Method for preparing adrenocorticotropic hormone (acth) analogs, sequence (4-10), possessing neurotropic activity and tetrapeptide for its preparing

FIELD: pharmaceutical chemistry, chemistry of peptides, hormones.

SUBSTANCE: invention relates to a method for preparing analogs of adrenocorticotropic hormone (ACTH) (4-10) possessing neurotropic activity. Method for preparing analogs of adrenocorticotropic hormone (ACTH), a sequence (4-10), of the general formula (I): A-Glu-His-Phe-Pro-Gly-Pro-OH (I) wherein A means hydrogen atom (H), Met, Met(O), Lys, Ser, Trp, Ala, Gly, Thr is carried out by liquid-phase method by step-by-step splicing peptide chain beginning from C-terminal protected tetrapeptide of the formula: H-Phe-Pro-Gly-Pro-OH (II) wherein X means a protective group and using corresponding fully protected amino acids in activated form followed by removal of protective groups at each step and purification of the end product by liquid chromatography. Method provides simplifying the process and to enhance the yield of the end product.

EFFECT: improved preparing method.

5 cl, 1 tbl, 5 ex

 

The invention relates to the field of physiologically active peptides, specifically to a new method of obtaining analogues of ACTH (4-10) of General formula I

where A=H, Met, Met(O), Lys, Ser, Trp, Ala, Gly, Thr,

and can find application in medicine.

Known heptapeptide the Peptide of the formula Met-Glu-His-Phe-Pro-Gly-Pro, which is a stimulant memory [1]. Currently, the Peptide used as a substance in obtaining nootropic drugs [2].

A method of obtaining analogues of ACTH (4-10) of General formula I, which is based on block-condensation of the protected C-terminal of Tripeptide H-Pro-Gly-Pro-OBzl with the Tripeptide Z-Glu(OBut)-His-Phe-N2H3azide method, and then attach to the Hexapeptide C-terminal N-protected amino acids and the removal of the protective groups [3].

A significant disadvantage of this method is the low yield of the final products and use in synthesis of the azide method, the condensation of peptide fragments, which requires careful observance of a temperature mode and use in the synthesis of highly toxic reagents such as hydrazine hydrate and tert-butylnitrite, which is especially difficult in industrial synthesis. In addition, the synthesis is complicated by undesirable side reactions [4].

Task invented the I is to increase the output, simplify the process of obtaining the peptides of formula I.

The problem is solved by the described method, according to which the peptides of General formula I are obtained by a stepwise increase of the peptide chain, starting with the C-terminal tetrapeptide General formula

The original C-terminal tetrapeptide formula II receive conventional methods of peptide synthesis, for example by block-step extension of the peptide chain in solution using activated esters of the corresponding protected amino acids and the method of mixed anhydrides [3].

List of abbreviations

Asón - acetic acid;

Boc - tert-butyloxycarbonyl;

Buttert-butyl;

Bzl is benzyl;

DCC is N, N'-dicyclohexylcarbodiimide;

DMF - N,N-dimethylformamide;

HONSu - N-hydroxysuccinimide;

HONp - p-NITROPHENOL;

TFA - triperoxonane acid;

Z - benzyloxycarbonyl;

TLC - thin layer chromatography.

Experimental part

In the synthesis used derivatives of L-amino acids company Bachem (Switzerland). DCC, TFA, HONSu, HONp company Fluka (Switzerland). DMF was purified by distillation over ninhydrin and barium oxide. Analytical HPLC was carried out on the chromatograph company Gilson (France).

TLC was performed on glass chromatographic plates Merck-Kiselgel (Germany) in the following solvent system:

And - chloroform:methanol:32% Asón15:4:1
In - chloroform:methanol:32% Asón5:3:1
With a chloroform:methanol:acetic acid ethyl ester6:3:1
D - N.-butanol:Asón:water3:1:1

1H-NMR spectra shoot spectrometer WH-500 Bruker 500 MHz (Germany) in DMSO-d6at 300 K. Chemical shifts (δ, ppm) measured relative to tetramethylsilane.

Mass spectra were recorded on a mass spectrometer Analytical Compact MALDI 4 firms Kratos (UK).

The described method is illustrated by the following examples:

Example 1.

Option A.

Stage I:

19.5 g (0,260 M) of glycine are dissolved in 260 ml of 1 N. NaOH, the resulting solution was added to a cooled to 0°With the solution 96,0 g (0,260 M) Z-Pro-ONp in 600 ml of DMF. The reaction mixture was stirred at room temperature for 16 hours, evaporated, the residue is dissolved in 300 ml of water and extracted three times with ether. The ether layer is separated, water layer, add 150 ml of 2 n HCl solution, the precipitated oil is extracted with 600 ml of ethyl acetate. The organic layer is separated and washed with water until neutral, dried over Na2SO4, evaporated, the residue is recrystallized from ether

In the end you get: 67,5 g (85%) IIIa: MP. 122-123°S; Rf0,67 (A); Rf0,81 (In), R f0,65 (S).

Stage II:

of 72.9 g (0.238 M) IIIa hydronaut in the presence of 7.0 g of 5% Pd/C in a mixture of 600 ml of ethanol and 238 ml of 1 N. NaOH. After hydrogenation (control by TLC in system (A) the catalyst is filtered off, washed on the filter with ethanol, the filtrate evaporated, the residue is dissolved in 700 ml of DMF. To the resulting solution at 0°add 86,0 g (0,238 M) Boc-Phe-ONp. The reaction mixture was stirred at room temperature for 16 hours, evaporated, to the residue add 280 ml of 1 n solution of H2SO4, the precipitated oil is extracted with 600 ml of ethyl acetate, the organic layer is washed with water until neutral, dried over Na2SO4and evaporated. The residue is recrystallized from a mixture of ethyl acetate-ether.

End up of 47.3 g (55%): MP. 139-141°S; Rf0,69 (A); Rf0,85 (B); Rf0,23 (S).

Option B.

34,6 g (0,300 M) Proline dissolved in 150 ml of 2 N. NaOH, the resulting solution was added to a cooled to 0°With the solution 115,9 g (0.30 M) of Boc-Phe-ONp in 900 ml of DMF. The reaction mixture was stirred at room temperature for 16 hours, evaporated, the residue is dissolved in 900 ml of water and extracted three times with ether. The ether layer is separated, the aqueous layer add 180 ml of 2 n solution of H2SO4, the precipitated oil is extracted with 700 ml of ethyl acetate, the organic layer washed the t water until neutral, dried over Na2SO4and evaporated. The residue is dissolved in 600 ml of DMF, the resulting solution was added to 42.0 g (0,330 M) p-NITROPHENOL. The reaction mixture is cooled to -15°and with stirring, add it to the pre-cooled to -15°With solution 63,0 g (0,310 M) DCC in 300 ml of DMF. The reaction mixture was kept at +4°C for 18 hours, evaporated to constant weight, the residue is washed three times with hexane and dissolved in 500 ml DMF. The resulting solution was cooled to -10°and gradually, so that the temperature of the reaction mixture did not rise above +5°C, add a solution of 22.5 g (0,300 M) glycine in 150 ml of 2 N. NaOH. The reaction mixture was stirred at room temperature for 16 hours, evaporated, the residue is dissolved in 500 ml of water and extracted three times with ether. To the aqueous layer add 200 ml of 2 n solution of N2SO4, japansee oil is extracted with 900 ml of ethyl acetate, the organic layer is washed with water until neutral, dried over Na2SO4, evaporated. The residue is recrystallized from ether.

In the end you get a 109 g (80%) III t square 138-141°S; Rf0,69 (A); Rf0,85 (B); Rf0,23 (S).

Stage III:

66,4 g (0,153 M) III dissolved in 500 ml of DMF, the resulting solution was added 17,0 ml (0,153 M) N-methylmorpholine. The reaction mixture with stirring, cooled to -15°and gradually (this is corotu, to the temperature of the reaction mixture did not rise above -10°C)add 19,85 ml (0,153 M) isobutylphthalate, stirred at -10°C for 10 minutes, then add cooled to -10°With a solution of 64.4 g (0,290 M) H-Pro-OBzl in 200 ml of DMF. After 30 minutes the reaction mixture is evaporated, the residue is dissolved in 600 ml of ethyl acetate, the resulting solution was washed sequentially with 5% solution of NaHCO3, water, 2% solution of N2SO4, water, dried over Na2SO4and evaporated. The remaining oil is dissolved in 250 ml of TFA, was incubated for one hour, evaporated. The residue is dissolved in 300 ml of water, add 300 ml of ether and kept at +4°C for 2 hours. Precipitated crystalline precipitate is filtered off, washed on the filter with water, ether and dried to constant weight.

In the end you get 88,58 g (92%) IIa 99.0% purity according to analytical HPLC: TR=25,81 minutes Column Ultropac TSK ODS-120T 4,6×250 mm, 5 μm. Buffer A: 0.05 M KN2PO4, pH 3.0; buffer B: 70% acetonitrile + 30% N2O; gradient: 10%-70% In 30 minutes. T square 167-169°, Rf0,60 (A); Rf0,85 (B); Rf0,68 (S).

Amino acid analysis: Phe 1,00 (1), Pro 1,93 (2), Gly 1,05 (1).

MALDI-MS m/z, found: 507,6. [M+H]+calculated: 506,6

1H-NMR spectrum (DMSO-d6that δ, ppm):

Phe - 8,1 (NH, 1H); 4,34 (αCH, 1H); 3,12/2,94 (β-CH, 2H)

Pro - 4,49 (α-CH, 1H); 1,90/2,05 (β-CH, 2H)

Gly - 8,02 (NH, 1H); 4.09 to/3,85 (α-H, 1H)

Pro - 4,42 (α-CH, 1H); 2,17/1,83 (β-CH, 2H)

Example 2.

Stage I:

126,9 g (0,200) IIa dissolved in 600 ml of DMF, the resulting solution was added to 22.2 ml (0,200) N-methylmorpholine and 95,47 g (0,200 M) Boc2-His-ONp, pH of the reaction mixture is maintained within the range of 8.0 to 8.5 by addition of N-methylmorpholine. Completeness of the reaction is controlled by TLC in system (A). After 16 hours the reaction mixture is evaporated, the residue is dissolved in 700 ml ethyl acetate, washed several times with 2% aqueous ammonia solution, water, 5% aqueous citric acid solution, water. The organic layer is dried Na2SO4, evaporated. The remainder periostat from a mixture of ether-hexane.

In the end you get 151,90 g (90%) IV with Rf0,80 (A); Rf0,88 (B); Rf0,81 (S).

Stage II:

168,78 g (0,200 M) IV is dissolved in 800 ml of TFA, incubated for 1 hour, evaporated, the residue is treated with ether, the precipitated precipitate is filtered off, washed on the filter with ether and dried. The obtained product is dissolved in 600 ml of DMF, the solution was added to 22.2 ml (0,200 M) N-methylmorpholine and of 86.9 g (0,200 M) Boc-Glu(OBzl)-ONSu., the pH of the reaction mixture is maintained within the range of 8.0 to 8.5 by adding, if necessary, additional amounts of N-methylmorpholine. Completeness of the reaction is controlled by TLC in system (A). After 16 hours the reaction mixture is evaporated, the residue is dissolved in 700 ml ethyl acetate, washed with water, 2% aqueous solution of H2SO4, water. The organic layer is dried over Na2SO4and evaporated. The residue (Rf0,74 (A); Rf0,92 (B); Rf0,79 (C)) are dissolved in 700 ml of ethanol and hydronaut in the presence of 14.0 g of 5% Pd/C. the hydrogenation Process control by TLC in systems a and B. After completion of the hydrogenation the catalyst is filtered off, washed on the filter with ethanol, the filtrate is evaporated to constant weight, the residue is dissolved in 300 ml of TFA, incubated for 1 hour, add 200 ml of 2.5 N. HCl in ethyl acetate, evaporated, the residue is treated with ether, the precipitated precipitate is filtered off, washed on the filter with ether and dried.

In the end you get 138,9 g (92%) Ia 97,2% purity according to analytical HPLC: TR=12,84 minutes Column Ultropac TSK ODS-120T 4,6×250 mm, 5 μm. Buffer A: 0.05 M KH2PO4, pH 3.0; buffer B: 70% acetonitrile + 30% H2O; gradient: 10%-70% In 30 minutes. MP. 118-121°S; Rf0,21 (A); Rf0,53 (B).

Amino acid analysis: Glu 1.00 m (1), His 0,98 (1), Phe of 1.03 (1), Pro 1,92 (2), Gly 1,08 (1). MALDI-MS m/z, found: 683,8 [M+H]+; calculated: 682,77

1H-NMR spectrum (DMSO-d6that δ, ppm):

Glu - 8,10 (NH, 1H); 3,82 (αCH, 1H); 1,89 (β-CH, 2H); 2.26 and (γ-CH 2N)

His - 8,70 (NH, 1H); 4,67 (α-CH, 1H); 3,00 (β-CH, 2H); 8,97/7,34 (SN)

Phe - 8,54 (NH, 1H); 4,65 (αCH, 1H); is 3.08/2,80 (β-CH, 2H)

Pro - to 4.41 (αCH, 1H); 1,96 β -CH 2N)

Gly - 7,79 (NH, 1H); 4,01/3,82 (αCH, 1H)

Pro - 4,23 (α-CH, 1H); 2.13 in (β-CH, 2H)

Example 3.

Stage I:

138,9 g (0,200 M) la dissolved in 400 ml of water, with 20% aqueous NaOH solution was adjusted pH of the reaction mixture to 7,06, the water evaporated, the residue is evaporated twice with isopropyl alcohol, dissolved in 600 ml of DMF, the solution was added 81,1 g (0,220 M) Boc-Met-ONp. 16 hours the reaction mixture is evaporated, the residue is treated with 400 ml of a mixture of ethyl acetate-hexane 1:1, decanted, the remaining oil is dissolved in 700 ml of n-butanol, three times washed with 2% aqueous solution of H2SO4, water until neutral, evaporated, precipitated with 500 ml of a mixture of ethyl acetate-hexane 1:1, the precipitation is filtered off and dried to constant weight.

End up with 128 g (70%) V in the form of amorphous powder with Rf0,42 (A); Rf0,52 (B).

Stage II:

60,0 g (0.07 M) V with stirring, treated with 250 ml of 3 n HCl in acetic acid. After complete dissolution of the precipitate, the reaction mixture was incubated for one hour, evaporated, to the residue add 400 ml of ethyl acetate, precipitated amorphous precipitate is filtered off, washed on the filter with ethyl acetate, acetonitrile, hexane, dried, dissolved in water and purified on a column of diasorb-130-ST,12 μm; 50×250 mm, manufactured by CJSC "Biochim the ARTICLE", Buffer A: 0.01 M NH4OAc (pH 4.5); buffer B: 40% isopropyl alcohol in water, gradient: 0% B 10% B 10 min, followed by 0.3% per minute. The flow of V=50 ml/min, detector: λ=254 nm.

End up (after purification by HPLC) 48,43 g (85%), IB in the form of amorphous powder that 98.9% purity according to analytical HPLC: TR=14,49 minutes Column Ultropac TSK ODS-120T 4,6×250 mm, 5 μm. Buffer A: 0.05 M KN2PO4, pH 3.0; buffer B: 70% acetonitrile + 30% H2O; gradient: 10%-70% In 30 minutes V=1 ml/min, detector: λ=204 nm.

MALDY-MS, m/z found: 814,94 [M+H]+calculated: 813,95.

Amino acid analysis: Met 1,09 (1), Glu 1,00 (1), His 1,13 (1), Phe 1,05 (1), Pro 1,93 (2), Gly 1,07(1).

1H-NMR spectrum (DMSO-d6that δ, ppm):

Met - 8,18 (NH, 1H); 3,88 (αCH, 1H); 1,95 (β-CH, 2H); 2,01 (S-CH3)

Glu - 8,62 (NH, 1H); 4,29 (α-CH, 1H); 1,74/1,85 (β-CH, 2H); 2.26 and (γ-CH 2N)

His compared to 8.26 (NH, 1H); br4.61 (α-CH, 1H); 3,01/2,94 (β-CH, 2H); 8,97 (C2H)

Phe - 8,42 (NH, 1H); with 4.64 (α-CH, 1H); 3,06/2.91 in (β-CH, 2H)

Pro - 4,42 (α-CH, 1H); 1,96 (β-CH, 2H)

Gly - 7,82 (NH, 1H); 4,01 (α-CH, 1H)

Pro - 4.25 in (α-CH, 1H); 2.13 in (β-CH, 2H)

Example 4.

191,2 g (0.300 M) IIa dissolved in 1.5 l DMF. To the resulting solution was added 36 ml of N-methylmorpholine and 147,0 g (0,300 M) Boc2HisONp. The resulting solution was incubated for 16 hours at room temperature, the solvent evaporated, the residue is dissolved in 1.5 liters of ethyl acetate, washed with diluted aqueous solution of ammonia, water, in the s ' solution of citric acid, water evaporated. The oil obtained is dissolved in 200 ml of ethyl acetate, precipitated with 800 ml of hexane, decanted. The latter procedure was repeated once more. The residue is dissolved in 600 ml of TFA, incubated for 1 hour, TFA evaporated, the remaining oil RUB clean in the ether, the precipitate is filtered off, washed on the filter with ether, dissolved in 1.5 l of ethyl acetate, the pH of the solution was adjusted to 8.0 by addition of N-methylmorpholine, then to the solution was added 120 g (0,300 M) Boc-Glu(OBut)-ONSu and incubated at room temperature for 16 hours, washed with water, dilute aqueous solution of ammonia, 2% H2SO4, water until neutral and evaporated. The residue is triturated in ether, the precipitate is filtered off, washed on the filter with ether, dissolved in 600 ml of TFA, incubated for 1 hour, TFA evaporated, the remaining oil RUB clean in the ether, the precipitate is filtered off, washed on the filter with ether, dissolved in 1 liter of water and hydronaut in the presence of 5% Pd/C. complete hydrogenation is controlled by means of thin layer chromatography in the system A. Upon completion of the reaction the catalyst is filtered off, washed on the filter with water, the filtrate is evaporated to half the original volume, adjusted pH to 7.0 with 20% NaOH, and evaporated to dryness, the residue is dissolved in 1.5 liters of DMF, the resulting solution was added 11 g (0,300 M) Boc-Met-ONp incubated for 16 hours at room temperature. DMF is evaporated, the residue is dissolved in 1.5 l N.-butanol, acidified with 2% H2SO4to pH 3, washed with water until neutral, evaporated. The residue is dissolved in 600 ml of ethyl acetate, planted 600 ml of hexane. The formed precipitate is filtered off, washed on the filter with 900 ml of a mixture of ethyl acetate - hexane 2:1, hexane and dried. The obtained product is dissolved in 700 ml of 3 N. HCl in acetic acid, incubated for one hour at room temperature, evaporated, the residue triturated in ethyl acetate, washed with hexane, dried, dissolved in water and purified on a column of diasorb-130-ST,12 μm; 50×250 mm, manufactured by CJSC "Biohimik ST", Buffer A: 0.01 M NH4SLA (pH 4.5); buffer B: 40% isopropyl alcohol in water, gradient: 0% B 10% B 10 min, followed by 0.3% per minute. The flow of V=50 ml/min, detector: λ=254 nm.

End up with 140 g (52%), IB in the form of amorphous powder of 98.5% purity according to analytical HPLC: TR=14,49 minutes Column Ultropac TSK ODS-120T 4,6×250 mm, 5 μm. Buffer A: 0.05 M KN2PO4, pH 3.0; buffer B: 70% acetonitrile + 30% N2About; gradient: 10% - 70% In 30 minutes V=1 ml/min, detector: λ=204 nm.

MALDY-MS, m/z found: 814,94 [M+H]+calculated: 813,95.

Amino acid analysis: Met 0,99 (1), Glu 1,00 (1), His 0,92 (1), Phe 1,10 (1),Pro 1,93 (2), Gly 1,03(1).

1H-NMR spectrum (DMSO-d6that δ, ppm):

Met - 8,18 (NH, 1H); 3,88 (αCH, 1H); 1,95 (β-CH, 2H); 2,01 (S-CH3)

Glu - 8,62 (NH, 1H); 4,29 (; -CH, 1H); 1,74/1,85 (β-CH, 2H); 2.26 and (γ-CH 2N)

His compared to 8.26 (NH, 1H); br4.61 (α-CH, 1H); 3,01/2,94 (β-CH, 2H); 8,97 (C2H)

Phe - 8,42 (NH, 1H); with 4.64 (α-CH, 1H); 3,06/2.91 in (β-CH, 2H)

Pro - 4,42 (α-CH, 1H); 1,96 (β-CH, 2H)

Gly - 7,82 (NH, 1H); 4,01 (α-CH, 1H)

Pro - 4.25 in (α-CH, 1H); 2.13 in (β-CH, 2H)

Example 5.

Stage I:

4.3 g (5 mm) IV is dissolved in 30 ml of TFA, incubated for 1 hour, evaporated, the residue is treated with ether, the precipitated precipitate is filtered off, washed on the filter with ether and dried. The resulting product was dissolved in 20 ml of DMF, the solution was added 0.6 ml (5.4 mm) N-methylmorpholine and 2.17 g (5 mm) of Z-Glu(OBut)-ONSu. Completeness of the reaction is controlled by TLC in system (A), the pH of the reaction mixture is maintained within the range of 8.0 to 8.5 by adding, if necessary, N-methylmorpholine. After 16 hours the reaction mixture is evaporated, the residue is dissolved in 70 ml of ethyl acetate, washed with water, 2% aqueous solution of H2SO4, water. The organic layer is dried over Na2SO4, evaporated and triturated with ether. The precipitation is filtered off, washed with ether and dried.

In the end you get a 4.1 g (86%) VI with Rf0,82(A); Rf0,65 (B); Rf0,79 (S).

Stage II:

3.3 grams (3,40 mm) (VI dissolved in 50 ml of 95% acetic acid and hydronaut over 5% Pd/C until the disappearance of starting compound, controls the UYa the course of the reaction by TLC in system A. The catalyst is filtered off, the filtrate is evaporated to a volume of 10 ml and add 60 ml of ether. The precipitation is filtered off, washed on the filter with ether, and dried. The obtained product is dissolved in 30 ml of DMF, add to 0.62 ml diisopropylethylamine and 1.68 g (3.6 mm) of Boc-Thr-OPcp. The progress of the reaction is controlled by TLC in system A. the Solution is evaporated, to the residue is added ether, the precipitate filtered off, washed with ether and dried.

In the end you get a 3.2 g (100%) VII, Rf0,70 (A).

Stage III:

3.2 g (3,40 mm) (VII dissolved in 20 ml of TFA, over 40 min, the reaction mixture was evaporated to a volume of 5 ml add 50 ml of ether, the precipitated precipitate is filtered off and purified by HPLC on a column of diasorb-130-ST,12 μm; 50×250 mm, manufactured by CJSC "Biohimik ST", Buffer A: 0.01 M NH4OAc (pH 4.5); buffer B: 40% isopropyl alcohol in water, gradient: 0%B 10% B 10 min, followed by 0.3% per minute. The flow of V=50 ml/min, detector: λ=254 nm.

End up with 2.5 g (94%) If Rf0,18 (A); 0,12 (D); 0,17 (); Rf0,5; HPLC: Rt 14,9 minutes Column Ultropac TSK ODS-120T 4,6×250 mm, 5 μm. Buffer A: 0.05 M KN2RHO4pH 3.0; buffer B: 70% acetonitrile + 30% H2O; gradient: 10%-70% In 30 minutes V=1 ml/min, detector: λ=204 nm.

Similarly receive other analogues of ACTH (4-10), physico-chemical characteristics are presented in the table.

The formula of the compoundValues of RfThe calculated molecular massMALDY-MS, m/z
H-Ser-Glu-His-Phe-Pro-Gly-Pro-OH0,25 (A);769,8770,6 [M+H]+
0,18 ();
0,12 (D)
H-Trp-Glu-His-Phe-Pro-Gly-Pro-OH0,45 (A);868,9870,0 [M+H]+
0,33 ();
0,27 (D)
H-Ala-Glu-His-Phe-Pro-Gly-Pro-OH0,25 (A);753,8754,5 [M+H]+
0,15 ();
0,12 (D)
H-Gly-Glu-His-Phe-Pro-Gly-Pro-OH0,20 (A);739,8740,9 [M+H]+
0,13 ();
0,12 (D)
H-Lys-Glu-His-Phe-Pro-Gly-Pro-OH0,08 (A);810,9811,7 [M+H]+
0,03 ();
0,05 (D)

As seen from the above examples, the claimed method allows you to scale the process to increase the yield of the target compounds.

Literature

1. Auth. mon. The USSR №939440, SS 103/52, 1981.

2. RF patent №2045958, AK 38/02, 1994.

3. RF patent №2206573, SC 7/06, 2003.

4. H.-Djobke, CheckIt. Amino acids, peptides, proteins: Per. s nem. - M.: Mir, 1985.

1. The method of obtaining analogues adenocorticotropic hormone (ACTH), sequence (4-10), the General formula I

where A=H, Met, Met(O), Lys, Ser, Trp, Ala, Gly, Thr,

characterized in that the synthesis is carried out by liquid-phase method by stepwise extension of the peptide chain, starting with the C-terminal protected tetrapeptide formula

where X is a protective group, using the corresponding fully protected amino acid in an activated form with subsequent removal of the protective groups at each stage and the purification of the target product using liquid chromatography.

2. The method according to claim 1, characterized in that as the original C-terminal tetrapeptide using the peptide of the formula

H-Phe-Pro-Gly-Pro-OBzl,

where Bzl is benzyl.

3. The method according to claim 1 or 2, characterized in that the recip is jut a peptide of the formula

H-Met-Glu-His-Phe-Pro-Gly-Pro-OH.

4. The method according to claim 1 or 2, characterized in that the peptide of the formula

H-Glu-His-Phe-Pro-Gly-Pro-OH.

5. The use of tetrapeptide formula

TFA·H-Phe-Pro-Gly-Pro-OBzl

as intermediate compounds for the synthesis of analogs of ACTH (4-10) the General formula

A-Glu-His-Phe-Pro-Gly-Pro-OH,

where A-H, Met, Met(O), Lys, Ser, Trp, Ala, Gly, The, according to claim 1 of the claims.



 

Same patents:

FIELD: bioorganic chemistry.

SUBSTANCE: disclosed is oligonucleotide-peptide conjugate of general formula 3'TTCTCTAGG5'-dRib-dRib-dRib-N-terminal-Leu-Arg-Leu-Arg-Leu-Arg-Arg-Gly-NH2-C-terminal (wherein T is thymine residue; C is cytosine residue; A is adenosine residue; G is guanosine residue; drib is deoxyribose residue; Leu is leucine residue; Arg is arginine residue; Gly is glycine residue). Said conjugate cleaves phosphodiester bonds in single-stranded RNA sites in 5'GpN3' (wherein G is guanosine; N is any ribonucleotide) sequences only.

EFFECT: new conjugate being functional analog of T1 ribonuclease.

3 dwg, 4 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to method for production of heptapeptide of general formula: H-Thr-Lys-Pro-Arg-Pro-Gly-Pro-OH having psychostimulating activity. Synthesis is carried out by condensation of C-terminal protected tripeptide of formula HCl.H-Pro-GlY-Pro-OX, wherein X is protective group, with protected N-terminal tetrapeptide of formula Y-Thr-Lys(Y1)-Pro-Arg-OH, wherein Y and Y1 are protective groups, and obtained protected heptapeptide of formula Y-Thr-Lys(Y1)-Pro-Arg-Pro-Gly-Pro-OX is treated with deprotecting reagents to remove protective groups and target product is isolated by chromatography method. Also described are tripeptide of formula HCl.H-Pro-GlY-Pro-OBzl and tetrapeptide of formula Boc-Thr-Lys-(Boc)-Pro-Arg-OH as intermediates for synthesis of heptapeptide of general formula: H-Thr-Lys-Pro-Arg-Pro-Gly-Pro-OH.

EFFECT: simplified process; increased yield of target product.

4 cl, 7 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention provides novel compounds of general formula (I) and pharmaceutically acceptable salts thereof, wherein G represents glycin; D aspartic acid; R1 group -(CH2)n- or -(CH2)n-C6H4-; n is integer from 1 to 10; h is 1 or 2; X1 represents amino acid residue having one functional deviation such as amine; X2 and X4 independently represent amino acid residue capable of forming disulfide bond; X3 represents hydrophobic amino acid such as phenylalanine; X6 represents group -NH-[CH-]-C(O)-; X7 is missing or represents biomodifying grouping consisting of monodisperse poly(ethylene glycol): (II), wherein is integer from 1 to 10; C-end unit represents amide group or 1-10 amino acid residues; Z1 chelating group or reporter group; and W1 is missing or represents spacing moiety derived from glutaric or succinic acid. Compounds of invention are designed as diagnostic agents for visualization or as therapeutic agents, the two agent types being directional vectors capable of binding to integrin receptors.

EFFECT: expanded diagnostic and therapeutic possibilities.

19 cl, 3 tbl, 5 ex

FIELD: chemistry of peptides, chemical technology.

SUBSTANCE: invention relates to a method for purification of nona- or decapeptide representing antagonist of luteinizing hormone releasing factor (LHRH) from residual organic solvent. Method involves dissolving nona- or decapeptide in a water-containing solvent and at least one (C1-C3)-alcohol followed by precipitation in a solvent under condition with intensive stirring wherein this solvent represents carboxylic acid alkyl ester comprising from 3 to 6 carbon atoms and one or some nonpolar compounds chosen from hexane, heptane, octane, cyclohexane and methylcyclohexane and, possibly, up to 5% of acetic or propionic acid. Then precipitate of nona- or decapeptide is washed out with a mixture of (C3-C5)-esters and dried. The water content in water-containing solvent used and at least one alcohol is less 8% (vol./vol.) and in the volume ratio of solvents mixture used for dissolving and solvents mixture used for precipitation is 1:10 or above.

EFFECT: improved purifying method.

8 cl, 1 tbl, 6 ex

FIELD: medicine, oncology.

SUBSTANCE: invention relates to immunomodulator representing pentapeptide of formula Val-Val-Tyr-Pro-Asp and drug in liquid and dry forms based on the same. Both pentapeptide and pharmaceutical composition containing the same have antitumor activity in small doses and have no side effects.

EFFECT: new low molecular peptide with immunomodulating activity and antitumor preparation based on the same.

2 cl, 6 ex, 4 tbl

FIELD: medicine, polypeptides.

SUBSTANCE: invention relates to fusion polypeptides with enhanced pharmacokinetic properties. Fusion polypeptides comprising enhancing peptide sequences associated with the core polypeptide possess with the enhanced pharmacokinetic properties, such as prolonged half-time period. Also, invention relates to methods for enhancing pharmacokinetic properties of any core polypeptide by binding the enhancer peptide sequences with the core polypeptide. Proposed core polypeptides can comprise any pharmacologically useful peptide that can be used, for example, the therapeutic or prophylactic agent. The advantage of invention involves the enhancing of pharmacokinetic properties of polypeptides.

EFFECT: enhanced pharmacokinetic properties of polypeptides.

52 cl, 18 dwg, 14 tbl, 11 ex

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing antibodies raised to human leukocyte differentiation factor (HLDF) or to HLDF fragment (31-38) representing peptide of the following structure: Arg-Arg-Trp-His-Arg-Leu-Glu-Lys possessing with antigenic and nucleic acids-hydrolyzing properties, and for diagnostic aims also. Antibodies are prepared from rabbit plasma blood immunized with three injections of antigens wherein synthetic HLDF factor or conjugate is used as antigens. Diagnosis of anaplastic state of human cells is carried out by using solutions of antibodies to HLDF factor or HLDF fragment (31-38) in the concentration 0.0013 mg/ml as biological markers. Invention provides carrying out the differential diagnosis of tumors and normal organs and effective detecting initial stages in cell differentiation disturbances.

EFFECT: improved preparing method of antibody, improved method for diagnosis.

6 cl, 21 dwg, 1 tbl

FIELD: chemistry of peptides, medicine.

SUBSTANCE: invention relates to preparing new peptides possessing immunomodulating, anti-proliferative, anti-tumor and antiviral activity. Invention proposes new peptides comprising up to 30 amino acid residues of the general structural formula: X1-Trp-Gly-Gln-X2 wherein X1 is taken among the following group: -His-Gly-Val-Ser-Gly-, -His-Gly-Gly-Gly-, -His-Val-Gly-Gly-, -His-Gly-Gly-Gly-Gly-, and -Gln-Gly-Gly-Gly-Gly, or absent; X2 is taken among the following group: -His-Gly-Thr-His-Gly, -Gly-Gly-Thr-His-Gly, -Pro-His-Val-Gly-Gly, -Pro-His-Gly-Gly-Gly, -Pro-His-Gly-Gly-Gly-Trp-Gly, -Gly-Gly-Gly-Thr-His-Ser, or absent.

EFFECT: valuable medicinal properties of peptides.

8 cl, 5 tbl, 5 dwg, 6 ex

FIELD: organic chemistry, peptides, pharmacy.

SUBSTANCE: invention relates to biologically active compounds as antagonists of somatostatin. Invention represents compound of the formula (I): A1-cyclo-{D-Cys-A2-D-Trp-A3-A4-Cys}-A-Y1 wherein A1 represents aromatic α-amino acid taken among the group: Cpa, Tfm, 1-Nal, 2-Nal and 3-Pal and substituted optionally with one or some substitutes taken among halogen atom and (C1-6)-alkyl; A2 represents aromatic amino acid taken among the group: 3-Pal and Tyr and substituted optionally with one or some substituted taken among halogen atom, (C1-6)-alkyl, OH-group; A3 represents Lys, Dab, Dap, Orn; A4 represents Thr, β-hydroxyvaline, Ser, Hser; A5 represents D- or L-aromatic α-amino acid taken among the group: Dip, 2-Nal, Trp; Y1 represents -NH2 or -MHMe wherein amine nitrogen in each amide peptide bond and amino-group A1 are substituted optionally with methyl group under condition that at least one abovementioned methyl group presents; or its pharmaceutically acceptable salt.

EFFECT: valuable biological properties of compounds.

11 cl, 1 dwg, 3 tbl, 16 ex

FIELD: organic chemistry, amino acids.

SUBSTANCE: invention proposes agonists of somatostatin of the formula (I): X-A1-cyclo-(D-Cys-A3-A4-Lys-A6-A7)-A8-Y or its pharmaceutically acceptable salt wherein X represents hydrogen atom (H); A1 represents L-Cpa, L-Phe, L-Trp or L-Nal; A3 represents L-3-Pal or L-4-Pal; A4 represents D-Trp; A6 represents -NH-(CHR1)n-CO- wherein n = 2, 3 or 4; A7 represents L- or D-Cys; A8 represents D- or L-isomer of amino acid taken among the group consisting of Nal, Phe, Cpa and Trp; Y represents NH2; R1 represents hydrogen atom (H), and Cys in A3 is bound by disulfide bong in A wherein this disulfide bond is formed by thiol groups of each Cys residue.

EFFECT: valuable biological properties of compounds.

9 cl, 2 ex

FIELD: biotechnology, medicine, oncology.

SUBSTANCE: invention proposes peptide of the structure Tyr-Ser-Leu and a pharmaceutical composition based on thereof that is used for stimulating antitumor immune response. Also, invention proposes methods for treatment of mammal and for modulation of the immune response. Proposed inventions expand assortment of agents used in treatment of cancer diseases.

EFFECT: valuable medicinal properties of peptide and pharmaceutical composition.

20 cl, 48 tbl

FIELD: medicine, chemistry of peptides, amino acids.

SUBSTANCE: invention relates to novel biologically active substances. Invention proposes the novel composition comprising peptides of the formula: H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys. The composition shows ability to inhibit proliferative activity of mononuclear cells, to induce suppressive activity and their ability for secretion of cytokines TNF-1β (tumor necrosis factor-1β) and IL-10 (interleukin-10 ).

EFFECT: simplified method for preparing composition, valuable medicinal properties of composition.

4 cl, 16 tbl, 9 ex

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

The invention relates to novel soluble synthetic polimersvarka the anthracyclines, exhibiting antitumor activity, to a method of receiving and containing pharmaceutical compositions

FIELD: biochemistry, medicine, allergology.

SUBSTANCE: invention relates to a method for preparing the hypoallergic birch pollen basic allergen r Bet v 1. Method involves one or more steps of chromatography purification using essentially non-buffered aqueous bases as an eluent and the following neutralization. Hypoallergic basic allergens from birch pollen distinct by absence or decreased binding of immunoglobulin E and simultaneous retaining therapeutically relevant stimulation of T cells. Therefore, prepared preparation can be used as a therapeutic agent with reduced adverse effects for carrying out the specific immunotherapy.

EFFECT: improved and valuable properties of allergen, improved preparing method.

27 cl, 6 dwg, 1 tbl, 6 ex

FIELD: biochemistry, medicine, allergology.

SUBSTANCE: invention relates to a method for preparing the hypoallergic birch pollen basic allergen r Bet v 1. Method involves one or more steps of chromatography purification using essentially non-buffered aqueous bases as an eluent and the following neutralization. Hypoallergic basic allergens from birch pollen distinct by absence or decreased binding of immunoglobulin E and simultaneous retaining therapeutically relevant stimulation of T cells. Therefore, prepared preparation can be used as a therapeutic agent with reduced adverse effects for carrying out the specific immunotherapy.

EFFECT: improved and valuable properties of allergen, improved preparing method.

27 cl, 6 dwg, 1 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to protein deposition by overcritical fluid medium as well as stabilization and protection from melting thereof. Method for codeposition of protein or polypeptide with stabilizing agent thereof by gas antisolvent process includes feeding overcritical fluid medium into vessel for particle formation individually or in mixture with modification agent and solution containing said protein or polypeptide and said modification agent dissolved in solvent differ from said modification agent. In such a manner than abovementioned solvent is extracted from solution with said overcritical fluid medium and substance and stabilizing agent (such a sugar) are simultaneously deposited. Device for such method and obtained product also are disclosed.

EFFECT: increased protein stability.

18 cl, 2 tbl, 7 dwg

FIELD: biotechnology and genetic engineering.

SUBSTANCE: in order to renature membrane protein, homogenous solution of the protein and phospholipid or phospholipid mixture in organic solvent blend or solvent/water mixture is prepared, after which solvents are removed and remaining mixture is hydrated. Method can also be applied in pharmaceutical industry.

EFFECT: increased yield of renatured membrane protein.

3 dwg, 3 ex

Up!