Synthesis of salicylic acid phenylethyl ester (SAPE) and its implication in immunomodulatory and anticancer roles

Abstract

Salicylic acid phenylethyl ester (SAPE) was synthesized by Zn(OTf)₂-catalyzed selective esterification of salicylic acid and phenylethyl alcohol and studied for its role as an immunomodulatory and anticancer agent. Low toxicity and favorable physical, Lipinski-type, and solubility properties were elucidated by ADME-tox studies. Molecular docking of SAPE against COX-2 revealed favorable MolDockscore, rerank score, interaction energy, internal pose energy, and hydrogen bonding as compared to ibuprofen and indomethacin. An average RMSD of ~ 0.13 nm for the docked complex with stable dynamic equilibrium condition was noted during the 20 ns MD simulation. A low band gap predicting a strong binding affinity at the enzyme's active site was further predicted by DFT analysis. The ester caused a reduction in the percentage of erythrocyte hemolysis and was shown to be non-cytotoxic against human lymphocytes, CaCo-2, and HepG-2 cells by the MTT assay. Moreover, it's in vitro efficacy in inhibiting COX-2 enzyme under both LPS stimulated intestinal cells and direct sequestration assays was found to be higher than salicylic acid and indomethacin. The anticancer activity of SAPE was tested on the breast cancer cell line MCF-7, and potential efficacy was exhibited in terms of decreased cell viability. Flow cytometry analysis exhibited the arrest of the cell cycle at G1/G0 and S phases, during which induction of autophagic vesicle formation and decrease in mitochondrial membrane potential was observed owing to increased ROS production. Furthermore, at these phases, the onset of apoptosis along with DNA damage was also observed. Pre-treatment with SAPE in colitis-induced Wistar rats displayed low disease activity index and reduction in the extent of intestinal tissue disruption and lipid peroxidation. A marked increase of anti-oxidative enzymes viz., catalase, GGT, and GST, and a decrease of pro-inflammatory cytokines IL-6 and TNF-α in the intestinal tissue extracts of the treated groups was noted. The results of this study have sufficient credence to support that the synthesised ester (SAPE) be considered as an anti-oxidative and anti-inflammatory compound with therapeutic potential for the effective management of cancer.

Figure 1

[i] (A) Protein–ligand interactions between SAPE and active site residues of COX-2 enzyme (B) Binding mode of SAPE at the active site residues of COX-2 enzyme (C) Protein–ligand interactions between ibuprofen and active site residues of COX-2 enzyme (D) Binding mode of ibuprofen at the active site residues of COX-2 enzyme (E) Protein–ligand interactions between indomethacin and active site residues of COX-2 enzyme (F) Binding mode of indomethacin at the active site residues of COX-2 enzyme. [ii] MD simulation showing the RMSD plot of the docked protein ligand complex showing that SAPE-COX-2 is more stable than the ibuprofen and indomethacin complex. [iii] (a) Molecular orbital depicting the HOMO (E = -6.29 eV) of SAPE calculated at DFT/B3LYP/6-31G level of theory. (b) Molecular orbital depicting the LUMO (E = -1.09 eV) of SAPE calculated at DFT/B3LYP/6-31G level of theory.

Figure 2

(A) Membrane stability assay of SAPE in erythrocytes and (B), (C) and (D) Results of Cell viability assay of SAPE at 48 h on cells of (A) PBMC, (B) Caco2 and (C) HepG2 cells with doses of (25, 50 and 100 µg/ml) or with 0.1% DMSO as the vehicle control measured by MTT based method. Graphs were presented in terms of mean ± SEM percentage of live, dead cells of the three independent sets. Data represented as mean ± S.E.M. Values were significant at ***P ≤ 0.001, **P ≤ 0.01 and *P ≤ 0.05 as compared to control.

Figure 3

(A) The inhibition of COX-2 production in inflamed CaCo-2 cells, and (B) the direct relative inhibition of COX-2 by phenyl ethyl alcohol (PEA), salicylic acid (SA), salicylic acid phenyl ethyl ester (SAPE) and indomethacin (IM).

Figure 4

(a) Results of cell viability assay of SAPE on MCF-7 cells measured by MTT based method. (b) Cell viability assay of SAPE at 48 h on cells of MCF-7 with doses of (25, 50 and 100 µg/mL) or with 0.1% DMSO as the vehicle control using trypan blue exclusion method. Graphs are presented in terms of mean ± SEM percentage of live and dead cells of the three independent sets. Values were significant at *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 as compared to control.

Figure 5

(A) Cell cycle analysis of SAPE on MCF-7 cells with doses of 25, 50, 100 µg/mL or with 0.1% DMSO as the vehicle control for 48 h of cell cycle phase distribution detected in a flow cytometer. Histogram display of DNA content viz. PI-fluorescence (x-axis) vs. counts (y-axis) and corresponding bar diagram is the representation of cell cycle phase distribution of G0/G1, S and G2/M phases. (B) Effect of SAPE on intracellular ROS levels in MCF-7 cells at 48 h were analyzed by flow cytometer with ROS specific fluorescence dye (DCFDA). Intracellular ROS levels were depicted through histogram and corresponding fold changes represented by bar graph were assessed by flow cytometry. (C) Effect of SAPE on MMP in MCF-7 cells. After that the fluorescent intensity of Rhodamine-123 was measured using flow cytometry at 488 nm (excitation) and 525–530 nm (emission) and data were depicted through histogram and corresponding fold changes represented by bar graph. Data represented as mean ± S.E.M. Values were significant at ***P ≤ 0.001, **P ≤ 0.01 and *P ≤ 0.05 as compared to control.

Figure 6

(A) Representative pictures of apoptosis assay of SAPE on MCF-7 with different doses (25, and100 µg/mL) or with 0.1% DMSO as the vehicle control for 48 h, determined by AO/EtBr dye staining; (B) Representative Comet images for DNA damage in of SAPE in MCF-7 cells detected by neutral comet assay, and quantification of the percentage of DNA damaged cells by measuring the (i) area of comet and (ii) length of comet tail with Open Comet software. Data represented as mean ± SEM. Values were significant at *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 as compared to control.

Figure 7

(A) Fluorescence microscopy for analysis of acidic vesicles and autophagosomes against SAPE treated MCF-7 cells (B) Analysis of the degree of acidity against SAPE (25, 50, 100 µg/mL) treated MCF7 cells for 48 h. (C) Analysis of autophagic vesicles formation against MCF7 cells treated with SAPE (25, 50, 100 µg/mL) for 48 h; (D) The mean fluorescent intensity of AO measured using flow cytometry at 488 nm (excitation) and 525–530 nm (emission). Data expressed as (Mean ± SEM, n = 3). Values were significant at *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 as compared to control.

Figure 8

Excised intestines and SEM images of the intestinal section from (A) RG-CF (colitis free control group without any treatment) rats, (B) RG-CI (colitis induced group without any treatment) rats, (C) RG-SP (colitis induced group treated with SAPE) rats, RG-IM (colitis induced group treated with indomethacin) rats.

Figure 9

(a) Malondialdehyde (MDA) content; (b) catalase (CAT) content; (c) γ-glutamyl transferase (GGT) activity; (d) glutathione S-transferase (GST) activity; (e) tumor necrosis factor alpha (TNF-α) content and (f) interleukin 6 (IL-6) content in the intestinal tissue extract for different treatment groups of rats (RG-CI: colitis induced group without any treatment; RG-CF: colitis free control group without any treatment; RG-SP: colitis induced group treated with SAPE; Group RG-IM: colitis induced group treated with indomethacin).