Synthesis of Halogenated 1,5-Diarylimidazoles and Their Inhibitory Effects on LPS-Induced PGE2 Production in RAW 264.7 Cells

A series of halogenated 1,5-diarylimidazole compounds were synthesized and their inhibitory effects on LPS-induced PGE2 production in RAW 264.7 cells were evaluated. A wide variety of 2,4-, 4-, and 2-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles were synthesized for SAR study via two different pathways. Overall, 4-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles, regardless of the species of halogen, exhibited very strong inhibitory activities of PGE2 production. Among them, 4-chloro-5-(4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazole (3, IC50 3.3 nM ± 2.93), and 4-chloro-5-(4-chlorophenyl)-1-(4-methylsulfonylphenyl)imidazole (13, IC50 5.3 nM ± 0.23) showed the best results.


Introduction
Cyclooxygenase (COX), is one of the key proinflammatory enzymes which catalyzes the conversion of arachidonic acid to prostaglandins (PGs). Cyclooxygenase exists in two isoforms, COX-1 and COX-2. Among them, COX-2 is inducible and known as a major isoform found in inflammatory lesions [1].
During the past two decades, extensive efforts have been made on the development of selective COX-2 inhibitors by modifying the central heterocycle scaffold of tricyclic lead compounds. A wide variety of 5-member heterocycle scaffolds can serve as scaffolds for COX-2 inhibitors, such as pyrazole (Celecoxib), thiazole (DUP 697), furanone (Rofecoxib), isoxazole (Valdecoxib), imidazole (Cimicoxib), and pyrrole ( Figure 1) [2][3][4][5][6][7][8][9][10]. It was well acknowledged from previous SAR studies that the nature of the central heterocycle scaffold is very important for the bioactivity as well as selectivity. Therefore, the design of new compounds based on alternative structural scaffolds has been demanded.
We previously reported a series of 1,5-diarylimidazole analogs, along with their inhibitory activity toward COX-2 enzyme [11][12][13]. Through a series of studies, we found that 1,5-diarylimidazole analogs with the 4-methylsulfonylphenyl group at 1-or 5-position are important for their inhibitory activities against COX-2 catalyzed PGE 2 production, but its position can be exchanged without significant reduction of bioactivity. We also observed that halogen substitution at the 2-or 4-position of 1,5-diarylimidazole ring significantly influenced bioactivity. These results led us to conduct a supplementary SAR study of 1,5diarylimidazoles halogenated at 2-or/and 4-position(s) of the imidazole ring ( Figure 2).

RAW 264.7 Cell Culture and Measurement of PGE2 Concentrations
RAW 264.7 cells obtained from the American Type Culture Collection (ATCC, VA, USA) were cultured in a petri dish in DMEM supplemented with 10% FBS and 1% antibiotics under 5% CO2 at 37 °C for 3 days based on the previously described procedures [15]. Briefly, cells were plated in 96-well plates (2 × 10 5 cells/well). After pre-incubation with the test compounds for 1 h, LPS (1 μg/mL) were added and incubated for 24 h. PGE2 concentration in the medium was measured using an ELISA kit for PGE2 (Cayman Chem. Co.) according to the manufacturer's recommendation.
Cell viability was assessed with MTT assay. All tested compounds showed no or less than 10% reduction of MTT assay at the tested concentrations, indicating that they were not significantly cytotoxic to RAW 264.7 cells in the presence or absence of LPS (Supplementary Material). Therefore, the inhibition of PGE2 production by halogenated 5-aryl-1-(methylsulfonylphenyl)imidazoles might not be associated with their cytotoxicity.

RAW 264.7 Cell Culture and Measurement of PGE 2 Concentrations
RAW 264.7 cells obtained from the American Type Culture Collection (ATCC, VA, USA) were cultured in a petri dish in DMEM supplemented with 10% FBS and 1% antibiotics under 5% CO 2 at 37 • C for 3 days based on the previously described procedures [15]. Briefly, cells were plated in 96-well plates (2 × 10 5 cells/well). After pre-incubation with the test compounds for 1 h, LPS (1 µg/mL) were added and incubated for 24 h. PGE 2 concentration in the medium was measured using an ELISA kit for PGE 2 (Cayman Chem. Co.) according to the manufacturer's recommendation.
Cell viability was assessed with MTT assay. All tested compounds showed no or less than 10% reduction of MTT assay at the tested concentrations, indicating that they were not significantly cytotoxic to RAW 264.7 cells in the presence or absence of LPS (Supplementary Material). Therefore, the inhibition of PGE 2 production by halogenated 5-aryl-1-(methylsulfonylphenyl)imidazoles might not be associated with their cytotoxicity.
To examine the selectivity of halogenated imidazole analogs towards COX-2 in comparison to COX-1, we conducted COX Inhibitor Screening Assay (Cayman, MI, USA) using compound 3, one of lead compounds. We evaluated its potency and selectivity of inhibition in vitro. It was found that compound 3 showed a significant inhibitory effect on COX-2 activity at 100 nM, whereas it did not show any inhibitory effect on COX-1 ( Figure  6). To examine the selectivity of halogenated imidazole analogs towards COX-2 in comparison to COX-1, we conducted COX Inhibitor Screening Assay (Cayman, MI, USA) using compound 3, one of lead compounds. We evaluated its potency and selectivity of inhibition in vitro. It was found that compound 3 showed a significant inhibitory effect on COX-2 activity at 100 nM, whereas it did not show any inhibitory effect on COX-1 ( Figure 6).

Conclusions
In our study, a series of halogenated 1,5-diarylimidazole compounds were d synthesized, and their inhibitory effects on LPS-induced PGE2 production in RA

General
All chemicals, solvents, and reagents were obtained from commercial suppliers and used without further purification, unless specified. All solvents used for reaction were freshly distilled from proper dehydrating agent under nitrogen gas. Reactions were monitored by thin-layer chromatography performed on glass-packed silica gel plates (60F-254) (Merck, Darmstadt, Germany) with UV light. Flash column chromatography was performed with silica gel (100-200 mesh). 1 H-NMR (300 MHz) was recorded on Bruker DPX 300 spectrometers, (Bruker, Billerica, MA, USA) and 13 C-NMR was recorded on Bruker Avance Neo 600 spectrometers (Bruker, Billerica, MA, USA), fully decoupled, and chemical shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane as an internal standard. Peak splitting patterns are abbreviated as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), and m (multiplet). Lowresolution mass spectra (LRMS) were recorded on an API 3200 MS system of AB SCIEX. (AB Sciex, Flamingham, MA, USA) High resolution mass spectra (HRMS) were recorded on JEOL JES-X320 electron spin resonance spectrometer. Analytical thin-layer chromatography (TLC) was performed using a commercial glass plate with silica gel 60F254 purchased from Merck. Chromatographic purification was carried out by flash chromatography using Kieselgel 60 (230-400 mesh, Merck, Darmstadt, Germany).

General Procedure for Synthesis of Intermediates Ia and Ib
To the solution of imine (4 mmol) in methanol (MeOH) and dimethoxyethane (20 mL, v/v = 1/2) was added anhydrous potassium carbonate (1.66 g, 12 mmol) and tosylmethyl isocyanide (0.94 g, 4.8 mmol). The reaction mixture was refluxed for 1 day. The solvent was removed, and the residue was extracted with dichloromethane (DCM). The organic layer was washed with brine, dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography with hexane-ethyl acetate (EtOAc) yielded the corresponding imidazole intermediates, respectively. To the solution of intermediates (Ia and Ib, 1 mmol) in DCM (10 mL) was added, at 0 • C, 3-chloroperbenzoic acid (0.56 g, 2.5 mmol). The mixture was stirred for 2 h, followed by the addition of more DCM, washing with aqueous Na 2 S 2 O 3 , NaHCO 3 , and brine, drying over magnesium sulfate, and concentration. The residue was purified by silica gel column chromatography with hexane-EtOAc.  To the solution of 5-aryl-1-(4-methylthiophenyl)imidazoles (Ia or I, 0.4 mmol) in tetrahydrofuran (THF) 3 mL of lithium bis(trimethylsilyl)amide (1 M in THF, 1.2 mL) was added dropwise at −20 • C. The mixture was stirred for 0.5 h, then solution of NCS or NBS (1.6 mmol) in THF (3 mL) was added. The reaction mixture was stirred for 0.5 h at −20 • C and 6 h at room temperature. Saturated aqueous NH 4 Cl was added to the mixture and extracted with ethyl acetate. The organic layer was washed with aqueous NaHSO 3 and brine, dried over magnesium sulfate, and concentrated under vacuum. Following oxidation of crude 2-halogenated 5-aryl-1-(4-methylthiophenyl)imidazole with 3-chloroperbenzoic acid followed by silica gel column chromatography yielded pure 2halogenated 5-(4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazoles.

MTT Assay for Cytotoxicity
RAW 264.7 cells, 2 × 10 5 cells/mL with 10% fetal bovine serum cell culture medium, and 1 mL of cell suspension were added to each hole of a 24-well plate for 24 h in a 37 • C incubator. Cytotoxicity was determined by MTT assay after treating various concentrations of SAMPLE (1, 10, 100 nM and 1, 10 µM) for another 24 h after overnight incubation. The formed formazan crystals in the cells were dissolved by DMSO, followed by measurement at 540 nm.

Measurement of NO and PGE 2 Production
RAW 264.7 cells (2 × 10 5 cells/mL) were seeded onto a 24-well plate and then incubated with/without LPS (100 ng/mL) in the presence or absence of SAMPLE (1, 10, 100 nM and 1, 10 µM) for 24 h. Nitrite levels of cellular supernatants were measured using the Griess reaction and estimated to reflect the concentration of NO. The absorbance was measured at the wavelength of 540 nm using the microplate reader. In all experiments, fresh culture media were used as blanks. The levels of nitrite in the samples were determined using the standard curve of sodium nitrite. PGE 2 concentration in the medium was measured using an ELISA kit for PGE 2 (R&D Systems, Minnesota, MN, USA).

Effects of Samples on the COX-1 and COX-2 Activity
Samples were evaluated for its potency and selectivity of inhibition in vitro using COX Inhibitor Screening Assay (Cayman, Michigan, MI, USA). Recombinant COX-1 (ovine) or COX-2 (human) proteins were pre-incubated with Compound 3 for 10 min at 37 • C. The reaction was started by the addition of 100 µM arachidonic acid and allowed to proceed for 2 min. The reaction was terminated by the addition of an HCl solution containing SnCl 2 . The COX activity assay directly measures PGF2α produced by SnCl 2 reduction of COX-derived PGH 2 . The prostanoid product is quantified via EIA. As control inhibitors for COX-1 or COX-2, SC-560 (100 nM) or Dup-697 (100 nM) were used.

Statistical Analysis
Results are expressed as the mean ± SD of triplicate experiments with similar patterns. Statistically significant values were compared using ANOVA and Dunnett's post hoc test, and P values of less than 0.05 were considered statistically significant. # p < 0.05 compared with the control group, and * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-stimulated group.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Acknowledgments:
The authors thank to Drug Development Research Institute and Central Laboratory of Kangwon National University for the use of analytical instruments and bioassay facilities.

Conflicts of Interest:
The authors declare no conflict of interest.