RU1825800C - 5-(tosylglycyl -l-prolyl -l-arginyl)aminonaphthalene -1-methylsulfonamide as a ansa-substrate for thrombin assay - Google Patents

Abstract

Usage: in bioorganic chemistry as ANSA substrate for the determination of thrombin. SUMMARY OF THE INVENTION: 5- (tosylglycyl-1-prolyl-1-arginip) -aminonaphthalene-1-methylsulfonamide. Reagent I: tosylglycyl-1-lroline. Reagent II: 5-arginylaminononaphthaline-1-methylsulfonamide dihydrochloride. The process is carried out in dimethylformamide in the presence of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide upon cooling, followed by neutralization with triethylamine, dilution with water, extraction, and evaporation. Yield 82%. C3iH4oNG07S2, mp 184-195 ° C. 20-8.7 (.СНзОН), Лпах No. (.СНзОН), Лпах254нм.

Description

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with

The invention relates to the field of bioorganic chemistry, and in particular to a new peptidyl derivative of aminonaphthalenesulfamides. specifically to 5- {tosylglycyl-1-prolyl-1-arginyl) aminonaphthalene-1-methylsulfonamide of the formula

ToS-Gly-PrO-Arg-NH

& §

S02NHCH,

which can be used in chemistry to produce an artificial chromogenic substrate, and in biochemistry to determine thrombin by ANSA analysis.

The aim of the invention is to search for a number of new compounds in a series of substituted peptidyl derivatives of aminonaphthalene sulfamides that allow thrombin to be determined when used as ANSA substrates.

The goal is achieved by the new 5-tosylglycyl-1-prolyl-b-arginylaminon-aftaline-1-methylsulfonamide as an ANSA substrate.

The invention is illustrated by examples.

Example 1. 5-Arginylaminaminaphthalene-1-methylsulfonamide dihydrochloride.

3.3 g (10 mmol) G of tert-butyloxycarbonyl arginine (monohydrate) is dissolved in 10 ml of dry DMF, 30 ml of dry

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ABOUT

toluene. The solution was evaporated under reduced pressure to 10 ml, cooled to 10 ° C, 0.8 m (10 mmol) of dry pyridine and 2.7 ml (15 mmol) of di-tert-butyl pyrocarbonate were added. After stirring for 1 hour, 2.6 g (11 mmol) of 5-aminonaphthalene-1-methylsulfonamide were added portionwise over 40 minutes. Stirred for 3 hours at 20 ° C, after which 1.3 ml (7 mmol) of di-tert-butyl pyrocarbonate was added and stirred for another 16 hours at 20 ° C. The reaction mixture is poured into 100 ml of dry ether, stirred for 10 minutes, the ether is decanted, the residue is poured into 50 ml of a mixture of trifluoroacetic acid - methylene chloride (1: 1) and stirred for 2 hours at 20 ° C, concentrated to 10 ml and poured into 100 ml of dry ether . The resulting product is filtered off with suction, dried, dissolved in 20 ml of water, and unreacted aminonaphthalene sulfamide is filtered off. The aqueous solution was extracted with 80 ml of n-butanol, the organic layer was separated, washed with 5% MAHC. PH 7-8 (water layer), then with water (2x10 ml), concentrated to 15 ml and saturated with dry HCI. The solvent was evaporated, the residue was triturated with ether, the precipitate formed was filtered off with suction and dried. Obtain 4.3 g (93%) of the target product, so pl. 181-190 ° C, 44 (BTsV 412), l l lD20 + 7.9 (CI; CHzON) 1 H-NMR (DMSO):

2.38 (CH3).

Found,%: C 43.58; H 5.75; N, 18.32; 57.03; CI 15.56.

S17N26Mb50zS12.

Calculated,%: C 43.87; H 5.63; N, 18.06; S6.89: CI 15.24.

Example 2. 5- (Toeylglycyl-prolyl-1-arginyl) aminonaphthalene-1-methylsulfonamide.

0.33 g (1 mmol) of tosylglycyl-1-proline, 0.14 g (1 mmol) of 1-hydroxybenzotriazole and 0.27 g (1.3 mmol) of dicyclohexylcarbodiimide are mixed, 7 ml of chilled to -20 ° are added From dry dimethylformamide, it is stirred in an ice bath for 0.5 hour. 0.47 g (1 mmol) of 5-erginylaminonaphthalene-1-methylsulfonamide dihydrochloride obtained in approx. 1, in 4 ml of dimethylformamide, the solution was added to the reaction mixture, stirred in an ice bath for another 0.5 hours. Triethylamine was added to pH 7, stirred for another 16 hours, the reaction mixture was allowed to warm to 20 ° C, and the precipitate was filtered off; the reaction mixture is poured into 100 ml of water. The resulting suspension was extracted four times with 10 ml of a mixture of ethyl acetate-butanol (1: 1). the organic layer was separated and washed three times with 5 mt of 5% aodic PaHCO :. 10% of KHSCu and milk yield of TJ

5

The reagent is aryuated, the remaining oily residue is triturated with dry ether, filtered off, washed with ether, and dried. Obtain 0.57 g (82%) of the target product, so pl., 184-195 ° C. Ryu, 46 (BUV 412). | | | P20-8.7 (CI: CHzON).

Found. %: C 55.21; H, 6.13; N, 12.18; S9.71.

CaiH4oNe07S2.

Calculated,%: C 55.34; H 5.99; N, 12.49: 59.53.

PMR (DMSO) Tos-Gly -Pro-ArgHN

foToi

SOjNHCHj

1.74 (m, 4H, 2CH2), 1.83 (m, 4H, 2CH2), 2.28 (s, 3H, CH3), 3.25 (m, 2H. CH2), 3.39 (m, 2H. CH2).

0 3.61 (d, 2H, CH2), 4.13 (m, 1H, CH), 4.46 (m, 1H. CH), 7.28 (g, 2H. 2CH-aromatic), 7.64 (q , 2H, 2CH-aromatic). 5.30-8.72 (14H, 6H-naphthalene. 8H-NH).

PRI me R 3. Analysis of thrombin.

5 to 0.3 ml of a substrate mixture containing 3.5 O.E. (nm) / ml: 5- (ghG-tosylglycyl-1-prolyl-1-arginyl) aminonaphthalene -1- (n-butyl) sulfonamide and methyl sulfonamide, 0.1 O.E. (A 340 nm) danzilvaline

0 (internal standard) in 0.05 M Tris-HCI buffer, pH 7.4 10, ikl thrombin aqueous solution, stirred, initiated for 2 min at 37 ° C, CH3COOH was added to pH 4.3, mixed; 0.5 ml of a mixture of ethyl acetate-hexane (2: 1) was added, again stirred, the top layer was separated, dried, the residue was dissolved in 0.05 M ammonium acetate in 35% acetonitrile, chromatographed on a Zorbax C-8 column (4.6x250 mm) at 40 ° C and

At a flow rate of 1 ml / min, 5-aminonaphthalene-1-sulfonamides were determined from the maximum absorption at 254 nm. Upon completion of the enzymatic reaction, the peak of the starting substrate disappears (99%) and at the same time peaks of the products of the enzymatic hydrolysis of 5-aminonaphthalene-1- (n-butyl) sulfonamide and -1-methyl sulfonamide appear. Retention times of 8.3 minutes and 2.3 minutes, respectively.

0 the table shows the experimental results confirming the linear dependence of the determined amounts of cleaved amine, it phthal n- (methyl l) sulfamide on the amount of thrombin 5

Stock solution of the farm g P 26 mg / ml bovine thrombin. Tramp incubation 12

5

min, temperature 37 ° C. In nm absorption

D TRACTION BY

t BOK-L-Arg-OH H20

93%

2HCI H-L-Arg-AHCA (Me)

Tos-Gly-L-Pro-OH, 1-hydroxybenzotriazole, dicyclohexylcarbodiimide, triethylamine

82%

Tos-Gly-L-Pro-L-Arg-AHCA (Me)

Claims (1)

The synthesis scheme for TLC is presented below: Silufol UV-254 plates, 1 H NMR spectra were recorded on a Hitachi P-22 spectrophotometer (90 MHz, Japan), rotation angles were measured on a Beckman polarimeter, and HPLC on a Du Pont chromatograph (USA). Claims 5- (Tosylglycyl-1-prolyl-1-arginyl) aminonaphthalene-1-methylsulfonamide of the formula (i.e.BOK) 20, ANSA (Me) HCI Tos-gly-pro-arg-NH OY S02NHCH b as an ANSA substrate for the determination of 10 thrombin.