RU2243972C1 - Nucleoside analog phosphoramidates as inhibitors of human immunodeficiency virus reproduction - Google Patents

Abstract

FIELD: organic chemistry, biochemistry, medicine.

SUBSTANCE: invention relates to phosphoramidates of nucleoside analogs comprising 2',3'-dideoxy-2',3'-didehydrothymidine 5'-phosphodimorpholidate of the formula (I)

and phosphoramidates of 3'-azido-3'-deoxythymidine of the formula (II)

and the formula (III)

that inhibit activity in reproduction of human immunodeficiency virus (HIV). Compounds are resistant to effect of dephosphorylating enzymes and able to penetrate into cells and elicit the selective activity in inhibition of DNA biosynthesis catalyzed by HIV-reverse transcriptase.

EFFECT: valuable medicinal and biochemical properties of nucleoside analogs.

4 dwg, 1 tbl, 5 ex

Description

The invention relates to new biologically active derivatives of 5'-phosphate 3'-azido-3'-deoxythymidine and 2 ', 3'-dideoxy-2', 3'-didehydrothymidine and can be used as antiviral agents, primarily against the virus human immunodeficiency (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.

Various compounds are currently known to inhibit the reproduction of the human immunodeficiency virus. The most effective known compounds are 3'-azido-3'-deoxythymidine (azidothymidine or AZT, zidovudine, “Retrovir”, “Timazide”), which is used in medical practice (Mitsuya, N .; Broder, S. Inhivition of the in vitro infectivity and cytiopathic effect of human T-lymphotropic virus type III / lymphoadenopathy-associated virus / HTLV-III (LAV) by 2 ', 3'-dideoxynucleosides. Proc. Nat. Acad. Sci. USA, 1986, 83, 1911- 1915), as well as 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 molecular mechanism of action of this compound includes its diffusion into the cell infected with HIV. Further, it undergoes triphosphorylation and specifically blocks DNA synthesis catalyzed by HIV reverse transcriptase. Other antiviral nucleosides used in medical practice for the treatment of AIDS are similar: 2 ', 3'-dideoxycytidine and, 2', 3'-dideoxyinosine (Mitsuya, H .; Broder, S. Inhibition of the in vitro infectivity and cytopatic effect of human T-lymphotropic virustype III / lymphoadenopathy-assosiated virus (HTLV-III / LAV) by 2 ', 3'-dideoxynucleosides. Proc. Nat. Acad. Sci. USA, 1986, 82, 1911-1915), 2', 3 ^1- dideoxy-2 ′, 3'-didehydrothymine (Herdewijn, P .; Balzarini, J .; DeClercq, E .; et al., 3'-Substituted 2 ', 3'-dideoxynucleoside analog as potential anti-HIV ( HTLV / LAV) agents. J. Med. Chem., 1987, 30, 1270-1278) and 2 ', 3'-dideoxy-3'-thiothymidine (Soudeyns, H .; Yao, Q .; et al., Anti -human immunodeficiency virus type 1 activity and in vitro toxicity of 2'-deoxy-3'-thiacytidine (BCH-189), a novel hetero cyclic nucleoside agents. Antimicrob. Agents Chemother., 1991, 35, 1386-1390).

However, phosphorylation of modified nucleosides by cellular enzymes occurs much less efficiently than natural nucleosides. The process of converting a nucleoside in the human body into the corresponding 5’-triphosphate takes about 1.5-2 hours. During this time, the virus that has penetrated the cells manages to integrate into the human genome in the form of proviral DNA. The use of nucleoside-5’-triphosphates with an unmodified triphosphate moiety as drugs 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. Thus, the drugs used, even if taken at the time of infection, cannot protect against HIV infection.

The closest technical solution (prototype) is the use of 3'-azido-3'-deoxythymidine derivatives containing a modified phosphate group in the 5'-position as an antiviral preparation, namely AZT H-phosphonate (US Patent No. 5043437, IPC C 07 N 19/00, published on 08.27.1991), as well as: N.B. Tarusova, A.A. Khorlin, A.A. Kraevsky, et al., Inhibition of human immunodeficiency virus in cell culture with 5'-phosphonates 3'- azido-2 ', 3'-dideoxynucleoside, Mol. Biol, 1989, 23, N6, 1716-1724). It is currently being used as a medicine called “Nikavir” to treat AIDS (HIV infection).

However, it was shown that in the body this compound is mainly subjected to dephosphorylation, turning into AZT (Kuznetsova E.V., Kukhanova M.K., et al. Reaction of 5'-H-phosphonates, 5'-fluorophosphates and 5'-phosphates modified thymidines in blood plasma. - Mol. Biol, 1995, 29, N2, 415-420).

The technical result of the present invention is the creation and use of new compounds that are resistant to dephosphorylation enzymes, are able to penetrate the cells and have selective activity in inhibiting DNA biosynthesis catalyzed by HIV reverse transcriptase.

The specified technical result is achieved by the fact that according to the invention, new compounds are claimed: phosphoramidates of nucleoside analogues, including 5'-phosphodimorpholidate 2 ', 3'-dideoxy-2', 3'-didehydrotimidine (formula I) and phosphoramidates 3'-azido-3 '-deoxythymidine (formulas II and III), inhibiting the activity of reproduction of human immunodeficiency virus and having the following formulas:

To obtain the compounds of formulas I-III, a single synthetic approach was used, consisting in the reaction of a nucleoside with phosphorus oxychloride in triethyl phosphate, followed by treatment of the resulting phosphodichloridate with various amines. The obtained phosphoramidates I-III can be isolated and purified by standard methods, for example, extraction, precipitation, chromatography, etc.

The invention is illustrated by the following graphic materials. Figure 1 shows comparative graphs of the anti-HIV action of the compounds of formula I against HIV-1 HBV 4046 when introduced with the virus. Figure 2 shows comparative graphs of the anti-HIV action of the compounds of formula I when introduced after adsorption of the virus. Figure 3 - the same anti-HIV effect of the compounds of formula II with simultaneous introduction of the virus. Figure 4 - the same anti-HIV effect of the compounds of formula III with simultaneous introduction of the virus

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

Example 1. Obtaining 5’-phosphodimorpholidate 2 ’, 3’-Dideoxy-2’, 3’-didehydrothymidine 5’-phosphodimorpholidate (formula I)

To a solution of 2 ', 3'-dideoxy-2', 3'-didehydrothymidine (235 mg, 1.05 mmol) in triethyl phosphate (3 ml), with rt, with shaking, tert-butanol (8.5 mg, 0.12 mmol) was added. and phosphorus oxychloride (0.35 g, 2.24 mmol). The reaction mixture was kept for 16 h at rt, cooled to 4 ° С, after which a solution of morpholine (1.3 g, 15 mmol) in methanol (5 ml) was added. Then the reaction mixture (suspension) was diluted with ethyl acetate (10 ml) and kept for 18 h at 4 ° C. The precipitate (morpholinium chloride) was filtered off, the mother liquor was evaporated, and the residue was separated by silica gel column chromatography (10 g of Silasorb in chloroform), eluting with a gradient (0-5%) of methanol in chloroform. The yield of the title compound was 0.87 mmol (82%, UV), the product contained about 2 mol of triethyl phosphate per mole of dimorpholidate. For repeated purification, half of this amount was taken. Purification was carried out on silica gel (5 g Silasorb in chloroform), eluting with 3% methanol in chloroform. Received 117 mg of the target compound (0.264 mmol, 50%).

¹ H-NMR (D ₂ O): 7.49 s (1H, H-6); 6.93 r.s. (1H, H-1 '); 6.54d (J = 6.2 Hz, 1H, H-3 '); 6.10 d (J = 6.2 Hz, 1H, H-2 '); 5.18 r.s. (1H, H-4 '); 4.22 and 4.07 2m (2H, H-5 '); 3.67 m (4H, CH ₂ O-morpholine); 3.13 m (4H, CH ₂ N-morpholine); 1.94 s (3H, Thymine Me).

³¹ P-NMR (D ₂ O): 16.38 s.

Example 2. Obtaining 3’-Azido-2 ’, 3’-dideoxythymidine 5’-phosphobis (methoxyamide) (formula II)

To a solution of 3'-azido-3'-deoxythymidine (82 mg, 0.31 mmol) in triethyl phosphate (1.7 g) with stirring, at rt, tert-butanol (5 mg, 0.06 mmol) and phosphorus oxychloride (86 mg, 0.56 mmol) were added. ) The reaction mixture was kept for 1 h at rt, and then 20 h at 4 ° С, after which methoxyamide chlorohydrate (309 mg, 3.7 mmol) and triethylamine (0.51 g, 5.05 mmol) in methanol (5 ml) were added to the cooled solution. The precipitate was filtered off, the mother liquor was evaporated and diluted with ethyl acetate (10 ml). The precipitate was again filtered off, the mother liquor was evaporated, and the product was isolated as previously described. The yield of the target compound is 58 mg (0.143 mmol, 46%)

¹ H-NMR (D ₂ O): 7.49 s (1H, H-6); 6.19 t (J = 6.4 Hz, 1H, H-1 '); 4.49 k (J = 6.7 Hz, 1H, H-3 '); 4.28-4.15m (3H, 2H-5 '+ H-4'); 3.62 d (J = 17 Hz, 6H, 2 CH ₃ ON); 2.43 t (J = 6.7 Hz, 2H, 2H-2 '); 1.86s (3H, Me thymine).

³¹ P-NMR (D ₂ O): 18.73 s.

Example 3. Obtaining 3’-Azido-2 ’, 3’-dideoxythymidine 5’-phosphomorpholidate (formula III)

To a solution of 3'-azido-3'-deoxythymidine (178 mg, 0.67 mmol) in triethyl phosphate (2.6 g) with stirring, at rt, tert-butanol (7 mg, 0.09 mmol) and phosphorus oxychloride (170 mg, 1.09 mmol) were added. ) The reaction mixture was kept for 1 h at rt, and then 20 h at 4 ° С, after which water (55 mg, 3.06 mmol) was added to the cooled solution, maintained for 40 min at 4 ° С, morpholine (0.33 g, 3.8 mmol) was added to methanol (3 ml). The reaction mixture was evaporated and diluted with ethyl acetate (10 ml). The precipitate was filtered off, the mother liquor was evaporated and the product was isolated as previously described, using the chloroform-methanol-25% aqueous ammonia system for elution (60: 35: 5). The yield of the target compound is 182 mg (0.420 mmol, 63%)

¹ H-NMR (D ₂ O): 7.49 s (1H, H-6); 6.16 t (J = 6.4 Hz, 1H, H-1 '); 4.48k (J = 6.7 Hz, 1H, H-3 '); 4.25-4.14m (3H, 2H-5 '+ H-4'); 3.72m (2H, CH ₂ O-morpholine); 3.01 m (2H, CH ₂ N-morpholine); 2.31 t (J = 6.7 Hz, 2H, 2H-2 '); 1.88s (3H, Me thymine).

³¹ P-NMR (D ₂ O): 7.81 s.

The following are studies of inhibition of HIV reproduction.

The study of inhibition of HIV reproduction involves the cultivation of primary infected lymphoid cells of the MT-4 line in the presence of the studied compounds, the final concentration of which in the culture medium is 0.0001-100 μg / ml, over one passage for 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 when staining 3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT).

Example 4. Assessment of cytotoxicity of compounds

The cytotoxicity of the drug is assessed by adding dilution in serum-free RPMI-1640 medium to the MT-4 cell suspension, placed in the wells of a 96-well plate ("Orange"), to a final concentration of 0.001-100 μg / ml (three wells per dose) followed by cultivation at 37 ° C for 4 days. The inoculum concentration is 0.5 × 10 ⁶ cell particles per milliliter. Cells are used as control without the addition of a drug, instead of which the same amount of serum free medium is added. Cell viability is counted on the 4th day of cultivation using the formazan method (intravital staining of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) cells [Leslie R. Bisset, Hans Lutz, Jurg Boni, Regina Hoffinann-Lehmann, Ruedi Luthy, Jorg Schupbach. Combined effect of zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) antiretroviral therapy in suppressing in vitro FIV replication. Antiviral Research 53 (2002) 35-45].

The toxicity of various doses of the drug is determined by the viability of the cells relative to the control, the dose-dependent curve is built from the obtained results and the concentration is determined that reduces cell viability by 50% (CD ₅₀ ). It should be noted that the studied compounds do not have toxic effects on MT-4 cells in effective concentrations: 50% toxic doses are 5-6 orders of magnitude higher than the effective doses for HIV-1 (table).

Table
Quantitative characteristics of the anti-HIV action of the compounds Compound CD ₅₀ , μM Simultaneous application Addition after adsorption of the virus ID_fifty
PD_fifty ID ₉₀ IS ID_fifty
PD_fifty ID ₉₀ IS d4T 314.0 0.01
2,4 0.3 31400 0.24
23.8 23.8 1308.3 I
(IMB-43) > 226 <0.0022
0.0022 <0.0022 > 102727 <0.0022
0.22 16.9 > 102727 AZT 37,4 0,037
18.7 1.87 1010 0.0037
18.7 0.09 10100 II
(IMB-94) 740.2 0.012
0.37 0.2 61683 0,025 0.22 29608 III
(IMB-101) > 230.76 0,023
- 1,0 10033 0,07 0.2 3296.6

Example 5. The study of the effect of the claimed compounds on the reproduction of HIV-1 in cell culture MT-4.

A study of the antiviral activity of compounds 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 immediately after application of the virus-containing material, and the test compounds diluted in RPMI-1640 medium without serum are immediately added , to final concentrations of 0.0001-100 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 for the adsorption of the virus, then the cells are diluted to a seed concentration (0.5 x 10 per milliliter) with RPMI-1640 nutrient medium with the addition of 10% fetal bovine serum pre-inactivated by heating at 56 ° C for 30 minutes 300 mg / ml L-glutamine and 100 μg / ml gentamicin. Then the tablet is placed in a thermostat at 37 ° 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 formazan method. According to the data obtained, graphs of the dependence of the growth of cell viability relative to control under the influence of increasing doses of drugs are constructed, i.e. determine the ability of drugs to protect infected cells from the cytopathogenic effect of the virus. Evaluation of the anti-HIV activity of the compounds is carried out using the quantification of virus-specific protein p24 by direct enzyme immunoassay, as described in [Leslie R. Bisset, Hans Lutz, Jurg Boni, Regina Hoffmann-Lehmann, Ruedi Luthy, Jorg Schupbach. Combined effect of zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) antiretroviral therapy in suppressing in vitro FIV replication. Antiviral Research 53 (2002) 35-451], and dose-dependent curves are constructed (FIGS. 1-4), which are used to calculate the concentrations that suppress the growth of the viral antigen by 50 and 90% (ID ₅₀ and ID ₉₀ ). The therapeutic index, or selectivity index (IS) is considered as the ratio of a 50% toxic concentration of a compound to its 50% effective dose. Compound I (2 ', 3'-Dideoxy-2', 3'-didehydrothymidine 5'-phosphodimorpholidate), when introduced into the culture of sensitive cells simultaneously with HIV, is an order of magnitude and more superior to the original d4T preparation by ID ₅₀ and by two orders of magnitude according to ID ₉₀ , when introduced after adsorption of the virus, it is two orders of magnitude superior to the original drug according to ID ₅₀ with almost the same level of 90% inhibition. Compounds II (3'-Azido-2 ', 3'-dideoxythymidine 5'-phosphobis (methoxyamide)) and III (3'-Azido-2', 3'-dideoxythymidine 5'-phosphomorpholidate) showed activity comparable to the activity of the original azidothymidine, approximately the same when added simultaneously with HIV and when added under post-adsorption conditions. Based on these quantitative indicators of inhibition, one can judge the effectiveness of the antiviral effect of the new compounds, which consists in a high degree of suppression of HIV-1 replication in MT-4 cell culture and exceeding the efficiency of the initial d4T and AZT.

Claims (1)

Phosphoramidates of nucleoside analogues, which are 5'-phosphodimorpholidate 2 ', 3'-dideoxy-2', 3'-didehydrothymidine (formula I) or phosphoramidates of 3'-azido-3'-deoxythymidine (formulas II and III), inhibiting reproduction activity human immunodeficiency virus and having the following formulas:

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