BENZOTRIAZOLE DERIVATIVES AND FLUORESCENCE-EMITTING REAGENTS CONTAINING THEM

Abstract: The invention relates to compounds represented by formula (I) and their salts: wherein R¹ represents hydrogen, lower alkyl, halogen, cyano or lower alkyloxycarbonyl, R² and R³ each represents hydrogen, lower alkyl, C₃-C₇ cycloalkyl or aryl or, when taken together, form piperadino or morpholino together with the adjacent nitrogen atom. These compounds scarcely emit fluorescence when present alone in an aqueous solution but, in the presence of a protein such as α₁-AG, they bind to the protein to emit strong fluorescence, thus being suitable reagents for determining proteins existing in a minute amount in blood, particularly α₁-AG.

Description: This invention relates to reagents for analysis of protein-binding, which are suitable for estimating the binding strength between a drug and a protein, especially between a basic drug and α₁-acid glycoprotein (hereafter referred to as α₁-AG). The drawing indicates the relationship of concentration versus fluorescence intensity, where the abscissa shows concentrations of the compound 1 (× 10⁻⁶M) and the ordinate shows fluorescence intensity (%). It is important to estimate quantitatively the binding strength of a drug to serum protein in the explication of drug behaviors in the living body. Recently much attention has been paid to α₁-AG as a protein bound to a basic drug in blood and reports on the interaction of α₁-AG with a wide variety of drugs are increasing. The fluorescence probe method, one of the methods for estimating protein-binding strength, is highly sensitive and easy to handle, however, one can hardly say that known fluorescence probes are suitable for the estimation of the binding strength to α₁-AG. For example, Auramin O and Phenprocoumon etc. are known and reported as fluorescence probes for α₁-AG. The former, which is disclosed in Y. Sugiyama et al, Biochem. Pharmcol. 34(6), 821-829 (1985), has a demerit of low sensitivity in determining a very small quantity of a protein, because the binding strength of it to α₁-AG is very weak. The latter, which is disclosed in M. Otagiri et al., J. of Pharmaceutical Sciences 76(8), 646-649 (1987), has also some demerits that it is easily affected by fluorescence of a protein itself, because the wavelength suitable for determination is in the short range. It is therefore the object of the present invention to provide a fluorescence probe which has a high affinity with a protein, especially α₁-AG, and can be determined at a long wavelength. This object is solved by the benzotriazole derivatives which are provided by this invention and shown by the following formula: wherein R¹, R² and R³ are as defined in claim 1, or salts thereof (hereafter referred to simply as the compounds of this invention). These compounds per se scarcely emit fluorescence in an aqueous solution, while in the presence of a protein such as α₁-AG they bind to the protein to emit strong fluorescence. The compounds of this invention compete with a wide variety of basic drugs for the same site on a protein and, thereby the change in fluorescence intensity can be observed. From this change of fluorescence intensity, an exact determination can simply be performed by estimating the binding strength of the drug to a protein by means of known fluorescence probe methods. Since the compounds of this invention have, as mentioned before, a very high binding affinity for a protein, the quantitative determination of a minute amount of a protein in blood, especially α₁ - AG, can also be performed easily. Benzotriazole derivatives provided by this invention can be prepared easily by reactions known in the field of organic chemistry. For example, as shown below, 5-substituted metaphenylenediamine (a) is subjected to a known diazo coupling reaction with a paraphenylenediamine derivative (b) (Step 1), then to ring-closing reaction (Step 2), to give the compounds of this invention. In the reaction scheme, R¹, R², and R³ each has the same meaning as defined above. The process above is explained below step by step. (Step 1) The reaction is usually carried out in an aqueous solution at a temperature from ice-cooling to 15°C. The compound (b) is dissolved or suspended in an inorganic acid, which is then reacted with nitrous acid added as sodium nitrite to obtain a highly reactive diazo compound. Sulfamic acid or urea is added thereto to remove the excessive nitrous acid. Next, the compound (a) is added thereto, whereby it is easily coupled with the diazo compound to give the compound (c). The reaction is completed in several minutes to several hours. (Step 2) The azo group of the compound (c) easily reacts with the adjacent amine intramolecularly to give the compounds (I) of this invention. The reaction is completed in several minutes to several ten hours at room temperature or under heating, if carried out in a water-containing organic solvent in the presence of a copper-ammonia complex. In this invention, halogen means fluoro, chloro, or bromo. C₁-C₅ alkyl means a straight or branched chain C₁-C₅ alkyl, including methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and tert-pentyl, etc. C₃-C₇ cycloalkyl means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, which may have one or two substituents such as halogen, C₁-C₅ alkyl, C₁-C₅ alkyloxy. Aryl means a phenyl group which may have one or two substituents, and includes, for example, phenyl, tolyl, xylyl, cumenyl, o-, m-, p-chlorophenyl. C₃-C₇ alkylene includes propylene, butylene, pentylene, hexylene, heptylene etc. The representative examples of the compounds of this invention are listed below, which do not limit the scope of this invention. (1) 7-chloro-2-(p-diethylaminophenyl)-2H-benzotriazolyl-5-amine, (2) 7-chloro-2-(p-methylaminophenyl)-2H-benzotriazolyl-5-amine, (3) 7-chloro-2-(p-anilinophenyl)-2H-benzotriazolyl-5-amine, (4) 7-chloro-2-(p-morpholinophenyl)-2H-benzotriazolyl-5-amine, (5) Ethyl 6-amino-2-(p-diethylaminophenyl)-2H-benzotriazole-4-carboxylate, (6) 2-(p-cyclohexylaminophenyl)-2H-benzotriazolyl-5-amine, (7) 7-cyano-2-(p-piperidinophenyl)-2H-benzotriazolyl-5-amine, (8) 7-bromo-2-(p-cyclopropylaminophenyl)-2H-benzotriazolyl-5-amine, (9) 7-chloro-2-(p-piperazinophenyl)-2H-benzotriazolyl-5-amine, and, (10) Methyl 6-amino-2-(p-diisopentylaminophenyl)-2H-benzotriazole-4-carboxylate. By using the compounds of this invention above, (1) the binding strength of a drug to a protein and (2) the amount of protein in blood can be determined easily and precisely. The procedures for analysis are explained in the following Examples. (1) Determination of Binding Strength of Drug to Protein (Binding Strength of Basic Drug to α₁-AG) The two different kinds of solutions, one containing α₁-AG and a compound of this invention listed above, the other containing α₁-AG, the compound of this invention, and a drug, are prepared to measure fluorescence intensity at the excitation·fluorescence wavelength, as shown in Table 2. According to the working curve made beforehand, the binding rate of the compound to α₁-AG in each solution is determined and the binding constant of a drug to α₁-AG is calculated from the difference between the binding rates in the presence and the absence of a drug. (2)Quantitative Determination of Protein in Blood (Quantitative Determination of Albumin) A solution containing a compound is added to a solution containing albumin and the mixed solution is subjected to determination of fluorescence intensity at the excitation·fluorescence wavelength as shown in Table 2. According to the working curve on the standard albumin solution made beforehand, the concentration of albumin in the sample solution is determined. (Quantitative Determination of α₁-AG) Sulfosalicylic acid is added to the sample solution containing α₁-AG to remove coexisting proteins, to which the solution containing a compound is added. The mixed solution is subjected to determination of fluorescence intensity at the excitation·fluorescence wavelength as shown in Table 2. According to the working curve on the standard solution of α₁-AG, the concentration of α₁-AG in the sample is determined. This invention is further explained in the following Examples and Experiments, which are not intended to limit the scope of this invention. Example The compounds of this invention 1-5 were prepared as shown in the following Examples 1-5. Physical constants of each compound are shown in Table 1. Example 1 Preparation of 7-chloro-2-(p-diethylaminophenyl)-2H-benzotriazolyl-5-amine(1) . A 10% aqueous solution (7ml) of sodium nitrite was added to a solution of N,N-diethyl-p-phenylenediamine dihydrochloride (10mmol) dissolved in 50ml of 10% hydrochloric acid, while being stirred under ice-cooling. Fifteen minutes later, 10% ammonium sulfamate (15ml) was added thereto and the mixture was stirred for 15 minutes. The reaction mixture was adjusted to about pH 5 with sodium acetate, then 5-chloro-m-phenylenediamine (10mmol) was added thereto and the resulting mixture was stirred for 2 hours. The reaction mixture was adjusted to about pH 9 with 1N sodium hydroxide, then extracted with ethyl acetate. The ethyl acetate layer was evaporated to leave a residue, which was dissolved in pyridine (40ml), then an ammoniacal cupric sulfate solution which was prepared by dissolving 10g of cupric sulfate pentahydrate in 60ml of 14% ammonia was added thereto and the resulting mixture was refluxed for 4 hours. Next, after cooling the reaction solution, the resulting mixture was extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to leave a residue, which was dissolved in ethyl acetate (5ml), then the resulting mixture was purified using silicagel chromatography with ethyl acetate as eluent, whereby the objevtive compound 1 was obtained. Example 2 Preparation of 7-chloro-2-(p-methylaminophenyl)-2H-benzotriazolyl-5-amine(2) The reaction was performed according to the same manner as in Example 1, except that N-methyl-p-phenylenediamine dihydrochloride was used in place of N,N-diethyl-p-phenylenediamine dihydrochloride, whereby the objective compound 2 was obtained. Example 3 Preparation of 7-chloro-2-(p-anilinophenyl)-2H-benzotriazolyl-5-amine (3) The reaction was performed according to the same manner as in Example 1, except that N-phenyl-p-phenylenediamine dihydrochloride was used in place of N,N-diethyl-p-phenylenediamine dihydrochloride, whereby the objective compound 3 was obtained. Example 4 Preparation of 7-chloro-2-(p-morpholinophenyl)-2H-benzotriazolyl-5-amine(4) The reaction was performed according to the same manner as in Example 1, except that p-morpholinoaniline was used in place of N,N-diethyl-p-phenylenediamine dihydrochloride, whereby the objective compound 4 was obtained. Example 5 Preparation of Ethyl 6-amino-2-(p-diethylaminophenyl)-2H-benzotriazole-4-carboxylate (5) The reaction was performed according to the same manner as in Example 1, except that Ethyl 3,5-diaminobenzoate dihydrochloride was used in place of 5-chloro-m-phenylenediamine, whereby the objective compound 5 was obtained. The reagents of this invention for analysis were prepared as shown in the following Examples 6-10. Example 6 (1×10⁻⁵M aqueous solution) To a solution of the compound 1 (31.6mg) dissolved in 0.1N hydrochloric acid (10ml) was added a necessary amount of water to make the whole 10l. This was distributed into brown vials by 5ml each, then sealed hermetically to give the reagent for analysis. Example 7 (1×10⁻⁵M methanol solution) To a solution of the compound 2 (27.4mg) dissolved in methanol (10ml) was added a necessary amount of water to make the whole 10l. This was distributed into brown vials by 5ml each, then sealed hermetically to give the reagent for analysis. Example 8 (1×10⁻⁵M ethanol solution) The reagent for analysis was obtained according to the same manner as in Example 7, except that 33.6mg of the compound 3 and ethanol were used. Example 9 (1×10⁻⁴M aqueous solution) The reagent for analysis was obtained according to the same manner as in Example 6, except that 33.0mg of the compound 4 was used. Example 10(1×10⁻⁴M methanol solution) The reagent for analysis was obtained according to the same manner as in Example 7, except that 35.3mg of the compound 5 was used. No Appearance IR (Nujol) m.p.(°C) Molecular Formula Elementary Analysis (Calcd./Found) C H N Cl 1 orange plates 3230, 3340, 3440 187-188 C₁₆H₁₈N₅Cl 60.85 5.75 22.18 11.23 61.00 5.86 22.02 11.06 2 light brown plates 3220, 3330, 3415 211-213 C₁₃H₁₂N₅Cl 57.04 4.42 25.59 12.95 57.10 4.52 25.35 12.84 3 brown plates 3210, 3360 3400, 3480 174-175 C₁₈H₁₄N₅Cl 64.38 4.20 20.86 10.56 64.62 4.36 20.61 10.60 4 yellow needles 3230, 3360, 3480 246-247 C₁₆H₁₆N₅OCl 58.27 4.89 21.24 10.75 58.35 4.98 21.11 10.75 5 dark prism 1713, 3230 3860, 3450 146-148 C₁₉H₂₃N₅O₂ 64.57 6.56 19.82 ― 64.85 6.74 19.51 ― No α₁-AG Human Serum Albumin Excitation Wavelength(nm) Fluorescence Wavelength(nm) Excitation Wavelength(nm) Fluorescence Wavelength(nm) 1 418 508 415 505 2 396 482 385 480 3 400 505 400 485 4 396 502 383 465 5 452 567 420 520 Example 11 Determination of the Binding Constant of Chloropromazine (Experiment 1) In a fluorescence cell made of quartz with a plug was placed a buffer solution of 0.1M phosphoric acid (2ml) pH 7.4. 2 µl of 1×10⁻⁴M α₁-AG and 2 µl of a 5×10⁻⁴M aqueous solution of the compound 1 which was prepared by dissolving about 3mg of the compound 1 in 1N hydrochloric acid (1ml) followed by diluting with water to make the total volume 200 times were added with a microsyringe. The resulting solution was thoroughly mixed and the fluorescence intensity was determined at 508nm (excitation wavelength: 418nm). In this case, all of the compound 1 bound to α₁-AG and the relationship of concentration versus fluorescence intensity is shown in the drawing as the straight line 1. (Experiment 2) The same procedure as in Example 1 was performed with a buffer solution of 0.1M phosphoric acid (2ml) pH 7.4 and containing 3×10⁻⁶M α₁-AG. In this case, the binding of the compound 1 to α₁-AG was under an equilibrium condition and the relationship of concentration versus fluorescence intensity is shown in the drawing as the curve 2. (Experiment 3) The same procedure as in Example 1 was performed with a buffer solution of 0.1M phosphoric acid (2ml) pH 7.4 and containing 1×10⁻⁶M chloropromazine and 3× 10⁻⁶M α₁-AG. In this case, the binding of the compound 1 to α₁-AG was inhibited by chloropromazine and the relationship of concentration versus fluorescence intensity is shown in the drawing as the curve 3. (Method of Calculation) The binding rate (X) of the compound 1 to α₁-AG was determined from the following formula (1) In this scheme, Fb is the fluorescence intensity at the concentration of the compound 1 which was obtained in Experiment 1, Fp is the fluorescence intensity at the same concentration of the compound 1 which was obtained in Example 2, and Fo is blank value. The value introduced from the binding rate at each concentration of the compound 1 was plotted according to the formula (2) of Scatchard, whereby the binding parameter of the compound 1 to α₁-AG was obtained. r / Df = nKa - rKa (2) In this scheme, r is the amount of binding of the compound 1 per α₁-AG (1mole), n is the number of binding sites, Ka is the binding constant of the compound 1, Df is the concentration of the compound 1 in the free form. The binding parameters of the compound 1 for α₁-AG were Ka=1.46× 10⁻⁶M⁻¹, n=0.40. The binding constant of chloropromazine was determined from the formula of Klotz et al. In this scheme, Ka and Kb each means the binding constant of the compound 1 and chloropromazine, Pt and Bt each means the total concentration of α₁-AG and chloropromazine, n means the number of binding sites. Df and Db each means the concentration of the free and the bound form of the compound 1, which were obtained when chloropromazine was added in Experiment 3. The binding constant of chloropromazine is shown in Table 3. Example 12 As for the other basic drugs shown in Table 3, binding constants were obtained according to the same manner as in Example 11. The results are shown together in Table 3. Drug log Kb Chloropromazine 6.46 Amitriptyline 5.58 Imipramine 5.61 Trimipramine 5.54 Desipramine 4.86 prochlorperazine 6.24 Levomepromazine 6.01 Promethazine 4.94 Quinidine 6.18 Diltiazem 5.40 Pindolol 5.33 Nicardipine 6.78 Lidocaine 5.12 propranolol 5.85 Dilevalol 4.69

Claims: A compound of the following formula: wherein R¹ is hydrogen, C₁-C₅ alkyl, halogen, cyano, or C₁-C₅ alkyloxycarbonyl, R² and R³ each is hydrogen, C₁-C₅ alkyl, C₃-C₇ cycloalkyl optionally substituted with one or two halogen atoms, C₁-C₅ alkyl or C₁-C₅ alkoxy groups or phenyl optionally substituted with a chloro atom, one or two methyl groups or an isopropyl group or taken together may form C₃-C₇ alkylene or may form piperazino or morpholino together with the adjacent nitrogen atom, or the salt thereof, except the compound 2-(p-dimethylaminophenyl)-2H-benzotriazolyl-5-amine. A reagent for analysis of protein-binding or determining a protein, comprising a compound according to claim 1.

Inventors: KITAGAWA TAKAYASU -F- KAMISHIN, NARITA SHIGERU - - SHIGINONISH, KITAGAWA, TAKAYASU 24-F-1306, KAMISHINDEN 1-CHOME, NARITA, SHIGERU 4-1-414, SHIGINONISHI 3-CHOME
Applicants: SHIONOGI & CO, SHIONOGI & CO., LTD.
Application year: 1989
Main classification: C07D249
IPC codes: G01N33, C07D249