Production of bitter principle derivatives

Abstract

It is an object of the present invention to introduce carboxylic acid-functionalities suitable for coupling into the denatonium structure by means of simple synthesis, namely the synthesis of bitter principle derivatives based on the denatonium structure according to formula 1: ##STR00001## For example, according to the invention, lidocaine derivatives may be reacted with carboxylated benzyl halogenides. The carboxylated denatonium derivatives of the present invention are especially applied in medicine, biology, medical engineering as well as cosmetics, the pharmaceutical, chemical, and foodstuff industry.

Claims

1. A compound of general formula 1 ##STR00005## wherein: (i) X.sup. represents halogenide, sulphate, benzoate, acetate, trifluoroacetate, hydroxide, saccharinate, or capsaicinate, (ii) R1-R10 independently represent: (a) hydrogen, (b) halogen, (c) C1-C5 alkyl, (d) C1-C4 alkoxy, (e) C1-C20 alkoxycarbonyl (f) NHP or OP, wherein P is a hydrogen or a residue consisting of 1-30 amino acids selected from the group consisting of D-amino acids, L-amino acids and unnatural amino acids, wherein said amino acids are optionally modified for coupling, (g) (Y).sub.nCOOM with M=Na, K, or [N(R12).sub.4].sup.+, or (h) (Y).sub.nC(O)NR14R15, further wherein Y represents an organic residue selected from the group consisting of: CH.sub.2, CH(CH.sub.3), CH.sub.2CH.sub.2, (CH.sub.2).sub.3, CHCH, CC, OCH.sub.2CH.sub.2, OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2, and NHCH.sub.2CH.sub.2 and n=0 or 1, (iii) R14 and R15 represent hydrogen, a C1-C12 alkyl, or a residue consisting of 1-30 amino acids selected from the group consisting of D-amino acids, L-amino acids and unnatural amino acids, and (iv) at least one of the residues R1-R10 is a (a) (Y).sub.nCOOM or (b) (Y).sub.nC(O)NR14R15, wherein Y, M, R14, and R15 are as defined above, and (v) R11 and R12 independently represent hydrogen or a C1-C10 alkyl residue.

2. The compound according to claim 1, wherein, in the general formula 1: (i) residue R11 is an ethyl group, (ii) residues R9 and R10 are methyl groups, (iii) residues R1, R4, R5, R6, R7, and R8 represent hydrogen, and (iv) one of residues R2 and R3 represents hydrogen and the other is selected from the group consisting of; (a) (Y).sub.nCOOM with M=Na, K, or [N(R12).sub.4].sup.+, and (b) (Y).sub.nC(O)NR14R15, or residues R2 and R3 independently represent: (a) (Y).sub.nCOOM with M=Na, K, or [N(R12).sub.4].sup.+, or (b) (Y).sub.nC(O)NR14R15, wherein Y, M, R14, and R15 are as defined in claim 1, further wherein: a. residues R14 and R15 independently represent hydrogen, a C1-C12 alkyl or a residue consisting of 1-30 amino acids selected from the group consisting of D-amino acids, L-amino acids and unnatural amino acids, residue R12 represents hydrogen or a C1-C10 alkyl residue.

3. A substrate comprising a compound according to claim 1, bound to a substrate surface fabricated of a metal, ceramic, glass, or polymeric material via one of residues R1-R10 of said compound.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) Therefore, the invention is based on the problem to produce bitter principles in a simple manner that contain at least one functional group, in the derivatization of which the bitter taste is maintained or the bitter taste occurs by suitable hydrolytic or enzymatic cleavage. According to the invention this problem is solved by suitably reacting 2-diethylamino-N-(2,6-dimethylphenyl)acetamide (lidocaine) or a functionalized lidocaine derivative with benzylhalogenide derivatives to compounds of general formula 1.

(2) ##STR00004##

(3) Here, X.sup. in formula 1 for example represents halogenide, pseudo-halogenide, sulphate, benzoate, acetate, trifluoroacetate, hydroxide, saccharinate, or capsaicinate. Preferably, X.sup. is a halogenide and particularly preferred Cl.sup., Br.sup., or I.sup..

(4) In formula 1 R1-R10H, halogen, a C1-C5 alkyl, C1-C4 alkoxy, C1-C20 alkoxycarbonyl, NHP, OP, wherein P is hydrogen, a peptide residue or a peptide residue consisting of 1-30 amino acids (modified and unmodified D-amino acids, L-amino acids as well as unnatural amino acids as defined in the World Intellectual Property Organization (WIPO) Handbook on Industrial Property Information and Documentation, Standard ST.25 (1998), including Tables 1 through 6 of Appendix 2), which is modified for coupling, (Y).sub.nCOOR13, (Y).sub.nCOOM with M=Na, K, [N(R12).sub.4].sup.+, or (Y).sub.nC(O)NR14R15 group, wherein R14 and R15H, a C1-C12 alkyl or a peptide residue consisting of 1-30 amino acids (WIPO), wherein at least one of residues R1-R10 is a group (Y).sub.nCOOM, (Y).sub.nCOOR13 or (Y).sub.nC(O)NR14R15. Y represents an organic residue such as for example CH.sub.2, CH(CH.sub.3), CH.sub.2CH.sub.2, (CH.sub.2).sub.3, CHCH, CC, OCH.sub.2CH.sub.2, OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2, NHCH.sub.2CH.sub.2, without limitation, wherein n=0 or 1. R11, R12, R13 independently represent H or a C1-C10 alkyl residue. Preferably, R1-R5, R6-R10H, a methyl, ethyl, methoxy, ethoxy, carboxyl, methoxycarbonyl, ethoxycarbonyl or (Y).sub.nC(O)NR14R15 group with R14H and R15C1-C12 alkyl or a peptide residue consisting of 1-30 amino acids (according to the previous definition), wherein one of residues R1-R5, R6-R10 represents a (Y).sub.nCOOR13, (Y).sub.nCOOM or (Y).sub.nC(O)NR14R15 group. Particularly preferred R1-R5H and one of residues R1-R3 is a group (Y).sub.nCOOR13, (Y).sub.nCOOM or (Y).sub.nC(O)NR14R15. Particularly preferred R6-R8H and R9-R10=methyl, if at least one of residues R1-R5 is a group (Y).sub.nCOOM, (Y).sub.nCOOR13 or (Y).sub.nC(O)NR14R15. Preferably, R11 is a C1-C8 alkyl residue such as e.g. a methyl, ethyl, n-propyl, i-propyl, n-butyl group. Particularly preferred R11 is a methyl, ethyl, or propyl group.

(5) Already known is to synthesize denatonium salts starting from lidocaine and benzylbromide in suitable solvents such as alcohols, water in a reaction of several hours of 20-24 hrs at an elevated temperature between 50-100 C. [WO2006062406 A2]. For the preparation of such compounds also an amide coupling of corresponding educts Ph-NH.sub.2 and Ph-CH.sub.2NR.sub.2CH.sub.2COX1 (X1=halogen, OH) or ammonium formation starting from Ph-NHCOCH.sub.2Hal (Hal=halogen) and Ph-CH.sub.2NR.sub.2 would be conceivable, but which in both variants is only possible via long synthesis pathways and low total yields.

(6) It was surprisingly found that carboxylated and carboxyalkyl-substituted (-alkylCOOR) benzylhalogenides with lidocaine in a molar ratio of 1:1 already at low temperatures of 20-80 C. in a suitable solvent react to the corresponding carbon acid derivatives of denatonium. Also, a microwave-assisted reaction in a suitable solvent for preparing substances according to formula 1 at temperatures up to 200 C. and reaction times of max. 60 min is suitable for the synthesis. Still more surprising was the result that the solid substances also react without using solvents after combination at room temperature and that the spontaneous reaction is completed with high yields with short reaction times of only a few minutes. In this way, carboxyl-functionalized denatonium derivatives are obtained that can be further reacted by means of suitable synthesis to salts, esters, and amides.

(7) Moreover, it was also surprisingly found that the carboxyl-functionalized denatonium derivatives have an intensive bitter taste, whereas changes in the denatonium basic structure lead to a loss of the bitter taste.

(8) Advantageous for the synthesis of the carboxyl-functionalized denatonium derivatives is the low technical and energetic expenditure, since either no solvents are needed and in general a reaction control at room temperature is sufficient or only short reaction times in the microwave-assisted reaction are needed. The products are characterized by a high purity, which eliminates the need for costly cleaning.

(9) The uncomplicated production of carboxylated denatonium derivatives makes it possible to provide such substances in larger quantities.

MODES FOR CARRYING OUT THE INVENTION

(10) In a preferred embodiment of the invention one of both educts (lidocaine or benzyl halogenide derivative) is added to the reaction vessel in a solid form. The second component is added at room temperature in a solid form. After short mixing, a spontaneous reaction starts, wherein the solid substances form a melt. The reaction is controlled by means of thin layer chromatography. In most cases, the reaction is completed within 10-120 min. The reaction can also be performed in a suitable solvent such as an organic solvent or water at room temperature or short-term moderate heating. Another option is the microwave-assisted reaction up to 200 C. with reaction times of max. 1 hr. Purification of the substances, if needed, can be performed by means of re-crystallization or column chromatography. For further derivatization suitable denatonium derivatives are reacted to salts, esters, and amides according to methods known to the skilled person.

(11) Testing the bitter taste of synthesized compounds compared to denatonium benzoate can be performed for example with an electronic tongue, wherein the electric potentials of aqueous substance solutions of a defined concentration are determined in accordance with methods known to the skilled person.

(12) Like amino acids denatonium derivatives can be coupled to an immobilized amino acid chain (bound to a polymeric carrier (resin)) of any length by means of solid phase peptide synthesis (SPPS). In the synthesis, the so-called Fmoc chemistry with HOBt (1-hydroxybenzotriazole) and DIPEA (N,N-diisopropylethylamine) as an auxiliary base is applied. First, HOBt forms an active ester from the carboxyl group of the Fmoc amino acid to be bound that then reacts with amines to a peptide bond. The Fmoc cleavage is performed with piperidine in dimethylformamide (DMF).

(13) After coupling, the protective groups and the construct consisting of denatonium derivative and peptide are simultaneously cleaved-off from the carrier with the addition of TFA. Reactive carbocations formed during the cleavage are quenched by the scavengers ethane dithiol, metacresol, and thioanisole.

(14) Subsequently, the denatonium derivative/peptide construct cleaved-off from the carrier is precipitated in ice-cold ether and after centrifugation the solution is removed from the construct pellet.

(15) Coupling can be verified by means of mass spectrometry (electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI)).

(16) The following examples are to explain the invention in more detail without limiting it.

EXAMPLES

Example 1

(17) Reaction of lidocaine with 4-(bromomethyl)benzoic acid methyl ester A small excess of 4-(bromomethyl)benzoic acid methyl ester (18 mg, 80 mol) was added to 17 mg of lidocaine (72.5 mol) and it was suspended in 3 ml of water. The suspension is left for 30 min at 200 C. in the microwave. Subsequently, the solution is concentrated to a small volume and taken up in 1 ml of THE. The white solid formed is filtered off and washed with THE. 2-(2,6-Dimethylphenylamino)-N,N-diethyl-N-(4-(methoxycarbonyl)benzyl)-2-oxoethane ammonium bromide is obtained.

Example 2

Reaction of Lidocaine with 4-(bromomethyl)benzoic Acid

(18) 1 g of lidocaine (4.3 mmol) was mixed with 0.92 g (4.3 mmol) of 4-(bromomethyl)benzoic acid while shaking. To complete the reaction it was heated for 20 Minutes to 50 C. Subsequently, 10 ml of THF were added and stirred. The white precipitate formed was filtered off, washed with THF and dried. The substance was separated from the by-product lidocaine-4-(bromomethyl)benzoate formed by column chromatography and N-(4-carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane ammonium bromide was obtained.

Example 3

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(3-(methoxycarbonyl)benzyl)-2-oxoethane Ammonium Bromide

(19) 1 g of lidocaine (4.3 mmol) is combined with 0.98 g (4.3 mmol) of 3-(bromomethyl)benzoic acid methyl ester and shaken several times at room temperature within 10 minutes after having been added. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)N,N-diethyl-N-(3-(methoxycarbonyl)benzyl)-2-oxoethane ammonium bromide is obtained. m.p.: 183-184 C.

(20) MS (ESI): m/z=383.233 Da (C.sub.23H.sub.31N.sub.2O.sub.3).sup.+

(21) .sup.1H-NMR (DMF-D7, ppm): 1.61-1.64 (t, 6H, 2CH.sub.3); 2.32 (s, 6H, 2CH.sub.3); 2.73-2.78; 2.91-2.95 (CH.sub.3, DMF-D7); 3.52 (H.sub.2O in DMF); 3.71-3.81 (m, 4H, 2CH.sub.2); 3.94 (s, 3H, OCH.sub.3); 4.68 (s, 2H, CH.sub.2); 5.20 (s, 2H, CH.sub.2); 7.13-7.16 (m, 3H, 3CH); 7.72-7.75 (t, 1H, CH); 8.03 (CH, DMF); 8.08-8.10 (d, 1H, CH); 8.16-8.18 (d, 1H, CH); 8.38 (s, 1H, CH); 10.87 (s, 1H, NH)

(22) .sup.13C-NMR (DMF-D7, ppm): 8.54 (CH.sub.3); 18.77 (CH.sub.3); 29.59-30.60 (DMF-D7); 34.73-35.73 (DMF-D7); 52.69 (OCH.sub.3); 55.39 (CH.sub.2); 56.66 (CH.sub.2); 62.11 (CH.sub.2); 127.80 (CH); 128.60 (CH); 129.46 (C); 130.26 (CH); 131.52 (C); 131.75 (CH); 134.55 (CH); 134.62 (C); 136.08 (C); 138.56 (CH); 162.60-162.92 (DMF-D7); 163.45 (C); 166.55 (C)

Example 4

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(4-(methoxycarbonyl)benzyl)-2-oxoethane Ammonium Bromide

(23) 1 g of lidocaine (4.3 mmol) is combined with 0.98 g (4.3 mmol) of 4-(bromomethyl)benzoic acid methyl ester and shaken several times at room temperature within 20 minutes after having been added. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)N,N-diethyl-N-(4-(methoxycarbonyl)benzyl)-2-oxoethane ammonium bromide.

(24) m.p.: 176-178 C.

(25) .sup.1H-NMR (DMF-D7, ppm): 1.61-1.64 (t, 6H, 2CH.sub.3); 2.31 (s, 6H, 2CH.sub.3); 2.73-2.78; 2.90-2.95 (CH.sub.3, DMF-D7); 3.53 (H.sub.2O in DMF); 3.74-3.82 (m, 4H, 2CH.sub.2); 3.96 (s, 3H, OCH.sub.3); 4.72 (s, 2H, CH.sub.2); 5.16 (s, 2H, CH.sub.2); 7.12-7.17 (m, 3H, 3CH); 7.97-7.98 (d, 2H, 2CH); 8.03 (CH, DMF); 8.10-8.11 (d, 2H, 2CH); 10.88 (s, 1H, NH)

(26) .sup.13C-NMR (DMF-D7, ppm): 8.83 (CH.sub.3); 18.95 (CH.sub.3); 29.76-30.76 (DMF-D7); 34.89-35.89 (DMF-D7); 52.89 (OCH.sub.3); 55.90 (CH.sub.2); 57.18 (CH.sub.2); 62.22 (CH.sub.2); 127.94 (CH); 128.74 (CH); 130.48 (CH); 132.40 (C); 133.80 (C); 134.51 (CH); 134.75 (C); 136.23 (C); 162.62-163.09 (DMF-D7); 163.55 (C); 166.73 (C)

Example 5

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(4-(methoxycarbonyl)benzyl)-2-oxoethane Ammonium Chloride

(27) 1 g of lidocaine (4.3 mmol) is combined with 0.79 g (4.3 mmol) of 4-(chloromethyl)benzoic acid methyl ester and shaken several times at room temperature within 20 minutes after having been added. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)N,N-diethyl-N-(4-(methoxycarbonyl)benzyl)-2-oxoethane ammonium chloride is obtained.

Example 6

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(4-(2-methoxy-2-oxoethyl)benzyl)-2-oxoethane Ammonium Bromide

(28) 0.3 g of lidocaine (1.3 mmol) are combined with 0.31 g (1.3 mmol) of methyl-2-(4-(bromomethyl)phenyl)acetate and shaken several times at room temperature within 20 minutes after having been added. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)N,N-diethyl-N-(4-(2-methoxy-2-oxoethyl)benzyl)-2-oxoethane ammonium bromide is obtained.

(29) .sup.1H-NMR (DMF-D7, ppm): 1.59 (m, 6H, 2CH.sub.3); 2.32 (s, 6H, 2CH.sub.3); 2.73; 2.91 (CH.sub.3, DMF-D7); 3.63-3.69; (m, 6H, 3CH.sub.2); 3.84 (s, 3H, OCH.sub.3); 4.70 (s, 2H, CH.sub.2); 5.06 (s, 2H, CH.sub.2); 7.14 (m, 3H, 3CH); 7.49-7.50 (d, 2H, 2CH); 7.76-7.77 (d, 2H, 2CH); 8.05 (CH, DMF); 10.99 (s, 1H, NH)

(30) .sup.13C-NMR (DMF-D7, ppm): 8.82 (CH.sub.3); 18.95 (CH.sub.3); 29.70-30.71 (DMF-D7); 34.86-35.87 (DMF-D7); 40.54 (CH.sub.2); 52.26 (OCH.sub.3); 55.43 (CH.sub.2); 56.87 (CH.sub.2); 62.52 (CH.sub.2); 127.24 (C); 127.83 (CH); 128.63 (CH); 130.84 (CH); 133.91 (CH); 134.65 (C); 136.09 (C); 137.89 (C); 162.53-163.0 (DMF-D7); 163.41 (C); 172.09 (C)

Example 7

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(4-(1-methoxy-1-oxopropane-2-yl)benzyl)-2-oxoethane Ammonium Bromide

(31) 0.4 g of lidocaine (1.7 mmol) are combined with 0.44 g (1.7 mmol) of methyl-2-(4-(bromomethyl)phenyl)propanoate and shaken several times. Simultaneously, to complete the reaction it is heated for 20 Minutes to 80 C. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)-N,N-diethyl-N-(4-(1-methoxy-1-oxopropan-2-yl)benzyl)-2-oxoethane ammonium bromide is obtained.

Example 8

Synthesis of 2-(2,6-dimethylphenylamino)-N-(4-(2-ethoxy-2-oxoethylcarbamoyl)benzyl)N,N-diethyl-2-oxoethane Ammonium Bromide

(32) 0.39 g of lidocaine (1.7 mmol) are added to 0.5 g of ethyl-2-(4-(bromomethyl)benzamido)acetate (1.7 mmol) and shaken several times at 80 C. within 20 minutes after having been added. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)N-(4-(2-ethoxy-2-oxoethylcarbamoyl)benzyl)-N,N-diethyl-2-oxoethane ammonium bromide is obtained.

(33) .sup.1H-NMR (D.sub.2O, ppm): 1.31-1.34 (t, 3H, CH.sub.3); 1.53-1.56 (t, 6H, 2CH.sub.3); 2.25 (s, 6H, 2CH.sub.3); 3.58-3.67; (m, 4H, 2CH.sub.2); 4.22 (s, 4H, 2CH.sub.2); 4.27-4.31; (q, 2H, CH.sub.2); 4.89 (s, 2H, CH.sub.2); 7.20-7.28 (m, 3H, 3CH); 7.68-7.69 (d, 2H, 2CH); 7.94-7.96 (d, 2H, 2CH); .sup.13C-NMR (D.sub.2O, ppm): 8.40 (CH.sub.3); 14.38 (CH.sub.3); 18.42 (CH.sub.3); 42.94 (CH.sub.2); 55.69 (CH.sub.2); 56.35 (CH.sub.2); 62.31 (CH.sub.2); 63.59 (CH.sub.2); 129.08 (CH); 129.37 (CH); 129.49 (CH); 131.86 (C); 132.98 (C); 134.11 (CH); 136.02 (C); 136.74 (C); 164.70 (C); 170.73 (C); 172.60 (C)

Example 9

Synthesis of 2-(2,6-dimethylphenylamino)-N-(4-(2-(2-ethoxy-2-oxoethylamino)-2-oxoethylcarbamoyl)benzyl)-N,N-diethyl-2-oxoethane Ammonium Bromide

(34) 0.23 g of ethyl-2-(2-(4-(bromomethyl)benzamido)acetamido)acetate (0.6 mmol) are combined with 0.15 g of lidocaine (0.6 mmol) and shaken several times at 80 C. within 20 minutes after having been added. Subsequently, 10 ml of THF are added and stirred. The white precipitate formed is filtered off, washed with THF and dried. 2-(2,6-Dimethylphenylamino)-N-(4-(2-(2-ethoxy-2-oxoethylamino)-2-oxoethyl-carbamoyl)benzyl)-N,N-diethyl-2-oxoethane ammonium bromide is obtained.

Example 10

Synthesis of N-(3-carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane Ammonium Bromide by Saponification with NaOH

(35) An excess of NaOH (3.2 mmol, 129.4 mg) in ethanol is added to 0.5 g of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(3-(methoxycarbonyl)benzyl)-2-oxoethane ammonium bromide (1.08 mmol) and heated for three hours to boiling. Subsequently, 25 ml of 0.1 N HCl are added. The solution is evaporated to dryness and the oily product is mixed with THF and stirred. The white precipitate formed is filtered off and washed with THE. N-(3-Carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane ammonium bromide is obtained.

(36) m.p.: 142-144 C.

(37) MS: (ESI) m/z=369.217 Da (C.sub.22H.sub.29N.sub.2O.sub.3).sup.+

(38) .sup.1H-NMR (D.sub.2O+NaOD, ppm): 1.50-1.55 (t, 6H, 2CH.sub.3); 2.13 (s, 6H, 2CH.sub.3); 3.44-3.60 (m, 4H, 2CH.sub.2); 4.89 (s, 2H, CH.sub.2+D.sub.2O); 4.91 (s, 2H, CH.sub.2); 6.94-6.97 (t, 1H, CH); 7.09-7.11 (d, 2H, 2CH); 7.59-7.62 (t, 1H, CH); 7.80-7.82 (d, 1H, CH); 8.00-8.01 (d, 1H, CH); 8.06 (s, 1H, CH)

(39) .sup.13C-NMR (D.sub.2O+NaOD, ppm): 10.41 (CH.sub.3); 20.56 (CH.sub.3); 56.35 (CH.sub.2); 63.53 (CH.sub.2); 70.66 (CH.sub.2); 125.54 (CH); 130.32 (CH); 130.51 (C); 131.92 (CH); 133.47 (CH); 133.88 (C); 135.68 (CH); 138.17 (CH); 140.00 (C); 149.15 (C); 163.75 (C); 177.55 (C)

Example 11

Synthesis of N-(3-carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane Ammonium Bromide by Saponification with Ba(OH).SUB.2

(40) 30 ml of a 0.05 M Ba(OH).sub.2 solution in ethanol is added to 0.5 g of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(3-(methoxycarbonyl)benzyl)-2-oxoethane ammonium bromide (1.08 mmol) and heated for three hours to boiling. Subsequently, 30 ml of a 0.05 M H.sub.2SO.sub.4 solution are added. The precipitate mainly consisting of BaSO.sub.4 is filtered off. Subsequently, the solution is evaporated to dryness and the crystalline product is washed with THE. N-(3-Carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane ammonium sulphate.

Example 12

Coupling of N-(3-carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane Ammonium Bromide with the Peptide (Fmoc)G-P-Q-G-I-A-G-Q-A(N3)-Q Resin (SEQ ID NO: 1)

(41) The peptide (Fmoc)G-P-Q-G-I-A-G-Q-A(N.sub.3)-Q resin (SEQ ID NO: 1) is gradually synthesized on a synthesis resin by means of common peptide synthesis (Fmoc strategy). To attach the bitter principle the Fmoc protective group of the N-terminal terminal amino acid glycine is cleaved-off by means of 40% and 20% piperidine solution in DMF. 83.5 mol of the denatonium derivative are dissolved in 500 l of a 0.5 M HOBt solution of H.sub.2O/DMF 1:4. To the resin there is added the solution and subsequently 20 l of diisopropylcarbodiimide (DIC) and shaken for one hour at room temperature. Subsequently, the resin is washed several times with DMF, dichloromethane, and diethyl ether. A resin-bound peptide-bitter principle coupling product is obtained.

(42) After coupling, the protective groups as well as the product consisting of denatonium derivative and peptide are simultaneously cleaved-off from the carrier with the addition of TFA. Reactive carbocations formed during the cleavage are quenched by the scavengers ethane dithiol, metacresol, and thioanisole.

(43) Subsequently, the denatonium derivative/peptide construct cleaved-off from the carrier is precipitated in ice-cold ether and after centrifugation the solution is removed from the pellet formed. Coupling can be verified by means of mass spectrometry (electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI)).

Example 13

Coupling of N-(3-carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane Ammonium Bromide with the Peptide (Fmoc)G-P-Q-G-I-A-G-Q-A(N.SUB.3.)-Q Resin (SEQ ID NO: 1)

(44) The peptide (Fmoc)G-P-Q-G-I-A-G-Q-A(N.sub.3)-Q resin (SEQ ID NO: 1) is stepwise synthesized on a synthesis resin by means of common peptide synthesis (Fmoc strategy). To attach the bitter principle the Fmoc protective group of the N-terminal terminal amino acid glycine is cleaved-off by means of 40% and 20% piperidine solution in DMF. 83.5 mol of the denatonium derivative are dissolved in 500 l of a 0.5 M HOBt solution of H.sub.2O/DMF 1:4. To the resin there is added the solution and subsequently a solution of 125 mol of N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in 100 l H.sub.2O/DMF 1:4 and shaken for one hour at room temperature. Subsequently, the resin is washed several times with DMF, dichloromethane, and diethyl ether. A resin-bound peptide-bitter principle coupling product is obtained.

(45) After coupling, the protective groups as well as the product consisting of denatonium derivative and peptide are simultaneously cleaved-off from the carrier with the addition of TFA. Reactive carbocations formed during the cleavage are quenched by the scavengers ethane dithiol and thioanisole.

(46) After an incubation time of 1.5 hours the denatonium derivative/peptide construct cleaved-off from the carrier is precipitated in ice-cold ether and after centrifugation the solution is removed from the pellet formed. Coupling can be verified by means of mass spectrometry (electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI)).

Example 14

Coupling of N-(3-carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane ammonium bromide with the peptide (Fmoc)NH-PEG(3)-Lys(Boc)-G-P-Q-G-1-A-G-Q-PEG(3)-Q resin (SEQ ID NO: 2)

(47) The peptide (Fmoc)NH-PEG(3)-Lys(Boc)-G-P-Q-G-1-A-G-Q-PEG(3)-Q resin (SEQ ID NO: 2) is gradually synthesized on a synthesis resin by means of common peptide synthesis (Fmoc strategy). To attach the bitter principle the Fmoc protective group of the terminal amino acid NH.sub.2PEG(3) COOH is cleaved-off by means of 40% and 20% piperidine solution in DMF. 83.5 mol of the denatonium derivative are dissolved in 500 l of a 0.5 M HOBt solution of H.sub.2O/DMF 1:4. To the resin there is added the solution and subsequently 20 l of diisopropylcarbodiimide (DIC) and shaken for one hour at room temperature. Subsequently, the resin is washed several times with DMF, dichloromethane, and diethyl ether. A resin-bound peptide-bitter principle coupling product is obtained.

(48) After coupling, the protective groups as well as the product consisting of denatonium derivative and peptide are simultaneously cleaved-off from the carrier with the addition of TFA. Reactive carbocations formed during the cleavage are quenched by the scavengers ethane dithiol, metacresol, and thioanisole.

(49) After an incubation time of 1.5 hours the denatonium derivative/peptide construct cleaved-off from the carrier is precipitated in ice-cold ether and after centrifugation the solution is removed from the pellet formed. Coupling can be verified by means of mass spectrometry (electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI)).

(50) MS (MALDI-TOF): m/z=1739

Example 15

Coupling of N-(3-carboxybenzyl)-2-(2,6-dimethylphenylamino)-N,N-diethyl-2-oxoethane Ammonium Bromide with the Peptide (Fmoc)NH-PEG(3)-Lys(Boc)-G-P-Q-G-1-A-G-Q-PEG(3)-A(N.SUB.3.)-Q Resin (SEQ ID NO: 3)

(51) The peptide (Fmoc)NH-PEG(3)-Lys(Boc)-G-P-Q-G-1-A-G-Q-PEG(3)-A(N.sub.3)-Q resin (SEQ ID NO: 3) is gradually synthesized on a synthesis resin by means of common peptide synthesis (Fmoc strategy). To attach the bitter principle the Fmoc protective group of the terminal amino acid NH.sub.2PEG(3)COOH is cleaved-off by means of 40% and 20% piperidine solution in DMF. 83.5 mol of the denatonium derivative are dissolved in 500 l of a 0.5 M HOBt solution of H.sub.2O/DMF 1:4. To the resin there is added the solution and subsequently 20 l of diisopropylcarbodiimide (DIC) and shaken for one hour at room temperature. Subsequently, the resin is washed several times with DMF, dichloromethane, and diethyl ether. A resin-bound peptide-bitter principle coupling product is obtained.

(52) After coupling, the protective groups as well as the product consisting of denatonium derivative and peptide are simultaneously cleaved-off from the carrier with the addition of TFA. Reactive carbocations formed during the cleavage are quenched by the scavengers ethane dithiol, metacresol, and thioanisole.

(53) After an incubation time of 1.5 hours the denatonium derivative/peptide construct cleaved-off from the carrier is precipitated in ice-cold ether and after centrifugation the solution is removed from the pellet formed. Coupling can be verified by means of mass spectrometry (electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI)).

(54) MS (MALDI-TOF): m/z=1852

Example 16

Enzymatic Peptide Cleavage 1

(55) The peptides listed in examples 12-15 can be specifically cleaved after having been cleaved-off from the resin by matrix metalloproteinases (MMP) such as MMP-8. For that, a 2-3 hour activation of the pre-MMP by p-aminophenyl mercury acetate (APMA 10 mM in 0.1 M NaOH) in a ratio of 10:1 (MMP:APMA v/v) takes place at 37 C. Subsequently, the activated MMP is added to the peptides dissolved in buffer (200 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl.sub.2, 1 M ZnCl.sub.2, 0.05% BRIJ 35, 0.05% NaN.sub.3, pH 7.0) in defined concentrations (9 ng/ml, 45 ng/ml, 90 ng/ml, 225 ng/ml, 450 ng/ml, and 900 ng/ml) and incubated for 1 h at 37 C. The reaction is stopped by separation/filtration of the MMP by centrifugation or addition of 10 equivalents of EDTA (250 mM). The cleavage products are analyzed by liquid chromatography with masse spectrometry coupling (LC-MS).

Example 17

Enzymatic Peptide Cleavage II

(56) The peptide NH2-G-p-q-G-I-A-G-q-QCOOH (small letters mean D-amino acids) (SEQ ID NO:4) modified by replacing L by D amino acids is specifically cleaved after having been cleaved-off from the resin by MMPs such as MMP-8. For that, a 2-3 hour activation of the pre-MMP by p-aminophenyl mercury acetate (APMA 10 mM in 0.1 M NaOH) in a ratio of 10:1 (MMP:APMA v/v) takes place at 37 C. Subsequently, the activated MMP is added to the peptides dissolved in buffer (200 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl.sub.2, 1 M ZnCl.sub.2, 0.05% BRIJ 35, 0.05% NaN.sub.3, pH 7.0) in defined concentrations and incubated for 1 h at 37 C. The reaction is stopped by separation/filtration of the MMP by centrifugation or addition of 10 equivalents of EDTA (250 mM). The cleavage products are analyzed by liquid chromatography with mass spectrometry coupling (LC-MS).

Example 18

Taste testing by means of an electronic tongue of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(3-(methoxycarbonyl)benzyl)-2-oxoethane Ammonium Bromide

(57) The potentials of aqueous solutions of the substance are compared with denatonium benzoate (in brackets) by means of an electronic tongue and are in a range of about: type of sensor SB2AC0: 0.05 mM=75 mV (75 mV); 0.1 mM=65 mV (61.5 mV); 0.5 mM=32 mV (30 mV); 1 mM=20 mV (24 mV); type of sensor SB2AN0: 0.05 mM=90 mV (90 mV); 0.1 mM=88.5 mV (85 mV); 0.5 mM=60 mV (60 mV); 1 mM=21.5 mV (26 mV); type of sensor SB2BT0: 0.05 mM=73 mV (72 mV); 0.1 mM=72 mV (54 mV); 0.5 mM=7 mV (2 mV); 1 mM=44 mV (47 mV). The electrical potentials measured indicate a strongly bitter substance.

Example 19

Taste Testing by Means of an Electronic Tongue of 2-(2,6-Dimethylphenylamino)N-(4-(2-ethoxy-2-oxoethylcarbamoyl)benzyl)-N,N-diethyl-2-oxoethane Ammonium Bromide

(58) The potentials of aqueous solutions of the substance are compared with denatonium benzoate (in brackets) by means of an electronic tongue and are in a range of about: type of sensor SB2AC0: 0.05 mM=93 mV (72 mV); 0.1 mM=88 mV (57 mV); 0.5 mM=52-mV (23 mV); 1 mM=42 mV (6 mV); type of sensor SB2AN0: 0.05 mM=70 mV (60 mV); 0.1 mM=60 mV (50 mV); 0.5 mM=30 mV (1 mV); 1 mM=10 mV (20 mV); type of sensor SB2BT0: 0.05 mM=95 mV (80 mV); 0.1 mM=94 mV (62 mV); 0.5 mM=35 mV (7 mV); 1 mM=5 mV (42 mV). The electrical potentials indicate a strongly bitter substance.

Example 20

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(3-methoxybenzyl)-2-oxoethane Ammonium Bromide

(59) 1 g of lidocaine (4.3 mmol) is combined with 0.86 g (4.3 mmol) of 3-methoxybenzyl bromide and shaken several times at room temperature within 20 minutes after having been added. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)-N,N-diethyl-N-(3-methoxybenzyl)-2-oxoethane ammonium bromide is obtained.

(60) m.p.: 144-151 C.

(61) .sup.1H-NMR (CDCl.sub.3. ppm): 1.52-1.55 (t, 6H, 2CH.sub.3); 2.27 (s, 6H, 2CH.sub.3); 3.45-3.52 (qd, 2H, CH.sub.2); 3.59-3.66 (qd, 2H, CH.sub.2); 3.81 (s, 3H, CH.sub.3); 4.65 (s, 2H, CH.sub.2); 4.85 (s, 2H, CH.sub.2); 7.02-7.11 (m, 6H, CH); 7.35-7.38 (m, 1H, CH); 10.48 (s, 1H, NH)

Example 21

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(3-hydroxybenzyl)-2-oxoethane Ammonium Bromide

(62) 0.5 g of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(3-methoxybenzyl)-2-oxoethane ammonium bromide (1.1 mmol) are dissolved in 10 ml of dichloromethane and stirred. A solution of borontribromide (2.3 mmol) in 10 ml of methylenechloride at 70 C. is added dropwise. When reaching room temperature 5 ml of water are added to the mixture. The organic phase is evaporated and purified by column chromatography over aluminumoxide (eluant:methanol/chloroform 1:9). 2-(2,6-Dimethylphenylamino)-N,N-diethyl-N-(3-hydroxybenzyl)-2-oxoethane ammonium bromide is obtained.

Example 22

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(tert-butyl-4-(bromomethyl)-phenylcarbamate)-2-oxoethane Ammonium Bromide

(63) 0.5 g of lidocaine (2.1 mmol) are combined with 0.61 g (2.1 mmol) of tert-butyl-4-(bromomethyl)phenylcarbamate and shaken several times. Simultaneously, to complete the reaction it is heated for 20 minutes to 80 C. Subsequently, 10 ml of THE are added and stirred. The white precipitate formed is filtered off, washed with THE and dried. 2-(2,6-Dimethylphenylamino)-N,N-diethyl-N-(tert-butyl-4-(bromomethyl)-phenylcarbamate)-2-oxoethane ammonium bromide is obtained.

Example 23

Synthesis of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-4-(aminobenzyl)-2-oxoethane Ammonium Bromide

(64) 0.5 g of 2-(2,6-dimethylphenylamino)-N,N-diethyl-N-(tert-butyl-4-(bromomethyl)-phenylcarbamate)-2-oxoethane ammonium bromide (0.96 mol) are stirred with 1.9 mol of trifluoroacetic acid in 10 ml of methylenechloride. The organic phase is evaporated and purified by column chromatography over aluminumoxide (eluant:methanol/chloroform 1:9). 2-(2,6-Dimethylphenylamino)-N,N-diethyl-N-4-(aminobenzyl)-2-oxoethane ammonium bromide is obtained.