CN112789286B - Carbon-terminal pentapeptide derivative of osteogenic growth peptide, preparation method and application thereof - Google Patents
Carbon-terminal pentapeptide derivative of osteogenic growth peptide, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an osteogenic growth peptide carbon-terminal pentapeptide derivative, a preparation method and application thereof. The carbon-terminal pentapeptide derivative of the osteogenic growth peptide comprises the following components: 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-Gly), cyclo (Gly-Gly-D-Phe-Gly-Tyr). The pharmacophore Tyr10 and Phe12 of the original pentapeptide are reserved in structure, and the structure characteristics of improving metabolic stability are achieved.
Description
Technical Field
The invention belongs to the technical field of polypeptides, and particularly relates to an osteogenic growth peptide carbon-terminal pentapeptide OGP (10-14) derivative, and a preparation method and application thereof.
Background
The osteogenic growth peptide (Osteogenic Growth Peptide) is a polypeptide growth factor and has the effects of promoting osteogenesis and stimulating hematopoiesis, and has a primary structure of ALKRQGRT LYGFGG. Wherein, the carbon-end pentapeptide OGP (10-14) is the minimum fragment for keeping the total activity of the osteogenic growth peptide, and the amino acid sequence is H-Tyr10-Gly11-Phe12-Gly13-Gly14-OH. A large number of experiments show that OGP (10-14) not only has the effect of improving and treating bone injury and bone metabolic diseases, but also has the effect of recovering the hematopoietic function of patients with radiotherapy and chemotherapy and bone marrow transplantation. Abiogen pharmaceutical company, italy, has reported submitting OGP (10-14) to European medicine administration (EMEA) as an orphan drug application for the treatment of Chronic Idiopathic Myelofibrosis (CIMF) diseases, and has been approved. The structure-activity relationship research shows that the side chains of the rest amino acids except Gly13 and Gly14 are pharmacophores which can influence the activity. On the basis, the positions 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 straight-chain OGP (10-14) without amino at nitrogen end by Bab et al. Because of the homology and high conservation of OGP (10-14) itself, it is only possible to modify the propeptide structure to obtain safer and more effective compounds.
In the technical field of osteogenic growth peptides, a technical problem to be solved urgently at present is to provide a compound with bioactivity and metabolic stability.
Disclosure of Invention
It is an object of the present invention to provide a novel OGP (10-14) pentapeptide derivative which increases the biological activity, increases the proliferative activity of MC3T3E1 osteoblasts and/or NIH3T3 fibroblasts, and/or improves pepsin and/or trypsin stability.
It is another object of the present invention to provide a process for the preparation of said OGP (10-14) pentapeptide derivatives.
It is another object of the present invention to provide pharmaceutical compositions comprising said OGP (10-14) pentapeptide derivatives.
It is another object of the present invention to provide the use of said OGP (10-14) pentapeptide derivatives and said pharmaceutical compositions for the preparation of a medicament for the treatment and/or prevention of bone damage.
It is another object of the present invention to provide the use of said OGP (10-14) pentapeptide derivatives for the preparation of a medicament for the treatment and/or prevention of bone metabolic diseases.
It is another object of the present invention to provide the use of said OGP (10-14) pentapeptide derivatives for the preparation of a medicament for the treatment and/or prevention of chronic idiopathic myelofibrosis diseases.
The aim of the invention is achieved by the following technical means:
in one aspect, the 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 has the same characteristics as the polypeptide drug as an active candidate. On one hand, the novel medicine has the advantages of simple structure, obvious activity, low immunogenicity and good safety, and has the advantages of new medicine development; on the other hand, the method is constrained by self-conformation, and has the defects of low oral utilization rate, high enzymatic degradability, short half-life and the like. The main reason for these instabilities is that the conformation of a particular amino acid in the structure, e.g., the order of attachment of the main chain and the nature of the side chain residues, affects the biological activity. Because the recognition effect of OGP and a substrate is not clear, the receptor cannot be simulated for drug screening, and therefore, 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. The research shows that compared with unmodified OGP (10-14) pentapeptide and other modified OGP (10-14) pentapeptide derivatives, the 7 OGP (10-14) pentapeptide derivatives structurally retain original pharmacophores (Tyr 10 and Phe 12) of the OGP (10-14), have the structural characteristics of improving metabolic stability, and have the biological activity and metabolic stability of the OGP (10-14) proved by cell activity experiments and enzymolysis experiments, and have stronger in-vitro proliferation activity than the OGP (10-14), higher stability to pepsin and trypsin, and can be widely applied to the aspects of treating or preventing fracture injury and regulating bone metabolic imbalance as an active ingredient.
On the other hand, the invention also provides a preparation method of the OGP (10-14) pentapeptide derivative, which comprises the following steps:
peptide chain ligation of OGP (10-14) linear peptide derivatives is performed by Fmoc-solid phase synthesis, or by using Fmoc-Tyr- (OAll) as an initial dichloro resin cyclization method;
the peptide resin obtained after the connection of peptide chains is cut by trifluoroacetic acid aqueous solution to obtain crude peptide;
purifying the crude peptide by reverse phase high performance liquid chromatography to obtain OGP (10-14) pentapeptide derivative.
The prior art reports that candidate derivatives for obtaining OGP (10-14) pentapeptide mainly comprise serial derivatives obtained by shortening the length of a peptide chain, modifying an amide bond, changing a side chain group, replacing D-amino acid, forming cyclic pentapeptide (clockwise or anticlockwise cyclizing) and the like, and performing cell activity screening and comparing with a protopeptide; the linear 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 upon which the osteogenic peptide acts is not clear, there is no specific basis for modification at a certain site. According to the invention, according to the residues shared by OGP (10-14) and daTyr10[ OGP (10-14) ] and other residues possibly being close to optimal pharmacophores, the original pentapeptide OGP (10-14) is directly modified, an Fmoc-solid phase method is adopted as a straight chain, and a dichloro resin cyclization method taking Fmoc-Tyr- (OAll) as an initial part is adopted as a cyclic peptide; on the basis, 7 OGP (10-14) pentapeptide derivatives are obtained by modification through pepsin and trypsin degradation tests with the aim of improving metabolic stability, and the purity of the OGP (10-14) pentapeptide derivatives obtained by the method is more than 97%.
In the above method, preferably, 2-CTC Resin (2-Chlorotrityl Chloride Resin) is used as a solid phase carrier in the solid phase synthesis method.
In another aspect, the invention also provides a pharmaceutical composition comprising the OGP (10-14) pentapeptide derivative and a pharmaceutically acceptable carrier.
In the above pharmaceutical compositions, 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 parenteral administration, or the like. Specifically, the composition can comprise stabilizer, wetting agent, solubilizer or other common auxiliary materials and/or additives, such as lactose, talcum powder, cellulose, polyvinylpyrrolidone, starch, pectin, tween-80, polyvinyl alcohol, etc.
In the above pharmaceutical composition, preferably, the pharmaceutical composition exists 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 various oral medicines in various dosage forms, and is orally taken by adults, and the dosage is usually 10 times per dose -9 ～10 -11 mg, taken after meals 1 time a day, and the dosage for children is reduced as appropriate. The 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 medicines 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 medicines 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 original pharmacophores (Tyr 10 and Phe 12) of the OGP (10-14), has the structural characteristics of improving metabolic stability, has the biological activity and metabolic stability of the OGP (10-14) through a cell activity experiment and an enzymolysis experiment, has stronger in-vitro proliferation activity than the OGP (10-14), and has higher stability to pepsin and trypsin; can be used as an active ingredient in the treatment or prevention of fracture injury and the regulation of bone metabolism imbalance, and is used in combination with solid or liquid auxiliary agents commonly used in pharmacy in a gastrointestinal administration or parenteral administration mode.
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 pepsin degradation experiments with OGP (10-14) pentapeptide derivatives.
FIG. 16 is a graph showing the results of an experiment of degradation of trypsin by OGP (10-14) pentapeptide derivatives.
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention. In the examples, experimental methods without specifying specific conditions are referred to conventional methods and conventional conditions well known in the art, or are operated according to conditions recommended by the instrument manufacturer.
Example 1
The structural formula of the OGP (10-14) pentapeptide derivative of this example is as follows:
H-Dopa-Gly-Phe-Gly-Gly-OH。
the synthesis method comprises the following steps:
reaction and detection: the solid phase carrier is 2-CTC Resin (1.0 mmol/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 1:1:4, obtaining Fmoc-Gly-2-CTC Resin, and then sequentially connecting Fmoc-Gly-OH, fmoc-Phe-OH, fmoc-Gly-OH and Fmoc-Dopa (tBu) -OH to the Resin, wherein each step of reaction is detected by adopting 0.05g/mL of ninhydrin ethanol solution, so that the forward detection or the reverse detection passes, and the molar ratio of the 2-CTC Resin to Fmoc-Gly-OH, fmoc-Phe-OH, fmoc-Gly-OH and Fmoc-Dopa (tBu) -OH is 1: the molar ratio of the 2,2-CTC 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, removing Fmoc in the peptide chain connection by using an N, N-dimethylformamide solution with the concentration of 20% of piperidine, washing 2 times by isopropanol, washing 3 times by N, N-dimethylformamide, washing 3 times by anhydrous methanol, and filtering and drying at normal temperature to obtain the peptide resin.
Cutting: the peptide resin is cut by using 95% trifluoroacetic acid aqueous solution, the reaction is carried out for 1.5 hours, the suction filtration is carried out, the filtrate is separated out by using cold diethyl ether to precipitate, the suction filtration is carried out, and the crude peptide is obtained by vacuum drying at normal temperature.
Purifying: purifying the crude peptide by reverse phase high performance liquid chromatography, and freeze drying to obtain the product. Chromatographic conditions: phase A (aqueous solution of trifluoroacetic acid with concentration of 0.1 percent) and phase B (aqueous solution of trifluoroacetic acid with concentration of 0.1 percent) are subjected to gradient elution for 30 minutes (10 to 90 percent of phase B), 14.69 minutes and freeze-dried, so that the product with purity of 94.55 percent is obtained. The structure is 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 chromatogram is shown in figure 2.
Example 2
The structural formula of the OGP (10-14) pentapeptide derivative of this example is as follows:
H-D-Tyr-Pro-D-Phe-Gly-Gly-OH。
the synthesis method comprises the following steps:
the solid phase carrier is 2-CTC Resin (1.0 mmol/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 1:1:4, fmoc-Gly-2-CTC Resin is obtained, fmoc-Gly-OH, fmoc-D-Phe-OH, fmoc-Pro-OH and Fmoc-D-Tyr (tBu) -OH are sequentially connected to the Resin respectively, and the reaction conditions, detection, cutting and purification steps are the same as those of the example 1, and the product is obtained by freeze-drying. Chromatographic conditions: phase A (aqueous solution of trifluoroacetic acid with concentration of 0.1%), phase B (acetonitrile solution of trifluoroacetic acid with concentration of 0.1%) and gradient elution for 30 minutes (10% -90% of phase B), 13.25 minutes and purity of 99.74%. The structure is 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 FIG. 3 and the mass chromatogram is shown in FIG. 4.
Example 3
The structural formula of the OGP (10-14) pentapeptide derivative of this example is as follows:
Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)。
the synthesis method comprises the following steps:
the solid phase carrier adopts 2-CTC Resin (1.0 mmol/g), fmoc-D-Tyr- (OAll) is firstly connected to the Resin in the presence of N, N-diisopropylethylamine, and the mole ratio of the 2-CTC Resin to the Fmoc-D-Tyr- (OAll) to the N, N-diisopropylethylamine is 1:1:4, fmoc-D-Tyr- (OAll) -2-CTC Resin is obtained, and Fmoc-Pro-OH, fmoc-D-Phe-OH, fmoc-Gly-OH and Fmoc-D-Tyr- (OAll) -2-CTC Resin are sequentially connected to the Fmoc-D-Tyr- (OAll) -2-CTC Resin respectively, wherein the molar ratio of the Fmoc-D-Phe-OH, fmoc-Gly-OH and Fmoc-Gly-OH to each of the Fmoc-Pro-OH and Fmoc-OH is 1: the molar ratio of the 2, fmoc-D-Tyr- (OAll) -2-CTC Resin to the 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1:2:2:2, each step of reaction is detected by adopting 0.05g/mL of ninhydrin ethanol solution, so that the positive detection or the negative detection is ensured to pass completely. Fmoc removal in peptide ligation was performed with a 20% piperidine in N, N-dimethylformamide and allyl removal in a piperidine solution with tetrakis (triphenylphosphine) palladium under nitrogen protection and ice salt bath. 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, 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: phase A (0.1% aqueous solution of trifluoroacetic acid), phase B (0.1% acetonitrile solution of trifluoroacetic acid), gradient elution for 30 minutes (10% -90% of phase B), 17.41 minutes, freeze-drying to obtain the product with the purity of 87.51%. The structure is 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 FIG. 5, and the mass chromatogram is shown in FIG. 6.
Example 4
The structural formula of the OGP (10-14) pentapeptide derivative of this example is as follows:
Cyclo(Tyr-Gly-D-Phe-Gly-Gly)。
the synthesis method comprises the following steps:
the solid phase carrier adopts 2-CTC Resin (1.0 mmol/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) and the N, N-diisopropylethylamine is 1:1:4, fmoc-Tyr- (OAll) -2-CTC Resin is obtained, and Fmoc-Gly-OH, fmoc-D-Phe-OH and Fmoc-Gly-OH are sequentially connected to the Resin, wherein the mole ratio of the Fmoc-Tyr- (OAll) -2-CTC Resin to the Fmoc-Gly-OH, fmoc-D-Phe-OH and Fmoc-Gly-OH is 1: the molar ratio of the 2, fmoc-Tyr- (OAll) -2-CTC Resin to the 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1:2:2:2, the detection, cyclization, cleavage, purification steps in the reaction were the same as in example 3. The chromatographic conditions were the same as in example 3 for 10.42 minutes and lyophilized to give the product with a purity of 93.87%. The structure is 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 FIG. 7, and the mass chromatogram is shown in FIG. 8.
Example 5
The structural formula of the OGP (10-14) pentapeptide derivative of this example is as follows:
Cyclo(D-Tyr-Gly-Phe-Gly-Gly)。
the synthesis method comprises the following steps:
the solid phase carrier adopts 2-CTC Resin (1.0 mmol/g), fmoc-D-Tyr- (OAll) is firstly connected to the Resin in the presence of N, N-diisopropylethylamine, and the mole ratio of the 2-CTC Resin to the Fmoc-D-Tyr- (OAll) to the N, N-diisopropylethylamine is 1:1:4, fmoc-D-Tyr- (OAll) -2-CTC Resin is obtained, fmoc-Gly-OH, fmoc-L-Phe-OH, fmoc-Gly-OH and Fmoc-D-Tyr- (OAll) -2-CTC Resin are respectively connected to the Resin in sequence, and the molar ratio of the Fmoc-D-Tyr- (OAll) -2-CTC Resin to Fmoc-Gly-OH, fmoc-L-Phe-OH and Fmoc-Gly-OH is 1: the molar ratio of the 2, fmoc-D-Tyr- (OAll) -2-CTC Resin to the 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1:2:2:2, the detection, cyclization, cleavage, purification steps in the reaction were the same as in example 3. The chromatographic conditions were the same as in example 3 for 10.48 minutes and lyophilized to give the product with a purity of 91.69%. The structure is 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 FIG. 9, and the mass chromatogram is shown in FIG. 10.
Example 6
The structural formula of the OGP (10-14) pentapeptide derivative of this example is as follows:
Cyclo(Gly-Gly-D-Phe-Gly-Tyr)。
the synthesis method comprises the following steps:
the solid phase carrier was prepared by using 2-CTC Resin (1.0 mmol/g), fmoc-L-Tyr- (OAll) was connected to the Resin in the presence of N, N-diisopropylethylamine, and the molar ratio of 2-CTC Resin to Fmoc-L-Tyr- (OAll), N-diisopropylethylamine was 1:1:4, fmoc-Tyr- (OAll) -2-CTC Resin is obtained, fmoc-Gly-OH, fmoc-D-Phe-OH, fmoc-Gly-OH and Fmoc-Gly-OH are respectively connected to the Resin in sequence, and the mole ratio of the Fmoc-Tyr- (OAll) -2-CTC Resin to Fmoc-Gly-OH, fmoc-D-Phe-OH, fmoc-Gly-OH and Fmoc-Gly-OH is 1: the molar ratio of the 2, fmoc-Tyr- (OAll) -2-CTC Resin to the 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1:2:2:2, the detection, cyclization, cleavage, purification steps in the ligation were the same as in example 3. The chromatographic conditions were the same as in example 3 for 11.82 minutes and lyophilized to give the product with a purity of 96.77%. The 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 chromatogram is shown in FIG. 12.
Example 7
The structural formula of the OGP (10-14) pentapeptide derivative of this example is as follows:
Cyclo(Gly-Gly-Phe-Gly-D-Tyr)。
the synthesis method comprises the following steps:
the solid phase carrier was prepared by attaching Fmoc-D-Tyr- (OAll) to a Resin using 2-CTC Resin (1.0 mmol/g) in the presence of N, N-diisopropylethylamine, wherein the molar ratio of 2-CTC Resin to Fmoc-D-Tyr- (OAll), N-diisopropylethylamine was 1:1:4, fmoc-D-Tyr- (OAll) -2-CTC Resin is obtained, fmoc-Gly-OH, fmoc-L-Phe-OH, fmoc-Gly-OH and Fmoc-D-Tyr- (OAll) -2-CTC Resin are respectively connected to the Resin in sequence, and the molar ratio of the Fmoc-D-Tyr- (OAll) -2-CTC Resin to the Fmoc-Gly-OH, fmoc-L-Phe-OH, fmoc-Gly-OH and Fmoc-Gly-OH is 1: the molar ratio of the 2, fmoc-D-Tyr- (OAll) -2-CTC Resin to the 1-hydroxybenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid and N, N-diisopropylethylamine is 1:2:2:2, the reaction and detection, cyclization, cleavage, purification steps in the ligation were the same as in example 3. The chromatographic conditions were the same as in example 3 for 12.58 minutes and freeze-dried to give the product with a purity of 94.90%. The structure is 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 FIG. 13, and the mass chromatogram is shown in FIG. 14.
Example 8
Taking 1000 tablets of the pharmaceutical tablet product of the invention as an example, the raw materials and auxiliary materials and the proportion thereof are as follows:
taking H-Dopa-Gly-Phe-Gly-Gly-OH 1000X 10 synthesized in example 1 -11 The addition of the starch to 300g was carried out according to the preparation method of the conventional tablet of the pharmacy. Each tablet weighs 0.3g and contains 10 pharmaceutically effective components -11 mg. Taken once a day after breakfast, 1 tablet each time.
Example 9
Taking 1000 tablets of the pharmaceutical tablet product of the invention as an example, the raw materials and auxiliary materials and the proportion thereof are as follows:
taking the H-D-Tyr-Pro-D-Phe-Gly-Gly-OH 1000X 10 synthesized in example 2 -11 The addition of the starch to 300g was carried out according to the preparation method of the conventional tablet of the pharmacy. Each tablet weighs 0.3g and contains 10 pharmaceutically effective components -11 mg. Taken once a day after breakfast, 1 tablet each time.
Example 10
Taking 1000 tablets of the pharmaceutical tablet product of the invention as an example, the raw materials and auxiliary materials and the proportion thereof are as follows:
the Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) synthesized in example 3 was taken to be 1000X 10 -11 The addition of the starch to 300g was carried out according to the preparation method of the conventional tablet of the pharmacy. Each tablet weighs 0.3g and contains 10 pharmaceutically effective components -11 mg. Taken once a day after breakfast, 1 tablet each time.
Example 11
Taking 1000 tablets of the pharmaceutical tablet product of the invention as an example, the raw materials and auxiliary materials and the proportion thereof are as follows:
the Cyclo (Tyr-Gly-D-Phe-Gly-Gly) synthesized in example 4 was taken to be 1000X 10 -11 The addition of the starch to 300g was carried out according to the preparation method of the conventional tablet of the pharmacy. Each tablet weighs 0.3g and contains 10 pharmaceutically effective components -11 mg. Taken once a day after breakfast, 1 tablet each time.
Example 12
Taking 1000 tablets of the pharmaceutical tablet product of the invention as an example, the raw materials and auxiliary materials and the proportion thereof are as follows:
the Cyclo (D-Tyr-Gly-Phe-Gly-Gly) synthesized in example 5 was taken to be 1000X 10 -11 The addition of the starch to 300g was carried out according to the preparation method of the conventional pharmaceutical tablets. Each tablet weighs 0.3g and contains 10 pharmaceutically effective components -11 mg. Taken once a day after breakfast, 1 tablet each time.
Example 13
Taking 1000 tablets of the pharmaceutical tablet product of the invention as an example, the raw materials and auxiliary materials and the proportion thereof are as follows:
the Cyclo (Gly-Gly-D-Phe-Gly-Tyr) synthesized in example 6 was taken to be 1000X 10 -11 The addition of the starch to 300g was carried out according to the preparation method of the conventional tablet of the pharmacy. Each tablet is heavy0.3g, each tablet contains medicinal effective component 10 -11 mg. Taken once a day after breakfast, 1 tablet each time.
Example 14
Taking 1000 tablets of the pharmaceutical tablet product of the invention as an example, the raw materials and auxiliary materials and the proportion thereof are as follows:
the Cyclo (Gly-Gly-Phe-Gly-D-Tyr) synthesized in example 7 was taken to be 1000X 10 -11 The addition of the starch to 300g was carried out according to the preparation method of the conventional tablet of the pharmacy. Each tablet weighs 0.3g and contains 10 pharmaceutically effective components -11 mg. Taken once a day after breakfast, 1 tablet each time.
Test experiment
In order to verify the beneficial effects of the invention, cell activity experiments and enzymolysis experiments are carried out by adopting the OGP (10-14) pentapeptide derivatives synthesized in the embodiments 1 to 7, and the various experimental conditions are as follows:
1. cell Activity assay
(1) Preparing a sample solution
Preparing a cell culture solution: mixing alpha-MEM liquid culture medium, inactivated fetal bovine serum and green streptomycin mixed solution according to the volume ratio of 89%:10%: mixing at a ratio of 1%, packaging and sealing to prepare a cell culture solution, and preserving at 4 ℃ for later use. The same culture medium without serum was prepared for activity test.
Preparing 5mg/mL phosphate buffer solution according to a conventional method, regulating the pH value to 7.2-7.4, sterilizing at high temperature under high pressure, packaging and sealing, and preserving at 4 ℃ for later use.
Preparing a thiazole blue (MTT) solution: 50.0mg of thiazole blue is weighed and dissolved in 10mL of phosphate buffer solution, the solution is stirred by a magnetic stirrer until the solution is completely dissolved, the solution is filtered and sterilized by a microporous filter membrane with the thickness of 0.22 mu m, the solution is packaged into a centrifuge tube with the thickness of 1.5mL, an aluminum box paper is sealed, and the solution is preserved in a dark place at the temperature of minus 20 ℃.
Sample test solution: taking 0.01mmol of each lyophilized sample of the OGP (10-14) pentapeptide derivative of 7 examples, respectively dissolving in 1mL of dimethyl sulfoxide to obtain 0.01mmol/mL sample solution, respectively diluting to 10 -7 mol/L and 10 -9 Two concentrations of mol/L.
(2) Cell culture
Cell resuscitation: respectively taking frozen productsThawing bone cells (MC 3T3E 1) and fibroblast cells (NIH 3T 3) in water bath at 37deg.C for 1-2 min, sucking cells into centrifuge tube 1500 rpm, centrifuging for 4 min, removing supernatant, blowing cells with preheated cell culture solution, homogenizing, and placing in 37deg.C and 5% CO 2 Culturing in an incubator for 24 hours, microscopic examination, and replacing the cell culture fluid once in 2-3 days if most cells are attached and normal in morphology.
Cell passage: and when the cells grow to 80-90% of fusion state, carrying out passage. And (3) carrying out passage: washing with 5mL phosphate buffer solution for 2 times, adding 1mL pancreatin with concentration of 0.25%, observing under microscope, shrinking most cells for 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 cell 1 into 3, placing at 37deg.C and 5% CO 2 Culturing in an incubator for 24 hours.
(3) Activity test
Collecting cells in exponential growth phase, washing twice with phosphate buffer solution, digesting the cells with pancreatin with 0.25% sodium ethylenediamine tetraacetate, repeatedly blowing with MEM culture medium to obtain cell suspension, and adjusting cell density to 2×10 7 Per liter to 5X 10 7 Inoculating to 96-well culture plate at a cell density of 5000/L/Kong 10000/L/well, and replacing fresh MEM culture medium (198 μL/well) without serum after 24 hr while adding 2 μL of 7 sample test solutions of each embodiment to each well of experimental group, wherein the concentration of each sample test solution is 10 -9 mol/L、10 -11 mol/L. Wherein the optimal test concentration in osteoblasts is 10 -11 mol/L, optimal test concentration in fibroblasts is 10 -9 mol/L. Setting a negative control hole and a blank zeroing hole, wherein the negative control hole is a culture solution containing 2 mu L of dimethyl sulfoxide; the blank zeroing holes are culture solutions without osteoblasts and fibroblasts, and each test solution group is provided with 3 parallel holes. After the sample addition was completed, the culture was continued for 24 hours. Fresh medium (180. Mu.L/well) without serum was changed, thiazole blue solution (20. Mu.L/well) was added, and the culture was continued for 4 hours, and the supernatant was aspirated with a pipette, and 150. Mu.L of dimethylene was added to each wellThe sulfone is shaken for 10 minutes at 37 ℃ until formazan crystals are fully dissolved. The absorbance A of each well was measured using an ELISA calibrator at 490nm as a blank Kong Diaoling. The proliferation activity was evaluated by comparing with the absorbance A value of the positive control group.
A rate of increase of less than 1 indicates lower activity than OGP (10-14), a rate of increase of greater than 1 indicates higher activity than OGP (10-14), and the experiment was repeated 3 times. The data were statistically analyzed using SPPS 13.0 software according to the instructions of the software, all expressed as x+ -s, and the group comparisons used t-test.
Cell proliferation rate = experimental group mean/positive control group mean
The test results are shown in Table 1.
TABLE 1 proliferation Activity of peptides of examples 1 to 7 in osteoblasts and fibroblast strainsn＝3)
Experimental results were statistically processed with P <0.05 and P <0.01. As can be seen from Table 1, the cell proliferation activity of the above compounds was comparable to or better than OGP (10-14), and the intensity of actions in both cell lines was correlated. These compounds retain the original pharmacophore of pentapeptides (Tyr 10, phe 12) structurally and have structures (D-configuration, cyclization) for improving metabolic stability.
2. Enzymolysis experiment
(1) Preparing a sample solution
7 example OGP (10-14) pentapeptide derivative solutions were prepared: 5mg of the freeze-dried powder of the OGP (10-14) pentapeptide derivative of 7 examples is weighed, dissolved in water, and 5mL of the freeze-dried powder is fixed in a volumetric flask to prepare 7 aqueous solutions with the concentration of 1 mg/mL.
Preparing pepsin solution: weighing 0.2g of sodium chloride, 0.32g of pepsin, adding 50mL of water for dissolution, adding 0.7mL of concentrated hydrochloric acid, fixing 100mL of volume with water, adjusting the pH to 1.2, and preparing a pepsin solution with the concentration of 3.2 mg/mL.
Preparing a trypsin solution: weighing KH 2 PO 4 0.68g, 25mL of water was added for dissolution; 19mL of a 0.2mol/L NaOH aqueous solution and 40mL of water, 1.0g of trypsin were added, the solution was mixed, the pH was adjusted to 7.5.+ -. 0.1 with a 0.2mol/L NaOH solution, and 100mL of water was used to prepare a trypsin solution having a concentration of 10 mg/mL.
(2) Experimental method
Pepsin degradation samples: taking a 5mL centrifuge tube, adding 1.5mL pepsin solution, keeping the temperature at 37 ℃ for 10 minutes, respectively adding 1.5mL of the peptide sample solutions of examples 1-7, sequentially taking 200 mu L of 7 peptide sample reaction solutions at 2 minutes, 4 minutes, 8 minutes, 16 minutes, 30 minutes, 60 minutes and 120 minutes, and respectively adding 50 mu L of 0.618mol/L Na 2 CO 3 The enzymolysis reaction is stopped by the solution, a sample to be detected is prepared, and the sample is detected by a high performance liquid chromatograph.
Preparing a trypsin degradation sample: taking a 5mL centrifuge tube, adding 1.5mL of trypsin solution, 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 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 enzymolysis reaction, preparing a sample to be detected, and detecting by a high performance liquid chromatograph.
(3) Detection conditions
7 samples to be detected are separated and detected by a Ri Li L-2455 full-automatic analysis liquid chromatograph, and related parameters are as follows:
mobile phase a: an aqueous solution of trifluoroacetic acid at a concentration of 0.1%; mobile phase B: acetonitrile solution of trifluoroacetic acid with concentration of 0.1%; wavelength: 215nm; the flow rate was 1mL/min.
H-Dopa-Gly-Phe-Gly-Gly-OH, H-D-Tyr-Pro-D-Phe-Gly-Gly-OH detection and analysis procedure: 16% constant current, run time 10 minutes.
Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) assay procedure: 30% constant flow, run 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 procedure: 20% constant current, run time 10 minutes.
(4) Detection result
The detection results are shown in fig. 15 and 16.
As can be seen from fig. 15 and 16, the effect of OGP (10-14) pentapeptide derivatives on cell proliferation activity was evaluated by performing in vitro activity screening assays on OGP (10-14) and its 7 examples using MC3T3E1 osteoblasts and NIH3T3 fibroblasts as test cell lines, with the activity of OGP (10-14) being shown as a positive control. The results show that the cell activity of the OGP (10-14) pentapeptide derivatives of 7 examples all retain even better activity than the original linear peptide OGP (10-14). The metabolic stability of each derivative in two enzymes was evaluated by performing an enzymolysis experiment on OGP (10-14) and its 7 example OGP (10-14) pentapeptide derivatives, using pepsin and trypsin as enzymolysis conditions, respectively, and using the enzymolysis reaction of OGP (10-14) as a blank control. The results show that the metabolic stability of the OGP (10-14) pentapeptide derivatives of the other 6 examples is better than that of the OGP (10-14) except that the metabolic stability of the H-Dopa-Gly-Phe-Gly-Gly-OH is totally similar to that of the OGP (10-14) without significant difference.
The results show that the OGP (10-14) pentapeptide derivatives of 7 examples structurally retain original pharmacophores (Tyr 10 and Phe 12) of the OGP (10-14), have the structural characteristics of improving metabolic stability, have the bioactivity and metabolic stability of the OGP (10-14), are applied as active ingredients in the aspects of treating or preventing fracture injury and regulating bone metabolism imbalance, and are combined with solid or liquid auxiliary agents commonly used in pharmacy to be used in a gastrointestinal administration mode or a parenteral administration mode.
Claims (9)
1. An OGP (10-14) pentapeptide derivative having the amino acid sequence as follows:
Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)。
2. a process for the preparation of OGP (10-14) pentapeptide derivatives as claimed in claim 1, comprising:
peptide chain ligation of OGP (10-14) cyclopeptide derivatives was performed using Fmoc-Tyr- (OAll) initiated dichloro resin cyclization;
the peptide resin obtained after the connection of peptide chains is cut by trifluoroacetic acid aqueous solution to obtain crude peptide;
purifying the crude peptide by reverse phase high performance liquid chromatography to obtain OGP (10-14) pentapeptide derivative.
3. A pharmaceutical composition comprising the OGP (10-14) pentapeptide derivative of claim 1 and a pharmaceutically acceptable carrier.
4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier is a solid excipient or a liquid excipient.
5. Use of an OGP (10-14) pentapeptide derivative according to claim 1 or a pharmaceutical composition according to any one of claims 3-4 for the preparation of a medicament for the treatment and/or prevention of bone damage.
6. Use of an OGP (10-14) pentapeptide derivative according to claim 1 or a pharmaceutical composition according to any one of claims 3-4 for the preparation of a medicament for the treatment and/or prevention of bone metabolic diseases.
7. Use of an OGP (10-14) pentapeptide derivative according to claim 1 or a pharmaceutical composition according to any one of claims 3-4 for the preparation of a medicament for the treatment and/or prevention of chronic idiopathic myelofibrosis diseases.
8. The use according to any one of claims 5 to 7, wherein the medicament is in the form of a tablet, granule, capsule, suspension, syrup or emulsion.
9. The use according to any one of claims 5 to 8, wherein the OGP (10-14) pentapeptide derivative according to claim 1 is as defined in claim 10 -9 ～10 -11 The mg/day/dose is used for preparing medicines.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1269242A (en) * | 1999-04-01 | 2000-10-11 | 上海医科大学附属中山医院 | Osteogenic growth peptide medicine composite and its preparation and application |
| CN1737009A (en) * | 2004-08-17 | 2006-02-22 | 中国医学科学院药物研究所 | Polypeptide compound and medicinal use thereof |
| CN1785424A (en) * | 2004-12-09 | 2006-06-14 | 兰州大学 | Bone growth penta peptide medicinal composition ant its preparation method |
| CN103665109A (en) * | 2013-11-15 | 2014-03-26 | 陕西东大生化科技有限责任公司 | Synthesis method of C-terminal pentapeptide of osteogenic growth peptide |
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| IL117426A (en) * | 1996-03-10 | 2005-09-25 | Yissum Res Dev Co | Synthetic pseudopeptides having osteogenic activity and pharmaceutical compositions containing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1269242A (en) * | 1999-04-01 | 2000-10-11 | 上海医科大学附属中山医院 | Osteogenic growth peptide medicine composite and its preparation and application |
| CN1737009A (en) * | 2004-08-17 | 2006-02-22 | 中国医学科学院药物研究所 | Polypeptide compound and medicinal use thereof |
| CN1785424A (en) * | 2004-12-09 | 2006-06-14 | 兰州大学 | Bone growth penta peptide medicinal composition ant its preparation method |
| CN103665109A (en) * | 2013-11-15 | 2014-03-26 | 陕西东大生化科技有限责任公司 | Synthesis method of C-terminal pentapeptide of osteogenic growth peptide |
Non-Patent Citations (1)
| Title |
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| "Bioactive Pseudopeptidic Analogues and Cyclostereoisomers of Osteogenic Growth Peptide C-Terminal Pentapeptide, OGP(10-14)";Yu-Chen Chen et al.;《Journal of Medicinal Chemistry》;20021231;第45卷(第8期);第1624-1632页 * |
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