CN101195653B - solid-liquid synthesizing method for leuprorelin - Google Patents
solid-liquid synthesizing method for leuprorelin Download PDFInfo
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- CN101195653B CN101195653B CN200610119341A CN200610119341A CN101195653B CN 101195653 B CN101195653 B CN 101195653B CN 200610119341 A CN200610119341 A CN 200610119341A CN 200610119341 A CN200610119341 A CN 200610119341A CN 101195653 B CN101195653 B CN 101195653B
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Abstract
The invention relates to a synthesis method of leuprorelin, which can resolve the problems that prior art uses solid-phase synthesis which cuts leuprorelin from resin unsafely, and has long liquid phase synthesis time. The invention comprises a, solid-phase synthesizing whole-protected peptide chain PGIuR-8, b, synthesizing material Pro-NHEt. HCI, c, condensing liquid-phase fractions to synthesize whole-protected PGIuP-9-NHEt via using DIC or BOP to react for 24-36h at 10-30DEG C, d, removing protection to obtain crude leuprorelin. The invention can synthesize polypeptide whose C end is amide.
Description
Technical field: the present invention relates to a kind of synthetic method of Leuprolide, particularly a kind of solid-liquid synthesis method of Leuprolide.
Background technology: leuprorelin acetate is the high reactivity derivative that short corpus luteum generates releasing hormone (LH-RH), and it is widely used in male patients such as female patients such as treating the preceding mammary cancer of precocity, endometriosis, hysteromyoma or menopause and prostate cancer.In report in the past, there is solid-phase synthesis to synthesize Leuprolide (ref:Tetrahedron.53,9,1997,3179-3194), promptly adopt class Merrifield resin, begin preceding four amino acid from carbon teminal and adopt the BOC method, back four amino acid adopt the Fmoc method to connect successively, finally use ethamine liquid to cut down from resin.Yet use the cutting of liquid ethamine, need under low temperature, band pressure sealing, carry out, inconvenient in suitability for industrialized production, be difficult to scale operation.We have researched and developed the synthetic method that this solid-liquid combines from this angle; polypeptide fragments by the synthetic full guard of Fmoc solid phase method; finish the synthetic of peptide chain by liquid phase segment synthesis method again; both kept the quick of solid phase synthesis; can under condition as mild as a dove, cut again, just with large-scale preparation.
Summary of the invention: the purpose of this invention is to provide a kind of method of synthetic Leuprolide, solving at present, the employing solid-phase synthesis uses liquid ethamine to cut inconvenient, dangerous and adopts the long problem of liquid phase segment generated time.This practical technical scheme comprises the steps:
1. selecting Arg (pbf) Trt (2-Cl)-Cl Resin for use is the full guard polypeptide of PGlu-His-Trp-Ser-Tyr-(D)-Leu-Leu-Arg by the solid-phase synthesis composition sequence in organic solvent.Organic solvent is methylene dichloride, N, a kind of in dinethylformamide and the N,N-dimethylacetamide.
2.BOC-Pro-OH be dissolved in the appropriate amount of organic, add NMM or Et
3N adds Vinyl chloroformate at-10 ℃～-15 ℃ and stirs 15～30min, adds ethylamine hydrochloride and NMM or Et
3N, the stirred overnight at room temperature aftertreatment obtains BOC-Pro-NHEt.Synthetic BOC-Pro-NHEt obtains H-Pro-NHEtHCl through 2mol/L dioxane hydrogen chloride gas deprotection.Organic solvent is tetrahydrofuran (THF) and N, a kind of in the dinethylformamide.
3. full guard polypeptide PGluR-8 and H-Pro-NHEtHCl are dissolved in the appropriate amount of organic; add HOBt and NMM or DIEA, ice bath stirs 5～15min down, adds BOP or DIC; remove ice bath behind 15～30min, 10 ℃-30 ℃ are stirred the PGluP-9-NHEt that obtains full guard after 24-36 hour.Organic solvent is N, a kind of in dinethylformamide and the N,N-dimethylacetamide solvent.
Full guard peptide PGluP-9-NHEt be dissolved in the cutting liquid (V/V%, trifluoracetic acid: p-cresol: water: thioanisole: mercaptan is 82.5: 5: 5: 5: 2.5) obtain the Leuprolide crude product through deprotection.
Some abbreviations commonly used have following implication among the present invention:
BOC-Pro-OH:N-tertbutyloxycarbonyl-proline(Pro)
DMF:N, dinethylformamide
The HOBt:1-hydroxybenzotriazole
The NMM:N-methylmorpholine
BOC-Pro-NHEt:N-tertbutyloxycarbonyl-proline(Pro) ethamine
H-Pro-NHEt: proline(Pro) ethamine
BOP: block special condensing agent
Et
3N: triethylamine
PGlu: Pyrrolidonecarboxylic acid
His: Histidine
Trp: tryptophane
Ser: Serine
Tyr: tyrosine
(D)-the Leu:D-leucine
Leu: leucine
Arg: arginine
Pbf:2,2,4,6,7-pentamethyl--2H-cumarone-5-alkylsulfonyl
Trt (2-Cl)-ClResin:2-chloro-triphenyl chlorine resin
Fomc-Leu-OH:N-fluorenylmethyloxycarbonyl-leucine
Collidine: trimethylpyridine
DIC: di-isopropyl carbodiimide
DIEA: diisopropylethylamine
Piperide: piperidines
Fmoc: fluorenylmethyloxycarbonyl
TFA: trifluoracetic acid
CH
2Cl
2: methylene dichloride
TBu: the tertiary butyl
Trt: triphenyl
PGluR-8: sequence is the abbreviation of PGlu-His-Trp-Ser-Tyr-(D)-Leu-Leu-Arg octapeptide
PGluP-9-NHEt: sequence is the abbreviation of PGlu-His-Trp-Ser-Tyr-(D)-Leu-Leu-Arg-Pro-NHEt nonapeptide
Synthetic route is as follows:
The invention has the beneficial effects as follows: the present invention had both avoided pure solid phase synthesis inconvenient, dangerous from the resin cutting by the synthetic Leuprolide of solid-liquid combination, and it is loaded down with trivial details to have simplified pure liquid phase segment synthetic again, has shortened generated time.
Embodiment:
The present invention is described in further detail hereinafter with reference to example, but the invention is not restricted to this specific examples.
Embodiment 1, with reference to synthetic route, 5g Arg (pbf) Trt (2-Cl)-Cl Resin (load:0.4mmol/g) places and connects the peptide bottle and be immersed in DMF solution, add Fomc-Leu-OH (mol ratio 1: 3), DIC (mol ratio 1: 1), HOBT (mol ratio 1: 1), collidine (mol ratio 1: 1), in shaking table, behind the concussion 40min, wash 3 times CH with DMF under 25 ℃
2Cl
2Wash 3 times, slough Fmoc with 20%piperide-DMF solution again, use the same method afterwards and react successively in the PGLu connection, use 1%TFA-CH
2Cl
2The solution cutting obtains 3gPGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-(D)-Leu-Leu-Arg (Pbf)-OH.
BOC-Pro-OH13g (60mmol) is dissolved in the 100ml tetrahydrofuran (THF), add NMM 6.6ml (60mmol), add Vinyl chloroformate 6ml (60mmol) at-10 ℃～-15 ℃, after stirring 15～30min, add ethylamine hydrochloride 4.8g (60mmol), stirred overnight at room temperature, aftertreatment obtain compd B OC-Pro-NHEt.Synthetic BOC-Pro-NHEt 6g is dissolved among the 50mlDMF through polypeptide PGluR-8 (1mmol) and the Pro-NHEtHCl (1.1mmol) that 2mol/L dioxane hydrogen chloride gas deprotection obtains the H-Pro-NHEtHCl. full guard; add HOBt (1mmol); NMM (2mmol); ice bath stirs 5～15min; add DIC (1mmol); remove ice bath behind the 20min; 25 ℃ of stirrings aftertreatment in 34 hours obtains full guard peptide PGluP-9-NHEt. full guard peptide PGluP-9-NHEt and is dissolved in cutting liquid (V/V%; trifluoracetic acid: p-cresol: water: thioanisole: mercaptan is 82.5: 5: 5: 5: 2.5) obtain Leuprolide crude product 650mg. Leuprolide crude product through deprotection and obtain leuprorelin acetate 200mg, HPLC98%. through high-efficient liquid phase chromatogram purification
Embodiment 2; the polypeptide PGluR-8 (2mmol) and the Pro-NHEtHCl (2.2mmol) of full guard are dissolved among the 100mlDMF; add HOBt (2mmol); DIEA (4mmol); ice bath stirs 5～15min; add BOP (2mmol); remove ice bath behind the 25min; 15 ℃ are stirred aftertreatment in 28 hours and obtain full guard peptide PGluP-9-NHEt; full guard peptide PGluP-9-NHEt is dissolved in cutting liquid (V/V%, trifluoracetic acid: p-cresol: water: thioanisole: mercaptan is 82.5: 5: 5: 5: 2.5) and obtains Leuprolide crude product 1400mg through deprotection.All the other are identical with embodiment 1.
Claims (1)
1. the solid-liquid synthesis method of Leuprolide may further comprise the steps:
The octapeptide PGluR-8 of a. synthetic full guard: selecting Arg (pbf) Trt (2-Cl)-Cl Resin for use is the full guard octapeptide PGluR-8 of PGlu-His-Trp-Ser-Tyr-(D)-Leu-Leu-Arg by the solid-phase synthesis composition sequence in the DMF solvent;
B. be raw material proline biosynthesis ethylamine hydrochloride with N-tertbutyloxycarbonyl-proline(Pro): N-tertbutyloxycarbonyl-proline(Pro) is dissolved in an amount of tetrahydrofuran solvent, add N-methylmorpholine or triethylamine, add Vinyl chloroformate at-10 ℃～-15 ℃ and stir 15～30min, add a kind of in ethylamine hydrochloride and N-methylmorpholine and the triethylamine, the stirred overnight at room temperature aftertreatment obtains N-tertbutyloxycarbonyl-proline(Pro) ethamine, and synthetic N-tertbutyloxycarbonyl-proline(Pro) ethamine obtains the proline(Pro) ethylamine hydrochloride through 2mol/L dioxane hydrogen chloride gas deprotection;
C. full guard octapeptide PGluR-8 and proline(Pro) ethylamine hydrochloride are dissolved in an amount of DMF solvent, add a kind of in I-hydroxybenzotriazole and N-methylmorpholine and the diisopropylethylamine, ice bath stirs 5～15min down, add BOP or DIC as condensing agent, remove ice bath behind 15～30min, 10 ℃-30 ℃ are stirred the nonapeptide PGluP-9-NHEt that obtains full guard after 24-36 hour;
D. full guard nonapeptide PGluP-9-NHEt is dissolved in cutting liquid and obtains the Leuprolide crude product through deprotection, and the volume ratio of cutting liquid each component is a trifluoracetic acid: p-cresol: water: thioanisole: mercaptan is 82.5: 5: 5: 5: 2.5.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101538315B (en) * | 2009-01-13 | 2012-11-28 | 深圳翰宇药业股份有限公司 | Method for preparing Leuprorelin by combination of solid phase method and liquid phase method |
| RU2527454C1 (en) * | 2013-05-23 | 2014-08-27 | Закрытое Акционерное Общество "Фарм-Синтез" | Method of producing l-proline ethylamide hydrochloride |
| CN106146622A (en) * | 2015-04-03 | 2016-11-23 | 中肽生化有限公司 | A kind of industrialized process for preparing of leuprorelin acetate |
| CN105330726A (en) * | 2015-11-05 | 2016-02-17 | 江苏诺泰生物制药股份有限公司 | Leuprorelin synthesis method |
Citations (4)
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|---|---|---|---|---|
| EP1207167A1 (en) * | 1999-06-30 | 2002-05-22 | Takeda Chemical Industries, Ltd. | Process for the preparation of lh-rh derivatives |
| CN1450906A (en) * | 1999-07-07 | 2003-10-22 | 特莱默里斯公司 | Methods and compositions for peptide synthesis |
| CN1513872A (en) * | 2003-06-10 | 2004-07-21 | 兰州大学 | Liquid phase sgnthesis method of thymus pentapeptide |
| CN1865280A (en) * | 2005-05-20 | 2006-11-22 | 周达明 | Solid phase polypeptide synthesis preparation method for leuprorelin |
-
2006
- 2006-12-08 CN CN200610119341A patent/CN101195653B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1207167A1 (en) * | 1999-06-30 | 2002-05-22 | Takeda Chemical Industries, Ltd. | Process for the preparation of lh-rh derivatives |
| CN1450906A (en) * | 1999-07-07 | 2003-10-22 | 特莱默里斯公司 | Methods and compositions for peptide synthesis |
| CN1513872A (en) * | 2003-06-10 | 2004-07-21 | 兰州大学 | Liquid phase sgnthesis method of thymus pentapeptide |
| CN1865280A (en) * | 2005-05-20 | 2006-11-22 | 周达明 | Solid phase polypeptide synthesis preparation method for leuprorelin |
Non-Patent Citations (8)
| Title |
|---|
| Ernesto Nicolas et al.The use of the Nbb-resin for the solid-phase synthesis ofpeptide alkylesters and alkylamides.synthesis of leuprolide.Tetrahedron53 9.1997,53(9),3179-3194. |
| Ernesto Nicolas et al.The use of the Nbb-resin for the solid-phase synthesis ofpeptide alkylesters and alkylamides.synthesis of leuprolide.Tetrahedron53 9.1997,53(9),3179-3194. * |
| 中国科学院上海生物化学研究所多肽激素组.固相法合成促黄体生成素释放激素及其类似物.生物化学与生物物理学报8 2.1976,8(2),121-128. |
| 中国科学院上海生物化学研究所多肽激素组.固相法合成促黄体生成素释放激素及其类似物.生物化学与生物物理学报8 2.1976,8(2),121-128. * |
| 于丽君等.奥曲肽的液相合成.内蒙古民族大学学报（自然科学版）20 1.2005,20(1),38-40. |
| 于丽君等.奥曲肽的液相合成.内蒙古民族大学学报(自然科学版)20 1.2005,20(1),38-40. * |
| 王德心.固相肽合成中保护-裂解方式的研究进展.化学通报 2.1991,(2),1-7. |
| 王德心.固相肽合成中保护-裂解方式的研究进展.化学通报 2.1991,(2),1-7. * |
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Inventor after: Xu Hongyan Inventor after: Zhu Qi Inventor after: Zhou Min Inventor after: Jiang Ting Inventor before: Xu Hongyan Inventor before: Zhu Qi |
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