Novel carbamazepine hapten analogues

Abstract: New carbamazepine hapten analogues of formula (I) are described comprising: (A) an active ester group; (B) a carbamazepine nucleus; and (C) a linking chain (i) linking the carboxamide group of the carbamazepine nucleus to the active ester group, the linking chain having 4 to 40 atoms.

Description: This invention relates to clinical chemistry, particularly immunoassays Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes (called ligands herein) include, for example, antigens, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, and so forth. In competitive binding immunoassays, a labeled ligand, including immunocompetent derivatives and analogs of the ligand, is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (that is, free) labeled ligand. The reaction proceeds as follows: ligand + labeled ligand + receptor ----> ligand-receptor + labeled ligand-receptor. Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, coenzymes, inhibitors and allosteric effectors. Consistent with the foregoing an immunoassay for carbamazepine in serum can be based on competition of an enzyme labeled carbamazepine analogue with carbamazepine in the patient serum for immobilized antibody binding sites. Specific requirements for the labeled carbamazepine analogue include: 1) at least 70 - 90% of the analogue can be bound by excess immobilized carbamazepine antibody; 2) affinity of the analogue for immobilized antibody is such that competition of a fixed amount of analogue with carbamazepine occurs in a therapeutically relevant concentration range; and 3) stability of the labeled carbamazepine analogue against hydrolysis of its enzyme label under storage conditions. Requirements imposed on the carbamazepine derivative include: 1) accessibility of the derivative to the immobilized antibody following conjugation with the enzyme label; 2) specific recognition of the analogue by the carbamazepine antibody; and 3) sufficient reactivity of the derivative with the enzyme label, either directly or following activation of the enzyme or derivative, under conditions that do not adversely affect enzyme activity. The present invention provides new carbamazepine analogues that meet the above requirements. The new analogues are according to claim 1. Several advantages are realized by use of the drug hapten analogues of this invention. The active esters of the carbamazepine analogues having short linking chains between the carbamazepine nucleus and the active ester group were sufficiently reactive with HRP to prepare labeled drug hapten analogues for use with useful immobilized antibodies. Haptens with a longer linker between the active ester group and the carbamazepine nucleus gave labels that could be bound by all immobilized antibodies tested. Stability of the linking groups containing amide bonds are stable against hydrolysis. Immunoassays comprising carbamazepine-derivatives are known from DE-A-3 245 854 and JP-P-57 109 724. We have prepared carbamazepine analogues containing active ester groups, especially succinimidoxycarbonyl groups, capable of rapidly and completely condensing with enzyme labels such as horseradish peroxidase (HRP). Other labels such as visible dyes, fluorescent dyes, radioactive materials, and so forth, can also be used by covalently bonding the labels to the analogues of this invention. The examples provided hereinafter illustrate the preparation of the new carbamazepine analogues of this invention. Preparation Procedure N-[3-(3-Succinimidoxycarbonylpropionamido)propyl]carbamazepine Step 1: N-[3-(Benzyloxycarbonylaminolpropyl]carbamazepine A mixture of N-benzyloxycarbonyl-1,3-propanediamine (8,0 g, 0.04 mole) and triethylamine (5.0 g, 0.05 mole) in chloroform (75 mL) was added dropwise over 15 minutes to 5-chlorocarbonyl-2,2'-iminostilbene (7.6 g, 0.03 mole) in chloroform (200 mL). The mixture was then heated at reflux for 1 hour and at ambient temperature for 16 hours. Dichloromethane (500 mL) was added, and the mixture was washed with 10% hydrochloric acid (2 x 100 mL), washed with saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator in vacuo. To the residue was added ethyl acetate (50 mL) and the solution placed in the freezer for 2 hours and filtered. Step 2: N-(3-Aminopropyl)carbamazepine Hydrobromide N-[3 -(Benzyloxycarbonylamino)propyl]carbamazepine (13.2 g, 0.03 mole) and 30-35% hydrogen bromide-acetic acid solution (70 mL) was allowed to stir at room temperature for 1 hour. This mixture was then poured into diethyl ether (3 L), and the solid which forms was triturated with fresh portions of ether (3 x 1L). The solid was filtered. Step 3: N-[3-(3-Carboxypropionamido)propyl]carbamazepine A mixture of N-(3-aminopropyl)carbamazepine hydrobromide (7.5 g, 0.02 mole), triethylamine (2.0 g 0.02 mole), and succinic anhydride (2.0 g, 0.02 mole) in chloroform (200 mL) was heated for 30 minutes at 50 - 60°C and allowed to stand at ambient temperature for 20 hours. Dichloromethane (500 mL) was added, and the mixture was washed with 10% hydrochloric acid (2 x 100 mL) and saturated sodium chloride solution (100 mL). then dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator in vacuo. To the residue was added dichloromethane (100 mL) and petroleum ether (100 mL), and it was placed in the freezer overnight. The solid was filtered. Step 4: N- [3- (3-Succinimidoxycarbonylpropionamido)propyl]carbamazepine A mixture of N-[3-(3-carboxypropionamido)propyl]carbamazepine (3.3 g, 0.01 mole), N,N'-dicyclohexylcarbodiimide (2.0 g, 0.01 mole), and N-hydroxysuccinimide (1.0 g, 0.01 mole) in chloroform (80 mL) was stirred at room temperature for 20 hours. The mixture was filtered and the solvent removed on a rotary evaporator in vacuo to give 4.7 g. The solid was dissolved in dichloromethane (20 mL), filtered and the solvent removed. This procedure was repeated an additional time to give 3.0 g (64% yield). Analytical calculated for C 26 H 26 N 3 O 6 : C, 65.54; H, 5.50; N, 8.82. Found: C, 62,38; H, 5.47; N, 11.02. Example 1 5-[4-(4-Succinimidoxycarbonylbutyryl)piperazinocarbonyl]-5H-dibenzo[b,f]azepine Step 1: 5-[4-(Benzyloxycarbonyl)piperazinocarbonyl]-5H-dibenzo[b,f]azepine This material was prepared using the procedures outlined in Preparation Procedure 2, step 1, except substituting benzyl 1-piperazinecarboxylate in place of the N-benzyloxycarbonyl-1,3-propanediamine. The compound was dissolved in ethyl ether (10 mL) and petroleum ether was added to the cloud point. The mixture was placed in a freezer and then filtered to give 9,3 g material. Step 2A: 5-(Piperazinocarbonyl)-5H-dibenzo[b,f]azepine Hydrobromide Step 2B: 5-[4-(4-Carboxybutyryl)piperazinocarbonyl]-5H-dibenzo[b,f]azepine These materials were prepared using the procedures outlined in steps 2 and 3 of Preparation Procedure 2, except starting with 5-[4-(benzyloxycarbonyl)-piperazinocarbonyl]-5H-dibenzo[b,f]azepine in place of the N-[3-(benzyloxycarbonylamino)propyl]carbamazepine in step 2, and thus the product 5-(Piperazinocarbonyl)-5H-dibenzo[b,f]azepine Hydrobromide in place of the product of step 2, and glutaric anhydride in place of succinic anhydride in the procedures of step 3. The residue (2B) was crystallized from ethyl acetate (10 mL) and petroleum ether (2 mL), placed in a freezer, and filtered to give the acid. Step 3: 5-[4-(3-Carboxypropionyl)piperazinocarbonyl]-5H-dibenzo[b,f]azepine This material was prepared using the procedures outlined in step 2B of Preparation Procedure 4, except using succinic anhydride in place of glutaric anhydride. A sample recrystallized from dichloromethane/ethyl acetate (1:1) gave pure material. Step 4: 5-[4-(4-Succinimidoxycarbonylbutyryl)piperazinocarbonyl]-5H-dibenzo[b,f]azepine This material was prepared using the procedure outlined in Preparation Procedure 2, step 4, except using 5-[4-(carboxybutyryl)piperazinocarbonyl]-5H-dibenzo-[b,f]azepine in place of the N-[3-(3-carboxypropion-amido)propyl]carbamazepine to give 4.6 g (100% yield). The material (3.0 g) was chromatographed using silica gel. Analytical calculated for C 28 H 28 N 4 O 6 : C, 65.09; H, 5.47; N, 10.85. Found: C, 64.87; H, 5.99; N, 10.62. Example 2 - 5-[4-(3-Succinimidoxycarbonylpropionyl)piperazinocarbonyl]-5H-dibenzo[b,f]azepine This material was prepared using the procedure outlined in step 4, Preparation Procedure 2, except using 5-[4-(3-carboxypropionyl)piperazinocarbonyl]-5H-dibenzo[b,f]azepine in place of the N-[3-(3-carboxypropionamido)propyl]carbamazepine. A sample was recrystallized from dichloromethane (35 mL) and ethyl acetate (8 mL) to give material melting at 135-140°C. Analytical calculated for C 27 H 26 N 4 O 6 : C, 64.33; H, 5.22; N, 11.15. Found: C, 62.46; H, 5.29; N, 10.82. Example 3 - N-{4-[4-(3-Succinimidoxycarbonylpropionyl)piperazinocarbonyl]butyl} - carbamazepine Step 1: N-[4-(4-Benzyloxycarbonylpiperazinocarbonyl)butyl]carbamazepine A mixture of N-(4-carboxybutyl)carbamazepine (3.4 g, 0.01 mole) and 1,1'-carbonyldiimidazole (2.1 g, 0.0125 mole) in tetrahydrofuran (100 mL) was stirred at ambient temperature for 30 minutes. To this mixture was added at room temperature over 30 minutes benzyl 1-piperazinecarboxylate (2.75 g, 0.0125 mole) in tetrahydrofuran (100 mL). After 20 hours, dichloromethane (300 mL) was added, and the organic solution was washed with 5% hydrochloric acid solution (3 x 100 mL), washed with saturated sodium carbonate solution (100 mL), washed with saturated sodium chloride solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator in vacuo. The material was used directly in the next step. Step 2A: N-(4-Piperazinocarbonylbutyl)carbamazepine Hydrobromide Step 2B: N- {4-[4-(3-carboxypropionyl)piperazinocarbonyl]butyl}carbamazepine These materials were prepared using the procedures outlined in steps 2 and 3 of Preparation Procedure 2, except starting with N-[4-(4-benzoyloxycarbonylpiperazinocarbonyl)butyl]carbamazepine in place of the N-[3-(benzoyloxycarbonylamino)propyl]carbamazepine in step 2A, and then using the product from step 2A of Example 3 in place of the product of step 2 of Preparation Procedure 2 in step 2B of Example 3 to give the acid. Step 3: N-{4-[4-(3-Succinimidoxycarbonylpropionyl)piperazinocarbonyl]butyl}carbamazepine This material was prepared using the procedure outlined in Preparation Procedure 2, step 4, except using N-{4-[4-(3-carboxypropionyl)piperazinocarbonyl]butyl}carbamazepine in place of the N-[3-(3-carboxypropionamido)propyl]carbamazepine. A sample was chromatographed using silica gel to give analytically pure material. Analytical calculated for C 32 H 35 N 5 O 7 : C, 63.88; H, 5.86; N, 11.64. Found: C, 63.13; H, 6.02; N, 11,06.

Claims: A new carbamazepine analogue conforming to the structure: wherein: R represents hydrogen or C 1 to C 6 alkyl; Y is 1,4-piperazinylene; 2,5-dimethyl-1,4-piperazinylene; 1,3-imidazolidinylene or 1,4-hexahydrodiazepinylene; R 2 represents ethylene, trimethylene, tetramethylene or pentylene; R 3 , R 4 and R 5 each independently represent alkylene groups; R 6 represents ethylene or o-phenylene; and each Z independently represents -O-, -S-, or -NR- wherein R is as previously defined; l represents 0, 1 or 2; m is 1 or 2; n is 0, 1, or 2; and the sum of carbon and hetero atoms in the linking chain, comprising the atoms in the brackets l, m and n, is 5 to 40. The drug hapten analogue derivatives of claim 1 according to structure I, wherein Y represents a 1,4-piperazinylene ring group; and R 2 represents ethylene, trimethylene, tetramethylene or pentylene; R 3 , R 4 and R 5 each independently, represents methylene, ethylene, trimethylene, tetramethylene or pentylene; and R 6 represents ethylene thereby forming succinimidoxycarbonyl. The analogue as claimed in claim 1, wherein structure I is selected from: 5-[4-(4-Succinimidoxycarbonylbutyryl)piperazino-carbonyl]-5H-dibenzo[b,f]azepine; 5-[4-(3-Succinimidoxycarbonylpropionyl)piperazino-carbonyl]-5H-dibenzo[b,f]azepine; N-{4-[4-(3-Succinimidoxycarbonylpropionyl)piperazinocarbonyl]butyl}carbamazepine;

Inventors: PONTICELLO IGNAZIO S, SAINI MOHAN SINGH, PONTICELLO IGNAZIO S., SAINI, MOHAN SINGH, Ponticello Ignazio S., c/o EASTMAN KODAK COMPANY, Saini, Mohan Singh, c/o EASTMAN KODAK COMPANY
Applicants: JOHNSON & JOHNSON CLIN DIAG, JOHNSON & JOHNSON CLINICAL DIAGNOSTICS, INC.
Application year: 1993
Main classification: C07D403
IPC codes: G01N33, C07D223, C07D403