Novel azo pyridone dyes based on dihydropyrimidinone skeleton: Synthesis, DFT study and anticancer activity

Highlights

• Novel azo pyridone dihydropyrimidinone based dyes are synthesized and characterized.

• Experimental and DFT approaches confirm that the most stable tautomer is hydrazone.

• New structures exhibited promising anticancer and antioxidant properties.

• Dyes show concentration dependent cytotoxicity against tested cancer cells.

• Novel dyes may serve for further development of new anticancer drugs.

Abstract

Seven novel azo dyes with 2-pyridone and dihydropyrimidinone moieties have been synthesized and thoroughly characterized. The azo-hydrazone tautomerism has been investigated by experimental and theoretical approaches. The optimizations of geometries have been performed with density functional theory (DFT). The vibrational and NMR spectra were calculated and correlated with experimental ones. Furthermore, quantum chemical descriptors were calculated and MEP maps were plotted to determine biological reactivity of dyes. The antioxidant assay evinced that 5, 6 and 7 are promising antioxidant candidates. In vitro cytotoxic activity was studied against three malignant cell lines: prostate adenocarcinoma (PC-3), lung carcinoma (A549) and chronic myelogenous leukemia (K562), as well as against human normal lung fibroblasts (MRC-5), using MTT assay. Examination of cytotoxic effects on human cancer cell lines showed the concentration dependent cytotoxicity of all investigated compounds. The K562 cells were the most sensitive to the cytotoxicity of the compounds 3, 5 and 6, wherein compound 5 was particularly prominent and selective in cytotoxic action between K562 (24.97 μM) and PC-3 (48.98 μM) cancer cells, and normal MRC-5 (91.11 μM) cells. Moreover, the cell cycle analysis of compound 5 was examined in K562 cells, by flow cytometry, to study its mechanism of anticancer action. Finally, in silico evaluation of physicochemical parameters, druglikeness and ADME properties showed that investigated compounds are orally bioavailable with no permeation to the blood brain barrier.

Introduction

Azo dyes are the most significant class of colored synthetic compounds. They have diverse application in various fields of science and industry, regarding their simple preparation and a broad spectrum of derivatives [1]. The synthesis of azo dyes based on heterocycle moieties has gained particular attention in last several decades [2,3]. Namely, the heterocyclic azo compounds exhibit antibacterial [4], antifungal [5], antioxidant [6], analgesic [7], anti-inflammatory [8], antitubercular [9] and anticancer properties [10,11]. Moreover, studies on new antitumor drugs have reviled that some azo compounds manifest cytotoxic action against EAC (Ehrlich-Lettre ascites carcinoma), MCF-7 and MDA-MB-231 (breast cancer), PC-3 (human prostate cancer) and K562 (myelogenous leukemia) tumor cells [12].

Among this class of azo dyes, those with 2-pyridone moiety in the structure are particularly significant. Besides their remarkable coloration properties [13], azo pyridone dyes exhibit antibacterial activity [14] and demonstrate the potency for cancer therapy [15]. One of the most investigated feature of azo pyridone dyes is the phenomenon of azo-hydrazone tautomerism [[16], [17], [18]]. The structural characterization of the dyes is crucial, since the tautomeric forms differ in physical properties and thus, biological activity [19].

On the other side, dihydropyrimidinone scaffold (DHPM or dihydropyrimidine-5-carboxylate core) is a heterocyclic system which has been synthesized via Biginelli reaction, including one-pot multicomponent reaction of an aromatic aldehyde, urea and ethyl acetoacetate [20]. The Biginelli synthesis is exceptionally attractive allowing development of numerous DHPM derivatives by simple variation of the starting components [21]. Furthermore, the compounds based on the dihydropyrimidine-5-carboxylate core have shown remarkable biological properties such as antibacterial, antiviral, antiinflammatory, antioxidant and antitumor activity [22]. Recently, DHPM based compounds have emerged as the calcium channel blockers, potential therapeutics against Alzheimer's disease, potential new AIDS therapy, the antihypertensive agents and potent breast cancer therapy [23].

In this manner, we merged dihydropyrimidinone based core with seven different 2-pyridones via reaction of diazo coupling, designing a novel series of azo pyridone dyes. Since 2-pyridone and DHPM moieties manifest biological activity, their conjugation can ameliorate biological properties of azo dyes. Contrary to the traditional arylazo dyes, where aniline-based compounds are the most commonly employed as diazotization agenesis, hereby, we use the Biginelli adduct. The substitution of aryl diazonium salts with DHPM-diazo moiety, leads to elimination of highly toxic degradation products of azo dyes, since Biginelli adducts exhibit negligible toxicity [24]. Besides, there are only few studies of DHPM-azo dyes [25,26], and according to our best knowledge, DHPM-azo pyridone dyes have been unexplored. Nevertheless, cancer is one of the main targets in therapeutic chemistry, so we made an effort to design azo molecules with potential anticancer activity.

Herein, the synthesis, structural characterization and azo-hydrazone tautomerism of seven novel DHPM-azo pyridone dyes have been reported. The structures of molecules have been confirmed by ATR-FTIR, NMR, UV–Vis, MS and elemental analysis. The calculated structural and spectroscopic properties of dyes have been investigated with density functional theory (DFT). The calculated vibrational spectrum of the hydrazone tautomer has been correlated with the experimental one. The nuclear magnetic resonance (NMR) chemical shifts of the hydrazone tautomer have been calculated and compared with experimental data. Molecular Electrostatic Potential (MEP) surfaces are plotted over the optimized geometry to brighten the reactivity of investigated molecules. Furthermore, the biological reactivity of novel azo molecules has been correlated with 14 quantum chemical descriptors. The antioxidant activity has been evaluated by ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay. In vitro antitumor action of dihydropyrimidinone (B2, Fig. 1) and DHPM-azo dyes (1–7, Fig. 1) was determined against following tumor cell lines: prostate adenocarcinoma (PC-3), lung carcinoma (A549), chronic myelogenous leukemia (K562), and human normal lung fibroblast (MRC-5) using microculture tetrazolium test (MTT) assay. Additionally, cell cycle analysis of the most active compound (5, Fig. 1) was examined in K562 cells in order to enlighten the antitumor properties. The effect of dye 5 on the cell cycle was measured by flow cytometry. Finally, physicochemical parameters, druglikeness and ADME properties of novel compounds were evaluated in silico by SwissADME and Vega 22.