Bis[3,5-bis(benzylidene)-4-oxo-1-piperidinyl]amides: a novel class of potent cytotoxins

Abstract

The principal objective of this study was the examination of the theory of cytotoxic synergism. In this exploratory study, we tested the hypothesis that doubling the number of sites available for thiol alkylation in a series of candidate cytotoxins increases potency more than two-fold. This concept was verified in one-third of our comparisons using human Molt 4/C8 and CEM T-lymphocytes and murine L1210 cells. In addition, the significant potencies of various members of our compound series justified further studies. Molecular modeling revealed that relative locations of the amidic groups correlate with cytotoxicity. A potent cytotoxic compound, 1,2-bis(3,5-dibenzylidene-4-oxo-piperidin-1-yl)ethane-1,2-dione (1a) inhibited the growth of a large number of human tumor cell lines and displayed greater toxicity toward certain non-adherent cells than toward adherent neoplasms or fibroblasts. The mode of action of 1a includes induction of apoptosis and necrosis.

Figure 1

General structure of series 1. * indicates the olefinic carbon atoms that are capable of interacting with different thiol groups of a protein.

Figure 2

Structures of compounds in series 2–5.

Figure 3

Various structural features of 1 a–j as determined by molecular modeling: a) olefinic carbon atoms C^A–C^D, piperidyl nitrogen atoms N1 and N2, and carbonyl oxygen atoms O1–O4; b) interatomic distances d₁ and d₂, and bond angle ψ; and c) interatomic distances d₃–d₅.

Figure 4

Growth inhibitory effect of 10 μM 1 a or 1 b toward a number of human tumor cell lines. Cells were exposed to 1 a (■) and 1 b ( ) for 48 h using a previously described procedure.^[20]

Figure 5

Cytocidal effects of 1 a (5 μM) as determined by flow cytometry using a previously reported method.^[25] Cells are non-adherent with the exception of the adherent HeLa, Hs27, and N1H-3T3 cell lines. Each bar represents the average value of triple measurements with error bars showing standard deviations. Cells were exposed to 1 a for 22 h.

Figure 6

Evaluation of 1 b–g, and 1 i against non-adherent (JURKAT, SUP-T1) and adherent (Hs27, HeLa) cells. Concentrations of compounds used were 2.5 μM (1 f, g, and i), 5 μM (1 e), and 10 μM (1 b–d). Cells were exposed to compounds for 22 h as previously described.^[25]

Figure 7

Flow cytometry analysis of the cytotoxic effect of 1a on four non-adherent cell lines following incubation for 8 and 24 h.^[25] The exact percentage of apoptotic ( ), necrotic (■), and viable ( ) cells is indicated at the top of each bar graph. Two concentrations of 1a are shown on the x-axis. Note that twofold higher concentrations were utilized for the most resistant cell lines, SUP-T and CEM.

Scheme 1

Synthesis of series 1. Reagents and conditions: a) AcOH, dry HCl_(g), 10 % aq K₂CO₃, RT, 12 h, 80 %; b) SOCl₂, 60–65 °C, 4–5 h, 90 %; c) Et₃N, ~ 20 °C, 12 h, 48–72 %.