Water-soluble tetrazolium salts

FIELD: chemistry.

SUBSTANCE: present invention relates to compounds of formula

, in which A is a counter ion, a=1-3, b=0-3, X=1-6C alkyl, R1=1-6C alkyl, one or R2 and R3 is 1-6C alkyl and the other is XN+Hb(R1)3-b, or R2 and R3 form a methylenedioxy group, one or R4 and R5 is a halogen and the other is a halogen-substituted 1-6C alkyl, or R4 and R5 are bonded to form a 6-10C aromatic ring or a substituted 6-10C aromatic ring in which the substitute is selected from 1-6C alkoxy, halogen and halogen-substituted 1-6C alkyl. The invention also relates to a method of measuring content of analysed substance capable of ensuring proportional colour change as a result of a reaction in a biological fluid, involving the following steps: ensuring availability of the disclosed tetrazolium salt as an indicator and determination of concentration of the said analysed substance in the biological fluid using the said tetrazolium salt which is used as an indicator.

EFFECT: agents are highly effective.

24 cl, 7 dwg, 1 tbl, 9 ex

 

The technical field to which the invention relates.

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

The level of technology

In the analysis of biological samples are often used such methods in which the resulting color is proportional to the amount of analyte in the sample. For example, to oxidize the analyte of interest can be used enzymes, and the degree of reaction is indicated by color change of the indicator compounds. In this regard, of particular interest is the family tetrazolium salts used as indicators. These salts are recovered by reducing agents to formisano dyes. For measurement of the analyte enzyme (for example, the enzyme NAD-dependent dehydrogenase) oxidizes an analyte with the formation of reduced forms (e.g., NADH), responsive to tetrazolium salt with the formation of colored formazan. To facilitate the reaction may require the mediator. Since the number formed in the reaction with the analyzed substance NADH is proportional to the quantity of received formazan, the amount of analyte may be indirectly determined by the resulting color.

Tetrazolium salts are used in various fields. In particular, they are applied in honey is the CIN for measuring the analyzed substances in various biological fluids, such as blood, urine, plasma and serum. These indicators are often used with systems of reagents located on the test strips, which are in contact with the sample liquid react with the analyzed substance with the formation of the color indicates the amount of analyte present. Although in some cases the color change can be defined visually by the color scale, more accurate results can be obtained spectrophotometrically by using intended for this purpose devices. As a rule, to determine changes color stripes on her direct light and produce a measurement of reflected light.

Tetrazolium salt should form formazan that absorb light at these wavelengths, which would rule out interference from other substances in a sample, such as hemoglobin in whole blood. To this end we obtained a family of thiazoleacetate salts forming formazan, which absorb light with a wavelength of more than approx. 640 nm. These wavelengths are characteristic to be used as the light sources are LEDs. LEDs create light in a narrow range, so that the wavelengths differ only by about ±5 nm. Such thiazoleacetate salts are disclosed in several U.S. patents, such as U.S. patent No. 5126275, 5322680, 5300637 and 5290536.

Most biological Ave is b are aqueous in nature, therefore, it is desirable that the indicators were soluble in the sample. However, many tetrazolium salts have low solubility. One of the suppliers tetrazolium salts is Dojindo Laboratories. The company offers a number of indicators that could be made more soluble by adding sulfoxylate groups to the molecule of the indicator. Cm. U.S. patent No. 6063587 and published patent application Japan JP 58113181 A2 and JP 58113182 A2. Their series WST indicators-tetrazolium salts often referred to in the patents disclosing analytical methods. One example can be found in U.S. patent No. 6586199. Specified in the report, the solubility tetrazolium salts series WST exceeds approx. 10 mg/ml of water. Solubility can also be increased by using some sulphonate and phosphonate counterions tetrazolium salts as described in U.S. patent No. 5250695.

Among other patents, which are considered tetrazolium salt, it should be noted EP 0476455 B1; US 2004/0132004 A1; WO 98/37157; U.S. patent No. 6183878 B1; U.S. patent No. 6207292 B1; U.S. patent No. 6277307 B1; DE 2147466; U.S. patent No. 5185450; U.S. patent No. 5196314.

The authors of the present invention has sought to improve the solubility thiazoleacetate salts, while maintaining the ability of their formisano to provide a relatively flat spectral response in the range of 600-640 nm incident on n is x light from the LEDs. As can be seen from the following description, they were able to offer thiazoleacetate salt having greater solubility while maintaining the desired spectral response.

Disclosure of inventions

The present invention relates to new thiazoleacetate salts. It was found that some of them have better solubility in water than the corresponding patented thiazoleacetate salt. Improvement of solubility facilitates their use in the test strips used to measure the amount of analyzed substances in biological samples (for example, glucose in the blood sample). Tetrazolium salt characterized by the presence of alkylammonium Deputy to increase the solubility. In accordance with one embodiment of the invention tetrazolium salt is determined by the following formula.

where

A=the counterion,

X=1-6C alkyl or heteroalkyl,

a=1-3,

b=0-3,

R1=1-6C alkyl,

one of R2and R3is alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy group,

one of R4or R5is halogen and the other represents a substituted halogen 1-6C alkyl,

one or both of R4and R5are XN+Hb(R1) 3-bor

R4and R5associated with the formation of aromatic or heteroaromatic rings or

substituted aromatic or substituted heteroaromatic ring.

The above formula represents a new thiazoleacetate salts, which contain a thiazole ring, attached to tetrazolium ring at the position of the nitrogen - 2. In addition, each of these compounds is phenyl rings, one of which is attached to tetrazole ring at the position of the nitrogen - 3, and the other in position 5. The increase in solubility compared to the patented disorderslava salt (designated here as STC-045) is achieved by attaching one or more of alkylammonium groups to disorderslava salt. Alkylammonium group can join thiazolidinone ring in the form of R4and/or R5or substituted on the aromatic or heteroaromatic ring formed by R4and R5. In preferred embodiments, the implementation of alkylammonium groups are substituents on the phenyl rings attached to tetrazolium ring. In more preferred embodiments, the implementation of alkylammonium group is dialkylaminoalkyl group. Benzothiazolylsulfenamide salts are the preferred connection of enemy, with increased solubility.

In another aspect of the invention thiazoleacetate salts are used as chromogenic indicators to determine the presence of reducing substances (e.g., NADH) in the analysis of biological samples with the use of enzymes for oxidation of the analyzed substances, for example, as in the determination of glucose in blood.

Brief description of drawings

Figure 1 shows a diagram of a first method of obtaining the compounds used in the examples.

Figure 2 shows a diagram of a second method of obtaining the compounds used in the examples.

Figure 3 shows the examples of the compounds for comparison.

On figa-b shows obtained in the examples of the compounds according to the present invention.

On figa and 5b shows absorption spectra tetrazolium salts.

Description of the preferred embodiments of the invention

Definition

The following definitions relate to the Deputy thiazoleacetate salts according to the invention.

"1-6" is a radical containing 1-6 carbon atoms

"alkyl" is an unbranched or branched hydrocarbon radicals of the General formula CnH2n+1

"heteroalkyl" is unbranched or branched hydrocarbon radicals containing heteroatoms, attached to osadnik carbon atoms

"alkoxy" is a radical-OR where R is alkyl

"methylenedioxy" - is a bivalent radical of formula-O-CH2-About-

"halogen" means fluorine, chlorine, iodine and bromine

"substituted with halogen 1-6C alkyl" is an unbranched or branched radical of General formula CnH2n-1Y2where Y is a halogen

"aromatic ring" means a benzene or naphthalene ring

"heteroaromatic ring" means a pyridine or quinoline ring

"counterion" is an ionic moiety of the reagents used to obtain tetrazolium salts (e.g., nitrite, phosphate, hydrogen phosphate, dihydrophosphate, hydrosulfate, sulfate, bicarbonate, carbonate, methanesulfonate, perborate, bromide, chloride, iodide, or a combination of them)

"tet-Sol" is the abbreviated name of tetrazolium salt

Thiazoleacetate salt

As disclosed in U.S. patent No. 5126275, formazane thiazoleacetate salts are characterized by a spectrum of reflection, with a long flat stretch in the region of wavelengths more than 600-650 nm. This quality can be used in equipment in which the light source LEDs are used. The sample is in contact with the reaction system, providing the appearance of a color that indicates the amount of analyte in the sample. Reflected from the test light p is isometsa and correlated with the analyte. As one of the most important examples should mention that glucosegalactose in the presence of NAD+catalyzes the oxidation of glucose in blood samples. Restored NADH is re-oxidized enzyme mediator, such as diaphorase, which catalyzes the restoration of tetrazolium salt to formazan. The observed color change is proportional to the amount of NADH formed during the oxidation of glucose, and indirectly proportional to the amount of glucose in the sample. Change the colors by turning thiazoleacetate salts with color formazan can be measured by directing light from a source. Reflected from the painted areas of the test light is received and converted in accordance with a suitable algorithm to the amount of analyte in the sample. Although tetrazolium salt according to the invention have been described in connection with the NAD-NADH, they actually have a wider application, including with dehydrogenase enzymes with other cofactors, such as PQQ-and FAD.

Because thiazoleacetate salts and their corresponding formazan must dissolve in the reaction mixture, applied to a test strip for the determination of the analyzed substances, the goal of the authors of the invention was to improve the solubility thiazoleacetate salts, while maintaining their ability obrotowy is the substance of formazane with a relatively smooth spectrum of reflection in the range of 600-640 nm. The inventors have discovered a new family thiazoleacetate salts include compounds corresponding to the set goal. These salts represented by the following General formula:

where

A=the counterion,

X=1-6C alkyl or heteroalkyl,

a=1-3,

b=0-3,

R1=1-6C alkyl,

one of R2and R3is alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy group,

one of R4or R5is halogen and the other represents a substituted halogen 1-6C alkyl,

one or both of R4and R5are XN+Hb(R1)3-bor

R4and R5associated with the formation of aromatic or heteroaromatic ring or substituted aromatic or substituted heteroaromatic ring.

Tetrazolium salt according to the invention can also be divided into substituted disorderisolate salt and substituted benzothiazolylsulfenamide salt, as shown in the following special interest formulas:

where

A=the counterion,

X=1-6C alkyl,

a=1-3,

b=0-3,

R1=1-3C alkyl,

one of R2and R3is 1-4C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3 connected with the formation of methylenedioxy group, R4=CHF2, R5= halogen.

A = the counterion,

X = 1-6C alkyl,

a=1-3,

b=0-3,

R1=1-3C alkyl,

one of R2and R3is 1-4C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3connected with the formation of methylenedioxy group,

R4and R5are H, or R4and R5associated with the formation of aromatic rings

R6=1-4C alkoxy, hydrogen or halogen.

The solubility of these compounds (I and II) will be shown in the examples below. Increasing the solubility thiazoleacetate salts should reduce the time required to obtain test results, and to provide greater linearity at high levels of analyte. In addition, increased solubility in water facilitates the formation of odniesieniu mixture that is easier and cheaper to produce.

Methods of obtaining tetrazolium salts according to the invention

In U.S. patent No. 5126275 describes how to get thiazoleacetate salts. The methods include carrying out the reaction of the hydrazone with diazotized aniline to form formazan which is then oxidised to tetrazolium salt. Described below are new ways of obtaining compounds with the according to the invention provide for the introduction of one (1) or two (scheme 2) polar groups in the preferred tetrazolium salts of compounds I and II. Figures 1 and 2 illustrate these two ways. The following examples illustrate in more detail the invention.

Scheme 1 is used to obtain tetrazolium salts, in which the phenyl substituent in the 5 position tetrazolium ring is methylendioxy Deputy. In figure 1 trimethylammonio group substituted on the phenyl substituent in the 3 position tetrazolium rings. Scheme 1 O-NITROPHENOL is reacted with 3-bromopropylamine with the formation of 3-(2-nitrophenoxy) propyltrimethylammonium, and then hereroense to the corresponding aniline. Reaction of this compound with hydrazone containing substituted thiazolidine ring and the phenyl ring with methylendioxy Deputy, gives formazan. Then formazan to oxidize tetrazolium salt. The hydrazone is obtained by reaction of the substituted benzaldehyde with substituted hydrazine containing required for tetrazolium salt substitutes.

Scheme 2 is used to obtain tetrazolium salts, in which the phenyl substituent in the 5 position tetrazolium ring is trimethylolpropane-group substituted in the 3 and/or 4 positions. As in figure 1, trimethylolpropane group substituted on the phenyl substituent in the 3 position tetrazolium rings. Figure 2 4-hydroxy-3 methoxybenzaldehyde reacts with 3-bromopropylamine what romida to add trimethylolpropane group in the benzaldehyde. The product is reacted with a substituted hydrazine to form a hydrazone. The hydrazone is reacted with 3-(2-aminophenoxy)propyltrimethylammonium obtained in the first step of scheme 1, with the formation of formazan, which then turned into tetrazolium salt.

The following results show that the solubility seems to be affected by the counterions associated with tetrazolium salts. Counterions come from the reagents used in the method, and you can change them to obtain the required counterions. In an alternative embodiment, the counterions can be replaced, for example, by using ion-exchange methods.

In the following non-limiting examples, the products at each stage identified using spectrophotometric methods, and the results were presented for each product.

Example 1

Getting 5-benzo[1,dioxol-5-yl]-3-[2-(3-ammonium)propoxy-phenyl]-2-(5-chloro-4-Stormer-thiazol-2-yl)-2H-Tetrachloromethane salt

As shown in figure 1, o-NITROPHENOL is reacted with 3-bromopropylamine with the formation of 3-(2-nitrophenoxy)propyltrimethylammonium, and then hereroense to the corresponding aniline. Aniline reacts with hydrazone containing substituted thiazolidine ring and the phenyl ring with methylendioxy Deputy, with the formation of formazan. Then formazan of Ocala the t to tetrazolium salt. The method is fully described in the following example: to a yellow solution of o-NITROPHENOL (Aldrich, 6.3 g, 45 mmol) in DMF (150 ml) in a dry 250 ml odnogolosy round bottom flask under nitrogen was added potassium carbonate (6.6 g, 48 mmol). The mixture was bought orange color. After 10 min stirring was added 3-bromopropylamine (Aldrich, 12,9 g, 45 mmol). The resulting mixture was heated at 125°C for 3 hours After cooling the reaction mixture to room temperature, the residue was filtered, and the residue washed with DMF (2×20 ml). The filtrate was slowly added to EtOAc (750 ml), the resulting precipitate. The resulting mixture was stirred for 30 minutes. The precipitate was filtered and washed with EtOAc solution:DMF, 5:1 (3×100 ml), then EtOAc (100 ml) and hexane (100 ml). After drying in air yellow solid within 5 minutes, the product was added to a solution of EtOAc:DMF, 10:1 (550 ml) and was stirred for 1 h, the Solid was filtered, washedEtOAc(100 ml) and hexane (100 ml). The obtained solid was filtered to obtain a light beige product (14,54 g, >100%), so square (178-181°C), which is identified by the following properties.13With NMR (400 MHz, DMSO-d6): δ 151, 139,45, 134,71, 125,14, 121, 115,39, 66,59, 62,84, 52,39, 22,481H NMR (400 MHz, DMSO-d6): δ of 7.90 (dd, J=8,1 Hz, J=1.7 Hz, 1H), 7,58 (dt, J=8,2 Hz, J=1.7 Hz, 1H), 7,39 (d, J=8,2 Hz, 1H), 7,15 (d, J=1.7 Hz, 1H), 4,25 (t, J=6 Hz, 2H), of 3.45 (m, 2H), 3,11 (s, 9H), of 2.23 (m, 2H). ESI-MS: m/z 239 (100%, M2 ).

A suspension of 3-(2-nitrophenoxy)propyltrimethylammonium (7 g, 21,94 mmol), 10% Pd/C (725 mg), Meon (70 ml) and water (70 ml) was first made at 35 psi for 2.5 h in a Parr apparatus Hydrogenation. After 30 minutes the pressure dropped to 22 psi. The resulting mixture was filtered through a layer of Celite 521. The black layer of catalyst/Celite was washed Meon (2×10 ml). The filtrate was concentrated on a rotary evaporator under reduced pressure at 40-45°C. When the distillate has ceased to form, light pink solution was transferred to another flask and concentrated there by freeze-drying for 2 days with the formation of a light beige aniline derivative (of 5.92 g, 93%)which was identified for the following spectral characteristics:13With NMR (400 MHz, DMSO-d6): δ 145,19, 137,76, 121,39, 116,15, 114,09, 111,86, 64,79, 63,27, 52,36, 22,781H NMR (400 MHz, DMSO-d6): δ is 6.78 (d, J=4 Hz, 1H), 6,66 (m, 2H), 6,53 (d, J=6 Hz, 1H), 4,82 (br s, 2H), 3,99 (t, J=6 Hz, 2H), 3,54 (m, 2H), 3,11 (s, 9H), 2,17 (m, 2H). ESI-MS: m/z 209 (100%, M2+).

To a solution of the above aniline derivative (7.7 g, 26,64 mmol) in water (80 ml) in an ice-water bath was added dropwise conc. HCl (7.2 ml). After stirring the solution for 5 min was added dropwise a solution of nitrite (2.2 g, 32 mmol) in water (16 ml). The color of the solution changed from light brown to Golden yellow. The resulting solution was stirred for 30 minutes. The solution of the hydrazone (Bayer, 9,8 g, 27,98 m is ol) in pyridine (400 ml) was stirred in a 3-necked 2-liter flask, equipped with a mechanical stirrer and a funnel for adding substances with a shirt on salt/ice/water bath (-15 to -20°C). Just received detractor was added dropwise to a solution of hydrazone through the funnel to add a shirt, filled with ice/water. After the first few drops of color of the reaction changed from yellow to dark blue. After 1/3 add the dark blue reaction mixture became very viscous. Upon completion of the addition the mixture was stirred for 30 minutes. Then the reaction mixture was heated up to 3-5°C by an ice/water bath. The reaction mixture was stirred for 1 hour. The mixture was transferred into a 2-liter round bottom flask with Meon (500 ml). The mixture was concentrated on a rotary evaporator under reduced pressure at 45°C to stop the formation of distillate. Newly added Meon (500 ml)and the mixture was concentrated. The process was repeated. The obtained solid substance was dried under vacuum overnight. Then the flask was washed Meon (100 ml) and dilutedEtOAc(500 ml). The suspension was stirred in an ice/water bath for 30 minutes. The product was filtered and washedEtOAc(100 ml), cold Meon (50 ml),EtOAc(150 ml) and hexane (150 ml). The obtained dark blue product was transferred into a bottle of yellow glass and dried under vacuum with the formation of the desired formazan (13, 11.3 g, 67%), which identificare the Ali for the following spectral properties: 19F NMR: (376 MHz, DMSO-d6): δ -177,77 (d, J=59,2 Hz),13With NMR (400 MHz, DMSO-d6): δ 169,57, 153,6, 148,1, 147,8, 144,81, 141,9, 137,1, 132,74, 128,82, 121,53, 121,5, 121,1, 115,89, 113,96, 108,8, 108,22, 106,68, 101,33, 66,21, 63,03, 51,99, 22,351H NMR (400 MHz, DMSO-d6): δ 11,78 (br s, 1H), with 8.05 (dd, J=8,1 Hz, J=1.5 Hz, 1H), 7,55 (dd, J=8,2 Hz, J=1.6 Hz, 1H), of 7.48 (dd, J=8,2 Hz, J=1.7 Hz, 1H), 7,43 (d, J=1.7 Hz, 1H), 7,32 (dd, J=8 Hz, J=<1 Hz, 1H), 7,16 (t, J=7.9 Hz, 1H), 7,15 (t, JHF=59,2, 1H),? 7.04 baby mortality (d, J=8,2 Hz, 1H), 6,11 (s, 2H), 4,32 (t, J=6 Hz, 2H), 3,49 (m, 2H), 3,05 (s, 9H), of 2.38 (m, 2H). ESI-MS: m/z 611 (100%, M-Ms), 258 (26%, M2+).

To the mixture above formazan (2.16 g, 3.4 mmol), water (1.4 ml) and THF (40 ml) was added methansulfonate (0.68 ml, 10,24 mmol). The mixture was stirred for 5 minutes. Then added in one portion sodium nitrite (720 mg, 10,24 mmol). The mixture was stirred for 3 days. The color of the suspension changed from dark blue to orange. The orange precipitate was filtered and washed with THF (2×10 ml). The product was dried under vacuum over night. Then the solid is suspended Meon (80 ml), filtered, and the residue was washed Meon (2×5 ml). The filtrate was concentrated on a rotary evaporator under reduced pressure at 40°C. the Obtained product was dried for 2 days under vacuum with the formation of orange tet-salt (2.86 g of >100%), which was identified for the following spectral properties:19F NMR: (376 MHz, DMSO-d6): δ - 181,13 (d, J=52.1 Hz),13With NMR (400 MHz, DMSO-d6): δ 165,1, 152,52, 152,27, 148,85, 146,68, 142,72, 142,45, 137,07, 135,95, 128,44, 123,87,121,87, 121,86, 121,39, 115,57, 114,68, 110,80, 109,84, 108,43, 107,14, 106,06, 102,78, 100,49, 66,92, 62,23, 52,21, 22,081H NMR (400 MHz, DMSO-d6): δ 7,98 (dd, J=8.0 Hz, J=1.5 Hz, 1H), to $ 7.91 (m, 2H), 7,82 (d, J=1.8 Hz, 1H), 7,53 (d, J=8,2 Hz, 1H), 7,37 (m, 1H), 7,31 (d, J=8,2 Hz, 1H), 7,25 (t, JHF=59,2, 1H), 6,27 (s, 2H), 4,17 (t, J=6 Hz, 2H), 3,49 (m, 2H), 2,93 (2, 9H), 2,02 (m, 2H). ESI-MS: m/z 663 (22%, M2++ TFA), 645 (17%, M+), 275 (100%, M2+).

Example 2

Getting 5-benzo[1,dioxol-5-yl]-3-[2-(3-ammonium)propoxy-phenyl]-2-(5-chloro-4-deformity-thiazol-2-yl)-2H-tetrachloroterephthalate salt

Sol was obtained according to example 1, except that used 48% tetrahydrocarbazol acid with the same formosanum to obtain the required orange tetrazolium salt.13With NMR (400 MHz, DMSO-d6): δ 137, 128, 124, 121, 114, 111, 110, 108, 107, 106, 103, 68, 63, 53, 2819F NMR: (376 MHz, DMSO-d6): δ - 181,13 (d, J=59,2 Hz),1H NMR (400 MHz, DMSO-d6): δ a 7.92 (m, 3H), of 7.82 (d, J=1.8 Hz, 1H), 7,52 (bd, J=8,2 Hz, 1H), 7,37 (m, 1H), 7,31 (d, J=8,2 Hz, 1H), 7,25 (t, JHF=59,2, 1H), 6,27 (s, 2H), 4,17 (t, J=6 Hz, 2H), 3,35 (m, 2H), 2,93 (2,9H), 2,02 (m, 2H). ESI-MS: m/z 637 (100%, M+-Br).

Example 3

Getting 5-benzo[1,dioxol-5-yl]-3-[2-(3-dimethylhydrazone)propoxy-phenyl]-2-(5-chloro-4-deformity-thiazol-2-yl)-2H-Tetrachloromethane salt

To the yellow solution of o-NITROPHENOL (Aldrich, 2.1 g, 45 mmol) in DMF (50 ml) in a dry 100 ml odnogolosy round bottom flask under nitrogen was added potassium carbonate (4.4 g, 32 mmol). The mixture was bought orange color. After 10 min paramesh the cation was added 3-chloropropionitrile (Aldrich, 2.4 g, 16.5 mmol). The resulting mixture was heated at 125°C for one day. After cooling the reaction mixture to room temperature, the residue was filtered, and the residue was washed Meon (2×10 ml). The filtrate was concentrated under vacuum at 40°C. to 5 ml was Then addedEtOAc(100 ml). Formed precipitate, which was filtered. To the filtrate was added water (100 ml) andEtOAc(150 ml), the layers were mixed and separated. The aqueous layer was extracted withEtOAc(3×25 ml). The UnitedEtOActhe extracts were washed with saturated aqueous sodium chloride (25 ml), dried over magnesium sulfate (5 g), filtered and concentrated with the formation of a yellow oil (2.55 g, 81%).1H NMR (400 MHz, DMSO-d6): δ 7,80 (dd, J=8,1 Hz, J=1.7 Hz, 1H), 7,58 (dt, J=8,2 Hz, J=1.7 Hz, 1H), 7,05 (d, J=8,2 Hz, 1H), 6,65 (d, J=1.7 Hz, 1H), 4,25 (t, J=6 Hz, 2H), 2,45 (m, 2H), 2,15 (s, 9H), and 1.9 (m, 2H). ESI-MS: m/z 225 (100%, M+1).

Alkilirovanny NITROPHENOL was treated in the same manner as described in example 1, to obtain the desired face-salt.13With NMR (400 MHz, DMSO-d6): δ 127, 124, 121, 115, 110, 109, 108, 107, 104, 102, 64, 48, 36, 2819F NMR: (376 MHz, DMSO-d6(d, J=59,2 Hz): δ - 119,13,1H NMR (400 MHz, DMSO-d6): δ to 7.95 (m, 3H), 7,80 (d, J=1.8 Hz, 1H), 7,50 (d, J=8,2 Hz, 1H), 7,35 (t, J=8,2 Hz, 1H), 7,31 (d, J=8,2 Hz, 1H), 7,25 (t, JHF=59,2, 1H), 6,27 (s, 2H), 4,20 (t, J=6 Hz, 2H), of 3.45 (m, 2H), 2,65 (d, j=1,8 Hz, 6H), to 1.98 (m, 2H). ESI-MS: m/z 535 (100%, M2+).

Example 4

Getting 5-benzo[1,dioxol-5-yl]-3-[2-(3-ammonium)propoxy-phenyl]-2(benzothiazol the-2-yl)-2H-Tetrachloromethane salt

Spent the reaction piperonal-benzothiazol-2-ingerson with 3-(2-aminophenoxy)propyltrimethylammonium as described in example 1, with the formation of formazan, which is then transformed into the corresponding tetrazolium salt.

Spent the reaction piperonal-benzothiazol-2-ingerson (Bayer, 2.5 g, 10.5 mmol) with aniline derivative from example 1 (2.85 g, 9.6 mmol) under the conditions similar to getting formazan in example 1, with the formation of a dark purple solid (4.7 g, 82%)which was identified for the following spectral properties:1H NMR (400 MHz, DMSO-d6): δ 11,78 (br s, 1H), 7,98 (dd, 2H), 7,73 (m, 1H), 7,60 (m, 2H), 7,50 (m, 1H), 7,41 (m, 2H), 7,29 (m, 1H), 7,05 (d, 1H), 6,14 (s, 2H), 4,39 (t, 2H), 3,55 (m, 2H), 3,05 (s, 9H), 2,48 (m, 2H). ESI-MS: m/z 517 (100%, M+).

The above formazan (600 mg, 1.01 mmol) was converted into tetrazolium salt (870 mg, >100%) under the same conditions as in example 1. Tetrazolium salt identified for the following spectral properties:13With NMR (100 MHz, DMSO-d6): δ 164,98, 152,59, 152,26, 148,91, 136,64, 132,74, 135,08, 129,27, 128,88, 128,24, 124,77, 123,97, 123,7, 122,6, 121,97, 115,81, 114,56, 109,86, 107,12, 102,82, 66,88, 62,26, 52,2, 22,02.1H NMR (400 MHz, DMSO-d6): δ 8,42 (dd, J=8,2 Hz, J=0.8 Hz, 1H), 8,03 (dd, J=7.7 Hz, J=1.6 Hz, 1H), 8,02 (dd, J=8,1 Hz, J=0.9 Hz, 1H), of 7.97 (dd, J=8,2 Hz, J=1.8 Hz, 1H), to 7.93 (m, 1H), 7,86 (d, J=1.7 Hz, 1H), 7,76 (m, 1H), 7,73 (m, 1H), 7,55 (dd, J=8.6 Hz, J=0.9 Hz, 1H), 7,39 (m, 1H), 7,32 (d, J=8,1 Hz, 1H), 6,28 (s, 2H), 4,12 (t, J=6 Hz, 2H), and 3.16 (m, 2H), 2.91 in (s, 9H). ESI-MS: m/z 561,2 (100%, M+).

Example 5

Receipt is 5-benzo[1,dioxol-5-yl]-3-[2-(3-ammonium)propoxy-phenyl]-2-(6-methoxynaphthalene-2-yl)2H-Tetrachloromethane salt

To obtain the desired hydrazone in example 4 instead of 6 hydrazino-benzothiazole used 6-methoxy-2-hydrazino-nurtitional. The hydrazone is then processed in the same conditions to obtain the desired formazan (44%).13With NMR (400 MHz, DMSO-d6): δ 135, 130, 125, 122, 121, 120, 115, 114, 113, 110, 65, 64, 55, 52, 38, 221H NMR (400 MHz, DMSO-d6): δ 8,61 (d, J=8,1 Hz, 1H), 8,29 (d, J=8,2 Hz, 1H), with 8.05 (dd, J=8,1 Hz, J=l,8 Hz, 1H), 7,82 (dt, J=8,2 Hz, J=1.7 Hz, 1H), 7,68 (m, 2H), to 7.61 (dd, J=8,1 Hz, J=1.6 Hz, 1H), 7,55 (d, J=1,8 Hz), 7,37 (dd, J=8,2 Hz, J=l,8 Hz, 1H), 7,31(dd, J=8,2 Hz, J=1.8 Hz, 1H), 7,27 (dt, J=8,1 Hz, J=1.7 Hz, 1H), was 7.08 (d, J=8,1 Hz, 1H), 6,12 (s, 2H), 4,45 (t, J=6 Hz, 2H), 4,10 (s, 3H), of 3.60 (m, 2H), 2.95 and (s, 9H), to 2.65 (m, 2H). ESI-MS: m/z 597 (100%, M+).

To a solution of acetic acid (5 ml) and tetrahydrofuran (5 ml) in an ice/salt/water bath was added sodium nitrite (143 mg, of 2.08 mmol). After stirring for 10 minutes was added to the above formazan (340 mg, 0,051 mmol). After 1 hour the reaction allowed to warm to room temperature and was stirred overnight. Dark red-brown mixture was filtered, and the solid was washed with acetic acid (3×5 ml), ethyl acetate (2×10 ml) and hexane (10 ml). The filtrate was concentrated on a rotary evaporator at 30°C, and then dried under vacuum over night. The residue is suspended with ethyl acetate (15 ml), allowed to settle and was saifali. The process was repeated. Then was added hexane (10 ml). The product was filtered and dried ove the terms nights getting the desired red-brown face-salt (260 mg, 75%).13With NMR (400 MHz, DMSO-d6): δ 129, 128, 126, 123, 121, 119, 115, 114, 112, 108, 105, 101, 98, 66, 63, 57, 51, 221H NMR (400 MHz, DMSO-d6): δ 8,32 (m, 1H), 8,02 (m, 4H), 7,87 (d, J=1.8 Hz, 1 Hz), 7,78 (m, 3H), 7,66 (d, 8.2 Hz, 1H), 7,47 (t, J=8,2 Hz, 1H), 7,33 (d, J=8,2 Hz, 1H), 6,28 (s, 2H), 4,13 (s, 3H), 3.15 in (m, 2H), 2,85 (s, 9H), 1,87 (m, 2H). ESI-MS: m/z 298 (100%, M2+), 709 (20%, M++ TFA).

Example 6

Getting 5-[4-(3-ammonium)propoxy-3-methoxy-phenyl]-3- [2-(3-ammonium)propoxy-phenyl]-2-(5-chloro-4-deformity-thiazol-2-yl)-2H-tetratriacontane salt

Spent the reaction of 4-hydroxy-3-methoxybenzaldehyde with 3-bromopropylamine for introduction of one of trimethylolpropane Deputy. The product was introduced in the reaction with 5-chloro-4-deformity-2-hydrazino-thiazole with the formation of the hydrazone. This hydrazone was introduced in the reaction with 3-(2-aminophenoxy)propyltrimethylammonium as described in example 1, with the formation of formazan, which is then transformed into the corresponding tetrazolium salt. 4-Hydroxy 3 methoxy-benzaldehyde (Vanillin, Aldrich, 4,56 g, 30 mmol) alkilirovanie 3-bromopropylamine (8.8 g, 34 mmol) under the conditions of example 1 to obtain a whitish product (9,73 g, 97%)which was identified for the following spectral properties:13With NMR (400 MHz, DMSO-d6): δ 192,5, 155, 150, 130, 125,75, 112,6, 110, 66,5, 63,4, 56, 25,5.1H NMR (400 MHz, DMSO-d6): δ 9,88 (s, 1H), 7,58 (dd, J=8,2 Hz, J=1.9 Hz, 1H), 7,41 (d, J=1.9 Hz, 1H), 7,22 (d, J=8,2 Hz, 1H), 4,35 (t, J=6.4 Hz, 2H), 3,49 (m, 2H), 3,14 (s, 9H), is 2.30 (m, 2H). ESI-MS: m/z 252,2 (100%, M+).

To a suspension of the above alkylated benzaldehyde (6.8 g, 20,5 mmol) and 5-chloro-4-deformity-2-hydrazino-thiazole (Bayer, and 3.72 g of 18.6 mmol) in Meon (60 ml) was added acetic acid (0,55 ml). Suspension almost completely dissolved within 5 minutes. The resulting mixture was heated at 80°C during the night. After cooling the reaction mixture to room temperature, the solution was slowly added inEtOAc(650 ml). Formed a light grey precipitate. The mixture was stirred for 30 minutes. The solid was filtered and washedEtOAcand hexane. Light grey product was transferred into a bottle of yellow and dried under vacuum over night with the formation of the desired hydrazone (8.7 g, 87%)which is identified by the following properties:19F NMR: (376 MHz, DMSO-d6): δ 111,08 (d, J=47 Hz),13With NMR (400 MHz, DMSO-d6): δ 143, 121, 114, 109, 108,5, 66, 63, 55, 53, 39, 22.1H NMR (400 MHz, DMSO-d6): 12,37 (s, 1H), 7,29 (d, J=1.9 Hz, 1H), 7,19 (dd, J=10 Hz, J=1.8 Hz, 1H), 7,06 (t, J=7.7 Hz, 1H), 7,18 (mm, 1H), 6,95 (t, JHF=47, 1H), 4.09 to (t, J=6,1 Hz, 2H), 3,83 (s, 3H), 3,47 (m, 2H), 3,47 (m, 2H), 3,29 (s, 9H), of 2.50 (m, 2), of 2.20 (m, 2H). ESI-MS: m/z 433,1 (100%, M+).

The above hydrazone (7,08 g, 13,86 mmol) were introduced into a reaction with an aniline derivative of example 1 (6 g, 20,76 mmol) under conditions analogous to example 1, with the formation of dark blue formazan (8,56 g, 76%), which identify what ovale by the following spectral properties: 19F NMR: (376 MHz, DMSO-d6): δ 177,77 (d, J=59,2 Hz),13With NMR (400 MHz, DMSO-d6): δ 153, 148,98, 148,63, 144,5,136,5, 132,73, 132,74, 127, 121,59, 119,53, 115,54, 113,96, 113,58, 110,98, 108,92, 66,10, 65,79, 62,77, 55,54, 52,15, 22,45, 22,25.1H NMR (400 MHz, DMSO-d6): δ 11,78 (br s, 1H), 8,03 (dd, J=8,1b Hz, J=1.5 Hz, 1H), 7,56 (m, 1H), 7,52 (dt, J=8.5 Hz, J=2 Hz, 1H), 7,50 (d, J=2 Hz, 1H), 7,35 (dd, J=8.5 Hz, J=1 Hz, 1H), 7,30 (d, J=7.9 Hz, 1H), 7,18 (mm, 1H), 7,17 (t, JHF=59,2, 1H), 4,35 (t, J=5.8 Hz, 2H), 4,13 (t, J=6,1 Hz, 2H), 3,88 (s, 3H), 3,55 (m, 2H), 3,50 (m, 2H), 3,14 (s, 9H), of 3.07 (s, 9H), of 2.50 (m, 2H), 2,30 (m, 2H). ESI MS: m/z 652,2 (45%, M+), 326,8 (100%, M+).

The above formazan (2,44 g, 3 mmol) was converted into tetrazolium salt in similar to example 1 conditions except that the reaction mixture was heated at 40°C for 15 hours. The product was identified for the following spectral properties:13With NMR (100 MHz, DMSO-d6): δ 136,7, 128,5, 121,9, 121,8, 114,5, 113,5, 110,2, 108,1, 105,7, 66,7, 66,1, 62,5, 62,1, 56,1, 52,1, 51,8, 21,8, 21,2.1H NMR (400 MHz, DMSO-d6): δ of 8.06 (dd, J=8,1 Hz, J=1.5 Hz, 1H), to 7.93 (m, 8H), to 7.77 (d, J=2 Hz, 1H), 7,53 (d, J=8.6 Hz, 1H), 7,38 (t, J=8.5 Hz, 2H), 7,26 (d, J=52,2 Hz, 1H), 4,23 (t, J=6H, 2H), 4,17 (t, J=6,1 Hz, 2H), 3,94 (s, 3H), 3,53 (m, 2H), 3,32 (m, 2H), 3,14 (s, 9H), 3,01 (s, 9H), and 2.26 (m, 2H), 2,03 (m, 2H), ESI MS: m/z 652,2 (45%, M+), 326,8 (100%, M4+), 326,8 (100%, M2+).

Example 7

Getting 5-[4-(3-ammonium)propoxy-3-methoxy-phenyl]-3-[2-(3-ammonium)propoxy-phenyl]-2-(6-ethoxy-benzothiazol-2-yl)-2H-tetratriacontane salt

6 Ethoxy-2-hydrazino-benzothiazole was introduced in the reaction with alkalinemanganese, obtained in example 6, to obtain the hydrazone which is then reacted with a substituted aniline obtained in example 1, obtaining formazan. Formazan transformed into the corresponding tetrazolium salt.

6 Ethoxy-2-hydrazino-benzothiazole (Acros, to 4.62 g, 22 mmol) were introduced in the reaction with 4-(3-trimethylammonio)-3-methoxy-benzaldehyde under conditions analogous to example 6, with the formation of a light gray product (10,23 g, 95%)which is identified by the following properties:13With NMR (400 MHz, DMSO-d6): δ 165,58, 153,82, 153,74, 149,24, 148,96, 142,42, 128,01, 120,16, 118,24, 113,62, 113,54, 108,66, 106,49, 101,33, 65,83, 63,51, 62,86, 55,32, 52,16, 22,51, 14,6.1H NMR (400 MHz, DMSO-d6): δ 11,98 (br s, 1H), 8,03 (s, 1H), 7,38 (d, J=2.2 Hz, 1H), 7,34 (d, J=8.7 Hz, 1H), 7,32 (d, J=1.8 Hz, 1H), 7,20 (dd, J=8,4 Hz, J=1.8 Hz, 1H), 7,07 (d, J=8,4 Hz, 1H), 6.89 in (dd, J=8.7 Hz, J=2.5 Hz, 1H), 4.09 to (t, J=6,1 Hz, 2H), a 4.03 (t, J=7 Hz, 2H), 3,84 (m, 3H), 3,49 (m, 2H), 3,12 (s, 9H), 2,22 (m, 2H), 1,33 (t, J=7 Hz). ESI-MS: m/z 561,2 (100%, M+). ESI-MS: m/z 443,1 (78%, M+), 222,1 (100%, M+2).

The above hydrazone (3.6 g, 6,93 mmol) were introduced in the reaction with aniline from example 1 (3 g, 10,38 mmol) under conditions analogous to example 1, with the formation of formazan, except that used was a mixture of 3:1 pyridine and DMF as solvent. There was obtained a dark blue product (3.8 g, 66%)which was identified for the following spectral properties:13With NMR (400 MHz, DMSO-d6): δ 130,5, 123,2, 121,8, 118,8, 116, 115,3, 114,1, 113,8, 109,8, 106,3, 67,1, 66,5, 63,6, 52,5, 22,6, 2,5, 14,2.1H NMR (400 MHz, DMSO-d6): δ 11,78 (br s, 1H), 8,01 (br d, J=8 Hz, 1H), 7,81 (br s, 1H=8,8 Hz, 1H), 7.62mm (m, 3H), of 7.48 (t, J=8 Hz, 1H), 7,33 (d, J=8,3 Hz, 1H), 7,16 (m, 1H), 7,10 (dd, J=8,8 Hz, J=2.4 Hz, 1H), to 4.38 (t, J=5.8 Hz, 2H), 4,13 (m, 2H), 3,91 (s, 3H), 3,55 (m, 2H), 3,50 (m, 2H), 3,13 (s, 9H), is 3.08 (s, 9H), of 2.54 (m, 2H, in), 2.25 (m, 2H), 1,38 (t, J=7 Hz, 3H). ESI MS: m/z 776 (12%, M+'+TFA) 662,2 (45%, M+), 331,8 (100%, M+2).

The above formazan (1.24 mg, 1.5 mmol) was converted into tetrazolium salt (2 g, >100%) similar to example 1 conditions. The product was identified for the following spectral properties:13With NMR (400 MHz, DMSO-d6): δ 136,8, 128,1, 125,4, 121,5, 121,4, 114, 113,5, 109,8, 106,8, 66,6, 66,2, 64,5, 62,5, 62,2, 56,1, 52,2, 52,1, 22,1, 21,5, 13,5.1H NMR (400 MHz, DMSO-d6): δ of 8.04 (dd, J=8 Hz, J=1.5 Hz, 1H), 7,95 (m, 4H), 7,78 (d, J=2 Hz, 3H), 7,54 (d, J=8.5 Hz, 1H), 7,40 (t, J=7.8 Hz, 1H), 7,38 (d, J=8.6 Hz, 1H), 4,23 (t, J=6,1 Hz, 2H), 4,1 (q, J=8 Hz, 2H), 4,12 (m, 2H), 3.96 points (s, 3H), 3,53 (m, 2H), 3,50 (m, 2H), 3,18 (m, 2H), 3.15 in (s, 9H), 2,92 (2, 9H), is 2.30 (m, 2H), 1,89 (m, 2H), 1,40 (t, J=7 Hz, 2H). ESI MS: m/z 220,8 (100%, M+3).

Example 8

Getting 5-[4-(3-ammonium)propoxy-3-methoxy-phenyl]-3-[2-(3-ammonium)propoxy-phenyl]-2-(6-bromo-benzothiazol-2-yl)-2H-tetratriacontane salt

To obtain the desired hydrazone in example 8 instead of 6-ethoxy-2-hydrazino-benzothiazole used 6-bromo-2-hydrazino-benzothiazole. The hydrazone is then processed in the same conditions to obtain the desired formazan (44%).1H NMR (400 MHz, DMSO-d6), δ of 7.90 (dd, J=8,1 Hz, J=1.7 Hz, 1 H), 7,58 (dt, J=8,2 Hz, J=1.7 Hz, 1H), 7,39 (, J=8,2 Hz, 1H), 7,15 (d, J=1.7 Hz, 1H), 4,25 (t, J=6 Hz, 2H), of 3.45 (m, 2H), 3,11 (s, 9H), 2,48 (m, 2H, in), 2.25 (m, 2H). ESI-MS: m/z 249 (100%, M2+).

Formazan (322 mg, the 0.375 mmol) were treated under the same conditions as in example 8, with the formation of tetrazolium salt, except that the reaction mixture was heated at 40°C during the night with the formation of the desired tetrazolium salt (478 mg, >100%),13With NMR (100 MHz, DMSO-d6): δ 151, 139,45, 134,71, 125,14, 121, 115,39, 66,59, 62,84, 52,39, 22,48: δ of 7.901H NMR (400 MHz, DMSO-d6), a total of 8.74 (d, J=2.1 Hz, 1H), 7,95 (m, 5H), 7,78 (d, J=2.1 Hz, 1H), 7,51 (d, J=7.8 Hz, 1H), 7,39 (m, 2H), 4,2 (m, 2H), 4,08 (m, 2H), 3.96 points (s, 3H), 3,53 (m, 2H), 3,19 (m, 2H), 3,11 (s, 9H), 2.91 in (s, 9H), of 2.24 (m, 2H), to 1.87 (m, 2H). ESI-MS: m/z 233 (100%, M3+).

Example 9

Getting 5 -[4-(3-ammonium)propoxy-3-methoxy-phenyl]-3-[2-(3-ammonium)propoxy-phenyl]-2-(6-ethoxy-benzothiazol-2-yl)-2H-tetrachlorophenol salt

To a suspension of formazan (from example 7, 1.18 g, 1,43 mmol) in acetonitrile (15 ml) was added n-bromosuccinimide (300 mg, was 1.69 mmol) at room temperature. After 1 hour all dissolved. After 3 hours was formed orange precipitate. The reaction was stirred over night. The precipitate was filtered and washed with acetonitrile (3x5 ml). Received the orange product was dried under vacuum over night. (1.07 g, 83%)13With NMR (100 MHz, DMSO-d6): δ 151, 139,45, 134,71, 125,14, 121, 115,39, 66,59, 62,84, 52,39, 22,481H NMR (400 MHz, DMSO-d6): δ 8,08 (d, J=8,2 Hz, 1H), 8,04 (d, J=l,7 Hz, 1H), 7,94 (m, 3H), 7,79 (bs, 1H), and 7.5 (d, J=8,2 Hz, 1H), 7,40 (d, J=8,2 Hz, 1H), 7,30 (dd, J=8,2 Hz, J=l,7 Hz, 1H), 4,23 (t, J=6 Hz, 2H), 4,15 (q, J=8,2 Hz, 2H), 4,10 (m, 2H), 3,95 (s, 3H), of 3.57 (m, 2H), up 3.22 (m, 2H), 3.15 in (s, 9H), 2,95 (s, 9H), is 2.30 (m, 2H), 1,90 (m, 2H), 1,4 (t, J=8,2 Hz). ESI-MS: m/z 221 (100%, M3+).

Solubility tetrazolium salts obtained in examples 1-9 was determined by adding 0.2m phosphate buffer, pH ~ to 7.5 at room temperature to known amounts of each compound to dissolve the connection. The results are shown in the table, where the results of their comparison with WST-4 - thiazoleacetate salt Dojindo Laboratories, NTS-045 - thiazoleethanol salt Bayer Corp. and the modified STC-045, in which the methoxy group is replaced by 3-sulfonatophenyl-group (see figure 3).

Example No.(Counterion)Solubility (mm)
1(pomnite)33
2(romatravellers)5
3(pomnite)9
4(pomnite)167
5(pomnite)5
6(dibromide)181
7(dibromide)169
8(dibromide)290
9(tribromide)260
WST-4118
NTS-04521
Modified STC-0458

Although improved solubility tetrazolium salts was achieved by the introduction of trialkylamines substituents in thiazoleacetate salt, this improvement was not obvious based on prior art. From the prior art, it can be assumed that the addition of 3-sulfonatophenyl groups in the molecule will increase the solubility of substances in water. Introduction sulfoxylates group in the molecule was carried out Dojindo Laboratories. However, the replacement of the methoxy group 3 solventproof groups who nd a phenyl group, attached to tetrazolium ring (see modified STC-045 figure 3) gave a lower solubility (8 mm)than the STC-045 (21 mm). Thus, in our proposed molecular structure of this phenomenon is not observed. We were able to determine that the solubility improves another polar group. However, there were other unexpected effects, which will be discussed below.

Replacement of the methoxy group trimethylolpropane group increased solubility with 21 mm (STC-045) to 33 mm (example 1), and adding the second trimethylolpropane-group (example 6) significantly increased the solubility of up to 181 mm. However, the replacement of nitrate tetrafluoroborates the counterion has led to a significant decrease in solubility. (Compare example 2 with example 1.) The solubility of tetrazolium salt in example 1 was reduced in example 3, when trimethylammonio group changed dimethylaminopropoxy group.

A significant increase in solubility is the result of adding two trimethylolpropane-groups (example 6) was also unexpectedly found in example 4 (167 mm), which was introduced only one trimethylammonio group, and the substituted thiazoline group has been replaced by unsubstituted benzothiazolyl group. However, further adding to thiazolino group, is illustrated in example 5, which used replaced by the th attitudeline group, gave substance with low solubility (5 mm). In example 7 was obtained another unexpected result: the addition of two ammonium groups and benzothiazole group, both of which improved solubility, did not give a complementary effect. The substance of example 7 has a solubility 167 mm.

Another significant increase in solubility was obtained when replacing ethoxypropan on benzothiazole group example 7 to braugruppe in example 8. The solubility increased from 169 mm to 290 mm, which is the best of these results. A similar result was obtained when the counterion in example 7 (nitrite ion) was replaced by brainy ion in example 9. The solubility increased from 169 mm to 260 mm. It seems that the introduction of bromine is effective to increase the solubility of this family tetrazolium salts.

Obtained and tested for solubility of the compounds had a slightly different spectral properties. Absorption spectrum of each formazan obtained from tetrazolium salt and tested for solubility (table), was measured by spectrophotometer Hewlett-Packard Model 8453 diode matrix, working in the field of UV and visible spectrum. Solutions of 100 μm tetrazolium salt was prepared in a buffer of 100 mm potassium phosphate. Spectra formazan was measured after addition of 5-fold from itca salt of ascorbic acid for the conversion of the tet-salts in appropriate formazane. The results are shown in figa and b.

As noted earlier, formazane thiazoleacetate salt should absorb light at wavelengths of more than 640 nm, preferably at a level characteristic response. On figa and b for comparison, the absorption spectra of the NTS-045 and WST-4. WST-4 has a peak absorption at about 560 nm, and STC-045 shows a relatively constant absorption in the range from about 460 nm to 660 nm. However, the solubility of the substance is less than the WST-4. Add trimethylolpropane-group led to the change curves of the spectrum. Connection examples 1, 2, 3, 4, 6, 7 and 8 have lower absorption than the STC-045, but their characteristics are relatively flat in the shown range. As you can see, tet-salt of example 5, including attitudeline group, not only have low solubility, but also rising with increasing wavelength characteristic absorption without smooth areas, as STC-045 or modified connection examples 1, 2, 3, 4, 6, 7 and 8. The compound of example 9 has a spectral response with a relatively flat area in the range of 400-640 nm, and high solubility.

In the ideal case, tetrazolium salt should form formazan with high absorption over a wide wavelength range that will allow you to easily and accurately measure produced by the reaction with the analysis of the generated substance color. Thus, the most desirable is relatively flat characteristic. As can be seen from the data on the solubility of formisano obtained from the tested one-salts, all of them (with the possible exception only of the compound of example 5) have good prospects for use as indicators. Tetrazolium salt of example 5 is the new connection and can be used as an indicator, however, it is less preferable than a connection with a more uniform spectral response and higher solubility.

Application tetrazolium salts

Although tetrazolium salt can be used as indicators in many areas, they are especially suitable for use in the test strips for measuring the content of the analyzed substances in biological samples. One of the important applications is the determination of glucose in blood.

Test strips usually contain the media and the composition of the reagents. Reagents (e.g., glucosegalactose) and cofactor (e.g., NAD or PQQ) react with the analyzed substance (such as glucose) in a biological sample, and the mediator (e.g., PMS or enzyme diaphorase) restores thiazoleacetate salt to the corresponding colored formazan. The colour is usually most accurately measured with the aid intended for e is th purpose measuring device. The light source (e.g., led) illuminates the test strip. Reflected from the test strip of light received by the transducer and is correlated with the number entered in the reaction of the analyte.

Thiazoleacetate indicators according to the invention provide a wide range of as and indicators disclosed in U.S. patent No. 5126275 and the other above-mentioned documents. However, as shown in the examples, many of the new thiazoleacetate indicators have greater solubility compared with NTS-045, providing, thus, a faster and more accurate testing.

The invention is summarized in the following alternative options for implementation.

An alternative implementation

The compound of the formula

where

A=the counterion,

a=1-3,

b=0-3,

X=1-6C alkyl or heteroalkyl,

R1=1-6C alkyl,

one of R2and R3is alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy group,

one of R4and R5is halogen and the other represents a substituted halogen 1-6C alkyl,

one or both of R4and R5are XN+Hb(R1)3-bor R4and R5connected with the formation of aromatic or heteroaromatic ring Il is a substituted aromatic ring or substituted heteroaromatic ring.

An alternative implementation

The connection according to the alternative implementation And having the formula

where

A=the counterion,

X=1-6C alkyl,

a=1-3,

b=0-3,

R1=1-3C alkyl,

one of R2and R3is 1-4C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy-group

R4=CHF2,

R5=halogen.

An alternative implementation

The connection according to alternative implementation, characterized in that

X is propylene,

b=0,

R1is stands,

R2and R3form methylenedioxy-group

R4CHF2,

R5is Cl.

An alternative implementation D

The connection according to alternative implementation, characterized in that

X is propylene,

b=1,

R1is stands,

R2and R3form methylenedioxy-group

R4CHF2,

R5is Cl.

An alternative implementation of E

The connection according to alternative implementation, characterized in that

X is propylene,

b=0,

R1is stands,

R2is trimethylammonio the sludge,

R3represents methoxy,

R4CHF2,

R5is Cl.

An alternative implementation of F

The connection according to the alternative implementation And having the formula

where

a=1-3,

A=the counterion,

X=1-6C alkyl

b=0-3,

R1=1-3C alkyl,

one of R2and R3is alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy-group

one of R4and R5is halogen and the other represents a substituted halogen 1-6C alkyl, or R4and R5are halogen,

R6=1-4C alkoxy, hydrogen or halogen.

An alternative implementation of the G

The connection according to the alternative implementation of F, characterized in that

X is propylene,

b=0,

R1is stands,

R2is trimethylammonium,

R3represents methoxy,

R4, R5and R6are hydrogen.

An alternative implementation N

The connection according to the alternative implementation of F, characterized in that

X is propylene,

R1is stands,

b=0,

R2is trimethylammonium,

R3is m tilam,

R4and R5form an aromatic ring,

R6represents methoxy.

An alternative implementation I

The connection according to the alternative implementation of F, characterized in that

X is propylene,

b=0,

R1is stands,

R2is trimethylammonium,

R3is stands,

R4and R5are hydrogen,

R6is ethoxy.

An alternative implementation J

The connection according to the alternative implementation of F, characterized in that

X is propylene,

b=0,

R1is stands,

R2is trimethylammonium,

R3is stands,

R4and R5are hydrogen,

R6is bromine.

An alternative implementation To

The connection according to the alternative implementation And, wherein at least one counterion is a nitrite, phosphate, hydrogen phosphate, dihydrophosphate, hydrosulfate, sulfate, bicarbonate, carbonate, methanesulfonate, perborate, bromide, chloride, iodide, or combinations thereof.

An alternative method L

The method of measuring the content of an analyte capable of providing proportional to the color change in the reaction, biologicheskii fluid, comprising the steps:

availability as an indicator of tetrazolium salt of the formula

where

A=the counterion,

a=1-3,

b=0-3,

X=1-6C alkyl or heteroalkyl,

R1=1-6C alkyl,

one of R2and R3is alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy-group

one of R4and R5is halogen and the other represents a substituted halogen 1-6C alkyl, one or both of R4and R5are XN+Hb(R1)3-bor R4and R5connected with the formation of aromatic or heteroaromatic ring or substituted aromatic ring or substituted heteroaromatic ring; and

determine the concentration of the specified analyte in a biological fluid using the specified tetrazolium salt used as an indicator.

An alternative method M

The method, according to an alternative method L, characterized in that said analyzed substance is glucose.

An alternative way N

The method, according to an alternative method M, characterized in that the specified biological fluid is blood.

An alternative way Of

The method, according to an alternative method M, featuring the the action scene, that said tetrazolium salt has the formula

where

A=the counterion,

X=1-6C alkyl,

a=1-3,

b=0-3,

R1=1-3C alkyl,

one of R2and R3is 1-4C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy-group

R4=CHF2,

R5=halogen.

An alternative method P

The method, according to an alternative method Of, wherein

X is propylene,

b=0,

R1is stands,

R2and R3form methylenedioxy-group

R4CHF2,

R5is Cl.

An alternative method Q

The method, according to an alternative method Of, wherein

X is propylene,

b=1,

R1is stands,

R2and R3form methylenedioxy-group

R4CHF2,

R5is Cl.

An alternative way R

The method, according to an alternative method Of, wherein

X is propylene,

b=0,

R1is stands,

R2is trimethylammonium,

R3represents methoxy,

R4CHF2,

R5is Cl.

An alternative way S

The method, according to an alternative method L in the region have the formula

where

a=1-3,

A=the counterion,

X=1-6C alkyl,

b=0-3,

R1=1-3C alkyl,

one of R2and R3is alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy-group

one of R4and R5is halogen and the other represents a substituted halogen 1-6C alkyl, or R4and R5are hydrogen,

R6=1-4C alkoxy, hydrogen or halogen.

An alternative way T

The method, according to an alternative method S, characterized in that

X is propylene,

b=0,

R1is stands,

R2is trimethylammonium,

R3represents methoxy,

R4, R5and R6are hydrogen.

An alternative way U

The method, according to an alternative method S, characterized in that

X is propylene,

R1is stands,

b=0,

R2is trimethylammonium,

R3is stands,

R4and R5form an aromatic ring,

R6represents methoxy.

Alternative V

The method, according to an alternative method S, characterized in that

X is propylene,

b=0,

R1is stands,

R2is trimethylamin what nipradilol,

R3is stands,

R4and R5are hydrogen,

R6is ethoxy.

An alternative method W

The method, according to an alternative method S, characterized in that

X is propylene,

b=0,

R1is stands,

R2is trimethylammonium,

R3is stands,

R4and R5are hydrogen,

R6is bromine.

An alternative method X

The method, according to an alternative method L, characterized in that at least one counterion is a nitrite, phosphate, hydrogen phosphate, dihydrophosphate, hydrosulfate, sulfate, bicarbonate, carbonate, methanesulfonate, perborate, bromide, chloride, iodide, or combinations thereof.

An alternative way Y

Method of increasing the solubility disorderisolate salts, in which thiazoline group attached to the third nitrogen atom tetrazolium rings, including the introduction in disorderisolate salt, at least one dialkylaminoalkyl group as Deputy.

An alternative method Z

The method according to alternative Y, wherein disorderslava salt is phenyl substituents at the third nitrogen atom and carbon atom tetrazolium rings.

An alternative way AA

The method according viola is nativename method Z, characterized in that at least one of dialkylammonium group is trimethylolpropane-group.

An alternative way BB

The method, according to an alternative method Z, wherein at least one of dialkylammonium group is the Deputy mentioned phenyl groups.

An alternative way SS

The method, according to an alternative method Z, wherein at least one of dialkylammonium group is Deputy both phenyl substituents.

An alternative way DD

The method, according to an alternative method Z, characterized in that the specified thiazoline group is benzothiazole group.

An alternative way of HER

The method, according to an alternative method Z, characterized in that the specified disorderslava salt has counterions nitrite, phosphate, hydrogen phosphate, dihydrophosphate, hydrosulfate, sulfate, bicarbonate, carbonate, methansulfonate, perborate, bromide, chloride, iodide, or combinations thereof.

An alternative way FF

The method, according to an alternative method, characterized in that the counterions are pomnite, dibrominated or tribromide.

1. The compound of the formula

where A=the counterion,
a=1-3,
b=0-3,
X=1-C alkyl,
R1=1-6C alkyl,
one of R2and R3is 1-6C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy,
one of R4and R5is halogen and the other represents a substituted halogen 1-6C alkyl, or R4and R5connected with the formation of 6-10C aromatic ring or substituted 6-10C aromatic ring, in which the Deputy is selected from 1-6C alkoxy, halogen and substituted with halogen 1-6S of alkyl.

2. The compound according to claim 1, having the formula

where A=the counterion,
X=1-6C alkyl,
a=1-3,
b=0-3,
R1=With 1-3 alkyl,
one of R2and R3is 1-4C-alkyl and the other represents N+Hb(R1)3-bor R2and R3form methylenedioxy,
R4=CHF2,
R5=halogen.

3. The compound according to claim 2, in which
X is propylene,
b=0,
R1is stands,
R2and R3form methylenedioxy,
R4CHF2,
R5is Cl.

4. The compound according to claim 2, in which
X is propylene,
b=1,
R1is stands,
R2and R3form methylenedioxy,
R4CHF2,
R5is Cl.

5. The compound according to claim 2, in which
X is propylene,
b=0,
R1 is stands,
R2is trimethylammonium,
R3represents methyl,
R4CHF2,
R5is Cl.

6. The compound according to claim 1, having the formula

where a=1-3,
A=the counterion,
X=1-6C alkyl,
b=0-3,
R1=1-3C alkyl,
one of R2and R3is 1-6C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy,
one of R7and R8is halogen and the other represents a substituted halogen 1-6C alkyl, or R7and R8are hydrogen, or R7and R8form an aromatic ring,
R9=1-4C alkoxy, hydrogen or halogen.

7. The connection according to claim 6, in which
X is propylene,
b=0,
R1is stands,
R2is trimethylammonium,
R3represents methyl,
R7, R8and R9are hydrogen.

8. The connection according to claim 6, in which
X is propylene,
R1is stands,
b=0,
R2is trimethylammonium,
R3is stands,
R7and R8form an aromatic ring,
R9represents methoxy.

9. The connection according to claim 6, in which
X is propylene,
b=0,
R1is stands,
R2alleycatallies,
R3is stands,
R7and R8are hydrogen,
R9is ethoxy.

10. The connection according to claim 6, in which
X is propylene,
b=0,
R1is stands,
R2is trimethylammonium,
R3is stands,
R7and R8are hydrogen,
R9is bromine.

11. The compound according to claim 1, in which at least one counterion is a nitrite, phosphate, hydrogen phosphate, dihydrophosphate, hydrosulfate, sulfate, bicarbonate, carbonate, methanesulfonate, perborate, bromide, chloride, iodide, or combinations thereof.

12. The method of measuring the content of an analyte capable of providing proportional to the color change in the reaction, in a biological fluid, comprising the steps:
availability as an indicator of tetrazolium salt of the formula

where A=the counterion,
a=1-3,
b=0-3,
X=1-6C alkyl,
R1=1-6C alkyl,
one of R2and R3is 1-6C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy,
one of R4and R5is halogen and the other represents a substituted halogen 1-6C alkyl, or R4and R5connected with the formation of 6-10C aromatic ring or substituted 6-10C ar is automatic ring in which the Deputy is selected from 1-6C alkoxy, halogen and substituted with halogen 1-6S of alkyl; and
determine the concentration of the specified analyte in a biological fluid using the specified tetrazolium salt used as an indicator.

13. The method according to item 12, which specified the analyzed substance is glucose.

14. The method according to item 13, in which the specified biological fluid is whole blood.

15. The method according to item 13, in which the specified tetrazolium salt has the formula

where A=the counterion,
X=1-6C alkyl,
a=1-3,
b=0-3,
R1=1-3C alkyl,
one of R2and R3is 1-4C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy,
R4=CHF2,
R5=halogen.

16. The method according to clause 15, where
X is propylene,
b=0.
R1is stands,
R2and R3form methylenedioxy,
R4CHF2,
R5is Cl.

17. The method according to clause 15, where
X is propylene,
b=1,
R1is stands,
R2and R3form methylenedioxy,
R4CHF2,
R5is Cl.

18. The method according to clause 15, where
X is propylene,
b=0,
R1is stands,
R2is trimethylammonium prom,
R3represents methyl,
R4CHF2,
R5is Cl.

19. The method according to item 12, in which the specified tetrazolium salt has the formula

where a=1-3,
A=the counterion,
X=1-6C alkyl,
b=0-3,
R1=1-3C alkyl,
one of R2and R3is 1-6C-alkyl and the other represents XN+Hb(R1)3-bor R2and R3form methylenedioxy,
one of R7and R8is halogen and the other represents a substituted halogen 1-6C alkyl, or R7and R8are hydrogen, or R7and R8form an aromatic ring,
R9=1-4C alkoxy, hydrogen or halogen.

20. The method according to claim 19, where
X is propylene,
b=0,
R1is stands,
R2is trimethylammonium,
R3represents methyl,
R7, R8and R9are hydrogen.

21. The method according to claim 19, where
X is propylene,
R1is stands,
b=0,
R2is trimethylammonium,
R3is stands,
R7and R8form an aromatic ring,
R9represents methoxy.

22. The method according to claim 19, where
X is propylene,
b=0,
R1is stands,
R2is trimethylammonium,
R3is stands,
R 7and R8are hydrogen,
R9is ethoxy.

23. The method according to claim 19, where
X is propylene,
b=0.
R1is stands,
R2is trimethylammonium,
R3is stands,
R7and R8are hydrogen,
R9is bromine.

24. The method according to item 12, in which at least one counterion is a nitrite, phosphate, hydrogen phosphate, dihydrophosphate, hydrosulfate, sulfate, bicarbonate, carbonate, methanesulfonate, perborate, bromide, chloride, iodide, or combinations thereof.



 

Same patents:

FIELD: medicine.

SUBSTANCE: cell material containing marrow stem cells in a liquid culture medium is placed over a prepared agar medium containing marrow cells producing homing factors. After incubation of the prepared two-layer culture, inadherent cells are transferred to a semi-viscous culture medium and incubated in a mode required for colony-formation. Then by recording the difference of stem cell count in the cell material in the reference, and after placing on the agar medium containing homing factors, stem cells migrated by stem cell homing factor are counted.

EFFECT: invention allows simplifying the method for determination of production of stem cell homing factors due to the use of the agar medium as a semipermeable membrane and the introduction of colony-forming ability change of the cell material as an evaluation criterion of production of stem cell homing factors.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to traumatology and orthopedics. In order to predict development of endoprosthesis instability after total endoprosthetics in connection with osteoarthrosis of hip joint, patient's blood serum is tested. Level of primary, secondary and final isopropanol-soluble products of lipid peroxidation (ISPP, ISSP, ISFP) and ascorbate-induced lipid peroxidation (AOA-1, AOA-2) is determined. ISPP, ISSP, ISFP levels are analysed on 10-14 day after operation, as well as 3 and more months after operation. AOA-1 and AOA-2 levels are analyzed only on 10-14 day after operation. Level of primary, secondary and final isopropanol-soluble products of lipid peroxidation and ascorbate-induced lipid peroxidation relative to norm on 10-14 day after operation and 3 and more months after operation is used to predict development of endoprosthesis instability after endoprosthetics of hip joint.

EFFECT: method is simple, available, efficient for long-term prediction of instability development.

4 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to traumatology and orthopedics. To prognosis progression of endoprosthesis instability after total endoprosthesis replacement on deforming coxarthrosis, seroscopy of the patient is conducted before surgery and 3 months after it. Additional seroscopy of the patient is conducted in 10-14 days after surgery. The level of ferritin and ceruloplasmin is detected. At increase of relative to normal levels of ferritin and ceruloplasmin before surgery, in 10-14 days and after 3 months or more, predict progression of endoprosthesis instability in the bone after hip endoprosthesis replacement.

EFFECT: method is inexpensive, simple, effective and a long-term prognosis of progression of endoprosthesis instability.

5 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to traumatology and orthopedics. For early diagnosis of progression of endoprosthesis instability after total endoprosthesis replacement on deforming coxarthrosis, the patient is examined. In addition, a study of ferritin and ceruloplasmin in the patient's blood serum is conducted. At level increase of ferritin in relation to the norm of 519.65% or more, and ceruloplasmin levels of 425.12% and more, endoprosthesis instability is diagnosed.

EFFECT: method is available, effective and has an independent diagnostic value.

3 ex

FIELD: medicine.

SUBSTANCE: there is offered a method of discrimination and calculation of at least two populations of biological elements - carriers of specific signs, probably presented in a sample. The method provides the use of three different probes, each of which is specifically fixed with one of the populations of biological elements which are required to be detected. Each probe itself becomes detectable due to its proper marker, and two different markers specified have two emission spectra containing at least one common part (overlapping emission spectra), and the third one has the emission spectrum which essentially contain no common parts with two others (nonoverlapping spectrum).

EFFECT: invention allows definitely detecting three populations of biological elements by using only two detection devices that assumes that two populations of biological elements will be detected by the same detection device.

9 cl, 2 ex, 1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns methods and compositions for improving effectiveness of antibody for medical application with using the compounds potentiating NK-cells. Substance of the invention covers a method of treating a disease and a pharmaceutical composition for treating a disease brought on or exacerbated partially by the cells which can be considered as targets and which can be eliminated by antibody for medical application, involving introduction to a patient of the first antibody blocking an inhibiting NK-cell receptor chosen of group: KIR2DL1, KIR2DL2, KIR2DL3 and NKG2A and then introductions of antibody for medical application.

EFFECT: advantage of the invention consists in higher clinical effectiveness.

40 cl, 2 ex, 1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: there is offered a method to assess of enzyme's ability to the level of phosphorylation of polypeptide that implies a reaction of the analysed enzyme and a substratum presented with a biotin-conjugated fragment of 516 to 777 residues of a human insulin 1 receptor substratum (hIRS-1-p30), binding of the reaction product and immobilised streptavidin and detection of the level of phosphorylation by antibodies specific to the phosphorylated polypeptide residues.

EFFECT: according to the invention, the method allows identifying tyrosine and serine proteinases and can be taken as a basis of a test system for new modulators of their activity.

9 cl, 8 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: immunological detection and quantitative analysis of sequential changes in protein levels of VEGF-165 in obtained patient's samples taken in course of time are conducted, where increasing levels of the protein VEGF-165 in course of time indicate progression of disease or adverse reaction to the therapy, and where decreasing levels of protein VEGF-165 in course of time indicate remission of the disease or a positive reaction to the therapy.

EFFECT: method enables to conduct noninvasive analysis of levels of circulating VEGF-165 which serves as a valuable prognostic indicator of disease outcome.

22 cl, 2 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: for diagnosis of feet mycosis in patients with recurrence of lower extremities erysipelatous inflammation content of zinc in blood plasma is determined. At reducing zinc content in blood plasma up to 0.3-0.5 mcg/ml patient undergoes mycological examination, on base of which the diagnosis is made.

EFFECT: method enables to select a group of patients with suspected feet mycosis for prescription of comprehensive mycological examination for the purpose of diagnosis of foot mycoses.

2 ex

FIELD: chemistry.

SUBSTANCE: in the method, a biological object is infused with ethylacetate twice for 30 minutes each time. Ethylacetate extractions are separated, combined and dehydrated. The combined extraction is first evaporated in an air current to a small volume and then in a nitrogen current until complete evaporation of the solvent. The residue is dissolved in a system of solvents, undergoes chromatography in a macro-column with sorbent using a multicomponent mobile phase. Eluate fractions containing 2-methoxy-4-allylhydroxybenzene are combined and evaporated. Before chromatography, the residue is dissolved in hexane, extracted, separated, acidified, saturated with sodium sulphate and extracted with diethyl ether. The ether extraction is separated, dehydrated, evaporated until complete removal of solvent. The residue is dissolved in a mixture of solvents - hexane - dioxane - proanol-2 (40:5:1). Determination is carried out through chromatography-mass spectrometry using a capillary macro-column. The amount of 2-methoxy-4-allylhydroxybenzene is determined from the area of the chromatographic peak.

EFFECT: increased sensitivity of determination.

3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula or its pharmaceutically acceptable salt, where R1 and R2 each independently denotes a hydrogen atom, a halogen atom, a lower alkyl, a hydroxyl group, a cyano group or a lower alkoxy; R3 independently denotes a hydrogen atom, a halogen atom, a lower alkyl, a lower alkoxy, a hydroxyalkyl, trifluoromethyl, lower alkenyl or cyano group; R4 independently denotes a hydrogen atom, a lower alkyl, a lower alkoxy, a halogen atom, trifluoromethyl, hydroxyalkyl optionally substituted with a lower alkyl, aminoalkyl optionally substituted with lower alkyl, alkanoyl, carboxyl group, lower alkoxycarbonyl or cyano group; Q denotes a nitrogen atom; R5 and R6 each independently denotes a hydrogen atom, a lower alkyl, a halogen atom, a lower alkylsulfonyl, a lower alkylsulfanyl, alkanoyl, formyl, aryl, mono- or di-(lower) alkylcarbamoyl or mono- or di-(lower) alkylsulfamoyl; and further as indicated in the formula of invention. The invention also relates to a glucokinase activator containing the compound in paragraph 1 and to a therapeutic agent based on said compounds.

EFFECT: novel compounds which can be useful in treating and preventing diabetes and obesity are obtained and described.

29 cl, 227 ex, 6 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I and their pharmaceutically acceptable salts. Disclosed compounds have inhibitory effect on CDK1 kinase and can be used to prepare medicinal agents for treating diseases associated with abnormal cell cycle development. In formula I , R1 is hydrogen, -C(O)OR9 or R2-(X)n-; X is (lower)alkylene, hydroxy(lower)alkylene, cyclised(lower)alkylne or mono- or dihalogen(lower)alkylene; R2 is a group, where denotes a phenyl or a 5-6-member heteroaromatic ring containing 1-2 heteroatoms selected from a group comprising oxygen, sulphur and nitrogen atoms; R5, R6 and R7 are independently selected from a group comprising hydroxy, hydrogen, (lower)alkyl, halogen and (lower)alkoxy; R4 is a halogen, , (O)k(CH2CH2O)y-R10, , -S-R12 or -O-(CH2)tR14, where denotes a phenyl, a cycloalkyl ring containing 3-6 carbon atoms, a 4-6-member heterocycloalkyl containing 3-5 carbon atoms and 1-2 heteroatoms selected from a group comprising oxygen, nitrogen and sulphur atoms; R9, R11, R15 and R16 independently denote (lower)alkyl; R10 and R12 denote (lower)alkyl; R14 denotes perfluoro(lower)alkyl or -NR15R16; R17 and R18 independently denote hydrogen, , F, OCH3 and -C(=O)CH3; n and k are equal to 0 or 1; m, w, y and z are equal an integer from 0 to 3; and t equals an integer from 0 to 6.

EFFECT: invention also relates to a pharmaceutical composition having antiproliferative activity, containing one or more of the disclosed compounds.

65 cl, 1 tbl, 49 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of general formula (I): wherein dashed lines present single or double bonds, and the values of radicals R1, R2, R3, R4 are described in cl. 1 of the patent claim. Besides the invention refers to application and a based pharmaceutical composition for prevention and treatment of neurodegenerative diseases and other diseases wherein cell dystrophy and/or cell loss (apoptosis) caused by free radicals act the main part.

EFFECT: production of new compounds and the based pharmaceutical composition which can find application in medicine for prevention and treatment of neurodegenerative diseases.

6 cl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to compounds of general formula I and to their pharmaceutically acceptable acid addition salts. The compounds of the present invention exhibit the properties of glycine carrier 1 (GlyT-1) inhibitors. In formula I , R1 represents -OR1', -SR1' or morpholinyl; R1' represents lower alkyl, halogen-substituted lower alkyl, or represents -(CH2)n-lower cycloalkyl; R2 represents -S(O)2-lower alkyl, -S(O)2NH-lower alkyl, NO2 or CN; X1 represents CR3 or N; X2 represents CR3' or N; R3/R3' independently represent hydrogen, halogen, lower alkyl, CN, NO2, -S(O)2-phenyl, -S(O)2-lower alkyl, -S(O)2-pyridine-2, 3 or 4-yl, phenyl optionally substituted with one or two substitutes specified from the group consisting of NO2 or halogen, or represent halogen-substituted lower alkyl, or represent -C(O)-lower alkyl; n has a value of 0, 1 or 2. The invention also concerns a drug containing one or more compounds of the invention and pharmaceutically appropriate excipients.

EFFECT: preparation of the compounds exhibiting the properties of glycine carrier inhibitors.

20 cl, 1 tbl, 133 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I) , where R1 is selected from group, including: phenyl, unsubstituted or mono-, di- or tri-substituted independently with lower alkyl, lower alkoxy group, halogen or lower halogenalkyl; naphtyl; tetrahydronaphtyl; C3-7cycloalkyl; -(CHR3)m-phenyl, where m stands for 1, 2, or 3; and phenyl is unsubstituted or mono-, di- or tri-substituted with lower alkoxy group, and where R3 is independently selected from hydrogen and lower alkyl; -(CH2)n-heteroaryl, where n stands for 1, 2 or 3; term "heteroaryl" relates to aromatic 5- or 6- member ring or bicyclic 9-member aromatic groups, which can include 1, 2 or 3 atoms, selected from nitrogen and/or sulphur; -(CH2)n-heteroaryl, where n stands for 1, 2 or 3; term "heteroaryl" relates to aromatic 5- or 6- member ring or bicyclic 9-member aromatic groups, which can include 1, 2 or 3 atoms, selected from nitrogen and/or sulphur, and heteroaryl is mono-, di- or tri-substituted independently with lower alkoxy group; and R2 is selected from group including: n-butyl; phenyl, unsubstituted or mono-, di- or tri-substituted independently with lower alkyl, halogen or lower alkoxy group; heteroaryl, where term "heteroaryl" relates to aromatic 5-member ring, which can include 1, 2 or 3 atoms, selected from nitrogen and/or sulphur; unsubstituted or mono-, di- or tri-substituted independently with lower alkoxy group; -C(O)-NR4R5; where R4 and R5 stand for lower alkyl or together with nitrogen atom, to which they are bound, form 5-member heterocycle, which can additionally contain heteroatom, selected from N or S, and to their pharmaceutically acceptable salts. Invention also relates to pharmaceutical composition.

EFFECT: obtaining novel biologically active compounds, able to inhibit DPP-IV.

13 cl, 43 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I)

, pharmaceutical compositions based on the said compounds, as well as methods of using said compounds in preparing medicinal agents.

EFFECT: obtaining compounds and a composition which can inhibit phosphatase cdc25, particularly phosphatase cdc25-C and can be particularly used for treating cancer.

12 cl, 56 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I and their pharmaceutically acceptable salts. The disclosed compounds have inhibitory effect on CDK1 kinase. In formula I , R1 is hydrogen or R2-(X)n-; X is a lower alkylene or cyclic lower alkylene; R2 denotes ; where denotes phenyl; cycloalkyl containing 3-6 carbon atoms; 4-6-member heterocycloalkyl ring having 3-5 carbon atoms and 1-2 oxygen atoms; R5, R6 and R7 are independently selected from a group containing hydrogen or halide; R4 is hydrogen or -(O)k(CH2CH2O)y-R10; R19 is hydrogen; R20 is hydrogen or -C(O)-R11; R10 and R11 is a lower alkyl; n and k are equal to 0 or 1; y is an integer from 0 to 3.

EFFECT: obtaining a pharmaceutical composition with inhibitory effect on CDK1 kinase, containing one or more of the disclosed compounds.

15 cl, 10 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry and specifically to compounds of formula I or to pharmaceutically acceptable salts thereof, where Ar is imidazole or pyrazole, where the said Ar can be substituted with substitute(s) selected from a group consisting of a C1-C6 alkyl group, a phenyl group and a halogen atom, each of Y1, Y2 and Y3 is a carbon ot nitrogen atom, A is an oxygen atom, a sulphur atom or a group of formula -SO2-, R1 is a hydrogen atom, a C1-C6 alkyl group which can be substituted with one phenyl group (where the said phenyl group can be substituted with one substitute selected from a group consisting of a halogen atom and a C1-C6 alkyl group), or a phenyl group, R2 is a C1-C6 alkyl group, R3 is (i) a C1-C18 alkyl group, (ii) C2-C8 alkenyl group, (iii) C2-C8 alkynyl group, (iv) C3-C8 cycloalkyl group, (v) C1-C6 alkyl group substituted with 1-3 substitutes selected from a group given in paragraph 1 of the formula of invention, or (vi) a phenyl group, a naphthyl group, a pyrazolyl group, a pyridyl group, an indolyl group, a quinolinyl group or an isoquinolinyl group, where each of the said groups can be substituted with 1-3 substitutes selected from a group given in paragraph 1, R4 is a hydrogen atom or a C1-C6 alkyl group, and R5 is (i) C1-C10 alkyl group, (ii) C1-C10 alkyl group which is substituted with one or two substitutes selected from a group given in paragraph 1, (iii) C2-C8 alkenyl group which can be substituted with a phenyl group, or (iv) phenyl group, naphthyl group, thienyl group, pyrrolyl group, pyrazolyl group, pyridyl group, furanyl group, benzothienyl group, isoquinolinyl group, isoxazolyl group, thiazolyl group, benzothiadiazolyl group, benzoxadiazolyl group, phenyl group, condensed with a 5-7-member saturated hydrocarbon ring which can contain one or two oxygen atoms as ring members, uracyl group or tetrahydroisoquinolinyl group, where each of the said groups can be substituted with 1-5 substitutes selected from a group given in paragraph 1, provided that when Ar is a group of formula 5, which can be substituted with a C1-C6 alkyl group, R5 is not a C1-C10 alkyl group, and the formula (I) compound is not 5-(3,5-dichlorophenylthio)-4-isopropyl-2-methane-sulfonylaminomethyl-1-methyl-1H-imidazole or 5-(3,5-dichlorophenylthio)-4-isopropyl-1-methyl-2-p-toluene-sulfonylaminomethyl-1H-imidazole. The invention also relates to a pharmaceutical composition based on the formula I compound and to formula II compounds, radicals of which are defined in the formula of invention.

EFFECT: obtaining novel compounds with inhibitory effect on the bond between S1P and its Edg-1 (SIP1) receptor.

32 cl, 43 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel benzene derivatives of general formula (I) or salts thereof: [Chem. 12]

(Symbols in the given formula have the following values X1:-NR12-C(=O)- or -C(=O)-NR12-, X2 : -NR13 -C(=O)-, Ring A is a 6-member ring, if necessary having 1 or 2 double bonds and if necessary having 1-3 heteroatoms selected from N, O, Ring B is a benzene ring or a 6-member heteroaryl ring having 1-3 heteroatoms selected from N, R is a hydrogen atom or a residue of β-D- glucopyranoside uronic acid; R1-R8 are identical or different and each denotes a hydrogen atom, a halogen atom, -O-(lower alkyl), R9-R11 are identical or different and each denotes a hydrogen atom, lower alkyl, -O-(lower alkyl), -(CH2)n-N(lower alkyl)2, -(CH2)n-NH(lower alkyl), -(CH2)n-N(lower alkyl) (if necessary substituted with -C=O; a 6-member heterocycle having 1-3 heteroatoms selected from N, S, O) -(CH2)n-(C=O)-N(lower alkyl)2, -(CH2)n-(C-O)-N(lower alkyl) (if necessary substituted with -C=O, alkyl, a 6-member heterocycle having 1-3 heteroatoms selected from N) -(CH2)n- if necessary substituted with alkyl, -COCH3, -SO2CH3, -COOCH3, -C=O, CF3, -OCH3, OH, halogen; 5-7-member heterocycle having 1-3 heteroatoms selected from N, S, O), -(CH2)n-O- (if necessary substituted with alkyl; 6-member heterocycle having 1-3 heteroatoms selected from N), n is an integer from 0 to 3, R12 and R13 denote a hydrogen atom, provided that in R1-R11, when two lower alkyls are bonded to a nitrogen atom, they can together form a 3-8-member nitrogen-containing heterocycle.) The invention also relates to benzene derivatives of general formula (II), to a pharmaceutical composition, as well as to use of the said compounds.

EFFECT: obtaining novel biologically active compounds which are active as inhibitors of activated blood-coagulation factor X.

16 cl, 365 ex, 42 tbl

FIELD: chemistry.

SUBSTANCE: formula (I) compounds, radicals of which are defined in the formula of invention, are described. A pharmaceutical composition containing formula (I) compounds is also described.

EFFECT: obtaining compounds which have inhibitory activity on protein kinase MEK1/2 and are meant for use as a therapeutically active substance which is useful for treating MEK1/2 mediated diseases.

13 cl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to compounds of general formula I and to their pharmaceutically acceptable acid addition salts. The compounds of the present invention exhibit the properties of glycine carrier 1 (GlyT-1) inhibitors. In formula I , R1 represents -OR1', -SR1' or morpholinyl; R1' represents lower alkyl, halogen-substituted lower alkyl, or represents -(CH2)n-lower cycloalkyl; R2 represents -S(O)2-lower alkyl, -S(O)2NH-lower alkyl, NO2 or CN; X1 represents CR3 or N; X2 represents CR3' or N; R3/R3' independently represent hydrogen, halogen, lower alkyl, CN, NO2, -S(O)2-phenyl, -S(O)2-lower alkyl, -S(O)2-pyridine-2, 3 or 4-yl, phenyl optionally substituted with one or two substitutes specified from the group consisting of NO2 or halogen, or represent halogen-substituted lower alkyl, or represent -C(O)-lower alkyl; n has a value of 0, 1 or 2. The invention also concerns a drug containing one or more compounds of the invention and pharmaceutically appropriate excipients.

EFFECT: preparation of the compounds exhibiting the properties of glycine carrier inhibitors.

20 cl, 1 tbl, 133 ex

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