Synthesis, anticancer, structural, and computational docking studies of 3-benzylchroman-4-one derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.10.026

A series of 3-Benzylchroman-4-ones were synthesized and screened for anticancer activity by MTT assay. The compounds were evaluated against two cancerous cell lines BT549 (human breast carcinoma), HeLa (human cervical carcinoma), and one noncancerous cell

Full text of DOI:10.1016/j.bmcl.2017.10.026

Accepted Manuscript
Synthesis, anticancer, structural, and computational docking studies of 3-ben-
zylchroman-4-one derivatives
Lalitha Simon, Abdul Ajees Abdul Salam, S. Madan Kumar, T. Shilpa, K.K.
Srinivasan, K. Byrappa
PII:
S0960-894X(17)31011-9
https://doi.org/10.1016/j.bmcl.2017.10.026
BMCL 25355
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 May 2017
12 October 2017
13 October 2017
Please cite this article as: Simon, L., Salam, A.A.A., Madan Kumar, S., Shilpa, T., Srinivasan, K.K., Byrappa, K.,
Synthesis, anticancer, structural, and computational docking studies of 3-benzylchroman-4-one derivatives,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.026
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Synthesis, anticancer, structural, and computational docking studies of
3-benzylchroman-4-one derivatives
Lalitha Simona,†, Abdul Ajees Abdul Salamb,†,*, Madan Kumar Sc, Shilpa Tb, Srinivasan,
K.Kd, Byrappa Ke
aDepartment of Chemistry, Manipal Institute of Technology, Manipal University,
Manipal 576 104, India.
bDepartment of Atomic and Molecular Physics, Manipal University, Manipal 576 104, India.
cPURSE Lab, Mangalagangotri, Mangalore University, Mangalore 574 199, India.
dDepartment of Chemistry, Shri Madhwa Vadiraja Institute of Technology and
Management, Vishwothama Nagar, Bantakal, Udupi - 576 115, India.
eDepartment of Material Science, Mangalagangotri, Mangalore University, Mangalore 574
199, India.
†These authors contributed equally to this study.
*Corresponding Author:
Department of Atomic and Molecular Physics,
Manipal University of Technology
Manipal University, Manipal 576 104,
Karnataka, India.
E-mail: abdul.ajees@manipal.edu
Phone: +91-8147966458.
Keywords: Benzylchroman-4-ones; Cytotoxicity; Single crystal; Molecular docking; MTT
assay
Abstract:
A series of 3-Benzylchroman-4-ones were synthesized and screened for anticancer activity by
MTT assay. The compounds were evaluated against two cancerous cell lines BT549 (human
breast carcinoma), HeLa (human cervical carcinoma), and one noncancerous cell line vero
(normal kidney epithelial cells). 3b was found to be the most active molecule against BT549
cells (IC50 = 20.1 µM) and 3h against HeLa cells (IC50 = 20.45 µM). 3b also exhibited
moderate activity against HeLa cells (IC50 = 42.8 µM). The molecular structures of 3h and 3i
were solved by single crystal X-ray crystallographic technique. Additionally, the molecular
Page 1 of 17

docking studies between the tumour suppressor protein p53 with the lead compound 3h,
which exhibited better anticancer activity against HeLa cells was examined.
Cancer is one of the most widespread and feared diseases in the world today. Cancer arises
from the transformation of healthy cells into tumour cells in a multistage process that
progresses from a pre-cancerous lesion to a malignant tumour. Cancer is the second leading
cause of death globally and was responsible for 8.8 million deaths in 2015. Globally, nearly 1
in 6 deaths is due to cancer (http://www.who.int/mediacentre/factsheets/fs297/en/). Many
cytotoxic drugs suffer from poor selectivity to target cells, leading to a high degree of toxicity
and potentially life-threatening side effects. To design drugs that specifically target cancer
cells is a major challenge. Flavonoids are shown to have anticancer activities.
3-Benzylchroman-4-ones (homoisoflavanones) are oxygen-containing heterocycles with a
sixteen carbon skeleton. They belong to the class of naturally occurring polyphenolic
flavonoids with limited occurrence in nature and are mainly found in families like
Hyacinthaceae, Liliaceae, Agavaceae, Fabaceae, Liliaceae, and Polygonaceae.1, 2 They have
been reported to possess antiinflammatory, antibacterial, antihistaminic, antimutagenic,
antiviral and angioprotective properties besides potent phosphodiesterase inhibition
property.3-5 Homoisoflavonoids, caesalpinianone, and 6-O-methylcaesalpinianone which
exhibited different levels of glutathione S-transferase inhibitory and antifungal activities were
isolated from the ethanolic extract of Caesalpinia bonduc (Fabaceae). Caesalpinanone and 6-
O-methylcaesalpinianone inhibited glutathione S-transferase with an IC50 = 16.5 µM and 17.1
µM respectively.6 Sappanone A, a homoisoflavanone isolated from the heartwood of
Caesalpinia sappan has been known to have antioxidant and anti-inflammatory effects. A
recent study showed that Sappanone A inhibited Cisplatin-induced kidney injury through
activating Nrf2 and inhibiting NF-κB activation.6 Pre-treatment with 5,7-dihydroxy-3-(3-
hydroxy-4-methoxybenzyl)-chroman-4-one inhibited the production of intracellular ROS
induced by UVB irradiation in HaCaT cells. Further analysis revealed a decrease in the level
of MAPK activation and down-regulation of COX2 expression.5,7-dihydroxy-3-(3-hydroxy-
4-methoxybenzyl)-6-methoxychroman-4-one inhibited vascular tube formation and new
vessel growth induced by basic fibroblast growth factor.7 Homoisopogon A (1), isolated from
Ophiopogon japonicus tubers exhibited potent cytotoxicity against human lung
adenocarcinoma LU-1, human epidermoid carcinoma KB, and human melanoma SK-Mel-2
Page 2 of 17

cancer cells with IC50 values ranging from 0.51 to 0.66 µM.8 However, reports on the
anticancer activity of synthetic 3-benzyl-chromone derivatives are scarce. As part of our
ongoing efforts to synthesize new flavonoids and explore their biological activities, we have
synthesized 15 derivatives of 3-benzylchroman-4-one. Their anticancer potential is evaluated
in vitro against two cancerous cell lines BT549 (human breast carcinoma), HeLa (human
cervical carcinoma), and one non-cancerous cell line Vero (normal kidney epithelial cells).
We also carried out the crystal structure of compounds 3h and 3i. Moreover, we performed
molecular docking studies of the tumour-suppressor protein p53 with a 3h molecule, which
shows better cytotoxic activity in HeLa cells.
3-Benzylchroman-4-one derivatives were synthesized as outlined in Scheme 1 and their
chemical structures are summarized. Chalcones were synthesized by condensation of
acetophenones/ methoxy/methyl substituted acetophenones and substituted aldehydes, using
40% w/v alcoholic KOH at room temperature.9 Reduction of chalcones to dihydrochalcones
was carried out using 10% Pd-C and ammonium formate.10 The dihydrochalcones were then
cyclized to the corresponding homoisoflavanones using paraformaldehyde and 50% v/v
aqueous diethylamine.11 The structures of derivatives 3a-o were confirmed using elemental
analysis, NMR spectroscopy and mass spectral analysis. For more details on experimental
and complete NMR, and mass spectra see the supplementary data. Because of their wide
range of pharmacological activity, we synthesized a series of 3-benylchroman-4-ones and
evaluated their cytotoxic potential against two cell lines, BT549, and HeLa.
CHO
OH
OH
(i)
R2
R1
R1
O
O
Acetophenone
1(a-o)
Aldehyde
(ii)
O
OH
R2
R2
(iii)
R1
R1
O
O
2(a-o)
3(a-o)
Page 3 of 17

Compound
R1
R2
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
H
H
H
H
H
4′-Cl
4′-OH
4′-F
4′-OCH3
2′,3′,4′-OCH3
2′,4′-OCH3
3′,4′-OCH3
3′-OH,4′-OCH3
4′-CH3
3j
6-CH3
4′-OCH3
3k
3l
5-OCH3
7-CH3
3′-OH,4′-OCH3
4′-OCH3
3m
3n
3o
7-CH3
7-OCH3
7-OCH3
4′-OH
4′-OH
3′-OH,4′-OCH3
Scheme 1. Synthesis of 3-Benzylchroman-4-one derivatives. Reagents and condition: (i) 40%
w/v alcoholic KOH, rt, 12-36 h; (ii) 10% Pd-C, HCOONH4, MeOH-THF (1:1), reflux, 90
min; (iii) 50% v/v aq. diethylamine, (HCHO)n, EtOH, reflux, 9 h.
The results of the in vitro anticancer activity of compounds 3a-o, against BT549, HeLa, and
Vero cells are listed in Table 1. 3b was found to be the most active molecule against BT549
cells (IC50 = 20.1 µM) and 3h against HeLa cells (IC50 = 20.45 µM). 3b also exhibited
moderate activity against HeLa cells (IC50 = 42.8 µM). The molecule 3b has hydroxyl
substitution at C-4’ of ring C, and 3h has hydroxyl and methoxy substitutions at C’3 at C’4 of
ring C respectively (Fig. 1). The compounds 3b, 3m, and 3n have the same substitution (OH
at C4’) in ring B, but 3m has a methyl group at C7 of ring A, and 3n has a methoxy group at
C7 of ring A. However the introduction of methyl group/methoxy group in ring A did not
enhance the activity, instead slightly reduced it. Comparing the activities of 3d, 3j and 3l we
observed that having OCH3 at C4’ of ring B and no substitution in ring A or OCH3 at C4’ of
ring C and methyl function at C-6 of ring A led to decreased anticancer activity (IC50 > 100
µM). However, the compound 3l with OCH3 at C4’ of ring C and methyl function at C7 of
ring A showed moderate activity with IC50 = 42.12 µM, 68.32 µM against BT549 and HeLa
cells respectively.
Page 4 of 17

Table 1
Anticancer activities of 3a-o. The values are mean SEM of three independent triplicates.
IC50 was determined by nonlinear regression using Graph Pad Prism v 5.03, CA, USA.
IC50 (µM)
Compound
BT 549
HeLa
Vero
3a
3b
3c
3d
3e
3f
69.30 1.2 61.9 4.1 >200
20.1 0.9 42.8 2.7 >200
>200
>100
>100
>200 >200
>100 >200
>100 >200
61.90 5.8 38.26 1.3 >200
>100 >100 >200
3g
3h
3i
>100 20.45 0.8 >200
42.16 1.6 48.90 1.2 >200
>100 76.3 2.5 >200
3j
3k
3l
>200
>200 >200
42.12 4.8 68.32 6.5 >200
28.7 07 43.01 4.0 >200
24.3 3.2 50.38 1.8 >200
30.6 1.4 50.12 1.1 >200
3m
3n
3o
For derivatives bearing OH at C3’ and OCH3 at C4’ of ring C (3h, 3k, 3o) 3h showed significant
activity against HeLa cells (IC50 = 20.45 µM). Furthermore, 3o having a methoxy function at C7
of ring A showed moderate activity against both cell lines (IC50 = 39.4µM BT549, IC50
=
50.12µM HeLa). However, a methoxy function at C5 of ring A (3k) substantially decreased the
activity (IC50 > 200µM). Considering halogen substitution at C4’ of ring C, 3a with chlorine at
C4’ was found to be active (IC50 = 69.3µM BT549, IC50 = 61.9µM HeLa) whereas 3c with
fluorine at C4’ was found to be inactive (IC50 > 200µM). Furthermore, the molecule having
methyl substitution at C4’ of ring C, 3i exhibited moderate anticancer activity against both cell
lines (IC50 = 42.16 µM BT549, IC50 = 48.9µM HeLa). By comparing the activities of 3d, 3e, 3f,
and 3g, we can infer that the number and positions of the methoxy groups in ring C influence the
anticancer activity. 3d has one methoxy at C4’, 3e has three methoxy groups at 2’,3’ and 4’
positions of ring C and 3g has two methoxy groups at 3’ and 4’ positions of ring C (Fig. 1). They
Page 5 of 17

were found to be inactive (IC50 > 100µM), whereas 3f with two methoxy groups at 2’ and 4’
positions of ring C was found to be moderately active (IC50 = 61.90 µM) against BT549, (IC50 =
38.26 µM) against HeLa. As can be seen from Table 1 none of the synthesized molecules
displayed cytotoxicity on normal cells (Supplementary Data Figs. S1-S2).
Single crystal X-ray diffraction experiments were carried out for the compounds 3h, and 3i
(Fig. 1) and the thermal ellipsoid diagrams of the molecules are shown in 3h and 3i
(Supplementary Data Fig. S3). Crystal data and structure refinement details are given in
Table 2, and hydrogen bonds are listed in Table 3-4 for 3h and 3i respectively. The atomic
coordinates of all the non-hydrogen atoms with their equivalent isotropic, anisotropic
displacements parameters, the positional and isotropic displacement of the hydrogen atoms,
the bond lengths, bond angles, and torsion angles involving all the atoms for 3h and 3i were
given in Supplementary Data (Table S1-S8). The relevant crystallographic data for the title
compound have been deposited at Cambridge Crystallographic Data Centre (CCDC) with
CCDC Nos. 1552499 (3h) and 1552500 (3i).
Fig. 1. Chemical diagram of 3h (a), and 3i (b)
The pyranone ring has adopted the envelope conformation.12 The ring puckering parameters
q2 = 0.2489 (7) Å, q3 = -0.1631 (7) Å, QT = 0.2976 (6) Å, and ϕ = -126.17(15)° for 3h are
indicative of an envelope conformation. Fig. 2 shows the molecular packing features of 3h. In
the crystal, the molecules are interlinked through strong O3-H3O…O3 [symmetry ½-x, ½+y,
½-z), O3-H3O…O4 [symmetry ½-x, ½+y, ½-z) hydrogen bonds (Table 3, Fig. 2a). Also two
weak C-H…O interactions (Fig. 2a) and C5’-H5’…π (C5-C10) (Fig. 2b) interactions with
H…Cg – 2.94 Å [symmetry code 1-x, 1-y, -z] play a role in stabilizing the molecules. There
are no π-π stacking interactions present in the structure.
Page 6 of 17

Table 2
Crystal data and structure refinement for 3h and 3i.
Identification code
Empirical formula
Formula weight
Temperature
3h
3i
C17H16O4
284.30
C17H16O2
252.30
293(2) K
0.71075 Å
Monoclinic
P 21/n
293(2) K
0.71075 Å
Monoclinic
P 21/n
Wavelength
Crystal system
Space group
Unit cell dimensions
a = 8.581(9) Å
b = 6.484(6) Å
c = 25.29(3) Å
β = 98.28(2) °
a = 4.6459(8) Å
b = 26.802(4) Å
c = 10.9485(19) Å
β = 101.876(17) °
3
3
Volume
1392(3) Å
4
1334.1(4) Å
4
Z
3
3
Density (calculated)
Absorption coefficient
F(000)
1.356 Mg/m
1.256 Mg/m
-1
-1
0.096 mm
600
0.081 mm
536
3
3
Crystal size
Theta range for data
collection
0.3 X 0.25 X 0.19 mm
3.167 to 25.324°.
0.28 X 0.20 X 0.18 mm
2.434 to 26.369°.
Index ranges
-10<=h<=8, -4<=k<=7, -
26<=l<=30
-5<=h<=5, -33<=k<=33, -
13<=l<=13
Reflections collected
Independent reflections
Completeness to theta =
25.242°
4929
14066
2493 [R(int) = 0.0381]
98.3 %
2721 [R(int) = 0.1420]
99.9 %
Refinement method
Full-matrix least-squares on Full-matrix least-squares on
2
2
F
F
Data / restraints / parameters 2493 / 0 / 207
2721 / 0 / 174
2
Goodness-of-fit on F
1.064
0.983
Final R indices[I>2sigma(I)] R1 = 0.0690, wR2 = 0.1402
R1 = 0.0863, wR2 = 0.2388
R1 = 0.1616, wR2 = 0.3192
0.005(5)
R indices (all data)
Extinction coefficient
Largest diff. peak and hole
CCDC
R1 = 0.1253, wR2 = 0.1670
0.0032(14)
-3
-3
0.172 and -0.207 e.Å
1552499
0.281 and -0.250 e.Å
1552500
Page 7 of 17

Fig. 2. The intermolecular interactions of 3h. (a) The O-H…O and (b) C-H…π hydrogen
bonds are represented by dashed lines. For the clarity purpose, C-H…O hydrogen bonds and
hydrogen atoms which are not involved in interactions are not shown. The O-H…O
hydrogen bonds forming a network of interactions along b direction is evident.
Table 3
Hydrogen bonds for 3h [Å and °].
___________________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
___________________________________________________________________________
O(3)-H(3O)...O(3)#1
O(3)-H(3O)...O(4)#1
C(2')-H(2')...O(3)#1
C(2A)-HC2A...O(2)#2
C(5')-H(5')...Cg1#3
0.95(4)
0.95(4)
0.93
2.52(4)
1.97(4)
2.63
3.291(3)
2.808(4)
3.245(4)
3.092(12)
3.788(4)
138(3)
146(4)
124.1
114.1
153.0
0.97
2.57
0.93
2.93
___________________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x+1/2,y+1/2,-z+1/2 #2 x,y-1,z #3 1-x,1-y,-z
Page 8 of 17

The ring B in 3i is not planar, as previously observed.13-15 The carbon C2 has the largest
offset from the plane defined by the adjacent benzene ring A. The ring puckering parameters
of pyronone ring of 3i is q2 = 0.3829 (1) Å, q3 = -0.2640(1) Å, QT = 0.4651(1) Å, and ϕ =-
107.21(1) ° are indicative of an envelope conformation. No classical inter-or intra molecular
hydrogen bonds are observed. The packing of the 3i is stabilized into a three-dimensional
network of weak C(2)-H(2B)...O(2) intermolecular interactions viewed along a direction,
which serve to link inversion-related sheets (Fig. 3 and Table 4), which is well supported by
the earlier investigations.16
Fig. 3. The intermolecular interactions of 3i viewed along a direction. The dashed lines
represent weak C-H…O hydrogen bonds. For the clarity purpose hydrogen atoms which are
not involved in interactions are not shown. The C-H…O hydrogen bonds are forming a
network of interactions along c direction, and overlay of molecules as sheet along b direction
is also shown.
Table 4
Hydrogen bonds for 3i [Å and °].
___________________________________________________________________________
D-H...A
___________________________________________________________________________
C(2)-H(2B)...O(2)#1 0.97 2.52 3.318(4) 139.8
d(D-H)
d(H...A)
d(D...A)
<(DHA)
___________________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
Page 9 of 17

#1 x-1/2,-y+1/2,z+1/2
In both the compounds, the bond lengths, bond angles are usual,17 and all 19 common non-
hydrogen atoms can be superimposed with Root Mean Square deviation (RMSD) of 0.23 Å.
Similarly, the geometric parameters are comparable with the analogue structures of rac-3-(4-
hydorxybenzyl)chroman-4-one,14 and 3-(3,4-dimethoxybenzyl)chroman-4-one15 reported
earlier. It is interesting to note that C2 and C3 atoms and its relevant hydrogen atoms are also
disordered in the earlier reported structure rac-3-(4-hydorxybenzyl)chroman-4-one14 and 2-
benzyl- and (E)-2-benzylidene-1-tetralones.13 Curiously either one of these atoms is involved
in stabilizing the crystal structures through weak C-H…O hydrogen bonding.
Superimposition of the chroman-4-one moiety (except C2, and C3 atoms) the RMSD is
varied from 0.014 Å to 0.019 Å, and C4’ atom of benzyl moiety is spanning within 2.08Å
(Fig. 4a). 3h and 3-(3,4-dimethoxybenzyl)chroman-4-one structures have methoxy moieties
at C4’ position. While 3h methoxy adopts a cis position with C5’-C4’-O4-C12 = 8.05°, 3-
(3,4-dimethoxybenzyl)chroman-4-one has a dihedral angle of 165.12° for the similar bond
with a methyl group is facing opposite each other (Fig. 4b). The conformation of the
molecule can also be explained systematically by the dihedral angle between the planes of the
two benzene rings (Fig. 1, A & C rings).18 In the reported molecules, it is found to be
82.6(2)° for 3h, and 69.6(2)° for 3i. This angle is 59.1(1)° for 3-benzylidene-4-chromanone19,
80.1(1) for rac-3-(4-hydorxybenzyl)chroman-4-one,14 and 87.0(1) for 3-(3,4-
dimethoxybenzyl)chroman-4-one molecules.15 This shows that the substitution at ring C, in
particular at C4’ position plays a significant role in the conformation of the 4-chromanone
molecules (Fig. 4).
Page 10 of 17

Fig. 4. Comparison of 3-benzylidene-4-chromanone molecules. 3h, 3i, 3-benzylidene-4-
chromanone, dimethoxybenzyl, and hydroxybenzyl chromanone molecules are superimposed
and shown in ball and stick. (a) All the atoms (except C2 and C3) in the rings A and B (are
superimposed to identify the effect of various substitutions at ring C. (b) All the common
atoms of 3h (cyan) and dimethoxybenzyl (orange) molecules are superimposed. Both the
molecules have overall similar conformations, and methoxy moieties at C4’ position adopt
opposite orientation.
Chroman-4-one derivatives are more often than not used as an inhibitor for the treatment of
21
cancer.20,
Chromone and chromanone moieties are also frequent targets for various
enzymes such as HIV-1,22 mTOR/PI3Kα,23 mycobacterium P450 DM,24 hepatitis C virus
NS5B polymerase,25 bovine serum albumin (BSA),26 β-site amyloid precursor protein
cleaving enzyme 1 (BACE1),27 COX1/COX2,28 and α-glucosidase.29,30 In particular,
benzylidenechroman-4-ones are tested as inhibitors of human monoamine oxidase isoforms A
and B,31 and its derivatives are identified as inhibitors of anticancer, anti-tubercular and
antioxidant agents.32
On the other hand, the tumour-suppressor protein p53 is one of the primary targets for cancer
therapy.33-35 p53 is described as “the guardian of the genome” due to its role in preserving the
genomic information, and it prevents cancer formation. The p53 human protein is a
homotetramer comprising 4 x 393 amino acids and primarily function as a transcription
Page 11 of 17

factor.36 The p53 protein is also responsible for repairing cellular DNA, and p53 mutants are
common and occur in almost 50% of all cancers. Mutations in p53, in particular at DNA
binding domain destabilize the activity of p53 and leads to a narrow opening in its surface.
Several mutations such as Y220C, V143A, D245S, R248Q, R249S, F270L, R273H, and
R282W are reported as responsible destabilizing the function of the protein thus cause 30% -
40% of cancer mutation functions. Any small molecule that binds to these positions may
reverse the stucture function relationship of this protein. Hietanen et al. showed that small
molecules could restore the function of p53 in the nucleus of HeLa, CaSki, and SiHa cells,
which leads to induction of the apoptotic death of the cells.37 Recently Joerger et al. have
proposed several small-molecule stabilizers for open and closed forms of the p53 cancer
mutant Y220C.36 As molecule 3h induced cytotoxicity in HeLa cells, docking study was
carreied out to explore the possibilites of 3-benzylchroman-4-one derivatives as inhibitors of
p53 at the Y220 active site of p53. In the docking model of p53-3h, ligand 3h is bound within
the active site of Y220C of p53 and it is surrounded by the Leu145, Trp146, Val147, Asp148,
Ser149, Thr150, Pro151, Cys220, Glu221, Pro222, Pro223, Asp228, Cys229, and Thr230
amino acids of p53 (Fig. 5a, Supplementary Data Table S9). All the oxygen atoms (except
O1) of 3h molecule make hydrogen bonds with Val147 (N-H…O, 3.29Å), Thr150 (O-H…O,
2.69 Å), and Thr230 (O-H…O, 3.57 Å) of p53. It also makes hydrophobic contacts (within 5
Å) with Lue145, Cys220, Pro222, Pro223, and Cys229 of p53 amino acid residues (Fig. 5b).
The binding affinity of ligand 3h for p53 (-7.0) is calculated, and the molecular interactions
of the final model are compared with the crystal structure of the p53 cancer mutant Y220C
(PDB id: 5AB9) crystallized with 7- ethyl-3-(piperidin-4-yl)-1H-indole (Fig. 5c). The small
molecule bound with p53 structure interact with ten residues of p53. Out of these ten
residues, eight residues were found to have common interactions with p53-3h docking model.
Page 12 of 17

Fig. 5. Molecular interactions of 3h with p53. (a) Surface representation of p53 with 3h is
shown. The amino acids in the active site are shown in stick (cyan) inside the transparent
surface of p53. The 3h molecule is shown in ball and stick model and colored green (carbon).
(b) In Silico docking results of p53 with 3h, and (c) molecular interactions of p53 crystallized
with 7-ethyl-3-(piperidin-4-yl)-1H-indole (right, PDB Id: 5AB9) is shown. The p53 amino
acids within 4.2Å proximity are shown, and the hydrogen bonds are depicted in dotted lines
and highlighted in a green ball and stick models. The hydrophobic contacts are shown in an
arc with spokes radiating towards the ligand atom in contact. Common amino acids
interacting with 3h and 7-ethyl-3-(piperidin-4-yl)-1H-indole are highlighted within the circle.
Recent studies on bio-inspired catechol synthesis showed that catechol derivatives are useful
building blocks for amino acids and pharmaceuticals.38-41 The 3h compound has a 4-
chromanone moiety connected with guaiacol moiety bridged through CH2. Guaiacol is a
universal substrate for all peroxidases. A simple search of Protein Data Bank yield five
structures bound with quaiacol (PDB id: 4QOQ – Bacillus pumilus catalase; PDB ids: 4A78
and 4A6Z – cytochrome c peroxidase; PDB id: 4G05 - Pleurotus eryngii versatile peroxidase;
and PDB id: 3HT9 – T4 lysozyme). In the case of Bacillus pumilus catalase structure,
guaiacol moiety stabilized the protein structure by reducing the disorder in the terminal
residues, and guaiacol exhibited the highest occupancy than catechol. The methyl group of
guaiacol moiety in 3h is located all the way inside of the binding pocket of p53 and surround
by the critical amino acids Trp146 (4.79 Å), Val147 (3.61 Å), and Cys220 (4.22 Å). It is
interesting to note that Cys220 p53 mutant is one of the potential therapeutic agents and
approximately 75000 tumours annually worldwide are found to have this common mutation.
This mutation destabilizes the protein by 4 kcal/mol and could potentially be a target by the
drug molecules.42
From the X-ray crystallographic structures, it is evident that benzyl moiety undergoes
conformational changes based on the substitutions and molecular interactions. The 3h
molecule has three rotatable bonds in between two phyenyl rings, and it may be possible that
either the amino acids of p53 or 3h molecule may reorient and come closer than the currently
proposed model and may make better contacts with Cys220 of p53 protein. As we discussed
earlier, the docking model of 3h-p53 forms three hydrogen bonds, and five hydrophobic
contacts studies, thus the ligand 3h may play a significant role with the receptor protein p53.
Though 3h molecule is different from the various small molecules proposed by Joerger et
Page 13 of 17

al.,33, 36 the interactions of 3h bear a resemblance to all these molecules co-crystallized with
p53. All amino acids (Leu145, Val147, Thr150, Cys220, Pro222, Pro223, and Cys229, and
Thr230) except Pro151 identified in the p53-3h interface (Fig. 5b) plausibly play a significant
role in cytotoxicity of the molecule. As we discussed earlier, p53 is a potential target for
anticancer immunotherapy, and our docking results provide an eviedence that 3-
benzylchroman-4-one derivatives may be poteintal inhibitors for Y220C p53.
In summary, a series of fifteen 3-benzylchroman-4-one derivatives were synthesized and
characterized. 3b (3-(4-Hydroxybenzyl)-chroman-4-one) exhibited maximum cytotoxicity
against BT549 cells (IC50 = 20.1 µM) and 3h (3-(3-Hydroxy-4-Methoxybenzyl)-chroman-4-
one) against HeLa cells (IC50 = 20.45 µM). It was observed that 3-benzylchroman-4-one
derivatives bearing OH substitution at 4’position of ring C displayed better cytotoxicity. X-
ray crystallographic structures of 3h and 3i provided an insight to understand the role of
substitution at benzyl moieties in stabilizing the structures. At the molecular level, the
structures in present study resemble well with the previously known structures of 3-
benzylidene-4-chromanone derivatives, but the crystal packing is different. The docking
studies of 3h revealed its specificity for p53 protein as a small molecule for the Y220C
mutant. These findings suggest that optimizing the structural elements of these new 3-
benzylidene-4-chromanone derivatives may enhance its anticancer properties further, and
provide a class of inhibitors with a plausible use in the treatment of cancer which warrants
future conclusive experiments.
Conflicts of Interest: None
Acknowledgement:
We would like to acknowledge the support of Department of Science and Technology,
Science and Engineering Research Board, India Grant SB/FT/LS-273/2012. Ms. Shilpa
thanks, Manipal University, India for the MU Ph.D. fellowship and we would like to thank
Professor Santhosh Chidangil for his encouragement and support. LS is thankful to Director,
Manipal Institute of Technology and Principal Manipal College of Pharmaceutical Sciences,
Manipal University for providing research facilities.
References
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1.
López, S. N.; Sierra, M. G.; Gattuso, S. J.; Furlán, R. L.; Zacchino, S. A. Phytochemistry 2006,
67, 2152.
2.
3.
Ata, A.; Gale, E. M.; Samarasekera, R. Phytochem. Lett. 2009, 2, 106.
Du Toit, K.; Elgorashi, E. E.; Malan, S. F.; Drewes, S. E.; van Staden, J.; Crouch, N. R.;
Mulholland, D. A. Bioorg. Med. Chem. 2005, 13, 2561.
4. Fusi, F.; Ferrara, A.; Koorbanally, C.; Crouch, N. R.; Mulholland, D. A.; Sgaragli, G. J. Pharm.
Pharmacol. 2008, 60, 489.
5.
Gaspar, A.; Matos, M. J.; Garrido, J.; Uriarte, E.; Borges, F. Chem. Rev. 2013, 114, 4960.
6.
Kang, L.; Zhao, H.; Chen, C.; Zhang, X.; Xu, M.; Duan, H. Int. Immunopharmacol. 2016, 38,
246.
7.
Hur, S.; Lee, Y. S.; Yoo, H.; Yang, J.-H.; Kim, T.-Y. J. Dermatol. Sci. 2010, 59, 163.
8.
Dang, N. H.; Chung, N. D.; Tuan, H. M.; Hiep, N. T.; Dat, N. T. Chem. Pharm. Bull. 2017, 65,
204.
9.
Bandgar, B. P.; Gawande, S. S.; Bodade, R. G.; Totre, J. V.; Khobragade, C. N. Bioorg. Med.
Chem. 2010, 18, 1364.
10.
11.
12.
13.
209.
14.
Ahmed, N.; van Lier, J. E. J. Chem. Res. 2006, 2006, 584.
Jaspal, S.; Grover, S. K. Indian J. Chem., Sect B 2004, 43B, 1782.
Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1354.
Valkonen, A.; Laihia, K.; Kolehmainen, E.; Kauppinen, R.; Perjési, P. Struct. Chem. 2012, 23,
Shalini, S.; Girija, C.; Simon, L.; Srinivasan, K.; Venkatesha, T. Acta Crystallogr., Sect. E: Struct.
Rep. Online 2013, 69, o1011.
15. Shalini, S.; Girija, C.; Simon, L.; Srinivasan, K.; Venkatesha, T.; Jotani, M. Acta Crystallogr.,
Sect. E: Struct. Rep. Online 2013, 69, o241.
16.
Oxford University Press on Demand, 2001.
17. Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.; Orpen, A. G.; Taylor, R. J. Chem. Soc.,
Perkin Trans. 2 1987, 0, S1.
Desiraju, G. R.; Steiner, T. The weak hydrogen bond: in structural chemistry and biology:
18.
19.
Abdul Ajees, A.; Manikandan, S.; Raghunathan, R. Acta Crystallogr., Sect. E 2002, 58, o535.
Katrusiak, A.; Ratajczak-Sitarz, M.; Kałuski, Z.; Orlov, V. Acta Crystallogr., Sect. C: Struct.
Chem. 1987, 43, 103.
20. Seifert, T.; Malo, M.; Kokkola, T.; Engen, K.; Fridén-Saxin, M.; Wallén, E. A.; Lahtela-
Kakkonen, M.; Jarho, E. M.; Luthman, K. J. Med. Chem. 2014, 57, 9870.
21.
22.
Emami, S.; Ghanbarimasir, Z. Eur. J. Med. Chem. 2015, 93, 539.
Ungwitayatorn, J.; Wiwat, C.; Samee, W.; Nunthanavanit, P.; Phosrithong, N. J. Mol. Struct.
2011, 1001, 152.
23.
Zhu, W.; Chen, C.; Sun, C.; Xu, S.; Wu, C.; Lei, F.; Xia, H.; Tu, Q.; Zheng, P. Eur. J. Med. Chem.
2015, 93, 64.
24.
25.
Ayati, A.; Falahati, M.; Irannejad, H.; Emami, S. Daru, J. Pharm. Sci. 2012, 20, 46.
Wei, Y.; Li, J.; Qing, J.; Huang, M.; Wu, M.; Gao, F.; Li, D.; Hong, Z.; Kong, L.; Huang, W. PloS
one 2016, 11, e0148181.
26. Ashok, D.; Madhuri, E. V.; Sarasija, M.; Kanth, S. S.; Vijjulatha, M.; Alaparthi, M. D.; Sagurthi,
S. R. RSC Adv. 2017, 7, 25710.
27.
28.
Seong, S. H.; Ali, M. Y.; Kim, H.-R.; Jung, H. A.; Choi, J. S. Bioorg. Med. Chem. 2017, 25, 3964.
Abolhasani, H.; Dastmalchi, S.; Hamzeh-Mivehroud, M.; Daraei, B.; Zarghi, A. Med. Chem.
Res. 2016, 25, 858.
29.
30.
Takao, K.; Yamashita, M.; Yashiro, A.; Sugita, Y. Chem. Pharm. Bull. 2016, 64, 1203.
Uesawa, Y.; Sakagami, H.; Kagaya, H.; Yamashita, M.; Takao, K.; Sugita, Y. Anticancer Res.
2016, 36, 5803.
31. Desideri, N.; Bolasco, A.; Fioravanti, R.; Proietti Monaco, L.; Orallo, F.; Yáñez, M.; Ortuso, F.;
Alcaro, S. J. Med. Chem. 2011, 54, 2155.
Page 15 of 17

32.
Das, U.; Lorand, T.; Dimmock, S. G.; Perjesi, P.; Dimmock, J. R. J. Enzyme Inhib. Med. Chem.
2015, 30, 259.
33. Joerger, A. C.; Bauer, M. R.; Wilcken, R.; Baud, M. G.; Harbrecht, H.; Exner, T. E.; Boeckler, F.
M.; Spencer, J.; Fersht, A. R. Structure 2015, 23, 2246.
34.
35.
36.
37.
Parrales, A.; Iwakuma, T. Front. Oncol. 2015, 5, 288.
Semenova, E. A.; Nagel, R.; Berns, A. Genes Dev. 2015, 29, 1447.
Joerger, A. C.; Fersht, A. R. Cold Spring Harbor Perspect. Biol. 2010, 2, a000919.
Hietanen, S.; Lain, S.; Krausz, E.; Blattner, C.; Lane, D. P. Proc. Natl. Acad. Sci. U. S. A. 2000,
97, 8501.
38. Wu, Q.; Yan, D.; Chen, Y.; Wang, T.; Xiong, F.; Wei, W.; Lu, Y.; Sun, W.-Y.; Li, J. J.; Zhao, J. Nat.
Commun. 2017, 8, 14227.
39. Xiong, F.; Lu, L.; Sun, T.-Y.; Wu, Q.; Yan, D.; Chen, Y.; Zhang, X.; Wei, W.; Lu, Y.; Sun, W.-Y.
Nat. Commun. 2017, 8, 15912.
40. Chen, Y.; Wang, D.; Duan, P.; Ben, R.; Dai, L.; Shao, X.; Hong, M.; Zhao, J.; Huang, Y. Nat.
Commun. 2014, 5, 4610.
41.
42.
Wu, Q.; Chen, Y.; Yan, D.; Zhang, M.; Lu, Y.; Sun, W.Y.; Zhao, J. Chem. Sci. 2017, 8, 169.
Wiman, K. G. Oncogene 2010, 29, 4245.
Page 16 of 17

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