The method of synthesis of des-gli 10, /d-s 6/ lh-rh-ethylamide

 

(57) Abstract:

The invention relates to organic chemistry, in particular to preparative peptide synthesis of GnRH analogues in solution. The purpose of the invention is the reduction and expansion of technological capabilities of the method. The method consists in the fact that the synthesis is carried out by condensation of amino acid derivatives by the method of series-parallel extension of the peptide chain according to the scheme:

< / BR>
The advantages of the proposed method are: the use of unprotected on guanidino group of arginine, the synthesis of fragments 1 - 2 and 3 - 4 unprotected C-terminal carboxyl functions. The proposed method allows for the preparative synthesis. 1 C.p. f-crystals.

The invention relates to organic chemistry, in particular to preparative peptide synthesis of GnRH analogues in solution.

Analogues of luliberin (luteinizing hormone releasing factor LH-RH) are widely used in medicine and veterinary medicine. In particular, the analogue containing d-leucine in position 6 and etelemed instead of the C-terminal glycine amide (trade names leuprolide and lupron) Pyr-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NH-C2H5used in the form of the acetate salt as the effect of the species of the synthesis of this compound is of great practical interest.

Known methods of synthesis of des-Gli10, [d-LEU6]-LH-RH-ethylamide [3, 4] the closest to the proposed method is the method of synthesis described in [4] the Method consists in the fact that the synthesis is carried out by condensation of amino acid derivatives by the method of series-parallel extension of the peptide chain according to the scheme I:

< / BR>
The disadvantages of this method of synthesis are: the use of a secure on guanidino group of arginine; use C-terminal protection in the synthesis of fragments 1 3 and 4 5; using a solution of hydrogen bromide in acetic acid to release the amine groups. These drawbacks are more expensive way and increase the number of stages, which limits the adaptability of the method.

The purpose of the invention, the reduction and expansion of technological capabilities of the method.

The method consists in the fact that the synthesis is carried out by condensation of amino acid derivatives by the method of series-parallel extension of the peptide chain according to scheme 2:

< / BR>
The advantages of the proposed method are: the use of unprotected on guanidino group of arginine, the synthesis of fragments 1 2 and 3 4 with unprotected C-terminal carboxyl functions.

10,[d-LEU6]-LH-RH-ethylamide.

P R I m e R 1. Synthesis Of Z-Leu-Arg.

19.32 g of Z-Leu-ONp (50 mmol) is dissolved in 100 ml of DMF, add 10,45 g (60 mmol) of arginine and the mixture is stirred on a magnetic stirrer 48 hours Upon completion of the reaction, the reaction mixture was evaporated to a thick oil, dissolved in chloroform and applied to a column h cm silica gel, balanced chloroform. The column was washed with chloroform until the disappearance of the yellow color and elute the product with a mixture of chloroform/methanol 40:60. A solution of the product in eluent evaporated to dryness, poured ether and stored in the refrigerator until the end of crystallization. The resulting crystals are filtered, washed on the filter with ether and air-dried. Output 18,116 g, 43 mmol, 86

P R I m m e R 2. Synthesis of Z-d-Leu-Leu-Arg.

30,33 g of Z-Leu-Arg (72 mmol) hydronaut in methanol with PD/C as catalyst. Upon completion of the hydrogenation the catalyst is filtered off, the filtrate is evaporated to dryness and the residue is dissolved in 100 ml of DMF. To the resulting solution was added a solution of 27,82 g (72 mmol) of Z-d-Leu-ONp in 50 ml of DMF. The reaction mixture was stirred days, evaporated to a thick oil, dissolved in 100 ml of methanol, add 200 ml of water and the methanol is distilled off on a rotary evaporator. The mixture was kept in holodilnik 100 ml of methanol and planted 300 ml of ether. The resulting crystals are filtered, washed on the filter with ether and air-dried. In case of incomplete separation of para-NITROPHENOL crystallization ether from methanol should be repeated. Output 34,22 g (64 mmol), 88.9

P R I m e R 3. Synthesis of BOC-Tyr(Bzl)-d-Leu-Leu-Arg.

30,48 g (57 mmol) of Z-d-Leu-Leu-Arg dissolved in 100 ml of DMF. add 350 ml Meon and hydronaut with PD/C as catalyst. Upon completion of the reaction, methanol and water are distilled off on a rotary evaporator, the residue is added 26.7 g of the BOC-Tight(Bzl)-OSu in 300 ml of DMF. The reaction mixture was stirred days, evaporated to dryness on a rotary evaporator, dissolved the residue in 100 ml of methanol, add 300 ml of water and the methanol evaporated on a rotary evaporator. The reaction mass to stand in the refrigerator overnight, the aqueous solution over the oily precipitate is drained, the operation is repeated and the residue is crystallized under hexane. The output of 39.8 g, 52,79 mmol, 92,6

P R I m e R 4. Synthesis of Z-Pro-NH-C2H5.

49,85 g of Z-Pro-OH (200 mmol) and 25.6 ml of N-ethylmorpholine (200 mmol) is dissolved in 400 ml of the WPPT. The mixture is cooled to -30oC and small portions over 2 min add to 27.3 ml (210 mmol) of isobutylacetate. After 2 min with vigorous stirring to the mixture in portions over 5 min 30 ml of 50 aqueous solution of ethyl the mixture was evaporated to dryness, dissolved in chloroform and washed successively 1 N. sulfuric acid in a saturated solution of sodium chloride, a saturated solution of sodium chloride, sodium carbonate in a saturated solution of sodium chloride, a saturated solution of sodium chloride and water, evaporated and the residue crystallized from isopropanol-hexane. The output of 43.4 g, 157,2 mmol, 78,6

P R I m e R 5. Synthesis of BOC-Tight(Bzl)-d-Leu-Leu-Arg-Pro-NH-C2H5.

5,713 g (20,7 mmol) of Z-Pro-NH-C2H5hydronaut in methanol with PD/C as catalyst. Upon completion of the hydrogenation the catalyst is filtered off, the filtrate is evaporated to dryness, add 10,41 g (to 13.8 mmol) of the BOC-Tight(Bzl)-d-Leu-Leu-Arg, 4.3 g t, cool and add 4.35 g DCC. The reaction mixture is stirred for 48 h, filtered off dicyclohexylphosphino, the filtrate is evaporated to dryness, add 200 ml of ether and cooled. Stand in the refrigerator overnight, the ether is poured, the residue is dissolved in a mixture of 400 ml of chloroform and 100 ml of butanol and washed successively 1 n NaOH in a saturated solution of sodium chloride, a saturated solution of sodium chloride, 1 N. Hcl in a saturated solution of sodium chloride, a saturated solution of sodium chloride until neutral and water. The organic phase is evaporated to dryness on UB>H5.

7,017 g (8 mmol) of the BOC-Tight(Bzl)-d-Leu-Leu-Arg-Pro-NH-C2H5dissolved in 50 ml FA and 10 ml of anisole. The reaction mixture is stirred for 30 min, evaporated, dissolved in 400 ml of chloroform and 100 ml of butanol, washed with 1 N. NaOH in a saturated solution of sodium chloride, 5-soda solution in a saturated solution of sodium chloride, evaporated to dryness, dissolved in 50 ml of DMF, added 8 mmol of the BOC-Trp-Ser-OH, obtained by decomposition 4,582 g (8 mmol) dicyclohexylammonium salt of the BOC-Trp-Ser-OH*, and 1.08 g t. The reaction mixture is cooled and added 1.68 g of DCC. The reaction mixture is stirred for 48 h, filtered off dicyclohexylphosphino, the filtrate is evaporated to dryness, add 200 ml of ether and cooled. Stand in the refrigerator overnight, the ether is poured, the residue is dissolved in a mixture of 400 ml of chloroform and 100 ml of butanol and washed successively 1 N. NaOH in a saturated solution of sodium chloride, a saturated solution of sodium chloride, 1 N. Hcl in a saturated solution of sodium chloride, a saturated solution of sodium chloride until neutral and water. The organic phase is evaporated to a thick oil and the residue crystallized from methanol-ether. Output 6,457, 5,44 mmol, 68

*Note: Dicyclohexylammonium salt VOS-Thr-Ser-OH since g (12 mmol) of L-histidine, dissolved in 6 ml of 2 N. NaOH and with stirring, add 3,843 g (10 mmol) of Z-Pyr-ONp in 20 ml of DMF. The reaction mixture was stirred days, evaporated to dryness, dissolved in 50 ml of 1 N. Hcl and extracted with 100 ml of ether. The aqueous phase is separated, alkalinized to a pH of 4.5 to 5.5 and leave for days in the refrigerator. The precipitate is filtered off and dried in air. Output 1,473 g, 3,68 mmol, 36,8

P R I m e R 8. Synthesis of Z-Pyr-His-Trp-Ser-Tyr(Bzl)-d-Leu-Leu-Arg-Pro-NH-C2H5.

5,104 g (4.3 mmol) of the BOC-Thr-Ser-Tyr(Bzl)-d-Leu-Leu-Arg-Pro-C2H5dissolved in 50 ml FA and 10 ml of anisole. The reaction mixture is stirred for 30 min, evaporated, dissolved in 400 ml of chloroform and 100 ml of butanol, washed with 1 N. NaOH in a saturated solution of sodium chloride, 5% solution of soda in a saturated solution of sodium chloride, evaporated to dryness, dissolved in 50 ml of DMF, add from 1,722 g (4.3 mmol) of Z-Pyr-His, 0,581 g t, cool and add 1.26 g DCC. The reaction mixture is stirred for 48 h, filtered off dicyclohexylphosphino, the filtrate is evaporated to dryness, add 200 ml of ether and cooled. Stand in the refrigerator overnight, the ether is poured, and the residue crystallized twice from methanol-ether and once from ethanol-water with the addition of a weak solution of sodium chloride. Output a 4.53 g, is 3.08 mmol, 71,7

P R I m e R 9. Synthesis Rog-Nmol) hydronaut in aqueous ethanol, containing 5 acetic acid with Pd/C as catalyst. Upon completion of the hydrogenation the catalyst is filtered off and the filtrate is applied to the column 2,h cm with sulfopropyl-Sephadex C-25, equilibrated with 0.2 M pyridine-acetate buffer, pH 5.0. The column was washed with the starting buffer and elute the product in the gradient of the ionic strength of 0.2 to 0.6 M (1 l). The fractions containing the product with a purity above 85 unite, evaporated and rechromatography in column 2,h cm with sulfopropyl-Sephadex in the gradient of the ionic strength of 0.3 to 0.5 M (2 l). After repeated chromatography combine the fractions containing the desired product purity and combined fractions lyophilized. The yield of product with a purity of 98 (according to HPLC) 2,385 g, 1.88 mmol, 61

P R I m e R 10. Synthesis Rog-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NH-C2H5(option 2).

4,53 g Z-Rog-His-Trp-Ser-Tyr(Bzl)-d-Leu-Leu-Arg-Pro-NH-C2H5(is 3.08 mmol) hydronaut in aqueous ethanol containing 2 acetic acid with PD/C as catalyst. Upon completion of the hydrogenation the catalyst is filtered off and the filtrate is applied to the column 2,h cm with sulfopropyl-Sephadex C-25, equilibrated with 0.2 M acetate-ammonium buffer, pH 5.0. The column was washed with the starting buffer and elute the product in the gradient of the ionic strength of 0.2 to 0.6 M (1 l). Faction is propyl-Sephadex, balanced 0.3 M ammonium acetate buffer, pH 5.0, in a gradient of ionic strength of 0.3 to 0.5 M After repeated chromatography combine the fractions containing the desired product purity, combined fractions evaporated to small volume and applied to a column (2,h cm) with Toyopearl HW-40F, balanced 2-Noah acetic acid. Elute the product with the same eluent, combine the fractions containing the desired product purity and combined fractions lyophilized. The yield of product with a purity of 99 + (according to HPLC) 2,111 g of 1.66 mmol, 54

List of abbreviations: DMF dimethylformamide, DCC - dicyclohexylcarbodiimide, FA triperoxonane acid, t - 1-hydroxybenzotriazole, NB N-hydroxy-5-Narbonne-2,3-dicarbonyl, DNP - dinitrophenyl, Z benzyloxycarbonyl, Bzl benzyl, BOC - tert-butoxycarbonyl.

1. The method of synthesis of des-gli10, [D-LEU6]-LH-RH-ethylamide: Pyr-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-ProNHC2H5including series-parallel extension of the peptide chain using active esters of protected amino acids and dicyclohexylcarbodiimide in the presence of nucleophilic additives, and as protective groups for the amino groups of amino acids using carbobenzoxy and tertbutyloxycarbonyl-; serine and GI purification of the final product, characterized in that the synthesis of fragments and their condensation is conducted according to the scheme 2+[2+(4+1)] as a nucleophilic additives used N-hydroxybenzotriazole, arginine is used in the synthesis of unprotected side function, the hydroxyl group of tyrosine benzyl protected by a protective group, a carboxyl group amino acids are not protected, and multi-step chromatographic purification of the final product is performed by the method of double ion-exchange chromatography on SP-Sephadex in the gradient peridiniaceae buffer.

2. The method according to p. 1, characterized in that the multi-step chromatographic purification of the final product is performed by the method of ion-exchange chromatography on SP-Sephadex in the gradient peridiniaceae buffer with subsequent ion exchange rechromatography on SP-Sephadex in the gradient AMMONIATING buffer and chromatography media Toyopearl HW-40 in environment 2% acetic acid.

 

Same patents:
The invention relates to medicine, in particular to medicinal chemistry and relates to the creation of new biological active agents with opioid activity

The invention relates to medicine, namely to Oncology, and can be used to create a drug used in cancer therapy

The invention relates to new biologically active compounds derived peptides or their salts accession acids and pharmaceutical compositions based on them, which can find application in medicine

The invention relates to an improved process for the preparation of derivatives peptidomics or their physiologically compatible acetates or hydrochloride, which can find application upon receipt of the peptides with C-terminal azide of esamination method for the synthesis in solid phase

The invention relates to a new Pentapeptide formula

pAad-Glv-Asp-Ser-Gly-OH

where pAad - acyl group Piro-alpha-amino-adipic acid, having a specific inhibitory effect on propitiatio epidermal cells, their salts, pharmaceutical compositions based on it and the way to get Pentapeptide and pharmaceutical compositions

The invention relates to the derivatives of Pentapeptide and especially to pentapeptidnogo compounds that exhibit inhibitory vozdeistvie on DNA synthesis and mitotic rate during regeneration of the liver, as well as on DNA synthesis and growth lines malignant hepatoma cells in vitro and in vivo

The invention relates to the processing of vegetable raw materials, namely the method of obtaining protein hydrolysates, for example, from soybean meal and algal waste agar production, which can be used for food or as supplements in animal feed
The invention relates to biologically active substances and can be used to obtain products with a high physiological activity as a form of application of amino acids in the preparations of clinical nutrition

The invention relates to a method for producing a protein hydrolysate improved quality

biomass of microorganisms" target="_blank">

The invention relates to biotechnology, and in particular to methods for proteins genetic engineering method, and more specifically to a technology for obtaining high-purity interleukin I-(IL-I)

The invention relates to biochemistry and biotechnology and is a method of isolation and purification of physiologically active substances from the producer strain containing a recombinant plasmid DNA encoding the polypeptide with properties of human lymphotoxin, and can be used for production of the polypeptide for a detailed study of its properties and application in medical practice

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.

EFFECT: improved preparing method, valuable biological and biochemical properties of system.

49 cl, 5 dwg, 2 tbl, 9 ex

Up!