Industrial preparation method of leuprolide acetate

A technology for leuprolide and amino acids, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve problems such as poor product quality stability, racemization, serious environmental pollution, etc., and reduce the possibility of racemization Sexuality and other side effects, suppression of by-products, and safety-enhancing effects

Inactive Publication Date: 2016-11-23
CHINESE PEPTIDE CO
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Patent CN 102464702 B discloses the Boc synthesis method of leuprolide acetate, which adopts multiple Boc amino acids without side chain protecting groups, such as Boc-Arg-OH, Boc-Tyr-OH, etc. The side chain reaction by-products caused by the partial nucleophilicity of the group, such as the by-products of the guanidinium amidation of the Arg side chain, etc.
The yield of leuprolide acetate synthesized by this process is low, and it is not suitable for industrialized large-scale production
[0004] Patent CN 1865280 B discloses the Fmoc synthesis method of leuprolide. This method needs to use a large amount of very toxic deprotection reagents such as hexahydropyridine, trifluoroacetic acid, ethanethiol and phenol, which seriously pollutes the environment. In addition, the amino acid linkage The reaction needs to be carried out at 20 to 30°C, which is prone to side reactions such as racemization, and the yield is low
[0005] Compared with the solid-phase synthesis method, the solid-liquid phase combination method and liquid-phase synthesis method of leuprolide reported in other invention patents and literatures have complicated processes and are not suitable for large-scale industrial production
[0006] In addition, in the prior art, leuprolide is usually synthesized with CTC resin, Wang resin and Cl-CH 2 Resin is the initial solid phase carrier, the quality stability of the obtained product is poor, and the optical rotation does not meet the requirements.

Method used

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  • Industrial preparation method of leuprolide acetate
  • Industrial preparation method of leuprolide acetate
  • Industrial preparation method of leuprolide acetate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Synthesis of Resin Boc-L-Pro PAM Resin

[0022] Put PAM Resin (12.5g, degree of substitution 0.80mmol / g) in the reactor, dissolve 4.3g Boc-L-Pro-OH with 100mL of DCM, then add it into the reactor, keep stirring, and then slowly add 6.5mL of pyridine , then slowly add 5.8 mL of 2,6-dichlorobenzoyl chloride, stir for 3-6 hours, drain the filtrate, add 2.5 times the volume of the resin in DCM, wash twice for 3 minutes each time, add 2.5 times the volume of the resin in DMF, Wash 2 times, 3 minutes each time, add 2.5 times the resin volume of MeOH, wash 2 times, 3 minutes each time. Vacuum dry, first air dry for 20-30 minutes, then vacuum dry for more than 12 hours, then it can be used in step 2.

Embodiment 2

[0024] Polypeptides were synthesized and purified using Boc-L-Pro-PAM Resin.

[0025] Step 1. Boc Pro-PAM Resin resin (15.4g; degree of substitution 0.65mmol / g) is placed in the reactor, swelled with 150mL of DCM for not less than 2h, then washed twice with DCM of 2.5 times the volume of the resin, each about 2 minutes each time. The solution was removed by suction filtration. It can be used for step 2.

[0026]Step 2. Add 2.5 times the resin volume of 50% TFA / DCM solution, stir for 30±5min, remove the reaction solution by suction filtration, add 2.5 times the resin volume of DCM to the above resin to wash for 2min, remove the reaction solution, add 2.5 times the resin Wash with 5% TEA / DCM by volume for 2 minutes, remove the reaction solution, add 2.5 times the volume of the resin to wash with MeOH for 2 minutes, remove the reaction solution, add 2.5 times the volume of the resin to wash with 5% TEA / DCM for 2 minutes, remove the reaction solution, add Wash with 2.5 times th...

Embodiment 3

[0056] Polypeptides were synthesized and purified using Boc-L-Pro-PAM Resin.

[0057] Step 1. Boc-Pro-PAM Resin resin (2155.3 g, 1250 mmol) was placed in the reactor, and 20 L of DCM was added to swell for 2 hours and 6 minutes. Wash 2 times with DCM, about 2 minutes each. Remove the solution by suction filtration, which can be used in step 2

[0058] Step 2. Add 30L of 50% TFA / DCM, react for 30 minutes, remove the reaction solution by suction filtration, and then wash repeatedly with DCM, 5% TEA / DCM, methanol, and DMF in sequence, and the ninhydrin test is positive.

[0059] Weigh 1216.6g of Boc-Arg(Mtr)-OH and 337.9g ​​of HOBt, then dissolve them in 2.3L of DMF, cool in an ice bath to less than 10°C, add 392mL of DIC to activate, activate for 23min, activate at a temperature of 14.8°C, and react for 2 hours and 40 Minutes later, the ninhydrin test was negative. The reaction solution was removed by suction filtration, and then washed repeatedly with DMF, methanol, and DCM ...

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Abstract

The invention discloses a method suitable for preparing leuprolide industrially on a large scale. According to the method, PAM resin is adopted as a solid-phase carrier, amino acid is sequentially coupled through a Boc and Fmoc combined solid-phase synthesis method, and fully-protected leuprolide precursor peptide resin is obtained. A low-temperature connecting method and side chain protection are adopted in the amino acid coupling process, and racemization and side reactions on a side chain are reduced. Through an ethylamine reaction on the peptide resin, fully-protected leuprolide crude peptide is directly obtained, a side chain protecting group is removed with a trifluoroacetic acid solution, and leuprolide crude peptide is obtained. Leuprolide acetate is obtained after purification through reversion phase chromatography, salt change and freeze drying. The method is easy and convenient to operate and high in synthesis efficiency, the total yield is 36%, the product purity reaches 99% or above, pollution to the environment is reduced, and the safety of the preparation process is improved.

Description

technical field [0001] The invention belongs to the technical field of pharmacy, and in particular relates to a new industrial preparation process of leuprolide and leuprolide acetate. Background technique [0002] Leuprolide is a highly active derivative of luteinizing-releasing hormone (LH-RH), which can produce pituitary-gonadal system stimulation (acute effect) immediately after administration, and then inhibit pituitary production and release of gonadotropins. It also further suppresses the response of the ovaries and testes to gonadotropins, thereby reducing the production of estradiol and testosterone (chronic effect). Therefore, it can effectively inhibit the function of the pituitary-gonadal system. Leuprolide acetate is widely used to treat female patients such as precocious puberty, endometriosis, uterine fibroids or premenopausal breast cancer and male patients such as prostate cancer. [0003] Patent CN 102464702 B discloses the Boc synthesis method of leuprol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/23C07K1/06C07K1/04
CPCY02P20/55
Inventor 李湘徐琪
Owner CHINESE PEPTIDE CO
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