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Synthesis method of bremelanotide acetate

A technology of bremelanotide acetate and its synthesis method, which is applied in the field of polypeptide production, can solve problems such as large steric hindrance of peptide chains, difficulty in forming rings, and restrictions on the application of bremelanotide, so as to improve the purification yield and purity, and achieve chromatographic Good symmetry of peak shape and improved separation selectivity

Active Publication Date: 2022-01-04
浙江湃肽生物股份有限公司南京分公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The cyclic peptide structure of bremelanotide makes its preparation difficult. Currently, the synthesis technology of bremelanotide can be divided into two categories according to the cyclization method. One is liquid phase cyclization, but it is easy to produce a large number of molecules in the liquid phase. Inter-coupling products produce polymers such as dimers and trimers, resulting in low synthesis yield and low purity. In order to suppress the occurrence of side reactions, it is necessary to reduce the reaction concentration, but this will generate a large amount of waste water. It is beneficial to environmental protection; the other is solid-phase cyclization. In the process of solid-phase cyclization, the peptide chain is difficult to fold to end-to-end due to too much steric hindrance, which leads to difficulty in cyclization and low synthesis efficiency.
[0004] Due to problems such as low synthesis yield and difficult purification in the synthesis process of bremelanotide, the application of bremelanotide is severely limited

Method used

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  • Synthesis method of bremelanotide acetate
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  • Synthesis method of bremelanotide acetate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] A preparation method of RinkAmide resin modified by 4-trifluoromethylmandelic acid:

[0077] 1) Rinse 100 g of RinkAmide resin (with a substitution degree of 0.45 mmol / g and a cross-linking degree of 1%) with DCM for 5 times, soak in 500 mL of DCM for 20 min to fully swell, and add the resin to 500 mL of piperidine / DCM mixed solution (the mass percentage of piperidine is 25%), stirred and reacted for 30min to de-Fmoc protect the resin;

[0078] 2) Add 22 g of 4-trifluoromethylmandelic acid and 25.4 g of HOBt into 300 mLDCM and stir to dissolve, pre-cool at 0°C for 10 min, add 15.2 g of DIC and stir for 10 min, then add resin, stir at room temperature for 2.5 h, filter , washed with DCM 6 times, that is.

[0079] The RinkAmide resin modified by RinkAmide resin and 4-trifluoromethylmandelic acid is carried out to infrared spectroscopic analysis, and the measured results are as follows: figure 1 shown.

[0080] observe figure 1 , A represents the unmodified RinkAmide ...

Embodiment 2

[0082] A preparation method of bremelanotide resin, said method comprising:

[0083] 1) Preparation of Fmoc-Lys(Boc)-resin:

[0084] Using the RinkAmide resin modified with 4-trifluoromethylmandelic acid obtained in Example 1 as a carrier, the resin was added to the piperidine / DCM mixed solution (the mass percentage of piperidine was 25%) and reacted for 20 min, rinsed with DCM 5 times; mix 70.2g Fmoc-Lys(Boc)-OH with 36g HOBt, 300 mLDCM, stir to dissolve, pre-cool at 0°C for 10 minutes, add 21.5g DIC and stir for 8 minutes, then add to the resin, stir and react for 10 minutes , add 2 gDMAP, stir and react at room temperature for 3.5h, add 46 g acetic anhydride, 35.5 g pyridine, react at room temperature for 40 min, wash 6 times with DCM, and obtain Fmoc-Lys(Boc)-resin shown in formula (1) ,

[0085] (1);

[0086] 2) Preparation of Fmoc-Trp(Boc)-Lys(Boc)-resin:

[0087] The Fmoc-Lys(Boc)-resin obtained in step 1) was added to the piperidine / DCM mixed solution (the mass ...

Embodiment 3

[0110] A kind of preparation method of bremelanotide crude product:

[0111] 1) Cracking: Mix TFA:TIS:deionized water according to the mass ratio of 93:3:4 to obtain a cracking agent, pre-cool at -20°C for 1 h, add the bremelanotide resin obtained in Example 2 to 1 L cracking agent solution, reacted at 0°C for 1 h, raised to room temperature and reacted for 3 h to remove the resin, filtered off the resin, and added the filtrate to 14 L pre-frozen methyl tert-butyl ether under stirring conditions, a large amount of white matter was precipitated, centrifuged, and collected The resulting solid was centrifuged, washed with methyl tert-butyl ether until neutral, and dried;

[0112] 2) Ester bond hydrolysis: Step 1) Dissolve the product in 20wt% acetonitrile aqueous solution, adjust pH=12 with 1 mol / L NaOH aqueous solution under ice-bath stirring condition, react at 0°C for 0.5 h, raise to 30°C to continue reaction 1 h, centrifuge to remove the precipitated white substance, adjust ...

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Abstract

The invention provides a synthesis method of bremelanotide acetate, and belongs to the technical field of preparation methods of polypeptides. The method particularly comprises the following steps: by adopting an Fmoc solid-phase polypeptide synthesis method, by using RinkAmide resin as a carrier, performing modifying with 4-trifluoromethyl mandelic acid, and according to a polypeptide sequence of the bremelanotide acetate under the action of a coupling agent, sequentially condensing amino acids with an Fmoc-protecting group from a C end to an N end to obtain linear peptide resin; and sequentially removing side chain protecting agents such as the Fmoc protecting group, Lys and Asp, then forming a ring on the resin under the action of the coupling agent, splitting the resin by using a splitting agent, breaking an ester bond under an alkaline condition to obtain crude bremelanotide, and finally, performing purifying to obtain refined bremelanotide. The method is simple, and the obtained bremelanotide acetate is high in yield and high in purification efficiency.

Description

technical field [0001] The invention belongs to the technical field of polypeptide preparation, and in particular relates to a synthesis method of bremelanotide acetate. Background technique [0002] Bremelanotide, also known as bremelanotide, is an alpha-melanocyte stimulating hormone analog with a cyclic heptapeptide structure. Bremelanotide is a drug for the treatment of female sexual dysfunction. The mechanism of action is to regulate sexual desire and sexual response by activating endogenous pathways in the brain, helping premenopausal women with hypoactive sexual desire disorder to maintain normal libido . Bremelanotide has completed Phase III clinical research and has broad market prospects. [0003] The cyclic peptide structure of bremelanotide makes its preparation difficult. Currently, the synthesis technology of bremelanotide can be divided into two categories according to the cyclization method. One is liquid phase cyclization, but it is easy to produce a large...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/64C07K1/08C07K1/06C07K1/04C07K1/16C07K1/18
CPCC07K7/64Y02P20/55
Inventor 刘志国刘慧敏张飞华
Owner 浙江湃肽生物股份有限公司南京分公司
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