RU2466136C1 - N-[3-oxo-lupano-28-yl]-piperidine - agent having anti-tumour, anti-metastatic, anti-inflammatory and cytoprotective activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel chemical compound and specifically to N-[3-oxo-lupano-28-yl]-piperidine of formula (1):

having complex activity - anti-tumour, anti-metastatic, anti-inflammatory and cytoprotective activity. The compound is synthesised from dihydrobetulonic acid.

EFFECT: obtaining compounds for reducing proliferation and dissemination of malignant cells and relieving necrobiotic disorders in normal cells, arising under the influence of paraneoplastic processes and cytotoxic chemotherapy.

1 cl, 11 ex, 7 tbl, 4 dwg

Description

The invention relates to a new chemical compound, namely to N- [3-oxo-lupano-28-yl] piperidine of the formula (1)

possessing complex activity - antitumor, antimetastatic, anti-inflammatory and cytoprotective. The compound can be used in medicine as a pharmaceutical agent to reduce the proliferation and dissemination of malignant cells and reduce the severity of necrobiotic disorders in normal cells that occur under the influence of paraneoplastic processes and cytotoxic chemotherapy.

The antitumor effect of most traditional chemotherapy drugs is not selective and causes severe disorders at the systemic and tissue levels. To increase the tolerability of antitumor therapy, biological response modifiers (MDBs) are included in treatment regimens. These additional therapies have a regulatory effect on vital processes in cells and tissues, which helps to increase the antitumor resistance of the body and reduce the side effects of highly toxic chemotherapy [Treschalina EM Antitumor activity of substances of natural origin. - M .: Practical medicine, 2005. - 227 p.]. The most commonly used ICBMs are recombinant forms of colony-stimulating factors, interferons, cytokines, which have mainly hemostimulating and immunomodulating effects. Side effects of these peptide preparations (the appearance of neutralizing antibodies, effects on the central nervous and cardiovascular systems, etc.) limit the duration and reduce the effectiveness of treatment [Kadagidze Z. G. Cytokines // Practical Oncology. 2003, vol. 4, No. 3, pp. 131-139]. Along with these agents, in plant medicine and clinical practice, plant-based low molecular weight compounds and their synthetic analogues, which have complex protective properties and have moderate antitumor effects, are successfully used as ICBMs. As a result of a wide pharmacological screening, the effectiveness of a number of plant extracts recommended as concomitant drugs for the chemotherapy of oncological diseases (extracts of shoots and leaves of sea buckthorn, sticky alder, plantain, frankincense, Rhodiola rosea, Scutellaria baicalensis, etc.) was established [E. Golberg, Zueva E.P. Preparations from plants in the complex treatment of malignant diseases. Tomsk, ed. Tomsk University, 2002, 130 pp.]. However, there remain problems associated with an increase in the antitumor and antimetastatic efficacy of herbal preparations; therefore, the search for new ICBMs among natural compounds of various classes is still relevant.

The objective of the present invention is to develop tools with antitumor, antimetastatic, anti-inflammatory and cytoprotective activity, to expand the arsenal of such funds.

The problem is solved by a chemical compound of the formula (1)

namely, N- [3-oxo-lupano-28-yl] piperidine,

- possessing antitumor and antimetastatic activity;

- the ability to reduce the severity of pathological changes in tissues caused by paraneoplastic syndromes;

- showing a pronounced cytoprotective effect in the conditions of cytostatic polychemotherapy;

- not stimulating the proliferation and dissemination of the tumor when combined with chemotherapy;

- possessing anti-inflammatory properties.

The claimed compound is new and relates to triterpenoids of the lupane series, namely, derivatives of dihydrobetulonic acid. The closest structural analogue - betulonic acid - has a high biological activity. In particular, she and her amides have antioxidant properties [IV Sorokina, TG Tolstikova, EB Bubnova and others. The study of the antioxidant properties of betulonic acid derivatives on a model of acute toxic hepatitis // Scientific Herald of the Tyumen Medical Academy . 2003, No. 1, pp. 60-61], as well as antitumor and antimetastatic effects [IV Sorokina, TG Tolstikova, N. A. Zhukova and others. Evaluation of the antitumor and antimetastatic effects of betulonic acid amides in mice with transplantable Lewis carcinoma // Bull. exp biol. and honey., 2006, No. 1, p.69-72]. Amides of betulonic acid containing fragments of caprylic, pelargonic and undecanoic acids inhibit the growth of tumor cells in culture [Pokrovsky AG, Shintyapina AB, N.V. Pronkina, BC Kozhevnikov, Plyasunova OA, Shults E. E., Tolstikov G.A. Activation of apoptosis by betulinic acid derivatives in human tumor cells in vitro // Doklady Akademii Nauk. 2006.V. 407. No. 5. S.698-701]. The betulonic acid dipeptide (N '{N- [3-Oxolup-20 (29) -en-28-oyl] -9-aminononanoyl} -3-amino-3-phenylpropionic acid) is patented as an antiviral (anti- HIV, anti-herpes) and immunostimulating activity [RF Patent 2211843 of January 25, 2002, by application 2002. 102338/04. BIPM. 2003. No. 25. 4.3. S.498-499].

Betulonic acid and its derivative [3-oxo-20 (29) -lupen-28-oil] -3-aminopropionic acid arrest necrobiotic lesions of the hepatic parenchyma in mice after cytostatic chemotherapy of malignant lymphosarcoma [N.A. Zhukova, D.E. Semenov, IV Sorokina, et al. Effect of betulonic acid and [3-oxo-20 (29) -lupen-28-oyl] -3-aminopropionic acid on liver morphology of mice with RLS lymphosarcoma after polychemotherapy // Bull. exp biol. and honey., 2005, T. 14, No. 9, p. 348-351].

High biological activity is also characteristic of derivatives of dihydrobetulinic acid. It was shown that the hydrogenation of betulinic acid increases the activity of its derivatives in comparison with similar compounds synthesized based on betulinic acid. For example, the anti-HIV activity of 3-O-glutaryldihydrobetulin increases by no less than three orders of magnitude compared with the usual betulin derivative [Kashiwada Y., Sekiya M., (I) keshiro Y., Fujioka T., Kilgore NR, Wild CT, Allaway GP, Lee K.-H. // Bioorgan.Med.Chem. Lett., 2004, V.14, P.5851-5853]. Thus, by hydrogenating derivatives of a series of betulin, potentially valuable agents for medicine can be obtained.

The claimed compound is a previously unknown dihydrobetulonic acid derivative, the creation of which is dictated by the goal of expanding the range of low molecular weight ICBMs based on triterpenoid natural compounds that increase the antitumor resistance of the body and stop necrobiotic disorders in cells that are not affected by malignant transformation.

The inventive compound (1) is obtained by the method shown in scheme 1. Betulin (4) obtained by extraction of birch bark with benzene was taken as the starting compound. Hydrogenation of betulin (4) with hydrogen in an autoclave over an N (1) -Raney gives dihydrobetulin (3), which is oxidized with Fizer solution. The acid chloride is obtained from dihydrobetulonic acid (2), which is further used without delay. The interaction of the dihydrobetulonic acid chloride (2) with a double excess of piperidine easily leads to the claimed compound of formula (1).

IR spectra of the compounds were recorded on a VECTOR 22 instrument in KBr. Specific rotation was determined on a polAAr 3005 spectrometer, the concentration of the solution is given in g / 100 ml. The melting point was determined on a Termosystem FP 900 instrument from Mettler Toledo and on a Kofler table S 30 A / G (Germany).

The gross formula was determined from the elemental analysis of the substance. ¹ H and ¹³ C NMR spectra were recorded on a Bruker DRX-500 spectrometer (500.13 MHz and 125.76 MHz, respectively) for solutions of substances in CDCl ₃ . Solvent signals (δ _H 7.24 and δ _C 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, LR JMD, ^2.3 J _{C, H} 10 Hz).

The study of the pharmacological activity of N- [3-oxo-lupano-28-yl] piperidine (1) included the determination of the antitumor, antimetastatic, and cytoprotective effects when administered to mice with solid Lewis lung carcinoma. We also studied its effect on the antitumor and antimetastatic effects of polychemotherapy when combined with the introduction of tumor-bearing mice and the possibility of correcting its cytotoxic effect on normal cells of internal organs that are not affected by malignant degeneration. In addition, the anti-inflammatory properties of the claimed compound were evaluated.

As a standard for antitumor and antimetastatic effects, the standard ASOR polychemotherapy scheme (cyclophosphamide, doxorubicin, vincristine and prednisolone) was used [Gershanovich M.L., Filov V.A., Akimov M.A., Akimov A.A. Introduction to the pharmacotherapy of malignant tumors. St. Petersburg: Sotis, 1999. P.105] as a modification [Greek O.R., Mishenina S.V., Pupyshev A.B. The protective effect of enterosgel on rat liver lysosomes with the introduction of a complex of cytostatic drugs // Bull. exp. biol. and honey. 2002.V. 134. No. 10. S.413-417]. The reference drug in the study of the anti-inflammatory effect was the substance indomethacin (Fluka) at a dose of 20 mg / kg. Statistical data processing was performed using parametric statistics methods using the Statistika 6.0 software package. The results were considered reliable at p <0.05 by Student's criterion.

The antitumor effect of compound (1) was studied on female C57BL / 6 female mice with intramuscularly injected Lewis lung carcinoma (2 × 10 ⁶ tumor cells in 0.1 ml of physiological saline). Compound (1) was administered 10 days after transplantation intragastrically at a dose of 20 mg / kg for 8 days. The comparison group underwent cytostatic polychemotherapy (PCT) according to the ASOR regimen in the regimen of single parenteral administration of cyclophosphamide (50 mg / kg), doxorubicin (4 mg / kg), vincristine (0.1 mg / kg) and prednisone (5 mg / kg). The control was animals with a tumor. The results were evaluated in dynamics by the percentage of inhibition of tumor growth. It was found that intragastric administration of compound (1) at a dose of 20 mg / kg for 8 days causes moderate inhibition of the growth of Lewis lung carcinoma, compared with the cytostatic PCT ACP (see Tables 1, 2).

The antimetastatic activity of compound (1) was determined in the same animals at the end of the period of administration of the agent by light microscopy of sections of both lung lobes. The area of metastases was calculated using the Video Test program. The intensity of the metastasis process was evaluated by the frequency of metastasis (the ratio of the number of animals with metastases to the total number of animals in the group) and the metastasis inhibition index (IIM). It was shown that compound (1) does not stimulate tumor dissemination, exhibiting a significant antimetastatic effect (IMI - 75.7%) (see Table 3).

The cytoprotective properties of compound (1) were evaluated by its effect on the liver cells of C57BL / 6 mice with Lewis lung carcinoma. For this, a pathomorphological study of liver tissue was performed by light microscopy. Compound (1) was administered as described above; the reference group was performed a single PCT according to the ASOR scheme; the control was animals with a tumor. At the end of the experiment, the animals were sacrificed, the liver was removed and subjected to standard histological processing. It was shown that the administration of compound (1) reduces the severity of necrobiotic damage to hepatocytes caused by tumor growth and improves the condition of the hepatic parenchyma. In the reference group with PCT, changes associated with increased degenerative and alternative processes were observed, as well as the presence of gross necrobiotic disorders in the liver structure was recorded.

The effect of compound (1) on the antitumor and antimetastatic effects of cytostatic polychemotherapy was studied in C57BL / 6 female mice with transplanted Lewis lung carcinoma. 10 days after inoculation, the animals were injected once parenterally with a complex of chemotherapeutic drugs according to the ASOR scheme: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg). One day after polychemotherapy, animals were injected intragastrically with compound (1) at a dose of 20 mg / kg for 8 days. The reference group was animals with PCT ASOR, the control was animals with a tumor. The antitumor effect was determined in dynamics by the index of inhibition of tumor growth (TPO), antimetastatic - at the end of the administration period by counting the area of metastases, the frequency of metastasis and the index of inhibition of metastasis. It was found that the administration of compound (1) under the conditions of PCT does not have a proliferating effect on Lewis lung carcinoma grafts (see Tables 5, 6) and does not significantly affect the antitumor efficacy of chemotherapy drugs.

With the combined administration of compound (1) and antitumor drugs that mimic polychemotherapy, the dissemination of the tumor does not increase, the antimetastatic effect of chemotherapy persists (see Table 7).

The cytoprotective properties of compound (1) were studied in the same animals by pathomorphological examination of the liver by light microscopy. It was found that under the action of compound (1), administered against a background of PCT, the degree of degenerative and necrotic lesions of hepatocytes caused by the development of the tumor decreases, while the morphological picture in the reference group is characterized by an intensification of alternative processes, accompanied by an increase in cell necrosis and the severity of dystrophy.

Administration of compound (1) at a dose of 50 mg / kg to Lewis carcinoma mice reduces the severity of necrobiotic tissue damage caused by tumor development, while the morphological picture in the reference group is due to an increase in the degree of all alterative processes (dystrophy, necrosis).

The anti-inflammatory activity of compound (1) was evaluated by reducing the inflammatory swelling of the paws of mice caused by subplanar administration of a 0.1% histamine solution. Compound (1) in the form of a water-twin suspension was introduced into the stomach at a dose of 20 mg / kg 1 hour before the phlogogen. The reference preparation indomethacin (Fluka) was administered similarly at the same dose. The magnitude of the edema was determined 6 hours after administration of the compound by the difference in the mass of the inflamed and healthy paws. It was found that the claimed compound has a moderate anti-inflammatory activity, which is 1.5 times less than that of the non-steroidal anti-inflammatory drug indomethacin (see table 4).

Thus, the new compound, N- [3-oxo-lupano-28-yl] piperidine of formula (1), can be considered as a promising agent with antitumor, antimetastatic, anti-inflammatory and cytoprotective effects aimed at reducing necrobiotic disorders in tissues caused by paraneoplastic processes and cytostatic polychemotherapy.

The advantages of the claimed compounds include:

- moderate antitumor and pronounced antimetastatic activity;

- the ability to reduce the severity of necrobiotic lesions of non-transformed cells caused by paraneoplastic processes;

- pronounced cytoprotective effect when applied against the background of cytostatic polychemotherapy;

- lack of a stimulating effect on tumor proliferation and dissemination during cytostatic polychemotherapy;

- the presence of anti-inflammatory activity.

The invention is illustrated by the following examples.

Example 1. Obtaining loop-20 (29) -en-3β, 28-diol-betulin (4).

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 decanted again. 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 E.W.H., Jordis U., Moche W., Sauter F. // Phytochem. 1989, 28, P.2229]). The yield of betulin to the original cortex is 11%.

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

Hydrogenation of betulin (4) 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 (4), 13.2 g of N (1) 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 (3) are obtained (yield - 93%). Mp 282-284 ° C (from alcohol). Lit.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 (2).

A suspension of 10 g (22.5 mmol) of dihydrobetulin (3) in 165 ml of glacial acetic acid and 110 ml of acetone is cooled at 0 ° C, and a freshly prepared solution of Fizera (11 g, 0.11 mol of chromic anhydride in 12 ml of ice cold) is added with stirring acetic 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 (2) 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 (2) is filtered off, washed with H ₂ O, and dried. As a result, 5.5 g (54% of theory) of dihydrobetulonic acid are obtained (2). 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 N- [3-oxo-lupano-28-yl] piperidine (1).

To a solution of 0.48 g (1.05 mmol) of dihydrobetulonic acid (2) in 40 ml of anhydrous CH ₂ Cl _{2 was} added 0.3 ml (3.44 mmol) of oxalyl chloride and kept at room temperature for 4 hours. After distillation of the solvent, 20 ml of CH _{2 were} added twice to the residue. Cl ₂ and evaporate as many times. The acid chloride is immediately introduced into a further reaction. To a solution of 0.5 g (1.05 mmol) of dihydrobetulonic acid chloride (2) in 40 ml of dry CH ₂ Cl ₂ , 0.19 g (2.2 mmol) of piperidine 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 was recrystallized in isopropyl alcohol to give 0.40 g (71%) of a product with mp. 254-255 ° C. Found (%): C, 80.26; H, 10.90; N, 2.73. C ₃₅ H ₅₇ NO ₂ . Calculated (%): S.80.25; H 10.97; N, 2.67. IR (KBr), v / cm ^-1 : 1629 (CON); 1708 (C = O). [α] ²² _D = -31.8 (C 0.18). ¹ H NMR spectrum (СОСl ₃ , δ, ppm,. J / Hz): 0.68 (d, 3Н, Н ²⁹ , J = 6.8); 0.78 (d, 3H, H ³⁰ , J = 6.8); 0.88 (s, 3H, H ²⁵ ); 0.89 (s, 3H, H ²⁷ ); 0.92 (s, 3H, H ²⁶ ); 0.96 (s, 3H, H ²⁴ ); 1.01 (s, 3H, H ²³ ); 1.02-1.12 (m, 3H, H ^12a , H ^15e , H ^{22a or 22β} ); 1.22 (dd, 1H, H ^18a , J _{18a, 13a} = 10.8, J _{18a, 19} = 10.8); 1.26 (dd, 1H, H ^5a , J _{5a, 6a} = 11.7, J _{5a, 6e} = 2.7); 1.58 (m, 2H, H ^{4 '} ); 1.63 (dm, 1H, H ^12e , J _{12e, 12a} = 11.0); 1.72 (septet d, 1H, H ²⁰ , J = 6.8, J _20.19 = 2.7); 1.82-1.90 (m, 1H, H ^{22α or 22β} ; 2.07 (ddd, 1H, H ^16e , J _{16e, 16a} = 13.0, J _{16e, 15a} = 3.6, J _{16e, 15e} = 3.0); 2.16 (dddd, 1H, H ¹⁹ , J _19.18a = 10.8, J _19.21a = 10.2, J _19.21β = 4.0, J _19.20 = 2.7); 2.34 (ddd, 1H, H ^{20a or 2e} , J _{2a, 2e} = 15.5, J _{2e, 1a} = 7.5, J _{2e, 1e} = 4.4); 2.44 (ddd, 1H, H ^{2a or 2e} , J _{2a, 2e} = 15.5, J _{2a, 1a} = 9.7, J _{2a, 1e} = 7.6); 2.94 (ddd , 1H, H ^13a , J _{13a, 12a} = 12.7, J _{13a, 18a} = 10.8, J _{13a, 12e} = 3.7); 3.43 (br.s, 2H, Н ^{2 '} ); 3.47 (br.s, 2Н, Н ^{6 '} ). The signals of the remaining protons (16H) are observed in the region of 1.28-1.52 ppm ¹³ C NMR spectrum, δ, ppm: 39.48 t (C ¹ ), 33.97 t (C ² ), 217.92 s (C ³ ), 47.14 s (C ⁴ ) 54.90 d (C ⁵ ), 19.49 t (C ⁶ ), 33.63 t (C ⁷ ), 40.45 s (C ⁸ ), 49.83 d (C ⁹ ), 36.74 s (C ¹⁰ ) , 21.57 t (C ¹¹ ), 27.02 t (C ¹² ), 36.57 d (C ¹³ ), 41.88 s (C ¹⁴ ), 29.73 t (C ¹⁵ ), 32.17 t (C ¹⁶ ), 54.89 s (C ¹⁷ ), 52.21 d (C ¹⁸ ), 42.67 d (C ¹⁹ ), 29.66 d (C ²⁰ ), 23.39 t (C ²¹ ), 35.98 t (C ²² ), 26.44 k (C ²³ ), 20.85 k (C ²⁴ ), 15.77 k (C ²⁵ ) , 15.77 k (C ²⁶ ), 14.27 k (C ²⁷ ), 173.27 s (C ²⁸ ), 14.52 k (C ²⁹ ), 22.77 k (C ³⁰ ), 24.62 t (C ^{4 '} ), 26.06 t (C ^{3' , 5 '} ). The signals (C ^{2 ', 6'} ) are greatly broadened and are observed in the region of 46.2 ppm.

Example 5. The study of the antitumor effect of the compound (1) in mice with transplantable Lewis lung carcinoma.

The antitumor effect of N- [3-oxo-lupano-28-yl] piperidine (1) was studied on 25-30 g C57BL / 6 female mice, which were injected intramuscularly with a Lewis lung carcinoma suspension in a volume of 2 × 10 ⁶ tumor cells in 0.1 ml of physiological saline. 10 days after inoculation, the mice were divided into 3 groups (10 animals each). The experimental group of mice was administered intragastrically daily for 8 days as a compound (1) in the form of a water-twin suspension at a dose of 20 mg / kg (0.2 ml per 10 g of body weight). The standard of effect was a group of animals that underwent cytostatic polychemotherapy (PCT) according to the standard ASOR scheme adapted for mice: single parenteral administration of cyclophosphamide (50 mg / kg), doxorubicin (4 mg / kg), vincristine (0.1 mg / kg) and prednisone (5 mg / kg). The control was animals with a tumor. The control and reference group with chemotherapy received an aqueous twin suspension for 8 days. During the period of administration of agents in all groups, the dynamics of tumor growth was evaluated by measuring its size with a caliper in three mutually perpendicular relationships. The magnitude of the antitumor effect was determined by the tumor growth inhibition index (TPO) as the difference between the average size of the tumors in the control and experimental groups, referred to the average size of the tumors in the control.

Table 1 shows the absolute and relative sizes of the tumor nodes during administration of the agent. The relative values of the tumor volumes (as a percentage of their background values in the group) allow us to more correctly compare the results between the experimental and reference groups, since the background sizes of the tumors (before the introduction of the agent) may differ. It was found that compound (1) causes moderate inhibition of tumor node growth: the maximum effect was observed at the end of the course of administration as a 1.5-fold decrease in the tumor relative to the control. In the reference group, a 2-fold difference with control was observed during these periods (see Table 1).

It was shown that in the experimental group the indicator of inhibition of tumor growth (TPO) at the beginning of the course administration of compound (1) lags, and in the end it approaches the indicator of the reference group (see Table 2).

It was concluded that intragastric administration of compound (1) for 8 days at a dose of 20 mg / kg causes moderate inhibition of the growth of Lewis lung carcinoma, compared with the cytostatic PCT ACP.

Example 6. The study of the antimetastatic effect of compound (1) in mice with transplantable Lewis lung carcinoma.

The antimetastatic properties of compound (1) were studied in mice of C57BL / 6 female mice weighing 25-30 g with solid Lewis lung carcinoma under the conditions of the experiment described in Example 5. At the end of the experiment, the animals were sacrificed, the lungs were removed, and after standard histological processing were examined by light microscopy . The antimetastatic effect was determined using light microscopy of sections of both lung lobes. The area of metastases was determined using the Video Test program. The intensity of the metastasis process was evaluated by the frequency of metastasis (FM) (the ratio of the number of animals with metastases to the total number of animals in the group) and the metastasis inhibition index (IIM)

,

,

where A _k is the frequency of metastasis in the control group, A is the frequency of metastasis in the experimental group, B _k is the area of metastases in animals of the control group, B is the area of metastases in animals of the experimental group.

It was found that compound (1) significantly reduces the area of tumor metastases in the lungs (4 times relative to the control). The introduction of chemotherapeutic drugs ASOR reduces the number of metastatic lesions by 6 times and slightly reduces the frequency of metastasis (see table 3). Despite the fact that the claimed compound is inferior in the activity of cytostatic chemotherapy, the severity of the antimetastatic effect in the experimental group indicates a significant antimetastatic effect of the compound (1). This is also indicated by the high value of the IIM index.

Thus, it was found that compound (1) does not stimulate the dissemination of the tumor, showing a significant antimetastatic effect.

Example 7. The study of the anti-inflammatory activity of compound (1) in a model of histamine inflammation.

The anti-inflammatory activity of compound (1) was evaluated by reducing the inflammatory swelling of the paws of mice caused by subplanar administration of a 0.1% histamine solution. Compound (1) in the form of a water-twin suspension was introduced into the stomach at a dose of 20 mg / kg 1 hour before the phlogogen. Control animals were injected in the same manner with a water-twin suspension. The reference drug was the substance indomethacin (Fluka) at a dose of 20 mg / kg. The magnitude of the edema was determined 6 hours after administration of the agent according to the mass difference of the inflamed and healthy paws. For each animal, the inflammation index was calculated as the ratio of this difference to the weight of a healthy paw. Differences were considered significant with a probability of p <0.05. For the experimental and reference groups, the magnitude of edema was calculated relative to the control, which was taken as 100%. The difference in the relative magnitude of edema in the control group and the corresponding value in the experimental groups was taken as an anti-inflammatory effect.

It was established (see table 4) that the claimed compound has moderate anti-inflammatory activity. According to the severity of the effect, it is 1.5 times inferior to indomethacin.

Thus, the claimed compound has moderate anti-inflammatory activity.

Example 8. The effect of compound (1) on paraneoplastic disorders in the liver of mice with transplantable Lewis lung carcinoma.

The effect of compound (1) on pathomorphological disorders caused by tumor growth was studied in 3 groups of mice of C57BL / 6 female mice, which were injected with intramuscularly solid Lewis lung carcinoma, as described above. 10 days after the inoculation, the experimental group was administered intragastrically for 8 days in the form of a water-twin suspension at a dose of 20 mg / kg, the reference group was administered a single PCT according to the ACOP scheme, as described above. The control was animals with a tumor; as a physiological norm, a group of intact animals without a tumor was taken. Control animals and the ASOR polychemotherapy group received at the same time an equivalent amount of water-twin suspension. At the end of the experiment, the animals were sacrificed, the liver was removed, it was fixed in 4% paraform, followed by standard histological processing on the MICROM complex. Sections 3-4 μm thick were stained with hematoxylin and eosin, followed by examination using transmitted light microscopy. The degree of liver damage was evaluated by the following criteria:

the presence of dystrophy (fatty, hyaline droplet, hydropic and balloon) and hepatocyte necrosis (monocellular, focal, confluent, bridge-like), their size and localization. The degree of plethora, the type of cell infiltration of sinusoids, portal tracts, the expansion of sinusoids, bile ducts, metastases, their size, and localization were also evaluated. Based on the analysis of the data, the index of morphological changes in the liver was calculated, corresponding to mild, moderate, severe degree of damage (Muratov A.V., Sukhorukov V.P., 2002).

Compared with intact animals, in almost all control animals, a moderately pronounced small-focal hydropic dystrophy was revealed mainly in the centrolobular zones. Focal coagulation cell necrosis developed mainly around small focal metastases in the periportal zones. All animals showed multiple diffuse cellular and small focal sinusoidal metastases without clear localization, as well as periportal small focal metastases that did not extend beyond the border plate. Tumor cells were in active mitotic division. Increased in size Kupffer cells with lipid granules, moderate lymphohistiocytic infiltration of portal tracts, indicating an inflammatory process, were revealed in the lumen of sinusoids against the background of moderate venous congestion. Also, all animals showed a slight zonal expansion of the bile ducts.

Thus, in control animals with Lewis lung carcinoma, non-specific reactive hepatitis with prevailing dystrophic lesion and macrophage infiltration over necrotic changes in hepatocytes developed on the background of metastatic liver damage.

In the reference group with PCT, signs of toxic damage were observed in the form of a pronounced regenerative-plastic deficiency syndrome. Compared with the control, in hepatocytes in all animals an increase in the area and degree of dystrophic lesion in the form of diffuse small and medium vesicular lipid infiltration, large focal hydropic and balloon dystrophy was noted.

Compared with the control group, an increase in the area of hepatocyte necrosis was also visually observed. Monocellular (coagulation) necrosis of hepatocytes was localized on the periphery of metastatic foci and in the centrolobular divisions of the lobules. Small focal hepatocyte necrosis was localized both in the periportal and in the centrolobular zones. Giant cells with foamy cytoplasm containing pale shadows of nuclei were detected periodically in all animals. In one animal, a combination of monocellular focal, large focal, bridge necrosis infiltrated by polymorphonuclear leukocytes and macrophages was noted.

In half of the animals, zonal, and in the rest, diffuse expansion of sinusoids was revealed. Enlarged Kupffer cells and cells with lipid inclusions in the cytoplasm, macrophages, cell detritus, as well as a homogeneous substance of a pale pink color (fibrin) were determined in the lumen of sinusoids. In all animals, as well as in the control, diffuse cellular metastases were localized mainly in sinusoids, and small focal in both centrolobular and periportal zones.

The introduction of the compound (1) to tumor-bearing animals reduced the severity of the plastic regenerative deficiency syndrome. In hepatocytes, focal small-vesicular lipid infiltration was predominantly detected, and only in individual animals, small-focal hydropic dystrophy. Monocellular coagulation necrosis of hepatocytes was localized mainly along the periphery of small focal metastases in the periportal zones. In sinusoids, against the background of slight venous congestion, mainly Kupffer cells and a small number of lipid-containing cells were detected. In all animals, as well as in the control, diffuse cellular metastases were localized in sinusoids, and small focal metastases in both centrolobular and periportal zones.

Thus, the administration of compound (1) at a dose of 50 mg / kg to mice with Lewis carcinoma reduces the severity of necrobiotic tissue damage caused by tumor development, while the morphological picture in the reference group is due to an increase in the degree of all alternative processes (dystrophy, necrosis).

Example 9. The study of the effect of compound (1) on the antitumor effect of cytostatic polychemotherapy of mice with transplantable Lewis lung carcinoma.

C57BL / 6 female mice were divided into 3 groups of 10 animals each. All animals were inoculated intramuscularly with a cell suspension of Lewis lung carcinoma, as described above. 10 days after inoculation, the two groups of animals were injected once parenterally with a complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) One of these groups, one day after polychemotherapy, was administered intragastrically with compound (1) in the form of a water-twin suspension of 0.2 ml per 10 g of body weight at a dose of 20 mg / kg, the second group with AST PCT was a reference. The control was animals with a tumor. The administration of compound (1) to experimental mice was continued for 8 days. The control and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween. The magnitude of the antitumor effect was determined by the tumor growth inhibition index (TPO) as the difference between the average size of the tumors in the control and experimental groups, referred to the average size of the tumors in the control.

Table 5 shows the dynamics of tumor growth during treatment. The absolute and relative (in% relative to the background) values of the volume of transplants are presented, which make it possible to level the difference in their sizes before treatment. It was found that compound (1), administered against a background of PCT, does not stimulate tumor development: in the experimental and reference groups, a significant antitumor effect was observed relative to the control.

As a result of a comparative assessment of the size of tumor transplants in the experimental and reference groups, it was shown that their absolute values did not have significant differences (see Table 5). The relative sizes of the tumors were slightly different due to differences in background values (Table 5).

The inhibition of tumor growth in the experimental and reference groups did not have significant differences (see table 6). The decrease in the TPO index detected on the 4th day in the reference group is associated with a high background value of the tumor size in the PCT group (Table 5).

The experimental results indicate that, when administered against a background of PCT, compound (1) does not stimulate tumor proliferation and does not significantly affect the antitumor efficacy of chemotherapy drugs.

Example 10. The study of the effect of compound (1) on the antimetastatic effect of cytostatic polychemotherapy of mice with transplantable Lewis lung carcinoma.

Antimetastatic properties were studied in C57BL / 6 female mice weighing 25-30 g with solid Lewis lung carcinoma under the conditions of the experiment described in Example 5. At the end of the experiment, the animals were sacrificed, the lungs were removed, and after standard histological processing they were examined by light microscopy. The antimetastatic effect was determined by analysis of sections of both lung lobes. The area of metastatic lesions was determined using the Video Test program. The intensity of the metastasis process was evaluated by the frequency of metastasis (FM) (the ratio of the number of animals with metastases to the total number of animals in the group) and the metastasis inhibition index (IIM) [Arkhipov S.A. et al., 1984]

where A _k is the frequency of metastasis in the control group, A is the frequency of metastasis in the experimental group, B _k is the area of metastases in animals of the control group, B is the area of metastases in animals of the experimental group.

It was found that in both groups with the introduction of cytostatic drugs, a significant decrease in the area of metastases was observed compared with the control (see Table 7). Chemotherapy alone causes a 6-fold decrease in the area of metastases and slightly reduces the frequency of metastasis relative to the control (Table 7). The introduction of compound (1) on the background of PCT leads to a more significant reduction in the area of metastatic lesions - by 9 times, but does not affect the frequency of metastasis. As a result, the severity of the antimetastatic effect in both groups is almost the same, as evidenced by close values of IMI.

Thus, with the combined administration of compound (1) and antitumor drugs that mimic polychemotherapy, tumor dissemination is not enhanced, the antimetastatic effect of chemotherapy is maintained.

Example 11. The study of the cytoprotective properties of compound (1) under the conditions of cytostatic polychemotherapy of mice with transplantable Lewis lung carcinoma.

The cytoprotective properties of compound (1) were studied by pathomorphological examination of the liver of mice with transplanted Lewis lung carcinoma after PCT. The experimental conditions are described in Example 5. At the end of the experiment, the animals were sacrificed, the liver was removed, it was fixed in 4% paraform, followed by standard histological processing on the MICROM complex. Sections 3-4 μm thick were stained with hematoxylin and eosin, followed by examination using transmitted light microscopy. The degree of liver damage was evaluated according to the following criteria: the presence of dystrophy (fatty, hyaline droplet, hydropic and balloon) and hepatocyte necrosis (monocellular, focal, confluent, bridge-like), their size and localization. The degree of plethora, the type of cell infiltration of sinusoids, portal tracts, the expansion of sinusoids, bile ducts, metastases, their size, and localization were also evaluated. Based on the analysis of the data, the index of morphological changes in the liver was calculated, corresponding to mild, moderate, severe damage (Muratov A.V., Sukhorukov V.P., 2002).

As a result of pathomorphological analysis in the liver of control animals under conditions of progressive tumor growth, the development of non-specific reactive hepatitis was noted, the characteristic features of which are a combination of regenerative-plastic deficiency syndrome (dystrophy and necrosis) with the presence of metastatic lesion and macrophage infiltration of sinusoids and portal tracts, as well as circulatory disorders (venous congestion and thickening of the sinusoidal walls).

In almost all animals, dystrophic lesions in the centrolobular zones were detected only in the form of small focal hydropic dystrophy. Focal coagulation cell necrosis developed primarily around small focal metastases in the periportal zones. All animals revealed multiple diffuse cellular and small focal sinusoidal metastases without clear localization, as well as periportal small focal metastases that did not extend beyond the border plate. Tumor cells were in active mitotic division. The walls of the sinusoids were thickened with signs of plasma impregnation. Increased cells with lipid granules in the cytoplasm, moderate lymphohistiocytic infiltration of portal tracts were detected in the lumen of sinusoids against a background of moderate plethora. Also, in all animals, a slight zonal expansion of the bile ducts was noted (see Fig. 1).

The combination of morphological changes in the liver in the group corresponds to a moderate lesion.

The introduction of a complex of cytostatics led to the development of toxic damage in the form of a pronounced regenerative-plastic deficiency syndrome. Compared with the control, in hepatocytes in all animals an increase in the area and degree of dystrophic lesion was observed in the form of diffuse small and medium vesicular lipid infiltration, focal hydropic and balloon dystrophy (see Fig. 2).

Compared with the control group, an increase in the area of hepatocyte necrosis was also visually observed. Monocellular (coagulation) necrosis of hepatocytes was localized on the periphery of metastatic foci and in the centrolobular divisions of the lobules. Small focal hepatocyte necrosis was localized both in the periportal and in the centrolobular zones. Giant cells with foamy cytoplasm containing pale nucleus shadows were detected periportally in all animals (see Fig. 2). In one animal, a combination of monocellular focal, large focal, bridge necrosis infiltrated by polymorphonuclear leukocytes and macrophages was noted.

In half of the animals, zonal, and in the rest, diffuse expansion of sinusoids was revealed. Enlarged Kupffer cells and cells with lipid inclusions in the cytoplasm, macrophages, cell detritus, as well as a homogeneous substance visually defined as fibrin (Fig. 2) were determined in the lumen of the sinusoids, which is an indicator of the disturbance of trans-sinusoidal metabolism. In all animals, as well as in the control, diffuse cellular metastases were localized mainly in sinusoids, and small focal in both centrolobular and periportal zones. Thus, a combination of morphological characters in the liver of animals of the reference group corresponds to a severe degree of damage, compared with the control.

The administration of compounds (1) to animals on the background of polychemotherapy had a pronounced positive effect on the course of the pathological process. Under the influence of (1), a decrease in the severity of regenerative-plastic insufficiency syndrome was noted: in hepatocytes isolated small foci of small-vesicular lipid infiltration and hyaline droplet dystrophy were detected, which were localized mainly in the centrolobular sections (see Figs. 3, 4). Single monocellular necrosis of hepatocytes and apoptotic bodies (Councilsilmen) were localized mainly around small focal metastases.

There was also a restoration of the lumen of sinusoids with the replacement of the infiltration of lipid-containing cells (Ito) with Kupffer and mononuclear cells (Fig. 3). Visually, there was no change in the volume of metastatic liver damage compared with the reference group.

Thus, the administration of compound (1) against the background of polychemotherapy leads to an improvement in the morphological parameters of the liver, compared with the control and reference groups.

The data obtained indicate that the claimed compound N- [3-oxo-lupano-28-yl] piperidine (1) can be used as a tool designed to inhibit the growth and dissemination of tumor cells and correct cytotoxic damage in tissues with malignant growth and cytostatic chemotherapy, it has the following advantages:

- shows antitumor and antimetastatic activity;

- reduces the severity of cytotoxic changes in tissues caused by paraneoplastic syndromes;

- exhibits a pronounced cytoprotective effect in the conditions of cytostatic polychemotherapy;

- does not stimulate the proliferation and dissemination of tumors when combined with chemotherapy;

- has anti-inflammatory properties.

Table 1 Lewis lung carcinoma growth dynamics and mouse survival during intragastric administration of compound (1) Group Tumor volume, cm ³ (and in% relative to background values) Day after chemotherapy 0 (background) four 6 8 The control 0.62 ± 0.06 2.05 ± 0.17 3.21 ± 0.17 3.64 ± 0.29 0% 230% 418% 487% PCT 0.79 ± 0.13 1.82 ± 0.28 2.42 ± 0.20 * 2.68 ± 0.18 * 0% 130% 206% 239% (1), 20 mg / kg 0.67 ± 0.10 1.94 ± 0.16 2.82 ± 0.26 2.85 ± 0.28 0% 190% 321% 325% * P <0.05; ** P <0.001; *** P <0.0001 differences with control are significant # P <0.05 differences with the comparison group are significant

table 2 Lewis lung carcinoma growth inhibition index (TPO) values in mice during the administration of compound (1) Group SRW,% (by tumor volume) Day after chemotherapy 0 (background) four 6 8 PCT 0 eleven 25 26 (1), 20 mg / kg 0 5 12 22

Table 3 Metastasis of Lewis lung carcinoma metastasis in the lungs of mice after 8-day administration of compound (1) Group Area of metastases (μm) Metastasis rate IMI% The control 319.78 ± 51.36 one hundred - PCT 50.66 ± 9.15 *** 88 86.0 (one) 77.62 + 25.37 *** one hundred 75.7 * P <0.05, *** P <0.001 differences with control are significant; FM - frequency of metastasis; IIM - metastasis inhibition index.

Table 4 Anti-inflammatory effect of compound (1) in a model of histamine paw edema Agent Inflammation index,% The magnitude of the edema relative to the control,% Anti-inflammatory effect,% The control 26, ± 1.8 one hundred 0 (one) 18.9 ± 1.4 * 72 28 Indomethacin 15.7 ± 0.77 * 59 41 ** P <0.01 relative to the control; ### P <0.001 relative to the reference drug

Table 5 Changes in the dynamics of the growth of Lewis lung carcinoma and the survival of mice under the influence of the administration of compound (1) on the background of cytostatic polychemotherapy (PCT) Group Tumor volume, cm ³ (and in% relative to background values) Day after chemotherapy 0 (background) four 6 8 The control 0.62 ± 0.06 2.05 ± 0.17 3.21 ± 0.17 3.64 ± 0.29 0% 230% 418% 487% PCT 0.79 ± 0.13 1.82 ± 0.28 2.42 ± 0.20 * 2.68 ± 0.18 * 0% 130% 206% 239% PCT + (1) 0.66 ± 0.09 1.63 ± 0.20 2.45 ± 0.27 * 2,50 ± 0,36 * 0% 147% 271% 279% * P <0.05; ** P <0.001; *** P <0.0001 differences with control are significant

Table 6 Lewis lung carcinoma growth inhibition index (TPO) values in mice during the administration of compound (1) during polychemotherapy (PCT) Group SRW,% Day after chemotherapy four 6 8 PCT eleven 25 26 PCT + (1), 20 mg / kg 21 24 31

Table 7 Metastasis of Lewis lung carcinoma metastasis in the lungs of mice after 8-day administration of compound (1) with polychemotherapy (PCT) Group Area of metastases (μm) Metastasis rate IMI% The control 319.78 ± 51.36 one hundred - PCT 50.66 ± 9.15 *** 88 86.0 PCT + (1) 34.78 + 3.68 *** one hundred 89.1

Claims (1)

N- [3-Oxo-lupano-28-yl] piperidine of the formula (I):

possessing antitumor, antimetastatic, anti-inflammatory and cytoprotective activity.

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