Synthesis, ABTS-Radical Scavenging Activity, and Antiproliferative and Molecular Docking Studies of Novel Pyrrolo[1,2-a]quinoline Derivatives

Abstract A new 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-radical scavenging and antiproliferative agents of pyrrolo[1,2-a]quinoline derivatives have been synthesized. An efficient method for the synthesis of 14 novel diversified pyrrolo[1,2-a]quinoline derivatives has been described using 4-(1,3-dioxolan-2-yl)quinoline and different phenacyl bromides in acetone and followed by reacting with different acetylenes in dimethylformamide/K2CO3. The structure of the newly synthesized compounds was determined by infrared, 1H NMR, 13C NMR, mass spectrometry, and elemental analysis. The in vitro antioxidant activity revealed that among all the tested compounds 5n exhibited maximum scavenging activity with ABTS. Compound 5b has showed good antiproliferative activity as an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. GRAPHICAL ABSTRACT


INTRODUCTION
Reactive oxygen species (ROS) play a critical role in cardiovascular diseases, inflammatory diseases, neurodegenerative disorders, cancer, and aging. [1] Antioxidants are compounds that detoxify ROS and prevent their damage through multimechanisms. [2] Cancer is one of the highest impact disease worldwide with significant morbidity and mortality rates. A key regulator of tissue homeostasis is the apoptosis or programmed cell death and the imbalances between cell death and proliferation, which may result in tumor formation. [3] The objective of using anticancer agents is to induce apoptosis-related signaling in cancer cells while disrupting their proliferation. [4] The time has come where synthesis of a newer class of anticancer agents is very much needed.
The structures of the new compounds (5a-n) were assigned on the basis of 1 H NMR, 13 C NMR, mass spectra, and elemental analysis. 1 H NMR spectrum of 5a showed a triplet at d 1.31 (3H) and a quartet at d 4.30 (2H) ppm corresponding to an ethyl ester group at position C-2 and the multiplet at d 4.13 (4H) ppm corresponding to the presence of a dioxalan ring. 13 C NMR spectrum of 5a has showed peaks at d 162.9 (COO-CH 2 -CH 3 ), 59.9 (O-CH 2 ), 14.21 (CH 3 ), 64.95 (O-CH 2 ), and 184.3 (CO-Ar) ppm, corresponding to ethyl ester, dioxalan ring, and carbonyl group in the COAr respectively. Infrared (IR) spectrum of 5a revealed that the appearance of the characteristic band at 1632 and 1748 cm À1 corresponds to carbonyl group in the COAr and for ethyl ester C=O stretch respectively. Additionally, the liquid chromatography-mass spectrometry (LC-MS) has supported through a peak at 415 corresponding to the molecular weight of the compound 5a. Finally, the elemental analysis also supported the compound 5a. Similarly, the remaining compounds (5b-n), shown in Tables 1 and 2, were confirmed. These compounds were then tested for antioxidant, antiproliferative, and molecular docking studies.
Solution I: ABTS (2 mM solution is prepared using distilled water). Solution II: Potassium persulfate (17 mM solution is prepared using distilled water).
An aliquot of 0.3 mL of solution II was added to 50 mL of solution. The reaction mixture was left to stand at room temperature overnight in the dark before use. Then, 1 mg of each of the drug samples was accurately weighed and dissolved in 1 mL of dimethylsulfoxide (DMSO). One mL of distilled DMSO was added to 0.2 mL of the drug samples or standard, and 0.16 mL of ABTS solution was added to make a final volume of 1.36 mL. Absorbance was measured spectrophotometrically after 7 min at 734 nm. A blank was maintained without ABTS. The radical scavenging activity (%) is calculated by the following formula: ABTS radical scavenging capacity assay is an electron-transfer-based assay, which measures the capacity of an antioxidant to reduce an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentration. The ABTS assay, which is applicable for both lipophilic and hydrophilic antioxidants, showed various radical scavenging activities between the derivatives. According to Table 3 values ranged from 27.14 to 92.88%, and compound 5n possessed the greatest ABTS radical scavenging activity (92.88%) followed by the 5i and 5l derivatives (89.02% and 87.44%) respectively, derivatives 5e, 5 m, 5 h, 5b, 5d, 5j, 5g, 5c, 5f, 5a (85. 72  Cell culture. A HeLa cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) medium (GIBCO) supplemented with 10% (v=v) heat-inactivated fetal bovine serum (FBS) and 1% antibiotic solution (penicillin 100 Uml À1 and streptomycin 100 µgml À1 ) at 37°C in a humidified atmosphere of 95% air=5% CO 2 . The medium was changed every second day, and cells were subcultured when confluency reached 95% by 0.25% trypsin containing 0.02% ethylene-diaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS) for 3 min at 37°C.
MTT assay. The MTT assay was carried out as described previously to measure cell viability. [25] Ten thousand cells in 100 µL of DMEM media were seeded in the wells of a 96-well plate. After 24 h, existing media was removed and 100 µL of various concentrations of complexes was added and incubated for 48 h at 37°C in a CO 2 incubator. Control cells were supplemented with 0.05% DMSO vehicle. At 48 h of incubation, MTT (3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide, supplied from Sigma, 10 µL of 5 mg=mL) was added to the plate. The contents of the plate were pipetted out carefully, the formazan crystals formed were dissolved in 100 µL of DMSO, and the absorbance was measured at 550 nm in a micro plate reader (Tecan, infinite F200 Pro). Experiments were performed in triplicate, and the results were expressed as mean of percentage inhibition. A graph of the concentration versus percentage growth inhibition was plotted, and the concentration at which 50% cell death occurred was considered as the IC50 value. Before adding MTT, bright field images (Olympus 1X81, cell Sens Dimension software) were taken for visualizing the cell death.
The values obtained demonstrated that all the compounds presented cytotoxic effects in a dose-dependent manner. The set of compounds showed excellent inhibitory activity, the results of which are shown in Fig. 2. Microscopy images representing the cell death caused by the compounds are as seen in Fig. 3. HeLa cells have been reported to contain human papilloma virus 18 (HPV-18) sequences, a low expression of p53, and normal expression of pRB (retinoblastoma suppressor). [26]  But the epidermal growth factor receptor is the first identified member of the type I receptor tyrosine kinase family and is a major regulator of several distinct, diverse cellular pathways. Selective compounds have been developed that target either the extracellular ligand-binding region of the EGFR or the intracellular tyrosine kinase region. This results in interference with the signalling pathways that modulate mitogenic and other cancer-promoting responses such as cell motility, cell adhesion, invasion, and angiogenesis. [27] It is important to correlate the structure of these compounds with their biological effect, which will be valuable to propose new lead compounds with better cytotoxic potential.

Molecular Docking Studies
The three-dimensional structure of target protein superoxide dismutase (SOD) and EGFR tyrosine kinase having keyword 1CB4 and 2J5 F was downloaded from the PDB (www.rcsb.org/pdb) structural database. This file was then opened in SPDB viewer and edited by removing the heteroatoms and adding C terminal oxygen. The active pockets on target protein molecule were found out using the CASTp server. [28] The ligands were drawn using ChemDraw Ultra 6.0 and assigned with proper twodimensional (2D) orientation (ChemOffice package). Three-dimensional (3D) coordinates were prepared using a PRODRG server. [29] Autodock V3.0 was used to perform automated molecular docking in AMD Athlon 2 Â 2 215 at 2.70 GHz, with 1.75 GB of RAM. AutoDock 3.0 was compiled and run under Microsoft Windows XP service pack 3. For docking, a grid map is required in AutoDock, and the size of the grid box was set at 82, 82, and 100 Å (R, G, and B) and grid center 16.191, 70.024, and 15.326 for x, y, and z coordinates for SOD. However, 102, 126, and 118 Å (R, G, and B) and grid center À58.865, À8.115, and À24.556 were used for x, y, and z coordinates for EGFR tyrosine kinase. All torsions were allowed to rotate during docking. The Lamarckian genetic algorithm and the pseudo-Solis and Wets methods were applied for minimization, using default parameters. [30] The newly synthesized compounds were taken as ligands and docked against target molecules.
To identify potential antioxidant lead among compounds 5a-n, docking calculations were performed using Autodock v3. After docking the synthesized molecule with superoxide dismutase, the compounds were bound exactly at the active site of superoxide dismutase, which is shown in Fig. 4. A careful inspection of the binding pocket indicated that the active site of SOD was similar in all synthesized compounds at the Cu-Zn domain of SOD (Fig. 5). Target information and docking details for the compounds are tabulated in Table 4, which may provide useful information for in-depth understanding of binding mechanism of the compound to the active site of the protein. Hydrogen bond formation also makes important contributions to the interaction between ligand and the receptor. All 14 molecules showed good binding energy and docking energy ranging from À6.27 kJ mol À1 to À10.28 kJ mol À1 and À8.87 kJ mol À1 to À13.61 kJ mol À1 respectively. Among the 14 molecules, docking of SOD with compound 5i followed by compound 5n revealed three hydrogen bonds and their binding energies and docking energies were À10.28, À9.88 kJ mol À1 and À13.61, À11.18 kJ mol À1 respectively, and these two compounds may be considered as the lead molecules to increase the activity of superoxide dismutase and reduce oxidative stress. In in vitro studies compound 5n has also emerged as an active molecule. Hence, from the study it has been proved that molecule 5n is one of the potent antioxidant molecules. Based on the antiproliferative activity results, it was worthwhile to perform docking studies for supportive coordination between in silico studies with the in vitro results. The 14 derivatives docked with EGFR tyrosine kinase domain revealed that our synthesized derivatives are having inhibitory potential and exhibiting interactions with one or the other amino acids in the active pockets  Table 5. Derivatives 5b, 5 h, and 5n showed À6.16, À5.93, and À6.11 kJ mol À1 binding energy with three hydrogen bonds respectively, whereas derivative 5l showed À5.61 kJ mol À1 binding energy with two hydrogen bonds with the target receptor, which indicates that among the nine derivatives 5l is the less efficient derivative. From the study it is evident that derivative 5b is a more efficient molecule for antiproliferative activity and a good inhibitor of EGFR tyrosine kinase.

EXPERIMENTAL
Chemicals were purchased from SD-fine and Sigma-Aldrich companies and were used without further purification. All products were characterized by comparison of their IR, 1 H NMR, and 13 C NMR spectra. All yields refer to the isolated products. The purity determinations of the substrates, products, and reaction monitoring were accomplished by thin-layer chromatography (TLC) on silica-gel polygram SIL=UV 254 plates. IR spectra were recorded on an infrared Fourier transform spectrometer. 1 H NMR (400 MHz) and 13  To a stirred solution of 1-[2-(4-chlorophenyl)-2-oxoethyl]-4-(1,3-dioxolan-2-yl) quinoliniumbromide (4b) (100 mg, 0.230 mmol) in dry DMF, ethyl propiolate (0.03 mL, 0.230 mmol) and K 2 CO 3 were added. Stirring was continued for 2 h at room temperature. Completion of reaction was monitored by TLC. The reaction mass was quenched by ice-cold water. Solids were separated out, filtered, and dried

CONCLUSIONS
The research work was focused on developing a simple and mild procedure for the synthesis of pyrrolo[1,2-a]quinoline derivatives from substituted quinoline. The reactions performed are ecofriendly as they are carried out at room temperature. The structures have been confirmed by spectroscopic techniques such as 1 H NMR, 13 C NMR, and mass. Based on the results of in silico studies and the in vitro activity, derivative 5n showed radical scavenging activity and 5b has been proved to be one of the potent antiproliferative agents. Hence, 5n and 5b can be used as efficient antioxidant and antiproliferative products.

ACKNOWLEDGMENTS
We are thankful to the authorities of Jain University for the support and encouragement and grateful to the NMR Research Center, IISc, Bangaluru, for providing NMR spectra.

SUPPLEMENTAL MATERIAL
Full experimental details, 1 H and 13 C NMR spectra, and LCMS spectra for this article can be accessed on the publisher's website.