WO2021042732A1

WO2021042732A1 - Osteogenic growth peptide carbon-terminal pentapeptide derivative, preparation method therefor, and use thereof

Info

Publication number: WO2021042732A1
Application number: PCT/CN2020/085363
Authority: WO
Inventors: 张腾; 张忠旗; 郭添; 刘少军; 李晨召; 李乾; 王昕�; 赵金礼
Original assignee: 陕西慧康生物科技有限责任公司
Priority date: 2019-09-06
Filing date: 2020-04-17
Publication date: 2021-03-11
Also published as: CN112789286A; CN112789286B

Abstract

The present invention provides an osteogenic growth peptide carbon-terminal pentapeptide derivative, a preparation method therefor, and use thereof. The osteogenic growth peptide carbon-terminal pentapeptide derivative includes one or more of H-Dopa-Gly-Phe-Gly-Gly-OH, H-D-Tyr-Pro-D-Phe-Gly-Gly-OH, Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr), Cyclo(Tyr-Gly-D-Phe-Gly-Gly), Cyclo(D-Tyr-Gly-Phe-Gly-Gly), Cyclo(Gly-Gly-D-Phe-Gly-Tyr), and Cyclo(Gly-Gly-Phe-Gly-D-Tyr). Said derivative retains pharmacophores Tyr10 and Phe12 of the original pentapeptide in the structure, and have the structural feature of improving metabolic stability.

Description

Osteogenic growth peptide carbon-terminal pentapeptide derivative and preparation method and application thereof

Technical field

The invention belongs to the technical field of polypeptides, and specifically relates to an osteogenic growth peptide carbon-terminal pentapeptide OGP (10-14) derivative, and a preparation method and application thereof.

Background technique

Osteogenic Growth Peptide (Osteogenic Growth Peptide) is a kind of polypeptide growth factor, which has the effect of promoting bone formation and stimulating hematopoiesis. The primary structure is ALKRQGRTLYGFGG. Among them, the carbon-terminal pentapeptide OGP (10-14) is the smallest fragment that maintains the full activity of the osteogenic growth peptide, and the amino acid sequence is H-Tyr10-Gly11-Phe12-Gly13-Gly14-OH. A large number of experiments have shown that OGP (10-14) not only improves the treatment of bone injury and bone metabolism diseases, but also restores the hematopoietic function of patients with radiotherapy and chemotherapy and bone marrow transplant patients. According to reports, the Italian pharmaceutical company Abiogen has submitted an application to the European Medicines Agency (EMEA) for OGP (10-14) as an orphan drug for the treatment of chronic idiopathic myelofibrosis (CIMF) disease, and it has been approved. Structure-activity relationship studies have shown that, except for Gly13 and Gly14, the other amino acid side chains are pharmacophores that may affect the activity. On this basis, adjusting the position and number of active sites and constructing cyclic peptides of different configurations can increase the biological activity of the pentapeptide structure or improve its metabolic stability.

At present, the only report on the structural modification of OGP(10-14) is the modification study of straight-chain OGP(10-14) with no amino group at the nitrogen end by Bab et al. Because OGP (10-14) itself has homology and high conservation, only by modifying the original peptide structure, it is possible to obtain safer and more effective compounds.

In the field of osteogenic growth peptide technology, a technical problem that needs to be solved urgently is to provide a compound with biological activity and metabolic stability.

Summary of the invention

An object of the present invention is to provide a new OGP(10-14) pentapeptide derivative, which improves the biological activity, improves the proliferation activity of MC3T3E1 osteoblasts and/or NIH3T3 fibroblasts, and/or improves pepsin and/ Or trypsin stability.

Another object of the present invention is to provide a method for preparing the OGP(10-14) pentapeptide derivative.

Another object of the present invention is to provide a pharmaceutical composition comprising the OGP(10-14) pentapeptide derivative.

Another object of the present invention is to provide the application of the OGP(10-14) pentapeptide derivative and the pharmaceutical composition in the preparation of a medicine for treating and/or preventing bone injury.

Another object of the present invention is to provide the application of the OGP(10-14) pentapeptide derivative in the preparation of medicines for the treatment and/or prevention of osteometastatic diseases.

Another object of the present invention is to provide the application of the OGP(10-14) pentapeptide derivative in the preparation of drugs for the treatment and/or prevention of chronic idiopathic myelofibrosis.

The purpose of the present invention is achieved by the following technical means:

In one aspect, the present invention provides an OGP(10-14) pentapeptide derivative, which includes one or more of the following compounds:

H-Dopa-Gly-Phe-Gly-Gly-OH;

H-D-Tyr-Pro-D-Phe-Gly-Gly-OH;

Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr);

Cyclo(Tyr-Gly-D-Phe-Gly-Gly);

Cyclo(D-Tyr-Gly-Phe-Gly-Gly);

Cyclo(Gly-Gly-D-Phe-Gly-Tyr);

Cyclo (Gly-Gly-Phe-Gly-D-Tyr).

As an active candidate, osteogenic growth peptide has the same characteristics as its polypeptide drugs. On the one hand, it has the advantages of simple structure, significant activity, low immunogenicity and good safety, and has the advantages of new drug development; on the other hand, due to its own conformation, it has the disadvantages of low oral utilization, high enzyme degradation, and short half-life. The main reason for these unstable factors is the conformation of specific amino acids in the structure. For example, the connection sequence of the main chain and the types of side chain residues will affect its biological activity. Since the recognition function of OGP and substrate is not clear, it cannot simulate the receptor for drug screening. Therefore, structural modification and structure-activity relationship study is an effective way to improve its physical and chemical properties and metabolic stability. Among the above-mentioned 7 kinds of OGP (10-14) pentapeptide derivatives provided by the present invention, 2 kinds of linear OGP derivatives and 5 kinds of cyclic OGP derivatives are respectively modified. Studies have found that the seven OGP(10-14) pentapeptide derivatives of the present invention retain their structure compared with unmodified OGP(10-14) pentapeptide and other modified OGP(10-14) pentapeptide derivatives. The original pharmacophore groups (Tyr10, Phe12) of OGP(10-14) have the structural characteristics of improving metabolic stability. The OGP(10-14) pentapeptide derivative of the present invention has undergone cell activity experiments and enzymatic hydrolysis experiments. It is confirmed that it has the biological activity and metabolic stability of OGP (10-14), has stronger in vitro proliferation activity than OGP (10-14), has higher stability to pepsin and trypsin, and can be used in treatment as an active ingredient Or it has a wide range of applications in preventing fracture damage and adjusting bone metabolism imbalance.

On the other hand, the present invention also provides a preparation method of the above-mentioned OGP(10-14) pentapeptide derivative, which includes:

Use Fmoc-solid phase synthesis method to link the peptide chain of OGP(10-14) linear peptide derivatives, or use Fmoc-Tyr-(OAll) as the starting dichloro resin cyclization method for OGP(10-14) Peptide chain connection of cyclic peptide derivatives;

The peptide resin obtained after the peptide chain is connected is cleaved with an aqueous solution of trifluoroacetic acid to obtain a crude peptide;

The crude peptide was purified by reversed-phase high performance liquid chromatography to obtain OGP(10-14) pentapeptide derivative.

It is reported in the prior art to obtain candidate derivatives of OGP(10-14) pentapeptide mainly by shortening the peptide chain length, modifying the amide bond, changing the side chain group, and replacing D-amino acid to form a cyclic pentapeptide (clockwise or counterclockwise). Cycling), etc. to obtain a series of derivatives, cell activity screening, and comparison with the original peptide; linear peptides use Boc solid-phase method, cyclic peptides use solution pseudo-dilution method or dirty resin solid-phase method; due to the role of osteogenic peptides The structure of the protein is not clear, so there is no specific basis for the modification of a certain site. In the present invention, based on the residues shared by OGP(10-14) and daTyr10[OGP(10-14)], all except Gly11 may be close to the optimal pharmacophore group, by comparing the original pentapeptide OGP( 10-14) Directly restructure, the straight chain adopts the Fmoc-solid phase method, and the cyclic peptide adopts the dichloro resin cyclization method starting from Fmoc-Tyr-(OAll); on this basis, it adopts pepsin and trypsin In the degradation test, the seven OGP(10-14) pentapeptide derivatives of the present invention were restructured for the purpose of improving metabolic stability, and the purity of the OGP(10-14) pentapeptide derivatives obtained by the method of the present invention was more than 97 %.

In the above method, preferably, 2-CTC Resin (2-Chlorotrityl Chloride Resin) is used as the solid-phase carrier in the solid-phase synthesis method.

On the other hand, the present invention also provides a pharmaceutical composition, which comprises the above-mentioned OGP(10-14) pentapeptide derivative and a pharmaceutically acceptable carrier.

In the aforementioned pharmaceutical composition, the pharmaceutically acceptable carrier may be a solid excipient or a liquid excipient, for example, an organic or inorganic solid or liquid excipient suitable for gastrointestinal administration. Specifically, it may include stabilizers, wetting agents, solubilizers or other commonly used auxiliary materials and/or additives, such as lactose, talc, cellulose, polyvinylpyrrolidone, starch, pectin, Tween-80, polyvinyl alcohol and the like.

In the above-mentioned pharmaceutical composition, preferably, the existence form of the pharmaceutical composition is a solid form and/or a liquid form; the solid form includes a tablet, a granule, or a capsule; the liquid form includes a suspension, a syrup Agent or emulsion.

The active ingredient OGP (10-14) pentapeptide derivative of the present invention is made into various dosage forms of oral drugs, which are taken orally for adults, and the usual dose is 10 ^-9 to 10 ^-11 mg once a day, taken after meals, and for children. Decrease as appropriate. Indications: treatment and prevention of bone injury, bone metabolism disease, chronic idiopathic myelofibrosis disease.

The present invention also provides the application of the above-mentioned OGP (10-14) pentapeptide derivative or pharmaceutical composition in the preparation of a medicine for treating and/or preventing bone injury.

The present invention also provides the application of the above-mentioned OGP(10-14) pentapeptide derivative or pharmaceutical composition in the preparation of a medicine for the treatment and/or prevention of bone metabolism diseases.

The present invention also provides the application of the above-mentioned OGP(10-14) pentapeptide derivative or pharmaceutical composition in the preparation of a medicine for the treatment and/or prevention of chronic idiopathic myelofibrosis.

The beneficial effects of the present invention:

The OGP(10-14) pentapeptide derivative provided by the present invention retains the original pharmacophore groups (Tyr10, Phe12) of OGP(10-14) in structure, and has the structural characteristics of improving metabolic stability. OGP(10-14) 10-14) The pentapeptide derivative has the biological activity and metabolic stability of OGP (10-14) through cell activity experiments and enzymatic hydrolysis experiments. It has stronger in vitro proliferation activity than OGP (10-14), and is resistant to pepsin. It has higher stability with trypsin; it can be used as an active ingredient in the treatment or prevention of fracture damage and adjustment of bone metabolism imbalance. Combined with solid or liquid adjuvants commonly used in pharmacy, it can be administered intragastrically or parenterally The way of administration is used.

Description of the drawings

Figure 1 is a liquid chromatogram of H-Dopa-Gly-Phe-Gly-Gly-OH synthesized in Example 1.

2 is a mass spectrum of H-Dopa-Gly-Phe-Gly-Gly-OH synthesized in Example 1.

3 is a liquid chromatogram of H-D-Tyr-Pro-D-Phe-Gly-Gly-OH synthesized in Example 2.

4 is a mass spectrum of H-D-Tyr-Pro-D-Phe-Gly-Gly-OH synthesized in Example 2.

Figure 5 is a liquid chromatogram of Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) synthesized in Example 3.

Figure 6 is a mass spectrum of Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) synthesized in Example 3.

Figure 7 is a liquid chromatogram of Cyclo (Tyr-Gly-D-Phe-Gly-Gly) synthesized in Example 4.

Figure 8 is a mass spectrum of Cyclo (Tyr-Gly-D-Phe-Gly-Gly) synthesized in Example 4.

Figure 9 is a liquid chromatogram of Cyclo (D-Tyr-Gly-Phe-Gly-Gly) synthesized in Example 5.

Figure 10 is a mass spectrum of Cyclo (D-Tyr-Gly-Phe-Gly-Gly) synthesized in Example 5.

Figure 11 is a liquid chromatogram of Cyclo (Gly-Gly-D-Phe-Gly-Tyr) synthesized in Example 6.

Figure 12 is a mass spectrum of Cyclo (Gly-Gly-D-Phe-Gly-Tyr) synthesized in Example 6.

Figure 13 is a liquid chromatogram of Cyclo (Gly-Gly-Phe-Gly-D-Tyr) synthesized in Example 7.

14 is a mass spectrum of Cyclo (Gly-Gly-Phe-Gly-D-Tyr) synthesized in Example 7.

Figure 15 is a graph showing the results of the degradation of pepsin by OGP(10-14) pentapeptide derivatives.

Figure 16 is a graph showing the experimental results of trypsin degradation of OGP(10-14) pentapeptide derivatives.

detailed description

In order to have a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are now described in detail below, but they should not be understood as limiting the scope of implementation of the present invention. In the examples, the experimental methods without specifying specific conditions refer to conventional methods and conventional conditions well-known in the art, or operate in accordance with the conditions recommended by the instrument manufacturer.

Example 1

The structural formula of the OGP(10-14) pentapeptide derivative in this example is as follows:

H-Dopa-Gly-Phe-Gly-Gly-OH.

The synthesis method is as follows:

Reaction and detection: 2-CTC Resin (1.0mmol/g) is selected as the solid phase carrier, in the presence of N,N-diisopropylethylamine, first connect Fmoc-Gly-OH, 2-CTC Resin and Fmoc to the resin The molar ratio of -Gly-OH and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Gly-2-CTC Resin, and then connect Fmoc-Gly-OH and Fmoc- to the resin in turn. Phe-OH, Fmoc-Gly-OH, Fmoc-Dopa(tBu)-OH, 0.05g/mL ninhydrin ethanol solution is selected for each step of the reaction to make the positive or negative test pass, 2-CTC Resin respectively The molar ratio with Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Dopa(tBu)-OH is 1:2, 2-CTC Resin and 1-hydroxybenzotriazole, The molar ratio of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate and N,N-diisopropylethylamine are both 1:2:2:2, peptide In the chain connection, use 20% piperidine in N,N-dimethylformamide solution to remove Fmoc, wash 2 times with isopropanol, wash 3 times with N,N-dimethylformamide, and wash with anhydrous methanol After 3 times, the peptide resin was obtained by suction filtration and drying at room temperature.

Cleavage: the peptide resin was cut with 95% trifluoroacetic acid aqueous solution, reacted for 1.5 hours, filtered with suction, the filtrate was then precipitated with cold ether, filtered with suction, and dried under vacuum at room temperature to obtain the crude peptide.

Purification: The crude peptide is purified by reverse-phase high performance liquid chromatography and freeze-dried to obtain the product. Chromatographic conditions: phase A (aqueous solution of trifluoroacetic acid with a concentration of 0.1%), phase B (a solution of trifluoroacetic acid with a concentration of 0.1% in acetonitrile), gradient elution 30 minutes (phase B 10% to 90%), 14.69 minutes, It was freeze-dried to obtain a product with a purity of 94.55%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd: 515.59, found: 516.33 ([M+H], 100%); 425.40 ([M-91], 100%). The liquid chromatogram is shown in Figure 1, and the mass spectrum is shown in Figure 2.

Example 2

H-D-Tyr-Pro-D-Phe-Gly-Gly-OH.

The synthesis method is as follows:

The solid phase carrier is 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-Gly-OH, 2-CTC Resin and Fmoc-Gly-OH to the resin The molar ratio of N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Gly-2-CTC Resin, and then connect Fmoc-Gly-OH and Fmoc-D-Phe- to the resin in turn. OH, Fmoc-Pro-OH, Fmoc-D-Tyr(tBu)-OH, the reaction conditions, detection, cutting, and purification steps are the same as in Example 1, and freeze-dried to obtain the product. Chromatographic conditions: phase A (aqueous solution with a concentration of 0.1% trifluoroacetic acid), phase B (a acetonitrile solution with a concentration of 0.1% trifluoroacetic acid), gradient elution 30 minutes (phase B 10% to 90%), 13.25 minutes, The purity is 99.74%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd:539.65,found:540.37([M+H],100%);541.50([M+H],20%). The liquid chromatogram is shown in Figure 3, and the mass spectrum is shown in Figure 4.

Example 3

Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr).

The synthesis method is as follows:

The solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-D-Tyr-(OAll) to the resin, 2-CTC Resin and Fmoc The molar ratio of -D-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-D-Tyr-(OAll)-2-CTC Resin, and then to Fmoc -D-Tyr-(OAll)-2-CTC Resin connected to Fmoc-Pro-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-D-Tyr-(OAll) -2-CTC Resin and Fmoc-Pro-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, respectively, the molar ratio is 1:2, Fmoc-D-Tyr-(OAll) -2-CTC Resin and 1-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, N,N-diisopropyl ethyl The molar ratio of amine is 1:2:2:2, and the ethanol solution of 0.05g/mL ninhydrin is selected for each step of the reaction to ensure that the positive or negative test is completely passed. In the peptide chain connection, Fmoc is removed with 20% piperidine in N,N-dimethylformamide solution, allyl removal is piperidine with tetrakis(triphenylphosphine)palladium under nitrogen protection and ice salt bath conditions In solution. Combine 1-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, N,N-diisopropylethylamine -The dimethylformamide solution is added to the peptide resin for cyclization, the peptide resin and 1-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoro The molar ratio of boric acid and N,N-diisopropylethylamine is 1:2:2:2. The cutting and purification steps are the same as in Example 1. Chromatographic conditions: phase A (0.1% trifluoroacetic acid in water), phase B (0.1% trifluoroacetic acid in acetonitrile), gradient elution 30 minutes (phase B 10% to 90%), 17.41 minutes, freeze-drying The product has a purity of 87.51%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd: 521.65, found: 522.41 ([M+H], 40%); 544.34 ([M+Na]+, 100%). The liquid chromatogram is shown in Figure 5, and the mass spectrum is shown in Figure 6.

Example 4

Cyclo (Tyr-Gly-D-Phe-Gly-Gly).

The synthesis method is as follows:

The solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-L-Tyr-(OAll) to the resin, where 2-CTC Resin and The molar ratio of Fmoc-L-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Tyr-(OAll)-2-CTC Resin, and then apply them to the resin in turn. Connect Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH, among which Fmoc-Tyr-(OAll)-2-CTC Resin and Fmoc-Gly-OH, Fmoc- The molar ratio of Gly-OH, Fmoc-D-Phe-OH and Fmoc-Gly-OH is 1:2, Fmoc-Tyr-(OAll)-2-CTC Resin and 1-hydroxybenzotriazole, O- The molar ratio of benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate and N,N-diisopropylethylamine is 1:2:2:2, the detection in the reaction The steps of cyclization, cleavage and purification are the same as in Example 3. The chromatographic conditions are the same as in Example 3, 10.42 minutes, and freeze-dried to obtain the product with a purity of 93.87%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd: 481.59, found: 482.35 ([M+H], 60%); 504.31 ([M+Na]+, 45%). The liquid chromatogram is shown in Figure 7, and the mass spectrum is shown in Figure 8.

Example 5

Cyclo (D-Tyr-Gly-Phe-Gly-Gly).

The synthesis method is as follows:

The solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-D-Tyr-(OAll) to the resin, 2-CTC Resin and Fmoc The molar ratio of -D-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-D-Tyr-(OAll)-2-CTC Resin, which are respectively applied to the resin in turn Connect Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH, Fmoc-D-Tyr-(OAll)-2-CTC Resin and Fmoc-Gly-OH, Fmoc respectively -Gly-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH have a molar ratio of 1:2, Fmoc-D-Tyr-(OAll)-2-CTC Resin and 1-hydroxybenzotriazole The molar ratio of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate and N,N-diisopropylethylamine is 1:2:2:2, reaction The steps of detection, cyclization, cleavage, and purification in are the same as those in Example 3. The chromatographic conditions are the same as in Example 3, 10.48 minutes, and freeze-dried to obtain the product with a purity of 91.69%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd: 481.59, found: 482.35 ([M+H], 40%); 504.31 ([M+Na]+, 45%). The liquid chromatogram is shown in Figure 9 and the mass spectrum is shown in Figure 10.

Example 6

Cyclo (Gly-Gly-D-Phe-Gly-Tyr).

The synthesis method is as follows:

The solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, connect Fmoc-L-Tyr-(OAll) to the resin, 2-CTC Resin and Fmoc- The molar ratio of L-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Tyr-(OAll)-2-CTC Resin, and then connect Fmoc- to the resin respectively. Gly-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr-(OAll)-2-CTC Resin and Fmoc-Gly-OH, Fmoc-D-Phe- The molar ratio of OH, Fmoc-Gly-OH and Fmoc-Gly-OH is 1:2, Fmoc-Tyr-(OAll)-2-CTC Resin and 1-hydroxybenzotriazole, O-benzotriazole The molar ratio of azole-N,N,N',N'-tetramethylurea tetrafluoroborate and N,N-diisopropylethylamine is 1:2:2:2. The detection, cyclization, The cutting and purification steps are the same as in Example 3. The chromatographic conditions are the same as in Example 3, 11.82 minutes, and freeze-dried to obtain the product with a purity of 96.77%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd:481.59,found:484.70([M+3H],100%). The liquid chromatogram is shown in Figure 11, and the mass spectrum is shown in Figure 12.

Example 7

Cyclo (Gly-Gly-Phe-Gly-D-Tyr).

The synthesis method is as follows:

The solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, connect Fmoc-D-Tyr-(OAll) to the resin, where 2-CTC Resin and Fmoc The molar ratio of -D-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-D-Tyr-(OAll)-2-CTC Resin, and then apply them to the resin respectively. Connect Fmoc-Gly-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-D-Tyr-(OAll)-2-CTC Resin and Fmoc-Gly-OH, Fmoc respectively -The molar ratio of L-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH is 1:2, Fmoc-D-Tyr-(OAll)-2-CTC Resin and 1-hydroxybenzotriazole The molar ratio of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate and N,N-diisopropylethylamine is 1:2:2:2, connected The reaction and detection, cyclization, cleavage, and purification steps are the same as those in Example 3. The chromatographic conditions were the same as in Example 3, 12.58 minutes, freeze-dried, and the product was obtained with a purity of 94.90%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd: 481.59, found: 482.35 ([M+H], 100%); 504.28 ([M+Na]+, 60%). The liquid chromatogram is shown in Figure 13, and the mass spectrum is shown in Figure 14.

Example 8

Taking the production of 1000 tablets of the pharmaceutical tablet product of the present invention as an example, the raw materials, auxiliary materials and their ratios used are:

Take the H-Dopa-Gly-Phe-Gly-Gly-OH 1000×10 ^-11 mg synthesized in Example 1, add starch to 300 g, and proceed according to the preparation method of conventional tablets in pharmacy. Each weighing 0.3g, each containing a pharmaceutically effective ingredient 10 ^-11 mg. Take 1 tablet once a day after breakfast.

Example 9

^{Take 1000×10 -11} mg of HD-Tyr-Pro-D-Phe-Gly-Gly-OH synthesized in Example 2, add starch to 300 g, and proceed according to the preparation method of conventional tablets in pharmacy. Each weighing 0.3g, each containing a pharmaceutically effective ingredient 10 ^-11 mg. Take 1 tablet once a day after breakfast.

Example 10

^{Take 1000×10 -11} mg of Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) synthesized in Example 3, add starch to 300 g, and proceed according to the preparation method of conventional tablets in pharmacy. Each weighing 0.3g, each containing a pharmaceutically effective ingredient 10 ^-11 mg. Take 1 tablet once a day after breakfast.

Example 11

^{Take 1000×10 -11} mg of Cyclo (Tyr-Gly-D-Phe-Gly-Gly) synthesized in Example 4, add starch to 300 g, and proceed according to the preparation method of conventional tablets in pharmacy. Each weighing 0.3g, each containing a pharmaceutically effective ingredient 10 ^-11 mg. Take 1 tablet once a day after breakfast.

Example 12

^{Take 1000×10 -11} mg of Cyclo (D-Tyr-Gly-Phe-Gly-Gly) synthesized in Example 5, add starch to 300 g, and proceed according to the preparation method of conventional pharmaceutics tablets. Each weighing 0.3g, each containing a pharmaceutically effective ingredient 10 ^-11 mg. Take 1 tablet once a day after breakfast.

Example 13

^{Take 1000×10 -11} mg of Cyclo (Gly-Gly-D-Phe-Gly-Tyr) synthesized in Example 6, add starch to 300 g, and proceed according to the preparation method of conventional tablets in pharmacy. Each weighing 0.3g, each containing a pharmaceutically effective ingredient 10 ^-11 mg. Take 1 tablet once a day after breakfast.

Example 14

^{Take 1000×10 -11} mg of Cyclo (Gly-Gly-Phe-Gly-D-Tyr) synthesized in Example 7, add starch to 300 g, and proceed according to the preparation method of conventional tablets in pharmacy. Each weighing 0.3g, each containing a pharmaceutically effective ingredient 10 ^-11 mg. Take 1 tablet once a day after breakfast.

Test experiment

In order to verify the beneficial effects of the present invention, the OGP (10-14) pentapeptide derivatives synthesized in Examples 1 to 7 of the present invention were used for cell viability experiments and enzymatic hydrolysis experiments. The various experimental conditions are as follows:

1. Cell viability experiment

(1) Prepare sample solution

Prepare cell culture fluid: mix α-MEM liquid medium, inactivated fetal bovine serum, and penicillin streptomycin mixture in a volume ratio of 89%: 10%: 1%, and seal them to prepare cell culture fluid. Store at 4°C for later use. Separately prepare the same culture medium without serum for the activity test.

Prepare a phosphate buffer solution with a concentration of 5mg/mL according to the conventional method, adjust the pH value to 7.2-7.4, autoclave and autoclave, seal in aliquots, and store at 4°C for use.

Prepare thiazole blue (MTT) solution: weigh 50.0 mg of thiazole blue, dissolve it in 10 mL of phosphate buffer, stir with a magnetic stirrer until it is completely dissolved, filter and sterilize with a 0.22 μm microporous membrane, and dispense into 1.5 mL centrifuge tubes. The aluminum box is sealed with paper and stored at –20°C and protected from light.

Sample test solution: Take the lyophilized samples of OGP(10-14) pentapeptide derivatives of 7 examples, each 0.01mmol, respectively, and dissolve them in 1mL dimethyl sulfoxide to obtain 0.01mmol/mL sample solution, and dilute them separately Into ^{two concentrations of 10 -7} mol/L and 10 ^-9 mol/L.

(2) Cell culture

Cell resuscitation: Take frozen osteoblasts (MC3T3E1) and fibroblasts (NIH3T3) separately, and thaw them in a 37°C water bath for 1 to 2 minutes. Suction the cells to a centrifuge tube at 1500 rpm and centrifuge for 4 minutes. The supernatant, use the preheated cell culture fluid to blow the cells evenly, place them in a 37°C, 5% CO ₂ incubator for 24 hours, and check by microscopy. If most of the cells have adhered to the wall and are in normal shape, 2～3 Change the cell culture medium once a day.

Passage of cells: Passage when the cells grow to 80%-90% confluent state. Passaging steps: Wash twice with 5mL phosphate buffer solution, add 1mL of 0.25% trypsin, observe under the microscope, most of the cells shrink in 2～3min, add 2mL cell culture medium to blow down the adherent cells, centrifuge for 1500 Centrifuge at rpm for 4 minutes to remove trypsin. Use the preheated cell culture fluid to blow the cells evenly, divide the cells into three, and place them in a 37°C, 5% CO ₂ incubator for 24 hours.

(3) Activity test

Collect the cells in the exponential growth phase, wash them twice with phosphate buffer, digest the cells with 0.25% sodium ethylenediaminetetraacetate trypsin, use MEM medium to repeatedly pipette to obtain a cell suspension, adjust the cell density 2×10 ⁷ cells/L～5×10 ⁷ cells/L, seeded on 96-well culture plate, so that the cell density is 5000 cells/L/well～10000 cells/L/well, after 24 hours, replace without serum Fresh MEM medium (198μL/well), and at the same time, add 2μL of the sample test solution of the 7 examples in each well of the experimental group. The concentration of each sample test solution is 10 ^-9 mol/L and 10 ^-11 mol/L. . Among them, the best test concentration in osteoblasts is 10 ^-11 mol/L, and the best test concentration in fibroblasts is 10 ^-9 mol/L. Set negative control wells and blank zero adjustment holes. The negative control well is the culture medium containing 2μL of dimethyl sulfoxide; the blank zero adjustment hole is the culture medium without osteoblasts and fibroblasts, and each test solution group is set 3 parallel holes. After adding the sample, continue to incubate for 24 hours. Replace with fresh serum-free medium (180μL/well), add 20μL/well of thiazole blue solution, incubate for 4 hours, aspirate the supernatant with a pipette, add 150μL dimethyl sulfoxide to each well, shake at 37°C for 10 minutes The chan crystals are fully dissolved. The blank hole at the wavelength of 490nm was adjusted to zero with an enzyme-linked immunoassay calibrator, and the absorbance A of each hole was measured. The absorbance A value of the positive control group was compared to evaluate the proliferation activity.

An increase rate of less than 1 indicates lower activity than OGP (10-14), and an increase rate greater than 1 indicates higher activity than OGP (10-14). The experiment was repeated 3 times. The data was statistically analyzed using SPPS 13.0 software according to the instructions of the software, all expressed as x±s, and the comparison between groups was performed by t test.

Cell proliferation rate = average value of experimental group/average value of positive control group

The test results are shown in Table 1.

Table 1 Proliferation activity of the peptides of Examples 1-7 in osteoblasts and fibroblast cell lines (

n=3)

The experimental results were processed by statistics, *P<0.05, **P<0.01. It can be seen from Table 1 that the cell proliferation activity of the above compounds is equivalent to or better than that of OGP (10-14), and the intensity of action in the two cell lines is correlated. These compounds retain the original pharmacophore groups (Tyr10, Phe12) of the pentapeptide in structure, and have structures (D-configuration, cyclization) that improve metabolic stability.

2. Enzymatic hydrolysis experiment

(1) Prepare sample solution

Prepare the OGP (10-14) pentapeptide derivative solutions of 7 examples: weigh 5 mg of the OGP (10-14) pentapeptide derivative lyophilized powder of 7 examples, add water to dissolve it, and make a volumetric flask with a constant volume of 5 mL. 7 kinds of aqueous solutions with a concentration of 1mg/mL.

Prepare pepsin solution: Weigh 0.2g of sodium chloride, 0.32g of pepsin, add 50mL of water to dissolve, then add 0.7mL of concentrated hydrochloric acid, dilute to 100mL with water, adjust pH to 1.2, and prepare pepsin with a concentration of 3.2mg/mL Solution.

Prepare trypsin solution: weigh _{0.68g of KH 2} PO ₄ , add 25mL of water to dissolve; add 19mL of 0.2mol/L NaOH aqueous solution and 40mL of water, 1.0g trypsin, mix the solution, and adjust with 0.2mol/L NaOH solution The pH is 7.5±0.1, the volume of water is 100mL, and the trypsin solution with a concentration of 10mg/mL is prepared.

(2) Experimental method

Pepsin degradation sample: Take a 5 mL centrifuge tube, add 1.5 mL of pepsin solution, keep at 37°C for 10 minutes, add 1.5 mL of the peptide sample solution of Examples 1-7, and count the time sequentially at 2 minutes, 4 minutes, 8 minutes, At 16 minutes, 30 minutes, 60 minutes, and 120 minutes, take 200μL of 7 kinds of peptide sample reaction solutions, add 50μL 0.618mol/L Na ₂ CO ₃ solution to terminate the enzymatic hydrolysis reaction, prepare the sample to be tested, and detect with high performance liquid chromatograph .

Prepare trypsin degradation samples: Take a 5mL centrifuge tube, add 1.5mL trypsin solution, keep at 37°C for 10 minutes, add 1.5mL of the 7 peptide sample reaction solutions, and count them in sequence at 2 minutes, 4 minutes, 8 minutes, and 16 minutes. , 30 minutes, 60 minutes, and 120 minutes, take 200 μL of 7 peptide sample reaction solutions, add 50 μL of 30% acetic acid solution to terminate the enzymatic hydrolysis reaction, prepare the sample to be tested, and detect it with high performance liquid chromatography.

(3) Testing conditions

Seven kinds of samples to be tested are separated and detected by Hitachi L-2455 automatic analysis liquid chromatograph, the relevant parameters are as follows:

Mobile phase A: an aqueous solution with a concentration of 0.1% trifluoroacetic acid; mobile phase B: an acetonitrile solution with a concentration of 0.1% trifluoroacetic acid; wavelength: 215 nm; flow rate 1 mL/min.

H-Dopa-Gly-Phe-Gly-Gly-OH, H-D-Tyr-Pro-D-Phe-Gly-Gly-OH detection and analysis program: 16% constant flow, running time 10 minutes.

Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) detection and analysis program: 30% constant current, running time 10 minutes.

Cyclo(Tyr-Gly-D-Phe-Gly-Gly), Cyclo(D-Tyr-Gly-Phe-Gly-Gly), Cyclo(Gly-Gly-D-Phe-Gly-Tyr), Cyclo(Gly-Gly) -Phe-Gly-D-Tyr) detection and analysis program: 20% constant current, running time 10 minutes.

(4) Test results

The test results are shown in Figure 15 and Figure 16.

It can be seen from Figures 15 and 16, that through the in vitro activity screening test of OGP(10-14) and its 7 examples of OGP(10-14) pentapeptide derivatives, MC3T3E1 osteoblasts and NIH3T3 fibroblasts were used as tests For cell lines, the activity of OGP (10-14) was used as a positive control to evaluate the effect of OGP (10-14) pentapeptide derivatives on cell proliferation activity. The results show that the cell activity of the OGP (10-14) pentapeptide derivatives of the 7 examples retains or even exceeds the activity of the original linear peptide OGP (10-14). Through enzymatic hydrolysis experiments on OGP(10-14) and the OGP(10-14) pentapeptide derivatives of 7 examples, pepsin and trypsin were used as enzymolysis conditions, and OGP(10-14) The enzymatic hydrolysis reaction is a blank control to evaluate the metabolic stability of each derivative in the two enzymes. The results show that, with the exception of H-Dopa-Gly-Phe-Gly-Gly-OH compared to OGP (10-14), the metabolic stability is generally similar, and there is no significant difference, the other 6 examples of OGP (10-14) five The overall metabolic stability of peptide derivatives is better than that of OGP (10-14).

The above results indicate that the OGP(10-14) pentapeptide derivatives of the 7 examples retain the original pharmacophore groups (Tyr10, Phe12) of OGP(10-14) in structure, and have a structure that improves metabolic stability. It has the biological activity and metabolic stability of OGP (10-14). It is used as an active ingredient in the treatment or prevention of fracture damage and adjustment of bone metabolism imbalance, combined with solid or liquid adjuvants commonly used in pharmaceutics, and used in gastrointestinal It is used by way of administration or parenteral administration.

Claims

An OGP(10-14) pentapeptide derivative, which includes one or more of the following compounds:

H-Dopa-Gly-Phe-Gly-Gly-OH;

H-D-Tyr-Pro-D-Phe-Gly-Gly-OH;

Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr);

Cyclo(Tyr-Gly-D-Phe-Gly-Gly);

Cyclo(D-Tyr-Gly-Phe-Gly-Gly);

Cyclo(Gly-Gly-D-Phe-Gly-Tyr);

Cyclo (Gly-Gly-Phe-Gly-D-Tyr).
The preparation method of the OGP(10-14) pentapeptide derivative of claim 1, which comprises:

Use Fmoc-solid phase synthesis method to link the peptide chain of OGP(10-14) linear peptide derivatives, or use Fmoc-Tyr-(OAll) as the starting dichloro resin cyclization method for OGP(10-14) Peptide chain connection of cyclic peptide derivatives;

The peptide resin obtained after the peptide chain is connected is cleaved with an aqueous solution of trifluoroacetic acid to obtain a crude peptide;

The crude peptide was purified by reversed-phase high performance liquid chromatography to obtain OGP(10-14) pentapeptide derivative.
The preparation method according to claim 2, wherein 2-CTC Resin is used as the solid phase carrier in the solid phase synthesis method.
A pharmaceutical composition comprising the OGP (10-14) pentapeptide derivative of claim 1 and a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable carrier is a solid excipient or a liquid excipient.
Use of the OGP(10-14) pentapeptide derivative of claim 1 or the pharmaceutical composition of any one of claims 4-5 in the preparation of a medicine for treating and/or preventing bone injury.
Use of the OGP(10-14) pentapeptide derivative of claim 1 or the pharmaceutical composition of any one of claims 4-5 in the preparation of a medicine for the treatment and/or prevention of bone metabolism diseases.
Use of the OGP(10-14) pentapeptide derivative of claim 1 or the pharmaceutical composition of any one of claims 4-5 in the preparation of a medicament for the treatment and/or prevention of chronic idiopathic myelofibrosis .
The use according to any one of claims 6-8, wherein the medicine is a tablet, granule, capsule, suspension, syrup or emulsion.
The use according to any one of claims 6-9, wherein the OGP(10-14) pentapeptide derivative of claim 1 is used in the preparation at a dose of 10 -9 to 10 -11 mg/day/time drug.
A method for treating bone injury, which comprises administering to a subject an effective amount of the OGP(10-14) pentapeptide derivative of claim 1 or the pharmaceutical composition of any one of claims 4-5.
A method for treating bone metabolism diseases, the method comprising administering to a subject an effective amount of the OGP(10-14) pentapeptide derivative of claim 1 or the pharmaceutical composition of any one of claims 4-5 .
A method for treating chronic idiopathic myelofibrosis, the method comprising administering to a subject an effective amount of the OGP(10-14) pentapeptide derivative of claim 1 or any one of claims 4-5 Pharmaceutical composition.