A cyclic peptide promoting bone formation and its modified products

Abstract

The present invention belongs to the field of biomedicine and relates to a cyclic peptide promoting bone formation and its modification. The cyclic peptide is shown as Formula I: where n = 0, 1 or 2. The half-life of the modified cyclic peptide compound of the present invention is prolonged, and the promoting effect on bone formation is further enhanced, having broad application prospects in the treatment of orthopedic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a cyclic peptide for promoting bone formation and a modified product thereof. Background Art

[0002] Bones are one of the most important supporting structures in the human body. They not only provide shape and protect internal organs, but also participate in movement and mineral metabolism. Osteoclasts and osteoblasts play key roles in bone physiology, working together to maintain the dynamic balance of bone, known as homeostasis. Osteoclasts are specialized cells responsible for bone resorption, secreting acids and enzymes to dissolve bone matrix, thereby removing aged or damaged bone tissue. Osteoblasts, on the other hand, are responsible for bone formation, synthesizing and mineralizing bone matrix to build new bone. The activities of these two cell types are mutually regulated: increased osteoclast activity leads to bone loss, while increased osteoblast activity promotes bone formation. In healthy conditions, their activities are balanced, but in certain disease states, such as osteoporosis, this balance can be disrupted, leading to decreased bone density and increased fracture risk. Therefore, understanding and regulating the activities of osteoclasts and osteoblasts is crucial for maintaining bone health.

[0003] Currently, the main treatments for osteoporosis focus on inhibiting bone resorption and promoting bone formation, increasing bone mass by increasing bone formation capacity over bone resorption capacity. Commonly used bone resorption inhibitors include bisphosphonates, anti-RANKL antibodies, calcitonin, and selective estrogen receptor modulators; bone formation promoters include parathyroid hormone analogs.

[0004] A journal article (Bone Res 10, 23(2022). http: / / doi.org / 10.1038 / s41413-022-00193-1) discloses AIB5P (a pentapeptide with the sequence ESSES). AIB5P has been shown to promote bone formation in various mouse models, such as osteoporosis, bone fractures, and implant osseointegration. For ease of reference in the patent, this compound will be referred to as Compound P1 in the following text. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to further modify the P1 compound in order to obtain a compound with better bone formation effect.

[0006] To achieve the above object, the present invention provides a cyclic peptide, which is shown in Formula I:

[0007] ,

[0008] Where n=0, 1 or 2.

[0009] A polypeptide, which is a linear polypeptide represented by a cyclic polypeptide represented by formula I with any peptide bond removed.

[0010] Preferably, the polypeptide is a cyclic polypeptide, n=0.

[0011] Preferably, the polypeptide is a cyclic polypeptide, and n=1.

[0012] Preferably, the polypeptide is a cyclic polypeptide, and n=2.

[0013] In a preferred embodiment of the present invention, the present invention further provides a cyclic peptide, which is shown in Formula II:

[0014] ;

[0015] Or the cyclic peptide is as shown in Formula III:

[0016] .

[0017] Preferably, the polypeptide is a cyclic polypeptide, n=0.

[0018] Preferably, the polypeptide is a cyclic polypeptide, and n=1.

[0019] Preferably, the polypeptide is a cyclic polypeptide, and n=2.

[0020] Preferably, the polypeptide is a linear polypeptide Ser-Asp-Ser-Asn-Asp.

[0021] Preferably, the polypeptide is a linear polypeptide:

[0022] Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp;

[0023] Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser;

[0024] Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp;

[0025] Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser;

[0026] Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser- Asp-Ser-Asn;

[0027] Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp;

[0028] Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly;

[0029] Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser;

[0030] Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly;

[0031] Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser;

[0032] Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp;

[0033] Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser;

[0034] Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn;

[0035] Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp;

[0036] Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly;

[0037] Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser;

[0038] Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly;

[0039] Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser;

[0040] Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp;

[0041] Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser; or

[0042] Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn。

[0043] Preferably, the polypeptide is a linear polypeptide:

[0044] Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp;

[0045] Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp;

[0046] Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser;

[0047] Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser;

[0048] Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn;

[0049] Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp;

[0050] Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gl;

[0051] Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser;

[0052] Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly;

[0053] Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly- Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Se;

[0054] Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp;

[0055] Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser;

[0056] Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn;

[0057] Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp;

[0058] Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly;

[0059] Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser;

[0060] Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly;

[0061] Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser;

[0062] Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp;

[0063] Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser;

[0064] Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn;

[0065] Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp;

[0066] Ser-Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly;

[0067] Gly-Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser;

[0068] Ser-Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly;

[0069] Asp-Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser;

[0070] Ser-Asn-Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp;

[0071] Or

[0072] Asp-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn-Asp-Gly-Ser-Gly-Ser-Asp-Ser-Asn.

[0073] In another preferred embodiment of the present invention, the present invention also provides a modified cyclic peptide.

[0074] Preferably, the modification of the cyclic peptide is selected from a methylation modification, an acetylation modification, a PEGylation modification, a biotin modification, a fluorescent labeling modification or a fatty acid modification.

[0075] Preferably, the modified cyclic peptide is as shown in Formula II-A:

[0076] .

[0077] Preferably, the modified cyclic peptide is as shown in Formula II-B:

[0078] .

[0079] In another preferred embodiment of the present invention, the present invention further provides a pharmaceutical composition, which comprises the cyclic peptide of the present invention and / or a modified substance of the cyclic peptide of the present invention, and a pharmaceutically acceptable carrier.

[0080] Preferably, the cyclic peptide of the present invention and / or the modified substance of the cyclic peptide of the present invention and / or the pharmaceutical composition of the present invention are used to promote bone formation.

[0081] In another preferred embodiment of the present invention, the present invention also provides the use of the cyclic peptide of the present invention and / or the modified product of the cyclic peptide of the present invention in the preparation of a drug for treating osteoporosis, wherein the treatment is by promoting bone formation.

[0082] The modified cyclic peptide compound of the present invention has a prolonged half-life and further enhanced bone formation promoting effect, and has broad application prospects in the treatment of orthopedic diseases.

[0083] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is the result of ALP staining experiment.

[0085] Figure 2 Micro-CT images of osteoporosis mouse model treated with P1 and P2 compounds. DETAILED DESCRIPTION

[0086] The following description introduces a plurality of preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied by many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned in the text. The present invention is further elaborated below in conjunction with specific examples. It should be noted that these examples are intended to help understand the content of the present invention rather than to limit the scope of the invention. The experimental methods for which specific conditions are not specified in the following examples are usually performed under conventional conditions or under the conditions recommended by the reagent manufacturer.

[0087] In one embodiment of the present invention, the promotion of bone formation 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.

[0088] As used herein, the term "polypeptide" refers to a compound composed of multiple amino acids linked by peptide bonds (-C(O)-NH-) formed through dehydration condensation. A "polypeptide" herein may be a linear polypeptide or a cyclic polypeptide. The means and methods for preparing polypeptides are well known in the art; for example, solid-phase synthesis can be employed. Generally, unless otherwise specified, solid-phase synthesis proceeds from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus).

[0089] In the present invention, the term "modification" of a polypeptide refers to any change made to the polypeptide. A modified polypeptide is also referred to as a modified polypeptide.

[0090] 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.

[0091] In the present invention, the term "room temperature" refers to 15-30°C.

[0092] 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."

[0093] 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.

[0094] As used herein, "bone disease" refers to any of those diseases that cause various abnormalities or deformities in one or more bones and / or bone cells.

[0095] The abbreviations and definitions of chemical reagents in the present invention are as follows:

[0096] 2-CTC resin 2-chlorotrityl chloride resin

[0097] DCM dichloromethane

[0098] DIEA N,N-Diisopropylethylamine

[0099] DMF N,N-dimethylformamide

[0100] THF Tetrahydrofuran

[0101] DMSO dimethyl sulfoxide

[0102] Fmoc-Ser-OH N-Fmoc-L-serine

[0103] Fmoc-Asp(OtBu)-OH N-Fluorenylmethoxycarbonyl- L-aspartic acid 4-tert-butyl ester

[0104] Fmoc-Gly-OH N-Fluorenylmethoxycarbonyl-L-glycine

[0105] Fmoc-Asn-OH N-Fmoc-L-asparagine

[0106] HOBT 1-Hydroxybenzotriazole

[0107] DIC N,N'-diisopropylcarbodiimide

[0108] Pd(PPh3)4Tetrakis(triphenylphosphine)palladium

[0109] Example 1: Preparation of cyclic peptide compound of formula II

[0110] In this example, a cyclic peptide was constructed by peptide solid phase synthesis based on a Fmoc / t-butyl protection strategy on 2-chlorotrityl chloride resin.

[0111] (1) Preparation of Fmoc-1-1 peptide resin

[0112] In solid-phase peptide synthesis, we selected 2-CTC resin with a degree of substitution ranging from 0.25 mmol / g to 1.0 mmol / g as the starting material for the synthesis.

[0113] Resin swelling: 10 g of dry 2-CTC resin (degree of substitution 0.59 mmol / g) was weighed and placed in 80 mM DCM for swelling for 0.5 h, after which the excess solvent was removed by filtration.

[0114] First amino acid loading: 17.7 mmol (7.28 g) of Fmoc-Asp(OtBu)-OH and 35.4 mmol (4.6 g) of DIEA were weighed and dissolved in 60 mL of DCM. The solution was then added to the reactor and reacted for 3.0 h, followed by filtration.

[0115] Capping and washing: After the reaction, wash with DCM and DMF alternately, and remove the washing liquid by filtration; prepare a capping liquid DCM / methanol / DIEA (volume ratio: 16 / 3 / 1) and add it to the reactor, cap the head for 0.5h; wash again with DCM and DMF alternately, and remove the washing liquid by filtration.

[0116] (2) Preparation of Fmoc-1-2 peptide resin

[0117] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0118] Activation and condensation: 17.7 mmol (6.27 g) of Fmoc-Asn-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0119] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0120] (3) Preparation of Fmoc-1-3 peptide resin

[0121] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0122] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0123] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0124] (4) Preparation of Fmoc-1-4 peptide resin

[0125] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0126] Activation and condensation: 17.7 mmol (7.28 g) of Fmoc-Asp(OtBu)-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0127] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0128] (5) Preparation of Fmoc-1-5 peptide resin

[0129] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0130] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0131] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0132] (6) Preparation of Fmoc-1-6 peptide resin

[0133] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0134] Activation and condensation: 17.7 mmol (5.26 g) of Fmoc-Gly-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0135] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0136] (7) Preparation of Fmoc-1-7 peptide resin

[0137] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0138] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0139] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0140] (8) Preparation of Fmoc-1-8 peptide resin

[0141] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0142] Activation and condensation: 17.7 mmol (5.26 g) of Fmoc-Gly-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0143] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0144] (9) Preparation of Fmoc-1-9 peptide resin

[0145] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0146] Activation and condensation: 17.7 mmol (7.28 g) of Fmoc-Asp(OtBu)-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0147] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0148] (10) Preparation of Fmoc-1-10 peptide resin

[0149] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0150] Activation and condensation: 17.7 mmol (6.27 g) of Fmoc-Asn-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0151] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0152] (11) Preparation of Fmoc-1-11 peptide resin

[0153] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0154] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0155] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0156] (12) Preparation of Fmoc-1-12 peptide resin

[0157] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0158] Activation and condensation: 17.7 mmol (7.28 g) of Fmoc-Asp(OtBu)-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0159] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0160] (13) Preparation of Fmoc-1-13 peptide resin

[0161] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0162] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0163] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0164] (14) Preparation of Fmoc-1-14 peptide resin

[0165] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0166] Activation and condensation: 17.7 mmol (5.26 g) of Fmoc-Gly-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0167] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0168] (15) Preparation of Fmoc-1-15 peptide resin

[0169] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0170] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0171] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0172] (16) Preparation of Fmoc-1-16 peptide resin

[0173] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0174] Activation and condensation: 17.7 mmol (5.26 g) of Fmoc-Gly-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0175] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0176] (17) Preparation of Fmoc-1-17 peptide resin

[0177] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0178] Activation and condensation: 17.7 mmol (7.28 g) of Fmoc-Asp(OtBu)-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0179] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0180] (18) Preparation of Fmoc-1-18 peptide resin

[0181] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0182] Activation and condensation: 17.7 mmol (6.27 g) of Fmoc-Asn-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0183] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0184] (19) Preparation of Fmoc-1-19 peptide resin

[0185] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0186] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0187] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0188] (20) Preparation of Fmoc-1-20 peptide resin

[0189] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0190] Activation and condensation: 17.7 mmol (7.28 g) of Fmoc-Asp(OtBu)-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0191] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0192] (21) Preparation of Fmoc-1-21 peptide resin

[0193] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0194] Activation and condensation: 17.7 mmol (5.79 g) of Fmoc-Ser-OH, 19.5 mmol (2.63 g) of HOBT, and 19.5 mmol (2.45 g) of DIC 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, followed by filtration.

[0195] Washing after condensation: After the reaction, use DCM and DMF to wash alternately, and remove the washing liquid by suction filtration.

[0196] (22) Preparation of 1-21 peptide resin

[0197] Removal of Fmoc and washing: Use a 20% Pip / DMF mixture to remove the Fmoc protecting group twice, wash alternately with DCM and DMF, and remove the washing solution by filtration.

[0198] Deallylation: Weigh 1.77 mmol (2.1 g) of Pd(PPh3)4 and dissolve it in a mixture of THF / DMSO / 0.5 M HCl. Weigh 295 mmol (25.67 g) of morpholine and stir thoroughly. Add the mixture to the reactor and react at 45°C for 3.0 h. After completion, filter the mixture. Wash the mixture three times with DCM and DMF, then remove the washings by filtration. Wash the mixture three times with 0.5% palladium detergent (sodium diethyltrithiocarbamate trihydrate), then filter the mixture. Wash the mixture alternately with DCM and DMF, then remove the washings by filtration.

[0199] (23) Preparation of 1-21 peptide resin cyclization

[0200] 17.7 mmol (9.22 g) of PyAoP, 17.7 mmol (2.41 g) of HOAT, and 35.4 mmol (4.6 g) of DIEA were weighed and dissolved in 60 mL of DMF. The mixed solution was added to the reactor and reacted for 3.0 h, followed by filtration.

[0201] Post-condensation washing: Alternately wash with DCM and DMF, and remove the washings by filtration. Add 80 mL of methanol for washing, then filter; repeat the methanol wash three times, and filter.

[0202] Drying: After washing with methanol, the peptide resin was placed at 35°C and vacuum dried for 8.0 h.

[0203] (24) Preparation of crude cyclic peptide

[0204] Prepare 140 mL of lysis buffer with trifluoroacetic acid, triisopropylsilane, and water in a ratio of 95:2.5:2.5. Precool the lysis buffer to -5°C. Slowly add the peptide resin to the precooled lysis buffer with stirring for 10 minutes. Then, warm the mixture to 30 ± 5°C and stir for 2.0 hours before filtering. Finally, wash the filter cake three times with 20 mL of trifluoroacetic acid and collect the filtrate.

[0205] Beating and drying: The filtrate was vacuum concentrated to 80 mL at 35 ° C. The concentrate was slowly added dropwise to 400 mL of methyl tert-butyl ether precooled to 0 ° C under 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 12.0 h to obtain 6.0 g of crude cyclic peptide.

[0206] (25) Purification and freeze-drying of crude cyclic peptides

[0207] Purification equipment: Chuangxin Tongheng DAC-100 (filler: particle size 10μm, C18-BP, 100A, Dacao)

[0208] Wavelength: 210nm Flow rate: 200ml / min Column temperature: room temperature

[0209] Mobile phase: A-0.1% trifluoroacetic acid aqueous solution; B-acetonitrile; methanol for later use

[0210] Gradient program: 0 min, 100% A + 0% B; 100 min, 80% A + 20% B

[0211] Before injection, the preparative column was equilibrated with 100% A + 0% B mobile phase for 8 min.

[0212] Dissolve crude product: Dissolve 1g of crude cyclic peptide in 10mL of trifluoroacetic acid. After the solution is clear, dilute to 50mL with water. Refer to this ratio for specific amounts. The single injection volume should not exceed 10g.

[0213] The solution is filtered using medium-speed filter paper to remove possible particulate matter, and the filtrate is then used directly for chromatographic analysis using a gradient elution procedure. Once the target component is successfully collected, we switch to a mixed solvent of 80% methanol and 20% water to clean the chromatographic column until the baseline of the chromatogram returns to a stable state, marking the end of the single purification process. The collected components are monitored by liquid chromatography to ensure that their purity meets the standard. Qualified components are combined to obtain a preparative purified solution of the target compound. Finally, an aqueous solution of the target compound is obtained through a vacuum concentration step.

[0214] (26) Structural verification

[0215] The molecular formula of the cyclic peptide compound of formula II is C68H100N24O44, the molecular weight is 1957.64 (M), and the theoretical mass-to-charge ratio is 1957.7; using mass spectrometry detection, the mass-to-charge ratio of the generated product is consistent with the theoretical mass-to-charge ratio.

[0216] The amino acid sequence of the target product was analyzed using electrospray ionization time-of-flight (ESI-TOF-MS / MS). The target product was confirmed to be correct by matching the theoretical sequence with the measured MS data.

[0217] By hydrolyzing and diffracting the peptide chain of the target product, data on the composition and configuration of different amino acids can be obtained; data analysis shows 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.

[0218] In summary, mass spectrometry accurately determined the molecular weight of the target product, and the peptide sequence was analyzed by secondary mass spectrometry. Diffraction confirmed the amino acid composition and configuration, completing the structural confirmation of the target product, which is the cyclic peptide compound of Formula II.

[0219] Example 2 Preparation of the Cyclic Peptide Compound of Formula III

[0220] In the "1-20 peptide resin cyclization preparation" of Example 1, the NH2 of the peptide bond and the COOH of the peptide chain itself are activated and cyclized to ultimately obtain the cyclic compound of Formula II. In the execution of Example 1, the cyclic peptide compound of Formula III can be obtained by repeating steps (9) to (16) before preparing the Fmoc-1-17 peptide resin in step (17). Subsequent purification and verification steps are the same as those in Example 1.

[0221] Example 3 In vitro study of the stability of the cyclic peptide compound of formula II and the cyclic peptide compound of formula III

[0222] For ease of expression, in Example 3, the cyclic peptide compound of formula II is named P2, and the cyclic peptide compound of formula III is named P3.

[0223] 0.5 mL of fresh human serum was added to the 2 mmol / L peptide, mixed thoroughly, and allowed to stand. A time series was established, with samples taken at 0 minutes at the start of the experiment and 15, 30, and 60 minutes thereafter. After each sampling, the peptide concentration in the mixed solution was accurately measured using high-performance liquid chromatography (HPLC) to assess the peptide's stability in serum and possible metabolic changes.

[0224] result

[0225] Compared with compound P1 disclosed in previous patent documents, P2 and P3 have a longer metabolism time in serum. In the concentration test of three-point sampling, P2 and P3 both showed better stability.

[0226]

[0227] Example 4 Verification of the osteogenic ability of cyclic peptides on mBMSCs cells by ALP staining

[0228] For ease of expression, in Example 4, the cyclic peptide compound of formula II is named P2, and the cyclic peptide compound of formula III is named P3.

[0229] Resuscitate and culture mouse BMSC cells. Place the cryovial in a 37°C water bath until the ice disappears. Transfer the cell suspension in the cryovial to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Collect the cell pellet, add complete culture medium, gently pipette and transfer it to a culture dish and shake well. Incubate at 37°C. Coat a 12-well plate with gelatin in advance. After digesting the BMSC cells, use 5×5 cells / cm 2 Cells were seeded onto 12-well plates at a density of 100 μg / mL. Observe every other day to confirm that the cell confluence reached 70%, then replace the culture medium with osteoblast induction medium and add the cyclic peptide compound to be tested (Formula II or Formula III). The medium was changed every 2-3 days. On the 14th day of differentiation induction, osteogenic differentiation ability was detected using ALP staining (BCIP / NBT alkaline phosphatase colorimetric kit, absin / abs9662).

[0230] ALP staining method:

[0231] 1. Prepare the color development solution: take 10 μL BCIP solution (100×);

[0232] 2. Cell pretreatment: Wash cells in 96-well plates three times with PBS; fix with 4% paraformaldehyde at room temperature for 15 minutes and then wash three times with PBS;

[0233] 3. Color reaction: remove the wash solution and add to each well; incubate at room temperature in the dark for 10 minutes;

[0234] 4. Stop color development: Remove the BCIP / NBT staining solution and wash three times with distilled water to stop the color development reaction. After washing, add 100 μL of distilled water to each well and take pictures under a microscope at 100× magnification.

[0235] Figure 1The results showed that compared with compound P1 (0.2 μmol / mL) reported in the previous literature, compounds P2 (0.2 μmol / mL) and P3 (0.2 μmol / mL) reported in this patent had stronger ALP staining. Compared with compound P1, P2 and P3 had significant osteogenic differentiation ability (stronger ALP staining).

[0236] Example 5: Verification of osteogenic capacity in osteoporosis mouse model by Micro-CT

[0237] This example aims to verify the osteogenesis ability of different intervention treatments on an osteoporosis mouse model using Micro-CT technology.

[0238] Twelve-week-old female C57BL / 6 mice were randomly divided into three groups, each consisting of eight mice. Group 1 served as an osteoporosis model (OVX), while groups 2 and 3, respectively, received the P1 compound and P2 compound treatments. The osteoporosis model was established by aseptic ovariectomy (OVX). Twelve weeks after surgery, the P1 and P2 compound groups were injected with 10 mg of the peptide P1 and P2 compounds, respectively, every two weeks for 12 weeks.

[0239] After the experiment, all mice were sacrificed under carbon dioxide anesthesia. First, the spinal column was completely dissected, and the lumbar region (L1-L6) was precisely located. The fifth lumbar vertebra (L5) was then isolated, and the attached muscles and ligaments were removed. The isolated L5 vertebra was then fixed in 4% paraformaldehyde solution (pH 7.4) for 48 hours.

[0240] Three-dimensional scanning was performed using a micro-CT scanner (micro-CT50, Scanco Medical, Switzerland) with the following scanning parameters: 12 μm resolution, 55 kV voltage, and 220 μA current. Data were reconstructed using NRecon software with a threshold of 180–220 to obtain high-quality cancellous bone imaging.

[0241] Micro-CT images will be used to analyze changes in cancellous bone structure, primarily assessing the following parameters: bone mineral density (BMD), trabecular bone volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). All data will be analyzed using SPSS statistical software, and differences between groups will be compared using one-way analysis of variance.

[0242] Micro-CT scanning analysis clearly demonstrated the effects of P1 and P2 compound treatment on mouse bone structure. In particular, in P Group 2, significant improvements in trabecular bone were observed compared to the model group and P1 Group 1, with increases in bone density and trabecular volume, demonstrating the potential of P2 peptide in the treatment of osteoporosis.

[0243] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A cyclic peptide, wherein the cyclic peptide is as shown in formula II: 。 2. A cyclic peptide, wherein the cyclic peptide is as shown in formula III: 。 3. A polynucleotide encoding the cyclic peptide according to any one of claims 1 to 2.

4. A modified substance containing the cyclic peptide according to any one of claims 1 to 2, characterized in that: The modifier is selected from a methylation modifier, an acetylation modifier, a PEGylation modifier, a biotin modifier, a fluorescent labeling modifier or a fatty acid modifier.

5. The modifier according to claim 4, characterized in that The modified substance is shown in Formula II-A: ； Or, the modified substance is as shown in formula II-B: 。 6. A pharmaceutical composition, comprising the cyclic peptide according to any one of claims 1 to 2 and / or the modified substance according to any one of claims 4 to 5, and a pharmaceutically acceptable carrier.

7. Use of the cyclic peptide according to any one of claims 1 to 2, the modified substance according to any one of claims 4 to 5, or the pharmaceutical composition according to claim 6 in the preparation of a drug for treating osteoporosis.

8. The use according to claim 7, characterized in that: The treatment is achieved by promoting bone formation.

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