2'-amino-2'-deoxynucleosides as inhibitors if measles and marburg virusus reproduction

FIELD: organic chemistry, biochemistry, medicine, virology.

SUBSTANCE: invention relates to derivatives of 2'=amino-2'-deoxynucleosides of the formula:

wherein R means hydrogen atom (H), alkyl, aminoalkyl; R1 means -(R2NR3) wherein R2 and/or R3 means H, -OH, -NH2, alkyl, benzyl under condition that R doesn't represent H or methyl when R2 and R3 mean H. Compounds elicit an antiviral activity with respect to measles and Marburg viruses exceeding that of ribavirin.

EFFECT: valuable properties of compounds.

4 tbl, 2 dwg, 18 ex

 

The invention relates to the field of molecular biology, Virology and medicine, namely to the use of new derivatives of nucleosides to inhibit reproduction of measles virus and Marburg.

Currently there are a number of compounds nucleotide analogues with antiviral activity, including those used in medical practice. The latter include acyclovir, azidothymidine, ribavirin, and other drugs. To treat severe cases of measles infection are mainly used ribavirin [1-(β-d-ribofuranosyl-1)-1,2,4-triazole-3-carboxamide] [1]. Pass through cell membranes, ribavirin is metabolized into mono - and triphosphate. Monophosphate is a competitive inhibitor insimenator dehydrogenase, which leads to inhibition of synthesis of viral RNA and DNA, weak acting on the host cells. In addition to the measles virus ribavirin suppresses the reproduction of influenza viruses type a and b, herpes, hepatitis a and b and others [2, 3].

However, in the case of Marburg virus no drugs with antiviral activity.

Known L-nucleosides and its pharmaceutically acceptable salts for the treatment of diseases of viral infections (patent RF №96117323, IPC 07 N 19/06, publ. Running from 10.12.1998) [4].

However, preparations based on these compounds effective for the treatment of diseases, call the data with the hepatitis b virus and Epstein-Barr and do not show activity against measles virus and Marburg.

The closest technical solution (prototype) are pharmaceutical compounds comprising substituted or unsubstituted nucleosides and their analogues, which are active against hemorrhagic fever caused by Dengue virus, and measles virus infection (international application number WO 00/15214, IPC a 61 K 31/19, publ. 23.03.2000) [5].

However, these pharmaceutical compounds are not active against Marburg virus and measles virus.

The technical result of the invention is the use of derivatives of nucleosides, effective against measles virus and Marburg, able to penetrate into the cell and having voting activity in suppressing the reproduction of these viruses.

This technical result is achieved by the fact that inhibitors of reproduction of viruses are derivatives of nucleosides according to the invention as derivatives of nucleosides use of pyrimidine 2'-amino-2'-deoxynucleoside with the chemical formula:

where R=H, halogen, haloalkyl, alkyl, aminoalkyl, aryl, arylalkyl and other R1=(R2NR3), R2or R3=H, HE, NH2, alkyl, aryl, arylalkyl etc. there are several Known methods for the synthesis of pyrimidine 2'-amino-'-deoxynucleosides, based on a modification of the carbohydrate residue of a nucleoside [6-9].

2'-Amino-2'-deoxyuridine and modified at the 5-position of the nucleic acid base derivatives were synthesized by the authors of the proposed technical solution according to the following scheme I with a total yield of 35-50%:

where R=H, halogen, haloalkyl, alkyl, aminoalkyl, aryl, arylalkyl and other

In the first stage of the synthesis result of the impact of diphenylcarbonate on commercial uridine (I, R=H) or modified at the 5-position of the nucleic base of uridine (I, R=halogen, alkyl, aminoalkyl, aryl, arylalkyl and others) were synthesized 2,2'-O-angidepressant (5-R)-uridine (II). The next stage of the synthesis sideropenia 2,2'-O-anhydrous-(5-R)-uridine (II). The last step of the synthesis was the restoration of 2'-sidegroup to the amino group. The selection of the target 2'-amino-2'-deoxy-(5-R)-uridine was carried out by ion exchange chromatography on a column of Dowex 50 (H+), followed by purification on a column with obremenitve silica gel LiChroprep RP-8 and lyophilisation of the product (IV).

2'-Amino-2'-deoxy-N4-(Rl,R2-citizen (VIII) and modified at the 5-position of the nucleic acid base derivatives were synthesized by the authors of the proposed technical solution of the respective 2'-azido-2'-deoxy-(5-R)-uridines (III) below scheme with a total yield 30-65%:

where R=H, halogen, haloalkyl, alkyl, aminoalkyl, aryl, arylalkyl and other

R1or R2=H, HE, NH2, alkyl, aryl, arylalkyl and other

The first stage of the synthesis was the acetylation of 2'-azido-2'-deoxy-(5-R)-uridine (III). Obtained 3',5'-di-O-acetyl-2'-azido-2'-deoxy-(5-R)-uridine (V) was treated with freshly prepared phospho-tristriata. After the reaction was conducted substitution triazolide group in compound VI to N4-(R1NR2-group by the action of NH4OH or the corresponding amine, followed by removal of protective groups. The last step of the synthesis was the restoration of 2'-sidegroup in (VII) to the amino group. The selection of the target 2'-amino-2'-deoxy-N4-(R1, R2)-(5-R)-cytidine (VIII) was carried out by ion exchange chromatography on a column of Dowex 50 (H+), followed by purification on a column with obremenitve silica gel LiChroprep RP-8 and lyophilisation of the product (VIII).

The structure of 2'-amino-2'-deoxy-(5-R)-uridine (IV) and 2'-amino-2'-deoxy-N4-(Rl,R2)-(5-R)-cytidine (VIII) was confirmed by mass-, UV - and NMR-spectra. The purity of the target substances was evaluated by HPLC and is 98%.

Inhibition of reproduction of measles virus and Marburg virus was investigated in initially infected cells green monkey kidney Vero in the presence of experimental the th drug the final concentration in culture medium was 0.1-400 mg/ml, for one passage within 5 days in the case of measles virus and 6 days in the study of virus Marburg. About the inhibition of the studied compound of reproduction of the virus in culture sensitive cells was judged to reduce virus-induced cytopathic effect in the presence of the drug compared to control. As the comparison drug used ribavirin.

Antiviral activity against Marburg virus was also investigated by the method of quantitative determination of the virus in cell culture Vero under semisolid agar surface.

In the drawings is shown a dose-dependent curves illustrating the inhibitory activity of the inventive compounds. Figure 1 shows graphs illustrating the antiviral activity of compounds (IVa), (VIIIa) and ribavirin against measles virus. Figure 2 shows graphs illustrating the cytotoxicity and antiviral activity of compound (VIIIa) in respect of the Marburg virus.

The following are specific examples that reveal the essence of the invention.

Example 1. Synthesis of 2'-amino-2'-dose irradiation on neurogenesis (compound IVa, R=H)

A solution of 1.95 g (8 mmol) uridine, of 1.93 g (9 mmol) of diphenylcarbonate and 100 mg NaHCO3in 5 ml of abs. of dimethylformamide (DMF) was heated at 120°prior to the termination allocated the gas I (5 hours), then was evaporated to 1/2 volume, cooled and the precipitate was filtered 2,2'-O-angeborene, washed it with 5 ml of cold methanol. The product is suspended in 3 ml of methanol and boiled under stirring for 3 hours, then cooled, the precipitate was filtered, washed it with 5 ml of cold methanol, 5 ml of ether and dried in a vacuum desiccator over KOH. The output of 2,2'-O-angeborene (II) 1.4 g of 78%.

To a solution of 0.9 g (4 mmol) of 2,2'-O-angeborene (II) in 10 ml of abs. DMF was added and 0.98 g (20 mmol.) LiN3, 0.54 g (10 mmol.) NH4Cl, and 0.61 g (10 mmol.) LiCl×H2O and 0.45 ml of hexamethylphosphoramide. The mixture was heated under stirring at 110°C for 8 hours., then was evaporated, to the residue was added 9 ml Meon and 18 ml of acetone and left for 18 h at 0°C. the Precipitate was filtered, washed with 10 ml of a mixture of Meon/acetone (1/3), the solution was evaporated, the residue was dissolved in 3 ml of acetone and filtered through a column of silica gel in acetone (3×10 cm). The filtrate was evaporated. The thick oil was dissolved in 3 ml of Meon and put on a column of silica gel (2×21 cm.) in chloroform. The target product was suirable system chloroform-methanol 8:2. 2'-Azido-2'-deoxyuridine (III) obtained with the yield of 0.59 g (55%).

To a solution of 0.54 g (2 mmol) of 2'-azido-2'-dose irradiation on neurogenesis (III) in 10 ml of a mixture of dioxane-water (1:1) was added triphenylphosphine 1.31 g (5 mmol.) and 2 ml of 25% NH4OH. After 18 h at 20°the mixture was evaporated, dissolved in 50 ml of water, the solution was Proektirovanie chloro what Orme (2× 10 ml) and applied onto a column of Dowex 50 (H+) (3×10 cm), which was washed with 100 ml of 30% Meon. The product was suirable 2% NH4OH in 30% Meon, the eluate was evaporated, the residue was dissolved in 2 ml of water and applied on the column with obremenitve silica gel LiChroprep RP-8 (3×24 cm. Was suirable in the concentration gradient Meon (0→5%) in the presence of 0.01 M NH4The HCO3. The target fraction was evaporated, parupalli with water (3×10 ml) and was liofilizovane. The yield of 2'-amino-2'-dose irradiation on neurogenesis (IVa) 0.39 g (80%).

2'-Amino-2'-deoxyuridine (IV, R=H), 2'-amino-2'-deoxyuridine C9H13N3O5Mr 243.2,

UV: λmax 261 (ε9600), [D2O; δ, ppm; J, Hz)]: of 7.90 d (1H, J5,68, H-6), 5,90 d (1H, J1',2'5,5, N-1'), 5,67 d (1H, H-5), or 4.31 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.90 m (1H, H-4'), 3,62 m (2H, H-5').

Example tintes 5-Methyl-2'-amino-2'-dose irradiation on neurogenesis (IVb, R=CH3)

5-Methyl-2'-amino-2'-deoxyuridine (IV, R=CH3) derived from thymidine, as in example 1, with a yield of 85%.

5-Methyl-2'-amino-2'-deoxyuridine (IV, R=CH3), UV: λmax 265 (ε9800), [D2O; δ, ppm; J, Hz)]: to $ 7.91 s (1H, H-6), of 5.92 d (1H, J1',2'5,5, N-1'), 4,35 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,07 t (1H, H-2'), 3.92 m (1H, H-4'), 3,65 m (2H, H-5'), 1.89 s (3H, CH3).

Example 3. Synthesis of 5-Bromo-2'-amino-2'-dose irradiation on neurogenesis (IVc, R=Br)

5-Bromo-2'-amino-2'-deoxyuridine (IVc, R=Br) was obtained from 5-bromo-2'-dose irradiation on neurogenesis, as in example 1, with a yield of 68%.

5-Bromo-2'-amino-2'-deoxyuridine (IVc, R=Br) 5-Bromo-2'amino-2'-deoxyuridine M-322.12, C9H12BrN3O5UV: λmax 265 (ε9800), [D2O; δ, ppm; J, Hz)]:8,09 s (1H, H-6), of 5.92 d (1H, J1',2'5,5, N-1'), 4,35 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,07 t (1H, H-2'), 3.92 m (1H, H-4'), 3,65 m (2H, H-5'), 1.89 s (3H, CH3).

Example 4. Synthesis of 5-Aminomethyl-2'-amino-2'-dose irradiation on neurogenesis (IVd, R=CH2NH2)

5-Aminomethyl-2'-amino-2'-deoxyuridine (IVd, R=CH2NH2) was obtained from 5-azidomethyl-2'-amino-2'-dose irradiation on neurogenesis[10], as in example 1, with a yield of 63%.

5-Aminomethyl-2'-amino-2'-deoxyuridine (IVd, R=CH2NH2) UV: λmax 265 (ε9800), [D2O δ, ppm; J, Hz)]: to $ 7.91 s (1H, H-6), 5,90 d (1H, J1',2'5,5, N-1'), 4,35 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,07 t (1H, H-2'), 3.90 br q (1H,J4',5'4 H-4'), 3,62 m (2H, H-5'), 2,77t (2H,J 6,CH2NH2).

Example 5. Synthesis of 5-Aminomethyl-2'-amino-2'-dose irradiation on neurogenesis (IVe, R=CH2-O(CH2)6NH2)

5-(6-aminohexanoate)-2'-amino-2'-deoxyuridine (IVe, R=CH2-O(CH2)6NH2) was obtained from 5-(6-acidogenicity)-2'-amino-2'-dose irradiation on neurogenesis [11], as in example 1, with a yield of 65%.

5-(6-aminohexanoate)-2'-amino-2'-deoxyuridine (IVe, R=CH2-O(CH2)6NH2), UV: λmax 265 (ε9800), [D2O δ, ppm; J, Hz)]: 7,89 s (1H, H-6), 5,90 d (1H, J1',2'5,5, N-1'), 4,35 br t (1H, J,2,3'. 5,5, J,3',4'5, H-3'), 4,15 s (2H,5-CH2), 4,07 t (1H, H-2'), 3.90 br q (1H, J4',5'4 H-4'), 3,62 m (2H, H-5'), 3,31 t (2H, J 6,OCH2C5H10), 2,77 t (2H, 6, CH2N), 1.41 m, 1.15 m [8H,(CH2)4].

Example 6. Synthesis of 2'-amino-2'-deoxycytidine (VIIIa, R=H)

To a solution of 2'-azido-2'-dose irradiation on neurogenesis (IIIa) 1.35 g (5 mmol) in 5 ml of abs. pyridine was added 1.2 ml (13.3 mmol) acetic anhydride and the mixture was stirred for 16 hours at 20°C, then evaporated, the residue of parupalli with nbutanol (2×5 ml) and toluene. 3',5'-Diacetyl-2'-azido-2'-deoxyuridine (Va) was obtained in the form of oily sludge with the release of 1.59 g (90%).

3',5'-Diacetyl-2'-azido-2'-deoxyuridine (Va, 2,46 mmol.) in 5 ml of abs. acetonitrile was cooled to -10°C. Triazole (1,43 g, 20 mmol) was dissolved in 5 ml of abs. acetonitrile and 2.9 ml of triethylamine (20 mmol), cooled to -10°and added with stirring POCl3, (0,64 ml and 6.9 mmol). The reaction mixture was stirred 30 min at -10°C, then filtered into a flask with (Va). The reaction mixture was stirred 2 hours at room temperature, then added 0.5 ml of water was evaporated, the residue dissolved in ethyl acetate, washed with water, us. NaHCO3, water, dried Na2SO4and was filtered through a column of silica gel in ethyl acetate (3×10 cm). The filtrate was evaporated. N4-Triazolyl-3',5'-diacetyl-2'-azido-2'-deoxyuridine (VIa) was obtained in the form of oily sludge with access to 0.78 (78%).

To a solution of N4-triazolyl-3',5'-diacetyl-2'-azido-2'-dose irradiation on neurogenesis (VIa, of 0.67 g of 1.64 mmol) in 6 ml of dioxane was added 25 mmol. the corresponding amine ostavil for 48 hours at room temperature, then added 2 ml conc. NH4OH and left for 18 h at room temperature. The reaction mixture was evaporated, the residue was dissolved in 50 ml of 30% Meon and put on a column of Dowex 50 (H+) (3×10 cm), which was washed with 100 ml of 30% Meon. The product was suirable 2% NH4OH in 30% Meon, the eluate was evaporated, the residue was dissolved in 2 ml of water and applied on the column with obremenitve silica gel LiChroprep RP-8 (3×24 cm. The product was suirable in the concentration gradient Meon (0→30%) in the presence of 0.01 M NH4HCO3. The target fraction persuasively with water (3×10 ml) and was liofilizovane. 2'-Azido-2'-deoxycytidine (VIIa) was obtained with the yield 0.33 g (75%).

To a solution of 2'-azido-2'-deoxycytidine (VI, of 0.54 g, 2 mmol) in 10 ml of a mixture of dioxane-water (1:1) was added triphenylphosphine (1.31 g, 5 mmol) and 2 ml of 25% NH4OH. After 18 h at 20°the mixture was evaporated, dissolved in 50 ml of water, the solution was Proektirovanie chloroform (2×10 ml) and applied onto a column of Dowex 50 (H+) (3×10 cm), which was washed with 100 ml of 30% Meon. The product was suirable 2% NH4OH in 30% Meon, the eluate was evaporated, the residue was dissolved in 2 ml of water and applied on the column with obremenitve silica gel LiChroprep RP-8 (3×24 cm. Was suirable in the concentration gradient Meon (0→30%) in the presence of 0.01 M NH4HCO3. The target fraction was evaporated, parupalli with water (3×10 ml) and was liofilizovane. The yield of 2'-amino-2'-deoxycytidine (VIIIa) 0.39 g (80%).

2'-Amino-2'-d is toxicitiy (VIII, R=R1=R2=H), M=242, UV: pH 7.5: λmax 271 (ε8200), pH 2: λmax 276 (ε11900), [DMSO-d6; δ, ppm; J, Hz)]: 7,79 d (1H, J5,6of 7.5, H-6), 7,12 br s (2H, NH2), 5,79 d (1H, H-5), 5,67 d (1H, J1',2'5,5, H-1'), or 4.31 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.90 br q (1H, J4',5'4 H-4'), 3,62 m (2H, H-5'). 2'-amino-2'-deoxycytidine.

Example 7. Synthesis of (5-Methyl)-2'-amino-2'-deoxycytidine (VIIIb, R=CH3, R1=R2=H))

(5-Methyl)-2'-amino-2'-deoxycytidine (VIIIb, R=CHC, R1=R2=H) is obtained from (5-methyl)-2'-azido-2'-dose irradiation on neurogenesis (IIIb), as in example 6, with a total yield of 52%

(5-Methyl)-2'-amino-2'-deoxycytidine (VIIIb, R=CH3, R1=R2=H), UV: pH 7.5: λmax 272 (ε8200), pH 2: λmax 278 (ε11900), [DMSO-d6; δ, ppm; J, Hz)]: to $ 7.91 s (1H, H-6), 7,14 br s (2H, NH2), 5,79 d (1H, J1',2'5,5, N-1'), 4,35 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,07 t (1H, H-2'), 3.92 m (1H, 4 H-4'), 3,65 m (2H, H-5'), 1.89 s (3H, CH3).

Example 8. Synthesis of (5-Aminomethyl)-2'-amino-2'-deoxycytidine (VIIIc, R=CH2NH2, R1=R2=H)

(5-Aminomethyl)-2'-amino-2'-deoxycytidine (VIIIc, R=CH2NH2, R1=R2=H) is obtained from (5-azidomethyl)-2'-azido-2'-dose irradiation on neurogenesis (IIIc), as in example 6, with a total yield of 43%.

(5-Aminomethyl)-2'-amino-2'-deoxycytidine (VIIIc, R=CH2NH2, R1=R2=H), UV: pH 7.5: λmax 274 (ε8200), pH 2: λmax 280 (ε11900), [DMSO-d6; δ, ppm; J, Hz)]: 7,89 s (1H, H-6), 7,14 br s (2H, NH2), of 5.82 d (1H, J1',2' 5,5, N-1'), 4,36 br t (1H, J,2,3'5,5, J3',4'5, H-3'), 4,07 t (1H, H-2'), 3.90 m (1H, H-4'), 3,62 m (2H, H-5'), 2,77 t (2H, J 6,CH2NH2).

Example 9. Synthesis of N4-hydroxy-2'-amino-2'-deoxycytidine (VIIId, R=R1=H, R2=OH)

N4-hydroxy-2'-amino-2'-deoxycytidine (VIIId, R=R1=H, R2=OH) was obtained from 2'-azido-2'-dose irradiation on neurogenesis (IIId), as in example 6, with a total yield of 51%.

N4-hydroxy-2'-amino-2'-deoxycytidine (VIIId, R=R1=H, R2=HE), UV: pH 7.5: λmax 271 (ε8200), pH 2: λmax 276 (ε11900), [DMSO-d6; δ, ppm; J, Hz)]: 7,79 d (1H, J5,67,5 H-6), 7,12 br s (2H, NH2), 5,79 d (1H, H-5), 5,67 d (1H, J1',2'5,5, N-1'), or 4.31 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.90 br q (1H, J4',5'4 H-4'), 3,62 m (2H, H-5').

Example 10. Synthesis of N4-amino-2'-amino-2'-deoxycytidine (VIIIe, R=R1=H, R2=NH2)

N4-amino-2'-amino-2'-deoxycytidine (VIIIe, R=R1=N, R2=NH2) was obtained from 2'-azido-2'-dose irradiation on neurogenesis (IIIe), as in example 6, with a total yield of 39%.

N4-amino-2'-amino-2'-deoxycytidine (VIIIe, R=R1=N, R2=NH2) M=257.25,9H15O4UV: pH 7.5: λmax 271 (ε8200), pH 2: λmax 276 (ε11900), [DMSO-d6; δ, ppm; J, Hz)]: 7,79 d (1H, J5,6of 7.5, H-6), 7,12 br s (2H, NH2), 5,79 d (1H, H-5), 5,67 d (1H, J1',2'5,5, H-1'), or 4.31 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.90 br q (1H, J,4',5'4 H-4'), 3,62 m (2H, N4-Amino-2'-amino-2'-deoxycytidine.

Example 11. Synthesis of N4-Benz is l-2'-amino-2'-deoxycytidine (VIIIf, R=R1=N, R2=CH2With6H5)

N4-benzyl-2'-amino-2'-deoxycytidine (VIIIf, R=R1=N, R2=NHCH2With6H5) was obtained from 2'-azido-2'-dose irradiation on neurogenesis (IIIe), as in example 6, with a total yield of 52%.

N4-benzyl-2'-amino-2'-deoxycytidine (VIIIf, R=R1=N, R2=NHCH2C6H5), M=332.36, C16H20N4O4UV: pH 7.5: λmax 271 (ε8200), pH 2: λmax 276 (ε11900), [DMSO-d6; δ, ppm; J, Hz)]: 7,79 d (1H, J5,6of 7.5, H-6), 7,25 m (5H,6H5) 7,12 br s (2H, NH2), 5,79 d (1H, H-5), 5,67 d (1H, J1',2'5,5, N-1'), 4,4 s (2H,CH2With6H5), or 4.31 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.90 br q (1H, J4',5'4 H-4'), 3,62 m (2H, H-5'). N4-Benzyl-2'-amino-2'-deoxycytidine

Example 12. Synthesis of N4-diethyl-2'-amino-2'-deoxycytidine (VIIIg, R=H, R1=R2=C2H5)

N4-diethyl-2'-amino-2'-deoxycytidine (VIIIg, R=H, R1=R2=C2H5) was obtained from 2'-azido-2'-dose irradiation on neurogenesis (IIIe), as in example 6, with a total yield of 58%.

N4-diethyl-2'-amino-2'-deoxycytidine (VIIIg, R=H, R1=R2=C2H5), M=298.34, C13H22N4O4UV: pH 7.5: λmax 271 (ε8200), pH 2: λmax 276 (ε11900), [DMSO-d6; δ, ppm; J, Hz)]: 7,79 d (1H, J5,6of 7.5, H-6), 7,12 br s (2H, NH2), 5,79 d (1H, H-5), 5,67 d (1H, J1',2'5,5, N-1'), or 4.31 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.50 m (5H, H-4' + (CH CH3)2), 3,62 m (2H, H-5'), 1.1 s (6H, (CH2CH3)2). N4-Diethyl-2'-amino-2'-deoxycytidine.

Example 13. Synthesis of N4-ethyl-2'-amino-2'-deoxycytidine (VIIIh R=R1=H, R2=C2H5)

N4-ethyl-2'-amino-2'-deoxycytidine (VIIIh R=R1=N, R2=C2H5obtained from 2'-azido-2'-dose irradiation on neurogenesis (IIIe), as in example 6, with a total yield of 53%. N4-ethyl-2'-amino-2'-deoxycytidine (VIIIh R=R1=N, R2=C2H5), M=270.29, C11H18N4O4UV: pH 7.5: λmax 271 (ε8200), pH 2: λmax 276 (ε11900), [DMSO-d6; 5, ppm; J, Hz)]: 7,79 d (1H, J5,6of 7.5, H-6), 7,12 br s (2H, NH2), 5,79 d (1H, H-5), 5,67 d(1H, J1',2'5,5, N-1'), 4,3 1br t(1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.90 br q (1H, J4',5'4 H-4'), 3,62 m (2H, H-5' (a, b), 3,21 m (2H,CH2CH3), of 1.03 m (3H, CH2CH3). N4-Ethyl-2'-ATTO-2'-deoxycytidine.

Example 14. Synthesis of N4-methyl-2'-amino-2'-deoxycytidine (IX, R=R1=N, R2=CH3)

N4-methyl-2'-amino-2'-deoxycytidine (IX, R=R1=N, R2=CH,) was obtained from 2'-azido-2'-dose irradiation on neurogenesis (IIIe), as in example 6, with a total yield of 58%. N4-methyl-2'-amino-2'-deoxycytidine (IX, R=R1=N, R2=CH3), M=256.26, C10H16N4O4UV: pH 7.5: λmax 271 (ε8200), pH 2: λmax 276 (ε11900), [DMSO-d6; 5, ppm; J, Hz)]: 7,79 d (1H, J5,6of 7.5, H-6), 7,12 br s (2H, NH2), 5,79 d (1H, H-5), 5,67 d (1H, J 1',2'5,5, N-1'), or 4.31 br t (1H, J,2,3'5,5, J,3',4'5, H-3'), 4,03 t (1H, H-2'), 3.90 br q (1H,.J4',5'4 H-4'), 3,62 m (2H, H-5'), 2,71 m (1H, CH3). N4-Methyl-2'-amino-2'-deoxycytidine.

Example 15. Evaluation of the cytotoxicity of 2'-amino-2'-deoxynucleosides

The cytotoxicity of the compounds evaluated by adding dilutions of each drug in the environment of the Needle MEM to a monolayer of Vero cell culture in the wells of 96-hole tablet ("Cel-Cult", England) to a final concentration of 0.1-400 µg/ml (three wells for each dose), followed by cultivation at 37°in CO2-the incubator for 5 days. The controls are cells without addition of the compound (1). Cell viability assessed by optical density (570 nm) after staining with crystal violet solution (1.3 g dye, 50 ml of ethanol, up to 700 ml of N2About 300 ml of 40% formalin) for 1.5 hours at room temperature. The toxicity of different doses of the compounds is determined by cell viability relative to control, according to the obtained results to calculate the dose by 50% reduce cell viability (CD50). The data are shown in table 1. It should be noted that 2'-amino-2'-deoxynucleoside have a low level of cytotoxicity.

Example 16. The study of the influence of 2'-amino-2'-deoxynucleosides on the reproduction of measles virus in cell culture Vero

The antiviral activity impact the mental preparations against measles virus carried out on transplantable lines sensitive cells of green monkey kidney Vero. For infection use of the supernatant of infected cells stored in liquid nitrogen, multiplicity of infection of 0.2 to 0.5 infectious units per cell. To the wells of the 96-hole tablet ("Cel-Cult", England) is placed a suspension of Vero cells with seed concentration of 150 thousand cells/ml, incubated for 2 days at 37°in CO2-incubator. After formation of the monolayer from the wells to remove the culture fluid, making the virus (15 ál) and at the same time the same volume of growth medium with a test compound to a final concentration of 0.1-400 µg/ml (three wells for each dose). The controls are infected Vero cells without addition of the drug instead of the drug make the same amount of MEM eagle medium without additives) and uninfected cells.

The adsorption of the virus carried out for 1.5 hours at 37°in CO2-incubator. Then in the wells contribute nutrient medium Needle MEM with the addition of 2% fetal cattle serum, previously inactivated by heating at 56°C for 30 minutes, 300 mg/ml L-glutamine, 100 µg/ml gentamicin, 100 μg/ml of lincomycin and investigational compound to a final concentration of 0.1-400 µg/ml In the control wells contribute nutrient medium with all the additives, but without the drug. After 5 days of cultivation remove the culture fluid contribute to 30 μl of a solution of crystal violet in to the introduced each hole, incubated 1.5 hours at room temperature and measure the absorbance on the spectrophotometer at a wavelength of 570 nm. The relative degree of protection of infected cells calculated by the formula:

Protection (%)=(A-O)/(O)·100%,

where a is the % viability of infected cells in the presence of the compounds for 5 days of culture, In % viability of control cells,% viability of infected cells without addition of the compound (control virus).

The data and build the graph of the protection of cells from increasing doses of compounds (compound VIIIa figure 1) and find the dose by 50% protect cells (ED50). Therapeutic index, or index of selectivity (IS), defined as the ratio of the concentration of the compound that is toxic to 50% of the cells (CD50), to a concentration of 50% protect cells from death (ED50):

IS=CD50/ED50

Table 1 presents the results for cytotoxicity and measles activity of 2'-amino-2'-deoxynucleosides. The data show that a pronounced antiviral activity of measles has compound (VIIIa), the results of which are shown in the table. 2. The compound (IVa) has the measles activity comparable to the antiviral action of ribavirin.

Table 1

Data on cytotoxicity and antiviral activity of 2'-amino-2'-deoxynucleosides against measles virus
ConnectionCD50that μMED50that μMIS
IVa8231405,9
IVc>620,9does not reach
VIIIa846,725,433,3
VIIId>774,6does not reach
VIIIf>601,9601,9>1
VIIIg>670,4670,4>1
IX>of 780.5

Table 2 shows that 50% effective dose of ribavirin is 57 μm or 12.4 µg/ml, which corresponds to the data of the literature [12]. 50% effective dose of the compound (VIIIa) was 25.4 microns or at 6.25 µg/ml 50% cytotoxic dose received for ribavirin and compound (VIIIa), differ slightly (846,7 and 823 μm, respectively), indices of selectivity, or therapeutic indexes, equal 14,37 and 33.3, respectively. Thus, the antiviral effect of the compounds (VIIIa) in respect of the measles virus prevoshodo the antiviral activity of ribavirin in vitro.

Example 17. The study of the influence of compounds (IVa) on the reproduction of measles virus in the culture of Vero cells in combination with ribavirin.

The antiviral activity of the claimed compounds in combination with ribavirin spend on transplantable cell line of green monkey kidney Vero. For infection use of the supernatant of infected cells stored in liquid nitrogen, multiplicity of infection of 0.2 to 0.5 infectious units per cell. To the wells of the 96-hole tablet ("Cel-Cult", England) is placed a suspension of Vero cells with seed concentration of 150 thousand cells/ml, incubated for 2 days at 37°in CO2-incubator. After formation of the monolayer from the wells to remove the culture fluid, making the virus (15 ml) and immediately the same volume of growth medium with the test compounds at a concentration of: ribavirin - of 3.125-25 mg/ml, the compound (IVa) - 8,750-70 mcg/ml (three wells for each combination of doses). The controls are infected Vero cells without addition of drugs (contributing the same amount of RPMI-1640 medium without additives) and uninfected cells.

The adsorption of the virus carried out for 1.5 hours at 37°in CO2-incubator. Then in the wells contribute nutrient medium RPMI-1640 with the addition of 2% fetal cattle serum, previously inactivated by heating at 56°C for 30 minutes, 300 mg/ml L-glutamine and 100 μg/ml of gentamicin, containing the studied combination of compounds at the desired concentration. In the control wells contribute nutrient medium with all the additives, but without connections. After 5 days of cultivation remove culture medium, contribute 30 µl per well of a solution of gentian Violeta, incubated 1.5 hours at room temperature and measure the absorbance on the spectrophotometer at a wavelength of 570 nm. The relative degree of protection of infected cells from destruction by various combinations of ribavirin and compound (IVa) are calculated according to the formula:

Protection (%)=(A-O)/(O)×100,

where a is the % viability of infected cells in the presence of compounds on the 4th day of cultivation, -% viability of control cells,% viability of infected cells without the addition of compounds (1) and / or ribavirin (control virus).

The data and construct graphs of the protection of cells from increasing doses of ribavirin and compound (IVa) (figure 2) and find the dose of ribavirin, which in combination with the compound (IVa) by 50% protects cells from death, and the dose of compound (IVa), which in combination with ribavirin 50% protects cells from death (ID50). Table 3 presents the values of ID50calculated for different combinations of ribavirin and compound (IVa).

From table 3 it can be seen that the use of the combination with ribavirin compound (IVa) in a concentration of 70 μm reduces the 50% effective dose of ribavirin 4.45 times.

Example 18. The influence of soedineniya (VIIIa) on the reproduction of Marburg virus in cell culture Vero.

The study was conducted using the quantitative determination of virus in cell culture under semisolid agar surface.

Transplantable line sensitive cells of green monkey kidney Vero contribute to peplace (seeding concentration of 200 thousand cells/ml) and incubated for 2 days at 37°C in an atmosphere of 5% CO2. After formation of the monolayer of replacenew remove the culture fluid, making the virus at a dose of 5.5×104The BATTLE in the amount of 0.1 ml Adsorption is carried out at 37°C. Then in bottles make 2 ml of semisolid agar coating (Wednesday Needle MEM with 2% siveroni, 10% of agar (2,4% gel), antibiotics, 100 U/ml and the drug) and incubated for 6 days at 37°C. Controls are infected Vero cells without addition of drug (control virus) and uninfected cells (control cells). After cultivation agar coating is removed, the monolayer of dye crystal violet for 1 hour, washed with water and count the number of plaques. The results are presented in table 4.

According to the data presented in table 4, determine the combination index, FIC, which is equal to:

FIC=ID50 substances And mixtures/ID50 substance And+ID50 substances in the mixture/ID50 substances In

Combinational index which allows you to assess the nature of the interaction of the drugs in the mixture: if the FIC is less than 1, conclude synergistic interaction of medicines, if the FIC is greater than 1, then antagonistic interaction [13]. The combination index for the combination of ribavirin and compound (IVa) is equal to:

FIC=12,8/57+49/140=0,575, i.e. the interaction of ribavirin and compound (IVa) synergistic.

Table 4

Inhibition of reproduction of the virus Marburg compound (VIIIa)
The drug concentration, µg/mlThe number of plaques
IMB-119The control virus
Total*x±sxTotal*x±Sx
100,04114,0±1,578,026,0±2,0
50,03812,6±2,8
25,03612,0±2,0
12,54414,6±1,2
6,257324,3±2,8
* the Sum of the three dimensions

Presented in the table 4 data show that inhibition of reproduction of the virus Marburg did not reach 100% even when the dose of 100 µg/ml, but concentrations of connection is possible (VIIIa), equal to 12.5 ág/ml and above, reduced the number virusinduced plaques by 50%.

Thus, it is shown that 2'-amino-2'-deoxyuridine and 2'-amino-2'-deoxycytidine have a weak cytotoxicity in cell culture Vero (CD50equal 823 and 846,7 μm, respectively) and a pronounced antiviral activity against measles virus (ID50equal to 140 mcg and 25.4 μm, respectively). In addition, 2'-amino-2'-deoxycytidine has antiviral activity against virus Marburg: at a concentration of 12.5 μg/ml or more, the compound (VIIIa) reduces virusinduced plaques or virusinduced cytopathic effect in cell culture Vero 50%.

2'-Amino-2'-deoxycytidine for its antiviral properties than the comparator drug ribavirin, and therefore, it seems promising drug development based on the compound (VIIIa) for the treatment of infections caused by measles virus or Marburg.

Sources of information

1. W.H. Mason Measles. // Adolesc. Med. - 1995. - v.6. - p.1-14.

2. Hosoya M., Shigeta, S., Nakamura K., De Clerq E. Inhibitory effect of selected antiviral compounds on measles (SSPE) virus replication in vitro. // Antiviral res. - 1989. - v.12. - p.87-98.

3. Shigeta, S., Mori, S., Baba, M., Honzumi K., Nakamura K., Numazaki Y., Matsuda, A., Obara T. Antiviral activities of ribavirin, 5-ethynyl-1-b-ribofuranosylimidazole-4-carboxamide, and 6'-(R)-methylneplanocin A against several ortho - and paramyxoviruses. // Antimicrob. Agents chemother. - 1992. - v.36. - p.435-9.

4. Application for patent of the RF No. 96117323, M Is To 07 N 19/06, publ. Running from 10.12.1998.

5. International application WO No. 00/15214, IPC a 61 K 31/19, publ. 23.03.2000

6. Verheyden. J.P.H., Wagner. D., Moffat. J.G. // Synthesis of some pyrimidine 2'-amino-2'-deoxynucleosides. // J. Org. Chem. V.36, pp.250-254, 1971.

7. Wagner. D., Verheyden. J.P.H., Moffat. J.G. // Preparation and synthetic utility of some organotin derivatives of nucleosides. // J. Org. Chem., V.39, pp.24-30,1974.

8. Matsuda. A., Yasuoka. J., Sasaki T., Ueda. So // Nucleosides and Nucleotides. 95. Improved synthesis of 1-(2-azido-2-deoxy-β-D-arabinofuranosyl)cytosine (Cytarazide) and thymine. Inhibitory spectrum of cytarazide on the growth of various human tumor cells in vitro. // J. Med. Chem., V.34, pp.999-1002, 1991.

9. McGee. D.P.C., Vaughn-Settle. A., Vargeese. C., Zhai. Y // 2'-Amino-2'-deoxyuridine via an intermolecular cyclization of a trichloroacetimidate // J. Org. Chem., V.61, pp.781-785, 1996.

10. Barwoiff, D., Langen, P. // in Townsend, L.B. and Tipson, R.S., (eds.) Nucleic Acid Chemistry, Wiley, NY. Vol.1, pp.359-366.

11. Alexandrova L.A., Skoblov monitoring computerized Jasko M.V., Victorova L.S., Krayevsky A.A., 2'-Deoxynucleoside 5'-triphosphates modified at α-, β- and γ-phosphates as substrates for DNA polymerases, Nucl. Acids Res., 1998, 26, No. 3, 778-786.

12. Watanabe W., K. Konno, Ijichi, K., Inoue H., Yokota T., Shigeta S. MTT colorimetric assay system for the screening of anti-orthomtxo - and anti-paramyxoviral agents. // J.Virol. Nethods. - 1994. - v.48. - p.257-65.

13. Suhnel J. Evaluation of synergism or antagonism for the combined action of antiviral agents. // Antiviral res. - 1990. - v.13. - p.23-40.

2'-Amino-2'-deoxynucleoside having the formula

where R = H, alkyl, aminoalkyl,

R1= (R2NR3), where R2and/or R3= H, HE, NH2, alkyl, benzyl,

provided that R is not H or methyl when R2and R3= N.



 

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