CN112789286A

CN112789286A - Osteogenic growth peptide carbon-terminal pentapeptide derivative and preparation method and application thereof

Info

Publication number: CN112789286A
Application number: CN202080004195.0A
Authority: CN
Inventors: 张腾; 张忠旗; 郭添; 刘少军; 李晨召; 李乾; 王昕�; 赵金礼
Original assignee: Shaanxi HuiKang Bio Tech Co Ltd
Current assignee: Shaanxi HuiKang Bio Tech Co Ltd
Priority date: 2019-09-06
Filing date: 2020-04-17
Publication date: 2021-05-11
Anticipated expiration: 2040-04-17
Also published as: WO2021042732A1; CN112789286B

Abstract

The invention provides an osteogenic growth peptide carbon-terminal pentapeptide derivative and a preparation method and application thereof. The osteogenic growth peptide carbon-terminal pentapeptide derivative comprises: 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-Gly-D-Tyr). The structure of the pentapeptide retains the pharmacodynamic groups Tyr10 and Phe12 of the original pentapeptide, and has the structural characteristic 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 polypeptide, and particularly relates to an osteogenic growth peptide carbon-terminal pentapeptide OGP (10-14) derivative, and a preparation method and application thereof.

Background

Osteogenic Growth Peptide (Osteogenic Growth Peptide) is a polypeptide Growth factor, has effects of promoting osteogenesis and stimulating hematopoiesis, and has a primary structure of ALKRQGRTLYGFGG. Wherein, the carbon-terminal pentapeptide OGP (10-14) is the minimum segment which keeps the whole activity of the osteogenic growth peptide, and the amino acid sequence is H-Tyr10-Gly11-Phe12-Gly13-Gly 14-OH. A large number of experiments show that OGP (10-14) not only has the effect of improving and treating bone injury and bone metabolism diseases, but also has the effect of recovering the hematopoietic function of patients with radiotherapy and chemotherapy as well as bone marrow transplantation. Abiogen, Italy, has reportedly filed applications for OGP (10-14) as an orphan drug for the treatment of Chronic Idiopathic Myelofibrosis (CIMF) diseases with the European drug administration (EMEA), and has passed approval. The structure-activity relationship research shows that the side chains of the other amino acids except Gly13 and Gly14 are all pharmacophores which can influence the activity. On the basis, the position and the number of active sites are regulated, and cyclic peptides with different configurations are constructed, so that the biological activity of the pentapeptide structure can be improved or the metabolic stability of the pentapeptide structure can be improved.

The only report on the structural modification of OGP (10-14) at present is the modification study of the nitrogen terminal straight-chain OGP (10-14) by Bab et al. Because OGP (10-14) has homology and high conservation, only by modifying the structure of the propeptide can a safer and more effective compound be obtained.

In the technical field of osteogenic growth peptides, one technical problem to be urgently solved at present is to provide a compound with biological activity and metabolic stability.

Disclosure of Invention

An object of the present invention is to provide a novel OGP (10-14) pentapeptide derivative which increases biological activity, increases the proliferative 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 derivatives.

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

The invention also aims to provide application of the OGP (10-14) pentapeptide derivative and the pharmaceutical composition in preparing medicines for treating and/or preventing bone injury.

The invention also aims to provide application of the OGP (10-14) pentapeptide derivative in preparing a medicament for treating and/or preventing bone metabolism diseases.

The invention also aims to provide application of the OGP (10-14) pentapeptide derivative in preparing a medicament for treating and/or preventing chronic idiopathic myelofibrosis diseases.

The purpose of the invention is realized by the following technical means:

in one aspect, the present invention provides an OGP (10-14) pentapeptide derivative comprising 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 osteogenic growth peptide as an active candidate has the same characteristics as the polypeptide drug. On one hand, the compound has the advantages of simple structure, remarkable activity, low immunogenicity and good safety, and has the advantages of new drug development; on the other hand, the oral administration utilization rate is low, the enzyme degradation is high, and the half-life period is short due to the self conformation constraint. The main reason for these instability factors is the conformation of the particular amino acids in the structure, for example, the order of attachment to the backbone and the type of side chain residues, which affect their biological activity. Since the recognition effect of OGP and a substrate is not clear and the OGP cannot simulate a receptor to carry out drug screening, the research on structural modification and structure-activity relationship is an effective way for improving the physicochemical property and metabolic stability of the OGP. Among the 7 OGP (10-14) pentapeptide derivatives provided by the invention, 2 modified linear OGP derivatives and 5 cyclic OGP derivatives are respectively provided. The research finds that the 7 OGP (10-14) pentapeptide derivatives have the structural characteristics of improving the metabolic stability compared with unmodified OGP (10-14) pentapeptide and other modified OGP (10-14) pentapeptide derivatives, retain the original pharmacodynamic groups (Tyr10 and Phe12) of OGP (10-14), and have the structural characteristics of improving the metabolic stability, and the OGP (10-14) pentapeptide derivatives have the biological activity and the metabolic stability of OGP (10-14) proved by cell activity experiments and enzymolysis experiments, have stronger in vitro proliferation activity than OGP (10-14), have higher stability to pepsin and trypsin, and can be widely applied to the aspects of treating or preventing fracture injury and regulating bone metabolism imbalance as active ingredients.

In another aspect, the present invention also provides a method for preparing the OGP (10-14) pentapeptide derivative, which comprises:

carrying out peptide chain connection of OGP (10-14) straight-chain peptide derivatives by adopting an Fmoc-solid phase synthesis method, or carrying out peptide chain connection of OGP (10-14) cyclic peptide derivatives by adopting a dichloro resin cyclization method initiated by Fmoc-Tyr- (OAll);

cutting the peptide resin obtained after peptide chain connection by using trifluoroacetic acid aqueous solution to obtain crude peptide;

and purifying the crude peptide by reverse phase high performance liquid chromatography to obtain the OGP (10-14) pentapeptide derivative.

The prior art reports that candidate derivatives of OGP (10-14) pentapeptide are obtained by mainly shortening the length of a peptide chain, modifying an amido bond, changing a side chain group, replacing D-amino acid, forming cyclic pentapeptide (forming a ring clockwise or anticlockwise), and the like to obtain a series of derivatives, performing cell activity screening, and comparing the derivatives with original peptide; the straight-chain peptide adopts a Boc solid-phase method, and the cyclic peptide adopts a solution pseudo-dilution method or a dirty resin solid-phase method; since the structure of the substrate protein for the action of the osteogenic peptide is not clear, there is no specific modification basis for a certain site. In the invention, according to the common residues of OGP (10-14) and DATyr10[ OGP (10-14) ], except Gly11, the original pentapeptide OGP (10-14) can be directly modified to be close to the optimal pharmacodynamic group, the straight chain adopts Fmoc-solid phase method, and the cyclic peptide adopts dichloro resin cyclization method which takes Fmoc-Tyr- (OAll) as the starting point; on the basis, 7 OGP (10-14) pentapeptide derivatives of the invention are obtained by reconstruction aiming at improving metabolic stability through degradation tests of pepsin and trypsin, and the purity of the OGP (10-14) pentapeptide derivatives obtained by the method of the invention is more than 97%.

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

In another aspect, the invention also provides a pharmaceutical composition, which comprises the OGP (10-14) pentapeptide derivative and a pharmaceutically acceptable carrier.

In the above-mentioned pharmaceutical composition, the pharmaceutically acceptable carrier may be a solid excipient or a liquid excipient, and may be, for example, an organic or inorganic solid or liquid excipient suitable for gastrointestinal administration. The composition may specifically comprise stabilizer, wetting agent, solubilizer or other common adjuvants and/or additives, such as lactose, pulvis Talci, cellulose, polyvinylpyrrolidone, starch, pectin, Tween-80, polyvinyl alcohol, etc.

In the above pharmaceutical composition, preferably, the pharmaceutical composition is present in a solid form and/or a liquid form; the solid form comprises a tablet, a granule or a capsule; the liquid form includes a suspension, syrup or emulsion.

The active ingredient OGP (10-14) pentapeptide derivative of the invention is prepared into oral drugs of various dosage forms, and the oral drugs are orally taken by adults, and the common dosage is 10 times^-9～10 ^-11mg, 1 time a day, taken after meals, and the dosage of children is reduced as appropriate. Indications are as follows: treating and preventing bone injury, bone metabolism diseases, and chronic idiopathic myelofibrosis diseases.

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

The invention also provides application of the OGP (10-14) pentapeptide derivative or the pharmaceutical composition in preparing medicines for treating and/or preventing bone metabolic diseases.

The invention also provides application of the OGP (10-14) pentapeptide derivative or the pharmaceutical composition in preparing a medicament for treating and/or preventing chronic idiopathic myelofibrosis diseases.

The invention has the beneficial effects that:

the OGP (10-14) pentapeptide derivative provided by the invention structurally reserves the original pharmacodynamic groups (Tyr10 and Phe12) of OGP (10-14), has the structural characteristic of improving metabolic stability, has the biological activity and metabolic stability of OGP (10-14) through cell activity experiments and enzymolysis experiments, has stronger in vitro proliferation activity than OGP (10-14), and has higher stability to pepsin and trypsin; can be used as active ingredient for treating or preventing fracture injury and regulating bone metabolism imbalance, and can be combined with solid or liquid adjuvant commonly used in pharmacy to be administered in a mode of gastrointestinal administration or parenteral administration.

Drawings

FIG. 1 is a liquid chromatogram of H-Dopa-Gly-Phe-Gly-Gly-OH synthesized in example 1.

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

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

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

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

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

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

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

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

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

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

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

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

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

FIG. 15 is a graph showing the results of experiments on pepsin degradation by the OGP (10-14) pentapeptide derivative.

FIG. 16 is a graph showing the results of experiments on trypsin degradation by OGP (10-14) pentapeptide derivatives.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention. In the examples, the experimental methods without specifying the specific conditions were carried out by referring to conventional methods and conventional conditions well known in the art or according to the conditions recommended by the instrument manufacturer.

Example 1

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

H-Dopa-Gly-Phe-Gly-Gly-OH。

the synthesis method comprises the following steps:

reaction and detection: the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), Fmoc-Gly-OH is firstly connected to the Resin in the presence of N, N-diisopropylethylamine, and the molar ratio of the 2-CTC Resin to the Fmoc-Gly-OH to the N, N-diisopropylethylamine is 1: 1: and 4, obtaining Fmoc-Gly-2-CTC Resin, sequentially and respectively connecting Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH and Fmoc-Dopa (tBu) -OH to the Resin, detecting by using 0.05g/mL ninhydrin ethanol solution in each reaction step, and enabling the positive detection or the negative detection to pass, wherein the molar ratio of the 2-CTC Resin to the Fmoc-Gly-OH, the Fmoc-Phe-OH, the Fmoc-Gly-OH and the Fmoc-Dopa (tBu) -OH is 1: the molar ratio of 2, 2-CTC Resin to 1-hydroxy benzotriazole, O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1: 2: 2: 2, removing Fmoc by using an N, N-dimethylformamide solution with the concentration of 20% piperidine in peptide chain connection, washing for 2 times by using isopropanol, washing for 3 times by using N, N-dimethylformamide, washing for 3 times by using anhydrous methanol, and performing suction filtration and drying at normal temperature to obtain the peptide resin.

Cutting: the peptide resin is cut by 95 percent trifluoroacetic acid aqueous solution, the reaction is carried out for 1.5 hours, the filtration is carried out, the filtrate is separated out by cold ether to precipitate, the filtration is carried out, and the vacuum drying is carried out at normal temperature to obtain crude peptide.

And (3) purification: and purifying the crude peptide by reverse phase high performance liquid chromatography, and freeze-drying to obtain the product. Chromatographic conditions are as follows: phase A (trifluoroacetic acid aqueous solution with the concentration of 0.1 percent) and phase B (trifluoroacetic acid acetonitrile solution with the concentration of 0.1 percent) are subjected to gradient elution for 30 minutes (phase B is 10 to 90 percent) and 14.69 minutes, and are subjected to freeze drying to obtain the product with the purity of 94.55 percent. Its structure is characterized by electrospray ionization mass spectrum, [ M ] calcd:515.59, found:516.33([ M + H ], 100%); 425.40([ M-91], 100%). The liquid chromatogram is shown in FIG. 1, and the mass spectrogram is shown in FIG. 2.

Example 2

H-D-Tyr-Pro-D-Phe-Gly-Gly-OH。

the synthesis method comprises the following steps:

the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), Fmoc-Gly-OH is firstly connected to the Resin in the presence of N, N-diisopropylethylamine, and the molar ratio of the 2-CTC Resin to the Fmoc-Gly-OH to the N, N-diisopropylethylamine is 1: 1: and 4, obtaining Fmoc-Gly-2-CTC Resin, then sequentially and respectively connecting Fmoc-Gly-OH, Fmoc-D-Phe-OH, Fmoc-Pro-OH and Fmoc-D-Tyr (tBu) -OH to the Resin, performing the same reaction conditions, detection, cutting and purification steps as those in the example 1, and freeze-drying to obtain the product. Chromatographic conditions are as follows: phase A (0.1% trifluoroacetic acid in water) and phase B (0.1% trifluoroacetic acid in acetonitrile) were gradient eluted for 30 min (phase B10% -90%), 13.25 min and 99.74% pure. Its structure is characterized by electrospray ionization mass spectrum, [ M ] calcd:539.65, found:540.37([ M + H ], 100%); 541.50([ M + H ], 20%). The liquid chromatogram is shown in FIG. 3, and the mass spectrogram is shown in FIG. 4.

Example 3

Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)。

the synthesis method comprises the following steps:

the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), Fmoc-D-Tyr- (OAll) is firstly connected to the Resin in the presence of N, N-diisopropylethylamine, and the molar ratio of the 2-CTC Resin to the Fmoc-D-Tyr- (OAll) to the N, N-diisopropylethylamine is 1: 1: and 4, obtaining Fmoc-D-Tyr- (OAll) -2-CTC Resin, then sequentially and respectively connecting Fmoc-Pro-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH and Fmoc-D-Tyr- (OAll) -2-CTC Resin with the Fmoc-Pro-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH and Fmoc-Gly-OH in a molar ratio of 1: the molar ratio of 2, Fmoc-D-Tyr- (OAll) -2-CTC Resin to 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate and N, N-diisopropylethylamine is 1: 2: 2: and 2, detecting each reaction by using 0.05g/mL ninhydrin in ethanol to ensure that a positive detection or a negative detection is completely passed. Fmoc removal in peptide ligation was performed with 20% piperidine in N, N-dimethylformamide and allyl removal was performed with tetrakis (triphenylphosphine) palladium in piperidine under nitrogen and ice salt bath conditions. Adding N, N-dimethylformamide solution of 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine into peptide resin for cyclization, wherein the molar ratio of the peptide resin to the 1-hydroxybenzotriazole, the O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and the N, N-diisopropylethylamine is 1: 2: 2: 2. the cleavage and purification steps were the same as in example 1. Chromatographic conditions are as follows: phase A (0.1% trifluoroacetic acid water solution) and phase B (0.1% trifluoroacetic acid acetonitrile solution), gradient eluting for 30 minutes (phase B10% -90%), 17.41 minutes, and freeze-drying to obtain the product with purity of 87.51%. Its structure is characterized by electrospray ionization mass spectrum, [ M ] calcd:521.65, found:522.41([ M + H ], 40%); 544.34([ M + Na ] +, 100%). The liquid chromatogram is shown in FIG. 5, and the mass spectrogram is shown in FIG. 6.

Example 4

Cyclo(Tyr-Gly-D-Phe-Gly-Gly)。

the synthesis method comprises the following steps:

the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), Fmoc-L-Tyr- (OAll) is firstly connected to the Resin in the presence of N, N-diisopropylethylamine, wherein the molar ratio of the 2-CTC Resin to the Fmoc-L-Tyr- (OAll) to the N, N-diisopropylethylamine is 1: 1: and 4, obtaining Fmoc-Tyr- (OAll) -2-CTC Resin, and then sequentially and respectively connecting Fmoc-Gly-OH, Fmoc-D-Phe-OH and Fmoc-Gly-OH to the Resin, wherein the molar ratios of the Fmoc-Tyr- (OAll) -2-CTC Resin to the Fmoc-Gly-OH, the Fmoc-D-Phe-OH and the Fmoc-Gly-OH are respectively 1: the molar ratio of 2, Fmoc-Tyr- (OAll) -2-CTC Resin to 1-hydroxy benzotriazole, O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1: 2: 2: the detection, cyclization, cleavage and purification steps in the reaction were the same as in example 3. The chromatographic conditions were the same as in example 3, 10.42 minutes, and lyophilization gave a product with a purity of 93.87%. Its structure is characterized by electrospray ionization mass spectrum, [ M ] calcd:481.59, found:482.35([ M + H ], 60%); 504.31([ M + Na ] +, 45%). The liquid chromatogram is shown in FIG. 7, and the mass spectrogram is shown in FIG. 8.

Example 5

Cyclo(D-Tyr-Gly-Phe-Gly-Gly)。

the synthesis method comprises the following steps:

the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), Fmoc-D-Tyr- (OAll) is firstly connected to the Resin in the presence of N, N-diisopropylethylamine, and the molar ratio of the 2-CTC Resin to the Fmoc-D-Tyr- (OAll) to the N, N-diisopropylethylamine is 1: 1: and 4, obtaining Fmoc-D-Tyr- (OAll) -2-CTC Resin, sequentially and respectively connecting Fmoc-Gly-OH, Fmoc-L-Phe-OH and Fmoc-Gly-OH to the Resin, wherein the mol ratios of the Fmoc-D-Tyr- (OAll) -2-CTC Resin to Fmoc-Gly-OH, Fmoc-L-Phe-OH and Fmoc-Gly-OH are respectively 1: the molar ratio of 2, Fmoc-D-Tyr- (OAll) -2-CTC Resin to 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate and N, N-diisopropylethylamine is 1: 2: 2: the detection, cyclization, cleavage and purification steps in the reaction were the same as in example 3. The chromatographic conditions were the same as in example 3, 10.48 minutes, and lyophilization gave a product with a purity of 91.69%. Its structure is characterized by electrospray ionization mass spectrum, [ M ] calcd:481.59, found:482.35([ M + H ], 40%); 504.31([ M + Na ] +, 45%). The liquid chromatogram is shown in FIG. 9, and the mass spectrogram is shown in FIG. 10.

Example 6

Cyclo(Gly-Gly-D-Phe-Gly-Tyr)。

the synthesis method comprises the following steps:

the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), Fmoc-L-Tyr- (OAll) is connected to the Resin in the presence of N, N-diisopropylethylamine, and the molar ratio of the 2-CTC Resin to the Fmoc-L-Tyr- (OAll) to the N, N-diisopropylethylamine is 1: 1: and 4, obtaining Fmoc-Tyr- (OAll) -2-CTC Resin, sequentially and respectively connecting Fmoc-Gly-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH and Fmoc-Gly-OH to the Resin, wherein the mol ratios of the Fmoc-Tyr- (OAll) -2-CTC Resin to the Fmoc-Gly-OH, the Fmoc-D-Phe-OH, the Fmoc-Gly-OH and the Fmoc-Gly-OH are respectively 1: the molar ratio of 2, Fmoc-Tyr- (OAll) -2-CTC Resin to 1-hydroxy benzotriazole, O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1: 2: 2: 2, the detection, cyclization, cleavage and purification steps in ligation were the same as in example 3. The chromatographic conditions were the same as in example 3, 11.82 minutes, and lyophilization gave a product with a purity of 96.77%. Its structure is characterized by electrospray ionization mass spectrometry, [ M ] calcd:481.59, found:484.70([ M +3H ], 100%). The liquid chromatogram is shown in FIG. 11, and the mass spectrogram is shown in FIG. 12.

Example 7

Cyclo(Gly-Gly-Phe-Gly-D-Tyr)。

the synthesis method comprises the following steps:

the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), Fmoc-D-Tyr- (OAll) is connected to the Resin in the presence of N, N-diisopropylethylamine, wherein the molar ratio of the 2-CTC Resin to the Fmoc-D-Tyr- (OAll) and the N, N-diisopropylethylamine is 1: 1: and 4, obtaining Fmoc-D-Tyr- (OAll) -2-CTC Resin, sequentially and respectively connecting Fmoc-Gly-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH and Fmoc-Gly-OH to the Resin, wherein the mol ratios of the Fmoc-D-Tyr- (OAll) -2-CTC Resin to the Fmoc-Gly-OH, the Fmoc-L-Phe-OH, the Fmoc-Gly-OH and the Fmoc-Gly-OH are respectively 1: the molar ratio of 2, Fmoc-D-Tyr- (OAll) -2-CTC Resin to 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate and N, N-diisopropylethylamine is 1: 2: 2: 2, the reaction and detection, cyclization, cleavage, purification steps in ligation were the same as in example 3. The chromatographic conditions were the same as in example 3, 12.58 minutes, and lyophilization gave a product with a purity of 94.90%. Its structure is characterized by electrospray ionization mass spectrum, [ M ] calcd:481.59, found:482.35([ M + H ], 100%); 504.28([ M + Na ] +, 60%). The liquid chromatogram is shown in FIG. 13, and the mass spectrogram is shown in FIG. 14.

Example 8

The raw materials and auxiliary materials used for producing 1000 tablets of the pharmaceutical tablet product of the invention and the mixture ratio thereof are as follows:

the H-Dopa-Gly-Phe-Gly-Gly-OH 1000X 10 synthesized in example 1 was taken^-11mg, starch to 300g, according to the preparation method of the conventional tablet in pharmaceutics. Each tablet is 0.3g in weight and each tablet contains 10 effective components^-11And (5) mg. It is taken 1 tablet each time after breakfast.

Example 9

the H-D-Tyr-Pro-D-Phe-Gly-Gly-OH 1000X 10 synthesized in example 2 was taken^-11mg, starch to 300g, according to the preparation method of the conventional tablet in pharmaceutics. Each tablet is 0.3g in weight and each tablet contains 10 effective components^-11And (5) mg. It is taken 1 tablet each time after breakfast.

Example 10

1000X 10 of the Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) synthesized in example 3 was taken^-11mg, starch to 300g, according to the preparation method of the conventional tablet in pharmaceutics. Each tablet is 0.3g in weight and each tablet contains 10 effective components^-11And (5) mg. It is taken 1 tablet each time after breakfast.

Example 11

1000X 10 of the Cyclo (Tyr-Gly-D-Phe-Gly-Gly) synthesized in example 4 was taken^-11mg, starch to 300g, according to the preparation method of the conventional tablet in pharmaceutics. Each tablet is 0.3g in weight and contains effective componentsPortion 10^-11And (5) mg. It is taken 1 tablet each time after breakfast.

Example 12

1000X 10 of the Cyclo (D-Tyr-Gly-Phe-Gly-Gly) synthesized in example 5 was taken^-11mg, starch to 300g, according to the preparation method of the conventional tablet in pharmacy. Each tablet is 0.3g in weight and each tablet contains 10 effective components^-11And (5) mg. It is taken 1 tablet each time after breakfast.

Example 13

1000X 10 of the Cyclo (Gly-Gly-D-Phe-Gly-Tyr) synthesized in example 6 was taken^-11mg, starch to 300g, according to the preparation method of the conventional tablet in pharmaceutics. Each tablet is 0.3g in weight and each tablet contains 10 effective components^-11And (5) mg. It is taken 1 tablet each time after breakfast.

Example 14

1000X 10 of the Cyclo (Gly-Gly-Phe-Gly-D-Tyr) synthesized in example 7 was taken^-11mg, starch to 300g, according to the preparation method of the conventional tablet in pharmaceutics. Each tablet is 0.3g in weight and each tablet contains 10 effective components^-11And (5) mg. It is taken 1 tablet each time after breakfast.

Test experiments

In order to verify the beneficial effects of the invention, cell activity experiments and enzymolysis experiments are carried out by using the OGP (10-14) pentapeptide derivatives synthesized in the embodiments 1-7 of the invention, and various experimental conditions are as follows:

1. cell viability assay

(1) Preparing a sample solution

Preparing a cell culture solution: mixing alpha-MEM liquid culture medium, inactivated fetal calf serum and streptomycin in 89% by volume: 10%: mixing at a ratio of 1%, packaging, sealing, and making into cell culture solution, and storing at 4 deg.C. The same culture medium without serum was prepared and tested for activity.

Preparing into 5mg/mL phosphate buffer solution by conventional method, adjusting pH to 7.2-7.4, sterilizing at high temperature under high pressure, packaging, sealing, and storing at 4 deg.C.

Preparing a thiazole blue (MTT) solution: weighing 50.0mg of thiazole blue, dissolving in 10mL of phosphate buffer solution, stirring by a magnetic stirrer until the thiazole blue is completely dissolved, filtering and sterilizing by a 0.22 mu m microporous filter membrane, subpackaging in 1.5mL of centrifuge tubes, sealing by aluminum box paper, and storing at minus 20 ℃ in a dark place.

Sample test solution: taking 0.01mmol of each of 7 example lyophilized OGP (10-14) pentapeptide derivative samples, dissolving in 1mL dimethyl sulfoxide to obtain 0.01mmol/mL sample solution, and diluting to 10^-7mol/L and 10^-9Two concentrations of mol/L.

(2) Cell culture

Cell recovery: respectively taking frozen osteoblasts (MC3T3E1) and fibroblasts (NIH3T3), thawing in water bath at 37 ℃ for 1-2 minutes, sucking the cells into a centrifuge tube at 1500 rpm, centrifuging for 4 minutes, removing supernatant, uniformly blowing the cells with preheated cell culture solution, placing at 37 ℃ and 5% CO₂Culturing for 24 hours in an incubator, performing microscopic examination, and replacing the cell culture solution once for 2-3 days if most cells are attached to the wall and have normal shapes.

Cell passage: and (5) when the cells grow to 80-90% of the fusion state, carrying out passage. A passage step: washing with 5mL phosphate buffer solution for 2 times, adding 1mL pancreatin with concentration of 0.25%, observing under microscope, most cells contract in 2-3 min, adding 2mL cell culture solution to blow off adherent cells, centrifuging at 1500 rpm for 4 min, removing pancreatin, blowing off cells uniformly with preheated cell culture solution, dividing cells into 3, standing at 37 deg.C and 5% CO for 4 min₂The culture was carried out in an incubator for 24 hours.

(3) Activity assay

Collecting cells in exponential growth phase, washing with phosphate buffer twice, and adding pancreatin with concentration of 0.25% sodium edetateDigesting, repeatedly blowing and beating with MEM medium to obtain cell suspension, adjusting cell density to 2 × 10⁷Per L to 5X 10⁷One cell per L was inoculated into a 96-well culture plate so that the cell density was 5000 to 10000 per L per well, and 24 hours later, fresh MEM medium (198. mu.L per well) containing no serum was replaced, and 2. mu.L of each of the sample solutions of 7 examples was added to each well of the experimental group, and the concentration of each sample solution was 10. mu.L^-9mol/L、10 ^-11mol/L. Wherein the optimal test concentration in osteoblasts is 10^-11mol/L, the optimum test concentration in fibroblasts is 10^-9mol/L. Setting a negative control hole and a blank zero-setting hole, wherein the negative control hole is a culture solution containing 2 mu L of dimethyl sulfoxide; the blank zero-setting hole is culture solution without osteoblasts and fibroblasts, and each test solution group is provided with 3 parallel holes. After the sample addition, the culture was continued for 24 hours. Fresh medium (180. mu.L/well) without serum was replaced, 20. mu.L/well of thiazole blue solution was added, the culture was performed for 4 hours, the supernatant was aspirated, 150. mu.L of dimethyl sulfoxide was added to each well, and the mixture was shaken at 37 ℃ for 10 minutes until formazan crystals were sufficiently dissolved. And (3) carrying out zero setting on blank holes at the wavelength of 490nm by using an enzyme-linked immunosorbent calibrator, and determining the absorbance A of each hole. The proliferation activity was evaluated by comparing the absorbance A value with that of the positive control group.

The increment rate of less than 1 means lower than the OGP (10-14) activity, the increment rate of more than 1 means higher than the OGP (10-14) activity, and the experiment is repeated for 3 times. Data were statistically analyzed using SPPS 13.0 software according to the software instructions, all expressed in x + -s, and comparisons between groups were performed using the t-test.

Cell proliferation rate-mean of experimental group/mean of positive control group

The test results are shown in Table 1.

TABLE 1 proliferative Activity of peptides of examples 1 to 7 in osteoblasts and fibroblast cell lines (S) ((S))

n＝3)

The results of the experiment were statistically processed, P <0.05, P < 0.01. As can be seen from Table 1, the cell proliferation activity of the above compounds was comparable to or superior to that of OGP (10-14), and the action strengths thereof were correlated with each other in the two cell lines. These compounds structurally retain the original pharmacophore of pentapeptide (Tyr10, Phe12) and have a structure (D-configuration, cyclization) that improves metabolic stability.

2. Enzymolysis test

(1) Preparing a sample solution

7 examples of OGP (10-14) pentapeptide derivative solutions were prepared: 7 OGP (10-14) pentapeptide derivative lyophilized powders of 7 examples were weighed (5 mg), dissolved in water, and prepared into 7 aqueous solutions having a concentration of 1mg/mL in a volumetric flask with a constant volume of 5 mL.

Preparing a pepsin solution: weighing 0.2g of sodium chloride and 0.32g of pepsin, adding 50mL of water for dissolving, then adding 0.7mL of concentrated hydrochloric acid, adding water to a constant volume of 100mL, adjusting the pH value to 1.2, and preparing into a pepsin solution with the concentration of 3.2 mg/mL.

Preparing a trypsin solution: weighing KH₂PO ₄0.68g, adding 25mL of water for dissolution; adding 19mL of 0.2mol/L NaOH aqueous solution, 40mL of water and 1.0g of trypsin, uniformly mixing the solutions, adjusting the pH to 7.5 +/-0.1 by using 0.2mol/L NaOH solution, and fixing the volume of water to 100mL to prepare 10mg/mL trypsin solution.

(2) Experimental methods

Pepsin degradation of the samples: adding 1.5mL of pepsin solution into a 5mL centrifuge tube, keeping the temperature at 37 ℃ for 10 minutes, adding 1.5mL of the peptide sample solution of examples 1-7 respectively, timing, sequentially taking 200 uL of 7 peptide sample reaction solutions at 2 minutes, 4 minutes, 8 minutes, 16 minutes, 30 minutes, 60 minutes and 120 minutes, and adding 50 uL of 0.618mol/L Na₂CO ₃The solution stops the enzymolysis reaction, and a sample to be detected is prepared and detected by a high performance liquid chromatograph.

Preparing a trypsin degradation sample: adding 1.5mL of trypsin solution into a 5mL centrifuge tube, keeping the temperature at 37 ℃ for 10 minutes, respectively adding 1.5mL of 7 peptide sample reaction solutions, timing, sequentially taking 200 mu L of the 7 peptide sample reaction solutions at 2 minutes, 4 minutes, 8 minutes, 16 minutes, 30 minutes, 60 minutes and 120 minutes, respectively adding 50 mu L of 30% acetic acid solution to terminate the enzymolysis reaction, preparing a sample to be detected, and detecting by using a high performance liquid chromatograph.

(3) Detection conditions

7 samples to be detected are separated and detected by a Hitachi L-2455 full-automatic analysis liquid chromatograph, and relevant parameters are as follows:

mobile phase A: aqueous solution of trifluoroacetic acid at a concentration of 0.1%; mobile phase B: 0.1% trifluoroacetic acid in acetonitrile; wavelength: 215 nm; the flow rate was 1 mL/min.

H-Dopa-Gly-Phe-Gly-Gly-OH, H-D-Tyr-Pro-D-Phe-Gly-Gly-OH assay program: 16% constant current, run time 10 min.

Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) assay program: 30% constant current, run time 10 min.

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) assay program: 20% constant current, run time 10 min.

(4) The result of the detection

The detection results are shown in FIGS. 15 and 16.

As can be seen from FIGS. 15 and 16, the effect of OGP (10-14) pentapeptide derivatives on cell proliferation activity was evaluated by performing an in vitro activity screening assay on OGP (10-14) and 7 examples of OGP (10-14) pentapeptide derivatives thereof, using MC3T3E1 osteoblasts and NIH3T3 fibroblasts as test cell lines, and expressing the activity of OGP (10-14) as a positive control. The results show that the cellular activity of the OGP (10-14) pentapeptide derivatives of 7 examples retains even better activity than that of the original linear peptide OGP (10-14). The metabolic stability of each derivative in two enzymes was evaluated by performing an enzymatic hydrolysis experiment on OGP (10-14) and its OGP (10-14) pentapeptide derivatives of 7 examples, using pepsin and trypsin as enzymatic hydrolysis conditions, respectively, and using the enzymatic hydrolysis reaction of OGP (10-14) as a blank control. The results show that the OGP (10-14) pentapeptide derivatives of the other 6 examples have better overall metabolic stability than OGP (10-14) except that the H-Dopa-Gly-Phe-Gly-Gly-OH has overall similar and no significant difference compared with the OGP (10-14).

The results show that the OGP (10-14) pentapeptide derivatives of 7 examples structurally reserve the original pharmacodynamic groups (Tyr10 and Phe12) of the OGP (10-14), have the structural characteristics of improving the metabolic stability, have the biological activity and the metabolic stability of the OGP (10-14), are used as active ingredients in the aspects of treating or preventing fracture injury and regulating bone metabolism imbalance, and are used in a mode of gastrointestinal administration or parenteral administration in combination with solid or liquid auxiliary agents commonly used in pharmaceutics.

Claims

An OGP (10-14) pentapeptide derivative comprising 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)。
a method for preparing the OGP (10-14) pentapeptide derivative of claim 1, comprising:

carrying out peptide chain connection of OGP (10-14) straight-chain peptide derivatives by adopting an Fmoc-solid phase synthesis method, or carrying out peptide chain connection of OGP (10-14) cyclic peptide derivatives by adopting a dichloro resin cyclization method initiated by Fmoc-Tyr- (OAll);

cutting the peptide resin obtained after peptide chain connection by using trifluoroacetic acid aqueous solution to obtain crude peptide;

and purifying the crude peptide by reverse phase high performance liquid chromatography to obtain the OGP (10-14) pentapeptide derivative.
The method of claim 2, wherein 2-CTC Resin is used as a 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 of claim 4, wherein the pharmaceutically acceptable carrier is a solid excipient or a liquid excipient.
Use of an OGP (10-14) pentapeptide derivative according to claim 1 or a pharmaceutical composition according to any one of claims 4-5 in the manufacture of a medicament for the treatment and/or prevention of bone injury.
Use of an OGP (10-14) pentapeptide derivative according to claim 1 or a pharmaceutical composition according to any one of claims 4-5 for the manufacture of a medicament for the treatment and/or prevention of a bone metabolic disease.
Use of an OGP (10-14) pentapeptide derivative according to claim 1 or a pharmaceutical composition according to any one of claims 4-5 for the manufacture of a medicament for the treatment and/or prevention of chronic idiopathic myelofibrosis disease.
Use according to any one of claims 6 to 8, wherein the medicament is a tablet, granule, capsule, suspension, syrup or emulsion.
The use according to any one of claims 6 to 9, wherein the OGP (10-14) pentapeptide derivative according to claim 1 is OGP 10^-9～10 ^-11The mg/day/time dose is used for preparing the medicament.
A method of treating bone injury, comprising administering to a subject an effective amount of an OGP (10-14) pentapeptide derivative of claim 1 or a pharmaceutical composition of any one of claims 4-5.
A method of treating a bone metabolic disease, the method comprising administering to a subject an effective amount of an OGP (10-14) pentapeptide derivative of claim 1 or a pharmaceutical composition of any one of claims 4-5.
A method of treating chronic idiopathic myelofibrosis, the method comprising administering to a subject an effective amount of an OGP (10-14) pentapeptide derivative of claim 1 or a pharmaceutical composition of any one of claims 4-5.