LV10943B - Method for preparation of 7-(n-benzyloxycarbonyl)arginylamido-4-methylcoumarine - Google Patents

Abstract

Invention applies to bio-organic chemistry, mainly the production of substrates used to evaluate activity of ferments and in the research of their activity mechanisms. The method for obtaining of N-benzyloxycarbonyl arginine 4-methylcoumaril-7 amide has been developed. This is substrate of fluorogenic ferments, the action of which is based on fermentative truncation of 7-amino-4-methylcoumarine and its ability to fluoresce at 440 nm.The method offered by the authors allows it to obtain N-benzyloxycarbonyl arginine 4 methylcoumaril-7 amide with high outcome (83%). The process is technologically easy. The result is achieved by mixing 7-amino-4-methylcoumarine suspension in pyridine together with phosphorous trichloride in pyridine for one hour after which hydrogen chloride of N-benzyloxycarbonyl arginine dissolved in pyridine is added. The mixture is heated for 3-4 hours at 40-50 degrees C temperature and kept for 10 hours in ambient temperature, pyridine is distilled off, the substance is treated with normal hydrochloric acid and crystallised from methanol/dimethylformamide (4:1).

Description

GB 10943 Λ T, 3LITZIL0KSIKAP301TILA3GGIETTA1 · · ODA LONG1TA3 I.Z3TOD2 7ILEULIAKIL-7-

The invention relates to the field of bioorganic chemistry, mainly to the production of enzyme substrates, which are used to determine the activity of enzymes and to study their mechanism of action. U-Benzyloxycarbonylarginine 4-Nettylcuaryl-7-smid (Z-Arg-LIKA) (I) belongs to a class of fluorogenic enzyme substrates whose function justifies the cleavage of " ermentativa 7-nmino-b-mc-tilcumarin and its fluorescence at 440 nm.

Several methods of entering Z-Arg-I.TIA (i) are described.

In the literature / 1 / Z-Arg-IBL- (I) was obtained with a 1J / 5-fold yield of Z-arginine and 7-amino-4-methylcunnarine by the mixed anhydrate method using isobutyl chloroformate.

The method proposed by Japanese scientists / 2,3 / uses IT, II'-dicyclohexylcarbodiimide to condensate the same compounds and raises the yield of compound I to 29.3.

With 32% yield, Z-Arg-I.IKU (I) was obtained from literature / 4 / using a modified phosphorus pentoxide method.

Pozdnew / 5 / proposes a general method for the preparation of II-substituted arginine aryl aldehydes in which di-tert-butyl irocarbonate is used as a condensing agent. Among other compounds, mention is made of Z-Arg-I.1IA (I), which has yielded (73 / and quality indices. Bad ORIGINAL gl 2. EN 10943 -atad - all methods described in the literature for - ^ 6j ^ iGlarn are used in Z- arginine hydrochloride, which is a g -G ro Z-arginine, and 7-amino-t-methylcunararin, a different condensation method and product I. The highest yield of Z-lrg-LIICA (I) (73.3) is reported in the literature / 5 /, but does not provide an experimental description of the synthesis, the method chemically closest to the method of the invention proposed in the literature / 4 /: 7-Anino-4-methylcoumarin (17 g, 0.09 11) in diatyl phosphite (92 ml) was chosen. of triethylamine (12.5 ml, 0.09 d) are added pre 110 ° C to a solution of phosphorus pentoxide (25.5 g, 0.09 1-1) in diethyl phosphite (92 ml) After 20 minutes at 90 ° C, Z is added. solution of arginine hydrochloride (27.7 g, 0.09 Ll) in diethyl phosphite (92 ml) containing 35 5 phosphoric acid (10.4 g, 0.09 II), evaporate for 2 hours at 110 ° C, evaporate (1 torso, bath temperature 60 ° C). to the oily residue add 910 ml of 1 II hydrochloric acid and stir at 90 ° C. Upon cooling, the product crystallizes. Maintain 13 hours at 4 ° C, filter. Dissolve in boiling methanol (2500 ml), decolorize with charcoal, filter and concentrate until crystallization begins. Yield: 25.4 g (52 3). K.p. 213 ° C (sat.) Io (j0 - 17.0 (c = 1; 11.11).

The main disadvantages of the prototype method are * 1) low product yield, 2) technologically complex solution, 3) use of toxic and deficient reagents. According to the prototype method, the yield of compound I is 52 ... i

In this method, d.ethyl phosphite, a highly toxic member of the class of organophosphorus compounds, is used both as a coumarin activating agent and as a solvent. Dictilfonf HH

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L LV 10943 requires an additional synthesis step - it is obtained from phosphorus trichloride and ethanol at -5 + 0 ° C with subsequent distillation in a lid (10 mm Hg: h).

The prototype method is complicated in technological terms: 1) the specific temperature at which a given operation has to be performed (addition of phosphorus pentoxide at 110 ° C; addition of Z-arginine hydrochloride at 90 ° C); 2) Evaporation of reaction mixture at 50 ° / l tors = 1 mm Hg h) is required.

The present invention overcomes all of the shortcomings of the prototype and allows the preparation of S-arginine 4-methylcunoyl-7-anide in a technologically simple process without the use of poisonous diethylphosphine. This was achieved by stirring a suspension of 7-amino-k-methylcoumarin in pyridine for 1 hour with phosphorus trichloride in pyridine, followed by the addition of a solution of 17-benzyloxycarbonylarginine hydrochloride in pyridine, heating for 3-4 hours at 40-50 ° C, refluxing for 10 hours, treatment with 1 7T hydrochloric acid, recrystallization from netanol / dine-t-1 R (4: 1). Preparation of Z-Arg-LIKii (I) from 7-e.mino-methylcumacumarin and Z-arginine hydrochloride using phosphorus trichloride and pyridine is not described in the literature.

The method provided by the invention provides a S, 3-fold, and I yield. The lintesc is carried out in a single step, with the exclusion of diethylformate at 0 C ~ - 0 and refluxing in a vacuum pump (gO-4C mm Hg st); prototype 1 mr. Hg st. ). BAD ORIGINAL L .. ..

LV 10943

The essence of the invention is illustrated by the inventive experimental protocol: 17.2 g of 17.5 £ (0.1 '*) of 7-smino-4-methylcunnarine pyridine are added to a suspension of 20 ml of phosphorus trichloride with stirring, 4.36 ml (0.05 of a solution of 20 ml). of pyridine, stir for 1 hour, then (0.005 liter) s-argimna The mixture is heated with 5-4 Hydrochloride solution in 50 ml of pyridine for 40 hours at 40-50 ° C, then left for 10 hours Distill the pyridine under reduced pressure (50-40 mm Hg) Dissolve 100-120 ml of ethanol in a bath, filter, add 70-100 ml of 1 L hydrochloric acid to the precipitate and stir on a magnetic stirrer for 1 hour. Crystallize from natanol / 4-dimethylamide (4: 1). ) mixture.

20.8 g (35%) of compound I.K.p. 212-214 ° C; (0 {J 17.0 (c = 2; DI.iF) · Chloroform is homogeneous in the system - chloroform: methanol: acetic acid: water = 20: 5: 0.5: 0.5).

LITERATURE 1. Zimmerman Γ.Ι., Asche 3. - Anal.3iochen., 1978, vol.78, p. 47-51. 2. CakaKHgapa c. 3aHBKa ΤΙποηηη .7 "54-3074; ΡΗΧ, 4,021 Π, 1980. " Πρ0Μ3Β0ΛΗΗβ πθπτι ^ οβ ”

Kanaoka 2., Takahashi Oalcakibara 0. - Chem, T., lTakayama H., Takaaa K., Kimura T Pharn. Buli., 1977, vol. 25, p.5126- * 4. Kharamungkhune 0., Sigler G. - Svnthecis, 1980, vol.8, p. 614-615. 5. ilosflHes B.sh. A.c. 1432983 CCCP. " Cnocotf noJivtreHHtf apigi- aMMflOB aam, M't8HHoro aprMHHHa "

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Claims (1)

LV 10943 Djojuru POEIkjL. '" r-? e n z i 1 c k? ikarbon1 arjinIna 4-e talku; •• aryl-P-sr.i.a. obtaining r:., T., Which comprises 7-nino-4-r, 2-cyclurarin, the cuts are subjected to activation and the activated compound: ε reaction with about enzyme: ε icaroneilar ginIna Hydroxychloride with a hopeful reaction-blown treatment with 1k-alcabab '7-sino-4-r ct ilkur.anna suspension p Laidina raises 1 stun with phosphorus trichloride in pyridine, add k-benzyl: cigars. 11- Reporting Hiccacchloride Oiri-'ma and Cilia -0-50 ° -2 Temperatures ^ -4 ε tuncas er se 'footprint •; 1 k 1 r ~ " λ - c. : ± cx 0 _ for over-counting from: ctanola / k i: oie * '; M ;: or: —nick 7 -: -: 1). BAD origin ^ l