CS260642B1 - Process for the preparation of etbylamide 10 desGly, 6-D-Tle LH-RH - Google Patents

Abstract

The solution concerns a method for producing ethylamide 10-desGly,6-D-Tle LH-RH of formula I Glu—His—Trp—Ser—Tyr—D—Tle—Leu— —Arg—Pro—iNHEt (I) by the solid phase method on a polystyrene support crosslinked with 1% divinylbenzene. The essence lies in the fact that the carboxy-terminal amino acid proline is anchored to a chloromethylated support functionalized with ethylamine and the peptide chain is exposed by gradual condensation. The peptide is cleaved from the resin by a two-phase action of liquid hydrogen fluoride in the presence of organic compounds of the p-cresol, p-thiocresol and dimethyl sulfide types, which limit undesirable side reactions. At the same time, all protecting groups are cleaved from the peptide. The crude peptide is purified by high-performance liquid chromatography. Thus, a very pure product is obtained in high yield, directly usable as a regulator of controlled reproduction in cattle.

Description

The invention relates to a process for the preparation of 10-desGly, 6-D-Tle LH-RH ethylamide of formula I

Glu-His-Trp-Ser-Tyr-D-Tle-Leu-Arg-Pro-NHEt (I)

The substance serves in veterinary medicine in controlled reproduction of cattle. Currently, the production of 10-desGly, 6-D-Tle LH-RH ethylamide is carried out by the method of peptide synthesis in solution according to U.S. Pat. AO No. 230 614 of April 25, 1984. This production has all the disadvantages of the classical solution synthesis method, namely high labor, higher demands on raw materials and energy, and is very lengthy.

(Disadvantages of this method are eliminated by the method for producing 10-desGly, 6-D-Tle LH-RH of formula I by the solid phase method on a polystyrene carrier crosslinked with 1% divinylbenzene, which consists in anchoring the carboxyterminal amino acid proline to chloromethylated carrier. and sequentially condensing the peptide chain, cleaving the carrier peptide and all of the protecting groups of the peptide with hydrogen fluoride in the presence of p-cresol, p-thiocresol, and dimethylsulfide organic species in one step, and then purifying the product by high performance liquid chromatography.

The polystyrene crosslinked with 1% DVB with chloromethyl functional groups was used as the polymer carrier. Chloromethyl groups were transformed with ethylamine into NEAM groups. By sequential condensation, protected amino acids were attached. The α-amino groups of the amino acids were temporarily protected with a Boc group and in each cycle the Boc group was cleaved acldolytically with a TFA solution. After attachment of the last amino acid, the peptide was simultaneously cleaved from the resin and side chain protecting groups by biphasic liquid HF digestion.

25% by volume of liquid HF, 65% by volume of DMS, 2.5% by volume of p-thiocresol and 7.5% by volume of p-cresol were used in the first phase, and 90% by volume of HF were used in the second phase , 2.5% by volume of p-cresol and 7.5% of p-cresol.

The presence of β-thiocresol, β-cresol and DMS is essential as they reduce unwanted side reactions when cleaving the side chain protecting groups. The lyophilized crude product was purified by high performance liquid chromatography. The pure product was characterized by amino acid analysis and comparative chromatography.

The process according to the invention, compared to the prior art process, saves raw materials, time and simplifies the synthesis of the desired product.

The process according to the invention has been successfully verified in practice. The following examples illustrate, but do not limit, the method of the invention.

Example 1

Polystyrene resin (2 g, 0.8 mmol Cl / g, crosslinked 1% DVB) was mixed with 10 ml ethylamine and allowed to react at 4 ° C for two days.

After removal of the ethylamine, the resin was washed with MeOH, DCM and again with MeOH. The obtained NEAM resin was dried and the content of free amino groups was determined by picnic test and elemental analysis at 0.7 mmol N / g. The resin was placed in the reactor and individual condensations of the following protected amino acids were performed:

Boc — For,

Boc — Arg (TOS)

Boc — Leu,

Boc — D — Tle,

Boc — Tyr (Z J,

Boc — Ser (Bzl)

Boc — Trp (For)

Boc-His (Boc) and pyroglutamic acids, cycles with the following synthetic steps:

min

1. Cleavage of the Boc-group 1X5

50% by volume of TFA in DCM and

X 30

2. Wash with DCM 3X1

3. neutralization of 5% by volume of DIE A in CHCl3 1X2

4. Wash with DCM 2X1

5. neutralization of 5% by volume of DIEA in CHCl3 1X2

6. Wash with DCM 3X1

7. Wash DMF 2X1

8. Attaching the activated protected amino acid with a solution of 4.5 mmol of HOBt ester in 30 mL of DMF 1 X 45

9. Wash MeOH 2X1

10. Wash with DCM 3X1

After the addition of the amino acid (step 8), a test for the presence of free amino groups was performed. In the case of a positive result, the reaction time was extended. steps 8-10 were repeated to attach the first amino acid Boc-Pro. It is not necessary to cleave the Bcc-group from the resin and therefore the cycle began with step 2.

After completion of the synthesis, the MeOH resin was washed and 3.5 g of dry peptidyl resin was obtained. One gram of this resin was subjected to two-phase cleavage of HF. In the first phase, 0.75 g of β-cresol, 0.25 g of β-thiocresol, 6.5 ml of DMS and 2.5 ml of HF were used. The cleavage mixture was stirred at 0 ° C for 2 h, HF and DMS were evaporated and condensed with 9 mL of HF. In this second phase the mixture was stirred for 4 h at temperature

5 ° C. After distillation of HF, the cresols were extracted with dry ethyl acetate and the peptide was dissolved in 50% v / v acetic acid. Lyophilization of the solution yielded 200 mg of crude peptide. This was purified by high performance liquid chromatography on silica gel sorbent with attached hydrophobic carbon residues (130 X 26 mm column, mobile phase composition 55% MeOH in water with 0.1% TFAj. Amino acid analysis:

For 0.99,

Arg 1,03,

Leu 0,97,

D — Tle 0,92,

Tyr 0,99,

Ser 0,86,

Trp 1,03,

His 0,94,

Glu 1.09.

Content determined by quantitative amino acid analysis of 76.3% peptide (calculated as Leu). The purity determined by high performance liquid chromatography was 96%. The yield was 110 mg of Glu-His-Trp-Ser-Tyr-D-Tle-Leu-Arg-Pro-NHEt.

Example 2

NEAM resin (250 mg; 0.7 mmol N / g) was placed in a flow through polypropylene reactor (40 X 8 mm, 1.4 mL internal volume). Reagents and wash solvents were pushed through the reactor with a slight nitrogen overpressure through the following wash cycles:

1. Boc group cleavage

10% by volume sulfuric acid in dioxane 60 min

2. Wash the DCM to neutral pH of the eluent for 5 min

3. neutralization with a 5% vol. Solution

DIEA in CHCl3 3 min

4. Wash DCM to neutral pH of eluent for 5 min

5. Attaching the activated protected amino acid with a triple excess

HOBt ester in DMF 30 min

Amino acids added:

Boc — For,

Boc — Arg (TOS)

Boc — Leu,

Boc — D — Tle,

Boc — Tyr (Z)

Boc — Ser (Bzl)

Boc — Trp (For)

Boc - His (Boc j and pyroglutamic acid.

At the end of each cycle, a test for free amino groups was performed. In the case of a positive test, a catalytic amount of DMAP was added to the active component and continued to be pushed through the reactor until the negative test. The peptide was cleaved from the resin and purified as described in Example 1.

Example 3

Following the procedure of Example 1, the desired product was prepared in satisfactory purity. Only in the first step was the cleavage agent replaced. Instead of a 50% by volume solution of TFA in DCM, a saturated solution of hydrogen chloride in DCM was used

Claims (1)

SUBJECT A process for the preparation of 10-desGly, 6-D-Tle LH-RH ethylamide of formula I Glu-His-Trp-Ser-Tyr-D-Tle-Leu-Arr-Pro-iNHEt (I) by a solid-phase method on a polystyrene support cross-linked with 1% divinylbenzene, characterized in that an carboxyterminal amino acid is anchored to the chloromethylated support Proline and sequential condensation expose the peptide chain, whereupon the peptide from the carrier and all protecting groups from the peptide are cleaved simultaneously with hydrogen fluoride in the presence of p-thiocresol, p-cresol and dimethylsulfide organic compounds and purified by high performance liquid chromatography.