Chemistry
The synthesis of TZD derivatives (1-20) were accomplished using the synthetic route depicted in Scheme 1. At first, 2-chloroacetic acid was treated with thiourea in conc. HCl to obtain TZD (I). Further, the reaction of (I) with terephthalaldehyde yielded 4-((2,4-dioxothiazolidin-5-ylidene) methyl) benzaldehyde (II). Intermediate-II on further treatment with substituted anilines/amines yielded final 5-((E)-4-((E)-(substituted aryl/alkyl)methyl)benzylidene)thiazolidine-2,4-dione derivatives (1-20). The physicochemical characterization and spectral analysis of the synthesized derivatives are given in Table 1.The molecular structures of the synthesized derivatives (1-20) were established using elemental analysis and spectral studies [FT-IR (KBr, cm-1), 1H-NMR (DMSO-d6, 400 MHz, δ ppm) and Mass spectra]. The 1H-NMR spectra designated that the presence of multiplet signals between 6.52 and 8.28 δ ppm reflected the presence of aromatic protons in synthesized molecules. The presence of singlet(s) between 7.62-7.84 δ ppm, 7.87-8.80 δ ppm and 12.12-12.70 δ ppm indicated the presence of -CH=, -CH=N and -NH groups, respectively. The compound 2 exhibited singlet (s) at 1.92 δ ppm due to the existence of H of -NH2 group. The appearance of singlet (s) at 2.08-2.33 δ ppm in compounds 7, 8, 9 and 10 revealed the existence of CH3 of Ar-CH3. The existence of OCH3 of Ar–OCH3 in the compounds, 15, 16 and 17 was confirmed by presence of singlet at 3.77–3.85 δ ppm. In compound 3 NH of Ar-NH existence was confirmed by appearance of singlet at 10.61 δ ppm. The compound 20 displayed multiplet at 1.23-1.69 δ ppm of CH2, triplet at 0.84 δ ppm of CH3 and multiplet at 3.66 δ ppm of CH2 adjacent to CH=N due to the existence of dodecyl group. The compound 6 showed doublet signal at 4.77 δ ppm of -CH2 adjacent to furan ring, doublet signal at 6.30 δ ppm of -CH of furan ring at 3rd position, triplet signal at 6.42 δ ppm of -CH of furan ring at 4th position and doublet signal at 7.47 δ ppm of -CH of furan ring adjacent to O due to the existence of furfuryl group. In case of IR spectrum, the presence of bands at 3437-3286 cm-1, 3048-2919 cm-1, 3197-3012 cm-1, 1615-1548 cm-1, 1522-1412 cm-1, 1702-1610 cm-1, 1747-1614 cm-1, and 618-594 cm-1 displayed the presence of N-H, C-H (aliphatic), C-H (aromatic), C=C (methylene), C=C (aromatic), C=N, C=O and C-S groups respectively in the synthesized analogues. The absorption bands around 1338-1224 cm-1 and 1165-1152 cm-1 corresponded to C-N and C-C stretching of compounds, respectively. Compounds 4, 5 and 14 displayed absorption bands of C-Cl around 774-750 cm-1. Mass of the synthesized compounds exhibited M++1, M+ and M+-1 peaks.
Antimicrobial activity
The in vitro antimicrobial screening studies of the synthesized TZD derivatives was evaluated by serial tube dilution procedure (Table 2, Fig. 2, 3 and 4). The antibacterial screening outcomes revealed that compounds 13 and 4 were moderately active against S. aureus with MICsa value of 17.9 µM and 18.2 µM, respectively. Further screening revealed that compounds 16 and 10 were moderately active against B. subtilis with MICbs value of 18.5 µM and 18.6 µM, respectively. Compound 13 (MICkp = 17.9 µM) and Compound 9 (MICkp = 18.6 µM) were found to be effective against K. pneumoniae. Compound 11 (MICec = 19.2 µM) possessed good activity against E. coli. Compound 5 (MICst = 18.5 µM) and compound 10 (MICst= 18.6 µM) exhibited promising activity against S. typhi. The antifungal screening results revealed that the compounds 13 (MICan = 17.9 µM) and 12 (MICca= 16.1 µM) had good activity against A. niger and C. albicans respectively. The antibacterial screening results were found to be comparable with the standard drug (cefadroxil), whereas antifungal results of synthesized molecules exhibited superior activity against both the fungal strains i.e. A. niger and C. albicans except compound 2 in comparison to the standard drug (fluconazole). So, these synthesized compounds can be taken as lead structures and may further be optimized to yield new antimicrobial agents with better activity.
Antioxidant evaluation
The antioxidant efficacy of the newly synthesized derivatives was assessed by applying 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method [21] using ascorbic acid as standard. 2, 2-diphenyl-1-picrylhydrazyl radical is a stable free radical which become a stable diamagnetic molecule by accepting an electron or hydrogen radical. A strong absorption band at 517 nm is observed by methanolic solution of DPPH due to its odd electron. DPPH radical reacts with appropriate reducing agent to produce new bond, which leads to change in the color of the solution. As concentration of antioxidant increases in the solution, more electrons are taken up by the DPPH radical from the antioxidant molecules leading to loss of color of the solution. Such reactivity has been used to test the ability of compounds that can act as free radical scavengers. Reduction of the DPPH radicals has been monitored at 517 nm absorbance spectrophotometrically indicated by decrease in the intensity of color (Purple color) [22]. The IC50 value in μg/mL was calculated for all the synthesized compounds. The antioxidant assay revealed all the synthesized compounds to be more potent than the standard drug. Further, amongst all the compound 6 (IC50 = 9.18 μg/mL) was found to be most active antioxidant compound. Results are displayed in Table 3 and Fig. 5.
Molecular Docking Results
DNA gyrase, a member of topoisomerases type II family has two genes i.e. GyrA and GyrB, which controls the topological state of DNA in cells [23]. During replication process DNA gyrase is required for maintenance of DNA topology during supercoiling of DNA through coupling of ATP hydrolysis by the GyrB subunit. DNA gyrase enzyme inhibition results in disruption of DNA synthesis in bacterial species leading to bacterial cell death [24].
Molecular docking study was carried out to analyze the binding affinity of the synthesized compounds with ATP binding pocket. The molecular docking study was carried out on GLIDE docking program. All the synthesized compounds were docked in the active site of the S. aureus GyrB ATPase domain (PDB: 3U2D) co-crystallized with 08B ligand. Binding affinity of compounds was compared using ATP as docking control. The results were investigated by comparing the docking score obtained from GLIDE (Table 4)..
Binding affinity of the compounds were demonstrated in terms of binding energy which were calculated in term of negative energy. Binding affinity is more when binding energy is less. Docking scores were shown as numerical value of interaction energy which is statistical evaluation function for displaying the results. Different visualization tools were used to visualize the 3D pose of the ligand interaction with receptor [25]. Molecular docking study revealed that the synthesized compounds exhibited good interaction with crucial amino acids of protein. Like if we look into the best-fitted compounds 4 and 7 showed the best dock score -4.73 and -4.61, respectively as compared to standard ofloxacin (dock score = -5.107) within the ATP binding pocket (Table 5). Ligand interaction diagram and binding mode of most active compounds 4 and 7 and ofloxacin in the active site of S. aureus GyrB ATPase domain co-crystallized ligand 08B is shown in (Table 5, Figs. 7, 8 and 9). Docking analysis studies revealed that more active antimicrobial which is comparable to the standard drug ofloxacin can be obtained by making more modifications in compounds.
ADME results
QikProp module of Schrodinger 2018-1 (Maestro version 11.5) was used for studying ADME parameters of the synthesized molecules. Around eleven pharmacologically significant and physically relevant parameters of the synthesized compounds (1-20) were studied. The ADME results of the synthesized TZD analogues 1-20 exhibited fairly good results within the recommended range of Qikprop module and in good agreement with Lipinski’s rule of five which includes Molecular weight of the molecule (mol. MW = <500), Predicted water/gas partition coefficient (QPlogKp = -8.0 to-1.0), Percent human oral absorption (0 to 100), donor HB (0.0 to -6.0), (QPlogPw = 4.0 to -45.0), Predicted octanol/water partition coefficient (QPlogPo/w = -2.0 to -6.5), human oral absorption (1, 2 or 3), Predicted brain/blood partition coefficient (QPlogBB = -3.0 to -1.2), accept HB (2.0 to -20.0) and hence found these analogues as suitable drug candidates. The results of ADME studies are expressed in the Table 6.
Structure activity relationship:
From the antimicrobial and antioxidant evaluation studies following structure activity relationship can be drawn (Fig. 6):
- The different substitution of amines/anilines used to synthesize the final derivatives of 5-((E)-4-((E)-(substitutedaryl/alkyl)methyl)benzylidene)thiazolidine-2,4-dione played an important role in improving the antimicrobial and antioxidant activities. Substitution of electron releasing group methyl (-CH3) at ortho and para position in the synthesized compound 9, increased the antibacterial potential against typhi and K. pneumoniae.
- Presence of Electron withdrawing group nitro (-NO2) at meta position enhanced antibacterial potential against pneumoniae and S. aureus as well antifungal activity against C. albicans and A. niger (Compound 4).
- Presence of electron withdrawing group fluoro (F) at ortho position of the synthesized compound 11, enhanced the antibacterial potential against coli whereas substitution of heterocyclic group furfuryl in the derived compound 6, improved the antioxidant potential. These molecules may further be used as lead compounds to derive more potent and less toxic novel antioxidant and antimicrobial agents.
Experimental part:
The chemicals of analytical grade were procured from commercial sources and were used for the synthesis without any purification. Open glass capillaries on a Stuart scientific SMP3 apparatus were used for determining melting point (m.p.) and reported uncorrected. Reaction progress was monitored by TLC glass plates of silica gel G for every synthetic step. KBr pellets were used for recording infrared (IR, KBr, cm-1) on Bruker 12060280 (Software: OPUS 7.2.139.1294) spectrophotometer. Bruker Avance III 400 NMR spectrometer was used to determine 1H spectra in appropriate deuterated solvents and using tetramethylsilane as internal standard and are expressed in parts per million (δ, ppm) downfield from internal standard. Mass spectra was obtained using Waters Micromass Q-ToF Micro instrument. CHN analyzer was used to perform elemental analysis.
Synthetic steps of Scheme 1:
Step 1: Synthesis of thiazolidin-2,4-dione TZD (I):
To a solution of chloroacetic acid (0.06 mol) in water (15 mL), thiourea (0.06 mol) in water (15 mL), acid was added and stirred till the occurrence of white precipitate. To the contents of flask, 6 mL of conc. HCl was added dropwise followed by refluxing for 10 h. On cooling, needle shaped crystals of TZD (I) were obtained which were filtered, dried and recrystallized using methanol as solvent [6].
Step 2: Synthesis of 4-((2,4-dioxo-1,3-thiazolidin-5-ylidene)methyl)benzaldehyde (II):
To a solution of (I) (0.03 mol) and terephthalaldehyde (0.03 mol) in ethanol (45 mL), 3 mL of piperidine (0.0188 mol) was added, stirred and refluxed for next 12 h. Contents of flask were then poured on ice followed by acidification with acetic acid (glacial). Yellow coloured product of 4-((2,4-dioxo-1,3-thiazolidin-5-ylidene)methyl)benzaldehyde (II) was obtained which was filtered, dried and further recrystallized using ethanol as solvent [26].
Step 3: Synthesis of various title compounds (1-20):
To the solution of compound II (0.01 mol) in methanol (50 mL), different substituted amines (0.01 mol) were added using catalytic amount of acetic acid (glacial) and refluxed for 4-18 h. The reaction mixture was then allowed to cool and finally recrystallized from methanol to give final compounds (1-20).
In vitro antimicrobial evaluation
The antimicrobial potential of the synthesized compounds was evaluated by serial tube dilution method [27] using fluconazole (antifungal) and cefadroxil (antibacterial) as standard drugs. Both Gram +ve {MTCC-3160 (S. aureus), MTCC-441 (B. subtilis)} and Gram -ve {MTCC-3231 (S. typhi), MTCC-9024, (K. pneumoniae) and MTCC-443 (E. coli)} bacterial species were used in the study. The antifungal potential was evaluated against MTCC-281 (A. niger) and MTCC-227 (C. albicans) strains. Nutrient broth double strength I.P. (for bacteria) or sabouraud dextrose broth I.P. (for fungi) [28] nutrient media were used for antimicrobial potential. Stock solutions of the test and reference compounds were prepared in dimethyl sulfoxide. A control set was also used at the same dilutions with the test medium supplemented with dimethyl sulfoxide. Results were recorded in MIC after incubating the samples at 25 ± 1 °C (7 days) for A. niger, at 37 ± 1 °C (24 h) for bacteria and at 37 ± 1 °C (48 h) for C. albicans, respectively. MIC was recorded for the tested compound as lowest concentration that showed no observable growth of microorganisms in the test tube.
In vitro antioxidant assay
The antioxidant evaluation of synthesized thiazolidine-2,4-dione derivatives was determined using stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging model [29]. The diluted solution of synthesized compounds in methanol of 25 μg/mL, 50 μg/mL, 75 μg/mL and 100 μg/mL were prepared and equal amount of methanolic solution of DPPH (0.0039 %) was added followed by vigorous shaking. The above solution was then kept in dark for 30 minutes and absorbance of the solution was measured spectrophotometrically at 517 nm using UV-visible double beam spectrophotometer. The mean of at least three observations was taken as mean IC50 value in the data presented.
Molecular Docking Study
The target protein for thiazolidine-2,4-dione derivatives was identified through the literature. S. aureus GyrB ATPase (PDB Id: 3U2D) co- crystallized with 08B ligand, an excellent target for docking against S. aureus strain [30] was retrieved from Protein Data Bank(http://www.rcsb.org/pdb/home/home.do) to dock synthesized thiazolidine-2,4-dione compounds. Docking score was obtained from GLIDE software through targeted the ATP binding site by creating active site grid. The active site grid possessed the important amino acids which interact with ATP [31].