Research paper
Design, synthesis and screening of 1, 2, 4-triazinone derivatives as potential antitumor agents with apoptosis inducing activity on MCF-7 breast cancer cell line

https://doi.org/10.1016/j.ejmech.2018.07.003Get rights and content

Highlights

  • The Aim of work is to design potential antitumor molecules over MCF-7 breast cancer cells.

  • Compound 4c with IC50 (0.36 μM) exhibits potent cytotoxic activity on MCF-7 cells by 6-fold higher than podo.

  • β-Tubulin polymerization inhibition displays that compound 6c is equipotent to podo at their IC50 over MCF-7 cells.

  • Triazinones 4c, 5e and 7c exhibit potent inhibitor of topo II-β enzyme with nano-molar concentration higher than etopsoide.

Abstract

Some triazinone derivatives are designed and synthesized as potential antitumor agents. Triazinone derivatives 4c, 5e and 7c show potent anticancer activity over MCF-7 breast cancer cells higher than podophyllotoxin (podo) by approximate 6-fold. DNA flow cytometry analysis for the compounds 3c, 4c, 5e, 6c and 7c show a potent inhibitory activity of cell proliferation and cell cycle arrest at G2/M phase. Compounds 4c, 5e and 7c exhibit low to moderate β-tubulin polymerization inhibition percentage. Meanwhile, compound 6c displayed excellent β-tubulin percentage of polymerization inhibition equivalent to that exhibited by podo. In addition, compounds 4c, 5e and 7c show strong topoisomerase (topo) II inhibitory activity in nano-molar concentration, compared to known topo inhibitor as etoposide. Finally, apoptotic inducing activity over MCF-7 of compounds 4c, 5e, 6c and 7c is due to up-regulation of p53, increased Bax/Bcl-2 ratio and caspase3/7 levels 2-fold higher than podo.

Introduction

Breast cancer cells are rapidly growing cells and are over-expressed in topoisomerase (topo) enzymes [1]. In addition, breast cancer cells have high mitotic rate due to excessive microtubule synthesis [2]. Therefore, DNA interfering agents, such as doxorubicin, and tubulin polymerization inhibitors, such as taxol, are frequently used combined, for breast cancer treatment to obtain synergistic cytotoxic effect [3]. Resistance of cancer cells to apoptosis induction by chemotherapy is one of the major barriers facing successful treatment of breast cancer [4]. The resistance usually occurs due to down regulation of p53 and pro-apoptotic proteins as Bax and up-regulation of anti-apoptotic protein, such as Bcl-2 protein, that prevents activation of terminal caspase 3/7 [5]. The therapeutic tactic to overcome this therapy obstacle is to use combination chemotherapy. Thus, lots of research were directed to discover cytotoxic agents with multimode mode of cytotoxic action [6]. Until now, nature has been the major source of cancer remedies [7]. Podophyllotoxin (podo) (Fig. 1) is a natural product that exhibits good in vitro cytotoxicity profile and is an apoptosis inducer against different types of cancer cells, however, the toxicity and side effects limit its use [8]. Podo and etoposide (Fig. 1) are two structurally related antitumor agents, having two different anti-proliferative mechanisms of action [9]. Podo exhibits a strong β-tubulin polymerization inhibition activity, leading to antimitotic effect, while etopsoide shows topo enzyme inhibition that prevents DNA synthesis [10,11]. Structure investigation of podo and etopsoide shows similar pharmacophoric fragments in their skeletons, as 3,4,5-trimethoxy phenyl (TMP) moiety and orthogonal tetrahydrofuronaphthodioxolone ring system. Both features are key pharmacophoric points responsible for interaction with colchicine binding site, leading to β-tubulin polymerization inhibition [12]. Moreover, etopsoide has a large structural skeleton allowing to act as topo interactive agents [13]. Triazinone ring may be used as an isostere for tetrahydrofuronaphthodioxolone ring system present in podo, as the nitrogen atoms have the ability to form hydrogen bond with the molecular receptor and anchor DNA by complex formation [14]. For example, triazinone 1 exhibits good cytotoxicity against breast cancer due to DNA damage (Fig. 1) [15]. Furthermore, some aryl triazinone derivatives 2 and 3 exhibit promising cytotoxic activity via good β-tubulin polymerization inhibition (Fig. 1) [16]. Based on foregoing findings, a mimic antitumor model is designed based on triazinone core ring. The model has the following structural outline, a diaryl ring system connected through a core moiety. One of the aryl rings attached to the core is composed of three aryl groups, including TMP, 3, 4-Dimethoxy phenyl (DMP) and phenyl group. The other aryl part is 3,5-dibromophenyl acetate and is isosteric to lactone ring (cyclic ester) in podo. Dibromo-substitution pattern maintains β-tubulin polymerization, as compounds 4 and 5display antitumor activity, due to β-tubulin polymerization inhibition [17,18]. The difference in structure between the synthesized compounds is summarized in the following modifications, starting with the free-rotatable hydrazine carbothioamide derivatives 3a-c; they are cyclized into rigid triazinone derivatives 4a-c. Rigid forms are subjected to substitution with phenylthiazole group at N-2 position, as derivatives 5a-c or monocylic ring 4a-c extended to triazolotriazinone 6a-c. Furthermore, C-3 position of the core ring contains different aryl groups (Fig. 2). illustrates the design strategy for the synthesis of compounds and the structural variations between target compounds.

Section snippets

Chemistry

The reaction sequences leading to the synthesis of titled compounds are outlined in Scheme 1, Scheme 2. The starting compounds 1a-c are synthesized according to published procedure [19]. The key intermediates 2a-c are prepared in one-step by Perkin condensation of N-benzoyl glycines, 3, 5-dibromosalicylaldehyde and acetic anhydride in the presence of anhydrous pyridine. IR spectra of compounds 2b and 2c reveal the presence of absorption band at 1803–1805 cm−1,characteristic of carbonyl group of

Conclusion

A series of novel 1,2,4-triazinone derivatives are designed and synthesized as potential antitumor agents. The synthetic protocol includes the following synthetic steps: hydrazine carbothioamides 3a-c, triazinone derivatives 4a–c and 7a-c are synthesized from their oxazolone precursor's 2a-c with thiosemicarbazide and phenyl hydrazine, respectively. Furthermore, thioamide group in 1,2,4-triazinone derivatives 4a-c is cyclized using either phenacyl bromide or acetic anhydride to afford

Apparatus and methods

Melting points were determined on a Gallen kamp melting point apparatus and are not corrected. IR spectra (KBr, cm−1) were recorded on a Bruker FT 8000 spectrometer. 1H NMR and 13C NMR spectra were measured in DMSO‑d6 on a Burker- Avance spectrometer at 400 MHz and 100 MHz, respectively. Chemical shifts are reported in ppm. Mass spectra (MS) were obtained on a gas chromatograph/mass spectrometer Schimadzu GCMS-QP2010 plus operating at 70 eV. Elemental analyses were performed using heraeus and

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