Abstract
In this work, some N-(9-Ethyl-9H-carbazole-3-yl)-2-(phenoxy)acetamide derivatives were synthesised and evaluated for their antimicrobial activity and cytotoxicity. The structural elucidation of the compounds was performed by IR, 1H-NMR, 13C-NMR and FAB+-MS spectral data and elemental analyses. The title compounds were obtained by reacting 2-chloro-N-(9-ethyl-9H-carbazole-3-yl)acetamide with some substituted phenols. The synthesised compounds were investigated for their antibacterial and antifungal activities against Micrococcus luteus, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Candida albicans. The compounds N-(9-Ethyl-9H-carbazole-3-yl)-2-(4-ethylphenoxy)acetamide (2c) and N-(9-Ethyl-9H-carbazole-3-yl)-2-(quinolin-8-yloxy)acetamide (2n) showed notable antimicrobial activity. The compounds were also studied for their cytotoxic effects using MTT assay, and it was seen that 2n had the lowest cytotoxic activity against NIH/3T3 cells.
Introduction
The alarming rates of emerging and reemerging microbial threats coupled with the rapid development of multi-drug-resistant microbial pathogens are major escalating concerns to the public health, particularly during the past decadesCitation1,Citation2. Despite a large number of antibiotics and chemotherapeutics available for medical use, at the same time, the emergence of old and new antibiotic resistance created revealed a substantial medical need for new classes of antimicrobial agentsCitation3. There is no doubt that the existing arsenal of antimicrobial agents we have in hand for the treatment of infectious diseases is insufficient to protect us over the long termCitation4–7. Thus there is a need to search for new and efficacious antimicrobial agentsCitation8,Citation9. Nevertheless, there is a continuing effort among the scientists especially in the pharmaceutical industry to develop new antimicrobial agents for the treatment of resistant infectionsCitation10.
The tricyclic carbazole nucleus is an important type of nitrogen-containing aromatic heterocyclic compound and possesses desirable electronic and charge-transport properties, as well as large p-conjugated system, and the various functional groups are easily introduced into the structurally rigid carbazole ring. These characteristics result in the extensive potential applications of carbazole-based derivatives in the field of chemistry and medicinal chemistryCitation11.
Carbazole and its derivatives are a considerable structural unit which have been isolated from different sources such as some genera of higher plants, blue green algae, actinomycetes and filamentous fungiCitation12–14. It is known that natural origin carbazoles especially for those complex carbazoles fusing with a heterocyclic fragment show well-known pharmacological activitiesCitation15,Citation16. Accordingly, to the present, a large number of natural and synthetic carbazole derivatives have been reported to exhibit diverse biological activities such as antituberculosisCitation17, antiproliferativeCitation14, antibacterialCitation18,Citation19, antiviralCitation20, antifungalCitation21,Citation22, antitumourCitation23, anti-inflammatoryCitation24, antioxidantCitation12 and antihistaminic activitiesCitation25.
It is known that the phenol and phenol derivatives are in use as potential antimicrobial agentCitation26. Several studies demonstrated the antimicrobial activity of phenols and/or phenolic compounds making them a good alternative to antibiotics and chemical preservativesCitation27. Also the alkoxy and aryloxy moiety, which is containing phenol residue, has low toxicity and active against the growth of various yeasts and bacteriaCitation28–30.
In the view of these observations, we designed and synthesised carbazole-based aryloxy compounds as potential antimicrobial agent.
Experimental
Chemistry
All chemicals were purchased from Sigma-Aldrich Chemical Co. All melting points (m.p.) were determined by Electrothermal 9100 digital melting point apparatus and are uncorrected. Spectroscopic data were recorded with the following instruments: 1H-NMR, Bruker 400 MHz spectrometer; 13C-NMR, Bruker 100 MHz spectrometer; and MS-FAB, VG Quattro Mass spectrometer. Elemental analyses were performed on a Perkin Elmer EAL 240 elemental analyzer.
General procedure for the synthesis of the compounds
2-Chloro-N-(9-ethyl-9H-carbazole-3-yl)acetamide (1)
9-Ethyl-9H-carbazole-3-amine (0.05 mol) and triethylamine (0.06 mol) were dissolved in THF with a constant stirring at 0–5°C, then chloroacetyl chloride (0.06 mol) was added dropwise gradually to this solution. The reaction mixture thus obtained was further agitated for 1 h at room temperature. After the solvent was evaporated to dryness, the solid was filtered and washed with water.
N-(9-Ethyl-9H-carbazole-3-yl)-2-(substituted phenoxy)acetamide derivatives (2a-n)
A mixture of 2-chloro-N-(9-ethyl-9H-carbazole-3-yl)acetamide (1) (1.65 mmol, 0.5 g), the appropriate phenol derivatives (1.98 mmol) and K2CO3 (1.98 mmol, 0.3 g) in acetonitrile was refluxed for 6 hours. The cooled mixture was filtered and recrystallised from alcohol. Some characteristics of the compounds are given in .
Table 1. Some characteristics of the synthesised compounds.
N-(9-Ethyl-9H-carbazole-3-yl)-2-(o-tolyloxy)acetamide (2a)
IR (KBr) νmax (cm−1): 3332 (amide N-H), 3050 (aromatic C-H), 2922, 2850 (aliphatic C-H), 1683 (amide C=O), 1604, 1556, 1442 (C=C), 1000–1300 (C-N), 1245 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t J = 6 Hz, CH3), 2.29 (3H, s, Ar-CH3), 4.43 (2H, q J = 7 Hz, N-CH2), 4.75 (2H, s, O-CH2), 6.91–7.60 (9H, m, Ar-H), 8.07 (H, d, J=8 Hz, carbazole C5-H), 8.44 (H, s, carbazole C4-H), 10.04 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.65 (CH3), 16.16 (CH3), 36.95 (CH2), 67.52 (CH2), 108.98 (CH), 109.16 (CH), 111.50 (CH), 111.88 (CH), 118.57 (CH), 119.39 (CH), 120.19 (CH), 120.85 (CH), 125.78 (CH), 130.23 (2C), 130.58 (2CH), 136.39 (2C), 139.96 (2C), 156.10 (C), 166.27 (C).
For C23H22N2O2 calculated: 77.07% C, 6.19% H, 7.82% N; found: 77.02% C, 6.18% H, 7.80% N.
MS (FAB) [M+1]+: m/z 359
N-(9-Ethyl-9H-carbazole-3-yl)-2-(m-tolyloxy)acetamide (2b)
IR (KBr) νmax (cm−1): 3299 (amide N-H), 3030 (aromatic C-H), 2904 (aliphatic C-H), 1665 (amide C=O), 1610, 1557 (C=C), 1000–1300 (C-N), 1247 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 8 Hz, CH3), 2.31 (3H, s, Ar-CH3), 4.43 (2H, q, J = 7 Hz, N-CH2), 4.71 (2H, s, O-CH2), 6.85–7.65 (9H, m, Ar-H), 8.07 (H, d, J = 8 Hz, carbazole C5-H), 8.44 (H, s, carbazole C4-H), 10.06 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.65 (CH3), 21.10 (CH3), 36.95 (CH2), 67.20 (CH2), 108.94 (CH), 109.16 (CH), 111.62 (CH), 115.47 (CH), 118.57 (3CH), 121.80 (C), 125.78 (C), 129.22 (2CH), 130.17 (2C), 136.42 (CH), 138.97 (2C), 139.96 (CH), 157.88 (C), 166.20 (C).
For C23H22N2O2 calculated: 77.07% C, 6.19% H, 7.82% N; found: 77.04% C, 6.19% H, 7.82% N.
MS (FAB) [M+1]+: m/z 359
N-(9-Ethyl-9H-carbazole-3-yl)-2-(4-ethylphenoxy)acetamide (2c)
IR (KBr) νmax (cm−1): 3354 (amide N-H), 3042, 3015 (aromatic C-H), 2987, 2889 (aliphatic C-H), 1691 (amide C=O), 1554, 1486 (C=C), 1000–1300 (C-N), 1230 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.15 (3H, t, J = 7.4, Hz, C-CH2CH3) 1.31 (3H, t, J = 7.1 Hz, CH3), 2.31 (2H, q, J = 7 Hz C-CH2), 4.43 (2H, q, J = 7.2 Hz, N-CH2), 4.71 (2H, s, O-CH2), 6.96–7.65 (9H, m, Ar-H), 8.07 (H, d, J = 7.5 Hz, carbazole C5-H), 8.44 (H, s, carbazole C4-H), 10.06 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.82 (CH3), 16.8 (CH3), 28.0 (CH2), 37.09 (CH2), 68.35 (CH2), 109.63 (CH), 111.51 (2CH), 115.72 (CH), 119.75 (CH), 120.94 (CH), 121.76 (2CH), 123.78 (C), 124.02 (CH), 128.81 (C), 132.42 (2CH), 134.34 (C), 137.17 (C), 141.63 (2C), 156.75 (C), 167.77 (C).
For C24H24N2O2 calculated: 77.39% C, 6.49% H, 7.52% N; found: 77.39% C, 6.47% H, 7.56% N.
MS (FAB) [M+1]+: m/z 373
N-(9-Ethyl-9H-carbazole-3-yl)-2-(2-chlorophenoxy)acetamide (2d)
IR (KBr) νmax (cm−1): 3413 (amide N-H), 3012 (aromatic C-H), 2989, 2876 (aliphatic C-H), 1698 (amide C=O), 1610, 1564 (C=C), 1000–1300 (C-N), 1218 (C-O-C),
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 6 Hz, CH3), 4.43 (2H, q, J = 6 Hz, N-CH2), 4.88 (2H, s, O-CH2), 7.01–7.60 (9H, m, Ar-H), 8.08 (H, d, J = 8 Hz, carbazole C5-H), 8.45 (H, s, carbazole C4-H), 10.14 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.65 (CH3), 36.95 (CH2), 67.80 (CH2), 109.06 (2CH), 109.16 (CH), 111.59 (CH), 114.11 (2C), 118.58 (CH), 120.20 (2C), 122.07 (CH), 125.81 (2CH), 128.25 (CH), 130.06 (CH), 130.22 (C), 136.38 (C), 139.97 (CH), 153.51 (C), 165.50 (C).
For C22H19ClN2O2 calculated: 69.75% C, 5.05% H, 7.39% N; found: 70.02% C, 5.03% H, 7.41% N.
MS (FAB) [M+1]+: m/z 379
N-(9-Ethyl-9H-carbazole-3-yl)-2-(4-chlorophenoxy)acetamide (2e)
IR (KBr) νmax (cm−1): 3249 (amide N-H), 3079 (aromatic C-H), 2908 (aliphatic C-H), 1683 (amide C=O), 1612, 1594 (C=C), 1000–1300 (C-N), 1274 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 8 Hz, CH3), 4.43 (2H, q, J = 7 Hz, N-CH2), 4.75 (2H, s, O-CH2), 7.09–7.61 (9H, m, Ar-H), 8.07 (H, d, J = 8 Hz, carbazole C5-H), 8.43 (H, s, carbazole C4-H), 10.11 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.65 (CH3), 36.95 (CH2), 67.42 (CH2), 108.95 (CH), 109.16 (2CH), 112.05 (2C), 116.53 (C), 118.58 (CH), 120.17 (2C), 124.84 (C), 129.24 (2CH), 130.10 (CH), 136.44 (2CH), 139.96 (2CH), 156.77 (C), 165.83 (C).
For C22H19ClN2O2 calculated: 69.75% C, 5.05% H, 7.39% N; found: 69.78% C, 5.06% H, 7.40% N.
MS (FAB) [M+1]+: m/z 379
N-(9-Ethyl-9H-carbazole-3-yl)-2-(2-nitrophenoxy)acetamide (2f)
IR (KBr) νmax (cm−1): 3317 (amide N-H), 3100 (aromatic C-H), 2980 (aliphatic C-H), 1687 (amide C=O), 1525, 1442 (C=C), 1000–1300 (C-N), 1239 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.30 (3H, t, J = 8 Hz, CH3), 4.42 (2H, q, J = 8 Hz, N-CH2), 4.99 (2H, s, O-CH2), 7.18–7.96 (9H, m, Ar-H), 8.08 (H, d, J = 8 Hz, carbazole C5-H), 8.44 (H, s, carbazole C4-H), 10.10 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.64 (CH3), 36.95 (CH2), 67.87 (CH2), 109.11 (CH), 109.17 (C), 111.55 (C), 115.44 (2C), 118.61 (2C), 121.20 (CH), 125.26 (CH), 125.84 (2CH), 130.05 (2CH), 134.57 (CH), 136.42 (CH), 139.46 (CH), 139.98 (CH), 150.92 (C), 165.04 (C).
For C22H19N3O4 calculated: 67.86% C, 4.92% H, 10.79% N; found: 67.85% C, 4.90% H, 10.75% N.
MS (FAB) [M+1]+: m/z 390
N-(9-Ethyl-9H-carbazole-3-yl)-2-(3-nitrophenoxy)acetamide (2g)
IR (KBr) νmax (cm−1): 3440 (amide N-H), 3012 (aromatic C-H), 2980 (aliphatic C-H), 1695 (amide C=O), 1602, 1543, 1551 (C=C), 1000–1300 (C-N), 1245 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J=8 Hz, CH3), 4.43 (2H, q, J = 8 Hz, N-CH2), 4.92 (2H, s, O-CH2), 7.19–7.89 (9H, m, Ar-H), 8.08 (H, d, J = 8 Hz, carbazole C5-H), 8.43 (H, s, carbazole C4-H), 10.20 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.66 (CH3), 36.96 (CH2), 67.46 (CH2), 108.99 (CH), 109.17 (CH), 109.52 (2CH), 112.11 (C), 116.01 (C), 118.60 (CH), 120.18 (2C), 121.91 (2C), 125.81 (CH), 130.02 (CH), 130.72 (CH), 136.48 (CH), 139.97 (CH), 148.62 (CH), 158.43 (C), 165.47 (C).
For C22H19N3O4 calculated: 67.86% C, 4.92% H, 10.79% N; found: 67.81% C, 4.90% H, 10.78% N.
MS (FAB) [M+1]+: m/z 390
N-(9-Ethyl-9H-carbazole-3-yl)-2-(4-nitrophenoxy)acetamide (2h)
IR (KBr) νmax (cm−1): 3298 (amide N-H), 3017 (aromatic C-H), 2980, 2875 (aliphatic C-H), 1669 (amide C=O), 1556, 1510 (C=C), 1000–1300 (C-N), 1236 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J=8 Hz, CH3), 4.43 (2H, q, J = 8 Hz, N-CH2), 4.95 (H, s, O-CH2), 7.19–8.42 (11H, m, Ar-H), 10.27 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.66 (CH3), 43.61 (CH2), 67.42 (CH2), 109.00 (CH), 109.09 (CH), 109.18 (CH), 111.60 (CH), 115.35 (C), 118.61 (C), 119.44 (CH), 121.86 (CH), 125.81 (CH), 130.05 (CH), 130.29 (CH), 136.45 (CH), 139.97 (2C), 141.22 (2C), 163.21 (CH), 164.21 (C), 165.16 (C).
For C22H19N3O4 calculated: 67.86% C, 4.92% H, 10.79% N; found: 67.82% C, 4.89% H, 10.80% N.
MS (FAB) [M+1]+: m/z 390
N-(9-Ethyl-9H-carbazole-3-yl)-2-(2,3-dimethylphenoxy)acetamide (2i)
IR (KBr) νmax (cm−1): 3356 (amide N-H), 3059 (aromatic C-H), 2932, 2879 (aliphatic C-H), 1689 (amide C=O), 1601, 1557, 1444 (C=C), 1000–1300 (C-N), 1215 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 8 Hz, CH3), 2.16 (3H, s, Ar-CH3) 2.21 (3H, s, Ar-CH3), 4.42 (2H, q, J = 8 Hz, N-CH2), 4.66 (2H, s, O-CH2), 6.97–7.76 (8H, m, Ar-H), 8.04 (H, d, J = 8.2 Hz, carbazole C5-H), 8.42 (H, s, carbazole C4-H), 10.02 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.56 (CH3), 18.82 (CH3), 19.65 (CH3), 35.54 (CH2), 67.12 (CH2), 108.92 (CH), 110.16 (CH), 115.18 (2C), 113.51 (2C), 117.26 (CH), 117.512 (CH), 121.18 (CH), 126.78 (CH), 128.67 (C), 132.54 (CH), 135.40 (2C), 137.58 (CH), 140.94 (2CH), 155.45 (C), 166.34 (C).
For C24H24N2O2 calculated: 77.39% C, 6.49% H, 7.52% N; found: 77.34% C, 6.48% H, 7.56% N.
MS (FAB) [M+1]+: m/z 373
N-(9-Ethyl-9H-carbazole-3-yl)-2-(3,4-dimethylphenoxy)acetamide (2j)
IR (KBr) νmax (cm−1): 3346 (amide N-H), 3013 (aromatic C-H), 2984, 2750 (aliphatic C-H), 1687 (amide C=O), 1650, 1556 (C=C), 1000–1300 (C-N), 1254 (C-O-C),
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 8 Hz, CH3), 2.16 (3H, s, Ar-CH3) 2.21 (3H, s, Ar-CH3), 4.43 (2H, q, J = 8 Hz, N-CH2), 4.66 (2H, s, O-CH2), 6.77–7.65 (8H, m, Ar-H), 8.07 (H, d, J = 8.1 Hz, carbazole C5-H), 8.43 (H, s, carbazole C4-H), 10.02 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.66 (CH3), 18.42 (CH3), 19.65 (CH3), 36.94 (CH2), 67.32 (CH2), 108.93 (CH), 109.16 (CH), 111.58 (2C), 112.03 (2C), 116.16 (CH), 118.57 (CH), 120.17 (CH), 125.78 (CH), 128.67 (C), 130.15 (CH), 136.40 (2C), 137.28 (CH), 139.94 (2CH), 155.94 (C), 166.34 (C).
For C24H24N2O2 calculated: 77.39% C, 6.49% H, 7.52% N; found: 77.35% C, 6.50% H, 7.53% N.
MS (FAB) [M+1]+: m/z 373
N-(9-Ethyl-9H-carbazole-3-yl)-2-(2,4-dimethylphenoxy)acetamide (2k)
IR (KBr) νmax (cm−1): 3381 (amide N-H), 3059 (aromatic C-H), 2927, 2874 (aliphatic C-H), 1694 (amide C=O), 1600, 1556, 1448 (C=C), 1000–1300 (C-N), 1224 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 8 Hz, CH3), 2.21 (3H, s, Ar-CH3) 2.26 (3H, s, Ar-CH3), 4.43 (2H, q, J = 8.3 Hz, N-CH2), 4.70 (2H, s, O-CH2), 6.83–7.59 (8 H, m, Ar-H), 8.07 (H, d, J = 8 Hz, carbazole C5-H), 8.43 (H, s, carbazole C4-H), 10.01 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.66 (CH3), 16.11 (CH3), 20.05 (CH3), 36.94 (CH2), 67.71 (CH2), 108.97 (C), 109.16 (C), 111.53 (C), 111.87 (CH), 118.57 (2C), 119.39 (2C), 120.19 (CH), 121.96 (CH), 125.78 (CH), 129.49 (CH), 130.22 (CH), 131.29 (2CH), 136.37 (CH), 139.94 (CH), 154.01 (C), 166.40 (C).
For C24H24N2O2 calculated: 77.39% C, 6.49% H, 7.52% N; found: 77.40% C, 6.51% H, 7.47% N.
MS (FAB) [M+1]+: m/z 373
N-(9-Ethyl-9H-carbazole-3-yl)-2-(3,5-dimethylphenoxy)acetamide (2l)
IR (KBr) νmax (cm−1): 3412 (amide N-H), 3053 (aromatic C-H), 2962, 2858 (aliphatic C-H), 1635 (amide C=O), 1504, 1482 (C=C), 1000–1300 (C-N), 1287 (C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 8.2 Hz, CH3), 2.26 (6H, s, 2CH3), 4.43 (2H, q, J = 8.1 Hz, N-CH2), 4.67 (2H, s, O-CH2), 6.64–7.65 (8H, m, Ar-H), 8.07 (H, d, J = 8 Hz, carbazole C5-H), 8.43 (H, s, carbazole C4-H), 10.02 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.66 (CH3), 21.05 (2CH3), 36.95 (CH2), 67.17 (CH2), 108.94 (CH), 109.16 (CH), 112.05 (CH), 112.46 (CH), 118.57 (2C), 119.54 (CH), 120.16 (C), 122.77 (C), 125.78 (C), 130.17 (2CH), 136.42 (2CH), 138.62 (CH), 139.96 (2C), 157.89 (C), 166.25 (C).
For C24H24N2O2 calculated: 77.39% C, 6.49% H, 7.52% N; found: 77.34% C, 6.46% H, 7.49% N.
MS (FAB) [M+1]+: m/z 373
N-(9-Ethyl-9H-carbazole-3-yl)-2-([1,1′-biphenyl]-4-yloxy)acetamide (2m)
IR (KBr) νmax (cm−1): 3346 (amide N-H), 3084 (aromatic C-H), 2978, 2880 (aliphatic C-H), 1673 (amide C=O), 1650, 1589, 1442 (C=C), 1000–1300 (C-N), 1238 (C-O-C),
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 7.2 Hz, CH3), 4.44 (2H, q, J = 7.6 Hz, N-CH2), 4.80 (2H, s, O-CH2), 7.14–7.67 (14H, m, Ar-H), 8.09 (H, d, J = 7.8 Hz, carbazole C5-H), 8.46 (H, s, carbazole C4-H), 10.20 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 14.24 (CH3), 37.95 (CH2), 67.87 (CH2), 109.11 (CH), 109.17 (CH), 112.55 (C), 116.44 (2C), 118.61 (2C), 121.20 (CH), 123.26 (CH), 125.84 (2CH), 127.05 (2CH), 128.57 (CH), 129.42 (CH), 130.46 (CH), 131.98 (CH), 134.92 (C), 137.04 (C), 141.04 (2CH), 141.27 (2CH), 158.96 (C), 167.72 (C).
For C28H24N2O2 calculated: 79.98% C, 5.75% H, 6.66% N; found: 79.96% C, 5.76% H, 6.68% N.
MS (FAB) [M+1]+: m/z 421
N-(9-Ethyl-9H-carbazole-3-yl)-2-(quinolin-8-yloxy)acetamide (2n)
IR (KBr) νmax (cm−1): 3411 (amide N-H), 3056 (aromatic C-H), 2987, 2850 (aliphatic C-H), 1659(amide C=O), 1652, 1587, 1498 (C=C), 1000–1300 (C-N), 1247(C-O-C).
1H NMR (400 MHz, DMSO-d6): 1.31 (3H, t, J = 8, CH3), 4.42 (2H, q, J = 8.3 Hz, N-CH2), 5.01 (2H, s, O-CH2), 7.19–9.04 (13H, m, Ar-H), 10.65 (H, s, NH).
13C NMR (100 MHz, DMSO-d6): 13.65 (CH3), 18.52 (CH2), 36.95 (CH2), 56.00 (CH), 70.12 (CH), 109.13 (CH), 111.40 (C), 113.06 (C), 118.58 (2C), 118.93 (2C), 120.25 (CH), 121.39 (CH), 125.83 (CH), 129.18 (CH), 130.24 (2CH), 136.24 (CH), 136.39 (CH), 139.97 (CH), 140.05 (CH), 149.55 (C), 154.21 (C), 166.40 (C).
For C25H21N3O2 calculated: 75.93% C, 5.35% H, 10.63% N; found: 75.92% C, 5.36% H, 10.60% N.
MS (FAB) [M+1]+: m/z 396
Antimicrobial activity
The antimicrobial activities of compounds (2a-n) were tested using the microbroth dilution methodCitation31. Tested microorganism strains were Micrococcus luteus (NRLL B-4375), Bacillus subtilis (NRS-744), P. aeroginosa (ATCC-254992), Staphylococcus aureus (NRRL B-767), Escherichia coli (ATCC-25922), Listeria monocytogenes (ATCC-7644) and Candida albicans (ATCC-22019). Microbroth dilution-susceptibility assay was used for antimicrobial evaluation of the compounds. Stock solutions of the samples were prepared in dimethylsulfoxide. Dilution series using sterile distilled water were prepared from 0.65–4 mg/mL to 0.000633–0.0039 mg/mL in micro test tubes that were transferred to 96-well microtitre plates. Overnight-grown bacterial and C. albicans suspensions in double-strength Mueller–Hinton broth were standardised to 108 CFU/mL using McFarland No: 0.5 standard solutions. 100 µL of each microorganism suspension were then added into the wells. The last well chain without a microorganism was used as a negative control. Sterile distilled water and the medium served as a positive growth control. After incubation at 37°C for 18–24 h, antimicrobial activity was detected by spraying of 0.5% TTC (triphenyl tetrazolium chloride, Merck) aqueous solution. MIC was defined as the lowest concentration of compounds that inhibited visible growth, as indicated by the TTC staining. Streptomycin was used as standard antibacterial agent, whereas ketoconazole was used as an antifungal agent.
Cytotoxicity
The cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayCitation32,Citation33. Mouse embryonic fibroblast (NIH/3T3) cells were cultured in 96-well flat-bottom plates at 37°C for 24 h (2 × 104 cells per well). All of the compounds were dissolved in dimethyl sulfoxide (DMSO) individually and added to culture wells at varying concentrations (0.5–500 µg/mL); the highest final DMSO concentration was under 0.1%. After 24-hour drug incubation at 37°C, 20 µl MTT solution (5 mg/mL MTT powder in PBS) was added to each well. Then 3-hour incubation period was maintained in the same conditions. Purple formazan was occurred at the end of the process which is the reduction product of MTT agent by the mitochondrial dehydrogenase enzyme of intact cells. Formazan crystals were dissolved in 100 µL DMSO, and the absorbance was read by ELISA reader (OD570nm). The percentage of viable cells was calculated based on the medium control.
Result, discussion and conclusion
Chemistry
In this study, a series of carbazole-based compounds were synthesised via an easy, convenient and efficient synthetic route. The synthesis of the compounds is shown in . To obtain final compounds, 3-amino-9-ethylcarbazole was reacted with chloroacetyl chloride to produce 2-chloro-N-(9-ethyl-9H-carbazole-3-yl)acetamide (1) which was then reacted with some phenol derivatives to get N-(9-ethyl-9H-carbazole-3-yl)-2-(substituted phenoxy)acetamide derivatives (2a-n).
Scheme 1. The synthetic protocol of the compounds (2a-n).
The structure elucidation of the compounds was determined by IR, 1H-NMR, 13C-NMR, FAB+-MS spectral data and elemental analyses results.
In the IR spectra of all compounds, characteristic amide function was observed in the region 1659–1698 cm−1 because of the amide C=O vibration. In addition, the amide N-H vibration of the compounds were seen at 3249–3440 cm−1 region as expected.
The 1H-NMR spectral data were also consistent with the assigned structures. In the 400 MHz 1H-NMR spectrum of compounds, the O-CH2 protons resonated at 4.6–5.01 ppm as a singlet and also N-H protons were observed at 10.01–10.65 ppm. For ethyl substitution, CH3 protons were observed at about 1.31 ppm as triplet and CH2 protons at 4.42–4.43 ppm as quartet. In aromatic region, the signal of the carbazole C4-H and carbazole C5-H protons was observed much further from upfield at about 8.06–8.46 ppm34 and the other characteristic aromatic protons were observed at expected regions.
In the 13C-NMR spectra of the compounds, the signal of characteristic carbonyl carbon appeared at 166.27 ppm and O-CH2 at 67.52 ppm, respectively.
In the MS spectra, the electron-spraying technique with positive polarity mode was applied and M+1 peaks were detected as base peak.
All compounds gave satisfactory elemental analysis results.
Antimicrobial activity and toxicity
Antimicrobial activity was investigated by finding minimum inhibitory concentration (MIC) of the synthesised compounds against S. aureus, L. monocytogenes, E. coli, Pseudomonas aeruginosa, M. luteus, B. subtilis and C. albicans comparing with streptomycin and ketoconazole as standard drug. The MIC value of the compounds and control drugs are summarised in .
Table 2. Antimicrobial activities of the compounds (µg/mL).
The MIC values were generally within the range of 31.25–500 μg/mL. Most of the compounds showed significant antifungal activity against C. albicans. In consideration of synthesised compounds’ MIC values with standard drug, compounds 2a, 2c and 2n had more and 2g had less antifungal activity than did ketoconazole and also 2b and 2f had similar antifungal activity to ketoconazole.
It was also observed that all compounds had antimicrobial activity against all tested bacteria according to standard drug streptomycin. Especially, compounds 2c and 2n are highly active against all of the evaluating bacterial strains. Compound 2n, which includes quinolinoxy moiety, exhibited equipotent activity to standard drug with an MIC value of 31.25 μg/mL against S. aureus.
Compounds 2a, 2c and 2b had satisfying activity against P. aeruginosa. Compound 2c was the most effective against this bacteria with an MIC value of 40.25 μg/mL, whereas streptomycin had an MIC value of 125 μg/mL.
Compounds were also studied for their cytotoxic properties using MTT assay. The IC50 (μg/mL) values of the compounds against NIH/3T3 cells are shown in . The biological study indicated that compounds 2a, 2c, 2e, 2j and 2l possessed the highest cytotoxicity, whereas the other compounds exhibited moderate cytotoxicity except 2n. The IC50 concentration of compound 2n was 300 μg/mL, which was greater than all MIC values observed against all tested bacteria and fungi for this compound.
Table 3. In vitro cytotoxicity of the compounds.
In comparison of the results of cytotoxicity and antimicrobial activity tests, it can be claimed that compound 2n possibly has antimicrobial activity not because of its general toxicity but because of its selective antimicrobial effect. The recognition of alkoxyquinoline’s antimicrobial activity supports this resultCitation35.
In conclusion, all synthesised compounds can be potential antimicrobial agent against different tested microorganisms.
Declaration of interest
The authors report no conflicts of interest.
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