Design, synthesis and evaluation of new methyl piperazine derivatives as anticancer agents

Background To overcome the problem of side effects and toxicity, development of new anticancer agents is needed. Recently, piperidine salicylanilide derivatives with nanomolar epidermal growth factor receptor (EGFR) inhibitory and cytotoxicity activity have been reported. In the present study effect of replacing piperidine in reported piperidine salicylanilide with N -methyl piperazine and changing substituent’s of phenyl ring at other end on anticancer activity have been explored. A series of sixteen methyl piperazine incorporated phenyl benzamide and phenyl methanone derivatives have been synthesized and tested in a panel of three cancer cell lines (adenocarcinomic human alveolar basal epithelial cells (A-549), human colon carcinoma (HCT-116) and human pancreatic carcinoma (MIAPaCa-2)), using gefitinib as standard. Further, to study the probable mechanism, due to their structural similarity with EGFR inhibitors, docking interactions with EGFR active site were observed using Schrodinger suite. Result The results indicated that most of the compounds showed promising activity; out of which, compound A-11 was most active having cytotoxicity much better than that of gefitinib. It showed IC 50 value of 5.71 µM against A-549 cell line, 4.26 µM against HCT-116 colon cancer line and 31.36 µM against MIAPaCa-2 cell line. Conclusion It was found that these compounds fit well in the active site and may be exhibiting anticancer activity via EGFR inhibition.


Background
Cancer results from uncontrolled cell growth, and remains one of the most potentially life threatening disease worldwide [1].As per reports of the International Agency for Research on Cancer, in 2012, 14.1 million new cancer cases were reported, along with 8.2 million cancer deaths worldwide.The global burden of cancer is expected to grow to 21.7 million as new cancer cases and 13 million cancer deaths in 2030 despite the presence of variety of anticancer medicines, development of anticancer agents has received more attention of medicinal chemists, due to occurrence of side effects [2,3].
EGFR amplification and overexpression are prevalent various cancers including NSCLC, occurring in up to 85% of patients with this type of cancer.Mutations typically arise in exons 18-21, which encompass the kinase domain of the EGFR gene.The majority, around 90%, of these mutations manifest as either exon 21 L858R point mutations or deletions within exon 21.These genetic alterations lead to heightened EGFR kinase activity, resulting in increased downstream signaling.Conversely, most exon 20 insertion mutations are associated with reduced sensitivity to EGFR TKIs.Hence, in order to address resistance, there is a critical need for the development of novel ligands that function as inhibitors of EGFR [4].
In literature, quinazoline and piperazine containing compounds have been reported to inhibit various types of cancers [4].Quinazoline derivatives are known for EGFR tyrosine kinase (TK) and other kinase inhibition in non small cell lung cancer (NSCLC) and other carcinomas.Gefitinib is one of the quinazoline derivatives which have been used for anticancer activity, especially in lung cancer.Unfortunately, resistance against quinazoline compounds develops within a short span of use [5].
Similarly, piperazine and benzothiazole piperazine derivatives have been evaluated for their anticancer potential against HCT-116 colon cancer cell line [6][7][8].Piperazine substituted adamantanes have been reported for colon and pancreatic cancer, whereas piperazine methanone shows good potency against human breast adenocarcinoma cell line, MCF-7 and mouse embryonic fibroblast cell lines, NIH3T3 [9,10].Moreover, various piperazine derivatives have been patented as lung cancer chemotherapy agents [11].Benzofuran with N-aryl piperazine derivatives have also been reported for anticancer activity as hybrid compounds [12].Recently, phenyl piperazine benzoxazole and benzhydryl piperazine derivatives have been shown to inhibit lung, breast and other carcinomas [13][14][15].
Hu et al. have described novel piperidine salicylanilide derivatives having nanomolar EGFR TK inhibitory and cytotoxicity activity (Fig. 1) [16].Using this structure as basis, scaffold I was considered wherein the piperidine was replaced with N-methyl piperazine and the phenyl ring on the amidic nitrogen was substituted with various electron donating and withdrawing substituent's, hypothesizing that these changes could enhance the anticancer potential.Further, in scaffold II, the amidic nitrogen was constrained in a piperazine ring and the effect of substituents on the phenyl ring on the other nitrogen was also evaluated.

Materials
For docking, Glide XP of Schrodinger suite was used and physicochemical properties of the compounds were predicted using Chemaxon Jchem for Excel.The docking protocol was validated to govern the reliability and reproducibility of the docking parameters used for the study.Reaction progress was monitored using analytical grade solvents and pre-coated Silicagel G TLC plates (Kieselgel 60 F254 , Merck) and visualized in UV light.Melting points were measured with Buchi 530 melting point apparatus and were uncorrected. 1H NMR spectra were recorded on a Bruker Avance-400 MHz system using CDCl 3 or DMSO as the solvent.Chemical shifts (d) are reported in parts per millions (ppm) relative to TMS as internal standard.FTIR spectra were performed on IR Prestige 21 Shimadzu using KBr as standard.MS were analyzed on MICROMASS Quattro-II LCMS system (Waters Corporation, Milford, USA).All reagents were obtained from commercial suppliers and used without further purification.Synthesized compounds were tested against A-549 (human lung carcinoma), HCT-116 (human colon cancrcinoma) and MIAPaCa-2 (human pancreatic carcinoma) for anticancer potential.

Method
The X-ray structure of EGFR bound with gefitinib as a cocrystallized ligand (pdb id: 2ITO) was obtained from the brookhaven protein database for docking purposes [17].Docking of the compounds was performed using Glide 5.9.Running on maestro version 9.4, to investigate their putative binding mode in EGFR binding pocket.The Protein preparation wizard within the Schrödinger suite was employed to prepare the designated protein.
The protein underwent distinct preprocessing stages, which involved the removal of the substrate co-factor and water molecules lacking hydrogen bonds, followed by the optimization of hydrogen bonds.Subsequently, a charge was assigned, and the energy was minimized, achieving a Root Mean Square Deviation (RMSD) value of 0.30 Å utilizing the Optimized Potentials for Liquid Simulations-2005 (OPLS-2005) force field.The structures of all compounds were drawn using ChemSketch and converted into 3D structures through a 3D optimization tool.The ligands, drawn previously, were geometry optimized using the LigPrep 2.6 module, with partial atomic charges computed through the OPLS-2005 force field.The prepared ligands were then subjected to docking with the prepared protein utilizing the Glide 5.9 module, operating in extra precision mode (XP) [18].The anticancer activity was measured by using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay and Gefitinib was used as positive control.

Biological evaluation: cell viability/MTT assay
The reduction of tetrazolium salts was used to examine cell proliferation and growth inhibition.Gefitinib was used as positive control and IC 50 (the concentration which resulted cytotoxicity in 50% cells) values are the mean of three independent experiments [19].
For in vitro evaluation, MTT assays for all compounds against three cell lines viz.A-549 human lung carcinoma, HCT-116 human colon cancer and MIAPaCa-2 human pancreatic carcinoma were performed.The DMEM, RPMI-1640, DMEM-F12, were used as culture mediums for A-549 human lung carcinoma, HCT-116 colon cancer and pancreatic MIAPaCa-2 cell lines, respectively.10 4 Cells per well were grown in 96-well plates and exposed to different concentrations of various test compounds for 48 h.After 44 h treatment, 20 µl of MTT solution (2.5 mg/ml) was added to each well and incubated at 37 °C for 4 h in a humidified atmosphere containing 5% CO 2 .In case of suspension cell lines, the plates were centrifuged at 1500 r.p.m. for 15 min, and the supernatant was discarded while in adherent cell lines, the media was removed without centrifugation.The MTT-formazan crystals were dissolved in 150 µl dimethyl sulfoxide.The absorbance was recorded at a wavelength of 570 nm in the microplate reader and cytotoxicity was calculated as % cell growth inhibition.where At, Ab and Ac are absorbance of test, blank and control, respectively.Concentrations 1 µM, 10 µM, 20 µM, 30 µM, and 50 µM were used for assay.

In silico studies (docking and physiochemical properties)
The series was prepared keeping similar features to that of piperidine salicylanilide, having EGFR inhibitory activity.Therefore, it was thought to study the in silico docking of active compounds with EGFR to suggest the possible mechanism of action.Glide score and physicochemical propoerties of active compounds are given in Table 2 [20][21][22][23].
In the HCT-116 colon cancer line, three compounds showed better activity than gefitinib.Compounds A1 (-H) and A4 (2,4 di-CH 3 ) displayed IC 50 value of   6.54 µM and 10.54 µM, respectively while compound A-11 having meta methoxy substitution was most active with IC 50 of 4.26 µM.Again, in this cell line, electron donating substituted compounds exhibited good inhibition and compounds with electron withdrawing groups were inactive.
Overall, compound A-11 was found to be the most active in two of the three cell lines.Also, by observing the IC 50 values of all the compounds, it can be said that compounds with electron-donating substituents on the aniline portion exhibited better inhibition as compared to compounds with electron withdrawing groups.

Docking
It was found that the docked ligand superimposed well with reference ligand (gefitinib, co-crystallized ligand) with root mean square deviation value of 0.458 Å.The gefitinib in active site of EGFR displayed hydrogen bonding interaction with MET793 of hinge region, which is essential.Additionally, it shows hydrophobic interactions with CYS797 and LEU792 of hinge region and LEU844 and MET766 in the C-helix with a glide score of − 7.31.
It was observed that compounds with electron-withdrawing substituents did not dock well in the active site and compounds with electron donating substituents fit well.Compound A-10 with p-fluoro substituent on scaffold I was the only compound with electron withdrawing substituent and weak in the MTT assay was found to have least activity glide score.Its glide score was less than the unsubstituted compound.It was observed that it does not take part in any hydrogen bonding interaction.
When all the compounds were docked, it was observed that hydrophobic interactions with CYS797, LEU792, MET793 of hinge region, LEU844 and MET766 in the C-helix were similar for all compounds.However, the compounds showed hydrogen bonding interaction with MET793 and those with better score also showed side chain hydrogen bonding with ASP855 in activation loop.This indicates that these compounds do show necessary interactions in active site and hence may exert anticancer activity by inhibiting as EGFR.
Docking interactions of representative compounds are shown in Fig. 2.

Conclusion
A range of 16 compounds consisting of methyl piperazine-incorporated phenyl benzamide and phenyl methanone derivatives were synthesized and assessed for their potential as anticancer agents against A-549 human lung carcinoma, HCT-116 human colon cancer, and MIA-PaCa-2 human pancreatic carcinoma cell lines.It was noted that compounds possessing electron-donating groups exhibited heightened cytotoxicity across the three cell lines.Additionally, a hypothesis was put forward suggesting that the observed anticancer effects may stem from the inhibition of EGFR.Previous literature indicates that hydrogen bond interactions with MET793 have been linked to EGFR inhibition, and the compounds exhibiting superior anticancer activity were found to interact with MET793 within the EGFR binding site.The results suggest that these derivatives function as anticancer agents; nevertheless, further investigations are necessary to ascertain their specificity as EGFR inhibitors.

Fig. 1
Fig. 1 Structures of reported piperidine salicylanilide, scaffolds I and II

Table 1
Results of MTT/cell viability assay

Table 2
Docking score and physiochemical properties of compounds MW: Molecular weight, HBA: hydrogen bond acceptor, HBD: hydrogen bond donar, PSA: polar surface area, GS: glide score, DL: drug likeliness