The effect of novel synthetic semicarbazone- and thiosemicarbazone-linked 1,2,3-triazoles on the apoptotic markers, VEGFR-2, and cell cycle of myeloid leukemia

Highlights

• Twelve 1,2,3-triazoles bearing semicarbazone and thiosemicarbazone fragments were designed and synthesized.

• Their cytotoxicity was evaluated against sixty human cancer cell lines.

• Myeloid leukemia cancer cells were the most sensitive toward their effect.

• Compounds 21, 26, and 30 exerted the most potent cytotoxic activity.

• Compound 30 further evaluated for its apoptosis induction and VEGFR-2 inhibition potential.

Abstract

Vascular Endothelial Growth Factor II (VEGFR-2) has been proved as a rational target in cancer therapy. Although currently prescribed VEGFR-2 inhibitors are showing potent antitumor activity, they are often causing serious unwanted effects, restricting their extensive use as chemotherapeutics. Herein, after analyzing the structures of the effective VEGFR-2 inhibitor molecules, we report the synthesis of a new set of semicarbazone- and thiosemicarbazone-linked 1,2,3-triazoles with expected potency of inhibiting the VEGFR-2 signaling. The design of new compounds considered maintaining the essential pharmacophoric features of sorafenib for effective binding with the receptor target. All compounds have been evaluated for their growth inhibition effect against a panel of sixty cancer cells at the National Cancer Institute. Leukemia cancer cells, especially HL-60 and SR, were shown to be the most sensitive to the cytotoxic effect of new compounds. Thiosemicarbazones 21, 26, and 30 exhibited the best activity against almost all tested cancer cells. Therefore, a set of subsequent in vitro biological evaluations has been performed to understand the mechanistic effect of these compounds further. They inhibited the VEGFR-2 with IC₅₀ values of 0.128, 0.413, and 0.067 µM respectively compared with 0.048 µM of Sorafenib. The probable mechanistic effect of 30 has been further evaluated on a number of apoptotic and antiapoptotic markers including BAX, BCL2, caspase-3, and caspase-9. Results revealed the potential of the thiosemicarbazone-linked triazole 30 to induce both the early and the late apoptosis, elevate BAX/BCL2 ratio, induce caspase-3 & caspase-9, and arrest the HL-60 cell cycle at the G2/M and G0-G1 phases. Molecular docking of new semicarbazones and thiosemicarbazones into the proposed biological target receptor has also been performed. Results of docking studies proved the potential of new semicarbazone- and thiosemicarbazone-linked 1,2,3-triazoles to effectively bind with crucial residues of the VEGFR-2 binding pocket.

Introduction

Myeloid leukemia (ML) is a blood cancer type that arises as a result of the uncontrolled generation of immature monocytes and granulocytes in the bone marrow [1]. In the adult population, it represents the most frequent leukemia and accounts for more than 75% of all cases [2]. Chemotherapy and bone marrow transplantation remain the main strategies for the treatment of such a malignant disorder. However, the cure rates with both strategies are still low (∼15% in elderly patients and ∼40% in young patients) [1]. The aberrant vascular endothelial growth factor (VEGF) signaling has been reported to induce the proliferation, survival, and resistance of myeloid leukemia cancer cells to chemotherapy [3]. Accordingly, VEGF-targeting with anti-angiogenic therapies has long been considered a valuable approach for the management of such a malignancy [4]. As well, apoptosis induction is another effective approach for the treatment of ML [5]. The dysregulation of apoptosis and apoptotic markers in myeloid leukemia plays a crucial role in the development of drug resistance [6]. So, investigating the biochemical changes in the levels of various apoptotic and antiapoptotic markers is helpful to determine the perfect anticancer medication to improve the sensitivity of ML cells to apoptotic stimuli.

Semicarbazone, thiosemicarbazone, urea, and thiourea fragments have long been incorporated as privileged linkers in a number of potent anticancer molecules with VEGFR-2 inhibition and apoptosis induction potentials [7], [8]. They played as hydrogen bond donors/acceptors to bind with the essential amino acid residues of the ATP or the allosteric binding sites of VEGFR-2. These linkers were attached with numerous nitrogenous heterocycles which include, but are not limited to, quinoxalines [9], phthalazines [10], quinazolines [11], [12], acridines [13], quinolines [14], thiazolidinediones [15], and triazoles [16], [17]. To the best of our knowledge, none of the previously developed semicarbazones- and thiosemicarbazone-linked heterocycles has been evaluated for their cytotoxicity against myeloid leukemia. However, two trials were adopted to evaluate profiles of anticancer candidates incorporating urea and thiourea fragments against myeloid leukemia over the last two decades [18], [19]. The earlier one described the potential anticancer effect of sorafenib (1; Fig. 1) in acute myeloid leukemia [18]. Findings of this study revealed the ability of sorafenib to inhibit acute myeloid leukemia blasts and confirmed the rationale for its use as a recommended therapy. Sorafenib inhibited the proliferation of acute myeloid leukemia cells via apoptosis induction and cell cycle arrest [18]. The latter study revealed the excellent activity as anticancer of some thiourea derivatives compared with cisplatin [19]. In particular, the biphenylthiourea derivative 2 induced the apoptotic cell death of myeloid leukemia [19]. On the other hand, the acridine-linked thiosemicarbazone derivative 3 presented a wide spectrum growth inhibition profile against many types of cancer cells, including renal, ovarian, and breast carcinomas [13]. Similarly, the Parthenolide-derived semicarbazone 4 showed exceptional anticancer activity against mice colon tumor in the in vivo experiments and induced the apoptotic cell death of U87-MG in a concentration-dependent manner. Cell cycle analysis of U87-MG revealed the potential of 4 to arrest the cell cycle at the G0/G1 phase [20].

Among the isomeric forms of triazoles, 1,2,3-triazole has been previously reported to possess a significant anticancer activity in various tumors [21]. Over the last two decades, numerous attempts have been conducted for the development of VEGFR-2 inhibitor and apoptosis inducer 1,2,3-triazoles [21]. The indolone-based 1,2,3-triazole (5; Fig. 2) had a better VEGFR-2 inhibitory activity than sunitinib (IC₅₀ = 26.38 nM compared with 83.20 nM) and presented lower toxicity to the human umbilical vein endothelial cells [22]. Additionally, the Curcumin–based 1,2,3-triazole hybrid molecules 6 and 7 showed remarkable anticancer effects on acute lymphoblastic leukemia (IC₅₀ = 3.13 & 3.95 μM, respectively) [23]. Among which, the hybrid 7 has been validated as an apoptosis inducer [24]. Furthermore, 1,2,3-Triazole-chalcone hybrid 8 demonstrated greater activity than gefitinib against many leukemia cell lines. Flow cytometry analysis of 8 revealed its ability to arrest the cell cycle of leukemia cancer cells at the G2/M phase and to induce its apoptotic death via the induction of Bax, downregulation of Bcl-2, and the activation of different caspases [21].

Based on the aforementioned proved scientific findings, and in continuation of our recent efforts for the development of bioactive molecules [25], [26], [27], [28], [29], we decided to design and synthesize a new set of novel derivatives by tethering the pharmacophoric privileged fragments semicarbazone and thiosemicarbazone to the 1,2,3-triazole ring (Fig. 3). Herein, the synthesis of twelve novel derivatives, their growth inhibition effects against a panel of sixty cancer cells, and their VEGFR-2 inhibitory potentials were reported. As well, we analyzed the effect of the most potent growth inhibitory compound on certain apoptotic and antiapoptotic markers and the cell cycle of myeloid leukemia cancer cells. The VEGFR-2 inhibitory effect of sorafenib was reported to be mediated through the presence of a number of pharmacophoric fragments and mediated through four main interactions with VEGFR-2 as presented in Fig. 3 [30]. The pharmacophoric groups include: (i) A peripheral aryl ring to occupy the ATP binding pocket of VEGFR-2. The acetamide side chain attached to this aryl ring in sorafenib has the ability to interact with Cys919 by hydrogen bonding interaction. (ii) A spacer to occupy the linker region and to interact through a hydrophobic interaction with one or more amino acid residues. (iii) A hydrogen bonding donor/acceptor linker (e.g., urea or thiourea) both to act as a pharmacophore and at the same time to bind with Glu885 and/or Asp1046; iv) A terminal hydrophobic tail to occupy the allosteric hydrophobic pocket. In our designed compounds, the pyridine ring of sorafenib was replaced with the bioisosteric phenyl ring while the spacer was replaced with the 1,2,3-triazole privileged fragment. Aryl rings in new compounds made attached with hydrogen bond acceptor groups (Br, Cl, F, and NO₂), to maintain their ability to interact with Cys919. Hydrogen bonding donor (HBD) and acceptor (HBA) linkers were tethered in the form of semicarbazone and thiosemicarbazone fragments which are reported as pharmacophores in numerous synthetic anticancer compounds [31], [32]. Accordingly, both the number and length of hydrogen bonding atoms in the spacer and HBD/HBA linkers were increased. This action is expected to improve both the hydrophobic and hydrogen bonding interactions with the receptor target. Another terminal aryl ring was attached to the hydrogen bond linker with a variety of substituents of different electronic environments to occupy the terminal allosteric hydrophobic pocket. This ring was replaced with aliphatic fragments in two derivatives (31 & 33) to assess its importance for effective occupation in the allosteric pocket.