.

[0214] (15) Containing C 18 Preparation of crude cyclic peptide

[0215] Prepare 125 mL of lysis solution (trifluoroacetic acid: triisopropylsilane: water = 95:2.5:2.5) and precool to -5°C; slowly add the peptide resin under stirring conditions and stir for 10 minutes; heat to 30±5°C, stir for 2 hours, and filter; wash the filter cake three times with 20 mL of trifluoroacetic acid each time and collect the filtrate.

[0216] Beating and drying: The filtrate was concentrated to 80 mL in vacuum at 35°C. The concentrate was slowly added dropwise to 400 mL of methyl tert-butyl ether precooled to 0°C under stirring for beating; the filter cake was collected by suction filtering; the filter cake was beating in 50 mL of methyl tert-butyl ether solution and filtered; the above operation was repeated twice, and the filter cake was vacuum dried at 30±5°C for 12 h to obtain C 18 4.8g of crude cyclic peptide.

[0217] (16)C 18 Purification and freeze-drying of crude cyclic peptides

[0218] Purification equipment: Chuangxin Tongheng DAC-100 (filler: particle size 10 μm, C18-BP, 100A, Dacao)

[0219] Wavelength: 214 nm Flow rate: 200 mL / min Column temperature: room temperature

[0220] Mobile phase: A-0.1% trifluoroacetic acid aqueous solution; B-acetonitrile; methanol for later use

[0221] Gradient program: 0 min, 95% A + 5% B; 10 min, 76% A + 24% B; 100 min, 41% A + 59% B

[0222] The preparative column was equilibrated with 100% A + 0% B mobile phase for 8 min before injection.

[0223] Dissolve the crude product: Crude product / trifluoroacetic acid (approximately 1g / 10mL). Once dissolved, dilute to 50mL with water. Refer to this ratio for specific amounts. The single injection volume should not exceed 10g.

[0224] The solution was filtered through medium-speed filter paper and injected directly into the sample, running a gradient. After the target fraction was collected, the column was washed with 80% methanol + 20% water, and eluted until the baseline was stable, completing the single-pass purification. Liquid phase monitoring was performed on the collected fractions, and qualified fractions were combined to obtain a preparative purified solution of the target product, which was then concentrated under reduced pressure to obtain an aqueous solution.

[0225] Freeze drying: The aqueous solution of the target compound obtained by preparative liquid phase purification is pre-frozen into a solid, and then freeze-dried in a freeze dryer to obtain a white solid, which is the target product.

[0226] (17) Structural confirmation

[0227] Contains C 18 The molecular formula of the cyclic peptide compound is C 62 H 104 N 12 O 26 The molecular weight is 1433.57 (M) and the theoretical mass-to-charge ratio is 1432.72. Mass spectrometry detection shows that the mass-to-charge ratio of the target product is consistent with the theoretical mass-to-charge ratio.

[0228] The amino acid sequence of the target product was analyzed using electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS / MS). The target product sequence was confirmed to be correct by matching the theoretical sequence with the measured MS data.

[0229] By hydrolyzing and derivatizing the peptide chain of the target product, data on the composition and configuration of different amino acids can be obtained; the results show that the amino acid composition of the target product is all L-type, which is consistent with the theoretical configuration, indicating that its amino acid composition and configuration are correct.

[0230] In summary, the molecular weight was determined by mass spectrometry, the polypeptide sequence was analyzed by secondary mass spectrometry, and the composition and configuration of the amino acids were determined by derivatization methods; the structure of the target product was confirmed, and the target product was the compound of formula V.

[0231] Example 4: Preparation of N-terminally acetylated linear polypeptide (b001)

[0232] (1) Preparation of Fmoc-1-1 peptide resin

[0233] Rink Amide-MBHA Resin with a substitution degree between 0.25 mmol / g and 1.0 mmol / g is selected as the starting material.

[0234] Resin swelling: Weigh 10 g of Rink Amide-MBHA Resin (degree of substitution 0.64 mmol / g) and add 80 mL of DCM. Swell for 0.5 h and filter.

[0235] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0236] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (12.8 mmol, 5.45 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and then filtered.

[0237] Washing after condensation: Use DCM and DMF to wash alternately and filter;

[0238] (2) Preparation of Fmoc-1-2 peptide resin

[0239] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0240] Preparation of activation solution and condensation: Fmoc-Ser(tBu)-OH (12.8 mmol, 4.91 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0241] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0242] (3) Preparation of Fmoc-1-3 peptide resin

[0243] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0244] Preparation of activation solution and condensation: Fmoc-Ser(tBu)-OH (12.8 mmol, 4.91 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0245] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0246] (4) Preparation of Fmoc-1-4 peptide resin

[0247] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0248] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (12.8 mmol, 5.45 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0249] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0250] (5) Preparation of Fmoc-1-5 peptide resin

[0251] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0252] Preparation of activation solution and condensation: Fmoc-Ser(tBu)-OH (12.8 mmol, 4.91 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0253] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0254] (6) Preparation of Fmoc-1-6 peptide resin

[0255] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0256] Preparation of activation solution and condensation: Fmoc-Lys(Boc)-OH (12.8 mmol, 6.0 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 10 min, the mixture was added to the reactor, reacted for 2.0 h, and filtered.

[0257] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0258] (7) Preparation of Fmoc-1-7 peptide resin

[0259] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0260] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (12.8 mmol, 5.45 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0261] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0262] (8) Preparation of Fmoc-1-8 peptide resin

[0263] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0264] Preparation of activation solution and condensation: Fmoc-Ser(tBu)-OH (12.8 mmol, 4.91 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0265] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0266] (9) Preparation of Fmoc-1-9 peptide resin

[0267] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0268] Preparation of activation solution and condensation: Fmoc-Ser(tBu)-OH (12.8 mmol, 4.91 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0269] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0270] (10) Preparation of Fmoc-1-10 peptide resin

[0271] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0272] Preparation of activation solution and condensation: Fmoc-Glu(OtBu)-OH (12.8 mmol, 5.45 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0273] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0274] (11) Preparation of Fmoc-1-11 peptide resin

[0275] Removal of Fmoc and washing: add 20% Pip / DMF to remove Fmoc twice, wash with DCM and DMF alternately, and filter.

[0276] Preparation of activation solution and condensation: Fmoc-Ser(tBu)-OH (12.8 mmol, 4.91 g), HOBT (14.08 mmol, 1.90 g), and DIC (14.08 mmol, 1.78 g) were weighed and dissolved in 60 mL of DMF. After activation for 15 min, the mixture was added to the reactor and reacted for 2.0 h, and filtered.

[0277] Washing after condensation: Use DCM and DMF to wash alternately and filter.

[0278] (12) Preparation of acetylated peptide resin

[0279] Removal of Fmoc and washing: Add 20% Pip / DMF to remove Fmoc three times, wash with DCM and DMF alternately, and filter.

[0280] Preparation of sealing liquid and reaction: Weigh acetic anhydride (19.2 mmol, 1.96 g) and pyridine (19.2 mmol, 1.52 g) and dissolve in 60 mL DMF, add to the reactor, react for 0.5 h, and filter.

[0281] Washing after reaction: Use DCM and DMF to wash alternately and filter.

[0282] Drying: After washing with methanol, the peptide resin was placed in a vacuum dryer at 35°C for 8 h;

[0283] Yield: Weigh the peptide resin to obtain 16.3 g.

[0284] (13) Crude product preparation

[0285] Prepare 125 mL of lysis solution (trifluoroacetic acid: triisopropylsilane: water = 95:2.5:2.5) and precool to -5°C; slowly add the peptide resin under stirring conditions and stir for 10 minutes; heat to 30±5°C, stir for 2 hours, and filter; wash the filter cake three times with 20 mL of trifluoroacetic acid each time and collect the filtrate.

[0286] Beating and drying: The filtrate was vacuum concentrated to 80 mL at 35 ° C, and the concentrate was slowly added dropwise to 400 mL of methyl tert-butyl ether precooled to 0 ° C with stirring for slurrying; filtration was performed to collect the filter cake; the filter cake was slurried in 50 mL of methyl tert-butyl ether solution and filtered; the above operation was repeated twice, and the filter cake was vacuum dried at 30 ± 5 ° C for 12 h to obtain 5.83 g of crude cyclic peptide.

[0287] (14) Crude product purification and freeze drying

[0288] Purification equipment: Chuangxin Tongheng DAC-100 (filler: particle size 10 μm, C18-BP, 100A, Dacao)

[0289] Wavelength: 214 nm Flow rate: 200 mL / min Column temperature: room temperature

[0290] Mobile phase: A-0.1% trifluoroacetic acid aqueous solution; B-acetonitrile; methanol for later use

[0291] Gradient program: 0 min, 95% A + 5% B; 10 min, 76% A + 24% B; 100 min, 41% A + 59% B

[0292] The preparative column was equilibrated with 100% A + 0% B mobile phase for 8 min before injection.

[0293] Dissolve the crude product: Crude product / trifluoroacetic acid (approximately 1g / 10mL). Once dissolved, dilute to 50mL with water. Refer to this ratio for specific amounts. The single injection volume should not exceed 10g.

[0294] The solution was filtered through medium-speed filter paper and injected directly into the sample, running a gradient. After the target fraction was collected, the column was washed with 80% methanol + 20% water, and eluted until the baseline was stable, completing the single-pass purification. Liquid phase monitoring was performed on the collected fractions, and qualified fractions were combined to obtain a preparative purified solution of the target product, which was then concentrated under reduced pressure to obtain an aqueous solution.

[0295] Freeze drying: The aqueous solution of the target compound obtained by preparative liquid phase purification is pre-frozen into a solid, and then freeze-dried in a freeze dryer to obtain a white solid, which is the target product.

[0296] (15) Structural confirmation

[0297] The molecular formula of the linear peptide compound is C 46 H 74 N 12 O 27 The molecular weight is 1227.155 (M) and the theoretical mass-to-charge ratio is 1226.478. Mass spectrometry detection shows that the mass-to-charge ratio of the target product is consistent with the theoretical mass-to-charge ratio.

[0298] The amino acid sequence of the target product was analyzed using electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS / MS). The target product sequence was confirmed to be correct by matching the theoretical sequence with the measured MS data.

[0299] By hydrolyzing and derivatizing the peptide chain of the target product, data on the composition and configuration of different amino acids can be obtained; the results show that the amino acid composition of the target product is all L-type, which is consistent with the theoretical configuration, indicating that its amino acid composition and configuration are correct.

[0300] In summary, the molecular weight was determined by mass spectrometry, the peptide sequence was analyzed by secondary mass spectrometry, and the composition and configuration of the amino acids were determined by derivatization methods; the structure of the target product was confirmed, and the target product was a linear peptide compound.

[0301] Example 5: Stability test

[0302] The stability test of the cyclic peptide compound of formula II and the cyclic peptide compound of formula V was carried out.

[0303] (1) Stability study of cyclic peptide compound of formula II

[0304] Test solution: Take appropriate amounts of samples of the cyclic peptide compound of formula II that have been placed at 40°C, 60°C, high humidity 92.5%, high humidity 75%, light exposure and dark protection for 0, 5, 10, and 30 days, respectively, and dissolve them in 0.5% ammonia solution to prepare solutions containing approximately 2 mg / mL of the cyclic peptide compound of formula II.

[0305] Blood test solution: Place approximately 100 mg of the cyclic peptide compound of Formula II, 10 mg of benzalkonium chloride, and 2 mL of blood in a 10 mL centrifuge tube and vortex to mix thoroughly. Weigh 8 blood samples, approximately 160 mg each, into 2 mL centrifuge tubes, seal, and heat in a 38°C oven. Prepare and analyze samples at 0 min, 5 min, 30 min, 1 h, 2 h, 6 h, 12 h, and 24 h to assess the stability of the cyclic peptide compound of Formula II in blood. Sample preparation: Add 0.5 mL of 0.5% ammonia solution to the blood sample at each time point, vortex to mix thoroughly, then add 1 mL of acetonitrile and vortex. Filter through a 0.45 μm filter membrane and collect the filtrate.

[0306] Blood test solution: Place approximately 50 mg of the cyclic peptide compound of Formula II, 5 mg of benzalkonium chloride, and 1 mL of blood in a 10 mL centrifuge tube and vortex to mix thoroughly. Weigh four blood samples, approximately 100 mg each, into 2 mL centrifuge tubes, seal, and heat in a 38°C oven. Process and analyze samples at 0 h, 36 h, 3 days, and 7 days to assess the stability of the cyclic peptide compound of Formula II in blood. Sample preparation: Add 0.5 mL of 0.5% ammonia solution to the blood sample at each time point, vortex to mix thoroughly, then add 1 mL of acetonitrile and vortex. Filter through a 0.45 μm filter membrane and collect the filtrate.

[0307] Blood and saliva test solution: Place approximately 50 mg of the cyclic peptide compound of Formula II, 5 mg of benzalkonium chloride, 0.5 mL of blood, and 0.5 mL of saliva in a 10 mL centrifuge tube and vortex to mix thoroughly. Weigh approximately 100 mg of each of the blood and saliva samples into four 2 mL centrifuge tubes, seal them, and heat them in a 38°C oven. Samples were processed and analyzed at 0 h, 36 h, 3 days, and 7 days to assess the stability of the cyclic peptide compound of Formula II in blood and saliva. Sample preparation: Add 0.5 mL of 0.5% ammonia solution to the blood and saliva samples at each time point, vortex to mix thoroughly, then add 1 mL of acetonitrile and vortex. Filter through a 0.45 μm filter membrane and collect the filtrate.

[0308] Chromatographic conditions: Octadecylsilane bonded silica gel as the filler (Welch Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm or equivalent), baseline stabilization column: Phenomenex baseline 5μ (4.6 mm × 50 mm) or Welch Ghost-Buster Columm (4.6 mm × 50 mm), sulfuric acid and hydrochloric acid aqueous solution (1 L of water is adjusted to pH 2.5 with dilute sulfuric acid, and then to pH 2.1 with dilute hydrochloric acid) as mobile phase A, acetonitrile as mobile phase B, linear gradient elution according to Table 3 below; flow rate, 1.0 mL / min; column temperature, 30°C; detection wavelength, 214 nm; injection volume, 10 μl.

[0309] Table 3 Chromatographic conditions

[0310] Determination method: Accurately measure each test solution, inject it into the liquid chromatograph respectively, and record the chromatogram.

[0311] Stability test results: The stability test results of each test solution are shown in Table 4-Table 7.

[0312] Table 4 Influencing factors of cyclic peptide compound of formula II - total impurity results (%)

[0313] As shown in Table 4, the cyclic peptide compound of formula II has good stability when placed under 40°C, 60°C, high humidity 92.5%, high humidity 75%, light and dark conditions for 30 days (data for the 5th and 10th days are not shown).

[0314] Table 5 Stability of cyclic peptide compound of formula II in blood

[0315] As shown in Table 5, the cyclic peptide compound of formula II has good stability in blood at 38°C within 24 hours, and the relative content is between 90% and 103%.

[0316] Table 6 Stability of cyclic peptide compound of formula II in blood

[0317] As shown in Table 6, the cyclic peptide compound of Formula II is stable in blood at 38°C for 7 days, with relative contents ranging from 100% to 123%. (The experimental operation of taking 100 mg of sample into a 2 mL centrifuge tube is not precise weighing / measurement)

[0318] Table 7 Stability of cyclic peptide compound of formula II in blood and saliva

[0319] As shown in Table 7, the cyclic peptide compound of Formula II has good stability in blood and saliva at 38°C for 7 days, and the relative content is between 100% and 129% (the experimental operation of taking 100 mg of the sample into a 2 mL centrifuge tube is not precisely weighed / measured).

[0320] (2) Stability study of the V-ring peptide compound

[0321] Test solution: Take samples of the V-cyclic peptide compound that have been placed at 40°C, 60°C, high humidity 92.5%, high humidity 75%, light and dark conditions for 0, 5, 10, and 30 days, and add appropriate amounts of 0.5% ammonia solution to dissolve them to prepare test solutions containing approximately 5 mg / mL of the V-cyclic peptide compound.

[0322] Blood test solution: Place approximately 50 mg of the Formula V cyclic peptide compound, 5 mg of benzalkonium chloride, and 1 mL of blood in a 10 mL centrifuge tube and vortex to mix thoroughly. Weigh approximately 100 mg of each blood sample into five 2 mL centrifuge tubes, seal them, and heat in a 38°C oven. Process and analyze the samples at 0 hours, 3 hours, 24 hours, 3 days, and 7 days to assess the stability of the Formula V cyclic peptide compound in blood. Sample preparation: Add 0.5 mL of 0.5% ammonia solution to the blood sample at each time point, vortex to mix thoroughly, then add 1 mL of acetonitrile and vortex. Filter through a 0.45 μm filter membrane and collect the filtrate.

[0323] Blood and saliva test solution: Place approximately 50 mg of the Formula V cyclic peptide compound, 5 mg of benzalkonium chloride, 0.5 mL of blood, and 0.5 mL of saliva in a 10 mL centrifuge tube and vortex to mix thoroughly. Weigh approximately 100 mg of each blood and saliva sample into five 2 mL centrifuge tubes, seal them, and heat them in a 38°C oven. Samples were processed and analyzed at 0 hours, 6 hours, 24 hours, 3 days, and 7 days to assess the stability of the Formula V cyclic peptide compound in blood and saliva. Sample preparation: Add 0.5 mL of 0.5% ammonia solution to the blood and saliva samples at each time point, vortex to mix thoroughly, then add 1 mL of acetonitrile and vortex. Filter through a 0.45 μm filter membrane and collect the filtrate.

[0324] Chromatographic conditions: Octadecylsilane bonded silica gel as the filler (Inertsil ODS-SP, 4.6 mm × 250 mm, 5 μm or equivalent), baseline stabilization column: Phenomenex baseline 5μ (4.6 mm × 50 mm) or Welch Ghost-Buster Columm (4.6 mm × 50 mm), sulfuric acid and hydrochloric acid aqueous solution (1 L of water is adjusted to pH 2.5 with dilute sulfuric acid, and then to pH 2.1 with dilute hydrochloric acid) as mobile phase A, acetonitrile as mobile phase B, linear gradient elution according to Table 8 below; flow rate, 1.0 mL / min; column temperature, 30°C; detection wavelength, 214 nm; injection volume, 2 μl.

[0325] Table 8 Chromatographic conditions

[0326] Determination method: Accurately measure each test solution, inject it into the liquid chromatograph respectively, and record the chromatogram.

[0327] Stability test results: The stability test results of each test solution are shown in Table 9-Table 11.

[0328] Table 9 Influencing factors of V-ring peptide compound - total impurity results (%)

[0329] As shown in Table 9, the cyclic peptide compound of formula V has good stability when placed under 40°C, 60°C, high humidity 92.5%, high humidity 75%, light and dark conditions for 30 days (data for the 5th and 10th days are not shown).

[0330] Table 10 Stability of V-ring peptide compound in blood

[0331] As shown in Table 10, the cyclic peptide compound of formula V has good stability in blood at 38°C for 7 days, and the relative content is between 94% and 123%.

[0332] Table 11 Stability of V-ring peptide compound in blood and saliva

[0333] As shown in Table 11, the cyclic peptide compound of formula V has good stability in blood and saliva at 38°C for 7 days, and the relative content is between 88% and 100%.

[0334] Example 6: ALP staining to investigate the ability of cyclic peptides to enhance the osteogenic differentiation of mBMSC cells

[0335] Recovery and culture of mouse BMSC cells ( C57BL / 6 mouse bone marrow mesenchymal stem cells (Saiye Bio, catalog number: MUBMX-01001) were pre-coated with gelatin in a 96-well plate. After digestion, BMSC cells were plated at a density of 2 × 10 4 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a 96-well plate. The next day, the cells were observed to confirm that the confluence reached 70% (i.e., not less than 70%) and the medium was replaced with osteoblast induction medium ( Mouse bone marrow mesenchymal stem cell osteogenic differentiation kit (Cell Bio, Catalog No.: MUXMX-90021) was added with 0.2 μg / mL of the cyclic peptide compound to be tested (Formula II or Formula III). The medium was changed every 2-3 days (i.e., fresh osteoblast induction medium was replaced and 0.2 μg / mL of the cyclic peptide compound to be tested was added). On the 7th day of induction, ALP staining was performed (BCIP / NBT alkaline phosphatase colorimetric kit, Absin, Catalog No.: abs9332). The ALP staining results are shown in Figure 1.

[0336] Figure 1 shows that compared to the uninduced group (a), the induced non-drug group (b) and the drug-treated groups (c, d) all showed stronger ALP staining. Compared to the induced non-drug group (b), the Formula II cyclic peptide compound (0.2 μg / mL, c) had a significant ability to enhance osteogenic differentiation (stronger ALP staining). Compared to the induced non-drug group (b), the Formula III cyclic peptide compound (0.2 μg / mL, d) had a significant ability to enhance osteogenic differentiation (stronger ALP staining).

[0337] The ALP staining method is shown in Table 12:

[0338] Table 12 ALP staining method

[0339] Example 7: ARS staining to investigate the ability of cyclic peptides to enhance osteogenic differentiation of MC3T3-E1 cells

[0340] 1. Experimental Materials

[0341] Cells: Mouse MC3T3-E1 cell line (Nanjing Kebai Biotechnology, catalog number: CBP60946);

[0342] Basal medium: MEMα + 10% (v / v) FBS;

[0343] Osteogenic differentiation induction medium: MEMα+10% FBS+5 mM sodium β-glycerophosphate+25 μM vitamin C+1% (w / v) penicillin-streptomycin.

[0344] 2. Experimental Methods

[0345] In a 24-well plate, 0.2% gelatin was pre-coated and 2 × 10 4cells / cm 2 MC3T3-E1 cells were seeded at a density of 100 μg / mL and cultured overnight. The next day, when the cell confluence reached 80%-90%, the basal medium was replaced with osteogenic differentiation induction medium, and 0.2 μM of the cyclic peptide compound to be tested (compound of Formula II) was added. Culture was continued in the incubator, and fresh induction medium was replaced every 2-3 days and 0.2 μM of the cyclic peptide compound to be tested (compound of Formula II) was added. ARS staining was performed on days 14 and 19 of induction, and the results are shown in Figure 2.

[0346] ARS staining method:

[0347] Washing: Wash cells twice with PBS to remove residual substances in the culture medium.

[0348] Fixation: Fix cells with 4% paraformaldehyde at room temperature for 10 minutes.

[0349] Washing: Wash cells twice with PBS to remove residual material.

[0350] Staining: Add 0.2% ARS staining solution to the cell culture plate, 300 μl / well, and stain at 37°C for 15 min.

[0351] Washing: Wash cells 3 times with water to remove unbound dye and take pictures with a camera.

[0352] 3. Experimental Results

[0353] Figure 2 shows that ARS staining was stronger in both the induced and untreated groups compared to the uninduced group. Compared to the induced and untreated group, the cyclic peptide compound of Formula II at a concentration of 0.2 μM significantly enhanced osteogenic differentiation (stronger ARS staining).

[0354] Example 8: Animal Experiment on Alveolar Bone Defect in Beagle Dogs

[0355] 1. Purpose of the study

[0356] To evaluate the effect of the cyclic peptide compound of formula II in a canine tooth extraction socket filling animal model, and to evaluate the operability, safety and efficacy of the cyclic peptide compound of formula II.

[0357] 2. Experimental Design

[0358] This study was conducted in accordance with the "Guidelines for Registration Review of Animal Experimental Research on Medical Devices Part II: Experimental Design and Implementation Quality Assurance". Twelve healthy ordinary beagle dogs (weighing about 15 kg, about 14 months old) were selected as experimental animals and divided into two observation time points, 1 month (1M) and 3 months (3M) after surgery; 6 experimental animals were set up at each time point. One tooth root of the mandibular bilateral molars (P2, P3) of each experimental animal was extracted. After the tooth extraction, the extraction socket was filled with materials (blank control, or bone powder, or cyclic peptide compound of formula II), and the material was covered with a biofilm after filling. X-ray and CT examinations were performed before surgery, immediately after surgery, 1M after surgery, 2M after surgery, and 3M after surgery to evaluate the morphological changes of the alveolar bone (6 animals in the 1M group only underwent X-ray and CT examinations before surgery, immediately after surgery, and 1M after surgery). The general design of the experiment is shown in Table 13:

[0359] Table 13 Trial groups and drug administration

[0360] 3. Test results

[0361] CT scans of Hu values ​​immediately after administration and one month after administration showed an increase in both the blank and experimental groups, with the experimental group showing higher values ​​than the blank group. The Hu value in the bone powder control group decreased. This is because the bone powder control group's filling material is inherently radiopaque, resulting in a high initial Hu value. Subsequently, as soft tissue and new bone are formed, the Hu value decreases relative to the filling material, resulting in a downward trend. Furthermore, changes in defect width and length indicate a decreasing defect size after one month.

[0362] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above disclosure, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A polypeptide according to (a) or (b), n = 0, 1, or 2; (a) a cyclic polypeptide represented by formula I; (b) A linear polypeptide formed by breaking one peptide bond of the cyclic polypeptide represented by formula I.

2. The polypeptide according to claim 1, characterized in that The polypeptide is a cyclic polypeptide.

3. The polypeptide according to claim 2, characterized in that The polypeptide is as shown in Formula II or Formula III, 4. The polypeptide according to claim 1, characterized in that The polypeptide is a linear polypeptide.

5. The polypeptide according to claim 4, characterized in that When n=0, the linear polypeptide is selected from: (b001)Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu; (b002)Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser; (b003)Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu; (b004)Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser; (b005)Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser; (b006)Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu; (b007)Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys; (b008)Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser; (b009)Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu; (b010) Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser; or (b011) Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser.

6. The polypeptide according to claim 4, characterized in that When n = 1, the linear polypeptide is selected from: (b101) Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu; (b102) Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser; (b103) Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu; (b104) Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser; (b105) Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser; (b106) Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu; (b107) Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys; (b108) Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser; (b109) Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu; (b110) Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser;or (b111) Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser-Glu-Ser-Glu-Ser-Ser-Glu-Lys-Ser-Glu-Ser-Ser.; 7. A modified form of the polypeptide according to any one of claims 1 to 6.

8. The modified article according to claim 7, wherein The modification is selected from a methylation modification, an acetylation modification, an amidation modification, a PEGylation modification, a biotin modification, a fluorescent labeling modification, or a fatty acid modification.

9. The modified article according to claim 7, wherein The modified compound is shown in Formula II-A, Preferably, the modified compound is as shown in Formula V, 10. The modified article according to claim 7, wherein The modified compound is shown in Formula II-B, 11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to any one of claims 1 to 6 and / or the modified substance according to any one of claims 7 to 10, and a pharmaceutically acceptable carrier.

12. Use of the polypeptide according to any one of claims 1 to 6 and / or the modified substance according to any one of claims 7 to 10 in the preparation of a medicament for promoting bone formation.

13. Use of the polypeptide according to any one of claims 1 to 6 and / or the modified product according to any one of claims 7 to 10 in the preparation of a medicament for preventing and / or treating a bone disease; preferably, the bone disease is a bone defect.

Images