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(2020) 14:23 Shaker et al. BMC Chemistry https://doi.org/10.1186/s13065-020-00675-5 RESEARCH ARTICLE BMC Chemistry Open Access Synthesis and biological evaluation of 2‑(4‑methylsulfonyl phenyl) indole derivatives: multi‑target compounds with dual antimicrobial and anti‑inflammatory activities Ahmed M. M. Shaker1*, Eman K. A. Abdelall2, Khaled R. A. Abdellatif2,3 and Hamdy M. Abdel‑Rahman1,4 Abstract 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 antiinflammatory 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. Keywords: Antimicrobial, Indomethacin analogues, COX-2 inhibitors, Nitric oxide, Anti-inflammatory 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 *Correspondence: ph.ahmedshaker@yahoo.com 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, Beni‑Suef 62517, Egypt Full list of author information is available at the end of the article inflammation through inhibition of cyclooxygenase (COX) enzymes [4–6]. 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 ­(PGI2) and an increase in the level of platelet activator thromboxane A ­ 2 ­(TxA2) [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]. A lot of biologically aryl hydrazone derivatives with antimicrobial activity are found in many literatures [15–17] which include nitrofurantoin IV [18, 19]. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shaker et al. BMC Chemistry (2020) 14:23 Page 2 of 15 Fig. 1 Chemical structures of selective cyclooxygenase-2 (COX-2) inhibitor drugs (I, II, III) 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 (­SO2NH2) or methylsulfonyl ­(SO2CH3) 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). Results and discussion Chemistry The compounds were synthesized through a series of reactions illustrated in Scheme 1, 2. The reaction of p-methylsulfonyl acetophenone (3) with 4-un/substituted phenylhydrazine HCl under Fischer indole synthesis conditions yielded indole derivatives (5a–c) that are converted to indole-3-carbaldehyde derivatives (6a–c) by Vilsmeir Haack’s formylation reaction using ­POCl3 and DMF (Scheme 1). IR spectra for compounds 6a–c showed significant bands at 3205–3320 cm−1 of indole NH, 1657–1670 cm−1 of C=O and 1150, 1300 cm−1 of S ­ O2. 1H NMR spectra showed a signal at δ 10.00–10.04 ppm of an aldehydic proton (H-C=O), 3.17–3.21 ppm of ­SO2CH3 and 12.92– 12.62 ppm of indole NH which is ­D2O exchangable. 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 13C 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. 1H 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 ­ D2O exchangeable, 12.00 ppm for NH indole which is ­D2O exchangeable and disappearance of an aldehydic proton at δ 10.00–10.04 ppm which confirm hydrazone formation. 13C 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. 1H 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 ­(D2O exchangeable) in addition to a signal at δ 12.04–12.18 ppm of indole NH ­(D2O 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). 1H NMR spectra showed a singlet signal at δ 8.32 ppm of azomethine proton H-C=N, 10.89 ppm of OH ­(D2O exchangeable) in besides to signal at δ 11.79– 12.04 ppm of indole NH ­(D2O exchangeable) and disappearance of an aldehydic proton at δ 10.00–10.04 ppm which confirm oxime formation. Biological evaluation 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 Shaker et al. BMC Chemistry (2020) 14:23 Page 3 of 15 Fig. 2 Hybridization of chemical structures of indomethacin V, celecoxib II, nitrofurantoin IV to design indole derivatives 7a–k, 8a–c, and 9a–c of Queensland (Australia). Evaluation of all synthesized compounds for their antimicrobial activities was done against five pathogenic bacteria, methicillin-resistant Staphylococcus aureus (ATCC 43300) as Gram-positive bacteria, Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Acinetobacter baumannii (ATCC 19606) and Pseudomonas aeruginosa (ATCC 27853) as Gram-negative bacteria and antifungal activity against two pathogenic fungal strains Candida albi‑ cans (ATCC 90028) and Cryptococcus neoformans var. grubii (H99; ATCC 208821) (Table 1). Results revealed that hydrazone derivatives 7c, 7e, 7f, 7 h, and 7j have moderate antibacterial activity against Gram-negative A. baumannii with growth inhibition 43.29, 43.64, 66.69, 51.82 and 46.23%, respectively. While the hydrazone derivatives 7a, 7g, and 7i have high antibacterial activity against MRSA bacteria and E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii with growth inhibition ranged from 85.76 to 97.76%. 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 Shaker et al. BMC Chemistry (2020) 14:23 Page 4 of 15 Scheme 1 Reagents and conditions: a acetic anhydride, A ­ lCl3; b Oxone, H ­ 2O, reflux, 18 h; c 4-substituted phenylhydrazine HCl, ethanol, reflux, 4 h; d PPA, water bath, 4 h; e ­POCl3, DMF, RT, overnight 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. bau‑ mannii) 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. bau‑ mannii (4 µg/mL), which is higher than the cytotoxic concentration (2.987 µg/mL). In vitro cyclooxygenase (COX) inhibition assay The in vitro assay evaluated the ability of compounds 7a– k, 8a–c, and 9a–c to inhibit Ovine COX-1 and human recombinant COX-2. All tested compounds have weak COX-1 inhibition activity ­(IC50 = 9.14–13.2 µM) in comparison with indomethacin ­(IC50 = 0.039 µM). They also exerted potent COX-2 inhibitory activity ­ (IC50 = 0.1– 0.31 µM) with high COX-2 selectivity (SI = 132–31.29) in comparison with reference drugs, indomethacin and celecoxib. Hydrazone derivatives 7a–k showed potent COX-2 inhibitory activity ­ (IC50 = 0.10–0.31 µM) with high selectivity (SI = 132–31.29) more than other compounds. Likewise, benzimidazole 8a–c and oxime derivatives 9a–c showed good COX-2 inhibitory activity ­(IC50 = 0.13–0.35 µM) in comparison with reference drugs. 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 ­SO2CH3 or ­SO2NH2 group. Shaker et al. BMC Chemistry (2020) 14:23 Page 5 of 15 Scheme 2 Reagents and conditions: a 4-chloro-o-phenylenediamine, ­Na2S2O5, DMF, reflux, 6 h; b 4-substituted phenylhydrazine HCl, ethanol, reflux, 4–6 h.; c hydroxylamine, ethanol, few drops of pyridine, reflux, 4–6 h In vivo anti‑inflammatory activity The results listed in (Table 4) showed that compounds 7a–k, 8a–c, and 9a–c offered good anti-inflammatory activity (56.4–93.5% reduction of inflammation) after 6 h in comparison with celecoxib and indomethacin (94.7, 96.6% reduction of inflammation, respectively) after 6 h. Hydrazone derivatives (7a–k) showed good antiinflammatory activity (66.3–93.5% reduction of inflammation) after 6 h, Compounds that contained two ­SO2CH3 groups or one ­SO2CH3 and one ­SO2NH2 group (7b, 7c, 7d, 7e, 7h, and 7i) showed a reduction of inflammation by 93.5, 82.5, 78.6, 79.9, 92.7 and 90.1% after 6 h, respectively, more than other derivatives. Also, benzimidazole and oxime derivatives (8a–c, 9a–c) showed good inhibition of inflammation ranged from 56.4 to 76.2% after 6 h. Compounds 7b, 7c, 7h and 7i that showed the highest COX-2 inhibitory activity ­(IC50 = 0.1, 0.11, 0.11 and 0.1 respectively) with high selectivity (S.I. = 124.2, 103.7, Shaker et al. BMC Chemistry (2020) 14:23 Page 6 of 15 Table 1 The antibacterial and antifungal activities (growth inhibition %) for compounds 7a–k, 8a–c and 9a–c at 32 µg/mL concentration Compound No. Saa Ecb Kpc Pad Abe Caf 7a 95.76 96.48 97.64 97.76 96.66 6.28 7b 25.62 − 8.09 − 5.34 3.8 35.54 9.55 − 5.77 4.86 7c 21.88 7d 15.6 7e 7.58 7f 8.33 7g 96.15 7h 30.26 7i 95.22 7j 30.59 7k 28.25 8a 13.6 8b 11.28 3.17 − 11.49 86.42 87.53 23.59 4.13 2.54 43.64 28.66 66.69 3.5 − 7.9 94.63 85.76 4.88 7.97 51.82 25.34 96.45 94.4 96.93 94.34 15.32 12.27 − 0.85 46.23 1.91 2.23 31.42 1.79 − 45.65 − 22.34 − 28.34 − 15.51 − 25.19 − 8.38 − 10 − 13.94 8.77 − 8.78 4.62 − 3.37 − 12.05 − 2.72 − 15.45 a 43.29 34.95 − 2.24 − 0.72 10.33 11.3 − 8.11 − 22.55 8.05 − 13.86 8c 9b − 14.6 − 9.43 9a 9c 12.8 6.56 14.46 14.84 − 5.88 2.49 Cng − 64.35 − 280.7 − 110.9 − 177.2 − 59.93 − 118.8 − 57.42 − 99 − 104.5 − 80.19 − 55.44 7.71 22.42 − 292.1 13.22 − 114.4 1.65 42.1 − 288.1 9.15 9.23 7.9 33.26 2.47 1.19 − 0.83 33.66 4.88 − 254 − 119.3 − 136.1 MRSA b E. coli c K. pneumoniae d P. aeruginosa e A. baumannii f C. albicans g C. neoformans var. grubii Table 2 Minimum inhibitory concentrations (MIC µg/mL) of most active compounds 7a, 7g, 7i and reference drugs, ceftriaxone and amphotericin B Compound No. Saa Ecb Kpc Pad Abe Caf Cng CCh50 HCi10 7a 8 7g 1 ≤ 0.25 7i 2 Ceftriaxone 32 Amphotericin B NT a 8 4 16 > 32 > 32 > 32 > 32 ≤ 0.25 1 1 4 > 32 > 32 4.2 > 32 ≤ 0.25 2 2 4 > 32 > 32 2.987 > 32 0.125 16 32 32 NTj NT NT NT NT NT NT NT 1.56 1.56 NT NT MRSA b E. coli c K. pneumoniae d P. aeruginosa e A. baumannii f C. albicans g C. neoformans var. grubii h CC50 is the concentration at 50% cytotoxicity i HC10 is the concentration at 10% hemolysis j Not tested 112.7 and 132 respectively) were found to have excellent anti-inflammatory activity (edema inhibition = 93.5, 82.5, 92.7 and 90.1%, respectively) after 6 h. In vitro nitric oxide release The NO-releasing properties of compounds 9a–c were assessed in phosphate buffer of pH 7.4 with Griess Shaker et al. BMC Chemistry (2020) 14:23 Page 7 of 15 Table 3 In vitro COX-1 and COX-2 inhibition for compounds 7a–k, 8a–c, 9a–c and reference drugs Compounds COX Inhibition ­(IC50 µM) COX-1 Celecoxib Indomethacin 7a 14.80 0.039 10.32 COX-2 0.05 0.49 0.11 Selectivity ­indexa (SI) 0.079 93.81 7b 12.41 0.10 124.10 11.41 0.11 103.72 7d 10.40 0.15 69.33 7e 9.70 0.31 31.29 7f 9.73 0.17 57.23 7g 7.90 0.20 39.50 7h 12.40 0.11 112.72 7i 13.20 0.10 132 98.18 7j 10.80 0.11 8.24 0.21 8a 10.64 0.13 8b 9.41 0.15 8c 11.23 0.12 9a 10.64 0.13 9b 9.42 0.21 9c 8.24 0.24 a 39.20 81.84 62.73 93.58 81.84 44.85 34.33 Selectivity index (COX-1 ­IC50/COX-2 ­IC50) reagent [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 Compound 296 7c 7k Table 4 Anti-inflammatory activities for compounds 7a–k, 8a–c, 9a–c and reference drug in carrageen-induced rat paw edema test 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 ­SO2CH3 groups or one ­SO2CH3 and one ­SO2NH2 group (7b, 7c, 7d, 7e, 7h, and 7i) has COX-2 selectivity more than other derivatives. (Edema inhibition %) Edema thickness (mm) ± SEMa 1h Control 2.624 ± 0.255 3h 2.232 ± 0.235 6h 1.875 ± 0.181 Indomethacin 70.7 Celecoxib 68.9 7a 74.1 7b 76.1 7c 62.7 7d 51.6 7e 53.9 7f 60.5 7g 58.5 7h 75.4 7i 73.1 7j 64.3 7k 55.7 8a 52.5 8b 52.1 8c 67.9 9a 77.2 9b 66.2 9c 55.2 68.6 61.7 0.886 ± 0.077 0.435 ± 0.033 0.504 ± 0.009 a 0.768 ± 0.050 0.810 ± 0.074 0.679 ± 0.03 0.627 ± 0.045 0.978 ± 0.071 1.270 ± 0.015 1.209 ± 0.11 1.036 ± 0.009 1.088 ± 0.090 0.645 ± 0.058 0.705 ± 0.047 0.936 ± 0.064 1.162 ± 0.088 1.246 ± 0.076 1.256 ± 0.074 0.842 ± 0.062 0.598 ± 0.050 1.175 ± 0.057 96 0.075 ± 0.007 95.5 0.100 ± 0.009 88.7 0.250 ± 0.021 91.2 0.196 ± 0.017 81.2 0.419 ± 0.028 77.5 0.502 ± 0.044 71.1 0.645 ± 0.01 72.7 0.609 ± 0.03 72.3 0.618 ± 0.010 89.4 0.236 ± 0.008 94.3 0.127 ± 0.009 78.6 0.477 ± 0.027 69.1 0.689 ± 0.011 58 0.937 ± 0.046 68.1 0.712 ± 0.066 71.9 0.627 ± 0.05 61.8 0.852 ± 0.073 80.5 0.700 ± 0.024 96.6 0.075 ± 0.004 94.7 0.100 ± 0.005 92 0.151 ± 0.007 93.5 0.123 ± 0.009 82.5 0.331 ± 0.011 78.6 0.405 ± 0.018 79.9 0.381 ± 0.007 79.2 0.394 ± 0.01 67.4 0.618 ± 0.045 92.7 0.138 ± 0.006 90.1 0.187 ± 0.015 71 0.549 ± 0.013 66.3 0.638 ± 0.051 56.4 0.826 ± 0.078 76.2 0.451 ± 0.013 74.2 0.489 ± 0.032 62.3 0.714 ± 0.052 73.4 0.726 ± 0.055 Each value represents mean ± SEM (n = 4) Replacement of acidic center ­(CH2COOH) moiety in position 3 in indomethacin by benzimidazole moiety Shaker et al. BMC Chemistry (2020) 14:23 Page 8 of 15 Table 5 The amount of NO released from tested compounds 9a–c in phosphate buffer pH = 7.4 (% mol/mol) Compound No. Amount of NO released (% mol/mol) ± standardization error (in phosphate buffer PH 7.4) 1h 9a 9b 9c 0.027 ± 0.002 0.086 ± 0.001 0.061 ± 0.001 2h 0.065 ± 0.002 0.147 ± 0.003 0.130 ± 0.002 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 docking results of compounds 7h, 7i, 7b, and celecoxib were presented in (Table 6). Hydrazone derivatives 7b, 7h, and 7i have been fully fitted within COX-2 active site with high affinity (− 17.19, − 16.71 and − 16.42 kcal/mol, respectively) in assessment with celecoxib (− 14.12 kcal/mol). Compounds 7b, 7h, and 7i contained one ­SO2CH3 and one ­SO2NH2 group or two ­SO2CH3 groups that formed hydrogen bonds with different amino acids (Leu338, Arg499, Ser339, Val335, Arg106, and His75). Besides, the indole ring of compound 3h 4h 0.194 ± 0.007 0.210 ± 0.002 0.187 ± 0.001 0.165 ± 0.002 0.198 ± 0.003 0.198 ± 0.003 5h 0.138 ± 0.004 0.218 ± 0.005 0.225 ± 0.002 7h and 7i offered hydrophobic interaction with Val509 (Fig. 3, 4). Thus, the molecular docking results ensure that compounds 7b, 7h and 7i bind to COX-2 active site with the same manner of celecoxib. Conclusion Three series of 2-(4-methylsulfonylphenyl) indole derivatives 7a–k, 8a–c, and 9a–c were evaluated for their antimicrobial and anti-inflammatory activities. 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%. Regarding anti-inflammatory activity, all synthesized compounds 7a–k, 8a–c and 9a–c showed potent anti-inflammatory (56.4–93.5% reduction of inflammation after 6 h.) and selective COX-2 inhibitory activity ­(IC50 = 0.1–0.31 µM, SI = 132–31.29) more than indomethacin. Besides, oxime derivatives 9a–c showed good selective COX-2 inhibitory activity with moderate Table 6 Molecular docking data for compounds 7b, 7h, 7i and celecoxib in COX-2 active site (PDB ID: 3LN1) Compound No. Celecoxib 7b 7h 7i COX-2 Affinity (kcal/mol) Affinity kcal/mol Distance (in A ­ o) from main residue Functional group Interaction − 14.12 − 2.7 − 0.8 − 17.198 − 16.71 − 16.42 3.07 Leu338 –NH2 H-donor − 1.6 2.99 Ser339 –NH2 H-donor 3.54 Arg499 –SO2 H-acceptor − 1.5 3.18 Leu338 –SO2CH3 H-donor − 0.7 2.70 Arg499 –SO2 H-acceptor − 2.3 2.84 Arg106 –SO2 H-acceptor − 1.4 3.23 Leu338 –SO2CH3 H-donor 2.83 Arg499 –SO2 H-acceptor − 0.6 4.71 Val509 –Ph-ring H-pi − 2.7 3.36 Val335 –NH H-donor − 0.6 − 0.9 3.47 Ser339 –SO2CH3 H-donor − 4.5 2.86 His75 –SO2 H-acceptor − 1.5 2.94 Arg106 –SO2 H-acceptor − 0.9 3.77 Val509 –Ph-ring H-pi Shaker et al. BMC Chemistry (2020) 14:23 Page 9 of 15 Fig. 3 Binding of celecoxib inside COX-2 active site. a 2D interaction, the most important amino acids are shown together with their respective numbers. b The 3D proposed binding mode inside the active site of COX-2 resulted from docking in vitro nitric oxide release, which can offer valuable drug design to decrease the cardiovascular problems. The molecular modeling study ensured in vitro COX-2 inhibition assay results. Compounds 7b, 7h, and 7i fitted to a COX-2 enzyme similar to celecoxib. 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. 1 H NMR and 13C NMR spectra were measured on a Bruker Avance III 400 MHz for 1H NMR and 100 MHz for 13C NMR (Bruker AG, Switzerland) with BBFO Smart Probe and Bruker 400 AEON Nitrogen-Free Magnet, Faculty of Pharmacy, Beni-Suef University, Egypt in DMSO-d6 with TMS as the internal standard, where J (coupling constant) values are estimated in Hertz (Hz) and chemical shifts were recorded in ppm on δ scale. Microanalyses for C, H, and N were carried out on Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA) at the Microanalytical unit of Al Azhar University, Egypt and all compounds were within ± 0.4% of the theoretical values. 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. General procedure for synthesis of 5‑substituted‑2‑(4‑(methylsulfonyl) phenyl)‑1H‑indole‑3‑carbaldehyde 6a‑c A mixture of phosphorous oxychloride P ­ OCl3 (1.53 g, 10 mmol) and DMF (0.73 g, 10 mmol) was stirred for 30 min at room temperature, the solution of respective indole (1 mmol) in DMF (5 mL) was added slowly to the mixture which allowed to stir overnight. The reaction mixture was poured into ice-cold water and neutralized with 40% NaOH. The separated solid was filtered, dried and recrystallized from ethyl alcohol (yield: 70–80%). 2‑(4‑(Methylsulfonyl)phenyl)‑1H‑indole‑3‑carbalde‑ hyde (6a) Yellow solid; Yield 70%; mp 232–235 ℃; IR (KBr, cm−1) 3205 (NH), 3065–3042 (CH aromatic), 2929–2871 (CH aliphatic), 1657 (C=O), 1305, 1150 ­(SO2); 1H NMR (DMSO-d6) δ (ppm): 3.21 (s, 3H, ­SO2CH3), 7.27–7.36 (m, 2H, indole H-5, H-6), 7.57 (d, 1H, J = 8 Hz, indole H-7), 8.08 (d, 2H, J = 8.4 Hz, phenyl H-2, H-6), 8.15 (d, 2H, J = 8.4 Hz, phenyl H-3, H-5), 8.26 (d, 1H, J = 7.6 Hz, indole H-4), 10.04 (s, 1H, aldehydic H), 12.64 (s, 1H, indole NH, D ­ 2O exchangeable). Anal. Calced for ­C16H13NO3S: C, 64.20; H, 4.38; N, 4.68. Found: C, 64.48; H, 4.40; N, 4.84. Shaker et al. BMC Chemistry (2020) 14:23 Page 10 of 15 Fig. 4 Binding of compound 7b inside COX-2 active site. a 2D interaction, the most important amino acids are shown together with their respective numbers. b The 3D proposed binding mode inside the active site of COX-2 resulted from docking 5‑Methyl‑2‑(4‑(methylsulfonyl)phenyl)‑1H‑indole‑3‑car‑ baldehyde (6b) Brown solid; Yield 80%; mp 244–246 ℃; IR (KBr, cm−1) 3279 (NH), 3059–3029 (CH aromatic), 2927–2856 (CH aliphatic), 1670 (C=O), 1301, 1148 ­(SO2); 1H NMR (DMSO-d6) δ (ppm): 2.45 (s, 3H, ­CH3), 3.17 (s, 3H, S ­ O2CH3), 7.17 (d, 1H, J = 8 Hz, indole H-6), 7.46 (d, 1H, J = 8 Hz, indole H-7), 8.06–8.14 (m, 5H, indole H-4, phenyl H-2, H-3, H-5, H-6), 10.00 (s, 1H, aldehydic H), 12.62 (s, 1H, indole NH, ­D2O exchangeable). Anal. Calced for ­C17H15NO3S: C, 65.16; H, 4.82; N, 4.47. Found: C, 65.27; H, 4.68; N, 4.52. 5‑Fluoro‑2‑(4‑(methylsulfonyl)phenyl)‑1H‑indole‑3‑car‑ baldehyde (6c) Yellow solid; Yield 72%; mp 195– 197 ℃; IR (KBr, ­cm−1) 3320 (NH), 3064–3027 (CH aromatic), 2928–2853 (CH aliphatic), 1661 (C=O), 1302, 1146 ­(SO2); 1H NMR (DMSO-d6) δ (ppm): 3.18 (s, 3H, ­SO2CH3), 7.2 (d, 1H, J = 8 Hz, indole H-6), 7.58 (s, 1H, indole H-4), 7.91 (d, 1H, J = 9.6 Hz, indole H-7), 8.09 (d, 2H, J = 8.4 Hz, phenyl H-2, H-6), 8.14 (d, 2H, J = 8.4 Hz, phenyl H-3, H-5), 10.00 (s, 1H, aldehydic H), 12.92 (s, 1H, indole NH, ­ D2O exchangeable). Anal. Calced for ­C16H12FNO3S: C, 60.56; H, 3.81; N, 4.41. Found: C, 60.73; H, 3.72; N, 4.62. General procedure for synthesis of 5‑substi‑ tuted‑3‑((2‑(4‑substituted‑ phenyl)hydrazono) methyl)‑2‑(4‑(methylsulfonyl)phenyl)‑1H‑indole 7a‑k A mixture of an ethanolic solution of respective indole3-carbaldehyde derivative (6a–c) (1 mmol) and 4-substituted phenylhydrazine HCl (1 mmol) was heated under reflux for 4–6 h in the presence of a few drops of glacial acetic acid. After cooling, the reaction mixture was poured into ice-cold water and the separated solid was filtered, dried and recrystallized from methanol (yield: 73–92%). 3‑((2‑(4‑Fluorophenyl)hydrazono) methyl)‑2‑(4‑(methyl sulfonyl)phenyl)‑1H‑indole (7a) Brown solid; Yield 73%; mp 204–206 ℃; IR (KBr, ­cm−1) 3282–3317 (indole NH, hydrazone NH), 3063 (CH aromatic), 2927–2843 (CH aliphatic), 1597 (C=N), 1302, 1148 ­(SO2); 1H NMR (DMSO-d6) δ (ppm): 3.26 (s, 3H, ­SO2CH3), 7.04–7.18 (m, 4H, phenyl hydrazone H-3, H-5, indole H-5, H-6), 7.44 (d, 1H, J = 8 Hz, indole H-4), 7.59 (d, 2H, J = 8.4 Hz, phenyl hydrazone H-2, H-6), 7.99 (d, 2H, J = 8.4 Hz, phenyl H-2, H-6), 8.12 (d, 2H, J = 8.4 Hz, phenyl H-3, H-5), 8.27 (s, 1H, CH), 8.4 (d, 1H, J = 8 Hz, indole H-7), 10.01 (s, 1H, hydrazone NH, D ­ 2O exchangeable), 11.79 (s, 1H, indole NH, D ­ 2O exchangeable); 13C NMR (DMSO-d6) δ (ppm): 43.0 (­SO2CH3), 110.4, 111.9, 112.7, 115.2, 120.3, 125.6, 126.2, 128.0, 129.7, 132.1, 135.7, 136.4, 137.3, 140.2, 143.6 (CH=N), 154.7, 157.1. Anal. Calced for ­C22H18FN3O2S: C, 64.85; H, 4.45; N, 10.31. Found: C, 65.08; H, 4.33; N, 9.95. 2‑(4‑(Methylsulfonyl)phenyl)‑3‑((2‑(4‑(methylsulfonyl) phenyl)hydrazono)methyl)‑1H‑indole (7b) Yellow solid; Yield 85%; mp 228–230 ℃; IR (KBr, c­m−1) 3262–3309 (indole NH, hydrazone NH), 3017 (CH aromatic), 2934– 2863 (CH aliphatic), 1593 (C=N), 1299, 1150 ­(SO2); 1H NMR (DMSO-d6) δ (ppm): 3.11 (s, 3H, ­SO2CH3), 3.33 (s, 3H, S ­ O2CH3), 7.17 (d, 2H, J = 8 Hz, phenyl hydrazone H-3, H-5), 7.24–7.33 (m, 2H, indole H-5, H-6), 7.51 (d, 1H, J = 8 Hz, indole H-4), 7.75 (d, 2H, J = 8 Hz, phenyl hydrazone H-2, H-6), 7.95 (d, 2H, J = 8 Hz, phenyl H-2, H-6), 8.13 (d, 2H, J = 8 Hz, phenyl H-3, H-5), 8.3 (s, 1H, CH), 8.4 (d, 1H, J = 8 Hz, indole H-7), 10.72 (s, 1H,