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Polypeptide liquid-phase synthesizing method of oxytocin

A technology of oxytocin and polypeptide liquid, which is applied in the field of polypeptide liquid phase synthesis, can solve the problems of difficult to control reaction conditions, low yield and titer of oxytocin products, and achieves low synthesis cost, high titer and high purity. Effect

Active Publication Date: 2017-07-21
LANZHOU KAIBO PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] In summary, there are still a series of challenges in the synthesis of oxytocin at home and abroad, such as the use of precursor chemicals such as piperidine and ether, highly toxic and expensive reagents such as thallium trifluoroacetate, and difficult-to-control reactions. conditions (such as metal sodium / liquid ammonia, HF, etc.), and the yield and potency of the synthetic oxytocin product are relatively low

Method used

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  • Polypeptide liquid-phase synthesizing method of oxytocin
  • Polypeptide liquid-phase synthesizing method of oxytocin
  • Polypeptide liquid-phase synthesizing method of oxytocin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Synthetic Fragment 1: Boc-Cys(Acm)-Tyr(tBu)-OH

[0084] Dissolve Boc-Cys(Acm)-OH (100mol, 29.2g, 1eq.), HOSu (110mmol, 12.6g, 1.1eq.) in 200ml of tetrahydrofuran, cool at -10°C for 10min, DCC (110mmol, 22.7g, 1.1eq.) was dissolved in 20ml THF, added to the above solution, reacted for 20min, weighed H-Tyr(tBu)-OH (110mmol, 26.1g, 1.1eq.) and NaHCO 3 (110mmol, 9.2g, 1.1eq.) was dissolved in 150ml of water and added to the above solution. React at 15-25°C, monitor the reaction by HPLC, adjust the pH to neutral with 0.5M hydrochloric acid, filter out the insoluble matter, concentrate to remove THF, adjust the pH to 2 with 0.5M hydrochloric acid to obtain a white solid, filter, wash with water until neutral, and dry in vacuo Fragment 1 was obtained with a purity of 99.1%, 47.1 g of the product, and a yield of 92%. Fragment 1: Boc-Cys(Acm)-Tyr(tBu)-OH RP-HPLC spectrum see attached figure 1 , its high acid cleavage sample (H-Cys(Acm)-Tyr-OH m / z calculated.355.4; found 356.3...

Embodiment 2

[0086] Synthetic Fragment 2: H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe

[0087] (1) Synthesis of Compound 2.1: Fmoc-Cys(Acm)-Pro-OMe:

[0088] Dissolve Fmoc-Cys(Acm)-OH (100mmol, 41.5g, 1eq.), HOBt (105mmol, 14.1g, 1.05eq.) in 200ml THF and 30ml DMF, cool at -10°C for 10min, DCC (105mmol, 21.6g, 1.05eq.) was dissolved in 20ml THF, added to the above solution, and after reacting for 20min, weigh H-Pro-OMe. , 1.05eq.), mix well and add to the above reaction solution. Reaction at 15-25°C, HPLC monitoring for complete reaction, filtration to remove insoluble matter, concentration, precipitation of solid with 0.1M hydrochloric acid, filtration, washing to neutrality, vacuum drying to obtain compound 2.1, purity 95.3%, product 50.9g, yield 97 % (yields are all calculated on the basis of 100 mmol, the same below). See the attached manual for the HPLC analysis chart image 3 .

[0089] (2) Synthesis of Compound 2.2: H-Cys(Acm)-Pro-OMe:

[0090] The dried compound 2.1 was dissolve...

Embodiment 3

[0104] Synthesis of Fragment 3: H-Leu-Gly-NH according to Scheme 2 2

[0105] Weigh Fmoc-Leu-OH (1eq., 100mmol, 35.3g), BOP (1.01eq., 101mmol, 44.7g) dissolved in DMF200ml, add DIPEA (1.2eq., 120mmol, 21ml), after 5min, called H-Gly -NH 2 .HCl (1.1eq., 110mmol, 12.2g) was dissolved in 100ml of DMF, added TEA (1.1eq., 110mmol, 15ml), poured into the above reaction solution, followed by TLC (DCM:MeOH:AcOH=100:6:1) After monitoring, after the reaction is complete, precipitate with 0.1M HCl solution, wash with water until neutral, and dry in vacuum to obtain the product Fmoc-Leu-Gly-NH 2 .HCl, purity 97%, product 43.7g, yield 98%, HPLC analysis spectrum see attached Figure 10 .

[0106] Dry Fmoc-Leu-Gly-NH 2 Add 400ml of diethylamine to HCl, track and monitor with TLC (DCM:MeOH:AcOH=100:6:1), after the reaction is complete, concentrate, precipitate petroleum ether, filter, wash with petroleum ether, and dry in vacuo to obtain Fragment 3 with a purity of 98.4% , product 17.9...

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Abstract

The invention discloses a liquid-phase synthesizing method of oxytocin for the first time. The method which is mild in reaction conditions is characterized in that three oxytocin fragments which include Boc-Cys(Acm)-Tyr(tBu)-OH, H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe and H-Leu-Gly-NH2 are synthesized for the first time, the fragments are assembled to synthesize an all-protected oxytocin amino acid sequence, iodine is used to remove Acm while cyclization is performed to form a disulfide bond to obtain protected cyclic oxytocin, trifluoroacetic acid is used to remove residual protecting groups to obtain crude oxytocin, ethyl acetate is used to perform recrystallization, and reversed-phase chromatography purification is performed to obtain high-purity (crude product purity reaches 95%) and high-titer oxytocin (588IU / mg). The liquid-phase synthesizing method has the advantages that a Boc polypeptide synthesizing method and an Fmoc polypeptide synthesizing method are used in a combined manner, all reactions are performed under mild conditions, and the ammonia-sodium-method decapping reaction reported in all domestic and abroad oxytocin liquid-phase synthesizing literature, the synthesizing cost of the oxytocin is lowered greatly, and a method is provided for the industrial production of the oxytocin.

Description

technical field [0001] The invention relates to the preparation of polypeptide drugs, in particular to a method for liquid-phase synthesis of oxytocin, which belongs to the technical field of polypeptide liquid-phase synthesis. Background technique [0002] Oxytocin, also called oxytocin, English name oxytocin, molecular formula C 43 h 66 N 12 o 12 S 2 , the molecular weight is 1007.2, and the chemical structural formula is as follows: [0003] [0004] Oxytocin is mainly distributed in the posterior lobe of the hypothalamus and pituitary in humans and mammals, and is clinically used for labor induction, postpartum hemorrhage and labor induction. With the gradual deepening of its research, in addition to the common effects of oxytocin on uterine contraction and lactation, it has been found that oxytocin also has many complex physiological functions, such as participating in learning and memory processes, affecting drug addiction and Social adaptive behavior, materna...

Claims

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Application Information

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IPC IPC(8): C07K7/16C07K1/20C07K1/06C07K1/02
CPCC07K7/16C07K5/06043C07K5/0606Y02P20/55
Inventor 孙鹏程胡碧煌
Owner LANZHOU KAIBO PHARMA
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