RU2430104C1 - Novel depot-forms of zidovudine and lamivudine based on phosphonic acid derivatives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to dinucleoside forms of phosphonic acids which can be used as antiviral agents, having formula

where Azt is a 3'-azido-3'-deoxythymidine residue, ZTS - 2',3'-dideoxy-3'-thiacitidine residue, R is an electron acceptor group, for example ClCH₂-, CH₃C(O)CH₂-, H₂NCO-, PhCH₂CH₂HNCO-,

PhOCH₂-, CH₃OCH₂-, N₃CH₂-.

EFFECT: obtaining novel antiviral agents.

1 cl, 3 ex, 2 tbl

Description

The invention relates to the field of molecular biology, virology and medicine, in particular to the use of new derivatives of nucleosides, namely dinucleoside esters of phosphonic acids to suppress the reproduction of viruses.

Currently, a number of compounds with antiviral activity against HIV are used in medical practice. Among them, nucleoside and non-nucleoside inhibitors are distinguished. Among the nucleoside derivatives, the most commonly used are 3'-azido-3'-deoxythymidine (AZT, Zidovudine®) and 2 ', 3'-dideoxy-3'-thiacytidine (3TC, Lamivudine®) [De Clercq, E., 2002. New development in anti-HIV chemotherapy. Biochim. Biophys. Acta, 1587 258-275].

The mechanism of action of these compounds is that after penetration into infected cells, they undergo triphosphorylation and specifically block DNA synthesis catalyzed by HIV reverse transcriptase. The high variability of HIV leads to the rapid emergence of resistant strains of the virus [Groschel, B., Cinatl, J.H., and Cinatl J. Jr., 1997. Viral and cellular factors for resistance against antiretroviral agents. Intervirology, 40, 400-407; Antonelli, G, Turriziani, O., Verri, A., Narciso, P., Ferri, F., D'Offizi, G., Dianzini, F., 1996. Long-term exposure to zidovudine affects in vitro and in vivo the efficiency of thymidine kinase. AIDS Res. Hum. Retrovir., 12, 223-228], and, therefore, to the need to change the drug. In addition, due to the low efficiency of intracellular transformations, the drugs used require high doses of application, which causes the appearance of pronounced toxic effects.

For example, the effects of toxicity of AZT are suppression of the activity of spinal cord cells, impaired liver function and myopathy [Chariot, P., Drogou, 1, De Lacroix-Szmania, I., Eliezer-Vanerot, MC, Chazaud, B., Lombes, A., Schaeffer, A., and Zafrani, ES, 1999. Zidovudine-induced mitochondrial disorder with massive liver steanosis, myopathy, lactic acidosis, and mitochondial DNA depletion. J. Hepatol. 30, 156-160; Kellam, P., Boucher, C.A., and Larder, B.A., 1992. Fifth mutations in HIV reverse transcriptase contributes to the development of high level resistance to zidovudine. Proc. Natl. Acad. Sci. U.S.A, 89, 1934-1938; Ren, J., Esnouf, RM, Hopkins, AL, Jones, EY, Kirby, I, Keeling, J., Ross, CK, Larder, BA, Stuart, DI, Stammers, DK, 1998. 3'-Azido-3 '-deoxythymidine drug resistance mutations in HIV-1 reverse transcriptase can induce long-range conformational changes. Proc. Natl. Acad. Sci. U.S.A, 95, 9518-9523]. The rapid removal of AZT from the body requires frequent administration of the drug. In addition, with prolonged use of AZT, resistant strains of the virus are formed quite quickly and the treatment loses its effectiveness. Other nucleoside antiviral drugs have similar side effects. Despite all of the above disadvantages, AZT remains the most widely used anti-HIV drug.

One of the ways to improve the pharmacological properties of antiviral agents is to create their depot forms (latent forms), that is, those derivatives that, after entering the body, undergo chemical or enzymatic biotransformations, releasing the active compound. Currently described various depot forms of nucleoside antiviral drugs [Parang K., Weibe LI, Knaus E.E. // Current Medical Chemistry. 2000. V.7. P.995-1039.]. In particular, AZT N-phosphonate (Nikavir®), approved in Russia for AIDS therapy, is less toxic than AZT [Intracellular metabolism and pharmacokinetics of 5'-hydrohenphosphonate of 3'-azido-2 ', 3'-dideoxythymidine, a prodrug of 3'-azido-2 ', 3'-dideoxythymidine. Antiviral Research 63 (2004), 107-113]. According to pharmacokinetic studies, the clinical benefits of Nikavir are explained by a slower and smoother increase in the concentration of AZT in the blood than when taking AZT itself; wherein C _max AZT from Nikavir <C _max AZT from Zidovudine, and T _1/2 AZT from Nikavir> T _1/2 AZT from Zidovudine [Y. Skoblov et al. / Antiviral Research 63 (2004) 107-113]. However, the toxicity of Nikavir remains quite high. Another disadvantage is the emergence of resistance to Nikavir.

Another way to increase the effectiveness of anti-HIV therapy is to use a combination (or cocktail) of antiviral drugs. The most famous of the “combined” preparations is combivir, the tablets of which contain 150 mg of lamivudine and 300 mg of zidovudine® as the active substance. Lamivudine is a synergist of zidovudine with respect to inhibition of HIV replication in cell culture. Clinical observations in vivo show that combination therapy with lamivudine and zidovudine slows down the development of zidovudine resistance in patients who have not previously received antiretroviral therapy.

This invention solved the problem of creating low toxic dinucleoside esters of phosphonic acids with the ability to gradually release active nucleosides in the body. This will allow to maintain a constant intracellular concentration of two drugs for a long time and thus reduce a single dose of the drug and / or frequency of administration and reduce side effects.

The first compounds of this structure, namely bis-azido-deoxy-midi-methylphosphonate and bis-dideoxycytidine methylphosphonate, were synthesized by the Imbach group in 1990 [Puech F, Gosselin G, Balzarini J, Good SS, Rideout JL, De Clercq E, Imbach JL. Synthesis and biological evaluation of dinuc-leoside methylphosphonates of 3'-azido-3'-deoxythymidine and 2 ', 3'-dideoxycytidine. Antiviral Res. 1990 Jul; 14 (1): 11-23], however, these methylphosphonates were found to be too stable and did not hydrolyze in either cell cultures or animal organisms. In 1995, Chris Meer et al. First showed that di-nucleoside alpha-hydroxyphosphonates bearing two identical nucleosides can act as an effective depot form [Meier C, Habel L, Laux W, De Clercq E, Balzarini J. Homo dinucleoside-ct-hydroxyphosphonate diesters as prodrugs of the antiviral nucleoside analogues 2 ', 3'-dideoxythymidine and 3'-azido-2', 3'-dideoxythymidine. Nucleosides & Nucleotides, 1995; 14 (3-5): 759-762]. These two examples illustrate the importance of the structure of R.

The problem is solved by creating compounds of the general formula:

, PhOCH₂-, СН₃ОСН₂- или N₃CH₂-.where Azt is the residue of 3'-azido-3'-deoxythymidine, 3TC is the residue of 2 ', 3'-dideoxy-3'-thiacytidine, R = electron-acceptor group, for example С1СН ₂ -, CH ₃ C (O) CH ₂ -, H ₂ NCO-, PhCH ₂ CH ₂ HNCO-,

, PhOCH ₂ -, CH ₃ OCH ₂ - or N ₃ CH ₂ -.

The new compounds inhibit the reproduction of the human immunodeficiency virus in a culture of transplantable MT-4 lymphocytes, protect the cells from the cytopathogenic effect of the virus, and do not show toxicity to host cells up to extremely high concentrations (Table 1). From the obtained experimental data, it can be seen that the tested compounds, without exerting toxic effects on cells at effective concentrations (50% toxic doses are 2-4 orders of magnitude higher than 50% inhibitory doses), inhibit the reproduction of the immunodeficiency virus in cell culture to a high degree MT-4. The therapeutic indices of the studied compounds (IS), defined as the ratio of the toxic dose of the drug to its effective dose, are comparable to those for AZT and Nikavir. Virological tests are carried out in accordance with the previously described protocols.

Target phosphonates were prepared as follows:

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

Example 1

General method for the synthesis of phosphonates (II)

To a solution of phosphonates (I) obtained by the method of [Shirokova EA, Yasko M.V., Khandazhinskaya A.L., Ivanov A.V., Yanvarev D.V., Skoblov Yu.S., Pronyaeva T. R., Fedyuk N.V., Pokrovsky A.G., Kukhanova M.K. New derivatives of 3'-azido-3'-deoxythymidine and phosphonic formic acid, Bioorgan. Chemistry, 2004, 30, No. 3, 273-280.] (0.2 mmol) in pyridine (3 ml) was added the corresponding nucleoside (0.4 mmol), cooled to 0 ° C and TPSC1 (0.6 mmol) was added. . The reaction mass was stirred for 16 hours at room temperature. The solvent was removed in vacuo. The residue was chromatographed on a column (2 × 25 cm) with silica gel, eluting with a gradient of methanol concentrations in chloroform (0 → 10%). Fractions containing the desired products (II) were evaporated to dryness in vacuo.

5'-Chloromethylphosphono-3'-azido-3'-deoxythymidylyl (5'-5 ') - L-2', 3'-dideoxy-3'-thiacytidine (IIa). Received in 29% yield. ¹ H-NMR (CD ₃ OD): 7.89 (1H, d, J 4.1, H-6 (3TC)), 7.48 (1H, 2 s, H-6 (Azt)), 6, 32 (1H, t, H-1 ', J 5.3 (3TC)), 6.15 (2H, t, J 6.6 H-1' (Azt)), 5.49 (1H, br.s , H-4 '(3TC)), 4.54-4.41 (5H, m, H-3', H-5 '(Azt) and H-5' (3TC)), 4.07 (1H, broad s, H-4 '(Azt)), 3.95 and 3.92 (2H, 2d, J 10.6 and 11.2, CH ₂ Cl), 3.60 and 3.12 (2H, 2 m, H-2 '(3TC)), 2.55-2.34 (2H, m, H-2' (Azt)), 1.89 and 1.88 (3H, 2 s, 5-CH ₃ ) . ³¹ P-NMR (CD ₃ OD): 23.5 s and 23.1 s.

5 '- (2-Oxopropyl) phosphono-3'-azido-3'-deoxythymidylyl (5'-5') - L-2 ', 3'-dideoxy-3'-thiacytidine (IIb). Received in 51% yield. ¹ H-NMR (CD ₃ OD): 7.87 (1H, d, J 4.1, H-6 (3TC)), 7.47 (2H, 2 s, J 1.2, H-6 (Azt )), 6.12 (2H, 2 t, J 6.5, H-1 '(Azt)), 5.47 (1H, m, H-4' (3TC)), 4.52-4.40 (3H, m, H-3 '(Azt) and H-5' (3TC)), 4.23 (2H, m, H-5 '(Azt)), 3.99 (2H, m, H-4 '), 3.58 (1H, m, H-2'a (3TC), 3.42 (2H, d, J 21.8, CH ₂ -P), 3.10 (2H, 2 m, H- 2'b (3TC)), 2.31-2.45 (4H, m, H-2 '), 2.21 (3H, s, CH ₃ C (O)), 1.78 (6H, s, 5-CH ₃ ). ³¹ P-NMR (DMSO-d ₆ ): 23.12 s and 23.34 s.

5'-Aminocarbonylphosphono-3'-azido-3'-deoxythymidinyl (5'-5 ') - L-2', 3'-dideoxy-3'-thiacytidine (IIc). Received in 27% yield. ¹ H-NMR (CD ₃ OD): 7.70 (1H, m, H-6 (3TC)), 7.47 (1H, 2 s, H-6 (Azt)), 6.23 (1H, t , H-1 ', J 5.6 (3TC)), 6.13 (2H, t, J 6.6, H-1' (Azt)), 5.76 (1H, d, J 7.4, H-5 '(3TC)), 5.38 (1H, m, H-4' (3TC)), 4.49 (1H, m, H-3 '(Azt)), 4.38-4.30 (4H, m, H-5 '(Azt) and H-5' (3TC)), 4.04 (1H, br.s, H-4 '(Azt)), 3.07-3.02 (2H m, H-2 '(3TC)), 2.39-2.34 (2H, m, H-2' (Azt)), 1.78 (3H, 2 s, 5-CH3). ³¹ P-NMR (CD ₃ OD): 1.99 s and 2.31 s.

5'-Phenylethylaminocarbonylphosphono-3, -azido-3'-deoxythymidinyl (5, -5 ') - L-2', 3'-dideoxy-3'-thiacytidine (IId). Received in 36% yield. ¹ H-NMR (CD ₃ OD): 7.85 (1H, m, H-6 (3TC)), 7.46 (1H, s, H-6 (Azt)), 7.29-7.18 ( 5H, m, Ph) 6.37 (1H, t, J 5.3, H-1 '(3TC)), 6.12 (1H, t, J 6.5, H-1' (Azt)), 5.94 (1H, d, J 7.5, H-5 '(3TC)), 5.47 (1H, m, H-4' (3TC)), 4.45-4.31 (6H, m , H-3 ', H-4' and H-5 '(Azt) and H-5' (3TC)), 4.04 (2H, m, CH ₂ N), 3.52 (1H, m, H -2'a (3TC)), 3.15 (1H, m, H-2'b (3TC)), 2.85 (2H, t, J 7.2, CH ₂ Ph, 2.45 (2H, m, H-2 '(Azt)), 1.88 (3H, s, 5-CH ₃ (Azt)). 31 P-NMR (CD ₃ OD): 1.21 s and 1.28 s.

5'-Morpholinocarbonylphosphono-3'-azido-3'-deoxythymidyl (5'-5 ') - L-2', 3'-dideoxy-3'-thiacytidine (IIe). Received in 47% yield. ¹ H-NMR (CD ₃ OD): 7.81 (1H, m, H-6 (3TC)), 7.25 (1H, 2 s, H-6 (Azt)), 6.32 (1H, t , J 5.3, H-1 '(3TC)), 6.06 (1H, t, J 6.6, H-1' (Azt)), 5.76 (1H, m, H-5 (3TC) )), 5.36 (1H, m, H-4 '(3TC)), 4.40 (1H, m, H-3' (Azt)), 4.38-4.30 (4H, m, H -5 '(Azt) and H-5' (3TC)), 4.02, 3.96, 3.71, 3.65 (9H, 4 m, morpholine and H-4 '(Azt)), 2, 53-2.38 (2H, m, H-2 '), 1.91 (3H, s, 5-CH ₃ ). ³¹ P-NMR (CD ₃ OD): 2.11 s and 1.96 s.

5'-Phenoxymethylphosphono-3'-azido-3'-deoxythymidylyl (5'-5 ') - L-2', 3'-dideoxy-3'-thiacytidine (IIf). Received in 39% yield. ¹ H-NMR (CD ₃ OD): 7.84 and 7.82 (1H, 2 d, J 5, H-6 (3TC)), 7.51 and 7.49 (1H, 2 d, J 0, 9, H-6 (Azt)), 7.34-7.26 (2H, m, o-PhO), 7.05-6.94 (3H, m, m- and p-PhO), 6.32 and 6.31 (1H, 2 t, J 6, H-1 '(3TC)); 6.14 (1H, t, J 7.5, H-1 '(Azt)), 5.86 and 5.85 (1H, 2 d, H-5), 5.44 (1H, m, H- 4 '(3TC)), 4.54-4.41 (7H, m, H-3' (Azt), H-5 '(Azt), PCH ₂ O and H-5' (3TC)), 4, 08 (1H, br.s, H-4 '(Azt)), 3.48 and 3.12 (2H, 2 m, H-2' (3TC)), 2.51-2.38 (2H, m , H-2 '(Azt)), 1.83 and 1.81 (3H, 2 d, 5-CH ₃ (Azt)). ³¹ P-NMR (CD ₃ OD): 24.1 s and 23.4 s.

5'-Methoxymethylphosphono-3, -azido-3'-deoxythymidylyl (5'-5 ') - L-2', 3'-dideoxy-3'-thiacytidine (IIg). Received in 31% yield. ¹ H-NMR (CD ₃ OD): 7.84 (1H, m, H-6 (3TC)), 7.48 (1H, s, H-6 (Azt)), 6.35 (1H, t, J 5.3, H-1 '(3TC)), 6.14 (1H, t, J 6.5, H-1' (Azt)), 5.92 (1H, d, J 7.5, H -5 (3TC)), 5.46 (1H, m, H-4 '(3TC)), 4.44-4.30 (5H, m, H-3' and H-5 '(Azt) and H -5 '(3TC)), 4.27 (1H, m, H-4' (Azt)), 4.07 and 4.05 (2H, 2d, J 8.7, CH ₂ -P), 3, 50 (1H, m, H-2'a (3TC)), 3.44 (3H, d, J 0.9, CH ₃ O), 3.14 (1H, m, H-2'b (3TC) ), 2.47 (2H, m, H-2 '(Azt)), 1.89 (3H, s, 5-CH ₃ (Azt)). ³¹ P-NMR (CD ₃ OD): 24.7 s and 24.1 s.

5'-Azidomethylphosphono-3'-azido-3'-deoxythymidylyl (5 ', 5') - L-2 ', 3'-dideoxy-3'-thiacytidine (IIh). Received in 65% yield. 1 H NMR (CD ₃ OD): 7.83 (1H, m, H-6 (3TC)), 7.49 and 7.45 (1H, 2 s, J 1.2, H-6 (Azt)) 6.34 (1H, t, J 5.4, H-1 '(3TC)), 6.13 and 6.07 (2H, 2 t, J6.5, H-1' (Azt)), 5 92 (1H, d, J 7.5, H-5 (3TC)), 5.44 (1H, m, H-4 '(3TC)), 4.35 (5H, m, H-3', H-5 '(Azt) and H-5' (3TC)), 4.06 (2H, m, H-4 '(Azt)), 3.93 and 3.90 (2H, 2d, J 11.8 , CH ₂ -P), 3.50 and 3.15 (2H, 2 m, H-2 '(3TC)), 2.41 (2H, m, H-2' (Azt)). ³¹ P-NMR (CD ₃ OD): 25.3 s and 24.9 s.

Example 2

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 0.001-100 μg / ml, over one passage for 4 days.

Inhibition of HIV reproduction in a culture of sensitive cells is determined 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 stained with 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT).

Assessment of cytotoxicity of compounds

The cytotoxicity of the drug is assessed by adding dilutions in serum-free RPMI-1640 medium to the MT-4 cell suspension, placed in the wells of a 96-well plate (Cel-Cult, UK), to final concentrations of 0.001-100 μg / ml (three wells each) 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 MTT cells). 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 constructed from the obtained results and the concentration is determined that reduces cell viability by 50% (CD ₅₀ ). The studied compounds do not have toxic effects on MT-4 cells in effective concentrations. It should also be noted that 50% toxic doses are 2-4 orders of magnitude higher than the doses effective against HIV-1 (Table 1).

The influence of the studied compounds on the reproduction of HIV-1 in the culture of MT-4 cells was studied by a known method.

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 (the results are presented in Table 1). Based on these quantitative indicators of inhibition, one can judge the effectiveness of the antiviral effect of the claimed compounds, which consists in a high degree of suppression of HIV-1 replication in MT-4 cell culture, comparable to the efficiency of Nikavir.

Example 3

A test compound (10 μg) was added to 1 ml of heparin stabilized dog blood. Incubated at 37 ° C for 6 hours. Samples were taken every hour, centrifuged (10 minutes, 2000 rpm), the supernatant was separated. Aliquots (0.25 ml) were taken from the supernatant, oxetane (0.25 μg as internal standard) and methanol (0.75 ml) were added. The resulting mixture was centrifuged for 3 minutes at 5000 rpm. The supernatant was separated and evaporated in a stream of air at 40 ° C, water (1 ml) was added to the residue. Aliquots (20 μl) were analyzed by HPLC on a liquid chromatograph Gynkotec, Germany; analytical column Ultrasphere ODC "Beckman" USA. Eluent: 6% acetonitrile in 0.1% H ₃ PO ₄ (pH 2.1) in the presence of 0.15% triethylamine. Detection at λ _max 265 nm, temperature 30 ° C. The data obtained as a result of the analysis are given in table. 2.

Table 1 Anti-HIV activity of drugs in the culture of MT-4 cells infected with HIV-1 GKV-4046 (addition of drugs after virus adsorption) Compound CD ₅₀ , mM PD ₅₀ , mm Inhibition of p24 accumulation ID ₅₀ , mm IS

>> 2.6 <0.0258 0.258 > 10016

> 2.1 <0.0465 0.536 > 3920 AZT 0.142 <0,0374 0,037 3838 Nikavir 0.184 <0.0262 0.131 1405

table 2 Data on the hydrolysis of the claimed compounds in blood plasma of a dog after adding 10 μg of test compounds in 1 ml of blood (products of hydrolysis of AZT, 3TC and the corresponding phosphonates I) compound T _1/2 hours IIa ~ 6 IIe 0.5 IIf > 6 Nikavir 3

Thus, it was shown that the studied compounds have low toxicity and the ability to effectively inhibit the reproduction of the immunodeficiency virus in cell culture MT-4. In addition, the claimed compounds are able to generate a mixture of AZT and 3TC in the body of mammals, providing a smooth increase in their concentrations in the blood.

Claims (1)

Dinucleoside esters of phosphonic acids having the general formula

where Azt is the remainder of 3'-azido-3'-deoxythymidine, 3TC is the remainder
2 ', 3'-dideoxy-3'-thiacytidine, R = electron-acceptor group, for example СlСН ₂ -, CH ₃ C (O) CH ₂ -, H ₂ NCO-, PhCH ₂ CH ₂ HNCO-,

, PhOCH ₂ -, CH ₃ OCH ₂ -, N ₃ CH ₂ -.