5′-Chloro-2,2′-dihydroxychalcone and related flavanoids as treatments for prostate cancer

Article abstract of DOI:10.1016/j.ejmech.2018.08.069

Several flavonoids and their biosynthetic precursor chalcones were designed and synthesized to improve the biological effects of the lead compound 2′-hydroxyflavonone against androgen receptor (AR)-dependent transcriptional stimulation. Newly synthesized chalcones 19 and 26 suppressed AR-dependent transcription as well as DHT-dependent growth stimulation at a low micromolar level. These compounds were also effective against ligand-independent constitutively active mutant AR derived from castration-resistant PCa (CRPC). Compounds 19 and 26 showed broad spectrum antiproliferative activity at 5–10 μM against multiple tumor cell lines including androgen-independent and taxane-resistant prostate cancer as well as a multidrug-resistant subline. Mode of action studies suggested that 19 induced sub-G1 accumulation in PC-3 cells by disrupting the microtubule network without affecting cell cycle progression. Furthermore, the in vivo effectiveness of chalcone 19 was confirmed in a xenograft model antitumor assay. Thus, chalcone 19 has the potential to be a bifunctional lead for treatment of AR-dependent PCa at lower doses as well as AR-independent PCa, including CRPC, at higher doses.

Full text of DOI:10.1016/j.ejmech.2018.08.069

Accepted Manuscript
5′-Chloro-2,2′-dihydroxychalcone and related flavanoids as treatments for prostate
cancer
Yohei Saito, Atsushi Mizokami, Hiroyuki Tsurimoto, Kouji Izumi, Masuo Goto, Kyoko
Nakagawa-Goto
PII:
S0223-5234(18)30741-4
10.1016/j.ejmech.2018.08.069
EJMECH 10680
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2018
Revised Date: 7 August 2018
Accepted Date: 25 August 2018
Please cite this article as: Y. Saito, A. Mizokami, H. Tsurimoto, K. Izumi, M. Goto, K. Nakagawa-Goto,
5′-Chloro-2,2′-dihydroxychalcone and related flavanoids as treatments for prostate cancer, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.069.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
5′-Chloro-2,2′-dihydroxychalcone and Related
Flavanoids as Treatments for Prostate Cancer
Yohei Saito,† Atsushi Mizokami,*,‡ Hiroyuki Tsurimoto,† Kouji Izumi,‡ Masuo Goto § and Kyoko
Nakagawa-Goto*,†,§
†School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Science,
Kanazawa University, Kanazawa, 920-1192, Japan
‡Department of Integrative Cancer Therapy and Urology, School of Medical Sciences, Kanazawa
University, Kanazawa, 920-1192, Japan
§Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of
North Carolina, Chapel Hill, North Carolina 27599, United States
Corresponding Authors
*E-mail: kngoto@p.kanazawa-u.ac.jp. Phone: 81-76-264-6305, mizokami@staff.kanazawa-
u.ac.jp. Phone: 81-76-265-2393.
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ABSTRACT
Several flavonoids and their biosynthetic precursor chalcones were designed and synthesized to
improve the biological effects of the lead compound 2′-hydroxyflavonone against androgen
receptor (AR)-dependent transcriptional stimulation. Newly synthesized chalcones 19 and 26
suppressed AR-dependent transcription as well as DHT-dependent growth stimulation at a low
micromolar level. These compounds were also effective against ligand-independent
constitutively active mutant AR derived from castration-resistant PCa (CRPC). Compounds 19
and 26 showed broad spectrum antiproliferative activity at 5–10 µM against multiple tumor cell
lines including androgen-independent and taxane-resistant prostate cancer as well as a multidrug-
resistant subline. Mode of action studies suggested that 19 induced sub-G1 accumulation in PC-3
cells by disrupting the microtubule network without affecting cell cycle progression.
Furthermore, the in vivo effectiveness of chalcone 19 was confirmed in a xenograft model
antitumor assay. Thus, chalcone 19 has the potential to be a bifunctional lead for treatment of
AR-dependent PCa at lower doses as well as AR-independent PCa, including CRPC, at higher
doses.
Keywords: Chalcone, Castration-resistant prostate cancer, Androgen receptor reporter assay,
PC-3, LNCaP
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1. Introduction
Flavonoid is a collective term for compounds containing a diphenylpropane skeleton
(C6C3C6) and is applied to flavonols, flavones, isoflavones, flavanones, flavanols, chalcones,
and other related compounds (Figure 1). Such compounds are found abundantly in plants,
including dietary vegetables and fruits [1]. Dietary flavonoids are generally harmless to humans
and exhibit various beneficial biological effects, such as anticancer [2], antioxidant [3], anti-
inflammatory, anti-allergic [4,5] and estrogenic [6]. A common basic skeleton of flavonoids,
phenylchromone, is similar structurally and biologically to some hormones, such as estrogen and
androgen. Thus, special attention has been paid to flavonoids in the discovery of agents to treat
hormone-related diseases, such as breast [6,7] and prostate cancers [7]. In our previous study, 2′-
hydroxyflavanone (2′-HF, 1) suppressed androgen receptor (AR) activity and prostate cancer
(PCa) cell proliferation [8].
Figure 1. Chemical structures of flavanone, flavone, and chalcone.
While the survival rate from PCa has improved remarkably due to early detection and
effective treatments, the incidence of PCa has increased recently in Asia, likely due to the
diffusion of Westernized diets [9]. PCa can be treated initially with hormonal therapy to suppress
AR activity; however, this regimen is not effective forever and the disease advances to
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castration-resistant PCa (CRPC) after several years of hormonal treatment. CRPC shows
resistance to hormonal therapy and causes severe problems, such as metastasis and recurrence.
CRPC escapes hormonal therapy through a mutation on the AR or the activation of an androgen-
independent pathway [10]. CRPC is treated generally with taxane-derived anticancer agents;
however, the development of drug resistance always eliminates the treatment’s success. Thus, a
solution to the problems of existing drugs is an urgent need.
Using the structure of 2′-HF as a template, we designed and synthesized 32 derivatives,
including flavanones 1–10, flavones 11 and 12, and chalcones 13–32 (Table 1). Our goal was to
improve the activity of the parent compound and to evaluate structure-activity relationships in
this structural class. Chalcones are known biosynthetic precursors of flavonoids and have shown
more potent anti-proliferative activity than related flavones and flavanones against human liver
cancer [11]. In addition, the chalcone structure mimics a portion of ASC-J9, which has been
reported to be a potent anti-CRPC agent [12]. We evaluated all synthetic compounds for activity
against AR, including wild-type and ligand-independent constitutively active mutant AR, and
chose the compounds with potent inhibitory activity against AR-related transcription to evaluate
for antiproliferative activity against various tumor cell lines, including taxane-resistant PCa cell
lines and a multi-drug resistant (MDR) subline. Furthermore, we performed mode of action
studies and in vivo anticancer evaluation. Our studies demonstrate the potential of chalcone
derivatives as a new class of drug candidate for the treatment of PCa, including CRPC.
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Table 1. Structures of synthetic compounds.
R1
R2
R3
R4
R5
R6
Yield (%)2
Flavanones
H
H
H
H
H
H
H
H
Br
H
H
H
OMe
OH
OH
H
H
H
H
H
H
OH
H
H
H
OMe
Cl
Br
Br
I
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
–
–
–
–
–
–
–
–
–
–
98
68
65
40
54
71
64
51
66
60
1
2
3
4
5
6
7
8
9
10
Flavones
H
OMe
H
Me
–
–
–
–
–
–
–
–
31
59
11
12
Chalcones
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
F
F
F
OMe
OMe
OH
OMe
OMOM
H
OH
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
85
53
66
64
69
76
57
80
52
41
68
70
79
78
64
31
80
78
66
82
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
I
H
OH
-OCH2O-
OH
OMe
H
H
H
H
H
H
H
H
-OCH2O-
OMe
OMe
H
H
H
H
H
H
H
OH
OMe
OEt
OiPr
OPr
OH
OH
OMe
29
30
31
32
H
H
H
I
H
a Overall yield from the related acetophenone.
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2. Results and discussion
2.1 Chemistry
Chalcones 13–32 were obtained via a Claisen-Schmidt condensation of a substituted 2-
hydroxyacetophenone and an appropriate benzaldehyde in the presence of aqueous KOH
followed, as needed, by removal of methoxymethyl (MOM) ether protecting groups under acidic
conditions (Scheme 1). To study the effect of a C ring, ring closure was accomplished via an
intramolecular oxa-Michael reaction of selected chalcones in the presence of aqueous Na2HPO4
or NaOAc. 2′,6-Dimethoxyflavanone 6 was obtained directly and subsequent deprotection of
MOM ethers provided 2′-hydroxyflavanones 1–5 and 7–10. Two flavones, 11 and 12, were also
synthesized from chalcones using a catalytic amount of I2 in DMSO. Table 1 contains the
structures of the synthetic target compounds. A primary focus in this investigation was
halogenated flavonoids. 2’-Hydroxyflavanones 1–5 [13-15], flavones 11 [16] and 12 [17], as
well as chalcones 14– 25 [18-23] were reported in previous literatures.
Scheme 1. Synthesis of flavonoidsa
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aReagents and conditions: (a) 40% KOH, EtOH, room temp.; (b) 0.1 M Na2HPO4, EtOH, room temp.; (c)
I2 (10 mol%), DMSO, 140°C.; (d) 1% H2SO4, AcOH, room temp.; (e) BBr3, CH2Cl2, -70°C.
2.2. Biological Evaluation
2.2.1. Suppression of AR-dependent transcriptional stimulation by compounds
All synthesized flavonoids were first evaluated in a reporter assay of AR-related
transcriptional activation. The assay was performed by our established method using AR-positive
LNCaP cells transfected with a luciferase expression plasmid driven by the prostate specific
antigen (PSA) promoter (PSA-Luc) [8]. In this system, the luciferase gene is transcribed by the
endogenous AR in the presence of a ligand, such as dihydrotestosterone (DHT). Compounds 1,
11, 13, 19, 26 and 28 showed potent inhibitory activity against AR-related transcription (Table 2).
Table 2. Inhibitory effect of flavonoids on AR-dependent transcription in androgen-dependent
LNCaP cells.
compounds
IC50 (µM)a
compounds
IC50 (µM)a
compounds
IC50 (µM)a
compounds
IC50 (µM)a
<1
3.57
4.26
<1
>10
1.51
<1
8.69
<1
1
2
3
4
5
6
7
8
9
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
2.73
>10
>10
>10
>10
1.68
1.77
10
11
12
13
14
15
16
5.27
<1
6.19
<1
>10
5.92
4.36
5.95
5.25
3.96
2.87
1.64
1.72
3.50
1.42
5.01
a
The concentration of compound that caused 50% reduction of AR-dependent transcriptional
activity relative to untreated cells determined by the luciferase assay. DHT was used at 1 nM.
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Regarding the flavanones, compounds 7–10 with halogen atoms on C-6 (IC50 2~4 µM)
displayed greater activity than compounds 3–6 with hydroxy or methoxy groups on C-5, -6 or -7
(IC50 >10 µM). 2′,6-Dihydroxyflavonone (2) was an exception (IC50 2.73 µM). However, all new
flavonones were less potent than the lead compound, 2′-HF (1, IC50 <1 µM). Like 1, 2′-
hydroxyflavone (11) displayed an IC50 value of less than 1 µM, while 2′,6-dimethoxyflavone
(12) showed reduced activity (IC50 6.19 µM). Among chalcones 13–32, compounds 13, 19, 26
and 28 exhibited the most significant suppression of AR activity (IC50 <1 µM), suggesting that
the combinations of substituents on ring-A, particularly at C-5′, and -B, particularly at C-2,
might be important for the inhibitory activity. In the case of 5′-halogenated chalcones, the
identity/properties of the halogen influenced the effect of a hydroxy or alkoxy group at C-2
(Figure 2). Namely, with a small, highly electronegative fluoride on C-5′, the 2-hydroxy
chalcone derivative exhibited greater potency than the 2-methoxy or 2-MOM ether (compare 15
vs 16 and 17). Similarly, with chloride on C-5′, the 2-hydroxy chalcone (19) was one of the most
potent compounds in the assay, while the 2-unsubstituted compound (18) was less active and the
2-methoxy compound (20) was much less active. In contrast, when the 5′-substituent was a
larger, less electronegative bromide, the reverse effect was found; the 2-hydroxy chalcone (25)
exhibited the lowest potency compared with 2-alkoxy substituted compounds (26–29). Indeed,
the IC50 values of 26 (2-methoxy) and 28 (2-isopropoxy) were less than 1 µM. The two 5′-
iodinated chalcones showed comparable potency (31, 2-OH, IC50 1.64 µM vs 32, 2-OMe, IC50
1.72 µM). From the viewpoint of a fixed substituent on C-2, the rank order of potency was 5′-Br
(25) < 5′-F (15) = 5′-I (31) < 5′-Cl (19), when the C-2 substituent was OH, but 5′-Cl (20) < 5′-F
(16) < 5′-H (14) < 5′-I (32) < 5′-Br (26) = 5′-OMe (13), when the C-2 substituent was OMe.
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Although only a few derivatives were synthesized with hydroxy or alkoxy groups on C-3 or C-4
of the B-ring, the potency was decreased by this modification (compare 21−24 with 18).
Figure 2. Preliminary SAR of halogenated chalcones for AR activity in LNCaP cells
Based on the above results from the reporter assay in LNCaP cells, the target chalcones
inhibited endogenously expressed wild-type AR-dependent transcription. Next, we determined
whether the chalcones could inhibit CRPC-derived mutant AR, such as AR-V7, which is
constitutively active ligand-independently [24]. For this assay, we used AR-negative PC-3 cells
transformed with wild-type or mutant (AR-V7) AR expressing vector together with PSA-Luc
encoding reporter plasmid. Wild-type AR-dependent transcription was stimulated by DHT in
PC-3 cells and, as expected, was suppressed by 2′-HF (1), 5′-chloro-2,2′-dihydroxychalcone (19),
and 5′-bromo-2′-hydroxy-2-methoxychalcone (26) (Figure 3A). Under the same conditions using
AR-V7, PSA-Luc was expressed DHT-independently and was not suppressed by an androgen
antagonist, enzalutamide (Enz), or compound 1. In contrast, compounds 19 and 26 suppressed
PSA-Luc expression dose-dependently (Figure 3B). Thus, compounds 19 and 26, especially 26,
directly inhibited the transcriptional activation of constitutively active AR-V7. These results
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strongly suggest that compound 26 is not an antagonist of the ligand, but rather targets the AR at
a different site from ligand binding.
Figure 3. Effect on AR activity in PC-3 cells. Luciferase reporter assay was conducted using
PC-3 cells expressing (A) wild-type AR or (B) mutant AR (AR-V7) were treated with
compounds without (-) or with (+) stimulation by DHT. AR-dependent transcriptional
stimulation was measured as luciferase activity. DHT was used at 1 nM. *P < 0.05 versus 10 µM
1 (DHT –). **P<0.005 versus 10 µM 1 (DHT +).
2.2.2. Inhibition of AR-related growth stimulation
Compounds 1, 19 and 26, which suppressed AR-dependent transcription (Table 2), were
investigated also for effects on AR-related growth stimulation using androgen-responsible
LNCaP (Figure 4). First, we observed that Enz suppressed the stimulatory effect of DHT on cell
growth, but did not show an antiproliferative effect, even at 10 µM, in the absence of DHT.
When compounds 1, 19 and 26 were used at lower concentrations (1 and 3 µM), DHT
stimulation was suppressed similarly. These observations suggest that these compounds function
as AR-antagonists at 1 and 3 µM in LNCaP cells. In contrast, treatment of cells with 10 µM of
19 and 26 led to an inhibitory effect on cell growth, greater than that of 1. The above
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observations suggest that these compounds suppress DHT/AR-dependent growth stimulation at 1
and 3 µM but exert antiproliferative effects at 10 µM by an AR-independent mechanism.
Figure 4. Antiproliferative activity against AR-dependent LNCaP. LNCaP cells were seeded on
12-well plated (2-layer chambers) with DMEM containing 5% charcoal-stripped fetal calf serum
(CCS). After 24 h, cells were treated with DHT. Cell count was calculated after culturing 4 days
with compound. DHT was used at 0.1 nM (+).
2.2.3. Antiproliferative activity of compounds against AR-responsive and AR-independent cells
Because compounds 19 and 26 showed DHT/AR-independent antiproliferative activity against
LNCaP cells, the IC50 values of selected compounds 1, 11, 13, 19, 26 and 28 were determined
against androgen-independent PCa cell lines PC-3 and DU145. The LNCaP cell line with or
without DHT stimulation was used as a control. As expected, compounds 19 and 26 showed AR-
independent antiproliferative activity with IC50 values of 1.5–4.5 µM and 1.8–6.1 µM,
respectively (Table 3). This androgen-independent antiproliferative activity was confirmed using
CRPC model LNCaP subline LNCaP-SF [25], demonstrating that compounds 1, 19 and 26 at 10
µM can overcome CRPC by inhibiting cell growth AR-independently (Figure 5). Additionally,
compounds 19 and 26 were tested against taxane-resistant sublines established from PC-3 (PC-
3/TxR) and DU145 (DU145/TxR) as well as cabazitaxel-resistant PC-3/TxR (PC-3/TxR/CxR)
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and DU145/TxR (DU145/TxR/CxR) sublines [26]. Compounds 19 and 26 exhibited potent
antiproliferative activity against all tested resistant PCa cell lines with IC50 values of 3.0–7.4 µM
and 3.7–5.2 µM, respectively (Table 4). Consequently, both chalcones displayed the same effect
against all PCa cell lines, whether chemosensitive or chemoresistant. Therefore, compounds 19
and 26 were further assayed against five human tumor cell lines, non-small cell lung (A549),
triple-negative breast (MDA-MB-231), estrogen-responsible breast (MCF-7), originally isolated
from epidermoid carcinoma (KB), and P-glycoprotein (P-gp) overexpressing multidrug resistant
KB subline (KB-VIN). We confirmed the KB and KB-VIN cell lines were identical to AV-3
(ATCC number, CCL-21) as a HeLa (cervical carcinoma) contaminant by short tandem repeat
(STR) profiling. The compounds showed broad spectrum against the five human tumor cell lines,
while 26 was somewhat less active against MCF-7, KB and KB-VIN (Table 5).
Table 3. Antiproliferative activity against LNCaP and androgen-independent prostate cancer cell
lines PC-3 and DU145.
Cell lines / IC50 (µM)a
compounds
LNCaP
(DHT–)
LNCaP
PC-3
DU145
(DHT+)b
6.40
>10
>10
3.74
4.87
9.11
5.45
>10
2.56
NDc
7.33
1.52
1.84
4.16
4.11
NDc
>10
4.50
6.13
5.03
1
11
13
19
26
28
7.25
3.52
4.96
4.20
a The concentration of compound that caused 50% reduction of cell growth relative to untreated
cells determined by cell counting. b DHT was used at 0.1 nM. c Not Determined.
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Figure 5. Antiproliferative activity against castration-resistant PCa model LNCaP-SF subline.
Cells were treated with or without DHT and cultured for 4 days with compound at indicated
concentration. DHT was used at 0.1 nM. *P < 0.05 versus Enz (10 µM). **P<0.005 versus Enz
(10 µM).
Table 4. Antiproliferative activity against docetaxel and cabazitaxel resistant prostate cancer cell
lines PC-3/TxR, PC-3/TxR/CxR, DU145/TxR and DU145/TxR/CxR.
Cell lines / IC50 (µM)a
compounds
PC-3/TxR
PC-3/ TxR/CxR
DU145/TxR
DU145/ TxR/CxR
<3
4.07
3.73
7.43
4.47
5.32
5.16
19
26
3.76
a The concentration of compound that caused 50% reduction of cell growth relative to untreated
cells determined by cell counting.
Table 5. Antiproliferative activity of compounds 19 and 26 against other tumor cell lines.
Cell linea (IC50 µM)b
compounds
A549
9.1
MDA-MB-231
5.1
MCF-7
4.9
KB
5.0
KB-VIN
4.9
19
26
8.5
6.8
9.7
9.6
14.1
11.5
16.2
5.9
11.1
paclitaxel (nM)
1727.4
aA549 (lung carcinoma), MDA-MB-231 (triple-negative breast cancer), MCF-7 (estrogen
receptor-positive and HER2-negative breast cancer), KB (originally isolated from epidermoid
carcinoma of the nasopharynx), KB-VIN (P-gp-overexpressing MDR subline of KB).
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bAntiproliferative activity 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.
2.2.4. Mode of action studies with compounds 19 and 26
Flow cytometric analysis was used to observe the impact of compounds 19 and 26 on the cell
cycle in PC-3 and A549 cells. Interestingly, the compounds exhibited different effects in the two
cell lines. Sub-G1 cell accumulation was induced significantly in PC-3 cells treated with the
compounds at twofold IC50 concentration without a notable impact on the cell cycle progression.
In A549 cells treated at threefold IC50 concentration, compounds 19 and 26 induced cell cycle
delay in G1 and G2/M stages, respectively. No significant sub-G1 accumulation was seen in
A549 cells, even though the compound concentration was higher than that used in PC-3 cells
(Figure 6). These observations imply that compounds may induce cell cycle delay in A549 cells
but induce obvious apoptosis in PC-3 cells in a cell cycle-independent manner.
Figure 6. Flow cytometric analysis of chalcones on (A) PC-3 and (B) A549 cell lines.
Cells were treated with compounds for 24 or 48 h as indicated at the 2-fold (PC-3) or 3-fold
(A549) concentrations of their IC50. CA-4 was used for 24 h at 200 nM for PC-3 or 20 nM for
A549 as a mitotic inhibitor arresting cells at G2/M by inhibiting tubulin polymerization. Cells
were fixed and stained with propidium iodide (PI) followed by flow cytometric analysis.
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Compounds 19 and 26 induced accumulation of sub-G1 in PC-3 cells (A), while no obvious sub-
G1 accumulation was observed in A549 cells (B).
The effects of compound 19 on cell morphology were also investigated
immunocytochemically. After treatment of PC-3 for 24 h with 19 at twofold IC50 concentration,
cells were stained with monoclonal antibody to α-tubulin for microtubules and 4′,6-diamidino-2-
phenylindole (DAPI) for DNA. Microtubules and mitotic spindles were clearly stained in
untreated PC-3 cells, while microtubules were depolymerized in cells treated with 19 (Figure 7).
These results indicated that compound 19 interfered with microtubule organization, which is
observed in cells treated with tubulin polymerization inhibitors [27]. Although tubulin
polymerization inhibitors, such as CA-4, induce notable cell cycle arrest at G2/M, compound 19
showed no significant impact on cell cycle progression (Figure 6A). Thus, the morphological
defects in microtubules induced by 19 might not be caused by directly targeting tubulin. We
predict that compound 19 caused sub-G1 induction in PC-3 cells by interfering indirectly with
the microtubule network.
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Figure 7. Effect of compound 19 on cell morphology. PC-3 cells were treated with 19 for 24 h
followed by labelling with antibody to α-tubulin (green) and DAPI (blue) for DNA. DMSO was
used a control. Immunostained cells were observed by confocal fluorescence microscope.
Interphase microtubules (white arrow) and bipolar mitotic spindle (yellow arrow) are clearly
observed in control cells (DMSO), while undetectable in cells treated with compound 19. Bar,
0.025 mm.
2.2.5. Antitumor effects of compounds in vivo
Because compounds 19 and 26 were effective against taxane-resistant AR-independent PCa as
well as multidrug resistant cells, these compounds might overcome the severe obstacles of
prostate cancer chemotherapy. These results encouraged us to further investigate antitumor
effects in vivo using a PC-3 tumor xenograft model. Compounds 19 and 26 were administered
intraperitoneally (i.p.) at 30 mg/kg every 4 days. Although the two compounds showed almost
identical antiproliferative activities in vitro against PC-3 cells, their effects in vivo were
considerably different (Figure 8). The tumor growth was significantly inhibited by chalcone 19
with no significant effect on weight loss compared with control, while no effect was shown by
26. Our antitumor studies demonstrated that 19 was effective against PC-3 in vivo, but
compound 26 may be detoxified quickly in vivo.
Figure 8. Effect of chalcones against PC-3 tumor xenograft in C.B-17 scid mice. Compounds
were intraperitoneally (i.p.) administrated at 30 mg/kg every 4 days (n = 5). (A) Tumor volume
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in SCID mice during treatment with the compounds. (B) Average body weights of mice during
treatment with the compounds. Data were presented as the average ± SD.
3. Conclusions
Among 32 synthesized flavonoid derivatives, we discovered two flavonoids and four
chalcones that inhibited directly AR-dependent transcriptional activation at a concentration less
than 1 µM. 5′-Chloro-2,2′-dihydroxychalcone (19) and 5′-bromo-2′-hydroxy-2-methoxychalcone
(26) showed broad spectrum antiproliferative effects at 10 µM against several tumor cell lines,
including androgen-independent PCa, lung, triple-negative breast, and taxane-resistant PCa as
well as a multidrug-resistant subline. Compounds 19 and 26 suppressed AR-dependent
transcription and growth stimulation at a low micromolar level and exhibited antiproliferative
activity AR-independently at 5–10 µM. Furthermore, chalcone 19 was demonstrated to be an
antitumor agent in an animal study. Thus, chalcone 19 has the potential to be a bifunctional lead
for treatment of AR-dependent PCa at lower doses as well as AR-independent PCa, including
CRPC, at higher doses.
4. Experimental section
4.1. 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 synthetic compounds. 1H and
17

ACCEPTED MANUSCRIPT
13C NMR spectra were recorded on JEOL JNM-ECA 600 or JNM-ECS 400 using CDCl3 as
solvent and referenced to TMS or residual solvent peak. Chemical shifts δ are reported in ppm.
Mass spectrometric analysis were performed using JEOL JMS-700 Mstation or JMS-T100TD.
Purity of all compounds was determined as >95% by 1H NMR or HPLC.
4.2. General procedures for chalcones
To a solution of 2-methoxybenzaldehyde (25 mg, 0.18 mmol) and 2’-hydroxy-5’-
methoxyacetophenone (30 mg, 0.18 mmol) in EtOH (0.2 mL) was added 40% aqueous KOH (0.2
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 layer was washed with brine, dried over MgSO4
and concentrated under reduced pressure. The residue was purified by column chromatography
on silica gel, eluting with hexane-EtOAc (15:1) to give 2′-hydroxy-2,5’-dimethoxychalcone (13)
(44 mg, 0.15 mmol) in 85% yield as an yellow solid. Chalcones 16, 17, 20, 26–29, and 32 were
produced by the same procedure.
1
4.2.1. 2
′
-Hydroxy-2,5
′
-dimethoxychalcone (13). H NMR (400 MHz, CDCl3): δ 12.5 (s, 1H),
8.23 (d, J = 15.6 Hz, 1H), 7.72 (d, J = 15.6 Hz, 1H), 7.65 (dd, J = 8.0, 1.6 Hz, 1H), 7.42 (m, 1H),
7.38 (d, J = 3.2 Hz, 1H), 7.14 (dd, J = 9.2, 3.2 Hz, 1H), 7.02 (dd, J = 7.6, 7.6 Hz, 1H), 6.98 (d, J
= 8.8 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 3.95 (s, 3H), 3.84 (s, 3H); 13C NMR (150 MHz, CDCl3):
δ 194.0, 159.0, 157.9, 151.6, 141.2, 132.2, 129.6, 123.7, 123.6, 120.81, 120.80, 119.8, 119.2,
112.9, 111.3, 56.0, 55.6. HRMS (FAB) m/z: [M+H]+ calcd for C17H17O4 285.1127, found
285.1134.
18

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4.2.2. 5
′
-Fluoro-2
′
-hydroxy-2-methoxychalcone (16). 1H NMR (400 MHz, CDCl3): δ 8.24 (d, J =
15.6 Hz, 1H), 7.69 (d, J = 15.6 Hz, 1H), 7.65 (dd, J = 8.0, 1.6 Hz, 1H), 7.59 (d, J = 8.8, 3.2 Hz,
13
1H), 7.43 (m, 1H), 7.24 (m, 1H), 7.05–6.97 (complex, 3H), 3.96 (s, 3H); C NMR (100 MHz,
CDCl3): δ 193.4, 159.7, 159.2, 154.8 (d, J = 236.4 Hz), 142.1, 132.5, 130.0, 123.6 (d, J = 23.8
Hz), 123.3, 120.8, 120.2, 119.7 (d, J = 7.6 Hz), 114.6 (d, J = 22.9 Hz), 111.3, 55.6. HRMS
(FAB) m/z: [M+H]+ calcd for C16H14FO3 273.0927, found 273.0916.
1
4.2.3. 5
′
-Fluoro-2
′
-hydroxy-2-(methoxymethoxy)chalcone (17). H NMR (400 MHz, CDCl3):
δ 8.31 (d, J = 15.6 Hz, 1H), 7.70 (dd, J = 8.0, 1.6 Hz, 1H), 7.65 (d, J = 15.6 Hz, 1H), 7.58 (dd, J
= 8.8, 3.2 Hz, 1H), 7.40 (ddd, J = 8.8, 7.6, 1.6 Hz, 1H), 7.25 (m, 1H), 7.21 (dd, J = 8.0, 0.8 Hz,
1H), 7.08 (dd, J = 7.6 Hz, 1H), 7.00 (dd, J = 8.8, 4.8 Hz, 1H), 5.32 (s, 2H), 3.54 (s, 3H); 13C
NMR (100 MHz, CDCl3): δ 193.3, 159.7, 156.7, 154.8 (d, J = 237.5 Hz), 141.6, 132.5, 128.9,
124.0, 123.7 (d, J = 22.9 Hz), 122.0, 120.1, 119.8 (d, J = 7.6 Hz), 119.6 (d, J = 6.7 Hz), 114.9,
114.5 (d, J = 23.8 Hz), 94.6, 56.4. HRMS (FAB) m/z: [M+H]+ calcd for C17H16FO4 303.1033,
found 303.1034.
1
4.2.4. 5
′
-Chloro-2
′
-hydroxy-2-methoxychalcone (20). H NMR (400 MHz, CDCl3): δ 12.9 (s,
1H), 8.26 (d, J = 15.2 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 15.2 Hz, 1H), 7.67 (dd, J =
8.0, 2.0 Hz, 1H), 7.46–7.41 (complex, 2H), 7.03 (dd, J = 7.6, 7.6 Hz, 1H), 6.99 (d, J = 8.4 Hz,
1H), 6.98 (d, J = 8.4 Hz, 1H), 3.96 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 193.3, 162.0, 159.1,
142.1, 135.9, 132.6, 129.7, 128.9, 123.4, 123.3, 120.8, 120.7, 120.1, 119.9, 111.3, 55.6. HRMS
(FAB) m/z: [M+H]+ calcd for C16H13ClNaO3 311.0451, found 311.0444.
4.2.5. 5′-Bromo-2′
-hydroxy-2-methoxychalcone (26). 1H NMR (600 MHz, CDCl3): δ 8.26 (d, J =
15.6 Hz, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.69–7.67 (complex, 2H), 7.56 (dd, J = 9.0, 2.4 Hz, 1H),
19

ACCEPTED MANUSCRIPT
7.43 (m, 1H), 7.03 (dd, J = 7.8, 7.8 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H),
3.96 (s, 3H); 13C NMR (150 MHz, CDCl3): δ 193.2, 162.4, 159.1, 142.1, 138.7, 132.6, 131.9,
129.7, 123.3, 121.4, 120.8, 120.5, 119.9, 111.3, 110.3, 56.6. HRMS (FAB) m/z: [M+H]+ calcd
for C16H14BrO3 333.0126, found 333.0134.
1
4.2.6. 5
′
-Bromo-2
′
-hydroxy-2-ethoxychalcone (27). H NMR (400 MHz, CDCl3): δ 8.18 (d, J =
15.6 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.86 (d, J = 15.6 Hz, 1H), 7.63 (dd, J = 8.0, 1.6 Hz, 1H),
7.56 (dd, J = 9.2, 2.4 Hz, 1H), 7.40 (m, 1H), 7.02 (dd, J = 7.6 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H),
6.94 (d, J = 9.2 Hz, 1H), 4.18 (q, J = 6.8 Hz, 2H), 1.59 (t, J = 6.8 Hz, 3H); 13C NMR (150 MHz,
CDCl3): δ 193.4, 162.4, 158.8, 142.7, 138.6, 132.5, 132.0, 130.9, 123.2, 121.5, 120.7, 120.5,
120.4, 112.1, 110.3, 64.1, 14.9. HRMS (FAB) m/z: [M+H]+ calcd for C17H16BrO3 347.0283,
found 347.0272.
1
4.2.7. 5
′
-Bromo-2
′
-hydroxy-2-isopropoxychalcone (28). H NMR (600 MHz, CDCl3): δ 12.9 (s,
1H), 8.18 (d, J = 15.0 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.82 (d, J = 15.0 Hz, 1H), 7.63 (dd, J =
7.8, 1.8 Hz, 1H), 7.56 (dd, J = 9.0, 2.4 Hz, 1H), 7.39 (m, 1H), 7.00 (dd, J = 7.8, 7.8 Hz, 1H),
6.98 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 4.72 (sept, J = 6.0 Hz, 1H), 1.47 (d, J = 6.0
Hz, 6H); 13C NMR (100 MHz, CDCl3): δ 193.5, 162.4, 157.8, 142.9, 138.6, 132.3, 132.0, 130.9,
124.0, 121.5, 120.6, 120.5, 120.2, 113.6, 110.3, 70.8, 22.2. HRMS (FAB) m/z: [M+H]+ calcd for
C18H18BrO3 361.0439, found 361.0455.
4.2.8. 5′-Bromo-2′
-hydroxy-2-propoxychalcone (29). 1H NMR (400 MHz, CDCl3): δ 12.9 (s, 1H),
8.15 (d, J = 15.2 Hz, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.88 (d, J = 15.2 Hz, 1H), 7.61 (dd, J = 7.6,
1.6 Hz, 1H), 7.55 (dd, J = 9.2, 2.4 Hz, 1H), 7.40 (m, 1H), 7.01 (dd, J = 7.6, 7.6 Hz, 1H), 6.96 (d,
J = 8.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 4.08 (t, J = 6.8 Hz, 2H), 1.99 (sext, J = 6.8 Hz, 2H),
20

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1.17 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 193.5, 162.4, 159.0, 142.8, 138.6, 132.4,
131.9, 131.4, 123.3, 121.5, 120.7, 120.5, 112.1, 110.3, 70.1, 22.7, 10.9. HRMS (FAB) m/z:
[M+H]+ calcd for C18H18BrO3 361.0439, found 361.0435.
1
4.2.9. 2
′
-Hydroxy-5
′
-iodo-2-methoxychalcone (32). H NMR (600 MHz, CDCl3): δ 12.9 (s, 1H),
8.25 (d, J = 15.6 Hz, 1H), 8.17 (d, J = 1.8 Hz, 1H), 7.72 (dd, J = 9.0, 2.4 Hz, 1H), 7.67 (dd, J =
7.8, 1.8 Hz, 1H), 7.66 (d, J = 15.6 Hz, 1H), 7.43 (m, 1H), 7.03 (dd, J = 7.8, 7.8 Hz, 1H), 6.97 (d,
13
J = 8.4 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H), 3.96 (s, 3H); C NMR (150 MHz, CDCl3): δ 193.2,
163.1, 159.1, 144.3, 142.1, 138.1, 132.6, 129.7, 123.4, 122.3, 121.0, 120.9, 120.0, 111.4, 79.6,
55.7. HRMS (FAB) m/z: [M+H]+ calcd for C16H14IO3 380.9988, found 380.9987.
4.3. General procedure for 2- or 4-hydroxychalcones
5′-Chloro-2′-hydroxy-4-(methoxymethoxy)chalcone (50 mg, 0.16 mmol) was dissolved in
acetic acid (3.0 mL) containing 1% H2SO4 (v/v) and stirred at room temperature. After the
reaction was complete, the mixture was extracted with EtOAc and the combined organic layer
was washed with water, saturated aqueous NaHCO3 and brine, dried over MgSO4 and
concentrated under reduced pressure. The residue was recrystallized with MeOH/H2O to give 5′-
fluoro-2′,2-dihydroxychalcone (23) (39 mg, 0.14 mmol) in 96% yield as a yellow solid. 2′-
Hydroxychalcones 19 [19], 25 [19], 30 and 31 as well as 4′-hydroxychalcones 21 [21] were
obtained by the same procedure.
4.3.1. 5′-Chloro-2′
,4-dihydroxy-3-methoxychalcone (23). 1H NMR (600 MHz, CDCl3): δ 12.9 (s,
1H), 7.90 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 9.0, 3.0 Hz, 1H), 7.41 (d, J
= 15.6 Hz, 1H), 7.28 (dd, J = 7.8, 1.8 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 6.989 (d, J = 8.4 Hz,
21

ACCEPTED MANUSCRIPT
1H), 6.987 (d, J = 9.6 Hz, 1H), 6.00 (s, 1H), 4.01 (s, 3H); 13C NMR (150 MHz, CDCl3): δ 192.6,
162.0, 149.0, 147.0, 146.9, 135.9, 128.7, 127.0, 124.3, 123.4, 120.7, 120.2, 116.7, 115.0, 110.2,
56.1. HRMS (FAB) m/z: [M+H]+ calcd for C16H14ClO4 305.0581, found 305.0590.
1
4.3.2. 3
′
,5′
-Dibromo-2
′
,2-dihydroxychalcone (30). H NMR (600 MHz, CDCl3): δ 8.20 (d, J =
15.6 Hz, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.88 (d, J = 2.4 Hz, 1H), 7.77 (d, J = 15.6 Hz, 1H), 7.62
(dd, J = 7.8, 1.8 Hz, 1H), 7.33 (m, 1H), 7.03 (dd, J = 7.8 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 5.41
(s, 1H); 13C NMR (150 MHz, CD3OD): δ 194.6, 160.1, 159.5, 144.8, 141.9, 133.9, 132.8, 131.2,
123.3, 122.7, 121.0, 120.0, 117.2, 113.8, 111.3. HRMS (FAB) m/z: [M+H]+ calcd for
C15H11Br2O3 396.9075, found 396.9057.
4.3.3. 2′,2-Dihydroxy-5′
-iodo-chalcone (31). 1H NMR (600 MHz, CDCl3): δ 12.9 (s, 1H), 8.19 (d,
J = 15.6 Hz, 1H), 8.18 (d, J = 1.8 Hz, 1H), 7.75 (d, J = 15.6 Hz, 1H), 7.72 (dd, J = 9.0-, 1.8 Hz,
1H), 7.63 (dd, J = 7.8, 1.8 Hz, 1H), 7.32 (m, 1H), 7.02 (dd, J = 7.2, 7.2 Hz, 1H), 6.85 (d, J = 8.4
Hz, 1H), 6.83 (d, J = 9.0 Hz, 1H), 5.57 (s, 1H); 13C NMR (150 MHz, CDCl3): δ 193.3, 163.1,
155.5, 144.4, 141.9, 138.1, 132.4, 130.5, 122.3, 121.8, 121.4, 121.0, 120.6, 116.5, 79.7. HRMS
(FAB) m/z: [M+H]+ calcd for C15H12IO3 366.9831, found 366.9836.
4.4. Preparation of 6,2′-dimethoxyflavanone (6).
A solution of 2′-hydroxy-2,5’-dimethoxychalcone (15 mg, 0.053 mmol) in 0.1 M aqueous
Na2HPO4-EtOH (1:4 v/v) (5 mL) was stirred at room temperature for 36 h. The reaction mixture
was concentrated under reduced pressure and extracted with EtOAc. The organic layer was
washed with water and brine, dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by preparative TLC, eluting with hexane-CH2Cl2-MeOH (6:1:0.1) to give
22

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1
6,2′-dimethoxyflavanone (6) (12.6 mg, 0.044 mmol) in 84 % yield as a colorless solid. H NMR
(400 MHz, CDCl3): δ 7.64 (dd, J = 7.6, 1.6 Hz, 1H), 7.37 (d, J = 3.6 Hz, 1H), 7.34 (m, 1H), 7.12
(dd, J = 9.2, 3.2 Hz, 1H), 7.06 (dd, J = 7.6 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 8.4 Hz,
1H), 5.81 (dd, J = 12.8, 3.2 Hz, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 2.97 (dd, J = 16.8, 3.6 Hz, 1H),
2.89 (dd, J = 16.8, 12.8 Hz, 1H); 13C NMR (150 MHz, CDCl3): δ 192.8, 156.8, 155.7, 154.1,
129.3, 127.6, 126.4, 125.2, 120.9, 120.8, 119.4, 110.5, 107.3, 74.8, 55.8, 55.3, 43.6. HRMS
(FAB) m/z: [M+H]+ calcd for C17H17O4 285.1127, found 285.1122.
4.5. General procedure for 2′-hydroxyflavanones
A solution of 5′-chloro-2′-hydroxy-2-(methoxymethoxy)chalcone (20 mg, 0.063 mmol) in 0.1
M aqueous Na2HPO4-EtOH (1:4 v/v) (7 mL) was stirred at room temperature for 36 h. The
reaction mixture was concentrated under reduced pressure and extracted with EtOAc. The
organic layer was washed with water and brine, dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by preparative TLC, eluting with hexane-CH2Cl2-
MeOH (6:1:0.1) to give 6-chloro-2′-(methoxymethoxy)flavanone (18 mg, 0.056 mmol) in 90%
yield as a colorless solid. A solution of 6-chloro-2′-(methoxymethoxy)flavanone (13 mg, 0.040
mmol) in acetic acid (0.5 mL) containing a catalytic amount (25 µL) of sulfuric acid was stirred
at room temperature. After the reaction was complete, water was added to the mixture and which
was extracted with EtOAc. The organic layer was washed with water three times, saturated
NaHCO3 and brine, dried over MgSO4 and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel, eluting with hexane-EtOAc (3:1) to give
6-chloro-2′-hydroxyflavanone (7) (10 mg, 0.037 mmol) in 93% yield as a colorless solid. 2′-
Hydroxyflavanones 1–5 [13-15] and 8–10 were obtained by the same procedure.
23

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1
4.5.1. 6-Chloro-2
′
-hydroxyflavanone (
7
). H NMR (400 MHz, CDCl3): δ 7.92 (d, J = 2.4 Hz,
1H), 7.47 (dd, J = 8.4, 2.4 Hz, 1H), 7.39 (dd, J = 7.6, 1.6 Hz, 1H), 7.27 (ddd, J = 8.0, 8.0, 1.6 Hz,
1H), 7.05 (d, J = 9.2 Hz, 1H), 7.01 (dd, J = 8.0, 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 5.91 (br,
1H), 5.78 (dd, J = 12.8, 3.6 Hz, 1H), 3.12 (dd, J = 17.2, 12.8 Hz, 1H), 3.03 (dd, J = 17.2, 3.6 Hz,
1H); 13C NMR (100 MHz, DMSO-d6): δ 191.1, 160.2, 154.3, 135.8, 129.5, 127.1, 125.5, 125.3,
124.6, 121.6, 120.4, 119.1, 115.5, 74.6, 42.0. HRMS (FAB) m/z: [M+H]+ calcd for C15H12ClO3
275.0475, found 275.0482.
1
4.5.2. 6-Bromo-2
′
-hydroxyflavanone (
8
). H NMR (400 MHz, CDCl3): δ 8.07 (d, J = 2.8 Hz,
1H), 7.60 (dd, J = 8.8, 2.8 Hz, 1H), 7.39 (dd, J = 8.0, 1.6 Hz, 1H), 7.27 (ddd, J = 8.0, 8.0, 1.6 Hz,
1H), 7.01 (m, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 5.81 (br, 1H), 5.77 (dd, J =
12.8, 3.2 Hz, 1H), 3.12 (dd, J = 17.2, 12.8 Hz, 1H), 3.01 (dd, J = 17.2, 3.2 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6): δ 191.0, 160.6, 154.3, 138.5, 129.5, 128.3, 127.1, 124.6, 122.1, 120.8,
119.1, 115.5, 113.0, 74.6, 42.0. HRMS (FAB) m/z: [M+H]+ calcd for C15H12BrO3 318.9970,
found 318.9971.
1
4.5.3. 6,8-Dibromo-2
′
-hydroxyflavanone (
9
). H NMR (600 MHz, DMSO-d6): δ 9.93 (s, 1H),
8.15 (d, J = 2.4 Hz, 1H), 7.85 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 7.8, 1.2 Hz, 1H), 7.22 (m, 1H),
6.92–6.89 (complex, 2H), 5.93 (dd, J = 13.2, 3.0 Hz, 1H), 3.28 (dd, J = 16.8, 13.2 Hz, 1H), 2.88
(dd, J = 16.8, 3.0 Hz, 1H); 13C NMR (150 MHz, DMSO-d6): δ 190.3, 157.1, 154.2, 140.3, 129.6,
128.1, 126.8, 124.3, 122.8, 119.2, 115.5, 112.94, 112.87, 75.4, 41.5. HRMS (FAB) m/z: [M+H]+
calcd for C15H11Br2O3 396.9075, found 396.9066.
4.5.4. 2′
-Hydroxy-6-iodoflavanone (10). 1H NMR (400 MHz, CDCl3): δ 8.25 (d, J = 2.0 Hz, 1H),
7.78 (dd, J = 8.8, 2.0 Hz, 1H), 7.38 (dd, J = 8.0, 1.6 Hz, 1H), 7.27 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H),
24

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7.01 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 6.89 (dd, J = 8.0, 0.8 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 5.77
(dd, J = 12.8, 3.2 Hz, 1H), 3.12 (dd, J = 16.8, 12.8 Hz, 1H), 3.01 (dd, J = 16.8, 3.2 Hz, 1H); 13C
NMR (150 MHz, DMSO-d6): δ 190.9, 161.1, 154.3, 144.0, 134.4, 129.5, 127.0, 124.6, 122.6,
120.8, 119.1, 115.5, 84.3, 74.5, 41.9. HRMS (FAB) m/z: [M+H]+ calcd for C15H12IO3 366.9831,
found 366.9813.
4.6. Cell proliferation assay using PCa cells
LNCaP cells (5×104) were seeded on 12-well plates (2-layer chambers) with DMEM including
5% charcoal-stripped fetal calf serum (CCS) (HyClone Laboratories, Logan, UT, USA). After 24
h, cells were treated with DHT (0, and 0.1 nM) 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.
4.7. Luciferase assay
The assay was performed as described previously [8]. Briefly, freshly trypsinized cells were
seeded on 12-well plates at the density of 50,000 cells per well. After 24 h, cells were transfected
by 0.5 ug/well of luciferase reporter plasmid (pGL3PSAp-5.8) using Lipofectamine (Invitrogen).
After 24 h of transfection, cells were treated with compounds with or without DHT for 24 h.
Harvested cells were lysed in lysis buffer (Promega) and luciferase activity was quantitated by a
luminometer (GENE LIGHT 55, Microtec).
4.8. Antiproliferative activity against multiple tumor cell lines
25

ACCEPTED MANUSCRIPT
Assay was performed as described previously [28]. Briefly, freshly trypsinized cell
suspensions were seeded in 96-well microtiter plates at densities of 4,000–11,000 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) operated by Gen5 software (BioTek)
after solubilizing the bound dye with 10 mM Tris base. The mean IC50 is the average from at
least three independent experiments of duplication for an assay and similar determinations.
4.9. Flow cytometric analysis
A549 (8 × 104 cells/well) and PC-3 (10 × 104 cells/well) cells were seeded in a 12-well plate
24 h prior to treatment with compounds 19 and 26 for 24 or 48 h. Compounds were used against
A549 or PC-3 at a concentration threefold or twofold of their IC50 value, respectively. Harvested
and 70% EtOH-fixed cells were stained with propidium iodide (PI) containing RNase (BD
Bioscience) subjected to flow cytometry (BD LSRFortessa, BD Biosciences). 20 nM (for A549)
or 200 nM (for PC-3) CA-4 was used as tubulin polymerization inhibitor arresting cells at G2/M.
4.10. Immunostaining
PC-3 cells were grown on an 8-well chamber slide (Lab-Tech) for 24 h prior to treatment
with reagents. Cells were treated with compound 19 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 α-tubulin (B5-1-2, Sigma) followed by
FITC-conjugated antibody to mouse IgG (Sigma) [27, 28]. Nuclei were labeled with DAPI
(Sigma). Fluorescence labeled cells were observed using confocal microscope (Zeiss, LSM700)
26

ACCEPTED MANUSCRIPT
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.
4.11. Tumor xenograft model in SCID Mice
Seven-week old SCID male mice (C.B-17 scid) were purchased from CLEA (Japan). PC-3
cells (2.0 × 106 cells) with 50% matrigel were xenografted subcutaneously into the dorsal side of
the mice at 8 weeks old. When tumor volume reached about 50 mm3, the mice were randomly
divided into three groups each containing 5 mice and compound administration started on the
second day. The test compounds were administrated intraperitoneally at a dose of 30 mg/kg body
weight every 4 days. Tumor size was measured at a 4-day interval using micrometer clipper, and
the volume (mm3) was calculated by width (mm)2 × length (mm) × 0.5. This animal study was
approved by the Institutional Animal Care and Use Committee (IACUC) of the Kanazawa
University (Kanazawa, Japan) and performed according to the institutional guidelines.
Author Contributions
Y.S., A.M. and K.N.G. designed this study. Y.S., H.T., and K.N.G. designed and performed
chemical experiments. A.M., K.I., and M.G. designed and performed biological experiments.
Y.S., A.M., K.I., M.G. and K.N.G. evaluated all results and wrote manuscript. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
Acknowledgements
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ACCEPTED MANUSCRIPT
We appreciate critical comments, suggestions, and editing on the manuscript by Dr. Susan L.
Morris-Natschke (UNC-CH). We thank the Microscope Service Laboratory (UNC-CH) for their
expertise in confocal microscope. This work was supported 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 the Eshelman Institute for Innovation, Chapel Hill, North Carolina, awarded to M.G.
Appendix A. Supplementary data.
Supplementary data related to this article can be found at supporting information, uploaded
file with the manuscript.
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