WO2021086234A1

WO2021086234A1 - Novel chemical compound l-lysine 9-oxoacridinyl-10-acetate

Info

Publication number: WO2021086234A1
Application number: PCT/RU2020/050289
Authority: WO
Inventors: Алексей Леонидович Коваленко; Андрей Юрьевич Петров
Original assignee: Общество С Ограниченной Ответственностью "Научно-Технологическая Фармацевтическая Фирма "Полисан"
Priority date: 2019-10-29
Filing date: 2020-10-22
Publication date: 2021-05-06
Also published as: CN114901643A; EA035914B1; MD20220026A2; EA202000071A1; RU2730530C1; BR112022008046A2; GEP20237566B; ECSP22041864A; CO2022007317A2

Abstract

The invention relates to chemistry, pharmaceuticals and medicine, and specifically to a new chemical compound of the class of derivatives of 9-oxoacridinyl-10-acetate: L-lysine 9-oxoacridinyl-10-acetate, which stimulates production of interleukin-24 (IL-24) and tumor necrosis factor beta (TNF-β) and has anti-tumor activity. The invention may be used in anti-tumor therapy, in particular, as an adjuvant in the complex treatment of oncological diseases. The invention addresses the problem of creating a novel chemical compound which stimulates production of the cytokines interleukin-24 and tumor necrosis factor beta and exhibits anti-tumor activity with low toxicity. The stated problem is solved by a novel chemical compound: L-lysine 9-oxoacridinyl-10-acetate of formula (I) or (II).

Description

[Established by ISA according to rule 37.2] NEW CHEMICAL COMPOUND L-LYSINE 9-OXOACRIDINYL-10-ACETATE

The invention relates to chemistry, pharmaceuticals and medicine, namely, to a new chemical compound from the class of derivatives of 9-oxoacridinyl-10-acetate: L-lysine 9-oxoacridinyl-10-acetate, which stimulates the production of interleukin-24 (IL-24) and tumor necrosis factor beta (TNF-β), and has antitumor activity. The invention can be used in anticancer therapy, in particular, as an adjuvant in the complex treatment of cancer.

Known chemical compounds belonging to the class of derivatives of 9-oxoacridinyl-10-acetates, obtained by directed chemical synthesis and exhibiting various types of biological activity. Considering that 9-oxoacridinyl-10-acetate is slightly soluble in water and exhibits low bioavailability, either special dosage forms in the form of microparticles with a large solubilization surface (RF patent 2545755) or various chemical derivatives with inorganic and organic grounds.

So, for example, for sodium 9-oxoacridinyl-10-acetate, interferonogenic activity was first shown (US patent 3681360, RF patent 2031650, RF patent 2080108) with the claimed antiviral effect (Grossberg SE // Antimicrob Agents Chemother. 1980 Jul; 18 (1 ): 20-26 .; Storch E. // Eur J Immunol. 1982 Sep; 12 (9): 793-796.).

Later, the sodium salt of 9-oxoacridinyl-10-acetate was found to act on estrogen and progestin receptors (RF patent 2126253), as well as to increase sensitivity to androgens (RF patent 2328285), which made it possible to develop drugs based on this molecule for use in gynecology.

For the lithium derivative of 9-oxoacridinyl-10-acetate (RF Patent No. 2202547), similar interferonogenic and antiviral activity was established and, in addition, antimicrobial and antifungal activity, apparently associated with the action of the lithium ion.

Later, to create parenteral drugs and increase the bioavailability and stability during storage by chemical synthesis, a number of 9-oxoacridinyl-10-acetate derivatives, new in chemical structure and manifested biological activity, were obtained: with a group of aliphatic 1-alkylamino-1-deoxypolyols (RF patent 2135474 ), possessing interferonogenic activity and activating populations of T- and B-lymphocytes.

For a derivative of 9-oxoacridinyl-10-acetate with 1-deoxy-N-methylamino-D-glucose (RF patent 2036198), an interferonogenic, antiparasitic and radioprotective effect was also shown, due to the induction of endogenous alpha-interferon.

To obtain derivatives of 9-oxoacridinyl-10-acetate with additional anti-inflammatory, antibacterial and antifungal effects, complex compounds of 9-oxoacridinyl-10-acetate were synthesized with a number of different biologically active amino derivatives of carbohydrates, for example, 2-deoxy-2-amino-D- glucose (chitosamine) (RF patent 2093510) and 3-O- (N, N-dimethylamino-n-propyl) -1,2: 5,6-di-O-isopropylidene-D-hexose (RF patent 2197248, RF patent 2080108, patent RF 2363466, patent RF 2325160, patent EA 005929).

The closest analogue of the claimed compound in terms of chemical structure is 1-deoxy-1-N - [- methyl- (2-acridon-9-one-10-yl-acetate)] - ammonium-D-glucitol, meglumine 9-oxoacridinyl-10- acetate (Eurasian patent 000382), the structural formula of which is shown in FIG. 1, which is an inducer of the prolonged action of endogenous alpha, beta and gamma interferons.

FIG. one

The biological activity of the known compound allows it to be used in the treatment of a wide range of diseases in humans and animals: treatment of therioiditis (RF patent 2306932), oral candidiasis (RF patent 2642053), recurrent vaginal candidiasis (RF patent 2309761), influenza (RF patent 2281097), viral hepatitis in children (RF patent 2154478), arthritis (RF patent 2417091, RF 2169563), purulent-destructive lesions of the mucous membranes and skin (RF patent 2414221, RF 2361626, RF 2295348), onychomycosis (RF patent 2391100), siloadenitis (RF patent 2413507 ), brucellosis (RF patent 2423143), tuberculosis (RF patent 2296996) viral infections (RF patent 2281097, RF 2526253, RF 2538083, RF 2228756, RF 2459629), secondary immunodeficiencies (RF patent 2364396, RF 2320293), choreoretinitis (RF patent 2275933), odontogenic maxillary sinusitis (RF patent 2460528), hyperplastic laryngitis (RF patent 2214249), tonsillitis (RF patent 2357732), psoriasis and neurodermatitis (RF patent 2292215), squamous cell carcinoma (RF patent 2332277), autologous cell transplantation (RF patent 2325934), burns (RF patent 2166960), staphylococcal infection (RF patent 2441656).

However, the property of this compound to stimulate the production of antitumor cytokines interleukin-24 (IL-24) and tumor necrosis factor TNF-β (TNF-β) in the body and its antitumor activity are not known from the available information sources.

The objective of the invention is to create a new chemical compound that stimulates the production of cytokines interleukin-24 and tumor necrosis factor beta and exhibits antitumor activity with low toxicity.

The task is implemented by a new chemical compound: L-lysine 9-oxoacridinyl-10-acetate of the formula:

FIG. 2

Currently, the scientific and technical literature does not describe new promising derivatives of 9-oxoacridinyl-10-acetate with such L-isomers of natural "basic" amino acids such as L-lysine, L-arginine and L-histidine, and their biological activity.

To obtain a number of new compounds of 9-oxoacridinyl-10-acetate and to study their biological activity, the chemical synthesis of new promising derivatives of 9-oxoacridinyl-1-acetate and three amino acids L-arginine, L-glutamine and L-lysine was carried out.

The reaction of 9-oxoacridinyl-10-acetate with L-arginine, which is a strong base (pKa ₃ = 12.48), unexpectedly led to the formation of compounds L- arginine 9-oxoacridinyl-10-acetate with a yield of almost 100%. Low solubility in water (0.01% at 100 ℃) makes this compound potentially unacceptable for creating effective parenteral dosage forms and potentially ineffective when used orally. However, this complex of 9-oxoacridinyl-10-acetate with L-arginine, due to its physicochemical properties, can be used for the isolation and purification of 9-oxoacridinyl-10-acetate.

An attempt to obtain a new derivative of 9-oxoacridinyl-10-acetate using another main natural alpha-amino acid L-histidine c led to a negative result - after the reaction, the starting compounds were found in the dried residue of the reaction mixture without signs of salt formation or the formation of coordination compounds.

The interaction of 9-oxoacridinyl-10-acetate, which is a weak carboxylic acid (pK = 3.7), and the aliphatic amino acid L-Lysine with pronounced base properties (pKa2 = 9.06, pKa3 = 10.54) led to the formation of a true aqueous solution L-lysine of 9-oxoacridinyl-10-acetate, from which pure L-lysine 9-oxoacridinyl-10-acetate was isolated by lyophilization in the form of an amorphous monohydrate. Additional vacuum drying resulted in the formation of a water-crystalline compound.

Thus, the only possible new derivative of 9-oxoacridinyl-10-acetate for all investigated natural "basic" amino acids L-arginine, L-lysine and L-histidine to create an effective drug for parenteral and oral administration was unexpectedly the derivative of an essential amino acid - L β-lysine 9-oxoacridinyl-10-acetate - the claimed chemical compound (hereinafter - "new compound") in this invention.

An example of obtaining L-lysine 9-oxoacridinyl-10-acetate:

0.01 mol of the amino acid L-lysine was dissolved in 100 ml of purified water, heated to 60 ℃, and 2.54 g (0.01 mol) of 9-oxoacridinyl-10-acetate were added in portions. The resulting reaction mass is intensively stirred for 60 minutes until 9-oxoacridinyl-10-acetate is completely dissolved, the resulting solution is filtered through a sterilizing filter (pore diameter 0.22 μm). The filtrate is dried in a laboratory freeze dryer model LP03 for 24 hours to constant weight.

According to the preparation example, the interaction of 9-oxoacridinyl-10-acetate, which is a carboxylic acid (pK = 3.7) and the aliphatic amino acid L-lysine with pronounced base properties (pKa2 = 9.06, pKa3 = 10.54) leads to the formation a true aqueous solution of L-lysine 9-oxoacridinyl-10-acetate, from which pure L-lysine 9-oxoacridinyl-10-acetate monohydrate was isolated by lyophilization. Additional vacuum drying makes it possible to obtain a new chemical compound of the formula shown in FIG. 1, free of crystalline hydrate water. 2.

The physicochemical properties of L-lysine 9-oxoacridinyl-10-acetate are shown in Table 1.

Thus, the good solubility in water and the physiologically acceptable pH value of aqueous solutions (7.0–7.2) of L-lysine 9-oxoacridinyl-10-acetate, in contrast to L-arginine 9-oxoacridinyl-10-acetate, makes it promising for creating a drug and suitable for obtaining true solutions, tablets, creams, ointments, suppositories and other dosage forms.

The study of the safety and biological activity of the new compound in comparison with the prototype compound (hereinafter referred to as the "prototype") was carried out in animal experiments (experiment 1). Keeping animals and carrying out all manipulations with their participation, including euthanasia, were carried out in compliance with national legislation and international GLP rules (Good Laboratory Practice, GOST R-53434-2009 "Principles of Good Laboratory Practice").

A comparative analysis of the safety of the use of the prototype and L-lysine 9-oxoacridinyl-10-acetate established that in terms of mortality, clinical presentation, lethal doses, the new compound is characterized by low toxicity, which is an important advantage in the creation of drugs for anticancer therapy, which implies long courses of treatment.

When studying the biological activity of the new compound (experiments 2, 3), its previously undescribed property was revealed to stimulate the production of cytokines interleukin-24 (IL-24) and tumor necrosis factor beta (TNF-β) in the body.

It is known that the manifestation of antitumor resistance of the organism is mediated by the cytokine system [Floros T. // Semin Oncol. 2015.V.42.N4.P.539-548] and natural killer cells EK (NK) [Li Y. // Chin J Cancer Res. 2018.V30.N2.P173-196 .; Weilei Hu. // Front Immunol. 2019.N10. P.1205.].

Interleukin-24 (IL-24) and tumor necrosis factor TNF-β (TNF-β) are among such significant cytokines that directly regulate apoptosis of tumor cells.

Interleukin-24 is produced by activated macrophages, monocytes and T-lymphocytes [Berezhnaya N.M. // Oncology (Ukraine). 2014.T.16. # 2. S. 130142 .; Rasslian M. // Expert Opin Biol Ther. 2019. V.19. N3. P.211-223 .; Menezes ME. // Adv. Cancer Res. 2018.N138. P.143-182 .; Menezes M.E. // Adv Exp Med Biol. 2014.N818.P.127-53.].

It is believed that IL-24 plays an essential role in the antiviral defense of the body through the mechanisms of apoptosis of the cells of the body infected with the influenza virus [Laengle J. // Antiviral Res. 2015.V. 123.P.93-104 .; Seong R.K. // J Microbiol. 2016. V.54.N10.P.695-700.].

Tumor necrosis factor TNF-β is produced by activated lymphocytes. [Tang H. // Cell Mol Immunol. 2017. V.14. N. 10. P. 809-818.]

TNF-β plays an essential role in the growth, differentiation and induction of apoptosis of tumor cells [Bjordahl R.L. // Curr Opin Immunol. 2013.V.25.N2.P222-229.

It is now known that some biological objects, in particular Pseudomonas aeroginosa, induces IL-24 induction in keratitis in mice [Ross B.X. // J. Immunol. 2017.S.1.V.198.N9.P.3536-3547.]. It was previously found that phytohemmaglutenin and lipopolysaccharides also induce IL-24 induction in peripheral blood mononuclear cells [Poindexter N.J. // J. Leukoc Biol. 2005. V.78. N3. P.745-752 .; Garn H. // Immunobiology. 2002.V.205. N3. P321-334.] And TNF-beta in T-lymphocytes of Ranneh Y. // Arch Immunol Ther Exp (Warsz). 2019.V 5.N.10. P.1007.]

Analysis of information sources showed that no chemical compounds from the class of derivatives of 9-oxoacridinyl-10-acetate: L-lysine 9-oxoacridinyl-10-acetate, which cause the simultaneous synthesis of both IL-24 and TNF-β, are currently known.

The results of studying the new compound in experiments 1-5 indicate its high antitumor activity and safety.

Experiment 6 showed a significant potentiating effect of the new compound as part of standard chemotherapy with cytostatics.

Experience 7 proves the antitumor and radiosensitizing effect of the new compound in combination with radiation therapy.

The results obtained in these experiments confirm the effectiveness of the use of the new compound as an adjuvant in the complex treatment of oncological diseases.

Thus, the new chemical compound stimulates the production of cytokines interleukin-24 and tumor necrosis factor-beta and has antitumor activity with low toxicity.

Experience 1. Comparative assessment of the safety (acute toxicity) of the prototype and the new compound with a single administration.

The study was conducted on 195 male albino laboratory mice (SHR). The choice of the animal species is due to the fact that laboratory albino SHR mice are a standard object of toxicological and pharmacological studies, their physiological and biochemical characteristics, neurological characteristics, behavioral and nutritional characteristics are comparable to humans, which makes the choice of such a test system optimal for solving the problems of this study.

The following chemical compounds were studied as test objects:

1.meglumine 9-oxoacridinyl-10-acetate (prototype),

2. L-lysine 9-oxoacridinyl-10-acetate (new compound).

The study compounds were administered to animals once intravenously, slowly, using an infusion pump, into the tail veins. The total volume of administration was 0.5-0.51 ml, bringing to the required volume was carried out by diluting the compounds in 0.9% sodium chloride solution.

The following doses of test compounds were used in the study: 0 mg / kg (control) 25 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg, 400 mg / kg, 600 mg / kg, 800 mg / kg , 1000 mg / kg, 1200 mg / kg, 1400 mg / kg, 1600 mg / kg, 1800 mg / kg, 2000 mg / kg, body weight of the animal.

The results of the experiment were processed by the least squares method using probit analysis in the program "GraphPadPrism 7.0"

The results of the influence of the prototype and the new compound on the lethality of animals are shown in Table 2.

The lethality of the animals occurred within 24 - 72 hours after the administration of the studied chemical compounds, depending on the dose. The clinical picture that preceded death was characterized by a drop in body weight in relation to the background data, pallor of the skin and cyanosis of the extremities, adynamia followed by muscle relaxation, and rapid breathing. The death of animals in all cases was due to the development of acute cardiovascular failure.

In general, when using the prototype in the dose range of 25 - 800 mg / kg and the new compound in the dose range of 25 - 1200 mg / kg, the state of the animals during 14 days of observation corresponded to those of healthy control animals. No deviations in the functioning of the life support systems and the cognitive sphere of the mice were recorded.

Deviations from the norm, consisting in the presence of a tousled coat and a decrease in response to stimuli, were observed in some of the animals from the groups receiving the prototype at doses over 800 mg / kg, and in the groups receiving the new compound over 1200 mg / kg during the first 10 those days of observation. There was an increase in the frequency of respiratory movements (up to 200%), dysfunction of the nervous system of mice, consisting in inhibition of locomotor activity, lethargy and a decrease in muscle tone when picked up. The above violations were transient, were observed in the first 2-3 hours after the administration of the compounds, resolved on their own and had no consequences during the observation period.

The calculation of lethal doses of the studied compounds showed a significant difference in toxicological parameters: LD _{50 of the} prototype and the new compound were 769.9 ± 74.0 and 1223.6 ± 181.4 mg / kg, respectively.

Thus, according to the key indicator LD _{50, the} toxicity of the new compound is 1.5 times lower than that of the compound according to the prototype.

To study the antitumor activity of the prototype and the new compound, the effect of their effect on the induction of IL-24 and tumor necrosis factor-beta (TNF-β) in healthy animals was investigated.

Experience 2. The influence of the prototype and the new compound on the production of IL-24 in healthy animals.

The study was conducted on 168 male albino laboratory mice. Analysis of the scientific literature indicates the possibility of determining IL-24 in the blood of animals of this species.

Compounds were investigated as test objects:

1. meglumine 9-oxoacridinyl-10-acetate (prototype);

2. L-lysine 9-oxoacridinyl-10-acetate (new compound).

Statistical processing was performed using the SPSS for Windows software package. The character of the data distribution was checked by calculating the Kolmogorov-Smirnov criterion. Comparison of mean data of independent samples using the Mann-Whitney U-test. Comparison of the mean data of dependent samples was carried out using the Friedman test. A significant level of differences was considered a probability of at least 95% (p <0.05), which is the standard in biomedical research.

The introduction of the prototype and the new compound to animals was carried out by intravenous injection into the tail veins. Were investigated the following doses of 10, 100 and 200 mg / kg of body weight of the animal. The assessment of the level of IL-24 in the blood of animals and the parameters of the blood system was performed 1, 3, 6, 10, 11 days after the start of the experiment.

Blood was taken in disposable syringe systems with an anticoagulant (EDTA), by a manual method, from the tail veins of the animal. After the end of the procedure for taking blood, it was processed for an enzyme-linked immunosorbent assay.

The method for studying the level of IL-24 in the blood is an enzyme-linked immunosorbent assay (ELISA, ELISA Kit for Interleukin 24 (IL24), according to the instructions of the manufacturer Cloud-Clone Corp.).

It was found that the prototype and the new compound at doses of 10 mg / kg, 100 mg / kg and 200 mg / kg of body weight per day significantly increased the production of interleukin-24 (IL-24) in the body of healthy animals. An increase in IL-24 production with both compounds was noted as early as 24 hours after the start of the experiment.

The highest values of the content of IL-24 in the blood of animals were noted 72 hours after the first administration of the compounds at all studied doses. The maximum values of the concentration of IL-24 in the blood of mice during this observation period were recorded in both groups of animals that received a dose of 200 mg / kg.

At the same time, with the introduction of L-lysine 9-oxoacridinyl-10-acetate on the third day of the experiment, the content of IL-24 in the blood of mice was statistically significantly higher than in the groups of animals receiving the prototype by 74% (p = 0.016), 89% (p = 0.004) and 58% (p-0.037), respectively. The results are presented in table. four.

Thus, the ability of both the prototype and the new compound to stimulate the production of IL-24 was established for the first time. At the same time, the significant advantage of L-lysine 9-oxoacridinyl-10-acetate over the prototype in terms of the effect on the production of IL-24 was experimentally confirmed. Thus, the new compound has a pronounced and intense effect on the production of IL-24, which consists in the earlier formation of high concentrations of this cytokine in the blood of animals, as well as in the higher absolute and average values of this indicator for 1-3 days in comparison with the prototype.

Experience 3. The influence of the prototype and the new compound on the production of tumor necrosis factor-beta (TNF-β) in healthy animals.

The study was conducted on 36 male albino laboratory mice. Analysis of the scientific literature indicates the possibility of determining TNF-β in the blood of animals of this species.

The following chemical compounds were studied as test objects:

1.meglumine 9-oxoacridinyl-10-acetate (prototype),

2. L-lysine 9-oxoacridinyl-10-acetate (new compound).

The introduction of the prototype and L-lysine 9-oxoacridinyl-10-acetate to animals was carried out in the tail veins. The test compounds were administered to mice once a day for 10 (ten) days of the experiment at a dose of 100 mg / kg of animal body weight.

The level of TNF-β in the blood of animals and the parameters of the blood system were assessed 1, 3, 6, 10, 11 days after the start of the experiment. Blood was taken in disposable syringe systems with an anticoagulant (EDTA), by a manual method, from the tail veins of the animal. After the end of the procedure for taking blood, it was processed for an enzyme-linked immunosorbent assay. The TNF-β enzyme immunoassay was performed according to the instructions of the manufacturer Cloud-Clone Corp.

Parenteral administration of the prototype and the new compound at a dose of 100 mg / kg of body weight per day for 10 days caused an increase in the production of TNF-β in the body of healthy animals in the first 5 days from the start of the course of administration. The highest levels of TNF-β in the blood of animals were observed 72 hours after the first administration of the test compounds.

24 hours after the first administration of the test compounds, the level of TNF-β in the blood of mice receiving L-lysine 9-oxoacridinyl-10-acetate was significantly higher than that in the group of mice receiving the prototype - by an average of 14% (p = 0.150).

72 hours after the first administration of the test compounds, the maximum level of TNF-β in the blood of mice in the experimental groups was recorded, which significantly differed from the control values. At the same time, with the introduction of L-lysine 9-oxoacridinyl-10-acetate, the content of TNF-β in the blood of mice was statistically significantly higher than that in animals receiving the prototype, on average by 24% (p = 0.025), respectively. The results are shown in Table 5.

As a result, significant advantages of the new compound in comparison with the prototype were experimentally established in relation to its effect on the production of TNF-β, which reflects a faster rise in the concentration of this cytokine in the blood of animals, higher absolute and average values of this indicator for 1-3 days compared to the prototype. ...

Thus, the established fact of simultaneous stimulation of the production of IL-24 and TNF-β using a new chemical compound is a new property of L-lysine 9-oxoacridinyl-10-acetate and the basis for the study of its antitumor activity.

To establish the effect of L-lysine 9-oxoacridinyl-10-acetate on the growth and development of primary malignant neoplasms, experiments were carried out to establish its effect on the development of the C6 glioma cell line (experiment 4) and on experimental animals on the development of metastases of Lewis lung epidermoid carcinoma (experiment five).

Experiment 4. Influence of the new compound on the growth and development of primary malignant neoplasms of the brain on the C6 glioma cell line.

To simulate the tumor process, a C6 glioma cell line obtained from the Shared Use Center of the Institute of Cytology of the Russian Academy of Sciences was used. The choice of the test system is due to its high biological equivalence to oncological diseases in humans (primary malignant neoplasms of the brain).

Reproduction of the neoplasm was performed in a conventional manner, by cultivation under controlled conditions. For a parallel assessment of the effect of the studied compounds on unchanged tissues, a cell line of untransformed human fibroblasts of the DF-2 line was used.

L-lysine 9-oxoacridinyl-10-acetate was added to the culture medium at doses of 1, 5, 10, and 100 μg / ml of the medium.

The following indicators of neoplasm development were assessed:

- Proliferation - cells were cultured in the presence of a prototype and a new compound, and after 48 hours their number was counted using a standard method.

- Colony formation: cells were plated into 6-well culture plates at a concentration of 250 cells per well and cultured under standard conditions. After 12 hours, test compounds were added to the cells. After 4 days, the resulting cell colonies were fixed, stained and counted. The analysis of the effect of the test compounds on proliferation was carried out on the basis of changes in the number of colonies.

- Cell death: the analysis of the number of dead cells is carried out by assessing the amount of the enzyme lactate dehydrogenase in the culture medium. When cells die, this enzyme enters the medium and its amount is a differential characteristic of the number of dead cells.

- Metabolism (MTT test): The MTT test is the most accessible and commonly used method for the analysis of cellular metabolism. Based on enzymatic reduction of non-colored tetrazolium salt (MTT) in living metabolically active cells.

The results of studies of the effect of the new compound on the proliferative activity of cells of the experimental C6 glioma revealed the absence of inhibition of colony formation at doses of 1 and 5 μg / ml of the medium and the ability to inhibit the estimated indicator at doses of 10 and 100 μg / ml of the medium - there was a pronounced positive dose dependence in relation to the inhibition of colony formation (Table . 6).

At the same time, the analysis of the effect of L-lysine 9-oxoacridinyl-10-acetate on the proliferative activity of untransformed fibroblasts of the DF-2 line did not reveal a negative effect on this indicator of cell development. Even at the maximum dose (100 μg / kg), the decrease in the degree of colony formation of non-transformed fibroblasts did not reach a statistically significant level (Table 7).

In addition, the clinically and statistically significant effect of the new compound on the rate of cell death in experimental C6 glioma after exposure for 24 hours was experimentally proved. In the cell populations of the experimental C6 glioma, which developed on the background of the use of L-lysine 9-oxoacridinyl-10-acetate at a maximum dose of 100 μg / ml of the medium, the level of cell lethality was significantly higher compared to the control values (p <0.001) (Table 8). ).

The results of the analysis of the effect of the new compound on the ability of experimental C6 glioma cells to survive by assessing the level of metabolic activity after exposure for 24 hours are shown in Table 9. The maximum, statistically significant inhibition of cell activity was observed when using high L-lysine 9-oxoacridinyl-10- acetate at a dose of 100 μg / ml of medium.

Thus, the new compound causes a statistically and clinically significant decrease in the rate of development of the C6 glioma cell line - it inhibits the proliferative and migratory activity of neoplastic cells 4 times higher compared to the control, reduces the metabolic rate and accelerates the processes of cell death. At the same time, no significant negative impact on the development of cell culture of non-transformed fibroblasts was noted, which indicates not only a high antitumor activity, but also the safety of the new compound.

Experience 5. Influence of a new compound on the development of metastases of Lewis lung epidermoid carcinoma.

The study was carried out on 22 male laboratory mice, line C57Bl6. Analysis of the scientific literature indicates the possibility of transplantation of Lewis carcinoma in animals of this species and line. Epidermoid carcinoma of the lung of Lewis, a solid transplantable (transplantable) neoplasm, was transplanted to experimental animals in a volume of 2 * 10 ⁶ cells / mouse, subcutaneously in the region of the last third of the right side.

Keeping animals and carrying out all manipulations with their participation, including euthanasia, were carried out in compliance with national legislation and international GLP rules (Good Laboratory Practice, GOST R-53434-2009 "Principles of Good Laboratory Practice").

The introduction of L-lysine 9-oxoacridinyl-10-acetate to animals was carried out into the tail veins, once a day, for 5 days at doses of 100 mg / kg of animal body weight.

Research method: morphological analysis of autopsy material (tissue).

The formula for calculating the metastatic inhibition index (IMI):

IMI (%) = (M _k - M _e / M _k ) x 100%,

where, M _to - the average number of metastases in the control group

M _e - the average number of metastases in the experimental group.

The results of the effect of the new compound on the inhibition of metastasis in animals are shown in Table 10.

Parenteral administration of L-lysine 9-oxoacridinyl-10-acetate at a dose of 100 mg / kg of body weight per day caused a clinically significant decrease in the intensity of metastasis. On the 14th day from the moment of neoplasm transplantation, the average number of metastases in the lungs in mice treated with L-lysine 9-oxoacridinyl-10-acetate was significantly lower than in the control group of animals, exceeding the clinically significant level of inhibition of metastasis (more than 25%).

Thus, the new compound has a significant effect on the process of metastasis and reduces the number of metastases by 47.5% compared to the control group of animals.

To evaluate the effectiveness of the use of L-lysine 9-oxoacridinyl-10-acetate as an adjuvant in antitumor therapy in conjunction with chemotherapy and radiation therapy, a number of experiments were carried out on its combined use with various chemotherapy drugs and radiation (experiments 6-7).

Experiment 6. Influence of a new compound on the development of C6 glioma during extracarnial transplantation in conjunction with standard chemotherapy in an animal experiment.

The study was carried out on 84 male albino gray rats. To simulate the tumor process, we used the C6 glioma strain obtained from the Center for Collective Use of the Institute of Cytology, Russian Academy of Sciences. The choice of the test system is due to its satisfactory rate of development in the animal body, as well as its high biological equivalence to oncological diseases in humans (primary malignant neoplasms of the brain).

Reproduction of the neoplasm was performed in a conventional manner, by direct transplantation of malignant cells obtained from tumor-bearing animals. C6 glioma development was investigated by subcutaneous transplantation to the lateral surface of the right femur in a volume of 10 ⁶ cells / animal.

The standard chemotherapy used was cytostatics with satisfactory efficacy against the modeled disease in the following dosage regimens:

- doxorubicin: 2 mg / kg body weight on days 2, 5 and 8 after tumor transplantation;

- cyclophosphamide: 35 mg / kg body weight on the 2nd day after tumor transplantation.

Intravenous administration of L-lysine 9-oxoacridinyl-10-acetate was performed at a dose of 100 mg / kg of animal body weight daily for 5 days.

To study the effect of 9-oxoacridinyl-10-acetate on the development of C6 glioma, the following experimental groups were identified:

Group 1 - animals with C6 glioma treated with 0.9% sodium chloride (Control), Group 2 - animals with C6 glioma treated with doxorubicin, 2 mg / kg,

Group 3 - animals with C6 glioma treated with Cyclophosphamide, 35 mg / kg,

Group 4 - animals with C6 glioma treated with L-lysine 9-oxoacridinyl-10-acetate, 100 mg / kg

Group 5 - animals with C6 glioma treated with doxorubicin, 2 mg / kg, L-lysine 9-oxoacridinyl-10-acetate, 100 mg / kg,

Group 6 - animals with C6 glioma treated with cyclophosphamide, 35 mg / kg, L-lysine 9-oxoacridinyl-10-acetate, 100 mg / kg.

In the study groups, the following indicators of neoplasm development were assessed:

- Average life expectancy (ALE) of animals (days).

- Increased life expectancy, according to the formula:

DL (%) = [(ALE (experience) - ALE (control)) / ALE (control)] × 100%.

- Clinically significant level of life expectancy> 25%.

- Tumor growth inhibition (TPO) according to the formula:

TPO (%) = [(Vcontrol-Vexperiment) / Vcontrol] × 100%, where V is the tumor volume in cm ³ .

- Clinically significant TPO level> 50%.

For this, the volume of the tumor node was preliminary estimated (change in the volume of the node at the control points of the study; three mutually perpendicular dimensions - a × b × c, cm ³ ).

Cytostatic treatment with standard cytostatics used in the treatment of C6 glioma (docosrubicin, cyclophosphamide) contributed to a statistically significant increase in the average life expectancy of animals with a tumor by 39.4% and 42.6%, respectively (p <0.05).

The use of chemotherapy using a new chemical compound also had an inhibitory effect on the rate of development of the tumor process, which was reflected in an increase in the lifespan of rats with C6 glioma by 28.9% (p <0.05), reaching a clinically significant level (more than 25%).

At the same time, the use of standard chemotherapy with doxorubicin or cyclophosphamide in combination with L-lysine 9-oxoacridinyl-10-acetate led to a significant increase in the life expectancy of animals (Table 11).

Chemotherapy with doxorubicin and cyclophosphamide was also effective in inhibiting the growth of the primary tumor node of C6 glioma during the first 2-3 weeks of the experiment (Table 12). Subsequently, a weakening of the pharmacological action of cytostatics was observed and, starting from the 30th day of the experiment, no statistically significant differences from the indicators of the control group were recorded.

Monotherapy of C6 glioma with a new compound at a dose of 100 mg / kg of animal body weight was unexpectedly also highly effective and comparable in most control points of the study with the indicators in the groups of rats receiving only standard cytostatics.

Adjuvant (joint) use of L-lysine 9-oxoacridinyl-10-acetate and standard chemotherapy has established a higher efficacy of therapy. Thus, in the groups of animals receiving the combined treatment, the lowest rates of increase in the volume of the tumor mass were recorded (Table 12), and on the 30th day of the experiment, statistically significant differences were observed in the evaluated trait not only with the control values, but also with the indices in the groups of rats. who received standard chemotherapy.

Thus, the use of a new compound causes a statistically significant decrease in the growth and development of experimental C6 glioma, which is manifested in a decrease in the growth dynamics of the primary tumor node and, as a consequence, in an increase in the lifespan of rats with C6 glioma by 28.9% compared to the control.

In addition, the use of a new compound in combination with docosrubicin and cyclophosphamide leads to a significant increase in the life expectancy of animals, which proves the effectiveness of complex therapy for C6 glioma.

Experiment 7. Comparative analysis of the radiosensitizing effect of the new compound during experimental radiation therapy of malignant neoplasms in a model of metastatic colorectal cancer in BALB / c mice.

The study was carried out on 104 male BALB / c mice; to simulate the tumor process, a colon adenocarcinoma strain obtained from the Shared Use Center of the Institute of Cytology of the Russian Academy of Sciences was used. The choice of the test system is due to its satisfactory rate of development and distribution in the animal body with the involvement of various organs and systems in the pathological process, as well as high biological equivalence to oncological diseases in humans (colorectal cancer).

Reproduction of the neoplasm was performed in a conventional manner, by direct transplantation of malignant cells obtained from animal tumor-bearing animals, in a volume of 0.2 ml of suspension of the tumor mass in 0.9% sodium chloride solution in a ratio of 1:10, subcutaneously into the right side area.

L-lysine 9-oxoacridinyl-10-acetate was used as an experimental chemotherapy against the background of radiation therapy.

4 experimental groups were identified:

Group 1 - animals to which adenocarcinoma of the colon was transplanted (n = 26).

Group 2 - animals that were transplanted with adenocarcinoma of the colon and received radiation therapy on the 5th day after transplantation (n = 26).

Group 3 - animals that were transplanted with adenocarcinoma of the colon and, starting from day 2, for 3 days from the beginning of the experiment, L-lysine 9-oxoacridinyl-10-acetate was administered (n = 26).

Group 4 - animals that were transplanted with adenocarcinoma of the colon and, starting from day 2, for 3 days from the beginning of the experiment, L-lysine 9-oxoacridinyl-10-acetate was injected and radiation therapy was carried out on day 5 (n = 26).

Each group of mice was divided into 2 subgroups, which evaluated:

1. life expectancy of mice and dynamics of growth of the primary tumor node (n = 6);

2.the growth of the tumor node and the dynamics of metastasis at the control points of the study (n = 20; 10 + 5 + 5).

Radiation therapy was performed on a standard gamma device PX-gamma-30, VO "Isotope". The radiation dose was 2 Gy.

The introduction of L-lysine 9-oxoacridinyl-10-acetate was carried out intravenously, in a solution of 0.9% sodium chloride at a dose of 100 mg / kg of animal body weight.

The following indicators of neoplasm development were assessed:

1. Inhibition of tumor growth (TPO), according to the formula:

SRW (%) = [(Vк-Vо) / Vк] × 100%,

where Vk is the tumor volume in the control group in cm ³ .

V is the volume of the tumor in the experimental group in cm ³ .

For this, the volume of the tumor node was preliminary estimated (change in the volume of the node at the control points of the study; three mutually perpendicular dimensions - a × b × c; cm ³ ),

Clinically significant TPO level> 50%.

2. Average life expectancy (ALE) of animals (days).

3. Increased life expectancy (life expectancy,%), clinically significant level of life expectancy> 25%.

4. Frequency of tumor metastasis (by organs) - the percentage of animals with metastases in relation to the total number of animals in the group.

5. Average number of metastases per animal in each group (by organs).

6. Index of inhibition of metastasis (IMI), according to the formula:

IMI (%) = [(Ak × Vk) - (A × V) / Ak × Vk] × 100%,

where, A is the frequency of metastasis in animals of the experimental group

Ak - the frequency of metastasis in animals of the control group

B - the average number of metastases in one animal in the experimental group.

Bk - the average number of metastases in one animal in the control group

Clinically significant level> 75%.

The control points of the study were the 14th, 28th and 42nd days from the moment of tumor transplantation. During the indicated periods, complete necropsy of the animal was performed and the size of the tumor and its prevalence in the body were assessed. Macroscopically altered lymph nodes and the liver were studied using a binocular magnifying glass (tenfold magnification) and, if there were grounds, histological analysis was carried out using a conventional method, with the preparation of microscope preparations and staining with hematoxylin-eosin in order to verify neoplasm metastases.

Statistical processing was performed using the SPSS for Windows software package. Data are presented as M ± m (arithmetic mean ± arithmetic mean error). The character of the data distribution was checked by calculating the Kolmogorov-Smirnov criterion. Comparison of the mean values of the quantitative data of independent samples was performed using the Student's t-test. Analysis of qualitative data was carried out by calculating the ^{Pearson χ 2} test. The exact confidence intervals (CIs) for the shares were calculated by the Clopper-Pearson method using the freely available Confint software. A significant level of differences was considered a probability of at least 95% (p <0.05), which is the standard in biomedical research.

With the above volume of transplanted tumor material, a neoplasm developed in all animals included in the study. At the same time, the time of detection of the primary tumor node in the experimental groups differed significantly. In general, colon adenocarcinoma was characterized by relatively slow tumor development, the dynamics of neoplasm growth is shown in Table 13.

Radiation therapy for tumors in mice was clinically effective in the early stages of colon adenocarcinoma growth. On the 14th day of the experiment in the group of animals treated with RT, a statistically significant inhibition of tumor growth was observed to 54% (p = 0.044).

At the same time, it was found that the effect of a single gamma-irradiation on the used model of tumor growth has a short-term character - the slowdown in the development of AKATOL under the influence of ionizing radiation was practically not traced in the long-term observation period on days 28 and 42 (Table 13).

Chemotherapy using the new compound had little effect on the tumor model compared to radiation therapy. However, radiation therapy against the background of the use of L-lysine 9-oxoacridinyl-10-acetate unexpectedly showed a significant and prolonged effect on the dynamics of tumor growth: on the 14th day, and on the 28th day, there was a significant decrease in the volume of the primary tumor node compared with a group of animals that did not receive treatment (Table 13). So, on the 14th day, the effect of inhibiting the development of neoplasms reached 62%, while there was no development of neoplasms at the level of the palpable node. On the 28th day of the study, the average tumor volume in animals of this group was statistically significantly different not only from the control values, but also from the parameters in the groups of mice receiving only radiation therapy; the inhibition of tumor node growth during this time period was 73%.

The high efficiency of the use of the new compound in combination with radiation therapy was reflected in the integral indicator of the therapeutic action - an increase in the average life span of animals (Table 14).

A single separate use of gamma radiation and a new compound in the treatment of colon adenocarcinoma practically does not have a significant effect on increasing the life expectancy of animals and is 7% and 3%, respectively.

At the same time, the combined use of gamma radiation and a new compound is therapeutically effective due to the radiosensitizing effect of L-lysine 9-oxoacridinyl-10-acetate, which is confirmed by the inhibition of the growth of the primary tumor node of experimental colon adenocarcinoma by 73% and an increase in the life expectancy of animals by 38 % compared with the control group (Table 14).

Thus, the results of the experiments confirm the effectiveness of the use of the new compound as an adjuvant in the combination therapy of oncological diseases.

Claims

The new chemical compound L-lysine 9-oxoacridinyl-10-acetate of the formula:

,
stimulating the production of interleukin-24 and tumor necrosis factor - beta.
A compound according to claim 1 exhibiting antitumor activity.
The use of a compound according to claim 1 as an adjuvant in the combination therapy of cancer.