Synthesis and biological evaluation of 2-(4-methylsulfonyl phenyl) indole derivatives: multi-target compounds with dual antimicrobial and anti-inflammatory activities

Three series of 2-(4-methylsulfonylphenyl) indole derivatives have been designed and synthesized. The synthesized compounds were assessed for their antimicrobial, COX inhibitory and anti-inflammatory activities. Compound 7g was identified to be the most potent antibacterial candidate against strains of MRSA, E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii, respectively, with safe therapeutic dose. Compounds 7a–k, 8a–c, and 9a–c showed good anti-inflammatory activity with excessive selectivity towards COX-2 in comparison with reference drugs indomethacin and celecoxib. Compounds 9a–c were found to release moderate amounts of NO to decrease the side effects associated with selective COX-2 inhibitors. A molecular modeling study for compounds 7b, 7h, and 7i into COX-2 active site was correlated with the results of in vitro COX-2 inhibition assays.


Introduction
Bacterial resistance reached a dangerous level due to the misuse of antibiotics thus searching for new antimicrobial agents is a significant issue [1]. Furthermore, the administration of multiple drugs to relieve inflammation associated with a bacterial infection may have some secondary health problems and may increase adverse effects [2]. Unfortunately, few drugs possessed these two activities in a single compound. Therefore, there are continuous trails to develop a monotherapy against inflammation due to microbial infection (dual antimicrobial/ anti-inflammatory agent) with minimal adverse effects and high safety margin [3].
The nonsteroidal anti-inflammatory drugs (NSAIDs) are used as the primary remedy for pain, fever, and inflammation through inhibition of cyclooxygenase (COX) enzymes [4][5][6]. Selective COX-2 inhibitor drugs like valdecoxib I, celecoxib II and rofecoxib III relieve inflammation without any gastric side effects [7] (Fig. 1). Despite less gastric irritation of selective COX-2 inhibitors, they showed a few cardiovascular issues consisting of myocardial infarction and high blood pressure [8,9], leading to the withdrawal of both rofecoxib and valdecoxib from the market [10]. The cause of cardiovascular issues may be due to inhibition of vasodilatory prostacyclin (PGI 2 ) and an increase in the level of platelet activator thromboxane A 2 (TxA 2 ) [11]. Nitric oxide (NO) showed vasodilator activity and inhibition of platelet aggregation [12]. Accordingly, attachment of NO donor moiety to selective COX-2 inhibitors may be beneficial to overcome the cardiovascular side effects [13,14].
Additionally, indole-based indomethacin V is a potent NSAID used for the treatment of inflammatory diseases such as rheumatoid arthritis and osteoarthritis [20]. Still, due to its high selectivity for COX-1 inhibition and its acidic nature, it had an apparent ulcerogenic effect [21].
Herein, we aimed to make molecular hybridization of the indole part of indomethacin with p-methylsulfonyl phenyl part of selective COX-2 inhibitors to match the overall structure of coxibs [presence of a diaryl heterocycle bearing one sulfonamide (SO 2 NH 2 ) or methylsulfonyl (SO 2 CH 3 ) group] [22]. Keep in mind the presence of arylhydrazone derivatives at position 3 in indole with the hope to get compounds with dual antimicrobial/antiinflammatory activity (Fig. 2).
Indole-3-carbaldehyde derivatives (6a-c) were reacted with 4-substituted phenylhydrazine HCl to give hydrazone derivatives (7a-k) in good yield. The structure elucidation of hydrazone derivatives (7a-k) was based on IR, 1 H NMR, and 13 C NMR spectral data. IR spectra showed bands at 1593-1597 cm −1 for C=N and disappearance of the carbonyl absorption band at 1657-1670 cm −1 which confirm hydrazone formation. 1 H NMR spectra showed a signal at δ 8.24-8.36 ppm of hydrazone proton (H-C=N), 10.03-10.73 ppm of hydrazone NH which is D 2 O exchangeable, 12.00 ppm for NH indole which is D 2 O exchangeable and disappearance of an aldehydic proton at δ 10.00-10.04 ppm which confirm hydrazone formation. 13 C NMR spectra showed a peak at 143-149 ppm of hydrazone carbon (C=N) which confirm hydrazone formation.
On the other hand, benzimidazole derivatives (8a-c) are synthesized from the reaction of Indole-3-carbaldehyde derivatives (6a-c) with 4-chloro-o-phenylenediamine in the presence of sodium metabisulphite. IR spectra showed bands at 3272-3382 cm −1 (indole NH, benzimidazole NH) and disappearance of the carbonyl absorption band at 1657-1670 cm −1 . 1 H NMR spectra showed the disappearance of an aldehydic proton at δ 10.00-10.04 ppm and the appearance of a signal at δ (12.37-12.45) ppm of benzimidazole NH (D 2 O exchangeable) in addition to a signal at δ 12.04-12.18 ppm of indole NH (D 2 O exchangeable).
Oxime derivatives (9a-c) resulted from the reflux of the reaction of Indole-3-carbaldehyde derivatives (6a-c) with hydroxylamine HCl. IR spectra lacked the carbonyl absorption band at 1657-1670 cm −1 and showed absorption bands at 3272-3382 cm −1 (NH, OH) and 1597 cm −1 (C=N). 1 H NMR spectra showed a singlet signal at δ 8.32 ppm of azomethine proton H-C=N, 10.89 ppm of OH (D 2 O exchangeable) in besides to signal at δ 11.79-12.04 ppm of indole NH (D 2 O exchangeable) and disappearance of an aldehydic proton at δ 10.00-10.04 ppm which confirm oxime formation.

Antimicrobial screening
The antimicrobial study was performed by CO-ADD (The Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and The University  Additionally, the oxime derivatives 9a showed moderate antibacterial activity against Gram-negative A. baumannii with growth inhibition 42.1%, while benzimidazole derivatives (8a-c) showed weak antibacterial activity.
On the other hand, all compounds have weak antifungal activity against C. albicans and C. neoformans var. grubii.
Minimal inhibitory concentrations (MIC µg/mL) measurements were performed for compounds with significant microbial growth inhibition (7a, 7g, and 7i) using ceftriaxone and amphotericin B as a reference drug for antibacterial and antifungal activity, respectively.
As shown in Table 2, compounds 7a, 7g and 7i have the best antibacterial activity comparable to that of ceftriaxone against MRSA, E. coli, K. pneumoniae, P. aeruginosa and A. baumannii, respectively.
The safety margin for the active compounds to human cells was determined through cytotoxicity against human embryonic kidney cell line and hemolysis of human red blood cells. The tested compounds 7a, 7g, and 7i were tolerated and non-toxic to human cells as the cytotoxic and hemolytic dose was higher than the therapeutic dose ( Table 2).
Compound 7a lacked general nonspecific toxicity, as the largest therapeutic dose (16 µg/mL against A. baumannii) was lower than the cytotoxic and hemolytic concentration (> 32, > 32 µg/mL respectively). Also, compound 7g showed safe therapeutic concentration against all tested microbes except for A. baumannii (4 µg/mL) which is near to cytotoxic concentration (4.2 µg/mL). Otherwise, the therapeutic concentration of compound 7i against all tested microbes was safe except for A. baumannii (4 µg/mL), which is higher than the cytotoxic concentration (2.987 µg/mL).
Generally, all tested compounds were more selective toward the COX-2 enzyme (SI = 31.29-132) than indomethacin (SI = 0.079) ( Table 3) because the size of synthesized compounds was too large to fit into the small COX-1 active site in addition to the presence of diaryl structure bearing SO 2 CH 3 or SO 2 NH 2 group.

In vitro nitric oxide release
The NO-releasing properties of compounds 9a-c were assessed in phosphate buffer of pH 7.4 with Griess Table 1 The antibacterial and antifungal activities (growth inhibition %) for compounds 7a-k, 8a-c and 9a-c at 32 µg/mL concentration   [23]. As shown in Table 5, compounds 9a-c were found to release moderate amounts of NO compared to the sodium nitrite standard solution, which may explain that the desired action of NO is mediated systemically in the biological system [24]. Therefore, the insertion of nitric oxide releasing group (oxime) can offer a method to decrease the cardiovascular side effects of selective COX-2 inhibitors.

Structure-activity relationship
Presence of arylhydrazone moiety 7a-k at position 3 of indole can possess antimicrobial activity against strains of Gram-positive MRSA bacteria and Gram-negative E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii beside their COX-2 inhibitory activity. Concerning the anti-inflammatory activity, replacement of methyl group in position 2 in indomethacin by p-methylsulfonyl phenyl moiety increased COX-2 selectivity through increasing the interaction with a hydrophobic residue of COX-2 active site [25]. In addition, the presence of two SO 2 CH 3 groups or one SO 2 CH 3 and one SO 2 NH 2 group (7b, 7c, 7d, 7e, 7h, and 7i) has COX-2 selectivity more than other derivatives.
Replacement of acidic center (CH 2 COOH) moiety in position 3 in indomethacin by benzimidazole moiety  8a-c, as a rigid isostere of p-chlorobenzoyl moiety of indomethacin, enhances the anti-inflammatory activity and COX-2 selectivity.

Molecular modeling
To understand the nature of the interaction of the most active synthesized compounds and COX-2 active site, a molecular docking study was performed using crystal structure data for COX-2 (PDB: ID 3LN1) active site obtained from protein data bank [26]. Molecular modeling of compounds 7h, 7i, 7b, and co-crystallized ligand, celecoxib was performed using MOE 2018.0101 modeling software.
The results showed that arylhydrazone derivatives 7a-k exhibited moderate to good levels of antimicrobial activity. In particular, compounds 7a, 7g, and 7i showed the highest antimicrobial activity against strains of MRSA bacteria and many species of Gram-negative with growth inhibition ranged from 85.76 to 97.76%.
These results suggested that the presence of methylsulfonyl moiety in the indole ring offered an increase in COX-2 selectivity more than the reference drug indomethacin. Also, hybridization of methylsulfonyl and arylhydrazone moiety with an indole ring, providing valuable design for the development of compounds with dual antimicrobial/anti-inflammatory activity. Many investigations are currently undergoing to determine the mechanism of action of these compounds.

Experimental
Chemistry A Thomas-Hoover capillary apparatus used to determine melting points. Infrared (IR) spectra were recorded as films on KBr plates using the FT-IR spectrometer.
Thin-layer chromatography (Merck, Darmstadt, Germany) was used for monitoring the reaction mixture, purity, and homogeneity of the synthesized compounds. UV was used as the visualizing agent. p-Methylthioacetophenone (2) and p-methylsulfonyl acetophenone (3) and 5-Un/substituted-2-(4-(methylsulfonyl) phenyl)-1H-indole (5a-c) were prepared according to a previous procedure [13]. The compounds were confirmed by matching their physical properties with the reported ones.