RU2086561C1 - Method for production of nonapeptide ethyl amide - Google Patents

Abstract

FIELD: production of nonapeptide ethyl amide having high LH-RH/FSH-RH activity. SUBSTANCE: compound having formula

is prepared by condensation of peptide fragments having formula

and formula H-Tyr-D-Ser(Bu^t)-Leu-Arg-Pro-NH-C₂H₅. The process is followed by purification and isolation. EFFECT: improved efficiency.

Description

 The invention relates to the chemistry of peptides, specifically to methods for producing nonapeptidethylamide having strong LH-RH / FSH-RH activity.

Нонапептидэтиламид представляет собой аналог гормона LH-RH [1] в котором глицин в положении 6 заменен на О-трет.бутил-D-серин, глицинамид в положении 10 отсутствует, а L-пролин в положении 9 представлен как L-пролинэтиамид

Нонапептидэтиламид формулы 1 (бусерелин) имеет овуляторную активность, в 120 раз большую, чем у гормона LH-RH [2] проявляет активность в отличие от гормона LH-RH при оральном применении, что позволяет использовать это соединение более широко в различных препаративных формах [3 8]
В патенте [1] указано, что аналоги гормона LH-RH могут быть получены либо конденсацией пептидных фрагментов согласно схеме
1-3 + 4-9(4-10) или 1-2 + 3-9(3-10)
либо постадийным синтезом.The releasing hormone LH-RH / FSH-RH, which causes the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), has the structure

Nonapeptidethylamide is an analogue of the hormone LH-RH [1] in which the glycine at position 6 is replaced by O-tert-butyl-D-serine, the glycine is at position 10, and the L-proline at position 9 is represented as L-prolinethiamide

Nonapeptidethylamide of formula 1 (buserelin) has an ovulatory activity 120 times greater than that of the hormone LH-RH [2] it is active in contrast to the hormone LH-RH when administered orally, which allows the use of this compound more widely in various formulations [3 8]
In the patent [1] it is indicated that the analogues of the hormone LH-RH can be obtained either by condensation of peptide fragments according to the scheme
1-3 + 4-9 (4-10) or 1-2 + 3-9 (3-10)
either stepwise synthesis.

с
H-Ser-Tyr-D-Ser(Bu^t-Leu-Arg-Pro-NH-C₂H₅ •2HCl
Целевой продукт очищают и выделяют в виде диацетатной соли.Nonapeptidethylamide of the formula (1) according to this patent is specifically obtained in the form of diacetate by condensation of peptide fragments according to the scheme
3 + 6,
namely, the interaction

from
H-Ser-Tyr-D-Ser (Bu ^t -Leu-Arg-Pro-NH-C ₂ H ₅ • 2HCl
The target product is purified and isolated in the form of a diacetate salt.

 The disadvantage of this method is the low yield (25%) of the target product at the last stage of synthesis, as well as the use of technologically complex azide condensation at this stage.

подвергают взаимодействию с гептапептидом формулы
H-Trp-Ser-Tyr-D-Ser(Bu^t)-Leu-Arg-Pro-NH-C₂H₅ •2HCl
в среде диметилацетамида в присутствии дициклогексилкарбодиимида и 3-гидроокиси-4-оксо-3,4-дигидро-1,2,3-бензотриазина (или 1-оксибензотриазола). Защитные группы удаляют и целевой продукт очищают и выделяют в виде моноацетатной соли [9]
Недостатком данного способа получения нонапептидэтиламида формулы (1) является низкий выход (30%) целевого продукта на последней стадии синтеза, кроме того, использование Dnp-защищенного гистидина увеличивает общее количество стадий синтеза.The closest in technical essence and the achieved result to the proposed method is a method for producing nonapeptide ethylamide of the formula [1] in the form of a monoacetate of condensation of peptide fragments according to scheme 2 + 7, according to which the dipeptide of the formula

subjected to interaction with a heptapeptide of the formula
H-Trp-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NH-C ₂ H ₅ • 2HCl
in dimethylacetamide in the presence of dicyclohexylcarbodiimide and 3-hydroxide-4-oxo-3,4-dihydro-1,2,3-benzotriazine (or 1-hydroxybenzotriazole). The protective groups are removed and the target product is purified and isolated in the form of a monoacetate salt [9]
The disadvantage of this method of obtaining nonapeptidethylamide of the formula (1) is the low yield (30%) of the target product in the last stage of synthesis, in addition, the use of Dnp-protected histidine increases the total number of synthesis stages.

 The technical result of the invention is to simplify the process and increase the yield of the target product (30 62%).

конденсирует с пентапептидом формулы (Ш),
H-Tyr-D-Ser(Bu^t)-Leu-Arg-Pro-NH-C₂H₅ (III),
полученным каталитическим гидрогенолизом защищенного пентапептида формулы (IV)
Z-Tyr(Bzl)-D-Ser(Bu^t)-Leu-Arg-Pro-NH-C₂H₅ (IV),
где Z карбобензоксигруппа;
Bzl бензильная группа.The proposed method that solves this problem is the condensation of peptide fragments of the compounds of formula (1) according to the scheme
4 + 5,
according to which the tetrapeptide of the formula (II)

condenses with a pentapeptide of the formula (III),
H-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NH-C ₂ H ₅ (III),
obtained by catalytic hydrogenolysis of a protected pentapeptide of the formula (IV)
Z-Tyr (Bzl) -D-Ser (Bu ^t ) -Leu-Arg-Pro-NH-C ₂ H ₅ (IV),
where Z is a carbobenzoxy group;
Bzl is a benzyl group.

 In the condensation of tetrapeptide (II) and pentapeptide (III), condensation methods common in peptide synthesis, for example, the carbodiimide method, can be used.

The tetrapeptide of formula (II) is known and is used in the synthesis of polypeptides in [10]
The known pentapeptide (III) [11] used in the method is obtained by catalytic hydrogenolysis of a new pentapeptide of the formula (IV). The pentapeptide (IV) can be obtained by conventional methods of peptide synthesis, for example, stepwise condensation of amino acids using activated esters.

 In this case, the pentapeptide of formula (IV) is prepared starting from free arginine by sequential addition of activated esters of L-leucine, D- (O-tert. Butyl) -serine, L- (O-benzyl) tyrosine in the form of the corresponding tertes . butyloxycarbonyl- or carbobenzoxy derivatives, N-protecting groups from the obtained di- and tripeptides are removed by catalytic hydrogenolysis and the obtained protected tetrapeptide is condensed with prolineethylamide.

 Through the use of readily available, obtained with a high yield of pentapeptide of the formula (IV) in the proposed method, it is possible to obtain the target product with a higher yield.

 Example.

a) Tert. butyloxycarbonyl-leucyl-arginine
To a solution of 24 g (68 mol) of p-nitrophenyl ether tert. butyloxycarbonyl-leucine in 70 ml of dimethylformamide add 11.3 g of arginine (65 mmol) and mix until the precipitate disappears. The reaction mixture was evaporated in vacuo, the residue was dissolved in 100 ml of isopropyl alcohol and precipitated into the product by adding 800 ml of ether. The precipitate formed is filtered off, washed with ether and dried. Yield 23.5 g (60.7 mmol) (93%). Rf = 0.78 (1).

b) Carbobenzoxy-O-tert-butyl-D-serial-leucyl-arginine
Dissolve 23 g (59.4 mmol) of tert-butyloxycarbonyl-leucyl-argenine in 250 ml of methanol, add 1.5 g of 10% palladium-carbon and hydrogenate until the starting product disappears (chromatographic control). The precipitate was filtered off, the solution was evaporated, the residue was dissolved in 100 ml of dimethylformamide, and 28.8 g (63 mmol) of carbobenzoxy-O-tert-butyl-D-serine N-hydroxynorbornendicarboximide ester were added. After 6 hours, add 2 ml of N, N-dimethyltrimethylenediamine, after 30 minutes the reaction mixture is evaporated, the residue is dissolved in 300 ml of chloroform, washed with 150 ml of 3% sulfuric acid, 200 ml of water, evaporated to dryness. The residue is triturated with ether, filtered, dried. Yield 29.1 g (51.6 mmol) (86.8%). Rf = 0.82 (1).

c) Carbobenzoxy-O-benzyl-tyrosyl-O-tert.butyl-D-serial-leucyl-arginine
28.5 g (50.5 mmol) of carbobenzoxy-O-tert-butyl-D-seryl-leucyl-arginine (b) are dissolved in 300 ml of methanol and hydrogenated with a palladium catalyst (see b). The precipitate was filtered off, the solution was evaporated to dryness, the residue was dissolved in 100 ml of dimethylformamide and 28.5 g (54 mmol) of carbobenzoxy-O-benzyl-tyrosine n-nitrophenyl ether was added. After a day, the reaction mixture is treated similarly to (b) and 34.1 g (41.7 mmol) (82.5%) of the product are isolated. Rf = 0.86 (1), 0.33 (2).

g) Carbobenzoxy-O-benzyl-tyrazyl-O-tert. butyl-D-seryl-leucyl-arginyl-proline-ethylamide
16.7 g (20.4 mmol) of product (c) are dissolved in 200 ml of dimethylformamide, 3.15 g (22.2 mmol) of proline ethylamide, 6.75 g (45 mmol) of N-hydroxybenzotriadol and 2.55 ml are added. 8M perchloric acid. About 30 ml of solvent is evaporated in vacuo and 6.3 g (30 mmol) of dicyclohexylcarbodiimide are added to the solution. The reaction mixture was stirred for 20 hours at room temperature, 80 ml of water was added, the precipitate was filtered off, the solution was evaporated to dryness in vacuo, the residue was stirred with 300 ml of ethyl acetate and 200 ml of water. The organic layer was separated, washed with 200 ml of water, evaporated and the residue was triturated with ether. The resulting precipitate was filtered off, washed on the filter with ether and dried in vacuo. Yield 15.7 g (16.7 mmol) (81.8%). Rf = 0.47 (2).

d) Carbobenzoxy-tryptophil-serine
6.12 g (58.3 mmol) of serine is dissolved in 29 ml of a 2N sodium hydroxide solution, 60 ml of dimethylformamide are added and a solution of 24.8 g (54 mmol) of carbobenzoxy-tryptophan p-nitrophenyl ether in 90 ml is added dropwise. dimethylformamide. The reaction mixture was stirred for another 4 hours, evaporated, 200 ml of ethyl acetate and 300 ml of water were added to the residue. The aqueous layer was separated, acidified with 10% sulfuric acid to pH 3 and extracted twice with 200 ml of ethyl acetate. The extract was washed with 100 ml of water, evaporated to dryness, the residue was crystallized from ether. The precipitated crystals are filtered off and dried. Yield 19.9 g (45.8 mmol) (86.7%).

e) Carbobenzoxy-histidyl-tryptophil-serine sodium salt
13.2 g (31.2 mmol) (d) are dissolved in 200 ml of methanol and hydrogenated with a palladium catalyst. 15.6 ml of 2N are added to the filtered solution. sodium hydroxide solution, the solution was evaporated and the residue was dissolved in 100 ml of dimethylformamide. A solution of carbobenzoxy-histidine azide in ethyl acetate is prepared according to the standard procedure from 11.5 g (38 mmol) of carbobenzoxy-histidine hydrazide and mixed with an amine component solution at 0 ^° C. After a day, the reaction mixture is evaporated, the residue is dissolved in 300 ml of ethyl acetate, washed 2 times 100 ml of 10% sodium sulfate, evaporated to a small volume and the sodium salt of the tripeptide precipitated by the slow addition of hexane. Yield 14.3 g (24.6 mmol) (78.8%). Rf = 0.41 (2).

g) Pyroglutamyl-histidyl-tryptophil-serine sodium salt
12.8 g (22 mmol) (e) are dissolved in 200 ml of methanol and hydrogenated with a palladium catalyst. The solution was evaporated, the precipitate was dissolved in 100 ml of dimethylformamide and 7.22 g (24.5 mmol) of pyroglutamic acid pentafluorophenyl ester were added. After 2 hours, the solution was evaporated in vacuo, 200 ml of ether and 150 ml of water were added, the organic layer was separated, the aqueous solution was saturated with sodium sulfate, the product was extracted three times with 200 ml of n-butanol. The combined organic extract was washed with 50 ml of a saturated sodium sulfate solution, dried, evaporated to dryness, the residue was triturated with ether. Yield 10.3 g (17.9 mmol) (81.3). Rf = 0.41 (1), 0.13 (2).

h) Pyroglutamyl-histidyl-tryptophil-seryl-tyrosyl-O-tert. butyl-D-seryl-leucyl-arginyl-proline-ethylamide acetate (buserelin acetate)
8.26 g (10.1 mmol) (g) are hydrogenated over a palladium catalyst in methanol, the precipitate is filtered off, the solution is evaporated to dryness, the residue is dissolved in 120 ml of dimethylformamide, 3.37 g (25 mmol) of N-hydroxybenzotriazole are added, 6.80 g (11.8 mmol) (g) and 1.37 ml of 8 N. chloric acid. About 20 ml of solvent are evaporated in vacuo, cooled to 0 ^° C., 3.78 g (18 mmol) of dicyclohexylcarbodiimide are added, the reaction mixture is stirred for 3 hours at 0 ^° C. and 1.66 ml (15 mmol) of N-methylmorpholine is added. The reaction mixture was kept at room temperature for one day, evaporated in vacuo to a small volume, 100 ml of water was added, the precipitate was filtered off, the solution was evaporated to dryness and triturated with ether. The crude product yield was 9.91 g. For purification, the product was dissolved in 0.05 M pyridine acetate buffer, applied to a 50x600 mm SP-Sephadex column and eluted in a 0.05-1 M pyridine acetate buffer gradient. The separated fractions were evaporated, dissolved in 2% acetic acid and lyophilized. Yield 8.27 g (62.3%). Rf = 0.53 (1). Amino acid analysis: Glu 0.87 (1), His 0.93 (1), Trp 0.76 (1), Ser 1.76 (2), Tyr 0.93 (1), Leu 1.00 (1) Arg 1.06 (1), Pro 1.11 (1).

Thin-layer chromatography is performed on Merck Kieselgel-60 plates in systems:
(1) Chloroform-methanol-acetic acid-water 60: 45: 6: 14
(2) Ethyl acetate-pyridine-acetic acid-water 120: 20: 6: 11
The manifestation is carried out by treatment with chlorine and then with a solution of tolidine.

Claims (1)

The method of obtaining nonapeptidethylamide of the formula I

condensation of peptide fragments, followed by purification and isolation in the form of monoacetate, characterized in that the tetrapeptide of formula II

subjected to condensation with a pentapeptide of the formula III
NTug-D-Ser (Bu ^t ) -Ley-Arg-Pro-NHC ₂ H ₅
obtained by catalytic hydrogenolysis of a protected pentapeptide of the formula IV
Z-Tyr (BZI) -Ser (in Y ^t) -Ley-Arg-Pro-NHC ₂ H _5.