Anti-Cancer Activity and Molecular Docking of Some Pyrano[3,2‑c]quinoline Analogues

Quinoline analogues exhibited diversified biological activities depending on the structure type. A number of natural products with pyrano[3,2-c]quinolone structural motifs and patented chromenes were reported as promising cytotoxic agents. A molecular docking study was employed to investigate the binding and functional properties of 3-amino pyranoquinolinone 2a-c as anti-cancer agents. The three 3-amino pyranoquinolinone 2a-c showed an interesting ability to intercalate the DNA-topoisomerase complex and were able to obtain energetically favorable binding modes (−8.3 - −7.5 kcal/mol). Compound 2c containing butyl chain superiority over the other two compounds 2a-b which appeared to be involved in arene-H interactions with the two dG13 aromatic centers. The butyl chain also appeared to be immersed into a side subpocket formed by the side chains of Asn520 and Glu522 and the backbone amide of Arg503, Gly504, Lys505 and Ile506. Hence, the 3-amino pyranoquinolinone 2c used as starting material to prepare derivatives of pyrano[3,2-c]quinolone containing 1,2,4-triazine ring 4a-b which will enhance the anti-cancer activity. Pyrano[3,2-c]quinoline-2,5-diones 2a-c and 4a-b were evaluated in vitro on cell lines Ehrlich Ascites carcinoma cells (EAC), liver cancer cell line Hep-G2 and breast cancer cell line MCF-7 for the development of novel anticancer agents. The screening results revealed that compounds 4a-b were found most active candidates as anticancer agents.


Introduction
Over the past few years, several quinoline containing compounds are reportable as potential antitumor agents. Further, Quinoline scaffold plays a crucial role in antitumor drug development as their derivatives have shown excellent results through different mechanism of action such as growth inhibitors by cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, andmo dulation of nuclear receptor responsiveness. The anti-cancer potential of many of these derivatives has been demonstrated on various cancer cell lines. The strength of quinoline motif in anticancer drug development is evident from clinically used anticancer drugs like Camptothecin, Topotecan and Irinotecan, etc. [1].
Heterocyclic scaffolds, particularly nitrogen-containing heterocyclic compounds, play an important role in the design of novel drugs because of their utility for various biological receptors with a high degree of binding affinity. Among the heterocycles, the triazines with their numerous biological profile, occupy a prominent position [11]. The 1,2,4-triazine ring as one of the most ubiquitous heterocycles in Nature, and it has been reported to possess a broad spectrum of biological properties, including anticonvulsant [12], neuroprotective [13], anxiolytic [14], antiparkinson [15], antidepressant [16], anti-inflammatory [17], antimicrobial [18] activities. In addition, there are also many reports indicating significant anticancer properties for the 1,2,4-triazine fragment. For example, tirapazamine (C, Figure 1) is currently in various clinical trial phases for the treatment of human non-small cell, cervical, ovarian, head and neck cancers.
Tirapazamine works by inducing DNA damage in poorly oxygenated tumor cells [19]. With this background and on the basis of that the pyrano [3,2-   Working two-fold serially diluted test material (µm) was prepared. Cells monolayers were washed with PBS (phosphate buffer saline pH = 7.2) and the additional serially diluted material was dispensed to the precultured plates for the determination of test material's toxicity [20].

MTT Assay for Cytotoxicity
MTT assay is a sensitive, quantitative and reliable colorimetric method that measures viability of cells. The assay is based on the ability of mitochondrial lactate dehydrogenase enzymes (LDH) in living cells to convert the water soluble substrate 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) used as 5 mg/mL in a dark blue formazan which is water-insoluble. Dimethyl sulfoxide is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring it using spectrophotometer at a wavelength usually between 500 and 600 nm [21].

Method
Test and standard materials were 2 fold serially diluted on precultuled cell lines

Anticancer Activity for EAC Cells
EAC cells were maintained by weekly intraperitoneal transplantation of 2.5 × 10 5 cells in mice. The tumor is characterized by a moderately rapid growth, which leads to the death of the mice in about 20 days due to the distal metastasis. Ascites was withdrawn under aseptic conditions from the peritoneal cavity of tumor bearing mice by needle aspiration after 7 days of EAC cells inoculation. To adjust the number of EAC cells/mL, tumor cells obtained were diluted several times with normal saline. EAC viable cells were counted by trypan blue exclusion method where 10 μl trypan blue (0.05%) was mixed with 10 μl of the cell suspension. Within 5 min, the mixture was spread onto haemocytometer, covered with a cover slip and then the cells were examined under microscope. Dead cells were stained blue, viable cells were not. Cell suspension was adjusted to contain 2.5 × 10 5 viable cells/ml. EAC cells, RPMI medium drugs, and DMSO were added in sterile test tubes according to trypan blue exclusion method [24]. The cells were incubated for two hours at 37˚C under a constant over lay of 5% CO2. EAC viable cells were counted by trypan blue exclusion using haemocytometer as mentioned above. The cell surviving fraction was calculated from the relation T/C; where T and C represent the number of viable cells in a unit volume and the number of total (viable + dead) cells in the same unit volume, respectively. The in vitro cytotoxicity was performed against two different human cancer cell lines namely: Liver Hep-G2 and Breast MCF-7. Comparison between new compounds is carried out to evaluate their toxicity against Liver Hep-G2 and Breast MCF-7 cancer cell lines. Cell viability assessment using the MTT method indicated a significant difference between the synthesized compounds and reference drug, 5-flurouracil.

Docking Analysis
The crystal structure of the Topoisomerase-II-DNA-inhibitor complex was obtained from the protein data bank (PDB: 4G0V [25]). The ternary complex was then checked for any missing atoms/residues via the protein preparation module in MOE [26]. All solvent molecules and all hetero ligands were removed. Using the Protein Preparation Wizard [26] the Topoisomerase-II-DNA complex was fully prepared via creating bonds and adding hydrogens. Subsequently, partial charges were assigned on all atoms and protonation states were predicted for ionizable groups. The binding pocket was identified by the co-crystallized ligand and a grid box was produced in Glide using the Receptor Grid Generation module [26] [27]. Ligands were prepared in Maestro [27] [28] via the LigPrep module using the default settings. The dominant ionization states at pH range of 7.0 ± 2.0 were generated for ionizable functional groups. Next, ligands were docked into the binding site of the previously prepared protein-DNA complex using the Glide software [26] [27], where the extra-precision (XP) Algorithm [29] was used for conformational sampling. Afterwards, generated poses were scored via the Glide_XP scoring function which includes terms for van der Waals, hydrogen bond, electrostatic interactions, desolvation penalty and penalty for intra-ligand contact [29].

Docking Study
A molecular modelling study was initiated in order to support the assumed mode of action for tested compounds 2a-c and optimize a reliable model for predicating novel effective antitumour hits. The design of the 3-amino pyranoquinolinone 2a-c compounds was based on the anticancer quinolone structure.
Voreloxin, a quinolone compound, is an antineoplastic agent that reached the clinical trial. [31] Voreloxin was observed to work through intercalating DNA and poisoning topoisomerase II via forming a protein-DNA-inhibitor complex [31]. Similar to the standard intercalating agent doxorubicin, the resulting ternary  complex will consequently cause DNA fragmentation and finally apoptosis [32] [33]. Based on structural similarities between our compounds and quinolones, it was just sensible to propose that the 3-amino pyranoquinolinone compounds exert their antitumor activity via binding to the topoisomerase-II-DNA complex through the intercalation mechanism. The 3-amino pyranoquinolinone 2a-c along with the reference intercalating agent doxorubicin were docked into the topoisomerase-II-DNA complex. The resultant docking scores of the test compounds and reference ligands along with their ligand efficiency score are shown in Table 1. Interestingly, the three 3-amino pyranoquinolinone 2a-c were able to obtain energetically favorable binding modes (−8.3 -−7.5 kcal/mol). The best scorer 2c was nicely placed on top of the co-crystalized ligand, intercalating the double stranded DNA (Figure 3(A)). Several stacking and hydrogen bonding interactions appear to contribute to the 3-amino pyranoquinolinone intercalation ability. The planar tricyclic system of these compounds seemed to give them a great capacity to slide in between the DNA bases and to make π-π interaction with the surrounding nitrogen bases, Figure 3(B). Additionally, the 3-amino pyranoquinolinone compounds were capable of forming two hydrogen bonds with the dT9 deoxyribose and phosphate groups through their amine and another electrostatic interaction with the topoisomerase Asn778 side chain through their aromatic hydrogen. Compound 2c has an extended alkyl side chain, compared to 2a and 2b, which appeared to be involved in arene-H interactions with the two dG13 aromatic centers. The butyl chain also appeared to be immersed into a side subpocket formed by the side chains of Asn520 and Glu522 and the backbone amide of Arg503, Gly504, Lys505 and Ile506. For these two reasons, the butyl chain seems to be the main responsible factor for 2c superiority over the   other two compounds 2a-b. As it can be noticed in Figure 3(B), this alkyl group is pointed towards the side chains of Asn520 and Glu522; meaning that extend-   Table 2 and Figure 4, that the three 3-amino pyranoquinolinone 2a-c showed significant activity (IC 50 = 31.1, 29.2 and 27.7 μM) respectively which is nearly as potent as the reference drug (doxorubicin, IC 50 = 39.5 μM). Compound 2c showed the higher activity among the 3-amino pyranoquinolinone serieswith IC 50 = 27.7 μM. The reason for the higher reactivity of the amines can be explained by the presence of the bioactive 3-aminopyrane and quinolinone moieties in one molecular frame, quinolinone is known with its activity towards tumor cells. It has been reported that quinolones are known as potent antitumor agents because they target topoisomerase II enzyme and are considered as therapeutic promise [34]. It has been reported also that the activity of quinolinone depends on attack of topoisomerease IV (DNA gyrase) in microbes and topoisomerease II in animals that in eukaryotes, DNA gyrase and topoisomerease IV is functionally replaced by two isoenzymes: topoisomerease IIa and IIb, a170 and b 180 kDa protein, respectively [35] [36]. Also interested are the 2-pyrones themselves, as some of the 2-pyrones with hydroxyl group at C4 position were reported to be biologically active [37]. In our study, the presence of the aliphatic chain beside the amino group in 3-amino pyranoquinolinone 2a-c is playing a vital role to improve the efficiency of the quinoline moiety as anti-tumor inhibitors, where it was found the presence of the butyl chain which seems to be the main responsible factor for 2c superiority over the other two compounds 2a-b and may give an idea about the possible importance of extended alkyl group in nitrogen N1 at quinolinone ring to enhance activity. Considerable evidence indicated that as longer as the alkyl group at position N-1 as higher as antitumor activity and increase affinity towards topoisomerease II [38].   Moreover, the most potent compound in this study was triazinopyranoquinolinone compound 4b (IC 50 = 24.4 μM) which was found to be more potent than the reference drug (doxorubicin) (IC 50 = 39.5 μM) and also they found to be more active than the starting material 2c (IC 50 = 27.7 μM) this may be attributed to the presence of triazine ring which enhance the anti-cancer activity.  which was found to be more potent than the reference drug 5-flurouracil (IC 50 = 11.2 μM). And 11.1 and 9.7 μM at 72 hr respectively against MCF-7 cell line compared with reference drug 5-flurouracil (IC 50 = 11.6 μM).

Conclusion
A series of the pyrano[3,2-c]quinolonine analogues were evaluated for its anticancer activity. The screening results revealed that compounds 4a-b were found as the most active candidates of the series against anticancer activity compared to the reference doxorubicin and 5-flurouracil against the three human tumour cell lines: showed a significant alteration of the bioactive conformer of the parent scaffold and outcome with compounds 4a-b as the most potent candidates of the series.
In summary, pyrano[3,2-c]quinolone compounds 4a-b have been confirmed as a useful lead compound which can be developed for the clinical trial for its therapeutic use.