METHOD FOR FLUORESCENCE LABELING OF BIOLOGICAL MATERIALS, THERMALLY REMOVABLE FLUORESCENT LABELS FOR THIS METHOD, AND METHODS OF THEIR PREPARATION AND USE

Abstract

A thermosensitive fluorescent label for labeling of biological material with fluorescent dyes used for biological, chemical or physical analyzes. A method for fluorescence labeling of nucleosides, nucleotides and oligonucleotides with those fluorescent dyes wherein a moiety of general formula 1 or 2 or 3 or 4 with the double bond configuration is attached to a nucleoside, nucleotide or oligonucleotide.

Claims

1. A method for fluorescence labeling of nucleosides, nucleotides and oligonucleotides with fluorescent dyes wherein a moiety of general formula 1 or 2 or 3 or 4 with the double bond configuration E is attached to a nucleoside, nucleotide or oligonucleotide ##STR00023## wherein: n is 1 or 2 —marks the bonding site with the nucleoside or nucleotide or oligonucleotide R.sub.1 means: hydrogen, methyl, amino group, carbonyl, benzyl, naphthyl, naphthylmethyl, phenyl, benzyl, quinolinemethyl, benzyl substituted with one or more the same or different substituents: chlorine, fluorine, methyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, saturated alkyl containing from 2 to 8 carbon atoms substituted with a phenyl, amino group, hydroxyl group or simultaneously phenyl and hydroxyl group or phenyl and amino group, R.sub.2 means: hydrogen, methyl, benzyl, phenyl, naphthyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, R.sub.3 are the same or different and represent hydrogen, methyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, amino group, nitro group, azido group, a group of general formula 5 ##STR00024## R.sub.4 means carbon or nitrogen R.sub.5 and R.sub.6 are the same or different and represent hydrogen, methyl, benzyl, phenyl, naphthyl, halogen (F, Cl, Br, I), hydroxyl, amino group, nitro group, carboxyl, saturated or unsaturated alkyl containing 2 to 8 carbon atoms and one double bond, saturated or phenyl substituted unsaturated alkyl containing 2 to 8 carbon atoms and one double bond, in a chemical reaction of a free primary hydroxyl group of the nucleoside, nucleotide or oligonucleotide with a compound of general formula 6 or 7 or 8 or 9 ##STR00025## wherein A represents a group of general formula 10 or 11 ##STR00026## wherein n and substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 have the same meanings as given above, with the compounds of the formulas 6 and 7 being obtained directly in the reaction medium as reaction products between an alcohol of general formula 12 or 13 ##STR00027## wherein n and substituents R.sub.1, R.sub.2, R.sub.3, EC, R.sub.5, and R.sub.6 have the same meanings as above, with a carbonyldimidazole of general formula 14 or a carbonyldi(1,2, 4-triazole) of general formula 15 ##STR00028## forming an intermediate of general formula 6 or 7, with the compound of formula 8 being added into the reaction medium or being obtained directly in the reaction medium by reacting an alcohol of general formula 12 or 13 wherein n and substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 have the same meaning as above, with N,N,N′,N′-bis(diisopropylamino)chlorophosphine of formula 16, ##STR00029## whereat the reaction proceeds in two steps, with first step in which an alcohol of general formula 12 or 13 reacts with N,N,N′,N′-bis(diisopropylamino)chlorophosphine and the second step in which 1-H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole is added in order to enable further metathesis resulting in a product that reveals rests A introduced in reaction with the alcohol of formula 12 or 13, and whereat the compound of formula 9 is injected into the reaction medium or is obtained directly in the reaction medium by reacting an alcohol of formula 12 or 13, wherein n and substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 have the same meaning as above, with 2-cyanoethyl-N,N,N′,N′-bis(diisopropylamino)phosphine of formula 17 ##STR00030## in the presence of 1H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole, wherein after the reaction between a nucleoside, nucleotide or oligonucleotide and the intermediate of formula 8 or 9 the reaction product is subjected to standard agents used in the oxidation of phosphorus (III) compounds, whereat in the case when CSO is used, methylamine or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or concentrated aqueous ammonia solution is added after the oxidation step is completed.

2. The method according to claim 1 wherein all reactions are carried out at a temperature not higher than 30° C.

3. The method according to claim 1 wherein the reaction between alcohol of formula 12 or 13 and carbonyldiimidazole (14) or karbonyldi(1,2, 4-triazole) (15) is carried out in an anhydrous polar aprotic organic solvent selected from the group consisting of acetonitrile, dimethyl sulfoxide, dimethylformamide, aliphatic ethers, in an inert atmosphere, wherein carbonyldimidazole or carbonylditriazole is used in a proportion of 1 to 1.5 equivalent, relative to the alcohol of formula 12 or 13.

4. The method according to claim 1 wherein after the reaction between alcohol of formula 12 or 13 and carbonyldiimidazole (14) or carbonyldi(1,2, 4-triazole) (15) into the reaction mixture a nucleoside, nucleotide or oligonucleotide is added in a ratio of 1-2 eq. relative to the alcohol of general formula 12 or 13.

5. The method according to claim 4 wherein the reaction is carried out with an addition of a non-nucleophilic organic base selected from the group consisting of guanidine derivatives having the same or different substituents selected from methyl, ethyl, isopropyl; in an amount of 0.3-7.8 eq., relative to the alcohol of general formula 12 or 13.

6. The method according to claim 1 wherein the reaction between the alcohol of formula 12 or 13 and N,N,N′,N′-bis(diisopropylamino)chlorophosphine (16) is carried out in anhydrous acetonitrile or methylene chloride in an atmosphere of inert gas, using N,N,N′,N′-bis(diisopropylamino)chlorophosphine (16) in an amount of 0.4 to 0.6 eq., relative to the alcohol 12 or 13, wherein the reaction is carried out in two steps whereat in the first step, N,N,N′,N′-bis(diisopropylamino)chlorophosphine reacts with one molecule of alcohol of formula 12 or 13 and then in the second step 1-H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole is added in an amount of 0.3 to 0.8 eq. relative to the alcohol of formula 12 or 13 as an activator which initiates the reaction with a second alcohol molecule of formula 12 or 13 and the reaction is continued until completion.

7. The method according to claim 1 wherein the reaction between the alcohol 12 or 13 and 2-cyanoethyl-N,N,N′,N′-bis(diisopropylamino)phosphine (17) is carried out in acetonitrile or methylene chloride under an inert gas atmosphere, in the presence of 1H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole used in an amount of 0.5 to 1 eq. and wherein 2-cyanoethyl-N,N,N′,N′-bis(diisopropylamino)phosphine (17) is used in an amount of 0.8 to 1.1 eq. relative to the alcohol.

8. The method according to claim 1 wherein the proportion of the mixture of compound 8 or 9 and 1H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole, relative to the nucleoside, nucleotide or oligonucleotide for reactions carried out in the liquid phase is 2 eq. to 1 eq.

9. The method according to claim 1 wherein the proportion of the mixture of compounds 8 or 9 and 1H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole relative to the nucleoside, nucleotide or oligonucleotide for solid-phase reactions is 20 eq. to 1 eq.

10. A method of thermal detachment of fluorescent labels from labeled nucleosides, nucleotides or oligonucleotides labeled according to a method revealed in claim 1, wherein the nucleoside, nucleotide or oligonucleotide labeled with a thermally removable label of formula 1 or 2 or 3 or 4 is dissolved in a phosphate buffer (pH in the range from 6.86 to 7.2) or a mixture of phosphate buffer and acetonitrile, and heated to a temperature in the range of 30-90° C.

11. A method of monitoring the progress of thermal detachment of fluorescent labels from a labeled nucleoside, nucleotide or oligonucleotide wherein a difference in fluorescence intensity at the wavelength range corresponding to the label emission is determined between the fluorescence of a dissolved heated sample of a labeled nucleoside or nucleotide or oligonucleotide and a respective sample at lower temperature, wherein both samples are measured at the same time intervals and then the data obtained are analyzed numerically.

12. Pyridin-2-yl-vinylopyridine derivatives of general formula 12 or 13 ##STR00031## wherein: n is 1 or 2 R.sub.1 means: hydrogen, methyl, amino group, carbonyl, benzyl, naphthyl, naphthylmethyl, phenyl, benzyl, quinolinemethyl, benzyl substituted with one or more the same or different substituents: chlorine, fluorine, methyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, saturated alkyl containing from 2 to 8 carbon atoms substituted with a phenyl, amino group, hydroxyl group or simultaneously phenyl and hydroxyl group or phenyl and amino group, R.sub.2 means: hydrogen, methyl, benzyl, phenyl, naphthyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, R.sub.3 are the same or different and represent hydrogen, methyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, amino group, nitro group, azido group, a group of general formula 5, ##STR00032## R.sub.4 means carbon or nitrogen, R.sub.5 and R.sub.6 are the same or different and represent hydrogen, methyl, benzyl, phenyl, naphthyl, halogen (F, Cl, Br, I), hydroxyl, amino group, nitro group, carboxyl, saturated or unsaturated alkyl containing 2 to 8 carbon atoms and one double bond, saturated or phenyl substituted unsaturated alkyl containing 2 to 8 carbon atoms and one double bond.

13. A preparation method of compounds of general formula 12 or 13, wherein n, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 have the same meaning as given above, consisting in a reaction between a compound of formula 26 or formula 27 ##STR00033## wherein R.sub.3, R.sub.4, R.sub.5, and R.sub.6 have the same above meaning as given above, with an aminoalcohol of general formula 28 ##STR00034## wherein n, R.sub.1, and R.sub.2 have the same meaning as given above, in the presence of a tertiary organic amine in an anhydrous aprotic polar organic solvent.

14. A preparation method of pyridin-2-yl-vinylpyridine derivatives of general formula 12 or 13 ##STR00035## wherein: n is 1 or 2 R.sub.4 means: hydrogen, methyl, naphthyl, naphthylmethyl, phenyl, benzyl, quinolinemethyl, benzyl substituted with one or more the same or different substituents: chlorine, fluorine, methyl, R.sub.2 means: hydrogen, methyl, benzyl, phenyl, naphthyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, R.sub.3 are the same or different and represent hydrogen, methyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, amino group, nitro group, azido group, a group of general formula 5, ##STR00036## R.sub.4 means carbon or nitrogen, R.sub.5 and R.sub.6 are the same or different and represent hydrogen, methyl, benzyl, phenyl, naphthyl, halogen (F, Cl, Br, I), hydroxyl, amino group, nitro group, carboxyl, saturated or unsaturated alkyl containing 2 to 8 carbon atoms and one double bond, saturated or phenyl substituted unsaturated alkyl containing 2 to 8 carbon atoms and one double bond, consists in a reaction between a compound of general formula 29 ##STR00037## wherein R.sub.1, R.sub.2, and R.sub.3 have the same meaning as given above and X is halogen (Br, Cl, I) with a compound of general formula 30 or 31 ##STR00038## wherein R.sub.5 and R.sub.6 have the same meanings as given above and R.sub.t means carbon or nitrogen in the presence of palladium (II) salts, wherein the reaction is carried out in a polar aprotic organic solvent selected from the group consisting of acetonitrile, dimethyl sulfoxide, dimethylformamide, aliphatic ethers.

15. The method according to claim 14 wherein into the polar aprotic organic solvent, a palladium (II) salt, in an amount of 0.01 eq. up to 0.1 eq. and a phosphine, in an amount of 0.03 eq. to 0.3, are added and subsequently a compound 30 or 31 in an amount of 0.6 eq. to 1.5 eq., or 0.6 eq. to 1.5 eq., respectively, a compound of formula 29, ##STR00039## and a base selected from the group consisting of tBuOLi, tBuOK, CS.sub.2CO.sub.3, K.sub.2CO.sub.3, TEA, DBU, KOH, NBu.sub.4OH, NaOAc, K.sub.3PO.sub.4, in an amount of 1.1 eq. up to 1.8 are added.

16. The method according to claim 15 wherein a ionic liquid, tetrabutylammonium acetate (TBAA), 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6], 1-butyl-3-methylimidazolium bromide [BMIM][Br], tetraheptylammonium bromide (THeptAB) in an amount of 0.03 eq. to 0.1 eq., relative to the palladium salt is applied.

Description

EXAMPLE 1

Conversion of 2-(pyridine-2-yl)vinyl]pyridine Amino Alcohols into Imidazole-Carbonyl Esters

[0101] (E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino) ethan-1-ol (1 eq., 0.1 mmol) was dissolved in anhydrous acetonitrile (2 ml) and placed in a flask containing a magnetic stir bar. To this solution, 1′1′-carbonyldiimidazole (2 eq., 0.24 mmol) was added and the reaction was carried out for 30 minutes at room temperature and its course was monitored by TLC (ethyl acetate/n-hexane 9/1). After 30 minutes, the active form of imidazole-carbonyl ester was obtained, which was further used in the next example without any isolation

EXAMPLE 2

Labeling of 3′-O-Acetylthymidine with an Imidazole-Carbonyl Form of a Fluorescent Dye

[0102] To the solution of the imidazole-carbonylderivative obtained in the previous example 3′-O-acetylthymidine (1 eq; 0.1 mmol) and 1,1,3,3-tetramethylguanidine (1.6 eq., 0.16 mmol) were added. The reaction mixture was stirred for 2 hours at room temperature. The course of the reaction was monitored by TLC (dichloromethane/methanol 95/5 v/v). The solvent was evaporated under reduced pressure and the thus obtained residue was purified by silica gel column chromatography (dichloromethane (DCM)/MeOH; 0-5% MeOH) to obtain a pure product which was characterized by spectroscopic methods.

[0103] .sup.1H NMR (400 MHz; Chloroform-d) δ 8.66-8.55 (m; 1H); 7.70-7.65 (m; 1H); 7.63 (d; J=5.3 Hz; 1H); 7.48 (d; J=8.6 Hz; 1H); 7.44 (d; J=8.0 Hz; 2H); 7.35 (d; J=2.2 Hz; 2H); 7.14 (dd; J=7.5; 4.9 Hz; 1H); 6.71 (d; J=7.3 Hz; 1H); 6.45 (d; J=8.5 Hz; 1H); 6.35 (dd; J=8.8; 5.7 Hz; 1H); 5.16 (dd; J=5.2; 3.2 Hz; 1H); 4.52 (dt; J=11.5; 5.9 Hz; 1H); 4.42 (q; J=4.6; 3.7 Hz; 1H); 4.39 (d; J=2.9 Hz; 1H); 4.33 (dd; J=11.8; 2.8 Hz; 1H); 4.17 (q; J=2.7 Hz; 1H); 4.02 (tq; J=14.7; 8.8; 7.4 Hz; 2H); 3.10 (s; 3H); 2.40-2.29 (m; 1H); 2.22-2.10 (m; 2H); 2.09 (s; 3H); 1.88 (s; 3H),

[0104] .sup.13C NMR (101 MHz; Chloroform-d) δ 169.89; 163.00; 157.25; 155.08; 154.11; 152.11; 149.93; 149.18; 137.44; 136.01; 134.34; 132.11; 130.24; 127.82; 122.10; 121.78; 112.34; 111.15; 104.99; 84.04; 81.62; 74.07; 66.76; 65.86; 47.87; 36.73; 36.64; 20.39; 12.13.

EXAMPLE 3

Conversion of 2-(pyridine-2-yl)vinyl]pyridinyl-aminoalcohols into [1,2,4]-triazole-carbonyl Esters

[0105] (E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino) ethan-1-ol (1 eq., 0.1 mmol) was dissolved in anhydrous acetonitrile (2 ml) and placed in a flask containing a magnetic stir bar. To this solution, 1′1′-carbonyldi[1,2,4]triazole (2 eq., 0.24 mmol) was added and the reaction was carried out for 10 minutes at room temperature and its course was monitored by TLC (ethyl acetate/n-hexane 9/1). After 10 minutes, active [1,2,4] triazole-carbonyl ester was obtained, which was further used in the next example without any isolation

EXAMPLE 4

Labeling of 3′-O-acetylthymidine with [1,2,4] triazole-carbonyl Ester of a Fluorescent Dye

[0106] To the solution of [1,2,4]triazole-carbonyl ester obtained in the previous example 3′-O-acetylthymidine (1 eq; 0.1 mmol) was added. The reaction mixture was stirred for 0.5 hours at room temperature. The course of the reaction was monitored by TLC (dichloromethane/methanol 95/5 v/v). The solvent was evaporated under reduced pressure and the thus obtained residue was purified by silica gel column chromatography (dichloromethane (DCM)/MeOH; 0-5% MeOH) to obtain a pure product which was characterized by spectroscopic methods. Because the product obtained is the same compound as in Example 2, the spectroscopic characteristics are given above in Example 2.

EXAMPLE 5

Label Detachment from 3′-O-acetyl-5′-(2-{methyl-[6-(2-pyridin-2-yl-vinyl)-pyridin-2-yl)-aminopropyloxycarbonylthymidine

[0107] The labeled nucleoside obtained in Example 2 was dissolved in phosphate buffer pH 6.86, in 5 portions of 1 mg each, and placed in sealable vials. Then, each sample was heated under shaking to the following temperatures: 30, 40, 50, 60 and 70° C. for the same period of time of 2 hours. Then the samples were quickly cooled down (in dry ice), from each sample an aliquot was taken and analyzed using a HPLC setup equipped with a DAD detector (diode array detector for the spectral range of 220-700 nm) and FLD detector (λ.sub.abs=370 nm, λ.sub.t=476 nm), using a Synergy Fusion—RP 80 Å column (together with Phenomenex pre-column), measuring 150×4.6 mm, using the gradient method: buffer A (0.01M triethylammonium acetate)/buffer B (0.01M triethylammonium acetate in 40% acetonitrile), flow rate 1 ml/min, analysis duration 30 min. The results of the kinetics of the label removal are presented on the graph: decay rate of the labeled species in function of temperature—FIG. 5. Complete removal of the label occurs at 70° C.

EXAMPLE 6

[0108] (E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)propan-1-ol (1 eq., 0.13 mmol, 35 mg) was dissolved in anhydrous acetonitrile (2 ml) and placed in a flask containing a magnetic stir bar together with 1′1′-carbonyldiimidazole (1 eq., 0.13 mmol, 38.09 mg). The reaction was carried out for 30 minutes at room temperature and was monitored by TLC (ethyl acetate/n-hexane 9/1). After 30 minutes, the active carbamate form of (E)-2-(methyl{6-[2-(pyridin-2-yl) vinyl] pyridin-2-yl} amino) propan-1-ol was obtained and used in the following example without any isolation.

EXAMPLE 7

[0109] To the solution of the carbamate derivative of (E)-2-(methyl{6-[2-(pyridin-2-yl) vinyl] pyridin-2-yl} amino)propan-1-ol obtained in the previous example 6 3′-O-acetyl-thymidine (1 eq.; 0.13 mmol, 38.09 mg) and 1,1,3,3-tetramethylguanidine (1.6 eq., 0.21 mmol, 20 uL) were added. The reaction mixture was stirred for 2 hours at room temperature. The course of the reaction was monitored by TLC (DCM/methanol 95/5 v/v). The solvent was evaporated under reduced pressure and the thus obtained residue was purified by silica gel column chromatography (DCM/MeOH; 0-5% MeOH) to obtain a pure product (19 mg) which was characterized by spectroscopic methods.

[0110] .sup.1H NMR (500 MHz, Chloroform-d) δ 8.67-8.57 (d, J=4.01 1H); 7.70-7.65 (m, 1H); 7.63 (d, J=5.3 Hz, 1H); 7.48 (d, J=8.6 Hz, 1H); 7.44 (d, J=8.0 Hz, 2H); 7.35 (d, J=2.2 Hz, 2H); 7.14 (dd, J=7.5, 4.9 Hz, 1H); 6.71 (d, J=7.3 Hz, 1H); 6.35 (d, J=8.5 Hz, 1H); 6.35 (dd, J=8.8, 5.7 Hz, 1H); 5.16 (dd, J=5.2, 3.2 Hz, 1H); 4.52 (dt, J=11.5, 5.9 Hz, 1H); 4.42 (q, J=4.6, 3.7 Hz, 1H); 4.39 (d, J=2.9 Hz, 1H); 4.33 (dd, J=11.8, 2.8 Hz, 1H); 4.17 (q, J=2.7 Hz, 1H); 4.02 (tq, J=14.7, 8.8, 7.4 Hz, 2H); 3.10 (s, 3H); 2.43-2.39 (m, 1H); 2.24-2.19 (m, 2H); 2.15-2.08 (m, 2H); 2.04 (s, 3H); 1.82 (s, 3H).

[0111] .sup.13C NMR (101 MHz, Chloroform-d) δ 170.40; 163.22; 157.86; 155.44; 154.64; 152.11; 149.93; 149.21; 137.78; 136.82; 134.87; 132.11; 130.24; 128.30; 122.10; 121.78; 112.57; 111.63; 105.60; 84.52; 81.62; 74.07; 66.76; 65.86; 47.87; 36.73; 36.64; 25.16; 20.39; 12.58.

EXAMPLE 8

Label Detachment from 3′-O-acetyl-5′-(2-{methyl-[6-(2-pyridin-2-yl-vinyl)-pyridin-2-yl)-aminopropyloxycarbonylthymidine

[0112] The labeled nucleoside obtained in Example 5 (1 mg) was dissolved in phosphate buffer pH 6.86 (0.5 mL, pH=7). The sample was heated (90° C.) and then in time intervals of 1 hour 50 μm (0.05 μL) aliquots were taken and quickly cooled to 0° C. After collecting 4 aliquots, TLC analysis showing the progress of the detachment reaction was performed. The heating was carried out for 24 hours after which a complete decay of the starting compound was determined. The reaction product was isolated from the TLC plates. A mass analysis revealed a molecular mass that corresponds to the bicyclic six-membered product 15.

[0113] MS-ESI: C.sub.16H.sub.18N.sub.3.sup.+ calculated 252.1495 [M].sup.+, observed 252 [M].sup.+;

EXAMPLE 9

Label Detachment from 3′-O-acetyl-5′-(2-{methyl-[6-(2-pyridin-2-yl-vinyl)-pyridin-2-yl)-aminopropyloxycarbonylthymidine

[0114] The labeled nucleoside obtained in Example 2 was dissolved in phosphate buffer pH 6.86 in a glass vial with a screw cap. Then, the sample was continuously shaken and heated for 45 minutes at 90° C. for 45 minutes. After 5, 15, 30, and 45 minutes, aliquots were taken from the reacting mixture. The aliquots were quickly cooled down and analyzed using a HPLC setup equipped with a DAD detector (diode array detector for the spectral range of 220-700 nm) and FLD detector (λ.sub.abs.=370 nm, λ.sub.t=476 nm), using a Synergy Fusion—RP 80 Å column (together with a Phenomenex pre-column), measuring 150×4.6 mm, using the gradient method: buffer A (0.01M triethylammonium acetate)/buffer B (0.01M triethylammonium acetate in 40% acetonitrile), flow rate 1 ml/min, analysis duration 30 min. The results of the kinetics of the label removal are presented on the graph: the decay rate of the labeled species plotted versus temperature—FIG. 6. Complete removal of the label occurs after 45 minutes.

EXAMPLE 10

Formation of a Cyclic Dye Species as a Result of the Herein Developed Label Detachment Method from 3′-O-acetyl-5′-(2-{methyl-[6-(2-pyridin-2-yl-vinyl)-pyridin-2-yl)-aminoethyloxycarbonylthymidine

[0115] The labeled nucleoside obtained in Example 2 was dissolved in phosphate buffer pH 6.86 in a glass vial with a screw cap. The sample was continuously shaken and heated for 45 minutes at 90° C. for 45 minutes until complete decay of the labeled biomolecule which was determined by means of a thin layer plate chromatography (TLC). The solvent was evaporated under reduced pressure and the thus obtained residue was purified by column chromatography on a silica gel column.

[0116] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.68 (dd, J=5.1, 1.7 Hz, 1H), 8.04 (dd, J=8.8, 7.6 Hz, 1H), 7.91 (td, J=7.7, 1.8 Hz, 1H), 7.82 (d, J=15.7 Hz, 1H), 7.72 (dt, J=7.9, 1.1 Hz, 1H), 7.59 (d, J=15.7 Hz, 1H), 7.48-7.44 (m, 1H), 7.43-7.39 (m, 1H), 7.12 (d, J=8.8 Hz, 1H), 4.80 (dd, J=10.6, 9.0 Hz, 2H), 3.95 (dd, J=10.6, 9.1 Hz, 2H), 3.10 (s, 3H), .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ 172.75; 171.48; 154.94; 152.61; 150.02; 145.44; 144.12; 138.44; 137.39; 124.78; 120.41; 109.65; 106.01; 49.21; 48.09.

[0117] HRMS-ESI: C.sub.15H.sub.16N.sub.3 calculated 238.1344 [M+H].sup.+. found 238.1343 [M+H].sup.+;

EXAMPLE 11

Conversion of (E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol into Carbamate

[0118] (E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol (1 eq., 25 mg, 0.098 mmol) was dissolved in anhydrous acetonitrile (2 ml) and placed in a flask containing a magnetic stir bar. Three portions of 1′1′-carbonyldiimidazole were added to the solution three times; each portion in an amount of 1.2 eq.; at 15 minute intervals (in total 3.6 eq; 57.10 mg; 0.36 mmol) and the reaction was carried out for a total of 1.5 hours at room temperature and its course was monitored by TLC (ethyl acetate/n-hexane 9/1). After 1.5 hours, the carbamate derivative of the aminoalcohol was obtained, which was further used in the next Example without any isolation.

EXAMPLE 12

Labeling of 3′-O-acetyl-N2-isobutyrylodeoxyguanosine with a Carbamate Derivative of a Fluorescent Dye

[0119] To the solution of carbamate aminoalcohol derivative obtained in Example 11 3′-O-acetyl-N2-isobutyrylodeoxyguanosine (1 eq.; 0.1 mmol) and 1,1,3,3-tetramethylguanidine (7.8 eq., 0.78 mmol) were added. The reaction mixture was stirred for 2 hours at room temperature. The course of the reaction was monitored by TLC (DCM/methanol 95/5 v/v). The solvent was evaporated under reduced pressure and the thus obtained residue was purified by silica gel column chromatography (DCM/MeOH; 0-5% MeOH) to obtain pure product with a yield of 30%.

EXAMPLE 13

Label detachment from 5′-[6-(2-pyridin-2-yl-vinyl)-pyridin-2-yl)-aminoethyloxycarbonyl N2-isobutyrylguanosine

[0120] The labeled N2-isobutyrylguanosine obtained according to the method described in Example 12 was dissolved in phosphate buffer pH 6.86, in 5 portions of 1 mg each, and placed in sealed vials. Then, each vial was heated under shaking to the following temperatures: 30, 40, 50, 60 and 70° C. for the same period of time of 2 hours. Then the samples were quickly cooled down (in dry ice) and analyzed using a HPLC setup equipped with a DAD detector (diode array detector for the spectral range of 220-700 nm) and FLD detector (λ.sub.abs=370 nm, λ.sub.n=476 nm), using a Synergy Fusion—RP 80 Å column (together with Phenomenex pre-column), measuring 150×4.6 mm, using the gradient method: buffer A (0.01 M triethylammonium acetate)/buffer B (0.01 M triethylammonium acetate in 40% acetonitrile), flow rate 1 ml/min, analysis duration 30 min. The results of the kinetics of the label removal are presented on the graph: the decay rate of the labeled species plotted versus temperature—FIG. 7. Complete removal of the label occurs at 70° C.

EXAMPLE 14

Conversion of 2-(pyridine-2-yl)vinyl]pyridinyl-aminoalcohols into Phosphoramidates

[0121] 3(E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol (80 mg; 0.313 mmol) was freeze-dried from benzene and dried under vacuum generated by an oil pump for 12 hours. The compound was then placed in a small round bottom flask flushed with argon and sealed with a septum. 1 ml of anhydrous dichloromethane was added to the flask to obtain a clear yellow solution. 40.9 mL (0.235 mmol) of N,N-diisopropylethylamine (DIPEA) was added in one portion and then the solution was cooled to about −5° C. To this clear solution, 41.75 mg (0.156 mmol) of N,N,N′,N′-bis(diisopropylamino)chlorophosphine was added. At the moment when a TLC analysis done in the developing system: hexane/DCM/triethylamine; v/v/v/; 6/3/1 revealed a half decay of the staring compound, 15 mg (0.078 mmol) of 5-benzylmercaptotetrazole (BMT) was added until complete disappearance of the starting compound. The solution was evaporated on a rotary evaporator and the product was purified by column chromatography (silica gel) applying a gradient elution in the system: DCM/hexane/triethylamine; v/v/v; from 1/8/1 to 2/7/1. Fractions containing the product were combined and evaporated on a rotary evaporator and then freeze-dried from benzene. Thus, 30 mg of bis[2-(pyridin-2-yl)vinyl]pirydynolo]-N,N-diisopropylphosphoramidite was obtained as yellow oil; with a yield of 30%.

[0122] The product was characterized by .sup.31P NMR spectroscopy

[0123] .sup.31P NMR (400 MHz; benzene): 146.46 (s).

EXAMPLE 15

Conversion of 2-(pyridine-2-yl)vinyl]pyridinyl-aminoalcohols into Phosphoramidates

[0124] (E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol (80 mg; 0.313 mmol) was freeze-dried from benzene and dried under vacuum generated by an oil pump for 12 hours. This compound was then placed in a small round bottom flask flushed with argon and sealed with a rubber cap. 1 ml of anhydrous dichloromethane was added to the flask to obtain a clear colorless solution. Then, 58.66 mL (1.3 eq; 0.177 mmol) of 2-cyanoethyl-N,N,N′,N′-bis(diisopropylamino)phosphine was added in one portion and the resulting solution was left for 5 minutes. Into this clear solution 5-benzyl-mercaptotetrazole (BMT) was added in portions of approximately 4.8 mg for one hour (in total 0.9 eq; 0.128 mmol, 23.7 mg). The resulting solution was left at room temperature for 1 hour. Then 70 μL of N,N-diisopropylamine (DIPA) was added to the solution in one portion and the whole was evaporated on a rotary evaporator. The product was purified by column chromatography (silica gel) using a gradient elution in the system DCM:hexane:triethylamine; v:v:v; 1:8:1 to 4:5:1. Fractions containing the product were combined and evaporated on a rotary evaporator; the resulting residue was immediately freeze-dried from benzene. Thus, approx. 25 mg of 2-(pyridin-2-yl)vinyl]pyridinyl-2-cyanoethyl-N,N-diizopropylphosphoramidite was obtained as fine powder.

[0125] The product was characterized by .sup.31P NMR spectroscopy

[0126] .sup.31P NMR (400 MHz; benzene-d6): 146.46 (s)

EXAMPLE 16

Oligomer Labeling

[0127] A 12-mer oligomer with the sequence 5′-TTT TTT TTT TTT TTT-3′ was synthesized on a solid phase using a Q-dT silicate substrate (Glen Research) and commercial thymidine phosphoramidite. The synthesis was carried out using an automated DNA synthesizer. For each nucleotide being attached, 4 reaction steps (detritylation, condensation, capping, and oxidation) were done repeatedly. For this purpose, the Q-dT fixed-bed columns were filled with respective solutions using for each step the following conditions: [0128] 1 detritylation: 3% solution of dichloroacetic acid in methylene chloride, reaction time 2 minutes [0129] 2 condensation: 0.1 molar solution of dT phosphoramidite in acetonitrile mixed in equal proportions with a 0.1 molar solution of 5-benzyl-mercaptotetrazole (BMT), reaction time 3 minutes [0130] 3. capping: a mixture of two solutions (A and B) in equal proportions, reaction time 1 minute. Solution A—10% solution of 1-N-methylimidazole in tetrahydrofuran. Solution B—10% solution of acetic anhydride in anhydrous tetrahydrofuran. [0131] 4. oxidation: 0.05 molar solution of iodine in aqueous pyridine, reaction time 1 minute.

[0132] After each such step, the column is rinsed with anhydrous acetonitrile and flushed with dry argon.

[0133] After attachment of the last oligonucleotide, the final dimethoxytrityl group was removed (detritylation) and the condensation step was carried out using a mixture of a dye phosphoramidite derivative solution (phosphoramidite derivative of the dye (12 mg) dissolved in 200 μl of dry ACN) and 0.1 molar BMT solution in equal proportions. The reaction time was 10 minutes. Subsequently, the oxidation step was carried out as described above.

[0134] After completion of the oxidation step, the column was washed with acetonitrile, flushed with dry argon, and the medium bed was placed into a concentrated ammonia solution at 22° C. for 2 minutes. The silicate support was then removed from the solution by decantation and the labeled oligomer in ammonia solution was applied at the top of a LGC MicroPure II column. Then the column was eluted with the following mixtures with each fraction being collected into separate tubes: [0135] 1. 97% 1M triethylammonium acetate (TEAA)+3% MeCN v/v [0136] 2. ultra-pure water with a conductivity of 18.2 MΩ.Math.cm at 25° C. (i.e. H.sub.2O MiliQ) [0137] 3. 20% solution of acetonitrile in MiliQ water

[0138] The labeled oligonucleotide was contained in the fraction eluted with H.sub.2O MiliQ as found spectrophotometrically (in an amount corresponding to 14 OD optical units). The oligonucleotide was evaporated without heating and was subjected to further analysis.

EXAMPLE 17

Purification of the Labeled Oligomer by HPLC

[0139] Preparation of the analyte: labeled oligonucleotide (0.4 OD) was dissolved in 500 mL of MiliQ water, mixed on a Vortex and centrifuged. OD was measured at 260 nm to determine the injection volume per chromatographic column. (OD260—0.528, Analyte volume—100 μl).

Conditions of the Analysis:

[0140] A Synergy Fusion column—RP 80 Å Phenomenex, 150×4.6 mm with pre-column gradient method, duration 30 min: [0141] flow rate 1 ml/min [0142] gradient analysis using a buffer A (0.01M triethylammonium acetate)/buffer B (0.01M triethylammonium acetate in 40% acetonitrile) system [0143] DAD diode detector 220-700 nm [0144] FLD detector (λ.sub.abs.=370 nm, λ.sub.em=476 nm)

[0145] The labeled oligomer 1, purified by high pressure HPLC chromatography under specified above conditions, was evaporated and then re-evaporated 3 times from MiliQ water (in order to completely remove the remaining triethylammonium acetate buffer) and freeze-dried. The resulting analyte was analyzed by mass spectrometry (MALDI).

[0146] Calculated mass of the labeled oligonucleotide: 3904.6258 g/mol.

[0147] Found mass of the labeled oligonucleotide: m/z 3905.561 Da.

EXAMPLE 18

[0148] Detachment of the Label Dye from the Oligomer

[0149] To remove the label from the oligonucleotide, the labeled oligonucleotide (in the amount of 10 OD) was dissolved in 1000 mL of MiliQ water, mixed on a Vortex and centrifuged.

[0150] Subsequently, OD was measured at 260 nm and the injection volume containing 6 OD of the oligomer was determined. The solution was placed in a screw cap vessel and heated for 4 hours at 90° C. After every hour, from the mixture a sample containing about 1 OD of the oligomer was taken and, after cooling (dry ice), each of them was subjected to an analysis with an HPLC set equipped with a DAD detector (diode array detector for the range 220-700 nm) and a FLD detector (λ.sub.abs.=370 nm, λ.sub.t=476 nm) using a Synergy Fusion—RP 80 Å column (together with a Phenomenex pre-column) measuring 150×4.6 mm, and using the gradient method: buffer A (0.01M triethylammonium acetate)/buffer B (0.01 M triethylammonium acetate in 40% acetonitrile), flow rate 1 ml/min, analysis time 30 min. The results of the dye removal analyzes are shown on HPLC chromatograms.

[0151] In the following: [0152] FIG. 9 shows chromatograms of unheated oligomer and oligomer heated oligomer for 2 and 4 hours, respectively, that were recorded using DAD detector, [0153] FIG. 10 shows chromatograms of unheated oligomer and the oligomer heated for 2 and 4 hours, respectively, recorded using Fluorescence detector at the excitation wavelength 370 and emission wavelength 480 nm.

TABLE-US-00002 TABLE 1 Retention time of individual compounds on the Synergy Fusion - RP 80 Å column during analyses using the HPLC kit performed under the conditions for the analyzed compounds as described above. Compound Retention time [min] Labeled oligonucleotide 14.49 Dye-free oligonucleotide 12.57 Cyclic derivative of the dye 22.64-24.52

EXAMPLE 19

Preparation of (E)-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}-aminoetan-1-ol

[0154] (E)-2-bromo-6-[2-(pyridin-2-yl)vinyl)pyridine (1.23 mmol, 320 mg) in 0.5 mL of anhydrous DMF, diisopropyl ethanolamine (1.8 mmol, 0.3 mL) and a large excess of ethanolamine (1.5 mL) were placed in a round bottom flask. The reaction was carried out in a microwave reactor (120° C., 300 W) for 3 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from dichloromethane (DCM)/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using DCM/methanol mixtures as eluents (gradient method, starting with one-component eluent (100% DCM) and ending with the mixture 95/5 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents the purified product was freeze-dried from benzene. Thus, 175 mg of (E)-{6-[2-(pyridin-2-yl)vinyl]-pyridin-2-yl}-aminoethan-1-ol was obtained as yellowish oil, with a yield of 60%.

[0155] The product was characterized by NMR spectroscopy:

[0156] .sup.1H NMR (500 MHz, Chloroform-d) δ 8.48 (dd, J=5.0, 1.8 Hz, 1H. H6), 7.52 (dd, J=7.7, 1.8 Hz, 1H, H4), 7.48 (d, J=15.7 Hz, 1H, H7), 7.39 (d, J=15.7 Hz, 1H, H8), 7.32 (d, J==7.8 Hz, 1H, H3), 7.21 (dd, J=8.3, 7.2 Hz, 1H, H11), 7.06-6.99 (m, 1H, H5), 6.58 (d, J=7.2 Hz, 1H, H10), 6.23 (d, J=8.3 Hz, 1H, H12), 5.55 (s, 1H, H17 (—OH)), 5.32 (t, J=5.7 Hz, 1H, H14 (—NH)), 3.74 (t, J=4.8 Hz, 2H, H16), 3.45 (q, J=5.1 Hz, 2H, H15)

[0157] .sup.13C NMR (126 MHz, Chloroform-d) δ 158.47 (C13), 155.05 (C2), 152.11 (C9), 149.28 (C6), 137.63 (C11), 136.44 (C4), 131.87 (C8), 130.28 (C7), 122.70 (C3), 122.20 (C5)), 113.39 (C10), 108.50 (C12), 63.26 (C16), 44.71 (C15)

[0158] ESI MS m/z; calculated: 242.1293 C.sub.14H.sub.15N.sub.3O [M+H].sup.+; found: 242.1277 [M+H].sup.+;

EXAMPLE 20

Preparation of (E)-2-(methyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}-amino)ethan-1-ol

[0159] (E)-2-bromo-6-[2-(pyridin-2-yl)vinyl)pyridine (0.61 mmol, 160 mg) in 0.5 mL of anhydrous DMF, diisopropyl ethanolamine (0.92 mmol, 159 μL), and a large excess of N-methylethanolamine (1.5 mL) were placed in a round bottom flask. The reaction was carried out in a microwave reactor (110° C., 300 W). After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/water/saturated aqueous solution of NaHCO.sub.3 (1/1/1; v/v/v). The product was then purified by silica gel column chromatography using DCM/methanol mixtures as eluents (gradient method, starting with one-component eluent (100% DCM) and ending with the mixture 95/5 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 131 mg of (E)-2-(methyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}-amino)ethan-1-ol was obtained as yellowish oil, with a yield of 70%. The product was characterized by NMR spectroscopy:

[0160] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.57 (dd, J=4.9, 1.7 Hz, 1H); 7.78 (td, J=7.7, 1.9 Hz, 1H); 7.60 (m, 1H); 7.56 (m, 1H); 7.52 (d, J=9.1 Hz, 1H); 7.50-7.46 (m, 1H); 7.26 (ddd, J=7.4, 4.7, 1.0 Hz, 1H); 6.75 (d, J=7.1 Hz, 1H); 6.59 (d, J=8.5 Hz, 1H); 4.73 (t, J=4.8 Hz, 1H); 3.63 (m, 4H); 3.10 (s, 3H)

[0161] .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 157.77; 154.83; 151.88; 149.51; 137.69; 136.77; 132.49; 130.20; 122.55; 122.48; 111.65; 105.93; 58.58; 51.88; 36.73

EXAMPLE 21

Preparation of (E)-2-(benzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol

[0162] (E)-2-bromo-6-[2-(pyridin-2-yl)vinyl)pyridine (0.76 mmol, 200 mg) in 0.5 mL DMF, diisopropylethanolamine (1.14 mmol, 0.19 mL), and a large excess of 2-(benzylamino)ethanol (1.5 mL) were placed in a round bottom flask. The reaction was carried out in a microwave reactor (120° C., 300 W) for 5 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from dichloromethane (DCM)/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 87 mg of (E)-2-(benzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol was obtained as yellowish oil, with a yield of 34%. The product was characterized by NMR spectroscopy.

EXAMPLE 22

Synthesis of (E)-2-(methyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl]amino)-1-phenylethane-1-ol

[0163] 100 mg (1 eq., 0.383 mmol) of (E)-2-bromo-6-[2-(pyridin-2-yl)vinyl)pyridine and 289.6 mg (5 eq., 1.915 mmol) of α-(methylamino-methyl) benzyl alcohol were transferred into a high pressure tube and dissolved in 3 ml of anhydrous N-methylpyrrolidone (NMP). Then, 667 μl (10 eq., 3.83 mmol) of N,N-diisopropylethylamine (DIPEA) dried over molecular sieves was added in a single portion. The tube was sealed with a rubber septum and purged with argon. The reaction was carried out in a microwave reactor for 2 h at given operation parameters: P.sub.max=100 W, T.sub.max=160° C.). After completion of the reaction, the solvent was evaporated on a rotary evaporator and the residue was dissolved in a small amount of dichloromethane and applied directly to a silica gel column. The reaction mixture was purified by column chromatography, by eluting with an isocratic system: CH.sub.2Cl.sub.2/CH.sub.3OH (99/1 v/v). The main product fractions were combined, evaporated with a rotary evaporator, and freeze-dried from benzene to remove residual organic solvents. 70 mg of (E)-2-(methyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl]amino)-1-phenylethan-1-ol (yield=55%) as yellow oil.

[0164] .sup.1H NMR (400 MHz; Chloroform-d) δ 2.87 (s; 3H); 3.91 (m; 2H); 5.13 (dd; J=2.7; 6.7 Hz; 1H; CH); 6.46 (d; J=8.5 Hz; 1H); 6.79 (d; J=7.2 Hz; 1H); 7.17 (ddd; J=0.6; 4.9; 7.3 Hz); 7.28 (t; J=7.3 Hz); 7.37 (t; J=7.3 Hz; 2H); 7.45-7.51 (m; 4H); 7.57 (d; J=15.7 Hz; 1H; HC═); 7.67 (d; J=15.7 Hz; 1H); 7.67 (d; J=1.7 Hz; 1H); 8.62 (d; J=4.2 Hz; 1H).

[0165] .sup.13C NMR (101 MHz, Chloroform-d) δ 38.37; 60.17; 75.03; 106.12; 113.30; 122.48; 123.36; 125.97; 127.17; 128.26; 131.00; 131.72; 136.56; 138.48; 143.37; 149.68; 152.32; 155.17; 159.12.

[0166] ESI MS m/z found: 332.354; calculated for C.sub.21H.sub.22N30 [M+H].sup.+ 332.4; C.sub.21H.sub.21N.sub.3ON [M+Na].sup.+ 354.4.

EXAMPLE 23

Synthesis of (E)-2-(methyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl]amino)ethan-1-ol

[0167] 110 mg (1 eq., 0.35 mmol) of (E)-2-bromo-6-[2-(pyridin-2-yl)-vinyl)pyridine and 140 μl (5 eq., 1.75 mmol) of 2-(methylamino)ethanol were transferred into a high pressure tube and dissolved in 3 ml of anhydrous N-methylpyrrolidone (NMP). Then, 610 μl (10 eq., 3.5 mmol) of N,N-diisopropylethylamine (DIPEA) dried over molecular sieves was added in a single portion. The tube was sealed with a rubber cap and purged with argon. The reaction was carried out in a microwave reactor for 2 h at given operation parameters: P.sub.max=100 W, T.sub.max=160° C.). After completion of the reaction, the solvent was evaporated on a rotary evaporator and the residue was dissolved in a small amount of dichloromethane and applied directly to a silica gel column. The reaction mixture was purified by column chromatography, by eluting with an isocratic system: CH.sub.2Cl.sub.2/CH.sub.3OH (99/1 v/v). The main product fractions were combined, evaporated with a rotary evaporator, and freeze-dried from benzene to remove residual organic solvents. 73 mg of (E)-2-(methyl-{6-[2-(quinolin-2-yl)vinyl]-pyridin-2-yl}-amino)ethan-1-ol was thus obtained (yield=68%) as yellow viscous oil.

[0168] .sup.1H NMR (400 MHz; Chloroform-d): 3.11 (s; 3H); 3.87 (t; J 4.9=Hz; 2H); 3.94 (t; J=5.0 Hz; 2H); 6.52 (d; J=8.5 Hz; 1H); 6.83 (d; J=7.2 Hz; 1H); 7.47-7.54 (m; 2H); 7.64 (d; J=15.9 Hz; 1H); 7.73 (td; J=8.2; 1.0 Hz; 1H); 7.79 (t; J=7.3 Hz; 2H); 8.20 (t; J=8.2 Hz; 2H).

EXAMPLE 24

Synthesis of (E)-2-(methyl-{6-[2-(3-methoxyphenyl)-vinyl]-pyridin-2-yl]amino)ethan-1-ol

[0169] (E)-2-bromo-6-[2-(3-methoxyphenyl)vinyl)pyridine (0.61 mmol, 176 mg) in 0.5 mL of anhydrous DMF, diisopropyl ethanolamine (0.92 mmol, 159 μL), and a large excess of N-methylethanolamine (1.5 mL) were placed in a round bottom flask. The reaction was carried out in a microwave reactor (110° C., 300 W). After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/water/saturated aqueous solution of NaHCO.sub.3 (1/1/1; v/v/v). The product was then purified by silica gel column chromatography using DCM/methanol mixtures as eluents (gradient method, starting with one-component eluent (100% DCM) and ending with the mixture 95/5 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 125 mg of (E)-2-(methyl-{6-[2-(3-methoxyphenyl)vinyl]pyridin-2-yl}-amino)ethan-1-ol was obtained as yellowish oil, with a yield of 72%.

EXAMPLE 25

Preparation of (E)-2-(methyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}-amino) propan-1-ol

[0170] (E)-2-bromo-6-[2-(pyridin-2-yl)vinyl)pyridine (0.61 mmol, 160 mg) in 0.5 mL of anhydrous DMF, diisopropyl ethanolamine (0.92 mmol, 159 μL), and a large excess of N-methylaminopropan-1-ol (1.2 mmol) were placed in a round bottom flask. The reaction was carried out in a microwave reactor (120° C., 300 W). After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/water/saturated aqueous solution of NaHCO.sub.3 (1/1/1; v/v/v). The product was then purified by silica gel column chromatography using DCM/methanol mixtures as eluents (gradient method, starting with one-component eluent (100% DCM) and ending with the mixture 97/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 125 ng of (E)-2-(methyl-{6-[2-(pyridin-2-yl)-vinyl]pyridin-2-yl}-amino)propan-1-ol was obtained as yellowish oil, with a yield of 67%.

[0171] .sup.1H NMR (500 MHz, Chloroform-d) δ 8.53 (d, J=4.1 Hz, 1H); 7.57 (td, J=7.7, 1.8 Hz, 1H); 7.45-7.41 (m, 2H); 7.41-7.36 (m, 2H); 7.07 (ddd, J=7.6, 4.8, 1.1 Hz, 1H); 6.67 (d, J=7.2 Hz, 1H); 6.40 (d, J=8.5 Hz, 1H); 3.85-3.79 (m, 2H); 3.46 (t, J=5.5 Hz, 2H); 2.92 (s, 3H); 1.76 (p, J=5.6 Hz, 2H); 1.66 (s, 1H).

[0172] .sup.13C NMR (126 MHz, CDCl.sub.3) δ 158.88; 155.41; 152.88; 149.77; 138.54; 136.60; 132.26; 131.06; 123.08; 122.53; 112.37; 105.44; 58.12; 45.24; 35.76; 30.20.

EXAMPLE 26

Synthesis of (E)-2-(methy-{6-[2-(quinolin-2-yl)vinyl]pyridin-2-yl]amino)propan-1-ol

[0173] 110 mg (1 eq., 0.35 mmol) of (E)-2-bromo-6-[2-(quinolin-2-yl)-vinyl)pyridine and 140 μl (5 eq., 1.75 mmol) of 3-methylaminopropan-1-ol were transferred into a high pressure tube and dissolved in 3 ml of anhydrous N-methylpyrrolidone (NMP). Then, 610 μl (10 eq., 3.5 mmol) of N, N-diisopropylethylamine (DIPEA) dried over molecular sieves was added in a single portion. The tube was sealed with a rubber cap and purged with argon. The reaction was carried out in a microwave reactor for 2 h at given operation parameters: P.sub.max=100 W, T.sub.max=170° C.). After completion of the reaction, the solvent was evaporated on a rotary evaporator and the residue was dissolved in a small amount of dichloromethane and applied directly to a silica gel column. The reaction mixture was purified by column chromatography, by eluting with an isocratic system: CH.sub.2Cl.sub.2/CH.sub.3OH (99/1 v/v). The main product fractions were combined, evaporated with a rotary evaporator, and freeze-dried from benzene to remove residual organic solvents. 65 mg of (E)-2-(methyl-{6-[2-(quinolin-2-yl)vinyl]-pyridin-2-yl}-amino)propan-1-ol was thus obtained (yield=57%) as yellow viscous oil.

EXAMPLE 27

Preparation of 2-((6-bromo-pyridin-2-yl)amino)-ethan-1-ol

[0174] 2,6-dibromopyridine (0.03 mol; 6.5 g) in 10 mL of pyridine, ethanolamine (0.033 mol, 2 mL) and diisopropylethylamine (0.045 mol, 7.7 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 120° C. in a microwave reactor for 5 hours (300 W), controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using dichloromethane (DCM)/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 4.4 g of 2-(6-bromo-(pyridin-2-yl)amino)-ethan-1-ol was obtained as colorless oil in a 73% yield. The product was characterized by NMR spectroscopy:

[0175] .sup.1H NMR (400 MHz, Chloroform-d) δ 7.19 (dd, J=8.2, 7.5 Hz, 1H, H4); 6.68 (dd, J=7.5, 0.6 Hz, 1H, H5); 6.32 (dd, J=8.3; 0.6 Hz, 1H, H3); 5.24 (t, J=5.7 Hz, 1H, —NH); 3.98 (s, 1H, —OH); 3.78 (t, J=4.9 Hz, 2H, H8); 3.41 (td, J=5.6; 4.4 Hz, 2H, H7).

[0176] .sup.13C NMR (101 MHz, Chloroform-d) δ 158.96 (C2); 139.68 (C6); 139.50 (C4); 115.74 (C5); 105.77 (C3); 61.98 (C8); 44.59 (C7),

[0177] ESI MS m/z; calculated: 216.9976 C.sub.7H.sub.9BrN.sub.2O [M+H].sup.+; found: 238.9802 C.sub.7H.sub.9BrN.sub.2ON [M+Na].sup.+.

EXAMPLE 28

Preparation of 3-((6-bromo-pyridin-2-yl)amino)-propan-1-ol

[0178] 2,6-dibromopyridine (0.03 mol; 6.5 g) in 10 mL of pyridine, propanolamine (0.033 mol, 2 mL) and diisopropylethylamine (0.045 mol, 7.7 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 120° C. in a microwave reactor for 5 hours (300 W), controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents the purified product was freeze-dried from benzene. 3.8 g of 3-((6-bromo-pyridin-2-yl) amino)-propan-1-ol were obtained as colorless oil in a 59% yield.

EXAMPLE 29

Preparation of 2-[(2,4-difluorobenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol

[0179] 2-((6-bromo-pyridin-2-yl)amino)ethan-1-ol (0.0046 mol, 1 g) in 5 mL DMF, 1-(bromomethyl)-2,4-difluorobenzene (0.0055 mol, 0.7 mL), and diisopropylethylamine (0.007 mol, 1.13 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 120° C. in an oil bath under reflux for 24 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 950 mg of 2-[(2,4-difluorobenzyl)-(6-bromo-pyridin-2-yl) amino]-ethanol was obtained as oil in a 63% yield. The product was characterized by NMR spectroscopy:

[0180] .sup.1H NMR (500 MHz, Chloroform-d) δ 7.30-7.26 (m, 1H, H4); 7.20 (td, J=8.7; 6.4 Hz, 1H, H13); 6.90-6.82 (m, 211, H10, 11); 6.79 (d, J=7.4 Hz, 1H, H5), 6.42 (d, J=8.4 Hz, 1H, H3); 4.71 (s, 2H, H9); 3.90 (dd, J=5.5; 4.5 Hz, 2H, H8); 3.80 (t, J=5.0 Hz, 2H, H7).

[0181] .sup.13C NMR (126 MHz, Chloroform-d) δ 162.51 (dd, J=194.7; 11.8 Hz, C14); 160.54 (dd, J=194.3; 11.9 Hz, C12); 158.54 (C2); 139.88 (C4); 139.37 (C6); 129.28 (dd, J=9.6; 5.9 Hz, C10); 119.96 (dd, J=14.9; 3.7 Hz, C15); 115.92 (C5); 111.45 (dd, J=21.1; 3.7 Hz, C11); 104.86 (C3); 104.61-103.48 (m, C13); 62.15 (C8); 52.28 (C7); 47.04 (C9),

[0182] ESI MS m/z; calculated: 343.0258 C.sub.14H.sub.13BRF.sub.2N.sub.2O [M+H].sup.+; found: 343.0255 [M+H].sup.+.

EXAMPLE 30

Preparation of 2-[(2,4-dimethylbenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol

[0183] 2-((6-bromo-pyridin-2-yl)amino)ethan-1-ol (0.0029 mol; 0.63 g) in 5 mL DMF, 1-(bromomethyl)-2,4-dimethylbenzene (0.0035 mol; 0.53 mL), and diisopropylethylamine (0.0045 mol; 0.71 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 110° C. in an oil bath under reflux for 48 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 560 mg of 2-[(2,4-dimethylbenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol was obtained as oil, with a yield of 58%. The product was characterized by NMR spectroscopy:

[0184] .sup.1H NMR (400 MHz, Chloroform-d) S 7.20 (dd, J=8.4, 7.5 Hz, 1H, H4), 7.04 (s, 1H, H13), 6.96 (dd, J=7.9, 1.7 Hz, 1H, H10), 6.91 (d, J=7.8 Hz, 1H, H1), 6.74 (d, J=7.4 Hz, 1H, H5), 6.27 (d, J=8.4 Hz, 1H, H3), 4.55 (s, 2H, H9), 3.87 (td, J=4.7, 1.0 Hz, 2H, H8), 3.82-3.72 (m, 2H, H7), 2.31 (s, 3H, p-CH3), 2.28 (s, 3H, m-CH3). .sup.13C NMR (101 MHz, Chloroform-d) δ 159.10 (C2), 139.75 (C4), 139.15 (C6), 136.87 (C14), 135.37 (C12), 131.49 (C13), 130.89 (C15), 126.82 (C10), 125.48 (C11), 115.43 (C5), 105.09 (C3), 62.81 (C8), 52.35 (C7), 51.46 (C9), 20.88 (C16), 18.88 (C17),

[0185] ESI MS m/z; calculated: 335.0759 C.sub.6H.sub.19BrN.sub.2O [M+H].sup.+; found: 335.0757 [M+H].sup.+.

EXAMPLE 31

Preparation of 2-[(2-fluorobenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol

[0186] 2-((6-bromo-pyridin-2-yl)amino)ethan-1-ol (0.0037 mol; 0.8 g) in 5 mL of DMF, 1-(bromomethyl)-2-fluorobenzene (0.0044 mol; 0.54 mL), and diisopropylethylamine (0.0055 mol; 0.90 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 130° C. in an oil bath under reflux for 48 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 640 mg of 2-[(2-fluorobenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol was obtained as oil in a 54% yield. The product was characterized by NMR spectroscopy:

[0187] .sup.1H NMR (500 MHz, Chloroform-d) δ 7.34-7.27 (m, 2H, H4, H14); 7.21 (td, J=7.7; 1.8 Hz, 1H, H12); 7.16-7.10 (m, 2H, H13, H11); 6.80 (d, J=7.4 Hz, 1H, H5); 6.43 (d, J=8.4 Hz, 1H, H3); 4.77 (s, 2H, H9); 3.92 (dd, J=5.5; 4.3 Hz, 2H, H8); 3.87-3.81 (m, 2H, H7); 3.39 (s, 1H, —OH),

[0188] .sup.13C NMR (126 MHz, Chloroform-d) δ 161.75 (d, J.sub.C10F=250 Hz, C10); 159.80 (C2); 158.76 (C4); 139.75 (C6); 139.36 (C14); 128.95 (C12) (d, J=7.9 Hz); 128.11 (C13); 124.31 (C15) (d, J=3.6 Hz); 115.68 (C5); 115.45 (C11) (d, J=21.2 Hz); 104.83 (C3); 62.38 (C8); 52.38 (C7); 47.44 (C9) (d, J=4.8 Hz),

[0189] ESI MS m/z; calculated: 325.0352 C.sub.14H.sub.14BrFN.sub.2O [M+H].sup.+; found: 325.0349 [M+H].sup.+.

EXAMPLE 32

Preparation of 2-[(2-chlorobenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol

[0190] 2-((6-bromo-pyridin-2-yl)amino)ethan-1-ol (0.0037 mol; 0.8 g) in 5 mL of DMF, 1-(bromomethyl)-2-chlorobenzene (0.0043 mol; 0.58 mL), and diisopropylethylamine (0.0055 mol; 0.8 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 120° C. in an oil bath under reflux for 48 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 460 mg of 2-[(2-chlorobenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol was obtained as oil with a yield of 57%. The product was characterized by NMR spectroscopy:

[0191] .sup.1H NMR (500 MHz, Chloroform-d) δ 7.30-7.19 (m, 4H); 7.11 (dt, J=6.9, 1.9 Hz, 1H); 6.77 (d, J=7.4 Hz, 1H, H5); 6.36 (d, J=8.4 Hz, 1H, H3); 4.68 (s, 2H, H9); 3.88 (t, J=5.0 Hz, 2H, H8); 3.78 (t, J=5.0 Hz, 2H, H7),

[0192] .sup.13C NMR (126 MHz, Chloroform-d) δ 158.68; 139.77; 139.46; 134.72; 130.09; 127.53; 126.56; 124.60; 115.78; 104.80; 62.18; 52.86; 52.16.

[0193] ESI MS m/z; calculated: 341.0056 C.sub.14H.sub.14BrClN.sub.2O [M+H].sup.+; found: 341.0055 [M+H].sup.+.

EXAMPLE 33

Preparation of 2-[(4-methylbenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol

[0194] 2-((6-bromo-pyridin-2-yl)amino)ethan-1-ol (0.0046 mol; 1 g) in 5 mL DMF, 1-(bromomethyl)-4-methylbenzene (0, 0069 mol; 1.26 g), and diisopropylethylamine (0.007 mol; 1.13 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 110° C. in an oil bath under reflux for 24 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 990 mg of 2-[(4-methylbenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol were obtained as oil with a yield of 68%. The product was characterized by NMR spectroscopy:

[0195] .sup.1H NMR (400 MHz, Chloroform-d) δ 7.22 (dd, J=8.4, 7.5 Hz, 1H, H4); 7.15 (d, J=8.0 Hz, 2H, H10, H14); 7.10 (d, J=8.0 Hz, 2H, H11, H13); 6.75 (d, J=7.4 Hz, 1H, H5); 6.40 (d, J=8.4 Hz, 1H, H3); 4.64 (s, 2H, H9); 3.88 (td, J=4.8; 1.1 Hz, 2H, H8); 3.84-3.77 (m, 2H, H7); 3.15 (s, 1H, —OH); 2.34 (s, 3H, p-CH3),

[0196] .sup.13C NMR (101 MHz, Chloroform-d) δ 158.50 (C2); 139.32 (C4); 138.56 (C6); 136.64 (C12); 133.24 (C15); 129.07 (C10, C14); 125.92 (C11, C13); 115.08 (C5); 104.80 (C3); 62.15 (C8); 52.88 (C9); 52.10 (C7); 20.57 (C16),

[0197] ESI MS m/z; calculated: 321.0602 C.sub.1H.sub.17BrN.sub.2O [M+H].sup.+; found: 321.0600 [M+H].sup.+.

EXAMPLE 34

Preparation of 2-[(6-bromo-pyridin-2-yl)benzylamino]ethane-1-ol

[0198] 2-((6-bromo-pyridin-2-yl)amino)ethan-1-ol (0.0046 mol; 1 g) in 5 mL DMF, benzyl bromide (0.0069 mol; 0.83 mL), and diisopropylethylamine (0.007 mol; 1.13 mL) were placed in a round bottom flask. The mixture was stirred using a magnetic stirrer and heated to 110° C. in an oil bath under reflux for 72 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 790 mg of 2-[(6-bromo-pyridin-2-yl)benzylamino]ethan-1-ol were obtained as oil with a yield of 55%. The product was characterized by NMR spectroscopy:

[0199] .sup.1H NMR (400 MHz, Chloroform-d) δ 7.38-7.20 (m, 6H, 2×H11, 12, 2×10.4); 6.76 (d, J=7.4 Hz, 1H, H5); 6.39 (d, J=8.4 Hz, 1H, H3); 4.69 (s, 2H, H9); 3.93-3.85 (m, 2H, H8); 3.84-3.77 (m, 2H); 3.17 (s, 1H, H7),

[0200] .sup.13C NMR (101 MHz, Chloroform-d) δ 158.53 (C2); 139.29 (C4); 138.74 (C6); 136.49 (C13); 128.38 (C11); 126.91 (C12); 125.95 (C10); 115.12 (C5); 104.66 (C3); 62.11 (C8); 53.05 (C9); 52.05 (C7),

[0201] ESI MS m/z; calculated: 307.0446 C.sub.14H.sub.15BrN.sub.2O [M+H].sup.+; found: 307.0443 [M+H].sup.+.

EXAMPLE 35

Preparation of 3-[(6-bromo-pyridin-2-yl)benzylamino]propan-1-ol

[0202] 3-((6-bromo-pyridin-2-yl)amino)propan-1-ol (0.0046 mol; 1 g) in 5 mL DMF, benzyl bromide (0.0069 mol; 0.83 mL), and diisopropylethylamine (0.007 mol; 1.13 mL) were placed in a round-bottomed flask. The mixture was stirred using a magnetic stirrer and heated to 110° C. in an oil bath under reflux for 72 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 710 mg of 3-[(6-bromo-pyridin-2-yl)benzylamino]propan-1-ol were obtained as oil with a yield of 51%.

EXAMPLE 36

Preparation of 2-[(6-bromo-pyridin-2-yl)naphthylamino]ethan-1-ol

[0203] 2-((6-bromo-pyridin-2-yl)amino)ethan-1-ol (0.0069 mol; 1.5 g) in 5 mL DMF, 1-(bromomethyl)-naphthalene (0.0076 mol; 1.67 g), and diisopropylethylamine (0.007 mol; 1.13 mL) were placed in a round-bottomed flask. The mixture was stirred using a magnetic stirrer and heated to 110° C. in an oil bath under reflux for 24 hours, controlling the reaction by means of a thin layer chromatography. After completion of the reaction, the solvents were evaporated and the product was extracted using DCM/saturated aqueous solution of NaHCO.sub.3 (1/1 v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 7/3 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 1.4 g of 2-[(6-bromo-pyridin-2-yl)naphthylamino]ethan-1-ol was obtained as oil with a yield of 58%. The product was characterized by NMR spectroscopy:

[0204] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.82 (d, J=8.6 Hz, 2H); 7.80-7.76 (m, 1H); 7.61 (s, 1H); 7.52-7.41 (m, 2H); 7.33 (dd, J=8.5; 1.8 Hz, 1H); 7.18 (dd, J=8.4; 7.4 Hz, 1H); 6.74 (d, J=7.4 Hz, 1H); 6.40 (d, J=8.4 Hz, 1H); 4.82 (s, 2H); 3.89 (t, J=5.2 Hz, 2H); 3.84 (dd, J==5.3, 4.1 Hz, 2H),

[0205] .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 158.98; 139.68; 139.34; 134.46; 133.34; 132.68; 128.69; 127.63; 126.30; 125.83; 124.85; 124.63; 115.50; 105.03; 62.43; 53.55; 52.28, ESI MS m/z; calculated: 357.0602 C.sub.18H.sub.17BrN.sub.2O [M+H].sup.+; found: 379.0422.

EXAMPLE 37

Preparation of (E)-2-(benzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol

[0206] Pd(OAc).sub.2 (0.065 mmol, 15 mg) and PPh.sub.3 (0.19 mmol, 51 mg) dissolved in 5 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.05 mmol; 16 mg), 2-[(6-bromo-pyridin-2-yl)benzylamino]ethan-1-ol (1.6 mmol; 500 mg), vinylpyridine (1.9 mmol; 0.21 mL), and Cs.sub.2CO.sub.3 (2.4 mmol, 760 mg) were added. The reaction was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 3 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 324 mg of (E)-2-(benzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol was obtained as oil with a yield of 60%. The product was characterized by NMR spectroscopy:

[0207] .sup.1H NMR (500 MHz, Chloroform-d) δ 8.55-8.49 (m, 1H, H6); 7.59 (td, J=7.7; 1.8 Hz, 1H, H4); 7.53 (d, J=15.8 Hz, 1H, H7); 7.44 (d, J=15.9 Hz, 1H, H8); 7.40 (dt, J=7.9; 1.0 Hz, 1H); 7.30 (dd, J=8.5; 7.3 Hz, 1H, H11); 7.25-7.22 (m, 1H); 7.19-7.13 (m, 4H); 7.08 (ddd, J=7.5; 4.8; 1.1 Hz, 1H); 6.67 (d, J=7.2 Hz, 1H); 6.33 (d, J=8.5 Hz, 1H); 4.62 (s, 2H); 3.85 (t, J=2.7 Hz, 4H),

[0208] .sup.13C NMR (126 MHz, Chloroform-d) δ 158.86 (C13); 155.05 (C2); 152.15 (C9); 149.32 (C6); 138.50 (C11); 137.52 (C18); 136.79 (C4); 131.94 (C8); 130.69 (C7); 128.81 (C19); 127.22 (C21); 126.34 (C20); 123.09 (C3); 122.50 (C5); 113.50 (C10); 107.06 (C12); 63.33 (C16); 53.52 (C17); 52.40 (C15),

[0209] ESI MS m/z; calculated: 332.1763 C.sub.21H.sub.21N.sub.3O [M+H].sup.+; found: 332.1758 [M+H].sup.+.

EXAMPLE 38

Preparation of (E)-3-(benzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)propan-1-ol

[0210] Pd (OAc).sub.2 (0.065 mmol, 15 mg) and PPh.sub.3 (0.19 mmol, 51 mg) dissolved in 5 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.05 mmol; 16 mg), 3-[(6-bromo-pyridin-2-yl)benzylamino]propan-1-ol (1.6 mmol; 485 mg), vinyl pyridine (1.9 mmol; 0, 21 mL), and Cs.sub.2CO.sub.3 (2.4 mmol, 760 mg) were added. The reaction was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 3 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 324 mg of (E)-3-(benzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)propan-1-ol was obtained as oil with a yield of 60%.

EXAMPLE 39

Preparation of (E)-2-(naphthyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol

[0211] Pd (OAc).sub.2 (0.21 mmol, 47 mg) and PPh.sub.3 (0.63 mmol, 165 mg) in 10 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.1 mmol; 32 mg), 2-[(6-bromo-pyridin-2-yl) naphthylamino] ethan-1-ol (4.2 mmol; 900 mg), vinylpyridine (5.0 mmol; 0.54 mL), and Cs.sub.2CO.sub.3 (6.3 mmol, 2 g) were added. The reaction was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 3 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 249 mg of (E)-2-(naphthyl-{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol was obtained as oil with a yield of 28%. The product was characterized by means of mass spectrometry:

[0212] ESI MS m/z; calculated: 382.1919 C.sub.25H.sub.23N.sub.3O [M+H].sup.+; found: 382.1915 [M+H].sup.+.

EXAMPLE 40

Preparation of (E)-2-(2,4-difluoro-6-{[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol

[0213] Pd (OAc).sub.2 (0.046 mmol, 10 mg) and PPh.sub.3 (0.14 mmol, 36 mg) in 10 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.1 mmol; 32 mg), 2-[(2,4-difluorobenzyl)(6-bromo-pyridin-2-yl)amino]-ethan-1-ol (2.33 mmol; 800 mg), vinylpyridine (2.8 mmol; 0.3 mL), and Cs.sub.2CO.sub.3 (2.99 mmol; 971 mg) were added. The reaction 1255 was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 4 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 412 mg of (E)-2-(2,4-difluorobenzyl){6-[2-(pyridin-2-yl) vinyl]pyridin-2-yl} amino) ethan-1-ol was obtained as oil in a yield of 48%. The product was characterized by NMR spectroscopy:

[0214] .sup.19F NMR (376 MHz, Chloroform-d) δ −111.77 (p, J=7.8 Hz), −114.25 (q, J=8.6 Hz), ESI MS m/z; calculated: 368.1574 C.sub.21H.sub.19F.sub.2N.sub.3O [M+H].sup.+; found: 368.1570 [M+H].sup.+.

EXAMPLE 41

Preparation of (E)-2-(4-methyl-6-{[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol

[0215] Pd (OAc).sub.2 (0.025 mmol; 5.6 mg) and PPh.sub.3 (0.075 mmol; 20 mg) dissolved in 5 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.05 mmol; 16 mg), 2-[(4-methylbenzyl)(6-bromo-pyridin-2-yl) amino]ethan-1-ol (0.6 mmol; 200 mg), vinylpyridine (0.74 mmol; 81 μL), and Cs.sub.2CO.sub.3 (0.9 mmol; 305 mg) were added. The reaction was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 4 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 90 mg of (E)-2-(4-methylbenzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol were obtained as oil in a yield of 42%.

EXAMPLE 42

Preparation of (E)-2-(2-chloro-6-{[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol

[0216] Pd (OAc).sub.2 (0.023 mmol; 5.3 mg) and PPh.sub.3 (0.07 mmol; 18 mg) dissolved in 5 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.05 mmol; 16 mg), 2-[(2-chlorobenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol (0.58 mmol; 200 mg), vinylpyridine (0.7 mmol; 76 μL), and Cs.sub.2CO.sub.3 (0.88 mmol; 287 mg) were added. The reaction was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 4 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 88 mg of (E)-2-(2-chlorobenzyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino) ethan-1-ol was obtained as yellow oil with a yield of 41%. The product was characterized by NMR spectroscopy:

[0217] .sup.1H NMR (500 MHz, Chloroform-d) δ 8.60 (dd, J=5.3, 1.5 Hz, 1H, H6); 7.67 (td, J=7.7; 1.9 Hz, 1H, H4); 7.61 (d, J=15.8 Hz, 1H, H7); 7.53 (d, J=15.8 Hz, 1H, H8); 7.48 (d, J=7.8 Hz, 1H, H3); 7.40 (dd, J=8.5, 7.2 Hz, 1H, H11); 7.27-7.21 (m, 3H, H21, 22, 23); 7.17 (ddd, J=7.5; 4.8; 1.1 Hz, 1H, H5); 7.13 (dt, J=7.1; 1.7 Hz, 1H, H2O); 6.77 (d, J=7.2 Hz, 1H, H10); 6.38 (d, J=8.5 Hz, 1H, H12); 4.69 (s, 2H, H17); 3.94 (dd, J=5.2; 3.8 Hz, 2H, H16); 3.90 (dd, J=7.5, 3.0 Hz, 2H, H15),

[0218] .sup.13C NMR (126 MHz, Chloroform-d) δ 158.54 (C13); 155.00 (C2); 152.31 (C9); 149.31 (C6); 140.07 (C18); 138.53 (C11); 136.76 (C4); 134.72 (C19); 131.96 (C8); 130.73 (C7); 130.08 (C23); 127.40 (C21); 126.48 (C22); 124.51 (C20); 123.04 (C3); 122.49 (C5); 113.68 (C10); 106.75 (C12); 62.91 (C16); 52.96 (C17); 52.18 (C15),

[0219] ESI MS in/z; calculated: 366.1373 C.sub.21H.sub.20ClN.sub.3O [M+H].sup.+; found: 366.1372 [M+H].sup.+.

EXAMPLE 43

Preparation of (E)-2-{(2,4-dimethylbenzyl)[6-((2-pyridin-2-yl)vinyl] pyridin-2-yl)amino}ethan-1-ol

[0220] Pd (OAc).sub.2 (0.021 mmol; 4.8 mg) and PPh.sub.3 (0.064 mmol; 17 mg) dissolved in 5 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.05 mmol; 16 mg), 2-{2,4-dimethylbenzyl-(6-bromopyridin-2-yl)}-aminoethan-1-ol (0.54 mmol; 180 mg), vinylpyridine (0, 64 mmol; 70 μL), and Cs.sub.2CO.sub.3 (0.8 mmol; 262 mg) were added. The reaction was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 4 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 83 mg of (E)-2-{(2,4-dimethylbenzyl)[6-((2-pyridin-2-yl)vinyl]pyridin-2-yl)amino}ethan-1-ol was obtained as oil in a yield of 43%. The product was characterized by mass spectrometry:

[0221] ESI MS m/z; calculated: 360.206 C.sub.23H.sub.25N.sub.3O [M+H].sup.+; found: 360.2071 [M+H].sup.+.

EXAMPLE 44

Preparation of (E)-2-{2-fluorobenzyl-[6-((2-pyridin-2-yl)vinyl)pyridin-2-yl)-aminoethan-1-ol

[0222] Pd(OAc).sub.2 (0.025 mmol; 5.6 mg) and PPh.sub.3 (0.075 mmol; 20 mg) dissolved in 5 mL of anhydrous DMF were placed in a round-bottom flask flushed with argon. After 10 min, TBAB (0.05 mmol; 16 mg), 2-[(2-fluorobenzyl)(6-bromo-pyridin-2-yl)amino]ethan-1-ol (0.61 mmol; 200 mg), vinylpyridine (0.73 mmol; 79 μL), and Cs.sub.2CO.sub.3 (0.9 mmol; 300 mg) were added. The reaction was carried out under an argon atmosphere in a microwave reactor (120° C., 300 W) for 4 hours. After completion of the reaction, DMF was evaporated and the row product was preliminarily purified by extraction from DCM/saturated aqueous solution of NaHCO.sub.3 (1/1; v/v). The product was then purified by silica gel column chromatography using hexane/ethyl acetate mixtures as eluents (gradient method, starting with one-component eluent (100% hexane) and ending with the mixture 1/1 v/v as the final eluent), and the solvents were evaporated on a rotary evaporator. To completely remove traces of the organic solvents, the purified product was freeze-dried from benzene. Thus, 75 mg of (E)-2-{2-fluorobenzyl-[6-((2-pyridin-2-yl)vinyl)pyridin-2-yl)aminoethan-1-ol was obtained as yellow oil in a 35% yield. The product was characterized by NMR spectroscopy:

[0223] .sup.1H NMR (500 MHz, Chloroform-d) δ 8.61 (dd, J=5.0, 1.7 Hz, 1H, H6); 7.66 (td, J=7.7; 1.8 Hz, 1H, H4); 7.60 (d, J=15.7 Hz, 1H, H7); 7.53 (d, J=15.7 Hz, 1H, H8); 7.46 (d, J=7.8 Hz, 1H, H3); 7.40 (dd, J=8.5; 7.3 Hz, 1H, H11); 7.27-7.22 (m, 1H, H21); 7.23-7.14 (m, 2H, H5, H23); 7.12-7.04 (m, 2H, H2O, 22); 6.77 (d, J=7.2 Hz, 1H, H10); 6.40 (d, J=8.5 Hz, 1H, H12); 4.76 (s, 2H, H17); 4.03-3.81 (m, 4H, H15, 16),

[0224] .sup.13C NMR (126 MHz, Chloroform-d) δ 160.81 (C19) (d, JF-C=245.5 Hz); 158.67 (C13); 155.12 (C2); 152.39 (C9); 149.51 (C6); 138.50 (C11); 136.60 (C4); 131.84 (C8); 130.87 (C7); 128.75 (C23); 127.87 (C21); 124.50 (C18); 124.31 (C22); 123.10 (C3); 122.46 (C5); 115.39 (C20); 113.57 (C10); 106.68 (C12); 63.28 (C16); 52.36 (C15); 47.52 (C17),

[0225] ESI MS m/z; calculated: 350.1169 C.sub.21H.sub.20FN.sub.3O [M+H].sup.+; found: 360.1164 [M+H].sup.+.

EXAMPLE 45

[0226] Labeling of 3-O-Acetylthymidine with a Fluorescent Dye Derivative

Step 1

[0227] Conversion of an Aminoalcohol Derivative of 2-(pyridine-2-yl) vinyl] Pyridine into a Carbamate Derivative

[0228] (E)-2-(methyl{6-[2-(pyridin-2-yl)vinyl]pyridin-2-yl}amino)ethan-1-ol (1 eq., 0.1 mmol) was dissolved in anhydrous acetonitrile (2 ml) and placed in a flask containing a magnetic stir bar. To this solution, 1′1′-carbonyldiimidazole (2 eq., 0.24 mmol) was added and the reaction was carried out for 30 minutes at room temperature and its course was monitored by TLC (ethyl acetate/n-hexane 9/1). After 1 hour, the active carbamate derivative of the amino alcohol was obtained, which was used in the next step.

Step 2

[0229] Labeling of 3′-O-Acetylthymidine with a Carbamate Derivative of a Fluorescent Dye

[0230] To the solution of carbamate derivative of (E)-2-(methyl(6-(2-(pyridin-2-yl)vinyl)pyridin-2-yl)amino)ethanol obtained in the previous step, 3′-O-acetylthymidine (1 eq; 0.1 mmol) and 1,1,3,3-tetramethylguanidine (1.6 eq., 0.16 mmol) were added. The reaction mixture was stirred for 2 hours at room temperature. The course of the reaction was monitored by TLC (dichloromethane/methanol 95/5 v/v). The solvent was evaporated under reduced pressure and the thus obtained residue was purified by silica gel column chromatography (DCM/MeOH; 0-5% MeOH) to obtain a 3′-O-acetylthymidine derivative labeled with (E)-2-(methyl (6-(2-(pyridin-2-yl) vinyl) pyridin-2-yl) amino) ethanol which was characterized by spectroscopic methods.

[0231] .sup.1H NMR (400 MHz; Chloroform-d) δ 8.66-8.55 (m; 1H); 7.70-7.65 (m; 1H); 7.63 (d; J=5.3 Hz; 1H); 7.48 (d; J=8.6 Hz; 1H); 7.44 (d; J=8.0 Hz; 2H); 7.35 (d; J=2.2 Hz; 2H); 7.14 (dd; J=7.5; 4.9 Hz; 1H); 6.71 (d; J=7.3 Hz; 1H); 6.45 (d; J=8.5 Hz; 1H); 6.35 (dd; J=8.8; 5.7 Hz; 1H); 5.16 (dd; J=5.2; 3.2 Hz; 1H); 4.52 (dt; J=11.5; 5.9 Hz; 1H); 4.42 (q; J=4.6; 3.7 Hz; 1H); 4.39 (d; J=2.9 Hz; 1H); 4.33 (dd; J=11.8; 2.8 Hz; 1H); 4.17 (q; J=2.7 Hz; 1H); 4.02 (tq; J=14.7; 8.8; 7.4 Hz; 2H); 3.10 (s; 3H); 2.40-2.29 (m; 1H); 2.22-2.10 (m; 2H); 2.09 (s; 3H); 1.88 (s; 3H). .sup.13C NMR (101 MHz; Chloroform-d) δ 169.89; 163.00; 157.25; 155.08; 154.11; 152.11; 149.93; 149.18; 137.44; 136.01; 134.34; 132.11; 130.24; 127.82; 122.10; 121.78; 112.34; 111.15; 104.99; 84.04; 81.62; 74.07; 66.76; 65.86; 47.87; 36.73; 36.64; 20.39; 12.13.

[0232] Fluorescence properties (Absorption and emission were measured in methanol; the analyte concentration was in the order of 10.sup.−5-10.sup.−6 M; the measurement error margin for ε is +/−20%)

TABLE-US-00003 ε λ.sub.abs λ.sub.em [M.sup.−1 cm.sup.−1] 371 485 16800