RU2188203C2 - 2',3'-didehydro-2',3'-dideoxythymidine-5'-[(ethoxy-carbonyl)(ethyl)phosphonate] as inhibitor of reproduction of human immunodeficiency virus - Google Patents

Abstract

FIELD: organic chemistry, molecular biology, virology, medicine. SUBSTANCE: invention relates to a novel derivative of 2',3'-dideoxy-2',3'-didehydrothymidine (d4T), i. e. 2',3'-didehydro-2',3'-dideoxy-thymidine-5'-[(ethoxycarbonyl)(ethyl)phosphonate] as an inhibitor of human immunodeficiency virus (HIV). An antiviral activity of the novel compound with respect to HIV exceeds that of d4T. EFFECT: enhanced antiviral activity. 1 dwg, 1 tbl

Description

 The invention relates to the field of molecular biology, virology and medicine, namely to the use of new derivatives of nucleosides to suppress the reproduction of the human immunodeficiency virus.

 Currently, there are a number of compounds with antiviral activity against HIV, including those used in medical practice. These include nucleoside derivatives such as 3-azido-3-deoxythymidine (AZT), 2'3'-dideoxycytidine, 2'3'-dideoxyinosine, 2'3'-dideoxy-2'3'-didehydrotimidine and 2'3 '-dideoxy-3'-thiotimidine [1-4]. The mechanism of action of these compounds consists in penetrating into infected cells, where they undergo triphosphorylation and specifically block DNA synthesis catalyzed by HIV reverse transcriptase.

 However, compared with natural nucleosides, the process of phosphorylation of modified nucleosides by cellular enzymes is much less efficient, which allows time for the virus that has entered the cells to integrate into the genome in the form of proviral DNA. In addition, the need to create new modified nucleoside derivatives is dictated by the rather high toxicity of the drugs used (AZT) and the emergence of resistant strains even in the case of combined treatment [5,6].

 An object of the invention is the creation of new nucleoside derivatives that are highly effective against HIV, resistant to dephosphorylation enzymes that can penetrate into the cell and have selective activity in inhibiting DNA synthesis catalyzed by HIV reverse transcriptase.

Ингибирование репродукции ВИЧ включает культивирование первично инфицированных лимфоидных клеток МТ-4 в присутствии исследуемого препарата, конечная концентрация которого в культуральной среде составляет 0,001-100 мкг/мл, на протяжении одного пассажа - в течение 4 суток.The problem is solved by the fact that according to the invention a new compound is claimed - a derivative of 2'3'-dideoxy-2'3'-didehydrothymidine (d4T) [prototype; 4] -2 ', 3'-didehydro-2', 3'-dideoxythymidine-5 '[(ethoxycarbonyl) (ethyl) phosphonate], with the chemical formula:

Inhibition of HIV reproduction involves the cultivation of initially infected MT-4 lymphoid cells in the presence of the test drug, the final concentration of which in the culture medium is 0.001-100 μg / ml, over a passage of 4 days.

 Inhibition of HIV reproduction in a culture of sensitive cells is judged by a decrease in the accumulation of the virus-specific protein p24 (according to enzyme-linked immunosorbent assay), as well as by an increase in cell viability in the presence of the drug compared to the control determined on the 4th day of cultivation by trypan blue exclusion.

 From the obtained experimental data shown in the table, it can be seen that the studied drug, in addition to the need to protect cells from death (> 50% at a dose of 0.28 μM / ml), highly inhibits the reproduction of type 1 immunodeficiency virus in MT-4 cell culture (> 50% at a dose of 0.0027 μM / ml and> 90% at a dose of 0.28 μM / ml), which is 100 and 10 times higher than the corresponding characteristics for d4T, respectively. The therapeutic index of the test compound (the ratio of the toxic dose of the drug to its effective dose) is 200 times greater than that of d4T (table).

- защита клеток; % по отношению к контролю вируса,

- ингибирование р24.FIG. 1. Anti-HIV action of 2 ', 3'-didehydro-2', 3'-dideoxythymidine-5 '[(ethoxycarbonyl) (ethyl) phosphonate]:
■ - toxicity of the drug; % cell viability MT-4 in relation to the control,

- cell protection; % in relation to the control of the virus,

- inhibition of p24.

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

 Example 1. Synthesis of 2 ', 3'-didehydro-2', 3'-dideoxythymidine-5 '[(ethoxycarbonyl) (ethyl) phosphonate].

), 1.88с (3Н, СН₃), 1.37 м, 1.32м (6Н,

). ³¹Р ЯМР (CD₃CN), δ ppm: -5.96 с, -6.25 с.To a solution of diethyl [(ethoxy) carbonyl] phosphonate (190 μl, 1 mmol) in absolute CCl ₄ (10 ml) was added PCl ₅ (210 mg, 1 mmol). Boil under reflux for 3 hours. The solvent is evaporated, the dry residue is dissolved in dimethylformamide (4 ml). The resulting solution was cooled to -50 ^{° C.} and a solution of 2, 3-didehydro-2, 3-dideoxythymidine (70 mg, 0.3 mmol) in dimethylformamide (2 ml) was added. Stirred overnight at room temperature. The solvent is evaporated. The residue is chromatographed on a silica gel column (2 x 25 cm), eluting with a 96: 4 chloroform-methanol system. Yield 89%. UV (H ₂ O, λ _max ): 266 nm. ¹ H NMR (CDCl ₃ ), δ ppm: 8.50 s (1H, NH), 7.28 s, 7.30 s (1H, H6,), 7.01 s (1H, H1 '), 6.32 m (1H, H3 '), 5.91m (1H, H2'), 5.00m (1H, H4 '), 4.42m (2H, H5'), 4.31m (4H,

), 1.88s (3H, CH ₃ ), 1.37 m, 1.32m (6H,

) ³¹ P NMR (CD ₃ CN), δ ppm: -5.96 s, -6.25 s.

Example 2. Evaluation of the cytotoxicity of the compound. The cytotoxicity of the drug is assessed by adding dilutions in RPMI-1640 medium to MT-4 cell suspension, placed in the wells of a 96-well plate (Cel-Cult, England), to final concentrations of 0.001-100 μg / ml (three wells per each dose), followed by cultivation at 37 ^o C for 4 days. The inoculum concentration is 0.5 • 10 ⁶ cell particles per milliliter. The control cells are without adding the drug. Cell viability is counted in a Goryaev chamber with preliminary trypan blue staining. The toxicity of various doses of the drug is determined by the viability of the cells relative to the control (see drawing), the dose calculated by the obtained results is calculated, which reduces the cell viability by 50% (CD ₅₀ ). The data are given in the table. It should be noted that the toxic effect on MT-4 cells, manifested in a change in their morphology, decreased reproduction and viability, is exerted only by high drug concentrations (> 150 μM / ml), which is more than two times higher than that of d4T and 50,000 times the effective dose for HIV-1 (table).

 Example 3. The study of the effect of the study drug on the reproduction of HIV-1 in cell culture MT-4.

The study of the antiviral activity of the drug against HIV-1 is carried out on the transplantable line of sensitive cells MT-4. For infection using the supernatant of infected cells stored in liquid nitrogen, the multiplicity of infection is 0.2-0.5 infectious units per cell. A suspension of MT-4 cells with a concentration of 2.0 • 10 ⁶ cell particles per milliliter and a viability of at least 90% is placed in the wells of a 96-well plate (Cel-Cult, England) immediately after application of the virus-containing material and the test preparations are added immediately diluted in serum-free RPMI-1640 medium to a final concentration of 0.001-100 μg / ml (three wells per dose). The controls are HIV-1 infected MT-4 cells without drug addition (instead of the drug, the same amount of RPMI-1640 medium without additives is added) and uninfected cells.

The plate is incubated for one hour at 37 ^° C to adsorb the virus, then the cells are diluted to a seed concentration (0.5 x 10 ⁶ per milliliter) with RPMI-1640 nutrient medium with the addition of 10% fetal bovine serum pre-inactivated by heating at 56 ^° C in for 30 minutes, 300 mg / ml L-glutamine and 100 μg / ml gentamicin. Then the tablet is placed in a thermostat at 37 ^o C in an atmosphere of 5% CO ₂ . On the 4th day of cultivation, the concentration and viability of the cells are calculated by the exclusion of trypan blue. Tween-80 is added to the wells of the plate to a final concentration of 0.1% and left for 24 hours at 4 ^{° C.} to inactivate HIV. According to the data obtained, a graph is built of the dependence of the increase in cell viability relative to the control under the influence of increasing doses of the drug (see drawing), i.e. determine the ability of the drug to protect infected cells from the cytopathogenic effect of the virus (table). Then, the anti-HIV activity of the compound is evaluated using the quantification of the virus-specific p24 protein by direct enzyme immunoassay as described in [10], and a dose-dependent curve is constructed that calculates concentrations that suppress the growth of the viral antigen by 50 and 90% (ID ₅₀ and ID ₉₀ ). The results 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 the multiplication of HIV-1 in MT-4 cell culture and exceeding the efficiency of d4T.

The relative degree of protection of infected cells from death by various doses of the drug is calculated by the formula:
Protection (%) = (A-O) / (B-O) * 100,
where A is the% viability of infected cells in the presence of the drug on the 4th day of cultivation, B is% viability in the control of cells, O is% viability of infected cells without adding the drug (virus control).

The relative degree of inhibition of viral protein reproduction is calculated by the formula:
% Ing. = (P-Ro) / (P-Ro) * 100,
where Px is the concentration of p24 protein on the 4th day of cultivation in the presence of the drug, Po is the concentration of p24 after 1 hour of incubation of HIV-1 with cells without adding the drug ("O" point), P is the concentration of p24 on the 4th day of cultivation of HIV-1 in cells without the addition of the drug (virus control).

The therapeutic index, or selectivity index (IS), is defined as the ratio of the concentration of the drug, toxic to 50% of the cells (CD ₅₀ ), to the concentration, 50% inhibiting the growth of viral antigen (ID ₅₀ ):
IS = CD ₅₀ / ID ₅₀
Thus, it was shown that the studied drug has the ability to inhibit the reproduction of type 1 immunodeficiency virus in MT-4 cell culture, without exerting a toxic effect on the cells, and its antiviral activity exceeds the activity of d4T. This suggests the promise of the proposed compounds for creating new dosage forms used in the treatment of AIDS.

List of references:
1. GJVeal, S. Agrawal. RA Byrn. Synergistic inhibition of HIV-1 by an antisense oligonucleotide and nucleoside analog reverse transcriptase inhibitors. Antiviral Research 1988, 38, 63-73.

 2. H. Mitsuya, K. J. Weinhold, P. A. Furman, M.N. St. Clair, S. Nusinoff-Lehrman. , R. C. Gallo, D. Bolognesi, D.W. Barry, S. Broder. 3'-Azido-3'-deoxythymidine; an antiviral agent that inhibits the inefectivity and cytopathetic effect of human T-lymphotropic virus type III / lymphoadenopathy-associated virus in vitro. Proc. Nat. Acad Sci. USA 1985, 82, 7096-7100.

 3. V. M. Cardona, A.I. Ayy, A.M. Aubertin, R. Guedj. Synthesis and anti-HIV activity of some novel arylphosphate and H-phosphonate derivatives of 3'-azido-2 ', 3'-dideoxythymidine and 2', 3'-didehydro-2 ', 3'-dideoxythymidine. Antiviral Res 1999, 42 (3), 189-196.

 4. P. Herdewijn, J. Balzarini, M. A. Baba, E. De Clercq et al. 3'-Substituted 2 ', 3'-dideoxynucleoside analogues as potential anti-HIV (HTLV / LAV) agents. J. Med. Chem. 1987, 30, 1270-1278.

 5. R.F.Schinazi, R.M. Lloyd, JR., C.S. Ramanathan and E.W. Taylor. Antiviral Drug Resistance Mutation in Human Immunodeficiency Virus T-type 1 Reverse Transcriptase Occur in Specific RNA Structural Regions. Antimicrobial Agents and Chemotherapy 1994, 38 (2), 268-274.

 6. V. I. Avramis, R. Kwock, M. M. Solorzano, and E. Gomperts. Evidense of In Vitro Development of Drug Resistanse to Azidothymidine in T-Lymphocytic Leukemia Cell Lines (Jurkat E6-1 / AZT-100) and in Pediatric Patients with HIV-1 Infection. Journal of Acquired Immune Deficiency Syndromes 1993, 6, 1287-1296.

 7. F. P. Svinarchuk, D.A. Konevetz, O.A. Pliasunova, A.G. Pokrovsky, V.V. Vlasov. Inhibition of HIV proliferation in MT-4 cells by antisense oligonucleotide conjugated to lipophilic groups. Biochimie 1993, 75, 49-54.

 8. A.G. Pokrovskii, O.A. Pliasunova, V.M. Blinov, V.V. Vlasov, F.P. Svinarchuk, T.V. Abramova, V.V. Gorn and D.A. Konevetz. Inhibition of Human Immunodeficiency Virus by Sense and Antisense Oligodeoxynucleotides. Molecular Biology 1993, 27 (5), N 1, 641-644.

 9. RF patent 2144956, cl. C 12 N 7/04, publ. 01/27/2000, BI 3.

 10. Enzyme-linked immunosorbent assay // Ed. TT. Ngo, G. Lenhoff. - M: World, - 1988.

Claims (1)

2 ', 3'-Didehydro-2', 3'-dideoxythymidine-5 '[(ethoxycarbonyl) (ethyl) phosphonate] having the formula

human immunodeficiency virus reproduction inhibitor.

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