Introduction

The quinoline scaffold is prevalent in a variety of pharmacologically active synthetic and natural compounds. A large variety of quinoline derivatives have been used as antimalarial, anti-inflammatory, antiasthmatic, antibacterial, antihypertensive (Dube et al., 1998; Maguire et al., 1994) and anticancer (Denny et al., 2006) and anti-HIV (Wilson et al., 1992). Coumarin derivatives on the other hand having wide applications as drugs and pharmaceuticals, such as antibacterial (Appendino et al., 2004; Khan et al., 2004), antioxidant (Nicolaides et al., 1998; Raj et al., 1998), anti-inflammatory (Litinadj et al., 2004; Ghate et al., 2005) and anticancer (Bhattacharyya et al., 2009). Keeping in view the biological importance of both quinoline and coumarin in a single molecule (Miri et al., 2011; Tabakovic et al., 1983, 1987; Emami et al., 2008), here, with which we are reporting the synthesis of 7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-one derivatives and their invitro antibacterial, antioxidant and molecular docking studies. The idea in molecular docking is to computationally design pharmaceuticals targeted against proteins. Docking methods not only add insights to the biological processes at the molecular level but also aid in the development of novel lead compounds (drugs) that can help to combat disease. Molecular docking algorithms seek to predict the bound conformations of two interacting molecules, such as protein–ligand and protein–protein complexes.

Results and discussion

Chemistry

2,4-Dichloroquinolines (2a–g) have been synthesized by the reaction of aniline on malonic acid in excess of phosphorus oxychloride (POCl3) (Rajesh et al., 2009). The reaction of 2a–g with 7-hydroxy-2H-chromen-2-one (1) at 60 °C for 15 h in the presence of K2CO3 as a catalyst afford the 7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-ones (3a–g) with 60–80 % yield. In continuation of our earlier interest (Balaji et al., 2012; Rajesh et al., 2012) on ultrasound assisted reactions, the above reaction has also been subjected to the ultrasonic irradiation at 60 °C for 20 min, which yield the product 3a–g with 80–94 %, and hence ultrasound-promoted synthesis can be the better approach to the synthesis 2-chloroquinolin-4-pyrimidine carboxylate derivatives Scheme 1.

The reactivity of the halogen atoms in the various quinolines varied widely, but the kinetic studies indicate that the chloro atom at C-4 of 2,4-dichloroquinolines is about two times more reactive towards nucleophiles and predominantly an addition elimination mechanism is observed. The reaction of 2,4-dichloro-6-methyl quinoline with sodium azide (1:1 molar ratio) in DMF lead to regioselective 4-azido-2-chloro-6-methylquinoline (Natarajan et al., 2009) also confirmed the reactivity at C-4 of 2,4-dichloroquinolines. 1H NMR, 13C NMR and mass spectra confirmed the formation of 3a–g. The 1H NMR spectrum of compound 3b exhibited two singlets at δ 2.47, 2.87 ppm which corresponds to the protons of methyl group at C-4 of coumarin and C-7 of quinoline, respectively. The singlet at δ 6.31 ppm and δ 6.65 ppm corresponds to the protons at C-3 of coumarin and quinoline, respectively. A singlet at δ 7.09 ppm and δ 7.12 ppm corresponds to the protons at C-8 of coumarin and quinoline, respectively. A doublet at δ 7.37 ppm and δ 7.62 ppm corresponds to the protons at C-8 of coumarin and quinoline, respectively, and the doublet at δ 7.71 ppm and δ 7.89 ppm corresponds to the protons at C-5 of coumarin, quinoline, respectively. Its 13C NMR spectrum shows chemical shift values at δ 18.76 ppm and δ 23.91 ppm corresponds to C-4 and C-7 on coumarin and quinoline, respectively, and the chemical shift values at δ 114.62, 116.21 and 118.20 ppm corresponds to C-8, C-3 and C-10 on coumarin. The chemical shift values at δ 129.63, 130.94 and 142.30 ppm corresponds to the carbons at C-8, C-6 and C-7 on quinoline. The chemical shift values at δ 160.18 and 163.78 ppm corresponds to C-4 and C-2 carbons on quinoline and coumarin, respectively. M/z value observed at 352.1 (M + 1) peak in ES-MS spectra also confirms the formation of target molecule (Table 1).

Scheme 1
scheme 1

Synthesis of 7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-ones (3a–g)

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Fig. 1
figure 1

Equilibrated molecular dynamics snap shot of docked compounds 3a–g, the aminoacids pertaining to the binding site or contacting to the ligands are shown in atom-coloured sticks, active site binding interactions are shown in dotted lines (Color figure online)

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Table 1 Synthesis of 7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-ones (3a–g)
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Biological evaluation

Antimicrobial studies

As part of our interest to find the new antibacterial agents (Venkatragavan et al., 2009, 2010, 2011; Sarveswari et al., 2011) all the newly synthesized compounds 3a–g were screened for their invitro antibacterial activity against gram(+)ve and gram(−)ve bacterial strains namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Meloidogyne litoralis and Bacillus subtilis. Compounds possessing methyl, methoxy and fused aryl rings such as 3c, 3d and 3g at C-8 of quinoline ring showed better activity than their standard drug Streptomycin against Escherichia coli, similarly compounds 3c and 3g showed better activity against P. aeruginosa. Compound 3f with bromine at C-6 showed better activity against M. litoralis, whereas 3a with methyl at C-6 showed better activity against S. aureus. No compound is having good activity against B. subtilis with the standard drug Streptomycin, respectively (Table 2).

Table 2 Antibacterial activity for the compounds 3a–g
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DPPH radical scavenging assay

Radical scavenging activity is very important due to the deleterious role of free radicals in foods and in biological systems. Diverse methods are currently used to assess the antioxidant activity. In the present study, DPPH (1,1-diphenyl-2-picryl-hydrazil) radical-scavenging method has been chosen to evaluate the antioxidant potential of the compounds 3a–g. DPPH radical scavenging activity has been determined spectrophotometrically by means of the literature method (Farhanullah et al., 2006; Brand-Williams et al., 1995). The percentage of inhibition was given in Table 3 and compared with that of commercial antioxidant (Tepe et al., 2006) butylated hydroxy toluene (BHT). The results in percentage are expressed as the ratio of absorbance decrease at 517 nm, and the absorbance of DPPH solution in the absence of compounds. The observed values given in Table 3 revealed that the radical scavenging activity of 7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-one on DPPH radicals increases with the increase in concentration. Compounds possessing chloro, bromo substituents at C-6 (3e, f) showed maximum activity at a concentration of 1,000 μg/mL. The radical scavenging activity of compounds possessing methyl at C-7 (3b) exhibited less potent than the standard.

Table 3 Antioxidant activity for the compounds 3a–g by DPPH Method
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Molecular Docking

In silico modeling is an upcoming facade to investigate promising therapeutics for their effective inclination as able leads towards specific pathologies. The docking analysis caters with the knowledge of the extent of plausible interaction between the target of interest and the drug under investigation. This in turn helps to procure a primary understanding of the viability of the drug or compound under scrutiny. The analysis etches an advantage over the in vitro or in vivo analyses in being faster, safer and having less infrastructural requirements (Garg et al., 2010). The compounds 3a–g synthesized in the present study revealed their respective abilities in successful binding to the pathogen (Plasmodium falciparum) erythrocyte membrane protein, consequently proving their merits towards being molded as an anti malarial agent. The Plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP 1) structure was obtained from protein data bank (PDB) (Hu et al., 2009). The ligands 3a–g were explored to test their effectiveness in binding with the receptor, PfEMP 1. The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a significant virulence factor which expresses itself on the surface of erythrocytes infected with the pathogen. Also, the protein is responsible for directly mediating adhesion to a plethora of host cells (Horrocks et al., 2005). Thus, analysing the effect of any compound on this specific protein could therefore lead towards a potential source by which the deleterious manifestations of this protein can be curbed. The approach would also help towards identifying potent lead towards developing antimalaria drugs. For all the compounds, 3a–g protein–ligand docking calculations (Hu et al., 2009) were carried out on plasmodium falciparum UCHL3 protein to compare the ligand binding energy with standard antimalarial drugs like amodiaquine and chloroquine (showing binding energy value of −6.48 and −6.59 kcal/mol) and are given in Table 4, Fig. 1. Blind docking was carried out in which the entire receptor was scanned for probable docking sites so as to facilitate maximum possible fits. Energy minimization for the 2D structure (drawn by chem sketch) of each of the isolated compounds was initiated by means of Chimera software. In principle, amongst a variety of ligands, the ones with the lowest binding energies are considered to be the most potential hits (Garg et al., 2010). Therefore, the analysis indicates towards 2-chlorobenzo(h)quinoline (3g) that exhibits binding energy of −9.65 kcal/mol. Moreover, the results obtained help to identify the efficacy of the isolated novel compounds as potent antimalarial drug.

Table 4 Comparison of binding energy value of compounds 3a–g with standard antimalarial drugs
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Experimental

Chemistry

The materials were purchased from Sigma–Aldrich, Merck and were used without any additional purification. All reactions were monitored by thin layer chromatography (TLC). Melting points were recorded on an Elchem digital melting point apparatus in open capillaries and are uncorrected. The ultrasound for the synthesis is generated with the help of ultrasonic instrument (Make: E-chrom Tech Co. Ltd., Taiwan. Operating frequency: 22 kHz, Rated output power: 800 W). The 1H NMR was measured on a Bruker Avance-400 MHz instrument at room temperature. The 1H NMR was measured for ~0.03 M solutions in CDCl3 using TMS as internal reference. The accuracy of the 1H shifts is considered to be 0.02 ppm. The coupling constants J are in Hertz. Mass spectra were obtained by ESI mass spectrometry.

General procedure for the synthesis of 7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-one derivatives (3a–g)

Conventional method

All the substituted 2,4-dichloroquinolines (2a–g) were prepared according to the method available in literature [Rajesh et al., 2009; Balaji et al., 2012]. To the solution of the appropriate 2,4-dichloroquinoline (5 mmol) in 20 mL of DMF, 7-hydroxy-2H-chromen-2-one (1) (5 mmol) and K2CO3 (15 mmol) was added and heated at 60 °C for 15 h. After the completion of reaction, the reaction mixture poured into ice cold water and the product was collected by filtration and recrystallized using ethanol.

Ultrasonic irradiation Method

To the solution of the appropriate 2,4-dichloroquinoline (5 mmol) in 20 mL of DMF, 7-hydroxy-2H-chromen-2-one (5 mmol) and K2CO3 (15 mmol) was added and kept under ultrasonic irradiation (Make: E-chrom Tech Co. Ltd., Taiwan. Operating frequency: 22 kHz, Rated output power: 800 W) at 50 % amplitude for 20 min with five intervals each for 4 min at 60 °C. After the completion of the reaction, the reaction mixture poured into ice cold water and the product was collected by filtration and recrystallized using ethanol. All the synthesized compounds were characterized by 1H NMR, 13C NMR, ESI-MS and Elemental analysis techniques. The spectral data of compounds 3a-g has been given below.

7-(2-Chloro-6-methylquinolin-4-yloxy)-4-methyl-2H-chromen-2-one ( 3a ) white powder; m.p. 155–157 °C; 1H NMR (400 MHz, CDCl3) δ: 2.49 (s, 3H, C4–CH3 of coumarin), 2.56 (s, 3H, CH3 at C6 of quinoline), 6.32 (s, 1H, –H at C3 of coumarin), 6.61 (s, 1H, –H at C3 of quinoline), 7.13 (d, 1H, J = 8.6 Hz, –H at C6 of coumarin), 7.18 (s, 1H, –H at C8 of coumarin), 7.62 (d, 1H, J = 8.6 Hz, –H at C7 of quinoline), 7.71 (d, 1H, J = 8.6 Hz, –H at C5 of coumarin), 7.92 (d, 1H, J = 8.6 Hz, –H at C8 of quinoline), 8.03 (s, 1H, –H at C5 of quinoline). ES-MS: m/z 352.0 (M+). Anal. Calcd. for C20H14ClNO3: C, 68.28; H, 4.01; N, 3.98. Found: C, 67.97; H, 4.13; N, 3.85.

7-(2-chloro-7-methylquinolin-4-yloxy)-4-methyl-2H-chromen-2-one ( 3b ) white powder; m.p. 142–144 °C; 1H NMR (400 MHz, CDCl3) δ: 2.47 (s, 3H, C4–CH3 of coumarin), 2.87 (s, 3H, CH3 at C6 of quinoline), 6.31 (s, 1H, –H at C3 of coumarin), 6.65 (s, 1H, –H at C3 of quinoline), 7.09 (s, 1H, –H at C8 of coumarin), 7.12 (s, 1H, –H at C8 of quinoline), 7.37 (d, 1H, J = 10.4 Hz, –H at C6 of coumarin), 7.62 (d, 1H, J = 9.1 Hz, –H at C6 of quinoline), 7.71 (d, 1H, J = 8 Hz, –H at C5 of coumarin), 7.89 (d, 1H, J = 8 Hz, –H at C5 of quinoline); 13C NMR (400 MHz, CDCl3) δ: 18.76, 23.91, 108.72, 114.62, 116.21, 117.56, 118.20, 120.14, 121.46, 126.55, 127.09, 129.63, 130.94, 142.30, 150.34, 151.75, 155.10, 156.94, 160.18, 163.75; ES-MS: m/z 352.0 (M+). Anal. Calcd. for C20H14ClNO3: C, 68.28; H, 4.01; N, 3.98. Found: C, 68.07; H, 4.23; N, 3.79.

7-(2-chloro-8-methylquinolin-4-yloxy)-4-methyl-2H-chromen-2-one ( 3c ) white powder; m.p. 174–176 °C; 1H NMR (400 MHz, CDCl3) δ: 2.48 (s, 3H, CH3 at C4 of coumarin), 2.78 (s, 3H, CH3 at C8 of quinoline), 6.31 (s, 1H, –H at C3 of coumarin), 6.64 (s, 1H, –H at C3 of quinoline), 7.12 (d, 1H, J = 8 Hz, ArH of coumarin), 7.16 (s, 1H, –H at C8 of coumarin), 7.45 (t, 1H, J = 6.8 Hz, –H at C6 of quinoline), 7.64 (d, 1H, J = 8 Hz, –H at C5 of coumarin), 7.70 (d, 1H, J = 8 Hz, –H at C7 of quinoline), 8.01 (d, 1H, J = 8 Hz, –H at C5 of quinoline); ES-MS: m/z 352.0 (M+). Anal. Calcd. for C20H14ClNO3: C, 68.28; H, 4.01; N, 3.98. Found: C, 68.23; H, 4.13; N, 3.93.

7-(2-chloro-8-methoxyquinolin-4-yloxy)-4-methyl-2H-chromen-2-one ( 3d ) white powder; m.p. 180–182 °C; 1H NMR (400 MHz, CDCl3) δ: 2.48 (s, 3H, CH3 at C4 of coumarin), 4.09 (s, 3H, CH3 of –OCH3 at C8 of quinoline), 6.32 (s, 1H, –H at C3 of coumarin), 6.67 (s, 1H, –H at C3 of quinoline), 7.17 (dd, 2H, J = 5.6 Hz, ArH of coumarin), 7.18 (s, 1H, –H at C8 of coumarin), 7.51 (t, 1H, J = 6.12 Hz, –H at C6 of quinoline), 7.71 (d, 1H, J = 8.6 Hz, –H at C5 of quinoline), 7.77 (d, 1H, J = 8.7 Hz, –H at C5 of coumarin); 13C NMR (400 MHz, CDCl3) δ: 18.76, 56.16, 106.93, 109.26, 109.99, 113.22, 114.64, 116.67, 117.91, 121.47, 126.59, 127.17, 140.66, 150.14, 151.77, 154.67, 154.78, 156.68, 160.19, 161.96; ES-MS: m/z 368.0 (M+). Anal. Calcd. for C20H14ClNO4: C, 65.31; H, 3.84; N, 3.81. Found: C, 65.50; H, 3.77; N, 3.68.

7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-one ( 3e ) white powder; m.p. 166–168 °C; 1H NMR (400 MHz, CDCl3) δ: 2.49 (s, 3H, CH3 at C4 of coumarin), 6.33 (s, 1H, -H at C3 of coumarin), 6.63 (s, 1H, –H at C3 of quinoline), 7.14 (d, 1H, J = 8.6 Hz, –H at C6 of coumarin), 7.23 (s, 1H, –H at C8 of coumarin),7.54 (t, 1H, J = 7.54 Hz, –H at C6 of quinoline), 7.62 (d, 1H, J = 8.6 Hz, –H at C5 of coumarin), 7.66 (t, 1H, J = 7.2 Hz, –H at C7 of quinoline), 7.72 (d, 1H, J = 8.6 Hz, –H at C8 of quinoline), 7.80 (d, 1H, J = 7.2 Hz, C5–H of quinoline); ES-MS: m/z 338.0 (M+). Anal. Calcd. for C19H12ClNO3: C, 67.56; H, 3.58; N, 4.15. Found: C, 67.35; H, 3.69; N, 3.97.

7-(2-chloro-6-bromoquinolin-4-yloxy)-4-methyl-2H-chromen-2-one ( 3f ) white powder; m.p. 160–162 °C; 1H NMR (400 MHz, CDCl3) δ: 2.47 (s, 3H, CH3 at C4 of coumarin), 6.40 (s, 1H, –H at C3 of coumarin), 6.61 (s, 1H, –H at C3 of quinoline), 7.14 (d, 1H, J = 8 Hz, –H at C7 of coumarin), 7.19 (s, 1H, –H at C8 of quinoline), 7.74 (d, 1H, J = 8 Hz, –H at C5 of coumarin), 7.86 (dd, 2H, J = 7.2 Hz, –H at C7 and –H at C8 of quinoline), 8.45 (s, 1H, –H at C5 of quinoline); 13C NMR (400 MHz, CDCl3) δ: 18.76, 106.38, 109.49, 114.89, 116.82, 118.28, 120.95, 121.38, 124.31, 126.76, 130.10, 135.04, 147.45, 151.46, 151.68, 154.99, 156.02, 160.05, 160.17; ES-MS: m/z 416.0 (M+ − 1). Anal. Calcd. for C19H11BrClNO3: C, 54.77; H, 2.66; N, 3.36. Found: C, 54.72; H, 2.76; N, 3.27.

7-(2-chlorobenzo(h)quinolin-4-yloxy)-4-methyl-2H-chromen-2-one ( 3g ) white powder; m.p. 208–210 °C; 1H NMR (400 MHz, CDCl3) δ: 2.50 (s, 3H, CH3 at C4 of coumarin), 6.31 (s, 1H, –H at C3 of coumarin), 6.82 (s, 1H, –H at C3 of quinoline), 7.14 (d, 1H, J = 8.6 Hz, –H at C6 of coumarin), 7.20 (s, 1H, –H at C8 of coumarin), 7.75 (m, 3H, ArH of quinoline), 7.88 (d, 1H, J = 8 Hz, –H at C6 of quinoline), 7.94 (d, 1H, J = 10 Hz, ArH of coumarin), 8.11 (d, 1H, J = 8 Hz, ArH of benzo), 9.22 (d, 1H, J = 10 Hz, ArH of benzo); ES-MS: m/z 388.0 (M+). Anal. Calcd. for C23H14ClNO3: C, 71.23.77; H, 3.64; N, 3.61. Found: C, 71.01; H, 3.57; N, 3.45.

Antibacterial activity

Sterile nutrient broth was prepared and inoculated with different species of bacteria (Escherichia coli, P. aeruginosa, S. aureus, M. litoralis and B. subtilis) and incubated at 37 °C for overnight. From the overnight culture, 1 % stock culture was prepared (99 mL of sterile nutrient broth + 1 mL of overnight culture). 25 mL of nutrient agar was poured in sterile Petri plates and allowed to cool. Each agar plate was inoculated with 200 μL of 1 % bacterial culture and spread using spreader. Using a sterile cork borer, 6-mm diameter of holes was made in the solidified agar plates containing 1 % of respective bacterial culture. A total volume of 20 μL of test sample of 3a-g was poured into the well. Streptomycin was used as a standard drug. The minimum inhibitory concentration (MIC) values are provided in Table 2.

Antioxidant activity

The synthesized compounds were used to prepare stock using ethanol (0.3 mM). The appropriate concentrations of the compounds were made by serial dilution in different concentrations, i.e. 50, 100, 500 and 1,000 μg/mL of test samples in AR grade ethanol. The samples (3 mL) of above concentrations were mixed with 1 mL of 0.15 mM of DPPH prepared in AR grade ethanol and incubated at room temperature for 30 min in dark. The absorbance of the incubated solutions and the blank (without sample) were recorded against BHT. The absorbance was measured at 517 nm using a UV–Visible (Systronics 118 model) spectrophotometer. Radical-scavenging capacity (RSC) in percent was calculated by the following equation:

$$ {\text{RSC }}\left( \% \right) \, = \, 100 \, \times \, \left( {A_{\text{control}} - \, A_{\text{sample}} } \right)/A_{\text{control}} $$

where RSC is the radical-scavenging capacity, A control the absorbance of control, A sample is the absorbance of sample.

Molecular docking

PDB coordinates of P. falciparaum UCHL3 structure (PDB code: 2WDT) were retrieved from PDB (http:/www.pdb.org/pdb/home/home.do) from the complex, the cocrystallized ligands were identified and removed from the structure and the protein was minimized by means of the off line software chimera. Water molecules were removed and H atoms were added to the structure. Biochemical compounds selected to perform this study was related to malarial diseases. All the chemical compounds were drawn with chem. sketch and optimized by means of chimera. The optimized structures were converted to Mol2 file format by means of chimera. For all the chemical compounds protein–ligand docking calculations were carried out on plasmodium falciparum UCHL3 protein. In all cases, binding affinities were reported (Table 4) and was compared with existing antimalarial drugs like amodiaquine and chloroquine.

Conclusion

In summary, K2CO3 proved to be an efficient catalyst to obtain the mono-substituted 7-(2-Chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-one with high regioselectivity from 2,4-dichloroquinoline and 7-hydroxy-2H-chromen-2-one. These compounds has been subjected to the antimicrobial screening against a panel of human pathogens that most of the them are found to be more active than the standard drugs, antioxidant activity for compound 3e shows moderately 78 % of inhibition. It is worth mentioning that the binding energy value of synthesized compounds, very less compare to standard antimalarial drugs like chloroquine and amodiaquine.