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ICST 2016

Microwave-Assisted Synthesis, Molecular Docking Study and


In Vitro Evaluation of Halogen Substituted Flavonols Against
P388 Murine Leukemia Cells
Ihsan Ikhtiarudin1, Neni Frimayanti2, Hilwan Y. Teruna2, and Adel Zamri2*
1
Sekolah Tinggi Ilmu Farmasi (STIFAR) Riau, Pekanbaru 28293 Indonesia
2
Jurusan Kimia, FMIPA, Universitas Riau, Pekanbaru 28293, Indonesia

ihsan@lecturer.unri.ac.id, neni.frimayanti@lecturer.unri.ac.id, hyteruna@lecturer.unri.ac.id and


adel.zamri@lecturer.unri.ac.id

*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.

Keywords: microwave-assisted synthesis; halogenated flavonol; molecular docking; cytotoxic assay

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.

3. Material & Methodology


3.1. Material
There are some materials used in this work: hydrogen peroxide 30%, potassium hydroxide,
hydrochloric acid, and some organic solvents, such as ethanol, n-hexane and ethyl acetate, were
produced by Merck. The halogenated chalcones 1a-1c are our laboratory collections that have been
synthesized by modification from previously method [20].
3.2. Method
The synthesis reaction of 2a-2c were performed in a Samsung ME109F domestic microwave
oven. Melting point was determined on a Fisher-Johns apparatus (Fisher Scientific) (uncorr). TLC
Analysis was performed using GF254 (Merck Millipore) under UV Lamp 254/366 nm (Camag TM). UV
spectrum were recorded on GenesysTM 10S UV-Visible spectrophotometer (Thermo Scientific TM),
FT-IR spectra were recorded in KBr powder on a Shimadzu  FT-IR Prestige-21 spectrophotometer
(Shimadzu Corporation), Mass spectral data were recorded on a Waters LCT Premier XE (ESI-TOF)
system in positive mode, 1H-NMR spectral data were recorded on an Agilent  (Agilent Technologies)
at 500 MHz.

Figure 1. Synthesis reaction of halogen substituted flavonols under microwave irradiation

Applied Science and Technology, Vol.1 No.1 2017 http://www.estech.org 376


Microwave-Assisted Synthesis, Molecular Docking Study and In Vitro Evaluation of Halogen Substituted Flavonols
Against P388 Murine Leukemia Cells
Microwave-assisted synthesis of halogen substituted flavonols (2a-2c). As much as 0.5 mmol
1a-1c were respectively dissolved in 5 mL ethanol and 1 mL KOH 3N was added. The mixture was
cooled to 10oC. Then, 0.25 mL H2O2 30% was added to the mixture and irradiated using a domestic
microwave (180 W) for 2 minutes. TLC Analysis was performed every 30 s of reaction. After reaction
was completed, 3-7 mL cold HCl 10% was added dropwise into the mixture to afford the precipitate.
The precipitate was filtered using Whatman 42 filter paper, washed by cold distilled water and n-
hexane and allowed to dry in a desiccator to get pure 2a-2c directly, without any purification.
Molecular docking study. Three ligands and macromolecules (download from PDB data base
www.pdb.org: PDB ID 3DU6) were prepared before the docking process. For the macromolecule was
added polar hydrogen atoms and its non-polar hydrogen atoms were merged. Kollman charges were
assigned and solvation parameters were added to this enzyme molecule. While, for the ligands, non-
polar hydrogen atoms were merged with Gasteiger charges assigned. All rotatable bonds of ligands
were set to be rotatable with 0.375 Å grids spacing around the catalytic triad. Docking was performed
through Genetic Algorithm using Autodock 4.0 software package [21], with a grid box 55, 17, 42
dimensions along x, y, z axes. Upon the completion of the docking process, confirmation with the
lowest energy was chosen. The hydrogen bonding, Van der Waals and any other interactions were
then analyzed using Discovery studio visualizer 3.0 (Accelrys) [22] for better insight.
In vitro evaluation. Cytotoxic assay was determined using MTT assay method [23].
Approximately 3 x 104 cell cm-3 of P388 murine leukemia cells were plated in 96 well culture dishes
and incubated at 37°C in humidified CO 2 incubator for 24 hours. After incubation, various
concentrations in DMSO solvent of the samples were added. Six desirable sample concentrations
were prepared using PBS (phosphoric buffer solution, pH = 7.30-7.65), except control. After 48 hours
incubation, assay was stop by adding MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] and the incubation continue for next 4 h before the addition of MTT-stop
solution containing sodium dodecyl sulphate (SDS), and the incubation continue for next 24 h.
Optical density was measured using microplate reader at 550 nm. IC 50 value obtained from the plotted
graph between percentage live cells compared to control (receiving only PBS and DMSO) against
various concentration of the compound tested.

4. Results and Discussion


4.1. Result
Compound 2-(2-chlorophenyl)-3-hydroxy-4H-chromen-4-one (2a). The compound was
obtained as pink solid in 72% yield; mp: 94-95°C; Rf: 0.36 (n-hexane:EtOAc = 85:15); UV (MeOH),
 (nm): 213, 236, 287, 326; FT-IR (KBr), ῡ (cm-1): 3363, 3062, 1608, 1566, 1134, 756; 1H NMR (500
MHz in CDCl3), δ (ppm), J (Hz): 8.30 (dd, 1H, Kr-5H, J = 8.0, 2.0), 7.71 (dt, 1H, J = 8.0, 2.0), 7.71
(dt, 1H, Kr-7H, J = 8.0, 2.0), 7.67 (dd, 1H, Kr-8H, J = 7.5, 2.0), 7.56 (dd, 1H, Ar-3’H, J = 8.0, 1.0),
7.54 (d, 1H, Ar-6’H, J = 8.5), 7.46 (dt, 1H, Kr-6H, J = 7.5, 2.0), 7.43 (m, 1H, Ar-4’,5’H), 6.56 (s, 1H,
Kr-3OH); HRMS (ES+): m/z [M+H]+ calcd for C15H10O3Cl: 273.0318, found: 273.0323.
Compound 2-(3-bromophenyl)-3-hydroxy-4H-chromen-4-one (2b). The compound was
obtained as pale yellow solid in 73 %; mp: 159-161°C; Rf: 0.46 (n-hexane:EtOAc = 85:15); UV
(MeOH),  (nm): 211, 242, 305, 343; FT-IR (KBr), ῡ (cm-1): 3298, 3082, 1631, 1577, 1130, 624; 1H
NMR (500 MHz in CDCl3), δ (ppm), J (Hz): 8.40 (s, 1H, Ar-2’H), 8.26 (d, 1H, Kr 5H, J = 8.0), 8.23
(d, 1H, Ar-6’H, J = 8.0), 7.73 (t, 1H, Kr-7H, J = 8.0), 7.61 (d, 1H, Kr-8H, J = 8.0), 7.61 (dt, 1H, Ar-
5’H, J = 8.0, 1.0), 7.44 (d, 1H, Ar-4’H, J = 8.0), 7.41 (t, 1H, Kr-6H, J = 8.0), 7.10 (s, 1H, Kr-3OH);
HRMS (ES+): m/z [M+H]+ calcd for C15H10O3Br: 316.9813, found: 316.9810.
Compound 2-(4-chlorophenyl)-3-hydroxy-4H-chromen-4-one (2c). The compound was
obtained as pale yellow solid in 85%; mp: 158-160°C; Rf: 0.48 (n-hexane: EtOAc = 85:15); UV
spectra (MeOH),  (nm): 206, 247, 308, 345; FT-IR (KBr), ῡ (cm-1): 3282, 3116, 1608, 1566, 1111,
759. 1H NMR (500 MHz in CDCl3), δ (ppm), J (Hz): 8.27 (d, 1H, Kr-5H, J = 8.0), 8.23 (d, 2H, Ar-
3’,5’H, J = 9.0), 7.52 (d, 2H, Ar-2’,6’H, J = 8.5), 7.74 (t, 1H, Kr-7H, J = 7.5), 7.60 (d, 1H, Kr-8H, J
= 8.5), 7.52 (d, 1H, Ar-8’H, J = 8.5), 7.44 (t, 1H, Kr-6H, J = 7.5), 7.08 (s, 1H, Kr-3OH). HRMS
(ES+): m/z [M+H]+ calcd for C15H10O3Cl: 273.0318, found: 273.0324.

Applied Science and Technology, Vol.1 No.1 2017 http://www.estech.org 377


Microwave-Assisted Synthesis, Molecular Docking Study and In Vitro Evaluation of Halogen Substituted Flavonols
Against P388 Murine Leukemia Cells
Molecular docking study and in vitro evaluation. The docking result showed the formation of
hydrogen bonds and van der Walls interactions between the ligands and protein. The binding
interactions of compound 2a, 2b, 2c to the binding site and in vitro evaluation for their cytotoxic
activity against P388 murine leukemia cells are presented in Table 1.

Table 1. Molecular docking study and in vitro evaluation of the synthetic compounds

Interactions observed IC50


Compounds
Hydrogen bonds van der Walls interactions (μM)
Between carbonyl and residue
2a Tyr519, between hydroxyl and residue Not observed 14.4
Tyr519 and Lys515
2b Between carbonyl and residue Lys515 Between the ligand and residue Asp440 1.23
Between the ligand and residue Arg511
2c Not observed 1.32
and Lys515

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.

Applied Science and Technology, Vol.1 No.1 2017 http://www.estech.org 378


Microwave-Assisted Synthesis, Molecular Docking Study and In Vitro Evaluation of Halogen Substituted Flavonols
Against P388 Murine Leukemia Cells
In silico docking studies were performed to study the effects of all three compounds against P388
cell line. From the docking results, compound 2a showed to have three hydrogen bonds (green dashed
line). Two hydrogen bonds were performed between hydroxyl of the ligand with residues Tyr519 and
Lys515, respectively. Another hydrogen bond was performed betwen carbonyl of the ligand and
residue Tyr519. Likewise, in the case of compound 2b only one hydrogen bond was observed
between carbonyl of the ligand and residue Lys515. In addition, van der Waals interaction was also
found betwen the ligand and residue Asp440. The best docking poses of compound 2c exhibited van
der Waals interaction between the ligand and residue Arg511 and Lys515, suggesting the importance
of this residue in the formation of the van der Waals specificity pocket. The binding interaction of all
three compounds (i.e. 2a, 2b, 2c) are presented in Figure 2.
In vitro assay was also performed to evaluate the cytotoxic activity of the compounds. All
compounds were tested for their cytotoxic activity against P388 murine leukemia cells. The result
showed that compounds 2a, 2b and 2c have good cytotoxic activity and potentially as anticancer agent
with IC50 value of 14.4, 1.23 and 1.32 µM, respectively. This result was supported by the reports from
previous workers. Some halogen substituted flavonols have been reported potentially as anticancer
agent based on their cytotoxic activity. Compound 2b and 2c were previously tested against HCT116
[4]. Meanwhile, compound 2a and 2c were also previously tested against HL60 cell line [5]. Based on
their works, the halogen substituted flavonols have ability to inhibit cancer cells growth because their
capacity to induce cell cycle arrest and apoptosis. The cytotoxic activity of compound 2a-2c against
various cell lines in previous and present works were presented in Table 2.

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.

Acknowledgement. This research is fully supported by a Research Grant, Contract No.


042.04.2.40087/2015, from Ministry of Research, Technology and Higher Education, Ministry of
Education and Culture, Indonesia.

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Microwave-Assisted Synthesis, Molecular Docking Study and In Vitro Evaluation of Halogen Substituted Flavonols
Against P388 Murine Leukemia Cells
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Against P388 Murine Leukemia Cells
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