Efficient lead finding, activity enhancement and preliminary selectivity control of nuclear receptor ligands bearing a phenanthridinone skeleton

Article abstract of DOI:10.3390/ijms19072090

Background: Nuclear receptors (NRs) are considered as potential drug targets because they control diverse biological functions. However, steroidal ligands for NRs have the potential to cross-react with other nuclear receptors, so development of non-steroi

Full text of DOI:10.3390/ijms19072090

International Journal of
Molecular Sciences
Article
Efficient Lead Finding, Activity Enhancement and
Preliminary Selectivity Control of Nuclear Receptor
Ligands Bearing a Phenanthridinone Skeleton
1
1
2
ID
, Yuichi Hashimoto 1
Yuko Nishiyama , Shinya Fujii , Makoto Makishima
1
,
ID
and Minoru Ishikawa *
1
Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032,
Japan; ynishiya@ncc.go.jp (Y.N.); fujiis@iam.u-tokyo.ac.jp (S.F.); hashimot@iam.u-tokyo.ac.jp (Y.H.)
Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan;
makishima.makoto@nihon-u.ac.jp
2
*
Correspondence: m-ishikawa@iam.u-tokyo.ac.jp; Tel.: +81-358-417-853
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 25 June 2018; Accepted: 17 July 2018; Published: 18 July 2018
Abstract: Background: Nuclear receptors (NRs) are considered as potential drug targets because
they control diverse biological functions. However, steroidal ligands for NRs have the potential to
cross-react with other nuclear receptors, so development of non-steroidal NR ligands is desirable to
obtain safer agents for clinical use. We anticipated that efficient lead finding and enhancement of
activity toward nuclear receptors recognizing endogenous steroidal ligands might be achieved by
exhaustive evaluation of a steroid surrogate library coupled with examination of structure-activity
relationships (SAR). Method: We evaluated our library of RORs (retinoic acid receptor-related
orphan receptors) inverse agonists and/or PR (progesterone receptor) antagonists based on the
phenanthridinone skeleton for antagonistic activities toward liver X receptors (LXRs), androgen
receptor (AR) and glucocorticoid receptor (GR) and examined their SAR. Results: Potent LXR
β, AR,
and GR antagonists were identified. SAR studies led to a potent AR antagonist (IC : 0.059
µM).
50
Conclusions: Our approach proved effective for efficient lead finding, activity enhancement and
preliminary control of selectivity over other receptors. The phenanthridinone skeleton appears to be
a promising steroid surrogate.
Keywords: phenanthridinone; steroid surrogate; antagonist
1
. Introduction
Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate DNA
transcription by binding small molecular agonists such as hormones. Forty-eight structurally conserved
kinds of NRs have been identified, and many of them recognize endogenous steroidal ligands (Figure 1).
Because NRs control diverse biological functions including reproduction, differentiation, homeostasis,
and the immune system, they were the targets of approximately 5% of FDA-approved drugs in 2011 [1].
However, not only a steroidal ligand, but also its metabolites, brings with it the potential to cross-react
with other nuclear receptors, which can result in unwanted side effects, potentially limiting the clinical
utility of these agents. Therefore, surrogates for the steroid skeleton are required to develop selective
non-steroidal NR ligands for clinical use [2].
Int. J. Mol. Sci. 2018, 19, 2090; doi:10.3390/ijms19072090
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 2090
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Figure 1. Chemical structures of endogenous steroidal ligands for NRs and phenanthridinone ligands
for target proteins that recognize endogenous steroidal ligands.
The number of fold structures of human proteins is at least 50 times smaller than the number
of human proteins [
structure might serve as a common scaffold for ligands that would interact specifically with more than
0 different human proteins, neglecting interactions of the peptide sequences of the proteins. In other
2]. This fact indicates that a scaffold that is spatially complementary to one-fold
5
words, the structures of ligands that bind to one member of fold structures may be useful for the
development of novel lead compounds for other proteins with the similar fold structure. However, lead
compounds obtained based on this approach are likely to possess polypharmacological character.
Therefore, chemical modification of polypharmacological lead compounds, aimed at optimizing
selectivity for a particular target, is required. Since the fold structures of NR ligand-binding domains
(
LBDs) are similar, we have used this strategy to create ligands bearing a diphenylmethane skeleton
for several NRs, including farnesoid X receptor [ ], liver X receptor (LXR) [ ], vitamin D receptor [ ],
3
4
5
androgen receptor (AR) [6], and estrogen receptor [7].
We have previously developed LXRs antagonist
1
[
8
], retinoic acid receptor-related orphan
10], and pharmacological
for Niemann-Pick disease type C1 (NPC1) [11] and Niemann-Pick type C1-like 1
NPC1L1) (Figure 1) [12]. These compounds all contain a phenanthridin-6(5H)-one skeleton as a cyclized
carba-analog of the skeleton of LXRs pan agonist T0901317 ( ). Because endogenous ligands for the
receptors (RORs) inverse agonist
chaperones and
2 [9], progesterone receptor (PR) antagonist 3 [
4
5
(
6
above proteins (LXRs, RORs, PR, NPLC1 and NPC1L1) all have a steroid skeleton, we hypothesized that
if the phenanthridin-6(5H)-one scaffold acts as a steroid surrogate, phenanthridinone derivatives would
also bind to other NRs that recognize endogenous steroidal ligands. The approach described here is
expected to be useful in the development of novel steroid surrogates, such as the phenanthridinone
skeleton, enabling efficient lead finding of ligands for target proteins that recognize endogenous
steroidal ligands, and the generation of more selective ligands by further molecular modifications of
new and convenient scaffolds (easy to synthesis, easy to introduce substitutent(s) at any position, etc.),
compared to the steroid skeleton. Here, we show that application of this approach to our small library
of phenanthridinone-based RORs inverse agonists and PR antagonists led to several selective LXR
AR and glucocorticoid receptor (GR) antagonists.
β,
2
. Results and Discussion
AR and GR, members of NRs, recognize endogenous steroidal ligands as shown in Figure 1.
In addition, because LXRs antagonistic activities of only limited numbers of phenanthridinone
analog have been reported [ ], precise structure-activity relationships (SAR) remain unclear.
Therefore, we exhaustively evaluated our small library of RORs inverse agonists and/or PR
antagonists bearing a phenanthridinone skeleton for LXR , LXR , AR and GR antagonistic activities,
and examined their SAR. LXRs regulate ATP-binding cassette proteins (ABCs), ApoE and glucose
8
α
β

Int. J. Mol. Sci. 2018, 19, 2090
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transporter 4 (GLUT4), are involved in lipid metabolism, reverse cholesterol transport, and glucose
transport, so LXRs agonists are likely to be of therapeutic value in the treatment of atherosclerosis,
hyperlipidemia, and metabolic syndrome. They are also involved in the upregulation of sterol
regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS), so LXRs antagonists
might have therapeutic value for hepatic steatosis [13]. AR is a receptor of androgens essential for the
development and maintenance of the male reproductive system and secondary male sex characteristics.
AR antagonists including hydroxyflutamide (OHF) are used for treatment of androgen-dependent
tumors, especially prostate tumors. GR is a receptor of cortisol, and selective antagonists of GR could
be useful in treating hypercortisolemia associated with Cushing’s syndrome and other conditions in
which the endogenous GR is hyperactivated either through higher glucocorticoid levels or increased
receptor sensitivity [14]. A steroidal GR antagonist, mifepristone (RU486), shows potent activities
toward other steroid receptors.
For this work, compounds
PR-antagonistic activity was evaluated by assay of PR-regulated alkaline phosphatase activity
in human breast cancer cell line T47D [10 16] while ROR , ROR and ROR inverse agonistic
activities, and LXR , LXR , AR and GR antagonistic activities were evaluated by reporter gene
assay in HEK293 cell line [ 10]. Concentration of the agonists were set as around their EC50
1–3, 7–42 and 44 were prepared as described previously [9,10,15].
,
α
β
γ
α
β
9,
values. Reproducibility of our assay systems is shown in Table S1. First, we focused on the SAR
at the 2-position of phenanthridinone. The results, including the activities of positive controls
T0901317 (
alkyl groups (
decrease of the PR-antagonistic activity [10] and RORs inverse agonistic activity [
a hexafluoropropanol moiety somewhat increased the RORs activity and PR activity. According to
the reported X-ray crystal structures of compound and human LXRs LBD, the hydroxyl group
of forms a hydrogen bond with a histidine residue (His421 for LXR and His435 for LXR ) in
helix 11 [17 18]. The SARs for RORs and PR are broadly consistent with those of LXRs, AR, and GR.
6
), RU486, and OHF, are shown in Table 1. We have reported that introduction of
10) or a hydroxymethyl group (11) at the 2-position of resulted in retention or
]. Introduction of
8–
7
9
6
6
α
β
,
For AR and PR, introduction of a hydroxymethyl group (11) resulted in retention of the activity,
whereas introduction of hexafluoropropanol (12) resulted in 6-fold and 9-fold increases of PR and
AR activity, respectively. On the other hand, for LXRs and RORs, 11 showed weaker activity than
7
whereas 12 showed stronger activity than , suggesting that the two trifluoromethyl groups might
7
be associated with more potent activity and the hydroxyl group might not form a strong hydrogen
bond with histidine in these receptors, in contrast to the other receptors. This idea is consistent with
the reported co-crystal structure of T0901317 (
compound 12 showed not only PR-antagonistic activity and RORs inverse agonistic activity, but also
LXR , LXR , AR and GR antagonistic activity (Table 1). These results support our view that the
6) complexed with RORγ [19]. Overall, we found that
α
β
phenanthridin-6(5H)-one scaffold acts as a steroid surrogate.
Next, SAR at the nitrogen atom is shown in Table 2. Similar to the SAR for RORs, introduction
of a longer-chain alkyl group on the nitrogen atom (12 and 15) resulted in enhancement of the GR
antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent
AR antagonistic activity with an IC50 value of 0.10 µM, and more than 200-fold selectivity for AR over
RORs and GR, and about 7.8- and 50-fold selectivity over PR and LXRs, respectively. The longest alkyl
analog 17 showed decreased activity toward all NRs, suggesting that the binding pocket hosting the
N-alkylated derivatives might not be able to accommodate a group larger than a hexyl group.
Next, SAR at the nitrogen atom is shown in Table 2. Similar to the SAR for RORs, introduction
of a longer-chain alkyl group on the nitrogen atom (12 and 15) resulted in enhancement of the GR
antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent
AR antagonistic activity with an IC50 value of 0.10 µM, and more than 200-fold selectivity for AR over
RORs and GR, and about 7.8- and 50-fold selectivity over PR and LXRs, respectively. The longest alkyl
analog 17 showed decreased activity towards all NRs, suggesting that the binding pocket hosting the
N-alkylated derivatives might not be able to accommodate a group larger than a hexyl group.

Int. J. Mol. Sci. 2018, 19, 2090
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Table 1. SAR at 2-position.
Compound
R
RORα 1
RORβ 1
RORγ 1 LXRα 2
LXRβ 2
PR 2,3
AR 2
GR 2
290 4
130 4
6
-
-
>20,000
>20,000
6500
RU486
0.073
2.0
OH-Flu
-
170
7
8
9
H
10,000
18,000
9000
12,000
>20,000
9800
13,000
17,000
7900
13,000
>20,000
6500
15,000
17,000
9200
10,000
>20,000
3900
19,000
17,000
17,000
>20,000
>20,000
8400
15,000
16,000
16,000
>20,000
>20,000
3700
7100
7900
7800
8300
8600
1200
17,000
11,000
19,000
17,000
9600
>20,000
>20,000
19,000
>20,000
>20,000
7400
Me
Et
t-Bu
10
11
12
CH OH
2
(CF ) COH
1800
3
2
1
Inverse agonistic activity (IC50: nM) [9 [
]. 2 Antagonistic activity (IC50: nM). 3 10]. 4 Agonistic activity (IC50: nM).
Table 2. SAR at nitrogen atom.
Compound
1
R
H
RORα 1
>20,000
>20,000
RORβ 1
>20,000
>20,000
RORγ 1 LXRα 2
LXRβ 2
5100 4
PR 2,3
780
AR 2
100
GR 2
5000 4
4100 4
>20,000
>20,000
>20,000
>20,000
>
20,000
1
3
Me
340
500
4
1
1
1
1
4
5
6
7
Et
n-Pr
n-Hex
>20,000
11,000
7600
>20,000
8200
5400
>20,000
4200
4700
14,000
12,000
16,000
>20,000
6600
6900
10,000
1400
3800
2500
1700
5600
4000
>20,000
17,000
>20,000
>20,000
n-C H
>20,000
>20,000
>20,000
>20,000
10,000
15,000
9
19
1
Inverse agonistic activity (IC50: nM) [9]. 2 Antagonistic activity (IC50: nM). 3 [10]. 4 [8].
Next, the effect of introduction of a methoxy group at every position (methoxy scanning) was
investigated (18 24; Table 3) because many types of NR ligands possess a methoxy group(s) [20].
We have reported that 3-methoxy and 4-methoxy analogs 19 and 20 showed more than 3-fold and
–
2
9
-fold enhanced PR activity compared with the unsubstituted analog 12, respectively [10]. In contrast,
-methoxy analog 23 showed 5-fold weaker PR activity than 12. In the case of RORs, we have reported
that introduction of a methoxy group at the 9-position (23) enhanced ROR activities [9]. The activities
toward LXRs, AR and GR also depended on the position of the methoxy group. 1-Methoxy analog 18
showed decreased activity for all receptors tested. Enhanced activity was observed when a methoxy
group was introduced at the 4-, 9- or 10-position for LXRα, 9- or 10-position for LXRβ, 3- and 8-position
for AR, and 3- or 4-position for GR. Especially, 3-methoxy analog 19 showed 5.7-fold improved GR
activity compared with 12. Overall, introduction of a methoxy group resulted in increased activity in
almost all cases except ROR
β, but the most suitable position depended on the NR (3-position: PR and
GR, 8-position: AR, 9-position: ROR
α
, ROR and LXR , 10-position: LXR ). The introduction of a
γ
β
α
substituent at every position is useful to investigate substituent effects. In this context, it is important
to note that phenanthridin-6(5H)-one analogs bearing substituent(s) at any position can be quite easily
synthesized, and this represents a considerable advantage compared to the steroid skeleton.
The results of methoxy scanning prompted us to investigate the effect of introduction of two
methoxy groups (25–31 Table 3). In the cases of RORα, PR, AR and GR, 3,4-dimethoxy analog 25
exhibited similar or weaker activity to the 3-methoxy and 4-methoxy analogs 19 and 20. A possible
explanation might be the direction of the two methoxy groups, that is, one or both might occupy a space

Int. J. Mol. Sci. 2018, 19, 2090
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not suitable for receptor interaction due to steric hindrance. This idea is supported by the fact that
,4-dioxolane analog 26 showed stronger activity than 25 toward PR, AR and GR. Compound 26 was
the most potent GR antagonist with an IC50 value of 0.76 M, although this compound also showed PR
and AR antagonistic activities with IC50 values of 0.23 and 0.54 M, respectively. When two methoxy
groups were introduced at distal positions, the activity was improved. Thus, 3,8-dimethoxy analog
and 4,8-dimethoxy analog 28 showed more potent PR-antagonistic activity than monomethoxy
analogs 19 20, and 22, and 3,8-dimethoxy analog 27 showed more potent AR antagonistic activity than
monomethoxy analogs 19 and 22.
3
µ
µ
2
7
,
Table 3. SAR of substitution effect of alkoxy groups.
Compound
R
RORα 1
RORβ 1
RORγ 1 LXRα 2
LXRβ 2
PR 2,3
AR 2
GR 2
18
19
20
21
22
23
24
25
26
27
28
29
30
31
1-OMe
3-OMe
4-OMe
7-OMe
8-OMe
9-OMe
10-OMe
3,4-diOMe
>20,000
8200
7500
12,000
12,000
6600
>20,000
8200
6900
>20,000
7500
5400
13,000
13,000
2700
8500
8500
>20,000
12,000
9700
>20,000
8400
6900
11,000
12,000
7000
>20,000
9200
5500
12,000
11,000
2400
4000
410
560
12,000
1100
5800
2700
510
230
300
340
4200
>20,000
9600
>20,000
1100
2400
9400
1000
>20,000
>20,000
6100
540
580
>20,000
1300
3900
14,000
8200
10,000
>20,000
>20,000
760
1700
5200
7900
>20,000
17,000
15,000
15,000
7200
>20,000
>10,000
>20,000
13,000
11,000
7700
7700
4600
8500
4300
9600
>10,000
>20,000
9500
11,000
7300
3,4-(OCH O)
>20,000
17,000
>10,000
3200
>20,000
8800
>20,000
18,000
>10,000
3200
>20,000
8900
2
3,8-diOMe
4,8-diOMe
7,8-diOMe
7,9-diOMe
8,9-diOMe
1
1700
4600
>20,000
1600
8000
>20,000
19,000
>20,000
18,000
>20,000
16,000
Inverse agonistic activity (IC50: nM) [9]. 2 Antagonistic activity (IC50: nM). 3 [10].
We next examined the SAR of fluoro derivatives 32–35 (Table 4) to investigate the effect of this
electron-withdrawing substituent. For PR, the preferences for fluorine substitution position were
roughly consistent with the results of the methoxy scanning, that is, 4- and 8-substitued analogs 32 and
3
4
showed enhanced PR activity, whereas 9-substituted analogs 35 showed weaker PR activity [10].
For AR, all fluoro analogs 32 35 showed enhanced activity compared with 12, suggesting that an
electron-withdrawing effect at these positions is important for AR antagonistic activity. For LXR
–
α
,
4
- and 7-fluoro analogs 32 and 33 showed enhanced activity compared with 12.
Table 4. SAR of substitution effect of fluorine atom.
Compound Position RORα 1
RORβ 1
RORγ 1 LXRα 2
LXRβ 2
PR 2,3
AR 2
GR 2
3
3
3
3
2
3
4
5
4
7
8
9
15,000
7900
17,000
9800
14,000
9800
11,000
8300
18,000
13,000
>20,000
5000
3100
3100
7900
6400
4600
3100
6000
5400
130
730
310
390
460
680
920
6900
9800
>20,000
7000
1000
1
Inverse agonistic activity (IC50: nM) [9]. 2 Antagonistic activity (IC50: nM). 3 [10].
We then examined various substituents at the 9-position (Table 5). We have reported that
introduction of a chlorine atom at the 9-position ( ) enhanced inverse agonistic activity toward
2

Int. J. Mol. Sci. 2018, 19, 2090
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RORγ
[9
]. The SARs of ROR
γ
were roughly consistent with the results for LXR
β, and
2
also exhibited
potent LXR
β
antagonistic activity with the IC50 value of 0.88
µM. Replacement of fluorine at the
9-position (35) with chlorine (2) caused enhanced activity toward RORs and LXRβ, but decreased
activity toward PR, AR, and GR, suggesting that molecular modification at the 9-position would be
important to improve selectivity for metabolic NRs over steroid receptors.
Table 5. SAR of substitution effect at 9-posiiton.
Compound
R
RORα 1
RORβ 1
RORγ 1 LXRα 2
LXRβ 2
PR 2,3
AR 2
GR 2
3
2
6
Me
Cl
7600
5600
5500
6400
7100
4900
5600
5500
1000
690
2600
4100
12,000
9200
7100
7000
3000
880
4500
5500
3700
4300
4200
5100
6400
5200
>20,000
>3000
16,000
10,000
>20,000
>20,000
3
3
7
8
CF3
OH
1
Inverse agonistic activity (IC50: nM) [9]. 2 Antagonistic activity (IC50: nM). 3 [10].
Next, we focused on the 4-position (Table 6), because 4-methoxy analog 20 showed enhanced
ROR , LXR and PR activity, and 4-fluoro analog 32 showed enhanced LXR , PR and AR activity.
Compounds 39 44 and showed weak activity toward RORs and GR, indicating that the alkyl group
on the nitrogen atom is important for activity toward these receptors. We have reported that 4-alkyl
analogs 40 42 showed greater PR activity than the unsubstituted analog , and 4-methyl analog 40
had an IC50 value of 0.15 M [10]. As for LXRs and AR, 4-alkyl analogs 40 42 showed decreased
activity compared with the unsubstituted analog , whereas 4-fluoro analog 32 showed greater
activity than . Based on the reported SARs of non-steroidal AR antagonists, an electron-withdrawing
α
α
α
–
2
–
1
µ
–
1
1
substituent neighboring the amide bond is expected to be important for potent AR antagonism [21].
Therefore, 4-chloro analog 43 was designed and synthesized as shown in Scheme 1. Interestingly,
4
3
showed greater AR activity and weaker RORs, LXRs, PR and GR activity than 4-fluoro analog 32
Compound 43 showed the IC50 value of 0.059 M, being 2.9-fold more potent than clinically used
OHF under our assay conditions. Compound 43 showed more than 340-fold selectivity over RORs
and LXR , and 90-fold and 6-fold selectivity over LXR and PR, respectively. This result indicates that
.
µ
β
α
molecular modification at the 4-position can increase selectivity for AR over the other NRs.
Table 6. SAR at 4-posiiton and other substitution effects.
Compound
R
RORα 1
RORβ 1
RORγ 1 LXRα 2
LXRβ 2
PR 2,3
AR 2
GR 2
3
4
4
4
4
9
0
1
2
3
4-OMe
4-Me
4-Et
4-n-Pr
4-Cl
4-Me, 8-F
-
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
11,000
>20,000
>20,000
>20,000
9600
>20,000
>20,000
>20,000
>20,000
5300
>20,000
>20,000
>20,000
>20,000
>20,000
20,000
>20,000
>20,000
>20,000
270
150
210
360
350
46
>20,000
>20,000
14,000
5400
59
1700
>20,000
>20,000
>20,000
16,000
>20,000
>20,000
>20,000
>20,000
>20,000
9900
>20,000
>20,000
>20,000
3
4
4
3
3
27
300
1
Inverse agonistic activity (IC50: nM) [9] 2 Antagonistic activity (IC50: nM). 3 [10].

Int. J. Mol. Sci. 2018, 19, 2090
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Scheme 1. Reagents and conditions: (a) CF COCF
·
1.5H O, p-TsOH H O, toluene, reflux, 27%;
·
3
3
2
2
◦
◦
(
b) 2-iodobenzoic acid, EDC, DMAP, DMF, 100 C; (c) SEMCl, NaH, DMF, 0 C to RT; (d) Pd(OAc) ,
2
◦
PCy ·HBF , Cs CO , DMA, 130 C, 24% in 2 steps; (e) TBAF, THF, reflux, 18%.
3
4
2
3
Chronic administration of AR antagonists often leads to the development of resistance.
Mutations in the LBD of AR, such as T877A, have been identified in patients treated with flutamide
(Figure 2), and the activated metabolite, hydroxyflutamide, is an agonist of AR bearing T877A [22].
In contrast, another AR antagonist, enzalutamide, was reported to antagonize AR mutant T877A [23].
Thus, development of a novel chemical class of AR antagonists, different from flutamide analogs
seems an attractive approach for the treatment of AR antagonist-resistant cancer, providing further
motivation to evaluate steroid surrogates. Therefore, we investigated the antiandrogenic activity of
several potent AR antagonists by means of PSA (prostate-specific antigen, an AR-regulated gene)
ELISA (enzyme-linked immunosorbent assay) in human prostate cancer cell line LNCaP, which has
T877A mutation in the AR LBD. Potent AR antagonist 43 did not show antiandrogenic activity in
LNCaP, whereas several other AR antagonists including 33 decreased the PSA level with an IC50 of
1
90 nM (Table 7). This result suggests that the new non-flutamide type AR antagonist 33 is a promising
candidate for antiandrogen therapy of prostate cancer.
Figure 2. Chemical structures of AR antagonists.
Table 7. Antiandrogenic activity toward AR antagonist-resistant cell line LNCaP.
Compound
1
AR Reporter Gene IC50 (nM)
LNCaP IC50 (nM) ± SEM
100
500
540
>10,000
>10,000
560 ± 260 (N = 2)
490 ± 260 (N = 2)
190 ± 95 (N = 2)
>10,000
13
26
32
33
34
35
43
390 ± 30 (N = 2)
460
680
920
550 ± 110 (N = 2)
59 ± 9.5 (N = 2)
>10,000
Mean IC50 values with standard error of mean (SEM) from 1 or N times independent experiments.
3
3
3
. Materials and Methods
.1. Chemistry
.1.1. General
1H NMR spectra were recorded on a JNM-ECA500 (500 MHz) spectrometer (JEOL Ltd., Tokyo,
Japan). Chemical shifts ( ) are reported in parts per million. Flash column chromatography was
δ

Int. J. Mol. Sci. 2018, 19, 2090
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performed on silica gel 60N (40–50 mm) (Kanto Chemical Co., Inc., Tokyo, Japan). HPLC analyses
to check purity were performed on an analytical column (GL Science Inc., Tokyo, Japan) Inertsil
ODS-4 reversed-phase column, 5
µ
m, 4.6 mm
×
150 mm) eluted with a mobile phase consisting of
◦
CH CN/water at a flow rate of 1.0 mL/min, with UV (ultraviolet) monitoring at 254 nm, at 37 C.
3
3
.1.2. 2-(4-Amino-3-chlorophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (46)
To a solution of 2-chloroaniline (45) (1.58 mL, 15.0 mmol) in toluene (11 mL) were added
hexafluoroacetone trihydrate (2.06 mL, 18.0 mmol) and p-TsOH (258 mg, 1.50 mmol). The mixture was
◦
stirred for 8.5 h at 110 C, and then cooled to room temperature. Water was added, and the resulting
mixture was extracted with AcOEt. The combined organic layer was washed with brine, dried over
Na SO , and concentrated under reduced pressure. The residue was purified by silica gel column
2
4
1
chromatography (n-hexane/AcOEt = 10:1-3:1) to give 46 as a pink solid (9%). H NMR (500 MHz,
CDCl ) δ: 7.58 (s, 1H), 7.35 (d, J = 8.6 Hz, 1H), (d, J = 9.2 Hz, 1H).
3
3
.1.3. N-(2-Chloro-4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)-2-iodobenzamide (47)
To a solution of EDC (742 mg, 3.87 mmol) and DMAP (473 mg, 3.87 mmol) in DMF (4.0 mL) was
added 2-iodobenzoic acid (640 mg, 2.58 mmol) under an Ar atmosphere. The mixture was stirred for
3
0 min at room temperature, then 46 (379 mg, 1.29 mmol) was added to it, and stirring was continued
◦
at 100 C for 4 h. The resulting mixture was cooled, diluted with water, and extracted with AcOEt.
The combined organic layer was washed with brine, dried over Na SO , and concentrated under
2
4
reduced pressure. The residue was purified by column chromatography (n-hexane/AcOEt = 4:1)
1
to give 47 (181 mg, 0.346 mmol, 27%) as a brown solid. H NMR (500 MHz, DMSO-d )
δ: 7.94 (d,
6
J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 1.7 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 4.6 Hz,
2
H), 7.24 (td, J = 3.9, 1.3 Hz, 1H).
3
.1.4. N-(2-Chloro-4-(1,1,1,3,3,3-hexafluoro-2-((2-(trimethylsilyl)ethoxy)methoxy)propan-2-yl)phenyl)-
-iodo-N-((2-(trimethylsilyl)ethoxy)methyl)benzamide (48)
2
Sodium hydride (41.6 mg, 1.04 mmol) was added to a solution of 47 (181 mg, 0.346 mmol) in
◦
◦
DMF (1.5 mL) at 0 C. The mixture was stirred for 1 h at 0 C, then SEMCl (184
µ
L, 1.04 mmol) was
◦
added at 0 C, and stirring was continued for 6 h at room temperature. The resulting mixture was
diluted with AcOEt, quenched with water, and extracted with AcOEt. The combined organic layer
was washed with water, dried over Na SO , and concentrated. The residue was purified by column
2
4
chromatography (n-hexane/AcOEt = 20:1 to 10:1) to give 48 (219 mg, 0.279 mmol) as a colorless oil.
The compound was used for the next reaction without further purification.
3
.1.5. 4-Chloro-2-(1,1,1,3,3,3-hexafluoro-2-((2-(trimethylsilyl)ethoxy)methoxy)propan-2-yl)-5-
(
(2-trimethylsilyl ethoxy)methyl)phenanthridin-6(5H)-one (49)
To a solution of 48 (219 mg, 0.279 mmol) in DMA (1.0 mL) were added PCy3 HBF (63.7 mg,
·
4
0
.173 mmol), Cs CO (620 mg, 1.90 mmol) and Pd(OAc) (12.0 mg, 0.0532 mmol) under an Ar
2 3 2
◦
atmosphere. The mixture was stirred for 3 h at 130 C, then cooled to room temperature, and water
was added. The resulting mixture was extracted with AcOEt. The combined organic layer was washed
with brine, dried over Na SO , and concentrated under reduced pressure. The residue was purified by
2
4
column chromatography (n-hexane/AcOEt = 20:1) to give 49 as a brown oil (45.2 mg, 0.0824 mmol,
1
2
4% in 2 steps). H NMR (500 MHz, CDCl )
δ
: 8.56 (s, 1H), 8.53 (dd, J = 8.0, 1.1 Hz, 1H), 8.26 (d, J = 7.7,
3
1
.5 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.47 (s, 1H), 5.85 (s, 2H), 4.95 (s, 2H), 3.88 (t, J = 8.6 Hz, 2H), 3.74 (t,
J = 8.3 Hz, 2H), 1.02 (t, J = 8.3 Hz, 2H), 0.98 (m, 2H), 0.02 (s, 9H), −0.05 (s, 9H).
3
.1.6. 4-Chloro-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenanthridin-6(5H)-one (43)
TBAF (1 M in THF, 0.779 mL, 0.779 mmol) was added to a solution of 49 (42.7 mg, 0.0779 mmol) in
THF (0.8 mL) at room temperature. The reaction mixture was stirred under reflux for 9 h, then diluted

Int. J. Mol. Sci. 2018, 19, 2090
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with water, and extracted with AcOEt. The combined organic layer was washed with brine, dried
over Na SO , and concentrated under reduced pressure. The residue was purified by column
2
4
chromatography (CHCl /MeOH = 40:1) to give 43 as a pale yellow solid (18%).
3
1
H NMR (500 MHz, DMSO-d6) δ: 8.35 (d, J = 8.6 Hz, 1H), 8.27 (dd, J = 8.0, 1.1 Hz, 1H), 7.86–7.83
(
(
m, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.41 (d, J = 8.6 Hz, 1H). HLPC purity 97.0%
area %).
3
.2. Biology
3
.2.1. T-47D Alkaline Phosphatase Assay
T-47D alkaline phosphatase assays were performed as described in the literature [10].
Briefly, human ductal breast epithelial tumor (T-47D) cells were treated with fresh medium containing
test compound plus progesterone (final concentration 1 nM), and incubated for 24 h. The fixed cells
were washed with PBS and assay buffer was added. After the reaction was terminated, the absorbance
at 405 nm was measured. All data points were measured in triplicate.
3
.2.2. Reporter Gene Assay
Human embryonic kidney (HEK) 293 cells (RIKEN BRC, Ibaraki, Japan) were maintained
in DMEM (Dulbecco’s Modified Eagle’s medium) containing 5% (v/v) fetal bovine serum,
◦
and penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO in air. Cells were
2
4
seeded in clear-bottomed white 96-well plates at a density of 1
h prior to transfection. Cells were co-transfected with 30 ng of a NR expression plasmid, 50 ng
×
10 cells/well and incubated for
6
of a luciferase reporter and 10 ng of CMX-β-galactosidase expression vector per well, using the
calcium phosphate co-precipitation method. After 24 h, transfected cells were treated with dimethyl
sulfoxide (DMSO) or DMSO solution of test compounds for 24 h. T0901317 (0.3 M), T0901317
0.1 M), dihydrotestosterone (0.3 nM), and dexamethasone (1 nM) were used as agonists for LXR
LXR , AR and GR, respectively. After addition of a luciferase substrate, luminescence was detected
with a luminometer. The luciferase activity of each sample was normalized by -galactosidase
activity: 2-nitrophenyl- -D-galactopyranoside was added, and the absorbance was measured at 405 nm.
µ
(
µ
α,
β
β
β
Each sample was carried out in triplicate. A six-point sigmoidal dose-response curve was generated
for each compound. The IC50 value for each compound was calculated by linear approximation of
two points adjacent to 50% inhibition with logarithmic scale. In Table S1, biological reproducibility are
reported as the mean IC50 ± SEM.
Nuclear receptor plasmids: GR (CMX-hGR), AR (CMX-hAR), ROR (pcDNA3.1(-)-hROR
α
1,
pcDNA3.1(-)-hRORβ1, pcDNA3.1(-)-hRORγ1), LXR (CMX-GAL4N-hLXRα-LBD, CMX-GAL4N-
hLXRβ-LBD).
Luciferase reporter plasmids: GR (MTV-Luc), AR (ARE-Luc), ROR (RORE-TK-Luc), LXR
TK-MH100x4-Luc).
(
Plasmids for AR, GR and LXRs were provided by Prof. Dr. Makishima, and plasmids for RORs
were provided by Itsuu Institute (Kanagawa, Japan).
3
.2.3. PSA Level in LNCaP Cell Line
The human prostate cancer cell line LNCaP (ECACC, London, UK) was routinely maintained
◦
in RPMI1640 containing 10% (v/v) fetal bovine serum, and penicillin/streptomycin at 37 C in a
humidified atmosphere of 5% CO in air. Cells were cultured in RPMI1640 without phenol red
2
◦
containing 10% (v/v) charcoal-stripped fetal bovine serum, and penicillin/streptomycin at 37 C in
a humidified atmosphere of 5% CO in air for 3 days. Then cells were plated in 96-well plates at
2
4
1
× 10 cells/well and incubated overnight. On the next day, cells were treated with fresh media
containing test compound plus dihydrotestosterone (final concentration 10 nM), and incubated for
8 h. Then, PSA levels were measured in the culture supernatants, after 100-fold dilution with the
4

Int. J. Mol. Sci. 2018, 19, 2090
10 of 12
medium, using ELISA (Immunospec Corp., CA, USA Free prostate-specific antigen cat. #E29-212)
according to the manufacturer’s instructions. All data points were carried out twice in triplicate.
4
. Conclusions
Development of non-steroidal NR ligands is desirable to obtain safer, more selective agents
for clinical use. Here, non-steroidal antagonists for multiple target proteins including LXR (IC :
.88 M), AR (IC : 0.10 M), GR (IC : 0.76 M) were efficiently identified by exhaustive evaluation
of a small steroid surrogate library. In addition, a potent AR antagonist 43 (IC : 0.059 M) belonging
β
5
0
0
µ
µ
µ
50
50
µ
50
to a new chemical class was developed. Further, AR antagonist 33 showed antiandrogenic activity
toward AR antagonist-resistant cell line LNCaP with the IC50 value of 190 nM. Structure-activity
relationship studies of phenanthridinone analogs revealed that (1) hydrogen at the nitrogen is
important for AR and PR activity, and a longer alkyl group is favorable for RORs and GR activity,
(2) molecular modification at the 9-position can increase selectivity for metabolic NRs over steroid
receptors, and (3) an electron-withdrawing substituent on the phenyl group is important for AR
antagonistic activity, while modification at the 4-position can increase selectivity for AR over other NRs.
The phenanthridinone skeleton is more readily modifiable than the steroid skeleton. Thus, our strategy
of efficient lead finding, activity enhancement and preliminary control of selectivity over other
receptors should expand the utility of the multi-template approach, even though the compounds
obtained here still require further optimization.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/7/
2
090/s1.
Author Contributions: Conceptualization, Y.H.; methodology, Y.N. and M.I.; validation, Y.N., S.F., Y.H. and
M.I.; organic synthesis, Y.N.; plasmid preparation, M.M.; data curation, Y.N. and M.I.; writing—original draft
preparation, M.I.; supervision, S.F., M.M., Y.H. and M.I.; funding acquisition, S.F., Y.H. and M.I.
Funding: The work described in this Letter was partially supported by Grants-in Aid for Scientific Research
from The Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Japan Society for the
Promotion of Science (KAKENHI Grant No. 17H03996 (Y.H.), No. 17H03997(S.F.) and No. 25293027 (M.I.)).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
NR
Nuclear receptors
SAR
PR
Structure-activity relationship
Progesterone receptor
LXR
AR
Liver X receptor
Androgen receptor
GR
Glucocorticoid receptor
Ligand-binding domains
retinoic acid receptor-related orphan receptors
Niemann-Pick disease type C1
Niemann-Pick disease type C1-like 1
Hydroxyflutamide
LBD
ROR
NPC1
NPC1L1
OHF
Ts
p-Toluenesulfonyl
EDC
DMAP
DMF
SEM
Cy
N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide
4-(N,N-Dimethylamino)pyridine
Dimethylformamide
2-(Trimethylsilyl)ethoxymethyl
Cyclohexyl
DMA
TBAF
THF
Dimethylacetamide
Tetrabutylammonium fluoride
Tetrahydrofuran

Int. J. Mol. Sci. 2018, 19, 2090
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©
2018 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 (http://creativecommons.org/licenses/by/4.0/).
(