RU2373218C2 - 5'-phosphorus-containing derivatives of 2',3'-dideoxy-3'-thiacytidine - new antiviral agents - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to 5'-phosphorus-containing derivatives of 2',3'-dideoxy-3'-thiacytidine with general structural formula given below

, where R=H, NH₂-C(O)-. The invented compounds can be used for inhibiting reproduction of the human immunodeficiency virus type 1 in an MT-4 cell culture.

EFFECT: increased antiviral activity of the compounds.

1 cl, 2 tbl, 4 ex

Description

The invention relates to the field of molecular biology, virology and medicine, in particular to new derivatives of nucleosides, namely to substituted 5'-phosphorus derivatives of 2 ', 3'-dideoxy-3'-thiacytidine. These compounds have a pronounced antiviral effect, primarily against human immunodeficiency virus, and can also be potentially used against human hepatitis B virus.

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®), 2 ', 3'-dideoxycytidine (ddC, Zalcitabine®), 2', 3'-dideoxyinosine (ddI, Didanosine®), 2 ', 3'-dideoxy-2', 3'-didehydrothymidine (d4T, Stavudin®) and 2 ', 3'-dideoxy-3'-thiacytidine (3TC, Lamivudin®) [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. High variability of HIV leads to the rapid emergence of resistant strains of the virus [Groschel, B., Cinati, J.H., and Cinati J. Jr., 1997. Viral and cellular factors for resistance against antiretroviral agents. Intervirology, 40, 400-407; Antonelli, G, Turrziani, O., Verri, A., Narciso, P., Feri, F., D'Offizi, G., and 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.

The main consequence of 3TC toxicity is lactic acidosis, which manifests itself in both mono- and combination therapy [Painter GR, Almond MR, Mao S. and Liotta DC, 2004, Biochemical and mechanistic basis for the activity of nucleoside analogue inhibitors of HIV reverse transcriptase . Curr. Top Med Chem, 4 (10), 1035-44; De Clercq E., Emerging anti-HIV drugs. 2005, Expert Opin Emerg Drugs. 10 (2), 241-73; Piliero PJ., 2004, Pharmacokinetic properties of nucleoside / nucleotide reverse transcriptase inhibitors. J Acquir Immune Defic Syndr. 37 (Suppl 1), S2-S12]. The rapid removal of 3TC from the body requires frequent administration of the drug. In addition, with prolonged use of 3TC, resistant strains of the virus quickly form, and treatment loses its effectiveness. Despite all of the above disadvantages, 3TC is still a widely used anti-HIV drug. 3TC is used both individually and in the form of combinations with other drugs. For example, the composition 3TC with AZT is called "Combivir®".

Of the related compounds, N-phosphonate AZT (Nikavir®), approved in Russia for the treatment of AIDS, is known. It is significantly less toxic than AZT [Nikavir (phosphazide) antiretroviral drug: anti-HIV activity, toxicology, pharmacokinetics and some prospects for clinical use [G. Galegov, 2004, 49 (7), 5-8. Antibiotics and Chemotherapy]. The action of Nikavir is based on the ability to release AZT, which, after intracellular conversion to AZT-5'-triphosphate, inhibits HIV replication. 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. Intracellular metabolism and pharmacokinetics of 5'-hydrogenphosphonate of 3'-azido-2 ', 3'-dideoxythymidine, a prodrug of 3'-azido-2', 3'-dideoxythymidine. Antiviral Research 63 (2004), 107-113].

This invention solves the problem of creating low toxic derivatives of 3TC, with the ability to penetrate into the cells and gradually release the active nucleoside (3TC). This allows you to maintain an intracellular concentration of the drug, sufficient for the manifestation of a therapeutic effect for a long time and, thus, reduce a single dose of the drug, as well as the frequency of administration and, as a result, reduce side effects.

The problem is solved by the creation of compounds 5'-phosphonyl-2 ', 3'-dideoxy-3'-thiacytidine of the General formula

where R = H, NH ₂ —C (O) -.

The new compounds inhibit the reproduction of type 1 human immunodeficiency virus in a culture of transplantable MT-4 lymphocytes, protect 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 studied compounds, without exerting a toxic effect on the cells in effective concentrations, highly inhibit the reproduction of type 1 immunodeficiency virus in the MT-4 cell culture. The therapeutic index of the studied compounds (IS), defined as the ratio of the toxic dose of the drug to its effective dose, is comparable to that for 3TC. Virological tests are carried out in accordance with the previously described protocols.

It was shown that the inventive phosphorus-containing derivatives of 3TC in the animal body slowly release 3TC, thus representing latent forms of 3TC (Example 4, Table 2). Studies show that for the claimed compounds, the only metabolic product detected in the blood of animals is 3TC. The pharmacokinetic parameters shown in Table 2 were determined by the resulting 3TC.

Since the claimed 5'-phosphorus derivatives of 2 ', 3'-dideoxy-3'-thiacytidine are effective depot forms of 3TC, which is also widely used against human hepatitis B virus, similar activity from new compounds should be expected.

The target compounds were obtained according to the following scheme in one or more stages:

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

Example 1

5'-Phosphite 2 ', 3'-dideoxy-3'-thiacytidine was prepared according to the following scheme:

For nucleoside phosphitylation, it is possible to use RCl ₃ , (PhO) ₂ P (O) H, phosphorous anhydride or free phosphorous acid with activation of 1,3-dicyclohexylcarbodiimide, 2,4,6-triisopropylbenzenesulfonyl chloride or pivaloyl chloride, as well as other methods.

5'-Phosphite 2 ', 3'-dideoxy-3'-thiacytidine

To a solution of 2 ', 3'-dideoxy-3'-thiacytidine (500 mg, 2.18 mmol) and phosphorous acid (268 mg, 3.27 mmol) in pyridine (2 ml) was added dicyclohexylcarbodiimide (674 mg, 3.27 mmol), the reaction mass was stirred at room temperature for 10 hours and diluted with water (5 ml). After 30 minutes of additional stirring, the precipitate was separated, the solution was evaporated, the residue was dissolved in water (100 ml). The solution was applied to a DEAE-Toyopearl column, eluting with a linear gradient of NH ₄ HCO ₃ (0 → 0.1 M). The desired fractions were evaporated and re-evaporated with water (3 times 5 ml), the residue was diluted with water (3 ml) and applied to a LiChroprep RP-18 column, eluting with water. The target fraction was lyophilized to give 546 mg (85%). ¹ H NMR (D ₂ O): 8.08 d (1H, J = 7.8 Hz, H-6), 6.77 d (1H, J = 642.4 Hz, H-P), 6.32 dd (1H, J = 4.4 and 5.0 Hz, H-1), 6.09 d (1H, J = 7.8 Hz, H-5), 5.43 dd (1H, J = 3.1 and 3.7 Hz, Н-4 '), 4.27 m (1Н, Н-5'а), 4.14 m (1Н, Н-5'б), 3.56 m (1Н, Н-2' a) 3.24 m (1H, H-2'b). ³¹ P NMR with suppression of the P-N interaction (D ₂ O): 6.98 s.

Example 2

5'-Aminocarbonylphosphonate 2 ', 3'-dideoxy-3'-thiacytidine was prepared according to the following scheme:

2 ', 3'-Dideoxy-3'-thiacytidine was condensed with ethoxycarbonylphosphonic acid in the presence of dicyclohexylcarbodiimide. Treatment of the obtained 5'-ethoxycarbonylphosphonate 2 ', 3'-dideoxy-3'-thiacytidine with a solution of 25% aqueous ammonia led to the formation of the target product.

5'-Ethoxycarbonylphosphonate 2 ', 3'-dideoxy-3'-thiacytidine

To a solution of the bis (pyridinium) salt of ethoxycarbonylphosphonic acid (203 mg, 0.65 mmol) in pyridine (5 ml) was added 2 ', 3'-dideoxy-3'-thiacytidine (115 mg, 0.5 mmol) and dicyclohexylcarbodiimide (436 mg, 2 mmol). The reaction mass was stirred for 18 hours at room temperature, and then diluted with water to 100 ml and the precipitate formed was filtered. The filtrate was applied to a DEAE-Toyopearl column; eluted with a linear gradient of NH ₄ HCO ₃ (0 → 0.15 M). The desired fractions were evaporated, re-evaporated with water (3 times 5 ml), the residue was diluted with water (3 ml) and applied to a LiChroprep RP-18 column, eluting with water.

The target fraction was lyophilized to give 152 mg (83%) of 5′-ethoxycarbonylphosphonate 2 ′, 3′-dideoxy-3′-thiacytidine. ¹ H NMR (D ₂ O): 8.16 d (1H, J = 7.8 Hz, H-6), 6.32 dd (1H, J = 4.4 and 4.6 Hz, H-1 '), 6.12 d (1H, J = 7.8 Hz, H-5), 5.47 m (1H, H-4 '), 4.42-4.31 m (2H, H-5'), 4 10 m (2H, CH ₂ CH ₃ ) 3.58 m (1H, H-2'a), 3.25 m (1H, H-2'b), 1.13 t (3H, J = 7, 1, CH ₂ CH ₃ ). ³¹ P NMR with suppression of the P-N interaction (D ₂ O): -5.02 s.

5'-Aminocarbonylphosphonate 2 ', 3'-dideoxy-3'-thiacytidine

To the obtained 5′-ethoxycarbonylphosphonate 2 ′, 3′-dideoxy-3′-thiacytidine (140 mg, 0.38 mmol) was added a solution of 25% aqueous ammonia (5 ml), the solution was stirred for 12 hours at a temperature of + 4 ° C, and then concentrated in vacuo. The residue was dissolved in water and chromatographed on DEAE-Toyopearl (HCO ₃ ^- ), eluted with a linear gradient of NH ₄ HCO ₃ (0 → 0.2 M), the desired product was eluted with 0.12 M NH ₄ HCO ₃ . The desired fractions were evaporated and re-evaporated with water (3 times 5 ml), the residue was diluted with water (3 ml) and applied to a LiChroprep RP-18 column, eluting with water. The target fraction was lyophilized to give 121 mg (71%) of 5′-aminocarbonylphosphonate 2 ′, 3′-dideoxy-3′-thiacytidine. ¹ H NMR (D ₂ O): 8.14 d (1H, J = 7.8 Hz, H-6), 6.30 dd (1H, J = 4.4 and 4.6 Hz, H-1 ' ), 6.11 d (1H, J = 7.8 Hz, H-5), 5.44 m (1H, H-4 '), 4.36 m (1H, H-5'a), 4, 23 m (1H, H-5'b), 3.56 m (1H, H-2'a), 3.24 m (1H, H-2'b). ³¹ P NMR with suppression of the P-N interaction (D ₂ O): -1.65 s.

Example 3

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 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- bromide (4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT).

The study of the cytotoxic effect of the drug

The studied drug in various concentrations is added to the cells. Cells are incubated at 37 ° C in an atmosphere with 5% CO ₂ and 98% humidity for 3-5 days. Analysis of results: determination of viability and cell number using dye.

The influence of the studied compounds on the reproduction of HIV-1 in cell culture MT-4

The test drug was added to the cells and infected with the virus at a dose of 0.01 TCID50 / cell. Cell cultures were incubated at 37 ° C in an atmosphere with 5% CO ₂ and 98% humidity for 4-5 days. The results were taken into account by staining the cells with a tetrazolium dye (MTT method) with spectrophotometry and light microscopy: investigation of the cytopathic effect of the virus (CPD) and virus-induced syncytium formation (syncytium is a conglomerate of several cells with a common cell membrane resulting from the fusion of their membranes).

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, the antiviral effect of the claimed compound can be judged.

Example 4

A dog weighing 12 kg was administered orally (mixed with cottage cheese) 190 mg of the test substance. At certain intervals, blood samples (1 ml) were taken from the femoral vein. Samples were centrifuged (10 minutes, 2000 rpm), the supernatant was separated. Aliquots (0.25 ml) were taken from the supernatant, oxetane (0.25 μg as an 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 pharmacokinetic parameters obtained by analyzing the data are given in table.2.

Table 1 Antiviral activity of 3TC and its phosphite against HIV-1 GB-4046 Compound ID ₅₀ , CD ₅₀ , IS µM µM 5'-phosphite of 2 ', 3'-dideoxy-3'-thiacytidine 42 ± 9 > 2200 > 52 5'-aminocarbonyl phosphonate of 2 ', 3'-dideoxy-3'-thiacytidine 67 ± 8 > 3700 > 55 3TC 1.1 ± 0.5 41 37

table 2 Pharmacokinetic parameters of 3TC after administration to a dog of 140 mg of substances 3TC and 5'-phosphite 2 ', 3'-dideoxy-3'-thiacytidine in an equivalent of 190 mg 3TC Compound T _1/2 hours AUC, mg.h / liter MRT watch CL, liter / hour T _max hours C _max mg / liter 5'-phosphite of 2 ', 3'-dideoxy-3'-thiacytidine 6.8 21.8 9.98 8.7 2.0 2.82 5'-aminocarbonyl-phospho-nat 2 ', 3'-dideoxy-3'-thiacytidine 6.5 18.5 10.54 9.1 2.7 1.79 3TC 5.9 30.7 8.62 6.2 1,5 5.17

Thus, it was shown that the claimed compounds have low toxicity, are able to effectively inhibit the reproduction of the human immunodeficiency virus of the first type in MT-4 cell culture and generate 3TC in the mammalian body, providing a smooth increase in its concentration in the blood.

Claims (1)

5'-Phosphorus-containing derivatives of 2 ', 3'-dideoxy-3'-thiacytidine having the general formula

,
where R = H, NH ₂ —C (O) -.