CS273184B2 - Method of resorufin's derivative glycoside production - Google Patents

Abstract

A method of production glycoside from resorufin derivatives of general formula Ia or Ib, where R<1> = H, R<2>, R<3> and R<5> are independent H, halogen or C1-C5 alkyl, R<4> and R <6> are independent H, halogen, cyano, alkyl, alkoxyl, carboxyl, alkoxycarbonyl, carboxyalkyl or alkoxycarbonylalkyl, where all alkyls and alkoxyls contain 1 to 5 C-atoms or the carboxamide group possibly the single or double substituted alkyl alkoxyalkyl, carboxyalkyl or alkoxycarbonylalkyl group, where the alkyl and alkoxy group always contain 1 - 5 C-atoms, whereas in the case of two substituents these may be linked causing a ring possibly interrupted also by O, N or S or the group -COO-(CH2CH2O)n-R<7>, where R<7> = H, C1-C5 alkyl and n = 1 - 4 and R<6> is sulphonyl or nitro- and Y = N or N -> O of the reaction of derivatives of resorufin of tautomeric formulas IIa and IIb with mono- or oligosaccharide with 2 to 10 monosaccharide units or its halogen derivative, where all OH groups are protected by cleavage of the protective groups from the occurring per-O-substituted glycosides. The compounds of general formula Ia and Ib are especially good as chromogenic and fluorogennic substrates for determining the activity of corresponding glycosidases or enzymes of splitting oligosaccharides.<IMAGE>

Description

The present invention relates to a process for the production of glycosides of resorufin derivatives which find use in determining glycosidase activity.

Gycosidases fulfill multiple physiological functions in human and animal organisms.

For example, β-D-galactosidase plays an important role in the metabolism of carbohydrates, since it acts to hydrolyze lactose. In addition, β-D-galactosidase is a key enzyme in the degradation of glycolipids, mucopolysaccharides and glycoproteins.

Other physiologically important glycosidases include n-O-gul kososulfide, οΟ-Ο and β-D-glucosidase, as well as α-D-mannosidase.

In addition to being physiologically important, glycosidases have gained importance in the field of diagnostics and biotechnology in recent years. Thus, for example, these enzymes are increasingly used as so-called indicator enzymes in enzymatic immunoassays (enzymimmunoassay). Particularly preferred in this context is β-D-galactosidase (see, for example, Annals of Clinical Biochemistry 16, 221-240 (1979)).

Consequently, the determination of glycosidase activity plays an increasing role in both clinical chemistry and diagnostics. In general, the glycosidase-containing sample is mixed with a suitable substrate. The substrate is cleaved by the enzyme. One of the cleavage products is then determined in an appropriate manner. It can be determined either by the action of the enzyme liberated glycone or by aglycone. As a rule, the aglycone is determined.

Natural substrates of proven enzymes are often suitable as substrates. However, it is particularly preferred to use glycosides in which the aglycone is a spectroscopically readily detectable residue.

A number of substrates for glycosidases are known in which, after cleavage by glycosidase, aglycone can be determined spectroscopically in the visible or ultraviolet region of the spectrum or fluorometrically.

Thus, β-D-galactosidase substrates are described in Biochem. Z. 333, 209 (1960) phenyl-β-D-galactoside and some of its other aromatic-substituted derivatives (e.g. o-nitrophenyl and p-nitrophenyl-β-D-galactoside). Phenols released by hydrolysis are determined photometrically in the ultraviolet region or, in the case of nitrophenols, in the short-wave visible region of the spectrum. An oxidative coupling with an aminoantipyrine may be added as an indicator reaction (Analytical Biochem. 40, 281 (1971)).

For histochemical assays, naphthyl- [beta] -D-galactoside is used, for example, the use of the 1-naphthyl compound is described in Histochemistry 35, 199 (1973), using the 6-bromo-2-naphthyl derivative in J. Biol. Chem. 195, 239 (1952) and the use of naphthol- [beta] -D-galactoside in Histochemistry 37, 09 (1973). For visualization, the resulting naphthols are reacted with various diazonium salts to form azo dyes.

Determination of enzyme activity using fluorogenic substrates is widespread, since the sensitivity of fluorometric assays is several decimal orders higher than photometric methods. In many cases, fluorogenic substrates have to be used, for example in the determination of enzymatic activity in cells using automatic devices for cell differentiation (cytofluorometry) and in the analysis of immobilized enzymes by flow microfluorometry. In other cases, for example in determining the enzymatic labeling of test systems (enzymatic immunoassays), the multiplier effect of enzymatic catalysis using fluorogenic substrates is greatly enhanced.

Hitherto known fluorogenic substrates for β-D-galactosidase and other glycosidases contain, as fluorophores, derivatives of fluorescein, indoxyl or methylumbelliferone. However, these compounds have serious disadvantages for the kinetic analysis of complex systems. Disubstituted fluorescein derivatives are hydrolyzed in a multistep series of reactions. Monosubstituted fluorescein glycosides already fluoresce themselves. Indoxyl derivatives undergo a number of chemical reactions following their enzymatic cleavage, which also complicate kinetic analysis.

CS 273105 B2

Methylumbelliferone derivatives must be excited by ultraviolet radiation. It can interfere with the intrinsic fluorescence of biological or synthetic substances. In addition, ultraviolet excitation, especially in the case of laser optics, is expensive. Most fluorogenic substrates provide reaction products that exhibit only low solubility so that they are not suitable for kinetic analysis of enzyme activity where good solubility of the substrate and product is needed.

Therefore, there remains a need for substrates through which glycosidases can be readily, rapidly and reliably determined, and such substrates should, as far as possible, be applicable to both photometric and fluorometric assay methods. The object of the invention was to satisfy the aforementioned demand.

The problem is solved by novel glycosides of resorufin derivatives which are cleaved by glycosidases into a sugar residue and a resorufin derivative. The latter are compounds with good water solubility, which exhibit well-measurable absorption in the visible region of the spectrum and can moreover be easily excited for fluorescence.

The present invention provides a process for the preparation of glycosides of resorufin derivatives of the formulas Ia and Ib, respectively

(Ib) _t wherein the glycoside is a glycoside residue of a monosaccharide or oligosaccharide containing 2 to 10 1 2 3 5 monosaccharide units, R is hydrogen, each of R, R and R, which are the same or different, is hydrogen, halogen or an alkyl group of 1 up to 5 carbon atoms, each of the symbols 4 6

R and R, which are the same or different, are hydrogen, halogen, cyano, (C 1 -C 5) alkyl, (C 1 -C 5) alkoxy, carboxy, alkoxycarbonyl, carboxyalkyl or alkoxycarbonylamino, the latter three; alkyl or alkoxy radicals each containing from 1 to 5 carbon atoms, or a carboxamido group, optionally mono- or di-substituted by alkyl, alkoxyalkyl, carboxyalkyl or alkoxycarbonylalkyl radicals, in which the alkyl and alkoxy groups each contain 1 to 5 carbon atoms, in the presence of two substituents may be joined together to form a ring, which is optionally also interrupted by oxygen, nitrogen or sulfur, or a group of the general formula

- C00 - _{(CH2 -CH2} -O) _n - R ^7, wherein R ⁷ is hydrogen or an alkyl group having 1-5 carbon atoms and n is an integer from 1 to 4, wherein in addition R ^a denotes a nitro or sulfonyl and Y represents nitrogen or N-O.

The glycosides of the resorufin derivatives of the formulas Ia and Ib respectively are novel compounds. According to the invention, they can be prepared by resorphine derivatives of the general tautomeric formulas IIa and IIb

wherein R ⁷ to R 6 and Y are as defined above, reacted with a mono- or oligosaccharide containing from 2 to 10 monosaccharide units or a 1-halogeno derivative thereof, each hydroxy group being substituted by a protecting group customary in carbohydrate chemistry to form a peroxy- O-substituted glycosides from which the glycosides of resorufin derivatives of formula I are obtained in a manner known per se by cleavage of protecting groups.

The reaction of the compounds of formula II with per-O-substituted 1-halosaccharides is preferably carried out in the presence of acid binders such as an alkali metal hydroxide or carbonate in aqueous acetone or (under interfacial transfer conditions) in water / benzene or water / chloroform.

In addition, this process can be carried out by first converting the resorufin derivatives of formula II to an alkali metal salt or alkali metal alkoxide or an optionally substituted amine to the ammonium salts and then reacting with per-O-substituted alkali metal salts. -halosaccharides in a dipolar aprotic solvent such as acetone, dimethylsulfoxide, dichloromethane, tetrahydrofuran or dimethylformamide.

The synthesis of per-O-substituted glycosides from resorphin derivatives of formula II and per-O-substituted 1-halosaccharides has also proven to be useful in the addition of individual silver salts or mixtures of silver salts (silver oxide, silver carbonate, stearate carbonate). units.

% on silver, Celite, silver triflate, silver salicylate) and / or individual mercury salts or mixtures of mercury salts (mercuric bromide, mercuric cyanide, mercuric acetate, mercuric oxide), optionally using desiccants such as calcium chloride, molecular sieve or Drierit, in solvents , such as methylene chloride, chloroform, benzene, toluene or dioxane. In the synthesis of glycosides bound in the t-position, it is expedient to melt the compound of formula II with a carbohydrate whose hydroxy groups are substituted with protecting groups, preferably acetyl, in the presence of a Lewis acid such as tin tetrachloride, aluminum chloride and preferably zinc chloride (cf. Chem. Ber. 66, 37B-383 (1933) and Methods in Carbohydr. Chem. 2, 345-347 (1967)). The temperature is set to between 80 and 150 ° C, preferably between 110 and 130 ° C.

The per-O-substituted glycosides of the resorphin derivatives of formula II thus obtained are also novel compounds.

The cleavage of protecting groups with per-O-substituted glycosides to form glycosides of formula I is carried out by methods common in carbohydrate chemistry (see, for example, Advances Carbohydrate Chem. 12, 157 (1975)), for acyl groups, for example, by sodium methoxide or barium methoxide, or ammonia in methanol.

Halogen in the definition of R ¹ to R means fluorine, chlorine, bromine and iodine, preferably chlorine and bromine.

The alkyl group in the definition of R 1 to R 1 preferably contains 1 to 3 carbon atoms, with methyl groups being particularly preferred.

The alkoxy groups in the definitions of R ⁴ and R ⁶ preferably contain 1 to 3 carbon atoms, with methoxy being most preferred.

The alkoxy or alkyl radicals of the alkoxycarbonyl, carboxyalkyl and alkoxycarbonylalkyl radicals in the definition of the substituents R @ ⁴ to R @ ⁴ also preferably contain from 1 to 3 carbon atoms, with methoxy or methylene being particularly preferred.

Acetyl, benzoyl, benzyl or trimethylsilyl are particularly suitable as the protective group customary in carbohydrate chemistry.

Alkyl-, alkoxyalkyl-, carboxyalkyl- and alkoxycarbonylalkyl groups are suitable as carboxamido substituents, preferably having 1 to 3 carbon atoms.

Any mono- and oligosaccharide containing from 2 to 10 monosaccharide units which can be cleaved off from the resorphin backbone by the corresponding glycosidase is suitable as the glycosidic residue which is attached to the resorphin derivatives of formula II to form the glycosides of formula I. Examples of glycosides which can be used according to the invention are? -D-galactopyranoside,? -D-galactopyranoside,? -D-glucopyranoside,? -D-glucopyranoside and? -D-mannopyranoside.

Suitable glycosidic residues are, inter alia, suitable oligosaccharide chains, which can be degraded by carbohydrate-chain enzymes to the degree of mono- or oligosaccharides, which can then be cleaved from the resorudin backbone by the corresponding glycosidases. These oligosaccharide chains are primarily understood to be chains built from 2 to 10 and a precursor of general tautomers.

Compounds of formula (II) include formulas (IIa) and (l'b)

(II'a) _;

(II 'bj

Wherein R ¹ 'to R ⁶ ' have the same meaning as the substituents R ¹ to R 6, wherein R ¹ 'to R ⁶ do not all represent simultaneously hydrogen atoms and Y is as defined above.

These compounds are novel and are particularly useful as intermediates for the preparation of glycosides of the resorufin derivatives of the invention of general formula (I).

Compounds of formula (II ') may be prepared in an analogous manner to that which is suitable for the preparation of the known resorufin (a compound of formula (II) in which all the substituents R 1 to R 2 denote hydrogen atoms).

The compounds of formula II are preferably prepared by reacting a nitroresorcin derivative of formula III.

(III) wherein R ¹ 'to R ³ ' are as defined above, with a resorcinin derivative of the general formula IV

wherein R ⁴ 'to R ⁶ ' are as defined above, in the presence of burel and sulfuric acid at low temperatures. The compounds of formula (II ') in which Y is N = 0 are initially formed. These compounds are readily converted to compounds of formula (II) in which Y is nitrogen by treatment with zinc dust in the presence of ammonia.

The reaction of the compounds of the formula III with the compounds of the formula IV is usually carried out at a temperature in the range from -10 to 50 ° C, preferably from 0 to 30 ° C. The reaction is particularly gentle when the compounds of the formulas III and IV are mixed at about 0 ° C and the reaction mixture is then allowed to warm to room temperature. The concentration of burel should conveniently be in the range of about 0.5. to about 5, preferably from about 1 to about 2 mol / L. The concentration of sulfuric acid should be in the range of 0.5 to 5, preferably 1 to 3 mol / l.

The reduction of compounds of formula II 'in which Y is N 0 to compounds of formula II * in which Y is nitrogen is preferably carried out with zinc dust in an ammonia solution (cf. Nietzki et al., Ber. Dtsch. Chem. Ges. 22, 3020 (1889)). Suitably, the solvent used is a mixture of water and an alcohol, preferably a mixture of 1 part of water with 0 to 4 parts of methanol. 1 to 20, preferably 1 to 5 moles of zinc dust is added in portions per mole of reduced substance. The temperature of the reaction solution is maintained in the range from -10 to +35 ° C, preferably from +5 to +10 ° C. For the reaction to proceed unambiguously, the temperature range mentioned above must be strictly observed. Without cooling, the exothermic reaction leads to the formation of by-products which are difficult to separate.

Under the mild conditions chosen, the reaction between the compound of formula III and the compound of formula IV is clearly in good yield. The synthesis method chosen may be varied. This opens up numerous possibilities for synthesis, particularly with respect to the preparation of unsymmetrically substituted resorufin or possibly resazurine derivatives.

The resorufin derivatives of the general formula II 'in which R' and / or R '' denote a lower alkoxycarbonyl group, a carboxamido group which is optionally mono- or di-substituted, or a group of the formula

- COO - _(CH2 - CH _of 0) _n - R ^7, wherein R ⁷ and n are as hereinbefore defined, are preferably prepared through the tri-acylated dihydro resorufin formula V

wherein R ^1, R ^2, R ³ and R ⁵ are as defined above, R ⁴ and R ^E is carboxy, and R ⁷ is hydrogen or lower alkyl.

The carboxylic acid functional group is converted into the acid chloride from a method known in the literature, for example by means of oxalyl chloride / DMF or thionyl chloride / DMF, from which the correspondingly substituted carboxylic acid esters or carboxamides are obtained by reaction with any alcohols and amines.

Treatment of the thus obtained acetylated derivatives of formula III with caustic, preferably 0.1 to 5 M sodium or potassium hydroxide or 1 to 15 M ammonia (aqueous solution) and an oxidizing agent, preferably potassium hexacyanoferrate, with the addition of water-soluble organic solvents such as 1, 4-dioxane or methanol gives the corresponding resorufin derivatives of formula II.

The alcohol component may in principle consist of any alcohol. Particular preference is given to diethylene glycol monoethyl ether, triethylene glycol monoethyl ether, triethylene glycol monoethyl ether or simple alcohols such as methanol or ethanol. The amine component can also be selected from any of the amines. Particularly preferred are amines containing a polar group, such as morpholine, methoxyethylamine or glycinamide or ammonia, and primary and secondary lower alkylamines. Furthermore, aminocarboxylic acids whose carboxyl group is protected in a conventional manner can be used, such as tert-butyl glycine ester, benzyl glycine ester or N 6 -BOC-lysine methyl ester. After cleavage of the protecting groups, resorphins of the formula II or glycosides of resorphine of the formula I with an aliphatic-linked carboxylic acid functional group are obtained.

The acetylated dihydroresorufins of formula V are obtained from the corresponding resorufin or resazurine by reaction with a strong reducing agent such as stannous chloride or chromium acetate or electrochemical reduction followed by acetylation. In the reduction, resorufin or resazurin is heated for 2 to 10 hours, preferably 2 to 6 equivalents of tin (II) chloride in 5 to 35% aqueous hydrochloric acid for 10 minutes to one hour. Upon cooling, the dihydro compound precipitates. The acetylation is carried out in the usual manner with acetic anhydride. Preferably, the compounds of formula V are prepared by a one-step process by reductive acetylation. The corresponding resorufin or resazurin is heated with 2 to 6 equivalents of stannous chloride for 5 minutes to 5 hours, preferably 10 minutes to 3 hours, in acetic anhydride at reflux or stirred at room temperature for 4 to 16 hours.

Synthesis of glycosides of resorufin derivatives of formula (IX) produces non-fluorescent compounds from which fluorescing resorufin derivatives are released by enzymatic cleavage using the corresponding glycosidases. The fluorogenic derivatives of resorufin according to the invention exhibit very advantageous properties in comparison with hitherto known glycosides from other chromogenic or fluorogenic groups. Oako monosubstituted derivatives show the kinetics of enzymatic hydrolysis, which can be described very simply by the Michaelis-Menton relationship. For resorufin (i-D-galactopyranoside, for example, the Michaelis constant K i = 0.38 mmol / l is suppressed. Suppression of hydrolysis by the natural substrate with lactose is competitive so that for certain assays the specific conversion of glycosides of resorufin derivatives can be defined and reversed by lactose in the case of β-D-galactosicase

The glycosides of the resorufin derivatives of the invention are practically unlimited in aqueous solution at +4 ° C. The solubility of resorufin glycosides with a backbone is sufficient for most kinetic purposes. By introducing carboxylic acid esters and carboxylic acid derivatives with polar or sulfo groups, the solubility of the corresponding glycosides is substantially improved.

The excitation and emission of the products of the invention lie in the visible range of the spectrum at a sufficient quantum yield. The maximum fluorescence intensity of resorufin is reached at pH values above 7 and the fluorescence intensity decreases only gradually as the pH decreases. Resorufin glycosides are mostly yellow in aqueous solution (Λ _max · at about 470 nm).

After the enzymatic reaction, the products are mostly colored red (Λ at about 570 nm), so they are also well suited for photometric and non-instrumental visual testing.

The novel glycosides of the resorufin derivatives of the formula I can be used for determining the activity of the corresponding glycosidases, for example .alpha.-D-glucosidase, .alpha.-D-galactosidase and .alpha.-D-mannosidase.

The resorphin derivatives of the formula I, in which the glycosidic residue is composed of an oligosaccharide chain, are also particularly suitable for demonstrating the presence of carbohydrate-chain-cleaving enzymes, for example e-amylase. In this case, the oligosaccharide chain is typically cleaved by the enzyme to be detected, preferably up to the monosaccharide stage, where further additional enzymes can be used if necessary. The product obtained is then cleaved into the resorufin backbone and the monosaccharide by treatment with the corresponding glycosidase.

The use of glycosides of resorufin derivatives of general formula I as substrates for glycosidases leads to significantly more sensitive test systems as compared to previously known systems. The novel substrates can be used to determine the activity of glycosidases, preferably in both the biochemical and biotechnological as well as the clinico-chemical field. They are more sensitive and consequently offer numerous benefits:

(a) Lower enzyme activities may be measured.

(b) Minor quantities of substances may be used as samples.

c) The activity will be determined in a much shorter time.

(d) Use of a small sample of other sample components for

(e) Conversion can be measured and favorable measurement wavelengths reduce the method susceptibility.

in immobilized enzyme carrier matrices.

The substrates of the invention have been shown to be suitable for determining glycosidase activity of any origin. The diagnostic compositions according to the invention containing substrates of the formula I react much more sensitively than the prior art test compositions.

The glycosides of resorufin derivatives of formula I are also useful in immunoassays in which glycosidases are used as indicator enzymes whose activity after immunological reaction has to be determined. These immunoassay methods using enzymatic indicator reactions are referred to as enzymimmunoassay (hereinafter, enzymatic immunoassays). These methods are used to determine the concentration of proteins, polysaccharides, hormones, drugs and other low molecular weight substances present at a concentration ranging from 10 to 10 mol / l. Depending on the phase separation requirements, a distinction is made between homogeneous and heterogeneous testing. The further distribution of the tests is based on whether they are conducted on a competitive or non-competitive basis.

However, all test principles work with enzyme-antigen or enzyme-antibody conjugates. The enzymatic indicator reaction is common to all enzymatic immunoassays. Suitable indicator enzymes for this purpose are, for example, glycosidases, in particular β-D-galactosidase. The determination of glycosidases in these enzymatic immunoassays is carried out in a conventional manner by adding a suitable substrate, which is enzymatically cleaved and then photometrically or fluorometrically measured in a conventional manner.

Consequently, the improvement of the glycosidase assay systems also results in substantial advantages in the following enzymatic immunoassays:

1. Higher sensitivity allows here to achieve a further reduction of the detection limit, furthermore to reduce the reaction time and reduce the amount of the sample, thereby also reducing the interference of the method with other sample components.

2. More favorable wavelengths reduce the susceptibility of the method to insoluble components, such as haze, in certain embodiments of the reaction.

The diagnostic composition comprises, in addition to one or more substrates of Formula I, a suitable buffer system and, optionally, other ingredients commonly used in such diagnostic agents as wetting agents, stabilizers, etc. The diagnostic composition may be in the form of a solution, lyophilisate, powder mixture, reagent tablet or test. a strip in which the substances are deposited in an absorbent carrier.

The solution formulation preferably contains all the reagents required for the assay. Suitable solvents are water or mixtures of water with water-soluble organic solvents such as methanol, ethanol, acetone or dimethylformamide. For reasons of shelf life, it may be advantageous to separate the reagents necessary for carrying out the test into two or more solutions which are only mixed together when the test is carried out.

For the preparation of a diagnostic composition in the form of a lyophilisate with a total weight of 5 to 20 mg, preferably about 10 mg, a solution is dried which, in addition to all the reagents required for carrying out the test, also contains conventional carriers such as polyvinylpyrrolidone and optionally other carriers such as mannitol. , sorbitol or xylite.

The diagnostic composition in the form of a powder mixture or reagent tablet can be prepared by mixing the components required to carry out the test with conventional galenic additives and granulating the mixture. Additives of this type are, for example, sugar alcohols such as mannitol, sorbitol or xylite or other soluble inert compounds such as polyethylene glycols or polyvinylpyrrolidone. Powder mixtures or reagent tablets usually have a final weight of about 30 to about 200 mg, preferably 50 to 80 mg.

In the manufacture of a test strip diagnostic device, the absorbent carrier, preferably filter paper, cellulose or synthetic fiber web, is impregnated with a solution of the required reagents commonly used in the test strip in a readily volatile solvent such as water, methanol, ethanol or acetone. The impregnation can be carried out in one step, but it is often expedient to perform the impregnation in several stages, using solutions which always contain part of the components of the diagnostic agent. Thus, for example, impregnation may be carried out in the first step with an aqueous solution containing a buffer and other water-soluble additives, and in the second step impregnated with a solution containing a glycosidase substrate. The finished test specimens may be used as such or may be glued into grooves in a known manner, or may preferably be glued between a plastic and a fine mesh as described in DE 2 118 455.

The following examples show several of the numerous process variants which lead to the synthesis of the compounds of the invention. The examples are illustrative only and do not limit the scope of the invention in any way.

Example 1

Resorufin- [beta] -D-galactopyranoside

a) Preparation of resorufin

Resorufin is prepared from its oxidation product, resazurin, which is commercially available in good purity, in a manner analogous to that described by Nietzki et al., Ber. Dtsch. Chem. Ges. 22 (1889), pages 3020 to 3038. In a beaker, 10 g (43 mmol) of resazurin (Fluka, Bucha, Switzerland) was heated in a boiling bath with 50 ml of a 25 ml aqueous ammonia solution, 25 ml 37¾ aqueous sodium hydrogen sulphite solution for 30 minutes. and 50 ml of water. Then 20 ml of ammonia solution, 7 ml of dibrogen sulphite solution and 20 ml of water were added and the mixture was heated for a further 30 minutes. The progress of the reaction can be monitored by color change from blue to red or by thin layer chromatography. After the reaction is complete, 10 ml of 32¾ hydrochloric acid is added to adjust the pH to 5. The reaction solution is allowed to stand in the refrigerator for one day. The brown pellet was filtered off with suction and washed with ice-cold hydrochloric acid, pH 4, and dried at 110 ° C. Yield: 7.8 g (36.7 mmol; 85%).

b) Glycosylation of resorufin to resorufin-γ-O-galactopyranoside

The glycosylation of the phenolic hydroxy group via intermediate tetraacetate is carried out as follows: 2.13 g (10 mmol) of finely powdered resorufin, 4.12 g (10 mmol) of acetobromo-D-D-galactose (Sigma, Munich), 1.16 g (5 mmol). mmol) of silver oxide (Fluka, Buchi, Switzerland), 5 g of calcium sulphate hemi hydrate (CaSO ^ .1 / 2 H ₂ 0, Drierite), a few grains of iodine and 50 ml of quinoline .mu.l for 40 hours at room temperature in 50 ml methylene chloride. In this process, 40 ¾ of resorufin is converted to resorufin-β-D-galactopyranoside tetraacetate. The reaction can also be monitored by thin layer chromatography. After separation of the solid on the pleated filter and centrifugation, the methylene chloride is drawn off in a rotary evaporator. Resorufin- [beta] -D-galactopyranoside tetraacetate is obtained as a brown-yellow syrup residue. Yield about 2 g (35 ¾ of theory).

g of resorufin- [beta] -D-galactopyranoside tetraacetate are taken up in 80 ml of dry methanol without further purification. 0.5 g of sodium is dissolved in small portions in 20 ml of dry methanol in an ice bath. While stirring in an ice bath, 6-8 mL of the methoxide solution is added in 1 mL portions over 30 minutes to the resorufin- / 9-O-galactopyranoside tetraacetate solution and the deacetylation is monitored by thin layer chromatography. The precipitated orange-colored resorufin- N -D-galactopyranoside is filtered off with suction and washed with a small amount of ice-cold methanol. The yield is about 1 g of crude material (70 ¾ of theory). Recrystallization from methanol followed by drying at a temperature not exceeding 60 ° C gave 0.2 g of pure product. All thin-layer chromatography tests performed to monitor the progress of the reaction can be performed using silica gel 60 (Merck, Darmstadt) and methylene chloride / methanol.

CS 273104 B2 (9/1) as eluent. For resorufin resazurin resorufin-β-D-galactopyranoside tetraacetate resorufin-β-D-galktopyranoside this system applies · following Rj values 0,4 0,35

0.9

0.1

Example 2

Resorufin- [1-D-glucoside]

2.13 g (10 mmol) of resorufin (prepared according to Example 1a), 4.11 g (10 mmol) of acetobromo-06-D-glucose and 3.64 g (10 mmol) of hexadecyltrimethylammonium bromide were refluxed in a mixture for 4 hours. 11.5 ml of 1 N sodium hydroxide and 50 ml of chloroform. The mixture was evaporated to dryness and digested with ethyl acetate. The ethyl acetate solution was again evaporated to dryness. The residue is taken up in 20 ml of ethanol. Tetraacetylresorufin-β-D-glucoside is recrystallized from methanol with the addition of activated carbon.

For deacetylation, the above product is taken up in 10 ml of methanol. After stirring at room temperature for 2 hours, the precipitated product is filtered off with suction and dried. Yield: 50 mg.

¹ H-NMR ([D] ₆ -DMSO): _δ 3.15-3.80 (m, 6H), 4.5-6.5 (broad, 4H), 5.12 (d, 3 = 6) 9 Hz, 1H), 6.29 (d, 3 = 2.0 Hz, 1H), 6.80 (dd, 3 = 9.6 and 2.0 Hz, 1H), 7.12 (dd, 3 =

8.5 and 2.0 Hz, 1H), 7.16 (d, 3 = 2.0 Hz, 1H), 7.55 (d, 3 = 9.6 Hz, 1H), 7.80 (d, Δ = 8.5 Hz, 1H).

Example 3

Resorifin-1-carboxylic acid methyl ester

50 g of nitroresorcin, 53.3 g of methyl 3,5-dihydroxybenzoate and 28.0 g of burel are dissolved or suspended in 500 ml of methanol. While cooling with ice, it is added dropwise at 5 to 10 ° C

34.4 ml of concentrated sulfuric acid. After the ice bath was removed, the mixture was further stirred for two hours. Then 200 ml of aqueous ammonia solution are added under cooling. The precipitated precipitate is filtered through a glass fiber filter. 10 g of zinc dust are added in portions to the filtrate at 5-10 ° C. The mixture was stirred at room temperature until reduction was complete (reaction was monitored by thin layer chromatography using ethyl acetate / methanol 4: 1 as eluent, reaction time about 1.5 hours). The reaction mixture was evaporated at 25 ° C (bath temperature) in a rotary evaporator to 1/3 of its volume. The desired product precipitates upon acidification with concentrated hydrochloric acid under cooling until color change. After washing with dilute hydrochloric acid and drying under vacuum over calcium chloride, methyl resorufin-1-carboxylic acid methyl ester is obtained. Yield: 30.9 g (36% of theory).

¹ H-NMR: ([D] ₆ DMSO): 3.98 (s, 3H), 6.47 (d, 3 = 2.2 Hz, 1H), 6.85-6.96 (m, 2H) 7.09 (d, 3 = 2.2 Hz, 1H), 7.60 (d, 3 = 9.6 Hz, 1H),

Fluorescence: absorption Λ ___ = 570 nm max emission A _mov = 588 nm.

III d a

In an analogous manner it is obtained from

(a) 3,5-dihydroxybenzoic acid and nitrosoresorcin via resazurine-1-carboxylic acid resorufin-1-carboxylic acid UV / VIS (0.1 M potassium phosphate buffer, pH 7.5: λ = 569 nm.

= 569 nm max

CS 273185 82

(b) 4-O-methylgallic acid methyl ester and nitrosoresorcin via 4-methoxyresazurine-1-carboxylic acid methyl ester 4-methoxyresorufin-1-carboxylic acid methyl ester UV / VIS (0.1 M potassium phosphate buffer, pH 7.5): A _raax = 592 nm,

c) 3,5-dihydroxybenzoic acid methyl ester and 4-chloro-6-nitrosoresorcinol via 8-chloro-resorufin-1-carboxylic acid methyl ester of B-chlororesazurine-1-carboxylic acid.

d) 4-O-methylgallic acid methyl ester and 4-bromo-6-nitrosoresorcinol via 8-bromo-4-methaxyresazurine-1-carbaxylic acid methyl ester 8-bromo-4-methoxyresorufin-1-carboxylic acid methyl ester.

e) 5-nitroresorcin and nitrosoresorcin via 1-nitroresazurine 1-nitroresorufin; and

f) resorcin-5-sulfonic acid and nitrosoresorcin via resazurin-1-sulfonic acid resorufin-i-sulfonic acid.

Example 4

Reasazurine-4-carboxylic acid

1.60 g (10.5 mmol) of nitrosoresorcin, 1.55 g (10 mmol) of 2,6-dihydroxybenzoic acid and 0.86 g (10 mmol of burel) are taken up in 200 ml of methanol and the mixture is cooled to 0 ° C. Concentrated sulfuric acid (1.06 ml) was added dropwise and the mixture was stirred for a further 2 hours without cooling.The precipitated red product was filtered off, washed with methanol and dried to yield 2.3 g (85% of theory).

UV / vrs (0.1 M potassium phosphate buffer, pH 7.5):

-1

Λ = 614 nm (E = 48 cm mol), λ 3X

-1 after acidification A = 522 nm (E = 32 cm mol).

ΓΠ3Χ

Example 5

Resorufin-4-carboxylic acid

2.3 g of resazurine-4-carboxylic acid (prepared according to Example 4) are dissolved in 20 ml of water and 5 ml of 25¾ ammonia solution. To the blue solution was added 5 g of zinc dust with stirring and ice cooling. The ice bath was removed and the solution was allowed to warm slowly to room temperature. The progress of the reduction can be recognized by the color change from blue to dark violet. Thin layer chromatography (methanol / ethyl acetate 1: 1) can also be used to monitor the reaction. Excess zinc dust is filtered off. The reaction solution was acidified with 5 ml of glacial acetic acid and concentrated hydrochloric acid. The precipitated product is filtered off, washed with dilute hydrochloric acid and dried in vacuo over calcium chloride. Yield: 1.8 g (82% of theory).

UV / VIS: (0.1 phosphate buffer, pH 7.5):

λ max 57 579.4 nm (E = 48.6 cm 2 mol -1), after acidification: λ _mg = 485.9 nm (E = 34.7 cm ² mol ^-1 ).

Fluorescence: absorption: A = 579 _nm max emission: Λ = 593 nm.

max

Example 6 1-Methylresorfinine-4-carboxylic acid

840 mg of 2,6-dihydroxy-4-methylbenzoic acid, 760 mg of nitrosoresorcin, 430 mg of burel and 0.53 ml of sulfuric acid are reacted together as described in Example 4 to give

1-methylresazurine-4-carboxylic acid. Yield: 0.8 g. The thus obtained 1-methylresazurine-4-carboxylic acid is reduced to 1-methylresorufin-4-carboxylic acid analogously to Example 5.

Yield: 0.4 g,

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5)

_Λmax = 571 nm.

'max

Example 7

Resorufin-4-carboxylic acid morpholide

1) N, O, O-triacetyldihydroresorufin-4-carboxylic acid

Variant a) g (19.4 mmol) of resorufin-4-carboxylic acid or 5.3 g (19.4 mmol) of resazurine-4-carboxylic acid are refluxed in 100 ml of 10¾ hydrochloric acid for 0.5 hour in the presence of 7 g (38 mmol) of stannous chloride. The solution turns green. The mixture was allowed to cool and the precipitated dihydroresorfin-4-carboxylic acid was filtered off under a nitrogen atmosphere and dried in vacuo over phosphorus pentoxide. The crude product thus obtained was refluxed for 30 minutes with 30 ml of acetic anhydride and 20 mg of sodium acetate. The reaction mixture was poured into 200 ml of ice water and stirred for 14 hours. The pellet was recrystallized from ethanol / water. 4.8 g (65%) of the title compound are obtained.

Melting point: 197-199 ° C.

Thin layer chromatography (silica gel, chloroform / methanol / glacial, acetic acid 9: 1: 0.1).

R _f = 0.33.

Vlrianta b)

5.1 g (20 mmol) of resorufin-4-carboxylic acid were stirred in 20 ml of acetic anhydride for 1 hour at 80 DEG C. with 11 g (60 mmol) of stannous chloride. The mixture is poured into 230 ml of ice-water, stirred for 1 hour, filtered and worked up analogously to variant a).

Yield: 5.4 g (71.

2) Ν, Ο, Ο-triacetyldihydroresorufin-4-carbonyl chloride

3.85 g (10 mmol) of N, O, O-triacetyldihydroresorufin-4-carboxylic acid are mixed with 5.4 ml (60 mmol) of oxalyl chloride and the mixture is cooled to -10 ° C. One drop of dimethylformamide was then added to the mixture and the reaction mixture was allowed to warm to room temperature with stirring. The adduct dissolves during the gas evolution. After gas evolution had ceased, the mixture was stirred for a further 1/2 hour, evaporated and taken up in 3 x 20 ml dry methylene chloride, whereupon the methylene chloride was evaporated to dryness. 4 g of crude product are obtained, which product is used without further purification.

Thin-layer chromatography; (silica gel, eluent: chloroform / methanol / glacial acetic acid 9: 1: 0.1).

R _f = 0.42, colorless spots, which, after several hours, a red color.

3) N, O, O-triacetyldihydroresorufin-4-carboxylic acid morpholide

11.3 g (31.4 mmol) of crude acid chloride are dissolved in 150 ml of dry methylene chloride. To the solution was added dropwise 8.7 ml (63 mmol) of triethylamine and then 3.3 ml (37.7 mmol) of morpholine. The mixture was stirred for two more hours, the solution was washed with 1, citric acid, sodium bicarbonate solution and water, the organic phase was dried over magnesium sulphate and evaporated. The residue was recrystallized from ethanol.

Yield: 8.1 g (63%), m.p. 133-135 ° C (dec.).

4) Resorufin-4-carboxylic acid morpholide

3.7 g (9 mmol) of triacetyldihydroresorphin-4-carboxylic acid morpholide are taken up in 250 ml of methanol and 250 ml of water. To the solution was added 36 mL of 1 N sodium hydroxide and 6.0 g (18 mmol) of potassium ferrocyanide, and the mixture was stirred at room temperature for 14 hours.

After acidification to pH 3 with hydrochloric acid, the mixture was evaporated to dryness and digested with acetone. The dye solution is filtered through 500 ml of silica gel, eluting with acetone. Evaporation of the dye-containing eluate gave 2.3 g (80%) of the product.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

= 575 nm E = 55,000 cm ² mol ^-1 .

max

Thin Layer Chromatography (eluent same as in Step 2).

R _f = 0.52.

¹ H-NMR (CDCl ₃ -DMSO): δ = 3.3-3.8 (m, 8H), 6.50 (d, 3 = 2 Hz, 1H), 6.64 (d, J = 10) Hz, 1H), 6.76 (dd, 3 = 10 and 2 Hz, 1H), 7.44 and 7.51 (each d, 3 = Hz, 2H).

Example 8 tetraacetylresorufin-1-carboxylic acid β-D-galactopyranoside methyl ester and tetraacetylresorufin-9-carboxylic acid β-O-galactopyranoside methyl ester

6.8. Resorufin-1-carboxylic acid methyl ester (produced according to Example 3).

5.75 g of silver oxide, 6.75 g of silver carbonate, 15 g of 4 S molecular sieve and 10 g of N-bromotetraacetylgalactose in 250 ml of absolute chloroform are stirred at room temperature for 4 hours. After addition of an additional 5 g of .gamma.-bromotetraacetylgalactose, the mixture was further stirred overnight. The reaction mixture was filtered through a glass fiber filter and the filtrate was evaporated. The oily crude product was chromatographed on 2 L of silica gel using 2: 1 chloroform / ethyl acetate as eluent. Elute with 2.5 g yellow fractions with an R _f = 0.28 (silica gel HPTLC same eluant). Mixing in methanol gave tetraacetyrresorufin-9-carboxylic acid β-D-galactopyranoside methyl ester as orange-colored crystals.

Yield: 1.5 g.

¹ H-NMR ([D] g-DMSO): δ = 1.95, 2.03, 2.04 and 2.14 (each s, 12H), 3.88 (s, 3H), 4.11 (d, J = 7Hz, 2H), 4.51 (t, 3 = 7Hz, 2H), 5.24 (m, 2H), 5.36 (m, 1H), 5.69 (m, 1H) 6.31 (d, 3 = 2 Hz, 1 H), 6.97 (d, 3 = 2 Hz, 1 H), 7.04 (dd, 3 = 8.8 and 2.4 Hz, 1 H), 7.14 (d, J =

2.4 Hz, 1H), 7.78 (dd, 3 = a, 8 Hz, 1H).

1.6 g of a yellow fraction with an Rj value of 0.24 are then eluted. Tetraacetylresorfin-1-carboxylic acid methyl ester β-D-galactopyranoside crystallizes from methanol in the form of yellow-orange crystals.

Yield: 1.2 g.

¹ H-NMR ([p] ₆ -DMSD): δ = 1.95, 2.02, 2.04 and 2.14 (each s, 12H), 3.90 (s, 2H), 4.09 ( m, 2H), 4.51 (m, 1 H), 5.24 (d, 3 = Hz, 1 H), 5.25 (m, 1 H), 5.36 (m, 1 H), 5.71 (m) (1H),

6.50 (d, 3 = 2 Hz, 1H), 6.83 (dd, 3 = 10 Hz, and 2 Hz, 1H), 7.20 and 7.24 (each d, 3 = 2 Hz,

2H), 7.47 (dd, 3 = 10 Hz, 1H).

Among the pure products, it is also possible to elute the mixed fraction from which 2.5 g of crystals of a mixture of the two isomeric compounds are obtained after recrystallization from methanol.

In an analogous manner, from o-bromotetraacetylgalactose a

(a) methyl 4-methoxyresorfinine-1-carboxylic acid methyl ester yields 3-D-galactopyranoside tetraacetyl-4-methoxyresorphin-1-carboxylic acid methyl ester and 3-D-galactopyranoside tetraacetyl-6-methoxyresorfin-9-carboxylic acid methyl ester,

b) methyl (β-D-galactopyranoside-1-carboxylic acid β-D-galactopyranoside methyl ester) and tetraacetyl-2-chloro-resorphin-9-carboxylic acid β-D-galactopyranoside methyl ester;

Example 9 Morpholide g -galactopyranoside · Tetraacetylresorufin-4-carboxylic acid β-galactopyranoside Morpholide tetraacetylresorufin-6-carboxylic acid β-galactopyranoside

3.26 g (10 mmol) of resorufin-4-carboxylic acid morpholide were galactosidated as described in Example 8. The crude product was chromatographed on 1 liter of silica gel using ethyl acetate / acetone as eluent. There was thus obtained tetraacetylresorfinine-6-carboxylic acid morpholide β-D-galactopyranoside (0.9 g).

Thin layer chromatography (silica gel, eluent see Example 7).

R _f = 0.71.

In addition, 0.4 g of tetraacetylresorfinine-4-carboxylic acid morpholide 3-D-galactopyranoside is obtained.

Thin layer chromatography (silica gel, same eluent).

R _f = 0.76.

There was also obtained a 1.2 g · mixed fraction of the two isomeric compounds.

Example 10 α-D-galactopyranoside resorufin-1-carboxylic acid methyl ester

1.2 g of tetraacetylresorphin-9-carboxylic acid β-D-galactopyranoside methyl ester were deacetylated with sodium methoxide in methanol as described in Example 2.

Yield: 0.8 g.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5): max ^{= nm =} 21.8 cm ^{1 2} mol ^-1 ).

After resolving with β-galectosidase, the anion of resorufin-1-carboxylic acid methyl ester is obtained.

Λ max = 572 nm ( ^λ = 65.3 cm ^from mole ^A ).

¹ H-NMR ([D] ₆ -DMSO): _δ 3.20-3.80 (m, 6H), 3.91 (s, 3H), 5.09 (d, 3 = 7.5 Hz, 1H), 6.30 (d, 3 = 2.1 Hz, 1H), 6.81 (dd, 3 = 9.8 and 2.1 Hz, 1H), 7.30 (m, 2H), 7, 51 (d, 3 = = 9.8 Hz, 1H), OH protons very wide at 5.

In an analogous manner, deacetylation of the corresponding tetraacetates gives the following substances:

a) N-galactopyranoside resorufin-1-carboxylic acid methyl ester ¹ H-NMR ((D) ₆ DMSO): δ 3.4-3.7 (m, 6H), 5.09 (d, 3 = 7.5) Hz, 1H), 6.34 (d, 3 =

Hz, 1H), 6.97 (d, 3 = 2 Hz, 1H), 7.08 to 7.17 (m, 2H), 7.75 (d, 3 = 10 Hz, 1H), OH: very broad at 5 ppm.

b) 4-methoxyresorufin-1-carboxylic acid methyl ester-D-galactopyranoside,

(c) 6-methoxyresorufin-9-carboxylic acid-D-galactopyranoside methyl ester,

d) β-D-galactopyranoside of 8-chloro-resorfin-1-carboxylic acid methyl ester,

(e) 2-chloro-resorphin-9-carboxylic acid β-D-galactopyranoside methyl ester,

f) Resorufin-6-carboxylic acid morpholide β-D-galactopyranoside, R f = 0.3 (ethyl acetate / isopropyl alcohol / water 9: 4: 2).

After β-galactosidase digestion:

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

^.Lambda.max = ^{574.6 nm}

Fluorescence emission = 593 nm and max

(g) Resorufin-4-carboxylic acid morpholide β-D-galactopyranoside, R f = 0.3 (ethyl acetate / isopropyl alcohol / water 9: 4: 2).

After β-galactosidase digestion:

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

^Λ max = ⁵⁷⁴ ' ^nm

Fluorescence emission: .lambda. = 593 nm.

max

Example 11

Resorufin-9-carboxylic acid 3-D-galactopyranoside triethylamine

266 mg of resorufin-9-carboxylic acid methyl ester β-D-galactopyranoside are taken up in 50 ml of water and 20 ml of 1,4-dioxane. A total of 5 ml of 0.1 N sodium hydroxide is added to the solution in portions of 0.5 ml, while the pH of the solution should not rise above 12.5. The reaction mixture is applied to 6 ml DEAE-Sephadex in carbonate form. The column is washed with 180 ml of water.

Next, the product was eluted with 0.1 M triethylammonium carbonate buffer at pH 7.5. The eluate was evaporated in vacuo and then evaporated several times with ethanol.

Yield: 110 mg of resorufin-9-carboxylic acid / 3-D-galactopyranoside triethylammonium salt.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

After β-galactosidase digestion:

max = 570 nm.

Example 12

Resazurin- [beta] -D-galactopyranoside

12.6 g of resazurin sodium are dissolved in 65 ml of 1 N sodium hydroxide and 80 ml of water.

To the solution was added. 20.6 g of acetobromo-6-D-galactose and 18.2 g of hexadecyltrimethylammonium bromide in 150 ml of chloroform. The mixture was refluxed for 3 hours, then evaporated to dryness and digested with ethyl acetate. The ethyl acetate solution is concentrated again and chromatographed on 500 ml of silica gel (eluent: methylene chloride / ethyl acetate 3: 1). There were obtained 2.13 g tetraacetylresazurin-β-D-galactopyranoside with a value R _f = 0.5 (silica gel HPTLC same eluant).

The deacetylation was carried out by stirring 2.13 g of tetraacetyl derivative with 0.1 g of sodium methoxide in 100 ml of absolute methanol for 1 hour at room temperature. The precipitated product is filtered off with suction and dried in vacuo over calcium chloride.

Yield: 1.0 g.

Rf = 0.62 (HPTLC on silica gel, eluting with ethyl acetate / isopropyl alcohol / / water 9: 4: 2).

^r H NMR (Ed] ₆ -DMS0): J 3.30 to 3.90 (m, 6H), 4.54 (br-d, 3 = 4.4 Hz, 1H), 4.67 (br t Δ = 4.4 Hz, 1H), 5.07 (d, δ = 7 Hz, 1H), 5.27 (broad d, δ = 4.4 Hz, 1H),

6.15 (d, 3 = 2 Hz, 1H), 6.63 (dd, 3 = 9.6 and 2 Hz, 1H), 7.10 (dd, 3 = 9 and 2 Hz, 1H), 7, 2 (m, 2H), 7.96 (d, J = 9.6 Hz, 1H), 8.07 (d, J = 9 Hz, 1H).

Example 13

8-Chloresazurine-4-carboxylic acid

4.3 g (30 mmol) of 4-chlororesorcin are dissolved in 20 ml of ethanol. To the solution was added

2.4 g of potassium hydroxide and the mixture was cooled to 5 ° C. While cooling, to this mixture was added dropwise 2.0.1 ml of isopentyl nitrite, and then the mixture was further stirred at room temperature overnight. The pellet is filtered off, dissolved in water and acidified. The yellow precipitate is filtered again and dried under vacuum at 40 ° C.

Yield: 2.5 -5 g (48%) of 4-chloro-6-nitrosoresorcin

_Rf = 0.28 (HPTLC silica gel eluting with methanol / ethyl acetate 1: 3).

1.83 g of 4-chloro-6-nitrosoresorcin, 1.55 g of 2,6-dihydroxybenzoic acid, 0.86 burel and 1.06 ml of sulfuric acid are added to 20 ml of methanol and the resulting mixture is stirred at 0 DEG for 2 hours. C and 14 hours at room temperature. A red deposit is obtained, which is filtered off and dried.

Yield: 2.45 g (80%) of 8-chlororesazurine-4-carboxylic acid.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

Λ = 621.5 nm. max ’

In an analogous way

a) 6-methylresazurine-4-carboxylic acid is obtained from 2-methylresorcin via 2-methyl-6-nitrosoresorcin,

(b) 8-methylresazurine-4-carboxylic acid is obtained from 4-methylresorcin via 4-methyl-6-nitrosoresorcin; and

c) from 4-bromo-resorcin via 8-bromo-6-nitrosoresorcin, 8-bromo-resazurine-4-carboxylic acid is obtained.

Example 14

8-Chlororesorufin-4-carboxylic acid g 8-Chloresazurine-4-carboxylic acid (prepared according to Example 10) was dissolved in 20 ml of water and 5 ml of a 25% ammonia solution. Under ice-cooling, zinc dust is added to the solution until the color changes to a reddish-violet hue. Excess zinc is filtered off, the filtrate is acidified, the precipitated product is filtered off and dried in vacuo over calcium chloride at 40 ° C.

Yield: 1.5 g (79%) of 8-chloro-resorfin-4-carboxylic acid.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

Λ = 585.5 nm max '

In an analogous way

a) 6-methylresazurine-4-carboxylic acid is obtained from 6-methylresazurine-4-carboxylic acid,

(b) from 8-methylresazurine-4-carboxylic acid there is obtained 8-methylresorufin-4-carboxylic acid; and

c) 8-bromo-resorphin-4-carboxylic acid is obtained from 8-bromo-resazurine-4-carbaxylic acid.

η

CS 273184 Β2

Example 15 Resorufin-9-carboxylic acid ού-D-galactopycanoside methyl ester

3.2 g of pentaacetylgalactose are mixed with 0.1 g of anhydrous zinc chloride and the mixture is mixed with 0.1 g of anhydrous zinc chloride and the mixture is heated to 125 [deg.] C. and then stirred at a pressure of 50 mmHg for 20 minutes. 1 g of resorulin-1-carboxylic acid methyl ester is added to the melt. The mixture was stirred at 41 ° C for 1 hour and then chromatographed as described in Example 7 on silica gel using 2: 1 chloroform / ethyl acetate as eluent.

Yield: 25 mg of tetraacetylresorfin-9-carboxylic acid ού-D-galactopyranoside methyl ester (yellow oil).

= 0.22 (HPTLC on silica gel, same eluent).

The deacetylation to resorufin-9-carboxylic acid (6-D-galactopyranoside methyl ester) was carried out as described in Example 10.

Example 16 Resorufin-9-carboxylic acid beta-D-galactopyranoside 3,6-dioxaoctyl ester

0.6 g of tetraacetylresorphin-9-carboxylic acid methyl ester-D-galactopyranoside are mixed and 50 ml of diethylene glycol monoethyl ether and a spatula tip of sodium hydride are added and the mixture is stirred at room temperature for 15 minutes. 100 ml of acetone are then added. The precipitated precipitate is filtered off and dried.

Yield: 56 mg of resorufin-9-carboxylic acid beta-D-galactopyranoside 3,6-dioxaoctyl ester.

_Rf = 0.54 (HPTLC on silica gel using ethyl acetate / isopropyl alcohol 9: 4 as eluent).

Example 17

Resorufin maltoheptoside

An amylase (E.C.2.4.1.19), for example an amylase obtained by Bacillus macerans, exhibits, in addition to the hydrolytic and cyclizing effect, also glycosyl transfer properties which can be used in the synthesis of oligosaccharides and their derivatives (Methods in Carbohydrate Chemistry II, 347).

680 mg of amylase obtained with Bacillus macerans DSM 24 (Liophilisate; 0.46 U / mg batch; protein content in batch 28.5%).

500 mg resorufinglucoside,

3.5 g ού-cyclodextrin and ml Soerensen phosphate buffer (pH 6.2, 0.01 M) are mixed together and the batch incubated for 24 hours at 37 ° C. For purification, the β- and β-cyclodextrin are first separated through the tetrachlorethylene addition compound. Chromatography on cross-linked dextran (Sephadex LH 20) yields 50 mg of resorufinyl maltoheptoside lyophilisate, which shows high activity in the amylase assay.

Example 19

a) Peracetyl- [beta] -D-maltopentoside

100 g (0.12 mol) of maltipentose and 81.8 g (1.0 mol) of anhydrous sodium acetate are suspended in 1.1 liter (11.7 mol) of acetic anhydride and the mixture is slowly heated until the reaction begins ( 110 ° C). The mixture was then cooled with ice water until the reaction subsided and refluxed for 1 hour to complete the reaction. The reaction mixture was cooled to about 70 ° C and poured into 4 L of ice water. After stirring for 60 minutes, the supernatant is decanted and the residue is dissolved in 500 ml of dichloromethane. The dichloromethane phase is successively shaken with water, saturated sodium bicarbonate solution and water and dried over sodium sulfate. After distilling off the solvent under reduced pressure, the residue is recrystallized from 1.5 l of ethanol-isopropyl alcohol (1: 1) with the addition of activated carbon.

Yield: 166.2 g (89.8% of theory) as colorless crystals of m.p. 120 DEG-125 DEG C. [.alpha.] D @ 20 = + 123.5 DEG.

Thin Layer Chromatography: Kieselgel 60 F ₂₃₄ (Merck) toluene / acetone (7: 4) silica gel plates.

R _f = 0.52, ¹ H-NMR (DMSO-d ₆ ): 1.8-2.2 (s, 51H, COOCH 3),

3.8 to 5.5 (m, 35H, H-1, -H-6).

Literature:

Herzfeld, Chem. Ber. 13 (1880), 267.

The following peracetyl-1H-D-maltosides are synthesized as described above using the same amount of maltose, maltotriose, maltotetrose, maltohexose and maltoheptose instead of maltopentose.

sugar temperature yield ²⁰ Kr _(CHCl3) Rf (Kieselgel 60 mp ° C % O taluene / acetone, 7/4 maltose • 159 to 160 90 + 62.5 0.64 maltotriosa - 85 + 100.3 0.59 maltotetrosa - 88 + 109.7 0.56 maltopentosa 120 to 125 90 + 123.5 0.52 maltohexosa 140 to 145 91 + 130.0 0.49 maltoheptosa 160 82 - 0.46

b) Hydroxy-6-D-peracetyl maltopentoside

150 g (0.096 mol) of peracetyl- (N-D-maltopentoside) are dissolved in 200 ml of absolute tetrahydrofuran, 33 ml (0.3 mol) of benzylamine are added with stirring, and the mixture is stirred at room temperature for 2 hours with exclusion of moisture. The mixture is then evaporated to dryness in vacuo, the residue is dissolved in 400 ml of dichloromethane, and the dichloromethane phase is washed successively with 400 ml of 5N hydrochloric acid, water, saturated sodium bicarbonate solution and water, and the organic phase is dried over sodium sulfate. After filtration of the activated carbon, the filtrate is evaporated to dryness under reduced pressure (140 g, 97% of theory) without further purification.

Thin layer chromatography: silica gel 60 F ₂₃₄ (Merck) toluene / acetone (7/4).

R _f = 0.42

CS 273104 B2 H-NMR (OMSQ-dg): 1.8 to 2.2 (s, 48H, COQH-j)

3.8 to 5.5 (m, 35H, H-1, -H-6)

5.7 (s, 1H, OH).

Literature:

Helferich B., Portz W., Chem. Ber. 86 (1953) 604

The following maltosides are synthesized in an analogous manner:

sugar yield Rj (Kieselgel 60, toluene / acetone (7: 4) maltose 96% 0.45 maltotriosa 96% 0.40

c) Peracetylmaltopentosyl-α-D-trichloroacetimidate g (0.05 mol) of hydroxy-δ-D-peracetylmaltopentoside and 25 ml (0.25 mol) of trichloroacetonitrile are dissolved in 150 ml of absolute dichloromethane. The solution is cooled to 0 ° C, 1.5 g (0.055 mol) of sodium hydride are added in portions with stirring, and the mixture is stirred at room temperature for 2 hours with the exclusion of moisture. The excess sodium hydride was collected on a glass frit, the filtrate was filtered through a silica gel column (Kieselgel 60, Merck, about 200 mL, 0 cm) and the column was washed with 2 L of ethyl acetate. The filtrate is heated to reflux with the addition of activated carbon. The activated carbon is filtered off and the filtrate is evaporated to dryness under vacuum.

Yield: 72 g (87.5% of theory) of a colorless foam

TLC: silica gel Kieselgal 60 F254 (Merck).

R _f = 0.58.

¹ H-NMR (DMSO-d 6): 1.8-2.2 (s, 48H, COOH-j),

3.8 to 5.5 (m, 35H, H-1, -H-6),

6.3 (s, 1H, NH).

Literature:

Schmidt R., Stumpp M., Liebigs Ann. Chem. 1983, 1249-1256.

Peracetyl-maltotriosatrichloroacetimidate is produced analogously:

Yield: 93%

R _f = 0.45.

d) Resorufinyl- [beta] -D-maltopentoside

72.0 g (0.044 mol) of peracetyl maltopentosyl-N-O-trichloroacetimidate and 4.4 g (0.02 mol) of resorufin are suspended in 1 liter of absolute dimethylformamide. 6.3 ml (0.05 mol) of boron trifluoride etherate (BFj.EEtj) was added dropwise to the suspension at room temperature and the mixture was stirred at 60 ° C for 8 hours to remove moisture. After cooling to room temperature, the mixture was filtered through alumina (about 500 mL) and this was additionally washed with 5 L of ethyl acetate. The filtrate is evaporated to dryness under reduced pressure, the residue is dissolved in 100 ml of absolute methanol and deacetylated with 2 g of sodium methoxide for 3 hours at room temperature, excluding moisture. The suspension is concentrated to dryness under vacuum, the residue is taken up in 100 ml of water, the pH of the solution is adjusted to 6 with 2N hydrochloric acid and then the solution is drawn onto a Diaion column (about 500 ml of 8 cm). Elution is carried out first with 4 liters of water and then with 3 liters of a 20% isopropyl alcohol solution. The eluate is concentrated to about 40 ml and then chromatographed on an RP-18-Flasch column (0 = 4 cm, h = 39 cm) using 15% isopropyl alcohol as eluent in 50 ml portions. The fractions containing resorufinmaltopentoside were combined, concentrated to about 20 ml under reduced pressure, and chromatographed on a RP-18-MPLC column in 10 ml portions using 15% isopropyl alcohol as eluent. Product containing fractions were combined, concentrated in vacuo and then lyophilized.

Yield: 2.8 g (14% of theory) of an orange lyophilisate

HPLC: RP-18 column, 1 mL / min, 17% isopropanol,

R t = 2.95 min.

Resorufin-G content: 97%.

⁷ H-NMR (DMSO-d ₆ ): 3.0-6.0 (m, 51K, OH, H 1 -H-6)

6.2 to 8.0 (m, 6H, ArH)

Literature:

Schmidt R., Grundler G., S. Synthesis 1981, 885-887.

Example 20

(a) Br-ού-D-maltoside:

mmol of peracetylated sugar is dissolved in 50 ml of glacial acetic acid. To the solution was added dropwise 50 ml of hydrobromic acid (30%) in glacial acetic acid, and the mixture was stirred at room temperature with exclusion of moisture. 300 ml of dichloromethane are then added and the mixture is poured into 1 liter of ice water. The organic phase is separated and shaken successively with water, saturated sodium bicarbonate solution and water. The organic phase is dried over sodium sulphate and further evaporated to dryness under reduced pressure. The product can be used in the next reaction without further purification.

sugar yield Rj (Kieselgel 60, toluene / acetone, (7: 4) maltose 88% 0.72 maltotriosa 88% 0.65 maltotetrosa 93% 0.61 maltopentosa 91% 0.57 maltohexosa 93% 0.53 maltoheptosa 95% 0.50

Literature:

Brauns, Am. Chem. Soc. 51 (1929), 1830.

CS 273105 B2

(b) Resorifin - /! - D-maltoside:

of resorufin and 25 mmol of silver oxide are suspended in 800 ml of absolute acetonitrile together with the molecular sieve (3A) and the mixture is refluxed for 4 hours under exclusion of moisture. 62.5 mmol of Br-cD-D-peracetyl maltoside in 200 ml of absolute acetonitrile are added and the mixture is refluxed for 6 hours. The mixture was stirred at room temperature for a further 18 hours, 10 g of activated carbon were added and the mixture was refluxed for a short time. The mixture is filtered through 100 g of alumina (activity level III / N) and this is additionally washed with 5 liters of ethyl acetate. The filtrate is evaporated to dryness under reduced pressure, the residue is dissolved in 1 liter of absolute ethanol and stirred for 18 hours with 5 g of sodium methoxide, excluding moisture. The suspension is evaporated to dryness under reduced pressure, the residue is dissolved in 200 ml of water, the pH of the solution is adjusted to 7 and the solution is applied to a 500 ml Diaion-HP-20 column. The column is washed with 15 liters of water and then eluted with 10 liters of a 15% isopropyl alcohol solution. The eluate is concentrated to 100 ml under reduced pressure and further chromatographed on an RP-18-MPLC column using an isopropanol solution and finally lyophilized as in the imidate method.

sugar yield % HPLC Rt /% isopropanol R _f (silica gel) butanol / led.kys.octová) water 50/15/25 maltotriosa 15 Dec 3.52 min / 17% 0.32 maltotetrosa 16 3.27 min / 17% 0.29 maltopentosa 14 2.95 min / 17% - maltoheptosa 10 2.60 min / 17% - maltose 15 Dec - 0.39

and)

25% isopropanol is used for resorphinyl-7H-D-maltotriose elution from the diaion column and 25% isopropanol for RP-18 column chromatography.

b) 40% isopropanol is used to elute resorufinyl- [beta] -D-maltoside from the diaion column. The product was evaporated to dryness under reduced pressure, then dissolved in dimethylformamide and precipitated with water. The pellet is suctioned off and dried under vacuum.

¹ H-NMR (DMSO-d 6):

Maltose: 3.1 to 3.9 (m, 11H, OH, H-6), 4.4 to 5.7 (m, 10H, H1-H-5), 6.2 to 8.0 (m, 6HArH),

Maltotriose: 3.0 to 5.9 (m, 32H, OH, H-1-H-6), 6.2 to 8.0 (m, 6H, ArH).

Claims (1)

SUBJECT OF THE INVENTION A process for preparing glycosides of resorufin derivatives of the general formula Ta or Ib wherein the glycoside denotes a glycoside residue of a monosaccharide or oligosaccharide containing from 2 to 10 1 2 3 5 monosaccharide units, R denotes hydrogen, each of R, R and R being the same or different denotes hydrogen, halogen or an alkyl group having 1 to 5 5 carbon atoms, each of R ⁶ and R ⁶ , which are the same or different, denote hydrogen; halogen; cyano; (C 1 -C 5) alkyl; (C 1 -C 5) alkoxy; carboxy; alkoxycarbonyl, carboxyalkyl or alkoxycarbonylalkyl; in the latter three groups the alkyl and alkoxy radicals each contain from 1 to 5 carbon atoms; or a carboxamido group, optionally mono- or di-substituted by alkyl, alkoxyalkyl, carboxyalkyl or alkoxycarbonylalkyl radicals, in which the alkyl and alkoxy groups each contain 1 to 5 carbon atoms, and in the case of the presence of two substituents they may be joined together to form a ring which optionally interrupted by oxygen, nitrogen or sulfur; or a group of formula - C00 - (CH ₂ - CH ₂ O) _n - R ⁷ 'where R ⁷ denotes hydrogen or alkyl of 1 to 5 carbon atoms and n denotes an integer of 1 to 4, in addition R 1 denotes a sulfonyl or nitro group and Y denotes nitrogen or the group N - * O, characterized in that resorufin derivatives of the general tautomeric formulas IIa and IIb are used. (IIa) (IIb) wherein R ⁷ to R ⁶ and Y are as defined above, reacted with a mono- or oligosaccharide containing 2 to 10 monosaccharide units or a halogenated derivative thereof, each hydroxy group being substituted by a protecting group customary in carbohydrate chemistry, to form per-O-substituted glycosides, from which the glycosides of resorufin derivatives of formula Ia or Ib are obtained by cleavage of protecting groups.