Abstract A64: Ester-protected hydroxybenzyl phosphates (EHBP) inhibit the growth of various cultured human cancer cells: Evidence of a tissue type-independent effect
Authors: Kodela Ravinder , Chattopadhyay Mitali , Nath Niharika , Cieciura Lucyna , Pospishill Liliya , Boring Daniel , Crowell James , Kashfi Khosrow 
Published: May 2012

Abstract
A64

Introduction
Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) consisting of a conventional NSAID to which an NO releasing moiety -ONO2 is covalently attached through a spacer have emerged as a new class of pharmaceutical agents. For several years we have been exploring the mechanism of action of NO-NSAIDs and in particular NO-aspirin (NO-ASA), with emphasis on their application to cancer. Our structure-activity studies with NO-ASA indicated that NO was pivotal for biological activity. However, careful re-examination regarding the contribution to the overall biological effect of each of the three structural components of NO-ASA, led to the surprising conclusion that the NO-releasing moiety was not required for the observed biological effects. Rather, it was the spacer that was responsible for the biological actions of NO-ASA. The NO-releasing moiety was acting as a leaving group leading to the generation of a reactive quinone methide (QM) intermediate which behaved as a powerful electrophile. The ASA component made no biological contribution (BBRC 2007:358, 1096-1101). We therefore synthesized a series of ortho, para, and meta EHBPs, in which the -ONO2 leaving group was replaced by a substituted phosphate as the leaving group and the ASA was replaced by an acetate. We also incorporated electron donating/withdrawing groups about the aromatic spacer in order to evaluate the effect of substitution on QM formation/stability and biological activity.

Methods
EHBPs as potential drugs: para-, ortho-, and meta-acetyloxybenzyl diethyl phosphate (Agents 1-3 respectively), p-acetyloxy-3-methylbenzyl diethyl phosphate (Agent 4), and p-acetyloxy-2-chlorobenzyl diethyl phosphate (Agent 5) were synthesized and purified at our lab with 1H-NMR verifying the structures. In some experiments we also used NO-ASA for comparison. Cell lines: Human colon, breast, leukemia, and pancreatic cancers. Growth inhibition: colorimetric MTT assay kit. Cell cycle phase distribution: flow cytometry. Apoptosis: subdiploid (sub-G0/G1) peak in DNA content histograms.

Results
Agent 1 was very effective in inhibiting the growth of all cell lines with IC50s of 0.6-0.8 µM, in contrast, p-NO-ASA had significantly higher IC50s, 10-20 µM. Agent 2 was less potent than agent 1 in all cell lines with IC50s of 63-155 µM, presumably because of steric interference between the diethyl phosphate and the acetyloxy groups making it difficult for the QM to form. Agent 3 was the least effective, with IC50s of 162-451 µM, this agent is comparable in potency to m-NO-ASA, both of which do not form QMs. Agent 4 containing an electron donating group, was extremely potent, IC50s being 0.3-0.5 µM. Agent 5 containing an electron withdrawing group had slightly higher IC50s, 0.5-1.0 µM. Agent 1 induced dose-dependent apoptosis, which was significantly higher compared to p-NO-ASA. Agent 1 dose-dependently altered the distribution of the cells in the cell cycle, causing an arrest in G0/G1.

Conclusions
EHBPs inhibited the growth of various human cancer cells, indicating a tissue type independent effect. They exercise pleiotropic effects involving cell death as well as cell cycle phase transitions. These results raise the possibility that EHBPs possess chemotherapeutic activity against a wide variety of human cancers.
Citation Information: Cancer Prev Res 2008;1(7 Suppl):A64.