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*Corresponding Author
Received: 10 October 2016, Accepted: 4 November 2016
Published online: 14 February 2017
Abstract: Three halogen substituted flavonols (2a-2c) have been synthesized via Algar-Flynn-Oyamada (AFO)
under microwave irradiation. The reactions took place in 2 minutes with 72-85% yield. The structures of all
compounds were characterized based on the interpretation of spectroscopic data include UV-Vis, FT-IR, 1H
NMR and HRMS. Molecular docking study was applied to ensure the biological activity of the compounds and
observe the interactions between all compounds and P388 cell line. Based on the docking result, the compounds
have a good potency as anticancer because they take up similar poses and similar binding orientation around the
protein. Higher number of hydrogen bonds and van der Waals interactions between all three ligands with amino
acid residues in the binding site which presumably cause all three compounds become active. In vitro evaluation
of their cytotoxic activity was also performed. The compound 2a, 2b and 2c were proved to have a good
cytotoxic activity against P388 murine leukemia cells with IC50 values of 14.4, 1.23 and 1.32 µM, respectively.
1. Introduction
Cancer is one of the leading causes of death worldwide. In Indonesia, the increased cancer deaths
reported annually started to 1.4% in 1972 to 4.4% in 1992 [1]. The World Health Organization
(WHO) estimated that 7.6 million people died of cancer in 2005 [2] and increased to 8.2 million in
2012 [3]. These figures are very disquieting. Therefore, anticancer discovery researches is currently
very active and growing.
The search for new molecules with the capacity to inhibit tumour initiation as well as tumour
progression is an important contribution to overcome cancer development [4]. Flavonol is one of the
heterocyclic compounds which known to have beneficial effects for health. They might be considered
as potentially protective or therapeutic agents against cancer [5]. Flavonols are commonly synthesized
by reacting 2'-hydroxychalcone analogues with hydrogen peroxide under alkaline conditions via AFO
reaction by stirring method [6,7]. This method usually required a long reaction time with an
unsatisfying yield. Therefore, an alternative method is needed for the workers to accelerate the
reaction rate and improve the yield. The microwave-assisted synthesis has been chosen as an
alternative method. This method has been widely applied to synthesize various heterocyclic
compounds. However, the application of microwave irradiation to synthesize flavonol has not been
widely reported. It was made be interesting to apply the microwave irradiation to synthesize some
flavonol analogues in a shorter time with a better yield.
Thus far, computational approaches can aid in drug design in many various ways. In this study,
we demonstrated molecular docking to ensure the biological activity of halogenated flavonols against
Microwave-Assisted Synthesis, Molecular Docking Study and In Vitro Evaluation of Halogen Substituted Flavonols
Against P388 Murine Leukemia Cells
P388 cell line and to observe the interactions between all three compounds with the cell line. From the
spatial arrangement, contributions of the ligands with amino acid residues of the active site were
determined. In vitro evaluation was also performed to determine the cytotoxic activity of the
compounds.
2. Related Works
Synthesis of flavonols by conventional method usually required a long reaction time, 3 hours to
overnight [8-10] with an unsatisfying yield. Some previously researchers have been reported yield of
40-60 % [8], 22-62 % [9], and 56-72 % [11]. Meanwhile, Britton et al. (2012) have been also reported
average yield less than 50 % [10]. Therefore, in this work, we were interested to apply another
synthesis method (microwave-assisted synthesis) to synthesize the halogen substituted flavonols.
Based on the literature search, this method has been widely applied to synthesize various heterocyclic
compounds, such us flavanone [12,13], flavon [14,15] and pyrazoline [16,17] in a shorter reaction
time and a better yield than a conventional method. Therefore, this method was also expected can
accelerate the reaction rate of flavonols synthesis and improve the yield of reaction.
Based on the previous reports, flavonols were known to have potent as anticancer agents
[4,10,18,19]. The anticancer properties of flavonols were influenced by the type of substituent
attached to the aromatic rings. Halogens were known as the cause of flavonols toxicity. Some of
halogen substituted flavonols have a good activity against several cancer cell lines, such as HCT116
cell line (colon cancer cells) [4] and HL60 cell line (promyelocytic leukemia cells) [5]. However, the
molecular docking studies of their cytotoxic activities have not been reported. Therefore, it was
interesting to synthesize and study the anticancer activity of the compounds. In this study, combined
of molecular docking and in vitro evaluation have been performed to determine the cytotoxic activity
of the halogen substituted flavonols against P388 murine leukemia cells.
Table 1. Molecular docking study and in vitro evaluation of the synthetic compounds
4.2. Discussion
In this work, we have successfully applied the microwave irradiation (180 W) from a
domestic microwave oven to synthesize three halogen substituted flavonols via AFO reaction.
The reactions took place in a shorter time (2 minutes) with a better yield (72-85%). The structures
of all flavonols were characterized based on the interpretation of spectroscopic data include UV-Vis,
FT-IR, 1H NMR and HRMS. All the spectroscopic data agreed with the structures of products that we
expected.
(a) (b)
(c)
Figure 2. Spatial arrangement of the binding site of compound 2a (a) compound 2b (b) and compound 2c
(c). The ligand is shown in stick and the binding site residues are shown in line.
Table 2. Cytotoxic activities of halogen substituted flavonols against various cell lines
IC50 (μM)
Cell lines References
Compound 2a Compound 2b Compound 2c
HCT116 ND* 7.5 6.8 [4]
HL60 10.7 ± 2.9 ND* 102 ± 7 [5]
P388 14.4 1.23 1.32 present work
*ND = not determined
The higher number of the hydrogen bond, may accounts for ligand is more active [24]. In our
case, with combined molecular docking study and in vitro assay suggested that in the presence of
carbonyl group may fill better into the adjunct pockets resulting hydrogen bonding with the relative
residues such as Lys515, Tyr519 and also the presence of van der Waals interaction with the residue
Asp440, which presumably cause all compounds have good cytotoxic activities.
5. Conclusion
We have successfully applied the microwave irradiation from a domestic microwave oven to
accelerate synthesis reaction of three halogen substituted flavonols. Molecular docking study was
performed to ensure the biological activity of the compounds and determine the contributions of
ligand with amino acid residues of the binding site. Combined molecular docking study and in vitro
evaluation suggested that in the presence of carbonyl group may fill better into the adjunct pockets
resulting hydrogen bonding with the relative residues such as Lys515, Tyr519 and also the presence
of van der Waals interaction with the residue Asp440, which presumably cause all three compounds
become active with IC50 values of 14.4, 1.23 and 1.32 µM, respectively. This study suggested that
three compounds 2a, 2b and 2c are promising as potential inhibitors for P388 murine cell line.