RU2537840C1 - A-seco-triterpenoids with methylketone fragment - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to A-seco-triterpenoids of general formula (I)

, where

or

, demonstrating inhibiting activity with respect to type I herpes simplex virus (HSV-1, strain 1C) and HIV-1. In addition, compounds (I) can be used as intermediates for other biologically active compounds.

EFFECT: increased efficiency of compound application.

2 cl, 1 tbl, 6 ex

Description

The invention relates to new chemical compounds of the class A-secotriterpenoids of 18αH-oleanane and lupane types.

Antiviral properties are characteristic of many natural and semi-synthetic A-secotriterpenoids, which makes this group of compounds promising for the development of new therapeutically significant agents [Y. Wei, C.-M. Ma, D.-Y. Chen, M. Hattori. Anti-HIV-1 protease triterpenoids from Stauntonia obovatifoliola Hayata subsp.intermedia // Phytochemistry. - 2008 .-- Vol.69. - P.1875-1879; Y. Wei, C.-M. Ma, M. Hattori. Synthesis of dammarane-type triterpene derivatives and their ability to inhibit HIV and HCV proteases // Bioorganic & Medicinal Chemistry. - 2009 .-- Vol.17. - P. 3003-3010; Y. Wei, C.-M. Ma, M. Hattori. Synthesis and evaluation of A-seco type triterpenoids for anti-HIV-1 protease activity // European Journal of Medicinal Chemistry. - 2009 .-- Vol.44. - P.4112-4120].

Among the compounds of this class are semisynthetic triterpenoids containing a highly reactive fragment of methyl ketone in the A-ring [T. Honda, G.W. Gribble Design and synthesis of 23.24-dinoroleanolic acid derivatives, novel triterpenoid-steroid hybrid molecules // Journal of Organic Chemistry - 1998. - Vol. 63. - P. 4846-4849; O.V. Kazakova, N.I. Medvedeva, O.S. Kukovinets, G.A. Tolstikov, E.F. Khusnutdinova, L. Zaprutko, B. Bednarczyk-Cwynar, Z. Paryzek. Chemoselective oxidation of oleanolic acid derivatives with ozone // Chemistry of Natural Compounds - 2010 .-- Vol. 46. - Number 3. - P.397-399; Y. Deng, J. K. Snyder. Preparation of a 24-nor-1,4-dien-3-one triterpene derivative from betulin: a new route to 24-nortriterpene analog // Journal of Organic Chemistry - 2002. - Vol. 67. - P.2864-2873], including those possessing antiparasitic and cytotoxic properties [S. Moiteiro, M.J.M. Curto, N. Mohamed, M. Bailen, R. Martinez-Diaz, A. Gonzalez-Coloma. Biovalorization of friedelane triterpenes derived from cork processing industry by products // Journal of Agricultural and Food Chemistry - 2006. - Vol. 54. - P.3566-3571; C. Moiteiro, C. Manta, F. Justino, R. Tavares, M.J.M. Curto, M. Pedro, M.S. Nascimento, M. Pinto. Hemisynthetic secofriedelane triterpenes with inhibitory activity against the growth of human tumor cell lines in vitro // Journal of Natural Products - 2004. - Vol. 67. - P.1193-1196].

The closest analogue (prototype) of the described compounds in structure is 3-nor-2,4-secofridelane-4-oxo-2-oic acid with antitumor (GI ₅₀ 10,19-17,6 µM) [C. Moiteiro, C. Manta, F. Justino, R. Tavares, MJM Curto, M. Pedro, MS Nascimento, M. Pinto. Hemisynthetic secofriedelane triterpenes with inhibitory activity against the growth of human tumor cell lines in vitro // Journal of Natural Products - 2004. - Vol. 67. - P.1193-1196] and insecticidal activity (EC ₅₀ 20,23 µg / ml) [C. Moiteiro, MJM Curto, N. Mohamed, M. Bailen, R. Martinez-Diaz, A. Gonzalez-Coloma. Biovalorization of friedelane triterpenes derived from cork processing industry by products // Journal of Agricultural and Food Chemistry - 2006. - Vol. 54. - P.3566-3571]. However, in the literature there is no evidence of the antiviral properties of 3-nor-2,4-secofridelane-4-oxo-2-oic acid.

The objective of the invention is the synthesis of new triterpene derivatives to expand the raw material base of antiviral agents, also promising as key intermediates for the preparation of new biologically active compounds.

1. To solve the problem synthesized A-secotriterpenoids of General formula (I)

,

,

или

.Where

or

.

проявляет ингибирующую активность в отношении вируса герпеса простого I типа (ВГП-1, штамм 1 С) и ВИЧ-1.2. Connection with

exhibits inhibitory activity against herpes simplex virus type I (VGP-1, strain 1 C) and HIV-1.

Synthesized compounds of General formula (I), where

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

The structure of compounds I and II was confirmed by IR and NMR spectroscopy. The spectral characteristics of compounds I, II and key intermediates are given in the table. ¹ H NMR spectra (δ, ppm; J, Hz) were recorded for the solution in CDCl ₃ on a Varian Mercury + 300 spectrometer (USA) at an operating frequency of the instrument of 300 MHz, and the internal standard was hexamethyldisiloxane. IR spectra (ν, cm ^-1 ) were recorded on an IFS 66 / S Bruker IR spectrometer (Germany) in a thin film obtained by evaporating a solution of a substance in CHCl ₃ on a NaCl glass surface. The threshold temperature at the melting point was determined on an OptiMelt MPA100 device (USA) at a rate of 1 ° C per min. The specific optical rotation value was measured for a solution in CHCl ₃ on a 341 polarimeter of the Perkin-Elmer model (USA) at a wavelength of 589 nm. Qualitative control of the substances was carried out by TLC on Sorbfil plates (Russia), the hexane-ethyl acetate 7: 3 system was used to determine the value of R _f , substances were detected by treatment with 5% H ₂ SO ₄ , followed by heating of the plate at 95-100 ° С within 2-3 minutes For column chromatography, silica gel of the Merck brand (60-200 μm) was used, eluent was a mixture of hexane-ethyl acetate (10: 1).

In vitro studies have shown the inhibitory activity of compound II in relation to the reproduction of herpes simplex virus type I (HHP-1) and human immunodeficiency virus type I (HIV-1).

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 1-6 and the research results shown in the table.

The synthesis of the claimed compounds was carried out in two ways.

The first method is the alkylation of 2-hydroxymino derivatives of betulonic acid or allobetulone with methyl magnesium iodide, followed by Beckman’s cleavage of the resulting 2-hydroxyimino-3β-hydroxy-3-methyl derivatives of betulonic acid or allobetulone [Synthesis of lupanoic and 19β, 28-epoxy-18α-2α-ol , 3-sec-derivatives based on betulin / I.A. Tolmacheva, A.V. Nazarov, O.A. Mayorova, V.V. Grishko // Chemistry of Natural Compounds. - 2008. - No. 5. - S. 491-494].

The second method consists in the alkylation with methyl magnesium iodide of methyl ester of 1-cyano-2,3-seco-2-norlup-20 (29) -en-3-al-28-oic acid or 1-cyano-19β, 28-epoxy-2, 3-seco-2-nor-18αH-oleanan-3-ala [Synthesis of lupanic and 19β, 28-epoxy-18α-oleananic 2,3-seco-derivatives based on betulin / I.A. Tolmacheva, A.V. Nazarov, O.A. Mayorova, V.V. Grishko // Chemistry of Natural Compounds. - 2008. - No. 5. - S.491-494] followed by oxidation with Jones reagent obtained 3-hydroxy-3-methyl-1-cyano-2-nor-2,3-secotriterpenoids.

Example 1. Obtaining methyl ester of 3-methyl-3-oxo-1-cyano-2,3-seco-2-norlup-20 (29) -en-28-oic acid (compound I, method 1).

To 3 ml of a freshly prepared solution of CH ₃ MgI (13.2 mmol) in diethyl ether, 6.6 mmol of 2-hydroxyimino-3-oxolup-20 (29) -en-28-acid acid methyl ester was added in small portions, then 13 ml was added dropwise. anhydrous diethyl ether. The reaction mixture was stirred under heating for 1 h, then 25 ml of ice water was added dropwise and 20 ml of twice diluted HCl was added. The mixture was stirred until the precipitate was completely dissolved for 1 h. The reaction product was extracted with ethyl acetate (3 × 20 ml). The organic layer was separated, washed with saturated NaHSO ₃ solution, then with 5% NaHCO ₃ solution and a small amount of water, dried over anhydrous MgSO ₄ . The solvent was evaporated, and the residue was purified by column chromatography.

(с 1,38; CHCl₃).The yield of the reaction product was 88%, R _f = 0.6 (chloroform-methanol 20: 1), so pl. 204.6 ° C (5: 1 hexane-ethyl acetate); $${[\alpha ]}_D^{25}+21,9$$

(c 1.38; CHCl ₃ ).

4.6 mmol of 3β-hydroxy-3-methyl-2-hydroxyimino-2-norlup-20 (29) -en-28-oic acid methyl ester of formula (II) thus obtained was dissolved in dry dichloromethane with stirring, was added 14.0 mmol of thionyl chloride. The reaction mixture was stirred at room temperature for 30 minutes. Checked the completeness of the reaction by TLC. After completion of the reaction, the solvent was evaporated in vacuo of a water-jet pump. The residue was washed twice with dry dichloromethane to get rid of traces of thionyl chloride. Compound I was purified by column chromatography.

(с 0,5; CHCl₃).Yield 83%; R _f = 0.6; t. pl. 229.8 ° C (5: 1 hexane-ethyl acetate); $${[\alpha ]}_D^{25}+\mathrm{29th},\mathrm{one}$$

(c 0.5; CHCl ₃ ).

(с 1,38; CHCl₃).Example 2. 3-Methyl-1-cyano-19β, 28-epoxy-2,3-seco-2-nor-18αH-oleanan-3-one (compound II, method 1) was synthesized according to the procedure described in example 1, using 2-hydroxyimino-19β, 28-epoxy-18αH-olean-3-one as the starting compound, from which the intermediate 3β-hydroxy-3-methyl-2-hydroxyimino-19β, 28-epoxy-2-nor-18αH was obtained -oleanane of the formula (III) with a yield of 86%, R _f = 0.6 (chloroform-methanol 20: 1), so pl. 193.7 ° C (5: 1 hexane-ethyl acetate); $${[\alpha ]}_D^{25}+43,\mathrm{four}$$

(c 1.38; CHCl ₃ ).

(с 0,5; CHCl₃).The yield of compound II is 84%; R _f = 0.4; so pl. 163.4 ° C (5: 1 hexane-ethyl acetate); $${[\alpha ]}_D^{25}+26,7$$

(c 0.5; CHCl ₃ ).

Example 3. Obtaining methyl ester of 3-methyl-3-oxo-1-cyano-2,3-seco-2-norlup-20 (29) -en-28-oic acid (compound I, method 2).

To 3 ml of a freshly prepared solution of CH ₃ MgI (4.4 mmol) in diethyl ether, 2.2 mmol of 1-cyano-2,3-sec-2-norlup-20 (29) -en-3-al methyl ester was added in small portions. -28-hydrochloric acid, then 13 ml of anhydrous diethyl ether were added dropwise. The reaction mixture was stirred under heating for 1 h, then 25 ml of ice water was added dropwise and 20 ml of twice diluted HCl was added. The mixture was stirred until the precipitate was completely dissolved for 1 h. The reaction products were extracted with ethyl acetate (3 × 20 ml). The organic layer was separated, washed with saturated NaHSO ₃ solution, then with 5% NaHCO ₃ solution and a small amount of water, dried over anhydrous MgSO ₄ . Ethyl acetate was evaporated in vacuo of a water-jet pump. The residue in an amount of 0.25 mmol was dissolved in 20 ml of acetone, 0.5 ml of Jones reagent was added. The reaction mixture was stirred for 6 hours. Monitoring of the reaction was carried out by TLC. Acetone was distilled off, the residue was diluted with water and extracted with 10 × 3 ml of ethyl acetate. The organic layer was dried over anhydrous MgSO ₄ . The solvent was evaporated, Compound I was purified by column chromatography.

The yield of compound I was 75%.

Example 4. 3-Methyl-1-cyano-19β, 28-epoxy-2,3-seco-2-nor-18αH-oleanan-3-one (compound II, method 2) were synthesized according to the procedure described in example 3. As the starting compound, 1-cyano-19β, 28-epoxy-2,3-seco-2-nor-18αH-oleanan-3-al was used.

The yield of compound II was 57%.

Example 5. The study of antiviral activity against herpes simplex virus type I.

The studies used the human rhabdomyosarcoma cell line (RD) and herpes simplex virus type I (HHP-1, strain 1C). A monolayer culture of RD cells grown in the wells of a plastic 96-well panel was washed from the growth medium, infected with 0.01-0.001 TTsID ₅₀ / VGP-1 cell by applying to the cells dilutions of the virus-containing suspension in a volume of 0.1 ml per 1 hour at 37 ° C. Then the liquid was removed and the cells were covered with support medium (DMEM medium) containing various concentrations of the test substances. Water-insoluble substances were previously dissolved in 10% ethanol. Subsequent dilutions used DMEM. The ethanol content in the final concentrations of the substances for the study did not exceed 1%. For each concentration of the studied substance, 2-4 wells with a cell culture infected with one dilution of the virus were used. To study the antiviral properties of each substance, 3-4 dilutions of the virus were used. After 48 h of incubation at 37 ° C, morphological changes in the cell monolayer were recorded (cytopathic effect (CPP) of the virus, magnification × 80). Based on the presence / absence of the VPC of the virus in the wells with different concentrations of the substance, the virus titer was calculated. The primary criterion for antiviral activity was considered the presence of differences in comparison with the control of the virus. Based on the values of the titer of the virus was calculated average effective concentration of the substance (EC ₅₀ ). The ratio of the maximum tolerated concentration (IPC) of the compound to the EC _{50 was} also calculated. IPC was determined as the maximum concentration of a substance that does not affect the morphology of an unpainted cell culture during the incubation period (48 h).

Example 6. Methodology for 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 TCID ₅₀ / ml was used as an infectious agent. The presence of anti-HIV activity was determined in the formazan test (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. A commercial preparation of azidothymidine was used in each series of experiments as a positive control for anti-HIV activity, 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-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium 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 IPC / EC ₅₀ ratio, which characterizes the wide spectrum of its non-toxic effective concentrations, were calculated.

It was found that compound II inhibits the reproduction of HBV-1 and HIV-1, the EC ₅₀ is 21.1 and 7.2 μg / ml, the ratio of IPC / EC ₅₀ is 9.5 and 9.4, respectively.

The resulting substances can be used as key intermediates for the preparation of new biologically active compounds, and compound II can be used directly for the development of antiviral agents.

Table Data IR, NMR ¹ H and ¹³ C spectra of compounds I, II Compound IR spectrum (ν, cm ^-1) Spectrum ¹ H-NMR (300.0 MHz, CDCl ₃ , δ, ppm, J / Hz) I 1705 (C = O); 1723 (COOSN ₃ ); 2236 (C≡N) 0.92, 0.94, 1.02, 1.14, 1.21 (each 3H, s, CH ₃ ), 1.68 (3H, s, H-30), 2.28 (3H, s, CH ₃ ), 2.42 and 2.55 (each 1H, d, J _AB = 18.2, H-1), 2.97 (1H, td, J = 12.0, 5.7, H-19 ), 3.65 (3H, s, COOCH ₃ ), 4.60 and 4.73 (each 1H, s, H-29) II 1695 (CO); 2237 (C≡N) 0.79, 0.92, 0.97, 1.15, 1.22 (each 3H, s, CH ₃ ), 0.96 (6H, s, 2CH ₃ ), 2.29 (3H, s, CH ₃ ), 2.44 and 2.59 (each 1H, d, J _AB = 18.0, H-1), 3.44 and 3.75 (each 1H, d, J _AB = 7.7, H- 28), 3.52 (H, s, H-19) 3β-hydroxy-3-methyl-2-hydroxyimino-2-norlup-20 (29) -en-28-oic acid methyl ester (formula II) 1719 (COOSN ₃ ); 1644 (C = N); ear. 3344 (OH) 0.74 (6H, s, 2CH ₃ ), 0.89, 0.94, 0.99, 1.32 (12H, 4s, 4CH ₃ ); 1.67 (3H, s, CH ₃ -30); 2.19 and 3.34 (2H, 2d, J _AB = 13.2, CH ₂ -1, AB system); 3.00 (1H, td, J = 4.5, 10.8, CH-19); 3.66 (H, s, COOSN ₃ ); 4.59 and 4.72 (2H, 2s, CH ₂ -29); 5.10 (1H, broad s. OH). 3β-Hydroxy-3-methyl-2-hydroxyimino-19β, 28-epoxy-2-nor-18αH-oleanan (formula III) 2931 (C = N), broad. 3238 (OH), 3460 (OH) 0.76, 0.78, 0.79, 0.95, 0.96, 1.37 (18H, 6s, 6CH ₃ ); 0.96 (6H, 2s, 2CH ₃ ); 1.67 and 3.39 (2H, 2d, J _AB = 13.2, CH ₂ -1, AB system); 3.45 and 3.78 (2H, 2d, J _AB = 8.0, CH ₂ -28, AB system); 3.54 (1H, s, CH-19); 5.50 (1H, broad s., OH); ¹ H NMR spectrum (300 MHz, DMSO-d ₆ , δ, ppm, J / Hz): 10.26 (1H, s, N OH ).

Claims (2)

1. A-secotriterpenoids of the general formula

,
Where

or

. 2. The compound according to claim 1, where

exhibiting inhibitory activity against herpes simplex virus type I (VGP-1, strain 1C) and HIV-1.