RU2444525C2 - Method for synthesis nonapeptide ethylamide and intemediate compounds for synthesis thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of nonapeptide ethylamide, having strong LH-RH/FSH-RH activity, of formula pGlu-His-Trp-Ser-Tyr-D-Ser(Bu^t)-Leu-Arg-Pro-NH-C₂H₅·2AcOH (I), and intermediate compounds for synthesis thereof. The nonapeptide ethylamide is obtained via condensation of a C-terminal tetrapeptide of formula H-D-Ser(Bu^t)-Leu-Arg-Pro-NH-C₂H₅·HCl (II) with a dipeptide of formula: X-Ser-Tyr-OH (IV), where X is a protective group. The obtained N-substituted hexapeptide ethylamide of formula X-Ser-Tyr-D-Ser(Bu^t)-Leu-Arg-Pro-NH-C₂H₅·HCl (III) is treated with an unblocking agent to remove the N-protective group, and then condensed with a tripeptide of formula pGlu-His-Trp-OH·HCl (V) and the end product is purified through chromatography and extracted in form of a monoacetate salt.

EFFECT: high output.

4 cl, 1 ex

Description

The invention relates to the field of pharmaceutical chemistry, namely to the chemistry of peptides and more specifically to a new method for producing nonapeptide ethylamide of the formula

and intermediates for its preparation.

Nonapeptidethylamide I is known as Buserelin and is a synthetic analogue of natural gonadotropin releasing hormone with strong LH-RH / FSH-RH activity (Sandow J., Konig W. Studies with Fragments a Highly active Analogues of Luteinizing Hormone - Releasing Hormone, J. Endocrinol, 1979, 81 (2), 175-182) [1].

From RU No. 2086561, 08/10/1997 (see [2]), a known method for producing nonapeptide ethylamide having strong LH-RH / FSH-RH activity, of the formula

Nonapeptidethylamide is obtained by condensation of peptide fragments 1-4 of the formula

and 5-10 formulas

H-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NH-C ₂ H ₅

followed by purification and isolation.

The method is carried out according to the following scheme 4 + 5, according to which the tetrapeptide of the formula (II)

condensed with a pentapeptide of the formula (III)

,

,

obtained by catalytic hydrogenolysis of a protected pentapeptide of the formula (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.

The known method allows to obtain the final product with a high yield, but does not provide a high yield of intermediate products in separate stages.

The closest in technical essence and the achieved result to the proposed method is a method for producing nonapeptide ethylamide of the formula (I) condensation of peptide fragments according to the scheme 3 + 6 by the azide method, according to which the tripeptide of the formula pGlu-His-Trp-NH-NH ₂ is reacted with a hexapeptide of the formula H -Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NH-C ₂ H ₅ · 2HCl in dimethylformamide at -30 ° C in the presence of tert-butyl nitrite and 6 N HCl. The target product is purified and isolated in the form of a monoacetate salt (Konig W, Geiger R., Sandow JK Peptides having LH-RH / FSH-RH activity. US Patent No. 4024248 (1977) [3].

A disadvantage of the known method for producing buserelin is the low yield of the target product and the need to use the azide method in the last stage. The azide method involves the use of highly toxic compounds such as tert-butyl nitrite. In addition, the synthesis of peptides by the azide method is a lengthy process that is carried out under strictly controlled low temperature conditions (see Peptides. The main methods for the formation of peptide bonds. M: Mir, 1983) [4].

All this makes the known method unsuitable for producing buserelin on an industrial scale.

The technical task of the claimed method is to simplify the process of producing buserelin and increase the yield of the final product. The problem is achieved by the claimed group of inventions.

The problem is achieved by the described method, according to which the synthesis of buserelin is carried out by condensation of the C-terminal tetrapeptide of the formula

with a dipeptide of the formula X-Ser-Tyr-OH,

where X is a protective group,

the resulting N-protected hexapeptide ethylamide of the formula

, treated with a deblocking agent to remove the N-protecting group, and then condensed with a tripeptide of the formula

,

 the final product is purified by chromatography and isolated as monoacetate (or monoacetate salt).

The invention also relates to intermediates used to produce nonapeptidethylamide by the claimed method.

So, the claimed invention also relates to an N-protected dipeptide of the formula (IV)

,

,

where Z is benzyloxycarbonyl,

as an intermediate for the synthesis of nonapeptidethylamide of the formula (I)

The group of the claimed invention also includes another intermediate compound - a tripeptide of the formula (V)

as an intermediate for the synthesis of nonapeptidethylamide of the formula (I)

Moreover, X in the formula of the dipeptide (IV) is benzyloxycarbonyl, in particular, it is Z in the formula of the N-protected dipeptide of the formula (IV).

The release of N-protected hexapeptidethylamide is carried out by catalytic hydrogenation in the presence of 5% Pd / C (palladium on charcoal).

List of abbreviations

Boc - tert-butyloxycarbonyl

Z - benzyloxycarbonyl

DCC - N, N'-Dicyclohexylcarbodiimide

DCCU - N, N'-Dicyclohexylurea

DMF - N, N-dimethylformamide

NMM - N-methylmorpholine

HONB - N-hydroxy-5-nonbornen-2,3-dicarboxyimide

THF - tetrahydrofuran

HOBt - 1-hydroxybenzotriazole

BSA - N, O-bis-trimethylsilylacetamide

MeOH - methanol

HPLC - High Performance Liquid Chromatography

TLC - thin layer chromatography

PE - petroleum ether

EA - ethyl acetate

experimental part

The synthesis uses derivatives of L-amino acids from Bachem (Switzerland), DCC, HONB, HOBt, BSA from Fluka (Switzerland). DMF is purified by distillation over ninhydrin and barium oxide. Analytical HPLC is carried out on a chromatograph company Gilson (France).

TLC was performed on Merck-Kiselgel glass chromatographic plates (Germany).

¹ H-NMR spectra were recorded on a WH-500 Bruker 500 MHz spectrometer (Germany) in DMSO-d ₆ at 300 K. Chemical shifts (δ, ppm) were measured relative to tetramethylsilane. Mass spectra were recorded on an Analytical Compact MALDI 4 mass spectrometer from Kratos (Great Britain).

The invention is illustrated by the following example.

.Example.

.

1. N ^α -tert-butyloxycarbonyl-L-leucyl-L-arginyl-L-prolineethylamide Boc-Leu-Arg-Pro-NHEt · HCl hydrochloride.

A 1 L three-necked flask equipped with a mechanical stirrer, thermometer and dropping funnel was charged with 74.4 g (0.19 mol) of Boc-Leu-Arg-OH, 35.7 g (0.2 mol) of H-Pro-NHEt · HCl, 44.1 g (0.29 mol) of HOBt · H ₂ O, 300 ml of DMF and stirred until complete dissolution. The reaction mixture is cooled in an ice bath to 0 ° C and, with vigorous stirring and a temperature of 0 - -5 ° C, a solution of 39.6 g (0.19 mol) of DCC in 50 ml is added dropwise, the reaction mass is stirred for 30 min at t = 0-5 ° C, left overnight, the precipitated DCCU was filtered off, washed with a filter of 50 ml of DMF. The filtrate was evaporated on a rotary evaporator. The residue was dissolved in 400 ml of water and left overnight in a refrigerator at t = 5 ° C, the precipitate was filtered off, the filtrate was extracted with ether (2 × 50 ml), evaporated on a rotary evaporator and 100 g of NaCl was added to the residue with stirring. The precipitated oil was extracted with chloroform (2 × 200 ml) and the extract was washed with water (3 × 40 ml). Chloroform was evaporated, the residue was dissolved in 200 ml of butanol. The solution was evaporated on a rotary evaporator, the residue was triturated with 1 L of ether, the product was filtered off, washed on the filter with 100 ml of ether and dried in air.

The result is 93.7 g (90%) of Boc-Leu-Arg-Pro-NHEt · HCl. The product is chromatographically homogeneous.

2. N-Carboxybenzoxy-O-tert-butyl-D-serine N-oxinorbornene-2,3-dicarboxyimide ester ZD-Ser (Bu ^t ) -ONB.

51.7 g (0.17 mol) of ZD-Ser (Bu ^t ) -OH, 34.4 g (0.19 mol) of HONB, 350 are charged into a 1-liter three-necked flask equipped with a mechanical stirrer, thermometer and dropping funnel. ml of ethyl acetate and stirred until a homogeneous suspension is formed. The reaction mixture is cooled on ice. bath to t = 0 ° C and with vigorous stirring, a solution of 41.3 g (0.2 mol) of DCC in 50 ml of ethyl acetate is added dropwise at a temperature of 0-5 ° C. After adding DCC, the reaction mass is stirred for 30 min at t = 0-5 ° C and left under stirring overnight. The precipitated DCCU was filtered off, washed with a filter of 50 ml ethyl acetate, the filtrate was evaporated to dryness on a rotary evaporator, 200 ml of isopropanol was added to the residue, and stirred at t = 60 ° С until complete dissolution (20 min). The solution is cooled to t = 0 ° C and stirred until a heavy precipitate forms, 200 ml of petroleum ether are added and the reaction mixture is placed overnight in a freezer at a temperature of -20 ° C. Then, the precipitate formed is filtered off, washed with 100 ml of petroleum ether on a filter, and dried in air. The result is 66.0 g (85%) of ZD-Ser (Bu ^t ) -ONB.

3. N-Carbobenzoxy-O-tert-butyl-D-seryl-L-leucyl-L-arginyl-L-prolineethylamide hydrochloride ZD-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl.

73.5 g (0.13 mol) of Boc-Leu-Arg-Pro-NHEt · HCl, 150 ml of dioxane are charged into a 500 ml single-necked flask, stirred until complete dissolution, 150 ml of 11% HCl in dioxane are added. The reaction mixture was kept for 1.5 h, stirring periodically, the precipitate formed was filtered off, washed with ether (2 × 100 ml) and dried in vacuo. The resulting product was loaded into a 500 ml two-necked flask equipped with a mechanical stirrer, 220 ml of DMF was added and stirred until complete dissolution. Add 30.8 ml (0.28 mol) of NMM, 63.9 g (0.14 mol) of ZD-Ser (Bu ^t ) -ONB, mix for 3 hours and put the reaction mixture overnight in a refrigerator at t = 5 ° C . The precipitated precipitate is filtered off with NMM · HCl. The filtrate was evaporated on a rotary evaporator to a volume of 70 ml, 70 ml of water was added, washed with ether (3 × 25 ml), (TLC 2) and evaporated on a rotary evaporator. The residue was dissolved in 150 ml of chloroform, washed with water (3 × 20 ml) and evaporated on a rotary evaporator. The residue was triturated in ether (500 ml), the ether was decanted, and the precipitate was dried in vacuo. The result is 91.4 g (96.3%) of ZD-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl. According to TLC (chloroform - methanol - acetic acid - water 30: 6: 2: 0.8), the product is chromatographically homogeneous.

4. O-tert-Butyl-D-serial-L-leucyl-L-arginyl-L-prolineethylamide hydrochloride (II).

HD-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl,

where Et is C ₂ H ₅ .

In a two-necked 1 L flask equipped with a mechanical stirrer, 91.4 g (0.13 mol) of ZD-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl were charged, 300 ml of abs. ethanol and stirred until complete dissolution. Add 0.1 g of Pd (OH) ₂ and bubble the reaction mass with gaseous hydrogen with stirring overnight. Then the catalyst is filtered off, the filtrate is evaporated on a rotary evaporator. The residue was triturated in ether (400 ml), the precipitate was filtered off, washed on the filter with 100 ml of ether and dried in air. The result is 73.9 g (96.5%) of HD-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl (II).

According to TLC (chloroform - methanol - acetic acid - water 30: 6: 2: 0.8) the product is homogeneous.

5. N-Carbobenzoxy-L-seryl-L-tyrosine (IV) Z-Ser-Tyr-OH (Z-L-Ser-L-Tyr-OH).

29.3 g (0.16 mol) of H-Tyr-OH, 109.8 g (0.54 mol) of BSA, 250 ml of methylene chloride are charged into a 1-liter three-neck flask equipped with a reflux condenser, and the reaction mixture is boiled 7 -8 hours until complete dissolution of H-Tyr-OH. The reaction mixture was cooled in an ice bath to 0 ° C and, with vigorous stirring, 70 g (0.16 mol) of Z-Ser-OPfp were sprinkled at 0-5 ° C. Then the reaction mass is stirred for 30 minutes at 0-5 ° C and left with stirring at room temperature overnight. The reaction control is carried out using TLC in the system: PZ: EA 1: 1. The reaction mixture is evaporated on a rotary evaporator (t≤40 ° C) and the residue is dissolved in 150 ml of EA, the solution is placed in an ice bath, cooled to t = 0 ° C and 50 ml of 5% H ₂ SO ₄ are added dropwise with vigorous stirring. After 10 minutes, the cooling was stopped and the reaction mixture was stirred at 30-35 ° C until a heavy, uniform precipitate formed and placed overnight in a refrigerator (5 ° C). Then the reaction mass is filtered, washed on a filter with 2 × 50 ml with a mixture of: aq. NaCl _(sat) : H ₂ O (1: 1), 50 ml of a mixture: PE: EA (3: 2) and dried in air. The result is 61.2 g (95%) of Z-Ser-Tyr-OH in the form of a chromatographically homogeneous product. TLC in the system: PE: EA 1: 1.

6. N-Carbobenzoxy-L-seryl-L-tyrosyl-O-tert-butyl-D-seryl-L-leucyl-L-arginyl-L-prolineethylamide hydrochloride (III).

H-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl, where Et is C ₂ H ₅ .

In a three-necked 1 L flask equipped with a mechanical stirrer, thermometer and dropping funnel, 73.9 g (0.12 mol) of HD-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl, 55.1 g ( 0.14 mol) Z-Ser-Tyr-OH, 27.3 g (0.18 mol) HOBt · H ₂ O, 500 ml THF, heated to 45 ° C and stirred until complete dissolution. Then the reaction mixture was cooled to room temperature and, with stirring, a solution of 28.3 g (0.14 mol) of DCC in 70 ml of THF was added dropwise over 4-5 hours. After adding DCC, the reaction mass is stirred for 1 h and placed overnight in a freezer at -20 ° C. The precipitate formed is filtered off, washed with 130 ml THF on a filter and used without drying.

7. L-Seryl-L-tyrosyl-O-tert-butyl-D-seryl-L-leucyl-L-arginyl-L-prolineethylamide hydrochloride.

H-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl, where Et is C ₂ H ₅ .

In a two-necked 1 L flask equipped with a mechanical stirrer, 105 g (0.12 mol) of Z-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl were charged, 350 ml of abs. ethanol, stirred to obtain a homogeneous suspension and filtered DCCU. Wash the filter cake with 70 ml abs. ethanol, add Pd (OH) ₂ and bubble the reaction mass with gaseous hydrogen with stirring overnight. Then the catalyst is filtered off, the filtrate is evaporated on a rotary evaporator. The residue was triturated with ethyl acetate (650 ml), filtered off, washed with a filter of 150 ml EA and dried in air. The result is 91 g (90%) of H-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl, where Et is C ₂ H ₅ .

8. L-Histidyl-L-tryptophan dihydrochloride H-His-Trp-OH · 2HCl.

124.6 g (0.23 mol) (Boc) ₂ -His-Trp-OH, 300 ml of dioxane are charged into a 1-necked 1-liter flask, and stirred until complete dissolution, the solution is placed on ice. bath and add 300 ml of 11% HCl in dioxane. The reaction mixture was kept for 1 hour on ice. bath, periodically shaking. The temperature of the reaction mixture was brought to room temperature, held for 1.5 hours, the precipitate formed was filtered off, the precipitate was triturated with 500 ml of ether, filtered off, washed with ether (2 × 100 ml) and dried in vacuo. The result is 80 g (84%) of H-His-Trp-OH · 2HCl.

9. L-Piroglutamyl-L-histidyl-L-tryptophan hydrochloride pGlu-His-Trp-OH · HCl (V).

In a two-necked 1 L flask equipped with a mechanical stirrer, 75.8 g (0.18 mol) of H-His-Trp-OH · 2HCl are charged, 400 ml of DMF are added and stirred until complete dissolution. The flask is placed on ice. bath, cooled to t = 0 ° C, 40.6 ml (0.37 mol) of NMM and 59.0 g (0.2 mol) of Pyr-Glu-OPfp are added with stirring. Stirred for 30 minutes at t = 0-5 ° C, then 2 hours at room temperature. Place the reaction mixture overnight in a refrigerator at t = 5 ° C, filter off the precipitated NMM · HCl and wash the precipitate with 100 ml of DMF. DMF was evaporated on a rotary evaporator. The residue was triturated in acetone (600 ml), filtered off, the product on the filter was washed with 100 ml of acetone and dried in vacuo. The result is 70 g (80%) of pGlu-His-Trp-OH · HCl.

10. L-Pyroglutamyl-L-histidyl-L-tryptophil-L-seryl-L-tyrosyl-O-tert-butyl-D-seryl-L-leucyl-L-arginyl-L-proline-N-ethylamide acetate

pGlu-His-Trp-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · AcOH (Buserelin Acetate).

In a two-necked 1 L flask equipped with a mechanical stirrer and a dropping funnel, 53.8 g (0.11 mol) of H-Pyr-His-Trp-OH · HCl, 84.1 g (0.1 mol) of H-Ser are charged -Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NHEt · HCl, 21.4 g (1.4 mol) of HOBt · H ₂ O, 400 ml of DMF are added and stirred until complete dissolution. 11.1 g (0.11 mol) of NMM are added and, with stirring, a solution of 35.1 g (0.17 mol) of DCC in 50 ml of DMF is added dropwise over 6-8 hours. Then the reaction mass is stirred at room temperature for 10 hours. The precipitated DCCU is filtered off, washed with 50 ml of DMF on a filter, and the filtrate is evaporated on a rotary evaporator. The residue was triturated with THF (900 ml), the precipitate was filtered off, washed with 100 ml THF and, without drying, it was subjected to chromatographic purification.

For purification, the product is dissolved in 0.05 M pyridine acetate buffer, applied to a 50 × 600 mm column with SP-Sephadex and eluted in a 0.05-1 M pyridine acetate buffer gradient. The separated fractions were evaporated, dissolved in 2% acetic acid and lyophilized.

After isolation of the product by preparative HPLC, 70.3 g (50%) of product with 98% purity was obtained.

The product is chromatographically homogeneous (TLC, system E), Rf = 0.53 (1). Amino acid analysis: Glu 0.91 (1), His 0.93 (1), Trp 0.83 (1), Ser 1.90 (2), Tyr 0.93 (1), Leu 1.00 (1) Arg 1.06 (1), Pro 1.11 (1). [α] _D ²⁰ = -45.3 ° (c = 1; water). TLC is carried out on Merck Kieselgel-60 plates in the following systems: D. The manifestation is carried out by treating the plates with chlorine and then a tolidine solution.

As can be seen from the above example, the proposed method can significantly simplify the process of producing buserelin by eliminating the use of the azide method at the stage of obtaining the final product and by refusing to use benzyl protection for the lateral function of tyrosine and to increase the yield of the target product.

Additional advantages of the claimed method are:

- according to the target stage — condensation of the tripeptide with the hexapeptide with the yield of the target product 70% versus 62% during the condensation of the tetrapeptide with the pentapeptide;

- yields of intermediate products increased (from 60 to 70% and from 70 to 80% for similar reactions), which affected the increase in the total yield of the target product;

- the number of protective groups decreased significantly: from 6 to 4 (previously 1 Boc protection and 5 Z-protection were used, in the present patent, respectively 2 and 2), which reduced the consumption of expensive Pd (OH) ₂ catalyst and increased process safety due to a decrease in the number stages of catalytic hydrogenation.

Information sources

1. Sandow J., Konig W. Studies with Fragments a Highly active Analogues of Luteinizing Hormone - Releasing Hormone, J. Endocrinol, 1979, 81 (2), 175-182.

2. Patent of the Russian Federation No. 2086561, published. 08/10/1997.

3. Konig W., Geiger R., Sandow J. K., Peptides having LH-RH / FSH-RH activity.

US patent No. 4024248 (1977) (the closest analogue).

4. Peptides. The main methods of formation of peptide bonds. - M .: Mir, 1983

Claims (4)

1. The method of producing nonapeptidethylamide of the formula:

,
consisting in the fact that the synthesis is carried out by condensation of the C-terminal tetrapeptide of the formula:

with a dipeptide of the formula:

,
where X is a protective group and the obtained N-protected hexapeptide ethylamide of the formula:

treated with a deblocking agent to remove the N-protecting group and the obtained hexapeptide of the formula:
H-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NH-C ₂ H ₅
condensed with a tripeptide of the formula:

and the final product is purified by chromatography and isolated as a monoacetate salt. 2. The method according to claim 1, characterized in that X represents benzyloxycarbonyl (Z), and the release of N-protected hexapeptidethylamide is carried out by catalytic hydrogenation in the presence of 5% Pd / C. 3. The use of N-protected dipeptide of the formula (IV)
ZL-Ser-L-Tyr-OH,
where Z is benzyloxycarbonyl (IV), as an intermediate for the synthesis of nonapeptidethylamide of the formula (I)

. 4. The tripeptide of the formula (V):

as an intermediate for the synthesis of nonapeptidethylamide of the formula:
pGlu-His-Trp-Ser-Tyr-D-Ser (Bu ^t ) -Leu-Arg-Pro-NH-C ₂ H ₅ · 2AcOH.