Method for producing tripeptide diacetate

FIELD: chemistry.

SUBSTANCE: invention refers to peptide chemistry and concerns producing tripeptide diacetate H-β-Ala-Pro-DabNHBzl referred to a biologically active compound used in cosmetic industry as an active component for cosmetic products, particularly for stimulating skin rejuvenation, tightening and prevention. The method is based on the 6-staged synthesis and is free from the stages of setting and releasing the protective groups. The method involves proline β-chlorpionyl chloride acylation followed by producing N-(3-chlorpropionyl)proline pentafluorphenyl ester in the presence of N,N'-dicyclohexyl carbodiimide. The above pentafluorphenyl ester is condensed thereafter with glumatic acid monomethyl ester to produce N-(β-chlorpropionyl)-Pro-Glu(δ-OMe)OH. That is followed by benzylamine amidation in a combination with ammonolysis and chlorine substitution by an amino group. That enables producing the tripeptide β-Ala-Pro-Glu(δ-NH2)NHBzl; Hofmann rearrangement is conducted with the use of iodo-benzene diacetate to produce a target product.

EFFECT: method is characterised by simplicity, effectiveness; it is cost-effective and uses more accessible and cheap agents.

6 ex

 

The invention relates to the field of peptide chemistry and specifically relates to the receipt of the diacetate of the Tripeptide H-β-Ala-Pro-DabNHBzl (Syn-Ake) related to biologically active compounds and used as an active ingredient for cosmetics.

Synthetic peptides are widely used in the pharmaceutical industry and in modern cosmetology. In particular, Syn-Ake stimulates rejuvenation of the skin by smoothing out wrinkles and belongs to the group of peptides botulu like actions (blockers of neuromuscular transmission) along with such synthetic peptides, such as Argireline (Argireline NP, acetyl Hexapeptide-3, acetyl Hexapeptide-8), SNAP-8 (acetyl glutamyl heptapeptide-1, EEMQRPA) (DERMAQUEST SKIN THERAPY, http://WWW.Dermaquestinc.Ru), Leuphasyl (Pentapeptide-18), Vialox (Pentapeptide-3), Inyline (acetyl Hexapeptide-25).

Features of synthesis of certain peptides of the structure are the result of bifunctionality source of amino acids and, consequently, the possibility of formation of peptide bonds between different and between identical molecules of amino acids. To prevent these complications it is necessary to protect the amino or carboxyl group. The protective group must be selectively installed it on the protected group, and retire to such conditions, which will not be affected other functions�tional groups of the molecules. In addition, it is desirable that the protection was staged and filmed with a high yield. For example, one of the most common ways of protecting (blocking) an amino group lies in the interaction of amino acids with benzylchloride or tertbutoxycarbonyl group (BOC) ("the Chemical synthesis of peptides", Kiev, Nauk. Dumka, 1992).

In peptide chemistry are widely used stepwise method of producing peptides by increasing the chain with the C-end of the original amino acids with the use of activated amino acid derivatives, for example panafcortelone esters (I. M., Klausner J. S. Activated esters and the strategy of peptide synthesis. Chemistry of polypeptides. Ed. by P. Katsoyannis. M., Mir, 1977, pp. 30-51).

In WO 2006047900, 11.05.2006 (or US 7964630) described compounds, in particular peptides H-Ala-Pro-Arg-Arg-NHBzl, H-β-Ala-Pro-Dab-NHBzl, H-Dap-Pro-Arg-NHBzl and acid-additive salts of these compounds, such as salts, for example, acetic acid, trifluoroacetic acid, etc. These compounds - peptides - suitable for smoothing fine lines and wrinkles in human skin. These compounds (peptides) can be obtained according to known methods in the chemistry of peptides, including a complete synthesis of these compounds, if necessary, cleavage (removal) of the protective group, the optional alkylation of the free amino group or its transformation into a funk�Oia guanidino and/or esterification or amidation of free carboxylic groups, and/or converting the obtained basic compound in acid-additiou salt, and/or receiving acid-additive salt to another salt.

So, in the case of the synthesis of H-β-Ala-Pro-DabNHBzl known method consists of 10 stages (including stage productions of protective groups) and as a source of amino acids uses L-Proline, L-2,4-diaminobutane acid (DabOH) and β-alanine. It should be noted that L-(DabOH) is not a natural amino acid and has high value.

The diacetate H-β-Ala-Pro-DabNHBz (Syn-Ake) - acetate salt of the synthetic peptide, the effect of which is aimed at immediate and long-term relaxation of facial wrinkles. This action is like the action of the protein contained in the venom of the temple copii (Tropidolaemus wagleri), which prevents the transmission of nerve impulses to the muscles. Its use in cosmetics allows you to not only smooth out fine wrinkles, but also prevents their reappearance (Fragrance Journal V34, N10, h.93-98 (2006). DE 202012002309, Anti-aging cosmetic foam, 2012)

The object of this invention is to simplify the process of obtaining, with the exception of reagents for the installation and removal of protective groups, reducing the cost of production of diacetate H-β-Ala-Pro-DabNHBzl (I).

The inventive method relates to the field of peptide chemistry, specifically to a method of producing diacetate H-β-Ala-Pro-DabNHBzl used as a bioactive component for cosmetics, formula I:

and has as its objective the development of a simple and convenient method for the synthesis of the target compounds. The invention consists in that the Tripeptide (I) is obtained 6-step synthesis without the use of stages of setting and removing the protective groups and comprising the following stages multi-stage peptide synthesis:

1. Acylation of Proline in dioxane β-chloropropionitrile with the use of an excess of Proline as an acceptor of hydrogen chloride gives N-(3-chloropropionyl)Proline (II);

2. Reaction (II) of pentafluorophenol in ethyl acetate in the presence of N,N'-dicyclohexylcarbodiimide (DCC) leads to plattenbau ether N-(3-chloropropionyl)Proline (III);

3. Condensation of III with the monomethyl ester of glutamic acid in DMF gives N-(β-Chloropropionyl)-Pro-Glu(δ-OMe)OH (IV);

4. The amidation IV benzylamine using HOBt and DCC with the formation of N-(β-chloropropionyl)-Pro-Glu(δ-OMe)NHBzl (V);

5. Simultaneous ammonolysis and chlorine substitution on the amino group of V are performed in a methanol solution of ammonia, leads to the Tripeptide hydrochloride of β-Ala-Pro-Glu(δ-NH2)NHBzl (VI);

6. Regrouping by Hoffmann a VI using the reagent of the diacetate of odensala gives trust I.

Used above and below the abbreviations indicate:

Pro - Proline;

Ala - alanine;

HOBt is 1-GI�moxibustion;

DCC - N,N'-dicyclohexylcarbodiimide;

Bzl, benzyl;

Dab - 2,4-diaminobutane acid;

AcOH - acetic acid;

β-Ala - beta-alanine (3-aminopropionic acid).

So, the solution of this problem is achieved by a 6-stage synthesis that do not use as a source of reagent L-DabOH and not containing the stage of setting and removing the protective groups:

As can be seen from the following examples claimed as the invention the method is characterized by simplicity, lack of stages of setting and removing the protective groups and uses more readily available and cheap reagents and raw products.

The developed method allows not only to simplify the process of obtaining the target compound (reduces the number of stages of synthesis), but also makes it more cost effective (more available and cheap raw materials, the elimination stages of setting and removing the protective groups).

In the first stage of acylation of L-Proline used as acceptor evolved hydrogen chloride an excess of Proline. The process is carried out at room temperature in dioxane. Eye-catching Proline hydrochloride is removed by filtration. The Proline residue is washed with an acidic solution of sodium chloride (pH 1) and after removal of the solvent in vacuo and washing the residue of sulfur and petroleum ether get II as a viscous about�Rechnoy liquid (purity 97.8%).

Panafcortelone ether III is obtained by reaction of II with pentafluorophenol in ethyl acetate. As the condensing agent used DNA. The reaction is carried out at room temperature for 2 h. After separation of dicyclohexylamine by filtration, removal of solvent in vacuo and trituration of the residue in petroleum ether get crystal III with the content of the basic substance of 95.1%.

Interaction III and monomethyl ether hydrochloride of glutamic acid in dimethylformamide for 20 hours at room temperature gives IV. N-Methylmorpholin used to bind hydrogen chloride. Dimethylformamide was removed under vacuum, the residue dissolved in ethyl acetate and washed main impurities citrate aqueous solution of sodium chloride. After distilling off the solvent in vacuo and trituration in the solvent system chloroform - diethyl ether 1:1 to receive IV amorphous content of the basic substance of 94.6%.

The amidation IV benzylamine is carried out in a solution of dimethylformamide at 5°C using HOBt and DCC for 18 h. increasing the temperature above 5°C leads to a decrease of the purity of the final product due to the side reactions: the formation of bis-benzylamine and nucleophilic substitution of the chlorine atom. The obtained amorphous V contains 95.9% of the basic substance.

In the interaction of V with ammonia in methyl alcohol�e (20% solution) simultaneously methyl ester ammonolysis and replacement of the chlorine by the amino group. The reaction is carried out in a sealed steel vessel (to prevent evaporation of ammonia) within 70-80 h. After removal of methanol in vacuo and rubbing in tetrahydrofuran of the residue obtained amorphous substance with VI contents 63%. Additional chromatographic purification on silica gel gives VI with the content of the basic substance of 96.8%.

Target I get a regrouping by Hoffmann a VI in a mixture of dioxane - pyridine using the diacetate of odensala at 5°C. the Fractional addition of diacetate of odensala allows reducing the adverse processes and increases the output I. the Optimal molar ratio of the diacetate of odensala/VI 4-5/1. Final cleaning and I translated it into diacetato form is carried out preparative HPLC.

The purity of the resulting diacetate H-β-Ala-Pro-DabNHBz and its intermediate products is confirmed by HPLC and NMR spectroscopy. Analytical HPLC was performed on chromatography Shimadzu. Column: Grom-Sil 12J ODS-4HE, 5 µm, 250*4.6 mm. Conditions: gradient 20%B (0 min) 60%B (10 min) 70%B (15 min) 70% B (30 min). A - phosphate buffer pH 3 (20,4 g KH2PO4was dissolved in 3 l of distilled water and pH was adjusted to 3 by addition of concentrated phosphoric acid), B - acetonitrile. The PMR spectra obtained on the instrument Bruker AM-360 (operating frequency 360.13 MHz). The NMR spectra of13C received on the instrument Bruker AM-360 (operating frequency 90.56 MHz). IR spectra were obtained on IR-fours�e spectrometer Nicolet 6700 (Thermo Scientific) in a tablet with KBr (2.5 mg/300 mg KBr). The specific angle of rotation was measured on an automatic polarimeter "OTAGO" AP-300. Spectra ESI-MS was register on the device "Agilent LC/MS 1200" during ionization of a sample by elektrorazpredelenie in the registration mode, the positive ions. Samples were prepared in the system acetonitrile/water 1/1, concentration 2 mg/ml. Conditions of analysis: flow 1 ml/min, pressure on nebulizer 20 psi, a temperature of 360°C, flow rate of drying gas 9 l/min, voltage 3500 (B, a target weight of 100 to 2000.

The described method is illustrated by the following examples.

Experimental part

Example 1. N-(3-Chloropropionyl)Proline (II)

Suspended 53 g (0.46 mol) of L-Proline in 300 ml of anhydrous dioxane at room temperature. With stirring dropwise 29.5 g (0.18 mol) of β-chloropropionitrile (heating up to 35-45°C). The reaction mixture was stirred at room temperature for 5 h. the precipitate was Filtered off Proline hydrochloride, washed on the filter with 50 ml of dioxane and the combined organic phase was distilled off in vacuo (45°C, 30 mm Hg.CT.) before the termination of the distillation. Dissolve the residue in 150 ml of ethyl acetate. The resulting solution is washed with 2 times 50 ml of a saturated aqueous solution of sodium chloride, acidified to pH 1 with concentrated hydrochloric acid. Diluted with 50 ml of hexane, dried with sodium sulfate and evaporated in vacuum until completely distilling off the solvent (50°C, 3 mm Hg.CT.). Viscous residues�to diluted with 50 ml of ether, stirred for 10 min and add 50 ml petroleum ether. The liquid phase was decanted, the oil washed with 100 ml of petroleum ether and dried in vacuum to constant weight (50°C, 3 mm Hg.CT.). Get 25.5 g (68.9%) of a colorless, very viscous liquid. The content of the basic substance 97.8% (HPLC).[α]D25=74.95(5% R-R, methanol); ESMS, m/z (I%): 206.5 [M+H]+(100), 228.2 [M+Na]+(28.4), 433.3 [2M+Na]+(94.0). The NMR spectrum1H (CDCl3d, M. D.): 1.84-2.27 (m, 4H, βCH2-Pro+γCH2-Pro), 2.76-2.84 (m, 2H, COCH2), 3.46-3.65 (m, 2H, δCH2-Pro), 3.66-3.83 (m, 2H, ClCH2), 4.42-4.54 (m, 1H, α-CH-Pro), 10.25 (s, 1H,COOH).

Example 2. Panafcortelone ether N-(3-chloropropionyl)Proline (III)

Dissolve 28.4 g (0.14 mol) of II and 28 g (0.15 mol) of pentafluorophenol in 250 ml of ethyl acetate at room temperature. Cooled to 10°C and at this temperature is added dropwise a solution of 31 g (0.15 mol) of dicyclohexylcarbodiimide in 80 ml. Warm the reaction mixture to room temperature and stirred for 2 h. Filtered off the precipitated dicyclohexylamine and ethyl acetate was distilled off under vacuum (50°C, 25 mmHg.CT.). The oily residue was triturated 4 times with 50 ml of petroleum ether until crystallization starts. The crystalline product was dried in vacuum to�a constant weight (30°C, 2 mm Hg.CT.). Get 30.5 g (58.6%) of a colorless low-melting crystals. The content of the basic substance 95.1% (HPLC). TLC: ethyl acetate/AcOH 100/1 (aluminum plate, a manifestation of the R-R potassium permanganate). Rf(II)=0.20, Rf(III)=0.75.[α]D24=78.8(5% R-R, chloroform). So a MP 51-53°C. ESMS, m/z (I%): 372.4 [M+H]+(100), 394.1 [M+Na]+(4.8). The NMR spectrum1H (CDCl3d, M. D.): 1.98-2.29 (m, 3H, βCH2-Pro+γCH-Pro), 2.33-2.54 (m, 1H, γCH-Pro), 2.82 (t, 2H, COCH2) 3.57-3.93 (m, 4H, δCH2-Pro+ClCH2), 4.77-4.86 (m, 1H, α-CH-Pro).

Example 3. N-(β-Chloropropionyl)-Pro-Glu(δ-OMe)OH (IV)

Dissolve 20 g (0.054 mol) of III and 12.8 g (0.065 mol) of hydrochloride of methyl ester of glutamic acid in 200 ml of dimethylformamide at room temperature. The resulting solution was cooled to 10°C and at this temperature is added dropwise within 15 min of 6.5 g (0.064 mol) of N-methylmorpholine. The reaction mixture was stirred at room temperature for 20 h. After the reaction (TLC), the dimethylformamide was distilled off in vacuo (45°C, 1 mm Hg.St.), the oily residue is dissolved in 500 ml of ethyl acetate and the resulting solution was washed with 100 and 50 ml of a saturated solution of sodium chloride with the addition of citric acid to pH 3. The organic phase is dried with sodium sulfate, evaporated in vacuum d� complete distillation of the solvent (45°C, 15 mm Hg.CT.). A semi-solid residue triturated 2 times with 100 ml of diethyl ether, 2 times 40 ml mixture of chloroform-diethyl ether 1/1. Amorphous IV was dried in vacuum to constant weight (40-50°C, 2 mm Hg.CT.). Get 15.1 g (80.2%) of white amorphous powder. The content of the basic substance 94.6% (HPLC). TLC: BuOH/AcOH/H2O 2/1/1 (aluminum plate, a manifestation of R-RA of potassium permanganate). Rf(III)=0.97, Rf(IV)=0.76.[α]D25=66.8(5% p-p, methyl alcohol). ESMS, m/z (I%): 349.3 [M+H]+(100). The NMR spectrum1H (d6-DMSO, δ, M. D.): 1.70-1.94 (m, 4H, βCH2-Pro+γCH-Pro), 1.96-2.12 (m, 2H, β-CH2-Glu), 2.33-2.41 (m, 2H, γCH2-Glu), 2.66-2.88 (m, 2H,CH2CO), 3.35-3.56 (m, 2H, δCH2-Pro), 3.59 (s, 3H, COOCH3), 3.68-3.81 (m, 2H,CH2Cl), 4.14-4.30 (m, 1H, α-CH-Glu), 4.32-4.45 (m, 1H, α-CH-Pro), 8.05+8.33 (dd, 1H, CONH), 12.58 (br. s, 1H, COOH).

Example 4. N-(β-Chloropropionyl)-Pro-Glu(δ-OMe)NHBz (V)

Dissolve 13.8 g (0.040 mol) of IV, 6.6 g (0.043) monohydrate of N-hydroxybenzotriazole and 5.3 g (0.049 mol) of benzylamine in 130 ml of dimethylformamide at room temperature. The resulting solution was cooled to 0°C and at this temperature is added dropwise 8.5 g (0.041 mol) of dicyclohexylcarbodiimide in 20 ml of dimethylformamide. The reaction mixture was stirred 18 h at 5°C. after completion of the reaction (TLC) Hotfile�revival dropped dicyclohexylamine, pour 500 ml of ethyl acetate. The resulting solution was washed sequentially 2 times with 250 ml of saturated sodium chloride solution, 2 times with 100 ml of 1% aqueous citric acid solution, 2 times with 150 ml of 2% aqueous sodium bicarbonate solution and 3 times with 70 ml of water. The organic layer was dried with sodium sulfate and distilled off the solvent under vacuum (50°C, 20 mm Hg.CT.). The residue was added 150 ml of diethyl ether, leaving the resulting suspension for 12 h, V filtered and dried in vacuum to constant weight (40°C, 2 mm Hg.CT.). Get 12.1 g (69.1%) of white amorphous powder V. the Content of the basic substance 95.9% (HPLC). TLC: ethyl acetate/MeOH 5/1 (aluminum plate, a manifestation of R-RA of potassium permanganate). Rf(V)=0.06, Rf(VI)=0.71.[α]D25=62.6(5% R-R, methyl alcohol). ESMS, m/z (I%): 438.4 [M+H]+(100), 460.0 [M+Na]+(1.6). The NMR spectrum1H (CDCl3d, M. D.): 1.94-2.18 (m, 6H, βCH2-Pro+γCH2-Pro+βCH2-Glu), 2.35-2.51 (m, 2H, γCH2-Glu), 2.67-2.88 (m, 2H,CH2CO), 3.45-3.76 (m, 4H, δCH2-Pro+CH2Cl), 3.62 (s, 3H, COOCH3), 4.24-4.32 (m, 1H, α-CH-Pro), 4.40-4.58 (m, 3H, α-CH-Glu+CH2Ph), 7.15-7.36 (m, 6H, CONHBz+Ph), 7.56 (d, 1H, CONH).

Example 5. β-Ala-Pro-Glu(δ-NH2)NHBz hydrochloride (VI)

Dissolved 7.8 g (0.018 mol) of V in 300 ml of 20% �of astora ammonia in methanol at 10°C. Pour the resulting solution in a sealed vessel (steel tube) and incubated at room temperature 70-80 hours until the reaction (TLC). The solvent is distilled off in vacuo (40°C, 25 mmHg.CT.) before the end of the distillation, the residue was added 20 ml of methyl alcohol and repeat the process again. Washed the oily residue 3 times with 20 ml of tetrahydrofuran to the solidification of the product. Dried raw VI in vacuum to constant weight (40°C, 2 mm Hg.CT.). Get 6.6 g of a white amorphous powder. The content of the basic substance 63% (HPLC). The resulting crude product VI was dissolved in 8 ml of methanol and chromatographically purified on silica gel (column: length 75 cm, diameter 3 cm, eluent methanol/chloroform 1/1). Target fractions (TLC) is evaporated under vacuum (40°C, 22 mm Hg.CT.) and finally dried at 30-35°C, 1 mm Hg.PT. Get 4.2 g (53%) of white amorphous VI. The content of the basic substance 96.8% (HPLC). TLC: methanol/acetic acid 50/1 (aluminum plate, a manifestation of the ninhydrin). Rf(V)=0.87, Rf(VI)=0.23.[α]D23=40.7(5% R-R, methyl alcohol). ESMS, m/z 404.3 [M+H]+(100), 426.2 [M+Na]+(12.5), 807.5 [2M+H]+(10.2), 829.4 [2M+Na]+(1.6). The NMR spectrum1H (DMSO-d6d, M. D.): 1.72-2.38 (m, 8H, βCH2-Pro+γCH2-Pro+βCH 2-Glu+γCH2-Glu), 2.53-2.75 (m, 2H, α-CH2-Ala), 2.91-3.05 (m, 2H, β-CH2-Ala), 3.36-3.61 (m, 2H, δCH2-Pro), 4.09-4.44 (m, 4H, α-CH-Glu+CH2Ph+α-CH-Pro), 4.87 (br. s, 3H,NH3+), 7.19-7.33 (m, 5H, Ph), 7.73 (br. s, 2H, CONH2). The NMR spectrum13C (D2O, δ, M. D.): 177.18, 173.72, 172.31, 170.27, 137.73, 128.12 (2C), 126.86, 126.60 (2C), 59.83, 53.51, 47.18, 42.33, 34.60, 30.54, 30.21, 28.99, 25.94. 23.60.

Example 6. H-β-Ala-Pro-DabNHBz diacetate (I)

Dissolved 8.1 g (0.0183 mol) of VI in 150 ml of water at room temperature. Add 100 ml of dioxane and 25 ml of pyridine. The resulting solution was cooled to 5°C and at this temperature dropwise 14.1 g (0.0438 mol) of diacetate of odensala in 180 ml of dioxane. The reaction mixture was stirred 3 h without removing the cooling, and again added dropwise 10.5 g (0.0327 mole) of diacetate of odensala in 180 ml of dioxane. Stirring was continued for 2 hours (control by TLC). Reaction mixture is sprinkled with 300 g of ice and pour 500 ml of ethyl acetate. The aqueous layer is separated and washed first with 2 times 300 ml of ethyl acetate and then 300 ml of a mixture ethyl acetate-butanol 4:1. The aqueous layer is evaporated (conversion 81% - HPLC) and chromatographic by preparative HPLC, and then translate the product into the diacetate. Get 4.92 g (54.3%) of oily I. the Content of the basic substance 98.1% (HPLC). TLC: methanol/acetic acid 20/1 (aluminum �Latinka, the manifestation of the ninhydrin). Rf(VI)=0.35, Rf(I)=0.11.[α]D24=81.3(1% R-R, methyl alcohol). ESMS, m/z (I%): 269.2 [M-BzNH]+(3.3), 376.2 [M+H]+(100), 751.1 [2M+H]+(33.2). The NMR spectrum1H (CD3OD, δ, M. D.): 1.94-2.12 (m, 4H, γCH2-Pro+βCH2-DIBA), 2.18-2.29 (m, 2H, β-CH2-Pro), 2.77 (t, 2H, α-CH2-Ala), 3.05 (t, 2H, γCH2-DIBA), 3.15 (t, 2H, β-CH2-Ala), 3.54-3.68 (m, 2H, δCH2-Pro), 4.38-4.43 (m, 3H,CH2Ph+α-CH-Pro), 4.48-4.54 (m, 1H, α-CH-DIBA), 7.21-7.34 (m, 5H, Ph). The NMR spectrum13C (D2O, δ, M. D.): 177.93, 171.43, 170.26, 137.06, 128.16 (2C), 126.92, 126.59 (2C), 59.47, 50.92, 47.23, 42.43, 35.81, 34.61, 30.34, 29.04, 28.04. 23.65. IR (KBr) 3060, 2212, 1961, 1651, 1557, 1410, 1341, 1276, 1203, 1181, 1137, 1049, 1015, 924, 888, 837, 800, 723, 701, 659, 621.

As can be seen from the above example, the described method is characterized by simplicity, lack of stages of setting and removing the protective groups and uses more readily available and cheap reagents and raw products.

A method of producing diacetate Tripeptide H-β-Ala-Pro-DabNHBzl (I) based on a 6-stage method of peptide synthesis without the use of stages of setting and removing the protective groups and comprising the following stages:
a) receive a first N-(3-chloropropionyl)Proline (II) by acylation of Proline (Pro) in dioxane β-chloropropionitrile;
b) continue to receive pentaf�orfamily ether compound (III) reaction of compounds (II) and pentafluorophenol in ethyl acetate in the presence of N,N'-dicyclohexylcarbodiimide;
b) carry out the condensation of the resulting panafcortelone ether compound (III) with the monomethyl ester of glutamic acid in dimethylformamide to produce a compound N-(β-chloropropionyl)-Pro-Glu-(δ-OMe)OH (IV);
g) carry out the amidation of the compound (IV) with benzylamine in the presence of 1-hydroxybenzotriazole and N,N'-dicyclohexylcarbodiimide with the preparation of the compound N-(β-chloropropionyl)-Pro-Glu-(δ-OMe)NHBzl (V);
d) conduct simultaneous ammonolysis of compound (V) and chlorine substitution on the amino group with obtaining the Tripeptide of formula (VI) β-Ala-Pro-Glu(δ-NH2)NHBzl;
(e) regrouped the Tripeptide of formula (VI) according to Hoffmann using a diacetate of iodobenzene and get the target product is the diacetate of the Tripeptide of formula (I).



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to prodrug preparations of glucagon superfamily peptides, in which the glucagon superfamily peptide is modified by binding a dipeptide with the peptide of the glucagon superfamily by an amide bond.

EFFECT: produgs, disclosed in the claimed invention, have an increased half-life and transfer into an active form in physiological conditions as a result of a non-enzymatic reaction, caused by chemical instability.

30 cl, 15 dwg, 8 tbl, 16 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutically acceptable crystalline or amorphous salts of D-isoglutamyl-D-tryptophan, methods of their obtaining, pharmaceutical compositions, containing them, and their application for obtaining pharmaceutical compositions for treatment of different conditions and/or diseases. In particular claimed invention relates to potassium salt of D-isoglutamyl-D-tryptophan (1:1) and magnesium salt of D-isoglutamyl-D-tryptophan (2:1).

EFFECT: obtaining pharmaceutically acceptable crystalline or amorphous salts of D-isoglutamyl-D-tryptophan.

22 cl, 15 dwg, 13 ex

FIELD: food industry.

SUBSTANCE: invention relates to fodder additives containing dipeptides or their salts; one amino acid residue of dipeptide is represented by DL-methionine residue; the other amino acid residue of dipeptide is represented by amino acid in L-configuration chosen from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

EFFECT: described are fodder mixtures containing such additives and the said dipeptides production method.

31 cl, 17 dwg, 10 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compounds, which can be used as inhibitors of protease of hepatitis C virus, pharmaceutical compositions, containing the said compounds, and methods of their application.

EFFECT: obtaining compounds which can be used as inhibitors of protease of hepatitis C virus.

41 cl, 10 dwg, 7 tbl, 26 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compounds, which can be used as inhibitors of protease of hepatitis C virus, to pharmaceutical compositions, which contain said compounds, and to methods of their application for treatment of diseases mediated by protease of hepatitis C virus.

EFFECT: obtaining compounds, which can be used as inhibitors of protease of hepatitis C virus.

37 cl, 22 dwg, 7 tbl, 34 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention described the macrocyclic compounds of formula wherein the radical values are presented in the patent claim. The above compounds are serine protease inhibitors wherein serine protease is hepatitis C virus (HCV) NS3 protease. This invention also discloses pharmaceutical compositions having antiviral activity against HCV, containing the claimed compounds, and one or more pharmaceutically acceptable carriers, as well as a method for preparing these compositions. The present invention describes a method of inhibiting the hepatitis C virus replication in a host, and a method of inhibiting activity of hepatitis C virus serine protease.

EFFECT: there are also presented a method of treating or preventing HCV infection, and a method of treating, preventing or ameliorating one or more symptoms of a liver disease or disorder associated with HCV infection in an individual.

69 cl, 39 ex, 8 tbl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula (I) a pharmaceutically acceptable salt thereof wherein each dash line (represented as ---) represents a double bond; X represents N or CH; R1a and R1b independently represent hydrogen or C1-6-jalkyl; L represents -O-; R2 represents hydrogen; R3 represents hydrogen or C1-6-alkyl; R4 represents quinolinyl substituted by one, two or three substitutes specified in C1-6-alkyl, C1-6-alkyloxy, thiazolyl or pyrazolyl, wherein said thiazolyl or pyrazolyl are substituted on any carbon atom by C1-6-alkyl; n is equal to 3, 4, 5 or 6; p is equal to 1 or 2. The invention refers to a pharmaceutical composition possessing the properties of KS3/4a-protease HCV inhibitors, containing a carrier, and an virally effective amount of the compound of formula (I) as an active ingredient. The method for preparing the compound of formula (I), wherein the above method involves forming an amide bond of an intermediate product (2a) and sulphonylamide (2b), as presented by the diagram, wherein G represents a group Also, the invention refers to alternative methods for preparing the compound of formula (I).

EFFECT: there are presented macrocyclic compounds possessing inhibitory activity on hepatitis C virus (HCV) replication.

13 cl, 1 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: application describes prodrugs being 2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)-phenyl]pyridine-3,5-dicarbonitryl derivatives, and a method for preparing them.

EFFECT: invention can find application in treating and/or preventing cardiovascular diseases.

8 cl, 4 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing pure crystalline D-isoglutamyl-D-trytophan which involves a step of removing protection from essentially pure N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan or diester thereof to yield essentially pure D-isoglutamyl-D- tryptophan. An amorphous ammonium alt of D-isoglutamyl-D- tryptophan (1:1) is also disclosed. Also disclosed is a method of producing a pure monoammonium salt of D-isoglutamyl-D-tryptophan from essentially pure N-tert-butoxycarbonyl-D- isoglutamyl-D-tryptophan. Disclosed is a compound H-D-Glu-(γ-D-Trp-OR2)-α-OR1 and pharmaceutically acceptable acid addition salts thereof. Disclosed is a solid pharmaceutical composition and use thereof as an immunodepressant or anti-psoriasis agent.

EFFECT: improved method.

51 cl, 14 ex, 8 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to organic chemistry, namely new 3,8-diaminotetrahydroquinoline derivatives of formula (1a) or to their pharmaceutically acceptable salts wherein X represents CH2, C=O or CH-OR; m is 1 or 2; Ar represents a phenyl group or a 5-merous or 6-merous aromatic heterocyclic group having one element specified in S and N, (wherein the phenyl group may be substituted by 1-2 halogen atoms); each R1 and R2 represents a hydrogen atom; R3 represents a C1-C6 alkyl group or indolyl-C1-4 alkyl group (the indolyl group is optionally substituted by a C1-C6 alkyl group or a halogen atom), n is 0; R4 and R5 which may be identical or different, each represents a hydrogen atom or a C1-C6 linear or branched alkyl group; each R6 and R7 represents a hydrogen atom; and R represents a hydrogen atom. Also, the present invention refers to a drug preparation and a pharmaceutical composition of the basis of the compound of formula (1a), to the compound of formula (F1), to a method for preparing an intermediate compound (e).

EFFECT: there are prepared new 3,8-diaminotetrahydroquinoline derivatives which possess high GHS-R antagonist activity.

10 cl, 1 tbl, 124 ex

FIELD: biotechnology.

SUBSTANCE: method of obtaining SSI comprises the following steps. The strain Yersinis pestis KM 1279 is grown on 1.5% agar LB, the bacteria are washed three times with cold buffered normal saline. The bacteria are pelleted by centrifugation, suspended in a solution of 5 mM NaOH, kept at 37°C for two hours and the cells are pelleted by centrifugation. The supernatant is selected and the procedure as repeated three times, three supernatants are combined and filtered through the nitrocellulose membrane. The filtrate is extracted three times with the mixture of chloroform-methanol-water in a ratio of 5:2:1. The chloroform fractions are separated by centrifugation, combined and freed from water-soluble impurities. The aqueous fraction is separated by centrifugation and removed, and the chloroform fraction is dried in a vacuum rotary evaporator and the dry preparation SSI is obtained. The proposed SSI is characterised with brown colouring of dry crystals, hydrophobic properties, fluorescence in ultraviolet, lipopeptide nature, the presence of iron ions, the molecular weight of 380.6 Da.

EFFECT: inventions enable to obtain the natural regulator of virulence of plague agent.

2 cl, 6 dwg, 6 ex

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