Derivatives of 5'-h-phosphonate 3'-azido-3'-deoxythymidine and pharmaceutical compositions based on them

 

(57) Abstract:

The invention relates to new antiviral derived 5'-H-phosphonate 3'-azido 3'-deoxythymidine General formula I

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where R represents isopropyl, neopentyl or cyclohexyl,

containing pharmaceutical compositions. The compounds of formula I is produced by interaction of the alcohol ROH with phosphorus trichloride and subsequent addition of the reaction mixture of 5'-H-phosphonate 3'-azido-3'-deoxythymidine or interaction of the 5'-H-phosphonate 3'-azido-3'-deoxythymidine and alcohol ROH in the presence of pivaloyl chloride as the activating agent. The compounds of formula I have low hydrophobicity and increased permeability through cell membranes. Pharmaceutical compositions containing the compounds I in the effective therapeutic dose and a pharmaceutically acceptable carrier, possess antiviral activity, including against HIV. 2 S. p. f-crystals, 2 tab.

The invention relates to new biologically active derivative 5'-H-phosphonate 3'-azido-3'-deoxythymidine formula I

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where R represents isopropyl, neopentyl or cyclohexyl, and containing pharmaceutical compositions.

The new compounds possess the local, that inhibit the reproduction of HIV can be 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 the reverse transcriptase of HIV, the first time the functioning of the enzyme in the chain replication of the virus, is the most attractive target for suppressing the propagation of the virus. Currently, the main group of drugs used to treat AIDS, are nucleoside reverse transcriptase inhibitors. These include 3'-azido-3'-deoxythymidine (AZT, AZT, zidovudine, Retrovir, Timated"), 2',3'-dideoxycytidine (ddC, zalcitabine, "GUID"), 2', 3'-dideoxyinosine (ddl, didanosine, videx"), 3'-deoxy-2',3'-didehydrothymidine (d4T, stavudine "stavudine, zerit) and 3'-thiacytidine (FTC, lamivudine, Epivir"). Their main disadvantage is the need of cellular phosphorylation to the corresponding triphosphates, the efficiency of which is very small. Thus, the process of transformation of AZT in humans in the corresponding 5'-triphosphate takes about 1.5-2 hours. During this time penetrated into the cells, the virus manages in the form of proviral DNA to integrate into the human genome. Since the output of the triphosphate is only a few hundredths of a share of izlenim side effects and rapid production of viral and cellular resistance.

Use as drugs nucleoside 5'-triphosphates with unmodified trifosfatnogo part is impossible due to their low stability to the action of enzymes hydrolysis and consequently a low ability to penetrate cells. Attempted synthesis of modified nucleoside 5'-triphosphates (WO 98/20017). However, the connection was insufficient to create a medicinal product.

More promising was such forms of active nucleosides, which has reduced the phosphorylation in the cell to the two stages were permeable to cell membranes and have been stable long enough in the bloodstream. One of such compounds is described in European patent 0 354 246 is 5'-H-phosphonate 3'-azido-3'-deoxythymidine (Phosphated) formulas II

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Currently Phosphated used as a medicine under the name "Nikavir" for treatment of AIDS (HIV infection).

Known derivatives of 5'-H-phosphonate 3'-azido-3'-deoxythymidine formula I, in which R is selected from the group: methyl, ethyl, hexyl, heptyl or octadecyl, which showed increased activity and less toxicity compared to ess toxic than the parent nucleosides. Antiviral Chem., Chemother., 1994, 5, 271-277).

In the work of V. M. Cardona F, Ayi A. I. and others (Antiviral Research, 1999, 42, 189-196) it was shown that the compound of the formula I, in which R is benzyl, active as anti-HIV drug trials in CEM-SS and MT-4 cells infected with HIV, and in the latter case activity 5 times higher than that of AZT. In CEM cells, deficient in timedancing, the connection is not active.

In accordance with the present invention offers derivatives of 5'-H-phosphonate 3'-azido 3'-deoxythymidine General formula I, where R represents isopropyl, neopentyl or cyclohexyl.

The compounds of formula I have low hydrophobicity and consequently increased permeability through cell membranes. The mechanism for the transformation of compounds of formula I in an active form inside cells are currently unknown. One of the proposed mechanisms is that due to the ability of the compounds I to the easy oxidation to phosphate AZT or its derivatives may flow reduced process intracellular phosphorylation. Another alternative mechanism for the transformation of compounds of formula I in the active form is their hydrolysis or until phosphazide, or to AZT.

To receive and alcohol ROH with phosphorus trichloride and subsequent addition of the reaction mixture of 5'-H-phosphonate 3'-azido-3'-deoxythymidine (method A). The second route involves the reaction of 5'-H-phosphonate 3'-azido-3'-deoxythymidine and alcohol ROH in the presence of pivaloyl chloride as the activating agent (method B).

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The compounds of formula I can be isolated and purified by conventional means, for example, extraction, precipitation, chromatography, etc.

According to the present invention the compounds of formula I can be used to obtain an antiviral pharmaceutical compositions with a suitable carrier. The pharmaceutical compositions can be obtained well-known in the art methods.

As carriers can be used various types of media commonly used in medicines, such as fillers, binders, loosening substances, lubricants, colorants, flavoring agents, fragrances, surfactants, etc.

Pharmaceutical compositions based on compounds of the formula I can be used as a drug in any dosage form. Dosage form can be properly selected in accordance with the object of treatment. Specific examples of dosage forms include oral drugs, the CLASS="ptx2">

The amount of the compounds of formula I of the present invention, which shall contain the above-described pharmaceutical composition, varies according to the type of composition, method of administration, the dosing scheme and therefore cannot be precisely defined, and is selected in accordance with the demand from a wide range. However, the composition may preferably contain about 1-80 wt.% the compounds of formula I by weight of the composition.

The present invention is illustrated by the following examples of the preparation of specific compounds, pharmaceutical compositions and pharmacological tests.

Example 1. 3'-Azido-3'-deoxythymidine, 5'-(2,2-dimethylpropyl)fosfat (Ia).

The solution l3(135 mg, 88 μl, 1 mmol) in dichloromethane (2 ml) cooled to 1-2oWith and with stirring, add neopentylene alcohol (88 mg, 1 mmol) and pyridine (80 μl, 1 mmol). A solution of AZT (130 mg, 0.5 mmol) in pyridine (80 μl) and acetonitrile (3 ml) is added at 4-5oC. the Mixture is stirred for 3 h at room temperature and diluted with cold saturated solution Panso3(3 ml) and chloroform (5 ml). The organic layer was washed with water, dried PA2SO4and evaporated. The remainder chromatographic on column>,max): 266 nm.1H NMR (CD3SP), , M. D.: 7.28, 7.31 (1H, N6), 6.87 d, 6.85 (1H, J706 Hz, P-H), 6.15 t (1H, J 6.0 Hz, H1'), 5.90 (1H, J 697 Hz, H-R), 4.35 (m 1H, H3'), 4.19 (m 2H, H5'), 4.01 m (1H, H4'), 3.73 dB, 3.70 (2H, J6 Hz), 2.38 t (2N, J 6.0 Hz, H2'), 1.85, 1.86 (3H, CH3), 0.92 (D, CH3C).31P NMR (CD3CN), ppm: 8.99, 8.40 C.

Example 2. 3'-Azido-3'-deoxythymidine, 5'-isopropylphenyl (IB).

An aqueous solution of Na-salt of 5'-H-phosphonate 3'-azido-3'-deoxythymidine (176 mg, 0.5 mmol) is passed through a column of Dowex-50 (PN+) (H cm), elwira water. The eluate evaporated to dryness and perevarivat with pyridine (32 ml). The residue is dissolved in MeCN (3.5 ml) and pyridine (2 ml) and added under stirring isopropyl alcohol (23 μl, 0.5 mmol). The mixture is cooled to -10oWith and add pivaloate (180 μl, 0.75 mmol). The cooling is removed and the mixture is stirred for 60 minutes the Mixture is diluted with chloroform (5 ml) and washed with saturated solution of NaHCO3(3 ml) and water (33 ml). The organic layer is dried Na2SO4, evaporated and perevarivat with toluene. Product chromatografic on a column of silica gel (152.5 cm), elwira system chloroform-methanol 96:4. Get the connection IB (156 mg). Yield 84%. UV (N2Omax): 266 nm.1H NMR (CD3CM), M. D.: 7.38, 7.39 (1H, N6), 6.86 (1H, J 700 Hz, P-H), 6.15 t is3).31P NMR (CD3CN), ppm: 10.09, 10.01 C.

Example 3. 3'-Azido-3'-deoxythymidine, 5'-cyclohexylmethyl (Ie).

An aqueous solution of Na-salt of 5'-H-phosphonate 3'-azido-3'-deoxythymidine (176 mg, 0.5 mmol) is passed through a column of Dowex-50 (PN+) (18 cm), elwira water. The eluate evaporated to dryness and perevarivat with pyridine (32 ml). The residue is dissolved in MeCN (3.5 ml) and pyridine (2 ml) and added dropwise with stirring cyclohexyloxy alcohol (60 μl, 0.55 mmol). The mixture is cooled to -10oWith and add pivaloate (180 μl, 0.75 mmol). The cooling is removed and the mixture is stirred for 60 minutes the Mixture is diluted with chloroform (5 ml) and washed with a saturated solution Panso3(3 ml) and water (33 ml). The organic layer is dried Na2SO4, evaporated and perevarivat with toluene. Product chromatografic on a column of silica gel (152.5 cm), elwira system chloroform-methanol 96:4. Get the connection In (128 mg). Yield 62%. UV (N2Omax): 266 nm.1H NMR (CD3CN). , M. D.: 7.38, 7.37 (1H, N6,), 6.86 (1H, J 700 Hz, P-H), 6.15 t (1H, J 6.0 Hz, H1'), 4.75 tons (1H, CH), 4.35 (m 1H, H3'), 4.19 (m 2H, H5'), 4.01 m (1H, H4'), 2.38 t (2N, J 6.0 Hz, H2'), 1.92 (3H, CH3), 1.90 t, 1.71 t, 1.50 t, 1.24, t (10H, cyclohexyl). 31P NMR (CD3CN), , M. D.: 7.60, 8.04 C.

Example 4. Tablet.

For prelolita microcrystalline - 40 mg Aerosil - 13 mg, calcium stearate - 12 mg.

Compound IB mixed with Aerosil and microcrystalline cellulose, sift and add calcium carbonate. The resulting mixture optivault the calcium stearate and mix to obtain a homogeneous mass. Received tabletting the mixture is pressed into briquettes on a tablet press. These briquettes are crushed in a ball mill. The granules are compressed on a tablet press to obtain tablets weighing 375 mg.

They studied the stability of the synthesized compounds of the formula I in aqueous solutions at pH 7.5 and in the serum of a person. The synthesized esters are hydrolyzed to the azidothymidine and qualitative composition of the products in both the investigated liquids is the same.

Example 5. Chemical hydrolysis of compounds of formula I.

The esters I (15-20 mg) was dissolved in acetonitrile (0.25 ml) and the solution is added to 0.1 M phosphate buffer (pH 7.5, 0.25 ml) at room temperature. After a certain period of time selected aliquots and explore them by TLC and HPLC. The HPLC conditions: column (1504 mm) Durasil C-8 (7 μm); the gradient of buffer B in buffer A. Buffer A: 5 mm potassium phosphate buffer (pH 6.2); buffer B: 80% Meon. 0.5 min with buffer A; 5 min - 40 min - 0-->100% buffer B; 40-45 min-is owaka.

To 100 mM solution of compounds I in formamide (0.5 ml) was added serum (100%, 99 μl) and the mixture is incubated at 37oC. After a certain period of time to aliquot (10 μl) was added acetone (40 ml), the mixture was incubated 20 min at -20oC and centrifuged. The supernatant evaporated, the residue is dissolved in water (50 μl) and analyzed by HPLC or TLC. The HPLC conditions as in example 5. The results are shown in table 1.

Was investigated the biological activity of the compounds of formula I, a is precisely their ability to inhibit the reproduction of human immunodeficiency virus and cytotoxicity.

Example 7. The antiviral activity of the compounds of formula I in relation to HIV.

The study was performed using HIV-1 strain GKV 4046 on transplantable lines sensitive cells MT-4. For infection used the supernatant of infected cells stored in liquid nitrogen, the multiplicity of infection was 0.2 to 0.5 infectious units per cell. The suspension of cells MT-4 with a concentration of 2,0106in the ml and the viability of at least 90% were placed in wells of 96-hole tablet ("l-Cult, UK) immediately after making vaccinated material and GPO three wells for each dose). Controls served infected with HIV-1 cells MT-4 without adding drugs (instead of the drug was made the same amount of RPMI-1640 medium without additives) and uninfected cells.

Tablet incubated for one hour at 37oFor adsorption of the virus, the cells are then diluted to seeding concentration (0,5106in milliliter) nutrient medium RPMI-1640 with the addition of 300 mg/ml L-glutamine and 100 μg/ml gentamicin and 10% fetal cattle serum, previously inactivated by heating at 56oC for 30 minutes. The tablet was placed in a thermostat at 37oC in an atmosphere of 5% CO2. For 3-4 days of cultivation was calculated concentration and cell viability by exclusion Trypanosoma blue. The data obtained was determined by the increase in cell viability relative to control under the influence of increasing doses of drugs.

In the wells was added tween-80 to a final concentration of 0.1% and were placed for 24 hours at 4oFor inactivation of HIV. Then evaluated the anti-HIV activity of drugs using the quantitative determination virousspecificakih protein P24 by direct enzyme immunoassay. Dose, 50 and 90% of suppressing p is the face 2. On the basis of these quantitative indicators of inhibition is possible to judge the efficacy of antiviral drugs, implying a high degree of suppression of the propagation of the HIV-1 in culture cells MT-4.

Example 8. Study the cytotoxicity of the compounds of formula I.

Evaluation of the cytotoxicity of the drugs was performed by adding their dilutions in medium RPMI-1640 cell suspension MT-4, placed in the wells of 96-hole tablet, to a final concentration of 0.001-100 µg/ml (three times), followed by cultivation at 37oWith in the next 3-4 days. Served as control cells without addition of drugs. The seeding concentration was 0,5106in milliliter. Viability of cells was counted in the Goryayev camera with advanced staining Trifanova blue. According to the obtained results were calculated dose by 50% reduce cell viability compared to control (CD50), the data are shown in table 2. It should be noted that the toxic effect on the cells MT-4, expressed in the change of their morphology, decreased reproduction and viability, provided only the maximum dose (100 µg/ml), 4-5 orders of magnitude greater than the effective who have the ability to inhibit the reproduction of human immunodeficiency virus type 1 in cell culture MT-4, moreover, the effectiveness of these drugs is comparable with the efficiency used in the clinical practice of medicine. It talks about the prospects of the compounds of formula I to create a new dosage forms, which can add to the Arsenal of tools used in therapy of AIDS.

1. Derivatives of 5'-H-phosphonate-3'-azido-3'-deoxythymidine General formula I

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where R represents isopropyl, neopentyl or cyclohexyl.

2. Pharmaceutical composition having antiviral activity, including the active ingredient and pharmaceutically acceptable carrier, characterized in that the active substance it contains a compound of General formula I on p. 1 in an effective therapeutic dose.

 

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