Design, synthesis and docking studies of flavokawain B type chalcones and their cytotoxic effects on MCF-7 and MDA-MB-231 cell lines

Article abstract of DOI:10.3390/molecules23030616

Flavokawain B (1) is a natural chalcone extracted from the roots of Piper methysticum, and has been proven to be a potential cytotoxic compound. Using the partial structure of flavokawain B (FKB), about 23 analogs have been synthesized. Among them, compounds 8, 13 and 23 were found in new FKB derivatives. All compounds were evaluated for their cytotoxic properties against two breast cancer cell lines, MCF-7 and MDA-MB-231, thus establishing the structure-activity relationship. The FKB derivatives 16 (IC₅₀ = 6.50 ± 0.40 and 4.12 ± 0.20 μg/mL), 15 (IC₅₀ = 5.50 ± 0.35 and 6.50 ± 1.40 μg/mL) and 13 (IC₅₀ = 7.12 ± 0.80 and 4.04 ± 0.30 μg/mL) exhibited potential cytotoxic effects on the MCF-7 and MDA-MB-231 cell lines. However, the methoxy group substituted in position three and four in compound 2 (IC₅₀ = 8.90 ± 0.60 and 6.80 ± 0.35 μg/mL) and 22 (IC₅₀ = 8.80 ± 0.35 and 14.16 ± 1.10 μg/mL) exhibited good cytotoxicity. The lead compound FKB (1) showed potential cytotoxicity (IC₅₀ = 7.70 ± 0.30 and 5.90 ± 0.30 μg/mL) against two proposed breast cancer cell lines. It is evident that the FKB skeleton is unique for anticancer agents, additionally, the presence of halogens (Cl and F) in position 2 and 3 also improved the cytotoxicity in FKB series. These findings could help to improve the future drug discovery process to treat breast cancer. A molecular dynamics study of active compounds revealed stable interactions within the active site of Janus kinase. The structures of all compounds were determined by ¹H-NMR, EI-MS, IR and UV and X-ray crystallographic spectroscopy techniques.

Full text of DOI:10.3390/molecules23030616

molecules
Article
Design, Synthesis and Docking Studies of
Flavokawain B Type Chalcones and Their Cytotoxic
Effects on MCF-7 and MDA-MB-231 Cell Lines
Addila Abu Bakar 1, Muhammad Nadeem Akhtar 1,*, Norlaily Mohd Ali 2, Swee Keong Yeap 3,
Ching Kheng Quah 4, Wan-Sin Loh 4, Noorjahan Banu Alitheen 5,*, Seema Zareen 1,
Zaheer Ul-Haq 6 and Syed Adnan Ali Shah 7
ID
ID
1
Faculty of Industrial Sciences & Technology, University Malaysia Pahang, Lebuhraya Tun Razak,
Kuantan 26300, Malaysia; addilaabubakar@gmail.com (A.A.B.); seema@ump.edu.my (S.Z.)
Faculty of Medicine and Health Sciences, University Tunku Abdul Rahman, Sungai Long 43400, Malaysia;
norlailyma@gmail.com
Chine-ASEAN College of Marine Sciences, Xiamen University Malaysia, Jalan Sunsuria, Bandar Sunsuria,
Sepang 43900, Malaysia; skyeap2005@gmail.com
X-ray Crystallography Unit, School of Physics, University Sains Malaysia, Penang 11800, Malaysia;
ck.quah@hotmail.com (C.K.Q.); wansin_loh@live.com (W.-S.L.)
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science,
University Putra Malaysia, Serdang, Selangor Darul Ehsan 43400, Malaysia
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi 75270, Pakistan; zaheer.qasmi@iccs.edu
Research Institute of Natural Products for Drug Discovery, Faculty of Pharmacy, University Teknologi
MARA, Puncak Alam Campus, Bandar Puncak Alam 42300, Malaysia; benzene301@yahoo.com
Correspondence: nadeemupm@gmail.com (M.N.A.); noorjahan@upm.edu.my (N.B.A.);
Tel.: +60-9-549-2393 (M.N.A.); Fax: +60-9-549-2766 (M.N.A.)
2
3
4
5
6
7
*
Received: 26 January 2018; Accepted: 21 February 2018; Published: 8 March 2018
Abstract: Flavokawain B (
has been proven to be a potential cytotoxic compound. Using the partial structure of flavokawain B
(FKB), about 23 analogs have been synthesized. Among them, compounds 13 and 23 were found in
1) is a natural chalcone extracted from the roots of Piper methysticum, and
8
,
new FKB derivatives. All compounds were evaluated for their cytotoxic properties against two breast
cancer cell lines, MCF-7 and MDA-MB-231, thus establishing the structure–activity relationship.
The FKB derivatives 16 (IC50 = 6.50
±
0.40 and 4.12
0.80 and 4.04
±
0.20
0.30
µ
g/mL), 15 (IC50 = 5.50
±
0.35 and
6.50 1.40 g/mL) and 13 (IC50 = 7.12
±
µ
±
±
µg/mL) exhibited potential cytotoxic
effects on the MCF-7 and MDA-MB-231 cell lines. However, the methoxy group substituted in
position three and four in compound
= 8.80 0.35 and 14.16 1.10 g/mL) exhibited good cytotoxicity. The lead compound FKB (
showed potential cytotoxicity (IC50 = 7.70 0.30 and 5.90 0.30 g/mL) against two proposed
2
(IC50 = 8.90
±
0.60 and 6.80
±
0.35 µg/mL) and 22 (IC50
±
±
µ
1)
±
±
µ
breast cancer cell lines. It is evident that the FKB skeleton is unique for anticancer agents, additionally,
the presence of halogens (Cl and F) in position 2 and 3 also improved the cytotoxicity in FKB
series. These findings could help to improve the future drug discovery process to treat breast cancer.
A molecular dynamics study of active compounds revealed stable interactions within the active site
of Janus kinase. The structures of all compounds were determined by 1H-NMR, EI-MS, IR and UV
and X-ray crystallographic spectroscopy techniques.
Keywords: chalcone synthesis; breast cancer cell lines; SARs; anti-cancer; flavokawain B derivatives
Molecules 2018, 23, 616; doi:10.3390/molecules23030616
www.mdpi.com/journal/molecules

Molecules 2018, 23, 616
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1. Introduction
Breast cancer (BC) is a complex disease, composed of several subtype receptors both at the
molecular and clinical level. BC is still the leading causes of death globally in women [ ]. Chalcones
are -unsaturated carbonyl compounds with two aromatic rings (ring A and B), conjugated double
bonds and a completely delocalized -electron system [ ]. Chalcones and their related analogues
display several pharmacological properties, such as anticancer [ 10], anti-inflammatory [11 14],
antimalarial [15 16], antileishmanial [17 18], antimicrobial [19], antifungal [20], antioxidant [21 23
and anticonvulsant activities [24]. Chalcone derivatives show acetylcholinesterase, tyrosinase [25 26],
1–3
α,β
π
4,5
6–
–
,
,
– ]
,
and monoamine oxidase (MAO) [27] enzyme inhibitory activities. Chalcones have gained the strong
attention from scientists searching for new pharmacological active analogs.
Flavokawain B (FKB) (
methysticum [28], which exhibited strong cytotoxicity against various cancer cell lines [29
In this study, we synthesized 23 FKB analogs by manipulating ring B with the various substituted
1), is a main bioactive chalcone isolated from the roots of Piper
–
31].
1–
benzaldehydes. The main purpose when designing this proposal was to observe the effects of ring B in
the breast cancer cell lines and to establish the structure–activity relationship, to eventually help future
drug discovery. Previously, we investigated the antinociceptive, cytotoxic and anti-inflammatory
activities of FKB (1) in vitro and in vivo [32–36].
2. Results and Discussion
2.1. Chemistry
Flavokawain B derivatives (1–23) were synthesized by Claisen–Schmidt condensation reaction
using the template of FKB (1) (Scheme 1). FKB comprises ring A (20-hydroxy-40,60-dimethoxyacetophenone)
and B (benzaldehyde). Among FKB derivatives, three chalcones (E)-3-(2,5-dimethoxyphenyl)-1-
(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (
8
), (E)-3-(2-fluorophenyl)-1-(20-hydroxy-40,60
0
-dimethoxyphenyl)prop-2-en-1-one (13) and (E)-3-(2-bromo-3-hydroxy-4-methoxyphenyl)-1-(2 -hydroxy
0
0
-4 ,6 -dimethoxyphenyl)prop-2-en-1-one (23) were found to be new FKB analogs (Figure 1). Previously,
compound 13 was synthesized by Bandgar and used as an intermediate for the preparation of
nitrogen-containing chalcones, however, the data were not published [37]. The structure of FKB
derivatives (
characteristic signals appeared in 1H-NMR at
were assigned to and protons, respectively. The presence of carbonyl groups was supported by the
signals at
191–193.45 in 13C-NMR and 1625–1634 cm−1 in the IR spectrum, respectively. The complete
assignments and spectral data of compounds 13 and 23 are given in Table 1, while the structure of
compounds 7 and 9 was confirmed by the single X-ray crystallographic technique (Figure 2).
1
–
23) were characterized by detailed spectroscopic techniques. The presence of
δ
7.66–7.84 (J = 15–16 Hz) and 7.86–8.0 (J = 15–16 Hz)
α
β
δ
8,
Scheme 1. Synthesis of flavokawain B derivatives 1–23.

Molecules 2018, 23, 616
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1
Table 1. H- (600 MHz) and 13C- (150 MHz) NMR data for compound 8, 13 and 23.
0
8
13
23
Carbon
1H (δ)
Multiplicity
13C (δ)
1H (δ)
Multiplicity
13C (δ)
1H (δ)
Multiplicity
13C (δ)
0
1
2
3
4
5
6
-
-
-
-
106.89
162.36
94.38
165.98
91.59
165.98
124.18
153.65
113.65
113.58
153.16
118.10
128.59
137.83
56.62
56.58
55.95
-
-
-
-
-
106.38
162.57
93.81
168.42
91.29
166.37
124.40
134.87
116.29
131.28
129.73
130.27
123.73
160.83
55.78
55.60
-
-
-
-
-
106.25
161.84
93.86
165.57
91.06
165.55
127.05
113.28
140.87
149.86
110.95
118.85
127.05
144.09
56.15
55.63
-
0
0
6.16
-
6.13
-
-
-
7.06
7.03
-
7.22
7.84
7.86
3.84
3.89
3.82
-
(d, J = 2.22 Hz, 1H)
6.11
-
5.96
-
-
-
7.11
7.18
7.59
7.35
7.86
8.00
3.84
3.91
-
(d, J = 2.40 Hz, 1H)
6.00
-
5.88
-
-
-
-
(d, J = 2.50 Hz, 1H)
0
-
-
-
0
(d, J = 2.46 Hz, 1H)
(d, J = 2.34 Hz, 1H)
(d, J = 2.35 Hz, 1H)
0
-
-
-
-
-
-
-
-
-
-
1
2
3
4
5
(d, J = 9.06 Hz, 1H)
(d, J = 9.06 Hz, 1H)
(m, 1H)
(td, J = 7.56 Hz, 7.50 Hz, 1H)
(td, J = 7.56 Hz, 7.68 Hz, 1H)
(m, 1H)
(d, J = 15.78 Hz, 1H, H-α)
(d, J = 15.78 Hz, 1H, H-β)
-
-
-
6.78
7.20
7.67
8.02
3.75
3.85
-
3.81
-
-
(d, J = 8.55 Hz, 1H)
(d, J = 8.60 Hz, 1H)
(d, J = 15.45 Hz, 1H, H-α)
(d, J = 15.45 Hz, 1H, H-β)
6
α
(brs, J = 2.76 Hz, 1H)
(d, J = 15.80 Hz, 1H, H-α)
(d, J = 15.80 Hz, 1H, H-β)
β
0
0
OCH3 (C4 )
OCH3 (C6 )
OCH3 (C2)
OCH3 (C4)
OCH3 (C5)
(s, 3H)
(s, 3H)
(s, 3H)
-
(s, 3H)
(s, 1H)
-
(s, 3H)
(s, 3H)
-
-
-
(s, 1H)
-
(s, 3H)
(s, 3H)
-
-
-
-
-
-
(s, 3H)
56.22
-
-
3.77
13.46
-
56.16
-
193.03
-
(s, 1H)
-
0
OH (C2 )
C=O
14.25
-
192.62
-
191.86

Molecules 2018, 23, 616
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Figure 1. The structure of compounds 8, 13 and 23.
7
9
Figure 2. ORTEP diagram of compound 7 and 9.
2.2. Structure–Activity Relationships
FKB derivatives 23 were screened against two breast cancer cell lines MCF-7 (human estrogen
receptor positive breast cancer cells) and MDA-MB-231 (human estrogen receptor negative) breast
cancer cell lines). FKB ( ) still possessed greater cytotoxicity (IC50 = 7.70 0.30 and 5.90 0.30 g/mL)
compared to the majority of FKB derivatives. Of the 23 tested compounds, thirteen FKB derivatives
recorded higher IC50 values than FKB ( ). On the other hand, FKB derivatives 13 15 16
and 22 were recorded as having potential cytotoxic effects on both MCF-7 and MDA-MB-231 cells,
compared to FKB ( ), see Table 2. Among the FKB derivatives, compounds 13 15 and 16 showed
slightly better cytotoxicity than FKB (1) and flavokawain A (FKA) (2) for MCF-7 cells.
Among the FKB derivatives with an IC50 value lower than 30 g/mL, only compound
1–
1
±
±
µ
1
2
–
4,
7,
9,
,
,
1
,
µ
3 and
22 showed higher sensitivity to MCF-7 cells than MDA-MB-231, indicating that the modification of
the FKB skeleton helps the selectivity between these two cell lines. In short, the FKB derivatives
FKB-2F (13), FKB-3Cl (15) and FKB-2Cl (16) exhibited greater sensitivity to both tested cell lines,
warranting further evaluation of their antitumor effects. It is well established that the anticancer
properties of chalcones are due to the presence of
tested chalcones, the 20 hydroxyl group in ring A is essential for the cytotoxicity, as has been studied
against several biological assays, predominantly its anticancer properties [38 39]. The presence of
electron-withdrawing and electron-donating groups effects the -unsaturated system [38 40],
α,β-unsaturated ketone moieties. Among the
,
α
,
β
,
which eventually effects the cytotoxicity. Generally, the electron-donating group in ring A improves
the cytotoxic properties, as it affects the acidity of the 20 hydroxyl group in ring A, while the
electron-donating group in ring B, as in compound
at 30 g/mL (Table 2), which possibly due to the effects of the electron-donating group on the
-unsaturated system. Since the -unsaturated system becomes more nucleophile and the Michael
5, 6, 8 and 10, did not improve the IC50 values
µ
α,
β
α,β

Molecules 2018, 23, 616
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receptor or protein cannot bind effectively thus activity decreases [38]. However, compounds
4
and
, which
α,β-unsaturated ketone. In contrast, compounds with electron-withdrawing
7
showed slightly better activity due to the selective position of the methoxy groups at
contributed less to the
3, 4 and 2, 3
groups in ring B possessing 2Cl (16), 3Cl (15) and 2F (13) exhibited better cytotoxicity, see Table 2.
The presence of the electron-withdrawing group in ring B, especially at position 2 and 3, as in 13 (2F),
15 (3Cl) and 16 (2Cl), contributed more to the enhancement of cytotoxicity against breast cancer cell
lines (38, 39). While the presence of electron-withdrawing groups in compounds 13
the electrons from the -unsaturated ketone make it more electrophilic, so the receptor (nucleophile)
possibly creates a strong interaction with the compound. While the electrons with the donating group,
such as methoxy groups—especially at position 3, 4 or 3, 5 and 2, 4, 6, as in compounds 10 11
cause rich in nucleophilicity and resonate the -unsaturated carbonyl [41], eventually, the structure
, 15 and 16 pulling
α,β
5
,
6,
8,
,
,
α,β
become less effective and binding or the interaction with the Michael receptor (nucleophile), and the
compounds reduce the cytotoxic effects [38,39].
Table 2. IC50 values (µg/mL) of FKB derivatives after 72 h treatment.
Compounds Substituted Groups
IC50 Values (µg/mL)
Compounds
R1
R2
R3
R4
R5
MCF-7
MDA-MB-231
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
H
H
H
CH3O
CH3O
CH3O
H
CH3O
H
H
H
CH3O
F
H
H
Cl
H
H
H
H
H
OH
Br
H
H
H
CH3O
H
H
CH3O
H
CH3O
CH3O
H
H
H
H
Cl
H
H
H
H
CH3O
SCH3
H
CH3O
CH3O
CH3O
H
H
H
CH3
H
H
H
H
H
H
H
H
H
CH3O
H
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
7.70 ± 0.30
8.90 ± 0.60
12.30 ± 1.40
25.00 ± 1.80
>30
5.90 ± 0.30
6.80 ± 0.45
18.10 ± 1.10
21.10 ± 1.20
>30
>30
>30
9.70 ± 0.70
8.30 ± 0.56
>30
>30
8.43 ± 0.40
>30
7.22 ± 0.70
>30
>30
>30
>30
9.50 ± 0.60
4.04 ± 0.30
>30
5.50 ± 0.40
4.12 ± 0.20
>30
7.12 ± 0.80
F
H
H
Cl
>30
5.50 ± 0.35
6.50 ± 0.40
>30
Br
OH
H
N(CH3)2
H
OCH3
>30
>30
>30
CH3O
NO2
H
H
OH
H
H
H
H
27.00 ± 1.50
17.10 ± 2.15
20.5 ± 1.60
14.16 ± 1.10
>30
H
H
Br
H
13.30 ± 3.10
>30
6.50 ± 0.35
>30
H
2.3. Computational Studies
The potential FKB derivatives
1, 13, 15 and 16 were subjected to rigid receptor docking, and
the resulting poses were analyzed visually. The crystal structure of JAK2 in complex with a potent
quinoxaline inhibitor, PDB: 3KRR, was downloaded in MOE and subjected to protein preparation.
The results of a PASS prediction highlight the probability of the activity of these compounds as
a caspase-3 stimulant, JAK2 expression inhibitor, and apoptosis agonist, as shown in Table S1.
Caspase-3 belongs to the cystine–aspartic-acid protease family of proteins [42], known to play
a fundamental role in the execution phase of cell death in apoptosis [43]. The activators of caspase-3,
such as MX-2060 [12
of cancers.
,44], Smac [45] and Leachianone A [46], induce apoptosis in various types
The high Pa values of the compounds (0.892–0.924) allowed us to conclude that
our newly-synthesized chalcone derivatives may exhibit anticancer effects by activating the
caspase-3-dependent apoptotic pathway. Moreover, the cell-specific response of the chalcone
derivatives can be explained partly by the fact that the human MCF-7 breast carcinoma cells completely
lack caspase-3 expression as a result of a frameshift mutation [43
has contributed to chemotherapeutic resistance in breast cancer [12
protein is targeted by many anti-cancer drugs to produce their response [45
,
47]. This loss-of-function mutation
44]. The Janus kinase-2 (JAK2)
48]. As presented in
,
,

Molecules 2018, 23, 616
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Table S1, our compounds may inhibit JAK2, with Pa values ranging from 0.821 to 0.878. We carried
out a molecular docking simulation to establish the binding mode of the potent FKB derivatives with
IC50 values in single digits, namely 1, 13, 15 and 16, with JAK2 (PDB:3KRR). All the compounds fit the
active site of JAK2 wells, depicted in Figure 3. The docking results revealed that all the compounds
mediated hydrophobic contacts with Val863 and Leu983. The methoxy group of the compounds
interacted via an acceptor–donor hydrogen bonding motif to the Leu932 backbone. A hydrogen bond
with Leu932 provided these compounds with anchorage in the cavity. In the case of compound 13 and
15, an additional hydrogen bond was observed with Leu855 and Asp944, respectively. Analysis of the
average duty cycle revealed that the most stable hydrogen bond pair was Arg938 and O2, and Leu932
and O3 in the 13 and 16 complexes, respectively. The binding profile of 13-JAK2 shows equally high
expression of three different host–guest pairs, Gly856, Leu855 and Lys857. Accurate characterization
of protein–ligand interactions is indispensable for the drug design. Molecular dynamic simulation is
a state-of-art technique that is used to characterize the time-dependent behavior for protein–ligand
complex (See Supplementary Material).
1
13
15
16
Figure 3. The visual presenting the interaction of FKB derivatives (1, 13, 15 and 16) with the ATP
binding site of human JAK2 tyrosine kinase.
3. Materials and Methods
3.1. Chemistry
Melting points were determined on a Fisher–Johns melting apparatus and are uncorrected.
UV spectra were recorded on UV-VIS spectrophotometer model of type Genesys 10s and expressed in
nm. FT-IR spectroscopic studies were carried out on a FTIR spectrophotometer 1000, model Perkin

Molecules 2018, 23, 616
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Elmer, at a room temperature of 25 ◦C. KBr pellets were dried in an oven and scanned for calibration
1
purposes. H-NMR spectra of compounds were recorded on a Bruker Ascend TM 600 MHz machine.
The chemical shifts (
δ) are presented with reference to CDCl3 (δ: 7.25) and tetramethylsilane (TMS)
(
δ
: 0.00) as the internal reference. Electron-spray ionization mass spectra in positive mode (ESI-MS)
data were recorded on a Bruker Esquire 3000+ spectrometer. Column chromatography purifications
were carried out on a Silica Gel 60 (Merck, 70–230 mesh). The purity of compounds was checked by
thin-layer chromatography (TLC) and 1H-NMR. All the chemicals were purchased from Sigma-Aldrich
(Saint Louis, MO, USA). Other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd.,
(Shanghai, China).
3.2. Docking Analysis Method
To rationalize the anti-cancer potential of FKB derivatives, the online web Prediction of Activity
Spectra for Substances (PASS) server was used to predict the biological activity of potential FKB
derivatives [49,50]. The information on the compounds was uploaded with the value of probability
of activity (Pa) set to 0.7. The results were then analyzed critically to understand the molecular basis
for the anti-cancer activity of the compounds. Molecular docking simulation of selected inhibitors
against JAK2 was carried out using MOE [51]. The crystal structure of JAK2 in complex with a potent
quinoxaline inhibitor, PDB:3KRR [52], was downloaded in MOE. The derivatives with potential IC50
values (Table 2.) for compound 1, 13, 15 and 16 were subjected to rigid receptor docking, and the
resulting poses were analyzed visually. All the graphics were rendered using MOE and Chimera [53].
To study the dynamic behavior of the proposed ligand–protein complex, we have performed in a short
production run of 10 ns using AMBER 14 [54]. The resulting trajectories were analyzed using the
CPPTRAJ module in AMBERTOOLS 16 [55,56].
3.3. Synthesis of Flavokawain B derivatives
KOH (20% w/v aqueous solution) was added to a stirred solution of the appropriate acetophenone
(1.0 eq) and a substituted benzaldehyde (1.25 eq.) in ethanol, and the mixture was stirred at room
temperature for 48–72 h. The reaction mixture was cooled to 0 ◦C (ice-water bath) and acidified with
HCl (10% v/v aqueous solution). In most cases, yellow precipitate was formed, filtered and washed
with 10% aqueous HCl solution. In the cases where an orange-yellow layer was formed, the mixture
was extracted with CH2Cl2 or ethyl acetate, the extracts was dried over Na2SO4 anhydrous and the
solvent was evaporated and crystalized with methanol or ethanol.
3.4. Characterization Data
The data of flavokawain B (
1
) and flavokawain A (
2
) have been published previously by us [26
,
57].
(E)-1-(20-Hydroxy-40,60-dimetho◦xyphenyl)-3-(4-(methylthio)phenyl)prop-2-en-1-one (
3). Orange crystal
(78.3% yield); m.p.: 129–131 C; UV-Vis (CHCl3) λ
max: 371–372 nm; IR (KBr, cm−1): 3469 (O-H),
2981–3014 (Ar C-H stretch), 1619 (C=O), 1548–1584 (C=C-C=O), 1431–1432 (Ar C=C), 1155–1218
(C-O) [36].
0
0
0
◦
(E)-3-(2,3-Dimethoxyphenyl)-1-(2 -hydroxy-4 ,6 -dimethoxyphenyl)prop-2-en-1-one (4). Yellow crystal (83.4%
yield); m.p.: 121–123 C (lit. [58] 120–122 C); UV-Vis (MeOH)λmax: 335–337 nm; IR (KBr, cm−1):
◦
3458 (O-H), 2839–2985 (Ar C-H stretch), 1627 (C=O), 1556–15810 (C=C-C=O), 1350–1442 (C=C),
1213–1268 (C-O); 1H-NMR (600 MHz, CDCl3)
δ: 14.34 (s, 1H, OH, C-2 ), 8.08 (d, 1H, H-β, J = 15.78 Hz),
7.96 (d, 1H, H-α, J = 15.78 Hz), 7.24 (d, 1H, H-6, J = 7.74 Hz), 7.08 (t, 1H, H-5, J = 8.04, 7.98 Hz),
6.95 (d, 1H, H-4, J = 8.04 Hz), 6.11 (d, 1H, H-30, J = 2.10 Hz), 5.96 (d, 1H, H-50, J = 2.10 Hz),
3.91 (s, 3H, OCH3, C-60), 3.90 (s, 3H, OCH3, C-2), 3.89 (s, 3H, OCH3, C-3), 3.84 (s, 3H, OCH3, C-40);
13C-NMR (150 MHz, CDCl3) : 192.93 (C=O), 168.40 (C-2), 166.18 (C-60), 162.55 (C-40), 153.24 (C-3),
δ
148.88 (C-1), 137.19 (C-20), 129.79 (C-5), 128.95 (C-6), 124.12 (C-4), 119.80 (C-50), 113.76 (C-30),

Molecules 2018, 23, 616
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106.47 (C-10), 93.70 (C-
β), 91.20 (C-
α
), 61.31 (OCH3), 55.92 (OCH3), 55.84 (OCH3), 55.58 (OCH3);
EI-MS: m/z = 344 (60), 329 (7), 313 (100), 207 (30), 181 (24), 180 (87), 164 (70), 152 (37), 149 (55), 137 (51),
121 (52), 91 (25), 77 (21), 69 (13); (Molecular formula C19H20O6).
(E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (
6
). Orange crystal
max: 386 nm; IR (KBr,
cm−1): 3464 (O-H), 2941–3010 (Ar C-H stretch), 1618 (C=O), 1414–1545 (C=C), 1122–1217 (C-O);
◦
◦
(82.8% yield); m.p.: 158–159 C (lit. [59] 151–153 C); UV-Vis (MeOH):
λ
0
1H-NMR (600 MHz, CDCl3)
δ; 14.76 (s, 1H, OH, C-2 ), 8.32 (d, 1H, H-β, J = 15.78 Hz), 8.25 (d, 1H, H-α,
J = 15.78 Hz), 6.13 (s, 2H, H-3 and H-5), 6.09 (d, 1H, H-30, J = 2.40 Hz), 5.94 (d, 1H, H-50, J = 2.40 Hz),
3.90 (s, 3H, OCH3, C-6 and C-60), 3.89 (s, 3H, OCH3, C-2), 3.85 (s, 3H, OCH3, C-40), 3.82 (s, 3H, OCH3,
C-4); EI-MS: m/z = 374 (2), 344 (44), 329 (8), 313 (100), 281 (10), 207 (50), 181 (25), 180 (54), 164 (44),
152 (21), 149 (37), 137 (37), 121 (33), 105 (12), 91 (26), 77 (23), 69 (15); (Molecular formula C20H22O7).
(E)-3-(2,5-Dimethoxyphenyl)-1-(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (
8). EI-MS: m/z = 344
(78), 327 (33), 282 (10), 207 (68), 191 (44), 181 (27), 164 (62), 152 (14), 151 (100), 137 (19); (Molecular
formula C19H20O6).
(E)-3-(2-Fluorophenyl)-1-(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (13). EI-MS: m/z = 302 (100),
301 (100), 285 (19), 282 (45), 207 (100), 181 (100), 166 (28), 152 (24), 149 (28), 137 (67), 121 (44), 101 (88),
95 (51), 69 (52); (Molecular formula C17H15FO4).
0
0
0
(E)-3-(4-Fluorophenyl)-1-(2 -hydroxy-4 ,6 -dimethoxyphenyl)prop-2-en-1-one (14). Yellow flat crystal (80.7%
yield); m.p.: 144–145 C (lit. [17] 140–141 C); UV-Vis (MeOH) λ
max: 342–343 nm; IR (KBr, cm−1):
◦
◦
3460 (O-H), 2854–3118 (Ar C-H stretch), 1632 (C=O), 1573–15920 (C=C-C=O), 1345–1507 (C=C),
1115–1216 (C-O); 1H-NMR (600 MHz, CDCl3)
δ: 14.28 (s, 1H, OH, C-2 ), 7.82 (d, 1H, H-β, J = 15.60 Hz),
7.74 (d, 1H, H-
α
, J = 015.60 Hz), 7.59 (m, 1H, H-2), 7.58 (m, 1H, H-6), 7.10 (m, 1H, H-3), 7.09 (m, 1H,
0
0
H-5), 6.11 (d, 1H, H-3 , J = 2.40 Hz), 5.96 (d, 1H, H-5 , J = 2.40 Hz), 3.92 (s, 3H, OCH3, C-6 ), 3.84 (s, 3H,
OCH3, C-40); GC-MS: m/z = 302 (84), 301 (100), 285 (12), 274 (14), 207 (99), 181 (41), 153 (12), 137 (23),
121 (13), 101 (17), 69 (11); (Molecular formula C17H15FO4).
(E)-3-(3-Chlorophenyl)-◦1-(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (15). Yellow solid (83.1%
◦
yield); m.p.: 105–107 C (lit. [60] 104–106 C); UV-Vis (MeOH) λ
max: 338–339 nm; IR (KBr, cm−1):
3460 (O-H), 2945–3014 (Ar C-H stretch), 1634 (C=O), 1567–15850 (C=C-C=O), 1342–1443 (C=C),
1213–1215 (C-O); 1H-NMR (600 MHz, CDCl3)
δ: 14.19 (s, 1H, OH, C-2 ), 7.86 (d, 1H, H-β, J = 15.60 Hz),
7.68 (d, 1H, H-
(d, 1H, H-30, J = 2.34 Hz), 5.97 (d, 1H, H-50, J = 2.34 Hz), 3.93 (s, 3H, OCH3, C-60), 3.85 (s, 3H, OCH3,
C-40); 13C-NMR (150 MHz, CDCl3) : 192.26 (C=O), 168.47 (C-20), 166.46 (C-40), 162.50 (C-60), 140.45
(C- ),
α, J = 15.60 Hz), 7.57 (brs, 1H, H-2), 7.46 (m, 1H, H-5) 7.35 (m, 2H, H-4 and H-6), 6.11
δ
β
), 137.45 (C-1), 134.83 (C-3), 130.12 (C-2), 129.84 (C-4), 128.86 (C-5), 127.88 (C-6), 126.62 (C-α
106.26 (C-10), 93.81 (C-30), 91.33 (C-50), 55.95 (OCH3), 55.65 (OCH3); EI-MS: m/z = 317 (1), 314 (90),
297 (10), 283 (68), 267 (7), 207 (100), 181 (65), 167 (16), 152 (19), 137 (27), 121 (22); (Molecular formula
C17H15ClO4).
(E)-3-(2-Chlorophenyl)-◦1-(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (16). Yellow crystal (80.7%
yield); m.p.: 132–134 C (lit. [18] 135–136 C); UV-Vis (MeOH) λ
max: 341–342 nm; IR (KBr, cm−1):
◦
3448 (O-H), 2957–3132 (Ar C-H stetch), 1632 (C=O), 1556–1585 (C=C-C=O), 1338–1435 (C=C),
1
1110–1219 (C-O), 745–748 (C-Cl); H-NMR (600 MHz, CDCl3)
δ
: 14.24 (s, 1H, OH, C-20), 8.12 (d,
1H, H-β, J = 15.60 Hz), 7.85 (d, 1H, H-α, J = 15.60 Hz), 7.67 (m, 1H, H-6), 7.40 (m, 1H, H-3), 7.28 (m, 2H,
H-4 and H-5), 6.08 (d, 1H, H-30, J = 2.34 Hz), 5.93 (d, 1H, H-50, J = 2.40 Hz), 3.88 (s, 3H, OCH3, C-60),
3.81 (s, 3H, OCH3, C-40); EI-MS: m/z = 318 (30), 301 (4), 283 (70), 267 (7), 207 (100), 181 (32), 165 (6),
152 (6), 137 (12), 101 (12), 69 (7); (Molecular formula C17H15ClO4).

Molecules 2018, 23, 616
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(E)-3-(4-Chlorophenyl)-◦1-(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (17). Yellow crystal (84.3%
yield); m.p.: 171–173 C (lit. [61] 173–175 C); UV-Vis (MeOH) λ
max: 342–343 nm; IR (KBr, cm−1):
◦
3420 (O-H), 2981–3018 (Ar C-H stretch), 1630 (C=O), 1488–1591 (C=C), 1217–1219 (C-O), 820–822 (C-Cl);
0
1H-NMR (600 MHz, CDCl3)
δ: 14.24 (s, 1H, OH, C-2 ), 7.85 (d, 1H, H-β, J = 15.60 Hz), 7.72 (d, 1H, H-α,
J = 15.60 Hz), 7.53 (d, 1H, H-2, J = 8.46 Hz), 7.52 (d, 1H, H-6, J = 8.46 Hz), 7.38 (d, 1H, H-3, J = 8.46 Hz),
7.37 (d, 1H, H-5, J = 8.46 Hz), 6.11 (d, 1H, H-30, J = 2.40 Hz), 5.96 (d, 1H, H-50, J = 2.40 Hz), 3.92 (s,
3H, OCH3, C-60), 3.84 (s, 3H, OCH3, C-40); EI-MS: m/z = 318 (57), 301 (7), 283 (2), 207 (100), 181 (34),
165 (13), 152 (19), 137 (25), 111 (6), 102 (20); (Molecular formula C17H15ClO4).
0
0
0
(E)-3-(4-Bromophenyl)-1-(2 -hydroxy-4 ,6 -dimethoxyphenyl)prop-2-en-1-one (18). Yellow flat crystal (80.5%
yield); m.p.: 165.8–167.5 C (lit. [17] 150–151 C); UV-Vis (MeOH) λ
max: 346 nm; IR (KBr, cm−1):
◦
◦
3447 (O-H), 2949–3010 (Ar C-H stretch), 1633 (C=O), 1568–1590 (C=C-C=O), 1338–1488 (C=C),
1
1215–1218 (C-O); H-NMR (500 MHz, CDCl3)
δ
: 7.85 (d, 1H, H-
β, J = 15.50 Hz), 7.68 (d, 1H, H-
α
,
J = 15.50 Hz), 7.53 (d, 1H, H-2, J = 8.50 Hz), 7.51 (d, 1H, H-6, J = 8.50 Hz), 7.45 (d, 1H, H-3, J = 8.50 Hz),
7.43 (d, 1H, H-5, J = 8.50 Hz), 6.09 (d, 1H, H-30, J = 2.00 Hz), 5.95 (d, 1H, H-50, J = 2.50 Hz), 3.90 (s, 3H,
OCH3, C-60), 3.83 (s, 3H, OCH3, C-40); 13C-NMR (150 MHz, CDCl3) : 192.31 (C=O), 168.45 (C-20),
δ
0
0
166.40 (C-4 ), 162.48 (C-6 ), 140.78 (C-
128.15 (C-
β), 134.53 (C-1), 132.10 (C-3), 132.10 (C-5), 129.68 (C-2), 129.68 (C-6),
α
), 124.19 (C-4), 106.29 (C-10), 93.85 (C-30), 91.32 (C-50), 55.89 (OCH3), 55.61 (OCH3); EI-MS:
m/z = 363 (48), 362 (48), 346 (5), 281 (5), 209 (16), 207 (100), 181 (25), 152 (9); (Molecular formula
C17H15BrO4).
(E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one (20). Yellow cotton (82.6%
◦
◦
yield); m.p.: 169–171 C (lit. [61] 169–170 C); UV-Vis (MeOH)
2851–3092 (Ar C-H stretch), 1638 (C=O), 1583 (C=C-C=O), 1427–1525 (C=C), 1223–1225 (C-O); 1H-NMR
(500 MHz, CDCl3) : 8.56 (brs, 1H, H-2), 8.30 (d, 1H, H-4, J = 8.20 Hz), 8.22 (d, 1H, H-6, J = 7.75 Hz),
λ
max: 336 nm; IR (KBr, cm−1): 3460 (O-H),
δ
8.15 (d, 1H, H-
β
, J = 15.70 Hz), 7.84 (d, 1H, H-
α
, J = 15.70 Hz), 7.78 (t, 1H, H-5, J = 7.95, 8.00 Hz), 6.17 (d,
0
0
0
0
1H, H-3 , J = 2.35 Hz), 6.14 (d, 1H, H-5 , J = 2.35 Hz), 4.04 (s, 3H, OCH3, C-6 ), 3.91 (s, 3H, OCH3, C-4 );
EI-MS: m/z = 329 (33), 301 (5), 281 (6), 254 (5), 207 (100), 181 (25), 152 (5), 95 (5), 73 (8); (Molecular
formula C17H15NO6).
(E)-3-(5-Bromo-2-hydroxyphenyl)-1-(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (22). Yellow crystal
(76.2% yield); m.p.: 166–168 ◦C (lit. [61] 177–178 ◦C); UV-Vis (MeOH) max: 359 nm; IR (KBr, cm−1):
λ
3435 (O-H), 2945–2994 (Ar C-H stretch), 1623 (C=O), 1419–1590 (C=C), 1220–1221 (C-O); 1H-NMR
: 14.19 (s, 1H, OH, C-20), 8.12 (d, 1H, H-
(600 MHz, CDCl3) , J = 15.70 Hz), 8.01 (d, 1H, H-α,
δ
β
J = 15.70 Hz), 7.78 (brs, 1H, H-6), 7.39 (d, 1H, H-4, J = 8.70 Hz), 6.97 (d, 1H, H-3, J = 8.64 Hz), 6.13 (brs,
1H, H-30), 6.12 (brs, 1H, H-50), 3.98 (s, 3H, OCH3, C-60), 3.88 (s, 3H, OCH3, C-40); EI-MS: m/z = 379 (1),
364 (14), 333 (5), 305 (3), 281 (9), 251 (3), 227 (9), 214 (17), 207 (60), 191 (10), 142 (13), 115 (8), 95 (10),
73 (14); (Molecular formula C17H15BrO5).
(E)-3-(2-Bromo-3-hydroxy-4-methoxyphenyl)-1-(20-hydroxy-40,60-dimethoxyphenyl)prop-2-en-1-one (23). Orange
◦
crystal (85.6% yield); m.p.: 201–203 C; UV-Vis (MeOH) λ
max: 366–369 nm; IR (KBr, cm−1): 3425 (O-H),
2838–3014 (Ar C-H stretch), 1634 (C=O), 1496–1596 (C=C), 1213–1259 (C-O); EI-MS: m/z = 409 (4),
408 (4), 403 (3), 229 (3), 207 (3), 202 (3), 181 (2), 165 (3), 141 (4), 112 (5), 87 (4), 59 (4); (Molecular formula
C18H17BrO6).
3.5. X-ray Crystallographic Analysis
X-ray analysis for all these samples was performed using Bruker APEX II DUO CCD
diffractometer, employing MoK
α radiation (λ = 0.71073 Å) with φ and ω scans, at room
temperature. Data reduction and absorption correction were performed using the SAINT and SADABS
programs [62]. All structures were solved by direct methods and refined by full-matrix, least-squares
techniques on F2 using the SHELXTL software package [63]. Crystallographic data for the reported

Molecules 2018, 23, 616
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structures have been deposited at the Cambridge Crystallographic Data Centre (CCDC) with the CCDC
deposition numbers of 1548733 and 1548734. Copies of available material can be obtained free of charge
on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223–336033 or
e-mail: deposit@ccdc.cam.ac.uk). The ORTEP (Oak Ride Thermal Ellipsoid Plot Program) of compound
7 and 9 are shown in Figure 2 and X-ray crystallographic data presented in Table 3.
Table 3. Crystal data and parameters for the structure refinement of 7 and 9.
Compound
7
9
CCDC Number
Molecular Formula
Molecular Weight
Crystal System
Space Group
a (Å)
1548734
C19H20O6
344.35
Triclinic
Pī
8.4560 (17)
8.4790 (17)
12.549 (3)
104.166 (3)
92.063 (3)
106.227 (3)
832.4 (3)
2
1548733
C18H18O5
314.32
Orthorhombic
Pbca
14.447 (3)
7.9755 (15)
26.203 (5)
b (Å)
c (Å)
◦
α ( )
90
90
90
◦
β ( )
◦
γ ( )
V (Å3)
3019.0 (10)
Z
8
−3
1.374
1.383
Dcalc (g cm
)
Crystal dimensions (mm)
0.58 × 0.18 × 0.11
0.55 × 0.27 × 0.14
−1
0.10
0.10
µ (mm
)
Tmin/Tmax
0.7665, 0.9584
28,367
−11→11; −11→11; −17→17
1.7−29.2
4491
0.8489, 0.9495
Reflections measured
Ranges/indices (h, k, l)
14,974
−16→16; −9→9; −30→28
◦
θ limit ( )
1.6−24.6
Unique reflections
Observed reflections (I > 2σ(I))
Parameters
2516
2352
234
1.02
1594
211
1.05
Goodness of fit on F2
R1, wR2 [I ≥ 2σ(I)]
0.055, 0.176
0.056, 0.156
3.6. Anticancer Activity
3.6.1. Sample preparation
Stock samples at 1 mg/mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) were prepared and
kept at 4 ◦C.
3.6.2. MTT Cell Viability Assay
Breast cancer MCF-7 and MDA-MB-231 cells were purchased from the American Type Culture
◦
Collection (ATCC, Manassas, VA, USA) and cultured at 37 C, 5% CO2 and 90% humidity using
RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher
Scientific, USA). The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method cell
viability assay was conducted according to our previous report [64]. In brief, MCF-7 and MDA-MB-231
were seeded overnight in 96-well plates at 8
×
104 cells/well. All compounds were dissolved in
dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) to get a stock solution of 1 mg/mL and
◦
stored at 4 C. Then, the compounds were serially diluted into the seeded cells at concentrations
ranging between 30–0.47
After 72 h of incubation, 20
were further incubated for another 3 hrs. Then, 170
and 100 L of DMSO (Sigma) were added to dissolve the purple crystals. The absorbance was then
µ
g/mL. Cells treated with 3% DMSO (Sigma) were used as a negative control.
L of MTT solution (5 mg/mL) was added into all the wells and the plates
L of solution from all the wells were discarded
µ
µ
µ
recorded using an ELISA plate reader (Biotek Instruments, Winooski, VT, USA) at the wavelength of
570 nm. All samples were tested for triplicates. The percentage of cell viability was calculated using
following formula:
Cell viability (%) = [OD sample at 570 nm/OD negative control at 570 nm] × 100%
(1)

Molecules 2018, 23, 616
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The IC50 value (concentration of compounds inhibited 50% of cell viability) was determined from
the graph of cell viability vs absorbance.
4. Conclusions
Twenty-three FKB derivatives were synthesized by Claisen–Schmidt condensation reaction.
Three FKB derivatives, namely 8, 13 and 23, were found to be new chalcones. The chalcones
with electron-withdrawing and electron-donating substituents had effects on cytotoxicity. The FKB
derivatives with electron-withdrawing groups in ring B, as in 13 (2F), 15 (3Cl) and 16 (2Cl),
exhibited potential cytotoxic effects on breast cancer cell lines, while the presence of the substituted
electron-donating groups, as in
MDA-MB-231 cell lines. The FKB derivatives 16 (IC50 = 6.50
1.40 g/mL) and 13 (IC50 = 7.12
were found to be the most active compounds among the FKB series chalcones. The structure–activity
relationship showed FKB chalcones with different substituents and effects on cytotoxicity. The FKB
skeleton is unique for anticancer agents, additionally, the presence of halogens (Cl and F) in position
4, 5, 9 and 10, showed lower cytotoxic effects on MCF-7 and
±
0.40 and 4.12
±
0.20
0.30
µ
g/mL),
15 (IC50 = 5.52 ± 0.35 and 6.50
±
µ
±
0.80 and 4.04
±
µg/mL)
two and three also improve the cytotoxicity of FKB (1). These findings could help to improve or find
new drugs for future anti-cancer agents.
Supplementary Materials: The following docking study is available online, Figure S1: the graph presenting the
trend of root mean square deviation (Å) of all the four systems in the present study, Figure S2 and Table S1:
the difference in the initial and final coordinates of FKB1-JAK2 complex. The cyan color illustrates the initial pose
(1 ns) while the final pose (10 ns) is presented in magenta. The ligand presented significant deviation from the
initial poses, which explains the abrupt hydrogen bonding profile of the system, Table S1: the selected biological
activities and their probabilities of activity (Pa) and (Pi) as obtained from the PASS online server.
Acknowledgments: We are grateful to the Universiti Malaysia Pahang (www.ump.edu.my), Ministry of Education
Malaysia FRGS [150109] and internal grant RDU 150349 and 150356. The authors thankful to USM for Fundamental
Research Grant Scheme (FRGS) (203/PFIZIK/6711411) and RUPRGS grant (1001/PFIZIK/846076). For the analysis
of HREI-MS, greatly thankful to HEJ Research Institute of Chemistry, University of Karachi, Pakistan.
Author Contributions: Addila Abu Bakar, Muhammad Nadeem Akhtar and Seema Zareen carried out the
literature, design and synthesis of chalcones (synthesis and purification), Swee Keong Yeap, Norlaily Mohd Ali
and Noorjahan Banu Alitheen contributed to the study of cancer cell lines, Ching Kheng Quah and Wan-Sin Loh
contributed to the X-ray analysis of the compounds. Zaheer Ul-Haq and Syed Adnan Ali Shah contributed to the
docking analysis. All the authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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