Analytical method using tetrazolium salt indicators having a reflectance plateau

Abstract: General classes of 2-thiazolyl tetrazolium salt compounds have been found to be characterized by a reflectance spectrum exhibiting an extended plateau above about 600-650 nm. Such compounds are useful as chromogenic indicators for reducing substances such as NADH. The reflectance plateau confers improved accuracy to analytical assays, particularly for the determination of analytes of medical diagnostic significance, in which a colorimetric response on a reagent carrier matrix is measured by reflectance.

Description: BACKGROUND OF THE INVENTION The present invention relates to chromogenic tetrazolium salt indicator compounds useful in the determination of reducing substances, particularly nicotinamide adenine dinucleotide (NADH). Tetrazolium salts are well known as chromogenic indicators responsive to reducing substances. Upon reduction, tetrazolium salts are converted into formazan dye products. These indicators have found use in a wide variety of fields, particularly the medical diagnostic field where they have been applied to, among others, cell staining and the determination of analytes in body fluids such as urine, milk, serum, and plasma. Commonly, the determination of body fluid analytes involves an NAD-dependent enzymatic reaction in which NADH is formed as a function of the amount of analyte present in the sample tested. The amount of NADH generated can then be determined by the reductive conversion of an appropriate tetrazolium salt indicator to its formazan dye product. Within the field of medical diagnostic tests, tetrazolium salt indicators are useful in a variety of different product types. One particular type is the reagent strip. This product is a solid state device comprising a paper or other porous carrier matrix which is impregnated or otherwise incorporated with chemical reagents responsive to a particular analyte, for example, glucose or cholesterol. The incorporated reagent system includes a chromogenic indicator which develops color, or changes color, as a function of the amount of analyte in a sample applied to the matrix. The resulting colorimetric response can be observed visually to give qualitative or semi-quantitative readings. Quantitative results can be obtained by reading the reflectance of the matrix surface at one or more defined wavelengths with an appropriate instrument (reflectance meter). There is a recognized need to develop tetrazolium indicators having strong absorbance at wavelengths longer than the absorbances of major interferants that can be present in the test sample. For instance, interference from hemoglobin coloration is a particular concern where the sample is whole blood. Indicators having significant absorption above about 640 nm are required in order to substantially overcome hemoglobin interference. The commonly used tetrazolium salt indicators are 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium chloride (MTT), and 2,2',5,5'-tetraphenyl-3,3'-(3,3'-dimethoxy-4,4'-diphenylene) ditetrazolium chloride (NBT). These compounds show maximum absorption (UVmax) in the range of 465 - 605 nm. Another shortcoming of the conventionally used prior art tetrazolium salt indicators relates to the evolution of the instrumentation used to measure their colorimetric response. Rapid advancements are being made in developing smaller, less expensive reflectance meters. One of the more costly components of such meters is the optical system which comprises a light source, a filter or other spectral element for selecting or limiting the wavelength of incident or reflect light, and a sensor. Significant cost savings could be realized by eliminating or combining functions of the optical system elements or by using less expensive components, e.g., LEDs as illuminating light sources. However, commercially available LEDs emit light having a center wavelength that can vary significantly due to manufacturing variances and temperature dependence. The conventionally used tetrazolium salt indicators INT, MTT, and NBT have reflectance spectra which are strongly sloped in the region above their UVmax. Accordingly, without individually calibrating both each instrument, to account for manufacturing variability in the LED, and each test run, to account for variance due to temperature, large errors can be introduced to the assay result. The following are representative of the prior art teachings concerning the use of various tetrazolium salts in colorimetric analysis. Tanaka et al, Japanese Kokai Tokkyo Koho JP 61000084 (Chem. Abst. 104:203469y) describes the detection of glucose using a formazan chelate obtained by the reduction of 2-(2-benzothiazolyl)-3-(carboxyphenyl)-5-phenyl-2H-tetrazolium halide in the presence of nickel (II). Limbach et al, German DE 3,247,894 (Chem. Abst. 101:125929v) relates to the use of INT in glucose assays. Rittersdorf et al, German DE 2,147,466 describes the use of seven 2-(2-benzothiazolyl)-3-phenyl-5-(4-[trimethylammonio]phenyl) tetrazolium salts in the determination of reducing substances such as reducing sugars, ascorbic acid, and ketosteroids. The variety of 2-thiazolyl tetrazolium salts and/or their corresponding formazans known in the literature are represented by the following. Serebryakova et al, Khim. Geterotsikl. Soedin. 10:1403-1405 (1970) describe the synthesis and chromatic properties of benzothiazolyl-3-phenyl-(methyl)-5-p-nitro(dimethylamino)phenylformazans. The authors state that both an electron-withdrawing nitro group at the para-position of the N-5 phenyl and a benzothiazolyl group at the N-1 position provides a bathochromic shift. Bednyagina et al, Khim. Geterotsikl. Soedin. 2:342-345 (1967) describe the synthesis of 1-benzothiazolyl- and 1-benzoxazolyl-3-methyl-(or phenyl) 5-arylformazans. The relationship between the depth of color and basicity of the heterocycle is also discussed. Gulemina et al, Khim. Geterotsikl. Soedin. 6:774-777 (1974) describe the effect of an o-nitro group on the structure and properties of benzothiazolyl formazans. The ionization constants and IR and UV spectra were determined and related to structure. Lipunova et al, Khim. Geterotsikl. Soedin. 4:493-499 (1974) describe the preparation of 3-(methyl, isopropyl, phenyl)-1-benzothiazolyl-5-nitrophenylformazans and their spectral properties. Gluemina et al, Zh. Prikl. Spektrosk. 22(5):941-943 (1975) describe the observation of both positive and negative UV thermochromism, depending upon solvent and conditions of temperature change, for 1-benzothiazolyl-, 1-thiazolyl-, and 1-(5-bromothiazolyl)-3-methyl-5-(p-nitrophenyl) formazans. Ponyaev et al, Zh. Obshch. Khim. 55(11):2615-2617 (1985) describe kinetic studies of interconversion of the photoinduced forms of 1-benzothiazolyl-3-methyl-5-phenylformazan analogs. It was shown that these forms are more deeply colored than triphenylformazan. Lipunova et al, Khim. Geterotsikl. Soedin. (1971) 831-835 compare the bathochromic effect of an N-5-naphthyl or o-tolyl group on the visible spectrum of N-1-benzothiazolylformazans. Johne et al., Pharmazie 34:790-794 (1979) describe the compounds 1-(4-methyl-5-carbethoxythiazol-2-yl-3-(3-pyridyl)-5-(2-carboxyphenyl) formazan and 1-(4-methyl-5-carbethoxythiazol-2-yl-3-(2-pyridyl)-5-(2-carboxyphenyl) formazan and certain 2-(4,5-diphenylthiazol-2-yl tetrazolium salts. Various 2-benzothiazolyl tetrazolium salts and related compounds have also been applied to the photographic field, as represented by the descriptions in U.S. Pat. Nos. 3,655,382 and 4,221,864. WO 90/12318, a citation under Art 158 (2) and Art 54 (3) and (4) EPC, describes an assay using albumin-tetrazolium interaction. WO 88/8882 is related to a method for the estimation of reduced pyrimidine nucleotides. JP 177 059 discloses an analytical element for determination of reduced type coenzyme. SUMMARY OF THE INVENTION It has now been found that 2-thiazolyl tetrazolium salts of Formula A below are characterized by a reflectance spectrum exhibiting an extended plateau above about 600 nm, preferably above about 650 nm. Such reflectance plateau confers improved accuracy to analytical assays in which a test sample is contacted with a reagent strip test device comprising a solid carrier matrix incorporated with a test composition comprising (a) reagents which react with said analyte to produce a reducing substance, e.g., NADH, and (b) an indicator compound which is reduced by such reducing agent to produce a chromogenic dye product, and in which the chromogenic dye product is measured by irradiating the carrier matrix with a light source having a central wavelength above about 600 nm and sensing the reflectance of the carrier matrix by measuring reflected light with a detector element. The reflectance plateau property of the present compounds is of particular importance in those situations wherein the light which reaches the detector element consists of a narrow wavelength band whose central wavelength is susceptible to variations of over about ± 5 nm, e.g., when using LED light sources. The object of the present invention is disclosed in the accompanying claims. The thiazolyl tetrazolium salts of the present invention are of the formula: In Formula A , R 3 is aryl or styryl, R 4 is aryl, and X is a suitable counteranion. With respect to the R 1 and R 2 substitutents, for convenience, the present compounds can be categorized into four principal groups: Group 1 Compounds - the unsubstituted benzothiazolyls wherein R 1 and R 2 together form a benzo ring which is unsubstituted, Group 2 Compounds - the substituted benzothiazolyls wherein R 1 and R 2 together form a benzo ring that is substituted as described hereinbelow, Group 3 Compounds - particular alkyl substituted thiazolyls wherein (i) R 1 is carboxyl, carbalkoxy, carbaryloxy, carbamoyl, or cyano, and R 2 is alkyl or chloro, or (ii) R 1 is alkyl or aryl, and R 2 is carboxyl, carbalkoxy, carbaryloxy, or carbamoyl or cyano, or (iii) R 1 is di- or trifluoroalkyl wherein the fluoro substituents are on the carbon adjacent to the thiazolyl residue in the formula, and R 2 is chloro, and Group 4 Compounds - the mono- and diphenyl substituted thiazolyls wherein one or both of R 1 and R 2 are substituted or unsubstituted phenyl, and if only one is substituted or unsubstituted phenyl, the other is hydrogen or alkyl. BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1 - 4 show the reflectance spectra of the formazans produced upon reduction of the prior art tetrazolium salts INT, MTT, NBT, and 2-(benzothiazol-2-yl)-3-(1-naphthyl)-5-phenyltetrazolium salt ( USSR ) at various concentrations of glucose. Figs. 5 - 16 show the corresponding spectra for the formazans from particular indicator compounds of the present invention (see list at the beginning of the Table in the Examples section). DESCRIPTION OF THE PREFERRED EMBODIMENTS The following definitions shall apply to the subject disclosure: "C 1-4 " - used to limit a residue, e.g., C 1-4 alkyl, to those forms which contain between 1 and 4 relevant atoms, e.g., carbon atoms, inclusive. "Alkyl" - linear and branched hydrocarbon residues of the general formula C n H 2n+1 , preferably "lower alkyl" such as the C 1-4 alkyls of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, and tert-butyl, as well as higher alkyls such as n-pentyl and n-hexyl. "Alkoxy" - the residue -OR wherein R is alkyl. "Alkylamido" - the residue -NRC(=O)R' wherein R and R', same or different, are alkyl. "Alkylthio" - the residue -SR wherein R is alkyl. "Amido" - the residue -NHC(=O)H. "Amino" - the residue -NRR' wherein R and R', same or different, are hydrogen or alkyl. "Aryl" - organic residues derived from an aromatic carbocyclic or heterocyclic ring or ring system by removal of a hydrogen atom attached to such ring or ring system, e.g., phenyl, naphthyl, pyridyl, oxazolyl, quinolyl, thiazolyl, thienyl, and furanyl. "Arylamido" - the residue -NRC(=O)R' wherein R and R', same or different, are aryl. "Aryloxy" - the residue -OR wherein R is aryl. "Arylthio" - the residue -SR wherein R is aryl. "Carbalkoxy" - the residue -C(=O)OR wherein R is alkyl. "Carbaryloxy" - the residue -C(=O)OR wherein R is aryl. "Carbamoyl" - the residue -C(=O)NRR' wherein R and R', same or different, are hydrogen or alkyl. "Carboxyl" - the residue -C(=O)OH. "Halo" - fluoro, chloro, and bromo. "Imidazo" - the divalent residue -N=CH-NH-. "Methylenedioxy" - the divalent residue of the formula -O-CH 2 -O-. "Phenylazo - the residue -N=N-phenyl. "Styryl" - the residue -CH=CH-R wherein R is aryl. "Sulfo" - the residue -SO 3 . "Sulfamido" - the residue -NRSO 2 R' wherein R and R', same or different, are alkyl, aryl, or hydrogen. "Sulfamoyl" - the residue -SO 2 NRR' wherein R and R', same or different, are alkyl, aryl, or hydrogen. "Trialkylammonio" - the residue -NR 3 + wherein R is alkyl. "Ureido" - the residue -NRC(=O)NR' wherein R and R', same or different, are alkyl, aryl, or hydrogen. It will be understood that, unless otherwise specifically stated, it is intended that the use of the above terms in the case of residues that can be substituted or unsubstituted, e.g., alkyl, aryl, phenylazo, and styryl, shall include the reasonably substituted forms of such residues as well as their unsubstituted forms. Reasonable substitutions which will produce useful compounds of the present invention will be evident to one of ordinary skill in the art, and will include such substituents, without limitation, as alkoxy, amino, alkylthio, carbalkoxy, carboxy, hydroxy, sulfo, and sulfamoyl, to name just a few. Plateau Compounds 2-Thiazolyl tetrazolium salts having the defined reflectance plateau property comprise both compounds which are known in the literature as well as novel compounds. Such compounds will find use in the method of the present invention. In general, such compounds are of Formula A having R 1 and R 2 substituents as defined above for the four principal groups. For the Group 2 (substituted benzothiazolyl) compounds, it is preferred that R 1 and R 2 together form a benzo ring to give a benzothiazolyl residue of Formula A1: wherein (i) R 5 and R 6 , or R 6 and R 7 , or R 7 and R 8 , together form a benzo or cyclohexyl ring that is unsubstituted or substituted with alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and wherein the others, same or different, are hydrogen, alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, or (ii) one or more of R 5 , R 6 , R 7 , and R 8 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and the others, if any, are hydrogen. For the Group 4 (phenyl-substituted thiazolyl) compounds, it is preferred that one or both of R 1 and R 2 be unsubstituted phenyl or phenyl substituted with, independently, alkoxy, aryloxy (preferably phenoxy), alkyl, amido, alkylamido, arylamido (preferably phenylamido), alkylthio, arylthio (preferably benzthio), amino, carbamoyl, carbalkoxy, carbaryloxy (preferably carbphenoxy), carboxy, cyano, halo, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and if only one is substituted or unsubstituted phenyl, the other is hydrogen, alkyl, or chloro. For each of Groups 1-4, R 3 and R 4 can vary widely. Essentially any tetrazolium salt of Formula A in which R 3 is an aryl group or a styryl group and R 4 is an aryl group will have the desired reflectance plateau property of the present invention. Selection of particular aryl residues at the R 3 and R 4 positions will, however, influence the intensity (extinction coefficient) of the formazan, and the position (bathochromicity) and flatness of the plateau. Preferred R 3 and R 4 Residues From the standpoints of synthesis and reflectance spectrum properties of the formazan, for each of Groups 1-4 as defined herein, R 3 will preferably be selected from: (a 1 ) residues of Formula B : wherein Q is a bond or -CH=CH-, and wherein (i) Y 1 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, or hydrogen, Y 2 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, alkylthio, amino, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, Y 3 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carbaryloxy, carboxyl, cyano, halo, hydrogen, hydroxyl, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and Y 4 is alkoxy, halo, or hydrogen, or (ii) Y 2 and Y 3 together form methylenedioxy or imidazo and Y 1 and Y 4 are hydrogen, (b 1 ) 2, 3, or 4-pyridyl, (c 1 ) 2 or 3-thienyl, and (d1) 2 or 3-furanyl; and R 4 will preferably be selected from: (a 2 ) residues of the Formula C : wherein Y 5 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, hydrogen, nitro, or ureido, Y 6 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, Y 7 is alkoxy, aryloxy, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, hydrogen, hydroxyl, nitro, phenylazo, sulfo, sulfonamido sulfamoyl, or ureido, and Y 8 is alkoxy, aryloxy, alkyl, halo, hydrogen or nitro, (b 2 ) residues of the Formula D : wherein Y 9 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, phenylazo, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, (c 2 ) 2, 4, 6, or 8-quinolyl, or 2-methylquinolyl, and (d 2 ) anthranyl. Most Preferred R 3 and R 4 Residues The most preferred compounds in Groups 1-4, those which have superior reflectance plateaus, are those wherein R 3 is selected from: (a 1 ) residues of Formula E : wherein (i) Y 2 , Y 3 , and Y 4 are each C 1-4 alkoxy, (ii) Y 4 is hydrogen and Y 2 and Y 3 are both C 1-4 alkoxy or together form methylenedioxy, or (iii) Y 2 and Y 4 are both hydrogen and Y 3 is C 1-4 alkoxy, C 1-4 alkyl, C 1-4 alkylamido, alkylthio, C 1-4 alkylthio, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, halo, hydrogen, nitro, tri(C 1-4 )alkylammonio, or ureido, and (b 1 ) 2 or 3-thienyl. Based on the properties and synthesis of compounds that have been prepared, it is even further preferred that R 3 be selected from: 3,4,5-trimethoxyphenyl, 3,4-dimethoxyphenyl, 3,4-methylenedioxyphenyl, 4-methoxyphenyl, 4-acetamidophenyl, 4-methylthiophenyl, 4-phenyl, 4-halophenyl, 4-cyanophenyl, 4-nitrophenyl, 2-thienyl, and 3-thienyl. The most preferred R 4 residues are: (a 2 ) residues of Formula C , supra , wherein (i) Y 5 is hydrogen and each of Y 6 , Y 7 , and Y 8 is C 1-4 alkoxy, (ii) Y 5 and Y 8 are both hydrogen and Y 6 and Y 7 are both C 1-4 alkoxy or together form methylenedioxy, (iii) Y 5 , Y 6 and Y 8 are each hydrogen and Y 7 is C 1-4 alkoxy, C 1-4 alkylamido, C 1-4 alkylthio, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, hydroxyl, nitro, phenylazo, sulfo, sulfonamido, sulfamoyl, or ureido, (iv) Y 5 is alkoxy or alkyl, Y 6 and Y 8 are both hydrogen, and Y 7 is C 1-4 alkoxy, C 1-4 alkylamido, C 1-4 alkylthio, benzamido, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, hydrogen, nitro, phenylazo, or ureido, (v) Y 5 and Y 8 are C 1-4 alkoxy, or (vi) Y 5 and Y 8 are C 1-4 alkoxy and Y 7 is C 1-4 -alkylamido or benzamido; (b 2 ) residues of Formula D , supra , wherein Y 9 is C 1-4 alkoxy, C 1-4 alkyl, C 1-4 alkylamido, C 1-4 alkylthio, benzamido, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, or ureido, and (c 2 ) 8-quinolyl. Based on the properties and synthesis of compounds that have been prepared, it is even further preferred that R 4 be selected from: 3,4,5-trimethoxyphenyl, 3,4-dimethoxyphenyl, 2,4-dimethoxyphenyl, 3,4-methylenedioxyphenyl, 4-methoxyphenyl, 4-acetamidophenyl, 4-methylthiophenyl, 4-carboxyphenyl, 4-nitrophenyl, 2-methoxyphenyl, 2-methoxy-4-carboxyphenyl, 2,5-dimethoxyphenyl, 2,5-dimethoxyphenyl-4-benzamidophenyl, 1-naphthyl, 4-nitro-1-naphthyl, 4-methoxy-1-naphthyl, 8-quinolyl, 2-methyl-4-carboxyphenyl, 4-carbmethoxyphenyl, 4-cyanophenyl, and 4-cyano-1-naphthyl. Novel Tetrazolium Salts The present invention also provides novel tetrazolium salts within Groups 1-4 as follows: Group 1 Compounds of Formula F : wherein R 3 and R 4 are selected from their preferred residues as described above. Group 2 Compounds of Formula G : wherein (1) R 6 , R 7 , and R 8 are all hydrogen and R 5 is alkoxy, aryloxy, alkyl, carbamoyl, carbalkoxy, or carboxy, or (2) R 5 , R 6 , and R 8 are all hydrogen and R 7 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, or (3) R 5 is hydrogen and R 6 , R 7 , and R 8 are independently alkoxy, aryloxy, or alkyl, or (4) R 6 and R 8 are both hydrogen, R 5 is alkoxy, aryloxy, alkyl, carbamoyl, carbalkoxy, or carboxy, and R 7 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, or cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, or (5) R 5 and R 6 together form a benzo ring, R 7 is alkoxy, aryoxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxy, cyano, halo, hydrogen, or ureido, and R 8 is hydrogen, or (6) R 7 and R 8 together form a benzo ring and R 5 and R 6 are both hydrogen, or (7) R 6 and R 7 together form a cyclohexyl ring, R 5 is alkyl, alkoxy, halo, or hydrogen, and R 8 is hydrogen, and wherein R 3 and R 4 are selected from their preferred residues as described above. Compounds which are most preferable are those wherein (i) all of R 5 , R 6 , R 7 and R 8 are hydrogen, (ii) R 5 , R 6 , and R 8 are hydrogen and R 7 is C 1-4 alkoxy (especially preferred), C 1-4 alkyl, carboxy, or halo, or (iii) R 5 and R 6 together form a benzo ring, R 7 is hydrogen or C 1-4 alkoxy, and R 8 is hydrogen. Group 3 Compounds of Formula A , supra , wherein (a) R 1 is carb(C 1-4 )alkoxy and R 2 is C 1-4 alkyl or chloro, or (b) R 1 is C 1-4 alkyl or phenyl, and R 2 is carb(C 1-4 )alkoxy, or (c) R 1 is di- or trifluoromethyl and R 2 is chloro, and wherein R 3 and R 4 are selected from their preferred residues as described above. Most preferred are those compounds wherein R 1 is di- or trifluoromethyl and R 2 is chloro. Group 4 Compounds of Formula A , supra , wherein (i) both of R 1 and R 2 are unsubstituted phenyl or phenyl para -substituted with the same group selected from alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carbaryloxy, carboxy, cyano, halo, trialkylammonio, and ureido, or (ii) one of R 1 and R 2 is unsubstituted phenyl or phenyl para -substituted with alkoxy or aryloxy and the other is hydrogen. The most preferred compounds of this Group are those wherein both of R 1 and R 2 are unsubstituted phenyl or one is unsubstituted phenyl and the other is phenyl para -substituted with C 1-4 alkoxy; or wherein R 1 is unsubstituted phenyl or phenyl para -substituted with C 1-4 alkoxy, C 1-4 alkyl, or halo, and R 2 is hydrogen. Further descriptions of preferred novel 2-thiazolyl tetrazolium salt indicators can be found in the commonly assigned U.S. patent applications filed on even date herewith entitled 2-Benzothiazolyl Tetrazolium Salt Indicators, Substituted 2-Thiazolyl Tetrazolium Salt Indicators, and Phenyl-Substituted 2-Thiazolyl Tetrazolium Salt Indicators, and identified by EP publication numbers EP 476 457, EP 476 456 and 476 455 respectively. Counteranion The selection of the counteranion will be based primarily on considerations of stability and solubility of the particular tetrazolium salt of interest. In general, one can select from such counteranions as the inorganic anions chloride, bromide, iodide, nitrate, fluroborate, perchlorate, and sulfate, as well as organic anions such as acetate, oxalate, tartrate, and aryl sulfonates (benzene sulfonate, tosylate). Synthetic Methods Tetrazolium salts are prepared by methods well known in the literature (Hooper, W.D., Rev. Pure and Appl. Chem., 1969, 19, 221; Putter, R., in Methoden der Organischen Chemie, Houben-Weyl-Muller ed., Thieme Verlag: Stuttgart, 1965, Bd. 10/3, p. 633; Nineham, A. W. Chem. Rev., 1955, pp. 355-483). In general, the tetrazolium salts of the present invention are prepared by first reacting a 2-hydrazinothiazole with an aldehyde and then treating the resulting hydrazone with a diazotized aniline. The resulting formazan is then oxidized to the tetrazolium salt by well known methods. Consequently, the synthesis involves three principal starting materials, the aldehyde, the aniline, and the 2-hydrazinothiazole. Preparation of 2-hydrazinothiazoles The Group 1 Compounds of the present invention are prepared from unsubstituted 2-hydrazinobenzothiazole (R 1 and R 2 together form an unsubstituted benzo ring). For the Group 2 Compounds, substituted 2-hydrazinobenzothiazoles are prepared from 1-halo-2-nitro arenes (1) by first displacement of the halide with thiocyanate to produce the nitrothiocyanate (2). This is then reduced with reducing agents such as tin, and the resulting aminothiocyanate (3) cyclized to the 2-aminobenzothiazole (4). The same aminothiocyanate (3) can also be synthesized by treating the amine with thiocyanogen (Metzger, J.V., in Comprehensive Heterocyclic Chemistry, A.R. Katritzky, C.W. Rees ed.; Peragamon: New York, 1984; Vol. 6, Part 4b, p. 324). The 2-hydrazinothiazole (5) is then prepared by reacting hydrazine with the 2-aminothiazole (4) in ethylene glycol at 140° C (Liu, K-C., Shih, B-J., Arch. Pharm., 1985, 18, 283). Other methods of preparing 2-aminobenzothiazoles (4) are reacting the aromatic amine (6) with mineral acid and thiocyanate to produce the thiourea (8) (Org. Synth., Coll. Vol. IV, 180) or reacting the aromatic amine with N-benzoylthiocyanate to form the N-benzoylthiourea (7) and then hydrolyzing the benzoyl group to produce the thiourea (Org. Synth., Coll. Vol., III, 635). Other methods of thiourea preparation can also be found in these references. These thioureas are then oxidized with reagents such as bromine to produce 2-aminobenzothiazoles [Bhargave, P.N., Baliga B.T., J. Ind. Chem., 1958, 5, 807). The 2-hydrazinobenzothiazolyls required for the various types of the Group 3 Compounds of the present invention are prepared as follows. Chlorination of the known 2-chloro-4-methylthiazole (J. Chem. Soc. 1919, pp. 1071-1090) at 100°C leads primarily to the 2,5-dichloro-4-dichloromethylthiazole (14) and at 160°C, 2,5-dichloro-4-trichloromethylthiazole (9). The 4-carbalkoxy-5-chlorothiazole are prepared by hydrolysis of 4-trichloromethyl-2,5-dichlorothiazole (9) to yield the carboxylic acid (10) (European Patent Publication No. 348,735). Treatment with reagents such as thionyl chloride yield the acid chloride which reacts with alcohols or amines to yield 4-carbalkoxy (11) or N-alkylcarbamoyl (12) thiazoles. Dehydration of 4-carbamoyl-2,5-dichlorothiazole is accomplished by methods well known in the literature to yield 4-cyano-2,5-dichlorothiazole (13). (March, J. Advanced Organic Chemistry Third Edition; John Wiley and Sons: New York, 1985, pp. 932-933). The preparation of 4-di and trifluoromethyl-5-chloro-2-hydrazinothiazoles uses the intermediates 4-dichloromethyl-2,5-dichlorothiazole (14) and the trichloromethyl compound (9), respectively. Treatment of each with hydrogen fluoride produces the fluoromethyl compounds (15 and 16) which when exposed to hydrazine yields the 2-hydrazine derivatives. The thiazoles, 4-alkyl or aryl-5-carbalkoxy, amido, or cyano-2-substituted thiazoles are prepared by halogenating the appropriate β-ketoester, amide or nitrile and reacting it with thiourea or thiosemicarbazone (Metzger, J.V., in Comprehensive Heterocyclic Chemistry, Katritzky, A.R., Rees, C.W., ed.; Peragamon: New York, 1985; vol. 6, Part 4 p. 297; Beyer, H., Bulka, E., Z. Chem., 1962, 2, 321-328). Thiazoles in the above scheme where R 1 is hydrogen and R 2 aryl may be prepared from the appropriate α-haloacetophenone (R 1 = H and R 2 = aryl). In the case of the Group 4 Compounds, the requisite starting materials are prepared as follows: The 4,5-diphenyl-2-hydrazinothiazoles are prepared by two methods. One is by first forming a benzoin condensation (17) product between two aldehydes (Ide, W.S., Buck, J.S., Org. React., 1948, Vol. 4, 269). Benzoins not available by this method are prepared from the condensation of a phenyl substituted "acyl anion equivalent" with another aldehyde followed by removal of the carbonyl protecting group (Bertz, S., J. Chem. Soc., Chem. Comm., 1980, 17, 831). A specific example is the condensation of the metallated O-trimethylsilylcyanohydrin (19) with an aryl aldehyde. The product (20) is deprotected using aqueous acetic acid to afford the benzoin product (17). The benzoin products are then converted into the halide (18) using conventional reagents such as thionyl chloride to produce the α-haloketones (18) which react with thiourea to produce 4,5-diphenyl-2-aminothiazoles (Traumann, V., Liebigs Ann. Chem., 1888, 250, 31, Dodson et al., J. Am. Chem. Soc., 1945, 67, 2442). These can be converted to the 2-hydrazino compounds with hydrazine as described for the benzo examples. The same α-haloketones react with thiocyanate to produce α-thiocyanoketones (21) which readily cyclize. For instance, when treated with hydrogen chloride gas, the 2-chlorothiazoles (22) are obtained which react with hydrazine to give the 2-hydrazinothiazoles. Alternatively, the α-haloketones may be used to directly yield the necessary hydrazone (24) by treating the haloketone with an N-arylthiosemicarbazone (23) (Johne, S., Schaks, A., Hartung, S., Scharf, K.-D., and Nover, L., Pharmazie, 1979, 34, 790). Preparation of aldehydes The aldehydes are obtained from commercial sources or can be prepared by methods familiar to one of ordinary skill in the art. For instance, aldehydes may be prepared by benzylic oxidation of an arylmethane (March, J., Advances Organic Chemistry Third Edition; John Wiley and Sons: New York, 1985; p. 1079), reduction of an aryl acid chloride (Ibid. p. 396) or aryl acid derivative, [Larock, R.C., Comprehensive Organic Transformations; VCH: New York, 1989; pp. 604-605). Aryl halides may also be used to synthesize aldehydes. In this method, a transmetallation reaction produces an arylmetallic species which can be treated with a variety of reagents, such as dimethylformamide, to produce the aldehyde (ibid, p. 681-683). The aforementioned aryl aldehydes, acids, methanes, and halides, may be derivatized with a variety of functional groups prior to their transformation into tetrazolium salts. This may be accomplished by aromatic nucleophilic substitution (March, J., Advanced Organic Chemistry Third Edition; John Wiley and Sons: New York, 1985; pp. 576-607), aromatic electrophilic substitution (ibid., pp. 447-511) or heteroatom-directed metallation reactions (Gschwend, H.W., Rodriguez, H.R., in Organic Reactions, John Wiley and Sons: New York, 1979; Vol. 26, 1). In cases where the aldehyde piece of the tetrazolium salt contains a phenol or amine, the groups must be protected so that there is not a reaction between these and the diazotized aniline or oxidizing reagent used to prepare the tetrazolium salt. This can be accomplished by protecting a hydroxyaryl aldehyde as an acetate, performing the reaction sequence to make the formazan, and then hydrolyzing the acetate at pH 10. Acidification to pH 5 and then filtration produces the desired formazan. Where the resulting phenol formazan reacts with oxidizing agent in the tetrazolium salt preparation, the phenol may be protected by an acid labile group such as dihydropyran (Greene, T.W., Protective Groups in Organic Synthesis, John Wiley and sons: New York; 1981, pp. 87-113) and which is removed by stirring the tetrazolium salt in acidic conditions. Similarly amines on the aldehyde piece must be protected to prevent their reaction. This is best accomplished by using an acid labile carbamate (ibid, pp. 218-247) which is later removed by stirring the tetrazolium salt under acidic conditions. Preparation of aryl amines Aryl amines may be prepared by reduction of the corresponding nitro or azide compound (Larock, P.C., Comprehensive Organic Transformations; VCH: New York, 1989; pp. 412-415 or 409-410), reaction between an arylmetallic compound and an electrophilic nitrogen reagent, (ibid., pp. 399-400), or rearrangement of acyl azides or oxidized amides (ibid., pp. 431-432). As in the aldehyde case, electrophilic and nucleophilic aromatic substitution can be used to introduce different functional groups into the aryl amine or synthetic precursor. Analytical Methods The present invention takes advantage of the finding that the 2-thiazolyl tetrazolium salt indicators described herein exhibit an extended plateau in their reflectance spectra above about 600 nm. The most preferred indicator compounds of the present invention have a plateau above about 650 nm (i.e., the flatest about 50 nm wide portion begins between 640 and 660 nm). Such a reflectance plateau confers improved accuracy to analytical tests based on the measurement of reflectance from a reagent strip. Reagent strips are known in the art as analytical devices comprising a solid carrier matrix incorporated with a test composition that produces a color change in response to contact with a liquid test sample containing the analyte of interest. Such test composition, in the case of the present invention, comprises (a) a reagent or reagents which react with the analyte to produce a reducing substance, and (b) a 2-thiazolyl tetrazolium salt as described herein which is reducable by such reducing substance to produce a chromogenic formazan dye product. The color response of such reagent strips can be observed visually to give semi-quantitative values, however, quantitative results are obtained by measuring the reflectance of the carrier matrix at a predetermined wavelength. Such measurements involve irradiating the reacted carrier matrix with a light source and sensing the reflectance of the carrier matrix by measuring reflected light with a detector element. The finding of tetrazolium salt indicators having a reflectance plateau is used to particular advantage where reflectance from a reagent strip is read using an instrument which is subject to variability in the central wavelength of its optical system (the combination of light source, detector element, spectral control elements, e.g., filters), and other components). Variability in the central wavelength of the optical system can be caused by a variety of factors, for example, variability in the central wavelength of the principal spectral control element such as the illuminating light source or filters. For instance, where light emitting diodes (LEDs) are used as the light source, the wavelength of emitted light will typically vary ± 4 nm within an instrument, and up to ± 8 nm between LEDs in different instruments, due to manufacturing variability. Moreover, LEDs are suceptible to variable central wavelength due to temperature effects as well. Where broad band light sources are used with filters to provide spectral control of the central wavelength, variability within an instrument is typically under 1 nm, however, between instrument variability can be as high as ± 6 nm. Thus, the present invention is applicable in those situations where the central wavelength of the light reaching the detector element in the instrument is susceptible to variations in the range of about ± 5 nm. In the making of a reagent strip for use in the present invention, selection of the carrier matrix, the test reagents which react with analyte to produce the reducing substance, and the method by which such reagents and the tetrazolium indicator are incorporated with the carrier matrix are matters well known in the art of reagent strips. For the sake of reciting just a few examples, typical carrier matrices are porous and absorbent paper, cloth, glass fiber filters, polymeric membranes and films, and the like. Incorporation methods include impregation of a formed carrier matrix with a solution, suspension, or other liquid form of the test composition, in one or more steps, followed by drying of the matrix; formation of a matrix in the presence of one or more of the components of the test composition, e.g., by casting or layering solutions of film or membrane forming formulations. In regard to the test reagents incorporated in the reagent strip, the tetrazolium salts of the present invention are particularly advantageous in the detection of NADH. Since NADH is produced in enzyme-catalyzed detection reactions specific for various biochemical substances, the present compounds are particularly useful in medical diagnostic tests, such as in the determination of glucose and cholesterol. However, in general other reducing substances besides NADH can also be detected, such as hydrogen sulfide gas, diborane, arsenic hydride, or phosphorus hydride. The present invention will now be illustrated, but is not intended to be limited by, the following examples. EXAMPLES A. Compound Synthesis Hydrazone Preparation A slurry of 25 mmole of the appropriate aldehyde and 25 mmole of the appropriate hydrazine in 125 mL of absolute ethanol, is refluxed for 3 hours. Water is removed with 3A molecular sieves in a Soxhlet extractor. The mixture is cooled to room temperature and then filtered to yield the hydrazone. Formazan Preparation The diazonium salt is first prepared by cooling a slurry or solution of 8.5 mmol of the amine in 60 mL of 3 N HCl to 5°C. Sodium nitrite (0.70 g, 10.15 mmol) in 5 mL of water is then added dropwise. After stirring for 30 minutes, the mixture added dropwise to a cold (-25°C) mixture of 8.5 mmol of the hydrazone in 120 mL of 1:1 (v/v) DMF-pyridine. The reaction is not allowed to warm beyond -15°C during the addition. The mixture is allowed to warm to room temperature while stirring for two hours. Filtration produces the formazan as a black solid. Impurities can be removed by repeated washing with methanol or refluxing the solid in methanol and filtering while hot. Tetrazolium Salt Preparation A slurry of 1.5 mmol of the formazan is stirred with 20 mL of acetic acid and 4 mL of isoamyl nitrite for a period of 16-48 hours. The mixture is then filtered to yield the tetrazolium salt. In cases where the salt does not precipitate, dilution with ether caused precipitation. 2,5-dichloro-4-dichloromethylthiazole A vigorous stream of chlorine was entered into 5888 g (42.9 mol) 97.3% 2-chloro-4-methyl-thiazole (J. Chem. Soc. 1919, pp. 1071-1090), initially at 80°C, after the exothermic reaction slowly ended at 100°C for about 100 hours. After cooling to room temperature and standing overnight, a crystalline precipitate formed of 2,5-dichloro-4-dichloromethylthiazole, which was filtered off and dried on ceramic clay. Yield 3907 g (38.4% of theory). The fluid fraction of the chlorination mixture was subjected to fractionated distillation in a 2-meter filled column. The distillate obtained at 101°C to 103°C/6 mbar crystallized to a large extent and then was dried on ceramic clay. Yield of 2,5-dichloro-4-dichloromethylthiazole by fractionated distillation : 2733 g (26.9% of theory.) Total yield 6640 g (65.3% of theory). Mp. 42-44°C (recrystallized from petroleum ether.) 1 H-NMR (inCDCl 3 ):δ = 6.78 ppm. 2,5-dichloro-4-trichloromethylthiazole Chlorine gas was passed into a mixture of 1093 g (8.19 mol) 2-chloro-4-methylthiazole and 4 liter methylene chloride in a three-neck flask equipped with a stirrer, thermometer, reflux condenser and gas tube, starting at room temperature. After the exothermic reaction died out, the methylene chloride was distilled off under a slow temperature increase with continued introduction of chlorine; the melt was slowly heated to about 160°C. At about 160°C, mostly excess chlorine gas (recognizable by the slight greenish color of the escaping gas) was bubbled in until a gas chromatogram showed almost solely the desired compound 2,5-dichloro-4-trichloromethylthiazole. Total duration of chlorination 40 to 50 hours. A crude distillation up to a head temperature of 150°C at 14 mbar gave 2057 g ca. 95% pure 2,5-dichloro-4-trichloromethylthiazole, corresponding to a yield of 88% of theory in pure product. 2,5-dichloro-4-trichloromethylthiazole was obtained pure fine distillation through a silver-coated, 220-cm filled column. Boiling point 123-125°C at 16 mbar. 2,5-dichloro-4-difluoromethylthiazole 1010 g (4.26 mol) 2,5-dichloro-4-dichloro-methylthiazole were fluorinated with 1500 ml anhydrous hydrofluoric acid in a VA-autoclave at 145°C/25 bar. The forming hydrochloric acid was continually removed. Excess hydrofluoric acid was drawn off under vacuum at room temperature at the end of the reaction. The residue was added to ice water, taken up in dichloromethane, dried over sodium sulfate and distilled. The yield was 753 g (86.6% of theory) 2,5-dichloro-4-difluoromethylthiazole, bp. 74-75°C/18 mbar; n 20 / D= 1.5171. 2,5-dichloro-4-trifluoromethylthiazole 750 g (2.76 mol) 2,5-dichloro-4-trichloromethyl-thiazole were fluorinated with 100 ml anhydrous hydrofluoric acid in a VA-autoclave at 130°C/19-20 bar. Formed HCl was continually removed. Excess hydrofluoric acid was drawn off under vacuum at room temperature at the end of the reaction. The residue was poured on ice water, taken up in dichloromethane, dried over sodium sulfate and distilled. The yield was 570 g (93% of theory) 2,5-dichloro-4-trifluoromethylthiazole; bp. 164°C; n 20 / D = 1.4774 5-chloro-2-hydrazino-4-trifluoromethylthiazole 75 g (1.5 mol) hydrazine hydrate were added to a mixture of 111 g (0.5 mol) 2,5-dichloro-4-trifluoromethylthiazole and 500 dioxane at such a rate that a reaction temperature of 25°C was not exceeded. After further stirring for 20 hours at room temperature, the reaction mixture was stirred into 2.5 liter ice water; then one filtered off, washed the residue with water and dried. The yield was 87.3 g (80.3% of theory) 5-chloro-2-hydrazino-4-trifluoromethyl-thiazol, mp. 136-137°C. 5-chloro-2-hydrazino-4-difluoromethylthiazole Similar to 5-chloro-2-hydrazino-4-trifluoromethylthiazole, one obtained from 2,5-dichloro-4-difluoromethylthiazole, 5-chloro-2-hydrazino-4-difluoromethylthiazole in 51.3% yield. Mp. 132°C (decomp.) (after recrystallization from cyclohexane). Smaller amounts of the compound can be sublimated at 70°C/0.1 mbar. B. Preparation of Reagent Strips Each indicator was impregnated into a reagent strip and tested with a solution containing a known quantity of glucose, cholesterol, or NADH. The reagent strip consists of a polystyrene handle onto which a single reagent pad is attached. The reagent pad was 0.5 cm x 0.5 cm (0.2 x 0.2") square and contains reagents allowing for a color change which was instrumentally read when an aliquot of sample containing the appropriate analyte was applied. The dry-phase reagent pad is a solid support composed of cellulosic fibers or a nylon membrane as examples. The reagent pad was impregnated first with a solution of the tetrazolium salt of interest (0.8M/L) and detergent (0.3%) in a solvent such as methanol. The second solution impregnated into the reagent pad contains the following components: Glucose Strips Glucose Dehydrogenase (GDH) 0.8U/L Diaphorase (DPH) 0.8U/L NAD 0.03 Mol/L PIPES Buffer 0.15 Mol/L Detergent 0.5% Cholesterol Strips Cholesterol Dehydrogenase (CDH) 0.3U/L Cholesterol Ester Hydrolase (CEH) 0.6U/L Diaphorase 0.3U/L NAD 0.04Mol/L Pipes Buffer 0.2Mol/L Detergent 1%v/v About 0.01 ml of several test solutions (serum, plasma, aqueous) containing at least five different analyte concentrations between 0 and 33 mM/L was applied to the center of the dried reagent pad. After a lag time of about 60 seconds, the reflectance spectra of each indicator was measured at 5 nm increments over the wavelength range of 400 to 1100 nanometers. C. Utility Data Following is a table of spectral and other analytical data pertaining to various synthesized tetrazolium salts of the present invention. The compounds are organized, in order, by the form of their benzothiazolyl residue, then by their R 4 substituent and finally by their R 3 substituent. For example, the first compound presented is A.1.a) and is of the Formula A wherein R 1 and R 2 together form an unsubstituted benzothiazolyl ring, R 4 is 4-nitrophenyl, and R 3 is 4-acetamidophenyl; the second compound, A.1.b), has the same R 1 , R 2 , and R 4 substituents as compound A.1.a) but with R 3 being phenyl; and so forth. The reflectance spectrum of tetrazolium salts is understood to be dependent upon the environment in which they are observed or measured. For purposes of comparison between individual tetrazolium salts, the data below include a measurement of the relative flatness of the flatest portion of the reflectance spectrum at wavelengths greater than 600 nm, which spectrum is generated using a glucose or cholesterol reagent strip prepared as described in Part B above. The relative flatness of the spectrum is expressed in the data in K/S units normalized for the level of analyte detected as defined below. K/S is defined by the equation (1-R) 2 2R wherein R is instrumentally read reflectance units. Percent change in K/S is the change, expressed as a percentage, over a 50 nm range divided by the average of the high and low K/S values over such range. The plateau property of the present compounds shall be understood, for the purposes of this invention, as a percent change in reflectance spectrum (expressed in terms of K/S as defined in the paragraph above) of less than about 17% over a 30-50 nm wavelength span beginning at a wavelength above about 600 nm. The more preferable compounds exhibit a plateau having a percent change in K/S of less than an about 10% over a 50 nm wavelength span. Most preferred are those tetrazolium salt indicators exhibiting a percent change in K/S of about 5% or less over a 50 nm wavelength span. With reference to the drawings, Figs. 1-4 show the reflectance spectra of the formazans produced upon reduction of the prior art tetrazolium salts INT, MTT, NBT, and USSR at various concentrations of glucose. For purposes of comparison, Figs. 5-16 show the corresponding spectra for selected compounds of the present invention. The presence of a plateau in the spectra of the formazans from the present compounds, and its absence from that of the formazans from the prior art compounds is readily apparent. The four above-mentioned prior art compounds exhibit percent changes in K/S over the wavelength range 650-700 nm as follows: INT 71% MTT 178% NBT 73% USSR 28% The lower the percent K/S value for the formazan, the more tolerant is the tetrazolium salt to variations in the central wavelength of the optical system used to measure reflectance, and hence to measure analyte concentration. The following non-standard abbreviations are used in the text below: "UV" - The wavelength in nanometers of maximum reflectance peak in the UV reflectance spectrum of the formazan. The extinction coefficient and solvent used during measurement are given in parentheses. "nm" - The position of the flatest portion of the reflectance spectrum of the formazan over a 50 nm wavelength span (expressed as the beginning and ending wavelengths in nanometers). "K/S" The percent change in K/S units over the above mentioned flatest 50 nm portion of the reflectance spectrum. The concentration of analyte used to generate the reflectance spectrum is given in parentheses. Mmol refers to the concentration in mmol/liter. Following is a list of the compounds whose reflectance spectra are shown in Figs. 5-16 of the drawings. Their shorthand reference number and location in the table that follows are given in brackets ahead of the compound names. Fig. 5 [A-2-a] 2-(Benzothiazol-2-yl)-3-(4-methoxyphenyl)-5-phenyl tetrazolium salt Fig. 6 [B-6-a] 2-(6-Ethoxybenzothiazol-2-yl)-3-(1-naphthyl)-5-(4-acetamidophenyl) tetrazolium salt Fig. 7 [G-1-a] 2-(Naphtho[1,2-d]thiazol-2-yl)-3-(2-methoxy-4-carboxyphenyl)-5-(3,4-methylenedioxyphenyl) tetrazolium salt Fig. 8 [G-17-a] 2-(Naphtho[1,2-d]thiazol-2-yl)-3-(4-carboxyphenyl)-5-(4-acetamidophenyl) tetrazolium salt Fig. 9 [G-23-a] 2-(Naphtho[1,2-d]thiazol-2-yl)-3-(4-methylthiophenyl)-5-(4-acetamidophenyl) tetrazolium salt Fig. 10 [H-1-a] 2-(8-Methoxynaphtho[1,2-d]thiazol-2-yl)-3-(3,4,5-trimethoxyphenyl)-5-(4-methoxyphenyl) tetrazolium salt Fig.11 [I-1-a] 2-(4-Difluoro-5-chlorothiazol-2-yl)-3-(4-methoxyphenyl)-5-[4-(2-(2-(2-ethoxy) ethoxy)ethoxy)phenyl] tetrazolium salt Fig. 12 [I-1-d] 2-(4-Difluoro-5-chlorothiazol-2-yl)-3-(4-methoxyphenyl)-5-(3,4-methylenedioxyphenyl) tetrazolium salt Fig. 13 [I-2-a] 2-(4-Difluoro-5-chlorothiazol-2-yl)-3-(3,4,5-trimethoxyphenyl)-5-(3,4-methylenedioxyphenyl) tetrazolium salt Fig. 14 [I-9-a] 2-(4-Difluoro-5-chlorothiazol-2-yl)-3-(2-methoxyphenyl)-5-(3,4-methylenedioxyphenyl) tetrazolium salt Fig. 15 [J-4-a] 2-(4-Trifluoromethyl-5-chlorothiazol-2-yl)-3-(3,4,5-trimethoxyphenyl) -5-(3,4-methylenedioxyphenyl) tetrazolium salt Fig. 16 [Q-1-a] 2-(4,5-Bis(4-methoxyphenyl) thiazol-2-yl)-3-(4-carboxyphenyl)-5-(2-thienyl) tetrazolium salt TABLE A. Benzothiazol-2-yl 1. R 4 = 4-nitrophenyl a) R 3 = 4-acetamidophenyl UV: 478 nm (7.8x10 3 , dioxane); nm: 620 - 670 nm; K/S: 9% (11.1 mmol) IR(KBr): 3441, 1691, 1684, 1614, 1600, 1535, 1486, 1460, 1425, 1385, 1318, 1262, 1183, 979, 853 cm -1 NMR (300 NHz, CDCl 3 ): 6 8:67 (d, 2H, J=8.9Hz, ArNO 2 H), 8.33 (d, 2H, J=9.0 Hz), 8.36 (d, 2H, J=9.3 Hz, ArH), 8.37 - 8.41 (m, 1H, ArH), 7.39 (d, 2H, J=8.7 Hz, ArH) 7.96 - 7.99 (m, 1H, benzothiazole H), 7.67 - 7.78 (m, 3H, benzothiazole H). Mass Spectrum FAB m/e: 120 (81.1), 162 (100) b) R 3 = phenyl UV: 540 nm (4.79 x 10 3 , dioxane); c) R 3 = phenyl UV: 576 nm (3.89 x 10 3 , dioxane) nm: 650 - 700 nm; K/S: 7.5% (11.1 mmol) d) R 3 = 4-nitrostyryl UV: 512 nm (11.2 x 10 3 , dioxane) e) R 3 = 4-methoxystyryl UV: 514 nm (7.17 x 10 3 , dioxane) f) R 3 = 4-nitrophenyl UV: 542 nm (2.1 x 10 3 , dioxane) g) R 3 = 4-phenylphenyl UV: 558 nm (14.1 x 10 3 , dioxane) h) R 3 = 3,4-methylenedioxyphenyl UV: 584 nm (4.8 x 10 3 , dioxane) nm: 570 - 620 nm; K/S: 16% (9.1 mmol) i) R 3 = 4-phenoxyphenyl UV: 564 nm (10.3 x 10 3 , dioxane) j) R 3 = 2-thienyl UV: 566 nm (6.3 x 10 3 , dioxane) k) R 3 = 5-nitro-2-thienyl UV: 514 nm (15.2 x 10 3 , dioxane) l) R 3 = 3,4-dimethoxyphenyl UV: 588 nm (2.3 x 10 3 , dioxane) nm: 600 - 650 nm; K/S: 14% (11.1 mmol) m) R 3 = 4-pyridyl UV: 516 nm (8.6 x 10 3 , dioxane) n) R 3 = 5-bromo-2-thienyl UV: 518 nm (6.1 x 10 3 , dioxane) o) R 3 = 3-thienyl UV: 512 nm (7.5 x 10 3 , dioxane) p) R 3 = 2-pyridyl UV: 478 nm (9.6 x 10 3 , dioxane) q) R 3 = 4-fluorophenyl UV: 546 nm (2.96 x 10 3 , dioxane) r) R 3 = 4-hydroxyphenyl UV: 582 nm (1.0 x 10 3 , dioxane) s) R 3 = 3,4,5-trimethoxyphenyl UV: 578 nm (1.0 x 10 3 , dioxane) nm: 560 - 610 nm; K/S: 10% (11.1 mmol) t) R 3 = 4-(methylthio)phenyl UV: 576 nm (1.5 x 10 3 , dioxane) u) R 3 = 1,4-benzodioxyl UV: 582 nm (4.8 x 10 3 , dioxane) 2. R 4 = 4-methoxyphenyl a) R 3 = phenyl UV: 468 (14.5 x 10 3 , dioxane) 476 (water) nm: 625 - 675 nm; K/S: 15% (22 mmol) b) R 3 = 4-acetamidophenyl nm: 665 - 715 nm; K/S: 10% (33 mmol) 3. R 4 = 4-methoxy-1-naphthyl a) R 3 = phenyl UV: 504 (7.8 x 10 3 , dioxane) 495 (water) 4. R 4 = 4-bromophenyl a) R 3 = 4-acetamidophenyl UV: 480 (7.7 x 10 3 , dioxane) B. 6-ethoxybenzothiazol-2-yl 1. R 4 = 4-nitrophenyl a) R 3 = 4-acetamidophenyl UV: 598 nm (3.2 x 10 3 , dioxane) nm: 625 - 675 nm; K/S: 9% (14 mmol) IR(KBr): 3447, 1601, 1535, 1459, 1255, 1183, 853 cm -1 NMR (CDCl 3 ): δ 8.42 - 8.70 (5H), 8.16 - 8.32 (2H), 8.03 - 8.12 (1H), 7.69 - 7.84 (2H), 7.16 - 7.27 (1H benzothiazol), 4.18 (q, 2H, J = 6.9 Hz), 2.56 (q, 2H, J = 7.4 Hz), 1.46 (t, 3H, J = 6.9 Hz), 1.17 (t, 3H, J = 7.4 Hz). b) R 3 = 4-hydroxyphenyl UV: 601 nm (13 x 10 3 , dioxane) c) R 3 = 4-(2,3-dihydroxypropylthio)phenyl UV: 604 nm (8.4 x 10 3 , dioxane) d) R 3 = 4-(trimethylammonium)phenyl UV: 604 nm (5.0 x 10 3 , dioxane) nm: 540 - 590 nm; K/S: 15% (11.1 mmol) 2. R 4 = phenyl a) R 3 = 4-(dihydroxypropylthio)phenyl UV: 530 nm (1.0 x 10 3 , dioxane) b) R 3 = 4-(trimethylammonium)phenyl UV: 530 nm (8.3 x 10 3 , dioxane) c) R 4 = 4-acetamidophenyl UV: 545 (water) nm: 620 - 670 nm; K/S: 18% (11.1 mmol) 3. R 4 = 4-nitro-1-naphthyl a) R 3 = 4-acetamidophenyl UV: 612 nm (3.4 x 10 3 , dioxane) nm: 675 - 725 nm; K/S: 5% (14.1 mmol) b) R 3 = (4-trimethylammonium)phenyl UV: 634 nm (4.6 x 10 3 , dioxane) nm: 640 - 690 nm; K/S: 15% (11.1 mmol) 4. R 4 = 8-quinolyl a) R 3 = 4-acetamidophenyl UV: 628 nm (12.1 x 10 3 , 420) nm: 650 - 700 nm; K/S: 8% (8.1 mmol) IR(KBr): 3436, 1684, 1600, 1531, 1458, 1423, 1385, 1318, 1258, 959, 939, 834 cm -1 NMR (DMSO-d 6 ): δ 10.8 (s, 1H, NH), 8.67 - 8.83 (m, 3H), 8.34 (d, 24, J = 8.8 Hz, pH), 8.11 (t, 1H, J = 7.8 Hz), 8.04 (d, 2H, J = 8.8 Hz, pH), 7.87 (d, 1H, J = 2.5 Hz), 7.74 (dd, 1H, J = 8.4 Hz, 4.3 Hz), 7.46 (1H, J = 9.1 Hz), 7.12 (dd, 1H, J = 9.0 Hz, 2.6 Hz), 4,10 (q, 2H, J = 7.0 Hz, -CH 2 -Me), 2.26 (s, 34, CH 3 -CO), 1.35 (t, 3H, J = 7.0 Hz, Me). Mass Spectrum (FAB) m/e: 508 (M + -1, 18.7), 353 (24.1), 128 (100). 5. R 4 = 3,4-methylenedioxyphenyl a) R 3 = 4-methylenedioxyphenyl UV: 504 nm (ε 5.9 x 10 3 , dioxane) nm: 670 - 720 nm; K/S: 14% (11.1 mmol) IR(KBr): 3465, 1602, 1540, 1501, 1462, 1384, 1254, 1179, 1117, 1037, 932, 813 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 8.01 (d, 1H, J = 9.08), 7.93 (dd, 1H, J = 8.14 Hz, 1.79 Hz), 7.92 (d, 1H, J = 2.46 Hz), 7.53 - 7.61 (m, 2H), 7.26 - 7.36 (m, 3H), 6.34 (s, 2H, -OCH 2 O-), 6.26 (s, 2H, -OCH 2 O-), 4.17 (q, 2H), J = 6.99 Hz, -CH 2 CH 3 ), 1.40 (t, 3H, J = 6.94 Hz, -CH 2 CH 3 ). Mass Spectrum (FAB) m/e: 93.1 (100), 185.2 (27.8), 340 (16.9), 488 (3.0, M + -1) 489 (1.4, M + ), 490 (7.6, M + +1). 6. R 4 = 1-naphthyl a) R 3 = 4-acetamidophenyl UV: 532 (13 x 10 3 , dioxane) 632 (water) nm: 660 - 710; K/S: 3% (11.1 mmol) 1 HNMR (300 MHz, 80:20 CDCl 3 /DMSO-d 6 ): 10.35 (s, 1H), 8.40 (d, J = 8, 1H), 8.40 (d, J = 8, 1H), 8.28 (m, 2H), 8.17 (d, J = 8, 1H), 8.03 (d, J = 8.5, 2H), 7.95 (d, J = 8, 1H), 7.8 - 7.6 (m, 3H), 7.58 (d, J = 7.5, 1H) 7.45 (d, J = 9, 1H), 7.08 (dd, J = 2.59, 9, 1H), 4.13 (q, J = 7, 2H), 2.2 (s, 3H), 1.43 (t, J = 7, 3H). b) R 3 = 4-nitrophenyl UV : 600 (8.4 x 10 3 , dioxane) 620 (water) nm: 645 - 695 nm; K/S: 7% (9.4 mmol) 7. R 4 = 2-methyl-4-carboxyphenyl a) R 3 = 4-acetamidophenyl UV: 600-630 (11 x 10 3 , dioxane) nm: 615 - 665; K/S: 5% (11.1 mmol) 8. R 4 = 4-carboxyphenyl a) R 3 = 4-acetamidophenyl UV : 592 (9.5 x 10 3 , dioxane) 610 (water) nm: 615 - 665 nm; K/S: 2% (8.1 mmol) 9. R 4 = 4-methoxyphenyl a) R 3 = phenyl UV: 488 (14.5 x 10 3 , dioxane) 10. R 4 = 4-chloro-1-naphthyl a) R 3 = 4-acetamidophenyl UV: 600 (water) nm: 610 - 660; K/S: 3% (8.3 mmol) 11. R 4 = 4-cyano-1-naphthyl a) R 3 = 4-acetamidophenyl UV: 632 (9.1 x 10 3 , dioxane) 606 (water) nm: 625 - 675; K/S: 11% (8.3 mmol) C. (5,6,7,8)-Tetrahydronaphtho[2,3-d]thiazol-2-yl 1. R 4 = 4-nitronaphthyl a) R 3 = 4-acetamidophenyl UV: 624 nm (5.1 x 10 3 , dioxane) nm: 635 - 685 nm; K/S: 14% (14 mmol) IR(KBr): 1635, 1540, 1500, 1470, 1440, 1390, 1260, 1120, 1040, 920 cm -1 NMR (300 MHz, CDCl 3 ): δ 8.99 - 9.12 (m, 1H, naphthalene), 8.60 (d, 1H, J = 8.6 Hz), 8.25 (d, 1H, J = 4.9 Hz, naphthalene) 7.72 - 7.94 (m, 6H, naphthalene and benzothiazole), 7.55 (d, 2H, J = 8.0 Hz, PhNHAC), 7.12 - 7.23 (m, 2H, naphthalene), 7.01 (d, 2H, J = 8.4 Hz, PhNHAC), 2.70 - 2.90 (broad S, 4H, -CH 2 -), 1.80 - 2.95 (m, 7H, CH 3 CO- and -CH 2 -). Mass Spectrum (E2) m/e: 121 (22.4), 149 (100, 165 (34.7), 165 (34.7), 164 (20.7), 205 (26.2), 349 (M + -1-C 10 H 2 NO 2 , 12.1) Mass Spectrum (FAB) m/e: 350 (M + - C 10 H 2 NO 2 , 100). b) R 3 = 4-methoxyphenyl UV: 590 nm (9.0 x 10 3 , dioxane) nm: 640 - 690 nm; K/S: 7.1% (14 mmol) c) R 3 = 3,4-methylenedioxyphenyl UV: 626 nm (1.2 x 10 3 , dioxane) nm: 660 - 710 nm; K/S: 5% (9.1 mmol) d) R 3 = 4-fluorophenyl UV: 612 nm (3.4 x 10 3 , dioxane) nm: 625 - 675 nm; K/S: 8.5% (14 mmol) 2. R 4 = 4-nitrophenyl a) R 3 = 4-acetamidophenyl UV: 596 nm (2.0 x 10 3 , dioxane) nm: 640 - 690 nm; K/S: 16% (14 mmol) b) R 3 = 4-methoxyphenyl UV: 590 nm (9.0 x 10 3 , dioxane) nm: 645 - 695 nm; K/S: 8% (14 mmol) c) R 3 = 3,4-methylenedioxyphenyl UV: 598 nm (4.4 x 10 3 , dioxane) nm: 640 - 690 nm; K/S: 20% (14 mmol) d) R 3 = 4-hydroxyphenyl UV: 598 nm (6.0 x 10 3 , dioxane) 3. R 4 = 1-naphthyl a) 4-(2-(2-(2-ethoxy)ethoxy)ethoxy)phenyl UV: 436 nm (12 x 10 3 , H 2 O) D. 6-methylbenzothiazol-2-yl 1. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 3,4-methylenedioxy UV: 492 nm (4.7 x 10 3 , dioxane) nm: 655 - 705 nm; K/S: 20% (11.1 mmol) IR(KBr): 1604, 1540, 1496, 1464, 1432, 1370, 1317, 1254, 1123, 1037, 993, 822, 770, 735 cm -1 NMR (300 MHz, DMSO-d6): δ 8.17 (5, 1H, PhH), 8.01 (d, 1H, J = 8.48 Hz). 7.95 (dd, 1H, J = 8.13 Hz, 1.76 Hz), 7.85 (d, 1H, J = 1.72 Hz), 7.57 (dd, 1H, J = 8.19 Hz), 1.53 Hz), 7.49 (s, 2H), 7.32 (d, 1H, J = 8.16 Hz), 6.30 (s, 24, -OCH 2 -O), 3.85 (s, 3H, -OMe), 3.79 (s, 6H, OMe), 2.54 (s, 3H, -CH 3 ) Mass Spectrum (FAB) m/e: 93.2 (100, 185 (62.8), 310 (25.9), 325 (4.1), 504 (10.9, M + -1) b) R 3 = 4-acetamidophenyl UV: 492 nm (3.6 x 10 3 , dioxane) nm: 650 - 700 nm; K/S: 10% (11.1 mmol) c) R 3 = 3,4,5-trimethoxyphenyl UV: 492 nm (4.9 x 10 3 , dioxane) nm: 655 - 705 nm; K/S: 8% (11.1 mmol) 2. R 4 = 4-methoxyphenyl a) R 3 = 4-acetamidophenyl UV: 488 nnm (3.9 x 10 3 , dioxane) nm: 650 - 700 nm; K/S: 8% (17.7 mmol) IR(KBr): 1690, 1599, 1531, 1501, 1459, 1422, 1370, 1317, 1265, 1181, 1164, 1021, 977, 837, 747, 695 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 10.45, (s, 1H, -NHAc), 8.30 (d, 2H, J = 8.75 Hz, Ph-H), 8.17 (s, 1H, PhH), 7.90 - 8.11 (m, 5H), 7.26 - 7.40 (m, 1H), 3.84 (s, 3H, -OMe), 2.50 (s, 3H, -Me), 2.14 (s, 3H, CH 3 ) Mass Spectrum (FAB) m/e: 93, 100, 149 (8.1), 163 (19.9), 185 (91.1), 323 (21.6) 457 (7.1, M + -1) E. 6-carboxybenzothiazol-2-yl 1. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 408 nm (4.9 x 10 3 , dioxane) nm: 665 - 735 nm; K/S: 10% (11.1 mmol) IR(KBr): 3405, 3200-2500 (broad), 1700, 1602, 1500, 1451, 1259, 1122, 1034, 990, 815, 771 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 8.57 (s, 1H, PhH), s, 7.95 dd, 1H, J = 8.09, 1.68 Hz), 7.85 (d, 1H, J = 1.70 Hz), 7.17 - 7.43 (m, 4H), 7.02 (d, 1H, J = 8.0 Hz), 6.11 (s, 2H), 3.90 (s, 3H, OMe), 3.74 (s, 6H, OMe) Mass Spectrum (FAB) m/e: 93.1 (57.1), 167.2 (100), 185.2 (50.8), 340.1 (48.1, M + - C 8 H 5 NSO 2 ) 2. R 4 = 4-nitrophenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 568 nm (0.6 x 10 3 , dioxane) F. 5,6,7-trimethoxybenzothiazol-2-yl 1. R 4 = 4-carboxyphenyl a) R 3 = 4-methoxyphenyl UV: 594 (dioxane) 516 (water) nm: 630 - 680; K/S: 19% (8.3 mmol) 1 H NMR (DMSOd 6 ): 8.33 (m, 3H), 8.20 (d, J = 7, 2H), 7.35 (m, 3H), 4.10 (s, 3H), 3.95 (s, 3H), 3.85 (s, 6H) G. naphtho[1,2-d]thiazol-2-yl 1. R 4 = 2-methoxy-4-carboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 568 nm (4.5 x 10 3 , 420) nm: 650 - 700 nm; K/S: 12% (11.1 mmol) IR(KBr): 1713, 1605, 1493, 1466, 1433, 1315, 1259, 1209, 1037, 741 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 8.42 (d, 1H, J = 9.0 Hz, -Ph), 8.17 - 8.17 (m, 3H), 8.00 - 7.96 (m, 2H), 7.87 - 7.66 (m, 5H), 7.33 (d, 1H, J = 8.1 Hz), 6.30 (s, 24, OCH 2 O), 3.82 (s, 3H, -OMe) Mass Spectrum (FAB) m/e: 93 (100, 185 (47), 346 (18), 524 (23, M + -1) b) R 3 = 4-(2-(2-(hydroxyethoxy)-ethoxy)-ethoxy)-phenyl UV: 566 nm (3.2 x 10 3 . H 2 O) nm: 650 - 700 nm; K/S: 6% (11.1 mmol) 2. R 4 = 4-acetamidophenyl a) R 3 = 4-cyanophenyl UV: 524 nm (9 x 10 3 , H 2 O) nm: 650 - 700 nm; K/S: 16% (8.1 mmol) IR(KBr): 3437, 3047, 1694, 1594, 1537, 1503, 1462, 1414, 1384, 1265, 1172, 985, 848 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 10.85 (s, 1H, NHAc), 8.59 (d, 24, J - 8.4 Hz), 8.41 (d, 1H, J = 9.0 Hz), 8.30 - 8.64 (m, 94), 7.79 - 7.76 (m, 2H), 2.18 (s, 3H, -NHCOCH 3 ) Mass Spectrum (FAB) m/e: 91 (100), 109 (28, 181 (56), 217 (45), 232 (13), 327 (8), 488 (10, M + -1), 487 (4, M + ) b) R 3 = 4-carbomethoxyphenyl UV: 558 nm (12 x 10 3 , 420) nm: 660 - 710 nm; K/S: 25% (8.3 mmol) c) R 3 = methylenedioxyphenyl nm: 685 - 735; K/S: 7% (8.1 mmol) 3. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 4-(3-trimethylammoniopropoxy)phenyl UV: 520 (11 x 10 3 , water) nm: 670 - 720; K/S: 13% (8.3 mmol) b) R 3 = 3,4-methylenedioxyphenyl UV: 565 nm (10.3 x 10 3 , H 2 O) nm: 675 - 725 nm; K/S: 20% (8.3 mmol) c) R 3 = 4-(2-(2-(2-hydroxy)ethoxy)ethoxy) phenyl UV: 592 nm (11.4 x 10 3 , H 2 0) 4. R 4 = 3,4-methylenedioxy a) R 3 = 3,4-methylenedioxy UV: 544 nm (10.7 x 10 3 , H 2 O) nm: 685 - 735 nm; K/S: 26% (8.3 mmol) 5. R 4 = 4-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 546 nm (7.9 x 10 3 , H 2 O) nm: 675 - 725 nm; K/S: 14% (8.3 mmol) 6. R 4 = 1-anthryl a) R 3 = 3,4-methylenedioxyphenyl UV: 644 nm (4.1 x 10 3 , H 2 O) nm: 750 - 800 nm; K/S: 32% (8.3 mmol) 7. R 4 = 8-quinolyl a) R 3 = 3,4-methylenedioxyphenyl UV: 640 nm (3.9 x 10 3 , H 2 O) nm: 650 - 700 nm; K/S: 5% (8.3 mmol) b) R 3 = 3,4,5-trimethoxyphenyl UV: 628 nm (12.1 x 10 3 , H 2 O) nm: 650 - 700 nm; K/S: 4% (8.3 mmol) 8. R 4 = 2,5-dimethoxy-4-nitrophenyl a) R 3 = 3,4-methylenedioxy UV: 654 nm (7.0 x 10 3 , H 2 O) nm: 655 - 705 nm; K/S: 17% (8.3 mmol) 9. R 4 = 2-methoxy-4-carbomethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 630 nm (6.3 x 10 3 , H 2 O) nm: 650 - 700 nm; K/S: 12% (8.3 mmol) 10. R 4 = 2-methoxy-5-acetamidophenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 584 nm (7.0 x 10 3 , H 2 O) nm: 660 - 710 nm; K/S: 11% (8.3 mmol) 11. R 4 = 2-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 618 nm (10.4 x 10 3 , H 2 O) nm: 650 - 700 nm; K/S: 10% (8.3 mmol) 12. R 4 = 2,4-dimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 675 - 725 nm; K/S: 33% (8.3 mmol) 13. R 4 = 2-methoxy-4-nitrophenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 650 nm (14 x 10 3 , H 2 O) nm: 650 - 700 nm; K/S: 14% (8.3 mmol) 14. R 4 = 2,5-dimethoxyphenyl a) R 3 = 3,4-methylenedioxy nm: 665 - 715 nm; K/S: 36% (8.3 mmol) 15. R 4 = 4-nitro-naphthyl a) R 3 = 4-fluorophenyl UV: 622 (15.6 x 10 3 , dioxane) 632 (water) nm: 625 - 725 nm; K/S: 12% (13.8 mmol) b) R 3 = 3,4-methylenedioxyphenyl UV : (13.1 x 10 3 , dioxane) nm: 605 - 655 nm; K/S: 4% (15 mmol) c) R 3 = phenyl UV: 618 (12.6 x 10 3 , dioxane) nm: 625 - 675; K/S = 8% (14 mmol) d) R 4 = 4-(2-(2-(2-hydroxyethoxy)ethoxy) phenyl UV: 628 nm (6.7 x 10 3 , H 2 O) e) R 4 = 4-(1'-(hydroxymethyl)-2',3'-dihydroxypropylthio)phenyl UV : 624 nm (5.18 x 10 3 , dioxane) nm: 635 - 685 nm; K/S: 15% (11.1 mmol) 16. R 4 = 4-nitrophenyl a) R 3 = 4-(2',3'-dihydroxypropylthio)phenyl uv: 604 nm (4.9 x 10 3 , dioxane) nm: 635 - 685 nm; K/S: 4% (11.1 mmol) b) R 3 = 4-(trimethylammonium)phenyl UV: 572 nm (14.7 x 10 3 , dioxane) 17. R 4 = 4-carboxyphenyl a) R 3 = 4-acetamidophenyl NMR(DMSO-d 6 ): δ 10.46 (s, 1H), 8.5 - 8.22 (m, lH), 8.17 (d, J = 8, 1H), 8.02 (d, J = 8.8, 2H), 7.85 (dd, J = 7.6, 0.9, 1H), 7.78 - 7.65 (m, 3H), 2.08 (s, 3H) UV: 598 (dioxane) 614 (10.5 x 10 3 , water) nm: 650 - 700 nm; K/S: 7.5% (13.8 mmol) b) R 3 = 4-(3-trimethylammoniopropoxy)phenyl UV: 632 (14.7 x 10 3 , H 2 O) nm: 640 - 690; K/S: 11% (8.6 mmol) c) R 3 = 4-(2',3'-dihydroxypropylthio)phenyl UV: 654 nm (1.0 x 10 3 , dioxane) nm: 645 - 695 nm; K/S: 11% (11.1 mmol) d) R 3 = 4-(2-(2-(2-hydroxyethoxy)ethoxy) ethoxy)phenyl nm: 625 - 675 nm; K/S: 6.5% (8.3 mmol) e) R 3 = carbmethoxyphenyl nm: 605 - 655; K/S: 11.5% (8.3 mmol) 18. R 4 = 2-methyl-4-carboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 620 - 640 (dioxane) 614 (8.4 x 10 3 , water) nm: 625 - 675 nm; K/S: 3% (14 mmol) 19. R 4 = 2-methyl-4-nitrophenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 644 (11.3 x 10 3 , dioxane) 625 (9.2 x 10 3 , water) nm: 665 - 715 nm; K/S: 6% (14 mmol) 20. R 4 = phenyl a) R 3 = 4-acetamidophenyl UV: 516 (7.1 x 10 3 , dioxane) 526 (7.2 x 10 3 , water) 21. R 4 = 4-methoxyphenyl a) R 3 = 4-acetamidophenyl UV: 502 (9.5 x 10 3 , dioxane) 500 (water) 22. R 4 = 4-phenylazophenyl a) R 3 = 4-methoxyphenyl UV: 614 (dioxane) 618 (water nm: 625 - 675 nm; K/S: 11% (8.3 mmol) 23. R 4 = 4-methylthiophenyl a) R 3 = 4-acetamidophenyl UV: 524 (dioxane) 526 (water) nm: 675 - 725 nm; K/S: 18% (8.3 mmol) 24. R 4 = 4-(3,4-dihydroxybutylamido)phenyl a) R 3 = 4-methoxyphenyl UV: 588 (9 x 10 3 , dioxane) 578 (12 x 10 3 , water) 25. R 4 = 4-carbmethoxyphenyl a) R 3 = 4-methoxyphenyl UV: 598 (9.4 x 10 3 , dioxane) 581 (9.1 x 10 3 , water) nm: 630 - 680 nm; K/S: 20% (8.3 mmol) 26. R 4 = 4-cyanophenyl a) R 3 = 3-methoxyphenyl UV: 602 (12.3 x 10 3 , dioxane) 608 (water) nm: 625 - 675 nm; K/S: 8% (8.3 mmol) 27. R 4 = 4-benzamido-5-methyl-2-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 690 - 740; K/S: 15% (10.3 mmol) 28. R 4 = 4-benzamido-2,5-dimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 715 - 765; K/S: 17% (10.3 mmol) H. 8-methoxynaphtho[1,2-d]thiazol-2-yl 1. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 4-methoxyphenyl UV : 466 (9.2 x 10 3 , dioxane) 530 (9 x 10 3 , water) nm: 670 - 720 nm; K/S: 12% (8.3 mmol) NMR (300 MHz, DMSO-d 6 ): δ 8.33 (m, 3H), 7.97 (m, 1H), 7.90 (s, lH), 7.75 (m, 2H), 7.55 (s, 2H), 7.35 (d, J = 9, 2H), 4.10 (s, 3H), 3.93 (s, 3H), 3.88 (s, 3H), 3.78 (s, 6H) I. 4-difluoromethyl-5-chlorothiazol-2-yl 1. R 4 = 4-methoxyphenyl a) R 3 = 4-(2-(2-(2-ethoxy)ethoxy)ethoxy) phenyl UV: 480 nm (12.8 x 10 3 , dioxane) nm: 645 - 695 nm; K/S: 6% (17.7 mmol) IR(KBr): 2942, 1612, 1504, 1464, 1451, 1261, 1178, 1126, 1061, 837, 658 cm -1 NMR (300 M Hz, DMSO-d 6 ): δ 8.24 (e, 2H, J = 8.8 Hz, Ph-OMe), 7.92 (d, 2H, J = 9.0 Hz, Ph-O-), 7.27 - 7.33 (m, 4H, PhH), 7.26 (d, 1H, J = 1.04 Hz, -CHF 2 ), 4.22 - 4.32 (m, 2H, -CH 2 O), 3.90 (s, 3H, -OMe), 3.20 - 3.68 (m, 10H, -OCH 2 CH 2 O-) Mass Spectrum (FAB): 107 (100, 135 (65.4), 568 (4.5, M + -H), 570 (1.9, M +1 b) R 3 = 4-(2-(2-(2-methoxy)ethoxy)ethoxy) phenyl UV: 468 nm (10.2 x 10 3 , dioxane) c) R 3 = 4-(2-(2-(2-hydroxyethoxy) ethoxy)ethoxy)phenyl UV: 466 nm (7.6 x 10 3 , dioxane) nm: 645 - 695 nm; K/S: 5% (11.1 mmol) d) R 3 = 3,4-methylenedioxyphenyl nm: 650 - 700 nm; K/S: 7% (11.1 mmol) 2. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 500 (5.66 x 10 3 , water) nm: 665 - 715 nm; K/S: 5% (17.7 mmol) NMR (300 M Hz, DMSO-d 6 ): δ 7.91 (dd, 1H), 7.82 (d, 1H), 7.35 (s, 3H), 7.31 (t, 1H, CHF 2 ), 7.30 (d, 1H), 6.28 (s, 2H), 3.79 (s, 9H) Mass Spectrum (FAB): 167 (71), 330 (34), 524 (m + , 100) b) R 3 = 4-(2-(2-(2-hydroxy)ethoxy)ethoxy) phenyl UV: 492 nm (3.62 x 10 3 , dioxane) nm: 660 - 710 nm; K/S: 14% (11.1 mmol) IR(KBr): 3436, 1611, 1456, 1384, 1309, 1241, 1180, 1126, 1053, 992 cm -1 NMR (DMSO-d 6 , 300 M Hz): δ 8.26 (d, 2H, J = 8.89, Ph), 7.40 (s, 2H, PhH), 7.33 (d, 2H, J = 8.90 Hz, Ph), 7.30 (t, 1H, J = 52.3, (HF 2 ), 4.29 (dd, 2H, J = 5.66, 4.0 Hz, CH 2 -OH), 3.91 (5.64, -OMe), 3.85 (s, 3H, OME), 3.64 - 3.41 (m, 10 H, -CH 2 O) Mass Spectrum (FAB) m/e: 628 (13.5), 630 (6.0) 3. R 4 = 1-naphthyl a) R 3 = 4-(2-(2-(2-hydroxyethoxy)-ethoxy) phenyl UV: 440 nm (6.32 x 10 3 , dioxane) 4. R 4 = 1-phenyl b) R 3 = 4-(2-(2-(2-ethoxy)ethoxy)ethoxy) phenyl UV: 474 nm (13.1 x 10 3 , dioxane) nm: 625 - 675 nm; K/S: 25% (11.1 mmol) IR(KBr): 3397, 3004, 2950, 1612, 1452, 1260, 1180, 1127, 1061, 842 cm -1 NMR (300 M Hz, DMSO-d 6 ): δ 8.25 (d, 2H, J = 8.73 Hz, Ph-O-), 8.00 (d, 2H, J = 7.87 Hz, PhH), 7.88 - 7.93 (m, 1H, PhH), 7.80 (d, 2H, J = 7.90 Hz, PhH), 7.32 (d, 2H, J = 8.81 Hz, PhO-), 7.22 (d, 1H, J = 104.3 Hz, -CHF 2 ), 4.26 - 4.30 (m, 2H, CHCH 2 -), 3,79 - 3,82 (m, 2H, CH 2 CH 2 OH), 3.31 - 3.62 (m, 8H, -OCH 2 CH 2 O-) Mass Spectrum (FAB): 301 (14.2, 434 (23.9), 538 (35.7, M + -1), 539 (11.5, M + ), 540 (15.8, M + +1). 5. R 4 = 4-(2-(2-(2-hydroxyethyl)ethoxy) ethoxy)phenyl a) R 3 = 3,4,5-trimethoxyphenyl UV: 516 nm (9 x 10 3 , H 2 O) b) R 3 = 3,4-methylenedioxyphenyl UV: 512 nm (13 x 10 3 , H 2 O) 6. R 4 = 4-(2-hydroxyethoxy)phenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 492 nm (12 x 10 3 , H 2 O) 7. R 4 = 4-(2-(2-hydroxyethyl)ethoxy)phenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 514 nm (11.3 x 10 3 , H 2 O) 8. R 4 = 3,4-dimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 410 nm (5.34 x 10 3 , water) nm: 675 - 725 nm; K/S: 15% (11.1 mmol) 9. R 4 = 2-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 610-640 (7.5 x 10 3 ) nm: 635-685 nm; K/S: 7% (8.2 mmol) J. 4-trifluoromethyl-5-chlorothiazol-2-yl 1. R 4 = 4-carboxyphenyl a) R 3 = 4-(2-(2-(2-(ethoxy)ethoxy)ethoxy) phenyl UV: 560 nm (5.9 x 10 3 , dioxane) nm: 630 - 680 nm; K/S: 18% (11.1 mmol) IR(KBr): 3461, 1612, 1455, 1385, 1263, 1178, 993 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 8.20 - 8.32 (m, 5H), 8.08 - 8.14 (m, 1H, Ph), 7.32 (3, 1H, J = 8.92, -PhH), 7.18 (d, lH, J - 8.89, -PhH), 4.16 - 4.32 (m, 2H, CH CH 3 ), 3.39 - 3.82 (m, 8H, ethylene glycol), 1.09 (t, 3H, J = 6.99, -CH 3 ) Mass Spectrum (FAB): 320 (15.6), 436 (18.80, 584 (13.9, M + -H) b) R 3 = 4-(2-(2-(2-hydroxyethoxy)ethoxy) ethoxy)phenyl UV: 492 nm (5.4 x 10 3 , dioxane) nm: 640 - 690 nm; K/S: 14% (11.1 mmol) IR(KBr): 3013, 1610, 1518, 1450, 1203, 1050, 928, 717, 664 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 8.24 - 8.33 (m, 4H, PhH), 8.12 (d, 2H, J = 8.8 Hz, PhCr2H), 7.32 (d, 2H, J = 8.9 Hz, PhCO 2 H), 4.27 - 4.29 (m, 2H, O CH CH 2 -), 3.79 - 3.82 (m, 2H, -O CH 2 CH 2 O), 3.40 - 3.63 (m, 8H, -O-CH 2 CH 2 -O-) Mass Spectrum (FAB): 121 (100), 146 (29.5), 149 (26.5), 600 (9.6, M + -H), 602 (4.2, M + +1) c) R 3 = methoxyphenyl nm: 630 - 680 nm; K/S: 10% (11.1 mmol) 2. Phenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 520 (water, 4.08 x 10 3 ) nm: 635 - 685 nm; K/S: 21% (11.1 mmol) b) R 3 = 4-(2-2-2-(ethoxy)ethoxy) ethoxy)phenyl UV: 580 nm (7.7 x 10 3 , dioxane) nm: 625 - 675 nm; K/S: 0.60 (11.1 mmol) IR(KBr): 3582, 3462, 1636, 1614, 1486, 1456, 1384, 1179, 1137, 1051, 993, 986, 848, 765, 745, 691 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 8.26 (3, 1H, J = 8.54 Hz, Ph-O-), 8.15 (d, 1H, J = 8.48, PhO-), 8.00 - 7.77 (m, 5H, -Ph), 7.32 (d, 1H, J - 8.73), 7.12 (d, 1H, J = 8.67, Ph-), 3.34 - 4.28 (m, 10H, ethylene glycol), 1.09 (t, 3H, J = 6.92, -CH 3 ) Mass spectrum (FAB): 540 (M-H) + c) R 3 = 4-(trimethylammonium)phenyl UV: 496 nm (11.4 x 10 3 , dioxane) 3. 4-nitrophenyl a) R 3 = 4-(trimethylammonium)phenyl UV: 590 nm (4.7 x 10 3 , dioxane) nm: 525 - 575 nm; K/S: 14% (11.1 mmol) IR(KBr): 3431, 3065, 1751, 1725, 1486, 1469, 1308, 1209, 1182, 1147, 990 cm -1 NMR (300 MHz, DMSO-d 6 ): δ 8.85 = 8.95 ) (m, 1H, Ph-), 8.59 (d, 1H, J = 8.8 Hz, PhNO 2 ), 8.47 - 8.55 (m, 1H), 8.44 (d, 1H, J = 8.9 Hz, PhNO 2 ), 8.22 - 8.35 (m, 2H, PhH), 7.96 - 8.05 (m, 1H, PhH), 7.80 - 7.87 (m, 1H) 3.75 (s, 9H) Mass Spectrum (FAB): 161 (100, NCPh + NMR 3 ), 185 (22.6), 511 (12.1, M + ) 4. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 500 (water, 6.68 x 10 3 ) nm: 675 - 725 nm; K/S: 5% (11.1 mmol) b) R 3 = 4-acetamidophenyl UV: 490 (water, 7.24 x 10 3 ) 5. R 4 =4-(3-dimethylammoniopropylamidophenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 400 (water) 6. R 4 = 2-methoxy-5-trimethylammoniophenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 540 (water) 7. R 4 = 4-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 500 (water) 8. R 4 = 4-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 500 (water) 9. R 4 = 3,5-dicarboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 635 - 685 nm; K/S: 16% (12.2 mmol) 10. R 4 = naphthyl a) R 3 = 3,4-methylenedioxyphenyl nm: 685 - 735 nm: K/S: 5% (9.4 mmol) K. 4-carboxyethyl-5-chlorothiazol-2-yl 1. R 4 = 4-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 490 (4.9 x 10 3 , water) L. 4-carbmethoxy-5-chlorothiazol-2-yl 1. R 4 = phenyl a) R 3 = 3-thienyl UV: 469 (7.66 x 10 3 , water) b) R 3 = 3,4-methylenedioxyphenyl nm: 620 - 670 nm; K/S: 21% (11.1 mmol) M. 4-carboxyisopropyl-5-chlorothiazol-2-yl 1. R 4 = phenyl a) R 3 = 3-thienyl UV: 468 (7.8 x 10 3 , water) 2. R 4 = phenyl a) R 3 = 2-thienyl UV: 490 (7.6 x 10 3 , water) 3. R 4 = phenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 625 - 675 nm; K/S: 6% (9.4 mmol) N. 4-phenyl-5-carbmethoxythiazol-2-yl 1. R 4 = 4-methoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 500 (9.82 x 10 3 , water) 2. R 4 = 3,4-dimethoxyphenyl a) R 3 = 3-thienyl UV: 510 (9.76 x 10 3 , water) 3. R 4 = carboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 500 (5.10 x 10 3 , water) nm: 660 - 710 nm; K/S: 13% (22 mmol) 4. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 4-methoxyphenyl UV: 500 (9.44 x 10 3 , water) nm: 670 - 720 nm; K/S: 20% (22 mmol) O. 4-methyl-5-carbmethoxythiazol-2-yl 1. R 4 = 4-carboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 510 (7.82 x 10 3 , water) nm: 670 - 720 nm; K/S: 14% (22 mmol) 2. R 4 = phenyl a) R 3 = 4-methoxyphenyl nm: 650 - 700 nm; K/S: 14% (9.4 mmol) b) R 3 = 3-thienyl UV: 480 (8.76 x 10 3 , water) c) R 3 = 3,4-methylenedioxyphenyl UV: 500 (6.18 x 10 3 , water) P. 4-carbmethoxy-5-ethylthiazol-2-yl 1. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 3-thienyl UV: 500 (7.38 x 10 3 , water) nm: 640 - 690 nm; K/S: 14% (8.3 mmol) 2. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 4-methoxyphenyl nm: 625 - 675 nm; K/S: 14% (8.3 mmol) b) R 3 = 3,4-methylenedioxyphenyl UV: 490 (7.96 x 10 3 , water) nm: 650 - 700 nm; K/S: 8% (8.3 mmol) 3. R 4 = 3,4-dimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 490 (5.26 x 10 3 , water) nm: 665 - 715; K/S: 5% (8.3 mmol) b) R 3 = 3-thienyl nm: 635 - 685 nm; K/S: 11% (8.3 mmol) Q. 4,5-bis(4-methoxyphenyl)thiazol-2-yl 1. R 4 = 4-carboxyphenyl a) R 3 = 2-thienyl UV: 623 (11.6 x 10 3 , water) nm: 660 - 710 nm; K/S: 9% (14 mmol) R. 4,5-diphenylthiazol-2-yl 1. R 4 = carboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 495 (12.6 x 10 3 , water) nm: 630 - 680 nm; K/S: 16% (15 mmol) b) R 3 = 4-methoxyphenyl UV: 613 (8.66 x 10 3 , water) nm: 630 - 680 nm; K/S: 17% (8 mmol) c) R 3 = 2-thienyl UV: 602 (8.22 x 10 3 , water) nm: 635 - 685 nm; K/S: 18% (9 mmol) d) R 3 = 3-thienyl UV: 599 (11.8 x 10 3 , water) e) R 3 = 4-fluorophenyl UV: 592 (13.6 x 10 3 , water) nm: 625 - 675 nm; K/S: 11% (33 mmol) f) R 3 = 4-hydroxyphenyl UV: 620 (11.2 x 10 3 , water) 2. R 4 = phenyl a) R 3 = 2-thienyl UV: 571 (9 x 10 3 , water) nm: 640 - 690; K/S: 4% (14 mmol) 3. R 4 = 3-pyridyl a) R 3 = 4-methoxyphenyl UV: 589 (11.8 x 10 3 , water) nm: 620 - 670 nm; K/S: 15% (15 mmol) 4. R 4 = 8-quinolyl a) R 3 = phenyl UV: 607 (14.4 x 10 3 , water) nm: 620 - 680 nm; K/S: 21% (15 mmol) 5. R 4 = 4-nitronaphthyl a) R 3 = 3-thienyl UV: 696 (10.4 x 10 3 , water) nm: 620 - 670 nm; K/S: 12% (14 mmol) 6. R 4 = 4-sulfophenyl a) R 3 = 2-thienyl UV: 610 (6.0 x 10 3 , water) 7. R 4 = 3,5-dicarboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl UV: 597 (5.2 x 10 3 , water) nm: 620 - 670 nm; K/S: 7% (14 mmol) S. 4-phenylthiazo-2-yl 1. R 4 = 3,4,5-trimethoxyphenyl a) 3,4-methylenedioxyphenyl nm: 645 - 695; K/S: 28% (8.3 mmol) 2. R 4 = 4-carboxyphenyl a) 3,4-methylenedioxyphenyl nm: 620 - 670; K/S: 11% (8.3 mmol) T. 4-(p-fluorophenyl)thiazol-2-yl 1. R 4 = 3,4,5-trimethoxyphenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 645 - 695; K/S: 19% (8.3 mmol) 2. R 4 = 4-carboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 625 - 675; K/S: 12% (8.3 mmol) U. 4-phenyl-5-methylthiazol-2-yl 1. 3,4,5-trimethoxyphenyl a) R 3 = 3,4-methylenedioxypnenyl nm: 625 - 675; K/S: 22% (8.3 mmol) 2. 4-carboxyphenyl a) 2-thienyl nm: 600 - 650; K/S: 10% (10.3 mmol) b) 4-methylphenyl nm: 580 - 630; K/S: 5% (10.3 mmol) V. 4-naphthyl-5-phenylthiazol-2-yl 1. R 4 = 4-carboxyphenyl a) R 3 = 3,4-methylenedioxyphenyl nm: 620 - 670; K/S: 10% (8.3 mmol) b) R 3 = 2-thienyl nm: 600 - 650; K/S: 4% (10.3 mmol) W. 4-styryl-5-phenylthiazol-2-yl 1. 3,4,5-trimethoxyphenyl a) 3,4-methylenedioxyphenyl nm: 670 - 720; K/S: 21% (10.3 mmol) The present invention has been particularly described and exemplified above. Clearly, other variations and modifications of the invention can be made without departing from the scope hereof.

Claims: A method for determining an analyte in a liquid sample wherein the sample is contacted with a test device comprising a solid carrier matrix incorporated with a test composition comprising (a) reagents which react with said analyte to produce a reducing substance and (b) an indicator compound which is reduced by said reducing substance to produce a chromogenic dye product, and where the chromogenic dye product is measured by irradiating the carrier matrix with a light source having a central wavelength above about 600 nm which wavelength will vary due to manufacturing variances and temperature dependence of the light source and sensing the reflectance of the carrier matrix by measuring reflected light with a detector element, characterized in that it comprises employing as said indicator compound a thiazolyl tetrazolium salt of the formula: wherein (a) R 1 and R 2 together form a benzo ring, said benzo ring being substituted or unsubstituted, or (b) R 1 is carboxyl, carbalkoxy, carbaryloxy, carbomyl or cyano, and R 2 is alkyl or chloro, or (c) R 1 is alkyl or aryl, and R 2 is carboxyl, carbalkoxy, carbaryloxy, carbamoyl or cyano, or (d) R 1 is di- or trifluoroalkyl wherein the fluoro substituents are on the carbon adjacent to the thiazolyl residue in the formula, and R 2 is chloro, or (e) one or both of R 1 and R 2 are substituted or unsubstituted phenyl, and if only one is substituted or unsubstituted phenyl, the other is hydrogen or alkyl; and wherein R 3 is aryl or styryl, R 4 is aryl, and X e is a counteranion wherein the tetrazolium salts are further characterized in that when they are reduced by the reducing substance to their corresponding formazan the reflectance of the formazan varies by less than 17% over a wavelength range of 50 nanometers beginning at a wavelength of above about 600 nanometers. The method of claim 1 wherein R 1 and R 2 together form a benzo ring which is unsubstituted. The method of claim 1 wherein R 1 and R 2 together form a benzo ring to give a benzothiazolyl residue having the formula: wherein (i) R 5 and R 6 , or R 6 and R 7 , or R 7 and R 8 , together form a benzo or cyclohexyl ring that is unsubstituted or substituted with alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and wherein the others, same or different, are hydrogen, alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, provided that where R 6 and R 7 together form a benzo or cyclohexyl ring, R 5 is not hydrogen, or (ii) one or more of R 5 , R 6 , R 7 , and R 8 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and the others, if any, are hydrogen. The method of claim 1 wherein (a) R 1 is carboxyl, carbalkoxy, carbaryloxy, carbamoyl, or cyano, and R 2 is alkyl or chloro, or (b) R 1 is alkyl or aryl, and R 2 is carboxyl, carbalkoxy, carbaryloxy, carbamoyl, or cyano, or (c) R 1 is di- or trifluoroalkyl wherein the fluoro substituents are on the carbon adjacent to the thiazolyl residue in the formula, and R 2 is chloro. The method of claim 1 wherein one or both of R 1 and R 2 are unsubstituted phenyl or phenyl substituted with, independently, alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, carbaryloxy, carboxy, cyano, halo, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and if only one is substituted or unsubstituted phenyl, the other is hydrogen, alkyl, or chloro. The method of any one of claims 1 to 5 wherein R 3 is selected from: wherein Q is a bond or -CH=CH-, and wherein (i) Y 1 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, or hydrogen, Y 2 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, alkylthio, amino, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, Y 3 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carbaryloxy, carboxyl, cyano, halo, hydrogen, hydroxyl, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and Y 4 is alkoxy, halo, or hydrogen, or (ii) Y 2 and Y 3 together form methylenedioxy or imidazo and Y 1 and Y 4 are hydrogen, (b 1 ) 2, 3, or 4-pyridyl, (C 1 ) 2 or 3-thienyl, and (d 1 ) 2 or 3-furanyl; and wherein R 4 is selected from: wherein Y 5 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, hydrogen, nitro, or ureido, Y 6 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, Y 7 is alkoxy, aryloxy, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, hydrogen, hydroxyl, nitro, phenylazo, sulfo, sulfonamido sulfamoyl, or ureido, and Y 8 is alkoxy, aryloxy, alkyl, halo, hydrogen or nitro, wherein Y 9 is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, phenylazo, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, (c 2 ) 2, 4, 6, or 8-quinolyl, or 2-methylquinolyl, and (d 2 ) anthranyl. The method of any one of claim 1 to 5 wherein R 3 is selected from: wherein (i) Y 2 , Y 3 , and Y 4 are each C 1-4 alkoxy, (ii) Y 4 is hydrogen and Y 2 and Y 3 are both C 1-4 alkoxy or together form methylenedioxy, or (iii) Y 2 and Y 4 are both hydrogen and Y 3 is C 1-4 alkoxy, C 1-4 alkyl, C 1-4 alkylamido, C 1-4 alkylthio, carbamoyl, carb(C 1-4 )-alkoxy, carboxyl, cyano, halo, hydrogen, nitro, tri(C 1-4 )alkylammonio, or ureido, and (b 1 ) 2 or 3-thienyl; and wherein R 4 is selected from: wherein (i) Y 5 is hydrogen and each of Y 6 , Y 7 , and Y 8 is C 1-4 alkoxy, (ii) Y 5 and Y 8 are both hydrogen and Y 6 and Y 7 are both C 1-4 alkoxy or together form methylenedioxy, (iii) Y 5 , Y 6 and Y 8 are each hydrogen and Y 7 is C 1-4 alkoxy, C 1-4 alkylamido, C 1-4 -alkylthio, benzamido, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, nitro, phenylazo, sulfo, sulfonamido, sulfamoyl, or ureido, (iv) Y 5 is alkoxy or alkyl, Y 6 and Y 8 are both hydrogen, and Y 7 is C 1-4 alkoxy, C 1-4 alkylamido, C 1-4 alkylthio, benzamido, carbamoyl, carb(C 1-4 )alkoxy, carboxyl, cyano, hydrogen, nitro, phenylazo, or ureido, (v) Y 5 and Y 8 are C 1-4 alkoxy, or (vi) Y 5 and Y 8 are C 1-4 alkoxy and Y 7 is C 1-4 -alkylamido or benzamido; wherein Y 9 is C 1-4 alkoxy, C 1-4 alkyl, C 1-4 -alkylamido, C 1-4 alkylthio, benzamido, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, or ureido, and (c 2 ) 8-quinolyl. The method of any one of claims 1 to 5 wherein R 3 is selected from: 3,4,5-trimethyoxyphenyl, 3,4-dimethoxyphenyl, 3,4-methylenedioxyphenyl, 4-methoxyphenyl, 4-acetamidophenyl, and 4-methylthiophenyl, and wherein R 4 is selected from: 3,4,5-trimethyoxyphenyl, 3,4-dimethoxyphenyl, 2,4-dimethoxyphenyl, 3,4-methylenedioxyphenyl, 4-methoxyphenyl, 4-acetamidophenyl, 4-methylthiophenyl, 4-carboxyphenyl, 4-nitrophenyl, 2-methoxyphenyl, 2-methoxy-4-carboxyphenyl, 2,5-dimethoxyphenyl, 2,5-dimethoxy-4-benzamidophenyl 1-naphthyl, 4-nitro-1-naphthyl, 4-methoxy-1-naphthyl, and 8-quinolyl. The method of any one of claims 1 to 8 wherein the light which reaches the detector element consists of a narrow wavelength band whose central wavelength is susceptible to variations of over about + 5 nm, and preferably wherein said light source is an LED. The method of any one of claims 1 to 9 wherein said reducing substance is NADH.

Inventors: HATCH ROBERT P, KOECHER JUERGEN, LIN NAN-HORNG, RUETTEN SCOTT, WEHLING KLAUS, HATCH, ROBERT P., KOECHER, JUERGEN, LIN, NAN-HORNG, RUETTEN, SCOTT, WEHLING, KLAUS, Köcher, Jürgen
Applicants: BAYER AG, BAYER CORPORATION
Application year: 1991
Main classification: G01N33
IPC codes: G01N21, G01N33, C12Q1, G01N31, C07D417