Novel non-cyclooxygenase inhibitory derivatives of naproxen for colorectal cancer chemoprevention

Abstract

A structure-based medicinal chemistry strategy was applied to design new naproxen derivatives that show growth inhibitory activity against human colon tumor cells through a cyclooxygenase (COX)-independent mechanism. In vitro testing of the synthesized compounds against the human HT-29 colon tumor cell line revealed enhanced growth inhibitory activity compared to the parent naproxen with 3a showing IC₅₀ of 11.4 μM (two orders of magnitude more potent than naproxen). Selectivity of 3a was investigated against a panel of three tumor and one normal colon cell lines and showed up to six times less toxicity against normal colonocytes. Compound 3a was shown to induce dose-dependent apoptosis of HT116 colon tumor cells as evidenced by measuring the activity of caspases-3 and 7. None of the synthesized compounds showed activity against COX-1 or COX-2 isozymes, confirming a COX-independent mechanism of action. Compound 3k was found to have no ulcerogenic effect in rats as indicated by electron microscope scanning of the stomach after oral administration. A pharmacophore model was developed for elucidating structure-activity relationships and subsequent chemical optimization for this series of compounds as colorectal cancer chemopreventive drugs.

Keywords: Apoptosis; Chemoprevention; Colorectal cancer; Cyclooxygenase; NSAIDs; Naproxen; Pharmcophore; Ulcerogenicity.

Fig. 1

2D representation of naproxen at the COX-2 enzyme pocket (PDB code: 3nt1) showing crucial hydrogen bonding between naproxen caroboxylic acid and residues at the enzyme pocket. Hydrogen bonds are shown as green arrows, hydrophobic amino acids are shown in green, and polar amino acids are shown in purple (Color figure online)

Fig. 2

Superimposition of 3a (green, ball and stick) and naproxen (yellow, ball and stick) both docked at the active site of COX-2 enzyme. Steric clashes were observed between 3a side chain and residues at the enzyme pocket are shown in red contour (Color figure online)

Fig. 3

Dose-dependent growth inhibitory activity of, a naproxen derivative 3a, b naproxen, and c celecoxib on human colon tumor cell lines, HT-29, SW480, and HCT116 and the normal human colonocyte cell line, NCM460 after 72 h of treatment. Growth inhibitory activity was determined by measuring viable cell number using the Cell Titer Glo ATP assay

Fig. 4

Apoptosis induction of HCT116 cells by 3a after 48 h of treatment

Fig. 5

Scanning electromicrographs of rat stomach specimen following a daily oral dose for 4 days: a naproxen; b compound 3k; and c control

Fig. 6

Top ranked HipHop pharmacophore model showing intra feature distances (Å). Features are color coded as follows: hydrogen bond acceptor; green and hydrophobic; cyan (Color figure online)

Fig. 7

Compound 3a (stick) mapped onto the generated pharmacophore model

Scheme 1

Synthesis of the designed naproxen amino acid derivatives (3a–m). Reagents and Conditions: (i) Ethyl chloroformate, TEA, DCM, 0 °C, 30 min; (ii) amino acid ester, DCM, RT, 24 h