I2/TBHP mediated domino synthesis of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-aryl/alkyl benzamides and evaluation of their anticancer and docking studies

A novel I2/TBHP mediated domino synthesis of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-phenyl benzamides by reaction of isatins with o-amino N-aryl/alkyl benzamides was described. This was the first application of o-amino N-aryl/alkyl benzamides participating in oxidative rearrangement with isatins for synthesis of desired products. The synthesized compounds contained amide and quinazoline units and their combination resulted in molecular hybridization of two important pharmacophores. In this study, the synthesized compounds 3a–r were screened for cytotoxicity against four cancer cell lines A549, DU145, B16-F10, and HepG2 and also non-cancerous cell line CHO-K1. The compounds 3c, 3l and 3o gave promising results. The in silico molecular docking studies (PDB ID 1N37) also validated the anticancer activity of these compounds showing good binding affinity with target DNA and by acting as DNA intercalators.


Introduction
Molecular hybridization has been a strategy in drug discovery research in recent years. 1 It involves blending or linking of two or more pharmacophore moieties to generate a single molecular entity which could exhibit better selectivity, and improved affinity and activity, and overcome the drug resistance due to a mixed mechanism of action. Cancer, a debilitating disease, is a worldwide major human health problem. Though many anticancer agents are available, still there is a call to identify effective and safe drugs for treatment of cancer. Numerous heterocyclic compounds have been researched to discover new anticancer agents. 2 Quinazoline pharmacophore is found in various pharmaceuticals including antimicrobials and cytotoxic agents. [3][4][5] Quinazolines exhibit a wide range of biological properties such as anti-hypertensive, 6 anti-HIV-1, 7 anti-cancer, 8 antitumor, 9 anti-inammatory, 10 progesterone receptor antagonists 11 and antibacterial activity. 12 Some small molecules containing quinazolines motif are also known to exhibit potent and specic puromycin-sensitive amino peptidase (PSA) inhibitory activity 13 and reversible inhibition of Bruton's tyrosine kinase (BTK). 14 Quinazoline scaffold is found also in several natural products. 15 Carboxamide scaffolds are of immense interest in medicinal chemistry. 16,17 The pharmaceutically important ergot alkaloids such as dehydro ergotamine and ergotamine contain amide linkage. There are many drugs like atorvastatin, valsartan, paracetamol and penicillin available in the market, which contains amide linkage in the nucleus and possesses varied therapeutic activities like antimycobaterial, antimicrobial, antifungal, cytotoxic, antiepileptic and analgesic properties. 2,18 The amide bond is planar, resonating and stable with large dipole moment, resembles the protein's peptide linkage. 19 Use of peptide-based therapeutics is steadily increasing, for example, cyclosporine A is a peptide which is a widely used powerful immunosuppressive agent. 20 Anticancer agents with amide pharmacophore 21 and quinazoline pharmacophore are shown in Fig. 1.
Hybrid drugs with multiple mechanism of action are of great interest for cancer treatment, for example, quinazoline-amide hybrid drug raltitrexed 1c known for potent anticancer activity. 22 In the present work, we aimed at synthesis of 2-(2,4dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-aryl/alkyl benzamides, which were new quinazoline-amide hybrid molecules. No literature is available for synthesis of these quinazoline-amide hybrid molecules. Some existing literature was available for synthesis of simple 2,4-quninozolinediones 23 but these methods had one or more disadvantages such as metal catalysts, harsh reaction conditions and limited substrate scope.
Under existing literature conditions were not promoted the synthesis of desired molecular hybrids 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-aryl/alkyl benzamides by reaction of isatins with 2-amino-(N-alkly/aryl) benzamides. Therefore, we were much interest to synthesize these quinazoline-amide hybrid molecules, because of their important applications in organic synthesis and medicinal chemistry. Moreover, I 2 /TBHP mediated oxidative coupling reactions are very important than those transition-metal-mediated oxidative reactions for they are greener and less expensive. It has been widely studied and applied for a variety of oxidation reactions. 24 Domino reactions have been one of the mainly constructive and powerful synthetic tools in organic synthesis. The advantage of these domino reactions as a methodology involve two or more bond constructions that takes place under the same reaction conditions in one-pot. In the past several years, authors have developed a variety of multicomponent domino reactions that can afford easy access to functionalized various heterocycles. 25 Here, we observed for the rst time a new domino reaction of isatins with 2-amino N-aryl/alkyl benzamides in the presence of I 2 /TBHP under reux in methanol to give 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-aryl/alkyl benzamides in high yields with broad functional group tolerance and short reaction time as shown in Scheme 1.
Furthermore, all the synthesized compounds were well characterized and assessed for their cytotoxicity by MTT assay against four cancer cell lines such as A549, DU145, B16-F10, HepG2 and non-cancerous cell line CHO-K1. In addition, to understand the structure-activity relationships, evaluate the possible intercalating potency of the synthesized compounds with DNA and validate the biological studies, molecular docking studies were conducted.
The results observed in the control experiments are shown in Scheme 2. In this study, formation of 3a was observed only when reaction was conducted using I 2 /TBHP under reux in methanol. This reaction did not proceed when TBHP used alone, and also no reaction was observed when conduct at cooling and ambient temperatures (Scheme 2, eqn (1) and (2)). However, when the reaction was carried out in presence of iodine without oxidant, it gave the intermediate (Z)-2-((2oxoindolin-3-ylidene)amino)-N-phenyl benzamide 4a (Scheme 2, eqn (3)). In our study, the intermediate 4a was found to react with TBHP and converted into intermediate (4)). The important observation noted in this study is, the intermediate 5a was converted into product 3a when treated with iodine under reux in methanol (Scheme 2, eqn (5)). The intermediate 4a also directly converted into 3a when treated with I 2 /TBHP or I 2 /H 2 O 2 under reux in methanol (Scheme 2, eqn (6) and (7)).
In view of the observations described above, the plausible reaction mechanism for the domino reaction of isatin 1a and 2amino-N-phenyl benzamide 2a in the presence of I 2 /TBHP is shown in Scheme 3. Here, the compound 4a is initially produced by the condensation of isatin 1a and 2-amino Nphenyl benzamide 2a under catalysis of iodine. Next 4a Scheme 3 Plausible mechanism for the formation of 2-(2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-N-phenylbenzamide (3a).  Bioactivity studies (i) Cytotoxicity assay. The synthesized derivatives were screened for their cytotoxicity towards cancer cell lines A549, B16-F10, DU145, and HepG2 and non-cancerous cell line CHO-K1 by MTT assay using doxorubicin as a standard drug. The cytotoxicity results expressed as IC 50 values are depicted in Table 5.
Interestingly all the synthesized compounds exhibited signicant cytotoxicity to cancer cell lines as compared to normal cell line. Predominantly, most of the compounds exhibited promising activity against DU145 and HepG2 cell lines. Notably, amongst all the derivatives, compounds 3o, 3c and 3l displayed the most potent activity against DU145 and HepG2. From the structure-activity relationship perspective, it was seen that type of substitutions on quinazoline and amide pharmacophores affects the cytotoxic activity. Different substitutions on quinazoline ring like -Cl, -H, -F and -NO 2 showed activity in the order of Cl > H > F > NO 2 . Whereas, substitutions on amide functionality like aromatic and aliphatic also inuenced the cytotoxicity. Here, N-aryl functionality showed high activity as compared to N-alkyl functionality. Type of substitutions on aromatic ring also imparted effect on cytotoxicity which follows the order F > Br > Me > OMe. Compound 3o having chlorine substitution on quinazoline pharmacophore with uorine on aromatic ring of amide functionality displayed highly potent cytotoxicity. The compounds 3c and 3l are the next potent derivatives amongst the series. Here, 3c has no substitution on quinazoline moiety and its amide functionality is substitution of N-(4-Me)Ph. In 3l, uorine substitution is present on quinazoline moiety and amide functionality is substituted with N-(4-Br)Ph. These observations suggest that presence of electron-withdrawing group on both the pharmacophores may be a promising factor for the high cytotoxicity.
(ii) Docking studies. Docking studies were implemented to validate the biological studies and to explore the plausible modes of interaction between the potent derivatives 3o, 3c, 3l and DNA [Protein Data Bank (PDB) 1N37]. 26 All the potent compounds were docked using AutoDock 4.2.6 against the promising anticancer target. For each ligand, the docking score was calculated in terms of kcal mol À1 . All the screened compounds, 3c, 3l and 3o were found to interact with DG B:13, DG A:5, DC B:12 and DC A:4. The molecular docking results are tabulated in Table 6.
Binding interactions like hydrogen bond and p-p interactions, binding energies and inhibition constants of the molecules are used to explain the affinity of a molecule with DNA and stability of the complex. Fig. 2(A-C) depicts the binding interaction of 3c, 3l, and 3o with DNA. From the gures it is clear that the molecules have been intercalated between the DNA base pairs. Docking studies showed that the compound 3c exhibits strong H-bond interactions with higher binding energy (À9.13 kcal mol À1 ) as compared to compounds 3l (À8.31 kcal mol À1 ) and 3o (À8.04 kcal mol À1 ).

Conclusions
We have developed a new and efficient metal-free oxidative domino reaction for preparation of 2-(2,4-dioxo-1,4-dihydro quinazolin-3(2H)-yl)-N-aryl/alkyl benzamides by reaction of an isatins with 2-amino N-aryl/alkyl benzamides using I 2 /TBHP under mild conditions. These 2-(2,4-dioxo-1,4-dihydro quinazolin-3(2H)-yl)-N-aryl/alkyl benzamides consisted of quinazolines and amide moieties, which are important pharmacophores in several anticancer agents. In this study, the synthesized compounds also were found to display excellent cytotoxicity towards tested cancer cell lines A549, B16-F10, DU145, and HepG2 and no or lesser cytotoxic effects towards normal CHO-K1 cells. Especially, compounds 3c, 3l and 3o exhibited promising bioactivity at micro molar concentration and these were found to be more potent than 5-ourouracil towards different cancer cell lines. Moreover, docking studies accomplished on these compounds also validated the results of cytotoxicity studies. The present study reveals that the compounds 3c, 3l and 3o have potent therapeutic applications for treatment of different types of cancers.

General information
Melting points of all the compounds were recorded on Veego programmable melting point apparatus and are uncorrected. IR spectra were recorded on a PerkinElmer FT-IR 240-C spectrophotometer using KBr optics. 1 H NMR and 13 C NMR spectra were recorded on Bruker AV 400 MHz in CDCl 3 and DMSO-d 6 using TMS as internal standard. High resolution mass spectra (HRMS) [ESI + ] were obtained using either a TOF or a double focusing spectrometer, micro mass VG 70-70H or LC/MSD trap SL spectrometer operating at 70 eV using direct inlet system. All the reactions were monitored by thin layer chromatography (TLC) on percolated silica gel 60 F254 (mesh); spots were visualized with UV light. Merck silica gel (60-120 mesh) was used for column chromatography.

MTT assay
All the synthesized compounds were tested for their cytotoxicity efficacy using MTT assay on both cancer and normal cell lines following the protocol of our earlier published literature. 27 For this assay, cancer lines A549 -Homo sapiens lung carcinoma (ATCC® CCL-185™); B16-F10 -Mus musculus mouse skin melanoma (ATCC® CRL 6475™); DU145 -Homo sapiens prostrate carcinoma (ATCC® HTB-81™); Hep G2 -Homo sapiens hepatocellular carcinoma (ATCC® HB-8065™); CHO-K1 -Cricetulus griseus Chinese hamster ovary cells (ATCC® CCL-61™) were used. All the cell lines used in this study were obtained from the ATCC (Bethesda, MD, USA) and grown in DMEM with 10% Fetal Bovine (FBS) along with 100 U mL À1 penicillin, 100 mg mL À1 streptomycin and 2 mM L-glutamine. Initially, each cell line was grown in the 96 well culture plate at 37 C at 5% CO 2 in an incubator and allowed to attach properly. Aer attaining the growth of cells, each test compound at different concentrations (from 1 to 100 mM) was treated to the cells and incubated for 24 h. Aer the incubation of the cells, MTT (0.5 mg mL À1 ) was added and further incubated for 3 h. About 100 ml DMSO was added to each well to dissolve the insoluble formazan crystals and nally, the absorbance reading was taken for each plate using a multi-mode plate reader, all the experiments were carried out in triplicate and 50% inhibitory concentration (IC 50 value) of each compound was calculated using Graphpad prism-6. Simultaneously, 5-uorouracil (5FU) and doxorubicin were used as positive control and standard drug respectively for the comparison of cytotoxicity activity of the test compounds.

Molecular docking
In order to explore plausible interaction models of compounds 3c, 3l and 3o, docking studies were performed with the antiparallel propeller type telomeric DNA and oncogene c-Myc promoter [Protein Data Bank (PDB) entry 1N37]. 26 The protein and ligand preparation were performed using Discovery studio 2021. 28 Molecular docking was carried out using AutoDock 4.2.6 version. 29 The structures of 3c, 3l and 3o were drawn and converted into 3D molecules using Discovery studio 2021. Water molecules were eliminated and the polar hydrogen atoms were added to the PDB system. To check the interactions of the ligands with all G-quadruplex DNA conformations, a docking experiment was done on the active site using AutoDock tools-1.5.7 and the best docked structure was taken using a dGscore (G score) function. The DNA-ligand interactions were visualized and the 3D & 2D gures were generated using the Discovery studio 2021. The protein-ligand interactions as gscore are tabulated in Table 6.

Conflicts of interest
The authors declare that they have no conict of interest.