Derivatives of 6-(d-leucyl-l-prolyl-l - arginyl/aminonaphthalene - 1 - intemationally as fluorescent substrates for the determination of factor ixa


C07C311/38 - having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton

 

(57) Abstract:

The invention relates to the field of Bioorganic chemistry, namely to new connections dibromide 6-(D-leucyl-L-prolyl-L-arginyl), aminonaphthalene-1-pentanitroaniline (1) and its benzyloxycarbonyl (Z) is the derivative (2). The essence of the invention lies in the fact that the new compounds are stable in buffer solutions. The time of hydrolysis of factor IXa is equal to 21 and 24 min, respectively. They detected the group has fluorescent properties. All these properties ensure the advantage of the proposed compounds in comparison with the prototype when used as substrates for the determination of factor IXa simplified analysis method of factor IXa by eliminating the need for additional expensive second component for detecting increases the reliability of the results of the analysis and committing a concentration 10 times lower than in the prototype, due to the use of fluorescent method (characteristic of the device).

The invention relates to the field of Bioorganic chemistry, namely to new connections dibromide 6-(D-leucyl - L-prolyl-L-arginyl)-aminonaphthalene-1-pentanitroaniline (I) and its benzilic the blood coagulation factor IXa.

Fluorogenic substrates for the determination of factor IXa with effective detektiruya group no.

Known complex thioesters of amino acids or peptides, for example, thiobenzoate N-carbobenzoxy-arginine or lysine, which are more often used in the analysis of factor IXa (McRae et al. Biochemistry 1980, v. 19, Nr. 17, p. 3973). During enzymatic hydrolysis they turn into thiols, for the detection of which by a spectrophotometric method using the second component is 4,4' -zitieren (1) or 5,5' -dithiobis-(2-nitrobenzoic acid) (Green, G. D. J. et al. Anal. Biochem. 1979, v. 93, Nr. 2, p. 223). The last connection is expensive and difficult to access.

Known complex thioethers have several disadvantages. First, they undergo spontaneous hydrolysis in buffer solutions. Even at high concentrations of factor IXa level of spontaneous hydrolysis is 6% (see example 8) from the enzymatic decomposition of the substrate. This distorts the results of enzymatic hydrolysis. In addition, their use in two-component system complicates the definition and makes it more expensive method of analysis.

The aim of the invention is the finding of peptide substrates to simplify and reduce the cost method opredeleniyami 6-(D-leucyl-L-prolyl-L-arginyl)aminonaphthalene-1-pentamethylenetetrazol (I) or its Z-derivative (II) as substrates for the direct determination of factor IXa. When using these compounds, there is no need to use the second component for the formation of the absorbing compounds (see example 7). The results of the analysis are more reliable, because the substrates I and II are stable in buffer solutions (see example 9). Detectable group proposed substrate is 6-aminonaphthalene-1-pentamethylbenzene, the possession - Mering fluorescent properties, which allows you to reliably capture its concentration on an "itachi MPF-4" to 0.05 μm. In the case of the prototype Z-Lys-SBzl using 4,4' -dicipline minimum detectable concentration (Cmin0.5 μm, and when using 5,5' -dithiobis-(2-nitrobenzoic acid)min= 0,74 ám. The minimum concentration is calculated by the formula given in the literature (M. Bulatov I. , Kalinkin I. P. a Practical guide photocolorimetric and spectrophotometric methods of analysis. L. , Chemistry, 1976, S. 39):

cmin= . Taking l= 1 cm and Dmin= 0,01 received: 4,4' -dicipline F= 19800 M-1cm-1when = 324 nm

cmin= = 5,0510-7= 0.5 µm, 5,5' -dithiobis-(2-nitrobenzoic acid)

E= 13600 M-1cm-1when = 412 nm

cmin= = 7,410-7= 0 is the R substrates, it is possible to reliably fix a concentration 10 times less than in the prototype, i.e., 0.05 to MK instead of (0,5-0,74) ám.

Synthesis of substrates, their stability and hydrolysis factor A are illustrated by examples.

P R I m e R 1. 6-phthalimidomethyl-1-pentamethylbenzene.

Dissolve 11 ml (0.11 mol) of piperidine and 14 ml (0.1 mol) of triethylamine in 500 ml of acetone, added dropwise within 10 min of 37.1 g (0.1 mol) of 6-phthalimidomethyl-1-sulphonylchloride and stirred at 20aboutWith 4 hours the Acetone is distilled off, the residue is poured 1 liter of water, the product is sucked off, washed with water, dried, recrystallized from methanol and get to 39.3 g (yield 94% ) of the product so pl. 165 to 169aboutC.

Found, % : C 65,88; N 4,84; N 6,61; S 7,41

WITH23H20N2SO4< / BR>
Calculated, % : FROM 65.7; H 4,79; N 6,66; S 7,62

P R I m m e R 2. 6-Aminonaphthalene-1-pentamethylbenzene.

Pour 42,0 g (0.1 mol) of 6-phthalimidomethyl-1-pentanitroaniline 450 ml of methanol, was added dropwise 5 ml (0.1 mol) of hydrazine hydrate is added and heated for 4.5 hours, the Methanol is distilled off, the residue is extracted with CH ml of boiling chloroform, the extract evaporated and the residue recrystallized from methanol. Get 23,5 g (yield 81% ) of product with so pl. 138-143aboutC. Rf= 0,78 (chloroform-ethyl acetate 1: 1). PMR (DMSO): 1,30 (CH2), 2,96 (CH2).

Neid Is R> P R I m e R 3. Bromohydrin 6-(N-benzyloxycarbonyl)-L-organisationally-1-pentamethylbenzene.

Dissolve to 3.89 g (0.01 mol) of bromhidrosis N-benzyloxycarbonyl-L-arginine and 2,90 g (0.01 mol) of 6-aminonaphthalene-1-pentanitroaniline in 15 ml of dry pyridine, add 30 ml of dry toluene and the latter is distilled off. The reaction mixture is cooled to -20aboutAnd add a solution 2,22 g (0.11 mol) of dicyclohexylcarbodiimide (DCC) in 6 ml of dry pyridine. The reaction mixture was kept at -20aboutC for 0.5 h, at 4aboutC for 1 h and at 20aboutWith over 20 hours Sucked off the fallen dicyclohexylamine, pyridine evaporated and the residue is dissolved in 40 ml of a mixture of chloroform-propanol (3: 1). The solution was washed with 15 ml of water, 15 ml of saturated aqueous NaCl, containing 1 ml of conc. HCl, 15 m 2% aqueous ammonia and 15 ml of water, dried over anhydrous Na2SO4. The solvent is distilled off, the residue is triturated with ether, sucked off, dried and recrystallized from propanol. Get 6,15 g (yield 93% ) of the product so pl. 117-123aboutS, Rf= 0,71 (butanol-acetic acid-water 4: 1: 2) (PDR 412), //D20by 12,8about(1; CH3IT), PMR (DMSO): 1,30 (CH2), 2,99 (CH2).

Found, % 2,10

P R I m e R 4. Bromohydrin 6-L-organisationally-1-pentanitroaniline.

Dissolve 6,62 g (0.01 mol) of bromhidrosis 6-(benzyloxycarbonyl)-L-organisationally-1-pentanitroaniline in 10 ml of 3N HBr in glacial acetic acid and incubated at 20aboutC for 1.5 h, the Reaction mixture was poured into 100 ml of dry ether, the precipitated precipitate is sucked off, washed with ether and dried. The dry residue is dissolved in 10 ml of water, add 50 ml of butanol and with shaking portions of 15 ml of 5% aqueous NaHCO3to pH 7.5 aqueous layer. The organic layer was washed with 2x10 ml of water, butanol evaporated, the residue triturated with ether, sucked off, washed with ether, dried and recrystallized from methanol. Get 4,91 g (yield 93% ) of the product so pl. 121-130aboutC, //D20-1,2about(1; DMSO), Rf= 0,48 (PDR 412). PMR (DMSO): 1,30 (CH2), 3,00 (CH2).

Found, % : C 47,93; N 6,01; N 15,81; S 5,76; Br 15,44

C21H31N6SBrO3< / BR>
Calculated, % : C 47,82; N. Of 5.92; N 15,93; S Between 6.08; Br br15.15

P R I m e R 5. 6-(Benzyloxycarbonyl-D-leucyl-L-prolyl-L-arginyl) aminonaphthalene-1-pentamethylbenzene (II).

Dissolve 0,86 g (2.5 mmol) of Z-D-Leu-L-Pro-OH, 0.35 g (2.5 mmol) of 1-hydroxybenzotriazole and 0.51 g (2.5 mmol) DCC in 18 ml) cooled to-P>about1 h and poured chilled to 4aboutTo a solution of 1.32 g (2.5 mmol) of bromhidrosis 6-L-organisationally-1-pentamethylene - lifemed in 10 ml of DMF. Stirred at 20aboutC for 20 h, sucked off the fallen dicyclohexylphosphino, the filtrate is poured into 125 ml of water, and the resulting suspension extracted with CH ml of a mixture of ethyl acetate: butanol (1: 1). The organic layer was washed with I ml of 5% aqueous NaHCO3, 10% aqueous SO4and water, the solvent evaporated, the residue triturated with ether, sucked off, washed with ether and dried. Get to 1.77 g (yield 92% ) of chromatographically pure target product with so pl. 167-172aboutC, //D20-9,9about(1; DMSO), Rf= 0,69 (PDR 412). PMR (DMSO): 1,30 (CH2), 3,00 (CH2), 5,01 (CH2, Z), 7,32 (C6H5, Z).

P R I m e R 6. Dihydrobromide 6-(D-leucyl-L-prolyl-L-arginyl)-aminonaphthalene-1-pentanitroaniline (I).

Dissolve 0,79 g (1 mmol) of the compound (II) in 5 ml of 3N HBr in glacial acetic acid and incubated at 20aboutC for 2 h, the Reaction mixture was poured in 50 ml of dry ether, the precipitated precipitate is sucked off, washed with ether and dried. Get 0,70 g (yield 85% ) of chromatographically pure target product with so pl. 210-222aboutC, //D20-20,9

Prepare a 1 μm solution of 6-aminonaphthalene-1-pentanitroaniline in 0.1 M Tris. -HCl buffer, pH 8.0, 0.1 M NaCl and 25aboutTo measure the fluorescencewosb.= 352 nm). In the cell of fluorimetry put 1 μm solution of the substrate 1 in 0.1 M Tris. HCl buffer. pH 8.0, 0.1 M NaCl, thermostatic at 25aboutC, add a solution of factor A in the same buffer at a concentration of 0.1 µm, and measure the increase in fluorescence at = 470 nwosb.= 352 nm). Record the time required for 10% hydrolysis, which is 21 minutes

Similarly conduct the reaction of compound II with factor A, fixed with 10% aqueous hydrolysis 24 minutes

P R I m e R 8. Check the stability of Z-Lys-SBzl (prototype).

A. a Solution of Z-Lys-SBzl, the concentration of which is 100 μm in 0.1 M Tris. -HCl buffer, pH 8.0, 0.1 M such as NaCl, is placed in the cuvette of the spectrophotometer and thermostatic at 25aboutC. Add a solution of 5,5' -dithiobis-(2-nitrobenzoic) acid (DTNB)2(concentration in the solution of 2 mmol/l) and measuring the increase in optical density at = 412 nm (D412in the course of time. The increase in D412for 1 h, is 0,156, i.e. D412/1min = = 0,0026.

B. In the same solution as in the experience And, instead of (DTNB)2add the factor Ha (concentration in solution of 0.1 μm) ,0412.

Thus, the spontaneous decomposition of the substrate Z-Lys-SBzl at 0.1 microns factor A is:

= 6.3% of its enzymatic decomposition.

P R I m e R 9. Check the stability of the compounds I and II.

Prepare 1 μm and 100 μm solutions of dibromide 6-(D-leucyl-L-prolyl-L-arginyl)-aminonaphthalene-1-pentane-Transylvania (I) in 0.1 M Tris. -HCl buffer, pH 8.0, 0.1 M NaCl, placed in a thermostated at 25aboutWith the cell of fluorimetry and measure the fluorescence at = 470 nmwosb.= 352 nm). Fluorescence is absent. The measurement is repeated for 10 h every 24 h for 10 days. The increase in fluorescence is not seen.

Similarly conduct a measurement of the stability of compound (II). Fluorescence is also not seen.

Examples 7-9 show that the proposed dibromide 6-(D-leucyl-L-prolyl-L-arginyl)aminonaphthalene-1-pentane - Transylvania or 6-(benzyloxycarbonyl-D-leucyl-L-prolyl-L-arginyl)amino - naphthalene-1-pentamethylbenzene are stable in buffer solutions. The time of hydrolysis factor A equal to 21 and 24 min, respectively. They detected the group has fluorescent properties.

These properties provide the following benefits proposed is a: simplified method of analysis of factor IXa by eliminating the need for additional expensive second component for detection; increase the reliability of analysis results; reliable commit concentration 10 times lower than in the prototype, due to the use of fluorescent method (characteristics of the device).

Derivatives of 6-/D-leucyl - L-prolyl-L-arginyl/ -aminonaphthalene-1-intemationally General formula

RDLeuLProhArgHN

where R = H, X = 2HBr,

R - benzyloxycarbonyl, x is absent. as fluorescent substrates for the determination of factor A.

 

Same patents:

FIELD: organic chemistry, medicine.

SUBSTANCE: invention represents ligands MC-4 and/or MC-3 of the formula (I): , wherein X means hydrogen atom, -OR1, -NR1R1' and -CHR1R1' wherein R1 and R1' are taken among the group: hydrogen atom, (C1-C6)-alkyl and acyl; (1) each R2 is taken independently among the group: hydrogen atom, (C1-C6)-alkyl; or (2) (a) R2 bound with carbon atom that is bound with X and Z1 and substitute R5 can be optionally bound to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; or (b) R2 bound with carbon atom that is bound with ring Ar can be bound with R7 to form ring condensed with ring Ar; each among Z1, Z2 and Z3 is taken independently from the following groups: -N(R3e)C(R3)(R3a)-, -C(R3)(R3a)N(R3e)-, -C(O)N(R3d)-, -N(R3d)C(O)-, -C(R3)(R3a)C(R3b)(R3c)-, -SO2N(R3d)- and -N(R3d)SO2- wherein each among R3, R3a, R3b and R3c, R3d, R3e when presents is taken independently among hydrogen atom and (C1-C6)-alkyl; p is a whole number from 0 to 5 wherein when p above 0 then R4 and R4' are taken among hydrogen atom, (C1-C6)-alkyl and aryl; R5 represents 5 substitutes in phenyl ring J wherein each R5 is taken among hydrogen atom, hydroxy-, halogen atom, thiol, -OR12, -N(R12)(R12'), (C1-C6)-alkyl, nitro-, aryl wherein R12 and R12' are taken among hydrogen atom and (C1-C6)-alkyl; or two substitutes R5 can be bound optionally to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; q = 0, 1, 2, 3, 4 or 5 wherein when q above 0 then R6 and R6' are taken among hydrogen atom and (C1-C6)-alkyl; Ar is taken among the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine; R7 are substitutes at ring Ar wherein each R7 is taken among hydrogen, halogen atom, -NR13R13', (C1-C6)-alkyl and nitro- wherein R13 and R13' are taken among hydrogen atom and (C1-C6)-alkyl; r is a whole number from 0 to 7 wherein when r is above 0 then R8 and R8' are taken among hydrogen atom and (C1-C6)-alkyl; B is taken among -N(R14)C(=NR15)NR16R17, -NR20R21, heteroaryl ring and heterocycloalkyl ring wherein R14-R17, R20 and R21 are taken independently among hydrogen atom and (C1-C6)-alkyl; s = 0, 1, 2, 3, 4 or 5 wherein when s is above 0 then R and R9' are taken among hydrogen atom and (C1-C6)-alkyl; R10 is taken among the group consisting of optionally substituted bicyclic aryl ring and optionally substituted bicyclic heteroaryl ring; D is taken among hydrogen atom, amino- and -C(O)R11 wherein R11 is taken among the following group: hydroxy-, alkoxy-, amino-, alkylamino-, -N(R19)CH2C(O)NH2 wherein R19 represents (C1-C6)-alkyl, -NHCH2CH2OH and -N(CH3)CH2CH2OH, or its isomers, salts, hydrates or biohydrolysable ester, amide or imide.

EFFECT: valuable medicinal properties of compounds.

18 cl, 107 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to macrocyclic peptides of the general formula (I): wherein W means nitrogen atom (N); R21 means hydrogen atom (H), (C1-C6)-alkoxy-, hydroxy-group or N-(C1-C6-alkyl)2; R22 means hydrogen atom (H), (C1-C6)-alkyl, CF3, (C1-C6)-alkoxy-group, (C2-C7)-alkoxyalkyl, C6-aryl or Het wherein het means five- or six-membered saturated or unsaturated heterocycle comprising two heteroatoms taken among nitrogen, oxygen or sulfur atom and wherein indicated Het is substituted with radical R24 wherein R23 means hydrogen atom (H), -NH-C(O)-R26, OR26, -NHC(O)-NH-R26, -NHC(O)-OR26 wherein R26 means hydrogen atom, (C1-C6)-alkyl; R3 means hydroxy-group or group of the formula -NH-R31 wherein R31 means -C(O)-R32, -C(O)-NHR32 or -C(O)-OR32 wherein R32 means (C1-C6)-alkyl or (C3-C6)-cycloalkyl; D means a saturated or unsaturated alkylene chain comprising of 5-10 carbon atoms and comprising optionally one-three heteroatoms taken independently of one another among oxygen (O), sulfur (S) atom, or N-R41 wherein R41 means hydrogen atom (H), -C(O)-R42 wherein R42 means (C1-C6)-alkyl, C6-aryl; R4 means hydrogen atom (H) or one-three substitutes at any carbon atom in chain D wherein substitutes are taken independently of one another from group comprising (C1-C6)-alkyl, hydroxyl; A means carboxylic acid or its alkyl esters or their derivatives. Invention relates to pharmaceutical compositions containing indicated compounds and eliciting activity with respect to hepatitis C virus and these peptides inhibit activity of NS3-protease specifically but don't elicit significant inhibitory activity with respect to other serine proteases.

EFFECT: valuable biochemical and medicinal properties of peptides.

106 cl, 9 tbl, 61 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to method for production of acetylamidiniophenylalanylcyclohexylglycilpypidinioalanin amides of formula I , wherein X anions are physiologically acceptable anions, and analogous thereof. Said compounds are effective inhibitors of fibrillation factor Xa and are useful, for example, in prevention of thrombosis. Claimed method includes coupling of 2-[2-acetylamino-3-(4-amidinophenyl)-propionylamino]-2-cyclohexylacetic acid, obtained from 2-[2-acetylamino-3-(4-cyanophenyl)acryloylamino]-2-cyclohexylacetic acid by assimetric hydration and converting of cyano group to amidine, or salt thereof with 3-(2-amino-2-carbamoylethyl)-1-methylpyridinic acid or salt thereof. Also are disclosed starting materials and intermediated used in this method, process for production the same and acetyl-(S)-4-amidiniophenylalanyl-(S)- cyclohexylglycil-(S)-(1-methyl-3-pypidinio)alanin amide in form of ditosylate.

EFFECT: simplified method; increased commercial availability of compounds with applicable anion.

14 cl, 16 ex

FIELD: synthesis of biologically active compounds.

SUBSTANCE: invention provides 1,5-benzothiazepines of general formula I (formulae presented below), in which Rv and Rw are independently selected from hydrogen and C1-C5-alkyl; one of Rx and Ry represents hydrogen or C1-C6-alkyl and the other hydroxy or C1-C6-alkoxy; Rz is selected from halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1 of invention; v is a number from 0 to 5; one of R4 and R5 represents group of general formula IA; R3 and R6 and the second from R4 and R5 are independently selected from hydrogen, halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1; R3 and R6 and the second from R4 and R5 being optionally substituted by one or several R16 groups at their carbon atoms; D represents -O-, -N(Ra)-, -S(O)b- or -CH(Ra)-, wherein Ra is hydrogen or C1-C6-alkyl; and b=0-2; ring A represents aryl or heteroaryl and is optionally substituted by one or several substituents selected from R17; R7 represents hydrogen, C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R18; R8 represents hydrogen or C1-C4-alkyl; R9 represents hydrogen or C1-C4-alkyl; R10 represents hydrogen or C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R19; R11 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORc)(ORd), -P(O)(OH)(ORc), -P(O)(OH)(Rd), or -(O)(ORc)(Rd), wherein Rc and Rd are independently selected from C1-C6-alkyl; or R11 represents group of general formula IB, in which X is -N(Rq)-, N(Rq)C(O)-, -O-, or -S(O)a, wherein a=0-2; and Rq is hydrogen or C1-C4-alkyl; R12 represents hydrogen or C1-C4-alkyl; R13 and R14 are independently selected from hydrogen, C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23 can be optionally independently substituted by one or several substituents selected from R20; R15 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORe)(ORf), -P(O)(OH)(ORe), -P(O)(OH)(Re), or -P(O)(ORe)(Rf), wherein Re and Rf are independently selected from C1-C6-alkyl; or R15 represents group of general formula IC, in which R24 is selected from hydrogen and C1-C4-alkyl; R24 is selected from hydrogen, C1-C4-alkyl carbocyclyl, heterocyclyl, and R27, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R27 can be optionally independently substituted by one or several substituents selected from R28; R26 is selected from carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORg)(ORh), -P(O)(OH)(ORg), -P(O)(OH)(Rg), or -P(O)(ORg)(Rh), wherein Rg and Rg are independently selected from C1-C6-alkyl; p=1-3; wherein meanings for R13 can be the same or different; q=0-1; r=0-3; wherein meanings for R14 can be the same or different; m=0-2; wherein meanings for R10 can be the same or different; n=1-3; wherein meanings for R7 can be the same or different; z=0-3; wherein meanings for R25 can be the same or different; R16, R17, and R18 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N,N-(di-C1-C4-alkyl)amino, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, and N,N-(di-C1-C4-alkyl)sulfamoyl; wherein R16, R17, and R18 can be optionally independently substituted by one or several of R21 at their carbon atoms; R19, R20, R23, R27, and R28 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N.N-(di-C1-C4-alkyl)amino, C1-C4-alkanoylamino, N-(C1-C4-alkyl)carbamoyl, N,N-(di-C1-C4-alkyl)carbamoyl, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, N,N-(di-C1-C4-alkyl)sulfamoyl, carbocyclyl, heterocyclyl, sulfo, sulfino, amidino, phosphono, -P(O)(ORa)(ORb), -P(O)(OH)(ORa), -P(O)(OH)(Ra), or -P(O)(ORa)(Rb), wherein Ra and Rb are independently selected from C1-C6-alkyl and wherein R19, R20, R23, R27, and R28 can be optionally independently substituted by one or several of R22 at their carbon atoms; R21 and R22 are independently selected from halogen, hydroxy, cyano, carbamoyl, mercapto, sulfamoyl, trifluoromethyl, trifluoromethoxy, methyl, ethyl, methoxy, ethoxy, vinyl, allyl, ethynyl, methoxycarbonyl, formyl, acetyl, formamido, acetylamino, acetoxy, methylamino, dimethylamino, N-methylcarbamoyl, N,N-dimethylcarbamoyl, methylthio, methylsulfinyl, mesyl, N-methylsulfamoyl, N,N-dimethylsulfamoyl; or pharmaceutically acceptable salt thereof, solvate, or salt solvate. Described are also method for preparing compounds of formula I, pharmaceutical compositions based on compounds I, and a method for achieving inhibiting effect relative to interscapular brown adipose tissue (IBAT), and intermediates. (I), (IA), (IB), (IC).

EFFECT: expanded synthetic possibilities in the 1,5-benzothiazepine series.

36 cl, 121 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: 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: biochemistry.

SUBSTANCE: invention relates to method for tripeptide production of formulae Ac-D-2Nal-D-4ClPhe-D-3Pal-OH and Boc-D-2Nal-D-4ClPhe-D-Pal-OH, which represent intermediates for synthesis of LHRH analogs in combination with acceptable heptapeptides in particular P1-Ser(P2)-NMeTyr(P3)-D-Lys(Nic)-Leu-Lys(iPr,P4)-Pro-D-AlaNH2 and P1-Ser(P2)-NMeTyr(P3)-D-Asn-Leu-Lys(iPr,P4)-Pro-D-AlaNH2 heptapeptides.

EFFECT: new synthetic intermediates for LHRH antagonists.

7 cl, 8 ex

FIELD: medicine, biochemistry.

SUBSTANCE: invention describes compounds that inhibit function of NS3-protease encoded by hepatitis C virus.

EFFECT: valuable medicinal properties of inhibitors.

6 cl, 2 tbl, 472 ex

FIELD: organic chemistry, biochemistry.

SUBSTANCE: invention describes heterocyclic compounds represented by the general formula (I): and possessing elastase-inhibitory activity, and intermediate compounds for synthesis of such compounds. In the formula (I) R1 represents heterocyclic group represented by the formula (II): wherein A represents presence or absence of benzene ring; X represents oxygen atom, sulfur atom or -NH; Y represents nitrogen atom or -CH. Indicated heterocyclic group can be substituted with 1-3 substitutes that can be similar or different and they are chosen from group consisting of lower alkyl, lower alkoxy group and phenyl that can be optionally substituted with halogen-containing lower alkyl, lower alkoxy group or halogen atom; each among R2 and R3 represents hydrogen atom or hydroxyl, or R2 and R3 can be combined to form oxo group under condition that both are not hydrogen atoms.

EFFECT: valuable biochemical property of compounds.

8 cl, 7 tbl

FIELD: chemistry, pharmaceutics.

SUBSTANCE: invention relates to immunology. Described is tripeptide-L-Tyrosyl-L-Seryl-L-Valine and its application in pharmaceutical composition and in production of food additive. Discovered is method of relieving state of human disease, where disease is selected from group, which consists of states which can be relieved by stimulation of T-lymphocytes transformation, and fault of cell proliferation, which includes introduction of described pharmaceutical composition. Claimed invention gives short peptides which possess biological activity.

EFFECT: obtaining short peptides which possess biological activity.

8 cl, 5 dwg, 1 tbl, 6 ex

FIELD: medicine, oncology, molecular pharmacology.

SUBSTANCE: invention relates to a method and set for identifying the individual subjected to risk for arising in it the vascular and cancer disease. Method involves stages for the quantitative determination of the analyte concentration, i. e. pepsinogen I (PGI), in serum sample taken in the personal individual; comparison of the analyte concentration determined by the proposed method with a method-specific boundary value for this analyte; determination of the homocysteine concentration in a serum sample taken in this individual. The set comprises the combination of separate components that are necessary for the quantitative determination of the PGI concentration. Method provides the early detection of the possibility for arising the vascular and cancer disease in the patient.

EFFECT: improved method for assay.

4 cl

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.

EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.

41 cl, 4 dwg, 17 ex

FIELD: medicine.

SUBSTANCE: according to the following stages, flow cytometry is used to detect living cells, damaged cells, VNC cells and dead cells of a microorganism in a tested sample: a) the stage of processing the tested sample with enzyme chosen from lipolytic enzymes and protease with activity to destruct the cells differing from those of the microorganism, colloid protein particles or lipids found in the analysed sample; b) the stage of processing the tested sample with topoisomerase inhibitor and/or DNA-gyrase inhibitor; e) the stage of processing the tested sample been processed at the stages a) and b) with a kernel-dyeing agent, and d) the stage of detecting microorganisms in the tested sample processed by the kernel-dyeing agent with using flow cytometry.

EFFECT: convenient and fast detecting of live microorganisms and identification of the damaged and dead cells in foodstuff and clinical samples.

19 dwg, 8 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there is offered a method for identification of an inhibiting candidate substance which enables inhibits hepsin-activated HGF. The method is based on comparing the degree of activation of pro-HGF substratum in a sample containing hepsin, pro-HGF and the analysed candidate substance, with the degree of activation of pro-HGF substratum in the reference sample who not containing the candidate substance. Also there is offered an agent for inhibiting hepsin and hepatocyte growth factor reaction containing a sequence of Kunitz domain which represents a KD-1 sequence of Kunitz domain HAI-1 or HAI-1B, or one or both Kunitz domains HAI-2.

EFFECT: invention allows identifying physiological hepsin modulators.

6 cl, 14 dwg, 1 tbl, 1 ex

FIELD: food industry.

SUBSTANCE: quantitative food proteins content determination method involves the following operations, sequentially performed: mixing test samples of the substrate and a fermentative substance coupled with a stabilising solution, incubation of the produced mixture at a temperature of 37°C, centrifugation of the fermentative-and-substrate complex and determination of quantitative food proteins content by way of calculation. The fermentative substance is represented by pancreatic juice preliminarily diluted with a stabilising solution down to 50% concentration, the ratio of the solution to the substrate test sample weight equal to 1:10. The prepared mixture incubation is performed during 5-15 minutes. Before quantitative food proteins content determination by way of calculation, the pure liquid fraction volume produced as a result of centrifugation is diluted with the stabilising solution at a ratio of 1:100-200. The food protein quantity is determined as equal to percentage expenditure of protease enzymes in comparison with a reference sample of pancreatic juice solution.

EFFECT: enhanced accuracy of determination of the quantitative food proteins content in food products.

3 tbl

FIELD: medicine.

SUBSTANCE: method of predicting efficiency of complex treatment of patients with nasopharyngeal cancer, which includes carrying out two-step course of radial therapy at the background of automyelochemical therapy, lies in the following: before treatment individual activity of enzyme kallikrein is determined in patient's blood plasma, and then on the 1-2 day after the end of the 1 step of complex treatment activity of enzyme kallikrein is determined in patient's blood plasma, and in case of its 2 and more fold reduction with respect to initial individual activity, efficiency of treatment is predicted, and in case if it reduces by less than 2 times, absence of clinical effect is predicted.

EFFECT: method ensures possibility of objective assessment of individual response to complex treatment and, in case of necessity, timely change of therapeutic mode.

3 ex

FIELD: medicine.

SUBSTANCE: there are presented a method and a kit for enzyme-linked thrombin-binding assay on C1 inhibitor functional activity. The method implies thrombin sorption in microplate wells, introduction of test samples containing the functionally active C1 inhibitor to be analysed, incubation that involves C1 inhibitor binding to thrombin, and measurement of the C1 inhibitor-bound amount by an enzyme conjugate with antibodies and a substrate of said enzyme. The kit comprises a flat-bottomed microplate with the sorbed preparation of thrombin, the enzyme conjugate with the human C1 inhibitor antibodies, a substrate buffer and a reference for measuring the active C1 inhibitor.

EFFECT: group of inventions provides the novel method and kit for enzyme-linked thrombin-binding assay on C1 inhibitor functional activity.

2 cl, 2 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to molecular biology and genetics. Disclosed is a method for qualitative and quantitative analysis of lipids that are strongly bound with genomic DNA, which comprises steps of isolating genomic DNA bound with lipids from cells using a detergent method, hydrolysis of DNA with a hydrolysing enzyme, separating lipids from the chloroform-methanol-water mixture at 37°C, evaporating the solvent, stabilising lipids with 2,6-di-tert-butyl-p-cresol, mixing the lipids with TMSH, chromatography with a gas chromatograph, followed by mass spectrometry.

EFFECT: method can be used to decode the lipid code of genomic DNA, ie, to determine the area of localisation of lipids that are strongly bound with genomic DNA for research and therapeutic purposes.

5 dwg

FIELD: measuring equipment.

SUBSTANCE: presented group of inventions refers to medicine. There are presented method and kit for the immune-enzyme assay of functional activity of human complement component C3. The method implies thrombin sorption in microplate wells, introduction of a test sample containing the human complement component C3 with unknown activity, incubation in the presence of ethylene diamine tetraacetate (EDTA), pouring out of the well contents, introduction of an enzyme conjugate with human complement component C3 antibodies and a substrate of the above enzyme. The component C3 activity is evaluated by the amount of the prepared product of enzymatic reaction. The kit comprises the flat-bottomed microplate with sorbed thrombin, conjugated enzyme and human complement component C3 antibodies, substrate buffer and donor blood serum with known activity C3 as a reference.

EFFECT: presented group of inventions enables evaluating the functional activity of human complement component C3 without the need of activation of the whole complement system, and possesses a good result reproducibility.

2 cl, 1 dwg, 2 ex

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