Liquid phase preparation method of thymopentin

Abstract

The invention discloses a liquid phase preparation method of thymopentin, and belongs to the technical field of chemical pharmacy. Carrying out condensation on the L-arginine active ester with protected amino and guanidyl and L-lysine with protected epsilon-amino, so as to obtain protected arginine-lysine dipeptide; the preparation method comprises the following steps: condensing L-Fmoc aspartic acid-delta-tert-butyl ester active ester with valine, continuously activating, and condensing with L-tert-butoxy tyrosine to obtain protected aspartic acid-valine-tyrosine tripeptide; carrying out condensation on the protected arginine-lysine dipeptide active ester and the Fmoc-removed tripeptide, so as to obtain a thymopentin precursor; and deprotecting the thymopentin precursor to obtain the thymopentin. The total reaction yield is more than 70%, and the HPLC purity is more than or equal to 99%.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a liquid phase preparation method of thymopentin. Background Art

[0002] Thymopentin is composed of five amino acids: arginine, lysine, aspartic acid, valine, and tyrosine. Its chemical name is N-〔N-〔N-〔NL-arginyl-L-lysyl〕-L-α-aspartyl〕-L-valyl〕-L-tyrosine. One of the functions of thymopentin is to induce T cell differentiation. It can selectively induce the transformation of Thy-1- prothymocytes into Thy-1+ T cells. Its T cell differentiation is mediated by the increase in intracellular cAMP levels. Another basic function of thymopentin is to bind to the specific receptors of mature peripheral blood T cells, increase the intracellular cAMP level, and thus induce a series of intracellular reactions, which is also the basis of its immunoregulatory function. Under normal body conditions, thymopentin exhibits an immunostimulatory effect, which can significantly increase the E rosette formation rate and conversion rate of splenic lymphocytes, and has an enhancing effect on different stages of the primary or secondary immune response, and can increase the number of IgM type and IgG or IgA type antibody-forming cells. Thymopentin can be used for patients with malignant tumors who have been treated with radiotherapy and chemotherapy and whose immune function is damaged; for the treatment of hepatitis B; for major surgical operations and serious infections; for autoimmune diseases such as rheumatoid arthritis and lupus erythematosus; for type 2 diabetes and menopausal syndrome; and for the elderly with weakened immune function.

[0003] At present, the main method for preparing thymopentin is solid phase synthesis. Solid phase synthesis generates a lot of waste by continuous protection and deprotection during the preparation process, and needs to use hydrofluoric acid to cut the product from the resin. The operation is very dangerous and requires very high equipment. Solid phase synthesis adopts a linear synthesis route, and each amino acid amine group needs to be protected by Fmoc. Solid phase synthesis is limited by scale, which is not conducive to industrialization and has no advantages in environmental protection, safety and cost. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a liquid phase preparation method of thymopentin, which has the advantages of simple operation, high directional conversion rate, environmental friendliness, high yield and high purity.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a liquid phase preparation method of thymopentin, the preparation method mainly comprises the following steps: Step 1), the L-arginine active ester with protected amine and guanidine groups and the L-lysine with protected ε-amine group are subjected to condensation reaction in a solvent, and the pH is controlled at 8.5-9.5 during the reaction process; a protected arginine-lysine dipeptide is obtained, and further activation is performed to obtain a protected arginine-lysine dipeptide active ester;

[0006] Step 2), L-Fmoc-aspartic acid δ-tert-butyl ester active ester and L-valine are subjected to condensation reaction in a solvent, and the pH is controlled at 8.5-9.5 during the reaction to obtain L-Fmoc-aspartic acid δ-tert-butyl ester-valine active ester; after further activation, it is condensed with L-tert-butoxytyrosine to obtain a protected aspartic acid-tripeptide, and Fmoc is removed by a secondary amine to obtain a de-Fmoc tripeptide;

[0007] Step 3), the protected arginine-lysine dipeptide active ester is subjected to a condensation reaction with the Fmoc-free tripeptide, and the pH is controlled at 8.5-9.5 during the reaction to obtain a thymopentin precursor;

[0008] Step 4), the thymopentin precursor is de-Fmoced by secondary amine (DEA, piperidine, etc.), and trifluoroacetic acid is used for deprotection to obtain thymopentin;

[0009] Furthermore, in step 1), the molar ratio of the L-arginine active ester with protected amine and guanidinium groups to the L-lysine with protected ε-amine group is 1:1 to 1:1.5.

[0010] Furthermore, in step 2), the molar ratio of the L-Fomc-aspartic acid δ-tert-butyl ester active ester to L-valine is 1:1~1:1.5; the molar ratio of the L-Fmoc-aspartic acid δ-tert-butyl ester-valine active ester to L-tert-butoxytyrosine is 1:1~1:1.5.

[0011] Furthermore, in step 3), the molar ratio of the protected arginine-lysine dipeptide active ester to the Fmoc-free tripeptide is 1:1 to 1:1.5.

[0012] Furthermore, the R substituent group in the L-arginine active ester and L-Fomc-aspartic acid δ-tert-butyl ester active ester with protected amine and guanidine groups includes any one of phthalimide (PHT), pentafluorophenyl oxide (PFP), succinimide (Su), and N-5-norbornene-2,3-dicarboximide (NB).

[0013] Furthermore, the solvent in step 1) and step 2) is one or more of DMF, DMSO, acetonitrile, chloroform, dichloromethane, dioxane, tetrahydrofuran, methyltetrahydrofuran, and water.

[0014] Furthermore, in step 1) and step 2), the pH adjusting reagent is an inorganic acid or base; the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, lithium carbonate, sodium bicarbonate, disodium hydrogen phosphate, and potassium bicarbonate; the inorganic acid includes one or more of hydrochloric acid, sulfuric acid, and phosphoric acid.

[0015] Furthermore, in step 3), the thymopentin precursor is refined by recrystallization.

[0016] The beneficial effects of the present invention are as follows: compared with the prior art, the liquid phase preparation method of thymopentin provided by the present invention adopts segmented synthesis, has simple operation, high directional conversion rate, high synthesis efficiency and high product purity; the liquid phase synthesis does not require the protection of the amine groups of all amino acids, but only requires the protection of the amine groups of the amino acids used as the starting materials, and has the advantages of simple operation, environmental protection, safety and cost; the scale of the liquid phase synthesis is not limited by the scale of the synthesis instrument, and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the mass spectrum of the thymopentin obtained in Example 1 of the present invention.

[0018] Figure 2 This is the HPLC chart of thymopentin obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0019] The present invention is further described below by specific examples, but these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0020] Example 1 At 5~15℃, under nitrogen protection, add 7.5g Fmoc-L-Arg(Pbf)-OSu (0.01mol) to a 250mL two-necked bottle, add 100mL DMF, stir, adjust the pH to 8.5~9.5, add 4.7g (0.01mol) L-Lys(Boc)-OH, and continue stirring for 2~3h. Obtain protected arginine-lysine dipeptide. Add 1.2g (0.01mol) N-hydroxysuccinimide, add 2.1g (0.01mol) DCC, continue stirring for 2~3h, and obtain protected arginine-lysine dipeptide active ester solution for standby use.

[0021] At 5~15℃, under nitrogen protection, add 5.1g Fmoc- L-Asp(Ot-Bu)-OSu (0.01mol) to a 250mL two-necked bottle, add 100mL DMF, stir, adjust the pH to 8.5~9.5 with sodium bicarbonate, add 1.17g (0.01mol) L-valine, and continue stirring for 2~3h. Add 1.2g (0.01mol) N-hydroxysuccinimide, add 2.1g (0.01mol) DCC, add 1.8g L- Tyr (t-Bu)-OH, stir for 2h to obtain a protected Asp-Val-Tyr tripeptide solution. Add 3mL piperidine, stir for 3h to obtain a de-Fmoc Asp-Val-Tyr tripeptide solution.

[0022] The Asp-Val-Tyr tripeptide solution after Fmoc removal was added dropwise to the reserved arginine-lysine dipeptide active ester, pH was controlled at 8.5-9.5, stirred for 3 hours, poured into pure water, solid was precipitated, dried, and recrystallized with acetonitrile: water = 3:1. 10.5 g of thymopentin precursor was obtained with a yield of 85.1%.

[0023] 10.5 g of thymopentin precursor was placed in a 100 mL three-necked flask, 50 mL of dichloromethane and 3 mL of diethylamine were added, and the mixture was stirred for 2 h. The solvent and excess diethylamine were removed by distillation under reduced pressure. 60 mL of trifluoroacetic acid and 1 g of phenol were added, and the mixture was stirred for 1 h. The mixture was added to 200 mL of methyl tert-butyl ether, and a solid was precipitated. The solid was washed twice with methyl tert-butyl ether to obtain 4.77 g of thymopentin with a yield of 82.5%. The purity was 99.5%. ESI-MS (m / z): 680.5 (M + +1), such as Figure 1 The HPLC results are shown in Figure 2 The total yield of the reaction was 70.5%.

[0024] Example 2 At 5-15°C, under nitrogen protection, add 7.5 g Fmoc-L-Arg (Pbf) -OSu (0.01 mol) to a 250 mL two-necked bottle, add 100 mL DMF, stir, adjust the pH to 8.5-9.5, add 5.94.7 g (0.01 mol) L-Lys (Fmoc) -OH, continue stirring for 2-3 hours to obtain a protected arginine-lysine dipeptide. Add 1.2 g (0.01 mol) N-hydroxysuccinimide, add 1.9 g (0.01 mol) EDCI, continue stirring for 2-3 hours to obtain a protected arginine-lysine dipeptide active ester solution for use.

[0025] At 5~15℃, under nitrogen protection, add 5.1g Fmoc-Asp(Ot-Bu)-OSu (0.01mol) to a 250mL two-necked bottle, add 100mL DMF, stir, adjust the pH to 8.5~9.5, add 1.17g (0.01mol) L-valine, and continue stirring for 2~3h. Add 1.2g (0.01mol) N-hydroxysuccinimide, add 1.9g (0.01mol) EDCI, add 1.8g L-Tyr (t-Bu)-OH, stir for 2h to obtain the protected Asp-Val-Tyr tripeptide. Add 3mL piperidine, stir for 3h to obtain the de-Fmoc Asp-Val-Tyr tripeptide solution.

[0026] The de-Fmoc Asp-Val-Tyr tripeptide solution was added dropwise to the reserved arginine-lysine dipeptide active ester, the pH was controlled at 8.5-9.5, stirred for 3 hours, poured into pure water, solid was precipitated, dried, and recrystallized with acetonitrile: water = 3:1. 10.7 g of thymopentin precursor was obtained with a yield of 85.3%.

[0027] 10.7 g of thymopentin precursor was placed in a 100 mL three-necked flask, 50 mL of dichloromethane and 3 mL of diethylamine were added, and the mixture was stirred for 2 h. The solvent and excess diethylamine were removed by vacuum distillation. 60 mL of trifluoroacetic acid and 1 g of phenol were added, and the mixture was stirred for 1 h. The mixture was added to 200 mL of methyl tert-butyl ether, and solids were precipitated. The solids were washed with methyl tert-butyl ether twice to obtain 4.81 g of thymopentin with a yield of 82.8%. The total yield of the reaction was 70.6%.

[0028] Example 3 At 5~15℃, under nitrogen protection, add 8.0g Fmoc-L-Arg(Pbf)-OPHT (0.01mol) to a 250mL two-necked bottle, add 100mL DMF, stir, adjust the pH to 8.5~9.5, add 4.7g (0.01mol) L-Lys(Boc)-OH, and continue stirring for 2~3h. Obtain protected arginine-lysine dipeptide. Add 1.7g (0.01mol) N-hydroxyphthalimide, add 2.1g (0.01mol) DCC, continue stirring for 2~3h, and obtain protected arginine-lysine dipeptide active ester solution for standby use.

[0029] At 5~15℃, under nitrogen protection, add 5.1g Fmoc-Asp(Ot-Bu)-OSu (0.01mol) to a 250mL two-necked bottle, add 100mL DMF, stir, adjust the pH to 8.5~9.5, add 1.17g (0.01mol) L-valine, and continue stirring for 2~3h. Add 1.2g (0.01mol) N-hydroxysuccinimide, add 2.1g (0.01mol) DCC, add 1.8g L-Tyr (t-Bu)-OH, stir for 2h to obtain the protected Asp-Val-Tyr tripeptide. Add 3mL piperidine, stir for 3h to obtain the de-Fmoc Asp-Val-Tyr tripeptide solution.

[0030] The de-Fmoc Asp-Val-Tyr tripeptide solution was added dropwise to the reserved arginine-lysine dipeptide active ester, the pH was controlled at 8.5-9.5, stirred for 3 hours, poured into pure water, solid was precipitated, dried, and recrystallized with acetonitrile: water = 3:1. 10.9 g of thymopentin precursor was obtained with a yield of 85.4%.

[0031] Place 10.9 g of thymopentin precursor in a 100 mL three-necked flask, add 50 mL of dichloromethane and 3 mL of diethylamine, stir for 2 h, and remove the solvent and excess diethylamine by vacuum distillation. Add 60 mL of trifluoroacetic acid, add 1 g of phenol, stir for 1 h, add to 200 mL of methyl tert-butyl ether, precipitate solid, wash twice with methyl tert-butyl ether, and obtain 4.76 g of thymopentin with a yield of 82.4%. The total yield of the reaction is 70.3%.

[0032] Example 4 At 5~15℃, under nitrogen protection, add 7.5g Fmoc-L-Arg(Pbf)-OSu (0.01mol) to a 250mL two-necked bottle, add 100mL DMF, stir, adjust the pH to 8.5~9.5, add 5.9g (0.01mol) L-Lys(Fmoc)-OH, and continue stirring for 2~3h. Obtain arginine-lysine dipeptide with protective group. Add 1.9g (0.01mol) pentafluorophenol (PFP), add 2.1g (0.01mol) DCC, continue stirring for 2~3h, and obtain the protected arginine-lysine dipeptide active ester solution for standby use.

[0033] At 5~15℃, under nitrogen protection, add 5.1g Fmoc-Asp(Ot-Bu)-OSu (0.01mol) to a 250mL two-necked bottle, add 100mL DMF, stir, adjust the pH to 8.5~9.5, add 1.17g (0.01mol) L-valine, and continue stirring for 2~3h. Add 1.9g (0.01mol) pentafluorophenol (PFP), add 2.1g (0.01mol) DCC, add 1.8g L- Tyr (t-Bu)-OH, stir for 2h to obtain the protected Asp-Val-Tyr tripeptide. Add 3ml diethylamine, stir for 3h to obtain the de-Fmoc Asp-Val-Tyr tripeptide solution.

[0034] The Asp-Val-Tyr tripeptide solution after Fmoc removal was added dropwise to the reserved arginine-lysine dipeptide active ester, pH was controlled at 8.5-9.5, stirred for 3 hours, poured into pure water, solid was precipitated, dried, and recrystallized with acetonitrile: water = 3:1. 10.8 g of thymopentin precursor was obtained with a yield of 85.4%.

[0035] 10.5 g of thymopentin precursor was placed in a 100 mL three-necked flask, 50 mL of dichloromethane and 3 mL of diethylamine were added, and the mixture was stirred for 2 h. The solvent and excess diethylamine were removed by vacuum distillation, 60 mL of trifluoroacetic acid and 1 g of phenol were added, and the mixture was stirred for 1 h. The mixture was added to 200 mL of methyl tert-butyl ether, and solids were precipitated. The solids were washed twice with methyl tert-butyl ether to obtain 4.77 g of thymopentin with a yield of 82.5%. The total yield of the reaction was 70.5%.

[0036] The above implementation modes are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention. The patent protection scope of the present invention should be defined by the claims.

Claims

1. A liquid phase preparation method of thymopentin, characterized in that: The preparation method mainly comprises the following steps: Step 1), the L-arginine active ester with protected amine and guanidine groups and the L-lysine with protected ε-amine group are subjected to condensation reaction in a solvent, and the pH is controlled at 8.5-9.5 during the reaction process; a protected arginine-lysine dipeptide is obtained, and further activation is performed to obtain a protected arginine-lysine dipeptide active ester; Step 2), L-Fomc-aspartic acid δ-tert-butyl ester active ester and L-valine are subjected to condensation reaction in a solvent, and the pH is controlled at 8.5-9.5 during the reaction to obtain L-Fmoc-aspartic acid δ-tert-butyl ester-valine active ester; after further activation, it is condensed with L-tert-butoxytyrosine to obtain a protected aspartic acid-tripeptide, and Fmoc is removed by secondary amine to obtain a de-Fmoc tripeptide; Step 3), the protected arginine-lysine dipeptide active ester is subjected to a condensation reaction with the Fmoc-free tripeptide, and the pH is controlled at 8.5-9.5 during the reaction to obtain a thymopentin precursor; Step 4), the thymopentin precursor is de-Fmoced by secondary amine and deprotected by trifluoroacetic acid to obtain thymopentin; 。 2. The liquid phase preparation method of thymopentin according to claim 1, characterized in that: In step 1), the molar ratio of the L-arginine active ester with protected amine and guanidinium groups to the L-lysine with protected ε-amine group is 1:1 to 1:1.

5.

3. The liquid phase preparation method of thymopentin according to claim 1, characterized in that: In step 2), the molar ratio of the L-Fomc-aspartic acid δ-tert-butyl ester active ester to L-valine is 1:1-1:1.5; the molar ratio of the L-Fmoc-aspartic acid δ-tert-butyl ester-valine active ester to L-tert-butoxytyrosine is 1:1-1:1.

5.

4. The liquid phase preparation method of thymopentin according to claim 1, characterized in that: In step 3), the molar ratio of the protected arginine-lysine dipeptide active ester to the Fmoc-free tripeptide is 1:1 to 1:1.

5.

5. The liquid phase preparation method of thymopentin according to claim 1, characterized in that: The R substituent group in the L-arginine active ester and L-Fomc-aspartic acid δ-tert-butyl ester active ester with protected amine and guanidine groups includes any one of phthalimide (PHT), pentafluorophenyl oxide (PFP), succinimide (Su), and N-5-norbornene-2,3-dicarboximide (NB).

6. The liquid phase preparation method of thymopentin according to claim 1, characterized in that: The solvent in step 1) and step 2) is one or more of DMF, DMSO, acetonitrile, chloroform, dichloromethane, dioxane, tetrahydrofuran, methyltetrahydrofuran and water.

7. The liquid phase preparation method of thymopentin according to claim 1, characterized in that: In the steps 1) and 2), the pH adjusting reagent is an inorganic acid or base; the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, lithium carbonate, sodium bicarbonate, disodium hydrogen phosphate, and potassium bicarbonate; the inorganic acid includes one or more of hydrochloric acid, sulfuric acid, and phosphoric acid.

8. The liquid phase preparation method of thymopentin according to claim 1, characterized in that: In step 3), the thymopentin precursor is purified by recrystallization.

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