Synthesis, antibacterial, anti-oxidant and molecular docking studies of imidazoquinolines.

Quinoline and imidazole derivatives have been playing a significant role in functional bioactivities and were potentially used as antibacterial, antifungal, anticancer, and anti-inflammatory drugs. Owing to the limitation of drug resistance, herein we synthesized thio-, chloro-, and hydroxyl-functionalized various imidazoquinolines by molecular hybridization approach. All the imidazoquinoline derivatives were examined for their antibacterial activity against selected bacterial pathogens by the agar well diffusion method. In addition, the anti-oxidant efficacy of imidazoquinolines was also tested using ferric reducing antioxidant power (FRAP). Among them, electron-withdrawing (-Cl) substituent containing imidazoquinoline 5f showed higher antibacterial and anti-oxidant activities than other imidazoquinolines and reached the effectiveness of the standard. In addition, compounds 4f, 5e, and 3f showed moderate antibacterial activity and other derivatives displayed weak activity against various pathogens. Molecular docking studies were also performed on selected imidazoquinoline derivatives (3f, 4f, and 5f), which showed high docking score and strong binding energy values. These results revealed that thio-imidazoquinoline could assist as a prototype for the designing of multidrug-resistant antibiotics against various microbial organisms.

Introduction

Bacterial infections have been vastly increased due to lack of potency with various antibiotics by strengthened antimicrobial resistance. Hence, the multidrug-resistant (MDR) bacteria is one of the major challenges in social health care [1, 2]. For example, ~2 million people acquire this MDR infection, and however, ~23,000 people decease per year [3]. At present, there is a wide range of antibacterial drugs available, even though it causes adverse side effects, such as high blood pressure, skin rashes, neuropathy, bone marrow diseases, etc [4, 5]. Also, the prolonged usage of antibiotics causes immunosuppresses of an individual. Thus, the progress of new antibiotics with enhanced toxicity is the crucial and worthwhile effort required to overcome the antibiotic resistance issue. Recently, various approaches have been implemented for the synthesis of novel antibacterial agents to overcome MDR bacteria [6, 7]. In particular, the molecular hybridization approach was implemented for the synthesis of the hybrid single molecular drug by attaching two or more bioactive pharmacophores, and thus produce fewer side effects with better efficacy. Inspired by this approach, researchers have been focusing on the synthesis of the hybrid single molecular drug by fusing both quinoline and imidazole-based pharmacophores [8]. Since quinolines and their various substituted functionalities have been stepping into the pharmacological drug activities from antiasthmatic to HIV-integrase inhibitors [9, 10, 11]. The imidazole derivatives have shown various pharmacological activities, including antihypertensive, anti-fungal, enzyme inhibition, cardiovascular activity, etc [12, 13]. These days imidazole-based antibacterial drugs, such as 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and 2-nitroimidazole (azomycin) have been used for trichomonacide applications. Also, some of the imidazoles containing vital anti-cancer drugs, such as metronidazole, clotrimazole, metrazole, and misonidazole are exploited to date. The fruitful combination of these two pharmacophores could achieve imidazoquinoline derivatives and be utilized for various applications, such as modulating the phosphodiesterase and adenosine A3 receptors, high toll-like receptor 7/8 (TLR7/TLR8) selective agonists, anticancer efficacy, etc. [14, 15]. Molecular docking studies are used as a potential tool to identify the various TLR and other residues interacting with small molecular imidazoquinoline derivatives [16, 17]. As a consequence, researchers are focusing on the robust design and synthesis of imidazoquinoline derivatives with suitable functionality and can be potentially used for pharmacological and biological fields.

On the other hand, antioxidants have been playing a crucial role to inhibit the pathogenesis of various chronic diseases. Such diseases were caused by the processes of generating various reactive oxygen species [ROS] and also the release of free radicals from oxidative injuries. These oxidative injuries are associated with radiation [18], food components, or pollution or which are produced endogenously by metabolic reactions in the human body [19] accountable for oxidative damage to proteins, DNA, and lipids [20]. Many experiments showed that antioxidants could be potent in inhibiting or overcoming such disorders [21]. Hence, the synthesized imidazoquinolines-based free radical scavenging activity occurs either from phenolic –OH or –SH groups of the quinoline moiety. Therefore, a free radical can abstract an H atom or can undergo electron transfer from these sites. Nevertheless, literature reports have supported it to the phenolic -OH group [22]. Based on the above interest, we have synthesized some new heterocyclic fused systems, which comprised of both quinolines and imidazoles together via the classical Vilsmeier-Haack reaction. This reaction is utilized for developing many synthetic methodologies as an efficient reaction for the reactive aromatic and heteroaromatic formylation, respectively [23]. Accordingly, we have synthesized the 2-chloro-3-formyl quinoline via the Vilsmeier-Haack reaction followed by cyclization in the presence of orthophenylenediamine (OPD) [24]. Herein, we synthesized the series of imidazoquinoline derivatives (Figure 1) and screened their antibacterial activity and molecular docking. In addition, the radical scavenging activity of the imidazoquinolines was studied by screening their reducing ability of FRAP assay.

Figure 1

Synthesis of various imidazoquinolines. (a) 4N HCl, reflux, 4h, (b) & (c) OPD, TEA, EtOH, reflux, 10 h, (d) Na2S, DMF, RT, Stir, 5 h.

Results and discussion

The precursor 2-chloro-3-formylquinoline (1a) was synthesized by the classical Vilsmeier-Haack reaction on acetanilide according to the earlier reported procedure [25]. The prepared compound was then treated with 4N HCl to obtain the 3-formyl-2-hydroxyquinoline 2a. This intermediate compound 2a was further treated with OPD in the presence of triethylamine in ethanol under reflux condition for 10 h obtained the compound 3a in 80% Yield (m.p. 251–253 °C). IR spectrum of 3a showed strong absorption peaks at 1606, 1654, and 3342 cm−1, which corresponds to C=N, amide, and –NH groups [26]. The 1H NMR spectrum of 3a showed that two singlets at δ 12.65 and 12.47 ppm accountable for the –OH of quinoline and –NH of the imidazole scaffolds, respectively. A fine singlet at δ 9.11 ppm was attributed to the C4–H of the quinoline. The rest of the eight aromatic protons resonances showed their signals between δ 7.73 and 7.18 ppm as multiplets. The elemental analysis was also corroborated with the proposed molecular formula C16H11N3O and the mass spectra displayed the molecular ion peak at m/z: [M++H]+ 262. All the above studies were supported that compound 3a as 3-(1H-benzo[d]imidazole-2-yl)quinolin-2-ol. Earlier, we aimed to obtain a dimer product by using an excess of 2-hydroxy-3-formyl quinoline in the reaction. Surprisingly, we end up only in the formation of imidazoquinolines even taking excess of 2-hydroxy-3-formyl quinoline for this reaction. Therefore, the same reaction procedures were extended to its derivatives 2(b-f) with OPD, and their products 3(b-f) were confirmed by various spectral and analytical studies.

After preparing the oxo-derivatives, we envisaged the preparation of its chloro- and thio-analogues. Accordingly, the same precursor 2-chloro-3-formylquinoline 1a was mixed with OPD in the presence of triethylamine in ethanol under reflux condition for 10 h obtained the chloro-derivative of the imidazoquinoline 4a in 84% Yield. (m.p. 201–203 °C) and its IR spectra showed three peaks at 741, 1615, and 3364 cm−1 corresponds to C–Cl, C=N, and –NH groups. Its 1H NMR spectrum represented a broad singlet at δ 10.59 ppm for the imidazole –NH proton. A fine singlet at δ 9.26 ppm was attributed to the C4–H of the quinoline. The rest of the quinoline and imidazole aromatic protons showed their multiplet between δ 8.03 and 7.38 ppm. The mass spectrum gave the molecular ion peak at m/z: [M+]+ 279 and the CHN analysis showed its molecular formula as C16H10ClN3. The above details including the analytical data confirmed that compound 4a as 3-(1H-benzo[d]imidazole-2-yl)-2-chloroquinoline. Subsequently, compound 4a was treated with sodium sulphide in the presence of dimethylformamide under room temperature stirring for 5 h to obtain the thio-derivative of imidazoquinoline 5a in 82% Yield as yellow colour solid. (m.p. 204–205 °C) and its IR spectra showed two peaks at 1658 (C=N) and 3444 cm−1 (N–H). The 1H NMR spectra of the compound showed a multiplet δ 8.00–7.27 ppm for the eight aromatic proton resonances for the quinoline and imidazole moieties. It also exhibited a sharp singlet and a broad singlet at δ 9.19 and 14.18 ppm for the quinoline C4–H and quinoline –SH protons, respectively. The molecular formula C16H11N3S was confirmed from its analytical data and the mass spectra showed its peak at m/z: [M++H]+ 278.07.

The same reaction conditions were performed on 1(b-f) with OPD and also 4(b-f) with sodium sulphide and got the expected products 4(b-f) and 5(b-f). All the newly synthesized imidazoquinolines were well characterized by the usual spectroscopic and analytical studies.

Biological evaluation

Antibacterial activity

The newly synthesized imidazoquinolines 3–5(a-f) were examined for their in vitro antibacterial activities against the seven bacterial strains namely, S. aureus, A. hydrophila, E. coli, K. pneumonia, S. paratyphi, S. typhi, and M. butyricum by the agar well diffusion method using Ofloxacin as the control. The nutrient agar was used to culture the bacterial strains and DMSO (solvent) was used as a negative control. The susceptibility was measured based on the zone of inhibition (in diameter) against various bacterial strains. The antibacterial activity results exhibited that most of the imidazoquinolines displayed diverging degrees of inhibition against various bacterial strains. The zone of inhibition measured in mm (diameter) is compared to the current antimicrobial drug Ofloxacin as shown in Figure 2. These results revealed that most of the imidazoquinoline derivatives flourished activity against almost all the pathogenic bacteria. Although all the thio-derivatives of imidazoquinolines 5(a-f) showed noteworthy inhibition effect on the bacterial growth. In particular, compound 5f showed high activity and almost reached the effectiveness of the control. Notably, a 10-fold excess concentration of 5f (100 mg/disc) displayed higher antibacterial activity to E. coli, K. pneumonia, S. paratyphi, S. typhi, M. butyricum than that of in standard (10 mg/disc). This may be due to the thio- and chloro-pharmacophores present in the 5f molecule. In the case of chloro-imidazoquinolines 4(a–f), 4f showed moderate activity against all bacterial strains, possibly due to the two electronegative chlorine atoms present in the quinoline ring of the 4f. Similarly, among the oxo-derivatives of imidazoquinolines 3(a-f), compound 3f exhibited moderate activity against all pathogens in comparison to other analogues. This may be due to the chlorine atom and the hydroxyl groups present in the 3f molecule. However, at low concentrations i.e., 10 mg/disc some of the derivatives of the synthesized imidazoquinolines (3(a-e) and 4(a-c)) had less significant activity. Finally, on comparing the oxo-, chloro-, and thio-derivatives of the imidazoquinolines, the thio-scaffolds showed prominent antibacterial activities and the pharmacore chloro-group plays a key role in improving their activity in dose-dependent.

Figure 2

In vitro antibacterial activity of imidazoquinoline derivatives by disc-diffusion assay. (a) 3(a–f) (mg/disc), (b) 4(a–f) (mg/disc), (c) 5(a–f) (mg/disc). Ofloxacin was used as a control and error bars represent the standard deviation (SD) from three independent analyses. Insets show the statistical analysis of the differences between zone of inhibitions of all compounds 3–5(a-f). Analyzed results are displayed as mean ± SD (∗p value: 0.05–0.01; ∗∗p value: <0.001; ∗∗∗p value: <0.0001).

Based on the above results, some of the selected imidazoquinolines (3f, 4e, 4f, 5e, and 5f) were further analyzed for their minimum inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC). In Figure 3, compounds 5f showed better MIC activity against S. aureus, A. hydrophila, and S. typhi. Similarly, 5f exhibited better MBC activity against S. aureus, E. coli, and K. pneumonia.

Figure 3

(a, b) MIC and MBC of the selected imidazoquinolines (3f, 4e, 4f, 5e, and 5f) were compared with standard Ofloxacin. Error bars represent the SD from three independent analyses. Insets show the MIC and MBC of selected bacterial strains activity against imidazoquinolines (3f, 4e, 4f, 5e and 5f). Results are presented as mean ± SD (∗p value: 0.05–0.01; ∗∗p value: <0.001; ∗∗∗p value: <0.0001).

To examine the SAR studies [27, 28]:

Electron withdrawing group (-Cl) on the quinoline ring of 5f showed maximum antibacterial activity than other imidazoquinolines and reached the effectiveness of the standard. At high concentration (100 mg/disc), 5f demonstrated the higher activity against E. coli, K. pneumonia, S. paratyphi, S. typhi, M. butyricum than that of in standard (10 mg/disc). In addition, other electron-withdrawing substituents containing compounds 4f and 3f also exhibited moderate activity.

Electron donating groups (-OCH3) on the quinoline ring of 5e displayed a moderate activity than other imidazoquinolines containing similar substituents (4e and 3e).

Chloro- and thio-imidazoquinolines of 4-5(a-d) also achieved moderate activity than that of in hydroxyl-imidazoquinolines of 3(a-e).

For overall comparison, the order of antibacterial activities of imidazoquinolines was shown here, 5f > 4f > 5e > 3f > 4e > 4d ≈ 4a ≈ 5b ≈ 5d > 5c > 5a ≈ 4c ≈ 4b > 3e > 3d > 3b ≈ 3c > 3a.

Antioxidant activity using FRAP assay

The reduction of ferric (Fe3+) to ferrous (Fe2+) ion in the presence of antioxidants could be measured by using FRAP assay. Based on the synthesized compounds reducing capacity, which could function as a substantial sign of its potent antioxidant activity [29]. Figure 4 reveals that compound 5f showed higher ferric reducing power than other compounds. This obtained value of the thio-functionalized imidazoquinoline 5f was comparable to that of the standard butylated hydroxytoluene (BHT). The remaining compounds in series 5a-e also displayed good to moderate activity. In general, the reducing power of the thio-functionalized derivatives (5(a-f)) was relatively higher than the hydroxyl-functionalized derivatives (3(a-f)). Though, 3 (a-f) showed a higher reducing power than the chloro-functionalized imidazoquinolines (4(a-f)). Therefore, the order of the reducing power of imidazoquinolines are 5(a-f) > 3(a-f) > 4(a-f). From these results, it is assumed that in this assay the substitutions at the C2 positions (-OH, -Cl, and –SH) and C6 position in the quinoline moiety, as well as the heteroatoms incorporated in the imidazole scaffold, might play substantial roles in suppressing the radicals.

Figure 4

Anti-oxidant activity of imidazoquinolines 3–5(a–f), (a) 1 mg/mL, (b) 10 mg/mL. BHT acts as a control and error bars represent the SD of three independent analyses. Insets shows the anti-oxidant activity of 3-5(a–f) against the FRAP. Values are mean of three determinations, the ranges of which are mean ± SD (∗p value: 0.05–0.01; ∗∗p value: <0.001; ∗∗∗p value: <0.0001).

Molecular docking studies

Imidazoquinoline derivatives (3f, 4f, and 5f) have a greater tendency to interact with taken seven proteins, including S. aureus tyrosyl-tRNA synthetase (1JIJ), E. coli-DNA gyrase B (1EI1), K. pneumoniae (4OR7), A. hydrophila PROAEROLYSIN (1PRE), S. paratyphi lipopolysaccharide acetyltransferase periplasmic domain (6SE1), S. typhi CDP-D-glucose 4, 6-dehydratase (1WVG), and M. tuberculosis MTB phosphotyrosine phosphatase B protein (2OZ5). These compound tendencies were evaluated based on the scoring values determined from the ligand-binding efficiency with protein. Particularly, 3f, 4f, and 5f showed the higher scoring values with 4OR7 (~-7 kcal/mol) and 2OZ5 (~-5 to -7 kcal/mol) proteins, respectively which is due to hydrogen bonding and π-π interactions. Conversely, these ligands exhibited the least scoring values with 1PRE (~-3.2 to -3.6 kcal/mol) and 6SE1 proteins (~-2.94 to -5.34 kcal/mol), respectively, even though docking scores are in the acceptable range. Especially, the docking energy and binding energy of these ligands with 1PRE and 6SE1 proteins showed higher values because of their hydrogen bonding and more numbers of π-π and π-cation interactions. The simulated binding energies revealed that all derivatives formed a stable complex with proteins. Briefly, 3f, 4f, and 5f displayed the strongest binding with 1JIJ, 1EI1, and 2OZ5, respectively based on its lowest energy values (~-40.00 kcal/mol). Also, the other proteins exhibited various binding energies ranged between ~ -25 to -30 kcal/mol, demonstrating that there was favorable binding with ligands. The detailed scoring values, hydrogen bonding, π-π, and π-cation interactions were shown in Table 1 and Figures 5, 6, and 7. Interestingly, the hydrogen bond, π-π interactions, and π-cation interactions were seen in 3f, 4f, and 5f bound with 1PRE, 6SE1, and 2OZ5, respectively.

Table 1

Molecular docking scores of 3f, 4f, and 5f with various proteins are obtained via Glide docking.

	1JIJ	1EI1	4OR7	1PRE	6SE1	1WVG	2OZ5
Docking Score (kcal/mol)
3f	-5.856	-5.75	-7.092	-3.254	-5.34	-5.681	-7.09
4f	-4.746	-5.62	-7.271	-3.527	-4.085	-4.522	-4.998
5f	-3.719	-5.50	-7.333	-3.658	-2.944	-5.036	-7.087
Docking Energy (kcal/mol)
3f	-42.631	-42.15	-38.027	-32.417	-21.946	-38.217	-30.993
4f	-38.106	-42.13	-34.736	-28.773	-20.645	-35.474	-34.333
5f	-47.912	-41.51	-36.696	-29.34	-9.108	-40.007	-31.662
Glide Emodel (kcal/mol)
3f	-60.326	-57.34	-46.985	-39.417	-5.063	-45.29	-41.582
4f	-54.606	-56.20	-44.643	-38.413	-3.656	-36.034	-43.70
5f	-62.649	-56.24	-47.257	-36.846	-2.327	-36.596	-44.246
Binding Energy (MM/GBSA) kcal/mol
3f	-27.94	-46.9	-32.86	-40.8	-27.04	-30.42	-40.60
4f	-40.16	-47.25	-33.43	-34.13	-24.9	-31.31	-40.22
5f	-40.16	-51.33	-31.24	-31.34	-25.44	-29.08	-45.01
Number of Hydrogen Bonds
3f	2	1	1	1	2	1	1
4f	1	0	1	1	1	0	0
5f	2	1	1	1	0	1	1
π-π and π-cation Interactions (Numbers)
3f	0	0	0	1	3	0	2
4f	0	0	0	1	3	0	0
5f	0	0	0	0	2	0	2

Figure 5

Docking interaction between imidazoquinoline derivative 3f and selected targets; a) 3f-1JIJ complex; b) 3f-1EI1 complex; c) 3f-4OR7 complex; d) 3f-1PRE complex; e) 3f-6SE1 complex; f) 3f-1WVG complex; g) 3f-2OZ5 complex. Hydrogen bonding (pink arrow), π-π stacking (green arrow), and π-cation (red arrow).

Figure 6

Docking interaction between imidazoquinoline derivative 4f and selected targets; a) 4f-1JIJ complex; b) 4f-1EI1 complex; c) 4f-4OR7 complex; d) 4f-1PRE complex; e) 4f-6SE1 complex; f) 4f-1WVG complex; g) 4f-2OZ5 complex. Hydrogen bonding (pink arrow), π-π stacking (green arrow), and π-cation (red arrow).

Figure 7

Docking interaction between imidazoquinoline derivative 5f and selected targets; a) 5f-1JIJ complex; b) 5f-1EI1 complex; c) 5f-4OR7 complex; d) 5f-1PRE complex; e) 5f-6SE1 complex; f) 5f-1WVG complex; g) 5f-2OZ5 complex. Hydrogen bonding (pink arrow), π-π stacking (green arrow), and π-cation (red arrow).

For comparative analysis, the ligands bound with the following crystal structure complexes (PDB ID's 1JIJ, 1EI1, 1WVG, 2OZ5, and 4OR7) were separated and re-docked with the same grid to ensure the quality, scoring, and molecular interactions (Table 2 and Figure 8). The imidazoquinoline derivatives showed prominent and strong binding interactions with low energy profiles compared to the ligands that existed in the crystal structures. These results indicated that the imidazoquinoline derivatives are the potential inhibitors to the selected targets.

Table 2

Re-docking profiles of crystal bound ligands taken for the study with XP docking methodology.

PDB ID	Name of the Co-crystal Structures	Docking Score (kcal/mol)	Docking energy (kcal/mol)	Glide Emodel (kcal/mol)	Binding energy (kcal/mol)	RMSD (nm)
1JIJ	SB-239629	5.412	-34.963	-48.822	-31.21	0.012
1EI1	ANP	-5.290	-38.067	-54.99	-50.07	0.009
1WVG	CXY	-4.302	-41.660	-38.739	-34.003	0.018
2OZ5	OMTS	-6.811	-32.392	-40.221	-41.020	0.093
4OR7	25U	-5.309	-41.720	-51.342	-34.293	0.037

Figure 8

Re-docking interactions of the selected targets with ligands in the respective structures: (a) SB-239629, (b) ANP, (c) CXY, (d) OMTS and (e) 25U.

Experimental section

Synthesis of imidazoquinolines

Compounds 1 and 2 were prepared according to the reported literature [30].

Synthesis of 3: Appropriate 2-hydroxyquinoline-3-carbaldehyde (2.89 mmol), o-phenylenediamine (3.17 mmol) were mixed with ethanol (20 mL) in the presence of triethylamine (4.33 mmol) and refluxed for ten hours. After that, the obtained precipitate was filtered and recrystallized from ethanol to give compound 3.

Compound 3a: Yellow colour Solid (80% yield). M.p.: 251–253 °C; 1H NMR (400 MHz, DMSO-d) δ: 12.65 (s, 1H, OH), 12.47 (s, 1H, NH), 9.11 (s, 1H, Ar), 7.95–7.97 (d, J = 8 Hz, 1H, Ar), 7.59–7.73 (m, 3H, Ar), 7.43–7.45 (d, J = 8 Hz, 1H, Ar), 7.29–7.32 (t, J = 10 Hz, 1H, Ar), 7.18–7.23 (m, 2H, Ar) ppm; 13C NMR (125 MHz, DMSO-d) δ: 113.2, 115.7, 118.7, 119.6, 120.4, 122.3, 122.7, 123.1, 129.5, 132, 134.9, 139.1, 139.5, 143.2, 148.1, 161.2 (C=N) ppm. Elemental analysis: Anal. calc. for: C16H11N3O: C, 73.55; H, 4.24; N, 16.08%. Found: C, 73.48; H, 4.21; N, 16.02%. LC-MS calcd. for C16H11N3O: [M+] 261, found [M++H]+ 262.

Compound 3b: (88% yield). M.p.: 235–237 °C; 1H NMR (400 MHz, DMSO-d) δ: 12.67 (s, 1H, OH), 12.42 (s, 1H, NH), 9.12 (s, 1H, Ar), 7.98–8.00 (d, J = 8 Hz, 1H, Ar), 7.45–7.72 (m, 3H, Ar), 7.16–7.26 (m, 3H, Ar), 2.41 (s, 3H, CH3) ppm; 13C NMR (125 MHz, DMSO-d) δ: 22.4, 114.3, 116.2, 117.4, 119.2, 120.5, 122.7, 122.3, 123.4, 129.6, 132.1, 134.6, 139.3, 139.8, 143.5, 148.6, 161.9 (C=N) ppm. Elemental analysis: Anal. calc. for: C17H13N3O: C, 74.17; H, 4.76; N, 15.26%. Found: C, 74.08; H, 4.82; N, 15.28%. LC-MS calcd. for C17H13N3O: [M+] 275, found [M++H]+ 276.

Compound 3c: (82% yield). M.p.: 231–233 °C; 1H NMR (400 MHz, DMSO-d) δ: 12.62 (s, 1H, OH), 12.43 (s, 1H, NH), 9.15 (s, 1H, Ar), 7.96–7.98 (d, J = 8 Hz, 1H, Ar), 7.52–7.74 (m, 3H, Ar), 7.19–7.24 (m, 2H, Ar), 2.46 (s, 3H, CH3), 2.42 (s, 3H, CH3) ppm; 13C NMR (125 MHz, DMSO-d) δ: 22.3, 22.7, 112.8, 115.2, 119.3, 119.8, 121.2, 122.8, 123.1, 123.4, 129.9, 131.9, 135.2, 139.3, 139.8, 144.2, 148.7, 161.9 (C=N) ppm. Elemental analysis: Anal. calc. for: C18H15N3O: C, 74.72; H, 5.23; N, 14.52%. Found: C, 74.83; H, 5.26; N, 14.48%. LC-MS calcd. for C18H15N3O: [M+] 289, found [M++2H] + 291.

Compound 3d: (78% yield). M.p.: 201–203 °C; 1H NMR (400 MHz, DMSO-d) δ: 12.62 (s, 1H, OH), 12.49 (s, 1H, NH), 9.13 (s, 1H, Ar), 7.97–7.99 (d, J = 8 Hz, 1H, Ar), 7.50–7.74 (m, 3H, Ar), 7.20–7.32 (m, 3H, Ar), 3.91 (s, 3H, OCH3) ppm; 13C NMR (125 MHz, DMSO-d) δ: 54.2, 113.6, 115.9, 118.5, 119.8, 120.6, 122.5, 122.6, 123.3, 129.6, 132.2, 135.2, 139.4, 139.7, 143.6, 148.3, 161.5 (C=N) ppm. Elemental analysis: Anal. calc. for: C17H13N3O2: C, 74.17; H, 4.76; N, 15.26%. Found: C, 74.12; H, 4.78; N, 15.30%. LC-MS calcd. for C17H13N3O2: [M+] 291, found [M++2H]+ 293.

Compound 3e: (79% yield). M.p.: 211–213 °C; 1H NMR (400 MHz, DMSO-d) δ: 12.64 (s, 1H, OH), 12.48 (s, 1H, NH), 9.14 (s, 1H, Ar), 7.94–7.96 (d, J = 8 Hz, 1H, Ar), 7.52–7.75 (m, 3H, Ar), 7.19–7.26 (m, 3H, Ar), 3.92 (s, 3H, OCH3) ppm; 13C NMR (125 MHz, DMSO-d) δ: 53.4, 114.1, 116.2, 119.3, 119.9, 121.3, 123.2, 123.1, 123.8, 130.2, 132.8, 135.1, 139.6, 140.2, 143.7, 148.7, 161.8 (C=N) ppm. Elemental analysis: Anal. calc. for: C17H13N3O2: C, 70.09; H, 4.50; N, 14.42%. Found: C, 69.89; H, 4.48; N, 14.48%. LC-MS calcd. for C17H13N3O2: [M+] 291, found [M++H]+ 292.

Compound 3f: (71% yield). M.p.: 202–204 °C; 1H NMR (400 MHz, DMSO-d) δ: 12.68 (s, 1H, OH), 12.52 (s, 1H, NH), 9.17 (s, 1H, Ar), 7.98–8.00 (d, J = 8 Hz, 1H, Ar), 7.55–7.78 (m, 3H, Ar), 7.21–7.32 (m, 3H, Ar) ppm; 13C NMR (125 MHz, DMSO-d) δ: 114.3, 116.2, 119.4, 120.1, 120.8, 122.9, 123.2, 123.7, 130.0, 132.6, 135.3, 140.3, 140.5, 143.9, 148.7, 161.8 (C=N) ppm. Elemental analysis: Anal. calc. for: C16H10N3O: C, 64.98; H, 3.41; N, 14.21%. Found: C, 65.06; H, 3.43; N, 14.18%. LC-MS calcd. for C16H1035ClN3O: [M+] 295, found [M++2H] + 297; calcd. for C16H1037ClN3O: [M+] 297, found [M++2H] + 299.

Synthesis of 4: o-phenylenediamine (1.15 mmol) treated with 2-chloroquinoline-3-carbaldehyde 3 (1.04 mmol) in ethanol (20 mL) and triethylamine (1.56 mmol), and refluxed for ten hours. The obtained precipitate was recrystallized from chloroform to afford compound 4.

Compound 4a: White solid in 84% yield. M.p.: 201–203 °C; 1H NMR (400 MHz, CDCl3) δ: 10.59 (bs, 1H, NH), 9.26 (s, 1H, Ar), 8.03–8.05 (d, 1H, Ar), 7.92–7.94 (d, J = 8 Hz, 1H, Ar), 7.79–7.83 (m, 2H, Ar), 7.60–7.64 (m, 2H, Ar), 7.27–7.38 (m, 2H, Ar) ppm. 13C NMR (125 MHz, CDCl3) δ: 110.7, 117.9, 119.4, 122.6, 123.3, 125.6, 125.7, 127.9, 128.1, 130.4, 139.3, 143.5, 147.1, 150.0, 159.8 (C=N) ppm. Elemental analysis: Anal. calcd. for: C16H10ClN3: C, 68.70; H, 3.60; N, 15.02%. Found: C, 68.64; H, 3.52; N, 14.98%. LC-MS calcd. for C16H1035ClN3: [M+] 279, found [M+] 279; calcd. for C16H1037ClN3: [M+] 281, found [M+] 281 [31].

Compound 4b: 88% yield. M.p.: 179–181 °C; 1H NMR (400 MHz, CDCl3) δ: 10.61 (bs, 1H, NH), 9.28 (s, 1H, Ar), 8.04–8.06 (d, J = 8 Hz, 1H, Ar), 7.94–7.96 (d, J = 8 Hz, 1H, Ar), 7.65–7.78 (m, 3H, Ar), 7.26–7.32 (m, 2H, Ar), 2.42 (s, 3H, CH3) ppm. 13C NMR (125 MHz, CDCl3) δ: 22.6, 110.2, 118.2, 119.6, 122.3, 123.1, 125.8, 125.9, 127.1, 128.5, 130.3, 139.6, 143.4, 147.3, 150.6, 159.9 (C=N) ppm. Elemental analysis: Anal. calcd. for: C17H12ClN3: C, 69.51; H, 4.12; N, 14.30%. Found: C, 69.56; H, 4.10; N, 14.36%. LC-MS calcd. for C17H1235ClN3: [M+] 293, found [M++H]+ 294; calcd. for C17H1237ClN3: [M+] 295, found [M++H]+ 296.

Compound 4c: 81% yield. M.p.: 209–211 °C; 1H NMR (400 MHz, CDCl3) δ: 10.63 (bs, 1H, NH), 9.28 (s, 1H, Ar), 8.02–8.04 (d, J = 8 Hz, 1H, Ar), 7.85–7.96 (m, 2H, Ar), 7.32–7.45 (m, 3H, Ar), 2.47 (s, 3H, CH3), 2.44 (s, 3H, CH3) ppm. 13C NMR (125 MHz, CDCl3) δ: 22.5, 22.9, 110.7, 118.4, 120.6, 122.8, 123.7, 125.9, 126.2, 127.4, 129.6, 130.4, 138.2, 144.6, 147.7, 150.9, 160.5 (C=N) ppm. Elemental analysis: Anal. calcd. for: C18H14ClN3: C, 70.24; H, 4.58; N, 13.65%. Found: C, 70.29; H, 4.53; N, 13.88%. LC-MS calcd. for C18H1435ClN3: [M+] 307, found [M++2H]+ 309; calcd. for C18H1437ClN3: [M+] 309, found [M++2H]+ 311.

Compound 4d: 89% yield. M.p.: 190–194 °C; 1H NMR (400 MHz, CDCl3) δ: 10.64 (bs, 1H, NH), 9.29 (s, 1H, Ar), 8.07–8.09 (d, J = 8 Hz, 1H, Ar), 7.76–7.89 (m, 2H, Ar), 7.32–7.45 (m, 4H, Ar), 3.92 (s, 3H, OCH3) ppm. 13C NMR (125 MHz, CDCl3) δ: 54.7, 111.2, 119.1, 121.5, 122.4, 123.9, 124.3, 126.1, 127.8, 129.7, 131.6, 138.7, 145.8, 147.5, 149.3, 160.1 (C=N) ppm. Elemental analysis: Anal. calcd. for: C17H12ClN3O: C, 65.92; H, 3.90; N, 13.57%. Found: C, 65.96; H, 3.87; N, 13.48%. LC-MS calcd. for C17H1235ClN3O: [M+] 309, found [M+] 309; calcd. for C17H1237ClN3O: [M+] 311, found [M+] 311.

Compound 4e: 86% yield. M.p.: 237–239 °C; 1H NMR (400 MHz, CDCl3) δ: 10.62 (bs, 1H, NH), 9.28 (s, 1H, Ar), 8.04–8.06 (d, J = 8 Hz, 1H, Ar), 7.83–7.95 (m, 2H, Ar), 7.42–7.65 (m, 4H, Ar), 3.94 (s, 3H, OCH3) ppm. 13C NMR (125 MHz, CDCl3) δ: 53.8, 111.8, 117.5, 119.6, 122.9, 123.4, 125.8, 126.2, 127.8, 128.7, 130.4, 138.6, 143.9, 147.7, 150.3, 159.2 (C=N) ppm. Elemental analysis: Anal. calcd. for: C17H12ClN3O: C, 65.92; H, 3.90; N, 13.57%. Found: C, 65.98; H, 3.87; N, 13.68%. LC-MS calcd. for C17H1235ClN3O: [M+] 309, found [M++H]+ 310; calcd. for C17H1237ClN3O: [M+] 311, found [M++H]+ 312.

Compound 4f: 72% yield. M.p.: 167–171 °C; 1H NMR (400 MHz, CDCl3) δ: 10.64 (bs, 1H, NH), 9.29 (s, 1H, Ar), 8.06–8.09 (d, J = 12 Hz, 1H, Ar), 7.83–7.95 (m, 2H, Ar), 7.32–7.46 (m, 4H, Ar) ppm. 13C NMR (125 MHz, CDCl3) δ: 111.2, 118.4, 119.7, 122.9, 123.6, 125.8, 126.4, 127.8, 128.6, 130.7, 139.8, 143.8, 148.7, 150.9, 159.3 (C=N) ppm. Elemental analysis: Anal. calcd. for: C16H9Cl2N3: C, 61.17; H, 2.89; N, 13.38%. Found: C, 61.23; H, 2.85; N, 13.45%. LC-MS calcd. for C16H935Cl2N3: [M+] 314, found [M++2H] 316; calcd. for C16H935Cl37ClN3: [M+] 316, found [M++2H] 318; calcd. for C16H937Cl2N3: [M+] 318, found [M++2H] 320.

Synthesis of 5: Chloroimidazoquinoline 4 (0.71 mmol) and sodium sulphide (1.07 mmol) were dissolved in dimethylformamide and stirred at room temperature for five hours. The reaction mixture was extracted by ethyl acetate and the crude product was purified by silica gel column chromatography with EA:hexane (1:1) to afford the thio-imidazoquinolines 5.

Compound 5a: yellow colour solid in 82% yield [31]. M.p.: 204–205 °C; 1H NMR (400 MHz, CDCl3) δ: 14.18 (bs, 1H, SH), 9.19 (s, 1H, Ar), 7.98–8.00 (d, J = 8 Hz, 1H, Ar), 7.78–7.80 (d, J = 8 Hz, 1H, Ar), 7.71–7.73 (m, 3H, Ar), 7.41–7.45 (t, J = 8 Hz, 1H, Ar), 7.24–7.27 (m, 2H, Ar) ppm; 13C NMR (125 MHz, DMSO-d) δ: 113.24, 115.71, 118.75, 119.62, 120.48, 122.36, 122.74, 123.12, 129.51, 132.06, 134.90, 139.15, 139.53, 143.25, 148.19, 161.26 (C=N) ppm. Anal. calcd for: C16H11N3S: C, 69.29; H, 4.00; N, 15.15%. Found: C, 69.15; H, 3.98; N, 15.18%. HR-MS calcd. for C16H11N3S: [M+] 277, found [M + H]+ 278.

Compound 5b: 85% yield. M.p.: 224–226 °C; 1H NMR (400 MHz, CDCl3) δ: 14.20 (bs, 1H, SH), 9.21 (s, 1H, Ar), 7.99–8.01 (d, J = 8 Hz, 1H, Ar), 7.70–7.79 (m, 3H, Ar), 7.31–7.46 (m, 3H, Ar), 2.32 (s, 3H, CH3) ppm. 13C NMR (125 MHz, DMSO-d) δ: 22.42, 113.26, 115.74, 118.77, 119.65, 120.49, 122.39, 122.78, 123.16, 129.54, 132.07, 134.97, 139.16, 139.58, 143.26, 148.23, 161.29 (C=N) ppm. Anal. calcd. for: C17H13N3S: C, 70.08; H, 4.50; N, 14.42%. Found: C, 70.18; H, 4.45; N, 14.36%. LC-MS calcd. for C17H13N3S: [M+] 291, found [M + H]+ 292.

Compound 5c: 79% yield. M.p.: 223–224 °C; 1H NMR (400 MHz, CDCl3) δ: 14.08 (bs, 1H, SH), 9.16 (s, 1H, Ar), 7.95–7.97 (d, J = 8 Hz, 1H, Ar), 7.71–7.82 (m, 2H, Ar), 7.22–7.36 (m, 3H, Ar), 2.48 (s, 3H, CH3), 2.41 (s, 3H, CH3) ppm; 13C NMR (125 MHz, DMSO-d) δ: 22.41, 22.82, 113.34, 115.81, 118.75, 119.68, 120.52, 122.38, 122.78, 123.16, 129.53, 132.09, 134.94, 139.17, 139.55, 143.29, 148.20, 161.36 (C=N) ppm. Anal. calcd for: C18H15N3S: C, 70.79; H, 4.95; N, 13.76%. Found: C, 70.68; H, 4.91; N, 13.83%. LC-MS calcd. for C18H15N3S: [M+] 305, found [M + H]+ 306.

Compound 5d: 85% yield. M.p.: 244–245 °C; 1H NMR (400 MHz, CDCl3) δ: 14.28 (bs, 1H, SH), 9.29 (s, 1H, Ar), 8.02–8.04 (d, J = 8 Hz, 1H, Ar), 7.75–7.82 (m, 3H, Ar), 7.27–7.38 (m, 3H, Ar), 3.93 (s, 3H, OCH3) ppm; 13C NMR (125 MHz, DMSO-d) δ: 54.86, 113.34, 115.81, 118.82, 119.65, 120.54, 122.46, 122.78, 123.22, 129.62, 132.12, 134.98, 139.23, 139.61, 143.32, 148.23, 161.29 (C=N) ppm. Anal. calcd for: C17H13N3OS: C, 66.43; H, 4.26; N, 13.67%. Found: C, 66.54; H, 4.28; N, 13.58%. LC-MS calcd. for C17H13N3OS: [M+] 307, found [M++2H]+ 309.

Compound 5e: 81% yield. M.p.: 217–220 °C; 1H NMR (400 MHz, CDCl3) δ: 14.29 (bs, 1H, SH), 9.27 (s, 1H, Ar), 7.99–8.01 (d, J = 8 Hz, 1H, Ar), 7.76–7.81 (m, 3H, Ar), 7.32–7.45 (m, 3H, Ar), 3.95 (s, 3H, OCH3) ppm; 13C NMR (125 MHz, DMSO-d) δ: 55.23, 113.32, 115.78, 118.79, 120.05, 120.98, 122.46, 122.84, 123.32, 129.71, 132.36, 134.98, 139.50, 139.59, 143.32, 148.29, 161.46 (C=N) ppm. Anal. calcd for: C17H13N3OS: C, 66.43; H, 4.26; N, 13.67%. Found: C, 66.35; H, 4.18; N, 13.49%. LC-MS calcd. for C17H13N3OS: [M+] 307, found [M++2H]+ 309.

Compound 5f: 74% yield. M.p.: 201–202 °C; 1H NMR (400 MHz, CDCl3) δ: 14.38 (bs, 1H, SH), 9.26 (s, 1H, Ar), 7.94–7.96 (d, J = 8 Hz, 1H, Ar), 7.74–7.83 (m, 3H, Ar), 7.27–7.41 (m, 3H, Ar) ppm; 13C NMR (125 MHz, DMSO-d) δ: 114.14, 116.73, 119.15, 119.92, 121.82, 122.86, 122.98, 124.13, 130.21, 133.09, 135.80, 139.75, 139.83, 144.35, 149.29, 163.16 (C=N) ppm. Anal. calcd for: C16H10N3S: C, 61.64; H, 3.23; N, 13.48%. Found: C, 61.85; H, 3.14; N, 13.38%. LC-MS calcd. for C16H1035ClN3S: [M+] 311, found [M + H]+ 312; calcd. for C16H1037ClN3S: [M+] 313, found [M + H]+ 314.

Biological studies

Antibacterial activity by agar well diffusion method

The antibacterial activity of the synthesized compounds 3–5(a-f) was performed on the agar well diffusion method. Each chemical compound was mixed with an equal amount of dimethyl sulfoxide (DMSO) i.e., 1 μl of DMSO contains 1 mg of chemical compound and is considered as the stock solution. The synthesized active chemical compounds were then subjected to the antibacterial activity against the human pathogenic bacteria isolated from Hospital clinical samples which were collected by The Rhizosphere Biology Laboratory, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. The procured pathogenic bacteria namely Staphylococcus aureus, Aeromonoas hydrophila, Escherichia coli, Klebsiella pneumonia, Salmonella paratyphi, Salmonella typhi, and Mycobacterium butyricum (MTCC 940) were cultured in nutrient agar medium and incubated at 37 °C for at 16 h. The medium was further used for the antibacterial activity with the synthesized imidazoquinoline derivatives by the agar well diffusion method. Each compound 3–5(a-f) was poured and checked for their antibacterial activity against the seven collected strains with different concentrations like 10 mg/10 μl, 50 mg/50 μl, and 100 mg/100 μl. The zone of inhibition was found after the incubation period of 24 h at 37 °C and the measurements were reported as diameters in millimeters. The higher zone of inhibition was considered as a significant activity of the synthesized chemical compound against the virulent human pathogenic bacteria [32, 33, 34].

The antioxidant potential of the imidazoquinolines was examined by using the ferric reducing ability of the plasma FRAP assay. A potent antioxidant could reduce Fe3+ to Fe2+ and forms a blue complex (Fe2+/TPTZ), which is monitored by UV-vis absorption studies (593 nm). In detail, FRAP reagent was prepared by mixing of acetate buffer (300 μM, pH 3.6), TPTZ (10 μM) in HCl (40 μM) and FeCl3 (20 μM) at 10:1:1 (v/v/v). The sample solutions (10 μl) and reagent (300 μl) were mixed and recorded the absorbance after 10 min. The standard curve was prepared by using Trolox. All the diluted solution results were (Trolox per 100 g dry weight (dw)) performed in triplicates expressed in μM. The stock solutions of each synthesized chemical compound individually mixed with double distilled water about 1 mg/mL and variant concentration about 10 mg/mL were prepared and proceeded the above procedure to react with the FRAP reagents and taken the Optical density level at 593 nm in UV visible spectroscopy. The Optical density was noted and the strength meant its antioxidant potential to control the free radical scavenging activity through standard curve [35, 36].

In silico environment preparation

For this study, the various protein structures from S. aureus tyrosyl-tRNA synthetase (1JIJ), E. coli -DNA gyrase B (1EI1), K. pneumoniae (4OR7), A. hydrophila proaerolysin (1PRE), S. paratyphi lipopolysaccharide acetyltransferase periplasmic domain (6SE1), S. typhi CDP-D-glucose 4, 6-dehydratase (1WVG), M. tuberculosis MTB phosphotyrosine phosphatase B protein (2OZ5) are taken for the study. For the desired molecular modeling calculations, the above-mentioned proteins are subjected to the protein preparation wizard implemented in Schrodinger maestro [37]. For the proteins in dimer form, the B chain is removed and the water molecules without interactions are also removed. Using the OPLS-3e forcefield, all the proteins are optimized for their hydrogen atoms and minimized till achieving the least conformation RMSD reaches 0.30Å [38]. Likewise, the ligands 3f, 4f, and 5f were also prepared using the LigPrep [39] molecules for ensuring the correctness of bond orders, ligand charges, and generating conformations up to 32 based on available rotatable bonds. Based on this approach both proteins and ligands are prepared for the molecular modeling environment [40].

GRID generation and molecular docking

For understanding the molecular interactions of a ligand inside the protein active site, each prepared protein is subject to grid generation using the GLIDE [41]. Before the grid generation, the protein was executed with a sitemap for the active site analysis, and the output of the sitemap generated white dots were manually picked for the Glide Grid generation. As the sitemap didn't predict the active site residues, so the white-colored dot regions and around 2Å radius were considered for active site regions. For each protein Grid generation, the methodology followed was the same, and for that, the white spheres from the sitemap output were manually picked for the XYZ axis. The position of the ligand to be docked was fixed from a 2 Å radius from the site predicted from the sitemap [42]. The generated grid file and prepared ligand files were docked using the XP ligand docking method and validated using Prime MM/GBSA method [43]. Among the ligands, best conformations were scrutinized based on docking score, docking energy, binding energy, and hydrogen bonds formed between the protein-ligand complex.

Statistical analysis

Statistical analysis was adopted with all parameters exploiting Graphpad prism 7.0 (GraphPad Software, San Diego, CA, USA). Data were represented as the mean ± SD. In column statistics, one sample t-test was performed to find the p-value (two tailed). The validity of the null hypothesis was verified with a significance level (α = 0.05). NS: not significant.

Conclusion

In conclusion, we have synthesized the various hydroxyl-, chloro-, and thio-derivatives of imidazoquinolines through a conventional approach in good to moderate yields from the classical intermediate 2-chloro-3-formylquinolines. All the hitherto synthesized imidazoquinolines were screened for their antibacterial activities. Among them, the electron-withdrawing substituent (-Cl) present in the compound 5f was found to be the most active derivative than other imidazoquinolines, which was comparable with that of the standard. Notably, at high concentration (100 mg/disc), 5f exhibited the higher activity against E. coli, K. pneumonia, S. paratyphi, S. typhi, M. butyricum than that of in standard (10 mg/disc). In addition, other electron-withdrawing and donating substituents containing compounds 4f and 3f and 5e also exhibited moderate activity. The antioxidant power of these compounds was also screened by FRAP. Furthermore, molecular docking studies were carried out for the selected imidazoquinoline derivatives (3f, 4f, and 5f) with seven types of different proteins. These preliminary results indicated that the antibacterial activity could be enhanced by electron-withdrawing substitution introduced by different positions of the quinoline scaffold. Therefore, these derivatives can be potentially used for MDR antibiotics-related applications.

Declarations

Author contribution statement

K. Velmurugan, Derin Don, S. VishnuPriya: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Rajesh Kannan: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

C. Selvaraj: Analyzed and interpreted the data; Wrote the paper.

G. Selvaraj: Analyzed and interpreted the data.

S.K. Singh: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

R. Nandhakumar: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.

Funding statement

This work was supported by RUSA-Phase 2.0 Policy (TNmulti-Gen), Dept. of Edn, Govt. of India (Grant No: F.24-51/2014-U).

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.