Development of androgen-antagonistic coumarinamides with a unique aromatic folded pharmacophore

Article abstract of DOI:10.3390/ijms21155584

First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide (2a) and bicalutamide (3), are effective for most prostate cancer patients, but resistance often appears after several years due to the mutation of AR. Second-generation A

Full text of DOI:10.3390/ijms21155584

International Journal of
Molecular Sciences
Article
Development of Androgen-Antagonistic
Coumarinamides with a Unique Aromatic
Folded Pharmacophore
Hitomi Koga 1, Mai Negishi 1, Marie Kinoshita 1, Shinya Fujii 2, Shuichi Mori 2,
Mari Ishigami-Yuasa 2, Emiko Kawachi 2, Hiroyuki Kagechika 2,
*
and Aya Tanatani 1,3,
*
1
Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku,
Tokyo 112-8610, Japan; Kogahitomi.ocha@gmail.com (H.K.); mai.negishi0903@gmail.com (M.N.);
aimer61@icloud.com (M.K.)
2
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU),
2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; fujiis.chem@tmd.ac.jp (S.F.);
s-mori.chem@tmd.ac.jp (S.M.); myuasa.chem@tmd.ac.jp (M.I.-Y.); e.k.sakuratomomochan@gmail.com (E.K.)
Institute of Human Life Innovation, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku,
Tokyo 112-8610, Japan
3
*
Correspondence: kage.chem@tmd.ac.jp (H.K.); tanatani.aya@ocha.ac.jp (A.T.);
Tel.: +81-3-5280-8032 (H.K.); +81-3-5978-2716 (A.T.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 July 2020; Accepted: 31 July 2020; Published: 4 August 2020
Abstract: First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide (2a
)
and bicalutamide (3), are effective for most prostate cancer patients, but resistance often appears
after several years due to the mutation of AR. Second-generation AR antagonists are effective
against some of these castration-resistant prostate cancers, but their structural variety is still limited.
In this study, we designed and synthesized 4-methyl-7-(N-alkyl-arylcarboxamido)coumarins as AR
antagonist candidates and evaluated their growth-inhibitory activity toward androgen-dependent
SC-3 cells. Coumarinamides with a secondary amide bond did not show inhibitory activity, but their
N-methylated derivatives exhibited AR-antagonistic activity. Especially, 19b and 31b were more
potent than the lead compound 7b, which was comparable to hydroxyflutamide (2b). Conformational
analysis showed that the inactive coumarinamides with a secondary amide bond have an extended
structure with a trans-amide bond, while the active N-methylated coumarinamides have a folded
structure with a cis-amide bond, in which the two aromatic rings are placed face-to-face. Docking study
suggested that this folded structure is important for binding to AR. Selected coumarinamide derivatives
showed AR-antagonistic activity toward LNCaP cells with T877A AR, and they had weak progesterone
receptor (PR)-antagonistic activity. The folded coumarinamide structure appears to be a unique
pharmacophore, different from those of conventional AR antagonists.
Keywords: prostate cancer; cell proliferation; coumarin; cis-amide
1. Introduction
Androgen receptor (AR) is a ligand-dependent transcription factor belonging to the nuclear
receptor superfamily [
(see Supplementary Materials) and dihydrotestosterone (DHT, 1b) (Figure 1). These androgens have
various roles in the differentiation, growth, and maintenance of the male reproductive organs [ ].
1,2], and its endogenous ligands, so-called androgens, are testosterone (1a)
3,4
Based on its physiological and pharmacological functions, AR is recognized as an important target for
drug discovery. For example, since androgen plays an important role in the progression of prostate
cancer, AR antagonists have been used clinically for the treatment of prostate cancer [5–7]. However,
Int. J. Mol. Sci. 2020, 21, 5584; doi:10.3390/ijms21155584
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AR antagonists bearing a steroid core structure have undesirable side effects due to their cross-activity
with other steroid hormone receptors. Consequently, nonsteroidal AR antagonists have been developed,
and some of them, such as flutamide (2a) [8] and bicalutamide (BIC, 3, Figure 1) [9,10], are in clinical
use to treat prostate cancer. These first-generation nonsteroidal AR antagonists are effective for most
prostate cancer patients, but castration-resistant prostate cancer (CRPC) often develops after a few
years, due to the mutation of AR [11–13]. For example, T877A is the most common mutation of AR,
and hydroxyflutamide (2b), an active metabolite of flutamide (2a), acts as an agonist toward T877A
AR [14]. Bicalutamide (3) acts as antagonist toward T877A AR, but as an agonist toward W741C
AR [15].
Figure 1. Structures of (a) endogenous androgens and (b) typical nonsteroidal AR antagonists.
Second-generation AR antagonists effective against CRPC have been developed, such as
enzalutamide (
4
) [16
,
17], apalutamide [18
,
19], and darolutamide [20]. Structurally, these AR antagonists
), and they
have the same pharmacophore as the lead compounds, flutamide (2a) and bicalutamide (
3
contain an anilide structure with electron-withdrawing functional groups on the phenyl ring (Figure 1).
Recently, non-anilide type AR antagonists have also been reported, but variation in the chemotype is
still limited [21].
We have reported 6-arylcoumarin derivatives such as
5 and 6 as novel nonsteroidal progesterone
receptor (PR) antagonists (Figure 2) [22 23]. In these compounds, the coumarin ring provides an
,
alternative hydrophobic core structure to the sterane ring of steroidal derivatives or to the bicyclic anilide
scaffold of known nonsteroidal PR ligands (i.e., benzoxadinone, and benzimidazolone derivatives
bearing a nitrogen atom in the bicyclic heterocyclic skeleton). Among them, compounds 5, 6b,
and 6c showed moderate AR-antagonistic activity in the androgen-dependent SC-3 cell growth
assay [24]. These results suggested that the coumarin ring could mimic the anilide scaffold of AR
antagonists. In addition, we reported 4-benzyl-1-(2H)-phthalazinone derivatives as AR antagonists
with a novel core structure [25]. With this background, we decided to search for compounds with
AR-antagonistic activity among our coumarin compound library, and we found that compound
7b bearing a 7-(N-methyl-benzoylamino) moiety showed AR-antagonistic activity with rather low

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PR-antagonistic activity (Figure 2). Compound 7b has a unique core structure that is different from
those of conventional AR antagonists. In this study, we examined the structure–activity relationship of
coumarinamide derivative 7b as an AR antagonist and identified a unique pharmacophore, consisting
of an aromatic folded core structure that can interact with amino acid residues in the ligand-binding
pocket of AR.
Figure 2. Structures of (a) 6-arylcoumarin derivatives 5 and 6 with progesterone receptor (PR)-antagonistic
activity and (b) androgen receptor (AR)-antagonistic coumarin 7.
2. Results and Discussion
Various 4-methyl-7-(N-methyl-arylcarboxamido)coumarins with different substituents on the
phenyl ring or amide nitrogen atom were synthesized from 7-amino-4-methylcoumarin by N-acylation,
followed by N-alkylation (Scheme 1); their structures are shown in Tables 1–3. In order to clarify the
electronic and steric properties of the amide bond, the biological activities of the derivatives bearing a
secondary amide bond (series a) were also examined.
Scheme 1. Synthesis of coumarinamides.
AR-agonistic and antagonistic activities of the synthesized coumarinamide derivatives were
evaluated in terms of growth-inhibitory activity toward SC-3 cells bearing wild-type (wt) AR [24].
These cells show androgen-dependent cell proliferation, which is one of the typical and reliable assays
for AR modulators [26]. None of the coumarinamide derivatives examined affected the proliferation
of SC-3 alone at a concentration below 10−5 M (data not shown), which means they do not act as AR
agonists. The antagonistic activity of the test compounds was examined in terms of their effect on 1 nM
DHT-dependent proliferation of SC-3 cells. The IC50 values of test compounds are shown in Table 1,
and the dose-dependency of the selected active compounds are shown in Figure 3. Compound 7b
the lead compound in this study, inhibited the DHT-induced proliferation of SC-3 cells with the IC50
value of 1.34 M, and its activity is nearly one-fifth that of hydroxyflutamide (2b; IC50 = 0.29 M),
the positive control.
,
µ
µ

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Table 1. AR-antagonistic activity of coumarinamide derivatives in SC-3 cell assay a.
Compound
N-Substituent
X
IC50, µM
0.29 ± 0.03
inactive c
inactive c
inactive c
inactive c
inactive c
inactive c
inactive c
inactive c
inactive c
7.66 ± 3.57
inactive c
inactive c
>10 b
inactive c
inactive c
inactive c
inactive c
inactive c
inactive c
Compound
N-Substituent
X
IC50, µM
Hydroxyflutamide (2b)
7a
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
none
p-CN
p-NO2
p-CF3
p-F
7b
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
none
p-CN
p-NO2
p-CF3
p-F
1.34 ± 0.21
inactive c
inactive c
inactive c
0.58 ± 0.06
inactive c
inactive c
inactive c
2.40 ± 0.39
0.60 ± 0.04
0.72 ± 0.12
0.73 ± 0.11
0.49 ± 0.04
0.67 ± 0.05
7.70 ± 0.45
inactive c
>10 b
8a
8b
9a
9b
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
20b
21b
22b
23b
24b
25b
p-Cl
p-Cl
p-Me
p-OMe
m-CN
m-CF3
m-F
p-Me
p-OMe
m-CN
m-CF3
m-F
m-Cl
m-Cl
m-Me
m-OMe
o-F
m-Me
m-OMe
o-F
o-Cl
o-Cl
o-OMe
3,5-Cl2
3,5-Me2
o-OMe
m,m’-Cl2
m,m’-Me2
1.04 ± 0.07
0.83 ± 0.07
a
Inhibitory activity of test compounds toward dihydrotestosterone (DHT)-induced proliferation of SC-3 cells was
b
examined [24,26]. ‘>10’ means that the test compound at 10 µM inhibited the proliferation of SC-3 cells by less
than 50% versus control cells. ‘Inactive’ means that the test compound did not inhibit the proliferation of SC-3
c
cells at concentrations up to 10 µM.
First, we examined the substituent effect on the phenyl ring of 7b. Among the synthesized
compounds, most of the coumarinamides with a secondary amide bond (series
cell growth, except for compounds 16a and 19a, which showed weak inhibitory activity only at 10
On the other hand, some of the N-methylated compounds (series ) dose-dependently inhibited the
a
) did not affect SC-3
µM.
b
DHT-induced proliferation of SC-3 cells. The introduction of a substituent at the para position of
the phenyl group was not effective, and only compound 11b bearing a small fluorine atom exhibited
inhibitory activity, being more potent than the lead compound without a substituent on the phenyl ring,
and comparable in potency to hydroxyflutamide (2b). The introduction of a substituent at the ortho
position was also ineffective, while compounds bearing a meta-substituent showed potent inhibitory
activity. The introduction of a meta-cyano group, which is an electron-withdrawing group common
to the structures of bicalutamide (
lead compound 7b. The introduction of a halogen atom or halogen-containing substituent at the meta
position increased the activity, and compounds 16b 17b, and 18b showed more potent activity than 7b
Among the compounds with a meta-substituent, compound 19b with the m-methyl group was most
active in SC-3 assay, showing an IC50 value of 0.49 M. Introduction of a second meta-substituent was
3) and enzalutamide (4), decreased the activity compared to the
,
.
µ
not effective, and compounds 24b and 25b with m,m’-dichloro or m,m’-dimethyl groups, respectively,
are slightly less active than the corresponding monosubstituted compounds 18b and 19b.

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Figure 3. Dose-dependent growth inhibition of selected coumarinamides in the presence of 1 nM
DHT. The vertical scale is SC-3 cell growth, normalized to that of DHT-treated cells without the test
compound, which is taken as 1.
Secondly, the effect of N-substituents was examined for the selected compounds 18b and 19b
(Table 2). In both cases, as the N-substituent became larger, the inhibitory activity decreased. Thus,
the
c and d compounds with a N-ethyl or N-n-propyl group showed moderate inhibitory activity,
while the e and f compounds with the N-substituent containing an aromatic ring were less active.
Table 2. Effect of N-substituents of coumarinamide derivatives on AR-antagonistic activity in SC-3 cell
assay a.
Compound
18b
X
N-Substituent
IC50, µM
0.73 ± 0.11
1.70 ± 0.20
2.88 ± 0.28
5.55 ± 0.40
Inactive b
Compound
19b
X
N-Substituent
IC50, µM
0.49 ± 0.04
2.46 ± 0.35
8.49 ± 0.18
1.83 ± 0.12
Inactive b
Me
Et
Me
Et
18c
19c
18d
n-Pr
19d
n-Pr
Cl
Me
18e
CH2Ph
19e
CH2Ph
18f
CH2-2-naphthyl
19f
CH2-2-naphthyl
a
Inhibitory activity of test compounds toward DHT-induced proliferation of SC-3 cells was examined [24
‘Inactive’ means that the test compound did not inhibit the proliferation of SC-3 cells at concentrations up to
,
26].
b
10 µM.
Thirdly, we replaced the phenyl group of compound 7b with other ring structures (Table 3).
Compound 26b with a saturated cyclic alkyl group instead of the phenyl ring was inactive, which was
probably due to the bulkiness of the nonplanar cyclohexyl ring. Interestingly, compound 27b with a
1-naphthyl group is more potent than the lead compound 7b, while compound 28b with a 2-naphthyl
group, an isomer of 27, exhibited weaker inhibitory activity. Thus, the orientation of the naphthyl
group is important for the activity. The derivatives of 7b with para-substituents were almost inactive
(Table 1), although the para position of compound 28b could be regarded as substituted by a benzo

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group. Replacement of the phenyl ring of 7b with the basic pyridine ring diminished the activity,
while replacement with 5-membered heterocyclic aromatics such as furyl and thiophenyl groups
increased the activity. Compound 31b had potent inhibitory activity with the IC50 value of 0.50
which was comparable to that of hydroxyflutamide (2b).
µM,
Table 3. AR-antagonistic activity of coumarinamide derivatives in SC-3 cell assay a.
Compound
7b
Ar
IC50, µM
1.34 ± 0.21
Inactive b
0.66 ± 0.04
7.81 ± 1.91
Inactive b
0.92 ± 0.18
0.50 ± 0.07
Ph
26b
c-C6H11
1-naphthyl
2-naphthyl
2-pyridyl
2-furyl
27b
28b
29b
30b
31b
2-thiophenyl
a
Inhibitory activity of test compounds toward the DHT-induced proliferation of SC-3 cells was examined [24
,
26].
b
‘Inactive’ means that test compound did not inhibit the proliferation of SC-3 cells at concentrations up to 10 µM.
Next, we examined the activity of the selected compounds 18b and 19b toward LNCaP cells
bearing mutated T877A AR (Figure 4) [27 28]. Bicalutamide ( ) inhibited the proliferation of LNCaP
cells, whereas hydroxyflutamide (2b) did not. Compounds 18b and 19b inhibited the DHT-dependent
,
3
cell growth of LNCaP cells, and their inhibitory activities were higher than that of bicalutamide (
3).
Thus, coumarinamide derivatives can act as AR antagonists toward mutated T877A AR.
Figure 4. Inhibitory activities of selected coumarinamide derivatives on the proliferation of LNCaP
cells with T877A AR. Cell viability was determined by the WST method and normalized to that of
vehicle-treated cells, taken as 1.

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As discussed above, the coumarinamide derivatives (series
a) with a secondary amide bond
(
R = H) showed little or no activity, while their N-methylated derivatives (series
AR-antagonistic activity. We have previously reported on the unique conformational properties of
aromatic amide compounds [28 29]. Thus, secondary amides such as benzanilide existed in trans
b) exhibited potent
,
form, while their N-methylated amides exist in cis form in the crystal and predominantly in cis
form in solution. The cis conformational preference is a general property of N-methylated amides,
including those bearing N-heterocyclic aromatics such as pyridine, pyrrole, and imidazole. Therefore,
we assumed that the difference in AR-antagonistic activity between secondary and N-methylated
compounds would result from the difference in their conformational properties. Then, we examined
the crystal structures of compounds 7a and 7b (Figure 5). As expected, compound 7a exists in the
extended structure with a trans-amide form, in which the phenyl and coumarin rings are located on
opposite sides of the molecule. On the other hand, compound 7b exists in a folded structure with
cis-amide, in which the two aromatic rings take a face-to-face position. Comparison of the 1H NMR
chemical shifts of 7a and 7b indicated that these compounds exist in solution in conformations similar
to those found in the crystal. Thus, compounds 7b shows aromatic proton signals at a higher field
than compound 7a in the 1H NMR spectra, due to the anisotropic effects between two aromatic rings.
A similar tendency was observed with the other coumarinamide derivatives. These results indicate
that the folded structure with a cis-amide bond is important for AR-antagonistic activity.
(a)
trans form
H
N
O
O
O
7a
(b)
Me
N
O
O
O
cis form
7b
Figure 5. Crystal structures of compounds (a) 7a and (b) 7b.
In order to analyze the binding features of coumarinamide derivatives with AR, we conducted
a docking study of compounds 7a and 7b with the AR ligand-binding domain (LBD) by using the
AutoDock program. For the docking, we used the crystal structure of the AR LBD in the complex with
DHT (1b) (PDB ID: 2AMA) and the crystal structures of 7a and 7b, as shown in Figure 6. Notably,
7b with the folded structure can dock with the AR LBD in a similar position to that of DHT (1b). In this
binding structure, the carbonyl group of the cis-amide bond of compound 7b lies in the same direction
as that of DHT (1b), and the phenyl group of 7b is placed in the hydrophobic region. On the other
hand, compound 7a with an extended structure can bind to the ligand-binding pocket, but there is no
strong interaction with the surrounding amino acid residues. The binding energy of 7a with AR LBD
is –7.55 kcal/mol, which is lower in energy than that of 7b
( 9.04 kcal/mol). Thus, the docking study
−
also supports the significance of the folded coumarin amide structure for AR-antagonistic activity.
Finally, we examined the activity of selected coumarinamide derivatives toward progesterone
receptor (PR), since some coumarin derivatives act as PR antagonists. The PR-antagonistic activities
were evaluated by means of alkaline phosphatase (AP) assay using the T-47D human breast carcinoma

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cell line (Figure 7) [30]. All the selected coumarinamide derivatives examined showed PR-antagonistic
activity only at 10 M, and they did not affect the progesterone (1 nM)-induced AP activity at
concentrations below 1 M. In particular, the PR-antagonistic activity of compound 31b was very
µ
µ
weak, and the compound only slightly inhibited AP activity, even at 10
derivatives show selectivity as AR antagonists over PR.
µM. Thus, the coumarinamide
(a)
(b)
Figure 6. Docking study of coumarin derivatives (
a)
7a and (b) 7b with the AR ligand-binding domain
(LBD) (PDB ID: 2AMA) using AutoDock 4.2, superposed with the structure of DHT (1b) in the crystal.
The CPK colored residue is Arg752. Coumarin derivatives and DHT (1b) are shown in green and
blue, respectively.
Figure 7. PR-antagonistic activities of selected coumarinamide derivatives, evaluated by means of
alkaline phosphatase (AP) assay using T-47D cells. The vertical axis is the activity of alkaline phosphatase
in the presence of 1 nM progesterone and test compound, normalized to progesterone alone as 1. MP:
mifepristone (PR antagonist).
3. Materials and Methods
3.1. Chemistry
3.1.1. General
All reagents were purchased from Sigma-Aldrich Chemical Co. (Tokyo, Japan), Tokyo Kasei
Kogyo Co. (Tokyo, Japan), Wako Pure Chemical Industries (Tokyo, Japan), or Kanto Kagaku Co.,
Inc. (Tokyo, Japan). Silica gel for column chromatography was purchased from Kanto Kagaku Co.,
1
Inc (Tokyo, Japan). H and 13C NMR spectra were recorded on a JEOL ECA 600 (Tokyo, Japan),

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or Bruker 600 spectrometer (Tokyo, Japan). Mass spectral data were obtained on a Bruker Daltonics
microTOF-2focus (Tokyo, Japan), Thermo Scientific Q-Exactive or Waters Q-TOF Premier in the positive
ion detection mode (Tokyo, Japan).
3.1.2. Synthesis of 23a (General Procedure for Coumarinamides with Secondary Amide Bond)
o-Anisoyl chloride (120 mg, 0.70 mmol) was added to a solution of 7-amino-4-methylcoumarin
(101 mg, 0.58 mmol) in pyridine (3 mL), and the mixture was stirred at 50 ◦C for 2 h. The mixture was
poured into 2 M HCl (30 mL), and the precipitates were collected, washed with water, and a mixture of
chloroform and methanol to give 23a (171 mg, 96%).
3.1.3. Synthesis of 23b (General Procedure for N-Alkylated Coumarinamides)
Sodium hydride (24 mg, 0.60 mmol) was washed with n-hexane. A solution of 23a (61 mg,
0.20 mmol) in DMF (1 mL) was added to a suspension of sodium hydride in DMF (2 mL). After 20 min,
iodomethane (83 mg, 0.58 mmol) was added to the mixture. After 5 h, the reaction mixture was poured
into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over
magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography
(ethyl acetate:hexane = 1:1) to give 23b (21.0 g, 32%).
3.2. SC-3 Growth Inhibition Assay
SC-3 cell growth inhibition assay was performed as the previous report [24
,26]. SC-3 cells were
seeded in 96-well plates at a concentration of 2000 cells per well in 100 L of MEM
supplemented with 2% FBS (fetal bovine serum), and the plates were incubated for 24 h at 37 C
µ
α
medium
◦
under 5% CO2. Then, 10 µL of medium was removed from each well and replaced with 10 µL of
drug solution supplemented with serial dilutions of test compound or DMSO as a dilution control
in the presence of 1 nM DHT, and the plates were incubated for 3 days. The cell number was
determined using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s
instruction. This parameter is proportional to the number of living cells in the culture. IC50 (Tables 1–3)
is the concentration of test compound that reduces DHT-induced cell growth to 50% of the control.
All experiments were performed in triplicate or more.
3.3. LNCaP Cell Proliferation Assay
LNCaP cell growth inhibition assay was performed as the previous report [27
LNCaP cells were seeded in 96-well plates at a concentration of 2000 cells per well in 100
RPMI-1640 medium supplemented with 2% charcoal-stripped FBS, and the plates were incubated for
24 h at 37 ◦C under 5% CO2. Then, 10
L of medium was removed from each well and replaced with
10 L of drug solution supplemented with serial dilutions of test compound or DMSO as a dilution
,28
,31]. Briefly,
µL of
µ
µ
control in the presence or absence of 10 nM DHT. Cells were incubated for 6 days, and half of the
medium was removed and replaced once after 3 days with medium containing test compound or
DMSO as a dilution control. At the end of the incubation, proliferation was evaluated using a Cell
Counting Kit-8 (Dojindo) according to the manufacturer’s instruction. This parameter is proportional
to the number of living cells in the culture. All experiments were performed in triplicate or more.
3.4. Alkaline Phosphatase Assay Using T-47D Cells
Alkaline phosphatase assay was performed based on the previous report [22
cells were plated in 96-well plates at 10,000 cell per well and incubated at 37 ◦C under 5% CO2 for
24 h. Then, 10 L of medium was removed from each well and replaced with 10 L of drug solution
,32]. Briefly, T-47D
µ
µ
supplemented with serial dilutions of test compound or DMSO as a dilution control in the presence or
absence of 1 nM progesterone, and incubation was continued for 24 h. Then, the medium was aspirated,
and the cells were fixed with 100 µL of 1.8% formalin in PBS. The fixed cells were washed with PBS,

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and 75
µL of assay buffer (1 mg/mL p-nitrophenol phosphate in diethanolamine water solution, pH 9.0,
2 mM MgCl2) was added. The mixture was incubated at room temperature with shielding from light
for 2 h, and then the reaction was terminated by the addition of 100
405 nm was measured with a microplate reader.
µL of NaOH. The absorbance at
4. Conclusions
We synthesized a series of 4-methyl-7-(N-alkyl-arylcarboxamido)coumarins as AR antagonist
candidates, using compound 7b as a lead compound. The AR-antagonistic activity of the synthesized
coumarinamide derivatives was examined in terms of growth-inhibitory activity toward SC-3 cells.
Coumarinamides with a secondary amide bond were inactive, but their N-alkylated derivatives
exhibited AR-antagonistic activity. Among them, compounds 19b and 31b showed more potent
AR-antagonistic activity than the lead compound 7b, and they were comparable in potency to
hydroxyflutamide (2b). Conformational analysis and docking study revealed that the folded structure
of coumarinamides associated with the cis-amide bond is important for binding to AR. The selected
coumarinamide derivatives showed AR-antagonistic activity toward LNCaP cells with T877A AR and
had weak PR-antagonistic activity. The detailed action mechanism of novel coumarinamides as AR
modulators including AR-binding ability and transcriptional regulation should be elucidated in the
future works, as the folded coumarinamide is a different pharmacophore from those of conventional AR
antagonists, and these compounds may be promising candidates for the treatment of prostate cancer.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/15/5584/s1.
Compound Data: Development of novel androgen antagonistic coumarinamides bearing unique aromatic
folded structure.
Author Contributions: H.K. (Hitomi Koga), M.N., M.K. synthesized the compounds. S.F., S.M., M.I.-Y., E.K.
examined the biological activities. H.K. (Hiroyuki Kagechika) and A.T. planed and supervised the scientific works,
and wrote the manuscript. All authors have read and edited the manuscript, and agreed to the published version
of the manuscript.
Funding: This work was partly supported by JSPS KAKENHI Grant No. 20K06963 (to A.T.), 17H03887 and
19K22486 (to S.F.), JSPS Core-to-Core Program, A. Advanced Research Networks, and Japan Agency for Medical
Research and Development (AMED) under Grant Number JP20am0101098 (Platform Project for Supporting Drug
Discovery and Life Science Research, BINDS). A part of this research involved a Cooperative Research Project of
the Research Center for Biomedical Engineering. A.T. thanks The Naito Foundation, the Cosmetology Research
Foundation, and the Tokyo Biochemical Research Foundation.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
AR
DHT
BIC
androgen receptor
dihydrotestosterone
bicalutamide
CRPC castration-resistant prostate cancer
PR
NMR
LBD
progesterone receptor
ligand-binding domain
keratinocyte growth factor
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