Docking Investigation, Synthesis and Cytotoxic Studies of Coumarin Substituted Azetidinone Derivatives
Deepika P. 1, Dhanya P. 1, Naseef PP2*
1Jamia Salafiya Pharmacy College, Pulikkal, Malappuram, Kerala, India.
2Moulana College of Pharmacy, Perinthalmanna, Malappuram, Kerala, India.
*Corresponding Author E-mail: drnaseefpp@gmail.com
ABSTRACT:
A new series of azetidin-2-one derivatives containing coumarin ring were designed and docked with the protein kinase C θ receptor (1XJD), the docking scores obtained for certain derivatives are better than the natural ligand and the standard. The compounds which showed good docking scores were synthesized and characterized by spectral data. The cytotoxic activity of the synthesized compounds was tested by MTT assay and the compounds showed good activity on HeLa cell lines.
KEYWORDS: Anticancer, Docking, Azetidinone, Coumarin, HeLa cell, MTT.
INTRODUCTION:
Coumarins have attracted intense interest in recent years because of their diverse pharmacological properties. It has been evidenced from literature survey that coumarin derivatives have shown dopaminergic antagonistic activity.6 Hydroxy derivatives of 4-methyl coumarin are important group of coumarin derivatives showing medicinal as well as other applications, as optical brightening agents, dispersed fluorescent and tunable dye lasers.7 7-Hydroxy-4-methyl coumarin derivatives possess diverse biological properties such as neuroleptic, antibacterial, antitubercular, antifungal, antineoplastic, anti HIV, and anthelmintic etc.8
The aim of the present work was to design and synthesize new azetidine-2-one derivatives containing coumarin moiety in order to find new biologically active compounds especially anticancer agents.
MATERIALS AND METHODS:
1. Drug Design15:
Target Identification and Retrieval:
The 3-D structure of the protein kinase C θ was obtained from PDB using their specific PDB ID (1XJD).
Protein Preparation:
The protein structure (PDB ID: 1XJD) was prepared using the protein preparation wizard in the Schrodinger software graphical user interface maestro version 9.3. The protein was preprocessed separately by deleting the substrate cofactor, adjusting the bond orders, removing the metal ions, as well as the crystallographically observed water molecules (water without H bonds); correcting the mistakes in PDB file and optimizing hydrogen bonds.
Ligand Preparation:
A set of derivatives of azetidinones were drawn using the workspace of maestro and was converted to 3D form. All the ligands were built using maestro build panel. The collected ligands were prepared by using lig prep.
ADME/T Studies:
The above prepared ligands were then neutralized and checked for their ADME/T properties using (Qikprop, Version 9.2, 2012). Qikprop helps in analyzing the pharmacokinetics and pharmacodynamics of the ligand by accessing the drug like properties.
Induced Fit Docking (IFD) Extra Precision (XP):
IFD XP was performed using the module induced fit docking of Schrodinger-maestro version 9.3 (2012). The entire receptor molecule constrained minimized with an RMSD cut off of 0.18 A0 was selected for generation of centroid of the residues and the box size was generated automatically.
2. Experimental:
Synthesis of 7-Hydroxy-4-Methyl Coumarin9:
To a beaker containing 100 ml of concentrated sulphuric acid kept in an ice -bath, added a solution of resorcinol (0.1moles) in ethylacetoacetate (0.1moles), with continuous stirring maintaining the temperature below 100C. The reaction mixture was kept at room temperature for 18 hrs and then poured onto the mixture of 200g of crushed ice and 300ml of distilled water, with vigorous stirring. The white precipitate formed was collected by vacuum filtration, washed with 350ml of cold water, dissolved in 150ml of 5% w/v sodium hydroxide solution and filtered. The filtrate was added with 55 ml of concentrated sulphuric acid with vigorous stirring until the solution was acidic. The crude 7-Hydroxy-4-methyl coumarin was collected by filtration, washed with cold water, dried, and purified by recrystallization from ethanol.
Synthesis of Ethyl 2-(2-Oxo-4-Methyl-2h-Chromen-7-Yloxy) Acetate10:
In a two necked 500ml R.B.F. take 50-60ml dry DMF. To this add 10gms 7-hydroxy-4-yl coumarin and ethylchloroacetate 6.8ml (0.056moles) and anhydrous potassium carbonate (7.7gm). The resultant mixture was stirred for 20hrs at 800C. Then after completion of reaction which was monitored by taking TLC, the reaction mixture was filtered and poured into large amount of water. The solid separated was filtered and washed with water. The solid was dried and recrystallized from ethanol.
Synthesis of (2-Oxo-4-Methyl-2h-Chromen-7-Yloxy) Acetic Acid:
A mixture of 1g ethyl 2-(2-oxo-4-methyl-2H-chromen-7-yloxy) acetate and 0.5M alcoholic potassium hydroxide (20ml) was taken in an RBF. It was refluxed for 1hr and the mixture was cooled to room temperature. The alcohol was evaporated and to this cooled solution 1:1 HCl was added dropwise until it became acidic with litmus. The precipitate obtained was filtered and washed with water. The solid was recrystallized using ethanol.
Synthesis of (2-Oxo-4-Methyl-2h-Chromen-7yloxy) Acyl Chloride:
To a solution of (2-oxo-4-methyl-2H-chromen-7-yloxy) acetic acid (1mole) in methylene chloride (15ml), DMF 0.5ml was added. To this mixture thionyl chloride (1.5mole) was added and it was refluxed for 1hr maintaining the temperature at 800C. The excess of thionyl chloride was removed by distillation. The product thus obtained was directly used for the next step.
General Procedure for the Synthesis of Schiff Bases11:
A solution of the amine (0.01mol) in absolute ethanol (10mL) was slowly added to a solution of the aldehyde (0.01mol) in absolute ethanol (10mL). To this mixture 2 drops of Conc. sulphuric acid was added and was refluxed for 3hrs. It was cooled to room temperature and the precipitate formed was collected by filtration then washed several times with cold ethanol and recrystallized from hot ethanol.
General Procedure for the Synthesis of Azetidinones12:
A mixture of Schiff base (0.002mol) and triethyl amine (0.004mol) was dissolved in 1,4-dioxane (50ml), cooled and stirred. To this well-stirred cooled solution 2-oxo4-methyl -2H-chromen-7-yloxy acyl chloride (0.004 mmol) was added drop wise within a period of 20 min. The reaction mixture was then stirred for an additional 3 hrs and left at room temperature for 48hrs. The resultant mixture was concentrated, cooled and poured into ice cold water, filtered and then dried. The product thus obtained was recrystallised using ethanol.
3. Scheme:
Fig 1: Synthetic scheme
Table 1: Compounds
Compound |
R1 |
R2 |
6a |
4-chloro |
4-chloro |
6b |
3-chloro-4-flouro |
4-chloro |
6c |
3-chloro-4-flouro |
3-nitro |
6d |
4-flouro |
2-hydroxy |
RESULTS:
Table 2: Docking results of synthesized compounds
Compound |
Glide score |
Compound 6a |
-5.62496 |
Compound 6b |
-5.77844 |
Compound 6c |
-6.84789 |
Compound 6d |
-8.29082 |
Table 3: Physicochemical parameters of synthesized compounds
Compound |
Molecular Weight |
Melting Point |
%Yield |
Colour |
TLC System (benzene: ethyl acetate) |
6a |
466.3 |
1450 C |
65w/w |
Violet crystals |
9:1 |
6b |
484.3 |
1480 C |
72w/w |
Light brown crystals |
9:1 |
6c |
494.85 |
1700 C |
78w/w |
Dark brown crystals |
9:1 |
6d |
431.4 |
1520 C |
61%w/w |
Brown crystals |
9:1 |
Cytotoxicity Study13,14:
MTT assay is the validated best method for screening cytotoxicity. The synthesized compound which showed highest dock score was evaluated for the activity at different concentrations 50-250μg/ml. The drug was administered as a solution in DMSO and a DMSO control was also used. The drug showed good cytotoxic behavior and could be considered being potent cytotoxic agent. (Table 4), (figure 2).
Table 4: Percentage Cell viability of compound 6d
Sample Concentration (µg/ml) |
MTT assay % viability |
Control |
97.25% |
50 |
80.15% |
100 |
65.23% |
150 |
52.12% |
200 |
40.02% |
250 |
36.25% |
Figure 2: Graph showing cell viability
DISCUSSION:
A preliminary drug design was initially carried out with Schrodinger software where the designed ligands were docked with protein kinase C θ (1XJD) and their docking score is shown in Table no.1. The docking score revealed that out of all the compounds docked (6a) showed highest affinity to bind with the protein (1XJD). The compound which showed highest dock score were taken for wet lab synthesis and invitro anticancer studies. Purity of the compounds was routinely checked by single spot TLC and melting points. IR Spectrum of each analogue showed characteristic peaks for the functional group present in it. The characteristic peaks at 1440-1490 of C-N, 1600-1680 of C=O of coumarin and 1700-1780 of C=O due to β lactam confirm the presence of both the nucleus. The presence of characteristic peak of protons in δ ppm found in 1H NMR spectra of selected 2-azetidinone analogues substantiates the formation of the designed compound.
CONCLUSION:
This research work was focused on the rational approach in design and development of azetidinone containing coumarin derivatives as novel anticancer drugs. These analogues also showed good binding with cancer molecular target, which was proved from the docking studies. The derivative which showed highest dock scores have shown good cytotoxic effects on Hela cancer cell lines. Hence we consider these derivatives may be future leads for anti cancer therapy. The above results establish the fact that azetidinone benzopyrans can be a rich source for exploitation. Therefore in search of new generation of active compounds, it may be worthwhile to explore the possibility in this area by fusing and substituting different moieties and thus increase the potency. All the synthesized compounds can be further explored for structural modifications to improve their activity so that they can be converted to prospective drugs.
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Received on 05.12.2019 Modified on 27.03.2020
Accepted on 07.05.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(12):6182-6185.
DOI: 10.5958/0974-360X.2020.01078.1