2-aminium-7-(diethylamino)-4-(4-methoxybenzo[d][1,3]dioxol-5-yl)-4h-chromene-3-carbonitrile n-acetyl-2-aminopentadioate, exhibiting antitumor activity

Abstract

FIELD: biologically active compounds.SUBSTANCE: 2-aminium-7-(diethylamino)-4-(4-methoxybenzo[d][1,3]dioxol-5-yl)-4H-chromene-3-carbonitrile N-acetyl-2-aminopentadiate (compound 1) is proposed.EFFECT: proposed compound exhibits antitumor activity.1 cl, 3 tbl, 4 ex

Description

[0001] Application area

[0002] The invention relates to medicine, to biologically active compounds, specifically to 2-aminium-7-(diethylamino)-4-(4-methoxybenzo[d][1,3]dioxol-5-yl)-4H-chromene-3-carbonitrile N-acetyl-2-aminopentadiate (compound 1), which exhibits antitumor activity.

[0003]

[0004] Technique level.

[0005] Currently used cancer treatments have a number of undesirable side effects and are also toxic. The development of low-toxicity, effective antitumor drugs that target the target organ and inhibit metastasis is urgently needed. A number of publications have been devoted to various 4-H-aminochromene derivatives.

[0006] In patent US 20050154015 A1 (2005) "Substituted 4H-chromenes and their analogs as caspase activators and apoptosis inducers and their use." More than 100 chromene analogs were synthesized and their antitumor activity was studied on two cancer cell lines (T47D, ZR-75-1 - breast cancer - ductal carcinoma), caspase activators and apoptosis accelerators. In the article Shestopalov AM, Litvinov YM, Rodinovskaya LA, Malyshev OR, Semenova MN, Semenov VV. Polyalkoxy substituted 4H-chromenes: synthesis by domino reaction and anticancer activity. ACS Comb Sci. 2012 Aug 13;14(8):484-90. doi: 10.1021 / co300062e. (2012) A series of 4H-chromenes were synthesized and it was shown that polyalkoxy-substituted 4H-chromenes may be promising for the further development of anticancer drugs. Compound 1 has not been described in available publications.

[0007] Disclosure of invention.

[0008] The aim of the invention is to synthesize a new compound 2-aminium-7-(diethylamino)-4-(4-methoxybenzo[d][1,3]dioxol-5-yl)-4H-chromene-3-carbonitrile N-acetyl-2-aminopentanedioate (compound 1), which exhibits antitumor activity, and to create a medicinal product with antitumor activity based on it.

[0009] The technical result is achieved by proposing a method for synthesizing compound 1 and conducting studies of its biological activity.

[0010] Example 1. Synthesis of 2-aminium-7-(diethylamino)-4-(4-methoxybenzo[d][1,3]dioxol-5-yl)-4H-chromene-3-carbonitrile NN-acetyl-2-aminopentanedioate.

[0011] 40.0 ml of isopropanol-2, 5.502 g (0.03 m) of 3-methoxy-4,5-methylenedioxybenzaldehyde, 1.995 g (0.03 m) of malonodinitrile, 4.950 g (0.03 m) of 3-diethylaminophenol and 1.005 g (0.0075 m) of triethylamine are loaded into a three-necked flask equipped with a stirrer, thermometer and reflux condenser. The mixture is heated to 80°C with stirring, boiled for 10-15 minutes, cooled with stirring to room temperature (20-25°C), crystallized at 2-3°C. The precipitate is filtered, washed with isopropanol-2, dried to constant weight. The resulting product is recrystallized from isopropanol-2 and crystallized at 0-+5°C. 5.98 g of 2-aminochromene are obtained, a crystalline powder of orange-yellow color, T ПЛ.=137-138°C. 4.96 g (0.0125 m) of 2-aminochromene and 2.36 g (0.0125 m) of N-acetyl-2-aminopentanoic acid are loaded into a homogenizer and the mass is thoroughly homogenized for 10-15 minutes. 60 ml of isopropanol-2 and the homogenized mixture of reagents are loaded into a flask equipped with a stirrer, thermometer and reflux condenser. The mass is heated to boiling with stirring for 15-20 minutes, filtered, isopropanol-2 is distilled off at 30 mm Hg in an amount of 2 / 3 of the loaded volume. The residue is crystallized at 5-8°C for 6-8 hours. The precipitate is washed with cooled isopropanol-2 and dried to constant weight. 6.83 g of almost white with a yellowish-orange tint crystalline powder are obtained, T ПЛ⋅ =152-153°С.

[0012] Found, %: C 59.69; H 5.98; N 9.53 C 29 H 34 N4O9

[0013] Calculated, %: C 59.79; H 5.88; N 9.62; About 24.71

[0014] IR spectrum, v, cm -1 : 3310, 3280 (NH), 2210 (CH); 2190 (CN), 1610,1630 (C=C), 1555, 1535 (NHCO).

[0015] TLC: in the n-butanol-acetic acid-water (4:15:10) system, one spot with R is observed in the main adsorption zone f 0.45.

[0016] The toxicity and pharmacological activity studies of compound 1 are presented in the following examples.

[0017] Example 2. Acute toxicity of compound 1 and the antitumor drug cyclophosphamide.

[0018] In a comparative experiment, acute toxicity was studied in experiments on white outbred mice (40 individuals of both sexes weighing 19-22 g, obtained from the nursery of the Andreevka branch of the Federal State Budgetary Scientific Institution Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency of Russia) with intragastric administration of compound 1 and the official solid dosage form of the prototype in a 1% solution of Tween-80 in a volume of 20 ml / kg in increasing concentrations. The LD50 value was calculated using the Litchfield-Wilcoxon method (Book E.V. Arzamastsev, I.V. Berezovskaya, O.V. Verstakova et al. Methodological recommendations for studying the general toxic effect of drugs / Guidelines for conducting preclinical studies of drugs. Part one. - Moscow: Grif i K, 2012. - pp. 25-35; Guskova T.A. Toxicology of drugs. - Moscow: Publishing house "Russian doctor". - 2003. - pp. 20-24.). The results of the study of compound 1, as well as the prototype cyclophosphamide, are presented in Table 1.

[0019] The data presented in Table 1 indicate that the claimed compound 1 is more than ten times less toxic than the prototype cyclophosphamide. The 2-aminochromene derivative compound 1 belongs to class 4 (non-toxic substances) of the toxicity classification of chemicals in accordance with GOST 12.1.007 - 76 (Berezovskaya I.V. Classification of chemical substances by acute toxicity parameters for parenteral administration / / Chemical and pharmaceutical journal. - 2003. - Vol. 37, No. 3. - Pp. 32-34).

[0020] Example 3. The antitumor activity of compound 1 was studied in a syngeneic Lewis lung carcinoma tumor model in mice.

[0021] The experiment was performed on 30 mice of both sexes (15 individuals of each sex) of the C57 B1 / 6 line obtained from the laboratory animal nursery - a branch of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Pushchino) with intragastric administration of compound 1 and cyclophosphamide (prototype) at a dose of 5% of the LD50 indicator for this route of administration, in a 1% Tween-80 solution in a volume of 20 ml / kg, 72 hours after tumor inoculation for 5 days. Antitumor activity was studied on a syngeneic tumor system - Lewis lung carcinoma (LLC) from the tumor strain bank of the N.N. Sechenov National Research Medical Center of Oncology. Blokhin National Research Center for Oncology and Microbiology of the Russian Ministry of Health. The tumor was reproduced by transplanting tumor strain cells (Treshchalina E.M., Zhukova O.S., Gerasimova G.K., et al.).Guidelines for preclinical studies of antitumor activity of drugs / Guidelines for conducting preclinical studies of drugs. Part one. - Moscow: Grif i K, 2012. - Pp. 640-654).

[0022] The antitumor effect was assessed in accordance with international recommendations for the study of the specific activity of antitumor drugs (Geran RL, Greenberg NH, MacDonald MM, Schumacher AM, Abbott BJ Protocols for screening chemical agents and natural products against tumor and other biological systems / / Cancer Chemother. Rep. - 1972. - Vol. 3. - P. 1-103).

[0023] According to the data in Table 2, repeated intragastric administration of compound 1 at a dose of 25 mg / kg in a Lewis lung carcinoma model resulted in statistically significant inhibition of tumor growth compared to both the control group and the group receiving an equitoxic dose of the prototype compound. A significant reduction in tumor volume, as well as a significant increase in the tumor growth inhibition index, were recorded on days 14 and 22 of the experiment.

[0024] Thus, in contrast to cyclophosphamide, compound 1 is characterized by greater antitumor efficacy against the primary tumor node formed at the site of tumor cell transplantation into the body of mice.

[0025] Example 4. Antimetastatic activity of compound 1 in an animal model using a xenograft of human tumor - non-small cell lung cancer.

[0026] The anti-metastatic activity of compound 1 was studied experimentally on 6-8-week-old 15 male mice of the nu / nu BALB / c atomic mouse line obtained from the laboratory animal nursery - a branch of the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Pushchino) with intragastric administration of compound 1 and cyclophosphamide (prototype) at a dose of 5% of the LD50 value for this route of administration, in a 1% Tween-80 solution in a volume of 20 ml / kg, 72 hours after tumor inoculation for 5 days. Antimetastatic activity was studied on a tumor system - a xenograft of non-small cell lung cancer from the tumor strain bank of the Federal State Budgetary Institution "National Research Medical Center of Oncology named after N.N. Blokhin Russian National Research Center for Oncology and Transplantology, Ministry of Health of the Russian Federation. The tumor was reproduced by transplanting tumor strain cells (Treshchalina E.M., Zhukova O.S., Gerasimova G.K., et al.Guidelines for preclinical studies of antitumor activity of drugs / Guidelines for conducting preclinical studies of drugs. Part one. - M.: Grif i K, 2012. - Pp. 640-654). Antimetastatic effect was assessed in accordance with international recommendations for the study of the specific activity of antitumor drugs (Geran RL, Greenberg NH, MacDonald M.M., Schumacher AM, Abbott BJ Protocols for screening chemical agents and natural products against tumor and other biological systems / / Cancer Chemother. Rep. - 1972. - Vol. 3. - Pp. 1-103).

[0027] As shown in Table 3, repeated intragastric administration of compound 1 at a dose of 25 mg / kg statistically significantly inhibited metastasis in a non-small cell lung cancer model. On day 31 of the experiment, the treatment group showed a significant reduction in both the multiplicity (number) and total volume of metastatic foci in the lungs compared to animals in the control group and the group receiving an equitoxic dose of cyclophosphamide.

[0028] Unlike cyclophosphamide, compound 1 has greater antimetastatic activity against non-small cell lung cancer xenograft transplanted into mice.

[0029] The studies were conducted in accordance with the ethical requirements for working with experimental animals (Rules for Conducting Work Using Experimental Animals (Order of the USSR Ministry of Health No. 755 dated 12.08.1987), the Federal Law "On the Protection of Animals from Cruelty" dated 01.01.1997, Order of the Ministry of Health of the Russian Federation dated 01.04.2016 No. 199n "On Approval of the Rules of Good Laboratory Practice", GOST 33216-2014 "Guidelines for the Maintenance and Care of Laboratory Animals. Rules for the Maintenance and Care of Laboratory Rodents and Rabbits" (came into force on 01.07.2016) and were approved by the Bioethics Commission.

[0030] Compound 1 demonstrates a number of advantages over the standard drug cyclophosphamide: a more favorable acute toxicity profile, increased efficacy in suppressing syngeneic tumor growth in mice, and pronounced antimetastatic activity in a non-small cell lung cancer model.

[0031]

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