RU2393165C2 - Triterpenic anti-tumour agent - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel chemical compound and specifically to methyl ether of 2-cyano-3-oxo-18,19-dehydroglycyrrhet-1-inic acid of formula (I):

.

EFFECT: obtaining compounds which can be used in medicine as a medicinal agent with anti-tumour activity.

11 ex, 1 tbl, 3 dwg

Description

The invention relates to a new chemical compound, namely to methyl ester of 2-cyano-3-oxo-18,19-dehydroglyceride-1-enoic acid of the formula (I):

which can be used in medicine as a drug with an antitumor effect.

Oncological diseases are one of the main problems of modern medicine, because even with a favorable course of the disease, in most cases it is not possible to achieve a complete recovery of the patient, significantly increase the duration and improve the quality of his life. The currently proposed treatment regimens for various types of malignant tumors use surgical methods in combination with high-dose aggressive therapy, a serious drawback of which is the high toxicity of modern antitumor drugs in relation to vital organs and body systems. Concomitant side effects reduce the effectiveness, and in some cases limit the use of antitumor agents. Another problem in the treatment of cancer is the problem of residual tumor clone. Tumor cells that have survived chemotherapy usually show drug resistance to a wide range of drugs and cause a relapse of the disease in a more severe form. In connection with all of the above, an urgent task is the search for new antitumor drugs that provide high selectivity and treatment effectiveness.

One of the important areas of medical chemistry, allowing to obtain new, effective antitumor drugs, is the use of synthetic transformations of plant metabolites. The most acceptable is the study of plant metabolites, on the biological activity of which there is reliable information and which are available at present or will become available in the near future as the raw material base is formed. This class of compounds includes triterpenic acids, a wide range of biological activity of which (anti-inflammatory, antiviral, antitumor, immunostimulating, etc.) attracts the close interest of researchers.

The objective of the invention is the creation of a new effective, low-toxic drug with antitumor activity and obtained from available plant materials.

The problem is solved by a new compound of triterpene nature, namely methyl ester of 2-cyano-3-oxo-18,19-dehydroglyceride-1-enoic acid of the formula (1), which can be used as an antitumor agent.

It is known from the literature that some derivatives of triterpenoids are promising as antitumor drugs [Honda T., Janosik T., Honda Y., Han J., Liby KT, Williams Ch.R., Couch RD, Anderson AC, Spom MB, Gribble GW / / J. Med. Chem. 2004, 47, 4923-4932]. For example, the synthetic triterpenoid CDDO (2-cyano-3,12-dioxoleolean-1,9-diene-28-oic acid) obtained from oleanolic acid exhibits antitumor activity against a wide range of cancer cells (IC ₅₀ 10 ^-6 -10 ^-9 M) [Suh N., Wang Y., Honda T., Gribble GW, Dmitrovsky E., Hickey WF, Maue RA, Place AE, Porter DM, Spinella MJ, Williams Ch.R., Wu G. , Dannenberg AJ, Flanders KC, Letterio JJ, MangelsdorfD. J., Nathan CF, Nguyen L., Porter WW, Ren RF, Roberts A.V., Roche NS, Subbaramaiah K., Spom M.V. // Cancer Res. 1999, V.59, P.336-341]. In particular, CDDO causes apoptosis of myeloid leukemia cells (IC ₅₀ ~ 10 ^-6 M) [Konopleva M., Tsao T., Estrov Z., Lee Ruey-min, Wang Rui-Yu, Jackson CE, McQueen T., Monaco G., Munsell M., Belmont J., Kantaqian H., Spom M. B., Andreeff M. // Canser Res. 2004, V.64, P.7927-7935], inhibits the growth of ovarian epithelial carcinoma cells (IC ₅₀ 1.1-3.0 × 10 ^-6 M) [Melichar B., Konopleva M., Hu Wei, Melicharova K., Andreeff M. , Freedman RS // Gynecologic Oncologe 2004, V.93, P.149-154]. A circumstance that increases the attractiveness of triterpenes is their widespread occurrence in nature and, in many cases, the relative simplicity of the technology for producing plant materials from large tonnage.

One of such accessible compounds with pronounced physiological properties is 18βH-glycyrrhetic acid of the formula (2), glycirrhizic acid aglycone, the predominant licorice root glycoside (~ 90% of the total amount of triterpene glycosides).

From a chemical point of view, 18βH-glycyrrhetic acid is unusual in that it is the only natural triterpene containing a carbonyl group in the 11th position [Tolstikov GA, Baltina LA, Grankina VP, Kondratenko PM, Tolstikova T. G. Licorice: biodiversity, chemistry, medical applications. Novosibirsk, Academic publication "GEO", 2007, 312 p.].

2-cyano-3-oxo-18,19-dehydroglycyrrhet-1-enoic acid methyl ester [11-cyano-2,4a, 6a, 6b, 9,9,12a-heptamethyl-10,13-dioxo-2 methyl ester , 3,4,4a, 5,6,6a, 6b, 7,8,8a, 9,10,12a, 12b, 13-hexadecahydrocycene-2-olic acid] of the formula (1) was obtained by us as a result of a combined ring modification A and E 18βH-glycyrrhetic acid of the formula (2):

The modification was carried out according to Scheme 1. As the starting compound, 18βH-glycyrrhetic acid acetate (3) was taken, obtained by deglycolization of glycyrrhizic acid in acetic acid [G. Tolstikov. Synthetic studies in the field of physiologically active higher terpenoids and steroids. Dis. work of Dr. chem. sciences. Ufa 1969]. 18βH-glycyrrhetic acid methyl ester (4) was prepared by reacting compound (3) with diazomethane. Initially, when compound (4) was reacted with bromine in acetic acid at 80 ° С and as a result of the bromination-dehydrobromination reaction, compound (5) containing an 18.19-double bond in ring E was obtained. Thus, 11-on-12 was retained -enovoy fragment (unique to glycyrrhetic acid), as well as elongated system of conjugation of multiple bonds.

The next step was to obtain a 2-cyano-3-oxo-1-ene fragment in ring A (Scheme 1). The hydroxyl group was oxidized to ketone with the formation of compound (7). An isoxazole derivative (9) was obtained by formylation of compound (7) with further condensation with hydroxylamine. Cleavage of the isoxazole ring with sodium methylate followed by oxidation with dichlorodicyanoquinone (DDQ) leads to compound (1).

The elemental composition of the obtained substances was determined from high resolution mass spectra recorded on a DFS (Double Focusing Sector) instrument from Thermo Electron Corporation.

In the work we used acetate of methyl ester of glycyrrhetic acid (3) of 94% purity (HPLC).

¹ H and ¹³ C NMR spectra were recorded on AM-400 spectrometers (operating frequencies 400.13 MHz for 1H and 100.61 MHz for ¹³ C) and DRX-500 (500.13 MHz and 125.76 MHz, respectively) from Bruker for solutions of substances in CDCl ₃ . Solvent signals (δ _H 7.24 and δ _C 76.9 ppm) were used as the internal standard. The structure of the obtained compounds was established on the basis of analysis of ¹ H NMR spectra using ¹ H- ¹ H double resonance spectra, as well as analysis of ¹³ C NMR spectra using standard methods for recording spectra in J-modulation mode (JMOD), with non-resonant and selective suppression of protons , two-dimensional spectra of heteronuclear ¹³ С- ¹ Н correlation at direct spin-spin coupling constants (С-Н COSY, ¹ J _{C, H} 135 Hz) and two-dimensional and one-dimensional spectra of heteronuclear ¹³ С- ¹ Н correlation at long-range spin-spin coupling constants (COLOC , LRJMD, ^2.3 J _{C, H} 10 Hz) with the use of literature data on spectra for structurally similar fragments of the studied molecules [Wehri FW, T. Nishida // Fortschritte Chem. Org. Naturst 1979, 3d, 98-99; Honda T., Rounds B.V., Bore L., Finlay H.J., Favaloro FG, Suh N. .. Wang Y., Spom M.V., Gribble GW // J. Med. Chem. 2000, 43, 423 3-4246].

The effect of the inventive 2-cyano-3-oxo-18,19-dehydroglyceride-1-enoic acid methyl ester (1) on the viability of human carcinoma cell lines, human neuroblastoma cells, and tumor cells of a kidney of a human embryo was investigated.

As a result, it was shown that the claimed compound (1) exhibits high antitumor activity in relation to all used tumor cell cultures, including the KV-8-5 carcinoma cell line, which has the multidrug resistance phenotype. In the study of the cytotoxicity of compound (1) with respect to the used tumor cells, IC ₅₀ values were obtained, the concentration of the compound at which 50% of the cells were killed. It was shown that the IC ₅₀ values for compound (1) have a similar order of magnitude for all tumor cells and range from 50 × 10 ^-6 (for the MCF-7 cell line) to 3 × 10 ^-6 (for the KB-3 cell line -one). The obtained data on the antitumor activity of the compound (1) allow us to consider it as a promising drug agent.

In addition, when comparing the claimed compound (1) with the well-known and widely tested antitumor agent CDDO, the following can be noted. With almost the same antitumor efficacy, the claimed compound is cheaper than the CDDO agent, since 18βH-glycerretic acid, the starting material in the synthesis of compound (1), is a much more affordable and cheaper compound than oleanolic acid, the starting compound for the synthesis of CDDO (cost of oleanolic acid more than 60 times the cost of 18βH-glycerol acid).

The invention is illustrated by the following examples.

Example 1. Obtaining methyl acetate 18βH-glycyrrhetic acid (4)

To a mixture of 18rH-glycerol acid acid acetate (3) (10 g, 19.0 mmol) in 200 ml of methanol was added an ether solution of diazomethane with stirring until the solution turned pale yellow. After the complete decomposition of diazomethane (the characteristic color disappeared), the solvent was distilled off on a rotary evaporator, and the obtained precipitate was recrystallized from methanol-chloroform mixture.

( ^* Hereinafter: chemical shifts (centers of multiplets) of hydrogen atoms enclosed in square brackets are obtained from two-dimensional spectra of ¹³ C - ¹ H correlations on the direct constants of CERs

^* Chemical shifts may need to be reversed.)

Example 2. Obtaining methyl acetate 18,19-dehydroglycyrrhetic acid (5)

To a solution of glycyrrhetic acid methyl ester acetate (4) (10.0 g, 19.0 mmol) in glacial acetic acid (400 ml) at 80 ° C, a 5% solution of bromine in glacial acetic acid (70.0 ml, 21.8 mmol). The reaction mixture was kept for 1 hour at the same temperature, cooled and poured into 1.5 L of cold water. The resulting precipitate was filtered off, washed with water and dried in air. Recrystallization from methanol-chloroform gave 6.5 g of product (5) (yield 65.3%). Mp 244-247 ° C. Found, m / z: 524.3490 [M] ⁺ . C ₃₃ H ₄₈ O ₅ .

Example 3. Obtaining methyl ester of 18.19-dehydroglycyrrhetic acid (6)

A solution of 18.19-dehydroglycererretic acid methyl ester acetate (5) (6.5 g, 12.4 mmol) and KOH (45 g, 775.8 mmol) in methanol (650 ml) was boiled for 1 hour. The reaction mixture was cooled and most of the methyl alcohol was distilled off in vacuo, then ethyl acetate (100 ml) and a 5% hydrochloric acid solution were added. It was extracted with a mixture of chloroform - ethyl acetate (1: 4) (3 × 750 ml). The combined extract was washed with sodium bicarbonate solution (3x25 ml) and saturated sodium chloride solution (3 × 25 ml). Dried with magnesium sulfate. The mass of the obtained product (6) is 5.9 g (yield 98.7%). To obtain an analytically pure sample, 100 mg of the product was recrystallized from methanol-chloroform. For further transformations, the product was used without further purification. Mp 208-210 ° C.

Example 4. Obtaining methyl ester of 3-oxo-18,19-dehydroglycyrrhetic acid (7)

To a solution of 18.19-dehydroglycererretic acid methyl ester (6) (5.8 g, 12.0 mmol) in acetone (400 ml) was added dropwise 6 ml of Jones reagent (sodium dichromate in 33% sulfuric acid). It was stirred for 2.5 hours, then 50 ml of ethyl alcohol was added and stirred for another 30 minutes to decompose the chromic acid. The reaction mixture was evaporated in vacuo to a volume of ~ 100-150 ml and poured into water (700 ml). The precipitate was filtered off and dried in air. For purification, the substance was dissolved in chloroform and filtered through a layer of alumina. The mass of the obtained compound (7) 5.2 g (yield 90.3%). To obtain an analytically pure sample, 100 mg of the product was recrystallized from methanol-chloroform. For further transformations, the product was used without further purification. T. pl.

Example 5. Obtaining methyl ester of 2-hydroxymethylene-3-oxo-18,19-dehydroglyceride acid (8)

To a stirred solution of 3-oxo-18,19-dehydroglyceride acid methyl ester (4.2 g, 8.8 mmol), ethyl formate (4 ml, 50.5 mmol) in benzene (32 ml) sodium methylate (3.0 g, 55.5 mmol) was added in portions. Stirred for 2 hours at room temperature. Then, benzene (50 ml) and a 5% hydrochloric acid solution were added to the reaction mixture. It was extracted with ethyl acetate (3 × 75 ml). The combined extract was washed with sodium bicarbonate solution (3 × 25 ml) and saturated sodium chloride solution (3 × 25 ml). Dried with magnesium sulfate. The mass of the obtained product (8) 4.3 g (yield 96.7%). To obtain an analytically pure sample, 150 mg was purified by silica gel column chromatography (eluent was hexane with a gradient of ethyl acetate 10-15%). For further transformations, the product was used without further purification.

Example 6. Obtaining methyl ester of isoxazolo [4,5-b] -18,19-dehydroglyceride acid (9)

A mixture of 2-hydroxymethylene-3-oxo-18,19-glycerol acid methyl ester (4.2 g, 8.3 mmol), ethyl alcohol (110 ml) and hydroxylamine hydrochloride (5.8 g, 83.5 mmol) in water (11 ml) was boiled with reflux for 2 hours. The reaction mixture was cooled, the alcohol was removed on a rotary evaporator, water was added, it was extracted with ethyl acetate (2 × 100 ml). The combined extract was washed with sodium bicarbonate solution (3 × 25 ml) and saturated sodium chloride solution (3 × 25 ml), dried with magnesium sulfate. Received 4.0 g (yield 95.4%) of the isoxazole derivative (9). To obtain an analytically pure sample, 100 mg was purified by silica gel column chromatography (eluent was hexane with a gradient of ethyl acetate 10–20%). For further transformations, the product was used without further purification. Found, m / z:

Example 7. The cleavage of the isoxazole ring in the compound (9)

To a stirred solution of compound (9) (3.9 g, 7.7 mmol), diethyl ether (250 ml) and methanol (120 ml), sodium methoxide (15 g) was added in portions while cooling in an ice bath. The mixture was stirred for 1 h at room temperature. Ethyl acetate and a 5% hydrochloric acid solution were then added. It was extracted with ethyl acetate (2 × 50 ml). The combined extract was washed with sodium bicarbonate solution (3 × 25 ml) and saturated sodium chloride solution (3 × 25 ml), dried with magnesium sulfate. The mass of the reaction mixture (mixture of tautomers) 3.8 g (yield 97%). To obtain an analytically pure sample, 100 mg was purified by silica gel column chromatography (eluent hexane with a gradient of ethyl acetate 10-20%). For further transformations, the product was used without further purification. Found, m / z:

Example 8. Obtaining methyl ester of 2-cyano-3-oxo-18,19-dehydroglyceride-1-enoic acid (1)

A solution of a mixture of tautomers (3.7 g, 7.3 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (1.91 g, 8.4 mmol) in benzene (200 ml) was refluxed for 4 hours Filtered, the filtrate was evaporated. Silica gel column chromatography (eluent benzene with an acetone gradient of 0-10%) produced a crystalline precipitate. Recrystallization of the latter from methanol-chloroform gave the desired product (1) weighing 3.0 g (yield 84%). T. pl. 214-217 ° C. Found, m / z 503.3025 [M] ⁺ . C ₃₂ H ₄₃ NO ₄ . Calculated M505.3181.

Example 9. The effect of methyl ester of 2-cyano-3-oxo-18,19-dehydroglyceride-1-enoic acid (1) on the viability of human carcinoma cell lines

Cells of the line KV-3-1 (epidermoid carcinoma of the oral cavity), KV-8-5 (line derived KV-3-1, with the phenotype of multiple drug resistance), HeLa (epitheloid carcinoma of the cervix uteri) and MCF-7 (mammary adenocarcinoma glands) were cultured in IMDM medium containing 10% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) and antimycotic amphotericin (0.25 μg / ml), in an atmosphere of 5% CO ₂ at 37 ° C. KV-8-5 cells were cultured under the same conditions in the presence of 200 nM vinblastine.

Cell viability after incubation with compound (1) was determined using the MTT test, which is based on the ability of living cells to convert tetrazole-based compounds (MTT) into brightly colored formazan crystals, which allows spectrophotometric estimation of the number of living cells in the preparation. For this, cells were plated in 96-well plates (15,000 cells per well for the KV-3-1, KV-8-5 and MCF-7 lines, 7000 cells per well for the HeLa line). After 24 hours, the medium was changed in the wells and a 0.1 M solution of compound (1) in DMSO was added to the cells to a final concentration in the medium from 10 ^-4 to 10 ^-9 M. Cells were incubated in the presence of compound (1) for another day in the same conditions. At the end of incubation, without changing the medium, an MTT solution (5 mg / ml) in phosphate-buffered saline was added to the cells to a concentration of 0.5 mg / ml and incubated for 3 hours under the same conditions. The medium was removed, 100 μl of DMSO was added to the cells, in which the formazan crystals formed in the cells were dissolved and the optical density was measured on a multichannel spectrophotometer at wavelengths of 570 and 630 nm, where A ₅₇₀ is the absorption of formazan and A ₆₃₀ is the background of the cells.

Data were presented as the number of living cells relative to the control. The number of cells in the control was taken as 100%, where the cells were incubated for 24 hours in the absence of compound, but in the presence of a DMSO solvent (Fig. 1). From the data shown in Fig. 1 it can be seen that treatment of cells of various carcinoma lines with compound (1) causes their effective death even at a concentration of 10 ^-6 M. IC ₅₀ values are the concentration of compound (1) at which 50% of the cells die - are given in the table.

IC ₅₀ values of 2-cyano-3-oxo-18,19-dehydroglycyrrhet-1-enoic acid methyl ester (1) for various human cell carcinoma lines Cell line IC ₅₀ (1) KB-3-1 3 × 10 ^-6 M KB-8-5 5 × 10 ^-6 M Hela 4.8 × 10 ^-6 M Mcf-7 50 × 10 ^-6 M

Example 10. The effect of incubation with methyl ester of 2-cyano-3-oxo-18,19-dehydroglyceride-1-enoic acid (1) on the viability of human neuroblastoma cells

SK-N-MC cells (neuroblastoma) were cultured in IMDM medium containing 10% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) and antimycotic amphotericin (0.25 μg / ml), a atmosphere of 5% CO ₂ at 37 ° C.

Cell viability after incubation with the compound was determined using the MTT assay as described in Example 9. For this, cells were plated in 96-well plates (30,000 cells per well). Further, as in example 9.

The data were presented as the number of living cells relative to the control after incubation in the presence of compound (1) for 24 hours. The number of cells in the control was taken as 100%, where the cells were incubated in the absence of compound, but in the presence of DMSO solvent (Fig. 2). From the data shown in figure 2 it is seen that the treatment of cells of the SK-N-MC line with compound (1) causes their effective death even at a concentration of 10 ^-6 M, which confirms the high antitumor potential of the compound (1). The IC ₅₀ value obtained for this cell line was 5 × 10 ^-6 M.

Example 11. The effect of incubation of tumor cells of the kidney of a human embryo with methyl ester of 2-cyano-3-oxo-18,19-dehydroglyceride-1-enoic acid (1) on their viability

HEK293 cells (human embryonic kidney) were cultured in IMDM medium containing 10% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) and antimycotic amphotericin (0.25 μg / ml) in atmosphere 5 % CO ₂ at 37 ° C.

Cell viability after incubation with the compound was determined using the MTT assay as described in Example 9. For this, cells were plated in 96-well plates (20,000 cells per well). Further, as in example 9.

The data were presented as the number of living cells relative to the control after incubation in the presence of compound (1) for 24 hours. The number of cells in the control was taken as 100%, where the cells were incubated in the absence of compound, but in the presence of DMSO solvent (Fig. 3). From the data shown in FIG. 3, it can be seen that treatment of HEK293 cells with compound (1) causes their effective death at a concentration of the compound above 1 × 10 ^-6 M, which indicates a high antitumor potential of compound (1). The IC ₅₀ value obtained for this cell line was 5 × 10 ^-6 M.

Claims (1)

2-cyano-3-oxo-18,19-dehydroglycyrrhet-1-enoic acid methyl ester of the formula (1):

possessing antitumor activity.

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