RU2448115C1 - Hydrogenated betulonic acid and amides thereof as triterpene anti-tumour agents - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel family of chemical compounds, specifically to hydrogenated betulonic acid of formula

and amides thereof of formula:

NR₁R₂=

which can be used in medicine as medicinal agents, having anti-tumour activity.

EFFECT: improved properties of the compound.

8 ex, 2 tbl, 23 dwg

Description

The invention relates to a new series of chemical compounds, namely, hydrogenated betulonic acid of the formula (1) and its amides of the formula (2-8):

which can be used in medicine as drugs with antitumor effects.

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 new effective, low toxic drugs with antitumor activity and obtained from available plant materials.

The problem is solved by new compounds of triterpene nature, namely, hydrogenated betulonic acid of the formula (1) and its amides of the formula (2-8), which can be used as antitumor agents.

From literary 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 // Bioorgan. Chemistry, 2006, 32, 291-307]. 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 agents in Medicinal Chemistry, 2006, V.6, P.271-279]. In the same work, there are examples of the fact that hydrogenated analogues often show greater activity compared to conventional derivatives of betulinic acid. The anti-HIV activity of 3-O-glutaryldihydrobetulin increases by at least three orders of magnitude when switching from a conventional derivative of betulin [Kashiwada Y., Sekiya M., Ikeshiro Y., Fujioka T., Kilgore NR, Wild CT, Allaway GP, Lee K.- H. // Bioorgan. Med. Chem. Lett., 2004, 14, P.5851-5853]. Derivatives of betulonic acid containing fragments of ω-amino acids are active inducers of apoptosis in leukemia and hepatocarcinoma cells in vitro [Shintyapina AB, Shults E.E., Petrenko NI, Uzenkova NV, Tolstikov G. A., Pronkina N.V., Kozhevnikov BC, Pokrovsky A.G. // Bioorgan. Chemistry, 2007, 33, 624]. 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 TG, Zhukova NA, Petrenko N.I., Schulz E.E., Tolstikov G.A. // Dokl. AN, 2004, 399, p.274]. A circumstance that increases the attractiveness of triterpenes is their wide distribution in nature and, in many cases, the relative simplicity of the technology for obtaining from large-tonnage plant materials.

One of the compounds of triterpene nature with pronounced physiological properties is dihydrobetulonic acid of the formula (1), which is obtained by the oxidation of dihydrobetulin of the formula (9). Dihydrobetulin of formula (9) is obtained by hydrogenation of betulin of formula (10), a widely available raw material isolated from the outer birch bark of the Betula family [Krasutsky P.A. // Nat. prod. rep., 2006, 23, P.919-942].

To achieve this goal, we carried out a number of chemical modifications, presented in scheme 1, Fig.22. Betulin (10) obtained by extraction of birch bark with benzene was taken as the starting compound. Hydrogenation of betulin (10) with hydrogen in an autoclave over Ni-Raney gave dihydrobetulin (9), which was oxidized with Fizer solution. The next step was to obtain the dihydrobetulonic acid chloride (1), which was further used without delay. The interaction of the dihydrobetulonic acid chloride (1) with a double excess of the corresponding amine easily led to the target compounds of the formula (2-8) with antitumor activity.

IR spectra were recorded on a VECTOR 22 instrument in KBr. Specific rotation was determined on a polAAr 3005 spectrometer; solution concentrations are given in g / 100 ml. Melting points were determined on a Termosystem FP 900 instrument from Mettler Toledo and on a Kofler table S 30 A / G (Germany).

The elemental composition of the obtained substances was determined from elemental analysis and from high-resolution mass spectra recorded on a DFS (Double Focusing Sector) device 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 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 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 С- ¹ H correlation at long-range spin-spin coupling constants (COLOC, LRJMD, ^2.3 J _{C, H} 10 Hz).

The effect of the inventive hydrogenated betulonic acid of the formula (1) and its amides (2-8) on the viability of human carcinoma cell lines was investigated. The CCID values of hydrogenated betulonic acid (1) and its amides (2-8) for various human cell carcinoma cell lines are shown in Table 1. Betulonic acid (BA) and its corresponding amides (BA1-BA7) were used as reference preparations (Fig.23 Scheme 2 Betulonic acid (BA) and its derivatives (BA1-BA7)).

As a result, it was shown that the claimed compounds (1-8) exhibit 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 compounds (1-8) have a similar order of magnitude for all tumor cells and lie in the range of 2.9-69.5 μM. The obtained data on the antitumor activity of the compounds (1-8) allow us to consider them as promising drug agents. The invention is illustrated by the following examples.

Example 1. Obtaining loop-20 (29) -en-3β, 28-diol (betulin, 10)

The crushed birch bark (344 g) is boiled in benzene (1.6 L) under reflux for 8 hours, then the hot solution is decanted. An additional 1 liter of benzene is added to the remaining bark, boiled for 1 hour, the hot solution is again decanted. The precipitate from benzene is filtered off, dried, and 52 g of crude betulin are obtained. After recrystallization in isopropanol, 38 g of betulin with melting point are obtained. 260-262 ° C (lit. mp 258-260 ° C [Hayek // Phytochem. 1989, 28, P.22.]). The yield of betulin to the original cortex is 11%.

Example 2. Obtaining lupan-3β, 28-diol (dihydrobetulin, 9)

Hydrogenation of betulin (10) is carried out similarly to the known method [Ruzicka L., Brener M., Rey E. // Helv. Chim. Acta, 1941, 24, P.515-529]. 132 g of betulin (10), 13.2 g of Ni-Raney and 2.5 l of ethanol are placed in a 5 liter autoclave with a stirrer, then everything is stirred in a hydrogen atmosphere (100 atm) at 180 ° C for 14 hours. They are freed from the catalyst by hot filtration in dioxane , after distillation of the solvent, 124 g of dihydrobetulin (9) are obtained (yield - 93%). Mp 282-284 ° C (from alcohol). Lit. so pl. 278-280 ° C [Ruzicka L., Brener M., Rey E. // Helv. Chim. Acta, 1941, 24, P.515-529].

Example 3. Obtaining 3-oxo-lupan-28-oic acid (dihydrobetulonic acid, 1)

A suspension of 10 g (22.5 mmol) of dihydrobetulin (9) in 165 ml of glacial acetic acid and 110 ml of acetone is cooled at 0 ° C and a freshly prepared Fizera solution (11 g, 0.11 mol of chromic anhydride in 12 ml of glacial acetic acid) is added with stirring acid and 15 ml of H ₂ O). It is kept at room temperature for 3 hours. At the end of the reaction, 50 ml of methanol are added, 300 ml of benzene and 300 ml of 10% NaCl solution are added. The benzene layer was separated, and the aqueous was extracted with 2 × 150 ml of benzene. The combined extracts are washed with 3 × 200 ml of 10% NaCl solution, dried with MgSO ₄ , the desiccant is filtered off, benzene is distilled off to approximately 100 ml and poured into 200 ml of 5% KOH solution. The precipitate of the potassium salt of dihydrobetulonic acid (1) is filtered off and dried. The dry salt is dissolved in 50 ml of ethanol, the insoluble part is filtered off, the filtrate is poured into 250 ml of a 5% HCl solution. The separated acid (1) is filtered off, washed with H ₂ O, and dried. As a result, 5.5 g (54% of theory) of dihydrobetulonic acid are obtained (1). The melting point and NMR spectra of ¹ H and ¹³ C correspond to the literature [Wahab A., Ottosen M., Bachelor FW // Can.J. Chem., 1991, 69, P.570-577].

Example 4. Obtaining 3-oxo-lupano-28-yl chloride (dihydrobetulonic acid chloride, 1)

To a solution of 5.7 g (12.48 mmol) of dihydrobetulonic acid (1) in 70 ml of anhydrous CH ₂ Cl _{2 was} added 2.3 ml (26.36 mmol) of oxalyl chloride and kept at room temperature for 4 hours. After the solvent was distilled off, the same amount of CH ₂ Cl _{2 was} added to the residue and reevaporated. The residue is taken up with anhydrous ether, the precipitate is filtered off, washed with ether, and 4.6 g of DHA chloride are obtained with a melting point of 225-232 ° C (yield - 91%). The acid chloride is immediately introduced into a further reaction.

Example 5. Obtaining amides of hydrogenated betulonic acid (2-8). General methodology

To a solution of 0.5 g (1.05 mmol) of dihydrobetulonic acid chloride (1) in 40 ml of dry CH ₂ Cl ₂ , 2.2 mmol of the corresponding amine are added with stirring. The reaction mixture was kept at room temperature for 18-20 hours, then 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. The residue is recrystallized or is divided by column chromatography on silica gel fractions 60-200 μm company Merk. Eluents are listed for each product. The data of ¹³ C NMR spectra for the synthesized dihydrobetuloic acid amides (2-8) in CDCl ₃ (δ, ppm) are shown in Table 2.

Notes: a) for C (2 ′, 6 ′) atoms, a wide signal is observed at 46.2 ppm, the CS of the C (4 ′) signal is 24.62 ppm; b) for C (2 ′, 6 ′) atoms, a wide signal is observed at 45.6 ppm; c) very wide signals of about 45 ppm are observed for C (2 ′, 6 ′) atoms of compounds (4-6); d) for C atoms (2 ′, 6 ′) of compound (7) and C atoms (2 ′, 7 ′) of compound (8), signals cannot be observed; for compound (7), the doublet signals of the aromatic part are as follows: 128.43 C (10 ′) and 126.94 C (11 ′) ppm

Example 6. The effect of hydrogenation of betulonic acid (1) and its amides (2-8) 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 compounds (1-8) 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 compounds (1-8) in DMSO was added to the cells to a final concentration in the medium of 0.1 to 100 μg / ml. Betulonic acid (BA) and its corresponding amides (BA1-BA7) were used as a comparison drug. Cells were incubated in the presence of compounds for another 3 days under 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 in the absence of compound, but in the presence of a DMSO solvent.

Data on the treatment of MT-4 leukemia cells with compounds (1-8) are shown in FIGS. 1-7.

Figure 1 shows the viability of cells of the MT-4 line after incubation with compound (2) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (2), was taken. As a comparison drug used a similar derivative of BC (BA1)

Figure 2 - the viability of cells of the MT-4 line after incubation with compound (3) for 72 hours. 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 (3), was taken as 100%. A similar derivative of BC (BA2) was used as a comparison drug.

Figure 3 - the viability of cells of the MT-4 line after incubation with compound (4) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (4), was taken. A similar derivative of BC (BA3) was used as a comparison drug.

Figure 4 - the viability of cells of the MT-4 line after incubation with compound (5) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (5), was taken. A similar derivative of BC (BA4) was used as a comparison drug.

Figure 5 - the viability of cells of the MT-4 line after incubation with compound (6) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO but without compound was taken (6). A similar derivative of BC (BA5) was used as a comparison drug.

Figure 6 - the viability of cells of the MT-4 line after incubation with compound (7) for 72 hours. The number of living cells was estimated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (7), was taken. A similar derivative of BC (BA6) was used as a comparison drug.

Figure 7 - the viability of cells of the MT-4 line after incubation with compound (8) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (8), was taken. A similar derivative of BC (BA7) was used as a comparison drug.

From the data shown in Fig.1-7, it is seen that treatment of MT-4 leukemia cells with compounds (1-8) causes their effective death even at a concentration of compounds of 10 ^-6 M. CCID ₅₀ values are the concentration of the compound at which death is observed 50% of the cells, as well as CCID ₈₀ and CCID ₉₀ (concentrations at which the death of 80 and 90% of the cells, respectively) are shown in table 1.

From the data given in table 1, it can be seen that compounds (1-8) have the same or more pronounced antitumor effect compared to the previously described similar derivatives of betulonic acid (BA1-BA7), with the exception of compound (4), whose antitumor effect on MT-4 cells, it is less pronounced compared with a similar derivative of betulonic acid.

Example 7. The effect of hydrogenated betulonic acid (1) and its amides (2-8) 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% - Nogo CO ₂ at 37 ° C.

Cell viability after incubation with compounds (1-8) 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 from 0.1 to 100 μg / ml. As a comparison drug, similar derivatives of betulonic acid were used (2). Cells were incubated in the presence of the test compounds for another 3 days under 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 h 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.

The calculation of CCID values was carried out as described in example 5. The CCID ₅₀ values are the concentration of the compound at which 50% cell death is observed, as well as CCID ₈₀ and CCID ₉₀ (the concentration at which 80 and 90% cell death is observed, respectively) are shown in the table 1. All investigated compounds (1-8) have the same or more pronounced antitumor effect compared to the previously described similar derivatives of betulonic acid (BA1-BA7), with the exception of compound (4), whose antitumor effect on CEM-13 cells is less pronounced in comparison a similar derivative of betulonic acid.

The viability of the cell line CEM-13 after incubation with compounds (2-8) is illustrated in Fig.8-14.

Fig. 8 shows the viability of CEM-13 cells after incubation with compound (2) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (2), was taken. As a comparison drug used a similar derivative of BC (BA1)

Figure 9 - cell viability of the CEM-13 line after incubation with compound (3) for 72 hours. 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 (3), was taken as 100%. A similar derivative of BC (BA2) was used as a comparison drug.

Figure 10 - cell viability of the CEM-13 line after incubation with compound (4) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (4), was taken. A similar derivative of BC (BA3) was used as a comparison drug.

11 - cell viability of the CEM-13 line after incubation with compound (5) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (5), was taken. A similar derivative of BC (BA4) was used as a comparison drug.

Figure 12 shows the viability of CEM-13 cells after incubation with compound (6) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO but without compound was taken (6). A similar derivative of BC (BA5) was used as a comparison drug.

Fig. 13 shows the cell viability of the CEM-13 line after incubation with compound (7) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (7), was taken. A similar derivative of BC (BA6) was used as a comparison drug.

Fig. 14 shows the cell viability of the CEM-13 line after incubation with compound (8) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (8), was taken. A similar derivative of BC (BA7) was used as a comparison drug.

Example 8. The effect of hydrogenated betulonic acid (1) and its amides (2-8) 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 compounds (1-8) 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 the studied compounds in DMSO was added to the cells to a final concentration in the medium from 0.1 to 100 μg / ml. Betulonic acid was used as a reference drug (2). Cells were incubated in the presence of compounds for another 3 days under 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.

The calculation of CCID values was carried out as described in example 5. The CCID ₅₀ values - the concentration of the compound at which 50% cell death is observed, as well as CCID ₈₀ and CCID ₉₀ (the concentration at which 80 and 90% cell death is observed, respectively) are shown in FIG. .15-22.

Fig. 15 shows the cell viability of the U-937 line after incubation with compound (2) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (2), was taken. A similar derivative of BC (BA1) was used as a comparison drug.

Figure 16 shows the cell viability of the U-937 line after incubation with compound (3) for 72 hours. 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 (3), was taken as 100%. A similar derivative of BC (BA2) was used as a comparison drug.

Fig - cell viability of the U-937 line after incubation with compound (4) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (4), was taken. A similar derivative of BC (BA3) was used as a comparison drug.

Fig. 18 shows the cell viability of the U-937 line after incubation with compound (5) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (5), was taken. A similar derivative of BC (BA4) was used as a comparison drug.

Figure 19 shows the cell viability of the U-937 line after incubation with compound (6) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO but without compound was taken (6). A similar derivative of BC (BA5) was used as a comparison drug.

Figure 20 shows the cell viability of the U-937 line after incubation with compound (7) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (7), was taken. A similar derivative of BC (BA6) was used as a comparison drug.

Fig - cell viability of the U-937 line after incubation with compound (8) for 72 hours. The number of living cells was evaluated using the MTT test. For 100%, the number of living cells incubated in the presence of DMSO, but without compound (8), was taken. A similar derivative of BC (BA7) was used as a comparison drug.

Thus, compounds (1-8) have a more pronounced antitumor effect in relation to the studied U-937 tumor cells compared with betulonic acid and its similar derivatives (BA1-BA8), for which the presence of a pronounced antitumor activity was previously shown.

Example 9. The effect of hydrogenated betulonic acid (1) and its amides (2-8) on the viability of tumor lines of human cells

The CCID values - the concentration of compounds (1-8) at which the death of carcinoma cells is observed - are shown in Table 1. CCID ₅₀ is the concentration of the compound at which 50% of the cells are dead, as well as CCID ₈₀ and CCID ₉₀ (concentrations at which death of 80 and 90% of cells is observed, respectively). Thus, compounds (1-8) have the same or more pronounced antitumor effect in relation to the studied tumor cells MT-4, CEM-13 and U-937 in comparison with betulonic acid and its similar derivatives (BA1-BA8), for which the presence of pronounced antitumor activity was previously shown.

Table 1 Hydrogenated betulonic acid and its amides as antitumor agents of triterpene nature CCID ₅₀ , µM Acid and its amides SEM-13 U-937 MT-4 (one) 4.4 3.8 3.5 (2) 17.2 6.0 8.2 (3) 14.3 22.8 2.9 3.8 26.6 (four) 13.0 7.8 33.4 (5) 6.9 6.0 7.2 (6) 12.4 23.5 5.9 7.7 16.8 (7) 26.0 36.2 69.5 10.9 26.0 (8) 10.6 11.9 11.9 CCID ₈₀ , μM (one) 186.1 87.6 100.7 (2) 101.2 > 190 61.1 (3) 121.7 190.2 123.6 (four) > 185.6 > 185.6 157.7 (5) 77.8 > 180.9 48.8 (6) > 167.7 21.8 115.7 (7) > 144.7 > 144.7 118.7 (8) > 185.9 > 185.9 66.9 CCID ₉₀ , µM (one) > 219 > 219 > 219 (2) 175.5 > 190 118.4 (3) > 190 > 190 > 190 (four) > 185.6 > 185.6 > 185.6 (5) 180.9 > 180.9 110.3 (6) > 167.7 > 167.7 > 167.7 (7) > 144.7 > 144.7 > 144.7 (8) > 185.9 > 185.9 117.1

table 2 Hydrogenated betulonic acid and its amides as antitumor agents of triterpene nature C atom amide (2) ^a amide (3) ^b amide (4) ^c amide (5) ^c amide (6) ^c amide (7) ^d amide (8) ^d S-1 39.48 t 39.48 t 39.48 t 39.46 t 39.45 t 39.50 t 39.50 t S-2 33.97 t 33.97 t 33.98 t 33.95 t 33.94 t 34.00 t 33.98 t S-3 217.92 s 217.83 s 217.91 s 217.85 s 217.82 s 217.97 s 217.91 s S-4 47.14 s 47.15 s 47.15 s 47.12 s 47.12 s 47.18 s 47.15 s S-5 54.90 d 54.90 d 54.90 d 54.88 d 54.86 d 54.93 d 54.90 d S-6 19.49 t 19.48 t 19.49 t 19.47 t 19.45 t 19.52 t 19.51 t S-7 33.63 t 33.64 t 33.62 t 33.60 t 33.60 t 33.67 t 33.65 t S-8 40.45 s 40.47 s 40.47 s 40.45 s 40.44 s 40.49 s 40.52 s S-9 49.83 d 49.78 d 49.80 d 49.79 d 49.76 d 49.83 d 49.88 d S-10 36.74 s 36.74 s 36.75 s 36.73 s 36.72 s 36.77 s 36.76 s S-11 21.57 t 21.53 t 21.54 t 21.53 t 21.51 t 21.56 t 21.59 t S-12 27.02 t 26.99 t 27.01 t 26.99 t 26.95 t 27.03 t 27.04 t S-13 36.57 d 36.55 d 36.54 d 36.51 d 36.55 d 36.56 d 36.51 d S-14 41.88 s 41.89 s 41.88 s 41.86 s 41.88 s 41.90 s 41.98 s S-15 29.73 t 29.65 t 29.65 t 29.61 t 29.63 t 29.65 t 29.80 t S-16 32.17 t 32.08 t 32.15 t 32.13 t 32.20 t 32.13 t 31.92 t S-17 54.89 s 54.74 s 54.79 s 54.75 s 54.87 s 54.80 s 55.21 s S-18 52.21 d 52.02 d 52.06 d 52.04 d 52.06 d 52.07 d 52.50 d S-19 42.67 d 42.59d 42.60 d 42.59 d 42.58 d 42.66 d 42.76 d S-20 29.66 d 29.61 d 29.63 d 29.61 d 29.60 d 29.65 d 29.73 d S-21 23.39 t 23.34 t 23.35 t 23.33 t 23.32 t 23.37 t 23.43 t S-22 35.98 t 35.91 t 35.97 t 35.95 t 35.99 t 35.96 t 36.23 t S-23 26.44 to 26.44 to 26.44 to 26.43 to 26.41 to 26.47 to 26.46 to S-24 20.85 to 20.86 to 20.86 to 20.84 to 20.84 to 20.90 to 20.84 to S-25 15.77 to 15.77 to 15.78 to 15.76 to 15.75 to 15.80 to 15.80 to S-26 15.77 to 15.77 to 15.77 to 15.76 to 15.74 to 15.80 to 15.74 to S-27 14.27 to 14.26 to 14.26 to 14.24 to 14.25 to 14.28 to 14.34 to S-28 173.27 s 173.69 s 173.50 s 173.41 s 173.85 s 173.47 s 173.88 s S-29 14.52 to 14.50 to 14.51 to 14.49 to 14.47 to 14.52 to 14.56 to S-30 22.77 to 22.76 to 22.77 to 22.75 to 22.73 to 22.79 to 22.82 to S-3 ′, 5 ′ 26.06 t 66.82 t 55.06 t 52.90 t 43.64 t 52.16 t C-7 ′ 45.76 to 52.08 to 155.34 s 76.12 d S-8 ′ 11.73 to 61.38 t 142.26 s S-9 ′ 14.45 to 126.66 d

Claims (1)

Hydrogenated betulonic acid of the formula (1) and its amides of the formula (2-8)

NR ₁ R ₂ =

possessing antitumor activity.

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