RU2846258C1 - USE OF ETHYL ETHER (E)-2-AMINO-4-OXO-5-(2-OXO-2-(4-FLUOROPHENYL)ETHYLIDENE)-1-(3-CYANO-4,5,6,7-TETRAHYDROBENZO[b]THIOPHEN-2-YL)-4,5-DIHYDRO-1H-PYRROLE-3-CARBOXYLIC ACID AS CYTOTOXIC AGENT FOR MELANOMA - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to organic and medical chemistry, particularly to biologically active substances of the class of substituted 4,5-dihydro-1H-pyrrole-3-carboxylic acids. Disclosed is use of ethyl ether (E)-2-amino-4-oxo-5-(2-oxo-2-(4-fluorophenyl)ethylidene)-1-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-4,5-dihydro-1H-pyrrol-3-carboxylic acid of formula 1 as an anti-cancer agent for melanoma.

EFFECT: wider range of cytotoxic agents having low toxicity and effective on melanoma.

1 cl, 1 tbl, 3 ex

Description

The invention relates to the field of organic and medicinal chemistry, to new biologically active substances of the class of substituted 4,5-dihydro-1 H -pyrrole-3-carboxylic acids, namely to ethyl ester ( E )-2-amino-4-oxo-5-(2-oxo-2-(4-fluorophenyl)ethylidene)-1-(3-cyano-4,5,6,7-tetrahydrobenzo[ b ]thiophen-2-yl)-4,5-dihydro-1 H -pyrrole-3-carboxylic acid - compound 1, having the formula:

which has anticancer activity against melanoma, which suggests its use in medicine as a drug with cytotoxic properties and low toxicity.

The closest analogue in structure for the claimed compound is ethyl ester ( E )-2-amino-4-oxo-5-(2-oxo-2-(p-tolyl)ethylidene)-1-(3-(ethoxycarbonyl)-5,6,7,8-tetrahydro-4 H -cyclohepta[ b ]thiophen-2-yl)-4,5-dihydro-1 H -pyrrole-3-carboxylic acid - compound 2, which has anticancer activity [European Journal of Medicinal Chemistry, 2023, Vol. 254, pp. 115325] of the formula:

Data on the anticancer activity of compound 2 are presented.

Compound LD ₅₀ mg/kg Anticancer activity (μM) IC ₅₀ (μM)
B16-F10 2 >1500 25.23

Vemurafenib with the formula: was chosen as the standard for comparison of biological activity:

which was approved by the FDA for the treatment of metastatic melanoma and is analogous in action [Geoffrey K. FDA approval summary: vemurafenib for treatment of unresectable or metastatic melanoma with the BRAFV600E mutation / K. Geoffrey // Clin Cancer Res. - 2014. - Vol. 20(19). - P. 4994-5000. - [Electronic resource]. - URL: DOI: 10.1158/1078-0432.CCR-14-0776], [HIGHLIGHTS OF PRESCRIBING INFORMATION // ZELBORAF® (vemurafenib) tablet for oral use Initial U.S. Approval: 2011. - [Electronic resource]. – URL: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/202429s012lbl.pdf].

The structural formulas of compound 1 are known from the prior art (Lipin, D.V., Parkhoma, K.Y., Shadrin, V.M. et al. Synthesis and antinociceptive activity of nitriles, esters, and amides of 2-amino-1-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-4-oxo-5-(2-oxo-2-arylethylidene)-4,5-dihydro-1H-pyrrole-3-carboxylic acids. Russ Chem Bull 72, 1913–1920 (2023). https://doi.org/10.1007/s11172-023-3976-x).

However, the patent and scientific literature did not reveal the use of compound 1 as a drug with cytotoxic properties and low toxicity against melanoma.

The objective of the invention is to expand the arsenal of cytotoxic agents with low toxicity that are effective against melanoma.

The set task is achieved by using ( E )-2-amino-4-oxo-5-(2-oxo-2-(4-fluorophenyl)ethylidene)-1-(3-cyano-4,5,6,7-tetrahydrobenzo[ b ]thiophen-2-yl)-4,5-dihydro-1 H- pyrrole-3-carboxylic acid, which has anticancer activity against melanoma.

The technical result is achieved by chemical synthesis and use of ethyl ester ( E )-2-amino-4-oxo-5-(2-oxo-2-(4-fluorophenyl)ethylidene)-1-(3-cyano-4,5,6,7-tetrahydrobenzo[ b ]thiophen-2-yl)-4,5-dihydro-1 H- pyrrole-3-carboxylic acid, which has anticancer activity against melanoma and low toxicity.

The claimed compound 1 is synthesized by the reaction of nitrile ( E )-2-((2-oxo-5-(4-fluorophenyl)furan-3(2 H )-ylidene)amino)-4,5,6,7-tetrahydrobenzo[ b ]thiophene-3-carboxylic acid with ethyl cyanoacetate in the presence of 1 equivalent of diisopropylethylamine. The reaction takes place in dichloromethane with stirring and room temperature for 30 minutes, followed by isolation of the target products by known methods according to the scheme:

The invention is illustrated by examples of studies of pharmacological properties.

Example 1. Obtaining compound 1.

To a solution of 3.52 g (0.01 mol) of nitrile ( E )-2-((2-oxo-5-(4-fluorophenyl)furan-3(2 H )-ylidene)amino)-4,5,6,7-tetrahydrobenzo[ b ]thiophene-3-carboxylic acid in 50 ml of anhydrous dichloromethane were added 1.13 g (0.01 mol) of ethyl cyanoacetate and 1.29 g (0.01 mol) of diisopropylethylamine. The resulting solution was kept at room temperature for 30 min with vigorous stirring. The mixture was cooled to 0 °C, the formed precipitate was filtered off and recrystallized.

Yield 78%, mp 278-279°C (dichloromethane). ^1H NMR spectrum (DMSO- d ₆ ), δ, ppm: 1.16 (t, J = 7.1 Hz, 3H), 1.84 (m, 4H), 2.78 (m, 2H), 2.91 (m, 2H), 4.03 (q, J = 7.1 Hz, 3H), 6.76 (s, 1H), 7.32 (m, 2H), 7.52 (br s, 1H), 8.01 (m, 2H), 8.99 (br s, 1H). Found, %: C, 61.98; H, 4.30; N, 8.98; S, 6.82. C ₂₄ H ₂₀ FN ₃ O ₄ S. Calculated, %: C, 61.93; H, 4.33; N, 9.03; S, 6.89.

The obtained compound 1 is a yellow crystalline substance, soluble in DMSO, when heated in toluene, acetone, insoluble in water and hexane.

Example 2. To evaluate the anticancer activity of compound 1, a standard resazurin test was used [Lancaster MV, Fields RD (1996) Antibiotic and cytotoxic drug susceptibility assays using resazurin and poisoning agents. US Patent No. 5,501,959]. The B16-F10 cell line was cultured using αMEM (Biolot) medium + 10% FBS (fetal bovine serum) (Biolot) at 37°C in a humidified atmosphere of 5% CO ₂ (PANASONIC (Sanyo) MCO-18AC (MCO-18AC-PE) air-jacketed CO ₂ incubator). They were passaged twice a week (at 80% competition) at a density of 4 x 10 ⁴ cells per cm ² .

Cells were added to a 96-well plate (TC-treated Culture Microplate, Corning) at a density of 10,000 cells per well + 100 μl of medium. Before adding to the cell cultures, all test compounds were dissolved in DMSO (500 μl - 10 mM); then diluted in the cell medium (40 μl in 960 μl medium) to obtain a 400 μM solution. After that, several concentrations of the test compounds (5, 10, 15, 20 μM) were added to the wells with cells (B16-F10). After 24 hours, the medium was replaced with a solution of medium + 10% resazurin (Resazurin sodium salt, Sigma-Aldrich) and incubated for 4 hours. Wells with cells without the addition of the substance were used as a positive control. As a negative control, wells without cells with the addition of medium with resazurin were used. After incubation, the medium with resazurin from all wells was transferred to a clean plate and assessed on a spectrophotometer at wavelengths of 570 nm and 600 nm. Viability was assessed using standard formulas.

Example 3. Acute toxicity (LD ₅₀ , mg/ml) of compound 1 was determined by the method of G. N. Pershin [Pershin G. N. Methods of experimental chemotherapy // Moscow, pp. 100, 109-117 (1971)]. Compound 1 was administered intraperitoneally to white mice weighing 16-18 g as a suspension in 2% starch mucus and the behavior and mortality of the animals were observed for 10 days. For the studied compound 1, LD ₅₀ is > 1500 mg/kg.

According to the classification of drug toxicity, compound 1 belongs to class V of practically non-toxic drugs [Izmerov N.F., Sanotsky I.V., Sidorov K.K. Toxicometry parameters of industrial poisons with a single exposure: Handbook. Moscow, 1977. – p. 196]. The test results are presented in the table:

Anticancer activity of compound 1, compound 2 and the comparison drug.

Compound LD ₅₀ mg/kg Anticancer activity ( μ M) IC ₅₀ ( μM ) B16-F10 1 >1500 14.69 2 >1500 25.23 Vemurafenib >2500 14.32

As can be seen from the table, the claimed compound 1 exhibits pronounced anticancer activity at the level of the reference drug (Vemurafenib) used in medical practice in relation to the therapy of melanoma. Thus, ethyl ester ( E )-2-amino-4-oxo-5-(2-oxo-2-(4-fluorophenyl)ethylidene)-1-(3-cyano-4,5,6,7-tetrahydrobenzo[ b ]thiophen-2-yl)-4,5-dihydro-1 H- pyrrole-3-carboxylic acid exhibits pronounced biological activity in comparison with the reference drug used in medicine, which makes it possible to use it to create new targeted drugs with low toxicity.

Claims (3)

Use of ethyl ester ( E )-2-amino-4-oxo-5-(2-oxo-2-(4-fluorophenyl)ethylidene)-1-(3-cyano-4,5,6,7-tetrahydrobenzo[ b ]thiophen-2-yl)-4,5-dihydro-1 H- pyrrole-3-carboxylic acid of formula 1 as an anticancer agent against melanoma.