Cytotoxicity and Molecular Docking Studies of a Novel Enaminone and its Cadmium (II) Complex

Rahila Huma1,2, Tariq Mahmud1,*, Rubina Munir2, Sana Javaid Awan3, Kiran Iftikhar4 and Mufakkira-Tul-Islam5

1Institute of Chemistry, University of the Punjab, Lahore-54590

2Department of Chemistry, Kinnaird College for Women, Lahore

3The University of Lahore, Lahore

4Department of Chemistry, University of Gujrat (UOG), Gujrat

5Department of Chemistry, Lahore College for Women University, Lahore

ABSTRACT

The anti-cancerous potential of novel Enaminone and its Cadmium complex was estimated by measuring its cytotoxicity for human breast cancer cell line (MCF-7). Both compounds PFA and PFA-Cd were prepared and subsequently characterized by FTIR, ICP-AES, UV-Vis, TGA, 1H NMR, 13C NMR and FAB-MS. The results were in great consistency with the structures of the both compounds. Furthermore, the ligand and its metal complex were screened for their cytotoxicity against human cancer cell lines. It was found that the compound PFA was more active anti-cancerous agent as compared to PFA-Cd. Molecular Docking studies were carried out to found the predominate binding mode of synthesized compound in the vicinity of double helical DNA structure.

Article Information

Received 02 August 2018

Revised 02 September 2018

Accepted 12 September 2018

Available online 26 February 2019

Authors’ Contribution

TM conceived and designed the experiments. RH executed the experiments and wrote the article. SA helped in performing cytotoxic activities. RM and MTI helped in the characterization of data. KI helped in Molecular docking studies.

Key words

Enaminone, Molecular docking, IC50 value, Cytotoxicity, Spectroscopy.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.2.697.702

* Corresponding author: tariqm06@yahoo.co.uk

0030-9923/2019/0002-0697 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

Cancer is a general term used for a huge group of diseases characterized by the growth of abnormal cells that can invade adjacent parts of the body and can spread to other organs. In 2015, 8.8 million deaths occurred due to cancer and it is the second leading cause of death worldwide. According to National Center for Health Statistics (based on Mortality data available through 2015) 1, 735,350 new cancer cases and 609,640 cancer deaths are expected to occur in the United States in 2018 (Madani et al., 2011; Rebecca et al., 2018). It is therefore vital to produce new drugs to counter the uncontrolled proliferation of cancer cells (Gupta et al., 2013; Gulzar et al., 2018).

Human body required several metals are in trace amount as these metal ions directly effect on the activity of various enzymes. If the quantity of trace metals increases in the body it may causes some toxic effects. One of the toxic effects is that these metals may act as pro-carcinogene.These metal ions become activated in the body and bind to the electron rich sites in DNA, which results in mutagenesis and finally causes cancer. Chelating agents can be used to remove controlled amount of undesirable metal ions from the body. Moreover, a large number of complexes are well-known to have antitumor activity. It is assumed that they deactivate either the carcinogenic metal or the enzymes essential for the fast growth of both healthy and malignant cells (Tripati et al., 2007). Enaminones is a class of compounds having the conjugated system (N-C=C-C=O) (Greenhill, 1997). Enaminone have great potential as reaction intermediates and medicinal compounds due to the presence of nucleophilic and electrophilic sites (Edafiogho et al., 1992). A literature survey reveals that enaminones and its derivatives shows a wide range of antimicrobial (Cindric et al., 2018), anticonvulsant (Jackson et al., 2012), anticancer (Shawali, 2010) and anti-inflammatory activities (Ahmed, 2015). On the other hand, enaminones have been commonly used as intermediates in the preparation of a large number of pharmacologically important compounds (Kalaria et al., 2014; El-Azab et al., 2016).

In view of these facts, the present work includes the synthesis of a novel enaminone and its Cd complex. The synthesized products were characterized by FTIR, NMR, Mass spectrometry, TGA and elemental analysis and screened for cytotoxicity against human cancer cell lines. The study was extended by the docking studies and the binding mode of best active compound was also predicted.

 

Materials and methods

The starting materials and solvents used for the synthesis of ligands and complexes were purchased from market and were used as such.

Synthesis of 3-chloro-4-(florophenyl) amino) pent-3-en-2-one

3-chloro-2,4-pentanedione (0.02 mole L-1,2.5 mL), p-floroaniline (0.02 mole L-1, 1.89mL), p-toluene sulfonic acid (1.0 g) and toluene (150 mL) were heated on heating mantle for12 h. The water produced as a result of condensation in reaction mixture was separated using a Dean–Stark apparatus. The reaction was monitored by TLC. Crystalline product was collected after solvent extraction using chloroform and sodium bicarbonate.

Molecular formula: C11H11ClFNO, off white crystalline solid, Yield: 60%, m.p: 59oC, Molecular weight: 227.66 g/mole, IR (cm-1): cm-1 3113(NH), 1591 (C=O), 1511 (C=C)), 1H NMR (300 MHz, CDCl3, δ ppm): 12.67 (s, 1H, -NH ), 7.07(d,J=6.3 Hz,4H), 2.37(s, 3H), 2.14(s, 3H).13C NMR (75 MHz, CDCl3) δ: 194.74, 162.56, 159.29, 134.45, 127.39, 116.28, 104.36, 28.67, 18.15, FAB-MS: m/z = 228.1[M+H] +, Anal. Calcd. for C11H11ClFNO: C, 58.03; H, 4.87; N, 6.15%. Found: C, 56.58; H, 5.92; N, 5.82%.

Synthesis of cadmium (II) complex of 3-chloro-4-(florophenyl) amino) pent-3-en-2-one (PFA-Cd)

A solution of (PFA) (0.02 mole) in 10 mL DCM was added drop wise with continuous stirring to a 10 mL of filtered ethanolic solution of the CdCl2. (0.01 mol) and refluxed for 2 h. The complex was obtained by filtration and evaporated slowly at room temperature. The synthesis of coordination compounds had good yields

Molecular formula: [C22H22Cl4CdF2N2O2], Light yellow Solid, Yield: 47%, M.p: Decomposed 240-244°C, Molecular weight: 638.64 g/mole, IR (cm-1): 3130 (NH), 1581 (C=O conjugated), 1533 (C=C stretching); 450(Cd-N stretching), 527(Cd-O stretching),1H NMR (300 MHz, CDCl3, δ ppm): 12.64 (s, 1H, -NH ), 7.32-7.21 (m, 4H, - Ar-H), 2.26(s, 3H), 2.12(s, 3H).Anal. Calcd. for [C22H22Cl4CdF2N2O2],: C, 41.37; H, 3.47; N, 4.39%. Found: C, 40.20; H, 2.75; N, 3.95%. % metal for [ML2Cl2]: Theoretical/experimental (17.60/16.56).

Elecro-analytical characterization

The IR spectrum was recorded on Agilent Technologies FT-IR instrument (in range of 4000-400 cm-1).The 1H-NMR spectrum was recorded on a Bruker Ascend NMR spectrometer. The (FAB) mass spectrum was recorded on a JEOL JMS-600H mass Spectrometer. Conductivity were measured with the inoLab Cond 720 conductometer using DMSO. Cytotoxicity were carried out and their IC50 values were compared by using standard drug.

Cultivation of cell lines

Cell lines were obtained from laboratory culture established in The University of Lahore. Cells were plated in the culturing flask in High glucose Dulbacco Modified Eagle medium (DMEM-HG) (GIBCO, USA) including 10 % serum, 100 U of penicillin and 100μg/ml streptomycin. When cell lines (MCF7) achieved 70-80% confluence, these were trypsinized by using 0.5ml of 0.05% trypsin and 0.53 EDTA (Thermo Scientific, USA). Medium of the cultured cells was changed after every three days. MCF-7 cell lines were cultured in 96-well plates (~2x104 per well) for cell proliferation assay. The treatments were given as 0.0 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 500 μg/ml of the PFA, PFA-Cd and Cis-platin in DMEM for 24 h.

In vitro growth inhibition test

Cytotoxic activity of the enaminone and its cadmium complex was determined by using 96 well level bottomed micro plates using the standard MTT colorimetric protocol (Mossman, 1983). Cell culture with the convergence of 6×104 cells/mL was arranged and presented (100 µL/well) into 96-well plates. Medium was removed after the incubation of one night, and 200 micro liter of new medium was incorporated with the different concentrations (0-500 µg/ml) of sample. After 24 h, 25µL MTT (5 mg/mL) was added to each well and further incubated for next 4 h. Accordingly, 100 µL of dimethyl sulphoxide was introduced into each well. The degree of MTT diminishment to formazan inside cells was computed by absorbance measurement at 570 nm, utilizing a smaller scale plate peruser (Spectra Max in addition, Molecular Devices, CA, USA). Cytotoxic results were obtained as their concentration responsible for 50% growth retardation (IC50). The % inhibition was computed by using the accompanying equation:

Molecular docking studies

To govern the predominate binding mode of synthesized compound in the vicinity of double helical DNA structure Molecular Docking studies was executed using Auto Dock v4.2 and Auto Dock tools v1.5.6 (Morris et al., 1998). The best possible conformation and the interaction of the most probable conformation were investigated. Crystal structure of DNA was taken from RCSB Protein Data Bank with PDB ID1BNA. Structures of both compounds were drawn and 3D optimized using Marvin sketch. Prior to docking DNA structure was prepared by adding hydrogen and a grid box of 80×80×120 dimensions was created. Additionally, Lamarckian genetic algorithm which is modified for flexible ligand-receptor docking was used and it consents to grip greater degrees of freedom. A total of 100 different confirmations were secured the utmost pose was then subjected to visualization for analysis of interactions between the DNA and the synthesized compound.

 

Table I.- Cytotoxicity of the new compounds against MCF-7 cell line.

Compound	Cell viability at concentrations (μg/ml)
	0	5	10	25	50	100	200	500	IC50
PFA	100	67	66	64	63	57	50	39	200
PFA-cd	100	191	104	76.3	69.7	65.3	57.3	44.0	334.5
Cis-platin	100	89	68	40	11	5.8	4.3	2.1	20

 

Results and discussion

The newly prepared enaminone and its cadmium complex were characterized by NMR, IR and elemental analysis. The molecular mass of enaminone was confirmed by MS (FAB). PFA showed peak at 228.1 as [M+H]+. 13C NMR of enaminone PFA showed peaks at: 194.74, 162.56, 159.29, 134.45, 127.39, 116.28, 104.36, 28.67, 18.15.These signals are in consistent with the proposed structure of enaminone PFA. Both compounds showed very low conductance values indicating their non-electrolyte nature.

FTIR

In the infrared spectra of the enaminone (PFA), band at 3113 cm–1 was allocated to NH stretching vibration. Two strong bands were observed at 1591 cm–1 and 1557 cm–1 that are assignable to ν (C=O, conjugated) and ν (C=C), respectively. The presence of ν (NH) band at 3130 cm–1 in the spectra of the PFA-Cd suggested that the hydrogen atom is not removed and the (NH) group is involved in the coordination (Souad et al., 2014). The (C=O) stretching vibration of conjugated carbonyl group of free ligand shifted to a Lower side 1581 cm_1 in metal complex, indicating the coordination of the ligand through the oxygen of Carbonyl group (Tyagi et al.,2015). Moreover new bands in the spectra of PFA-Cd appeared below 600 cm–1 assignable to (M-N) and (M-O) bonds (Kavitha et al., 2017).

 

Proton NMR

1H-NMR spectrum of synthesized compounds was recorded. 1H-NMR of ligand (PFA) revealed the presence of a single proton at 12.66 ppm due to the enamine N–H group (Shi et al., 2005). Aromatic protons were found as a doublets at 7.07 (d,J=6.3). Moreover methyl protons also show two singlets at 2.36 and 2.14 ppm. In metal complex, the slight shifting of chemical shift values of all protons confirms the formation of metal ligands bonds. In addition the presence of NH signals in the spectra of complexes indicated that N forms coordinate covalent bond with metal ions while its hydrogen remains intact. So enaminone ligand acts as a bidentate ligand by bonding through Nitrogen and oxygen (Jeragh and Abdel-Zaher, 2015). These observations are in consistent with the interpretation of IR spectra.

In vitro growth inhibition test

The cytotoxic effects of compounds PFA and PFA-Cd against human breast cell line (MCF-7) was evaluated using Cis-platin as a standard sample with an IC50 of 20µg/ml. The analysis of the data obtained indicated that the values of IC50 for PFA and PFA-Cd compounds against MCF-7 cell line are 200 μg/ml (Fig. 2A) and 334.6 μg/ml (Fig. 2B), respectively. The values of IC50 indicated that the tested compounds have cytotoxic activities at higher concentrations (200μg/ml and 334μg/ml) as compared to the reference drug (20μg/ml).

 

Table II.- Binding free energy of compounds.

S. No	Compound	Binding free energy (Kcal mol-1)
1.	Standard drug Cis-platin	-4.17
2.	PFA	-6.03
3.	PFA-Cd	-5.86

 

Molecular docking simulations

Enaminone PFA and its cadmium complex (PFA-Cd) has been synthesized and evaluated for cytotoxicity. Standard drug Cis-platin was found most effective. It basically interferes with DNA replication. Generally anticancer agents intermingle with the DNA by four bonding modes such as interaction with minor groove or major groove, surface binding and intercalation. There are different targets for anticancer activity so this era was of great interest for medicinal chemists.

To evaluate anticancer potential of both compounds, molecular docking studies was performed. Both compounds were found in major and minor grove. No surface binding and intercalation was observed. Least binding energy values are given in Table II. The Compound PFA was found to have the highest binding affinity with a value of -6.03 Kcal mol-1as compared with the complex of cadmium as well as with reference Cis-platin.

Visualization of this compound using Discovery Studio Visualizer v4.0 cleared that it binds to the minor groove of DNA helix. Several interactions were observed with the DNA such as hydrogen acceptor interaction with DA20 nucleotide and hydrogen donor with DA18 nucleotide. Moreover electrostatic interactions were also observed between ligand PFA and nucleotide of DNA such as DC9. Molecular docking also supports the IC50 values obtained from anticancer activity performed that PFA binds with highest affinity to DNA double helix. Carefully selected view of PFA inside DNA helix is shown in Figure 3.

 

Conclusion

Two novel compounds were synthesized, characterized electro-analytically and evaluated for their cytotoxic activities. All the spectroscopic results confirmed the formation of the targeted compounds. From the cytotoxic results of the synthesized compounds, it is concluded that the compound PFA having electron withdrawing ‘Group at its para position, was proved to be moderate cytotoxic agent as compared to the cadmium complex PFA-Cd. Molecular Docking studies showed that PFA binds with highest affinity to DNA double helix.

 

Acknowledgement

The authors are grateful to HEC, Govt. of Pakistan for access to Scientific Instrumentation and Institute of Chemistry, University of the Punjab, Lahore-54590, Pakistan, for providing laboratory facilities.

 

Statement of conflict of interest

The authors declare no conflict of interest.

 

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