Synthesis, inverse docking-assisted identification and in vitro biological characterization of Flavonol-based analogs of fisetin as c-Kit, CDK2 and mTOR inhibitors against melanoma and non-melanoma skin cancers

Article abstract of DOI:10.1016/j.bioorg.2020.104595

Due to hurdles, including resistance, adverse effects, and poor bioavailability, among others linked with existing therapies, there is an urgent unmet need to devise new, safe, and more effective treatment modalities for skin cancers. Herein, a series of flavonol-based derivatives of fisetin, a plant-based flavonoid identified as an anti-tumorigenic agent targeting the mammalian targets of rapamycin (mTOR)-regulated pathways, were synthesized and fully characterized. New potential inhibitors of receptor tyrosine kinases (c-KITs), cyclin-dependent kinase-2 (CDK2), and mTOR, representing attractive therapeutic targets for melanoma and non-melanoma skin cancers (NMSCs) treatment, were identified using inverse-docking, in vitro kinase activity and various cell-based anticancer screening assays. Eleven compounds exhibited significant inhibitory activities greater than the parent molecule against four human skin cancer cell lines, including melanoma (A375 and SK-Mel-28) and NMSCs (A431 and UWBCC1), with IC₅₀ values ranging from 0.12 to < 15 μM. Seven compounds were identified as potentially potent single, dual or multi-kinase c-KITs, CDK2, and mTOR kinase inhibitors after inverse-docking and screening against twelve known cancer targets, followed by kinase activity profiling. Moreover, the potent compound F20, and the multi-kinase F9 and F17 targeted compounds, markedly decreased scratch wound closure, colony formation, and heightened expression levels of key cancer-promoting pathway molecular targets c-Kit, CDK2, and mTOR. In addition, these compounds downregulated Bcl-2 levels and upregulated Bax and cleaved caspase-3/7/8 and PARP levels, thus inducing apoptosis of A375 and A431 cells in a dose-dependent manner. Overall, compounds F20, F9 and F17, were identified as promising c-Kit, CDK2 and mTOR inhibitors, worthy of further investigation as therapeutics, or as adjuvants to standard therapies for the control of melanoma and NMSCs.

Full text of DOI:10.1016/j.bioorg.2020.104595

Bioorganic Chemistry 107 (2021) 104595
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, inverse docking-assisted identification and in vitro biological
characterization of Flavonol-based analogs of fisetin as c-Kit, CDK2 and
mTOR inhibitors against melanoma and non-melanoma skin cancers
,
Tithi Roya, Samuel T. Boatenga, Sergette Banang-Mbeumia 1, Pankaj K. Singhd,
,
Pratik Basneta c, Roxane-Cherille N. Chamcheua, Federico Ladud, Isabel Chauvinc,
,
Vladimir S. Spiegelmane, Ronald A. Hilla, Konstantin G. Kousoulasf g, Bolni Marius Nagaloh,
,
*
Anthony L. Walkerb, Jean Fotiei, Siva Murruc, Mario Sechid, Jean Christopher Chamcheua
a School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana-Monroe, Monroe, LA 71209-0497, USA
b School of Clinical Sciences, College of Pharmacy, University of Louisiana-Monroe, Monroe, LA 71209-0497, USA
c Department of Chemistry, University of Louisiana-Monroe, Monroe, LA 71209-0497, USA
d Department of Chemistry and Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari, Italy
e Department of Pediatrics, Division of Pediatric Hematology/Oncology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey,
Pennsylvania 17033-0850, USA
f Division of Biotechnology and Molecular Medicine, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
g Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
h Division of Hematology and Medical Oncology, Mayo Clinic Hospital, 5777 E Mayo Blvd, Phoenix, AZ 85054, USA
i Department of Chemistry and Physics, Southeastern Louisiana University, SELU, Hammond, LA 70402-0878, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Due to hurdles, including resistance, adverse effects, and poor bioavailability, among others linked with existing
therapies, there is an urgent unmet need to devise new, safe, and more effective treatment modalities for skin
cancers. Herein, a series of flavonol-based derivatives of fisetin, a plant-based flavonoid identified as an anti-
tumorigenic agent targeting the mammalian targets of rapamycin (mTOR)-regulated pathways, were synthe-
sized and fully characterized. New potential inhibitors of receptor tyrosine kinases (c-KITs), cyclin-dependent
kinase-2 (CDK2), and mTOR, representing attractive therapeutic targets for melanoma and non-melanoma
skin cancers (NMSCs) treatment, were identified using inverse-docking, in vitro kinase activity and various
cell-based anticancer screening assays. Eleven compounds exhibited significant inhibitory activities greater than
the parent molecule against four human skin cancer cell lines, including melanoma (A375 and SK-Mel-28) and
Anticancer activity
Apoptosis
Flavonols
Melanoma
Non-melanoma skin cancer
Fisetin-analogs
Inverse docking
Kinase activity
NMSCs (A431 and UWBCC1), with IC50 values ranging from 0.12 to < 15 μM. Seven compounds were identified
as potentially potent single, dual or multi-kinase c-KITs, CDK2, and mTOR kinase inhibitors after inverse-docking
and screening against twelve known cancer targets, followed by kinase activity profiling. Moreover, the potent
compound F20, and the multi-kinase F9 and F17 targeted compounds, markedly decreased scratch wound
closure, colony formation, and heightened expression levels of key cancer-promoting pathway molecular targets
c-Kit, CDK2, and mTOR. In addition, these compounds downregulated Bcl-2 levels and upregulated Bax and
cleaved caspase-3/7/8 and PARP levels, thus inducing apoptosis of A375 and A431 cells in a dose-dependent
Abbreviations: BCA, bicinchoninic acid assay; BSA, bovine serum albumin; FBS, dialyzed fetal calf serum; DMEM, dulbecco’s modified eagle’s medium; DMSO,
dimethyl sulfoxide; DTI, defined trypsin inhibitor; TN, trypsin neutralizing solution; IC50, half maximal inhibitory concentration where 50% cells are dead; EtOAc,
ethyl acetate; FBS, fetal bovine serum; FDA, food and drug administration; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GC–MS, gas chromatography-mass
spectrometry; KOH, potassium hydroxide; LC-MS, liquid chromatography-mass spectrometry; MS, mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide; NMR, nuclear magnetic resonance; SAR, structure activity relationship; SDS, sodium dodecyl sulphate; SDS-PAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; TBS, tris buffered saline; TLC, thin layer chromatography; PBS, phosphate buffered saline.
* Corresponding author at: School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana-Monroe, 1800 Bienville Drive,
Room 362, Monroe, LA 71209-0497, USA.
E-mail address: chamcheu@ulm.edu (J.C. Chamcheu).
1
Current address: School of Nursing and Allied Health Sciences, Louisiana Delta Community College, Monroe, LA 71203, USA.
https://doi.org/10.1016/j.bioorg.2020.104595
Received 24 September 2020; Received in revised form 30 November 2020; Accepted 22 December 2020
Available online 30 December 2020
0045-2068/Published by Elsevier Inc.

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
manner. Overall, compounds F20, F9 and F17, were identified as promising c-Kit, CDK2 and mTOR inhibitors,
worthy of further investigation as therapeutics, or as adjuvants to standard therapies for the control of melanoma
and NMSCs.
1. Introduction
structurally related classes from this family of compounds include fla-
vones, flavonols, flavans, flavanones, flavanols, chalcones and dihy-
Hyper-proliferation and compromised cell death machinery are
universal features of cancer cells, as these impairments functionally
underlie the resistance mounted to apoptotic pointers resulting from
diverse therapeutic regimens [1–3]. In spite of recent advances in cancer
treatment, achieving durable complete responses for systemic metastatic
cancer patients is challenging, as cancer cells, such as melanoma, evade
eradication due to phenotypic complexity and the activation of cancer
cells’ pro-survival machinery, which thwarts apoptotic stimuli and en-
hances tumor development and progression [4,5]. Skin cancers consti-
tute the highest-incidence among all forms of cancers in the United
States [6]. There are two predominant classifications, melanoma and
non-melanoma skin cancers (NMSCs), the latter including basal cell
carcinoma (BCC) and squamous cell carcinoma (SCC). Melanoma is the
most aggressive culprit of the skin cancers, with a rising incidence of
new cases exceeding 7.7% in the U. S. [6,7], in association with a high
number of deaths, while BCC and SCC are the most prevalent forms of
NMSCs, with over 5.3 million yearly cases in the US [6,8,9]. Amongst
other skin-cancer related diseases, the prognosis of malignant melanoma
alone remains dismal despite recent advances in therapeutic strategies
[6,10–12]. In this context, to overcome the urgent treatment hurdles
associated with increased cancer progression and mortality, the identi-
fication of novel, more effective, safe, and (ideally) low-cost synthetic
analogs of natural bioactive compounds, with known anticancer health-
benefits, represents a suitable alternative [13–16]. Flavonoids are
attractive chemotypes for drug discovery due to their molecular di-
versity. These compounds belong to a large family of secondary me-
tabolites from plants, comprised of about 12 classes, harbouring a very
drochalcones, isoflavones, aurones, anthocyanins and anthocyanidins,
and catechins, differing from each other primarily by the degree of
oxidation of the C ring [19]. Flavonoids are widely recognized to exhibit
various pharmacological effects, including antioxidative, anticancer,
cardiovascular ion channel modulator, antiprotozoan and antimalarial
́
́
[13,14,17] and [20–23]. One group member, namely 3,4,7-trihydroxy-
flavonol, also known as fisetin (F0, Fig. 1), is well known to possess a
range of biological and pharmacological activities, including anti-cancer
properties [24–32]. Fisetin has shown efficacy in multiple in vitro and in
vivo preclinical animal models of human cancers, including melanoma,
colon and prostate cancers [14,25,29,33–46]. Fisetin’s induction of
cytotoxicity in metastatic human melanoma cells appears to involve the
activation of both extrinsic and intrinsic apoptotic pathways, at least in
part arising from the inhibition or beneficial modulation of kinases
involved in melanoma growth and progression [28–35]. However, one
of the major limitations to fisetin’s clinical efficacy has been associated
with the presence of the 7-hydroxyl substituent on ring A [47], which is
prone to a rapid extensive first-pass biotransformation, resulting in
biologically less-active and/or less-bioavailable metabolites upon oral
administration [47]. Moreover, the catechol moiety (ring B) is also
susceptible to rapid oxidation, generating a reactive ortho-quinone
metabolite [48–51]. Due to fisetin’s high susceptibility to first-pass
elimination, exacerbated by its relatively poor aqueous solubility
resulting in a limited dissolution absorption rate, this molecule suffers
from compromised oral bioavailability that reduces its therapeutic ef-
ficacy [43,44,46]. Moreover, fisetin’s three hydroxyl moieties may be
responsible for sub-optimal lipophilicity for transdermal or gut wall
absorption and access to intracellular targets [47]. Therefore, it was
deemed imperative to investigate anticancer activity of the fisetin
characteristic phenyl-benzopyran core, built around
a C6-C3-C6
arrangement, with C6 being benzene ring [17,18]. Major
a
R1
R2
R3
H
3C
O
H
O
R2
O
=
=
R1
R3
R4
,
,
=
=
=
=
1
1
:
:
F8
F9
R
R
:
:
R3 H R2
l 58
C
%
( )
*
%
R3 H R2
H
OC
49 62
/
OC
O
(
H
)
3
,
=
=
=
=
*
R2 R3
R2 H R3
54 5
1
r
F10
F11
7
R
R
B
/
%
(
)
3
-
,
=
=
*
36/58
1
R
u
B
t
%
)
(
H
O
H
O
O
3CO
O
=
R2
=
=
=
=
=
=
=
=
=
=
=
*
R2 R3 R4
H
7
2
7
H
8
1
1
1
1
1
1
1
:
:
:
:
:
:
:
F1
F2
F3
F4
F5
F6
F7
R
R
R
R
R
R
/
%
)
(
,
,
=
=
R3 R4
H
68
(
OC
%
)
3
H
O
F12
F13
=
=
SC
R2 R4 H R3
F
65
%
=
=
:
:
r
B
(
)
3
H
3
O
R
R
B
73
%
(
)
-
,
,
=
=
=
l R2 R4 H R3
B
7
4
%
( )
r
'
u
C
t
7
8
%
(
)
'
'
H
O
4
2
,
=
=
=
=
=
R3
F
C
R2 R4
H 58
%
( )
63/65
n
B 55
(
R2
3
1
O
B
'
,
8
=
=
=
*
R3 H R2 R4
F
'
3
R1
R3
R4
C
O
R2
%
H
O
2
(
)
5
'
1
,
=
=
R2 R4 H R3
7
6
R
:
%
6
)
%
A
C
3
,
=
=
=
R3 R4
H
OC
H
82
(
1
R
O
O
F23
R
)
3
H
O
Fi
4
5
se n
t
i
r
F0
B
(
)
O
N
O2
R1
H
O
O
R2
R3
,
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
*
R
R
R
R
R1 R2 R3 R4
H
68 63
:
r
F14
B
B
C
C
/
%
)
(
,
,
,
,
R
R
=
R1 R3 R4 H R2
H
64
%
( )
3
:
:
:
r
F15
F21
F24
OC
H
3C
H
O
H
O
,
,
=
=
l R1 R4 H R2 F R3 OE
t
7
6
%
( )
O
3C
O
,
=
=
R2
H
*
l R1 R3 R4
H
OC
7
1
%
(
)
3
F20
46 52
/
%
)
(
H
O
O
l
C
H
O
,
,
=
=
=
=
=
=
=
,
=
*
:
:
:
r
B
F16
F17
F22
R
R
R
R1 H R2 R3 OCH3 5 /64
7
%
)
O
-
,
=
=
F R1 R3 H R2
l
68
=
=
C
%
:
:
(
)
u
54
%
( )
F18
F19
t
R
R
B
,
=
=
F R1 R2 R3
OC
7
H 5
(
3
n
%
)
B
5
7
%
O
(
)
Fig. 1. List of fisetin analogs designed and synthesized (*% yields from the one-pot reactions).
2

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
analogs, having improved lipophilicity, and in which the above-listed
impediments deriving mainly from the presence of the 7-hydroxy
group on ring A and the 3′,4′-dihydroxy moiety of ring B, are cor-
rected. To achieve that objective, 24 fisetin-based derivatives (Fig. 1)
bearing a wide range of substituents at different positions of ring A and B
of the 2-phenyl-4H-chromenone scaffold were synthesized, and their
antitumor potentials were investigated. All the synthesized compounds
were first tested for their cytotoxicity and antiproliferative efficacy
against four human skin tumor cell lines, including two melanoma and
two non-melanoma lines. In comparison, two normal primary and non-
cancerous skin cell lines were used as controls. The most active com-
pounds were further subjected to an inverse docking protocol to identify
putative molecular targets from among a selection of prospectively and
commonly dysregulated ones in melanoma and NMSCs. Finally, by
employing in-vitro enzymatic, cell culture, immunocytochemical, and
Western-blot assessment assays, the most potent fisetin analogs were
evaluated against the identified target proteins for their inhibitory po-
tential and mechanistic effects.
These 2′-hydroxychalcone intermediates were subsequently con-
verted into flavonols via a base-promoted cyclization, using an aqueous
solution of hydrogen peroxide in the presence of KOH (Scheme 2),
following Algar-Flynn-Oyamada (AFO) reaction conditions [66,67] as a
starting point. However, under these initial conditions, the reaction
failed to produce a good yield at low temperature, prompting a quick
optimization through the variation of reaction parameters including
temperature, solvent, and the equivalents of H2O2 and KOH. The best
yield was obtained in methanol, using 1.5 equivalents of KOH and 6
equivalent of 30% aqueous H2O2 at room temperature. These reaction
conditions appeared to tolerate a number of substituents on both rings A
and B, and produced isolated yields of between 36 and 78% (Scheme 2).
It should be mentioned that a microwave-based, one-pot (two-step)
sequential addition approach through which the chalcone is oxidatively
cyclized in situ was also explored (Scheme 3). Even though reaction
times were much shorter, and the yields comparable to those obtained
with the two-pot approach, the number of unidentified side products
increased, resulting in complex mixtures that complicated the isolation
of the desired product. As such, this latter approach was used only in the
preparation of a few compounds, which could not be as efficiently
prepared using the two-pot approach. In fact, with the exception of F14,
the others compounds, namely F1, F6, F9-11, and F16 were produced in
relatively higher yields with the one-pot approach (Fig. 1). All of the
final products were purified and fully characterized by a combination of
spectroscopic and spectrometric techniques, including 1H NMR, 13C
NMR, IR, GC–MS and HR-ESIMS, with the detailed 1H NMR, 13C NMR
and HR-ESIMS spectra data provided as in supplementary dataset (68).
2. Results and discussion
2.1. Chemistry
The overarching goal for this project was to synthesize and investi-
gate the anticancer/anti-proliferative activities of fisetin (F0) analogs
lacking both the 7-hydroxy group on ring A as well as the 3′,4′-dihy-
droxy moiety on ring B. In the process, it was also set out to investigate
the electronic and steric effects of various groups at different positions of
the 3-hydroxy-2-phenyl-4H-chromen-4-one skeleton on the cytotoxic
activity of the resulting analogs, with a primary goal of modifying their
physicochemical characteristics, and eventually improve their pharma-
cokinetic and pharmacodynamic properties [52,53]. Balancing the lip-
ophilicity of the resulting analogs was also an important parameter in
this design, as it has been shown that replacing the hydroxy groups on
the flavonol moiety with substituents that are less susceptible to
oxidation, increases the bioavailability and reduces the metabolic
lability [54]. As such, a number of substituents, including alkyl, alkoxy,
halogen, trifluoromethyl, and nitro groups were introduced at the 6, 7,
and/or 8 positions on the A-ring, as well as at positions 2′, 3′, 4′, and/or
5′ on the ring B of the core flavonol, resulting in an array of 24 analogs as
illustrated in Fig. 1.
2.2. In vitro assessment of cytotoxic activity
The growth-suppressive and cytotoxic activities of all synthesized
target compounds, (F1-F24), alongside fisetin (F0, the parent flavonol),
and doxorubicin, co-assayed as positive controls, were investigated on
human solid skin cancer cells: two melanoma (A375 and SK-Mel-28) and
two NMSC (A431 and UWBCC1) cell lines, and for comparison, two
normal control cells (melanocytes and HaCaT keratinocytes), using in
vitro MTT and trypan blue dye exclusion assays. The screened anti-
proliferative and cytotoxicity activities were assessed in terms of the
concentration capable of reducing the measured viability of the cell
population to 50% of maximal (expressed as IC50) for the above-listed
six human skin-derived cell lines in culture, as described previously
[38].
The synthesis of flavonols is well defined and documented [55–61].
The target compounds listed in Fig. 1 were synthesized via a two-step
(two-pot) reaction sequence proceeding through the corresponding 2′-
hydroxychalcone intermediate, followed by an oxidative cyclization to
produce the expected flavonol derivatives. To enable a good control over
the reaction conditions, a microwave-assisted approach was utilized to
prepare the chalcone intermediates. Microwave-assisted reactions are
often much faster, cleaner, and thus more likely to produce higher yields
than conventional heating procedures [62,63]. In fact, a microwave-
assisted Claisen-Schmidt condensation [64] of suitably substituted 2′-
hydroxyacetophenones with benzaldehyde derivatives bearing the
appropriate substituents at the desired positions, afforded the corre-
sponding 2′-hydroxychalcones (Scheme 1), which were isolated in good
to excellent yields, and characterized by TLC and GC–MS analyses. It
should be mentioned that all the chalcone intermediates were synthe-
sized using microwave irradiations, except those used in the preparation
of compounds F3, F7, and F19, which were prepared following a pre-
viously reported procedure [65].
The obtained data (summarized in Table 1) indicates that several of
the tested analogs exhibited variable degrees of anti-proliferative ac-
tivities and potency against the human-skin-cancer cell lines as
compared to the control lines. Eleven analogs (F1, F3, F6, F8, F9, F11,
F14, F17, F19, F20 and F21) were more potent than fisetin, with the
most potent compound F20, displaying the lowest IC50 values (0.12 and
0.20 µM against non-melanoma cell lines UWBCC1 and A431, respec-
tively), which represents a 233– and 130-fold dose-advantage over
fisetin, respectively. F20 was also more selective on cancer cells
(UWBCC1 and A431) over normal (HaCaT) cells, with selectivity in-
dexes of about 283 and 170, respectively, as compared to both fisetin
(1.2) and doxorubicin (0.2). In melanoma cells, F20 displayed some
level of potency against A375 cells (IC50 value of 6.3 µM), but was only
marginally potent against SK-Mel-28 cells (IC50 value of 17 µM). F19,
bearing a 6-Cl, 7-CH3 and 4′-OBn, respectively, also exhibited strong
O
O
H
O
.
.
e
1 5 q KOH
R2
R2
R1
,
,
O
O
H
r
H
2O2
t 2h
O
,
H
O
ano
,
R1
KOH Eth
,
l
O
MW 250W 65 o
C
R2
R2
R1
+
R1
-
m n
i
15 20
HO
HO
Scheme 2. Synthesis of substituted flavonols via oxidative cyclization of 2′-
Scheme 1. Microwave assisted synthesis of 2′-hydroxychalcones.
hydroxychalcones at room temperature.
3

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
O
O
Table 1
O
H
.
.
HO
,
,
Cytotoxicity of flavonol analogs F1-F24, parent fisetin and doxorubcine against
human melanoma (A375 and SK-Mel-28), and non-melanoma skin cancer (A431
and UWBCC1) cells relative to normal control HaCaT and Melanocytes.
ano
,
1
KOH Eth
l
65 o
C
R2
,
m n
i
MW 250W 20
O
R2
R1
+
.
,
e
1
v
q
R1
H2O2
2
KOH
(
)
HO
,
MW 150W
Compounds
ID
Cell lines, IC50 values in
μ
M, and (selectivity index)a
Scheme 3. Microwave-assisted one-pot synthesis of substituted flavonol
Melanoma
Control
Non-Melanoma
Control
HaCaT
derivatives.
A375
SK-
Melanocyte
A431
UWBCC1
Mel-
28
activity against both UWBCC1 and A431 cells (IC50s 1.1 and 1.5 µM,
respectively), but with much lower selectivity indices (25 and 19,
respectively).
F1
14 ±
0.80
(1.9)
28 ±
2.3
21 ±
1.0
27 ± 0.90
15 ±
0.04
(2.5)
21 ±
16
12 ± 0.80
38 ±
(3.2)
0.02
F6 (IC50s 8.6 and 6.6 µM), F8 (IC50s 8.3 and 6.7 µM), F9 (IC50s 9.2
and 9.4 µM), F11 (IC50s 4.7 and 9.7 µM) and F14 (IC50s 5.3 and 3.3 µM),
which also displayed some potency against A431 and UWBCC1 cell
lines, but the selectivity was generally poor. Although F6 (IC50 7.0 µM)
and F8 (IC50 8.4 µM) also exhibited noteworthy potency against cells of
the melanoma A375 line, they were essentially inactive against SK-Mel-
28 cells. On the other hand, in melanoma cells, F9, F11, and F14 were
either marginally active against A375 cells or totally inactive against SK-
Mel-28 cells. It is worth noting that while both F6 (4′-CF3) and F8 (4′-Cl)
bear an electron-withdrawing group at the 4′-position on ring B, F14
carries a Br at the position 7 of ring A, with F9 (4′-OCH3) and F11 (4′-t-
Bu) bearing rather an electron-donating group at the 4′ position. Other
analogs that displayed some activity against at least one of the non-
melanoma cell lines include F3 (A431, IC50 9.7 µM), bearing a 4′-
SCF3, F17 (UWBCC1, IC50 8.1 µM), bearing 6-F and 4′-Cl, and F23
(UWBCC1, IC50 9.4 µM), which carries electron-donating groups (OCH3)
at the 2′, 4′ and 5′ positions. Among these, only F3 displayed some ac-
tivity against SK-Mel-28 cells (IC50 of 5.2 µM). Therefore, the respective
selective indexes in all cancer cell-lines (except for F6 in SKMEL-28)
were>1 (1.1–25), indicating a possible low toxicity profile when
administered (though warranting further confirmation) as compared to
doxorubicin.
(1.2)
ND
F2
F3
ND
ND
ND
ND
14 ±
0.20
(2.2)
>100
5.2
31 ± 0.74
9.7 ±
2.3
27 ±
± 1.7
(6.0)
ND
0.70
(2.8)
61 ±
30
F4
F5
F6
ND
ND
ND
ND
ND
95 ±
12
ND
ND
>100
7.0 ±
0.32
(3.7)
88 ±
35
27 ±
4.1
26 ± 1.3
8.6 ±
0.50
6.6 ±
0.79
(4.7)
ND
31 ±
1.9
(1.0)
ND
(3.6)
>100
F7
F8
ND
ND
8.4 ±
0.4
22 ±
3.0
26 ± 3.8
8.3 ±
2.1
6.7 ± 1.5
22 ±
(3.3)
0.20
(3.1)
12 ±
0.9
(1.1)
17 ±
1.0
(2.7)
9.2 ±
0.70
(2.9)
36 ±
0.10
4.7 ±
0.9
F9
26 ± 1.6
9.4 ±
0.30
27 ±
0.30
(2.2)
31 ±
13
(1.5)
ND
(2.9)
F10
F11
ND
11 ± 5.0
ND
11 ±
2.0
18 ±
2.0
26 ± 1.1
9.7 ±
0.60
(2.3)
ND
25 ±
0.78
(2.4)
>100
28 ±
24
(1.4)
ND
(4.7)
>100
>100
F12
F13
ND
ND
ND
ND
In the light of the structure activity relationship (SAR), it was
generally observed that compounds with a methoxy group at any posi-
tion on the A or B ring or on both (F2, F10, F12, F13, F15, F16, F22, F23
and F24), were mostly marginally active or inactive against all the tested
cell lines, except for F9. Though these observations are fairly consistent
with previous reports on substituted flavonols [68–70], it is yet unclear
how the position and the electronic profile of the different substituents
actually affect the activity of these analogs against the tested melanoma
or the NMSC cells. For example, compounds F18 and F19 both have the
same substitution profile on ring A (6-Cl and 7-CH3) and differ merely by
nature of the electron donating substituent (t-Bu and OBn) at position 4′,
but displayed a difference in activity. On the other hand, F7, which bears
a 4′-OBn like F19, but without 6-Cl and 7-CH3 substituents on ring A,
was completely inactive, compared to F19, which was substantially
active against the non-melanoma cells.
ND
ND
F14
15 ±
0.08
(1.4)
>100
79.5
± 9.0
13 ±
1.0
22 ±
24 ± 0.84
5.3 ±
2.1
3.3 ± 1.3
24 ±
4.3
(7.3)
0.20
(4.5)
>100
>100
F15
16
ND
ND
ND
ND
ND
ND
ND
40 ± 0.89
F17
25 ±
32 ± 0.72
10 ±
2.6
8.1 ± 1.2
38 ±
3.4
(4.7)
0.80
(2.5)
>100
(3.8)
24 ±
5.6
F18
F19
ND
ND
ND
ND
13 ±
2.6
19 ±
3.0
27 ± 1.0
1.5 ±
0.30
(19)
1.1 ±
28 ±
0.40 (25)
0.31
(1.7)
6.3 ±
1.0
(1.2)
17 ±
3.0
F20
F21
F22
F23
35 ± 0.23
27 ± 0.10
24 ± 1.6
22 ± 1.0
0.20
± 0.10
(170)
8.2 ±
0.20
(3.2)
13 ±
2.6
0.12 ±
0.11
34 ±
0.60
(5.6)
8.6 ±
0.40
(3.2)
15 ±
1.9
(2.1)
12 ±
3.0
(283)
ND
Given that no clear SAR emerged from this initial series of assays,
more insight into the relationship between the structural patterns and
the anticancer activities of this family of compounds was coveted and
explored through an inverse-docking analysis platform. Other 3-hydrox-
ychromen-4-one derivatives, with potent anticancer properties, have
previously been identified as CDK2 inhibitors [69]; and considering that
various oncogenic pathways involve drug targets, including EGFR, cKit,
26 ±
0.40
(2.3)
ND
11 ± 2.0
23 ±
(2.1)
1.9
(1.6)
13 ±
3.1
(1.8)
12 ±
1.9
19 ±
3.0
9.4 ± 1.4
24 ±
(2.6)
1.4
Akt, MET, MEK1, VEGFR, MAPK, MTOR, PI3K, PIP5K1α, CDK2, and
(1.7)
>100
21 ±
1.0
(1.6)
ND
(2.0)
>100
26 ±
1.0
FGFR, prospectively enabling the design of active agents for the man-
agement of melanoma (14 for a review, [71]), the eleven hits, obtained
from the anti-proliferative assays, were computationally screened on the
above-listed proteins.
F24
ND
ND
ND
cFisetin (F0)
29 ±
4
33 ± 2.7
28 ± 0.20
(1.1)
30 ±
1.1
(1.6)
0.1 ±
0.01
(1.1)
0.23
±
(1.2)
0.37
± 0.02
(0.2)
cDoxorubicin
ND
ND
0.07 ±
Molecular targets involving these pathways are usually associated
with the abnormal cell proliferation, malignant/carcinogenic trans-
formation and cell-cycle aberrations observed in cancer progression
[72–80]. Nonetheless, the development of resistance by human cancer
cells to established chemotherapeutic regimens is often associated with
0.01
0.06
(a): The selectivity index was determined as the ratio of IC50 value in the
respective normal cells (melanocytes or HaCaT keratinocytes) divided by the
IC50 for the corresponding specified cancer (melanoma or non-melanoma) cell
4

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
lines. IC50: compound concentration required to inhibit tumor cell proliferation
(assessed as cell viability) by 50%. cFisetin (the parent flavonoid) and doxoru-
bicin (a well-known anticancer agent) were employed as positive controls. Data
are expressed as the mean ± SD from the dose–response curves of at least five
independent experiments performed in octuplicates. ND: not determined.
compounds bind with the catalytic domain in the same pose as fisetin,
with the carbonyl on the flavone core moiety interacting as a hydrogen
–
bond acceptor, forming a hydrogen bond with Leu83 backbone N H. In
addition, the 3-hydroxy substituent of the flavonol skeleton acts as a
hydrogen bond donor, forming a hydrogen bond with the Glu81 side-
chain carboxyl, while the 2-phenyl group (ring B) is predicted to
mutations affecting the apoptotic machinery or signalling pathways
involving these processes [14]. Moreover, combination therapies (“drug
cocktails”) of agents with multiple and definite molecular mechanisms
of action (MMOAs) are often or would be key to improving outcomes for
patients, ideally by preventing the emergence of resistant cancerous
cells and tumors, often at distant sites, which is particularly common
and problematic with melanoma [72–80]. Consequently, it is expected
that computational screening via an inverse docking analysis of the
eleven hits obtained from the above antiproliferative assays on the listed
proteins could provide key parameters essential for future design of
more potent and metabolically less-labile analogs of fisetin to carry
forward to in vivo assays and other preclinical studies leading to clinical
candidacy.
consistently create π-π interactions with Phe80 throughout all of these
analogs (Fig. 2). Against c-Kit, these compounds are also predicted to
adopt binding poses consistent with that of fisetin, again with the
flavone carbonyl interacting as a hydrogen bond acceptor, consistently
forming a hydrogen bond with the backbone amide nitrogen of Cys673.
The data also predict ring B to maintain a π-π interaction with Phe811
across these analogs. Pertaining to mTOR, it was found that ring B
consistently maintains a interaction with Tyr2225. Overall, the re-
π-π
sults of the computational evaluation revealed that analogs with a single
and small substituent on ring B were able to easily maintain poses
similar to that of fisetin with almost all of the target proteins, whereas a
similar binding pose was not always maintained with analogs bearing
multiple or bulky substituents on ring B.
One interesting finding from in-silico studies was that F20 (the most
potent compound from the MTT assay) was not predicted to conserve a
similar binding pose and orientation as fisetin, which appears to be
primarily due to the presence of a nitro group at position 8 on ring A,
which forces the core scaffold out of the pocket in most of the cases.
Nonetheless, the computed interaction energies for F20 predicted
binding affinity comparable to fisetin’s for mTOR, CDK2, c-Kit and MET.
It is also worth mentioning that although many compounds maintained
key interactions within the catalytic domain of Akt, the obtained Glide
docking scores were low, translating to predicted poor binding affinities
with the protein, and thus, Akt was dropped from further studies. Also,
although VEGFR and MET displayed better docking binding scores with
most of the analogs than with fisetin itself, they were not predicted to
maintain the key H-bonding interactions observed with fisetin. Based on
these observations and taking into account the balance of both the
binding energy (docking score) and essential H-bonds, only CDK2, c-KIT
and mTOR were selected as possible molecular targets for further in-vitro
enzymatic evaluation of the inhibitory potential of these compounds.
2.3. Inverse-docking analysis
All the eleven fisetin derivatives (F1, F3, F6, F8, F9, F11, F14, F17,
F19, F20, and F21) identified via MTT assay, as potent anti-tumor
molecules, were subjected to an exhaustive inverse docking protocol
to identify the putative target for these hit molecules. In this technique, a
molecule is attempted to dock to the ligand-binding domain of proteins
associated with the potential activity. The term “inverse” is used because
the method identifies proteins that can fit a specific ligand, rather than
identifying ligands that fit within a specific protein [81]. For the current
inverse docking protocol, a pool of 12 proteins (EGFR, cKit, Akt, MET,
MEK1, VEGFR, MAPK, MTOR, PI3K, PIP5K1α, CDK2 and FGFR), re-
ported as key molecular targets for the management of melanoma, were
utilized for the exhaustive docking analysis against the eleven hits ob-
tained after MTT assay. The docking protocol was validated by re-
docking the co-crystallized ligand within the catalytic domain of spe-
cific selected target protein. It was observed that the re-docked pose of
co-crystallized ligand retained the binding conformation to the co-
crystallized pose with the RMSD value of<1 Å. Key interactions re-
ported in previous studies as essential for the inhibition of the selected
proteins were considered as screening parameters along with the ob-
tained docking score for each hit against corresponding proteins. For
molecular docking, the 3D structures of the target proteins were selected
from a data bank, using a cross-docking protocol with the available
Protein Data Bank (PDB) number associated with each selected protein
[82–85], also see supplemental dataset (68). The collected data indi-
cated that in the case of EGFR, most of the analogs maintained the key H-
bond interaction with Met769 as seen in PDB1M17 for the 4 anilino-
quinazoline EGFR kinase inhibitor erlotinib (Table 2). However, the
estimated interaction energies (based on Glide docking scores) for the
interaction poses predicted to be optimal within the ꢀ 7.41 to ꢀ 6.05
Kcal/mol range, as compared to ꢀ 8.41 Kcal/mol for fisetin. Against c-
Kit, however, these molecules, when optimally docked per Glide scoring,
maintained the required key interactions with Cys673 alone (F1, F3, F6,
F11, F14 and F20) and/or Glu671 as seen for AZD3229 in PDB 6GQJ,
and showed good docking scores (-11.04 to ꢀ 8.54 Kcal/mol) in com-
parison to fisetin (-11.51 Kcal/mol). Thus, each of these 11 fisetin an-
alogs studied are predicted to undergo stronger binding interactions
with c-Kit than with the EGFR kinase domain (–11.04 to –8.54 Kcal/
mol). The detailed results of docking scores for each of the fisetin ana-
logs interacting with each of the proteins selected for inverse docking
studies are summarized in Table 2, and the 3D interactions diagram of
fisetin and its analogs with the 12 selected proteins are shown in Fig. 2.
Based on the collective analysis, CDK2, c-Kit and mTOR were predicted
as the most likely targets for these analogs among the proteins screened.
The inverse docking analysis with CDK2 revealed that most of the
2.4. In-vitro enzymatic assay: c-KIT, CDK2, and mTOR kinase activity
assays
With the aim of further elucidating the mechanism(s) of the observed
anti-proliferative activities of these title analogs of continued interest,
nine compounds (see below) selected from the initial cell-culture
viability screens were further assessed for their inhibitory activities of
kinases chosen based on the results of the inverse-docking studies, as
discussed in the previous section. The four inverse-docking identified
targets CDK2 (cyclin A, and cyclin E), c-Kit and mTOR are commonly
associated with cancer progression [73–80] as already noted, and thus
represent attractive targets for anticancer drug discovery. Consequently,
F6, F8, F9, F11, F14, F17, F19, F20, and F21, as well as F0 were
evaluated via an in vitro kinase-profiling platform from Reaction Biology
Corporation. Rapamycin, staurosporine and PI-103 (at 1 and 10 μM), as
well as the parent flavonol (fisetin, F0), which has been previously re-
ported to inhibit mTOR/Akt and p70S6K1 as components of its observed
anti-melanoma activity [28,29], were co-assayed as positive control
drugs. For the initial screening, the activities were measured in a 10-
dose IC50 mode for three-fold serial dilutions of the compounds at a
starting concentration of 100 μM. The compounds were re-tested to
exclude false positives whenever inhibition of a target kinase was seen to
be pronounced, whereupon confirmed actives were further evaluated by
kinase activity assay to derive IC50 values from response-vs.-
concentration curves for c-Kit, CDK2/Cyclin A, CDK2/Cyclin E , and
mTOR kinase inhibition, as displayed in detail in the supplementary
report (68).
The obtained results summarized in Table 3 indicate that fisetin and
5

Table 2
Detailed results of inverse docking protocol studies for the selected flavonol analogs.
S.
Target Enzymes
F1
F3
F6
F8
F9
F11
F14
F17
F19
F20
Fisetin (F0)
Specific Co-
crystallized
ligand
No
1.
2.
3.
EGFR
c-Kit
Akt
Docking
ꢀ 7.07
ꢀ 7.17
ꢀ 6.25
ꢀ 7.31
ꢀ 7.31
ꢀ 6.05
ꢀ 7.19
ꢀ 7.41
ꢀ 7.41
ꢀ 7.13
ꢀ 8.46
ꢀ 8.96
Score
H-bond
Met769,
Lys721
Met769
Asp831
Met769,
Gln767
Met769,
Gln767
Met769
Met769
Met769
Met769
Met769
Met769,
Lys721,
Asp831
ꢀ 11.51
Met769
Docking
Score
ꢀ 9.86
ꢀ 10.08
ꢀ 8.54
ꢀ 10.42
ꢀ 10.62
ꢀ 10.48
ꢀ 10.20
ꢀ 10.39
ꢀ 11.04
ꢀ 8.65
ꢀ 8.52
H-bond
Cys673
Cys673
Cys673
Glu671,
Cys673
Glu671,
Cys673
Cys673
Cys673
Glu671,
Cys673
Glu671,
Cys673,
Asp810
ꢀ 5.15
Cys673
Cys673,
Asp810
Glu671, Cys673,
Asp810
Docking
Score
ꢀ 4.46
ꢀ 2.57
ꢀ 4.09
ꢀ 4.55
ꢀ 4.70
ꢀ 3.96
ꢀ 2.79
ꢀ 4.46
ꢀ 2.12
ꢀ 6.26
ꢀ 7.63
H-bond
Glu17, Ile19,
Arg23,
Lys14,
Arg86
Glu17,
Ile19,
Arg23,
Tyr18
ꢀ 7.81
Glu17, Ile19,
Arg23,
Glu17, Ile19,
Arg23,
Glu17, Ile19,
Arg23,
Glu17, Ile19,
Arg23,
Glu17, Ile19,
Arg23,
Glu17, Ile19,
Arg23, Asn53
Glu17, Ile19,
Arg23,
Lys14, Glu17,
Ile19, Arg23,
Tyr18
Lys14, Glu17,
Ile19, Arg23,
Tyr18
Tyr18
Tyr18
Tyr18
Tyr18
Tyr18
Tyr18
Tyr18
4.
5.
6.
MET
Docking
Score
ꢀ 7.86
Met1160
ꢀ 5.61
–
ꢀ 8.45
Met1160
ꢀ 5.29
–
ꢀ 8.05
Met1160
ꢀ 6.05
–
ꢀ 7.78
ꢀ 8.16
Met1160
ꢀ 6.20
–
ꢀ 7.72
ꢀ 8.08
ꢀ 8.25
ꢀ 8.22
ꢀ 8.18
ꢀ 9.26
H-bond
Arg1208
ꢀ 5.56
Met1160
ꢀ 6.28
Met1160
ꢀ 5.57
Met1160
ꢀ 6.09
–
Met1160
ꢀ 4.85
Met1160,
Tyr1159
ꢀ 8.45
Met1160,
Asp1222
ꢀ 9.12
MEK1
VEGFR
Docking
Score
ꢀ 6.03
H-bond
Gln153
ꢀ 9.12
Met146
ꢀ 6.37
Gln153
ꢀ 8.92
Ser150
ꢀ 8.52
–
Ser194,
Asp152
ꢀ 4.89
Glu144,
Glu144, Met146,
Lys192
Gln153, Ser194
ꢀ 6.81
Docking
Score
ꢀ 8.04
Cys912
ꢀ 8.84
Cys912
ꢀ 6.05
ꢀ 9.11
Cys912
ꢀ 8.76
ꢀ 10.15
H-bond
Glu878,
Ile1019,
Arg1021
Ile1019,
Arg1021
Cys912
Cys912
–
Arg1021
Asp807,
Glu878,
Ile1019,
Arg1021
ꢀ 8.10
Glu878, Cys912,
Asp1040
Asp1040
7.
MAPK
Docking
Score
ꢀ 4.58
ꢀ 5.45
ꢀ 5.84
ꢀ 5.28
ꢀ 5.95
ꢀ 5.34
ꢀ 6.47
ꢀ 6.01
ꢀ 4.61
ꢀ 6.39
ꢀ 7.26
H-bond
Lys114
Asp111,
Lys114
Met108
Asp111,
Lys114
Met108
Lys114
Met108
Met108
–
Met108,
Lys114
Asp106,
Met108,
Asp111
ꢀ 8.46
Gln105, Asp106,
Met108
8.
mTOR
PI3K
Docking
Score
ꢀ 8.36
ꢀ 7.59
ꢀ 7.72
–
ꢀ 8.93
ꢀ 9.68
ꢀ 8.19
Val2240
ꢀ 8.96
Val851
ꢀ 4.58
Lys171
ꢀ 6.68
Ala564
ꢀ 7.78
Leu83
ꢀ 8.67
ꢀ 9.02
Val2240
ꢀ 9.49
Val851
ꢀ 6.59
Leu224
ꢀ 7.13
Ala564
ꢀ 8.81
ꢀ 8.95
ꢀ 7.98
–
H-bond
Gly2238
Val2240
ꢀ 8.11
Val2240
ꢀ 8.65
Gly2238
Val2240
ꢀ 7.86
Gly2238
Val2240
ꢀ 8.82
Gly2238
Val2240
ꢀ 7.16
Val2240,
Asp2357
ꢀ 8.44
Val2240
ꢀ 8.53
Lys2187,
Asp2195
ꢀ 9.79
–
9.
Docking
Score
ꢀ 7.73
Val851
ꢀ 4.45
Lys171
ꢀ 6.98
Ala564
ꢀ 7.19
ꢀ 10.41
H-bond
Val851
ꢀ 6.20
Leu224
ꢀ 7.11
Val851
ꢀ 5.00
Val851
ꢀ 6.32
Leu224
ꢀ 7.36
Ala564
ꢀ 8.96
Val851
ꢀ 6.39
Leu224
ꢀ 7.78
Ala564
ꢀ 9.14
Tyr836,
Asp933
ꢀ 6.21
Val851
ꢀ 4.21
Thr257
ꢀ 7.43
Val851
ꢀ 4.33
Lys802,
Val851, Gln859
ꢀ 8.94
Tyr836, Val851
ꢀ 7.49
10.
11.
12.
PIP5K1
FGFR
CDK2
α
Docking
Score
H-bond
Leu224
ꢀ 7.55
Leu224
Leu224
ꢀ 7.55
Lys171, Leu224
ꢀ 7.91
Asn222, Leu224,
Asp238
Docking
Score
ꢀ 6.82
ꢀ 8.38
H-bond
Glu562,
Ala564
ꢀ 8.42
Glu562,
Ala564
ꢀ 8.47
Glu562,
Ala564
ꢀ 8.62
Glu562,
Ala564
ꢀ 6.93
Glu562,
Ala564
ꢀ 8.39
Glu571, Ala564
ꢀ 9.97
Asp541, Ala564
ꢀ 7.22
Docking
Score
H-bond
Leu83,
Glu81,
Leu83
Asp145,
Thr14
Leu83,
Glu81,
Leu83,
Glu81,
Asp145
Leu83,
Glu81,
Leu83,
Glu81,
Phe80,
Asp145
Leu83,
Leu83,
Leu83, Asp145
Asp145
Asp86, Asp145
*pdb selected for mTOR (2NPU) did not have any co-crystallized ligand

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
Fig. 2. 3-D interaction representations for the flavonol analogs superimposed over fisetin within the binding cavity of target enzymes a) EGFR b) cKIT c) Akt d) MET
e) MEK1 f) VEGFR g) MAPK h) mTOR i) PI3K j) PIP5K1 k) CDK2 l) FGFR.
α
several of the synthesized analogs exhibited potent inhibition of CDK2,
c-Kit and mTOR kinase activities. In reference to fisetin used as a posi-
tive control, the respective IC50 values obtained for c-Kit, CDK2 (cyclin
A, and cyclin E), and mTOR were determined to be 1.34, 2.62, 3.34 and
μM), whereas compounds F14 and F17 displayed IC50 values of 5.38 and
5.70 μM, respectively (cf. Table 3). The detailed analysis of the kinase
activities of these analogs are displayed in the supplementary report
(68).
28.7
μ
M, respectively, indicating that the in vitro kinase assays reliably
Compounds F9 and F19 emerged as the most potent inhibitors
reproduced previous reports [28,29,38]. Among the tested analogs, F20,
against mTOR, with IC50 values of 1.42 and 1.64 μM, respectively; an-
showed the most potent kinase inhibitory activity against c-Kit (IC50
alogs F8 and F17 showed mTOR kinase inhibitory activities (IC50 values
of 23.9 and 11.5 M, respectively) comparable to that of fisetin. How-
0.09
μ
M); F9 and F14 were comparably potent (IC50 0.12 and 0.14
μM,
μ
respectively); whereas, F8, F19, and F21 exhibited IC50 values of 0.54,
0.33, and 0.26 M, respectively, and F17 (IC50 1.74 M) displaying a
ever, analogs F11, F20 and F21 did not substantively inhibit cyclin E
and mTOR kinase activity. Not all of the identified active anti-
proliferative analogs exhibited strong inhibitory effects against these
kinases. This was particularly true for F6 and F11, with neither com-
pound showing detectable kinase inhibitory activity against any of the
tested targets (particularly surprising with respect to lack of c-Kit inhi-
bition given the Glide docking scores), despite their noteworthy anti-
proliferative and cytotoxic properties (see Table 1). These latter obser-
vations suggest the participation of some other yet-to-be-identified
cellular targets and signalling pathways mediating the anti-
proliferative activities of F6 and F11.
μ
μ
lesser activity comparable to fisetin’s. Coincidentally, F20 also appears
to be the most potent anti-proliferative and cytotoxic compound, though
its molecular docking score did not predict increased binding potency
vs. fisetin’s. Docking scores for F14 and F19 were similar to that of
fisetin and predictive of their observed increased potencies.
With respect to the CDK2 kinase activities, five analogs (F8, F9, F14,
F17, and F20) for CDK2/cyclin A, and three (F9, F14, and F17) for
CDK2/cyclin E, showed IC50 values below 10
μ
M. For cyclin A inhibi-
M), with F8, F14, F17,
M,
tion, F9 was seen to be most potent (IC50 2.95
μ
and F20 exhibiting IC50 values of 7.12, 6.64, 4.57, and 4.77
μ
Taken together, the kinase inhibition data suggest that F8, F9, F17
and F19 may exert their antiproliferative activities at least in part
respectively. F9 was also the most potent inhibitor of cyclin E (IC50 1.78
7

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
Fig. 2. (continued).
inhibitor, with some effects on cyclin A. F21 seems to have a significant
effect only on c-Kit among the kinases investigated. Reasonably, full-
kinome screening would be needed to reach any further conclusions,
and certainly, given the spectrum of biological activities of flavonols
seen in the totality of the literature for this class of compounds (and for
flavonoids more generally), other as-yet-to-be-identified non-kinase
targets may likely contribute to the observed antiproliferative actions.
Most importantly, it was noted that the multi-kinase activities observed
in the current studies corresponded with the anti-cancer effects exhibi-
ted by these compounds on skin cancer cells, and that the kinase inhi-
bition data for analogs F8, F9, F14, F17, F19, F20 and F21. However,
noting both observations might provide valuable information regarding
the anticancer mechanism(s) of actions of these compounds and for
treatment of these types of cancers, further mechanistic and phenotypic
functional studies were pursued.
Table 3
IC50 values for c-Kit, CDK2, and mTOR inhibition by active compounds.
Compound
c-Kit
CDK2
mTOR/FRAP1
IC50 (µM)
IC50 (µM)
IC50 (µM)
Cyclin A
Cyclin E
F0
1.34
>100
0.54
0.12
101
2.62
3.34
ND
28.69
ND
F6
>100
7.16
F8
17.12
1.78
ND
23.93
1.42
ND
F9
2.95
F11
F14
F17
F19
F20
F21
32.46
6.64
0.13
1.74
0.33
0.09
0.26
5.38
5.70
ND
>100
11.45
1.64
ND
4.57
12.74
4.77
>100
ND
30.10
ND
through their various degrees of actions as multi-kinase (c-Kits, CDK2/
cyclin A, CDK2/cyclin E, or mTOR) inhibitors (Table 3). Meanwhile,
F20, the most-potent and selective anti-proliferative hit for cancerous
vs. non-cancerous cells, may be acting primarily as a c-Kit kinase in-
hibitor, with some inhibitory effects on cyclin A. F14 displayed dual c-
Kit/CDK2 kinase inhibition, while F19 is a potent dual c-Kit/mTOR
2.5. Potent multi-kinase-inhibiting fisetin analogs modulate cellular
biochemical parameters to induce apoptosis in A375 and A431 skin cancer
cells.
To explore the mechanisms by which the synthesized flavonols
induce cell death, compounds F8, F9, F14, F17, F19, F20, and F21 were
8

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
selected based on their observed in vitro phenotypic efficacies (anti-
proliferative activities) and target-level potencies(kinase(s) inhibition).
Because the induction of apoptosis in cancer cells is a vital component
for successful cancer therapy, as it is linked to the efficacy of the vast
majority of anticancer agents, it was further investigated whether the
above-listed chosen analogs could induce or promote apoptotic pro-
cesses selectively in cancerous cells. Initially, their effects on nuclear
morphology were assessed via 4′,6-diamidino-2-phenylindole (DAPI)
staining, monitoring the effects and percentage of apoptotic cells by
immunofluorescent microscopy analysis. We found that the nuclei of
A375 and A431 cells treated with compounds F8, F9, F14, F17, F19,
F20, and F21 exhibited highly shrunken/condensed and fragmented
chromatin with punctuation, clearly distinguishing drug-treated
apoptotic from the normally cobblestone-like appearances of non-
apoptotic skin cancer cells (data not shown). As summarized
(Table 4), the percentage of apoptotic cells was significantly increased
by each of the seven best hit compounds at concentrations of ½ IC50 and
IC50 in both A375 and A431 cells. Following this initial pro-apoptotic
screening assay, only three among the seven tested analogs, namely
F20, F9, and F17, were retained for further experiments, based on their
aggregate anti-proliferative, kinase inhibitory and pro-apoptotic
potencies.
expression level of the pro-apoptotic proteins, Bax, whereas the level of
anti-apoptotic protein Bcl-2 was decreased (suppressed) after treatment
as compared with the control. Similar results were obtained from in-
depth analysis of these markers upon treatment with compounds F9
and F17, and are displayed in the supplementary report (68). Treatment
with these compounds increased the ratio of Bax/Bcl-2 in
a
concentration-dependent manner (see Fig. 4). Taken together, these
data further support the hypothesis that compounds F20, F9 and F17
can induce the apoptosis of skin cancer cells, specifically cells of A375
and A431 lines, through activation of the intrinsic mitochondrial
apoptotic signaling pathway, leading to cleavage of and caspases 3, 7
and 8 as well as of PARP activation.
2.6. Potent fisetin analogs inhibit proliferation and induce apoptosis
through downregulation of the expression of CDK2, and suppression of
phosphorylated c-Kit and downstream targets in A375 and A431 cells.
Aberrant cell proliferation and dysregulation of the cell cycle, hall-
marks of malignant tumors, are dependent on the coordination of cyclins
and cyclin-dependent kinases (CDKs), particularly the CDK2 which
amongst others, crucially regulates G1/S and modulates S/G2 cell cycle
progression [93]. However, CDK2 activity exceeding a certain threshold
has been suggested to worsen outcomes for “CDK2-low” cancers, thus
predictive that inhibitors targeting CDK2 could represent promising
anticancer strategy [93]. Melanoma and squamous cell carcinoma
represent the two such cancers, and together account for a major portion
of skin cancer-related deaths, with the incidence and mortality rate
having increased globally over the past few decades [94,95]. Addi-
tionally, alterations in c-KIT, a proto-oncogene that encodes the corre-
sponding transmembrane receptor tyrosine kinase, has been reported to
be mutated and aberrantly expressed in melanomas and SCC (95 and for
reviews see 94,96). Due to its important association with cell survival
and differentiation, c-KIT inhibition has been therapeutically targeted in
several advanced melanoma clinical trials using inhibitors, including
imatinib, dasatinib, nilotinib, and sunitinib, all with problematic side-
effects [94]. Even so, the data from these trials and subsequent clin-
ical experience provides endorsement of c-KIT as an opportune anti-
cancer target, particularly when co-targeted with other pathways
[94–97]. Moreover, it has been documented that the downstream ef-
fectors of c-KIT driven melanoma are the MAPK/ERK and PI3K/AKT
pathways [94,96,97]. In this study, since most of these compounds,
particularly F20, bind strongly c-KIT and/or CDK2 and inhibit their
kinase activities, A375 and A431 cells were treated with various doses
(0, IC50 and 2 × IC50) of F9, F17, and F20 for 48 h to evaluate their
biological effects on CDK2, activated c-KIT and the downstream targets
by Western blot analysis. As shown (Fig. 5), compound F20 significantly
suppress the protein expression levels of cyclin A2, cyclin E2 and CDK2
as compared to untreated control group, though somewhat disparately
between the two cell lines. A similar trend was observed for F9 and F17
treated cells as detailed in supplementary report (68).
Apoptotic caspases belong to the family of cysteinyl aspartate spe-
cific proteases, often classified into sets including initiators (caspases 8,
9 and 10) and executioners (caspases 3, 6 and 7) [90,91]. Employing
both A375 and A431 cells, the modulatory effects of compounds F20, F9,
and F17 treatment were examined on the intrinsic and extrinsic
apoptosis signaling pathways, by probing the protein expression levels
of a number of key apoptosis-related marker proteins using Western blot
analysis. Thereto, expression levels of apoptotic markers including the
pro- and cleaved (activated) forms of initiator (caspase 8) and execu-
tioner (caspase ꢀ 3 and ꢀ 7) caspases, and activation of Poly (ADP-
ribose) polymerase (PARP) cleavage, were assessed. Treatment of A375
and A431 cells with F20, resulted in significant activation of apoptosis,
as evidenced by the elevated protein expression levels of cleaved cas-
pases ꢀ 3, ꢀ 7, and ꢀ 8 (Fig. 3), and PARP (Fig. 4) in a concentration-
dependent manner when matched with untreated controls. In-depth
data displaying the effects of treatment with F9 and F17, indicating
similar patterns on these cells, are presented in the supplementary
dataset (68). The cleavage of caspase ꢀ 3, ꢀ 7, ꢀ 8, and of PARP, was also
present in UWBCC1 cells similarly treated with each of these three hit
compounds (data not shown).
The expression level changes for Bcl-2-family proteins functioning as
important regulator proteins in the mitochondrial pathways of apoptosis
induction, namely Bax, (a pro-apoptotic protein) and Bcl-2 (an anti-
apoptotic protein) [92], in A375 and A431 cells was studied upon
treatment with compounds F9, F17 and F20. Besides the increased
number of DAPI positively stained apoptotic cells, treatment of both
A375 and A431 cells with F20 (Fig. 4), significantly increased the
Moreover, the protein expression levels of total c-Kit were not
significantly altered by treatment with any of these drugs at indicated
concentrations. In contrast, the protein expression levels of the phos-
phorylated form of c-Kit were significantly suppressed by F20 (Fig. 5).
Detailed results on the effects of F9 and F17, which showed similar
activities, are presented in the supplementary report (68). Importantly,
these compounds also modulated the downstream effector targets
(signature) of c-Kit signalling, including those of the phosphorylated
Stat3, PI3K/p90RSK/Akt/mTOR, and MAPK (ERK1/2) pathways
(Figs. 5 and 6) and, as detailed in the supplementary report (68).
Western blot analysis for A375 and A431 cells treated with these com-
pounds showed that along with activated c-Kit, phosphorylated p90RSK
(Ser380), Akt (Ser473), ribosomal protein S6, and mTOR (Ser2448) were
significantly suppressed when compared to untreated controls (Fig. 5);
see detailed results of F9 and F17 in the supplementary dataset (68).
Similarly, these compounds notably suppressed the expression level of
Table 4
Summary of percentage of apoptotic A375 and A431 cells as derived from
relative numbers of DAPI-stained nuclei of cultured cells treated for 36 h with
selected flavonols at ½IC50 and IC50 antiproliferative concentrations (cf.
Table 1).
DAPI (+) apoptotic cells (%)
Cell line
A375
A431
Compound ID
Control
½IC50
IC50
Control
½IC50
IC50
F8
ND
3.4
2.9
3.9
3.3
3.0
3.2
6.5
7.5
6.2
6.9
5.6
8.2
5.4
11.3
18.2
10.9
17.7
13.3
20.4
ND
4.1
3.6
3.5
3.8
3.6
4.2
3.9
5.84
7.93
6.92
8.81
6.71
14.6
8.8
12.8
19.1
ND
F9
F14
F17
F19
F20
F21
18.4
14.2
27.7
14.4
9

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
Fig. 3. Potent flavonol F20 induces apoptosis
through activation of the extrinsic and intrinsic
apoptotic pathway caspases in melanoma and
non-melanoma cancer cells. The blots show the
effect of F20 at IC50 and 2 × IC50 concentrations on
the protein expression levels of markers of
apoptosis, including pro- and-cleaved caspase-3, ꢀ 7
and ꢀ 8, components of the intrinsic apoptosis
pathway, after 48-h treatment, comparing with un-
treated controls. A375 and A431 cells were incu-
bated in the absence or presence of F20 (0, IC50, 2
× IC50; 48 h), and whole-cell lysates were subjected
to SDS-polyacrylamide gel electrophoresis. The
Western blot data shown are representative immu-
noblots from three independent experiments with
similar results. Equal protein loading was confirmed
by re-probing for β-actin or GAPDH as loading
control, and the actual protein levels were normal-
ized to the loading control to obtain % Expression
values shown on the bar graphs (mean ± SD of
relative quantitative density values are plotted). The
data are expressed as the percentage to which de-
rivatives F20 suppressed or induced the protein
expression levels of pro- and-cleaved caspases in
A375 and A431 cells as compared with controls.
Statistical significance was assessed using one-way
ANOVA and Dunn’s multiple comparison tests, and
p < 0.05 (*) was considered significant.
phosphorylated Stat3 (Tyr705) (see Fig. 5 and supplementary dataset
report (68)), and mechanistically downregulated the corresponding in-
creases in protein expression levels of phosphorylated p44/42-MAPK
(ERK1/2; Thr202/Try204) in A375 and A431 cancer cells (see Fig. 6
and supplementary dataset report (68)). These results demonstrate that
the selected compounds, particularly F20, effectively inhibit cell pro-
liferation via targeting CDK2, c-Kit, and associated signalling pathways
(see Figs. 5-6 and supplementary dataset report (68)).
were assessed by measuring the area of cell wound closure area into the
initial cell-free areas, expressed as percentage of cell-free areas for
comparisons. After 48 h of incubation, the test compounds (particularly
F20) significantly decreased the cultured cells scratched wound area in a
dose-dependent manner (Fig. 7A-B). A similar trend was observed for
compounds F9 and F17; detailed results are displayed in the supple-
mentary dataset report) (68), which exhibited 28–72% inhibition of
wound closure, as compared to untreated controls.
To further assess the long-term effect of these selected analogs,
including F9, F17, and F20, on the A375 and A431 cells growth inhi-
bition, colony formation assays were performed in the presence or
absence of different concentrations of the compounds (½IC50, IC50 and 2
× IC50). As summarized in Fig. 8A-B, flavonol F20, significantly reduced
the percentage of colonies in a concentration-dependent manner as
compared to respective control groups. The significant reduction in
these parameters was similar upon treatment with F9 and F17, and the
in-depth results are displayed in supplementary dataset (68). Taken
together, these data from anti-proliferative/cytotoxic, scratch-wound
healing, and colony formation assays, together with altered levels of an
aggregation of protein markers of disease, indicate that the fisetin ana-
logs prepared and investigated in these studies inhibit growth, migration
and proliferation of cutaneous carcinoma cells.
2.7. Favonol analogs inhibit scratch wound healing and colony formation
in 2D cultures of A375 and A431.
The selected flavonols of interest, F9, F17 and F20 were further
investigated with respect to modulation of cell migration and were
evaluated using scratch wound healing assay with plated aggressive
melanoma (A375) and non-melanoma (A431) cells. Cells were plated
and grown to confluence, scratch wounds were created as described in
the Methods section 4.5.7, and were subsequently treated with or
without different doses (½IC50, IC50, and 2 × IC50) of the compounds,
with a goal of analysing their impacts on the migration of cells into the
wounded cell-free area after 48 h of scratch-wounding. Through imaging
analysis, the invasion of cells into the wounded area or migration rates,
10

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
Fig. 4. Potent flavonol F20 induces apoptosis
through activation of mitochondrial pro-
apoptotic pathways involving Bcl-2 family pro-
teins and of PARP cleavage in melanoma and
non-melanoma cancer cells. The Western blot
data show the effects of F20 at IC50 and 2 × IC50
concentrations for 48 h on the protein expression
levels of intact and cleaved PARP (85 kDa), as well
as Bcl-2 family of proteins (Bax and Bcl-2), compo-
nents of the intrinsic apoptosis pathway, in A375
and A431 cells. A375 and A431 cells were incubated
in the absence or presence of F20 (0, IC50, 2 × IC50
;
48 h), and whole-cell lysates were subjected to SDS-
polyacrylamide gel electrophoresis. The blots shown
are representative of immunoblots from three inde-
pendent experiments with similar results. The data
are expressed as the percentage suppression of the
protein expression levels of Bcl-2 and cleaved PARP
(116 kDa), and percentage increase in levels of
cleaved PARP ((85 kDa)) and of Bax, in treated
A375 and A431 cells as compared with controls.
Equal protein loading was confirmed by re-probing
for β-actin as loading control, and the actual pro-
tein levels were normalized to the loading control to
obtain % Expression values shown on the bar graphs
(mean ± SD of relative quantitative density values).
Statistical significance was assessed using one-way
ANOVA and Dunn’s multiple comparison tests, and
p < 0.05 (*) was considered significant.
3. Conclusions
fisetin. Finally, it should also be noted here that the effects of various
substituents on lipophilicity, and thus, cell penetrability, might also
matter and could perhaps be thought to confound the SAR studies to
some degree; the equilibrative nature of these cell culture studies should
have mostly averted that issue, but in that same vein, close attention was
paid to any prospective constraints as might be imposed by solubility
limits of compounds in the cell culture media. Taken together, the study
data identify F20, as well as F9 and F17, as promising c-Kit, CDK2 and
mTOR inhibitor prototypes that warrant further investigation as single
agents or in combination with, or as adjuvants to standard therapies for
the control of melanoma and NMSCs.
Growing evidence indicates that the potential high therapeutic index
of fisetin is significantly reduced due to bioavailability, variable activity,
toxicity and biotransformation into less active metabolites following
oral administration in vivo. Therefore, new fisetin derivatives designed
to overcome these issues may lead to the development of new com-
pounds exhibiting high therapeutic indexes. In this study, the anticancer
activity of 24 synthesized fisetin analogs were investigated, and the
results indicate that some of the compounds displayed comparable to
better anti-proliferative/cytotoxic properties compared to fisetin. A
selected number of these analogs exhibited significant inhibition of the
activation of deregulated disease molecular targets including specific
kinases, activation of apoptosis, and suppression of colony formation
and wound closure in a cell culture model of skin carcinogenesis.
Therefore, these data indicate that the synthesized flavonol-analogs of
fisetin lacking the 7-hydroxy group on ring A and the 3′,4′-dihydroxy
groups on ring B, are capable of displaying potent anti-proliferative/
cytotoxic properties up to 233-fold higher than the parent fisetin’s,
with selectivity index of 283, over normal cell line as shown for com-
pound F20. These fisetin derivatives likely mediate their actions through
several parallel pathways, including direct and indirect effects on pro-
survival and/or apoptotic pathways. Importantly, some of these ana-
logs (i.e. F9, F17 and F20) exert their cytotoxicity against cancer cells by
significantly inducing apoptosis markers; cleaved caspases ꢀ 3, ꢀ 7, ꢀ 8,
PARP, Bax/Bcl-2 ratio. In addition, they inhibited single or multi
deregulated molecular cancer targets such as CDK2, phosphorylated c-
Kit and its downstream effectors including Akt, mTOR, p70S6K,
p90RSK, Stat3, and MAPK (ERK1/2), which are mostly hyper-activated
in melanoma and NMSCs. Although extended cytotoxicity or in vivo
studies are needed to assess whether the broad activity is specific and
not connected to general cytotoxic effects, the current study provides
information and an available model to develop chemotherapeutic agents
with improved therapeutic index on the basis of phytochemical pro-
totypes. However, the main takeaway from these studies is the fact that a
number of derivatives lacking the aforementioned key impediments to
fisetin’s clinical efficacy, have proven to display better potency against
cells of both non-melanoma and melanoma lines when compared to
4. Materials and experimental methods
4.1. Chemistry
4.1.1. Synthesis and characterization of flavonols
General information
For the chemistry portion of this study, all solvents and other
chemicals were purchased from Sigma-Aldrich, and TCI except for 2′-
hydroxy-4′-methoxyacetophenone and m-anisaldehyde, which were
obtained from Alfa Aesar. All the reagents were commercial grade, and
were used without further purification unless otherwise indicated.
Organic extracts were dried over anhydrous sodium sulfate, and solvents
were removed under a reduced pressure on a Buchi Rotavapor equipped
with a vacuum controller and vacuum pump. Reaction mixtures were
monitored by TLC using silica gel 60 F254 plates, with a fluorescent
indicator, and silica gel (60–120 mesh size) was used for column chro-
matography. NMR spectra were recorded on JEOL 400 MHz FT spec-
trometer in the indicated solvent. Chemical shifts are expressed in δ
units (ppm) from tetramethylsilane (TMS) used as the internal standard
for 1H NMR (400 MHz) and for 13C NMR (100 MHz). Infrared spectra
(IR) were run on a SpectrumOne FT-ATR spectrophotometer (Perkin
Elmer). Band position and absorption ranges are given in cmꢀ 1. GC–MS
analysis carried out on an Agilent GC–MS (7890A – 5975C VL MSD)
system. High Resolution Mass Spectrometry (HRMS) was performed on
an Agilent 6230, and spectra recorded by electro-spray ionization (ESI)
with a Q-TOF. Microwave-assisted reactions were performed on
11

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
Fig. 5. Flavonol analogs inhibit the protein expression levels of cyclin A/
E, CDK2 and phosphorylated c-Kit and Stat3 in A375 melanoma and A431
non-melanoma cancer cells. (A) Western blot data showing the effect of
different concentrations of F20 on protein expression levels of cyclin A2, cyclin
E2, CDK2, c-Kit, and phosphorylated c-Kit and Stat3 in 48 h-treated A375 (left
panel) and A431 (right panel) cells. A375 and A431 cells were incubated with/
without F20; 0, IC50, 2 × IC50; µM, 48 h), and western blotting performed as
described in the method section. Equal protein loading was confirmed by
reprobing for GAPDH as loading control, and protein levels were normalized to
the loading control and expressed as percentage. The blot data shown are
representative of immunoblots of three independent experiments with similar
results. (B) The data expressed in the bar graphs represent mean ± SD of
relative quantitative normalized density values in percentage with an internal
loading control from three independent experiments. Analog F20 significantly
suppressed the protein expression levels of these in A375 and A431 as
compared with untreated control cells. Bar graphs, the statistical significance
was determined using one-way ANOVA and Dunn’s multiple comparison test,
and p < 0.05 (*) was considered significant.
Fig. 6. Potent flavonol analogs inhibit the protein expressions of acti-
vated c-Kit and downstream effectors levels, including phosphorylated
mTOR/ ribosomal protein S6/ MAPK (ERK1/2) and p90RSK in A375 mel-
anoma and A431 non-melanoma cells. (A) Western blots displays are rep-
resentatives of c-Kit downstream effectors the protein expression levels of
phosphorylated mTOR, ribosomal protein S6, MAPK p44/42(ERK1/2) and
p90RSK, in 48 h treated A375 (left panel) and A431 (right panel) cells. A375/
A431 cells were incubated with/without test compounds (F20; 0, IC50, 2 × IC50
;
µM, 48 h), and western blotting was carried out as described in the Methods
section. Equal protein loading was confirmed by reprobing for Rab11 as loading
control, and protein levels were normalized to the loading control and
expressed as percentage. The data shown are representative of immunoblot of
three independent experiments with similar results. (B) The data expressed in
the bar graphs represent mean ± SD of relative quantitative normalized density
values in percentage with an internal loading control. The analogs F20 signif-
icantly suppressed the protein expression levels of the phorphorylated form of
the targets in A375 and A431 as compared with untreated control cells. Sta-
tistical significance was determined using one-way ANOVA and Dunn’s multi-
ple comparison test, and p < 0.05 (*) was considered significant.
Discover S-Class (CEM) single mode microwave reactor, with the in-
strument settings controlled by PC-running Synergy 1.4 software. All
experiments were carried out in microwave reaction vials using a stir-
ring bar (length 20 mm, diameter 6 mm). Stirring, temperature, irradi-
ation power, PowerMAX (in situ cooling during the microwave
irradiation), ramp and hold times were set as indicated, and the tem-
perature of the reaction was monitored by a built-in infrared sensor.
maintained for 20 min at the same temperature, then cooled to room
temperature. The mixture was extracted with ethyl acetate and evapo-
rated to get the crude product. The solid product mixture was recrys-
tallized using acetonitrile to yield the product.
4.2. General synthetic procedures
4.2.1. Microwave assisted synthesis of chalcones
4.2.2. Room temperature synthesis of flavonols from chalcones
To a solution of chalcone intermediate (1 mmol) in methanol (5 mL),
3 M aqueous solution of KOH (1.4 mL, 3 mmol) was added and stirred for
10 min. Then a 30% aqueous solution of H2O2 (0.35 mL, 3 mmol) was
added slowly under a continuous stirring for 2 hrs at room temperature.
The resulting suspension was treated with 10% aqueous solution of HCl
(5 mL), extracted with dichloromethane, dried with sodium sulfate, and
A 10 mL microwave reaction vial, equipped with a stir bar, charged
with the substitued 2-hydroxyacetophenone (5 mmol), the appropriate
aldehyde (5 mmol), ethanol (6 mL) and potassium hydroxide (1.5 eqv.),
was stirred at room temperature. The vial was then sealed with Teflon-
lined silicone cap and placed in the microwave reactor. Microwave
irradiation of 250 W was used and the temperature ramped from 25 ◦C
to 80 ◦C. Once it reached 80 ◦C in about 1 min, the reaction was
12

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
evaporated. The products were purified by column chromatography
(using hexane–ethyl acetate) or recrystallization (using 80:20 acetoni-
trile/ethanol solvent system).
4.2.3. Microwave assisted One-Pot synthesis of flavonols
A 10 mL microwave reaction vial, equipped with a stir bar, charged
with substituted 2-hydroxyacetophenone (5 mmol), the appropriate
aldehyde (5 mmol), ethanol (6 mL) and potassium hydroxide (1.5
equiv.) was stirred at room temperature. The vial was the sealed with
Teflon-lined silicone cap and placed in the microwave reactor. Micro-
wave irradiation of 250 W was used and the temperature ramped from
◦
◦
25 C to 80 C, then maintained for 20 min at the same temperature
while stirring, and cooled to room temperature. A potassium hydroxide
(0.45 mL, 1 mmol) solution in methanol and 30% solution of hydrogen
peroxide (0.35 mL, 3 mmol) were added, and placed back into the mi-
crowave reactor. The mixture was stirred at 40 ◦C for 10 min and cooled
to room temperature, extracted with ethyl acetate, dried over magne-
sium sulfate, and evaporated to get the crude product. The products
were purified by column chromatography using hexane–ethyl acetate as
eluent.
Fig. 7. The most-potent analog, F20, significantly inhibited Scratch
wound healing in 2D cultures of A375 and A431 in a dose-dependent
manner. Effect of the flavonol analog F20 on the closure/migration into the
initial cell-free scratch-wound areas compared to the percentage of control cell-
free areas (A) (Upper panel) A431 and (lower panel) A375. (B) The data pre-
sented indicate significant dose-dependent decrease in cultured cells repopu-
lating the scratch wound healing area in the presence of F20, as compared to
untreated control cells, after 48 h of incubation. The data in the bar graphs
represent mean ± SD of scratched-wound area values, expressed as percentage,
compared to 0 h control and 48 h controls from three independent experiments.
F20 significantly decreased wound healing area A431 (left panel) and A375
(right panel) as compared with untreated controls. Statistical significance was
assessed using one-way ANOVA and Dunn’s multiple comparison test, and p <
0.05 (*) was considered significant.
4.2.4. Characterization of the flavonols synthesized
3-Hydroxy-2-phenyl-4H-chromen-4-one (F1): 1H NMR (400 MHz,
CDCl3): 7.41 (t, 1H, J = 7.8 Hz, ArH), 7.46 (d, 1H, J = 7.3 Hz, ArH), 7.52
(t, 2H, J = 7.3 Hz, ArH), 7.57 (d, 2H, J = 8.7 Hz, ArH), 7.68 (t, 1H, J =
6.8 Hz, ArH), 8.24 (d, 2H, J = 8.2 Hz, ArH). 13C NMR (100 MHz, CDCl3):
118.4, 120.7, 124.6, 125.5, 127.8, 128.7, 130.3, 131.1, 133.8, 138.5,
145.1, 155.5, 173.5. IR (KBr): 3258, 3153, 1619, 1607, 1562, 1481,
1471, 1415, 1211, 1129, 756, 705, 689 cmꢀ 1. HRMS (ESI) calcd for
C
15H10O3 [M + H] 239.07027; found, 239.0702.
3-Hydroxy-2-(3-methoxyphenyl)-4H-chromen-4-one (F2): 1H NMR
(400 MHz, CDCl3): 3.88 (s, 3H, OCH3), 7.09 (s, 1H, ArH), 7.01 (dd, 1H, J
= 8.0 and 1.2 Hz, ArH), 7.24 (s, 1H, ArH), 7.39 – 7.45 (m, 2H, ArH), 7.57
(d, 1H, J = 8.2 Hz, ArH), 7.69 (t, 1H, J = 8.0 Hz, ArH), 7.80–7.85 (m, 2H,
ArH), 8.22 (d, 1H, J = 6.8 Hz, ArH), 10.70 (brs, 1H, OH). 13C NMR (100
MHz, CDCl3): 55.5, 113.4, 116.1, 118.3, 119.5, 120.5, 124.8, 125.5,
129.7, 130.9, 132.3, 133.9, 136.6, 155.4, 159.6, 162.2, 174.1. IR (KBr):
3252, 3098, 1621, 1605, 1563, 1482, 1473, 1288, 1204, 1168, 903, 774
cmꢀ 1. HRMS (ESI) calcd for C16H12O4 [M + H] 269.08084; found,
269.08060.
3-Hydroxy-2-(4-((trifluoromethyl)thio)phenyl)-4H-chromen-4-one
(F3): 1H NMR (400 MHz, CDCl3): 7.24 (brs, 1H, OH)7.42 (t, 1H, J = 7.3
Hz, ArH), 7.58 (d, 1H, J = 8.7 Hz, ArH), 7.72 (t, 1H, J = 7.1 Hz, ArH),
7.79 (d, 2H, J = 8.2 Hz, ArH), 8.24 (dd, 1H, J = 7.2 and 1.3 Hz, ArH),
8.30 (d, 2H, J = 8.7 Hz, ArH). 13C NMR (100 MHz, CDCl3): 118.4, 120.6,
124.9, 125.6, 126.3, 128.6, 133.5, 134.2, 136.1, 139.2, 143.3, 155.5,
173.6. IR (KBr): 3243, 3012, 1622, 1606, 1495, 1478, 1422, 1286, 1213,
1127, 1100, 836, 758, 500 cmꢀ 1. HRMS (ESI) calcd for C16H9F3O3S [M
+ H] 339.02973; found, 339.02945.
2-(4-Bromo-2-chlorophenyl)-3-hydroxy-4H-chromen-4-one (F4): 1H
NMR (400 MHz, CDCl3): 6.58 (brs, 1H), 7.43 (t, 1H, J = 7.8 Hz, ArH),
7.50–7.54 (m, 4H, ArH), 7.72 (m, 1H, ArH), 8.26 (dd, 1H, J = 7.8 and
1.2 Hz, ArH). 13C NMR (100 MHz, CDCl3): 118.2, 121.9, 124.2, 125.3,
129.4, 129.8, 132.5, 132.8, 133.4, 134.6, 139.5, 142.1, 155.3, 173.3. IR
(KBr): 3261, 3019, 1625, 1611, 1468, 1292, 1215, 1130, 762, 664 cmꢀ 1
.
Fig. 8. Potent analog F20 significantly inhibits colony formation in 2D
cultures of A375 and A431 cells in a dose-dependent manner. (A-B) Long-
term effect of the most potent flavonol, F20, on clonogenic potential in A375,
and A431 cells. Treatment with derivative F20 (½IC50, IC50 and 2 × IC50) of the
respective cell lines, significantly reduced/suppressed the percentage of col-
onies formed in a dose-dependent manner when compared to the respective
control untreated cutaneous carcinoma cells. (B) The data expressed in the bar
graphs represent mean ± SD of values in percentage control from three-
independent experiments. Statistical significance was assessed using one-way
ANOVA and Bonferoni’s multiple comparison test; p < 0.05 (*) was consid-
ered significant.
HRMS (ESI) calcd for C15H8BrClO3 [M + H] 350.94181; found,
350.94151.
2-(2,4-Bis(trifluoromethyl)phenyl)-3-hydroxy-4H-chromen-4-one (F5):
1H NMR (400 MHz, CDCl3): 6.79 (brs, 1H, OH), 7.26–7.52 (m, 2H, ArH),
7.72–7.76 (m, 1H, ArH), 7.96–8.03 (m, 2H, ArH), 8.12 (s, 1H, ArH), 8.28
(dd, 1H, J = 8.4 Hz and 1.6 Hz, ArH). 13C NMR (100 MHz, CDCl3): 118.4,
121.1, 125.0, 125.6, 128.6, 128.7, 132.8, 134.3, 138.8, 143.1, 155.7,
173.4.. IR (KBr): 3285, 2978, 1625, 1606, 1350, 1274, 1175, 1119, 904,
754, 661 cmꢀ 1. HRMS (ESI) calcd for C17H8F6O3 [M + H] 375.04504;
found, 375.04475.
2-(3,5-Bis(trifluoromethyl)phenyl)-3-hydroxy-4H-chromen-4-one (F6):
13

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
1H NMR (400 MHz, CDCl3): 7.46 (t, 1H, J = 8.0 Hz, ArH), 7.66 (d, 1H, J
= 8.2 Hz, ArH), 7.77 (t, 1H, J = 6.8 Hz, ArH), 7.95 (s, 1H, ArH), 8.26 (d,
1H, J = 6.8 Hz, ArH), 8.72 (brs, 2H, ArH). 13C NMR (100 MHz, CDCl3):
118.4, 120.6, 121.9, 123.3, 124.6, 125.2, 125.8, 127.6, 130.4, 132.0,
132.4, 133.3, 134.6, 139.6, 141.5, 155.5, 173.7. IR (KBr): 3215, 3187,
3023, 1617, 11451, 1368, 1281, 1126, 901, 754, 683 cmꢀ 1. HRMS (ESI)
calcd for C17H8F6O3 [M + H] 375.04504; found, 375.04440.
MHz, CDCl3): 7.46–7.54 (m, 4H, ArH), 7.78 (d, 1H, J = 1.2 Hz, ArH),
8.09 (d, 1H, J = 8.4 Hz, ArH), 8.20 (d, 2H, J = 7.2 Hz, ArH). 13C NMR
(100 MHz, CDCl3) 121.4, 126.9, 127.8, 128.2, 128.4, 128.5, 128.7,
130.5, 130.7, 138.8, 145.5, 155.4, 173.1. IR (KBr): 3268, 2922, 1622,
1603, 1451, 1398, 1268, 1230, 1209, 1130, 766, 732, 697 cmꢀ 1. HRMS
(ESI) calcd for C15H9BrO3 [M + H]± 316.98078; found, 316.98099.
7-Bromo-3-hydroxy-2-(3-methoxyphenyl)-4H-chromen-4-one (F15): 1H
NMR (400 MHz, CDCl3): 3.88 (s, 3H, OCH3), 7.00–7.02 (m, 2H, ArH),
7.42 (t, 1H, J = 8.2 Hz, ArH), 7.51 (dd, 1H, J = 8.8 and 1.8 Hz, ArH),
7.78 (t, 3H, J = 8.2 Hz, ArH), 8.08 (d, 1H, J = 8.6 Hz, ArH). 13C NMR
(100 MHz, CDCl3): 55.5, 113.1, 116.3, 119.5, 120.2, 121.4, 126.8,
128.2, 128.4, 129.8, 131.9, 138.7, 144.9, 155.3, 159.7, 173.0. IR (KBr):
1
2-(4-(Benzyloxy)phenyl)-3-hydroxy-4H-chromen-4-one (F7): H NMR
(400 MHz, CDCl3): 5.09 (s, 2H, ArCH2), 7.01–7.07 (m, 3H, ArH),
7.39–7.44 (m, 3H, ArH), 7.49–7.51 (m, 2H, ArH). 13C NMR (100 MHz,
CDCl3): 70.1, 73.6, 76.8, 77.1, 83.6, 115.1, 118.2, 122.1, 127.5, 128.1,
128.7, 129.0, 137.0, 159.6, 161.8. IR (KBr): 3188, 3086, 1619, 1603,
1565, 1478, 1282, 1165, 901, 773 cmꢀ 1. HRMS (ESI) calcd for C22H16O4
[M + H] 345.11214; found, 345.11155.
3271, 2932, 1601, 1574, 1440, 1388, 1325, 1265, 1136, 896, 775 cmꢀ 1
.
HRMS (ESI) calcd for C16H11BrO4 [M + H]± 346.99135; found,
346.99080.
2-(3-Chlorophenyl)-3-hydroxy-7-methyl-4H-chromen-4-one (F8): 1H
NMR (400 MHz, CDCl3): 2.50 (s, 3H, CH3), 7.21 (dd, 1H, J = 8.0 and 1.2
Hz, ArH), 7.37–7.44 (m, 2H, ArH), 8.10 (d, 1H, J = 8.4 Hz, ArH),
8.13–8.15 (m, 1H, ArH), 8.20 (t, 1H, J = 2.0 Hz, ArH). 13C NMR (100
MHz, CDCl3): 22.1, 118.0, 118.3, 125.2, 125.9, 126.5, 127.4, 129.9,
130.0, 133.0, 134.8, 138.7, 145.6, 155.6, 173.4. IR (KBr): 3264, 2982,
6-Bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one
(F16): 1H NMR (400 MHz, CDCl3): 3.474 (s, 3H, OCH3), 3.478 (s, 3H,
OCH3), 6.54 (d, 1H, J = 8.7 Hz, ArH), 7.08 (d, 1H, J = 8.7 Hz, ArH), 7.30
(dd, 1H, J = 9.2 and 2.7 Hz, ArH), 7.36 (d, 1H, J = 1.8 Hz, ArH), 7.42
(dd, 1H, J = 8.7 and 2.1 Hz, ArH), 7.81 (d, 1H, J = 2.7 Hz, ArH). 13
C
2889, 1606, 1561, 1478, 1402, 1269, 1211, 1170, 1136, 775 cmꢀ 1
.
NMR (100 MHz, CDCl3): 56.0, 56.1, 111.2, 111.3, 117.2, 120.9, 122.1,
123.9, 127.4, 136.1, 139.0, 146.4, 148.7, 150.7, 153.7, 172.1. IR (KBr):
3299, 3012, 2918, 1627, 1598, 1553, 1469, 1386, 1259, 1146, 1017,
809, 677 cmꢀ 1. HRMS (ESI) calcd for C17H13BrO5 [M + H]± 377.00191;
found, 377.00179.
HRMS (ESI) calcd for C16H11ClO3 [M + H] 287.04695; found,
287.04639.
1
3-Hydroxy-2-(3-methoxyphenyl)-7-methyl-4H-chromen-4-one (F9): H
NMR (400 MHz, CDCl3): 2.50 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 7.00 (dd,
1H, J = 8.4 and 2.8 Hz, ArH), 7.21 (d, 1H, J = 8.2 Hz, ArH), 7.37 (s, 1H,
ArH), 7.42 (t, 1H, J = 7.8 Hz, ArH), 7.79–7.84 (m, 2H, ArH), 8.10 (d, 1H,
J = 8.2 Hz, ArH). 13C NMR (100 MHz, CDCl3): 22.1, 55.5, 113.1, 115.9,
118.0, 118.4, 120.2, 125.2, 126.4, 129.7, 132.5, 138.5, 144.4, 145.3,
155.6, 159.7, 173.4. IR (KBr): 3233, 2905, 1606, 1438, 1392, 1256,
1200, 1160, 1128, 1034, 868, 819, 776 cmꢀ 1. HRMS (ESI) calcd for
C17H14O4 [M + H] 283.09649; found, 283.09629.
2-(3-Chlorophenyl)-6-fluoro-3-hydroxy-4H-chromen-4-one (F17): 1H
NMR (400 MHz, CDCl3): 7.41–7.46 (m, 3H, ArH), 7.58 (dd, 1H, J = 9.1
and 4.1 Hz, ArH), 7.83 (dd, 1H, J = 8.2 and 3.2 Hz, ArH), 8.14 (m, 1H,
ArH), 8.18 (t, 1H, J = 1.8 Hz, ArH). 13C NMR (100 MHz, CDCl3): 109.9,
110.2, 120.5, 120.6, 122.5, 122.8, 126.0, 127.6, 129.9, 130.3, 132.5,
134.8, 138.6, 143.9, 151.7, 157.9, 171.3. IR (KBr): 3260, 3092, 1617,
1609, 1577, 1487, 1399, 1261, 1171, 1128, 1102, 887, 819, 792, 732,
686 cmꢀ 1. HRMS (ESI) calcd for C15H8ClFO3 [M + H]± 291.02188;
found, 291.02187.
2-(2-Bromo-3,4-dimethoxyphenyl)-3-hydroxy-7-methyl-4H-chromen-4-
one (F10): 1H NMR (400 MHz, CDCl3): 2.49 (s, 3H, CH3), 3.89 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 7.09 (s, 1H, ArH), 7.16 (s, 1H, ArH), 7.24 (s,
1H, ArH), 7.32 (s, 1H, ArH), 8.14 (d, 1H, J = 7.76 Hz, ArH). 13C NMR
(100 MHz, CDCl3): 22.0, 56.3, 56.4, 113.8, 116.1, 118.2, 119.0, 125.3,
126.5, 138.4, 145.3, 145.9, 148.2, 151.0, 156.0, 173.3. IR (KBr): 3270,
2978, 1622, 1617, 1510, 1447, 1373, 1262, 1212, 1165, 1014, 869, 847,
773 cmꢀ 1. HRMS (ESI) calcd for C18H15BrO5 [M + H] 391.01756;
found, 391.01684.
2-(4-(Tert-butyl)phenyl)-6-chloro-3-hydroxy-7-methyl-4H-chromen-4-
one (F18): 1H NMR (400 MHz, CDCl3): 1.36 (s, 9H, C(CH3)3), 2.50 (s, 3H,
CH3), 7.44 (s, 1H, ArH), 7.53 (d, 2H, J = 8.7 Hz, ArH), 8.12–8.16 (m, 3H,
ArH). 13C NMR (100 MHz, CDCl3): 21.0, 31.2, 35.0, 119.8, 120.0, 124.9,
125.7, 127.6, 128.1, 131.4, 138.2, 142.9, 145.5, 153.7, 153.9, 172.3. IR
(KBr): 3225, 2966, 1598, 1459, 1432, 1381, 1304, 1230, 1202, 1106,
883, 840, 781 cmꢀ 1. HRMS (ESI) calcd for C20H19ClO3 [M + H]+
343.1095; found, 343.1091.
2-(4-(Tert-butyl)phenyl)-3-hydroxy-7-methyl-4H-chromen-4-one
(F11): 1H NMR (400 MHz, CDCl3): 1.36 (s, 9H, C(CH3)3), 2.49 (s, 3H,
CH3), 7.21 (dd, 1H, J = 9.2 and 2.4 Hz, ArH), 7.36 (s, 1H, ArH), 7.54 (d,
2H, J = 8.7 Hz, ArH), 8.10 (d, 1H, J = 7.8 Hz, ArH), 8.16 (d, 2H, J = 8.2
Hz, ArH). 13C NMR (100 MHz, CDCl3): 22.0, 31.2, 35.0, 117.9, 118.5,
125.2, 125.7, 125.4, 127.6, 128.4, 138.1, 145.1, 153.7, 155.6, 173.1. IR
(KBr): 3284, 3094, 2928, 2856, 1611, 1596, 1473, 1425, 1248, 1165,
1098, 776 cmꢀ 1. HRMS (ESI) calcd for C20H20O3 [M + H] 309.14852;
found, 309.14871.
2-(4-(Benzyloxy)phenyl)-6-chloro-3-hydroxy-7-methyl-4H-chromen-4-
one (F19): 1H NMR (400 MHz, CDCl3): 2.48 (s, 3H, CH3), 5.12 (s, 2H,
ArCH2), 7.07 (d, 2H, J = 8.7 Hz, ArH), 7.39–7.43 (m, 6H, ArH), 8.15 (t,
3H, J = 8.7 Hz, ArH). 13C NMR (100 MHz, CDCl3): 21.0, 70.1, 115.0,
119.8, 119.9, 123.6, 124.9, 127.6, 128.3, 128.7, 129.6, 131.3, 136.4,
137.6, 142.7, 145.4, 153.5, 160.4, 172.0. IR (KBr): 3226, 2892, 1599,
1483, 1384, 1202, 1096, 834, 779, 734, 696 cmꢀ 1. HRMS (ESI) calcd for
C23H17ClO4 [M + H]± 393.0888; found, 393.0881.
2-(4-Bromophenyl)-3-hydroxy-7-methoxy-4H-chromen-4-one (F12): 1H
NMR (400 MHz, CDCl3): 3.89 (s, 3H), 6.89 (d, 1H, J = 2.2 Hz, ArH), 6.95
(dd, 1H, J = 9.2 and 2.4 Hz, ArH), 7.58 (d, 2H, J = 8.7 Hz, ArH),
8.06–8.08 (m, 3H, ArH). 13C NMR (100 MHz, CDCl3): 55.9, 99.8, 114.6,
115.1, 124.3, 126.8, 129.0, 130.2, 131.8, 138.4, 143.2, 157.3, 164.5,
172.8. IR (KBr): 3237, 2983, 1614, 1595, 1446, 1286, 1202, 1167, 1124,
879, 831, 791 cmꢀ 1. C16H11BrO4 [M + H]± 346.99135; found,
346.99113.
2-(4-Bromophenyl)-3-hydroxy-6-methyl-8-nitro-4H-chromen-4-one
(F20): 1H NMR (400 MHz, CDCl3): 1.22 (s, 3H, CH3), 7.13 (brs, 1H, OH),
7.65 (d, 2H, J = 8.2 Hz, ArH), 8.22 (d, 2H, J = 8.2 Hz, ArH), 8.26 (s, 1H,
ArH), 8.31 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3): 20.6, 123.1, 124.5,
129.4, 129.7, 130.9, 131.1, 131.6, 133.9, 139.8, 172.0. IR (KBr): 3228,
3094, 1627, 1605, 1563, 1482, 1420, 151, 1288, 1213, 1132, 899, 776,
759, 690 cmꢀ 1. HRMS (ESI) calcd for C16H10BrNO5 [M + H]± 375.9815;
found, 375.9814.
2-(4-(Tert-butyl)phenyl)-3-hydroxy-7-methoxy-4H-chromen-4-one
(F13): 1H NMR (400 MHz, CDCl3): 1.36 (s, 9H, C(CH3)3), 3.93 (s, 3H,
OCH3), 6.94–6.97 (m, 2H, ArH), 7.54 (d, 1H, J = 8.2 Hz, ArH),
8.11–8.16 (m, 3H, ArH). 13C NMR (100 MHz, CDCl3): 31.2, 35.0, 55.9,
99.9, 114.7, 115.0, 125.5, 125.7, 126.8, 127.4, 128.4, 137.9, 153.5,
157.4, 164.3, 172.7. IR (KBr): 3278, 3092, 2889, 1624, 1606, 1563,
1442, 1349, 1286, 1146, 887, 772, 687 cmꢀ 1. GC–MS m/z 324.3.
7-Bromo-3-hydroxy-2-phenyl-4H-chromen-4-one (F14): 1H NMR (400
7-Chloro-2-(4-ethoxy-3-fluorophenyl)-3-hydroxy-4H-chromen-4-one
(F21): 1H NMR (400 MHz, CDCl3): 1.50 (t, 3H, J = 6.8 Hz, CH3), 4.20 (q,
2H, J = 6.8 Hz, OCH2), 7.06 (t, 2H, J = 8.4 Hz, ArH), 7.37 (dd, 1H, J =
8.4 and = 1.6 Hz, ArH), 7.59 (d, 1H, J = 1.6 Hz, ArH), 7.98 (m, 2H, ArH),
8.15 (d, 1H, J = 8.4 Hz, ArH). 13C (100 MHz; CDCl3): 14.7, 64.8, 113.9,
115.4, 115.6, 118.1, 119.1, 123.2, 123.3, 124.4, 124.5, 125.6, 126.7,
138.0, 139.8, 144.1, 144.2, 148.7, 148.8, 150.9, 153.4, 155.1, 172.5. IR
(KBr): 3234, 2919, 1605, 1518, 1452, 1282, 1108, 1040, 771 cmꢀ 1
.
14

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
HRMS (ESI) calcd for C17H13ClFO4 [M + H]± 35.0481; found, 335.0477.
6-Fluoro-3-hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(F22): 1H NMR (400 MHz, CDCl3): 3.92 (s, 3H, OCH3), 3.94 (s, 6H,
2xOCH3), 7. 10 (brs, 1H, OH), 7.40–7.45 (m, 1H, ArH), 7.49 (s, 2H,
ArH), 7.58–7.61 (m, 1H, ArH), 7.83–7.86 (m, 1H, ArH). 13C NMR (100
MHz, CDCl3): 56.3, 61.1, 105.5, 109.9, 110.1, 120.4, 120.5, 122.1,
126.0, 138.0, 140.1, 145.3, 151.6, 153.3, 157.9, 160.3, 172.7. IR (KBr):
3252, 2953, 2889, 1606, 1560, 1490, 1391, 1261, 1169, 1126, 1098,
779, 665 cmꢀ 1. HRMS (ESI) calcd for C18H15FO6 [M + H]± 347.0925;
found, 347.0924.
precast Tris-Glycine Gels (TGX) were from Bio-Rad (Bio-Rad Labora-
tories Inc., Hercules, California, USA). The SuperSignal™ West Pico
PLUS Chemiluminescent substrate detection system was from Thermo-
Fisher Scientific (Waltham, Massachusetts, USA). A 2% (w/v) Aqueous
Solution of Gentian Violet was from Ricca Chemical Company (Arling-
ton, TX, USA). Dulbecco’s modified Eagle’s medium (DMEM) were from
Corning (Corning, Manassas, VA, USA). Cascade BiologicsR PMA-free
Human Melanocyte Growth Supplement (HMGS-2, # S0165), and me-
dium 254 (# Mꢀ 254ꢀ 500) from GibcoTM Invitrogen Cell culture
(ThermoFisher Scientific, Illinois, USA). RIPA buffer and Pierce BCA™
protein assay kit was purchased from ThermoFisher Scientific (Rock-
ford, Illinois, USA). EpiLife® Medium with 60 µM calcium was from Life
Technologies. The quick coating solution (cAP-01) was purchased from
Angio-Proteomie (Boston, MA, USA). The Dulbecco’s phosphate-
buffered saline (DPBS) and phosphate buffer saline (PBS) 1X, defined
trypsin inhibitor (DTI) 1X (R007-100), trypsin EDTA 0.25%, 1X
(R25200-072), Trypsin neutralizer (TN) 1X (R-002–100), penicillin
streptomycin (Pen Strep,15140–122) (PEST) 100X were purchased from
Gibco, ThermoFisher Scientific (Rockford, IL, USA) and human kerati-
nocyte growth supplement (HKGS) 100X (S-001–5) from Cascade Biol-
ogist. USDA approved Origin Fetal Bovine Serum (FBS) was from VWR
Seradigm Life Science (Missouri, Texas, USA). The organic solvents
including, ethanol (6183–10) and methanol (BDH 1135-4LP) were ac-
quired from Macron Chemicals (Radnor, Pennsylvania, USA) and VWR
(Missouri, Texas, USA) respectively.
3-Hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one (F23): 1H
NMR (400 MHz, CDCl3): 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.96 (s,
3H, OCH3), 6.55 (brs, 1H, OH), 6.65 (s, 1H, ArH), 7.22 (s, 1H, ArH), 7.39
(t, 1H, J = 7.2 Hz, ArH), 7.51 (d, 1H, J = 8.4 Hz, ArH), 7.64–7.68 (m, 1H,
ArH), 8.26 (d, 1H, J = 8.0 Hz, ArH). 13C (100 MHz; CDCl3): 56.1, 56.5,
57.0, 97.9, 110.8, 113.2, 118.29, 118.35, 121.3, 124.3, 125.40, 125.44,
133.2, 138.5, 143.1, 145.7, 151.9, 152.4, 155.8, 173.1. IR (KBr): 3284,
2922, 2876, 1606, 1566, 1469, 1386, 1282, 1210, 1163, 1105, 1029,
776 cmꢀ 1. HRMS (ESI) calcd for C18H17O6 [M + H]± 329.1020; found,
329.1021.
7-Chloro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
(F24): 1H NMR (400 MHz, CDCl3): 3.86 (s, 6H, 2xOCH3), 3.94 (s, 3H,
OCH3), 6.63 (s, 1H, ArH), 7.08 (s, 1H, ArH), 7.33–7.35 (m, 1H, ArH),
7.52 (d, 1H, J = 1.6 Hz, ArH), 8.17 (d, 1H, J = 8.72 Hz, ArH). 13C NMR
(100 MHz, CDCl3): 56.2, 56.6, 57.1, 97.9, 110.5, 113.2, 118.4, 120.0,
125.4, 126.9, 138.8, 139.4, 143.2, 146.2, 152.1, 152.6, 155.9, 172.6. IR
(KBr): 3288, 2921, 2886, 1603, 1513, 1441, 1272, 1211, 1163, 1036,
789 cmꢀ 1. HRMS (ESI) calcd for C18H15ClO6 [M + H]± 363.063; found,
363.0626.
4.4.2. In-vitro enzymatic assays
4.4.2.1. In-vitro CDK2, c-Kits, and mTOR kinase activity assays. The
ability of the fesitin analogs to inhibit c-Kits, CDK2, and mTOR kinase
enzymatic activity was assessed using an in vitro profiling platform, and
performed at Reaction Biology Corporation (Malvern, PA, USA). Briefly,
the specific kinase and substrate pairs alongside with their necessary
cofactors were mixed in a reaction buffer consisting of (20 mM Hepes pH
7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM
Na3VO4, 2 mM DTT, 1% DMSO). Fisetin (F0), and 10 best inverse
docking identified hit compounds, alongside respective positive acti-
vator molecules including rapamycin, staurosporine, and PI-103 at (1
4.3. Inverse-docking analysis
All of the inverse docking analysis was performed on a personal
Macbook installed with IOS operating system. For the docking analysis
of all the selected proteins, “Glide” was used (85). Glide consisted of
multiple modes for docking processing. For the present study, the XP
mode of docking was used. Eleven flavonol hits obtained after MTT
assay were sketched and cleaned by utilizing the “builder tool” option
available in Maestro. The molecules were then optimized with OPLS3
force-field using the “Ligprep” module of Maestro at a pH range of 7 ±
2.0. For each selected target (Table 2), the co-crystallized structures
available in the RCSB database were retrieved and subjected to a well-
established cross docking protocol to select the optimum structure (see
supplemental data [68]) (81–85). All of the selected protein structures
were then optimized using the “protein preparation wizard” module
implemented in the Maestro program and the optimization included
completion of bond order, addition of hydrogen atoms, construction of
missing side chains and loops. Finally, the complexes were minimized
using an OPLS3 force field to RMSD of 0.15 Å employing the “minimize”
option of the protein preparation wizard.
and 10 μM) dissolved in DMSO were conveyed into their respective ki-
nase reaction mixtures. The reaction was then initiated by the addition
of 32P-ATP (specific activity 10 nCi/nl), and incubated for 2 h at rt, and
the reactions later spotted onto P81 ion exchange filter paper (Whatman
Inc., Piscataway, NJ), and the unbound phosphate was eliminated by
extensively washing the filters in 0.75% phosphoric acid. Background
derived from control reactions containing inactive enzyme was sub-
tracted, and the residual kinase activity data expressed as percentage of
the outstanding kinase activity in the test samples compared to vehicle
(DMSO) reactions. Curve fitting and IC50 values were obtained using
GraphPad Prism software version 8.1 (GraphPad Prism Inc., San Diego,
CA, USA).
4.4. Biological evaluation
4.4.3. Cell lines and culture
Human epidermoid carcinoma A431, human melanoma A375 and
SK-Mel-28, and Human immortalized keratinocytes (HaCaT) cell lines
were purchased from American Type Culture Collection (ATCC; Mana-
ssas, VA, USA). The GFP-Expressing A431 (GFP-A431) and A375 (GFP-
A375) cells were purchased from Angio-Proteomie (Boston, MA, USA).
Primary normal human epidermal keratinocytes (NHEK) [86] and HEKa
were procured from ThermoFisher Scientific (Waltham, Massachusetts,
USA), respectively. Primary normal human melanocytes were kindly
donated by Dr. I. Caroline Le Poole, from Loyola University Chicago (IL),
USA, and were cultured in Medium 245 supplemented with HMGS-2, 1%
antibiotic and antimycotic. All immortalized cell lines were grown in
DMEM supplemented with FBS; 5% for A431 cells while A375 and
HaCaT cells were grown in 10% FBS containing DMEM supplemented
4.4.1. Antibodies, chemicals and reagents
MTT dye (3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide, 98% TLC) was purchased from Sigma–Aldrich Chemical Company
(St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from
MP Biomedicals (Irvine, California, USA). The antibodies for immuno-
blotting Bcl-2 (2876S), Bax(2772S), Caspase-3(9662S), mTOR, β-Actin,
pS6 (Ser235/236), S6, TSC2, p4E-BP1 (Thr37/46), 4EBP1, Akt, pAkt
(Ser473), phospho-mTOR(Ser2448) D9C2 (5536S), Phospho-p90RSK,
Phospho-Akt, Phospho-p44/42 MAPK and Phospho-S6 Ribosomal Pro-
tein Detection Cocktail
I (5301S), and horseradish peroxidase-
conjugated (HRP) anti-mouse and anti-rabbit secondary antibodies
were all obtained from Cell Signaling Technologies (Beverly, MA, USA).
Antibody to peIF4E (Ser209) (76256) was from Abcam. Mini-protean
15

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Bioorganic Chemistry 107 (2021) 104595
with 1% PEST (100X; 100U/ml penicillin, 100
μ
g/ml streptomycin).
4.4.6. Preparation of cellular protein lysates and western blotting/
immunoblotting:
NHEK and HEKa were seeded unto collagen coated tissue culture flasks
grown in low calcium EpiLife Medium supplemented with HKGS and 1%
PEST. All cultured cell lines were maintained at 37˚C in a 95% humidified
atmosphere with 5% CO2. For all experiments in their logarithmic
growth phase, the growth media were routinely changed every 2–3 days
until cell reach desired confluence 65–80% prior to treatment. For sub-
culturing, the cells were harvested after trypsin/EDTA treatment at 37
◦C. For harvest, primary cells grown in serum-free medium were tryp-
sinized, and trypsin was inhibited using DTI and/or TN. In A431 and
A375 cells, the effect of trypsin was inhibited with two-fold volume of
10% FBS containing growth medium. A 10 mM stock solution of parent
(fisetin) and each of the synthesized flavonol derivatives were prepared
in DMSO, and further serially diluted in respective growth media during
test treatment. Control cells were treated with equal volume of vehicle
alone at a final concentration of 0.1% (v/v) DMSO, concentration that
showed no effect on cell viability.
For cell lysates and immunoblot analyses, cell were plated in 10 cm2
plates at 3 × 105 cells/well in respective media overnight. After cell
monolayers were seeded overnight media were removed, and then cells
were treated with or without appropriate test hit drug dilutions (<0.1%
DMSO final) and incubated for 48 h. Cells were harvested by aspirating
media and washing the cells with cold 1xPBS (pH 7.4). The whole cell
lysates were prepared with cell lysis RIPA buffer supplemented with
protease and phosphatase inhibitors;
1
mM PMSF (phenyl-
methylsulfonyl fluoride), 10 mM RIPA buffer, 1 mM Protease inhibitor
cocktail (1 mM EDTA, 20 mM NaF, 0.5% NP-40, 1% Triton X-100)
(Roche) and 0.5% Na3VO4 at pH 7.4. The cells were incubated for 15
min in added ice-cold lysis RIPA buffer lysates, prepared as described
previously [38,87]. The protein concentration of the cleared lysates
were determined using a BCA protein assay kit (Thermo Scientific,
Pierce) according to the manufacturer’s protocol assayed using Eppen-
dorf BioPhotometer D30, and were aliquoted and stored at ꢀ 80 ◦C for
further analysis as earlier described [38].
4.4.4. Cell growth and death by trypan blue dye exclusion assay
To estimate cell growth/viability, melanoma, non-melanoma
alongside normal control cells were seeded (105 cells/well) in 6-well
plates with in 2 mL of their respective FBS containing growth medium
at 37 ◦C in a 5% CO2 incubator overnight. Cells were refed with fresh
medium, and treated with different doses of fisetin or various derivatives
For Western blotting analyses, experiments were performed essen-
tially as previously described [38], by denaturing in 2X Laemmli sample
buffer and resolving equal amount of protein (approximately 20–40 µg
of protein) loaded per lane on 8–12% sodium dodecyl sulfa-
te–polyacrylamide gels (SDS-PAGE) or on 4–12% polyacrylamide ready
mini-PROTEAN TGX gels electrophoresis to separate proteins based on
molecular weight. After running, the protein separated were transferred
onto nitrocellulose membrane using Trans-Blot Turbo Transfer Pack
according to manufacturer’s instructions (Bio-Rad). Followed by
washing, the membrane was blocked using 5% Bovine serum albumin
(BSA) for phosphorylated-proteins, or 5–7% non-fat dry milk in 1%
Tween-twenty in 1% Triton-X100-1xPBS (PBST) at room temperature
for 45 min. Briefly, following blocking membranes were incubated with
appropriate monoclonal or polyclonal primary antibody diluted at 1:250
to 1:1000 in the blocking buffer for 2 h at RT or overnight at 4 ◦C, fol-
lowed by two x10min washes. The blots were washed three times with
PBS: 0.1% Tween 20 (PBST) and incubated for 2 h at room temperature
(RT), on a shaker with 1:2000 diluted anti-mouse or anti-rabbit horse-
radish-peroxidase conjugated (HRP) secondary antibody obtained from
Amersham Life Science Inc. (Arlington Height, IL, USA). The blots were
then rewashed, exposed to enhanced chemiluminescence (ECL) (Amer-
sham life science incorporation), plus west pico and subjected to auto-
radiography. The intensity of each band was measured using Bio-Rad
ChemiDoc™ detection and imaging analysis software systems as earlier
described [38]. The blots were developed after 5 min of incubation with
ECL and auto exposure. Equal loading of protein was confirmed by
stripping the blotted membranes and re-probing with appropriate
loading controls; for β-actin, GAPDH or vinculin, and the results dis-
played are representative of more than two independent experiments
each performed in triplicate with comparable results. All primary anti-
bodies were used at 1:250 to 1:1,000 dilutions except for β-actin,
GAPDH, vinculin, which were at 1:2,000 dilution. Densitometric ana-
lyses of the visualized protein bands were performed using the BioRad
digitized scientific software program Quantity One. Bands were scanned
and processed with Adobe Photoshop CC 8.0 (Adobe Systems, San Jose,
CA, USA). Linear range of band densities was ensured by performing
multiple exposures and final data were analysed by one-way ANOVA.
(0, 1, 10, or 25 μmol/L). After 24 or 48 h of treatment, total cells were
harvested by brief trypsinization, and washed with 1xPBS. Total cell
number was determined by counting each sample in duplicate using a
hemocytometer under an inverted microscope and by using analysis by
an automated countess II cell counter from ThermoFisher Scientific
(Waltham, Massachusetts, USA). Cell viability was determined using the
trypan blue exclusion method. The data shown in this study are the
mean of 3 independent experiments.
4.4.5. Evaluation of cytotoxicity and cell viability by MTT assay
To estimate cell viability and cytotoxicity, proliferation of the
different melanoma and non-melanoma cells and cytotoxicity of the
different agents tested in diverse concentrations were assessed by using
MTT assay. Seeded cells in 5–10% FBS/DMEM were incubated at 37 ◦C
in a 5% CO2 incubator and were re-fed with fresh medium after 24 h.
Briefly, cells were seeded in 200 μl of growth medium in a density of
5x103 cells per well on a 96-well plate and grown in their respective
media in log growth phase until 70% confluent prior to treatment. The
different cells, each in 8 to 10 repeats were treated with or without the
different test compounds dissolved in DMSO (maximal incubation con-
centration of DMSO was 0.1%) at varied concentrations, and incubated
as above for 48 h, and the cells viability was measured by using MTT
assay as previously described [38]. The tested compounds were pre-
pared according to their solubility in DMSO, at the incubation concen-
trations of two-fold-serial dilution with maximum concentration as
indicated (1–80 μmol/L). The treatment was carried out in a humidified
atmosphere with 5% CO2 at 37 ◦C, for 48 h. At harvest, the individual
plates were vigorously shaken for 10–15 min to solubilize formazan
crystals and absorbance was recorded at 562 nm on a SynergyLX, multi-
mode microplate reader (BioTek Instruments Inc., Winooski, VT, USA).
The effect of each agent on growth inhibition was assessed, as a per-
centage cell viability where DMSO-treated cells (untreated controls) was
considered as 100% viable [87].
Cell proliferation inhibitory curves were constructed for each com-
pound, plotting incubation concentrations vs. percentage of viability
relative to untreated control. The standard toxicological parameter IC50
was calculated by a nonlinear regression analysis of the inhibitory
curves using GraphPad Prism software version 9.1, (GraphPad Software,
Inc., La Jolla, CA, USA).
4.4.7. Scratch wound healing assay
The cells (A431 and A375), were seeded in 24 well plates and
cultured to confluence in DMEM complete media. Two parallel straight
scratched wounds were made across the centre of the cell monolayer in
each well using a sterile 200-µL Gilson pipette tip, followed by extensive
washed. Their respective growth media with or without test compounds
at varied concentrations were replenished and incubated for 24 – 48 h
(Maximum of 0.1% DMSO containing medium served as control) as
previously described [88]. Briefly, phase contrast images were taken
16

T. Roy et al.
Bioorganic Chemistry 107 (2021) 104595
with a 10x, 20x objective lenses at regular intervals starting from 0 h
when the wounds were created, to 24 h and 48 h post-treatment using an
Olympus IX51 phase-contrast microscope. Cell migration and wound
healing was determined using ImageJ and calculated as a percentage of
initial cell-free areas vs percentage of cell-free areas after treatment
[88].
Funding
The research work reported in this publication was supported in part
by a start-up fund from the University of Louisiana at Monroe (ULM)
College of Pharmacy (to JCC), a ULM College of Pharmacy Faculty
Research Seed grant 5CALHN-260615 (to JCC), LBRN Pilot Awards (to
JCC and SM) from an Institutional Development Award (IDeA) Networks
of Biomedical Research Excellence (INBRE), award from the National
Institute of General Medical Sciences of the National Institutes of Health
(NIGMS/NIH) grant number P2O GM103424-18 (to KGK), NIH NCI
grants CA191550 and CA243167 (to VSS), and an LBRN-INBRE
Administrative Supplement Award (to JCC) from NIH/NIGMS grant
3P20GM103424-18S1 (to KGK). MS thanks the Fondazione di Sardegna
for its partial support.
4.4.8. Colony formation assay
To investigate the effects of flavonol derivatives on clonogenic po-
tential of A375 and A431cells, colony formation assay was performed as
previously described [88]. Briefly, cells were seeded in T-25 tissue cul-
ture flasks overnight, and 65% confluent log phased cells were treated
with compounds F6, F9, F11, F19, F20 at various doses corresponding
to 0, ½IC50, IC50 and 2xIC50 supplemented in respective growth media
for 48 h, with the exception of F6 and F9 which concentrations were 7.5,
15 and 30 μM in both cell lines (supplementary data (68)). After treat-
ment and incubation period, cells were trypsinized, enumerated and
then, low number of cells (~1000 cells) were seeded per 10-cm2 plate in
fresh media, and cells were grown for 10–14 days with normal drug-free
media. The media were replenished after every 2–3 days until control
cell colonies were confluent prior to harvest as describe previously [88].
As a vehicle control, control cells were treated with DMSO (≤0.1% v/v),
and were maintained under standard cell culture conditions at 37 ◦C and
5% CO2 in a humidified atmosphere. At harvest, cell colonies were
stained with a cell fixative solution containing 0.5% Gentian violet
staining agent in ≈ 64% (v/v) methanol and 4% paraformaldehyde in
protocol as previously described [88]. After staining, the plates were
immersed three-consecutive times in running tap water and air-dried
overnight. The photomicrographs of colonies were counted accord-
ingly using a phase-contrast Olympus IX51 microscope at 10x magnifi-
cation or using count PHICS analysis tool [89]. Using the PHICS, the
least counts for colonies was 22 for 10 cm plate and 21 for 6 well plate
with a range of 0.1 to 1.4 mm.
Author Contributions
J. C. C., conceived the research idea, designed and administered the
research study; S. M. and M. S., contributed in the study design; T. R., S.
T. B., S. B., P. K. S., P. B., R. C., F. L., J.F., R. A. H., B. M. N., A. L. W., S.
M., M. S., and J. C. C., performed Chemistry, Computational and Bio-
logical investigations; T. R., S. T. B., S. B., P. K. S., V. S. S., R. C., P. B., F.
L., A. L. W., B. M. N., and J. C. C. acquired data; T. R., S. B., S. T. B., J. F.,
S. M., B. M. N., K. G. K., M. S., and J. C. C., analyzed and interpreted the
data; T. R., P. S. K., M. S. and J. C. C., drafted the original manuscript and
figure presentation;V. S. S., R. A. H., A. L. W., J. F., B. M. N., K. G. K., S.
M., M. S., and J. C. C., wrote, edited and critically revised the manuscript
and provided important intellectual input; R. A. H., V. S. S., S. M., K. G.
K., M. S., and J. C. C., provided guidance and key insights in study
planning and funding acquisition; S. M., J. F., M. S. and J. C. C., su-
pervised the study; All authors revised, edited, read, and approved the
final version of the manuscript submitted.
4.4.9. Immunofluorescence analysis of GFP tagged cells for morphology
and apoptosis:
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