RU2445317C1 - N-ethylpiperazylamide of betulinic acid as triterpenic antitumour agent - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel chemical compound and specifically to N-ethylpiperazylamide of betulinic acid of formula (I):

which can be used in medicine as a medicinal agent with antitumour activity.

EFFECT: high efficiency of the compounds.

1 cl, 6 ex, 3 tbl, 2 dwg

Description

The invention relates to a new chemical compound, namely to N-ethylpiperazylamide betulonic acid of the formula (I):

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

Modern 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 survive chemotherapy usually show drug resistance to a wide range of drugs and cause a relapse of the disease in a more severe form. In this regard, the urgent task is the search for new antitumor drugs that provide high selectivity and treatment effectiveness.

An important area 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, N-ethylpiperazylamide betulonic acid of the formula (1), which can be used as an antitumor agent.

From literature sources it is known that derivatives of triterpenes, in particular, the lupane series are promising as antiviral and antitumor drugs [Tolstikova TG, Sorokina IV, Tolstikov GA, Tolstikov AG, Flekhter OB // Biorgan. Chemistry, 2006, 32, 291-307] [1]. The relationship between structure and antitumor activity against a wide range of cancer cells ( IC ₅₀ 10 ^-6 -10 ^-9 M) [ Mukherjee R., Kumar V., Srivastava SK, Agarwal SK, Burman AC // Anti-cancer is shown using betulinic acid as an example. agents in Medicinal Chemistry, 2006, V.6, P.271-279] [2]. It was shown that derivatives of betulonic acid containing fragments of co-amino acids are active inducers of apoptosis in leukemia and hepatocarcinoma cells in vitro [Shintapina AB, Shults E.E., Petrenko NI, Uzenkova N.V., Tolstikov G.A., Pronkina N.V., Kozhevnikov BC ., Pokrovsky A.G. // Biorgan. Chemistry, 2007, 33, 624] [3] . An important feature of betulonic acid amides is that an amide, for example, containing a β- alanine fragment, exhibits antioxidant activity and has the ability to reduce the organotoxic effect of antitumor drugs [Sorokina IV, Tolstikova TT., Zhukova NA, Petrenko N. I., Schulz E.E., Tolstikov G.A. // Dokl. AN, 2004, 399, S. 274] [4]. 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 the compounds of triterpene nature with pronounced physiological properties is betulonic acid of formula (2), which is easily obtained by oxidation of betulin of formula (3), a widely available raw material isolated from the outer bark of the birch family Betula [Krasutsky PA // Nat. prod. rep., 2006, 23, P.9 19-942] [5].

To achieve this goal, we carried out a number of chemical modifications, presented in the diagram - Figure 1. Betulin (3) obtained by extraction of birch bark with benzene was taken as the starting compound. Betulin (3) was oxidized with Fizer solution. The next step was to obtain betulonic acid chloride (2), which was then used without isolation. The interaction of betulonic acid chloride (2) with N-ethylpiperazine easily leads to the target compound of formula (1) with antitumor activity.

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.

¹ H and ¹³ C NMR spectra were recorded on AV - 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 8 with 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 C- ¹ H 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 C- ¹ H correlation at long-range spin-spin coupling constants ( COLOC , LRJMD, ^2.3 j _{c, h} 10 Hz).

The effect of the claimed N-ethylpiperazylamide betulonic acid of the formula (1) on the viability of various human tumor cells 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, namely CEM- 13, U- 937, MT-4. It was shown that the CCID ₅₀ values for compound (1) have a similar order of magnitude for all tumor cells and lie in the range of 5.8–10.5 µM . The obtained data on the antitumor activity of the compound (1) allow us to consider it as a promising drug agent. The invention is illustrated by the following examples.

Example 1. Obtaining lup-20 (29) -en-3 β , 28-diol (betulin, 3). The crushed birch bark (344 g) was boiled in benzene (1.6 L) under reflux for 8 h, then the hot solution was decanted. An additional 1 L of benzene was added to the remaining bark, boiled for 1 h, the hot solution was again decanted. The precipitate from benzene was filtered off, dried, and 52 g of crude betulin were obtained. After recrystallization in isopropanol, 38 g of betulin with melting point were obtained. 260-262 ° C (lit. mp 258-260 ° C [Hayek // Phytochem. 1989, 28, P. 22.] [6] ). The output of betulin to the original cortex was 11%.

Example 2. Obtaining 3-oxo-lup-20 (29) -en-28-oic acid (betulonic acid, 5).

A suspension of 10 g (22.6 mmol) of betulin (3) in 165 ml of glacial acetic acid and 110 ml of acetone was cooled at 0 ° C and a freshly prepared solution of Fizer (11 g (0.11 mol) of chromic anhydride in 12 ml of ice-cold was added over 1 h with stirring acetic acid and 15 ml of H ₂ O). It was held at room temperature for 3 hours. At the end of the reaction, 50 ml of methanol was added, 300 ml of benzene and 300 ml of 10% NaCl solution were added. The benzene layer was separated, and the aqueous was extracted with 2 × 150 ml of benzene. The combined extracts were washed with 3 × 200 ml of 10% NaCl solution, dried with MgSO ₄ , the desiccant was filtered off, benzene was distilled off to approximately 100 ml and poured into 200 ml of a 5% KOH solution. The precipitate of potassium salt of betulonic acid was filtered off and dried. The dry salt was dissolved in 50 ml of ethanol, the insoluble part was filtered off, the filtrate was poured into 250 ml of a 5% HCl solution. The separated acid (2) was filtered off, washed with H ₂ O , and dried. As a result, 7.2 g (69% of theory) of betulonic acid were obtained (2). The melting point and NMR spectra correspond to the literature [ Barthel A., Stark S., Csuk R. // Tetrahedron, 2008, 64, P.9225-9229] [6].

Example 3. Obtaining N - [3-oxo-loop-20 (29) -sio-28-yl] -4 ' -ethyl piperazine (N-ethylpiperazylamide betulonic acid, 1).

Oxalyl chloride (0.5 ml, 5.7 mmol) and two drops of DMF were added to a solution of betulonic acid (2) (1 g, 2.2 mmol) in 50 ml of CH ₂ Cl ₂ , and the solution was kept under stirring for 4 h at room temperature. The solvent was evaporated, 20 ml of CH ₂ Cl _{2 was} added and evaporated again, at the end 20 ml of CH ₂ Cl _{2 was} added. The resulting solution of betulonic acid chloride (2) was added dropwise to a solution of N-ethylpiperazine (0.55 g, 4.8 mmol) in 30 ml of CH ₂ Cl ₂ . The reaction mass was left at room temperature for 18 h, diluted with CH ₂ Cl ₂ to 100 ml, washed with 3 × 20 ml of H ₂ O , dried with MgSO ₄ , the desiccant was filtered off, and the solvent was distilled off. A small amount of methanol was added to the residue and allowed to precipitate. The precipitate was filtered off and recrystallized from methanol. The mass of the obtained product (1) 0.88 g ( 73% yield on acid). Mp 232-234 ° C. Found, m / z: 550.4488 [M ] ⁺ . C ₃₆ H ₅₈ N ₂ O ₂ . Calculated: M 550.4493. ¹ H NMR spectrum of compound (1), δ , ppm ( J , Hz): 0.87 s (C ²⁵ H ₃ ), 0.91 s (C ²⁷ H ₃ ), 0.92 s (C ²⁶ H ₃ ), 0.96 s (C ²⁴ H ₃ ), 1.00 s (C ²³ H ₃ ) , 1.13 t (С ^{8 '} Н ₃ , J _{8', 7 '} 7.2), 1.62 br. s (C ³⁰ H ₃ ). 0.90 m ( H ^12a ), 1.12 m ( H ^15e , J _{15e, 15a} 13.3), 1.26 d . d (H ^5a , J _{5a, 6a} 11.5, J _{5a, 6e} 2.3 Hz), 1.47 M ( H ^16a ), 1.50 d . d (H ^18a , J _{18a, 13a} 11.2, J _{18a, 19} 11.2 Hz), 1.68 dm ( H ^12e , J _{12e, 12a} 12.5), 1.76-1.88 m (2H, H ^21α , H ¹ ), 1.93 d .d (H ^22α , J _{22α, 22β} 10.2, J _{22α, 21} 7.6 Hz), 2.06 dd ( H ^16e , J _{16e, 16a} 13.3, J _{16e, 15a} , 3.7, J _{16e, 15a} 3.0 Hz) , 2.34 m (5H, H ² , 2H ^{3 '} , 2H ^5' ), 2.36 k (2H, H ^{7 '} , J _{7', 8 '} 7.2), 2.43 dd ( H ^2a , J _{2a, 2e} 15.7, J _{2a, 1a} 9.6, J _{2a, 1e} 7.2 Hz), 2.87 dd ( H ^13a , J _{13a, 12a} 12.8, J _{13a, 18a} 11.2, J _{13a, 12e} 3.4), 2.93 dd d (H ¹⁹ , J _19.18a 11.2, J _19.21α 11.2, J _{19.21 β} 3.4), 3.56 br.s (4H, 2H ^{2 '} , 2H ^6' ), 4.52 m and 4.66 m (2H, 2H ²⁹ ) The signals of the remaining protons are observed in the region of 1.23-1.47 ppm. (11H). ¹³ C NMR spectrum of compound (1), δ , ppm: 39.49 t (C ¹ ), 33.96 t (C ² ), 217.91 s (C ³ ), 47.13 s (C ⁴ ), 54.90 d (C ⁵ ) , 19.47 t (C ⁶ ), 33.57 t (C ⁷ ), 40.43 s (C ⁸ ), 50.05 d (C ⁹ ), 36.76 s (C ¹⁰ ), 21.50 t (C ¹¹ ), 25.50 t (C ¹² ), 36.77 d (C ¹⁵ ), 41.75 s (C ¹⁴ ), 29.58 t (C ¹⁵ ), 32.26 t (C ¹⁶ ), 54.32 s (C ¹⁷ ), 52.44 d (C ¹⁸ ), 45.44 d (C ¹⁹ ), 151.12 s (C ²⁰ ), 31.14 t (C ²¹ ), 35.73 t (C ²² ), 26.43 k (C ²³ ), 20.83 k (C ²⁴ ), 15.81 k (C ²⁵ ), 15.73 k (C ²⁶ ), 14.41 k (C ²⁷ ), 173.16 s (C ²⁸ ), 108.9 t (C ³⁰ ), 19.50 k (C ³⁰ ), 52.87 t (C ^{3 ', 5'} ), 52.08 t (C ^{7 '} ), 11.74 k (C ^{8 '} ). The signals (C ^{2 ', 6'} ) are greatly broadened and are observed in the region of 45 ppm.

Example 4. The effect of N-ethylpiperazylamide betulonic acid (1) on the viability of tumor cells of human T-cell leukemia MT-4

MT-4 cells (human T-cell leukemia cells) were cultured in RPMI 1640 medium containing 10% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) in an atmosphere of 5% CO ₂ at 37 ° C.

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 (100 μl of cells with a concentration of 500 thousand cells / ml. Then, a solution of compound (1) in DMSO was added to the cells to a final concentration in the medium of 0.1 to 100 μg / ml. betulonic acid (2). Cells were incubated in the presence of compound (1) for 3 more days under the same conditions. At the end of the incubation, without changing the medium, 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. Then, 100 μl 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 in the absence of compound, but in the presence of a DMSO solvent.

In FIG. 2. The data on the viability of MT-4 cells after incubation with compound (1) for 72 hours are presented. The number of living cells was evaluated using the MTT test. The number of living cells incubated in the presence of DMSO, but without compound (1), was taken as 100% . Betulonic acid was used as a comparison drug (2)

From the data shown in FIG. 1, it is seen that treatment of MT-4 leukemia cells with compound (1) causes their effective death even at a concentration of 10 ^-6 M. The CCID ₅₀ values are the concentration of compound (1) at which 50% of the cells are dead , as well as CCID ₈₀ and CCID ₉₀ (concentrations at which the death of 80 and 90% of the cells is observed, respectively) are shown in table 1. From the data shown in table 1, it is seen that compound (1) has a greater antitumor effect compared to the previously described compound (2) . Treatment of MT-4 cells with compound (1) causes 50% death at a concentration of 5.8 μM, and 80% death at a concentration of 14.5 μM , in contrast to the comparison compound (2), for which treatment of cells at the used concentrations is not achieved 80% cell death, and 50% cell death is observed at a higher concentration of 8.2 μm.

Table 1. CCID N-ethylpiperazylamide betulonic acid (1) CCID values for human MT-4 tumor cell lines Compound CCID ₅₀ uM CCID ₅₀ uM CCID ₅₀ uM Betulonic acid N-ethylpiperazylamide 5.8 14.5 30.9 Betulonic acid 8.2 > 200 > 200

Example 5. The effect of N-ethylpiperazylamide betulonic acid (1) on the viability of tumor cells of human T-cell leukemia CEM- 13

Cells of the CEM- 13 line (cell line of human T-cell leukemia) were cultured in RPMI 1640 medium containing 10% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) in an atmosphere of 5% solution of CO ₂ at 37 ° C.

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 (100 μl of cells with a concentration of 500 thousand cells / ml. Then, a solution of compound (1) in DMSO was added to the cells to a final concentration in the medium of 0.1 to 100 μg / ml. betulonic acid (2). Cells were incubated in the presence of compound (1) for 3 more days under the same conditions. At the end of the incubation, without changing the medium, 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. Then, 100 μl 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.

The calculation of CCID values was carried out as described in example 4. The CCID ₅₀ values are the concentration of compound (1) at which 50% of the cells are dead , as well as CCID ₈₀ and CCID ₉₀ (the concentrations at which 80 and 90% cells are dead, respectively) are shown in table 2. Treatment of CEM- 13 cell lines with compound (1) causes 50% death at a concentration of 10.5 μM, and 80% death at a concentration of 42.7 μM , in contrast to the comparison compound (2), for which 50% cell death It is observed at a comparable concentration 11.6 mM, but 80% cell death is not even dostigaeitsya m ksimalnoy used concentration of 200 uM.

Table 2. The CCID values of betulonic acid N-ethylpiperazylamide (1) for tumor cell lines of human cells CEM- 13 Compound CCID ₅₀ uM CCID ₈₀ , μM CCID ₉₀ uM Betulonic N-ethylpiperazylamide 10.5 42.7 147 Betulonic acid 11.6 > 200 > 200

Example 6. The effect of N-ethylpiperazylamide betulonic acid (1) on the viability of human tumor cells U-937.

U-937 cells (human monocyte tumor line) were cultured in RPMI 1640 medium containing 10% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) in an atmosphere of 5% CO ₂ at 37 ° C.

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 (100 μl of cells with a concentration of 400 thousand cells / ml. Then, a solution of compound (1) in DMSO was added to the cells to a final concentration in the medium of 0.1 to 100 μg / ml. As a comparison drug, we used betulonic acid (2). Cells were incubated in the presence of compound (1) for 3 more days under the same conditions. At the end of the incubation, without changing the medium, 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. Then, 100 μl 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.

The calculation of CCID values was carried out as described in example 4. The CCID ₅₀ values are the concentration of compound (1) at which 50% of the cells are dead , as well as CCID ₈₀ and CCID ₉₀ (the concentrations at which 80 and 90% cells are dead, respectively) are shown in table 2. The CCID ₅₀ values are the concentration of compound (1) at which 50% of the cells die, as well as CCID ₈₀ and CCID ₉₀ (the concentrations at which 80 and 90% cells die, respectively) are shown in Table 3. Cell processing line U -937 compound (1) causes 50% mortality at a concentration of 6 microns And 80% mortality at a concentration of 30.9 .mu.M, unlike the comparative compound (2), for which 50% cell death is observed at a concentration of 18.4 uM comparable, but 80% cell death is not achieved even in a maximum concentration of 200 uM used. Thus, compound (1) has a more pronounced antitumor effect in relation to the studied tumor cells (MT-4, CEM- 13, U-937) compared with betulonic acid, for which the presence of a pronounced antitumor activity was previously shown.

Table 3. CCID N-ethylpiperazylamide Betulonic Acid (1) CCID Values for Human U-937 Tumor Cell Lines Compound CCID ₅₀ uM CCID ₈₀ , μM CCID ₉₀ μm Betulonic acid N-ethylpiperazylamide 6.0 30.9 > 100 Betulonic acid 18.4 > 200 > 200

Information sources

1. Tolstikova T.G., Sorokina I.V., Tolstikov G.A., Tolstikov A.G., Flekhter O.B. // Biorgan. Chemistry, 2006, 32, 291-307].

2. Mukherjee R., Kumar V., Srivastava S.K., Agarwal S.K., Burman A.C. // Anti-cancer agents in Medicinal Chemistry, 2006, V.6, P.271-279.

3. A Shintyapina. B., E Schultz. E., Petrenko H. And., Uzenkova H. In., TOLSTIKOV D. A., Pronkina H. In., The BC Kozhevnikov, Pokrovsky A. R. // Biorgan . Chemistry , 2007, 33, 624.

4. Sorokina I.V., Tolstikova T.G., Zhukova N.A., Petrenko N.I., Schulz E.E., Tolstikov G.A. // Dokl. AN, 2004, 399, p. 274.

5. Krasutsky PA // Nat. prod. rep., 2006, 23, R. 919-942.

6. Hayek // Phytochem. 1989, 28, P.22.

Claims (1)

Betulonic N-Ethylpiperazylamide of Formula (1)

possessing antitumor activity.

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