RU2106353C1 - 5-h-phosphonate-3-azido-3-desoxyttemidine salts which are specific inhibitors vich-1 and vich-2 - Google Patents

Abstract

FIELD: virology. SUBSTANCE: present invention describes new biologically active compounds, more particularly 5-H- phosphonate 3-azido-3- desoxytimidine or general formula given in the specification. New compounds are capable of specifically inhibiting human deficiency virus production. This makes it possible to use them for medical purposes for treating AIDS. Said compounds are prepared by neutralization of 5-H-phosphonate 3-azido-3- desoxytimidine with hydroxides of corresponding metals in water to adjust pH to 7-7.2. The resulting soluble salts are evaporated to small volumes. Spontaneous crystallization then occurs and the whole is kept at 4 C for 5-6 hours till crystallization is completed. Precipitants are filtered off, washed with small amounts of water cooled to 4 C and dried. Yield of the desired substances is 95-98%. New compounds inhibit multiplication of VICH-1 and VICH-2 in human lymphocyte cultures H9, MT-4 and PBL and are low or nongygroscopic and thus zacilitate preparation of drugs based thereon. EFFECT: improved properties of the title compounds. 3 tbl

Description

где R - атом щелочного или щелочноземельного металла.The invention relates to virology and relates to new biologically active compounds, namely 5'-H-phosphonate 3'-azido-3'-deoxythymidine of the General formula:

where R is an alkali or alkaline earth metal atom.

 New compounds have the ability to specifically inhibit the production of human immunodeficiency virus, which suggests the possibility of their use in medicine for the treatment of AIDS.

 Various compounds are currently known to inhibit the production of human immunodeficiency virus. The greatest distribution of them was 3'-azido-3'-deoxythymidine (AZT) [1].

 5'-phosphonates of 3'-azido-2 ', 3'-dideoxynucleosides [2], including 5'-H-phosphonate of 3'-azido-3'-deoxythymidine, which have inhibitory activity against HIV-1, are also known and HIV-2, but less toxic than AZT. However, the creation of a dosage form based on 5'-phosphonates of 3'-azido-2 ', 3'-dideoxynucleosides is extremely difficult due to their high hygroscopicity.

 The claimed salts of 5'-H-phosphonate of 3'-azido-3'-deoxythymidine are not inferior in activity and toxicity to the acid form of 5'-H-phosphonate of 3'-azido-3'-deoxythymidine, but at the same time they are characterized by significantly lower hygroscopicity . In addition, new salts expand the range of compounds with inhibitory activity against HIV-1 and HIV-2.

 New substances are white crystalline substances, highly soluble in water, sparingly soluble in alcohols, chloroform and methylene chloride.

Their preparation is carried out by neutralization of 5'-H-phosphonate of 3'-azido-3'-deoxythymidine with hydrates of the oxides of the corresponding metals in water to a pH of 7-7.2. The resulting soluble salts are evaporated to small volumes, from which spontaneous crystallization begins and left at 4 ^° C for 5-6 hours until crystallization is complete, precipitated precipitates are filtered off, washed with small volumes of water cooled to 4 ^° C and dried. The yield of target substances is 95-98%.

 The purity and structure of the new compounds are confirmed by HPLC and UV and NMR spectroscopy.

 The following are examples of the preparation of specific salts of 5'-H-phosphonate 3'-azido-3'-deoxythymidine.

Example 1. Synthesis of the sodium salt of 5'-H-phosphonate 3'-azido-3'-deoxythymidine
To a solution of 166 g (0.5 moles) of 5'-H-phosphonate 3'-azido-3'-deoxythymidine in 200 ml of distilled water, 100 ml (0.5 mol) are added while cooling with ice-water and stirring for 20 min. 5n a solution of sodium hydroxide hydrate (qualification hch) to a pH of 7.2. The resulting clear white solution is evaporated on a rotary evaporator at a bath temperature of not more than 45 ^o C to 150-170 ml of the total volume and left for 6 hours at 4 ^o C. The precipitated fine crystalline precipitate is separated on a glass filter, washed with 40 ml cooled to 4 ^o C distilled water and dried in a vacuum desiccator. Yield 164 g (95.4%), mp. > 300 ^o C (carbonization). The substance is low-hygroscopic.

The retention time on a Nucleosil 120-7 NH ₂ column in a linear gradient of KH ₂ PO ₄ (0.05-1 M) was 7.2 minutes, flow rate 1 ml / min.

UV spectrum (water) λ _max 266 nm (ε 9600), 1H NMR spectrum (D ₂ O, δ), ppm: 1.93 d (3H, CH ₃ , J _CH3-H6 0.5 Hz ); 2.55 m (2H, H2 '); 4.60-3.95 m (4H, H3 '+ H4' + 2H5 '); 6.15 t (1H, H1 ', J _H1'H2' 6 Hz); 7.66 d (1H, H6, J _{H6, CH3} 1 Hz). ³¹ P-NMR spectrum (D ₂ O, δ), ppm: 6.5 d (1H, H ^P , J _{H, P} 626 Hz; J _{P-H5 '} 6.3 Hz, J _{P-H4 '} 1.6 Hz).

 Example 2. The synthesis of the potassium salt of 5'-H-phosphonate 3'-azido-3'-deoxythymidine.

To a solution of 166 g (0.5 moles) of 5'-H-phosphonate 3'-azido-3'-deoxythymidine in 200 ml of distilled water, 100 ml (0.5 mol) are added while cooling with ice-water and stirring for 20 min. 5n a solution of potassium oxide hydrate (qualification of hch) to a pH of 7.2. The resulting clear white solution is evaporated on a rotary evaporator at a bath temperature of not more than 45 ^o C to 150-170 ml of the total volume and left for 6 hours at 4 ^o C. The precipitated fine crystalline precipitate is separated on a glass filter, washed with distilled with 40 ml cooled to 4 ^o C water and dried in a vacuum desiccator. Yield 172 g (97.1%), mp. > 300 ^o C (carbonization). The substance is not hygroscopic.

The retention time on a Nucleosil 120-7 NH ₂ column in a linear gradient of KH ₂ PO ₄ (0.05-1 M) was 7.2 minutes, flow rate 1 ml / min.

UV spectrum (water) λ _max 266 nm (ε 9600), 1H NMR spectrum (D ₂ O, δ), ppm: 1.93 d (3H, CH ₃ , J _CH3-H6 0.5 Hz ); 2.55 m (2H, H2 '); 4.60-3.95 m (4H, H3 '+ H4' + 2H5 '); 6.15 t (1H, H1 ', J _H1'H2' 6 Hz); 7.66 d (1H, H6, J _{H6, CH3} 1 Hz). ³¹ P-NMR spectrum (D ₂ O, δ), ppm: 6.5 d (1H, H ^P , J _{H, P} 626 Hz; J _{P-5H '} 6.3 Hz, J _{P-H4 '} 1.6 Hz).

 Example 3. Synthesis of the calcium salt of 5'-H-phosphonate 3'-azido-3'-deoxythymidine.

To a solution of 166 g (0.5 moles) of 5'-H-phosphonate 3'-azido-3'-deoxythymidine in 200 ml of distilled water, 1000 ml (0.25 mol) are added while cooling with ice and water for 20 minutes. 0.5n pre-filtered solution of calcium oxide hydrate (qualification of chemically pure) to a pH of 7.0. The resulting clear white solution is evaporated on a rotary evaporator at a bath temperature of not more than 45 ^o C to about 200 ml of the total volume and left for 6 hours at 4 ^o C. The precipitated fine crystalline precipitate is separated on a glass filter, washed with 40 ml of distilled water cooled to 4 ^o C and dried in a vacuum desiccator. Yield 174 g (97.1%), mp. > 300 ^o C (carbonization). The substance is not hygroscopic.

The retention time on a Nucleosil 120-7 NH ₂ column in a linear gradient of KH ₂ PO ₄ (0.05-1 M) was 7.2 minutes, flow rate 1 ml / min.

UV spectrum (water) λ _max 266 nm (ε 9600), 1H NMR spectrum (D ₂ O, δ), ppm: 1.93 d (3H, CH ₃ , J _CH3-H6 0.5 Hz ); 2.55 m (2H, H2 '); 4.60-3.95 m (4H, H3 '+ H4' + 2H5 '); 6.15 t (1H, H1 ', J _H1'H2' 6 Hz); 7.66 d (1H, H6, J _{H6, CH3} 1 Hz). ³¹ P-NMR spectrum (D ₂ O, δ), ppm: 6.5 d (1H, H ^P , J _{H, P} 626 Hz; J _{P-5H '} 6.3 Hz, J _{P-H4 '} 1.6 Hz). Example 4. Synthesis of the barium salt of 5'-H-phosphonate 3'-azido-3'-deoxythymidine.

To a solution of 166 g (0.5 moles) of 5'-H-phosphonate 3'-azido-3'-deoxythymidine in 200 ml of distilled water, 1000 ml (0.25 mol) are added while cooling with ice and water for 20 minutes. 0.5 n pre-filtered solution of barium oxide hydrate (qualification of chemically pure) to a pH of 7.0. The resulting clear white solution is evaporated on a rotary evaporator at a bath temperature of not more than 45 ^o C to about 3300 ml of the total volume and left for 6 hours at 48 ^o C. The precipitated fine crystalline precipitate is separated on a glass filter, washed with 40 ml of distilled water cooled to 4 ^o C and dried in a vacuum desiccator. Yield 226 g (98.5%), mp. > 300 ^o C (carbonization). The substance is not hygroscopic.

The retention time on a Nucleosil 120-7 NH ₂ column in a linear gradient of KH ₂ PO ₄ (0.05-1 M) was 7.2 minutes, flow rate 1 ml / min.

UV spectrum (water) λ _max 266 nm (ε 9600), 1H NMR spectrum (D ₂ O, δ), ppm: 1.93 d (3H, CH ₃ , J _CH3-H6 0.5 Hz ); 2.55 m (2H, H2 '); 4.60-3.95 m (4H, H3 '+ H4' + 2H5 '); 6.15 t (1H, H1 ', J _H1'H2' 6 Hz); 7.66 d (1H, H6, J _{H6, CH3} 1 Hz). ³¹ P-NMR spectrum (D ₂ O, δ), ppm: 6.5 d (1H, H ^P , J _{H, P} 626 Hz; J _{P-5H '} 6.3 Hz, J _{P-H4 '} 1.6 Hz).

NMR spectra were recorded on a 250 MHz Brucker spectrometer. UV spectra were determined on a Specord UV-vis M-40 spectrophotometer. HPLC was performed using a Nucleosil 120-7 NH ₂ column.

 The biological properties of the new compounds were studied, namely their ability to specifically inhibit the production of human immunodeficiency virus and toxicity. Studies were carried out on human lymphocyte cultures — transplantable lymphoblastoid cell lines of human H9 / IIIB, MT-4 and PBL. Cell production of the virus was monitored in indirect immunofluorescence and enzyme immunoassay reactions. The activity of the compounds was characterized by the value of the ED-effective concentration, which suppresses the production of HIV-1 and HIV-2 by 50%, toxicity - by the value of the TD-concentration, which suppresses cell growth by 50%. The selectivity index was calculated as the ratio of TD to ED. For comparison, the acid form of 5'-H-phosphonate of 3'-azido-3'-deoxythymidine was used. The experimental data are given in table. 1-3.

 Thus, new substances — salts of 3′-azido-3′-deoxythymidine 5′-H-phosphonate salts — inhibit the reproduction of HIV-1 and HIV-2 in human lymphocyte cultures H9, MT-4, and PBL, with approximately the same activity with which this was also observed for the acid form of 5'-H-phosphonate 3'-azido-3'-deoxythymidine. The toxicity of the claimed salts of 5'-H-phosphonate 3'-azido-3'-deoxythymidine is also within the toxicity of the acid form of 5'-H-phosphonate 3'-azido-3'-deoxythymidine. At the same time, new compounds are low- or non-hygroscopic, which facilitates the creation of dosage forms based on them.

Claims (1)

Salts of 3'-azido-3'-deoxythymidine 5'-H-phosphonate of the general formula:

where R is an alkali or alkaline earth metal atom, which are specific inhibitors of the production of human immunodeficiency virus HIV-1 and HIV-2.

Images