Materials and methods
1,2,4-Triazole-3,5-diamine and tetrabromophthalic anhydride purchased from Sigma Aldrich Chemicals Pvt. Ltd. and 2,6-diaminopyridine, o-tolidine, 2,6-diamino-4-hydroxypyrimidine, and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) from Merck, India. All the reagents were used as received without further purification. For thin-layer chromatography, precoated aluminum sheets (silica gel 60 F254, Merck, Germany) were used to monitor the reaction by using methanol:dichloromethane (1:4) as a solvent system and for the spots visualization UV light cabinet was used.
To perform elemental analysis, Vario Micro Elementar Analyzer was used. Electronic spectra were recorded on Perkin Elmer Lamda 40 UV-visible spectrophotometer. IR spectra were recorded on Agilent Cary 630 FTIR spectrometer as neat sample. 1H NMR spectra were recorded on Bruker DPX-300 NMR spectrometer operating at 400 MHz using DMSO-d6 as solvent with TMS as internal standard. Chemical shift values are given in ppm. Cyclic voltammetric measurements were performed by using DY2312 potentiostat. The antibacterial experiment was performed against Streptococcus mutans (MTCC 3224), and Escherichia coli (ATCC 25922) bacterial strains. To perform molecular docking study Gaussian 03 software was used. The antioxidant potential of the phthalimide derivatives was also estimated using DPPH free radical and hydrogen peroxide assay.
Synthesis of phthalimide derivatives
Similar procedure was employed according to our earlier reported method (Arif et al. 2016). The color solids that obtained were filtered, washed with distilled water, and finally dried under vacuum on fused calcium chloride and recrystallized in chloroform.
4,5,6,7-tetrabromo-2-[3-(4,5,6,7-tetrabromo-1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-1H-1,2,4-triazol-5-yl]-2,3-dihydro-1H-isoindole-1,3-dione (1):
White solid; mp > 300 °C; C18HN5O4Br8: yield 60%; IR (cm−1): νC=O(asym) 1786, νC=O(sym) 1736, νNH 3453. 1H NMR (300 MHz, DMSO-d6), δH 9.46 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ in ppm 166.63, 145.02, 138.41, 133.29, 131.51, 129.19, 128.38, 126.22, 118.93, 116.30. Anal. Calcd. for C18HN5O4Br8: C, 21.82; H, 0.11; N, 7.05; found: C, 21.87; H, 0.11; N, 7.05.
4,5,6,7-tetrabromo-2-[6-(4,5,6,7-tetrabromo-1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl) pyridin-2-yl]-2,3-dihydro-1H-isoindole-1,3-dione (2):
Brown solid; mp > 300 °C; yield: 62%; IR (cm−1): νC=O(asym) 1780, νC=O(sym) 1730. 1H NMR (300 MHz, DMSO-d6): δH 7.46 (d, 2H, Ar-H); δH 9.84 (s, 1H, Ar-H). 13C NMR (100 MHz, DMSO-d6) δ in ppm 169.33, 132.59, 128.29, 129.23, 128.67, 124.88, 122.50, 119.29, 112.79. MS (m/z): 1000.35 (M+1). Anal. Calcd. for C21H3N3O4Br8: C, 25.20; H, 0.30; N, 11.03; found: C, 25.24; H, 0.34; N, 11.22.
4,5,6,7-tetrabromo-2-[4-hydroxy-6-(4,5,6,7-tetrabromo-1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)pyrimidin-2-yl]-2,3-dihydro-1H-isoindole-1,3-dione (3):
Off-white solid; mp > 255 °C; yield: 69%; IR (cm−1): νC=O(asym) 1783, νC=O(sym) 1720, νOH 3190. 1H NMR (300 MHz, DMSO-d6): δH 9.54 (s, 1H, −OH); δH 5.02 (s, 1H, Ar-H). 13C NMR (100 MHz, DMSO-d6) δ in ppm 169.08, 162.06, 160.07, 131.80, 131.53, 131.11, 127.87, 127.65, 126.42. MS (m/z): 1017.75 (M+1). Anal. Calcd. for C20H2N4O5Br8: C, 23.60; H, 0.19; N, 5.50; found: C, 23.25; H, 0.34; N, 5.87.
4,5,6,7-tetrabromo-2-{2-methyl-4-[3-methyl-4-(4,5,6,7-tetrabromo-1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl) phenyl] phenyl}-2,3-dihydro-1H-isoindole-1,3-dione (4):
Mud color solid; mp > 300 °C; yield 71%; IR (cm− 1): νC=O(asym) 1777, νC=O(sym) 1719, νCH 2985; 1H NMR ((δ, ppm in DMSO-d6): 2.18 (s, 6H, −CH3); 7.84–7.87 (3H, Ar-H), 8.06–8.09 (3H, Ar-H). 13C NMR (100 MHz, DMSO-d6) δ in ppm: 168.12, 167.26, 147.55, 136.87, 135.94, 131.36, 129.14, 126.97. 28.22. MS (m/z) 1104.5 (M+1). Anal. Calcd. for C30H12N2O4Br8: C, 32.64; H, 1.09; N, 2.53; found: C, 32.66; H, 1.11; N, 2.49.
DNA binding studies
Absorption measurements
UV-visible spectroscopy is very suitable and significant method to evaluate the binding nature of small molecules and DNA. All the experiments were performed in tris-HCl buffer. The absorption ratio of Ct-DNA in buffer was 1.9:1 at 260 nm which reveals that DNA was possibly absolutely free from protein contamination (Raja et al. 2016). Absorption intensity was determined at 260 nm for the investigation of DNA concentration of stock solution using a molar absorption coefficient ε260 = 6600 L mol−1 cm−1 by UV-visible spectrophotometer (Kumar et al. 2016). Test samples were allowed to equilibrate at room temperature for 10 min before recording the absorption spectrum, carried out in the range of 190–500 nm by varying the concentration of the DNA (1.2–3.0 × 10−5 M) and adapting the constant concentration of compound (1 × 10− 4 M).
Viscosity measurements
The viscosity was determined in the presence of increasing concentration of test compound (0.4–2.0 × 10−5 M) and fixing the DNA concentration (2.5 × 10−5 M) in the 5 mM Tris–buffer (pH = 7.2). All the experiments were carried out using an Ostwald capillary viscometer maintained at 25 ± 0.1 °C. The flow time of the test compounds through the viscometer was determined in triplicate to get the average and accurate value (Patel et al. 2014). The obtained data ploted as (η/ηo)1/3 versus [compound]/[DNA], where η is the viscosity of Ct-DNA in the presence of compound and ηo is the viscosity of Ct-DNA alone. Relative viscosity of the test compound 1 was determined from the observed flow time of DNA solution (t) corrected for the flow time of tris-buffer alone (t0), using the expression 1.
$$ {\eta}_{\mathrm{o}}=\left(t-{t}^0\right)/{t}^0 $$
(1)
Electrochemical study
Cyclic voltammetry (CV) was performed to investigate the redox behavior of test compound 1 using DY2312 potentiostat. The electrochemical experiments were carried out with and without DNA in tris-buffer (pH = 7.5). All the experiments were done at a scan rate 0.2 Vs−1 in the potential range + 1.2 to − 2.0 V at room temperature (Mazhabi and Arvand 2014). Experiment was performed in a electrochemical cell consisting three-electrode, platinum wire auxiliary electrode, glassy carbon working electrode, and silver/silver nitrate as a reference electrode. Alumina powder was used to polish the electrode surface, and for the deoxygenation of the test compounds, nitrogen gas had been used 20 min before the experiments.
Antibacterial activity
All the synthesized compounds were screened for antibacterial activity against gram-negative bacterium E. coli and gram-positive bacterium S. mutants using Kirby Bauer methods (1953) and broth dilution method (Boufas et al. 2014) which conformed to the recommended standards of the Clinical and Laboratory Standards Institute (CLSI). Ampicillin was used as a standard antibacterial drug. A diluted series with 10 mL nutrient broth medium containing 50–200 μg/mL of synthesized phthalimide derivatives were prepared. One hundred microliters of respective bacterial suspension (approximately 106 CFU/mL) was used to inoculate the each set. The bacteria were plated onto solid nutrient agar plates. MIC was defined as the lowest concentration for the inhibition of bacterial growth. All the experiments were done in triplicate and average was reported.
Molecular docking study
Phthalimide derivative 1 has been optimized using B3LYP method in conjunction with 6-31G** basis set utilizing Gaussian 03 (Yadava et al. 2015a, b). Absence of imaginary frequency modes for each molecule indicates that the true minima were achieved. Conformations of the molecule were generated and prepared through LIGPREP wizard of the SCHRODINGER suite (Yadava et al. 2013). The three-dimensional structure of the DNA duplex was retrieved from the protein data bank (PDB ID: 1BNA) which was prepared using protein preparation wizard. The electron affinity grid map was generated around the center of the DNA, and the docking of molecule was carried out using XP (extra precision) mode of GLIDE (Yadava et al. 2015a, b). During docking, ligand was treated as flexible while DNA was taken as rigid. Docking complex was considered for the calculation of glide energy and glide scores.
Antioxidant activity
DPPH free radical scavenging assay
Antioxidant activity of all the phthalimide derivatives was evaluated against DPPH free radical according to the method reported by the Miliauskas et al. which was the best method based on electron transfer (Miliauskas et al. 2004). Test compound (0.5–3.0 mg/mL) and ascorbic acid (0.2–1.4 mg/mL) in DMSO was added to 0.1 mM DPPH (3 mL) in ethanol. All test compounds were incubated at 60 °C for 2 h, and the decrease in absorbance was noted at 510 nm using UV-Vis spectrophotometer against a blank of ethanol and DMSO in 1:1. Absorbance of DPPH (control) solution was also recorded at same wavelength for comparative study. The IC50 in (mg/mL) was calculated from the graph between % antioxidant activity vs concentrations. For each of the tests, compound experiment was done in triplicate and antioxidant property of the compounds was measured by using Eq. 2:
$$ \%\kern0.5em \mathrm{Inhibition}\kern0.5em =\kern0.5em \frac{A_{\mathrm{Control}}-{A}_{\mathrm{Sample}}}{A_{\mathrm{Control}}}\kern0.5em \times \kern0.5em 100 $$
(2)
where Acontrol = absorbance of DPPH free radical in methanol without an antioxidant and Asample = absorbance of DPPH free radical in the presence of an antioxidant.
Hydrogen peroxide scavenging activity
The antioxidant ability of the phthalimide derivatives was also estimated by hydrogen peroxide using standard method (Ruch et al. 1989). To the different concentrations of test compounds, 1.8 mL of a 2 mM H2O2 solution prepared in phosphate buffer (50 mM, pH 7.4) was added and the samples were incubated for 10 min. We record the decrease in absorbance against phosphate buffer as a blank by UV-Vis spectrophotometer. The absorbance of test samples was noted at 240 nm and compared with hydrogen peroxide which was taken as a control. The antioxidant ability of hydrogen peroxide was calculated using following equation:
$$ \%\mathrm{Inhibition}=\frac{A_{\mathrm{B}}-{A}_{\mathrm{T}}}{A_{\mathrm{B}}}\times \kern0.5em 100 $$
(3)
where AB was the absorbance of blank (without compounds) and AT was the absorbance of tested samples.