Synthesis and biological evaluation of prodrugs for nitroreductase based 4-β-amino-4′-Demethylepipodophyllotoxin as potential anticancer agents

A series of prodrugs for nitroreductase (NTR) based 4-β-amino-4′- Demethylepipodophyllotoxin as potential anticancer agents were synthesized, and their antiproliferative activities in vitro showed compounds 2b (IC50 = 0.77, 0.83 and 1.19 μM) and 2d (IC50 = 0.98, 0.91 and 1.58 μM) were greatly selectively toxic to tumor cells A-549, HeLa and HepG2, respectively, and lower damage to normal WI-38 cells in comparison with positive agent Etoposide and Demethylepipodophyllotoxin, and induced cell cycle arrest in the G2/M phase with a concomitant decrease in the population of G1 phase in HeLa cells, which were accompanied by apoptosis. Furthermore, Molecular docking model showed that compounds 2b and 2d appeared to form stable bonds with NTR 1DS7. Taken together, these conjugates have the potential to be developed as antitumor drugs. Graphical abstract Graphical abstract


Introduction
With the rapid growth of cancer cases across the globe, a great number of approaches are being investigated to develop an effective cure for this deadly disease [1]. Among various approaches, naturally occurring substances account for the abundant sources to identify novel hits and leads [2]. However, a number of antitumor naturally occurring substances do not show target specificity, which may even show adverse toxicity to non-carcinoma cells. In order to reduce the cytotoxicity of the natural compounds, appropriate structural modification is the vital way and has been adopted to develop new drug [3].
Podophyllotoxin is a natural occurring aryltetralin lignin with pronounced cytotoxic activity. However, its clinical application for cancer treatment has been blocked due to its poor water solubility and selectivity [4]. For reducing cytotoxicity and increasing water solubility of the bioactive compound, a great number of structural modification have been adopted to address these limitations and obtain better therapeutic agents [5][6][7][8][9][10][11], and these efforts have led to the structure-activity relationships (SAR) unambiguously demonstrate that C4 is the major molecular area tolerable to significant structural diversification [12]. Therefore, most of the current derivatization on Podophyllotoxin focuses on C-4 position. Recently, A series of 4β-N substituted 4′-O-demethyl-4deoxyp-odophyllotoxins have been synthesized, and exhibited significant anticancer activity [13][14][15][16][17], Among these, the substituent of p-nitrobenzylpiperazine markedly affected the activity profiles of this compound class, and may serve as a potential anticancer drug [18].
NTR are a family of bacterial enzymes used in genedirected enzyme prodrug therapy (GDEPT) that selectively activate prodrugs containing aromatic nitro groups to exert cytotoxic effects [19]. It can catalyze nitro-substituted aromatic compounds into aromatic amines and undergoes internal cyclization to a non-toxic derivative, which reduces serious side effects on healthy cells and form toxic metabolites after selective reduction by enzymes in tumor tissues [20]. In recent years, Efforts for optimization of NTR-based GDEPT have focused on the key aspects of the technology, namely the delivery system, the NTR-enzyme and the prodrug. The improved prodrug is expected to boost the clinical application of NTR-based GDEPT in future [21]. Prodrug strategies based on NTR have been extensively studied and remarkable efforts employing NTR and a library of different nitro-containing scaffolds has led to promising in vitro prodrug candidates [22][23][24][25][26]. O-nitrophenoxyacetyl acid which containing aromatic nitro group has known as potential inhibitor of vascular endothelial growth factor in cancer chemotherapy [27][28][29]. NTR can catalyze nitro substituent into aromatic amines and undergoes internal cyclization to a non-toxic derivative, which reduces serious side effects on healthy cells and increases concentration of drugs at cancer cells is remarkable [21]. Furthermore, identification of structurally diverse prodrug will undoubtedly prove beneficial to design and synthesis of novel nitroaromatic prodrugs [22]. Inspired by the above background, herein, we present the design, synthesis, of a novel series of 4β-N-(4-Nitrophenyl piperazinyl)-4′-O-demethyl-4-deoxypodophyllotoxin and four different O-nitrophenoxyacetyl acids(substituent of electron-donating groups, electron-withdrawing groups, steric groups, comprehensive groups, respectively) hybrids and evaluation of their cytotoxic activity against several cancer cell lines.

Synthesis
The synthetic route to the target compounds is shown in Scheme 1. The O-nitrophenoxyacetyl acids were prepared according to our previous report [27]. 4β-N-(4-Nitrophenyl piperazinyl)-4′-O-demethyl-4-deoxypodophyllotoxin was according to the procedure described previous [18]. The chemical structures of the final compounds were fully characterized by 1 H NMR, 13 C NMR and HRMS spectroscopic techniques.

Cytotoxicity
The in vitro cytotoxicities of conjugates 2a-2t were evaluated against a panel of three human cancer cell lines (lung carcinoma A-549, cervical carcinoma HeLa, hepatocellular carcinoma HepG2, and human embryonic lung fibroblasts WI-38 cells), with Etoposide and Demethylepipodophyllotoxin as reference compounds. In addition, their toxicity evaluation was tested on normal embryonic lung fibroblast WI-38 cells. The screening procedure was based on the standard MTT growth inhibition assay, and the results are summarized in Table  1. As shown, most of the compounds were more potent against three tumor cell, these compounds were most effective in A549 cells and HeLa cells, and had lowest potency in HepG2 cells. The IC 50 value of compounds 2b (0.77, 0.83 and 1.19 μM) and 2d (0.98, 0.91 and 1.58 μM), and which were comparable to positive agent Etoposide and more potent than the positive agent Demethylepipodophyllotoxin (9.64, 15.29 and 12.73 μM) for HeLa, HepG2 and A549 cells, respectively. In comparison, compounds 2b (IC 50 = 688.54 μM) and 2d (IC 50 = 477.96 μM) showed greater selectivity, and were relatively less active toward normal WI-38 cells in comparison with positive agent Etoposide (IC 50 < 100 μM) and Demethylepipodophyllotoxin (IC 50 < 100 μM). Based on these results, it was possible to deduce some preliminary SAR. First of all, O-nitrophenoxyacetyl acids with electron-donating group substitution (2a-2e) appeared to be more potent than those with electron-withdrawing (2f-2k), steric (2l-2o), and comprehensive (2p-2t) groups.

Cell cycle arrest and apoptotic assay
The data above reveled that compounds 2b and 2d the most potent and selective were further investigate the effects on cell cycle progression by means of fluorescence-activated cell sorting analysis of HCT-116 cells stained with propidium iodide.
As shown in Fig. 1, treatment with compounds 2b and 2d led to a dose-dependent accumulation of cells in the G2/M phase with a concomitant decrease in the population of G0/G1 phase cells respectively, and showed significantly difference compared with untreated control group (P < 0.05 and P < 0.01). Similar effects also occurred with the Annexin V/PI apoptosis detection. It was illustrated from Fig. 2 that, following the treatment, the apoptotic cell percentage was as low as 0.14% of that of control group. After incubation using 2.5 and 50 μmol/ L of compounds 2b and 2d for 24 h, there were 38.83% and 37.81% (70.23% and 74.55%) apoptotic cells at the later stage, and showed significantly difference compared with untreated control group (P < 0.05 and P < 0.01). As suggested by the above findings, compounds 2b and 2d could efficiently induce HCT-116 cell apoptosis (Fig. 2).
These results demonstrated that compounds 2b and 2d were interfering with cell proliferation by arresting the cell cycle and induced G2/M arrest accompanied by apoptosis in HeLa cells.

Molecular docking assay
To prove the anticancer mechanism of the action, a molecular docking investigation was undertaken. As shown in Fig. 3, which revealed that compounds 2b and 2d were well inserted into the two active pockets of NTR 1DS7, Hydrogen bonds were shown with yellow dash lines and dark green spheres. The hydrophobic interactions which are van der Waals were represented by light green spheres, these results reveal that compounds 2b and 2d having hydrophobic property, good membrane permeability and specifically targeting NTR. The binding forms are demonstrated that the para nitro and carbonyl groups in phenyl ring were able to affect the interaction on NTR significantly. These data provide certain theoretical support for experimental results.

Conclusions
In conclusion, we have designed and synthesized a number of prodrugs for nitroreductase based 4-β-amino-4′-Demethylepipodophyllotoxin as potential anticancer agents were synthesized, and their antiproliferative activities in vitro showed compounds 2b and 2d were greatly selectively toxic to tumor cells A-549, HeLa and HepG2, respectively, and lower damage to normal WI-38 cells in comparison with positive agent Etoposide and Demethylepipodophyllotoxin, and induced cell cycle arrest in the G2/M phase with a concomitant decrease in the population of G1 phase in HeLa cells, which were accompanied by apoptosis. Furthermore, Molecular docking model showed that compounds 2b and 2d appeared to form stable bonds with NTR 1DS7. Taken together, these conjugates have the potential to be developed as antitumor drugs. Further studies on the relevant action mechanisms and structural modification of identified hits are on-going.