Appended Aromatic Moieties in Flexible Bis-3-chloropiperidines Confer Tropism against Pancreatic Cancer Cells

Abstract

Nitrogen mustards (NMs) are an old but still largely diffused class of anticancer drugs. However, spreading mechanisms of resistance undermine their efficacy and therapeutic applicability. To expand their antitumour value, we developed bis-3-chloropiperidines (B-CePs), a new class of mustard-based alkylating agent, and we recently reported the striking selectivity for BxPC-3 pancreatic tumour cells of B-CePs bearing aromatic moieties embedded in the linker. In this study, we demonstrate that such tropism is shared by bis-3-chloropiperidines bearing appended aromatic groups in flexible linkers, whereas esters substituted by aliphatic groups or by efficient DNA-interacting groups are potent but nonselective cytotoxic agents. Besides, we describe how the critical balance between water stability and DNA reactivity can affect the properties of bis-3-chloropiperidines. Together, these findings support the exploitation of B-CePs as potential antitumour clinical candidates.

Keywords: DNA alkylation; anticancer agents; nitrogen mustards; pancreatic cancer; spheroids.

Figure 1

Chemical structures of a) chlorambucil and melphalan and of the analysed bis‐3‐chloropiperidines bearing b) linear alkyl and c) Lys ester/amide linkers.

Scheme 1

Synthesis of new bis‐3‐chloropiperidine derivatives 6 and 8. a) Boc₂O, 1 M NaOH, H₂O/dioxane (1 : 1), RT, 12 h; b) ClCO₂Et, NEt₃, benzyl amine, dioxane, RT, 12 h; c) TFA, CH₂Cl₂, 0 °C to RT, 3 h; d) 2,2‐dimethoxypropane, conc. HCl, MeOH, reflux to RT, 15 h; e) 2,2‐dimethylpent‐4‐enal, ^[5] NaBH(OAc)₃, AcOH, dry CH₂Cl₂, 0 °C to RT, 12 h; f) NCS, dry CH₂Cl₂, 0 °C to RT, 2.5–3.5 h; g) cat. TBAI, dry CHCl₃, 60 °C (oil bath temperature), 2 h, (inseparable diastereomeric mixture).

Figure 2

Hydroxylation of B‐CePs. Time‐followed formation of B‐CePs reaction species at 37 °C in BPE buffer pH 7.4 as detected by ESI‐MS. Graphs report the relative percentages of reaction species (whose symbols and structures are schematized in the legend) resulting from the incubation test B‐CePs in BPE buffer pH 7.4 at 37 °C for 0, 1, 5 h and overnight (O.N.) detected by ESI‐MS.

Figure 3

DNA cleavage activity of bis‐3‐chloropiperidines a) 5 versus 6 and b) 7 versus 8. The supercoiled pBR322 plasmid was incubated with increasing concentrations of test compounds at 37 °C for 3 h in BPE buffer. Lower band: supercoiled plasmid form, upper bands: open circular plasmid species. C: supercoiled pBR322 plasmid control.

Figure 4

DNA cleavage activity of bis‐3‐chloropiperidines 8–9 after overnight preincubation of compounds at 37 °C in BPE buffer (pH 7.4). Preincubated compounds were added at increasing concentrations to the pBR322 plasmid, and the mixture was incubated at 37 °C for 3 h. C: supercoiled pBR322 plasmid control.

Figure 5

Denaturing polyacrylamide gel showing concentration‐dependent cleavage of a 22‐mer double‐stranded oligonucleotide caused by bis‐3‐chloropiperidine alkylation of guanines. The 5’‐FAM‐labelled scrambled duplex oligonucleotide (4 μm), GGA TGT GAG TGT GAG TGT GAG G, was treated with indicated compounds at 37 °C in BPE buffer pH 7.4 at 5 and 50 μm for 24 h. Products were run on denaturing polyacrylamide gel 20 % in TBE buffer. C: untreated duplex oligonucleotide control.

Figure 6

BxPC‐3 cell viability upon exposure to increasing concentrations of selected compounds 7, 9 and 10 for 5 h at 37 or 4 °C, followed by a gentle rinse of wells with PBS and addition of fresh RPMI medium. The MTT assay was performed after 72 h. Average viability percentages with associated SDs from three experimental replicates are reported.