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CN-122011092-A - Method for preparing Pal-GQPR by LPPS

CN122011092ACN 122011092 ACN122011092 ACN 122011092ACN-122011092-A

Abstract

The present invention relates to a method for preparing palmitoyl tetrapeptid-7 by liquid-phase peptide synthesis, comprising: step 1, obtaining palmitoyl chloride from palmitic acid through acyl chlorination, and then generating Pal Gly OH with glycine through substitution reaction; Step 1 ', synthesizing N-tert-butoxycarbonyl-glutamyl-proline benzyl ester from N-tert-butoxycarbonyl-glutamine and proline benzyl ester hydrochloride through dipeptide condensation, and obtaining glutamyl-proline benzyl ester hydrochloride (H-Gln-Pro-OBzl. HCl) through deprotection; Step 2: Pal Gly OH is condensed with H Gln Pro OBzl HCl to obtain palmitoyl glycyl glutamyl proline benzyl ester (Pal Gly Gln Pro OBzl) through tripeptide condensation; Step 3, remove the protective group through hydrolysis reaction to prepare palmitoyl glycidyl glutamyl proline (Pal Gly Gln Pro OH); And step 4, through ester condensation, Pal Gly Gln Pro OH is prepared into the active ester Pal Gly Gln Pro OSu, and then through tetrapeptide condensation, Pal Gly Gln Pro OSu is combined with arginine to obtain palmitoyl tetrapeptid-7.

Inventors

  • CHEN YANLI
  • LIU YULONG
  • WANG DAYANG
  • SUN LIQUAN
  • WANG SIDA
  • FENG YANSHU
  • GAO HANRONG

Assignees

  • 富乐马鸿凯（大连）医药有限公司

Dates

Publication Date
20260512
Application Date
20260122

Claims (9)

  1. 1. A method for preparing palmitoyl tetrapeptide-7 and salts thereof by Liquid Phase Polypeptide Synthesis (LPPS), wherein the method comprises: Step 1, obtaining palmitoyl chloride (Pal-Cl) from palmitic acid (Pal) through acyl chlorination reaction, and then generating palmitoyl glycine (Pal-Gly-OH) with glycine (Gly) through substitution reaction; Step 1'. N-t-butoxycarbonyl-glutaminyl-prolinyl benzyl ester (Boc-Gln-Pro-OBzl) is synthesized from N-t-butoxycarbonyl-glutamine (Boc-Gln-OH) and prolinyl benzyl ester hydrochloride (H-Pro-OBzl. HCl) by dipeptide condensation reaction, and glutaminyl-prolinyl benzyl ester hydrochloride (H-Gln-Pro-OBzl. HCl) is obtained by deprotection; Step 2, obtaining palmitoyl-glycyl-glutaminyl-proline benzyl ester (Pal-Gly-Gln-Pro-OBzl) from Pal-Gly-OH and H-Gln-Pro-OBzl.HCl by tripeptide condensation reaction; step 3, removing the protecting group by hydrolysis reaction to prepare palmitoyl-glycyl-glutaminyl-proline (Pal-Gly-Gln-Pro-OH), and Step 4, preparing Pal-Gly-Gln-Pro-OH into active ester palmitoyl-glycyl-glutaminyl-proline succinimidyl ester (Pal-Gly-Gln-Pro-OSu) by ester condensation reaction, and obtaining palmitoyl tetrapeptide-7 (Pal-Gly-Gln-Pro-Arg-OH) from Pal-Gly-Gln-Pro-OSu and arginine (Arg) by tetrapeptide condensation reaction.
  2. 2. The process according to claim 1, wherein the acylation reaction is carried out in the presence of an acylating agent selected from one or more of thionyl chloride, oxalyl chloride, phosphorus oxychloride and phosphorus pentachloride, preferably thionyl chloride, and at a first temperature of from 20 ℃ to 60 ℃, preferably from 40 ℃ to 50 ℃, and wherein the first solvent is selected from one or more of toluene, n-heptane and methyl tert-butyl ether, preferably n-heptane.
  3. 3. The process according to any one of claims 1 to 2, wherein the substitution reaction is carried out at a pH of 9 to 12, preferably 10 "11, at a temperature of 0 to 30 ℃, preferably 0 to 10 ℃, and the molar ratio of palmitoyl chloride (Pal-Cl) to glycine (Gly) is 1 (1.0 to 2), preferably the molar ratio is 1:1.5.
  4. 4. A process according to any one of claims 1 to 3, wherein the dipeptide condensation reaction is carried out in the presence of a first condensation reagent, and the first condensation reagent is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu and DSC, preferably HBTU.
  5. 5. The process according to any one of claims 1 to 4, wherein the deprotection is carried out in the presence of trifluoroacetic acid, an aqueous solution of hydrogen chloride, an acetonitrile solution of hydrogen chloride, an ethyl acetate solution of hydrogen chloride, preferably in the presence of an acetonitrile solution of hydrogen chloride.
  6. 6. The process according to any one of claims 1 to 5, wherein the tripeptide condensation reaction is performed in the presence of a second condensing agent and the second condensing agent is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu and DSC, preferably HBTU.
  7. 7. The process according to any one of claims 1 to 6, wherein the hydrolysis reaction is carried out in the presence of one or more of lithium hydroxide, sodium hydroxide and potassium hydroxide, preferably the hydrolysis reaction is carried out in the presence of lithium hydroxide.
  8. 8. The process according to any one of claims 1 to 7, wherein the ester condensation reaction is carried out in the presence of a third condensing agent, and the third condensing agent is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu and DSC, preferably DSC.
  9. 9. The process according to any one of claims 1 to 8, wherein the tetrapeptide condensation reaction is carried out in the presence of a base and a second solvent selected from one or more of 2-methyltetrahydrofuran, tetrahydrofuran and acetonitrile, preferably acetonitrile, the molar ratio of H-Arg-OH to base being 1 (1.0-3.0), preferably 1:3.0.

Description

Method for preparing Pal-GQPR by LPPS Technical Field The invention belongs to the field of polypeptide compound synthesis, and in particular relates to a preparation method of palmitoyl tetrapeptide Pal-Gly-Gln-Pro-Arg-OH for avoiding self-condensation and epimerization. Background Palmitoyl tetrapeptide-7 (Pal-GQPR, the structure of which is shown in the following figures) is a lipopeptide developed by sederm, intended to mimic the effects of DHEA, restoring cytokine balance in mature skin. With age, DHEA levels decrease, leading to an increase in IL-6, an inflammatory mediator that accelerates extracellular matrix degradation, ultimately leading to wrinkles, skin laxity and decreased elasticity. Studies show that palmitoyl tetrapeptide-7 can effectively reduce IL-6 production by 40% and has a remarkable anti-inflammatory effect in ultraviolet-induced inflammation. Comparable to the anti-inflammatory capabilities of DHEA, it helps reduce skin roughness, fine lines and wrinkles, improve skin tone irregularities, and helps repair and regenerate skin. The component can be used alone or combined with palmitoyl tripeptide-1 to promote collagen generation of skin, accelerate skin repair, and improve compactness and smoothness. Therefore, the palmitoyl tetrapeptide-7 is taken as a powerful anti-inflammatory and anti-aging component, is widely applied to anti-aging skin care products, and can obviously improve the appearance and texture of skin. In the prior art, the preparation of the peptide is widely dependent on a solid phase synthesis (SPPS) process, wherein the technology is based on a solid phase carrier, peptide chains are gradually prolonged from a C end through repeated steps of amino acid fixation, deprotection, carboxyl activation and peptide bond formation, and finally the carrier connection is deprotected and cut off to obtain crude peptide, and then the crude peptide is further purified by reverse phase chromatography to meet the purity requirement. The primary SPPS process developed by early Li Qian, zhang Zhongqi and the like lays a foundation for preparing palmitoyl tetrapeptide-7, but the polypeptide has extremely poor water solubility, is easy to separate out and has low sample concentration during reversed phase purification, so that the purification efficiency is low, the purity is difficult to improve, the scale is limited, the cost of the traditional C18 filler is high, the service life is short, the production investment is promoted, the CN109748948B replaces C18 with polystyrene divinylbenzene-based filler (such as PS 10-300), the purification and salt conversion are synchronously realized, the purity is improved to more than 98 percent, the yield reaches about 70 percent, the filler cost is reduced, but the problem of water-soluble core is not solved, the sample loading quantity can only be enlarged to 10 g grade, the scale potential is limited, the purity of CN112110984A can reach 99.66 percent by temporarily coupling hydrophilic fragments consisting of 3-6 lysines and p-hydroxybenzoic acid, the special solid phase carriers such as RINK AMIDE RESIN are needed to be additionally increased, and the cleavage resin removal process is needed, and the m-cresol sulfide (such as 2.5:2.5) is required to be used, the complex solid phase coupling and the complex process has the environmental protection risk of 2.5:2.5. In the Liquid Phase Polypeptide Synthesis (LPPS) method, intramolecular or intermolecular condensation (such as cyclization of dipeptide-dipeptide to generate DKP impurity) is easy to occur during the synthesis of Pal-Gly-Gln-OH, which results in reduced yield, and in the condensation process, configuration inversion occurs, epimerization (racemization) is generated, which seriously affects the optical purity of the product. For example, CN112830956A discloses a process for preparing palmitoyl tetrapeptide-7 (Pal-GQPR). The core is that the preparation of the target product is realized through 7 key synthesis steps (including synthesis of Boc-Pro-OH, synthesis of Pal-Gly-Gln-ONp and the like) and the refining process. However, in this method, pal-Gly-Gln-ONp is synthesized first and then condensed with H-Pro-Arg-OH. Since the Gln residue directly participates in the amide bond formation reaction, the α -position configuration is easily inverted under the activation and basic conditions, thereby causing racemization, and thus the racemization risk of the condensation stage of the Gln residue cannot be avoided. Therefore, a new synthetic route is highly demanded which can simultaneously inhibit self-condensation and racemization of Gln site in Pal-Gly-Gln-OH stage and has industrial feasibility. Disclosure of Invention In order to solve the problems, the application aims to provide an economic and environment-friendly method for commercially synthesizing Pal-GQPR. In the present application, the first amino acid to be used as a carboxyl group in the condensation step is either not ra