α-Trifluoromethyl Chalcones as Potent Anticancer Agents for Androgen Receptor-Independent Prostate Cancer

Article abstract of DOI:10.3390/molecules26092812

α-Trifluoromethyl chalcones were prepared and evaluated for their antiproliferative activities against androgen-independent prostate cancer cell lines as well as five additional types of human tumor cell lines. The most potent chalcone 5 showed superior antitumor activity in vivo with both oral and intraperitoneal administration at 3 mg/kg. Cell-based mechanism of action studies demonstrated that 5 induced cell accumulation at sub-G1 and G2/M phases without interfering with microtubule polymerization. Furthermore, several cancer cell growth-related proteins were identified by using chalcone 5 as a bait for the affinity purification of binding proteins.

Full text of DOI:10.3390/molecules26092812

molecules
Article
α-Trifluoromethyl Chalcones as Potent Anticancer Agents for
Androgen Receptor-Independent Prostate Cancer
1
2,
2
2
3
Yohei Saito , Atsushi Mizokami * , Kouji Izumi , Renato Naito , Masuo Goto
and
Kyoko Nakagawa-Goto 1
,3,
*
1
2
3
School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Science,
Kanazawa University, Kanazawa 920-1192, Japan; saito-y@staff.kanazawa-u.ac.jp
Department of Integrative Cancer Therapy and Urology, School of Medical Sciences, Kanazawa University,
Kanazawa 920-1192, Japan; kouji1974@staff.kanazawa-u.ac.jp (K.I.); thealfuu@yahoo.co.jp (R.N.)
Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599, USA; goto@med.unc.edu
*
Correspondence: mizokami@staff.kanazawa-u.ac.jp (A.M.); kngoto@p.kanazawa-u.ac.jp (K.N.-G.);
Tel.: +81-76-265-2393 (A.M.); +81-76-264-6305 (K.N.-G.)
Abstract: α-Trifluoromethyl chalcones were prepared and evaluated for their antiproliferative ac-
tivities against androgen-independent prostate cancer cell lines as well as five additional types of
human tumor cell lines. The most potent chalcone
both oral and intraperitoneal administration at 3 mg/kg. Cell-based mechanism of action studies
demonstrated that induced cell accumulation at sub-G1 and G2/M phases without interfering
5 showed superior antitumor activity in vivo with
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
5
ꢀ
ꢁꢂꢃꢄꢅꢆ
with microtubule polymerization. Furthermore, several cancer cell growth-related proteins were
Citation: Saito, Y.; Mizokami, A.;
Izumi, K.; Naito, R.; Goto, M.;
Nakagawa-Goto, K.
identified by using chalcone 5 as a bait for the affinity purification of binding proteins.
Keywords: chalcone; trifluoromethyl group; prostate cancer; antitumor activity; target proteins
α-Trifluoromethyl Chalcones as
Potent Anticancer Agents for
Androgen Receptor-Independent
Prostate Cancer. Molecules 2021, 26,
1. Introduction
2
812. https://doi.org/10.3390/
A small change in a chemical structure sometimes induces a big change in a biological
molecules26092812
profile. The features of substituents on a bioactive molecule influence the various interac-
tions between it and its target protein and subsequently affect its selectivity and potency,
Academic Editor: Maria Emília de
Sousa
as we have previously reported [1,2]. Fluorine’s unique properties, such as small atomic
size and high electronegativity, impact pKa, molecular conformation, binding affinity
with target molecules, membrane permeability, metabolic pathways, and pharmacokinetic
properties of bioactive molecules. Accordingly, fluorine attracts attention in drug discovery
Received: 19 April 2021
Accepted: 6 May 2021
Published: 10 May 2021
and development [3]. In our continuing structure–activity relationship (SAR) study of
chalcone derivatives as anti-prostate cancer agents [4,5
of fluorine and/or a fluorinated functional group into synthetic chalcones would improve
their activity. Chalcone, a biosynthetic intermediate of various flavonoids, is composed of
], we explored whether the insertion
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
two aromatic rings connected by
α
,
β
-unsaturated carbonyl unit. Initially, we introduced a
-position of
the olefin. This group might accelerate Michael addition of a nucleophile in biomolecules,
such as the SH in cysteine [ ]. CF has been also used as an isostere of a methyl group or
amide C=O bond with the aim of improving metabolic stability and hydrophobicity in drug
development [ ]. Secondly, we introduced electron withdrawing groups on the aromatic
ring connected to the olefinic double bond of the -CF chalcones. The synthesized -CF3
analogues were evaluated for anti-prostate cancer activity.
trifluoromethyl (CF ) group, a known electron withdrawing group, at the
α
3
6
3
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
7
α
α
3
In many cases, prostate cancer progresses slowly and is generally controllable by
hormone therapy and/or castration. However, the cancer can reoccur after several years
and then is termed castration-resistant prostate cancer (CRPC). Currently, no effective
treatment exists for CRPC. Prostate cancer is associated with androgen-receptors (ARs),
4
.0/).
Molecules 2021, 26, 2812. https://doi.org/10.3390/molecules26092812
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2812
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and prostate cancer cells grow in response to androgen. While hormone therapy suppresses
androgen secretion and restrains cancer cell growth, continued treatment for several years
often leads to diminished effectiveness due to the induction of mutations on the AR, which
then can be activated by only a small amount of androgen or independently of constitutive
active ligand [
8]. In addition, the emergence of prostate cancer that lacks AR expression
accelerates its malignancy. In this study, the synthesized
α
-CF chalcones were assayed
3
against DU145 and PC-3 cell lines, which do not express ARs, to evaluate their effectiveness
against CRPC. The most potent derivative was further investigated in mode of action
studies, including identification of target proteins, and for in vivo antitumor effects. The
differences of intracellular targets of previously reported
were also discussed.
α-substituted chalcones [9–11]
2. Results and Discussion
2.1. Chemistry
The novel
from the related chalcones [13
Claisen-Schmidt condensation of aryl methyl ketones and aromatic aldehydes. In target
compounds , the aromatic aldehyde was either unsubstituted or substituted with one
of three electron withdrawing groups (F, CF , or NO ) or an electron donating group
α
-trifluoromethyl chalcones (
2–7) and known 1 [12], were synthesized
–
16] including novel 10 and 11, which were obtained by
1–7
3
2
(
NMe ) for comparative purposes. The CF moiety was inserted at the α-position of the
2
3
chalcones by using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent) [17] in
◦
the presence of CuI at 80 C using a reported method (Scheme 1) [18]. In
6
, the chalcone
, the chalcone ring-B
was replaced with benzothiophene, since potent antiproliferative effects were observed
with other flavonoids containing 10 -electron aromatic ring systems [ 19 20]. It should be
noted that the conformation of the olefin in the resulting -CF chalcones is cis rather than
ring-A was changed from benzene to naphthalene and, in derivative
7
π
1
,
,
α
3
trans as in the natural chalcone, which has been confirmed with X-ray crystal structure
analysis by Bi et al. [18] and might also affect the biological activities. Spectroscopic data of
all newly synthesized compounds are in Supplementary Materials (Figures S1–S40).
a
a
Scheme 1. Synthesis of
1
α
-CF chalcones . Reagents and conditions: (
a) 40% KOH, EtOH, room temp. (b) 1-Trifluoromethyl-
3
◦
,2-benziodoxol-3(1H)-one, CuI, DMF, 80 C.
2
.2. Biological Evaluation
Antiproliferative Activity of Compounds against AR-Independent Cells
The antiproliferative activities of the synthesized -CF chalcones against AR-independent
cell lines, DU145 and PC-3 were evaluated (Table 1). 5 -Chloro-2,2 -dihydroxychalcone
Cl-DHC), which was found as a potent antiproliferative chalcone by our group [ ], was
used as a control. All -CF chalcones showed potent activity; especially 4-NO chalcone
and 3,4-difluorochalcone
values of less than 0.2 M. These results indicated the insertion of CF at the
beneficial to the antiproliferative activity, since most of the potent compounds among our
previously synthesized chalcones without an -CF3 [ ] exhibited IC50 values of over 5 M.
α
3
0
0
(
5
α
2
3
2
5
strongly inhibited the growth of both tumor cell lines with IC50
-position was
µ
α
3
α
4,
5
µ

Molecules 2021, 26, 2812
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Regarding the chalcone ring-A, although the
α
-CF chalcone
6
with a naphthyl ring-A was
3
active, it was threefold less potent than the analogous chalcone
Among this limited compound set, electron withdrawing groups on ring-B resulted in
slightly improved antiproliferative activity, as the non-substituted chalcone and 4-NMe2
chalcone 3 were less potent than 4-NO 2, 4-CF 4, and 3,4-difluoro 5.
5 with a phenyl ring-A.
1
2
3
Table 1. Antiproliferative activity against androgen-independent prostate cancer cell lines, DU145
and PC-3.
Cell Lines/IC50 (µM) a
Cell Lines/IC50 (µM) a
Compounds
DU145
PC-3
Compounds
DU145
PC-3
1
2
3
4
0.69 ± 0.30
0.19 ± 0.01
0.94 ± 0.27
0.45 ± 0.05
0.44 ± 0.06
0.15 ± 0.02
0.44 ± 0.03
0.46 ± 0.01
5
6
7
0.19 ± 0.04
0.54 ± 0.05
1.44 ± 0.18
4.50 ± 0.89
0.15 ± 0.03
0.44 ± 0.12
1.07 ± 0.18
1.52 ± 0.25
Cl-DHC b
a
The concentration of compound that caused 50% reduction of cell growth relative to untreated cells determined by
cell counting. The values are average
which was previously synthesized by our group [5].
±
SD of three independent experiment. b 5 -Chloro-2,2 -dihydroxychalcone,
0 0
All synthesized chalcones were also assayed against five additional human tumor cell
lines, non-small cell lung (A549), triple-negative breast (MDA-MB-231), estrogen-responsible
breast (MCF-7), cervical cancer cell line HeLa derivative (KB), and P-glycoprotein (P-gp)
overexpressing multidrug resistant KB subline (KB-VIN). Again, the chalcones containing
electron withdrawing group on ring-B (2, 4, and 5) showed more potent antiproliferative ac-
tivity than the remaining compounds (Table 2). In addition, KB and KB-VIN cells displayed
similar susceptibility to the tested chalcones. This finding suggested that the compounds
might target specific proteins that are critical for cell growth and overexpressed in KB and
KB-VIN cells.
Table 2. Antiproliferative activity of compounds 1–7 against other tumor cell lines.
Cell Line a (IC50 µM) b
Compounds
A549
MDA-MB-231
MCF-7
KB
KB-VIN
1
2
3
4
5
6
7
4.41 ± 0.27
0.52 ± 0.01
4.29 ± 0.12
2.97 ± 0.35
0.33 ± 0.03
3.71 ± 0.07
1.32 ± 0.16
4.90 ± 0.05
3.82 ± 0.24
0.47 ± 0.02
3.94 ± 0.30
0.42 ± 0.06
0.46 ± 0.01
3.14 ± 0.61
0.62 ± 0.01
6.78 ± 0.20
4.21 ± 0.14
0.68 ± 0.04
4.93 ± 0.10
1.49 ± 0.21
0.60 ± 0.01
4.07 ± 0.14
0.89 ± 0.05
10.94 ± 0.16
4.47 ± 0.07
0.55 ± 0.01
4.20 ± 0.41
0.88 ± 0.12
0.58 ± 0.02
4.24 ± 0.00
4.61 ± 0.10
5.24 ± 0.08
4.56 ± 0.16
0.84 ± 0.00
4.93 ± 0.10
2.45 ± 0.38
0.67 ± 0.35
4.33 ± 0.12
4.29 ± 0.20
1843.5 ± 29.9
paclitaxel (nM)
a
A549 (lung carcinoma), MDA-MB-231 (triple-negative breast cancer), MCF-7 (estrogen receptor-positive and
HER2-negative breast cancer), KB (cervical cancer cell line HeLa derivative), KB-VIN (P-gp-overexpressing
MDR subline of KB). The values are average
expressed as IC50 values for each cell line, the concentration of compound that caused 50% reduction relative to
untreated cells determined by the SRB assay.
b
±
SD of three independent experiment. Antiproliferative activity
The resistance of tumor cells to drugs is always a severe obstacle to effective chemother-
apy. As shown in Table 2, all tested compounds showed similar antiproliferative activity
against KB and KB-VIN, suggesting that our compounds were not affected by the drug
transporter P-gp. The most promising chalcone
5 was further tested against four kinds
of taxane-resistant prostate cancer cell lines, DU145/TxR (docetaxel resistant DU145),
DU145/TxR/CxR (docetaxel and cabazitaxel resistant DU145), PC-3/TxR (docetaxel re-
sistant PC-3), and PC-3/TxR/CxR (docetaxel and cabazitaxel resistant PC-3) [21]. Chal-
cone
.14–0.28
overcomes castration and taxane resistances.
5
showed significant antiproliferative activity against these cells with IC50 values of
0
µ
M (Table 3). Taken together, the results suggesting that chalcone potentially
5

Molecules 2021, 26, 2812
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Table 3. Antiproliferative activity against docetaxel and cabazitaxel resistant prostate cancer cell
lines DU145/TxR, DU145/TxR/CxR, PC-3/TxR and PC-3/TxR/CxR.
Cell Lines/IC50 (µM) a
Compounds
5
DU145/TxR
DU145/TxR/CxR
PC-3/TxR
PC-3/TxR/CxR
0.14 ± 0.03
0.21 ± 0.01
0.28 ± 0.08
0.25 ± 0.02
a
The concentration of compound that caused 50% reduction of cell growth relative to untreated cells determined
by cell counting. The values are average ± SD of three independent experiment.
To estimate the in vivo antitumor effects of chalcone
mor model assay using PC-3. As anticipated, the tumor growth was efficiently suppressed
with both intraperitoneal and oral administration of without significant weight loss com-
5, we tested it in a xenograft antitu-
5
pared with control (Figure 1). Notably, a dose of only 3 mg/kg was used in this study, even
though many reported studies have used much larger doses of test compounds.
Figure 1. Effect of
5
against PC-3 tumor xenograft in C.B-17 scid mice. Compounds were intraperitoneally administrated
at the indicated doses twice a week (n = 4) (
a
,
b
) or orally administrated at the indicated doses three times a week (n = 5)
) Average body weights of mice during
SD. Significance was defined as * p < 0.05, ** p < 0.005
(
c,d
). ( ) Tumor volume in SCID mice during treatment with the compounds. (b,d
a
,
c
treatment with the compounds. Data were presented as the average
using Student’s t test.
±
The identification of target proteins is an important step in drug discovery and devel-
opment. Accordingly, to study the mode of action of our new chalcones, we performed
affinity labeling using a specifically designed and synthesized chemical probe (
9
) from
0
the novel
α
-CF chalcone
8. 4 -Amino-3,4-difluorochalcone [22] was selected for the intro-
3
duction of propargyl group, which would work as a tether to immobilize on azide beads.
0
Trifluoromethylation of 4 -amino-3,4-difluorochalcone gave
8
and propargylation of the
(Scheme 2). The antiproliferative activities
M, respectively (Table 4). Thus, chalcone
M for DU145 and 2.48 M for PC-3). In
showed insignificant effects against KB-VIN despite
amino group on
of against DU145 and PC-3 were 1.43 and 1.34
was 1.9–2.3 fold more potent than
flow cytometric analysis, both and
8 provided the target chalcone 9
9
µ
9
8
9
(IC50 3.25
µ
µ
8

Molecules 2021, 26, 2812
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using a three-fold concentration of their IC50 value. However, the treated MDA-MB-231
cells clearly showed accumulations of sub-G1 phase, a typical apoptotic pattern, and G2/M
phase cells (Figure 2a).
a
a
Scheme 2. Synthesis of chemical probe for the identification of target proteins . Reagents and conditions: (a) 1-
◦
◦
Trifluoromethyl-1,2-benziodoxol-3(1H)-one, CuI, DMF, 80 C. (b) 3-Bromo-1-propyne, K CO , MeCN, 80 C.
2
3
Compared with the parent molecule
5
, chalcones
8
and 9 showed diminished antipro-
liferative effects against all tested human tumor cell lines (Tables 3 and 4). However, all
three compounds displayed the same distribution pattern using flow cytometric analysis
(
Figure 2a), indicating the same intracellular targets. The tubulin polymerization inhibitor
combretastatin A-4 (CA-4) was also evaluated in this study, since antiproliferative chal-
cones often target tubulin [23 24]. However, the effects of chalcone on the cell cycle
,
5
progression were clearly distinguishable from those of CA-4 (Figure 2a). Because G2/M
phase accumulation often results from treatment with tubulin inhibitors [25], we evaluated
the effects of chalcone
cytochemical studies with an antibody to
images indicated that chalcone did not significantly affect either tubulin polymerization
or cell morphology. The above cell-based mode of action studies suggested that chalcone
targets proteins related to the cell cycle progression in G2/M and apoptotic induction.
Therefore, we next conducted affinity purification of chalcone -binding proteins using
5
on tubulin polymerization and cell morphology by using immuno-
α
-tubulin (Figure 2b). The resulting confocal
5
5
5
beads conjugated to compound 9 as a bait to identify the target proteins.
Table 4. Antiproliferative activity of compounds 8 and 9 against other tumor cell lines.
Cell Line (IC50 µM)
Compounds
DU145 a
PC-3 a
A549 b
b
MCF-7 b
KB b
KB-VIN b
MDA-MB-231
8
9
3.25 ± 0.90
2.48 ± 0.60
4.10 ± 0.40
4.81 ± 0.05
4.89 ± 0.05
4.56 ± 0.09
4.98± 0.01
1.43 ± 0.13
1.34 ± 0.07
4.20 ± 0.01
4.53 ± 0.26
4.88 ± 0.05
3.97 ± 0.09
4.51 ± 0.06
a
The concentration of compound that caused 50% reduction of cell growth relative to untreated cells determined by cell counting. The
values are average
cells determined by the SRB assay.
b
±
SD of three independent experiment. The concentration of compound that caused 50% reduction relative to untreated
Since
cation of the intracellular binding proteins of
propargylated probe of . Compound was immobilized on azide beads via a click re-
5
and
9
likely have identical intracellular mechanisms of action, affinity purifi-
5
was carried out using compound , the
9
5
9
action, and the obtained beads were reacted with PC-3 cell lysate followed by elution of
binding proteins using SDS-PAGE sample buffer. The eluted fractions were separated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Five main bands were detected by
staining with Coumassie Brilliant Blue (CBB) dye, corresponding to proteins with molecular
weights between 100 and 40 kDa (Figure 3). Since all bands became undetectable when the
lysate was preincubated with compound 5 as a competitive inhibitor, the bands were cut out
and the proteins were digested by adding trypsin to the gel. The obtained trypsin-digested
peptide sequences were identified by LC-MS/MS analysis. HSP90 and MICOS complex
subunit MIC60 were detected from band No. 1. HSP70 (band No. 2), HSP60 (band No. 3),
pyruvate kinase (band No. 3), T-complex protein 1 subunits (band No. 3), alpha-enolase

Molecules 2021, 26, 2812
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(band No. 4), actin (band No. 5), and proliferation-associated protein 2G4 (band No. 5)
were also identified. The list of identified proteins is shown in Supplementary Materials
Table S1). Heat shock proteins (HSPs) are well known not only as significant factors for
(
cancer development but also targets for cancer therapy [26]. Glycolytic enzymes, including
pyruvate kinase and alpha-enolase, are also related to tumor growth [27] and reported as
diagnostic targets [28]. The unique target MIC60 plays an important role in the maintenance
of mitochondrial crista structure [29] and PA2G4 isoforms display opposing functions, i.e.,
suppressive and promoting effects, in cancer [30]. Compound
cell proliferation mainly by interacting with these proteins. Further studies are merited to
demonstrate the clear target of chalcone followed by the elucidation of druggable targets
5 might suppress cancer
5
for drug development against CRPC. We are currently performing gene knockdown studies
of the expressions of the identified proteins using siRNA in PC-3 cells.
Figure 2. Effect of
5,
8, and
9
on cell cycle progression and of
5
on immunochemical staining. (a) Triple negative breast
cancer MDA-MB-231 and multidrug-resistant KB-VIN cells were treated with
5,
8
, and for 24 h. DMSO was used as a
9
vehicle control (CTRL).
and 2.01 M (3 IC ) for KB-VIN, respectively.
for KB-VIN, respectively.
Combretastatin A-4 (CA-4) was used at 0.2
5
was used at 0.46
µ
M
(1× IC ) and 1.38
µ
M
M (3
(3× IC ) for MDA-MB-231, and 0.67
µ
M
M
(1× IC )
5
0
50
50
µ
×
8
was used at 14.4
µ
×
IC ) for MDA-MB-231, and 14.9
50
µ
(3× IC )
50
50
9
was used at 13.6
µ
M (3
M (3
×
×
IC50) for MDA-MB-231, and 13.5
µM (3
×
IC50) for KB-VIN, respectively.
µ
IC ). Tubulin polymerization inhibitor CA-4 was used as a control for
5
0
mitotic inhibitor (G2/M). Cell cycle distributions of treated cells were analyzed by flow cytometry (LSRFortessa operated by
FACS Diva software, BD Bioscience) after staining with propidium iodide (PI) in the presence of RNase. (
b
) MDA-MB-231
for 24 h at 1.38
-tubulin (green) and
4
cells were seeded in an 8-well chamber slide (2.4
×
10 cells/well) 24 h prior to treatment with
5
α
µM
(
3× IC ). 0.02% DMSO was used for negative control. Fixed cells were labeled with antibodies to
5
0
DAPI for DNA (blue). 14–18 confocal images were stacked and merged. Bar, 25 µm.

Molecules 2021, 26, 2812
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Figure 3. Target identification of compound
purification of binding proteins was performed using compound
detected by CBB staining after SDS-PAGE.
5
. PC-3 cell lysate was preincubated with (+) or without (
−
) compound
5. Affinity
9-immobilized beads. Five main bands (No. 1 to 5) were
3. Materials and Methods
3.1. Chemistry
All solvents and chemicals were used as purchased without further purification. The
progress of reactions was monitored on Merck Millipore precoated silica gel glass plates
TLC Silica gel 60 F254). Column chromatography was performed with Kanto chemical
(
silica gel 60 N (spherical, neutral) or preparative TLC was carried out with Merck Millipore
precoated silica gel glass plates (PLC Silica gel 60 F254, 1 mm) for the purification of
1
13
synthetic compounds. H and C NMR spectra were recorded on JEOL JNM-ECA 600
or JNM-ECS 400 using CDCl as solvent and referenced to TMS or residual solvent peak.
3
Chemical shifts
δ are reported in ppm. Mass spectrometric analysis were performed using
JEOL JMS-700 Mstation or JMS-T100TD. UV spectra were recorded on a Tecan SPARK 10 M
in MeCN-H O (1:1). IR spectra were measured on Shimadzu IRSprit in neat. Melting points
2
were measured on an AS ONE ATM-02. The purity of all compounds was determined as
1
>
95% by H NMR.
3
.2. General Procedures for Chalcones
0
To a solution of 1 -acetonaphthone (120 mg, 0.70 mmol) and 3,4-difluorobenzaldehyde
(100 mg, 0.70 mmol) in EtOH (1.0 mL) was added 40% aqueous KOH (0.5 mL), and the
mixture was stirred at room temperature. After the reaction was complete, ice-water was
added to the reaction mixture, which was then neutralized with 1N HCl. The mixture was
extracted with EtOAc and the resultant organic phase was washed with brine, dried over
MgSO , and concentrated in vacuo. The residue was purified by column chromatography
4
on SiO , eluting with hexane-EtOAc (15:1) to give (E)-3-(3,4-difluorophenyl)-1-(naphthalen-
2
1-yl)prop-2-en-1-one (172 mg, 0.58 mmol) in 83% yield as a colorless solid. All chalcones
were produced by the same procedure.

Molecules 2021, 26, 2812
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3
.2.1. (E)-3-(3,4-difluorophenyl)-1-(naphthalen-1-yl)prop-2-en-1-one (10)
◦
Rf 0.25 (hexane/EtOAc, 10:1); mp 81 C; UV (MeCN/H O, 1:1) λmax (nm) 220, 302;
2
−
1
1
IR (neat) νmax (cm ) 3050, 1652, 1583, 1516, 1271, 1097, 980, 796, 772; H NMR (600 MHz,
CDCl3): 8.33 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 6.6 Hz, 1H), 7.77 (d,
J = 6.6 Hz, 1H), 7.59–7.51 (m, 4H), 7.40 (ddd, J = 10.2, 8.4, 1.8 Hz, 1H), 7.30 (ddd, J = 6.6, 4.2,
δ
1
1
.8 Hz, 1H), 7.22 (d, J = 16.2 Hz, 1H), 7.19 (m 1H); 13C NMR (150 MHz, CDCl3):
51.7 (dd, J = 252.8, 12.9 Hz), 150.7 (dd, J = 248.6, 12.9 Hz), 143.2, 136.7, 133.9, 132.0, 131.9
δ 194.9,
(
1
dd, J = 4.4, 4.4 Hz), 130.4, 128.5, 127.8, 127.6, 127.3, 126.6, 125.5, 125.3 (dd, J = 5.7, 2.9 Hz),
24.5, 117.9 (d, J = 18.6 Hz), 116.6 (d, J = 18.6 Hz); HRMS (FAB) m/z: [M+H] calcd for
+
C H F O 295.0934, found 295.0941.
19
13 2
3.2.2. (E)-3-(benzo[b]thiophen-3-yl)-1-phenylprop-2-en-1-one (11)
The chalcone was prepared from acetophenone and benzo[b]thiophene-3-carboxaldehyde
◦
as a yellow solid. Yield: 70%. Rf 0.3 (hexane/EtOAc, 10:1); mp 94–95 C; UV (MeCN/H O,
2
−
1
1
1
:1) λmax (nm) 226, 266, 356; IR (neat) νmax (cm ) 3091, 3055, 1653, 1586, 1575, 1495, 1367,
1
202, 1010, 971, 780, 729, 690; H NMR (400 MHz, CDCl ):
δ 8.15–8.05 (m 4H), 7.93 (s, 1H),
3
7
.92 (d, J = 7.2 Hz, 1H), 7.66 (d, J = 16.0 Hz, 1H), 7.61 (m, 1H), 7.56–7.49 (m, 3H), 7.44 (dd,
1
3
J = 7.2, 7.2 Hz, 1H); C NMR (150 MHz, CDCl3):
δ
190.4, 140.6, 138.2, 137.3, 136.4, 132.8,
+
132.3, 128.7, 128.7, 128.5, 125.2, 125.1, 123.1, 122.4, 122.2; HRMS (FAB) m/z: [M+H] calcd
for C H OS 265.0687, found 265.0684.
17
13
3
.3. General Procedures for a-CF Chalcones
3
To a mixture of 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (170 mg, 0.22 mmol) and
copper iodide (2.7 mg, 0.014 mmol) was added chalcone (30 mg, 0.14 mmol) in DMF (0.2 mL)
◦
under N atmosphere and the mixture was stirred at 80 C. After the reaction was complete,
2
the mixture was concentrated. The residue was purified by column chromatography on
silica gel, eluting with hexane-EtOAc (50:1) to give (E)-1,3-diphenyl-2-(trifluoromethyl)prop-
2
-en-1-one (
1) [9] (9.4 mg, 0.034 mmol) in 24% yield as a pale, yellow oil. α-CF chalcones
3
2–8 were produced by the same procedure.
3.3.1. (E)-3-(4-nitrophenyl)-1-phenyl-2-(trifluoromethyl)prop-2-en-1-one (2)
Compound
2
was prepared from 4-nitrochalcone [10] as a colorless solid. Yield: 9%.
◦
Rf 0.25 (hexane/EtOAc, 10:1); mp 119–120 C; UV (MeCN/H O, 1:1) λmax (nm) 262; IR
2
−
1
1
(
(
1
neat) νmax (cm ) 3079, 2933, 2859, 1667, 1596, 1520, 1346, 1267, 917, 824, 687; H NMR
600 MHz, CDCl3):
δ
8.06 (d, J = 8.4 Hz, 2H), 7.87 (dd, J = 8.4, 1.2 Hz, 2H), 7.56 (tt, J = 7.2,
.2 Hz, 1H), 7.54 (s, 1H), 7.43 (d, J = 9.0 Hz, 2H), 7.40 (t, J = 7.8 Hz, 2H); 1 C NMR (125 MHz,
3
CDCl3):
δ
192.7, 148.3, 138.2, 135.0, 134.9, 134.1 (q, J = 5.9 Hz), 130.1, 129.6, 129.1, 123.9;
+
HRMS (FAB) m/z: [M+H] calcd for C H F NO 322.0691, found 322.0678.
16
11
3
3
3
.3.2. (E)-3-[4-(dimethylamino)phenyl]-1-phenyl-2-(trifluoromethyl)prop-2-en-1-one (3)
Compound was prepared from 4-(dimethylamino)chalcone [11] as a yellow oil.
Yield: 26%. Rf 0.2 (hexane/EtOAc, 10:1); UV (MeCN/H O, 1:1) λmax (nm) 254, 336, 398; IR
3
2
−
1
(
neat) νmax (cm ) 3060, 2909, 2861, 2816, 2359, 1660, 1593, 1526, 1278, 1147, 1108, 814, 696,
7.96 (dd, J = 8.4, 1.2 Hz, 2H), 7.52 (tt, J = 7.2, 1.2 Hz, 1H),
.39 (t, J = 8.4 Hz, 2H), 7.34 (d, J = 1.2 Hz, 1H), 7.11 (d, J = 9.2 Hz, 2H), 6.44 (d, J = 9.2 Hz,
193.9, 151.3, 137.3 (q, J = 5.7 Hz), 136.0,
34.0, 131.8, 129.7, 128.8, 119.3, 111.4, 39.9; HRMS (FAB) m/z: [M+H] calcd for C H F NO
1
6
7
2
87; H NMR (400 MHz, CDCl ):
δ
3
H), 2.91 (s, 6H); 13C NMR (100 MHz, CDCl3):
δ
+
1
1
8
17 3
320.1262, found 320.1262.
3
.3.3. (E)-1-phenyl-2-(trifluoromethyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4)
Compound was prepared from 4-(trifluoromethyl)chalcone [12] as a pale-yellow oil.
Yield: 9%. Rf 0.4 (hexane/EtOAc, 10:1); UV (MeCN/H O, 1:1) λmax (nm) 254; IR (neat)
4
2
−
1
1
νmax (cm ) 3067, 2928, 2853, 1670, 1323, 1269, 1163, 1067, 834, 687; H NMR (400 MHz,
CDCl3): 7.89 (dd, J = 8.4, 1.2 Hz, 2H), 7.56 (tt, J = 7.2, 1.2 Hz,1H), 7.51 (s, 1H), 7.47 (d,
δ

Molecules 2021, 26, 2812
9 of 13
J = 8.4 Hz, 1H, H-Ar), 7.42–7.36 (m, 4H); 13C NMR (150 MHz, CDCl3):
δ
192.0, 135.4, 135.1,
1
1
35.0 (q, J = 5.7 Hz), 134.7, 131.8 (q, J = 33.0 Hz), 131.4 (q, J = 30.2 Hz), 129.6, 129.6, 129.0,
25.8 (q, J = 4.2 Hz), 123.5 (q, J = 270.0 Hz), 121.9 (q, J = 274.4 Hz); HRMS (FAB) m/z: [M+H]+
calcd for C H F O 345.0714, found 345.0716.
17
11 6
3
.3.4. (E)-3-(3,4-difluorophenyl)-1-phenyl-2-(trifluoromethyl)prop-2-en-1-one (5)
Compound was prepared from 3,4-diifluorochalcone [13] as a colorless oil. Yield:
9%. Rf 0.3 (hexane/EtOAc, 10:1); UV (MeCN/H O, 1:1) λmax (nm) 254; IR (neat) νmax
5
2
2
−
1
1
(
cm ) 3064, 2918, 1667, 1517, 1263, 1119, 917, 686; H NMR (600 MHz, CDCl ):
δ
7.89 (dd,
3
J = 8.4, 1.2 Hz, 2H), 7.57 (t, J = 7.2 Hz, 1H), 8.16 (t, J = 7.8 Hz, 2H), 7.39 (s, 1H), 7.10–7.07 (m,
1
1
1
H), 7.03–6.98 (m, 2H); 13C NMR (150 MHz, CDCl3):
δ 192.1, 151.2 (dd, J = 252.8, 12.9 Hz),
50.1 (dd, J = 249.9, 12.9 Hz), 135.1, 134.8, 134.4 (q, J = 5.7 Hz), 130.1 (q, J = 30.2 Hz), 129.8,
29.6, 129.0, 126.2, 122.0 (q, J = 273.0 Hz), 118.4 (d, J = 18.6 Hz), 117.9 (d, J = 18.8 Hz); HRMS
+
(
FAB) m/z: [M+H] calcd for C H F O 313.0652, found 313.0653.
16
10 5
3.3.5. (E)-3-(3,4-difluorophenyl)-1-(naphthalen-1-yl)-2-(trifluoromethyl)prop-2-en-1-one (6)
Compound
6
was prepared from (E)-3-(3,4-difluorophenyl)-1-(naphthalen-1-yl)prop-
◦
2
-en-1-one 10 as a yellow solid. Yield: 16%. Rf 0.35 (hexane/EtOAc, 10:1); mp 71–72 C;
−
1
UV (MeCN/H O, 1:1) λmax (nm) 212, 332; IR (neat) νmax (cm ) 3087, 3063, 2919, 1655,
2
1
1
516, 1272, 1243, 1229, 1122, 1110, 1001, 920, 776; H NMR (600 MHz, CDCl ):
δ
9.05 (d,
3
J = 9.0 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 7.2 Hz, 1H), 7.88 (d, J = 7,8 Hz, 1H), 7.73
t, J = 7.8 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.43 (s, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.12–7.09 (m,
H), 7.04–7.01 (m, 1H), 6.87–6.83 (m, 1H); 13C NMR (150 MHz, CDCl3):
193.3, 151.0 (dd,
J = 252.8, 12.9 Hz), 150.1 (dd, J = 248.6, 12.9 Hz), 135.6, 135.2 (q, J = 5.7 Hz), 133.9, 132.7,
(
1
δ
1
32.2 (q, J = 30.2 Hz), 132.0, 130.9, 129.4, 129.3, 128.7, 127.0, 126.0, 125.4, 124.2, 122.2 (q,
+
J = 272.9 Hz), 118.3 (d, J = 18.6 Hz), 117.6 (d, J = 17.3 Hz); HRMS (FAB) m/z: [M+H] calcd
for C H F O 363.0808, found 363.0793.
20
12 5
3
.3.6. (E)-3-(benzo[b]thiophen-3-yl)-1-phenyl-2-(trifluoromethyl)prop-2-en-1-one (7)
Compound was prepared from (E)-3-(benzo[b]thiophen-3-yl)-1-phenylprop-2-en-1-
one 11 as a colorless oil. Yield: 20%. Rf 0.4 (hexane/EtOAc, 10:1); UV (MeCN/H O, 1:1)
7
2
−
1
λmax (nm) 228, 266, 316; IR (neat) νmax (cm ) 3064, 3031, 1663, 1335, 1272, 1169, 1120, 910,
7
1
57, 732, 686, 589; H NMR (600 MHz, CDCl ):
δ
7.90–7.87 (m, 3H), 7.78 (d, J = 7.8 Hz,
3
1
H), 7.74 (s, 1H), 7.48 (dt, J = 7.2, 1.2 Hz, 2H), 7.40 (t, J = 7.8 Hz, 1H), 7.37 (s, 1H), 7.33 (t,
J = 7.2 Hz, 2H); 13C NMR (150 MHz, CDCl3):
δ
192.8, 139.5, 137.6, 135.2, 134.4, 129.6, 129.4,
+
128.8, 128.1 (q, J = 5.7 Hz), 127.6, 125.2, 125.0, 122.9, 121.2; HRMS (FAB) m/z: [M+H] calcd
for C H F OS 333.0561, found 333.0574.
18
12 3
3
.3.7. (E)-1-(4-aminophenyl)-3-(3,4-difluorophenyl)-2-(trifluoromethyl)prop-2-en-1-one (
Compound was prepared from (E)-1-(4-aminophenyl)-3-(3,4-difluorophenyl)prop-
-en-1-one [19] as a yellow solid. Yield: 53%. Rf 0.2 (hexane/EtOAc, 3:1); mp 94–95 C;
8)
8
◦
2
−
1
UV (MeCN/H O, 1:1) λmax (nm) 248, 350; IR (neat) νmax (cm ) 3422, 3345, 3232, 3963,
2
2
1
919, 1636, 1580, 1558, 1523, 1300, 1256, 1173, 1116; H NMR (400 MHz, CDCl ):
δ
7.73
3
(
2
d, J = 8.8 Hz, 2H), 7.15–7.10 (m, 1H), 7.07–6.97 (m, 1H), 6.56 (d, J = 8.8 Hz, 2H), 4.27 (brs,
H); C NMR (150 MHz, CDCl3): δ 189.4, 152.7, 151.0 (dd, J = 252.8, 12.9 Hz), 149.1 (dd,
13
J = 248.4, 12.9 Hz), 132.9 (q, J = 4.4 Hz), 132.5, 130.6 (q, J = 30.2 Hz), 129.4, 126.2, 125.5,
1
22.2 (q, J = 273.0 Hz), 118.3 (d, J = 18.8 Hz), 117.7 (d, J = 17.1 Hz), 113.9; HRMS (FAB) m/z:
+
[
M+H] calcd for C H F NO 328.0761, found 328.0750.
16
11 5
3
.4. (E)-3-(3,4-difluorophenyl)-1-[4-(prop-2-yn-1-ylamino)phenyl]-2-(trifluoromethyl)prop-2-en-
1
-one (9)
To a solution of compound
8
(67 mg, 0.2 mmol) in MeCN (0.2 mL) were added 3-
bromo-1-propyne (23
µL, 0.3 mmol) and K CO (85 mg, 0.6 mmol) and the mixture was
2
3
◦
stirred at 80 C. After stirring overnight, the reaction mixture was concentrated. The residue

Molecules 2021, 26, 2812
10 of 13
was purified by preparative TLC eluting with toluene-EtOAc (10:1) to give compound
9
(13.4 mg, 0.037 mmol) in 18% yield as a yellow oil. Rf 0.35 (hexane/EtOAc, 3:1); UV
−
1
(MeCN/H O, 1:1) λmax (nm) 248, 354; IR (neat) νmax (cm ) 3369, 3306, 3058, 2922, 1585,
2
1
1
516, 1257, 1173, 1115, 915, 833; H NMR (400 MHz, CDCl ):
δ
7.79 (d, J = 8.4 Hz, 2H),
3
7
(
.19–7.11 (m, 2H), 7.08–6.98 (m, 2H), 6.58 (d, J = 8.4 Hz, 2H), 4.56 (t, J = 5.6 Hz, 1H), 3.98
dd, J = 5.6, 2.4 Hz, 2H), 2.27 (t, J = 2.4 Hz, 1H); 13C NMR (150 MHz, CDCl3):
189.5, 152.1,
51.0 (dd, J = 252.8, 12.9 Hz), 150.1 (dd, J = 248.4, 12.9 Hz), 132.9 (q, J = 5.9 Hz), 132.3,
δ
1
1
30.6 (q, J = 30.2 Hz), 129.4, 126.2, 125.5, 122.2 (q, J = 272.9 Hz), 118.3 (d, J = 18.8 Hz), 117.7
+
(d, J = 18.8 Hz), 112.4, 79.2, 72.2, 32.9; HRMS (FAB) m/z: [M+H] calcd for C H F NO
19
13 5
366.0917, found 366.0913.
3
.5. Cell Proliferation Assay Using PCa Cells
4
DU145 cells (5
×
10 ) were seeded on 12-well plates (2-layer chambers) with DMEM
including 5% charcoal-stripped fetal calf serum (CCS) (HyClone Laboratories, Logan,
UT, USA). PC-3 cells (5
4
×
10 ) were seeded on 12-well plates (2-layer chambers) with
RPMI-1640 including 5% charcoal-stripped fetal calf serum (CCS) (HyClone Laboratories,
Logan, UT, USA). After 24 h, cells were treated with compounds and cultured for 4 days.
Medium was replaced once, at day 2 of treatment. To determine cell proliferation, cells
were trypsinized and counted in triplicate using a hemocytometer. The data represent the
means±SD of three replicates.
3.6. Antiproliferative Activity against Non-Prostate Cancer Cell Lines
Assay was performed as described previously [20]. Briefly, freshly trypsinized cell
suspensions were seeded on 96-well microtiter plates at densities of 4000–11,000 cells per
well with compounds. After 72 h in culture with test compounds, attached cells were fixed
in 10% trichloroacetic acid and then stained with 0.04% sulforhodamine B. The absorbance
at 515 nm was measured using a microplate reader (ELx800, BioTek, Winooski, VT, USA)
operated by Gen5 software (BioTek, Winooski, VT, USA) after solubilizing the bound
dye with 10 mM Tris base. The mean IC50 is the average from at least three independent
experiments in duplicate.
3.7. Xenograft Model in Mice
Six-week-old male severe combined immunodeficient (SCID) mice were purchased
6
from CLEA Japan (Tokyo, Japan). After an acclimatization period, 2
×
10 PC-3 cells were
implanted with 50% Matrigel (Corning, NY, USA) in the dorsal subcutaneous region of the
SCID mice. When the tumors were large enough for their length to be measured, the mice
were divided into two groups (five mice in each group) so that the mean tumor size in each
group was approximately equal. For the intraperitoneal administration, chalcone
administered intraperitoneally in 20 L of DMSO at a concentration of 3 mg/kg, while the
control group received 20 L of DMSO only. For the oral administration study, chalcone
was administered orally in 100 L of corn oil at a concentration of 3 mg/kg, while the
L of corn oil only. The intraperitoneal group was administered
5 was
µ
µ
5
µ
control group received 100
µ
twice a week, and the tumor size and body weight were measured at the same time. The
oral group was administered three times a week, and the tumor size and body weight
were measured simultaneously using a vernier caliper and a scale, respectively. Tumor
size was calculated using the following formula (length
animal protocol was approved by the Institutional Animal Care and Use Committee of the
Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan.
×
width
×
width
×
0.5). This
3
.8. Flow Cytometric Analysis
4
MDA-MB-231 or KB-VIN (7
×
10 cells/well) cells were seeded in a 12-well plate 24 h
prior to treatment with compounds for 24 h. Compounds were used against MDA-MB-231
or KB-VIN at a concentration threefold of their IC50 value. Harvested and 70% EtOH-fixed
cells were stained with propidium iodide (PI) containing RNase (BD Bioscience, San Jose,

Molecules 2021, 26, 2812
11 of 13
CA, USA) and subjected to flow cytometry (BD LSRFortessa, BD Biosciences, San Jose, CA,
USA). CA-4 at 200 nM was used as a control tubulin polymerization inhibitor arresting
cells at G2/M.
3
.9. Immunostaining
4
MDA-MB-231 cells (2.4
×
10 cells/well) were grown on an 8-well chamber slide (Lab-
Tech, Waltham, USA) for 24 h prior to treatment with reagents. Cells were treated for 24 h
with compound or DMSO as a control for 24 h. Cells were fixed in 4% paraformaldehyde
in PBS and permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were labeled with
mouse monoclonal antibody to -tubulin (B5-1-2, Sigma, St. Louis, MO, USA) followed
by FITC-conjugated antibody to mouse IgG (Sigma) [31]. Nuclei were labeled with DAPI
Sigma). Fluorescence labeled cells were observed using confocal microscope (LSM700,
5
α
(
Zeiss, White Plains, NY, USA) controlled by ZEN (black edition) software (Zeiss). Confocal
images were stacked and merged using ZEN (black edition) software. Final images were
prepared using Adobe Photoshop.
3
.10. Affinity Purification of Binding Proteins
Compound was immobilized with Azide magnetic beads (TAS8848N1160, Tama-
gawa Seiki Co., Ltd. Iida, Japan) at a concentration of 125 M according to the manufac-
turer’s method. PC-3 cell lysates in soluble buffer [10 mM HEPES, 150 mM Na SO , 1 mM
9
µ
2
4
EDTA, 2% CHAPS, protease inhibitor (cOmplete Mini, Roche, Basel, Switzerland)] were
◦
incubated with or without compound
incubated with the compound
were washed five times with soluble buffer (200
with SDS-PAGE sample buffer (10
5
(5 mM) at 37 C for 1 h. The lysates (30
µ
L) were
◦
9
-immobilized beads (10
µ
L) at 37 C for 1 h. The beads
◦
µ
L each) and boiled at 95 C for 5 min
L). After the beads were removed using a magnetic
µ
stand, samples were analyzed by SDS-PAGE. The resultant gel was stained with CBB
and the stained bands were cut out. The small portions of gel were treated with usual
methods including reductive alkylation and in-gel digestion, and the resultant samples
were analyzed with LC-MSMS (Orbitrap QE plus, Thermo Fisher, Waltham, MA, USA). The
obtained peptide sequences were identified using Proteome Dicoverer software (Thermo
Fisher, Waltham, MA, USA).
4
. Conclusions
We developed seven
erative activity against androgen-independent prostate cancer cell lines. Many of them
showed potent activity with submicromolar IC50 values. The most effective chalcone
α
-trifluoromethyl chalcones and evaluated them for antiprolif-
5
also displayed significant antiproliferative activity against taxane-resistant cell lines and
antitumor activity in vivo with only 3 mg/kg administration in a mouse xenograft model.
To investigate the intracellular target molecules, we prepared the chemical probe
confirmed that its mode of action was the same as that of chalcone by using flow cy-
tometry. Affinity purification of binding proteins from PC-3 cell lysates using compound
-immobilized beads revealed several candidates as the ligands for chalcone . Some of
them are known to stimulate cancer related cell growth.
9 and
5
9
5
Supplementary Materials: The following are available online. Figures S1–S40: 1H NMR, 13C NMR, FT-
IR, and UV-VIS spectra of compounds 2–11, Table S1: Detailed information of detected binding proteins.
Author Contributions: Y.S., A.M. and K.N.-G. designed this study; Y.S. and K.N.-G. designed and
performed chemical experiments; A.M., K.I., R.N. and M.G. designed and performed biological
experiments; Y.S., A.M., K.I., R.N., M.G. and K.N.-G. evaluated all results and wrote manuscript. All
authors have given approval to the final version of the manuscript.
Funding: This research was funded by JSPS KAKENHI, grant number 25293024 awarded to K.N.-G.
and 17H04325 awarded to A.M. This work was also supported in part by IBM junior faculty grant
awarded to M.G.

Molecules 2021, 26, 2812
12 of 13
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Institutional Review Board of Kanazawa University.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding authors.
Acknowledgments: We appreciate critical comments, suggestions, and editing on the manuscript by
Susan L. Morris-Natschke (UNC-CH). We thank the Microscope Service Laboratory (UNC-CH) for
their expertise in confocal microscope. We also thank the Institute for Gene Research (KU) for their
expertise in identification of the binding proteins.
Conflicts of Interest: The authors declare no competing financial interest.
Sample Availability: Samples of the compounds 1–9 are available from the authors.
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