Novel Oxadiazole-Based Bioisostere of Caffeic Acid Phenethyl Ester: Synthesis, Anticancer Activity, and Inhibition of Lipoxygenase Product Biosynthesis

Abstract

Caffeic acid phenethyl ester (1), a honeybee propolis component, possesses many bioactive properties, making it a useful scaffold for drug research. Further, CAPE (1) is a more effective inhibitor of the biosynthesis of 5-lipoxygenase (5-LO) products compared to Zileuton, the only clinically-approved direct 5-LO inhibitor. However, CAPE (1) suffers from a poor metabolic profile, being rapidly metabolized to caffeic acid (CA). In this study, we synthesized and performed several biological assays on a new bioisostere of CAPE (1) possessing a 1,2,4-oxadiazole ring. The new bioisostere (OB-CAPE (5)) has a similar antiproliferative effect to CAPE (1) on NCI-60 cancer cell lines and maintains the activity of CAPE (1) as an inhibitor of the biosynthesis of 5-, 12- and 15-LO products and as an iron chelator. In human polymorphonuclear leukocytes, OB-CAPE (5) inhibits the biosynthesis of 5-LO products with an IC₅₀ of 0.93 µM compared to 1.0 µM for CAPE (1). Both compounds have similar antioxidant activity, with IC₅₀ values of 1.2 µM for OB-CAPE (5) and 1.1 µM for CAPE (1). The new hydrogen bond predicted for the oxadiazole ring and the GLN363 amino acid in the 5-LO active site may explain the small improvement in the affinity of OB-CAPE (5) for the protein compared to CAPE (1). Finally, stability studies in human plasma reveal that OB-CAPE (5) is 25% more stable than CAPE (1). Therefore, the increase in stability associated with the replacement of the ester function with its bioisostere, while maintaining the anti-inflammatory and anticancer properties of CAPE (1), suggests that OB-CAPE (5) may be a comparable yet more stable candidate for in vivo studies in disease models.

Keywords: anti‐leukotriene therapy; bioisosteres; caffeic acid phenethyl ester; inflammation.

FIGURE 1

Structures of compounds used as reference throughout this study.

SCHEME 1

Synthesis of compound OB‐CAPE (5). i) HBTU, DIEA, DMF, rt, 24 h.

FIGURE 2

NCI60 Cancer cell score of CAPE (1) or OB‐CAPE (5) after exposure to a single dose (10 µM) of each compound compared to an untreated control. For each cancer cell line, viability results are presented as mean values of duplicate analyses.

FIGURE 3

Inhibition of the biosynthesis of 5‐LO products by Zil (3), CAPE (1) and OB‐CAPE (5) (1 μM) in HEK293 cells transfected with 5‐LO/FLAP. Values are the means ± SD of three independent experiments, each performed in duplicate. The graph shows values that are normalized to diluent (DMSO) controls. Values without at least one common superscript letter are significantly different (p < 0.05) as determined by two‐way ANOVA of the non‐normalized data with subsequent Tukey's multiple comparisons test.

FIGURE 4

Inhibition of the biosynthesis of 12‐LO (left) and 15‐LO (right) products by Baic, CAPE (1) and OB‐CAPE (5) (1 μM) in 12‐LO and 15‐LO transfected HEK293 cells. Values are the means ± SD of three independent experiments, each performed in duplicate. The graph shows values that are normalized to diluent (DMSO) controls. Values without at least one common superscript letter are significantly different (p < 0.05) as determined by two‐way ANOVA of the non‐normalized data with subsequent Tukey's multiple comparisons test.

FIGURE 5

π‐π interactions (left) and H bond (right) of OB‐CAPE (5) with Gln363 amino acid within the 5‐LO active site.

FIGURE 6

Absorbance ratios of compounds and Fe (III) adjusted to background signal of each respective compound at 400 nm. Data are represented as the mean ± SD of three independent experiments. For clarity, error bars for CAPE point upwards, whereas error bars for OB‐CAPE point downwards. Variation was small in CA samples, such that no error bars were generated. Two‐way ANOVA analysis with Tukey's multiple comparisons test was performed; curves that do not share a common letter are considered statistically different (p < 0.0001).

FIGURE 7

Stability of compounds in human plasma. Data are presented as the means ± SD of four independent experiments, each performed in duplicate. A 5 µM dose of each compound was incubated with human plasma for up to 24 h. Following 2‐way repeated measures ANOVA analyses, there were significant effects of time (F (2, 6) = 8.135; p = 0.02), compounds (F (1, 3) = 57.32; p = 0.005) and their interaction (F (2, 6) = 58.21; p = 0.0001). At different time points, values without common superscripts within compounds are considered statistically significant, and values with an asterisk * are different from OB‐CAPE (5) at the same time point as determined using Tukey's multiple comparisons test (p < 0.05).