Structure optimization of new tumor-selective Passerini α-acyloxy carboxamides as Caspase-3/7 activators

Selective elimination of tumors has always been the mainstay of oncology research. The on-going research underlying the cellular apoptotic mechanisms reveal caspases activation, especially the key effector caspase-3, as a personalized tumor-selective therapeutic strategy. Our continued research protocol has exploited new optimized Passerini α-acyloxy carboxamides as efficient apoptotic inducers via caspase-3/7 dependent mechanism with highly selective anticancer profiles. The adopted design rationale relied on excluding structural alerts of previous leads, while merging various pharmacophoric motifs of natural and synthetic caspase activators via optimized one-pot Passerini reaction conditions. The prepared compounds resulting from Passerini reaction were screened for their cytotoxic activities against colorectal Caco-2 and liver HepG-2 cancer cells compared to normal fibroblasts utilizing MTT assay. Notably, all compounds exhibited promising low-range submicromolar IC50 against the studied cancer cell lines, with outstanding tumor selectivity (SI values up to 266). Hence, they were superior to 5-fluorouracil. Notably, 7a, 7g, and 7j conferred the highest potencies against Caco-2 and HepG-2 cells and were selected for further mechanistic studies. Caspas-3/7 activation assay of the hit compounds and flow cytometric analysis of the treated apoptotic cancer cells demonstrated their significant caspase activation potential (up to 4.2 folds) and apoptotic induction capacities (up to 58.7%). Further assessment of Bcl2 expression was performed being a physiological caspase-3 substrate. Herein, the three studied Passerini adducts were able to downregulate Bcl2 in the treated Caco-2 cells. Importantly, the mechanistic studies results of the three hits echoed their preliminary MTT antiproliferative potencies data highlighting their caspase-3 dependent apoptotic induction. Finally, the in silico predicted physicochemical and pharmacokinetic profiles, as well as ligand efficiency metrics were drug-like.


Materials and Equipment.
Commercially available solvents and reagents were purified according to the standard procedures. Dried glassware was used for all reactions. 1 H-NMR data were recorded by JEOLJNM ECA 500 MHz. The deuterated solvent was used as an internal deuterium lock. 13 C-NMR spectra were measured using 125 MHz UDEFT pulse sequence and broad-band proton decoupling. Chemical shifts were reported in parts per million (δ/ppm).
Internal reference for NMR spectra is tetramethylsilane at 0.00 ppm. Coupling constants are recorded in Hertz

MTT assay
Normal human lung fibroblast Wi-38 cell line was used to detect cytotoxicity of the studied compounds.
Wi-38 cell line was cultured in DMEM medium-contained 10% fetal bovine serum (FBS), seeded as 5x10 3  The anticancer effect of the above-mentioned compounds was assayed using human colon cancer cell line (Caco-2) and liver cancer cell line (HepG-2) that were cultured in DMEM (Lonza, USA) supplemented with 10% FBS. All cancer cells (5x10 3 cells/well) were seeded in sterile 96-well plates. After 24h, serial concentrations of the tested compounds and 5-fluorouracil (Fu) were incubated with two cancer cell lines for 72 h at 37ºC in 5% CO 2 incubator. MTT method was done as described above. The half maximal inhibitory concentration (IC 50 ) values were calculated using the Graphpad Instat software. Furthermore, cellular morphological changes before and after treatment with the most effective and safest anticancer compounds were investigated using phase contrast inverted microscope with a digital camera (Olympus, Japan). S17

Caspase-3/7 activation assay
The caspase 3/7 activation was assessed, as a key indicator in caspase-dependent apoptosis, using ApoONE® Caspase-3/7 kit following the manufacturer's instructions. This kit used a nonfluorescence substrate that was cleaved by caspases of the most active-treated cancer cells, relative to the untreated cells, resulting in the generation of the fluorescence signals of Rhodamine 110. Thesesignal were measured at 490 nm excitation and 520 nm emission using the fluorescence microplate reader (BMG LabTech, Germany).

Real-time Quantitative PCR Analysis of Bcl2
Colon cancer cell line (Caco-2) was incubated with the most effective anticancer compounds, at 0.06 µM, for 72 h in 5% CO 2 incubator. RNAs of untreated and treated cancer cells were extracted using Gene JET RNA purification kit (Thermo Scientific, USA). Then cDNAs were synthesized using cDNA Synthesis Kit (Thermo Scientific, USA). Real time PCR was performed using SYBR green master mix and specific primers (Forward/Reverse) as shown in Table 1. The thermal cycling parameters were pre-denaturation, followed by 40 amplification cycles of 1 min at 95°C and 30 s at 60 °C and at 72°C for 30 sec. GAPDH mRNA was quantified to adjust the amount of mRNA in each sample. The 2 −ΔΔCT equation was used to estimate change in gene expressions before and after treatment of cancer cells [2,3]. Reverse: 5′-TCTTCAGAGACAGCCAGGAGAAAT-3′

Flow cytometric analysis of apoptosis
The most effective compounds were selected to investigate their proapoptotic effect by incubation with human colon cancer cell line (Caco-2) for 72 h, at their minimum IC 50 doses. After trypsinization, the untreated and treated cancer cells were stained with fluorescein isothiocyanate (FITC)-annexin V/ propidium iodide (PI) for 15 min. After washing with PBS, the annexin-stained apoptotic population was quantified using flow cytometry at FITC signal detector (FL1) against the phycoerythrin emission signal detector (FL2) [4].