DE3248043A1 - Fluorogenic phosphoric esters, process for their preparation, and process and composition for the detection and fluorometric determination of phosphates - Google Patents

Abstract

Novel fluorogenic phosphoric esters of 7-hydroxycoumarine derivatives, which are also cleaved by acidic phosphatases, forming a highly fluorescent, coloured anion, and can be used in an improved determination method for phosphatases.

Description

Fluorogenic phosphoric acid esters, process for their production

position as well as methods and means for detection and for fluorometric Determination of phosphatases The invention relates to new fluorogenic phosphoric acid esters, which under the influence of phosphatases a strongly fluorescent colored anion and can be used to improve the determination of phosphatases can.

Fluorimetric method for the determination of phosphatase activities have long been known (Guilbault, Enzymatic Methods of Analysis, Pergamon Press 1970). The fluorimetry is so sensitive that it is also extremely slight Enzyme concentrations can be detected and determined. Known fluorometric Phosphatase reagents used are phosphoric acid esters of umbelliferone (G.G.Guilbault et al., Anal. Letters 1 (1968) 333), 4-methylumbelliferone (H.N. Fernley, P.G.Walker, Biochem. J. 97 (1965) 95), of flavonol (D.B. Land, E. Jakim, Anal. Biochem. 16 (1966) 481), des .t-naphthol (D.W. Moss, Clin. Chim.

Acta 5 (1960) 283), B-naphthol (L.J. Greenberg, Biochem Biophys. Res. Comm. 9 (1962) 430) and 3-0-methyl-fluorescein (H.D. Hill et al., Anal. Biochem. 24 (1968) 9).

However, these known detection methods are for various reasons not completely satisfactory: the phosphoric acid esters of the prior art mentioned are only split rapidly enough in the alkaline pH range to be in clinical Analytics to be used. For diagnosis, for example for early detection However, from prostate carcinoma, it would be important to also have acid phosphatase activities to determine. With many of the-known fluorogenic phosphoric acid ester occurs over it In addition, there is a disruptive overlap between the fluorescence of the ester and that of the hydrolysis resulting anion. Otherwise, the reagents of the prior art require in some cases also a considerable outlay in terms of equipment.

In US 3 772 340 bis-coumarinyl phosphates are described, which for fluorimetric determination of phosphodiesterases should be used, as they are in bacteria-containing Urine occur. A disadvantage of these compounds is their poor solubility in water, which limits their practical applicability.

The object of the present invention is to provide reagents for the fluorometric Provide detection of phosphatases which of those mentioned above Disadvantages of the prior art are free. This object is achieved by means of the inventive Phosphoric acid ester dissolved.

The invention relates to new phosphoric acid esters of the formula wherein M represents hydrogen, an alkali metal or ammonium ion which can be substituted by 1-4 C1-C2-alkyl radicals optionally containing a hydroxyl group, X for -0- or -NQ-, Q for hydrogen, optionally by 1-2 OH -C1-C4-alkyl substituted groups or for C1-C4-alkoxycarbonyl, R for hydrogen, chlorine, bromine, cyano or carbamoyl, A for cyano, C1-C4-alkoxycarbonyl, carbamoyl optionally substituted by 1-2 C1-C4-alkyl radicals or sulfamoyl, C1-C4-alkylsulfonyl, benzylsulfonyl, phenylsulfonyl, p-tolylsulfonyl, p-chlorophenylsulfonyl, sulfo, nitro, phenyl, p-tolyl, sulfophenyl or for one optionally with 1 - 2 C1-C4-alkyl, 1 - 2 chlorine, C1-C4-alkoxy, carboxyl, C1-C4-alkoxycarbonyl, cyano, trifluoromethyl, C1-C4-alkylsulfonyl, cyclohexyl, phenyl or sulfo-substituted benzoxazol-2-yl-, benzthiazol-2-yl-, thiazol-2 -yl-, benzimidazol-2-yl-, quinazol-4-on-2-yl-, 5-phenyl-1,3,4-thiadiazol-2-yl-, 5-phenyl-1,3,4-oxadiazole -2-yl, 2- or 4-pyridyl radical and R1 represent hydrogen or a sulfonic acid group.

Preferred are those compounds of the general formula (I) in which A for one optionally substituted by 1-2 C1-C4-alkyl, 1-2 chlorine, C1-C4-alkoxy, C1-C4 -Alkylsulfonyl, carboxyl, C1-C4-alkoxycarbonyl, cyclohexyl, phenyl or sulfo-substituted Benzoxazol-2-yl-, benzthiazol-2-yl-, thiazol-5-yl-, benzimidazol-2-yl-, quinazol-4-on-2-yl-, 5-phenyl-1,3,4-thiadiazol-2-yl or 4-pyridyl radical.

Particularly preferred are those compounds of the formula (I) in which A for one optionally represented by methyl, ethyl, chlorine, methoxy, ethoxy or sulfo substituted benzthiazol-2-yl or benzoxazol-2-yl radical and R. stand for CN.

The phosphoric esters of the formula (I) according to the invention can be prepared by adding hydroxy compounds of the formula in which X, A, R and R1 have the abovementioned meaning, initially reacted with a halide of pentavalent phosphorus (preferably in the presence of an inorganic or organic acid scavenger) and the dihalophosphonyloxy compound obtained is then hydrolyzed.

Examples of halides of pentavalent phosphorus are phosphorus oxide trichloride, Phosphorus oxide tribromide or phosphorus pentachloride into consideration, the latter is preferably used when the molecule contains a sulfo group (temporary Conversion into the corresponding sulfochloride).

Inorganic and organic bases are suitable as acid scavengers, for example anhydrous potassium or sodium carbonate, magnesium oxide, triethylamine, collidine, Picoline mixtures, pyridine and dimethylaniline.

The reaction is preferably carried out in the presence of a solvent or diluent in the temperature range from -5 to 1000 ° C., preferably in the case of the phosphorus oxide trihalides carried out at 0-300C, in the case of PCl5 preferably at 20-900C.

Suitable solvents and diluents under the reaction conditions are inert organic liquids such as toluene, chlorobenzene, dichlorobenzene, dichloromethane, Chloroform, carbon tetrachloride, dichloroethane, dichloropropane, trichlorethylene, acetonitrile, Dioxane, tetrahydrofuran or one of the above-mentioned organic bases such as pyridine.

About the reaction of the pentavalent phosphorus halide with 2 molecules To avoid (II), it is advisable to ensure an excess during the reaction of phosphorus halide by gradually adding (II) as the last component, optionally pre-dissolved in one of the abovementioned solvents.

The hydrolysis of the intermediate dichlorophosphonyloxy compounds to (I) can be done more gently, e.g. by carefully warming up with water to about 50 ° C but at temperatures of 0 - 250C by neutralization with alkali hydroxide or Alkali carbonate solution or aqueous ammonia can be carried out. Then steams the alkali metal or ammonium salts obtained are brought to dryness in vacuo or recovered by acidification with a mineral acid such as sulfuric acid the free acid (I with M = H) by filtering off.

The above method for the O-phosphorylation of (II) essentially agrees with that in Houben-Weyl, Methods of Organic Chemistry, Volume XII / 2 (1964) pp.

172-177 described general method for the phosphorylation of aromatic hydroxy compounds.

The starting compounds of the formula (II) are known (US Pat. No. 3,521 187 compound 11; DE-OS 27 02 337; DE-OS 30 44 128, DE-OS 21 00 295 and DE-OS 3 229 301) or according to the methods mentioned there.

The new compounds of formula (I) are water-soluble, slightly blue (R = H, Cl, Br) or greenish (R = CN, CONH2) fluorescent and mostly only slightly colored substances, which in the aqueous medium of alkaline or acid phosphatase to strong fluorescent, deep yellow to red colored mesomeric anion of the formulas be split.

The new phosphatase reagents of the formula (I) are therefore suitable for direct phosphatase detection in biological materials. Particularly beneficial can you for the quantitative fluorometric determination of the alkaline or acid phosphatases can be used in clinical analysis.

Compared to the prior art phosphatase reagents the new phosphoric acid esters of the formula (I) have significant advantages: 1. None disturbing overlap of the fluorimetrically to be followed in the enzymatic cleavage of (I) resulting, strong fluorescence of the hydroxylate (IIIa - IIIb) with the weak fluorescence of the reagent (I) used (large Stoke's shift).

2. Use of the simplest filter fluorimeter possible, as neither one Excitation by UV light, which usually causes the background fluorescence of the biological material makes disturbing noticeable, nor the use of complex monochromators or interference filters, as they are when using 3-O, methylfluorescein phosphate are needed.

3. Formation of the deeply colored fluorescent hydroxylate anion (IIIa - IIIb) surprisingly even under weakly acidic conditions (e.g. at pH 5.2). This makes it possible for the first time to fluorimetrically determine the activity of acid phosphatases to be followed in continuous measurement with such a high detection sensitivity, as previously only known in more time-consuming non-kinetic processes.

The phosphoric esters according to the invention can be used to determine the Phosphatase activity in various body fluids (serum; cerebrospinale Liquid; Urine). However, it is also possible to use the inventive Reagents analogous to the teaching of US Pat. No. 3,772,340 for measuring the bacterial concentration to be used in liquids (e.g. bacterially infected urine). To this end are initially in a manner known per se (e.g. osmotic shock; spheroplast formation) the bacterial enzymes, including phosphatases, are released. From the means the phosphatase activity determined by the compounds according to the invention can then be Draw conclusions about the number of bacteria present.

When determining the phosphatase activity, the inventive Compounds because of their good water solubility in the form of aqueous solutions, if appropriate buffered to the desired pH value (e.g. approx. 4.5 -7 for acid phosphatases; approx. 7-10 for alkaline phosphatases). However, it is also possible to apply the new phosphoric acid esters to a carrier in a manner known per se and to allow the detection reaction to proceed in a heterogeneous phase.

Suitable carrier materials are e.g. filter paper or on a Plastic (e.g. polystyrene) applied with an organic binder Silicon dioxide. In this way, test strips can be produced that are after the application of the liquid to be examined begin to fluoresce and their Fluorescence by means of the common fluorimeter can be measured. The quantitative determination of phosphatase activity in a unknown sample is possible if one looks at the time course of the fluorescence intensity in the aqueous solution or

on the test strip with that of standards with known phosphatase content compares.

In the tests described below, the following phosphatase preparations from Sigma Chemical Co. are used: a) alkaline Phosphatase (EC 3.1.3.1): Type I-S (P7640) (from bovine testicular mucosa) b) acid phosphatases (EC 3.1.3.2): Type III (P6760) Type IV-S (P1146) Type V (P1267) (from wheat seedlings, Potatoes, milk and beef testicles).

EC is the name for the internationally valid enzyme catalog.

Example 1 800 ml of dry pyridine are mixed with 39 g (23 ml; 0.25 mol) of phosphorus oxychloride dropwise with cooling and stirring at 0 ° -50 ° C. and then at the same temperature within about 30 minutes in portions with 80 g (0.25 mol ) 3-Benzthiazolyl-4-cyano-7-hydroxy-coumarin was added and the mixture was stirred at 0 ° -50 ° C. for 3 h. It is poured onto 2 l of ice water, 66 g (45 ml; 0.75 mol) of 45% strength by weight sodium hydroxide solution are added dropwise with stirring, a pH of approx. 7 being established, the mixture is allowed to stir for 10 h, evaporate it to dryness with the help of a rotary evaporator at 400C in vacuo and dry the residue further at 400C in vacuo to constant weight. The residue (185 gy consists of 44 g of sodium chloride and 141 g (96% of theory) of the compound of the formula For purification, the residue is recrystallized from ethanol-water (4: 1). Orange-colored crystals with a melting point of 3250 ° C. (decomp.) Are obtained.

The compound is soluble, e.g. in water, methanol, dimethylformamide, Dimethyl sulfoxide and ethylene glycol monomethyl ether.

Selects for fluorimetric monitoring of enzymatic hydrolysis excitation light with a maximum at 510 nm and the fluorescence measured at 595 nm. At this wavelength, only the fluorescence of the enzymatically cleaved Substrate detected.

In an analogous manner, using the appropriate starting compounds, the following phosphoric acid esters of the general formula are obtained manufactured: Example MRAX melting point (Decomposition) ° C 2 Na H # O 301 ° 3 K CN # O 309 ° 4 Na H # O 302 ° 5 Na CN # O 310 ° 6 NH4 H # O 318 ° 7 K CN # O 304 ° 8 Na CN # O 308 ° Example MRAX melting point (Decomposition) ° C 9 Li H # O 300 ° 10 Na CN # O 330 ° 11 K CN # O 317 ° 12 Na CN # O 315 ° 13 NH4 H # N-C4H9-n 300 ° 14 KH # N-CH3 313 ° 15 Na H # N-COOC2H5 322 ° Example MRAX melting point (Decomposition) ° C 16 H2N (C2H4OH) 2 CN # O 262 ° 17 HN (C2H5) 3 H # O 278 ° 18 Na CN # O 307 ° 19 K CN # O 316 ° 20 Na CN CN O 327 ° 21 K CN COOCH3 O 304 ° 22 Na CN SO2CH3 O 312 ° 23 Na CN SO2-C6H5 O 326 ° 24 K CN # O 305 ° EXAMPLE 25 160 ml of dry pyridine are mixed with 11.5 g of phosphorus pentachloride (55 mmol) and then with 22.6 g (50 mmol) of 3- (5-methyl-7) over the course of 30 minutes at 0-10.degree. C. with cooling and stirring -sulfo-benzoxazol-2-yl) -7-hydroxycoumarin-pyridinium salt is added and the mixture is heated to 80 ° C. for 1 hour, a clear solution being formed temporarily. It is poured onto 400 ml of ice water, 30 g (20 ml; 335 mmol) of 45% strength by weight sodium hydroxide solution are added dropwise with stirring, a pH of about 7 being established, and the mixture is allowed to stir for 10 h. The filtrate is evaporated to dryness with the aid of a rotary evaporator and further dried at 50 ° C. in vacuo to constant weight. In addition to 16 g of sodium chloride and approx. 1 g of Na2HPO4, the residue contains 25 g (95% of theory) of the compound of the formula To follow the enzymatic hydrolysis by phosphatases, excitation is carried out in the fluorimeter at approx. 450 nm and the emission is measured at approx. 495 nm. In practice, only the fluorescence of the enzymatically cleaved substrate is recorded.

In an analogous manner, using the corresponding starting compounds, the following sulfo-containing phosphoric acid esters of the general formula are obtained manufactured: Example MRA 26 Na CN ßX 27 KH know OCH3 28 Na CN ß> 29 Na H tt Cl N Cl 30 Na CN EXAMPLE 31 In a fluorimeter, the cuvette is filled at 230C with 3 ml of a 0.1 mmolar aqueous citrate-buffered (0.05 mol citrate / liter) solution of the compound of Example (1) at pH 5.2, the excitation wavelength is set to 510 nm and the Emission wavelength to 595 nm, add 0.1 ml of the body fluid to be determined for acidic phosphatase activity (serum, cerebrospinal fluid), the content of which should be in the order of 0.1 mg phosphatase per ml, and tracks the change over time Fluorescence intensity over a period of about 1 - 3 minutes in comparison to previously prepared calibration curves. The initially linear increase in fluorescence intensity is a direct measure of the enzyme activity. The detection limit for acid phosphatases in the arrangement described above is approx. 1x10 -5 enzyme units per ml. With known phosphatase reagents, a kinetic measurement of the acid phosphatase activity with such sensitivity has not been possible until now. Non-kinetic processes are similarly sensitive, but more tedious and complex.

Acid phosphatase from potatoes (enzyme catalog no. 3.1.3.2, Type IV-S), commercially available from Sigma Chemical Co. under No. P-1146, with an activity of 1.9 enzyme units / mg can be used (defined is the Enzyme unit as that amount of the enzyme which 1; iMol p-nitrophenyl phosphate per Minute at pH 4.8 and 370C).

Claims (10)

Claims Compounds of the general formula wherein M represents hydrogen, an alkali metal or ammonium ion which can be substituted by 1-4 C1-C2-alkyl radicals optionally containing a hydroxyl group, X for -0- or -NQ-, Q for hydrogen, optionally by 1-2 OH -C1-C4-alkyl substituted by groups or for C1-C4-alkoxycarbonyl, R for hydrogen, chlorine, bromine, cyano or carbamoyl, A for cyano, C1-C4-alkoxycarbonyl, carbamoyl optionally substituted by 1-2 C1-C4-alkyl radicals or sulfamoyl, C1-C4-alkylsulfonyl, benzylsulfonyl, phenylsulfonyl, p-tolylsulfonyl, p-chlorophenylsulfonyl, sulfo, nitro, phenyl, p-tolyl, sulfophenyl or for one optionally by 1 - 2 C1-C4-alkyl, 1 - 2 chlorine , C1-C4-alkoxy, carboxyl, C1-C4-alkoxycarbonyl, cyano, trifluoromethyl, C1-C4-alkylsulfonyl, cyclohexyl, phenyl or sulfo-substituted benzoxazol-2-yl-, benzthiazol-2-yl-, thiazol-2-yl -, benzimidazol-2-yl-, quinazol-4-on-2-yl-, 5-phenyl-1,3,4-thiadiazol-2-yl-, 5-phenyl-1,3,4-oxadiazol-2 -yl, 2- or 4-pyridyl radical and R1 f stand for hydrogen or a sulfonic acid group. 2) Compound according to claim 1, characterized in that A is for one optionally with 1 - 2 C1-C4-alkyl groups, 1 - 2 chlorine, C1-C4-alkoxy, C1-C4-alkylsulfonyl, Carboxyl, C1 -C4 alkoxycarbonyl, cyclohexyl, phenyl or sulfonic acid radicals substituted Benzoxazol-2-yl-, benzthiazol-2-yl-, thiazol-5-yl-, benzimidazol-2-yl-, quinazol-4-on-2-yl-, 5-phenyl-1,3,4-thiadiazol-2-yl or 4-pyridyl radical. 3) Compound according to claim 1, characterized in that R is CN and A for one optionally by methyl, ethyl, chlorine, methoxy, Ethoxy or sulfo-substituted benzothiazol-2-yl or benzoxazol-2-yl radical. 4) Process for the preparation of compounds according to Claims 1 to 3, characterized in that there are hydroxy compounds of the formula in which X, A, R and R1 have the meaning given in claims 1 to 3, initially reacted with a halide of pentavalent phosphorus and the resulting dihalophosphonyloxy compound is then hydrolyzed. 5) Method according to claim 4, characterized in that the Reaction with a halide of pentavalent phosphorus in the presence of an inorganic one or organic acid scavenger. 6) Method according to claim 4 or 5, characterized in that one as halide of pentavalent phosphorus phosphorus oxide trichloride, phosphorus oxide tribromide or phosphorus pentachloride is used. 7) Method according to claim 5, characterized in that as Acid scavenger anhydrous potassium carbonate, sodium carbonate, magnesium oxide, triethylamine, Collidine, picoline mixtures, pyridine or dimethylaniline are used. 8) Test agents containing fluorogenic phosphoric acid esters for the fluorimetric Detection of phosphatases, characterized in that they are used as fluorogenic phosphoric acid esters Compounds according to claim 1 to 3 contain. 9) test agent according to claim 8, characterized in that the phosphoric acid ester are contained in a carrier material. 10) Method of determining phosphatase activity in a liquid Sample, characterized in that the sample to be examined is mixed with a compound according to claim 1 to 3 or a test agent according to claim 8 or 9 brings into contact and followed the time course of fluorescence activity in the sample.