RU2850428C1 - Sodium 1-amino-1,6-dioxo-6-(p-tolyl)-2-cyano-4-(4-phenyl-(3-(ethoxycarbonyl)thiophen-2-yl)amino)hexa-2,4-diene-3-olate possessing anticancer activity in therapy of colorectal cancer - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to organic and medical chemistry. Disclosed is sodium 1-amino-1,6-dioxo-6-(p-tolyl)-2-cyano-4-(4-phenyl-(3-(ethoxycarbonyl)thiophen-2-yl)amino)hexa-2,4-dien-3-olate of formula given below.

EFFECT: extending the range of products possessing anticancer activity with respect to colorectal cancer.

1 cl, 1 tbl, 3 ex

Description

The invention relates to the field of organic chemistry, to new biologically active substances of the class of substituted 2-amino-4-aryl-4-oxobutanoic acids, namely to sodium 1-amino-1,6-dioxo-6-(p-tolyl)-2-cyano-4-(4-phenyl-(3-(ethoxycarbonyl)thiophen-2-yl)amino)hexa-2,4-dien-3-olate of the formula:

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

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

which was approved by the FDA for the treatment of colorectal cancer [Fluorouracil. – [Electronic resource]. – URL: https://www.vidal.ru/drugs/fluorouracil], Somayeh Handali et al. A novel 5-Fluorouracil targeted delivery to colon cancer using folic acid conjugated liposomes // Biomedicine & Pharmacotherapy. 2018. Vol. 108, p. 1259-1273. DOI: 10.1016/j.biopha.2018.09.128].

The objective of the invention is to search for substances with pronounced anticancer activity against colorectal cancer and low toxicity in a series of substituted derivatives of 2-amino-4-dioxobutanoic acids in order to expand the arsenal of cytotoxic agents.

The technical result is achieved by chemical synthesis of sodium 1-amino-1,6-dioxo-6-(p-tolyl)-2-cyano-4-(4-phenyl-(3-(ethoxycarbonyl)thiophen-2-yl)amino)hexa-2,4-dien-3-olate, which has antitumor activity against colorectal cancer and low toxicity.

The claimed compound 1 is synthesized from known starting reagents in 2 stages.

Stage 1. Ethyl 2-((2-oxo-5-(p-tolyl)furan-3(2H)-ylidene)amino)-4-phenylthiophene-3-carboxylate 3 is obtained by a reaction that takes place with slow heating to 140 °C of a solution of 2-((3-(ethoxycarbonyl)-4-phenylthiophen-2-yl)amino)-4-oxo-4-(p-tolyl)but-2-enoic acid 2 in propionic anhydride for 2 hours, followed by isolation of compound 3 by known methods.

Step 2. Sodium 1-amino-1,6-dioxo-6-(p-tolyl)-2-cyano-4-(4-phenyl-(3-(ethoxycarbonyl)thiophen-2-yl)amino)hexa-2,4-dien-3-olate 1 is synthesized by the reaction of ethyl 2-((2-oxo-5-(p-tolyl)furan-3(2H)-ylidene)amino)-4-phenylthiophene-3-carboxylate 3 with cyanoacetamide in the presence of 1 equivalent of sodium carbonate. The reaction takes place in anhydrous dioxane with stirring at 50°C for 3 hours, followed by isolation of the target product by known methods according to the scheme:

Compound 1 has not been described in the patent and scientific literature, and the use of compound 1 as an agent with cytotoxic properties effective against colorectal cancer has not been identified.

The invention is illustrated by examples of obtaining the compound and studying its pharmacological properties.

Example 1. Preparation of compound 1: 0.89 g (0.01 mol) of cyanoacetamide and 1.06 g (0.01 mol) of sodium carbonate were added to a solution of 4.17 g (0.01 mol) of ethyl 2-((2-oxo-5-(p-tolyl)furan-3(2H)-ylidene)amino)-4-phenylthiophene-3-carboxylate in 50 ml of anhydrous dioxane. The resulting mixture was maintained for 3 hours at 50 °C with vigorous stirring. Then the mixture was cooled to 0 °C, the formed precipitate was filtered off and recrystallized. Yield 4.66 g (89%), yellow crystals, mp 231.9-233.0 °C. ^1H NMR spectrum (DMSO- _d6 ) δ, ppm: 1.04 t (3H, _CH3 , J = 7.1 Hz), 2.36 s (3H, _CH3 ), 4.14 q (2H, _OCH2 , J = 7.1 Hz), 6.01 s (1H, =CH), 6.86 s (1H, =CH), 7.26–7.31 m (4H, H _Ar ; 1H, _NH2 ), 7.32–7.37 m (3H, H _Ar ), 7.66 br s (1H, _NH2 ), 7.78–7.82 m (2H, H _Ar ), 13.33 s (1H, NH). ¹³ C NMR spectrum (DMSO- _d6 ) δ, ppm: 13.6, 21.0, 60.0, 93.2, 113.8, 114.6, 114.7, 116.2, 126.8, 127.0, 127.6, 128.4, 129.0, 136.7, 137.2, 139.7, 141.4, 151.6, 159.0, 163.2, 164.0, 165.1, 188.7. Found, %: C, 61.99; H, 4.29; N, 8.00; S, 6.08. C ₂₇ H ₂₂ N ₃ NaO ₅ S. Calculated, %: C, 61.94; H, 4.24; N, 8.03; S, 6.12.

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

Example 2. Acute toxicity (LD₅₀, mg/ml) of compound 1 was determined by the method of G. N. Pershin [Pershin G. N. Methods of experimental chemotherapy // M., pp. 100, 109-117 (1971)]. Compound 1 was administered intraperitoneally to white mice weighing 16-18 g in the form of a 2% suspension starch mucus and observed the behavior and mortality of animals for 10 days. For the studied compound, 1 LD₅₀is > 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. Toximetry parameters of industrial poisons with a single exposure: Handbook. Moscow, 1977. – p. 196].

Example 3. To evaluate the antitumor 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 CT26 cell line was cultured using RPMI (Biolot) + 10% FBS (fetal bovine serum) (Biolot), the MEF NF2 line – DMEM (Biolot) + 10% FBS at 37 °C in a humidified atmosphere of 5% CO ₂ (CO ₂ incubator with an air jacket Panasonic (Sanyo) MCO-18AC (MCO-18AC-PE)). Passage was twice a week (at 80% competition) at a density of 4 x 10 ⁴ cells per cm ² .

Cells were placed in a 96-well plate (TC-treated Culture Microplate, Corning) at a density of 10,000 cells per well plus 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 this, several concentrations of the test compounds (0.8, 1.6, 3.2, 6.3, 12.5, 25, 50 μM) were added to the wells with the cells (CT26 and MEF NF2). After 48 hours, the medium was replaced with a solution of the 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. Wells without cells and resazurin-containing medium served as a negative control. After incubation, the resazurin-containing medium from all wells was transferred to a clean plate and analyzed spectrophotometrically at 570 nm and 600 nm. Viability was assessed using standard formulas.

Anticancer activity of compound 1 and the comparison drug.

Compound LD ₅₀ mg/kg Anticancer activity (μM) IC ₅₀ (μM)
CT26 IC ₅₀ (μM)
MEF NF2 1 >1500 8.92 57.2 Fluorouracil 981 26.91 -

As can be seen from the table, the claimed compound 1 exhibits pronounced anticancer activity and exceeds that of the reference drug used in medical practice for the treatment of colorectal cancer by more than 3 times. At the same time, the proposed compound 1 is more than 1.5 times less toxic than the reference drug. Thus, sodium 1-amino-1,6-dioxo-6-(p-tolyl)-2-cyano-4-(4-phenyl-(3-(ethoxycarbonyl)thiophen-2-yl)amino)hexa-2,4-dien-3-olate exhibits higher activity compared to the reference drug used in medicine, which makes it possible to use it in the development of new targeted drugs with low toxicity.

Claims (2)

Sodium 1-amino-1,6-dioxo-6-(p-tolyl)-2-cyano-4-(4-phenyl-(3-(ethoxycarbonyl)thiophen-2-yl)amino)hexa-2,4-dien-3-olate formula