Method for synthesizing leuprorelin by solid phase and liquid phase

A leuprolide and liquid-phase technology, applied in the field of synthesizing leuprolide, can solve the problems of secondary conformation change, low yield of HPLC chromatographic purification product, influence on molecular activity, etc., and achieve stable reaction and high yield , the effect of less side effects

Inactive Publication Date: 2016-06-01
WUXI ASIAPEPTIDE BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] The purpose of the present invention is to provide a high yield, low cost, mild reaction conditions, less environmental pollution, leuprolide synthetic method that is conducive to the realization of industrialization, mainly to solve the existing tryptophan in the synthesis of leuprolide The serine fragment will have a secondary conformation change, which will affect the molecular activity and lead to the technical problem of low yield of HPLC chromatographic purification products

Method used

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  • Method for synthesizing leuprorelin by solid phase and liquid phase
  • Method for synthesizing leuprorelin by solid phase and liquid phase
  • Method for synthesizing leuprorelin by solid phase and liquid phase

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Experimental program
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Embodiment 1

[0036] Embodiment 1: refer to figure 1 , Fmoc-Pro-resin synthesis:

[0037] Put 1000g of 2-CTC resin with a substitution degree of 0.4mmol in the reactor, wash with DCM for 2-3 times, and filter to dry; dissolve 1.2mol of Fmoc-Pro-OH and 2mol of DIEA in DMF, stir and vibrate for 10min, and add to the reactor , mixed with the resin in the reactor, and shaken for 2 hours; after the end, add anhydrous methanol and continue to shake for 30 minutes; after the reaction, filter the reaction solution, and wash the resin alternately with DCM, DMF and anhydrous methanol for 2 to 3 times; The product, Fmoc-Pro-resin, was obtained.

Embodiment 2

[0038] Embodiment 2: refer to figure 1 , Fmoc-Pro-resin synthesis:

[0039] Put 1000 g of 2-CTC resin with a substitution degree of 0.5 mmol into the reactor, wash with DCM for 2 to 3 times, and filter to dry; dissolve 1.5 mol of Fmoc-Pro-OH and 2.5 mol of DIEA in DMF, stir and vibrate for 10 min, and add to the reaction Mix with the resin in the reactor and shake for 2 hours; after the end, add anhydrous methanol and continue to shake for 30 minutes; after the reaction, filter the reaction solution, and wash the resin alternately with DCM, DMF and anhydrous methanol for 2 to 3 times ; Obtain the product namely Fmoc-Pro-resin.

Embodiment 3

[0040] Embodiment 3: refer to figure 1 , the growth of the peptide chain

[0041] Remove the Fmoc protecting group from the Fmoc-Pro-resin twice, each time with 500mL of 20% (V / V) piperidine / DMF for 5 and 15 minutes, and finally wash the resin alternately with DCM, DMF and anhydrous methanol for 2-3 Again, using HBTU / HOBt / DIEA as condensation reagent, repeat the two-step reaction of condensation and deprotection according to the sequence of amino acids in leuprolide from carboxy-terminus to amino-terminus, and assemble Fmoc-Arg(Pbf)-OH sequentially at room temperature , Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Trp(Boc)-Ser(Psi(Me,Me)pro)-OH, Fmoc-His(Trt) -OH, H-Pyr-OH amino acid excess multiple is 1.5 times. During the condensation process, the qualitative color development (chloranil method) was used to detect the progress of the reaction and the Fmoc absorbance method was used to quantitatively detect the condensation efficiency.

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Abstract

The invention relates to synthesis of leuprorelin, in particular to a method for synthesizing leuprorelin by a solid phase and a liquid phase, and mainly solves the technical problem that as tryptophan serine fragments in the conventional leuprorelin synthesis process has the probability of secondary conformation changes, the molecular activity is influenced to result in low yield of HPLC chromatography purification products. The method comprises the following steps: (1) enabling reaction between Fmoc-Pro-OH and 2-chlorotrityl choloride resin with the substitution degree of 0.4-0.6 mmol to produce Fmoc-Pro-resin; (2) sequentially coupling amino acids to form a solid-phase polypeptide; (3) cutting to obtain an all-protected polypeptide sequence; (4) performing liquid-phase condensation on all-protected polypeptides and ethylamine, closing the polypeptide carboxyl terminal, and performing side chain removal protection to obtain a leuprorelin crude peptide; (5) adopting efficient counter-current chromatography extraction, and then freeze-drying to obtain leuprorelin pure peptide.

Description

technical field [0001] The invention belongs to the technical field of pharmacy, and in particular relates to a process for synthesizing leuprolide combined with a solid phase and a liquid phase. Background technique [0002] Leuprorelin, English name: Leuprorelin, CAS: 53714-56-0, molecular formula: C 59 h 84 N 16 o 12 , molecular weight: 1209.42. [0003] The structural formula is as follows: [0004] [0005] Leuprolide is a GnRH analogue. The effect is the same as that of buserelin. Repeated administration of large doses of luteinizing-releasing hormone (LH-RH) or its highly active derivative leuprolide acetate can produce a transient excitatory effect on the pituitary-gonadal system immediately after the first administration (acute effect), It then inhibits the production and release of gonadotropins by the pituitary gland. It also further suppresses the response of the ovaries and testes to gonadotropins, thereby reducing the production of estradiol and test...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/23C07K1/06C07K1/04C07K1/02
CPCC07K7/23
Inventor 徐红岩冯庆贺
Owner WUXI ASIAPEPTIDE BIOTECH
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