RU2475476C1 - 3-hydroximino derivatives of 2,3-secolupane type, demonstrating inhibiting activity with respect to influenza a and hiv-1 virus - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: described are 3-hydroximino derivatives of 2,3-secolupane type of general formula:

, where R=H or CH₃, demonstrating inhibiting activity with respect to influenza A virus. Compound with R=H combines antiviral activity with respect to influenza A virus with anti-HIV activity.

EFFECT: compounds are promising for elaboration of antiviral preparations and as key intermediates for obtaining new biologically active compounds.

3 cl, 2 tbl, 4 ex

Description

The invention relates to new chemical compounds of the class of lupane 2,3-secotriterpenoids, which exhibit inhibitory activity against influenza A virus (H7N1) and HIV-1.

It is known that in the development of new antiviral agents, natural polycyclic terpenoids are actively used as base platforms. For example, on the basis of lupane triterpenoids (betulin, betulinic and betulonic acids), which in their native form exhibit moderate antiviral properties, a wide range of semisynthetic derivatives with high inhibitory activity against enveloped viruses (HIV-1, influenza A, herpes simplex and Epstein-viruses) were obtained. Barra) [Terpenoids of the lupane series as promising biologically active agents for medicine. Part 2. Semi-synthetic derivatives of lupane / T.G. Tolstikova, I.V. Sorokina, G.A. Tolstikov, A.G. Tolstikov, O.B. Flekhter // Bioorganic chemistry. - 2006. - T.32. - Number 3. - S.291-307]. A-sec derivatives of the lupane type, which have higher biological activity compared to the initial triterpenoids, can be considered as alternative base compounds for the synthesis of new antiviral agents [Microbial transformations of two lupane-type triterpenes and anti-tumor-promoting effects of the transformation products / T. Akihisa, Y. Takamine, K. Yoshizumi, H. Tokuda, Y. Kimura, M. Ukiya, T. Nakahara, T. Yokochi, E. Ichiishi, H. Nishino // Journal of Natural Products - 2002. - V .65. - P.278-282; Synthesis of dammarane-type triterpene derivatives and their ability to inhibit HIV and HCV proteases / Y. Wei, C.-M. Maa, M. Hattori // Bioorganic & Medicinal Chemistry. - 2009. - V.17. - P.3003-3010].

The closest analogues of the described compounds in structure are 1-cyano-2,3-secolup-20 (29) -en-3-al-28-oic acid [Synthesis and antiviral activity of 2,3-sec-derivatives of betulonic acid / I.A. Tolmacheva, V.V. Grishko, E.I. Boreko, O.V. Savinova, N.I. Pavlova // Chemistry of Natural Compounds. - 2009. - No. 5. - S.566-568; 2,3-sec-derivatives of betulinic acid / I.A. Tolmacheva, V.V. Grishko, E.I. Boreko, O.V. Savinova, N.I. Pavlova // Patent No. 2410390. - 03.08.2009], showing weak activity against influenza A virus and inactive against HIV-1, and methyl 1-cyano-2,3-secolup-20 (29) -en-3-al-28-oic acid [Synthesis of lupanic and 19β, 28-epoxy-18α-oleanan 2,3-seco derivatives based on betulin / I.A. Tolmacheva, A.V. Nazarov, O. A. Mayorova, V. V. Grishko // Chemistry natural compounds. - 2008. - No. 5. - S.491-494], the antiviral properties of which are not described.

The objective of the invention is the synthesis of new 2,3-secotriterpene derivatives with inhibitory activity against influenza A and HIV-1, promising to expand the raw material base of antiviral agents and as key intermediates for the preparation of new biologically active compounds.

To solve the problem synthesized

1. 3-Hydroxyimino derivatives of 2,3-secolupane type of the General formula:

where R = H or CH ₃ .

2. The compounds according to claim 1, exhibiting inhibitory activity against influenza A.

3. The compound according to claim 1, where R = H, exhibiting inhibitory activity against HIV-1.

Compounds of the general formula are obtained where R = H (compound I); R = CH ₃ (compound II).

The synthesized compounds are white crystalline substances, readily soluble in chloroform, dichloromethane, carbon tetrachloride, ethyl alcohol, benzene, toluene, dimethyl sulfoxide. These compounds are poorly soluble in hexane and insoluble in water.

The compounds of the general formula were synthesized by a method involving the interaction of 1-cyano-2,3-secolup-20 (29) -en-3-al-28-oic acid or its methyl ester with hydroxylamine hydrochloride in ethanol in the presence of pyridine.

The structure of compounds I and II was confirmed by IR and NMR spectroscopy. The spectral characteristics of compounds I, II are given in table 1. The IR spectrum (ν, cm ^-1 ) was recorded on an IFS 66 / S Bruker IR spectrometer (Germany) in a paste with liquid paraffin. A ¹ H- and ¹³ C-NMR spectrum (δ, ppm; J, Hz) was recorded for the solution in CDCl ₃ on a Varian Mercury + spectrometer (USA) at an operating frequency of the instrument of 300 or 75.5 MHz, and the internal standard was hexamethyldisiloxane. The threshold temperature at the melting point was determined on an OptiMelt MPA100 instrument (USA) at a rate of 1 ° C per min. The specific optical rotation was measured for a solution in chloroform on a 341 polarimeter of the Perkin-Elmer model (USA) at a wavelength of 589 nm. The substance was detected with 5% sulfuric acid by heating (100-120 ° C) for 1 min. For column chromatography, Merck silica gel (60-200 μm) was used, the ratio of the substance and sorbent was ≈ 1:50, and the eluent was a mixture of hexane: ethyl acetate (7: 1).

The antiviral activity of the compounds with respect to the reproduction of influenza A virus (H7N1) and human immunodeficiency virus type I (HIV-1) was studied in cell cultures.

Descriptions of the claimed compounds and their properties in the information sources were not found.

The essence of the proposed solution and the possibility of its implementation is confirmed by examples 1-4 and the research results shown in tables 1 and 2.

Example 1. Obtaining 3-hydroxyimino-1-cyano-2,3-secolup-20 (29) -en-28-oic acid (compound I).

(с 0,25; CHCl₃).A mixture of 1 mmol of 1-cyano-2,3-secolup-20 (29) -en-3-al-28-oic acid and 3 mmol of hydroxylamine hydrochloride was dissolved in 50 ml of a mixture of ethanol-pyridine (5: 1). The reaction mixture was boiled for 2-3 hours. The progress of the reaction was monitored by TLC. At the end of the reaction, the solvent was evaporated, the residue was diluted with water and extracted with ethyl acetate, the organic layer was separated, dried over anhydrous magnesium sulfate. The solvent was evaporated, the residue was purified by column chromatography, the yield of the obtained compound was 63%; R _f 0.44 (hexane: ethyl acetate - 7: 3); so pl. 208.7 ° C (hexane: ethyl acetate - 7: 1);

(c 0.25; CHCl ₃ ).

(с 0,3, CHCl₃).Example 2. 3-Hydroximino-1-cyano-2,3-secolup-20 (29) -en-28-acid acid methyl ester (compound II) was prepared according to the procedure described in Example 1. Yield 52%; R _f 0.3 (hexane: ethyl acetate - 7: 3); so pl. 109.6 ° C (hexane: ethyl acetate);

(c 0.3, CHCl ₃ ).

Example 3. The study of antiviral activity against influenza A.

During the tests, a cell culture of primary fibroblasts of chicken embryos (FEC) was used, as well as the influenza virus A / FPV / Rostock / 34 (H7N1). Studies were performed by reducing the number of plaques under an agar coating. A monolayer culture of FEC grown in vials was washed from the growth medium, infected with various dilutions of the virus in the range of 0.01-0.0001 PFU / cell by applying to the cells a virus-containing suspension in a volume of 0.1 ml for 1 h at 37 ° C. Then the liquid was removed and the cells were covered with a melt of a nutrient medium based on a 199 medium concentrate with the addition of 1% Bactagar and 0.005% neutral red (all reagents were Sigma) and various concentrations of the studied compounds. After the coating solidified, the vials were placed in a thermostat for 36 h and then the number of plaques visible due to the presence of vital dye in the coating was counted.

The virus titer in the presence of the analyte and in the control was calculated in lg PFU (plaque forming units) in 1 ml. The primary criterion for antiviral activity was considered the presence of differences in comparison with the control of the virus. Processing of the obtained data (calculation of the virus titer and the arithmetic mean error, Sx) was performed by statistical methods for small values of n in an ungrouped data series. The concentrations of substances that inhibit the multiplication of the virus by 50% (average effective concentration, EC ₅₀ ) were determined on the basis of probit analysis and weighted linear regression [A computer program in BASIC for estimation of ED ₅₀ and LD ₅₀ / KPFung. // Computers in Biology and Medicine. - 1989. - V.19. - No. 2. - P.131-135]. The ratios of the maximum tolerated concentration (IPC) of substances to the EU _{50 were} also calculated. IPC was defined as the maximum concentration of a substance that does not affect the morphology of unpainted cell culture and impaired absorption of vital dye (neutral red).

Example 4. The methodology of the study of antiviral activity against HIV-1.

The tests were carried out on transplantable suspension T-lymphoblastoid cell line of human MT-4 (density 4-5 × 10 ⁵ cells / ml) on a nutrient medium RPMI-1640 Sigma-Aldrich (USA). A highly _replicate HIV-1 _{zmb isolate} with a titer of 6.0 lg TCD _{50 was} used as an infectious agent. The presence of anti-HIV activity was determined using the formazan test in the MTT variant. A stock solution of the preparation (5.0 mg / ml) was prepared ex tempore by dissolution in 10% ethanol. The stock solution was titrated in the wells of a 96-well panel in 5-fold increments, after which cells and virus were sequentially added to the wells (therapeutic regimen). The final reaction volume was 200 μl / well. The untreated HIV-infected cells (virus control) and the untreated uninfected cells (cell control) served as controls. The panels were incubated in an atmosphere containing 5% CO ₂ at 37 ° C. The results were taken into account after 72 hours. As a positive control for anti-HIV activity, a commercial azidothymidine preparation was used in each series of experiments, which was titrated in parallel with the test sample.

The technique is based on determining the formation rate of the formazan product when MTT reagent (3- (4,5-dimethyl-thiazol-2) -2,5-diphenal-tetrazolium bromide) is introduced into the cell culture. After a 3-hour exposure at 37 ° C, the supernatant was removed, and the formed formazan product was dissolved in dimethyl sulfoxide, then the intensity of the developed staining was measured on a Plate Reader DAS A3 spectrophotometer (Italy) at a wavelength of 550/630 nm. The test results were taken into account by determining the cell protection index, which in positive cases should be at least 50%. The EC _{50 of the} preparation and the MPK / EC ₅₀ ratio, which characterizes the wide spectrum of its non-toxic effective concentrations, were calculated.

It was found that compound I combines two types of antiviral activity and has the ability to inhibit the reproduction of influenza A and HIV-1 in the concentration range of 50-12.5 and 1.5-0.016 μg / ml, respectively. Compound II has the ability to inhibit the reproduction of influenza A virus in a concentration range of 24-11 μg / ml. The calculated EU ₅₀ and IPC / EU ₅₀ ratios are shown in Table 2.

Table 1 Data IR, NMR ¹ H and ¹³ C spectra of compounds I, II Compound-
nie IR spectrum (ν, cm ^-1 ) Spectrum ¹ H-NMR (300.0 MHz, CDCl ₃ , δ, ppm, J / Hz): ¹³ C-NMR spectrum (75.5 MHz, CDCl ₃ , δ, ppm) I 1688 (C = N), 1720 (C = O), 2237 (C≡N), 3188 (NOH) 0.90, 0.95, 1.00, 1.13, 1.17 (15H, 5s, 5CH ₃ ), 1.67 (3H, s, CH ₃ ), 2.48 and 2.59 (2H, 2d, J = 18.2, CH ₂ ), 3.01 (1H, td, J = 10.8, 4.5, CH), 4.57 and 4.69 (2H, 2s, CH ₂ ), 7 74 (1H, s, CH), 10.26 (1H, s, NOH), 11.63 (1H, ss, COOH) 13.31, 14.74, 16.85, 18.15, 18.90, 20.68, 21.03, 24.47, 27.77 (2C), 28.20, 28.43, 29.33 , 30.86, 32.12, 35.71, 36.95, 39.76, 41.19, 41.60, 43.49, 45.59, 47.73, 50.34, 54.76, 108 , 49, 117.42, 149.27, 156.95, 176.87 II 1644 (C = N), 1728 (COOCH ₃ ), 2244 (C≡N), 3388 (NOH) 0.89, 0.92, 1.00, 1.13, 1.18 (15H, 5s, 5CH ₃ ), 1.67 (3H, s, CH ₃ ), 2.44 and 2.57 (2H, 2d, J = 18.6, CH ₂ ), 2.93-3.10 (1H, m, CH), 3.65 (3H, s, COOCH ₃ ), 4.60 and 4.72 (2H, 2s) , CH ₂ , 7.53 (1H, s, CH), 10.56 (1H, s, NOH) 14.50, 15.80, 18.11, 19.21, 20.05, 21.90, 21.94, 25.55, 28.59, 29.50, 29.62, 30.46, 31, 91, 33.27, 36.82, 38.26, 40.64, 41.01, 42.46, 42.79, 44.66, 46.88, 49.18, 51.28, 51.48, 56.51, 109.83, 118.42, 150.26, 160.08, 176.62

table 2 Antiviral properties of compounds I, II Compound-
nie Toxicity to cell cultures, MIC (RD / MT-4), μg / ml Antiviral properties influenza A virus HIV-1 EC ₅₀ μg / ml IPC / EU ratio ₅₀ EC ₅₀ μg / ml IPC / EU ratio ₅₀ I 50 / 1,5 8.8 5.7 0,03 fifty II 400/10 11.3 35,4 On. On. On. - the connection is not active.

Claims (3)

1. 3-Hydroxyimino derivatives of 2,3-secolupane type of the General formula:

where R = H or CH ₃ . 2. The compounds according to claim 1, exhibiting inhibitory activity against influenza A. 3. The compound according to claim 1, where R = H, exhibiting inhibitory activity against HIV-1.