RU2461563C1 - N-[3-oxo-lupano-28-yl]-morpholine - agent for correction of cytotoxic liver damages with antitumour and antimetastatic activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a chemical compound, namely N-[3-oxo-lupano-28-yl]-morpholine of formula (1):

possessing antitumour and antimetastatic activity and representing a corrector of hepatotoxic damages caused by a tumour process and cytostatic polychemotherapy, and may be applicable in medicine as a pharmaceutic agent for reducing manifestation of morphological liver damages accompanying oncological diseases, including as a part of the integrated antitumour therapy.

EFFECT: preparation of the chemical compound possessing antitumour and antimetastatic activity.

1 cl, 10 ex, 6 tbl, 3 dwg

Description

The invention relates to a new chemical compound, namely to N- [3-oxo-lupano-28-yl] -morpholine of formula (I):

possessing antitumor and antimetastatic activity and being a corrector of hepatotoxic injuries caused by the tumor process and cytostatic polychemotherapy. The compound can be used in medicine as a pharmaceutical agent to reduce the severity of morphological disorders of the liver, concomitant oncological diseases, both when used alone, and as part of complex antitumor therapy.

As you know, most traditional antitumor drugs do not have a selective effect on the tumor and cause severe disorders at the systemic and tissue levels, sometimes incompatible with life. To increase the tolerance of antitumor drugs, biological response modifiers (MBRs) are included in treatment regimens, which reduce the toxic effects of chemotherapy and increase the antitumor resistance of the body [EM Treschalina. Antitumor activity of substances of natural origin. - M .: Practical medicine, 2005. - 227 p.].

Currently, as MBRs, the most commonly used are recombinant forms of colony-stimulating factors, interferons, cytokines, which have mainly hemostimulating and immunomodulating effects. The disadvantages of protein drugs are their own side effects, including possible allergic reactions [Clinical hematology. Ed. Volkova M.A. - M .: Medicine, 2001, p.77-85]. Along with these agents, plant-based low molecular weight compounds and their synthetic analogues, having complex protective properties and having moderate antitumor effects, have great potential as MBRs. Various plant extracts (sea buckthorn, Rhodiola rosea, Scutellaria baicalensis, etc.) are usually used as herbal preparations in concomitant therapy [Razina TG, Zueva EP, Amosova EN et al. Preparations from medicinal plants as a means of additional therapy in experimental oncology // Questions of Oncology. - 2000, t. 63, No. 5, s 554-556]. However, their lack of effectiveness stimulates the search for new ICBMs in various classes of natural compounds.

The present invention is to develop a means of correcting cytotoxic liver damage with antitumor and antimetastatic 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] -morpholine having pronounced antitumor and antimetastatic activity; the ability to reduce the severity of pathological changes in the liver caused by paraneoplastic syndromes; exhibiting a pronounced hepatoprotective effect under the conditions of cytostatic polychemotherapy and not stimulating the proliferation and dissemination of the tumor.

The claimed compound is new and relates to triterpenoids of the lupane series, namely, derivatives of dihydrobetulonic acid. The closest structural analogue, betulonic acid, is known for its high biological activity. In particular, antioxidant properties were found in betulonic acid and some of its amides [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 Tyumen Medical Academy, 2003, No. 1, pp. 60-61], antitumor and antimetastatic effect [I.V. Sorokina, TGTolstikova, N.A. Zhukova and others. Evaluation of the antitumor and antimetastatic effects of betulonic acid amides on mice with transplantable Lewis carcinoma // Bu l exp biol. and honey., 2006, No. 1, pp. 69-72], as well as the ability to reduce necrobiotic lesions of the hepatic parenchyma caused by cytostatic antitumor chemotherapy [N.A. Zhukova, D.E. Semenov, I.V. Sorokin, etc. The effect of betulonic acid and [3-oxo-20 (29) -lupen-28-oyl] -3-aminopropionic acid on the liver morphology of mice with RLS lymphosarcoma after polychemotherapy // Bull. exp biol. and honey., 2005, T. 14, No. 9, p. 348-351].

On the other hand, it was reported that hydrogenation of betulin can increase the biological activity compared to that of the starting compound. In particular, it was shown that the anti-HIV activity of 3-O-glutaryl dihydrobetulin 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, v. 14, p. 5851-5853].

The claimed compound is a previously unknown derivative of dihydrobetulonic acid, the creation of which is dictated by the goal of expanding the range of non-toxic means of correcting cytotoxic liver damage that occurs against the background of malignant growth and antitumor therapy.

The inventive compound (1) is obtained by the method shown in scheme 1. As the starting compound, betulin (4) obtained by extraction of birch bark with benzene is used. Hydrogenation of betulin (4) with hydrogen in an autoclave over Ni-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 morpholine 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 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 ¹³ C- ¹ H heteronuclear correlation on direct spin-spin coupling constants (С-C COSY, ¹ J _{C, H} 135 Hz) and two-dimensional and one-dimensional ¹³ C- ¹ H heteronuclear correlation spectra on long-range spin-spin coupling constants (COLOC, LR JMD, ^2.3 J _{C, H} 10 Hz).

The study of the activity of N- [3-oxo-lupano-28-yl] -morpholine (1) included the determination of its antitumor, antimetastatic, and cytoprotective effects upon administration to mice with solid Lewis lung carcinoma. We also studied its effect on the antitumor and antimetastatic effects of polychemotherapy with their combined administration to tumor-bearing mice and the possibility of correcting its cytotoxic effect on normal cells of internal organs not affected by malignant degeneration.

As a reference, the standard ACOP polychemotherapy regimen (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]. 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 ACOP 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). Control animals were tumor. The results were evaluated in dynamics by the percentage of inhibition of tumor growth. It was found that compound (1) has a high antitumor activity comparable to the activity of cytostatic PCT ACP (see Table 1, 2).

Antimetastatic activity (1) was determined in the same animals at the end of the administration period 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 and exhibits a pronounced antimetastatic effect (IMI - 86%) (see Table 3).

In a pathomorphological study using light microscopy, the effect of compound (1) on the liver condition of C57BL / 6 mice with intramuscular Lewis lung carcinoma was determined. 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 (1) on the antitumor and antimetastatic effects of cytostatic polychemotherapy was studied in female mice of the C57BL / 6 line 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 chemotherapy, the animals were administered intragastrically (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 PCT does not have a proliferating effect on Lewis lung carcinoma transplants (see Tables 5, 6). The introduction of compound (1) against a background of PCT enhances its antimetastatic effect (1.6 times), which indicates the absence of stimulation of tumor dissemination under standard polychemotherapy (see table 7).

Hepatoprotective properties (1) were studied in the same animals by pathological examination of the liver by light microscopy. It was revealed that under the influence of (1), administered against the background of PCT, the degree of degenerative and necrotic lesions of the liver decreases.

Thus, the new compound, N- [3-oxo-lupano-28-yl] -morpholine of formula (1), can be considered as a promising remedy with corrective properties for paraneoplastic liver damage and hepatotoxic effects of cytostatic polychemotherapy with antitumor and antimetastatic effects .

The advantages of the claimed compounds include:

- pronounced antitumor and antimetastatic activity;

- the ability to reduce the severity of necrobiotic damage to hepatocytes caused by paraneoplastic processes;

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

- the absence of a stimulating effect on the proliferation and dissemination of the tumor during cytostatic polychemotherapy.

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, 25, p. 229.]). The output of betulin (4) 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., M. Brener, Rey E. // Helv. Chim. Acta, 1941, 24, p. 515-529]. 132 g of betulin (4), 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 (3) 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-one 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 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 (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 ¹ H and ¹³ C NMR spectra 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] -morpholine (1).

(С 0.20). Спектр ЯМР ¹Н (СDСl₃, δ, м.д., J/Гц): 0.69 (д, 3Н, Н²⁹, J=6.8); 0.79 (д, 3Н, Н³⁰, J=6.8); 0.89 (с, 3Н, Н²⁵); 0.90 (с, 3Н, Н²⁷); 0.92 (с, 3Н, Н²⁶); 0.97 (с, 3Н, Н²⁴); 1.02 (с, 3Н, Н²³); 1.25 (дд, 1Н, Н^18a, J_18a,13a=10.8., J_18a,19=10.8); 1.27 (дд, 1Н, Н^5a, J_5а,6а=11.4, J_5а,6е=2.6); 1.65 (дм, 1Н, Н^12e, J_12e,12a=11.0); 1.74 (септет д, 1Н, Н²⁰, 7=6.8, J_20,19=2.6); 1.82 (дд, 1Н, Н^{22α или 22β}, J_22а,22е=12.0, J_22а,21е=6.2); 2.02 (ддд, 1Н, Н^16e, J_16e,16a=13.2, J_16e,15a=3.6, J_16e,15e=3.0); 2.14 (дддд, 1Н, Н¹⁹, J_19,18a=10.8, J_19,21α=10.2, J_19,21β=3.7, J_19,20=2.6); 2.35 (ддд, 1Н, Н^{2a или 2е}, J_2а,2е=15.5, J_2e,1а=7.5, J_2e,1e=4.4); 2.45 (ддд, 1Н, Н^{2a или 2е}, J_2а,2е=15.5, J_2а,1а=9.7, J_2а,1е=70.6); 2.90 (ддд, 1Н, Н^13a, J_13a,12a=12.5, J_13a,18a=10.8, J_13a,12e=3.7); 3.49-3.62 (м, 8Н, Н^2',3',5',6'). Сигналы остальных протонов (12Н) наблюдаются в областях 1.03-1.15 и 1.28-1.49 м.д. Спектр ЯМР ¹³С, δ, м.д.: 39.48 т (С¹), 33.97 т (С²), 217.83 с (С³), 47.15 с (С⁴), 54.90 д (С⁵), 19.48 т (С⁶), 33.64 т (С⁷), 40.47 с (С⁸), 49.78 д (С⁹), 36.74 с (С¹⁰), 21.53 т (С¹¹), 26.99 т (С¹²), 36.55 д (С¹³), 41.89 с (С¹⁴), 29.65 т (С¹⁵), 32.08 т (С¹⁶), 54.74 с (С¹⁷), 52.02 д (С¹⁸), 42.59 д (С¹⁹), 29.61 д (С²⁰), 23.34 т (С²¹), 35.91 т (С²²), 26.44 к (С²³), 20.86 к (С²⁴), 15.77 к (С²⁵), 15.77 к (С²⁶), 14.26 к (С²⁷), 173.69 с (С²⁸), 14.50 к (С²⁹), 22.76 к (С³⁰), 66.82 т (С^3',5'). Сигналы (С^2',6') сильно уширены и наблюдаются в области 45.6 м.д.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 evaporated 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 morpholine is 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 separated by silica gel column chromatography of a 60-200 μm fraction from Merk. Eluent CH ₂ Cl ₂ -MeOH (9: 1). 0.45 g (81%) of the product are isolated with mp. 257-258 ° C. Found (%): C, 77.64; H, 10.46; N, 2.75. C ₃₄ H ₅₅ NO ₃ . Calculated (%): S.77.66; H 10.54; N, 2.66. IR spectrum (KBr), ν / cm ^-1 : 1634 (CON); 1706 (C = O).

(C 0.20). ¹ H NMR spectrum (CDCl ₃ , δ, ppm, J / Hz): 0.69 (d, 3H, H ²⁹ , J = 6.8); 0.79 (d, 3H, H ³⁰ , J = 6.8); 0.89 (s, 3H, H ²⁵ ); 0.90 (s, 3H, H ²⁷ ); 0.92 (s, 3H, H ²⁶ ); 0.97 (s, 3H, H ²⁴ ); 1.02 (s, 3H, H ²³ ); 1.25 (dd, 1H, H ^18a , J _{18a, 13a} = 10.8., J _{18a, 19} = 10.8); 1.27 (dd, 1H, H ^5a , J _{5a, 6a} = 11.4, J _{5a, 6e} = 2.6); 1.65 (dm, 1H, H ^12e , J _{12e, 12a} = 11.0); 1.74 (septet d, 1H, H ²⁰ , 7 = 6.8, J _20.19 = 2.6); 1.82 (dd, 1H, H ^{22α or 22β} , J _{22a, 22e} = 12.0, J _{22a, 21e} = 6.2); 2.02 (ddd, 1H, H ^16e , J _{16e, 16a} = 13.2, J _{16e, 15a} = 3.6, J _{16e, 15e} = 3.0); 2.14 (dddd, 1H, H ¹⁹ , J _19.18a = 10.8, J _19.21α = 10.2, J _19.21β = 3.7, J _19.20 = 2.6); 2.35 (ddd, 1H, H ^{2a or 2e} , J _{2a, 2e} = 15.5, J _{2e, 1a} = 7.5, J _{2e, 1e} = 4.4); 2.45 (ddd, 1H, H ^{2a or 2e} , J _{2a, 2e} = 15.5, J _{2a, 1a} = 9.7, J _{2a, 1e} = 70.6); 2.90 (ddd, 1H, H ^13a , J _{13a, 12a} = 12.5, J _{13a, 18a} = 10.8, J _{13a, 12e} = 3.7); 3.49-3.62 (m, 8H, H ^{2 ', 3', 5 ', 6'} ). The signals of the remaining protons (12H) are observed in the regions of 1.03-1.15 and 1.28-1.49 ppm. ¹³ C NMR spectrum, δ, ppm: 39.48 t (C ¹ ), 33.97 t (C ² ), 217.83 s (C ³ ), 47.15 s (C ⁴ ), 54.90 d (C ⁵ ), 19.48 t ( C ⁶ ), 33.64 t (C ⁷ ), 40.47 s (C ⁸ ), 49.78 d (C ⁹ ), 36.74 s (C ¹⁰ ), 21.53 t (C ¹¹ ), 26.99 t (C ¹² ), 36.55 d (C ¹³ ), 41.89 s (C ¹⁴ ), 29.65 t (C ¹⁵ ), 32.08 t (C ¹⁶ ), 54.74 s (C ¹⁷ ), 52.02 d (C ¹⁸ ), 42.59 d (C ¹⁹ ), 29.61 d (C ²⁰ ), 23.34 t (C ²¹ ), 35.91 t (C ²² ), 26.44 k (C ²³ ), 20.86 k (C ²⁴ ), 15.77 k (C ²⁵ ), 15.77 k (C ²⁶ ), 14.26 k (C ²⁷ ) , 173.69 s (C ²⁸ ), 14.50 k (C ²⁹ ), 22.76 k (C ³⁰ ), 66.82 t (C ^{3 ', 5'} ). The signals (C ^{2 ', 6'} ) are greatly broadened and are observed in the region of 45.6 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] -morpholine (1) was studied in mice of C57BL / 6 female mice weighing 25-30 g, which were injected intramuscularly with a suspension of Lewis lung carcinoma 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). An experimental group of mice was administered intragastrically daily for 8 days (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). Control animals were 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. After the introduction of the agents, the number of surviving animals was determined (as a percentage of their number before the introduction).

Table 1 shows the absolute and relative sizes of the tumor nodes during administration of the agent. The relative values of volumes, expressed as a percentage of their background values in each group, allow more correct comparison of treatment results in the experimental and reference groups, since the background sizes of the tumors (before the introduction of the agent) in them were slightly different. It was shown that during the entire observation period, compound (1) showed a significant antitumor effect, delaying the increase in the volume of tumor transplants relative to the control. In the comparison group (PCT), almost the same dynamics of tumor growth was observed. However, the introduction of (1) did not affect the survival of mice (see table 1).

The data of the SRW index given in table 2, taking into account the revealed differences in the background values, show a high antitumor activity of compound (1) during the entire observation period.

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 on mice of C57B L / 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, 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 _to is the frequency of metastasis in the control group, A is the frequency of metastasis in the experimental group, B _to 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 established that compound (1) reduces the area of tumor metastases in the lungs by 7.2 times relative to the control. The introduction of chemotherapeutic drugs ASOR reduces the number of metastatic lesions 6.3 times and slightly reduces the frequency of metastasis (see table 3). The severity of the antimetastatic effect in the experimental group almost coincides with the reference group, which indicates a high antimetastatic activity of the compound (1).

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

Example 7. The effect of compound (1) on morphological disorders of 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, and after standard histological treatment, its pathomorphological examination was performed by light microscopy.

For histological examination, the liver was fixed in 4% paraform, subjected to standard processing on a histological complex "MICROM". 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.

As a result of pathomorphological analysis in the liver of control animals under conditions of progressive tumor growth, the development of nonspecific 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.

Compared with intact animals, in almost all control animals, a moderately pronounced (hydropic) small focal 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 developed with dystrophic lesion and macrophage infiltration over necrotic changes in hepatocytes against 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, an increase in the area and degree of dystrophic lesion in the form of diffuse small and medium vesicular lipid infiltration, focal hydropic and balloon dystrophy was observed in all animals.

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 in the control, diffuse cellular metastases were localized in sinusoids, and small-focal in both centrolobular and periportal zones.

Thus, the administration of compound (1) at a dose of 50 mg / kg to Lewis carcinoma mice reduces the severity of necrobiotic tissue damage caused by the tumor. The morphological picture in the reference group is characterized by changes associated with increased degenerative and alterative processes, as well as the presence of gross necrobiotic disorders in the structure of the liver.

Example 8. 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. After the introduction of the agents, the number of surviving animals was determined (as a percentage of their number before the introduction).

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. It was noted that under the influence of (1) the relative sizes of the tumor additionally decreased by 1.2-1.4 times as compared with the reference group, but this effect was not statistically significant (see table 4).

The values of the SRW indicator show a noticeable antitumor effect in the experimental and reference groups (see Table 5). Given the differences in the background values, it can be noted that compound (1) is able to slightly enhance the antitumor effect of PCT.

The experimental results indicate that the administration of compound (1) under PCT does not have a proliferating effect on Lewis lung carcinoma transplants. The claimed compounds in the conditions of combined administration with chemotherapeutic drugs revealed a tendency to enhance their antitumor effect. Introduction (1) increased the survival of mice compared to the control and reference groups.

Example 9. 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 _to is the frequency of metastasis in the control group, A is the frequency of metastasis in the experimental group, B _to 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, as well as a slight decrease in the number of mice without metastatic lesions (see Table 5). The introduction of compound (1) on the background of PCT leads to a more significant reduction in the area of metastatic lesions - 10 times, while chemotherapy itself causes only a 6-fold decrease in the rate. The potentiating effect of (1) on the antimetastatic effect of PCT is evidenced by an increase in IMI in the group with combined administration of the agent and antitumor drugs compared with the reference group (91.4 versus 86%).

Thus, the administration of compound (1) against the background of standard polychemotherapy enhances its antimetastatic effect (a 1.6-fold decrease in the area of metastases).

Example 10. The study of the hepatoprotective properties of compound (1) under the conditions of cytostatic polychemotherapy of mice with transplantable Lewis lung carcinoma.

The hepatoprotective properties of compound (1) were studied in C57B L / 6 female mice 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 liver was removed, and pathomorphological examination was performed using light microscopy after standard histological processing.

For histological examination, the liver was fixed in 4% paraform, subjected to standard processing on a histological complex "MICROM". Sections 3-4 μm thick were stained with hematoxylin and eosin, followed by light microscopy examination in transmitted light. The degree of liver damage was evaluated according to the following criteria: the presence of dystrophy (hyaline droplet, fatty, hydropic and balloon), necrosis (monocellular, focal, confluent, bridge-like), their localization. The degree of plethora, cellular infiltration of sinusoids, portal tracts, expansion of sinusoids, bile ducts, metastases, their size, localization were also evaluated.

The development of nonspecific reactive hepatitis was noted in the liver of control animals with an inoculated tumor, 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. 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 sinusode walls 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, all animals showed a slight zonal expansion of the bile ducts (Fig. 1).

The nature of morphological changes in the group corresponds to moderate lesions.

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 degenerative lesion was observed in the form of diffuse small and medium vesicular lipid infiltration, focal hydropic and balloon dystrophy (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 (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, presumably fibrin, were pale pink in color in the lumen of sinusoids (Fig. 2), which is an indicator of trans-sinusoidal metabolism disturbance. 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. According to the totality of pathomorphological signs of liver damage in this group, they correspond to a more severe degree compared to the control group.

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 action of the morpholine derivative, a decrease in the severity of regenerative-plastic insufficiency syndrome was noted: in hepatocytes, single small foci of small-vesicular lipid infiltration and hyaline droplet dystrophy were detected, localized mainly in the centrolobular sections (Fig. 3). Single monocellular necrosis of hepatocytes was localized mainly around small focal metastases.

There was also a restoration of the lumen of sinusoids with predominant diffuse infiltration by their Kupffer cells and a decrease in their lumen of lipid-containing cells (Ito), the appearance of apototic bodies (Councilsilmen) (Fig. 3). Visually, there was no change in the volume of metastatic liver damage compared with the reference group.

Thus, the introduction of compound (1) on the background of polychemotherapy led 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] -morpholine (1) as a tool designed to correct liver damage in malignant growth and cytostatic chemotherapy, has the following advantages:

- pronounced antitumor and antimetastatic activity in conditions of malignant growth;

- the ability to reduce the severity of cytotoxic paraneoplastic syndromes in the liver;

- the ability to reduce the hepatotoxic effect of antitumor chemotherapy and at the same time not to stimulate the proliferation and dissemination of the tumor.

Table 1 Changes in the dynamics of growth of Lewis lung carcinoma and survival of mice under the influence of intragastric administration of compound (1) Group Tumor volume, cm ³ (and in% relative to background values) Survival rate,% Day after chemotherapy 0 (background) four 6 8 8 the control 0.62 ± 0.06 0% 2.05 ± 0.17 230% 3.21 ± 0.17 418% 3.64 ± 0.29 487% 70 PCT 0.79 ± 0.13 0% 1.82 ± 0.28 130% 2.42 ± 0.20 * 206% 2.68 ± 0.18 * 239% 80 (1), 20 mg / kg 0.63 ± 0.07 0% 1.39 ± 0.07 ** 121% 1.75 ± 0.17 *** # 178% 2.20 ± 0.26 ** 249% 70 * P <0.05; ** P <0.001; *** P <0.0001 differences with control are significant # P <0.05 differences with control 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 eleven 24 19 Thus, it was shown that agent (1) has antitumor activity comparable to the activity of cytostatic PCT ACP.

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) 44.43 ± 4.15 *** one hundred 86.1 * P <0.05, *** P <0.001 differences with control are significant; FM - frequency of metastasis; IIM - metastasis inhibition index.

Table 4 Changes in the dynamics of the growth of Lewis lung carcinoma and mouse survival 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) Survival,% of initial Day after chemotherapy 0 (background) four 6 8 8 the control 0.62 ± 0.06 0% 2.05 ± 0.17 230% 3.21 ± 0.17 418% 3.64 ± 0.29 487% 70 PCT 0.79 ± 0.13 0% 1.82 ± 0.28 130% 2.42 ± 0.20 * 206% 2.68 ± 0.18 * 239% 80 PCT + (1) 0.86 ± 0.08 * 0% 1.62 ± 0.19 88% 2.31 ± 0.17 ** 169% 2.83 ± 0.14 * 229% 90 * P <0.05; ** P <0.001; *** P <0.0001 differences with control are significant

Table 5 Lewis lung carcinoma growth inhibition index (TPO) values in mice during the administration of compound (1) with polychemotherapy (PCT) Group SRW,% Day after chemotherapy 0 (background) four 6 8 PCT 0 eleven 25 26 PCT + (1), 20 mg / kg 0 21 28 22

Table 6 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) 32.09 ± 4.95 *** 86 91.4

Claims (1)

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

possessing antitumor and antimetastatic activity and being a corrector of hepatotoxic injuries caused by the paraneoplastic process and cytostatic polychemotherapy.

Images