CS269070B1 - Process for the preparation of N-aoylaoinoacids 2-naphthylamides and their 4-methoxy derivatives - Google Patents

Abstract

There are a number of homogemonic and fluorogemonic substrates for the study of proteolytic enzymes & in particular for the determination of protease activity in tissues and body fluids, which provides information on the range of various diseases. These substances include 2-naphthylamides and 4-ethoxy-2-naphthylamides of N-acylaamino acids, the new method of preparation of which consists in treating a mixture of N-acylamino acid and 2-naphthylamine or a mixture of N-acylamino acid and 4-ethoxy-2-naphthylamine with papain in an aqueous buffer with a pH in the range of 4.1 to 5.5, preferably pH 4.8, for a period of 2 hours to 48 hours at a temperature of 25 to 45 C, thereby forming an adiodic bond. The advantage of this process is that it provides chemically and optically pure products, is simple, single-step, economical and minimizes the amount of waste and harmful substances.

Description

The invention relates to a process for the preparation of 2-naphthylamides, N-aminomethylaminomethyl and their 4-methoxy derivatives.

2-Naphthylamides of N-acetylamino acids and 4-methoxy-2-naphthylamides of N-acetylamino acids are the first synthetic steps in the preparation of 2-naphthylamides and 4-methoxy-2-naphthylamides of amino acids and peptides. The above-mentioned substances are used as chromogenic or fluorogenic substrates for the study of proteolytic enzymes. They are most widely used in immunochemistry for the detection of protease activity in tissues or in blood fluids. From a practical point of view, this involves determining the extent of various disease processes, such as malignant diseases. These substances are synthesized from the corresponding! N-acylamino acids and 2-naphthylamine or 4-methoxy-2-naphthylamine by the mixed anhydride method (Green M.M., Tsou K.C., Bressler R., Seligman A.M.: Arch. Biochem. Biophya. 57, 458 /1955/, Folk J.E., Burstone M.S.: Proc. Soo. Exp. Biol. Med. 89, 473 /1955/, Plapinger R.E., Neohlas M.M., Seligman M.L., Seligman A.M.: J. Org. Chem. 30, 1781 /1965/) or by means of dicyclohexylcarbodiimide (Glenner G.G., Cohen L.A., Folk J.E.: J. Hist. Cytochem. 13, 57 /19^5/, Goldstein T.P., Plapinger R.E., Nachlas M.M., Seligman A.M.: J. Med. Pharm. Chem. 5, 852 /1962/, Nesvadba H. : Monatah. Chem. 93, 386 /1962/)·

It is known that proteolytic enzymes, whose function in nature is the cleavage of peptide bonds, can under certain conditions be used for the opposite reaction, i.e. for the formation of peptide bonds (for a review, see: Brtník F., Jolt K.: Chem. listy 74, 951 /1980/, Fruton J.S.: Adv. Enzymol, 53, 239 /1982/, Jakubko H.-D., Kuhl P., Konneoke A.: Angev. Chem. 24, 85 /1985/). The synthesis of anilides and substituted anilides of N-acylamino acids using the proteolytic enzyme papain is also described in the literature (Valdsohmidt-Leitz E., Kuhn K.: Hoppe-Soyler's Z. physiol. Chem. 285, 23 /1950/).

It has now been found that papain can also be used to synthesize 2-naphthylamides and 4-ethoxy-2-naphthylamides of several N-amino acids with the exception of proline. This method of synthesis provides chemically and optically pure products and is simple both in terms of the reaction and the processing of the resulting product. Expensive chemical condensation reagents are replaced by a much smaller amount of cheap papain and some organic solvents by aqueous buffer, which makes this method advantageous both economically and ecologically. The synthesis is a single-step process, even in the case of amino acids with functional groups in the side chain, which eliminates the protection of these groups necessary for chemical synthesis. The synthesis of 2-naphthylamides and 4-methoxy-2-naphthylamides using papain is in principle feasible even for some non-coding N-aminoyl-L-amino acids.

The essence of the invention is that a mixture of N-acyl-L-amino acid and 2-methoxy-part or a mixture of N-acylamino acid and 4-methoxy-2-naphthylamine is treated with papain in an aqueous buffer with a pH in the range from 4.1 to pH 5.5, preferably with a pH of 4.8, for a period of 2 hours to 48 hours at a temperature of from 25 to 45 °C, thereby forming an amide bond.

Both types of compounds are schematically represented by formulas I and II.

(I)

Formula I represents the 2-naphthylamide of N-aylamino acid; formula XX represents the 4-methoxy- -^"naphthylamide of N-aylamino acid, where Ao is benzyloxycarbonyl, tert-butyloxycarbonyl or other protecting group, Xaa is L-amino acid. The arrow indicates papain-catalyzed formation of the amide bond.

The method for preparing 2-naphthylamides and 4-methoxy-2-naphthylamides of N-ylamino acids using papain is further explained in the examples.

CS 269 070 Bl

Example 1

2-Naphthylamide of benzyloxycarbonylbacon

To a solution of benzyloxycarbonylalanine (223 “«) and 2-naphthylamine (215 mg) in a mixture of dimethylformamide (3 ml) and 0.2 mol.l ^-1 acetate buffer pH 4.8 (7 ml) were added ethylenediaminetetraacetic acid (3 mg) and cysteine hydrochloride (10 mg) and the pH of the solution was adjusted to 4.8. After the addition of papain (40 mg), the mixture was incubated for 48 h at 38 °C. The precipitate formed was filtered off, washed with 1 mol.l ^-1 HCl, water, 5% NaHCO^, water * and then dried. ~170 ®β (IřO-Jí) of the product with a mp of 191>-192 °C was obtained. - 33.4° (o 0.3, dimethylformamide). For Ο,^Η^Ν.^ (383.4) calculated: 72.39 C 0, 5.79 H, 8.04 N; found: 72.5 C 0, 5.79 H, 7.90 N. Literature reports mp 193.5 °C. (Goldstein TP, Pla pinger RE, Nachlas HM, Seligman AM: J. Med. Pharm. Chem. 1, 852 (1962)).

Example 2

Benzyloxycarbonylleuoin 2-Naphthylamide

To a solution of benzyloxycarbonylleucoin (1.33 β) and 2-naphthylamine (1.1 g) in a mixture of dimethylformamide (17 ad.) * 0.2 mol.l ^-1 acetate buffer pH 4.8 (33 ml) was added ethylenediaminetetraacetic acid (15 mg) and cysteine hydrochloride (50 mg) and the pH of the solution was adjusted to 4.8. After the addition of papain (200 mg), the mixture was incubated for 24 h at 38 °C. The product, which precipitated, was extracted from the reaction mixture into ethyl acetate, the ethyl acetate solution was washed with 1 mol.l ^-1 HCl, water, 5% NaHCO^, water, dried over NaHSO^ * evaporated. The residue was crystallized from a mixture of ethyl acetate and petroleum ether. 1.72 g (88 µl) of product were obtained, mp 163°-163° C. Recrystallization from ethyl acetate did not reveal the product, [d]- 54.7° (o 1, chloroform). For C24^6^2°3* ^O, 5 ¾ ⁰ ^99.5) calculated: 72.15 Jí 0, 6.81 JÍ H, 7.01 JÍ Ni found: 72.52 JÍ C, 6.83 JÍ H, 6.72 JÍ N, Literature* gives mp 157 ai 158 °C, [o(J _n - 61.6° (o 1.98, chloroform). (Green HN, Tsou KC, Bressler R., Seligjaan AM 1 Arch. Bioohea, Bíophys, 57. 458 /1955/).

Example 3

Benzyloxycarbonylphenylalanine 2-Naphthylamide

To a solution of benzyloxycarbonylphenylalanine (300 mg) and 2-naphthylamine (215 mg) in a mixture of dimethylformamide (3 ml) and 0.2 mol.l ^-1 acetate buffer pH 4.8 (7 ml) was added ethylenediaminetetraacetic acid (3 mg) and cysteine hydrochloride (10 mg) and the pH of the solution was adjusted to 4.8. After the addition of papain (40 mg), the mixture was incubated for 24 h at 38 °C.

The precipitate was filtered off, washed with 1 mol.l ^-1 HCl, and then dried. 295 mg (70 Jí) of product with mp 180 °C was obtained, 5jí NaH30_, water and ⁺ °' ³ ' dimethylformamide). For Cg^H^NgO^ (424.5) calculated:' 76.39 jí C, 5.70 Jí H, 6.60 Jí N; found; 76.02 JÍ C, 5.71 JÍ H, 6.56 JÍ N. The literature gives mp 175 °C»Z?/j _D - M** (o 1.4, ethyl acetate). (Ooldsteln TP, Plaplixiger RE , Neohlas MM , Seligman Λ.Μ. :

J. Hed. Pharm. Chem. 1962, 852).

Example 4

2-Naphthylamide tert. butyloxycarbonyltyrosine To a solution of tert. butyloxycarbonyltyrosine (282 g) and 2-naphthylamine (215 “β) in a mixture of dimethylformamide (3 ml) * 0.2 mol.l ^-1 acetate buffer pH 4.8 (7 ml) was added ethylenediaminetetraacetic acid (3 mg) * cysteine hydrochloride (10 mg) and the pH of the solution was adjusted to 4.8. After addition, papain (40 mg) and the mixture was incubated for 24 h

CS 269 070 B1 at 38 °C. The product was extracted into ethyl acetate, the ethyl acetate solution was washed with HSO^ buffer pH 2, water, 5% NaHCO^ solution, water, dried over Na2SO4 and evaporated. The residue was triturated with petroleum ether and the product was recovered from a mixture of ethyl acetate and petroleum ether. 220 mg (53 µl) of product was isolated at mp 104 to 105 ° ^C (±0.3, dimethylformamide). For ^4^6^2^ ^K 2° (±5.3) calcd: 69.37 C, 6.55 H, 6.76 N; found: 69.83 C, 6.35 H, 6.75 % N. '

Example 5

2-Naphthylamide benzyloxycarbonyl-S-benzyloxyl

To a solution of benzyloxycarbonyl-S-benzyloxystein (346 mg) and 2-naphthylamine (215 mg) in a mixture of dimethylformamide (3 ml) and 0.2 mol.l ^-1 acetate buffer pH 4.8 (7 ml) was added ethylenediaminetetraacetic acid (3 mg) and cysteine hydrochloride (10 mg) and the pH of the solution was adjusted to 4.8. After addition of papal (20 mg), the mixture was incubated for 24 h at 38 °C.

The precipitate formed was filtered off, washed with 1 mol.l ^-1 HCl, water, 5 ml NaHCO-, water and . . . dried. 328 mg (70 ml) of product were obtained, mp 170-171 C. The product was extracted from a mixture of ethyl acetate and petroleum ether without stirring _.

Recrystallization for + 22° (o 0.3, dimethylformamide). For Cjglijg^OjS, (470.6) calcd: 71.46 1° C, found: 71.06 Jj C, 5.60 H, 6.06 N.

5.57 H, $5.95 N;

Example 6

2-Naphthylamide of benzyloxycarborylglutamic acid

To a solution of benzyloxycarbonylglutamic acid (282 mg) and 2-naphthylamine (215 ®β) in dimethylformamide (2 ml) and 0.2 mol.l ^-1 acetate buffer pH 4.8 (8 ml) were added ethylenediaminetetraacetic acid (3 mg) and oestein hydrochloride (10 mg) and the pH of the solution was adjusted to 4.8. After addition of papain (40 mg), the mixture was incubated for 24 h at 38 °C. The precipitate formed was filtered off, washed with 1 mol.l ¹ UCl, water and dried. After recrystallization from a mixture of ethyl acetate and petroleum ether, 130 mg (32 µl) of the product was obtained, mp 217 «ž 218 °C. Ceó J & - 6.8° (o 0.3, dimethylformamide). For Cj^H^NjO^ (406.4) calculated: 69.97 í C, 5.46 í H, 6.89 í Nj found,· 67.67 í C, 5.43 í H, 6.77 £ ». The literature gives τ 22 1 _D - 0.71° (o 1.7, dimethylformamide). (Goldstein TP, Plapinger RE , Naohlas MM , Seligman Λ.Μ. : J. Med. Pharm. Chem. 2, ⁸ 52 /1962/).

Example 7

-Me th oxy —2 -nat ylamide benzyloxycarbonylIf onylalanine

To a solution of benzyloxycarbonyl phenylalanine (30 mg) and 4-methoxy-2-naphthylamine (25 mg) in dimethylformamide (0.3 ml) and 0.2 mol.l” ¹ acetate buffer pH 4.8 (0.7 ml) were added ethylenediaminetetraacetic acid (0.3 mg) and cysteine hydrochloride (1 mg) <

The pH of the solution was adjusted to 4.8. After the addition of pepain (4 mg), the mixture was incubated for 24 h at 38 °C. The precipitate formed was filtered off, washed with 1 mol.l“ ¹ HCl, water, 199

C. Recrystallization of the product from ethyl acetate and petroleum ether gave a m.p.

198 AŞ did not mention.+ 45.7° (o 0.3, dimethylformaaid)« For ^C 2g^^2^4 ^^9^) calculated: 73.99 c, 5.77 t H, 6.17 JÍ N ₍ found: 73.54 JÍ C, 5.73 JÍ H, 5.95 % N.

Example 8

4-Methoxy-2-naphthylamide target, butyloxycarbonylleuoin

EN 269 070 B1

To a solution of tert-butyloxycarbonylleucoline (25 mg) and 4-methoxy-2-naphthylamine (23 mg) in a mixture of dimethylformamide (0.3 “l) and 0.2 mol.l ^- ' acetate buffer pH 4.8 (0.7 “l) was added ethylenediaminetetraacetic acid (0.3 mg) and cysteine hydrochloride (1 mg) and the pH of the solution was adjusted to 4.8. After the addition of papain (4 mg), the mixture was incubated for 24 h at 38 °C. The product was extracted from the reaction mixture into ethyl acetate, the ethyl acetate solution was washed with cooled 1 mol.l ^- ' HCl, water, 55· NaHCO^, water,' dried over Na2SO4 and evaporated. The residue was crystallized from a mixture of methanol and water. 28 mg (72) of product were obtained, mp 79-81°C. For ^c 22H ^{20 n} 2°4 (386.5) calcd.: 68.36% C, 7.82% H, 7.25% N. Found: 68.30% C, 7.80% H, 7.01% N.

Claims (1)

A method for preparing 2-naphthylamides N-aloylaminocyano