Method, tool, test system colorimetric determination of analyte and nitrosoaniline connection

 

(57) Abstract:

The invention can be used to detect and identify substances in a variety of test materials. Method of determining analyte includes enzymatic oxidation of the analyte using a PQQ-dependent dehydrogenase in the presence of the electron acceptor. Electron acceptor - rich electrons of aromatic nitroso compounds that restore to aminosidine the oxidation of the analyte. Restored acceptor find by color development. If the acceptor is not capable of color expression, detection is carried out in the presence of non-oxidizing dye indicator for aminosidine or in the presence of leucogranites. Thus leucocrystal oxidized in the presence of aminosidine to dye. Means for a method contains PQQ-dependent dehydrogenase and direct electron acceptor is enriched with electrons of aromatic nitroso compounds, non-oxidative dye as an indicator for aminosidine or leucocrystal. The test system for the method is an inert carrier containing the reagents of the proposed tools. Nitrosoaniline connection (new) formula

- N, - saturated or unsaturated three-membered chain with one N, or S atom and two carbon atoms, or two nitrogen atoms and one carbon atom, the carbon atoms may be substituted or form alkylenes chain. These compounds can be used as a chromogenic electron acceptors in the determination of the analyte. The invention makes possible the detection and identification of substances in a variety of test materials. The method is simple. He is the one that has less noise sensitive. 4 C. and 18 h.p. f-crystals, 4 tab., 7 Il.

The invention relates to a method colorimetric determination of the analyte through enzymatic oxidation using PQQ-dependent dehydrogenase (PQQ - pyrrolopyridine) in the presence of the electron acceptor group is enriched with electrons of aromatic nitrosoguanidine and determine the recovered electron acceptor due to the appearance of color as a measure of the amount of the analyte. Further, the invention relates to an agent for the colorimetric determination of an analyte by enzymatic oxidation, containing PQQ-dependent dehydrogenase, enriched with electrons aromatic nitrosoguanidine and coupler neocal is soedineniya during recovery. Further, the invention relates to new nitrosoaniline compounds, their receipt, and also to their use for the colorimetric determination of the analyte.

Enzymatic oxidation Analytics makes it possible to detect and identify substances in a variety of test materials. Thus oxidizing enzyme in the presence of host electrons oxidation reaction acceptor affects the appropriate enzyme substrate. The restoration of the electron acceptor indicates the presence of enzyme substrate. If this is still the least preferred was that the recovered electron acceptor can be detected due to the formation of painting, because it is possible not only when the need for expensive measuring devices, but if necessary can be made visually.

In known methods for the colorimetric determination of substances through okikawa existing enzymes used oxidase or dehydrogenase. Both enzymes belong to the main group of oxidoreductases (Rmpps Chemielexikon, Francksche Verlagsbuchhandlund, Stuttgart, 8th ed., 1985, so 4, 2952 S.; Lexikon Biochemie, Herausgeber H. D. Jakubke, Verlag Chemie, Weinheim, 2nd ed. , 1981, S. 194), h is elektronov for oxides is molecular oxygen (Rmpps Chemielexikon, Francksche Verlagsbuchhandlung, Stuttgart, 8th ed., 1985, so 4, S. 2946). In the case of the definitions of an analyte when the analyte is oxidized by glycerol phosphate oxidase and O2. The formed hydrogen peroxide with peroxidase is used for oxidation of leucogranites. In the prior art regarding the use of oxidase for the colorimetric determination of an analyte should be quoting A. Kunst and others in "Methods of enzymatic analysis", Hrsg.H.U.Bergmeyer, Verlag Chemie, Weinheim, 3rd ed., 1984, T. 6, S. 178-185. They determine glucose in serum, plasma or deproteinizing blood by entering into interaction with the glucose oxidase and oxygen that is formed during this reaction due to oxygen reduction, hydrogen peroxide is recovered in the presence of peroxidase and thereby causes staining is also present in the reaction mixture of phenol and 4-aminophenazone. High redoxpotential (redox potential) of hydrogen peroxide and neselektivno and instability peroxidase often lead to limitations of such a test. Mecause act, for example, ions of transition metals or gem or hemoprotein that can easily travel to get from the blood samples, as they decompose the hydrogen peroxide. Ingredients samples, as naprimerova samples or urine, can lead to staining with hydrogen peroxide and peroxidase, and thus to erroneous results, but they can also renew the already formed dye and thus discoloring.

Especially when making the above determination methods on solid media, the so-called dry test, additional oxygen demand oxidase appeared to be detrimental. First of all, when you require a lot of oxygen for oxidation of high concentrations of enzyme substrate, diffusion of oxygen from the air in the reaction medium can become the defining speed stage and lead to long reaction times or in particular when the kinetic methods of determination - to erroneous results.

Dehydrogenase in General can be divided into those that are necessary for the enzymatic oxidation of substrates nicotinamide-adenine-dinucleotide (NAD) or nicotinamide-adenine-dinucleotide-phosphate (NADP) as a direct natural electron acceptors, and those that are independent from NAD or NADP and who also use substances other than natural direct electron acceptors, in enzymatic reactions of atrogenes.

The use of NAD-dependent dehydrogenases for colorimetric measurements is known, for example, from the patent of Germany And 2147466. There is described that the lactate during the catalysis of the lactate dehydrogenase with nicotinamide-adenine-dinucleotide is converted into pyruvate and restored nicotinamide-adenine-dinucleotide. The formed NADH reacts then, for example in the presence of the enzyme diaphorase, with salts of tetrazole in the formation of NAD and painted formisano, the concentration of which can be determined photometrically. Instead diaphorase also can be called N-methylbenzene-methosulfate as a catalyst recovery for electron transfer from NADH to the salt of tetrazole.

The disadvantages of this method you should see that instead of NADH also other possibly present in a biological sample, such as blood, serum, plasma or urine, restorative active substances, such as glutathione, or medicine, as methyldiethanolamine or dobezilata, in the presence of non-specific catalysts recovery, as diaphorase or N-methylbenzene-methosulfate, transfer salts of tetrazole in the appropriate formazane and thus lead to false positive results, although they are not as fast re is P-A-0354441 known oxidizing, enzymatic detection of analytes by flavin-dependent oxidase or NAD-independent dehydrogenases, as PQQ-dependent dehydrogenase, using enriched with electrons of aromatic nitrosoguanidine. In the case of these detection methods, aromatic nitrosoguanidine enzyme is restored to the corresponding enriched with electrons, an aromatic amine, which is either detected using precipitated, insoluble heteroalicyclic due to the formation of heteropoly blue, or in the presence of an oxidant is introduced into the reaction mix with the color-forming reagent to form the dye. Blue-gray color gradations in the formation of heteropoly blue, however, not suitable for accurate visual assessment. For colorimetric detection in General unpainted or only very faintly colored aromatic amines using color-forming reagent negative is that additional oxidant. As such an oxidizing agent prevents enzymatic recovery of aromatic nitroso compounds to aromatic amine, the reaction detection should be carried out in two separate stages; the first stage of aromatic nitrosoguanidine f is Italy for oxidizing a combination of an aromatic amine with a color-forming reagent.

Another disadvantage of this method of detection is that to restore each equivalent of aromatic nitroso compounds to aromatic amine is necessary to oxidize two equivalent analyte. This is especially in the case of low concentrations of analyte can lead to poor sensitivity of this method of detection.

Therefore, the objective of the invention was to eliminate the above disadvantages of the prior art and the development of more simple, with less noise sensitive and visually better estimate of the way and finding the means to oxidative detection of analytes, which in particular can be done in one stage, which gives in the entire visible region of wavelengths visually well ocenivaemuyu color (color).

The problem is solved by the invention, which is characterized in the claims.

Accordingly, the method has been found colorimetric determination of the analyte through the enzymatic oxidation of the analyte with an oxidoreductase in the presence of a direct electron acceptor group is enriched with electrons of aromatic nitrosoguanidine, in which the definition in the tives such as those which is enriched with electrons aromatic nitrosoguanidine the oxidation of the analyte in the presence of PQQ-dependent dehydrogenase is restored to aminosidine it without further enzymatic recovery to aromatic amine is detected by the appearance of color.

The subject of the invention, in particular, is a method for the colorimetric determination of the analyte with the oxidoreductase in the presence of a direct electron acceptor group is enriched with electrons of aromatic nitrosoguanidine, in which the definition of reconstructed electron acceptor carry out due to the appearance of color as a measure of the amount of analyte, which differs in that it is enriched with electrons aromatic nitrosoguanidine the oxidation of the analyte in the presence of PQQ-dependent dehydrogenase is restored to aminosidine and it, instead of enzymatic further recovery to the aromatic amine, by interacting with non-oxidative color-forming indicator is determined colorimetrically.

Also, found a remedy for the colorimetric determination of an analyte by enzymatic oxidation of the analyte containing PQQ-dependent degidro the / establishment, which differs it either is enriched with electrons aromatic nitrosoaniline that enzymatic recovery forms the painted aminosidine, it further contains a non-oxidative color-forming reagent (indicator) for aminosidine formed from enriched in electrons of aromatic nitroso compounds by recovery.

According to the invention, the term "analyte" understand the matter that is oxidized enzyme. In many cases, the analyte is the substance to be detected directly or quantified in the test sample. For example, glucose can be directly oxidize using PQQ-dependent glucosegalactose (glucose-dye-oxidoreductase) and colorimetrically to determine. However, it is also possible that the analyte is formed only by one or more preliminary reactions of other substances, so that by colorimetric determination of the analyte can indirectly deduce the concentration of the original substance.

Analyte in the invention is a substance which accepted (recognized) as a substrate used PQQ-dependent dehydrogenase.

PQQ-dependent ejahn and other in PQQ and hemoprotein", Kluver Academic Publ. Dordrecht, The Netherlands, 1989. Examples used according to the invention the enzymes are PQQ-dependent glucosegalactose [glucose-dye-oxidoreductase, E. C. 1.1.1.50/1.1.1.91/1.1.1.97], spirodihydrofurans or lactate dehydrogenase. In the proposed invention the method is in particular possible to use PQQ-dependent glucosegalactose preferably for kolorimetricheskogo determination of glucose.

As a direct electron acceptors that accept electrons from the enzyme system /system dehydrogenase cofactor/ PQQ use enriched with electrons of aromatic nitroso compounds, which are known as electron acceptors for oxidase and NADH-independent dehydrogenases from European patent A-0-354441 and European patent EP-A-0441222 and in the case of which nitrosopropane directly connected with enriched electrons of the aromatic nucleus. The term "direct" electron acceptors means that the electrons directly catalyzed by enzymes, without the need for catalyst recovery, are accepted by the system enzyme/ cofactor.

In the sense of the invention, used as the recovered enriched with electrons of aromatic glenii corresponding to used PQQ-dependent the dehydrogenases of the substrate, from the enzyme and form aminosidine. Appropriate aminosidine contains aminogroup = NH, which through its double bond associated with the initial aromatic nucleus, and paired with its electrons of the double bond. Enriched with electrons of aromatic nitroso compounds contain one or more electron-donating residues or groups, or aromatic nucleus, which favor the formation of aminosidine the fact that they come in pair with aminogroups due to recoil electrons through the initial aromatic nucleus.

This refers to the substituents that have a +M effect on the aromatic nucleus. For example,

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Examples related to the aromatic nucleus residues R c +M-effect are such substituents as hydroxy, CNS, aryloxy, alkylthio, aaltio-, amino-, monoalkylamines, monoarylamino, dialkylamino and diarylamino.

In the case of derivative nitrosobenzene these substituents, for example, is particularly manifest their effect when they are in ortho - and/or paraprotein to nitrosopropane.

Groups with electron-donating and conducive to aminostyrene effect can also be sostav the practical nitroso compounds with an excess of electrons, in which the aromatic cyclic system is so enriched with electrons that external +M is the Deputy is unnecessary for the formation of stable due mezomerii aminogroup after recovery. The excess electrons formed due to the fact that there is more aromatic electrons than give the ring atoms through which can be distributed electrons. Such heterocycles are known to the specialist.

Examples of such aromatic heterocycles are antipyrine, pyrazol heterocycles and pyrazoles.

The excess of electrons used according to the invention, enriched with electrons of aromatic nitrosoguanidine has an additional effect, namely, that these nitroso compounds to a certain extent through keto-enol the tautomerism are in equilibrium with equivalent oximes limit structure

R-Ar-N=O____ R= Ar = N-OH

Both tautomeric limiting patterns in the framework of the invention should be covered by the term "aromatic nitroso compounds" as used according to the invention substances.

In the case of the above +M-substituents are meant CNS, alkylthio, monoalkylamines and dialkylaminoalkyl, to whom Eden hydroxyl group, if necessary substituted one or more times by alkyl with 1-6 C-atoms of the amino group, PO3H2, SO3H or CO2H. Acid residues PO3H2, SO3H and CO2H can be as such, or to be in salt form, as salts of ammonium, alkali or alkaline-earth metals.

Aryloxy and artioscad contain aromatic residues with 6-10 C atoms, and especially preferred phenoxy and penaltiestitle.

Ammonium salts are those which contain ammonium ion, or those that contain cations of ammonium, one - or multi-substituted alkyl, aryl or Uralkalij remains. The alkyl in the alkyl or Kilkenny residues, is a hydrocarbon residue with 1-6 C-atoms. Aryl in aryl and kalkilya residues is 6-10-u C-atoms of the aromatic cyclic system, preferably phenyl. Preferred Uralkali balance is benzyl.

Salts of alkali metals are preferably salts of lithium, sodium or potassium. Salts of alkaline-earth metals are preferably a salt of magnesium or calcium.

The above examples +M-substituents do not want what stiteler and in what place he has this effect on the aromatic system, or any aromatic heterocycles cyclical contain the appropriate grouping and how all these residues may be possible substituents in the aromatic nitrosoguanidine that can be used according to the present invention.

Aromatic basic structure used aromatic nitroso compounds preferably represents enriched with electrons of the aromatic ring with 5 to 7, especially preferably 5 to 6, carbon atoms or heteroatoms, which may be bellrowan with one or two aromatic and/or alicyclic rings. For aromatic analyoung rings while taking into account the carbon of the aromatic system and also heteroaromatic systems with the number of carbon atoms or heteroatoms 5-7, preferably 5-6.

Under alicyclic rings understand saturated or unsaturated cycloaliphatic compound with 5-7 C atoms or heteroatoms, preferably 5 - or 6 carbon atoms. Under the heteroatoms understand nitrogen, oxygen or sulfur.

Preferred to be used according to the invention by nitrosoguanidine are derived nitrazepam or more aromatic and/or alicyclic rings. As aromatic rings while taking into account the carbon of the aromatic system and also heteroaromatic compounds with the number in the cycle 5-7, preferably 5 or 6. Examples are bellerophone benzene or naphthalene cycles or bellerophone pyridine ring.

Especially preferred nitrosobenzene derivatives of General formula I

< / BR>
where R1is hydrogen, hydroxyl; alkyl, if necessary substituted by hydroxyl, COOH, PO3H2or SO3H; alkoxyl, alkylthio, aryloxy, aaltio, halogen, or amino group, in which case you need one - or multi-substituted by alkyl, if necessary substituted by hydroxyl, PO3H2, dialkylphosphinate, SO3H or CO2H; and

R2- hydroxyl group, CNS, aryloxy, aaltio or allylthiourea, and the alkyl residue, if necessary on its part is substituted by a hydroxyl group, CNS group, in case you need one - or multi-substituted alkyl amino group, PO3H2, SO3H or CO2H as such or in salt form, in the form of ammonium salts, alkali or alkaline-earth metals the mean hydrogen, aryl or alkyl group, which on its part can be substituted by hydroxyl, alkoxyl, hydroxyalkoxy, if necessary substituted by hydroxyl polyalkoxyalkyl group, PO3H2, SO3H, COOH as such or in salt form or, if necessary, one - or multi-substituted alkyl amino group; or

in which R3and R4can be alkilinity residue, which if necessary is interrupted by oxygen, sulfur or nitrogen, the nitrogen is substituted by an alkyl, hydroxyalkyl, hydroxyethoxyethyl, alkoxylalkyl, alkoxycarbonylmethyl, deoxynilvalenonum or polyalkoxyalkyl the remainder, which, for its part, depending on the circumstances, in the alkyl part may be substituted by a hydroxyl residue; or,

when R1is orthopaedie to NR3R4, R3or R4also with R1can be alkilinity the rest.

This halogen denotes fluorine, chlorine, bromine or iodine. Especially preferred fluorine and chlorine.

The alkyl in the alkyl, CNS or alkylthiophene is a hydrocarbon residue with 1-6 C-atoms, and about the Noah part hydroxyalkyl, dialkylaminoalkyl, hydroxyethoxyethyl, alkoxyalkyl, polyalkoxyalkyl, alkoxylalkyl and deoxynivalenol residues. Diaxonically residue is a residue that dioxane is a cyclic system is associated with the alkyl residue. Preferably it is about 1,4-dioxane cyclic system

< / BR>
Polyalkoxyalkyl residue is a residue of an alkyl(alkoxy)n-alkoxy, in which n = 1-10. Preferably n = 1-4. Particularly preferably n is 1-3. Alkilinity the remainder represents a linear or branched, preferably linear, saturated or unsaturated, preferably saturated, hydrocarbon chain of 2 to 5, preferably 2-4 C-atoms, with two empty seats in the binding.

Aryl in aryl and uralkaliem residues represents a containing 6-10 C atoms of the aromatic cyclic system, preferably phenyl.

Ammonium salts are those which contain the ammonium ion NH+4or those that contain cations of ammonium, one - or multi-substituted alkyl, aryl or Uralkalij remains.

Salts of alkali metals, preferably avli calcium.

Preferred residues R1are hydrogen and alkyl, preferably hydrogen.

Preferred residues R2are CNS residues and the amino group NR3R4.

The value of an educated R1and R3interrupted by oxygen, sulfur or nitrogen alkalinous residue is preferable formed by inclusion of the nitrogen atom according to the General formula I moholynagy, respectively thiomorpholine, respectively pieperazinove balance. Especially preferred pieperazinove the rest.

The value of an educated R1and R3alkalinous residue is preferable formed by incorporating an aromatic ring according to the General formula I indolinyl or 1,2,3,4-tetrahydroquinolines the rest.

To obtain salt proposed according to the invention nitrosoaniline derivative of General formula I are preferred in particular such a strong acid, in particular inorganic acids as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid. Particularly preferred hydrochloride, which are the salts of hydrochloric acid.

The preferred nitrosoguanidine General formula is-N'-/4-nitrosophenol/-piperazine, N-/2-hydroxyethyl/5-nitrosoaniline, 2,4-dimethoxy-nitrosophenol, N,N'-bis-/2-methoxyethyl/-4-nitrosoaniline, N-/4-nitrosophenol/-morpholine, N/2,2-diethoxy-ethyl/-N-/4-nitrosophenol/-piperazine, n-nitrosophenol, 3-methoxy-4-nitrosophenol.

Rich electrons heteroaromatic nitrosoguanidine, aromatic cyclic system which is so enriched with electrons that is excessive external +M-Deputy for nitroso-/oximetery and for the formation of imine, proposed according to the invention the method is especially suitable substituted nitrosopropane pyrazolones, pyrazoles and especially nitrosubstituted pyrazol compounds that, for example, described in Ullmann's Enzyclopedia of Industrial Chemistry, 5th ed., so And 20, S. 72-74. While the preferred 3-nitrosoproline compounds of General formula II

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These compounds mostly known as prestudy for preparative synthesis of the corresponding 3-aminopyrazole compounds of European patent application EP-A-0433854.

In formula II, X-Y - NR5CO or N = CR6;

R5is hydrogen; alkyl, if necessary substituted by hydroxyl, carboxyla, SO3H, PO3H2, dialkylphosphinate;

R6is hydrogen, unesenny by hydroxyl, carboxyla, SO3H, PO3H2, one salt of these acid residues and/or alkoxycarbonyl; or amino group, which, if necessary substituted by one or two, containing if need be one or more hydroxyl, carboxyl and/or alkoxycarbonyl alkyl residue remains, and when the amino group is substituted by two alkyl residues, these residues can also be cyklinowanie up ring which, in addition to the N atom of the amino group, if necessary, can also be interrupted by oxygen, sulfur or another nitrogen atom; or amino group, which, if necessary substituted by one or two acyl groups, alkoxy - and/or alcoxycarbenium groups, H2N-CO -, alkyl-, aralkyl and/or arylcarbamoyl groups; or carboxyl, alkoxycarbonyl, carboxamido or halogen; and

R7- alkyl, thioalkyl or aralkyl, if necessary substituted by hydroxyl, carboxyla, SO3H or PO3H2or amino group, which, if necessary substituted by one or two alkyl groups, which on its part can again be substituted by hydroxyl, carboxyla, SO3H, dialkylphosphinate or PO3H2; kalkilya group, which if necessary can be substituted by hydroxyl, carboxyla, SO3H or PO3H2; or

R7and R8together form a saturated or unsaturated 3 - or 4-membered chain of nitrogen atoms or carbon atoms and, if necessary, one or more nitrogen atoms or sulfur, and carbon atoms, if necessary, substituted alkyl, alkoxyl, alkylthio, hydroxyl, aralkyl, aryl, carboxyla, carboxamido, alkoxycarbonyl, cyano, halogen, amino group, which, if necessary substituted by one or two, if necessary containing one or more hydroxyl, carboxyl and/or alkoxycarbonyl residues, alkyl residues; and the nitrogen atoms, which are not bound via a double bond, substituted alkyl or aralkyl, if necessary substituted by hydroxyl, SO3H, PO3H2, carboxyla or dialkylphosphinate; or two adjacent substituent chains, if necessary, form alkylenes group, which on its part if necessary substituted by aryl or bellrowan with him;

and also, if necessary, the corresponding tautomeric forms and their salts.

"Ay or branched alkyl residue with 1-6, preferably 1-4 C-atoms. Examples are methyl, ethyl, through isobutylene or tert-bucilina group.

If the amino group is substituted by two alkyl residues, these residues can also be cyklinowanie in the ring so that, in General, they are interrupted by a nitrogen atom ring. While such preferred amino groups, which are generally 5 - or 6-membered ring and which, from its side, if necessary interrupted by oxygen, sulfur or nitrogen. Especially preferred moholynagy the rest.

"Alkoxy - alkoxy and alcoxycarbenium residues denotes a linear or branched CNS residue with 1-6, preferably 1-4 C-atoms. Examples are methoxy, ethoxy, propyloxy-, ISO -, Butylochka - or tert-butylacrylate.

"Aryl" is also in arylcarbamoyl groups denotes a carbon aromatic or heteroaromatic residue, preferably one with 6-10 atoms in the ring, particularly phenyl or naftalina group, which optionally can be substituted by alkyl, alkoxyl and/or halogen. Particularly preferred phenyl residue.

"Uralkaliy balance" - t is shekarchian aryl residue. The preferred benzyl group.

"Uralkaliy" residue, for example in alcoxycarbenium groups, refers to a residue in which the above-mentioned CNS group substituted vysokorelevatnym aryl residue. Preferred benzyloxy.

"Halogen" denotes residues: fluorine, chlorine, bromine and iodine. Preferred fluorine and chlorine,

"Acyl group" refers to a carboxylic acid residue, which may contain alkyl, kalkilya or aryl residues. The preferred acetyl, phenylacetylene or benzoline remains.

Under alkalinous group understand linear or branched, saturated or unsaturated hydrocarbon chain of 3 to 5, preferably 3 or 4 C-atoms with two empty seats in the binding.

Examples are: -CH2-CH= CH-, -(CH2)4- or-CH=CH-CH=CH-.

Preferred buteventually balance (-CH=CH-CH=CH-) and tetramethylenebis residue [-(CH2)4-].

Alkanniny the remainder represents a linear or branched hydrocarbon residue of 2-5 C-atoms with at least one double bond. Preferred, for example, vinyl residue. Under dialkylphosphinate group ponyatiyniy the rest.

As salts of SO3H, PO3H2- and carboxyl residues can be used salts of alkaline or alkaline-earth metal or ammonium. Under the salts of alkali metals understand salts of lithium, sodium, potassium, rubidium and cesium, preferably lithium salts, sodium and potassium, particularly sodium and potassium salts. Salts of alkaline-earth metals are salts of beryllium, magnesium, calcium, strontium or barium. Preferred salts of magnesium and calcium, particularly preferred calcium salt. As the ammonium salts can be used such unsubstituted ammonium ion, NH+4. However, it is also possible to use ammonium salts in which the ammonium ion is substituted by 1-4 two alkyl, aryl or Uralkalij remains. For these residues have the meaning of the above definition, and as the alkyl residue is particularly preferred methyl, ethyl, n-propyl, and as the aryl residue is particularly preferred phenyl group and as Arakelova residue is particularly preferred benzyl group.

As carboxamide residue understand the balance CONH2however , these residues, where the substituted amino group in the case of the selected and/or alkoxycarbonyl alkyl residue remains.

Used according to the invention of nitrosoguanidine General formula II are preferred such compounds in which R7and R8form a saturated or unsaturated chain, as described above. Particularly preferably in this case, when this circuit ninasimone and the electrons of the double bond and the free electron pair of nitrogen unsaturated chain conjugated with a double bond or colophony N-atom of the General formula II so that the result is bellerophone aromatic ring.

In case of necessity for substances of General formula II are also possible tautomeric forms. They must also covered the General formula II.

According to the invention preferred nitroso compounds of General formulas III - XII

< / BR>
When this X-Y has the same meaning as previously stated. R9, R10, R11and R12which are identical or different, denote hydrogen, hydroxyl, alkyl, alkoxyl, alkylthio, aralkyl, aryl, carboxyl, alkoxycarbonyl, carboxamido, cyano, amino group, which, if necessary substituted by one or two, if necessary containing one or more hydroxyl, carboxyl and/or alkoxycarbonyl residues, alkyl is, the which on its part if necessary substituted by aryl or bellrowan with him, and

R13represents an alkyl or aralkyl, which if necessary can be substituted by hydroxyl, carboxyla, SO3H, PO3H2or dialkylphosphinate. Determination of residues correspond to compounds of General formula II.

For proposed according to the invention apply particularly preferred substances of the General formulae III, IV, V, VII, VIII and IX, if necessary, the corresponding tautomeric forms and their salts. Especially preferred are such compounds where X-Y is set to N=CR6and R6can have a value as specified for the General formula II.

As especially suitable for use according to the invention has proved particularly such compounds as 3-nitroso-2-methyl-pyrazole-(1,5)-pyridine, 3-nitroso-pyrazolo(1,5-a) pyridine and 3-nitroso-pyrazolo(3.2-C]-S-triazole and their salts, in particular hydrochloride.

As described above, according to the proposed invention the method of aromatic nitroso compounds is introduced into contact with the test sample and PQQ-dependent dehydrogenases. In the European patents EP-A-0354441 and EP-A-0441222 describes x-electrons of aromatic amino compounds, which, on the other hand, can be detected using a color-forming reagent and oxidant through oxidative combinations.

It was unexpectedly found that in the presence of non-oxidative color-forming indicator for quinoid aminosidine and PQQ-dependent dehydrogenase, aromatic nitrosoguanidine the oxidation of the analyte does not pass through the stage of full, catalyzed PQQ-dependent dehydrogenases, enzymatic recovery to enriched electrons of the aromatic amine, and may stop at an intermediate stage formed after partial enzymatic recovery imine due to non-oxidative color-forming indicator and can be detected.

Even more unexpected was that, if you choose certain aromatic nitroso compounds which already contain chromogenic residue, these aromatic nitroso compounds in the presence of PQQ-dependent dehydrogenase, however, restored to the painted aminosidine, enzymatic further recovery of aromatic amine in essence, however, is not so well painted aminosidine, even without the need for reaction with the actual pigment x is s, chromogenic nitrosoguanidine below.

Although the suitable range, enriched with electrons of aromatic nitrosoguanidine very large, they all acceptinput PQQ-dependent dehydrogenase as direct electron acceptors. Significant, apparently, is only nitrosopropane, which is associated with enriched electrons aromatic residue.

It is suggested that the reaction can be represented by the following reaction scheme, for example nitrosoaniline

< / BR>
Aromatic nitrosoguanidine (1) the oxidation of the analyte is restored to an aromatic hydroxylamine (2), the latter spontaneously it water, thus a quinoid intermediate kinostudija (3). Before she recovers further enzymatic thanks PQQ-dependent the dehydrogenases to the aromatic amine, as it is known from European patent A-0354441 and A-0441222, it can, for example, by color-forming reagent (HP) to catch in the form of a colored product combinations (4) colorimetrically to decide whether or not it contains chromogenic residue product combinations already in the initial compound (5) and, thus, after enzymatic recovery can kolorimetricheskoe PQQ-dehydrogenases can be captured or to directly detect the intermediate kinostudio (3) according to the invention with the indicator, not realizing further known from European patent A-0354441 enzymatic recovery to Amin. When using flavin-dependent oxidase, which according to European patent A-0354441 also restore the aromatic nitroso compounds, it is possible to detect the intermediate kinostudio only in very small quantities, while a large part of the intermediate iminoctadine the oxidation of the analyte is very quickly restored enzymatic next to the aromatic amine.

According to the invention the detection of the quinoid aminosidine can also be performed using non-oxidizing coloring reagent. The term "dye" means that the color-forming reaction of the reagent with aminosidine obtained colored substance, the maximum absorption that is different from that used nitroso compounds and color-forming reagent to the reaction. Under non-oxidative dye indicator realize that the indicator reacts with the detected connection when there is color, and the light is not valid okikawa to find the connection (for example, due to contained in the auxiliary oxidizer).

It's also possible to combination of both principles detecting the fact that the color-forming reagent is combined with a molecule detectable aminosidine to obtain unpainted molecules leucogranites, which oxidize another molecule aminosidine to dye.

Such coloring indicators for aminosidine known in large numbers. Take into account all substances that react with oxidized derivatives of p-phenylenediamine with the emergence of color.

Examples of the dye by the reaction of a combination of indicators are aromatic compounds, preferably phenolic and naftalie compounds that are substituted for a good delete groups, and thus these groups are easily removed and can very quickly be replaced detected by aminosidine with the appearance of color. Preferred examples include 1-naphthol-4-acid, 2,4,6-tribromo-3-hydroxybenzoic acid, 2,4-dinosaurian also used indicators, which can not be combined with aminosidine, and which when restoring aminosidine to amine to form a colored compound. This occurs most often by dimerization of leucogranites. Suitable leucocrystal well-known specialist of the method for detecting hydrogen peroxide or peroxidase. Examples of such leucogranitee are 1-naphthalenesulfonate, triarylmethane (patent Germany 2735690), diariomotor (U.S. patent 4-919890), aminocarbonyl (patents Germany 2205733, 2338932), oksazolov, thiazole (European patent A-0167973), etc.

Fit all leucocrystal that on the basis of their redoxpotential able to restore aminosidine.

Finally, you can combine the principles of detection is the combination with the indicator and the oxidation of leucogranites is the fact that as a color-forming components for aminosidine used reagent, which aminosidine forms colourless lycosoidea that only through the second molecule of aminosidine when it was restored to the corresponding aromatic amine is oxidized to dye. The most common, well known in the color pictures of the so-called "color components about the Review about this kind of color components manifestations given by T. H. James in "theory of the Photographic Process", 3rd ed., Mc Millan, new York, 1966, Chapter 17, S. 385-390.

Proposed according to the invention the method is carried out in such a way that the sample is simultaneously in contact with PQQ-dependent dehydrogenases, one or more of the above, enriched with electrons, aromatic nitrosoguanidine and indicator for aminosidine. If the sample contains the analyte, which is oxidized PQQ-dependent dehydrogenases, aromatic nitrosoaniline enzymatic recovery reacts to the formation of the corresponding aminosidine. The latter reacts with the color-forming indicator so that the resulting color can be adjusted with the concentration of the analyte in the sample. Colorimetry can be accomplished visually through comparative colourings or photometrically.

A particularly simple way of oxidative detection of analytes takes place when formed due to the recovery of aromatic nitroso compounds aminosidine itself is colored and it is not necessary to detect the first, by combining with the dye indicator or by oxidation of leucogranites. Painted aminosidine, as described above, by themselves well-known specialist of color fotografico detection of the analyte due to the direct determination colored quinoid aminosidine as particularly suitable was nitrosoaniline the compounds of formula XIII as direct chromogenic electron acceptors, being restored to painted hinanden formula XIV

< / BR>
where R1(in the General formula (XIII) is hydrogen, hydroxyl, halogen, alkoxyl, alkylthio, aryloxy or aaltio; alkyl, if necessary substituted by hydroxyl, carboxyla, PO3H2or SO3H; amino group, in case you need one - or multi-substituted by alkyl, which in turn, if necessary, can be substituted by hydroxyl, PO3H2, dialkylphosphinate, SO3H or carboxyla;

X - Y - NR5-CO or N=CR6and R5and R6have the same meaning as specified in the General formula II;

A is a saturated or unsaturated three-membered chain with one atom of nitrogen or sulfur and two carbon atoms or two nitrogen atoms and one carbon atom, and carbon atoms, if necessary, substituted alkyl, alkoxyl, alkylthio, hydroxyl, aralkyl, aryl, carboxyla, carboxamido, alkoxycarbonyl, cyano, halogen, amino group, which, if necessary substituted by one or two, containing if need be one or more hydroxyl, carboxyl and/or alkoxycarbonyl residues, alkyl residues; and the atoms is SO3H or dialkylphosphinate, or aralkyl; or two adjacent substituent chains, if necessary, form alkylenes group, which on its part if necessary substituted by aryl or bellrowan with him;

and also, if necessary, the corresponding tautomeric forms and their salts.

The importance of certain substituents at this is the same as in the compounds of General formula II. Preferred residues R1are hydrogen or alkyl, and X Is Y preferably represents a group-N=CR6.

Particularly preferably forms A neighboring heterocycle imidazole, triazole, benzimidazole, thiazole or dihydroimidazole ring carbon atoms which can carry specified in the General definition for the value of A Deputy. Exclusively preferably imidazole ring.

Particularly preferred compounds are:

1) [2,4-dimethyl-pyrazole-(1,5-a)-imidazol-3-yl]-(4-nitrophenyl)- amine;

2) [4-methyl-pyrazole-(1,5-a)-imidazol-3-yl]-(4-nitrophenyl)- amine;

3) [4-(dimethylphosphino)-2-methyl-pyrazole-(1,5-a)imidazole-3 - yl] -(4-nitrodiphenylamine);

5) [5,6-dihydro-4-dimethylphosphino-2-methyl-pyrazole-(1,5-a) imidazole-3-the Ying;

7) [2,6-dimethyl-4-dimethylformamid-pyrazole(3,2-C)-S-triazole - 3-yl] -(4-nitrophenyl)-amine.

If proposed in the invention process is carried out so that by restoring the aromatic nitroso compounds are formed directly painted financiamento compound of formula XVII, the analyzed sample introduced simultaneously into contact with PQQ-dependent dehydrogenases and one or more enriched in electrons aromatic nitrosoguanidine General formula XVI. If the sample contains the analyte, which is oxidized PQQ-dependent dehydrogenases, enriched with electrons aromatic nitrosoguanidine reacts to the formation of the corresponding colored hinanden. Additional excess electrons recondensing on chromogenic enriched with electrons pyrazol ring A ring contributes to the fact that suddenly formed financiamento connection then no longer restores the enzyme to the corresponding amine, and painted financiamento connection right can be defined in the form of a measurement of light absorption and to correlate with the concentration of the analyte in the sample. In order to achieve a rapid enzymatic recovery chromogenic aromatics is positive to offer in the invention method using chromogenic nitrosoamine compounds of formula XIII is the case, when their concentration in the solution is at least 10-3mol/l, preferably at least 10-2mol/L.

Good solubility can be achieved in particular by the fact that the chromogenic nitrosoguanidine supplied hydrophilic groups.

Accordingly, exceptionally well suitable in the method of the invention, the chromogenic nitrosoguanidine are, for example, the above compounds 3, 4 and 7.

The method can be carried out by the so-called wet test, for example in a ditch, or in the form of so-called dry test on the corresponding carrier reagent, and the necessary test reagents are on the test media, in particular by absorbing or swelling material. Such test carriers are known, for example, from the patent of Germany A-3247608, European patent EP-A-0262445 or European patent EP-A-0256806.

Another object of the invention is an agent for the colorimetric determination of an analyte by enzymatic oxidation, which is characterized in the claims. This tool contains, along with the necessary enzymatic oxidation of the designated analyte PQQ-dependent dehydrogenases and along at least one or enzymatic oxidation of the analyte electrons from the system PQQ/dehydrogenase, then additionally give a non-oxidizing colouring indicator for aminosidine.

As PQQ-dehydrogenases, aromatic nitrosoguanidine coupler and indicators for quinoid aminosidine use described above for the proposed invention in the way of substance. If used in the proposed according to the invention the method is enriched with electrons of aromatic nitroso compounds of formula XIII, which already contain associated with aniline nitrogen chromogenic residue, the tool preferably contains no color-forming indicator for aminosidine, as are formed when restoring kinondoni formula XIV is already painted.

For establishing suitable for implementing the method pH-value, which is recommended especially for the used enzyme and indicator for aminosidine proposed according to the invention, the tool contains a buffer system. Preferably the medium contains a buffer system, which in the test solution sets pH 4-9. Particularly preferably slightly acidic pH value of 5-6,5.

Proposed according to the invention the tool may be in the form of solution are applied to the method of the invention, the reagents. The solvent is preferably water is concerned, however, about the mixture with a water-soluble organic solvents, such as methanol, ethanol, acetone or dimethylformamide. For reasons of stability, it may be preferable distribution required for the test reagents on two or more solutions, which are mixed only with the direct examination. However, you should pay attention to the fact that aromatic nitrosoguanidine and indicator for aminosidine before the beginning of the test was in the solution. In the case of concentrations used aromatic nitrosoguanidine are governed by the concentration of the designated analyte.

Typical concentrations determined according to the method of the invention of analytes represent 10-6- 10-2mol/l, in particular 10-5- 10-3mol/l, Respectively, typical concentrations used nitrosoguanidine be 10-4- 10-1mol/l to achieve a sufficiently rapid enzymatic recovery, especially preferred concentration nitrosoguanidine, especially chromogenic nitrosoguanidine formula XIV above 10-3mol/l if the concentration used in the concentrations of the enzyme are 1 mU/ml - 1 U/ml for the test in a ditch.

Indicators for aminosidine used in at least stoichiometric ratio to nitrosoguanidine, preferably 1.5 - 2-fold excess.

Proposed according to the invention, the tool also can be in the form of a test strip. These test strips are known in a variety of forms runtime, for example, from European patent EP-A-016387, the Federal Republic of Germany patent EP-A-3247608, European patents EP-A-0262445 or A-0256806. In the test strips (indicator paper) necessary to implement the method of determining the reagents are solid layers of media. As layers carriers take into account particularly absorbent and/or swellable materials that are investigated liquid. Examples are gelatin, cellulose or other fibers. Inside or on those media are reagents in solid form. When applying the liquid under study for a test strip or by dipping the test strip into the test liquid on the strips is formed a liquid medium in which the reaction takes place detection. Caused by the reaction of the formation of color can be evaluated visually or photometrically, for example reflective/photometrically.

Usually 10-4- 10-1< / BR>
Nitrosoguanidine - 10-3- 1

Non-oxidizing indicator - 10-3- 1

Enzyme units on the test field of 0.1 - 100

The present invention has the advantage that there is no need for any non-specific catalysts recovery, as diaphorase or methylpentene-methosulfate, to restore the electron acceptor, and its restoration is directly due to the specific analyte/enzyme. Thus, it is possible to avoid interfering side reactions. Through the use of nitrosoaniline as electron acceptors more there is no restriction of the electron acceptor, for example, due to slow diffusion, as in the case of oxygen as electron acceptor. One equivalent of detectable intermediate iminoctadine oxidized only one equivalent of the analyte. This contributes to the high sensitivity detection of the analyte. There is no need in any oxidizer to react with the coloring reagent. Therefore, all the reaction detection can be performed without interference in one stage and in the overall solution. Large assortment of used color-forming indicator, among other things such costulata measurement, and the extinction coefficient, which determines the sensitivity of the measurement.

Especially easy is proposed in the invention method when formed directly painted aminosidine and there is no need for reaction with the coloring reagent.

The next subject of invention is a new chromogenic nitrosoaniline compound of formula XIII

< / BR>
where R1is hydrogen, hydroxyl, halogen, alkoxyl or alkylthio, aryloxy or aaltio, alkyl, if necessary substituted by hydroxyl, carboxyla, PO3H2, dialkylphosphinate or SO3H, amino group, in which case you need one - or multi-substituted by alkyl, which in turn, if necessary, can be substituted by hydroxyl, PO3H2, SO3H or carboxyla; and NR5-CO or N=CR6,

X-Y - NR5-CO or N=CR6and R5is hydrogen, alkyl, if necessary substituted by hydroxyl, carboxyla, SO3H, PO3H2, dialkylphosphinate, and R6is hydrogen, alkyl, if necessary, depending on the circumstances, substituted by hydroxyl, dialkylphosphinate, carboxyla, SO3H, PO3H2, salt of one of these Kadoma, containing, if necessary, one or more hydroxyl, carboxyl and/or alkoxycarbonyl alkyl residues residues; and, when aminooctanoic substituted by two alkyl residues, these residues can also be cyklinowanie to form a ring, which in addition to the N atom of the amino group, if necessary, can also be interrupted by oxygen atom, sulfur or another nitrogen atom, or amino group, which, if necessary substituted by one or two acyl groups, alkoxy - and/or alcoxycarbenium groups, H2N-CO -, alkyl-, aralkyl and/or arylcarbamoyl groups; or hydrogen, carboxyl, alkoxycarbonyl, carboxamido or halogen; and

A is a saturated or unsaturated three-membered chain of one atom of nitrogen or sulfur atom and two carbon atoms or two nitrogen atoms and one carbon atom, and carbon atoms, if necessary, substituted alkyl, alkoxyl, alkylthio, hydroxyl, aralkyl, aryl, carboxyla, carboxamido, alkoxycarbonyl, cyano, halogen, amino group, which, if necessary substituted by one or two, if necessary containing one or more hydroxyl, carboxyl and/or Ala is, substituted by hydrogen, alkyl, if necessary substituted by hydroxyl, SO3H, carboxyla, PO3H2or dialkylphosphinate; or aralkyl, or two adjacent substituent chains, if necessary, form alkylenes group, which on its part if necessary substituted by aryl or bellrowan with him;

and also, if necessary, the corresponding tautomeric forms and their salts.

The importance of certain substituents at this is the same as in the compound of General formula II.

Preferred residues R1are hydrogen and unsubstituted or substituted alkyl. X-Y preferably forms a group N=CR6and R6- preferably hydrogen or unsubstituted or substituted alkyl.

Preferably A together with the neighboring heterocycle forms imidazole, triazole, benzimidazole, thiazole or dihydroimidazole ring, in which the substituents of the ring corresponds to those of the General formula III.

Exclusively preferably forms A imidazole ring. Particularly preferred compounds are:

1) [2,5-dimethyl-pyrazole-(1,5-a)-imidazol-3-yl]-(4-nitrophenyl)-amine;

2) [4-methyl-pyrazole-nitrosoproline);

5) [5,6-dihydro-4-dimethylphosphino-2-methyl-pyrazole-(1,5-a) imidazole-3-yl]-(4-nitrophenyl)-amine;

6) (4-(dimethylphosphino)-2-methyl-pyrazole-[1,5-a] imidazol-3-yl)-(4-nitrophenyl)-amine;

7) (2,6-dimethyl-4-dimethylformamid-pyrazole[3,2-c]-s-triazole-3-yl)-(4-nitrosamine)-amine.

Getting proposed according to the invention compounds of formula XIII exercise in itself known methods, which are described in the publication J. T. Hays and others in J. Org.Chem. 32, 158 (1967). For this purpose, ethers, preferably simple methyl esters, p-nitrosoaniline derivatives with the proton catalysis enter into interaction with the corresponding 3-aminoheterocycles. When replacing metoxygroup formed secondary amines of formula XIII.

Used heteroaromatic or described in the literature or can be obtained analogously to known methods. In particular, in European patent A-0433854 described amino compounds, which are the main structures contain pyrazol heterocycles.

Required as intermediate products of amino compounds usually for reasons of stability are in the form of salts of strong acids, for example inorganic acids. To interact with p-nitrosatable, for example, by dissolving the salt in water, adding a base until a pH of 8-10 and extraction of the free base with an organic solvent, for example acetic ether or methylene chloride. An alternative for this purpose, particularly if it is difficult extractable amino compounds, you can do the following: aminosidine dissolved in methanol, then add a base, for example a solution of NaHCO3, triethylamine and so on, until a pH-value of the methanol solution according to wet pH indicator paper about 5-6. Then add the second reaction component, p-nitrosoamines.

Example 1. Determination of glucose using PQQ-dependent glucose-dye-oxidoreductase, and 1-naphthol-sulfonic acids as components of a color display.

In a dish mix ml:

Citrate buffer/pyrophosphate buffer (50 mmol) of pH 7.5 - 0,38

N,N-Bis-(2-hydroxyethyl)-4-nitrosoaniline (5 mmol, buffer) - 0,40

1-Naphthol-4-acid (10 mmol in H2O) as a reagent colored manifestations - 0,20

Solution samples (0-40 mmol glucose) - 0,01

The test starts by adding 0.01 ml of an enzyme solution (glucose-dye-oxidoreductase, EC 1.1.99.17, 900 u/mg, 800 u/ml) and measuring the 5 min (Fig.1).

Example 2. Detection of glucose using PQQ-dependent glucose-dye-oxidoreductase, and 2,4,6-tribromo-3-hypoxantine acid as indicator.

The measured mixture (final concentration):

Citrate buffer, pH 5.8, mmol - 100

2,4,6,-tribromo-3-hydroxybenzoic acid, mmol - 10

N,N-bis-(2-hydroxyethyl)-4-nitrosoaniline, mmol - 1

Glucose-dye-oxidoreductase, u/ml - 10

Sodium nitrate, mol - 0,1

Glucose, mmol - 100-500

With increasing concentration of glucose see increasing education green dye (max = 700 nm, Fig.2).

Example 3. In the same way you can use the table. 1 other reagents color display for Iminov. In table. 1 shows absorption maxima obtained after combination of N,N-bis-(2-hydroxyethyl)-p-hinanden with the specified color components manifestations.

Example 4. Analogously to example 1 using various listed in table. 2, enriched with electrons of aromatic nitroso compounds. In table. 2 presents staining, respectively, the absorption maxima obtained after development with 1-naphthol-2-acid or 1-naphthol-4-acid.

Example 5. The comparison determining the glucose-dye-oxidoreductase and glucose-oxidase.

In the cuvette is placed, mmol:

Phosphate buffer, pH 7.5 - 100

N,N-bis-(2-hydroxyethyl)-4-nitrosoaniline - 1

1-Naphthol-4-acid - 10

Glucose-dye-oxidoreductase, units/ml - 100

By adding glucose (0-60 mmol) receive the appropriate amount of the dye (Fig. 3).

If glucose-dye-oxidoreductase replace the same number of glucose-oxidase, you get, on the contrary, only 8 times smaller than the output of the dye, as a large portion formed intermediate stage hinanden restored the next oxidase at the rate of glucose formation phenylenediamine (Fig. 4).

Example 6. Determination of glucose using PQQ-dependent glucose-dye-oxidoreductase through remedial education painted aminosidine.

The reaction equation

< / BR>
Spectra of compound 3 and compound 3A shown in Fig. 5.

The measured mixture (final concentration):

Citrate buffer, pH 6, mmol - 200

p-Nitrosoaniline (N 3 of table. 3), mmol - 1

CaCl2mmol - 1

Mutarotase, units/ml - 20

Glucose-dye-oxidoreductase, units/ml - 10

After the addition of different amounts of glucose between 0 and end continenza of the table. 3. In table. 3 shows absorption maxima used nitrosoguanidine and obtained colored financiamento.

Example 7. Determination of enzyme activity of PQQ-dependent glucose-dye-oxidoreductase by remedial education painted aminosidine of aromatic nitroso compounds.

Described in example 6 reaction can also be used to measure enzyme activity of PQQ-dependent dehydrogenase.

The measured mixture (final concentration), mmol:

Citrate buffer, pH 5.8 - 200

p-Nitrosoaniline (3) of example 6 - 1

Calcium chloride - 1

Glucose - 30

Kinetic measurement start using 0-1 mU glucose-dye-oxidoreductase and is measured in time staining (Fig. 7).

Example 8. (4-(Dimethylphosphino)-2-methyl-pyrazole-(1,5-a)-imidazol-3-yl) -(4-nitrophenyl)-amine (3).

of 13.7 g of 3-Amino-2-methyl-4-(dimethylphosphino)-pyrazole-[1,5-a]-imidazole-dihydrochloride are dissolved in 350 ml of methanol. The solution is cooled to about 5oC and mixed with a concentrated aqueous solution of sodium bicarbonate until wet pH indicator paper shows the pH-value of about 6. Then to the mixture during the course the Oh temperature and by adding further quantities NaHCO3maintain the pH value is 6.

The reaction mixture is filtered, mixed with approximately 150 ml of silica gel and evaporated to dryness. Silica gel contribute in a column and the product produce by elution using a mixture of toluene with methanol = 2:1. Obtain 10.3 g black-brown mass, which again chromatographic on silica gel using a mixture of methylene chloride with methanol = 12:1. Obtain 4.9 g of the target compound with so pl. 204oC (decomposition).

Rf(silica gel) toluene/methanol (2: 1) = 0,3; CH2Cl2/methanol (12:1) = 0.21 in.

< / BR>
Getting the original product.

a) 4-Dimethylphosphino-2-methyl-pyrazole-(1,5-a)-imidazole.

37 g of 2-Methyl-pyrazole-(1,5-a)-imidazole (J. Het. Chem. 10, 441/(1973)) was dissolved in 370 ml of anhydrous dimethylformamide and mixed with a 54.2 g of chlorotrimethylsilane and 119 g of potassium carbonate. The mixture is stirred for 10 h at 115oC (bath temperature). The precipitate is sucked off and the filtrate is evaporated. The remainder chromatographic on silica gel (eluting agent ethyl acetate/methanol = 2: 1). Generally get 36,6 g of target compound in the form of a mixture of brown crystals with brown butter.

RCU (silica gel, ethyl acetate/methanol = 2:1): Rf=0,2.

c). 3-Avarayr in 25 ml of concentrated hydrochloric acid and 50 ml of water. Then to the resulting solution at 0oC was added dropwise a solution of 6.2 g of sodium nitrite in 25 ml of water. After 30 min at 0oC solution slightly alkalinized by adding sodium bicarbonate solution and then added in several portions with 21.4 g of dithionite sodium. The mixture is stirred for another 30 min and mix it with a solution of 17 g of di-tert-butyl-dicarbonate in 100 ml of dioxane. The reaction mixture was stirred over night at room temperature, the dioxane is distilled off and the residue is repeatedly extracted with a mixture of n-butanol with acetic acid ethyl ester in a ratio of 3:1. Remaining after drying and evaporation of the organic phase the residue is dissolved in 320 ml of saturated hydrogen chloride methanol. Stirred for further 2 h, cooled in an ice bath and filtered off the crystals formed. In General gain of 18.1 g of the target compound.

TLC (silica gel, toluene/methanol = 3:1): Rf= 0,1.

Example 9. (4-Nitrophenyl)-2-methyl-(4-sulfopropyl-pyrazole-(1,5-a)-imidazol-3-yl)-amine (4).

Analogously to example 7 3-amino-4-sulfopropyl-pyrazole-(1,5-a)-imidazole enter into interaction with p-nitrosoaniline in methanol. The reaction mixture is filtered and the filtrate is evaporated. The remainder chromatographic on silica gel using a mixture of toluene/blueroot using stepwise gradient of methanol/water = 1:9 to 2: 8. Containing the product fractions unite, concentrate, process a small amount of ethanol and the product is precipitated by adding ether. Get the target connection in the form of a brown powder.

TLC (silica gel, ethanol: Rfor = 0.6, a mixture of isopropanol/butyl acetate/water = 5:3:2 Rf= 0,48).

< / BR>
Getting the original product.

a). 2-Methyl-4-sulfopropyl-pyrazole-(1,5-a)-imidazole.

2-Methyl-pyrazole-(1,5-a)-imidazole is administered in cooperation with the salt phenyldiazonium, which is obtained in the usual manner by diazotization of aniline at pH 2 to 5. The obtained 2-methyl-3-phenylazo-pyrazole-(1,5-a)-imidazole (5.6 g) was dissolved in 60 ml of dimethylformamide, mixed with 3.4 g of propanesultone and 7 g of potassium carbonate. The mixture is stirred for 6 h at 110oC, again add 5 g of propanesultone and 7 g of potassium carbonate and stirred for the next 8 h at 110oC. After cooling is filtered off and the residue is well washed twice with methanol. The solution is concentrated and the residue is chromatographically separated on silica gel (eluting agent ethyl acetate/methanol/water = 75:15:10). Output: 5.3v g of yellow crystals-korichnevogo color.

TLC (silica gel, ethyl acetate/methanol/water = 75:15:10): Rf= 0,3.

b). 3-Amine glacial acetic acid for 1 h portions mixed with 4 g of zinc powder. The mixture is stirred for another 30 min, sucked off and evaporated. The residue is triturated with 30 ml of ethyl acetate, sucked off and the filtrate discarded. Obtain 10.2 g of gray-brown powder which was sufficiently pure for further processing.

TLC (silica gel, ethyl acetate/acetone/glacial acetic acid/ water=5:2:2: 1): Rf=0,14.

Example 10. Analogously to example 8 or 9 receive connection table.4. The original synthesis of amino compounds is as follows:

a) Obtaining a source connection for the connection 5 of table.4: 3-amino-5,6-dihydro-, 4-dimethylphosphino-2-methyl-pyrazole-(1,5-a)- imidazole.

of 3.9 g of 2-methyl-pyrazole-(1,5-a)-imidazoline, 2.7 g of sodium acetate and 7.4 g of tetrafluoroborate p-methoxybenzothiazole dissolved in 40 ml of glacial acetic acid and the solution for 1 h, heated at 40-50oC. the Reaction mixture is evaporated, the residue is treated with sodium bicarbonate solution and ethyl acetate. Extracted with ethyl acetate, dried and evaporated. The residue is purified by chromatography on silica gel (eluting agent: ethyl acetate/ligroin= 1: 1 to 2:1, ethyl acetate, ethyl acetate/methanol = 95:5). Obtain 4.12 g of the corresponding 3-azo compounds (TLC: silica gel, ethyl acetate/methanol = 95:5: Rf= 0,5), which al the work carried out by recovery athropy analogously to example 8,b).

For isolation and purification, the crude amino compound is dissolved in a little water, mixed with solid sodium bicarbonate and add a solution of a threefold molar amount of di-tert-butyl-dicarbonate in dioxane. The mixture is stirred overnight, evaporated, extracted first with ether to remove by-products, and then 5 times with methylene chloride, to highlight the product. Get crystalline tert-butyloxycarbonyl connection (TLC: ethyl acetate/acetone/glacial acetic acid/water= 50:25:12: 5: Rf=0,5), which for the removal of tert-butyloxycarbonyl group dissolved in 50 ml of methanolic solution of hydrogen chloride. After 1 h at room temperature, concentrated to half the product of precipitation with ether. Get the target compound as hydrochloride.

TLC (n-butanol/glacial acetic acid/water = 2:1:1): Rf=0,3.

b). Getting the parent compound to compound 6 in table.4: 3-amino-4-dimethylphosphino-2-methyl-pyrazole-(1,5-a) benzimidazole.

The target connection receive analogously to example 10 by reacting 2-methyl-pyrazole-(1,5-a) benzimidazole (J. prakt.Chem. 326, 829 (1984) salt phenyldiazonium, N-alkylation using chloromethyl-dim is unity in the form of trihydrochloride-dihydrate with so pl. 192oC (decomposition).

TLC (silica gel; isopropanol/butyl acetate/water = 50:30:20):Rf= 0,38.

in). Getting the parent compound to compound 7 in table.4: 3-amino-2,6-dimethyl-4-dimethylphosphinic-pyrazole-(3,2-c)-s-triazole.

6 g of 2,6-Dimethyl-pyrazole-(3,2-c)-c-triazole are dissolved in 65 ml of dimethylformamide and mixed solution of 3.9 g of chloromethyl-dimethylpropanamide and 8 g of potassium carbonate. The mixture is stirred for 2 h at 100oC, sucked off hot and the filtrate is evaporated. The remainder chromatographic on silica gel (eluting agent: ethyl acetate/methanol = 4:1). For saponification and decarboxylation of the product is refluxed for 7 h in concentrated hydrochloric acid. The reaction mixture is evaporated. Get a light brown oil.

TLC (silica gel: ethyl acetate/methanol = 2:1): Rf=0,37.

Turning videolooking product 3-amino-connection is carried out analogously to the method described in example 8 for the corresponding 3-aminopyrazole-(1,5-a)-imidazole (8,b). Get the target compound in hydrochloride with so pl. 255oC (decomposition).

TLC (silica gel; ethyl acetate/methanol = 3:1): Rf=0,2.

1. The colorimetric method of determination and the second electron acceptor group is enriched with electrons of aromatic nitrosoguanidine and detection of reconstructed electron acceptor by a color display as measures of the quantity of the analyte, characterized in that as the oxidoreductase used PQQ-dependent dehydrogenase, while the electron acceptor restore to aminosidine by oxidation of the analyte, and if the electron acceptor is not capable of color expression, detection is carried out in the presence of non-oxidizing dye indicator for aminosidine or in the presence of leucogranites, which is oxidized in the presence of aminosidine to dye.

2. The method according to p. 1, characterized in that the aromatic nitroso compound is used as a compound of the formula I

< / BR>
where R1means hydrogen, hydroxyl, alkyl, if necessary substituted by hydroxyl, COOH or PO3H2, SO3H, alkoxy, alkylthio, aryloxy, aaltio, halogen or amino group, in which case you need one - or multi-substituted by alkyl, substituted if necessary by hydroxyl, PO3H2, dialkylphosphinate, SO3H or COOH;

R2means a hydroxyl group, alkoxyl, aryloxy, aaltio or allylthiourea, and the alkyl residue, if necessary on its part is substituted by a hydroxyl group, alkoxygroup, in case you need one in the first form, as ammonium salts, alkali or alkaline earth metal, or denotes an amino group, NR3R4where TO3and R4may be the same or different and represent hydrogen, aryl or alkyl group, which on its part can be substituted by hydroxyl, CNS, hydroxyalkoxy, if necessary, replacement by polyalkoxy, PO3H2, SO3H,COOH as such or in salt form, if necessary, one - or multi-substituted by alkyl, amino group, or in which R3and R4can be alkilinity residue, which if necessary is interrupted by oxygen, sulfur or nitrogen, the nitrogen is substituted by an alkyl, hydroxyalkyl, hydroxyethoxyethyl, alkoxyalkyl, alkoxycarbonylmethyl, deoxynilvalenonum or polyalkoxyalkyl residue, which, on its part, as the case may be, in the alkyl part may be substituted by hydroxyl, or if R1is orthopaedie to NR3R4, R3or R4also with R1can be alkilinity the rest.

3. The method according to PP.1 and 2, characterized in that the arc is>and R5is hydrogen, alkyl, if necessary substituted by hydroxyl, carboxyla, SO3H, PO3H2, dialkylphosphinate;

R6means hydrogen, alkyl, alkenyl, alkoxyl, alkylthio, aryl, aaltio, aralkyl, if necessary, depending on the circumstances, substituted by hydroxyl, dialkylphosphinate, carboxyla, SO3H, PO3H2, one salt of these acid residues and/or alkoxycarbonyl, or denotes an amino group, which, if necessary substituted by one or two, if necessary, bearing one or more hydroxyl, carboxyl and/or alkoxycarbonyl residues, alkyl residues, and if the amino group is substituted by two alkyl residues, these residues can also be closed to form a ring which, in addition to the N atom of the amino group, if necessary, can also be interrupted by oxygen, sulfur or another nitrogen atom, or amino group, which, if necessary substituted by one or two alkyl groups, alkoxy - and/or alcoxycarbenium groups, H2N-CO -, alkyl-, aralkyl and/or arylcarbamoyl groups, or denotes hydrogen, carboxyl, alkoxycarbonyl, carboxamido or halogen; the Silom, SO3H or PO3H2or amino group, which, if necessary substituted by one or two alkyl groups, which, in turn, can be substituted by hydroxyl, carboxyla, SO3H, dialkylphosphinate or PO3H2and at least R6and/or R7represents an amino group;

R8denotes alkyl or aracelio group, which if necessary can be substituted by hydroxyl, carboxyla, SO3H or PO3H2,

or B7and R8denote saturated or unsaturated three - or four-membered chain of nitrogen atoms or carbon atoms and, if necessary, one or more nitrogen atoms or sulfur atoms, and carbon atoms, if necessary, substituted alkyl, alkoxyl, alkylthio, hydroxyl, aralkyl, aryl, carboxyla, carboxamido, alkoxycarbonyl, cyano, halogen, amino group, which, if necessary substituted by one or two, if necessary, bearing one or more hydroxyl, carboxyl and/or alkoxycarbonyl residues, aryl residues, and nitrogen atoms that are not bound via a double bond, substituted by alkyl or Aral the group, that from their side if necessary substituted by aryl or bellrowan with him,

and also, if necessary, the corresponding tautomeric forms and their salts.

4. The method according to PP.1 to 3, characterized in that the aromatic nitroso compounds is used as a compound in which R7and R8form a saturated or unsaturated chain.

5. The method according to PP.1 to 4, characterized in that the aromatic nitroso compounds is used as a compound of formula III - XII

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
where R9, R10, R11and R12which are identical or different, denote hydrogen, hydroxyl, alkyl, alkoxyl, alkylthio, aralkyl, aryl, carboxyl, alkoxycarbonyl, carboxamido, cyano, amino group, which, if necessary substituted by one or two carriers, if necessary, one or more hydroxyl, carboxyl or/and alkoxycarbonyl residues, alkyl residues, or halogen, and two neighboring residue, if necessary, form alkylenes group, which on its part if necessary substituted by aryl or bellrowan with him;

R13denotes alkyl or aralkyl that opinion.

6. The method according to PP.1 to 5, characterized in that as non-oxidative coloring of the indicator uses a derivative of phenol, naphthol, aniline or naphtylamine.

7. The method according to PP.1 - 6, characterized in that as non-oxidative dye indicator use of 2,4,6-tribromo-3-hydroxybenzoic acid, 2,4-dibromo-1-naphthol or 1-naphthol-4-acid.

8. The method according to PP.1 to 7, characterized in that as leucogranites use diariomotor, triarylmethane or naphthylamines.

9. The method according to PP.1 to 8, characterized in that the use of aromatic nitroaniline compound of General formula XIII

< / BR>
where R1denotes hydrogen, hydroxyl, halogen, alkoxyl, alkylthio, aryloxy or aaltio, if necessary substituted by hydroxyl, carboxyla, PO3H2or SO3H, the amino group, if necessary, one - or multi-substituted by alkyl, which, in turn, if necessary, can be substituted by hydroxyl, PO3H2, dialkylphosphinate, SO3H or carboxyla;

X-Y represents NR5-CO or N = CR6and R5denotes hydrogen, alkyl, if necessary substituted by hydroxyl, carbox is kiltie, aryl, aaltio, if necessary, depending on the circumstances, substituted by hydroxyl, carboxyla, SO3H, PO3H2salt of one of these acid residues and/or alkoxycarbonyl, or denotes an amino group, which, if necessary substituted by one or two bearing one or more hydroxyl, carboxyl and/or alkoxycarbonyl residues, alkyl residues, and when the amino group is substituted by two alkyl residues, these residues can also be snapped to the ring, which, in addition to the N atom of the amino group, if necessary, can also be interrupted by oxygen, sulfur or another nitrogen atom, or amino group which, if appropriate, substituted by one or two acyl groups, alkoxy - and/or alcoxycarbenium groups, H2N-CO -, alkyl-, aralkyl and/or arylcarbamoyl groups, or denotes carboxyl, alkoxycarbonyl, carboxamido or halogen,

A represents a saturated or unsaturated three-membered chain with one atom of nitrogen or sulfur and two carbon atoms or two nitrogen atoms and one carbon atom, and carbon atoms, if necessary, substituted alkyl, alkoxyl, alkylthio, hydroxyl, aralkyl, Ari is nitrosoaniline the compound of formula XIII with A neighboring heterocycle forms a benzimidazole, the thiazole, imidazole, dihydroimidazole or triazole.

11. The method according to p. 9 or 10, characterized in that the aromatic nitrosoguanidine used in concentrations higher than 10-3mol/L.

12. The method according to PP.1 - 11, characterized in that for the determination of glucose as a PQQ-dependent dehydrogenase using glucose-dye-oxidoreductase.

13. Agent for the colorimetric determination of an analyte in a liquid by means of enzymatic oxidation, including the oxidoreductase and direct acceptor of electrons from the number of enriched electrons of aromatic nitrosoguanidine, characterized in that it further comprises a non-oxidizing dye as an indicator for aminosidine or leucocrystal, oxidizable in the presence of aminosidine to dye, and as the oxidoreductase is a PQQ-dependent dehydrogenase.

14. Means on p. 13, characterized in that the aromatic nitroso compounds it contains a compound of General formula I, characterized in p. 2.

15. Means under item 13 or 14, characterized in that the aromatic nitroso compounds it contains a compound of General formula II, as described in paragraph 3.

16. The tool contains leucocrystal.

17. Tool for PP.13 to 16, characterized in that the aromatic nitrosoguanidine is a compound of formula XIII, as described in paragraph 9.

18. Tool for PP.13 to 17, characterized in that the aromatic nitroso compounds it contains a compound of formula XIII, as described in paragraph 9, in which a has the meanings given in paragraph 10.

19. Test-system for detection of the analyte in the fluid, representing an inert carrier containing the reagents, characterized in that as the reagent it contains a tool according to any one of paragraphs.13 - 18.

20. Nitrosoaniline the compounds of formula XIII

where R1is hydrogen, hydroxyl, halogen, C1-C6-alkyl, C1-C6-alkoxyl;

X-Y - N=CR6and R6is hydrogen;

And denotes a saturated or unsaturated three-membered chain with one atom of nitrogen or sulfur and two carbon atoms or two nitrogen atoms and one carbon atom, and carbon atoms, if necessary, substituted C1-C6-alkyl, C1-C6-alkoxyl, C1-C6-alkylthio, hydroxyl, halogen, and nitrogen atoms that are not bound via a double bond, possibly substituted C1-C6-AA carbon in this three-membered chain form alkylenes chain.

21. Connection on p. 20, characterized in that together with the neighbouring heterocycle to form a benzimidazole, thiazole, imidazole, dihydroimidazole, triazole.

22. Connection on p. 20 or 21 as a chromogenic electron acceptors during enzymatic colorimetric determination of the analyte.

 

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The invention relates to the field of chemistry of biologically active substances, which may have application in medicine

The invention relates to new derivatives of pyrazolo/4,3-d/pyrimidine-7-it formula I, where R1- H, CH3C2H5, R2- CH3CH2OH, CH2OCH3or n - C3H7, R3- C2H5CH2= CH - CH2, R4together with the nitrogen atom to which it is attached is 4-(R5)-piperidino - or 4-N (R6)-piperazino group, R5- H, N(CH3)2, CONH2, R6- H, CH3i - C3H7CH2CH2OH, CSNH2C(NH)NHCH3or C(NH)S CH3and their pharmaceutically acceptable salts, pharmaceutical compositions showing inhibitory activity against cyclic guanosin-31,51-monophosphatase (CGMP), which contains 1-400 mg per single dose of the compounds of formula (I) in a mixture with a pharmaceutically acceptable diluent or carrier; the method of treatment or prevention of conditions caused by the activity of CGMP, the essence of which consists in assigning to the person an effective amount of the compounds of formula (I) or its pharmaceutically acceptable salt or above compositions

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The invention relates to compounds corresponding to the following formula I:

< / BR>
where R1- represents a group of formula

< / BR>
where n = 1 or 2, R3represents hydroxyl, lower alkoxygroup, aryl (lower) alkoxygroup, amino group, lower alkylamino or di (lower alkyl) amino group, R4represents hydrogen, lower alkyl or aryl (lower) alkyl , R5represents hydrogen, lower alkyl, aryl (lower) alkyl or lower alkylsulphonyl, R6represents hydrogen or lower alkyl, provided that when R6is lower alkyl, then R6replaces one of the methylene hydrogen atoms, R2represents a group of formula

< / BR>
where X is hydrogen, halogen, lower alkyl or lower alkoxygroup,

R7represents lower alkyl or aryl (lower) alkyl,

R8represents hydrogen or lower alkyl,

R9represents hydrogen, lower alkyl, lower alkenyl, lower quinil, aryl (lower) alkyl, formyl, lower alkyl carbonyl, aryl (lower alkyl) carbonyl Il is kilcarbery,

R11represents hydrogen, lower alkyl or aryl (lower) alkyl,

and such compounds are applicable for alleviating various memory disorders, characterized by a cholinergic deficit such as disease Alzheimer

FIELD: organic chemistry, chemical technology, herbicides.

SUBSTANCE: invention describes a method for preparing compounds of the formula (I):

wherein each R1, R2, R3 means independently of one another (C-C6)-alkyl; R can represent also pyridyl; R4 and R5 in common with nitrogen atoms to which they are joined form unsaturated 5-8-membered heterocyclic ring that can be broken by oxygen atom; G means hydrogen atom. Method involves interaction of compound of the formula (II):

wherein R1, R2 and R3 have above given values; R6 is a group RR9N-; R7 is a group R10R11N-; each among R8, R, R10 and R11 means independently of one another hydrogen atom or (C1-C6)-alkyl in inert organic solvent being optionally with the presence of a base with compound of the formula (IV) ,

(IVa)

or (IVb) ,

wherein R4 and R have above given values; H x Hal means hydrogen halide. The prepared compound of the formula (I) wherein G represents ammonium cation is converted to the corresponding compound of the formula (I) by treatment with Brensted's acid wherein G represents hydrogen atom. Also, invention describes compound of the formula (II) wherein R1, R2, R3, R6 and R7 have above indicated values.

EFFECT: improved preparing method.

9 cl, 12 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to derivatives of benzodiazepine. Invention describes a derivative of benzodiazepine of the formula (I): wherein dotted lines show the possible presence of a double bond; R1, R2, R3, R4 and R5 are given in the invention claim; n represents 0, 1, 2, 3 or 4; X represents sulfur atom (S) or -NT wherein T is give in the invention claim; A represents hydrogen atom, (C6-C18)-aryl group substituted optionally with one or more substitutes Su (as given in the invention claim) or (C1-C12)-alkyl; or in alternative variant R4 and R5 form in common the group -CR6=CR7 wherein CR6 is bound with X and wherein R6 and R7 are given in the invention claim, and their pharmaceutically acceptable salts with acids or bases. It is implied that compounds corresponding to one of points (a)-(e) enumerated in the invention claim are excluded from the invention text. Also, invention describes methods for preparing compounds of the formula (I) and a pharmaceutical composition eliciting the hypolipidemic activity. Invention provides preparing new compounds eliciting the useful biological properties.

EFFECT: improved preparing method, valuable medicinal properties of compounds.

20 cl, 6 tbl, 192 ex

FIELD: organic chemistry, pharmacy.

SUBSTANCE: invention relates to a new derivative of bicyclic heteroaromatic compound of the general formula (I) or its pharmaceutically acceptable salt eliciting agonistic activity with respect to luteinizing hormone (LH). Compounds can be used for preparing medicinal agents for control ability for conception. In compounds of the general formula (I) R1 represents R7 wherein R7 represents (C6-C10)-aryl optionally substituted with halogen atom at ortho- and/or meta-position; NHR8, OR8 wherein R8 means (C1-C8)-alkyl that can be substituted with halogen atom, (C1-C8)-alkylcarbonyl, (C1-C8)-alkylcarbonyloxy-group, phenyl, (C6-C10)-arylcarbonylamino-group, 5-methyl-2-phenylimidazol-4-yl, (C6)-heterocycloalkyl wherein 1-2 heteroatoms are taken among nitrogen and oxygen atoms, ethyloxycarbonylmethylthio-(C1-C4)-alkoxy-group, amino-group, (C6-C7)-heteroaryl; or (C5-C6)-heteroaryl comprising nitrogen, oxygen or sulfur atom as a heteroatom; R2 represents (C1-C8)-alkyl or (C6-C10)-aryl optionally substituted with one or more substitutes taken among (C1-C8)-alkoxy-group; or (C5-C6)-heteroaryl comprising nitrogen, oxygen or sulfur atom as a heteroatom; R3 represents (C1-C8)-alkyl possibly substituted with (C6-C14)-aryl possibly substituted with halogen atom, (C1-C4)-alkoxy-group, (C1-C4)-alkoxycarbonyl, mono- or tri-(C6-C10)-cycloalkyl, (C6-C10)-aryl, (C5-C6)-heteroaryl comprising nitrogen, oxygen or sulfur atom as a heteroatom; (C5-C7)-heterocycloalkyl comprising 2 heteroatoms taking among nitrogen or oxygen atom; (C3-C8)-cycloalkyl, (C2-C7)-heterocycloalkyl comprising 2 heteroatoms taking among nitrogen or oxygen atom; or (C6-C10)-aryl optionally substituted with one or more substitutes taken among (C1-C8)-alkoxy-group; X represents sulfur atom (S) or N(R4); Y represents nitrogen atom (N); R4 represents (C1-C8)-alkyl, phenyl-(C1-C8)-alkyl; or X represents sulfur atom (S), and Y represents CH; Z represents NH2 or OH; A represents sulfur (S), oxygen atom (O) or a bond. Also, invention relates to a pharmaceutical composition.

EFFECT: valuable properties of compounds and composition.

14 cl, 1 tbl, 119 ex

FIELD: organic chemistry.

SUBSTANCE: invention relates to improved synthesis method of pyrlindone hydrochloride having formula (I) 1. Method features intramolecular cyclization of 6-methyl-1-(2-chloroethyl-imino)-1,2,3,4-tetrahydrocarbazole hydrochloride of formula IV 2 at 80°-140°C with alkali agent in presence of phase transfer catalyst to provide 1,2,5,6-tetrahydro-8-methyl-pyrazine[3,2,1-j,k]-4H-carbazole of formula VI 3 followed by reduction at 80°-120°C. Method of present invention makes in possible to produce compound of formula I with yield nearly 70 % and purity more than 99 %.

EFFECT: method of high yield with reduced amount of alkali agent and phase transfer catalyst.

7 cl, 2 ex

FIELD: organic chemistry, pharmaceutical compositions.

SUBSTANCE: invention relates to novel pyrasolbenzodiazepines of formula I 1 (in formula R1 is hydrogen, -NO2, -CN, halogen, -OR5, -COOR7, -CONR8R9, -NR10R11, NHCOR12, NHSO2R13; each R2 and R4 independently of one another are hydrogen, halogen, -NO2, -CF3; R3 is hydpegen, C3-C8-cycloalkyl, aryl, in particular C6-C10-aromatic group having 1 or 2 rings, 5-10-membered heteroaryl, having 1 or 2 rings and1-3 heteroatoms, selected from N, O, and S, -COOR7, CN, C2-C6-alkenyl, -CONR8R9 or C1-C6-alkyl optionally substituted with OR9-group, F or aryl as mentioned above; R5 is C1-C6-alkyl; R7 is hydrogen or C1-C6-alkyl; each independently of one another are hydrogen or C1-C6-alkyl optionally substituted with hydroxyl or NH2, or alternatively R8 and R9 together form morpholino group; each R10,R11 and R12 independently of one another are hydrogen or C1-C6-alkyl; R13 is C1-C6-alkyl optionally substituted with halogen or -NR14R15; each R14 and R15 independently of one another are hydrogen or C1-C6-alkyl optionally substituted with halogen; or alternatively -NR14R15 is morpholino group) or pharmaceutically acceptable salts thereof, as well as to certain pyrasolbenzodiazepine derivatives, thiolactam intermediates for production of compound (I) and pharmaceutical compositions containing the same. Compound and pharmaceutical composition of present invention are cycline-dependent kinase (CDK2) inhibitors and antiproliferation agents used in treatment or controlling disorders associated with cell proliferation, in particular breast, colon, lung and/or prostate tumors.

EFFECT: new antiproliferation agents.

20 cl, 12 tbl, 8 ex

FIELD: organic chemistry, pharmaceutical composition.

SUBSTANCE: compounds satisfying the formula I 1 are disclosed, wherein each R1 and R2 independently to one another are H, OH, OA or Hal; or R1 and R2 together are -O-CH2-O- or -O-CH2-CH2-O-; R3 and R4 are A-group; X - group monosubstituted with R8, R5 or R7; R5 is linear or branched C1-C10-alkylene, wherein one or two CH2-groups may be substituted with oxygen atom; R7 is phenyl or phenylmethyl; R8 is COOH, COOA, CONH2, CONHA, CON(A)2 or CN; F is C1-C6-alkyl; and Hal is F, Cl, Br, or I, as well as physiologically acceptable salts or solvates thereof. Methods for production of claimed compounds (I) and pharmaceutical composition containing the same also are disclosed. Said compounds and pharmaceutical composition have activity as phosphodiesterase V inhibitors and are useful in treatment of cardiovascular diseases and potency disorders.

EFFECT: pharmaceutically applicable compounds and compositions.

7 cl, 16 ex

FIELD: organic chemistry, medicine, gastroenterology, pharmacy.

SUBSTANCE: invention relates to a pyrrolopyridazine derivative of the following formula: wherein R1 represents (C3-C7)-cycloalkyl-(C1-C6)-alkyl group that can be substituted optionally with (C1-C6)-alkyl group; R2 represents (C1-C6)-alkyl group; R3 represents hydroxymethyl group, (C2-C6)-aliphatic acyloxymethyl group, (C6-C10)-arylcarbonyloxymethyl group, (C1-C6)-alkoxycarbonyloxymethyl group, formyl group, carboxyl group, (C1-C6)-alkoxycarbonyl group or (C6-C10)-aryloxycarbonyl group; R4 represents (C6-C10)-aryl group that can be substituted optionally with substitutes taken among the group consisting of (C1-C6)-alkyl groups, halogen-(C1-C6)-alkyl groups, (C1-C6)-alkoxy-groups, halogen-(C1-C6)-alkoxy-groups and halogen atoms; A represents imino-group, oxygen or sulfur atom, or its pharmaceutically acceptable salt. Pyrrolopyridazine derivatives elicit inhibitory activity with respect to gastric juice secretion and protective activity with respect to stomach mucosa and can be useful as a curative agent for prophylaxis or treatment of ulcer disease. Except for, invention relates to a pharmaceutical composition based on compounds of the invention and to a method for prophylaxis and treatment of ulcer disease.

EFFECT: valuable medicinal properties of compound.

25 cl, 1 tbl, 11 ex

FIELD: organic chemistry of heterocyclic compounds, medicine, pharmacy.

SUBSTANCE: invention describes bicyclical nitrogen-containing heterocycles of the general formula (I): , wherein R1 means hydrogen atom, (C1-C7)-alkyl, (C3-C7)-cycloalkyl, (C3-C7)-cycloalkyl-(C1-C4)-alkyl, pyridyl, naphthyl, furyl-(C1-C4)-alkyl, phenyl optionally substituted with di-(C1-C7)-alkylamino-(C1-C7)-group, halogen atom, (C1-C7)-alkoxy-group or hydroxy-(C1-C7)-alkyl, or phenyl-(C1-C7)-alkyl optionally substituted with (C1-C7)-alkoxy-group, amino-(C1-C7)-alkyl, amino-group or di-(C1-C7)-alkylamino-(C1-C7)-alkoxy-group; R2 means (C1-C7)-alkyl, (C3-C7)-cycloalkyl, furyl-(C1-C4)-alkyl, pyridyl or its N-oxide; phenyl optionally substituted with halogen atom, (C1-C7)-alkyl, (C1-C7)-alkoxy-group, hydroxy-group or trifluoromethyl, or phenyl-(C1-C7)-alkyl optionally substituted with (C1-C7)-alkoxy-group; R3 means hydrogen atom, (C1-C7)-alkyl, (C3-C7)-cycloalkyl-(C1-C4)-alkyl, (C3-C7)-cycloalkenyl, pyridyl-(C1-C4)-alkyl, naphthyl, phenyl optionally substituted with phthalimido-(C1-C4)-alkyl, amino-(C1-C7)-alkyl, hydroxy-(C1-C7)-alkyl, (C1-C7)-alkylamino-(C1-C7)-alkyl, di-(C1-C7)-alkylamino-(C1-C7)-alkyl, morpholino-(C1-C4)-alkyl or piperazinyl-(C1-C4)-alkyl, or phenyl-(C1-C7)-alkyl optionally substituted with (C1-C7)-alkoxycarbonyl or carboxy-group. Also, invention relates to pharmaceutically acceptable salts of compounds of the formula (I) as a base with acids or pharmaceutically acceptable salts of compounds of the formula (I) as acid with bases, and pharmaceutical composition based on thereof. Compounds described above show inhibitory activity with respect to tyrosine kinase and can be used in treatment or prophylaxis of inflammatory, immunological, oncological, bronchopulmonary, dermatological and cardiovascular diseases, for treatment of asthma, disorders in the central nervous system or complications associated with diabetes mellitus, or for prophylaxis against transplant rejection after surgery transplantation.

EFFECT: valuable medicinal properties of compounds and composition.

14 cl, 1 tbl, 92 ex

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