RU2293739C2 - 3'-azido-3'-deoxythymidine 5'-choline phosphate as antiviral agent - Google Patents

Abstract

FIELD: organic chemistry, virology.

SUBSTANCE: invention relates to a novel compound, namely 3'-azido-3'-deoxythymidine 5'-choline phosphate

possessing antiviral activity. Indicated compound shows the enhanced penetration through cellular membranes.

EFFECT: improved and valuable property of agent.

1 tbl, 5 ex

Description

The present invention relates to a new biologically active derivative of 3'-azido-3'-deoxythymidine, namely, its 5'-cholinophosphate.

This compound has an antiviral effect, primarily against human immunodeficiency virus (HIV).

It is known that it is possible to inhibit the reproduction of the immunodeficiency virus at different stages of its life cycle, but it is obvious that it is advisable to use inhibitors of the earliest processes. That is why HIV reverse transcriptase, the first enzyme to function in the cycle of virus replication, is the most attractive target for suppressing virus reproduction. Currently, the main group of drugs used to treat AIDS are the nucleoside precursors of reverse transcriptase inhibitors. These include 3'-azido-3'-deoxythymidine (AZT, AZT, zidovudine, Retrovir, Timazide), 2 ', 3'-dideoxycytidine (ddC, zalcitabine, Guid), 2', 3 ' dideoxyinosine (ddI, didanosine, Videx), 3'-deoxy-2 ', 3'-didehydrothymidine (d4T, stavudine, Zerit) and 3'-thiocytidine (3TC, lamivudine, Epivir). The main disadvantage of these compounds is the need for their phosphorylation in the cell to the corresponding triphosphates, the effectiveness of which is extremely low. So the process of converting AZT in the human body into the corresponding 5'-triphosphate takes about 1.5-2 hours. Moreover, the first phosphorylation to AZT monophosphate is the limiting stage. Since the yield of triphosphate reaches only a few hundredths of a percent, large doses of the drug must be used for treatment. This, in turn, leads to numerous side effects and the rapid development of viral and cellular resistance.

The use of nucleoside-5'-triphosphates with unmodified triphosphate moieties as medications is impossible because of their low stability to the action of hydrolysis enzymes and, as a result, their low ability to penetrate into the cell. Attempts have been made to synthesize modified nucleoside 5'-triphosphates (WO 98/20017). However, the compounds were not active enough to create a drug.

More promising were those forms of active nucleosides that made it possible to reduce the phosphorylation process in the cell to two stages, were permeable to cell membranes, and were stable in the bloodstream for a long time. One of these compounds described in European Patent No. 0,354,246 is 5′-H-phosphonate of 3′-azido-3′-deoxythymidine (Phosphazide) having the following formula:

Phosphazide is currently used as a drug called Nikavir for the treatment of AIDS (HIV infection).

Another approach is to create an AZT monophosphate ester having an enzymatically hydrolyzable choline group. This approach allows you to bypass the first stage of coking, and the positive charge of the choline residue compensates for the charge of phosphate. Thus, AZT choline phosphate is a practically uncharged compound that can efficiently penetrate cell membranes.

In accordance with the present invention, there is provided 5'-choline phosphate of 3'-azido-3'-deoxythymidine of formula I:

The mechanism by which a compound of formula I is converted into an active form within a cell is currently unknown. The main proposed mechanism is that enzymatic cleavage of the choline group occurs with the release of AZT phosphate.

Two synthetic approaches can be used to produce 3'-azido-3'-deoxythymidine 5'-cholinphosphate.

The first of these consists in the condensation of AZT (II) with choline phosphate, and in the second AZT, monophosphate (III) reacts with choline. Using both methods, compound I is obtained, which can be isolated and purified by conventional methods, for example, extraction, precipitation, chromatography, etc.

The present invention is illustrated by the following examples of the preparation of this compound and the results of biochemical tests.

Example 1

To an emulsion of choline phosphate (0.353 g, 1.6 mmol) in 0.62 ml of dimethylformamide, methanesulfonyl chloride (244 mg, 2.13 mmol) was added with stirring at room temperature. After dissolution (about 15 minutes), 3'-azido-3'-deoxythymidine (175 mg, 0.64 mmol) was added to the resulting viscous solution. The reaction mixture was stirred for 24 hours at 37 ° C and then the reaction was stopped by adding 25 ml of 0.5 M NH ₄ HCO ₃ . Unreacted starting and non-polar products are extracted with ethyl acetate (2 × 10 ml), the aqueous solution is evaporated. The residue was dissolved in 1 ml of water and choline phosphate was precipitated by the addition of 10 ml of ethanol. The mother liquor is evaporated, the residue is dissolved in 2 ml of water and chromatographed on an RP-8 column (2 × 20 cm), eluting with a linear gradient (0 → 10%) of methanol in 0.05 M NH ₄ HCO ₃ (400 ml). After purification, 71 mg (26%) of compound (I) is isolated as an internal salt.

холина); 4.10-3.97 м (3Н, 2Н-5'+Н-4'); 3.60т_N (j_N,H=4.4 Гц, 2Н,

холина); 3.12с (9Н, Ме₃ холина) 2.47 ~ т (J=6.4 Гц, 2Н, 2Н-2'); 1.82т (3Н, 5-Ме). ¹ H-NMR: 7.58s (1H, H-6); 6.16 s (J = 6.5 Hz, 1H, H-1 '); 4.38 ~ k (J = 5.7 Hz, 1H, H-3 '); 4.21 br.s (2H,

choline); 4.10-3.97 m (3H, 2H-5 '+ H-4'); 3.60 t _N (j _{N, H} = 4.4 Hz, 2H,

choline); 3.12s (9H, Me ₃ choline) 2.47 ~ t (J = 6.4 Hz, 2H, 2H-2 '); 1.82t (3H, 5-Me).

³¹ P-NMR: -0.33 s.

холина); 68.1д (J_С,Р=4.8 Hz, С-5'); 63.3 (С-3'); 62.4д (J_С,Р=4.8 Гц,

холина); 57.1 т_N (J_N,C=3.8, Ме₃ холина); 39.2 (С-2'); 14.6 (5-Ме). ¹³ C-NMR: 169.3 + 154.6 (C-2 &C-4); 140.3 (C-6); 114.6 (C-5); 88.1 (C-1 '); 85.8 d (J _{C, P} = 8.8 Hz, C-4 '); 69.1 (

choline); 68.1d (J _{C, P} = 4.8 Hz, C-5 '); 63.3 (C-3 '); 62.4d (J _{C, P} = 4.8 Hz,

choline); 57.1 t _N (J _{N, C} = 3.8, Me ₃ choline); 39.2 (C-2 '); 14.6 (5-Me).

UV (ethanol): λ _max = 265 (8440), λ _max / λ _min 3.3.

MS: (FAB> 0): 433 (MH), (FAB <0): 431 (M-H ⁺ ).

HRMS: calculated for MH ⁺ C ₁₅ H ₂₆ N ₆ O ₇ P - 433.1601, found - 433.1676.

Example 2

Methanesulfonyl chloride (0.232 g, 2.03 mmol) was added to an emulsion of choline phosphate (340 mg, 1.52 mmol) in 0.45 ml of dimethylformamide with stirring at room temperature. After dissolution of the choline phosphate, 3'-azido-3'-deoxythymidine (0.187 g, 0.70 mmol) was added. The reaction mixture was stirred for 3 hours at 50 ° C. A solution of 2,4,6-triisopropylbenzenesulfonyl chloride (520 mg, 1.62 mmol) in 1.5 ml of pyridine is added to the resulting viscous mass. The reaction mixture was stirred for 4 hours at 50 ° C. and then for 24 hours at room temperature. Processing of the reaction mixture and isolation of AZT choline phosphate (I) is carried out analogously to example 1. After purification, 136 mg (45%) of compound (I) is isolated in the form of an internal salt.

Example 3

To a solution of 3′-azido-3′-deoxythymidine 5′-phosphate (185 mg, 0.53 mmol) in 5 ml of dimethylformamide was added choline bromide (190 mg, 1.03 mmol) and 2,4,6-triisopropylbenzenesulfonyl chloride (780 mg, 2.43 mmol) . The reaction mixture was stirred for 24 hours at 37 ° C, then dimethylformamide was evaporated and the reaction mixture was treated as in Example 1. After purification, 74 mg (32%) of compound (I) was isolated as an internal salt.

Example 4. The study of the antiviral activity of 5'-cholinophosphate 3'-azido-3'-deoxythymidine against HIV.

The study was performed using strain HIV-1 HBV 4046 on the transplantable line of sensitive cells MT-4. For infection, the supernatant of infected cells stored in liquid nitrogen was used; the multiplicity of infection was 0.2-0.5 infectious units per cell. A suspension of MT-4 cells with a concentration of 2 × 10 ⁶ per milliliter and a viability of at least 90% was placed in the wells of a 96-well plate (Cel-Cult, United Kingdom) immediately after the introduction of the virus-containing material and the test preparation diluted in the medium was immediately applied RPMI-1640 without additives to final concentrations of 0.001-100 μg / ml (three wells per dose). The controls were HIV-1 infected MT-4 cells without drug addition (instead of the drug, the same amount of RPMI-1640 medium without additives was added), as well as uninfected cells.

The plate was incubated for one hour at 37 ° C to adsorb the virus, then the cells were diluted to a seed concentration (0.5 × 10 ⁶ per milliliter) of RPMI-1640 nutrient medium supplemented with 300 mg / ml L-glutamine and 100 μg / ml gentamicin, as well as 10% fetal cattle serum, previously inactivated by heating at 56 ° C for 30 minutes. The tablet was placed in a thermostat at 37 ° C in an atmosphere of 5% CO ₂ . On the 3-4th day of cultivation, the concentration and cell viability were calculated by the exclusion of trypan blue. According to the data obtained, the increase in cell viability relative to control under the influence of increasing doses of drugs was determined.

Tween-80 was added to the wells of the plate to a final concentration of 0.1% and placed for 24 hours at 4 ° C to inactivate HIV. Then, the anti-HIV activity of the preparation was evaluated using a quantitative determination of the virus-specific protein p24 by direct enzyme immunoassay. Doses of the drug that suppress the increase in viral antigen by 50 and 90% (ID ₅₀ and ID ₉₀ ) were determined according to the schedule. The data obtained are presented in the table. Based on these quantitative indicators of inhibition, one can judge the effectiveness of the antiviral effect of the drug, which consists in a high degree of suppression of HIV-1 multiplication in MT-4 cell culture.

Example 5. The study of the cytotoxicity of 5'-choline phosphate 3'-azido-3'-deoxythymidine.

Evaluation of the cytotoxicity of the drug was carried out by adding dilutions in RPMI-1640 medium to MT-4 cell suspension, placed in the wells of a 96-well plate, to final concentrations of 0.001-100 μg / ml (in triplicate), followed by cultivation at 37 ° C in within 3-4 days. The cells served as control without the addition of the drug. The inoculum concentration was 0.5 × 10 ⁶ per milliliter. Cell viability was counted in a Goryaev chamber with preliminary trypan blue staining. Based on the results obtained, the doses of the drug were calculated, which reduce cell viability by 50% compared to the control (CD ₅₀ ), the data are shown in the table. It should be noted that the toxic effect on MT-4 cells, manifested in a change in their morphology, decreased reproductive capacity and viability, was exerted only by the maximum doses of the preparations (100 μg / ml), 4-5 orders of magnitude higher than the concentrations effective against HIV-1. The selectivity of the action of 5'-cholinophosphate 3'-azido-3'-deoxythymidine was judged by the selectivity index IS = CD ₅₀ / ID ₅₀ .

Thus, it has been shown that 5'-choline phosphate of 3'-azido-3'-deoxythymidine has the ability to inhibit the reproduction of human immunodeficiency virus type 1 in MT-4 cell culture, and the effectiveness of this drug exceeds the effectiveness of the AZT drug used in clinical practice. This suggests the promise of 5'-cholinophosphate 3'-azido-3'-deoxythymidine to create new dosage forms that can replenish the arsenal of drugs used in the treatment of AIDS.

Table.
Antiviral activity of AZT 5'-cholinphosphate against HIV-1 GKV-4046 Connection Method CD ₅₀ μm ID ₅₀ μm ID ₉₀ , μM IS after virus adsorption > 231.5 0.0003 0.0175 > 770,000 Simultaneous > 231.5 0.0002 0.025 > 1150000

Claims (1)

5'-Cholinphosphate 3'-azido-3'-deoxythymidine of the formula

as an antivirus agent.