Behind the Mirror: Chirality Tunes the Reactivity and Cytotoxicity of Chloropiperidines as Potential Anticancer Agents

Abstract

The pressing demand for sustainable antitumor drugs prompted us to investigate 3-chloropiperidines as potential mustard-based anticancer agents. In this study, an explorative set of variously decorated monofunctional 3-chloropiperidines (M-CePs) was efficiently synthesized through a fast and affordable route providing high yields of pure racemates and enantiomers. Consistently with their reactivity, M-CePs were demonstrated to alkylate DNA in vitro. On a panel of carcinoma cell lines, M-CePs exhibited low nanomolar cytotoxicity indexes, which showed their remarkable activity against pancreatic cancer cells and in all cases performed strikingly better than the chlorambucil control. Very interestingly, stereochemistry modulated the activity of M-CePs in unexpected ways, pointing to additional molecular mechanisms of action beyond the direct damage of genomic DNA. This encouraging combination of efficacy and sustainability suggests they are valid candidates for anticancer agent development.

Figure 1

Chemical structures of (a) the racemic monofunctional 3-chloropiperidines and (b) the enantiomerically pure compounds.

Scheme 1. Synthesis of 3-Chloropiperidine 3 (I) and Enantiomers d-1 and l-1 (II)

Conditions: (a) benzylic amine, NaBH(OAc)₃, CH₂Cl₂, 0 °C; (b) NCS, CH₂Cl₂, 0 °C; (c) catalytic Bu₄NI, CHCl₃, 60 °C; (d) LiAlH₄, THF; (e) butyl iodide, K₂CO₃, THF, reflux; (f) SOCl₂, pyridine, CH₂Cl₂, 0 °C.

Figure 2

DNA cleavage activity of 3-chloropiperidines after incubation for (a) 2 h and (b) 18 h. The supercoiled pBR322 plasmid (120 ng) was incubated with increasing concentrations (5 and 50 μM) of the compounds (1, 2, 3, d-1, l-1, and chlorambucil) at 37 °C for 2 and 18 h in BPE buffer. Cleavage of supercoiled DNA (SC) into its open circular (OC) and linearized (L) forms was analyzed by 1% agarose electrophoresis in TAE buffer. DNA was stained with GelRed. C, supercoiled plasmid control; Chl, chlorambucil.

Figure 3

ESI-MS spectra of the reaction mixture obtained by incubating the duplex dsDNA with compounds 1 and 2 both at 50 μM for 2 h (a and b, respectively) and 18 h (c and d, respectively) at 37 °C. The analyses were performed in 150 mM ammonium acetate. Lower-intensity signals near free/bound species consist of sodium and ammonium adducts. For the sake of clarity, the spectra show only the region containing the 6– charge state of the dsDNA. dsDNA, double bonds (=); adduct, red angles; base hydrolysis, winged V’s.