HK1174910B - Water-soluble tetrazolium salts - Google Patents

Description

Water soluble tetrazolium salts

The application is a divisional application of Chinese patent application No. 200680004439.5(PCT/US2006/001307), namely 'water-soluble tetrazolium salt' on 2006, 1 month and 13 days.

Technical Field

The present invention relates to the analysis of biological samples for medical purposes.

Background

The analysis of biological samples generally involves a method in which the color is displayed in proportion to the amount of analyte in the sample. For example, enzymes may be used to oxidize the analyte of interest and indicate the extent of reaction by a color change of the indicator compound. Of particular interest herein is a family of tetrazolium salts useful as indicators. Such salts are reduced to formazan by reducing substances(formazan) dye. To measure an analyte, an enzyme (e.g., an NAD-dependent dehydrogenase) oxidizes the analyte to produce a reduced form (e.g., NADH) that reacts with a tetrazolium salt to produce a colored formazan. A mediator may be required to facilitate the reaction. Because the amount of NADH produced by the analyte reaction and the formazan producedIs proportional, so the amount of analyte can be measured indirectly by the color formed.

Tetrazolium salts have been used in a variety of applications. In particular, however, they have been used in the medical field for measuring analytes in various biological fluids such as blood, urine, plasma and serum. These indicators are typically used as reagent systems placed on the test strip, which when contacted with a fluid sample react with the analyte of interest and display a color that indicates the amount of analyte present. While in some cases the color change may be read visually with reference to a color chart, a more accurate reading may be made spectrophotometrically by an instrument designed for this purpose. Typically, light is directed onto the test strip and the reflected light is measured to determine the effect of the color change on the test strip.

Tetrazolium salts should produce formazanSaid A isLight is absorbed at wavelengths that avoid interference due to substances in the sample, such as hemoglobin in whole blood. Thus, a class of thiazolyl tetrazolium salts have been developed that produce formazans that absorb light having wavelengths greater than about 640nm (such as those generated by LEDs used as light sources). LEDs provide a narrow wavelength range that varies only about ± 5 nm. Such thiazolyl tetrazolium salts are disclosed in several U.S. patents such as U.S. Pat. nos. 5,126,275; 5,322,680, respectively; 5,300,637 and 5,290,536.

Most biological samples are aqueous in nature, so it is desirable that the indicator be soluble in the sample. However, many tetrazolium salts are not readily soluble. One supplier of tetrazolium salts (dojindo laboratories) has a range of indicators that are made more soluble by the addition of sulfonic acid groups to the indicator molecule. See U.S. patent No. 6,063,587 and published japanese patent applications JP58113181a2 and JP58113182a 2. Their WST series tetrazolium salt indicators are commonly mentioned in patents disclosing analytical methods. See, for one example, U.S. patent No. 6,586,199. The solubility of the WST series tetrazolium salts was reported to be about 10mg/mL water. Solubility can also be increased when certain sulfonate and phosphonate counter ions of tetrazolium salts are used, such as disclosed in U.S. patent No. 5,250,695.

Other patents that discuss tetrazolium salt indicators include EP 0476455B 1; US2004/0132004A 1; WO 98/37157; U.S. patent nos. 6,183,878B 1; U.S. patent nos. 6,207,292B 1; U.S. patent nos. 6,277,307B 1; DE 2147466; U.S. patent nos. 5,185,450; U.S. patent No. 5,196,314.

The inventors of the present invention wanted to improve the solubility of thiazolyl tetrazolium salts while maintaining their formazan in response to incident light provided by the LEDThe ability to make a more gradual spectral response in the 600-640nm range. As will be appreciated from the following description of the invention, they have been successful in providing thiazolyl tetrazolium salts with greater solubility while maintaining the desired spectral response.

Disclosure of Invention

The present invention includes novel thiazolyl tetrazolium salts. Some of these have been found to have better solubility in water than the related proprietary thiazolyl tetrazolium salts. Improving solubility makes them easier to apply to test strips used to measure analytes in biological samples, such as glucose in blood samples. The tetrazolium salts are characterized by having an alkylammonium alkoxy substituent that increases solubility. According to one embodiment, the tetrazolium salt is defined by the following general formula.

Wherein:

a is a counterion

X is 1-6C alkyl or heteroalkyl

a＝1-3

b＝0-3

R₁1-6C alkyl

R₂And R₃One is alkyl and the other is XN + H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₄and R₅One being halogen and the other being halogen-substituted 1-6C alkyl, R₄And R₅One or two of them being XN + H_b(R₁)_3-bOr is or

R₄And R₅Linked to form aromatic or heteroaromatic compoundsA cyclic or substituted aromatic ring or a substituted heteroaromatic ring.

The general formula given above represents novel thiazolyl tetrazolium salts, i.e. they comprise a thiazole ring attached to the tetrazole ring at the 2 nitrogen position. In addition, they each have a phenyl ring attached at the 3 nitrogen position on the tetrazole ring and another phenyl ring attached at the 5 nitrogen position. Increased solubility relative to the patented thiazolyl tetrazolium salts, herein designated by HTC-045, is achieved by attaching one or more alkyl ammonium alkoxy groups to the thiazolyl tetrazolium salt. The alkyl ammonium alkoxy group may be as R₄And/or R₅To a thiazolyl ring, or at the position represented by R₄And R₅Substitution is made on the aromatic or heteroaromatic ring formed. In a preferred embodiment, the alkylammonium alkoxy group is a substituent on a phenyl ring attached to a tetrazole ring. In a more preferred embodiment, the alkyl ammonium alkoxy group is a trialkyl ammonium propoxy group. Benzothiazolyl tetrazolium salts are preferred compounds with increased solubility.

For another aspect of the invention, the thiazolyl tetrazolium salts are used as chromogenic indicators for detecting the presence of reducing substances (e.g., NADH) in biological sample assays that oxidize analytes using enzymes, such as in the determination of glucose content of blood.

Brief Description of Drawings

FIG. 1 is a schematic of a first method of making the compounds of the examples.

FIG. 2 is a schematic of a second method of making the compounds of the examples.

Figure 3 shows the comparative compounds reported in the examples.

Figures 4a-b show compounds of the invention prepared in the examples.

FIGS. 5a and b are plots of tetrazolium salt absorbance versus light wavelength.

Detailed Description

Definition of

The following definitions apply to the substituents of the thiazolyl tetrazolium salts of the present invention.

"1-6C" -refers to a residue containing 1-6 carbon atoms

"alkyl" -means a group of formula C_nH_2n+1Linear and branched hydrocarbon residues of

"Heteroalkyl" -refers to linear and branched hydrocarbon residues containing heteroatoms bonded to adjacent carbon atoms

"alkoxy" -means a residue-OR where R is alkyl

"methylenedioxy" -means a compound of the formula-O-CH₂Divalent residue of-O-

"halogen" -means fluorine, chlorine, iodine and bromine

"halogen-substituted 1-6C alkyl" -refers to a compound having the formula C_nH_2n-1Y₂Wherein Y is halogen

"aromatic ring" -means a benzene or naphthalene ring

"heteroaromatic ring" -means a pyridine or quinoline ring

"counterion" -refers to the ionic residue from the reactants used to prepare the tetrazolium salt (e.g., nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen sulfate, hydrogen carbonate, methane sulfonate, fluoroborate, bromide, chloride, iodide, or combinations thereof)

"tet salt" is an abbreviation for tetrazolium salt

Thiazolyl tetrazolium salts

Formazans from thiazolyl tetrazolium salts as disclosed in U.S. Pat. No. 5,126,275Characterized by a reflection spectrum exhibiting an extended plateau at greater than about 600-650nm, which is useful in devices using LEDs as light sources. The sample is contacted with a reaction system that produces a color that is indicative of the amount of analyte present in the sample. Light returning from the test zone is detected and correlated with the analyte level. In an important embodiment, glucose dehydrogenase catalyzes the oxidation of glucose in a blood sample in the presence of NAD +. Reoxidation of reduced NADH by an enzyme mediator, such as diaphorase, which catalyzes the reduction of tetrazolium salts to formazan. The resulting color change is proportional to the amount of NADH produced by oxidation of glucose and indirectly to the amount of glucose present in the sample. Conversion from thiazolyl tetrazolium salts to colored formazansCan be measured by exposing it to a light source. The light returning from the coloured test zone is detected and converted by a suitable algorithm to the amount of analyte in the sample. Although described herein with respect to NAD-NADH, the tetrazolium salts of the present invention have broader applications, including their use with dehydrogenases having other cofactors, such as PQQ and FAD.

Since thiazolyl tetrazolium salts and their corresponding formazansShould be soluble in the reaction mixture applied to the test strip used to detect the analyte, it is therefore an object of the inventors to improve the solubility of thiazole tetrazolium salts while maintaining them providing formants having a relatively slow reflectance spectrum in the 600-640nm rangeThe ability of the cell to perform. The inventors have discovered a novel class of thiazolyl tetrazolium salts that include compounds that meet their objectives.These salts are represented by the general formula:

wherein:

a is a counterion

X is 1-6C alkyl or heteroalkyl

a＝1-3

b＝0-3

R₁1-6C alkyl

R₂And R₃One is alkyl and the other is XN + H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₄and R₅One being halogen and the other being halogen-substituted 1-6C alkyl, one or both of R4 and R5 being XN + H_b(R₁)_3-bOr is or

R₄And R₅Linked to form an aromatic or heteroaromatic ring or a substituted aromatic or substituted heteroaromatic ring.

The tetrazolium salts of the present invention may also be separated into substituted thiazolyl tetrazolium salts and substituted benzothiazolyl tetrazolium salts, as shown in the formulas specifically contemplated below.

Wherein:

a is a counterion

X is 1-6C alkyl

a＝1-3

b＝0-3

R₁1-3C alkyl

R₂And R₃One being 1-4C alkyl and the other being XN + H_b(R₁)_3-bOr R is₂And R₃The bond is a methylene-dioxy group,

R₄＝CHF₂

R₅halogen ═ halogen

A is a counterion

X is 1-6C alkyl

a＝1-3

b＝0-3

R₁1-3C alkyl

R₂And R₃One being 1-4C alkyl and the other being XN + H_b(R₁)_3-bOr R is₂And R₃The bond is a methylene-dioxy group,

R₄and R₅Is H, or R₄And R₅Are linked to form an aromatic ring

R₆1-4C alkoxy, hydrogen or halogen.

The solubility of these compounds (I and II) will be illustrated in the examples below. Improving the solubility of thiazolyl tetrazolium salts should reduce the time required to obtain test results and provide better linearity at high analyte levels. Furthermore, the improved water solubility should facilitate the formulation of a mixture of reactants that will be easier and cheaper to manufacture.

The preparation method of the tetrazolium salt

In U.S. Pat. No. 45,126,275, a process for the preparation of thiazolyl tetrazolium salts is described. The process comprises reacting a hydrazone with a diazotized aniline to produce aThen the nail is putOxidized to form tetrazolium salts. The novel process to be described for the manufacture of the compounds of the invention was developed, introducing one (scheme 1) or two (scheme 2) polar groups into the preferred tetrazolium salts of compounds I and II. Fig. 1 and 2 illustrate these two methods. The following examples further illustrate the invention.

Scheme 1 was used to prepare tetrazolium salts in which the phenyl substituent at the 5 position on the tetrazolium ring has a methylenedioxy substituent. In scheme 1, the trimethylammonium propoxy group is substituted on the phenyl substituent at the 3-position on the tetrazole ring. In scheme 1, o-nitrophenol is reacted with 3-bromopropyltrimethylammonium bromide to form 3- (2-nitrophenoxy) propyltrimethylammonium bromide which is then hydrogenated to form the equivalent aniline. The compound is reacted with a hydrazone containing a substituted thiazoly ring and a phenyl ring having a methylenedioxy substituent to produce a methyl group. Then the nail is putOxidized to form tetrazolium salts. The hydrazone is formed by the reaction of a substituted benzaldehyde with a substituted hydrazine containing the substituents required for the tetrazolium salt.

Scheme 2 is used to prepare tetrazolium salts in which the phenyl substituent at the 5 position on the tetrazole ring has a trimethylammonium propoxy group substituted at the 3 or 4 position (or both). As in scheme 1, the trimethylammonium propoxy group is substituted on the phenyl substituent at the 3-position on the tetrazole ring. In scheme 2, 4-hydroxy-3-methoxybenzaldehyde is reacted with 3-bromopropyltrimethylammonium bromide toTrimethylammonium propoxy group is added to the benzaldehyde. The product is reacted with a substituted hydrazine to produce a hydrazone. The hydrazone is reacted with 3- (2-aminophenoxy) propyltrimethylammonium bromide prepared in the first step of scheme 1 to produce a formazanThe nail is put onTo a tetrazolium salt.

As will be seen from the results below, solubility appears to be affected by the counterion associated with the tetrazolium salt. The counterions are generated from the reactants used in the process and can be varied to provide preferred counterions. Alternatively, the counterions can be replaced, for example by ion exchange.

In the following non-limiting examples, the products of each step were identified by spectrophotometric analysis and the results were given for each product.

Example 1

5-benzo [1, 3-dioxol-5-yl]-3- [2- (3-trimethylammonium) propoxy-benzene Base of]Preparation of (E) -2- (5-chloro-4-difluoromethyl-thiazol-2-yl) -2H-tetrazole bromonitrite

As shown in scheme 1, o-nitrophenol is reacted with 3-bromopropyltrimethylammonium bromide to form 3- (2-nitrophenoxy) propyltrimethylammonium bromide, which is then hydrogenated to form the equivalent aniline. Reacting the aniline with a hydrazone containing a substituted thiazolyl ring and a phenyl ring to produce a methylThe phenyl ring has a methylenedioxy substituent. Then the nail is putBy oxidation to form tetrazolium salts. The process will be more fully described in the following examples: to a yellow solution of o-nitrophenol (Aldrich, 6.3g, 45mmol) in DMF (150mL) in a 250mL dry one-neck round bottom flask under nitrogen was added potassium carbonate (6.6g, 48 mmol). The mixture turned orange. After stirring for 10 minutes, 3-bromopropyltrimethylammonium bromide (Aldrich, 12.9g, 45mmol) was added. The resulting mixture was heated at 125 ℃ for 3 hours. After the reaction mixture was cooled to room temperature, the residue was filtered and the precipitate was washed with DMF (2X 2. phi. mL). The filtrate was slowly added to EtOAc (750mL), causing a precipitate to form. The resulting mixture was stirred for 30 minutes. The precipitate was filtered and washed with a solution of EtOAc: DMF, 5: 1 (3X 100mL), then EtOAc (100mL) and hexane (100 mL). After the light yellow solid was air dried for 5 minutes, the product was added to a solution of EtOAc: DMF, 10: 1(550mL) and stirred for 1 hour. The solid was filtered and washed with EtOAc (100mL) and hexane (100 mL). The resulting solid was filtered to give a pale beige product (14.54g, > 100%) mp (178 ℃ -181 ℃).¹³C NMR(400MHz，DMSO d₆)：151，139.45，134.71，125.14，121，115.39，66.59，62.84，52.39，22.48 ¹H NMR(400MHz，DMSO-d₆)：7.90(dd，J＝8.1Hz，J＝1.7Hz，1H)，7.58(dt，J＝8.2Hz，J＝1.7Hz，1H)，7.39(d，J＝8.2Hz，1H)，7.15(d，J＝1.7Hz，1H)，4.25(t，J＝6Hz，2H)，3.45(m，2H)，3.11(s，9H)，2.23(m，2H)。ES I-MS：m/z 239(100％，M²⁺)。

A slurry of 3- (2-nitrophenoxy) propyltrimethylammonium bromide (7g, 21.94mmol), 10% Pd/C (725mg), MeOH (70mL), and water (70mL) was hydrogenated in a Parr hydrogenation unit at 35psi for 2.5 hours. After 30 minutes, the pressure dropped to 22 psi. The resulting mixture was filtered through 521 a Celite pad. The black catalyst/Celite pad was washed with MeOH (2 × 10 mL). The filtrate was concentrated on a rotary evaporator under reduced pressure at 40-45 ℃. When no more distillate was formed, the pale pink solution was transferred to another flask and concentrated by freeze-drying for 2 days to yield a pale beige aniline derivative identified by the following spectral propertiesOrganisms (5.92g, 93%).¹³C NMR(400MHz，DMSO-d₆)：145.19，137.76，121.39，116.15，114.09，111.86，64.79，63.27，52.36，22.78¹H NMR(400MHz，DMSO-d₆)：6.78(d，J＝4Hz，1H)，6.66(m，2H)，6.53(d，J＝6Hz，1H)，4.82(br s，2H)，3.99(t，J＝6Hz，2H)，3.54(m，2H)，3.11(s，9H)，2.17(m，2H)。ESI-MS：m/z 209(100％，M²⁺)。

To a solution of the above aniline derivative (7.7g, 26.64mmol) in water (80ml) was added concentrated HCl (7.2ml) dropwise in an ice-water bath. After stirring the solution for 5 minutes, a solution of sodium nitrite (2.2g, 32mmol) in water (16mL) was added dropwise. The color of the solution changed from light brown to golden yellow. The resulting solution was stirred for 30 minutes. A solution of hydrazone (Bayer, 9.8g, 27.98mmol) in pyridine (400mL) was stirred in a 3-neck 2-liter flask equipped with a mechanical stirrer and a jacketed addition funnel in a salt/ice/water bath (-15 ℃ to-20 ℃). The diazo solution just prepared was added dropwise to the hydrazone solution via a jacketed addition funnel filled with ice/water. After the first few drops, the color of the reaction changed from yellow to dark blue. After the addition of 1/3, the dark blue reaction mixture became very viscous. When the addition was complete, the mixture was stirred for 30 minutes. The reaction was then warmed to 3-5 ℃ via an ice/water bath. The reaction was stirred for 1 hour. The mixture was transferred to a2 liter round bottom flask containing methanol (500 mL). The mixture was concentrated on a rotary evaporator under reduced pressure at 45 ℃ until no more distillate was formed. MeOH (500mL) was added again and the mixture was concentrated. The process is repeated. The resulting solid was dried under vacuum overnight. The flask was then rinsed with MeOH (100mL) and diluted with EtOAc (500 mL). The slurry was stirred in an ice/water bath for 30 minutes. The product was filtered and washed with EtOAc (100mL), cold MeOH (50mL), EtOAc (150mL) and hexanes (150 mL). The resulting dark blue product was transferred to a light yellow bottle and dried under vacuum to obtain the desired formazan identified by the following spectral properties(13，11.3g，67％)。¹⁹F NMR：(376mHz，DMSO d₆)：-177.77(d，J＝59.2Hz)，¹³C NMR(400MHz，DMSO-d₆)：169.57，153.6，148.1，147.8，144.81，141.9，137.1，132.74，128.82，121.53，121.5，121.1，115.89，113.96，108.8，108.22，106.68，101.33，66.21，63.03，51.99，22.351HNMR(400MHz，DMSO-d6)：11.78(br s，1H)，8.05(dd，J＝8.1Hz，J＝1.5Hz，1H)，7.55(dd，J＝8.2Hz，J＝1.6Hz，1H)，7.48(dd，J＝8.2Hz，J＝1.7Hz，1H)，7.43(d，J＝1.7Hz，1H)，7.32(dd，J＝8Hz，J＝＜1Hz，1H)，7.16(t，J＝7.9Hz，1H)，7.15(t，J_HF＝59.2，1H)，7.04(d，J＝8.2Hz，1H)，6.11(s，2H)，4.32(t，J＝6Hz，2H)，3.49(m，2H)，3.05(s，9H)，2.38(m，2H)。ES I-MS：m/z 611(100％，M-Ms)，258(26％，M²⁺)。

To the above-mentioned firstTo a mixture of (2.16g, 3.4mmol), water (1.4mL) and THF (40mL) was added methanesulfonic acid (0.68mL, 10.24 mmol). The mixture was stirred for 5 minutes. Then, sodium nitrite (720mg, 10.24mmol) was added in one portion. The mixture was stirred for 3 days. The slurry color changed from dark blue to orange. The orange precipitate was filtered and washed with THF (2X 10 mL). The product was dried under vacuum overnight. The solid was then slurried with MeOH (80mL), filtered and the residue washed with MeOH (2X 5 mL). The filtrate was concentrated on a rotary evaporator under reduced pressure at 40 ℃. The resulting product was dried under vacuum for 2 days to yield an orange tet salt (2.86g, > 100%) identified by the following spectral properties.

¹⁹F NMR：(376MHz，DMSO-d₆)：-181.13(d，J＝52.1Hz)，¹³C NMR(400MHz，DMSO-d₆)：165.1，152.52，152.27，148.85，146.68，142.72，142.45，137.07，135.95，128.44，123.87，121.87，121.86，121.39，115.57，114.68，110.80，109.84，108.43，107.14，106.06，102.78，100.49，66.92，62.23，52.21，22.08¹H NMR(400MHz，DMSO-d₆)：7.98(dd，J＝8.0Hz，J＝1.5Hz，1H)，7.91(m，2H)，7.82(d，J＝1.8Hz，1H)，7.53(d，J＝8.2Hz，1H)，7.37(m，1H)，7.31(d，J＝8.2Hz，1H)，7.25(t，J_HF＝59.2，1H)，6.27(s，2H)，4.17(t，J＝6Hz，2H)，3.49(m，2H)，2.93(2，9H)，2.02(m，2H)。ES I-MS：m/z 663(22％，M²⁺+TFA)，645(17％，M⁺)，275(100％，M²⁺)。

Example 2

5-benzo [1, 3-dioxol-5-yl]-3- [2- (3-trimethylammonium) propoxy-benzene Base of]Preparation of (E) -2- (5-chloro-4-difluoromethylthiazol-2-yl) -2H-tetrazole bromotetrafluoroborate And (4) preparing.

Salts were prepared as in example 1 except 48% of tetrahydrofluoroboric acid was used with the same formazanTo yield the desired orange tetrazolium salt.¹³C NMR(400MHz，DMSO d₆)：

¹³C NMR(400MHz，DMSO d₆)：137，128，124，121，114，111，110，108，107，106，103，68，63，53，28 ¹⁹F NMR：(376MHz，DMSO-d₆)：-181.13(d，J＝59.2Hz)，¹H NMR(400MHz，DMSO-d₆)：7.92(m，3H)，7.82(d，J＝1.8Hz，1H)，7.52(bd，J＝8.2Hz，1H)，7.37(m，1H)，7.31(d，J＝8.2Hz，1H)，7.25(t，J_HF＝59.2，1H)，6.27(s，2H)，4.17(t，J＝6Hz，2H)，3.35(m，2H)，2.93(2，9H)，2.02(m，2H)。ESI-MS：m/z 637(100％，M⁺-Br)。

Example 3

5-benzo [1, 3-dioxol-5-yl]-3- [2- (3-dimethylhydroammonium) propoxyBase- Phenyl radical]Preparation of (E) -2- (5-chloro-4-difluoromethylthiazol-2-yl) -2H-tetrazole bromonitrite Prepare for

To a yellow solution of o-nitrophenol (Aldrich, 2.1g, 45mmol) in DMF (50mL) in 100mL dry one-neck round bottom flask under nitrogen was added potassium carbonate (4.4g, 32 mmol). The mixture turned orange. After stirring for 10 minutes, 3-chloropropyldimethylammonium chloride (Aldrich, 2.4g, 16.5mmol) was added. The resulting mixture was heated at 125 ℃ for one day. After the reaction mixture was cooled to room temperature, the residue was filtered and the precipitate was washed with MeOH (2 × 10 mL). The filtrate was concentrated under vacuum at 40 ℃ until 5 mL. EtOAc (100mL) was then added. A precipitate formed and was filtered. Water (100mL) and EtOAc (150mL) were added to the filtrate and the layers were mixed and separated. The aqueous layer was extracted with EtOAc (3X 25 mL). The combined EtOAc extracts were washed with saturated aqueous sodium chloride (25mL), dried over magnesium sulfate (5g), filtered, and concentrated to give a yellow oil (2.55g, 81%).

¹H NMR(400MHz，DMSO-d₆)：7.80(dd，J＝8.1Hz，J＝1.7Hz，1H)，7.58(dt，J＝8.2Hz，J＝1.7Hz，1H)，7.05(d，J＝8.2Hz，1H)，6.65(d，J＝1.7Hz，1H)，4.25(t，J＝6Hz，2H)，2.45(m，2H)，2.15(s，9H)，1.9(m，2H)。ESI-MS：m/z 225(100％，M+1)。

The alkylated nitrophenol was treated in the same manner as in example 1 to give the desired tet salt.

¹³C NMR(400MHz，DMSO d₆)：127，124，121，115，110，109，108，107，104，102，64，48，36，28 ¹⁹F NMR：(376MHz，DMSO-d₆)(d，J＝59.2Hz)：-119.13，¹H NMR(400MHz，DMSO-d₆)：7.95(m，3H)，7.80(d，J＝1.8Hz，1H)，7.50(d，J＝8.2Hz，1H)，7.35(t，J＝8.2Hz，1H)，7.31(d，J＝8.2Hz，1H)，7.25(t，J_HF＝59.2，1H)，6.27(s，2H)，4.20(t，J＝6Hz，2H)，3.45(m，2H)，2.65(d，j＝1.8Hz，6H)，1.98(m，2H)。ESI-MS：m/z 535(100％，M²⁺)。

Example 4

5-benzo [1, 3-dioxol-5-yl]-3- [2- (3-trimethylammonium) propoxy-benzene Base of]Preparation of (E) -2- (benzothiazol-2-yl) -2H-tetrazole bromo-nitrite

Piperaldehyde-benzothiazol-2-ylhydrazone was reacted with 3- (2-aminophenoxy) propyltrimethylammonium bromide as in example 1 to prepare aAnd then converting it to the corresponding tetrazolium salt.

Preparation of a nail in example 1Piperaldehyde benzothiazol-2-ylhydrazone (Bayer, 2.5g, 10.5mmol) was reacted with the aniline derivative of example 1 (2.85g, 9.6mmol) under similar conditions as used to obtain a dark purple solid (4.7g, 82%) identified by the following spectral properties.

¹H NMR(400MHz，DMSO-d₆)：11.78(br s，IH)，7.98(dd，2H)，7.73(m，1H)，7.60(m，2H)，7.50(m，1H)，7.41(m，2H)，7.29(m，1H)，7.05(d，1H)，6.14(s，2H)，4.39(t，2H)，3.55(m，2H)，3.05(s，9H)，2.48(m，2H)。ESI-MS：m/z 517(100％，M⁺)。

The above-mentioned nail was treated under the same conditions as in example 1(600mg, 1.01mmol) was converted to the tetrazolium salt (870mg, > 100%). The tetrazolium salt was identified by the following spectral properties.¹3C NMR(100MHz，DMSO-d₆)：164.98，152.59，152.26，148.91，136.64，132.74，135.08，129.27，128.88，128.24，124.77，123.97，123.7，122.6，121.97，115.81，114.56，109.86，107.12，102.82，66.88，62.26，52.2，22.02。¹H NMR(400MHz，DMSO-d₆)：8.42(dd，J＝8.2Hz，J＝0.8Hz，1H)，8.03(dd，J＝7.7Hz，3＝1.6Hz，1H)，8.02(dd，J＝8.1Hz，J＝0.9Hz，1H)，7.97(dd，J＝8.2Hz，J＝1.8Hz，1H)，7.93(m，1H)，7.86(d，J＝1.7Hz，1H)，7.76(m，1H)，7.73(m，1H)，7.55(dd，J＝8.6Hz，J＝0.9Hz，1H)，7.39(m，1H)，7.32(d，J＝8.1Hz，1H)，6.28(s，2H)，4.12(t，J＝6Hz，2H)，3.16(m，2H)，2.91(s，9H)。ESI-MS：m/z 561.2(100％，M⁺)。

Example 5

5-benzo [1, 3-dioxol-5-yl]-3- [2- (3-trimethylammonium) propoxy-benzene Base of]Preparation of (E) -2- (6-methoxy naphthyl thiazole-2-yl) -2H-tetrazole bromo nitrite

The appropriate hydrazone was prepared using 6-methoxy-2-hydrazino-naphthyl thiazole instead of 2-hydrazino-benzothiazole in example 4. The hydrazone is then treated under the same conditions to produce the desired formazan(44% total).

¹³C NMR(400MHz，DMSO-d₆)：135，130，125，122，121，120，115，114，113，110，65，64，55，52，38，22 ¹H NMR(400MHz，DMSO-d₆)：8.61(d，J＝8.1Hz，1H)，8.29(d，J＝8.2Hz，1H)，8.05(dd，J＝8.1Hz，J＝1.8Hz，1H)，7.82(dt，J＝8.2Hz，J＝1.7Hz，1H)，7.68(m，2H)，7.61(dd，J＝8.1Hz，J＝1.6Hz，1H)，7.55(d，J＝1.8Hz)，7.37(dd，J＝8.2Hz，J＝1.8Hz，1H)，7.31(dd，J＝8.2Hz，J＝1.8Hz，1H)，7.27(dt，J＝8.1Hz，J＝1.7Hz，1H)，7.08(d，J＝8.1Hz，1H)，6.12(s，2H)，4.45(t，J＝6Hz，2H)，4.10(s，3H)，3.60(m，2H)，2.95(s，9H)，2.65(m，2H)。ESI-MS：m/z 597(100％，M⁺)。

To a solution of acetic acid (5mL) and tetrahydrofuran (5mL) in an ice/salt/water bath was added sodium nitrite (143mg, 2.08 mmol). After stirring for 10 minutes, the above formazan was added(340mg, 0.051 mmol). After 1 hour, the reaction was allowed to warm back to room temperature and stirred overnight. The dark chestnut colored mixture was filtered and the solid washed with acetic acid (3X 5mL), ethyl acetate (2X 10mL) and hexane (10 mL). The filtrate was concentrated on a rotary evaporator at 30 ℃ and then dried under vacuum overnight. The residue was slurried with ethyl acetate (15mL), deposited and decanted. The process is repeated. Hexane (10mL) was then added. The product was filtered and dried overnight to give the desired castanea tet salt (260mg, 75%).

¹³C NMR(400MHz，DMSO-d₆)：129，128，126，123，121，119，115，114，112，108，105，101，98，66，63，57，51，22 ¹H NMR(400MHz，DMSO-d₆)：8.32(m，1H)，8.02(m，4H)，7.87(d，J＝1.8Hz，1Hz)，7.78(m，3H)，7.66(d，8.2Hz，1H)，7.47(t，J＝8.2Hz，1H)，7.33(d，J＝8.2Hz，1H)，6.28(s，2H)，4.13(s，3H)，3.15(m，2H)，2.85(s，9H)，1.87(m，2H)。ES I-MS：m/z 298(100％，M²⁺)，709(20％，M⁺+TFA)，

Example 6

5- [4- (3-trimethylammonium) propoxy-3-methoxy-phenyl]-3- [2- (3-trimethylammonium) Propoxy-phenyl]-2- (5-chloro-4-difluoromethyl-thiazol-2-yl) -2H-tetrazole dibromo Preparation of nitrate

4-hydroxy-3-methoxybenzaldehyde is reacted with 3-bromopropyltrimethylammonium bromide to introduce another trimethylammonium propoxy substituent. The product is reacted with 5-chloro-4-difluoromethyl-2-hydrazino-thiazole to produce hydrazone. The hydrazone is reacted with bromine as described in example 1Reacting 3- (2-aminophenoxy) propyltrimethylammonium to produce a formazanThe nail is put onConverted to the corresponding tetrazolium salt. 4-hydroxy-3-methoxy-benzaldehyde (vanillin, Aldrich, 4.56g, 30mmol) was alkylated with 3-bromopropyltrimethylammonium bromide (8.8g, 34mmol) under the same conditions as used in example 1 to prepare an off-white product (9.73g, 97%) identified by the following spectral properties.

¹³C NMR(400MHz，DMSO-d₆)：192.5，155，150，130，125.75，112.6，110，66.5，63.4，56，25.5。¹H NMR(400MHz，DMSO-d₆)：9.88(s，1H)，7.58(dd，J＝8.2Hz，J＝1.9Hz，1H)，7.41(d，J＝1.9Hz，1H)，7.22(d，J＝8.2Hz，1H)，4.35(t，J＝6.4Hz，2H)，3.49(m，2H)，3.14(s，9H)，2.30(m，2H)。ESI-MS：m/z 252.2(100％，M⁺)。

To a slurry of the above alkylated benzaldehyde (6.8g, 20.5mmol) and 5-chloro-4-difluoromethyl-2-hydrazino-thiazole (Bayer, 3.72g, 18.6mmol) in MeOH (60mL) was added acetic acid (0.55 mL). After 5 minutes the slurry was almost dissolved. The resulting mixture was heated at 80 ℃ overnight. After cooling the reaction to room temperature, the solution was slowly added to EtOAc (650 mL). A light gray precipitate formed. The mixture was stirred for 30 minutes. The solid was filtered and washed with EtOAc and hexanes. The pale gray product was transferred to a light yellow bottle and dried under vacuum overnight to give the desired hydrazone (8.7g, 87%) identified by the following properties.

¹⁹F NMR：(376MHz，DMSO-d₆)：111.08(d，J＝47Hz)，¹³C NMR(400MHz，DMSO-d₆)：143，121，114，109，108.5，66，63，55，53，39，22。¹H NMR(400MHz，DMSO-d₆)：12.37(s，1H)，7.29(d，J＝1.9Hz，1H)，7.19(dd，J＝10Hz，J＝1.8Hz，1H)，7.06(t J＝7.7Hz，1H)，7.18(mm，1H)，6.95(t，J_HF＝47，1H)，4.09(t，J＝6.1Hz，2H)，3.83(s，3H)，3.47(m，2H)，3.47(m，2H)，3.29(s，9H)，2.50(m，2)，2.20(m，2H)。ESI-MS：m/z433.1(100％，M⁺)。

The above hydrazone (7.08g, 13.86 mmol) was reacted with the aniline derivative of example 1 (6g, 20.76mmol) under conditions similar to those of example 1 to obtain a deep blue formazan identified by the following spectral properties(8.56g，76％)。

¹⁹F NMR：(376MHz，DMSO-d₆)：177.77(d，J＝59.2Hz)¹³C NMR(400MHz，DMSO-d₆)：153，148.98，148.63，144.5，136.5，132.73，132.74，127，121.59，119.53，115.54，113.96，113.58，110.98，108.92，66.10，65.79，62.77，55.54，52.15，22.45，22.25。¹H NMR(400MHz，DMSO-d₆)：：11.78(br s，1H)，8.03(dd，J＝8.1bHz，J＝1.5Hz，1H)，7.56(m，IH)，7.52(dt，J＝8.5Hz，J＝2Hz，1H)，7.50(d，J＝2Hz，1H)，7.35(dd，J＝8.5Hz，J＝1Hz，1H)，7.30(d，J＝7.9Hz，1H)，7.18(mm，1H)，7.17(t，J_HF＝59.2，1H)，4.35(t，J＝5.8Hz，2H)，4.13(t，J＝6.1Hz，2H)，3.88(s，3H)，3.55(m，2H)，3.50(m，2H)，3.14(s，9H)，3.07(s，9H)，2.50(m，2H)，2.30(m，2H)。ES I MS：m/z652.2(45％，M⁺)，326.8(100％，M⁺)。

The above formazan was treated under similar conditions as used in example 1(2.44g, 3mmol) was converted to the tetrazolium salt except that the reaction mixture was heated at 40 ℃ for 15 hours. The product was identified by the following spectral properties.

¹³C NMR(100MHz，DMSO-d₆)：136.7，128.5，121.9，121.8，114.5，113.5，110.2，108.1，105.7，66.7，66.1，62.5，62.1，56.1，52.1，51.8，21.8，21.2。¹H NMR(400MHz，DMSO-d₆)：8.06(dd，J＝8.1Hz，J＝1.5Hz，1H)，7.93(m，8H)，7.77(d，J＝2Hz，1H)，7.53(d，J＝8.6Hz，1H)，7.38(t，J＝8.5Hz，2H)，7.26(d，J＝52.2Hz，1H)，4.23(t，J＝6H，2H)，4.17(t，J＝6.1Hz，2H)，3.94(s，3H)，3.53(m，2H)，3.32(m，2H)，3.14(s，9H)，3.01(s，9H)，2.26(m，2H)，2.03(m，2H)，ESI MS：m/z 652.2(45％，M⁺)，326.8(100％，M⁺)，326.8(100％，M²⁺)。

Example 7

5- [4- (3-trimethylammonium) propoxy-3-methoxy-phenyl]-3- [2- (3-trimethylammonium) Propoxy-phenyl]-2- (6-ethoxy-benzothiazol-2-yl) -2H-tetrazole dibromo nitrous acid Preparation of salts

6-ethoxy-2-hydrazino-benzothiazole was reacted with the alkylated benzaldehyde prepared in example 6 to form hydrazone, which was then reacted with the substituted aniline prepared in example 1 to prepare a. The nail is put onConverted to the equivalent tetrazolium salt.

6-ethoxy-2-hydrazino-benzothiazole (Acros, 4.62g, 22mmol) is reacted with 4- (3-trimethylammonium propoxy) -3-methoxy-benzaldehyde under similar conditions as in example 6 to give the product (10.23g, 95%) as a pale gray identified by the following properties.

¹³C NMR(400MHz，DMSO-d₆)：165.58，153.82，153.74，149.24，148.96，142.42，128.01，120.16，118.24，113.62，113.54，108.66，106.49，101.33，65.83，63.51，62.86，55.32，52.16，22.51，14.6。¹H NMR(400MHz，DMSO-d₆)：11.98(br s，1H)，8.03(s，1H)，7.38(d，J＝2.2Hz，1H)，7.34(d，J＝8.7Hz，1H)，7.32(d，J＝1.8Hz，1H)，7.20(dd，J＝8.4Hz，J＝1.8Hz，1H)，7.07(d，J＝8.4Hz，1H)，6.89(dd，J＝8.7Hz，J＝2.5Hz，1H)，4.09(t，J＝6.1H，2H)，4.03(t，J＝7Hz，2H)，3.84(m，3H)，3.49(m，2H)，3.12(s，9H)，2.22(m，2H)，1.33(t，J＝7Hz)。ESI-MS：m/z 561.2(100％，M⁺).ES I-MS：m/z 443.1(78％，M⁺)，222.1(100％，M⁺²)。

The hydrazone (3.6g, 6.93mmol) prepared above was reacted with the aniline (3g, 10.38mmol) of example 1 under similar conditions to those used in example 1 to prepare a formazanExcept that a 3: 1 mixture of pyridine and DMF was used as the solvent to yield the desired dark blue product (3.8g, 66%) as identified by the following spectral properties.

¹³C NMR(400MHz，DMSO-d₆)：130.5，123.2，121.8，118.8，116，115.3，114.1，113.8，109.8，106.3，67.1，66.5，63.6，52.5，22.6，22.5，14.2。¹H NMR(400MHz，DMSO-d₆)：11.78(br s，1H)，8.01(br d，J＝8Hz，1H)，7.81(br s，1H＝8.8Hz，1H)，7.62(m，3H)，7.48(t，J＝8Hz，1H)，7.33(d，J＝8.3Hz，1H)，7.16(m，1H)，7.10(dd，J＝8.8Hz，J＝2.4Hz，1H)，4.38(t，J＝5.8Hz，2H)，4.13(m，2H)，3.91(s，3H)，3.55(m，2H)，3.50(m，2H)，3.13(s，9H)，3.08(s，9H)，2.54(m，2H)，2.25(m，2H)，1.38(t，J＝7Hz，3H)。ESI MS：m/z 776(12％，M⁺′+TFA)662.2(45％，M⁺)，331.8(100％，M⁺²)。

The above formazan was treated under similar conditions as used in example 1(1.24g, 1.5mmol) was converted to the tetrazolium salt (2g, > 100%). The product was identified by the following spectral properties.

¹³C NMR(400MHz，DMSO-d₆)：136.8，128.1，125.4，121.5，121.4，114，113.5，109.8，106.8，66.6，66.2，64.5，62.5，62.2，56.1，52.2，52.1，22.1，21.5，13.5。¹H NMR(400MHz，DMSO-d₆)：8.04(dd，J＝8Hz，J＝1.5Hz，1H)，7.95(m，4H)，7.78(d，J＝2Hz，3H)，7.54(d，J＝8.5Hz，1H)，7.40(t，J＝7.8Hz，1H)，7.38(d，J＝8.6Hz，1H)，4.23(t，J＝6.1H，2H)，4.1(q，J＝8Hz，2H)，4.12(m，2H)，3.96(s，3H)，3.53(m，2H)，3.50(m，2H)，3.18(m，2H)，3.15(s，9H)，2.92(2，9H)，2.30(m，2H)，1.89(m，2H)，1.40(t，J＝7Hz，2H)。ES I MS：m/z 220.8(100％，M⁺³)。

Example 8

5- [4- (3-trimethylammonium) propoxy-3-methoxy-phenyl]-3- [2- (3-trimethylammonium) Propoxy-phenyl]Process for producing (E) -2- (6-bromobenzothiazol-2-yl) -2H-tetrazole dibromo-nitrite Preparation of

The appropriate hydrazone was prepared using 6-bromo-2-hydrazino-benzothiazole instead of 6-ethoxy-2-hydrazino-benzothiazole in example 8. The hydrazone is then treated under the same conditions to produce the desired formazan(44% total).

¹H NMR(400MHz，DMSO-d₆)，7.90(dd，J＝8.1Hz，J＝1.7Hz，1H)，7.58(dt，J＝8.2Hz，J＝1.7Hz，1H)，7.39(d，J＝8.2Hz，1H)，7.15(d，J＝1.7Hz，1H)，4.25(t，J＝6Hz，2H)，3.45(m，2H)，3.11(s，9H)，2.48(m，2H)，2.25(m，2H)。ES I-MS：m/z 249(100％，M²⁺)。

The nail was treated under the same conditions as in example 8(322mg, 0.375mmol) to form the tetrazolium salt except that the reaction was heated at 40 ℃ overnight to yield the desired tetrazolium salt (478mg, > 100%).

¹³C NMR(100MHz，DMSO d₆)：151，139.45，134.71，125.14，121，115.39，66.59，62.84，52.39，22.48：7.90 ¹H NMR(400MHz，DMSO-d₆)，8.74(d，J＝2.1Hz，1H)，7.95(m，5H)，7.78(d，J＝2.1Hz，1H)，7.51(d，J＝7.8Hz，1H)，7.39(m，2H)，4.2(m，2H)，4.08(m，2H)，3.96(s，3H)，3.53(m，2H)，3.19(m，2H)，3.11(s，9H)，2.91(s，9H)，2.24(m，2H)，1.87(m，2H)。ESI-MS：m/z 233(100％，M³⁺)。

Example 9

5- [4- (3-trimethylammonium) propoxy-3-methoxy-phenyl tribromide]-3- [2- (3-trimethyl) Aminonium) propoxy-phenyl]Preparation of (E) -2- (6-ethoxy-benzothiazol-2-yl) -2H-tetrazolium salt Prepare for

At room temperature, to the nail(from example 7, 1.18g, 1.43mmol) to a slurry in acetonitrile (15mL) was added n-bromosuccinimide (300mg, 1.69 mmol). After 1 hour, all materials dissolved. After 3 hours, an orange precipitate formed. The reaction was stirred overnight. The precipitate was filtered and washed with acetonitrile (3X 5 mL). The resulting orange product was dried under vacuum overnight. (1.07g, 83%)

¹³C NMR(100MHz，DMSO d₆)：151，139.45，134.71，125.14，121，115.39，66.59，62.84，52.39，22.48¹H NMR(400MHz，DMSO-d₆)：8.08(d，J＝8.2Hz，1H)，8.04(d，J＝I.7Hz，1H)，7.94(m，3H)，7.79(b s，1H)，7.55(d，J＝8.2Hz，1H)，7.40(d，J＝8.2Hz，1H)，7.30(dd，J＝8.2Hz，J＝I.7Hz，1H)，4.23(t，J＝6Hz，2H)，4.15(q，J＝8.2Hz，2H)，4.10(m，2H)，3.95(s，3H)，3.57(m，2H)，3.22(m，2H)，3.15(s，9H)，2.95(s，9H)，2.30(m，2H)，1.90(m，2H)，1.4(t，J＝8.2Hz)。ES I-MS：m/z 221(100％，M³⁺)。

The solubility of the tetrazolium salts prepared in examples 1-9 was determined as follows: 0.2M phosphate buffer (pH 7.5) was added to a known amount of each compound at room temperature until the compound was dissolved. The results are shown in Table 1, where they are compared to WST-4 (thiazolyl tetrazolium salt of Dojindo laboratories), HTC-045 (thiazolyl tetrazolium salt of Bayer Corp.), and modified HTC-045 (where the methoxy group is replaced by a 3-sulphonate propoxy group) (see FIG. 3).

TABLE 1

Example number (counterion)	Solubility (mM)
1 (Bromoino nitrite)	33
		2 (bromotetrafluoroborate)	5
3 (Bromoino nitrite)	9
		4 (Bromoino nitrite)	167
5 (Bromoino nitrite)	5
		6 (dibromo nitrite)	181
7 (dibromo nitrite)	169
		8 (dibromo nitrite)	290
9 (tribromide ion)	260
		WST-4	118
HTC-045	21
		Modified HTC-045	8

Although an improvement in the solubility of tetrazolium salts is achieved by the addition of trialkylammonium propoxy substituents to thiazolyl tetrazolium salts, such improvements are not readily predictable based on the prior art. The prior art would predict that the addition of a 3-sulfonate propoxy group to the molecule would increase the water solubility of the molecule. Dojindo Laboratories have used the introduction of sulfonic acid groups into molecules. However, substitution of the 3-sulfonate propoxy group for the methoxy group on the phenyl attached to the tetrazole ring (see modified HTC-045, FIG. 3) resulted in lower solubility (8mM) than HTC-045(21 mM). This phenomenon is therefore not evident in our molecular structure. We have found that different polar groups will improve water solubility. However, other unexpected effects are observed, which will be discussed below.

The replacement of methoxy by trimethylammonium propoxy increased the solubility from 21mM (HTC-045) to 33mM (example 1) and the addition of a second trimethylammonium propoxy group (example 6) significantly increased the solubility to 181 mM. However, when nitrate was replaced with tetrafluoroborate counterion, the solubility decreased significantly. (comparative example 2 with example 1). The solubility of the tetrazolium salt of example 1 decreased when the trimethylammonium propoxy group was changed to the dimethylammonium propoxy group (example 3).

Surprisingly, a significant increase in solubility due to the addition of two trimethylammonium propoxy groups (example 6) was also found in example 4(167mM) where only one trimethylammonium propoxy group was included, but the substituted thiazolyl group was replaced by unsubstituted benzothiazolyl group. However, the further addition of thiazolyl was found to have poor solubility (5mM), as shown in example 5 where substituted naphthyl thiazolyl was used. In another unexpected result, as shown in example 7, the addition of two trimethylammonium groups and one benzothiazolyl group (both of which result in a significant increase in solubility) appears to have no complementary effect. Example 7 had a solubility of 167 mM.

Another significant increase in solubility was obtained when the ethoxy group on the benzothiazolyl group of example 7 was replaced with the bromo group in example 8. The solubility increased from 169mM to 290mM, with the highest values shown here. Similar results were found when the counterion (nitrite ion) from example 7 was replaced by bromide ion from example 9. The solubility increased from 169mM to 260 mM. It appears that the inclusion of bromine appears to be an effective means of improving the solubility of such tetrazolium salts.

The compounds prepared and tested for their solubility had slightly different spectral properties. Each of the formazans in Table 1, which were formed from tetrazolium salts and tested for solubilityThe absorption spectrum of (a) was measured with a Hewlett packard Model 8453 diode array UV visible spectrophotometer. A solution of 100. mu.M tetrazolium salt was prepared in 100mM potassium phosphate buffer. After addition of a 5-fold excess of ascorbate to convert tet salts to their formazansThen, the nail is measuredSpectrum of light. The results are given in fig. 5a and b.

As indicated previously, formazan derived from thiazolyl tetrazolium saltsLight with a wavelength greater than 640nM is intended to be absorbed and a flat response is desired. For comparison, the absorbance curves for HTC-045 and WST-4 have been plotted in FIGS. 5a and b. WST-4 showed peak absorbance in the range of about 560nM, while HTC-045 showed a more gradual absorbance between about 460nM and 660 nM. However, its solubility is less than WST-4. Addition of trimethylammonium propoxy group changed the spectral curve. Examples 1, 2, 3, 4, 6, 7 and 8 have lower absorbance than HTC-045, but the curves are flatter throughout the range shown. It can be seen that the tet salt of example 5, which includes a naphthyl thiazolyl group, not only has low solubility, but also shows an ascending absorbance versus wavelength curve, indicating no flat areas comparable to HTC-045 or the modified compounds of examples 1, 2, 3, 4, 6, 7 and 8. Example 9 shows a spectral curve with a more gradual response between 400-640nM and shows to be highly soluble.

Ideally, the tetrazolium salt should produce formazan with high absorbance over a broad range of wavelengthsSo that the color displayed when the analyte reacts can be easily and reliably measured. Therefore, a relatively flat curve is highly desirable. From nails obtained from the tet salts testedAs can be seen, they all (with the possible exception of example 5) show promise as useful indicators. The tetrazolium salt of example 5 is a novel compound and may be used as an indicator, but it may not be preferred compared to those with a more uniform spectral profile and greater solubility.

Use of tetrazolium salts

While tetrazolium salts are useful indicators for many applications, they are particularly valuable when used in test strips for measuring analytes in biological samples. One important application is the measurement of the glucose content of blood.

The test strip typically comprises a carrier and a reactant composition. The reactant (e.g., glucose dehydrogenase) and a cofactor (e.g., NAD or PQQ) are reacted with the analyte (e.g., glucose) in the biological sample and a mediator (e.g., PMS or diaphorase) reduces the thiazolyl tetrazolium salt to its corresponding colored formazan. The resulting color is typically most accurately measured using a gauge designed for that purpose. A light source (e.g., an LED) provides incident light onto the test strip. The light reflected from the test strip is measured by a photodetector and correlated with the amount of analyte that has reacted.

The thiazolyltetrazole indicators of the present invention provide a broad spectrum that can be obtained from the indicators disclosed in U.S. Pat. No. 5,126,275 and other references mentioned earlier. However, as shown in the examples, many of the novel thiazolyl tetrazole indicators have improved solubility compared to HTC-045, thereby providing faster test times and more accurate results.

The invention will be summarized in the following alternative embodiments.

Alternative embodiment A

A compound having the formula

Wherein:

a is a counterion

a＝1-3

b＝0-3

X is 1-6C alkyl or heteroalkyl

R₁1-6C alkyl

R₂And R₃One is alkyl and the other is XN + H_b(R₁)_3-bOr R is₂And R₃Formation of methylenedioxy

R₄And R₅One being halogen and the other being halogen-substituted 1-6C alkyl, R₄And R₅One or two of them being XN + H_b(R₁)_3-bOr R is₄And R₅A linked form aromatic or heteroaromatic ring or a substituted aromatic or substituted heteroaromatic ring.

Alternative embodiment B

Compounds of alternative embodiment A having the general formula

Wherein:

a is a counterion

X is 1-6C alkyl

a＝1-3

b＝0-3

R₁1-3C alkyl

R₂And R₃One being 1-4C alkyl and the other being XN + H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₄＝CHF₂

R₅halogen.

Alternative embodiment C

A compound of alternative embodiment B wherein

X is propyl

b is 0

R₁Is methyl

R₂And R₃Formation of methylenedioxy

R₄Is CHF₂

R₅Is Cl.

Alternative embodiment D

A compound of alternative embodiment B wherein

X is propyl

b is 1

R₁Is methyl

R₂And R₃Formation of methylenedioxy

R₄Is CHF₂

R₅Is Cl.

Alternative embodiment E

A compound of alternative embodiment B wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is a methoxy group

R₄Is CHF₂

R₅Is Cl.

Alternative embodiment F

Compounds of alternative embodiment A having the general formula

Wherein:

a＝1-3

a is a counterion

X is 1-6C alkyl

b＝0-3

R₁1-3C alkyl

R₂And R₃One is alkyl and the other is XN + H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₄and R₅One of which is halogen and the other is halogen-substituted 1-6C alkyl, or R₄And R₅Is hydrogen

R₆1-4C alkoxy, hydrogen or halogen.

Alternative embodiment G

A compound of alternative embodiment F wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is a methoxy group

R₄、R₅And R₆Is hydrogen.

Alternative embodiment H

A compound of alternative embodiment F wherein

X is propyl

R₁Is methyl

b is 0

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄And R₅Formal aromatic ring

R₆Is methoxy.

Alternative embodiment I

A compound of alternative embodiment F wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄And R₅Is hydrogen

R₆Is an ethoxy group.

Alternative embodiment J

A compound of alternative embodiment F wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄And R₅Is hydrogen

R₆Is bromine.

Alternative embodiment K

The compound of alternative embodiment a wherein the at least one counterion is nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen sulfate, hydrogen carbonate, methanesulfonate, fluoroborate, bromide, chloride, iodide, or a combination thereof.

Alternative Process L

Method for measuring an analyte contained in a biological fluid, said analyte being adapted to produce a proportional color change after reaction, said method comprising the following measures:

providing as an indicator a tetrazolium salt having the general formula:

wherein:

a is a counterion

a＝1-3

b＝0-3

X is 1-6C alkyl or heteroalkyl

R₁1-6C alkyl

R₂And R₃One is alkyl and the other is XN + H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₄and R₅One being halogen and the other being halogen-substituted 1-6C alkyl, R₄And R₅One or two of them being XN + H_b(R₁)_3-bOr R is₄And R₅Linked to form an aromatic or heteroaromatic ring or a substituted aromatic or substituted heteroaromatic ring; and

determining the concentration of the analyte in the biological fluid by means of the tetrazolium salt as an indicator.

Alternative method M

The method of alternative method L, wherein the analyte is glucose.

Alternative method N

The method of alternative method M, wherein the biological fluid is whole blood.

Alternative Process O

The method of alternative Process M, wherein the tetrazolium salt has the general formula

Wherein:

a is a counterion

X is 1-6C alkyl

a＝1-3

b＝0-3

R₁1-3C alkyl

R₂And R₃One being 1-4C alkyl and the other being XN + H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₄＝CHF₂

R₅halogen.

Alternative Process P

Process of alternative Process O wherein

X is propyl

b is 0

R₁Is methyl

R₂And R₃Formation of methylenedioxy

R₄Is CHF₂

R₅Is Cl.

Alternative method Q

Process of alternative Process O wherein

X is propyl

b is 1

R₁Is methyl

R₂And R₃Formation of methylenedioxy

R₄Is CHF₂

R₅Is Cl.

Alternative Process R

Process of alternative Process O wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is a methoxy group

R₄Is CHF₂

R₅Is Cl.

Alternative Process S

Process having alternative Process L of the general formula

Wherein:

a＝1-3

a is a counterion

X is 1-6C alkyl

b＝0-3

R₁1-3C alkyl

R₂And R₃One is alkyl and the other is XN + H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₄and R₅One of which is halogen and the other is halogen-substituted 1-6C alkyl, or R₄And R₅Is hydrogen

R₆1-4C alkoxy, hydrogen or halogen.

Alternative method T

Method of alternative method S, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is a methoxy group

R₄、R₅And R₆Is hydrogen.

Alternative method U

Method of alternative method S, wherein

X is propyl

R₁Is methyl

b is 0

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄And R₅Formal aromatic ring

R₆Is methoxy.

Alternative method V

Method of alternative method S, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄And R₅Is hydrogen

R₆Is an ethoxy group.

Alternative method W

Method of alternative method S, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄And R₅Is hydrogen

R₆Is bromine.

Alternative Process X

The method of alternative process L, wherein the at least one counterion is nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen sulfate, hydrogen carbonate, methane sulfonate, fluoroborate, bromide, chloride, iodide, or a combination thereof.

Alternative Process Y

A method for increasing the solubility of a thiazolyl tetrazolium salt wherein the thiazolyl group is attached to the third nitrogen atom of the tetrazolium ring, comprising adding at least one trialkylammonium alkoxy group as a substituent to the thiazolyl tetrazolium salt.

Alternative Process Z

The method of alternative Process Y, wherein the thiazolyl tetrazolium salt has a phenyl substituent at the third nitrogen atom and at a carbon atom of the tetrazolium ring.

Alternative Process AA

The process of alternative process Z, wherein the at least one trialkylammonium alkoxy group is a trimethylammonium propoxy group.

Alternative method BB

The method of alternative process Z, wherein the at least one trialkylammonium alkoxy group is a substituent of the phenyl group.

Alternative method CC

The method of alternative method Z, wherein at least one trialkylammonium alkoxy group is a substituent of the two phenyl substituents.

Alternative method DD

The method of alternative process Z, wherein the thiazolyl is benzothiazolyl.

Alternative Process EE

The method of alternative method Z, wherein the thiazolyl tetrazolium salt has a counterion to nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen sulfate, hydrogen carbonate, methane sulfonate, fluoroborate, bromide, chloride, iodide, or a combination thereof.

Alternative method FF

The method of alternative method EE, wherein said counterion is a bromonitrite, dibromonitrite, or tribromo ion.

Claims (16)

1. A compound having the general formula:

wherein:

a is a counterion

X ═ l-6C alkyl

a＝1-3

b＝0-3

R₁Is ═ 3C alkyl

R₂And R₃One of which is 1-4C alkyl and the other is XN⁺H_b(R₁)_3-b，

R₄＝CHF₂

R₅Halogen.

2. The compound of claim 1, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄Is CHF₂

R₅Is Cl.

3. A compound having the general formula:

wherein:

a＝1-3

a is a counterion

X is 1-6C alkyl

b＝0-3

R₁1-3C alkyl

R₂And R₃One of which is alkyl and the other is XN⁺H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₇and R₈One of which is halogen and the other is halogen-substituted 1-6C alkyl, or R₇And R₈Is a hydrogen atom, and is,

R₉1-4C alkoxy, hydrogen or halogen.

4. The compound of claim 3, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₇、R₈And R₉Is hydrogen.

5. The compound of claim 3 which is 5-benzo [1, 3-dioxol-5-yl ] -3- [2- (3-trimethylammonium) propoxy-phenyl ] -2- (benzothiazol-2-yl) -2H-tetrazole bromonitrite.

6. The compound of claim 3, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₇And R₈Is hydrogen

R₉Is an ethoxy group.

7. The compound of claim 3, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₇And R₈Is hydrogen

R₉Is bromine.

8. The compound of any one of claims 1-7, wherein the at least one counterion is nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen sulfate, hydrogen carbonate, methane sulfonate, fluoroborate, bromide, chloride, iodide, or a combination thereof.

9. Use of a tetrazolium salt for the preparation of a product for the determination of an analyte contained in a biological fluid, said analyte being adapted to undergo a proportional colour change after reaction, wherein said tetrazolium salt is used as an indicator and has the general formula

Wherein:

a is a counterion

X ═ l-6C alkyl

a＝1-3

b＝0-3

R₁Is ═ 3C alkyl

R₂And R₃One of which is 1-4C alkyl and the other is XN⁺H_b(R₁)_3-b，

R₄＝CHF₂

R₅(ii) a halogen element selected from the group consisting of,

and determining the concentration of the analyte in the biological fluid by means of the tetrazolium salt as an indicator.

10. The use of claim 9, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₄Is CHF₂

R₅Is Cl.

11. Use of a tetrazolium salt in the manufacture of a product for use in the determination of an analyte contained in a biological fluid, said analyte being adapted to produce a proportional color change after reaction, wherein said tetrazolium salt acts as an indicator and has the general formula:

wherein:

a＝1-3

a is a counterion

X is 1-6C alkyl

b＝0-3

R₁1-3C alkyl

R₂And R₃One being alkyl and the other being XN⁺H_b(R₁)_3-bOr R is₂And R₃The formation of a methylenedioxy group,

R₇and R₈One being halogen and the other being halogen-substituted 1-6C alkyl, or R₇And R₈Is a hydrogen atom, and is,

R₉1-4C alkoxy, hydrogen or halogen,

and determining the concentration of the analyte in the biological fluid by means of the tetrazolium salt as an indicator.

12. The use of claim 11, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₇、R₈And R₉Is hydrogen.

13. The use according to claim 11 wherein the tetrazolium salt is 5-benzo [1, 3-dioxol-5-yl ] -3- [2- (3-trimethylammonium) propoxy-phenyl ] -2- (benzothiazol-2-yl) -2H-tetrazole bromonitrite.

14. The use of claim 11, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₇And R₈Is hydrogen

R₉Is an ethoxy group.

15. The use of claim 11, wherein

X is propyl

b is 0

R₁Is methyl

R₂Is a trimethylammonium propyl group

R₃Is methyl

R₇And R₈Is hydrogen

R₉Is bromine.

16. The use of any one of claims 9-15, wherein the at least one counterion is nitrite, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen sulfate, hydrogen carbonate, methane sulfonate, fluoroborate, bromide, chloride, iodide, or a combination thereof.