Structure-activity relationship-guided development of retinoic acid receptor-related orphan receptor gamma (RORγ)-selective inverse agonists with a phenanthridin-6(5H)-one skeleton from a liver X receptor ligand

Article abstract of DOI:10.1016/j.bmc.2014.03.007

Retinoic acid receptor-related orphan receptors (RORs), which belong to the nuclear receptor superfamily, regulate many physiological processes, including hepatic gluconeogenesis, lipid metabolism, immune function and circadian rhythm. Since RORs resemble liver X receptors (LXRs) in the fold structure of their ligand-binding domains, we speculated that ROR-mediated transcription might be modulated by LXR ligands, in line with the multi-template hypothesis. Therefore, we screened our LXR ligand library for compounds with ROR ligand activity and identified a novel ROR ligand with a phenanthridin-6(5H)-one skeleton. Structure-activity relationship studies aimed at separating ROR inverse agonistic activity from LXR-agonistic activity enabled us to develop a series of ROR inverse agonists based on the phenanthridin-6(5H)-one skeleton, including a RORγ-selective inverse agonist.

Full text of DOI:10.1016/j.bmc.2014.03.007

Bioorganic & Medicinal Chemistry 22 (2014) 2799–2808
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship-guided development of retinoic acid
receptor-related orphan receptor gamma (RORc)-selective inverse
agonists with a phenanthridin-6(5H)-one skeleton
from a liver X receptor ligand
Yuko Nishiyama a, Masahiko Nakamura a, Takashi Misawa a, Madoka Nakagomi b, Makoto Makishima c,
Minoru Ishikawa a, , Yuichi Hashimoto a
⇑
a Institute of Molecular & Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Department of Biology, Research Foundation Itsuu Laboratory, 2-28-10 Tamagawa, Setagaya-ku, Tokyo 158-0094, Japan
c Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Retinoic acid receptor-related orphan receptors (RORs), which belong to the nuclear receptor superfam-
ily, regulate many physiological processes, including hepatic gluconeogenesis, lipid metabolism, immune
function and circadian rhythm. Since RORs resemble liver X receptors (LXRs) in the fold structure of their
ligand-binding domains, we speculated that ROR-mediated transcription might be modulated by LXR
ligands, in line with the multi-template hypothesis. Therefore, we screened our LXR ligand library for
compounds with ROR ligand activity and identified a novel ROR ligand with a phenanthridin-6(5H)-
one skeleton. Structure–activity relationship studies aimed at separating ROR inverse agonistic activity
from LXR-agonistic activity enabled us to develop a series of ROR inverse agonists based on the phenan-
Received 21 January 2014
Revised 4 March 2014
Accepted 6 March 2014
Available online 17 March 2014
Keywords:
ROR
Phenanthridinone
Inverse agonist
LXR
thridin-6(5H)-one skeleton, including a ROR
c-selective inverse agonist.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
isoforms, ROR
adipose tissue, kidney, small intestines, and skeletal muscle.4
Recent studies indicate that ROR 1 is related to insulin resistance,
and the loss or potentially the inhibition of ROR 1 might protect
against insulin resistance and type 2 diabetes.4 On the other hand,
ROR t is exclusively expressed in cells of the immune system, and
is considered to play critical roles in differentiation of Th17 cells
and secretion of inflammatory cytokines such as IL-17.5–7 Th17
cells have been identified as key mediators of immune responses
in autoimmune diseases such as multiple sclerosis (MS), Crohn’s
disease and rheumatoid arthritis (RA). Therefore, novel inverse
c1 and RORct (RORc2). RORc1 is expressed in liver,
Nuclear receptors have highly conserved DNA-binding and
ligand-binding domains, and regulate DNA transcription by
binding transcription factors such as hormones. However, many
of these receptors are still classified as orphan receptors, since their
natural ligands have not been identified. Retinoic acid receptor-re-
lated orphan receptor (ROR) is the one of these orphan receptors,
and it is thought to be involved in immunity, cellular metabolism
c
c
c
and circadian rhythm.1 There are three subtypes, ROR
and ROR , which are generated by alternative splicing or alterna-
tive transcriptional initiation.1 These subtypes have different tissue
expression patterns and different functions. ROR is highly
expressed in brain, particularly cerebellum and thalamus, and it
a, RORb
c
agonists targeting RORc are considered to be candidates for treat-
ment of autoimmune diseases and diabetes.
a
Several studies have been directed at development of ROR in-
has been reported that ROR
a
-deficient mice exhibit ataxia and
verse agonists. The synthetic liver
T0901317 was the first reported inverse agonist for both ROR
and ROR
,8 but T0901317 also binds to several other nuclear recep-
tors.9 Development of this compound led to a variety of selective
ROR ligands, such as ROR
-selective inverse agonist SR1001,10
ROR c-selective
-selective inverse agonist SR3335,11 and ROR
X receptor (LXR) agonist
severe cerebellar atrophy.2 RORb is highly expressed in the central
nervous system, especially brain and retina,3 and is thought to be
involved in the processing of sensory information,1 although its
a
c
precise function is not yet fully established. ROR
c
has two different
a/c
a
modulator SR2211.12 Other compounds such as SR9805,13 and
digoxin14 were also identified as ROR-specific inverse agonists
⇑
Corresponding author. Tel.: +81 3 5841 7853; fax: +81 3 5841 8495.
E-mail address: m-ishikawa@iam.u-tokyo.ac.jp (M. Ishikawa).
http://dx.doi.org/10.1016/j.bmc.2014.03.007
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
(Fig. 1). Nevertheless, structure–activity relationships (SAR) of ROR
ligands are generally not well established. However, a recent SAR
study of compounds derived from high-throughput screening of
the GSK in-house compound library led to the discovery of potent
structures of nuclear receptor ligand-binding domains (LBDs) are
similar, we have used the multi-template approach to create li-
gands for several nuclear receptors, including farnesoid X recep-
tor,17 LXR,18 vitamin D receptor,19,20 androgen receptor,21 and
estrogen receptor.22 We have also reported improvement of selec-
tivity versus other target proteins.18,21,22 Based on those results, we
hypothesized that ROR ligands would be found among ligands of
other nuclear receptors possessing similar fold structure to ROR.
Our group has developed LXR ligands based on the phenanthridin-
6(5H)-one scaffold, which is a cyclized carba-analog of T0901317
(Fig. 2).23 As shown in Figure 3, RORs are similar to LXR in the fold
structure of the LBD. In addition, LXR agonist T0901317 binds to
indole-based RORc
t inverse agonists.15
Here, to obtain highly subtype-specific ROR ligands, we focused
on screening LXR ligands for ROR activity, and then set out to sep-
arate ROR inverse agonistic activity from LXR agonistic activity. We
describe here the identification and SAR study of ROR inverse ago-
nists based on the phenanthridin-6(5H)-one skeleton, and the
development of ROR subtype-specific ligands.
RORa and RORc. Therefore, we hypothesized that our LXR ligands
2. Results and discussion
might bind to the T0901317-binding pocket of ROR, and would be
candidate ligands for RORs. To test this idea, we used a full-length
receptor linked to an ROR response element (RORE) driving luciferase
gene expression. Screening of our compound library23–25 identified
compound 1 (Fig. 2) as a weak inverse agonist of ROR (IC50 values:
2.1. Identification of lead compound
The multi-template approach to drug discovery is based on the
fact that the number of three-dimensional spatial structures (fold
structures) of human proteins is at least 50 times smaller than
the number of human proteins (50,000–70,000).16 Therefore, in
principle, a template/scaffold structure that is spatially comple-
mentary to onefold structure might serve as a multi-template for
structural development of ligands that would interact specifically
with more than 50 different human proteins, neglecting physical/
chemical interactions. In other words, the structures of ligands that
bind to one member of a class of fold structures may be used for
the development of novel lead compounds for other members of
the same fold structure class. However, lead compounds obtained
on the basis of this approach are likely to possess polypharmaco-
logical character. Therefore, structural modification of polyphar-
macological lead compounds, aimed at optimizing selectivity for
the particular target, is required. Based on the fact that the fold
RORa; 9.9
lM, RORb; 10.1
lM, RORc; 9.0
lM). For comparison, the
IC50 values of T0901317 were ROR
a; >20
l
M, ROR ; 6.5 M. Next,
c
l
we examined the effects of N-substituents. The ROR transcriptional
activity of a series of N-alkylated derivatives was measured by lucif-
erase reporter gene assay.
As shown in Table 1, introduction of a longer-chain alkyl group
on the nitrogen atom resulted in enhancement of ROR inverse ago-
nistic activity. However, the longest alkyl analog (2j) showed de-
creased activity, suggesting that the binding pocket hosting the
N-alkylated derivatives might not be able to accommodate a group
larger than a hexyl group. In this series, compound 2h showed the
most potent effect on RORc activity, and therefore we selected the
N-butylphenanthridinone skeleton (2h) as a lead structure for fur-
ther structural development studies.
T0901317
SR1001
SR3335
SR2211
SR9805
Digoxin
Figure 1. Chemical structure of reported ROR inverse agonists.

Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
2801
Compound 1
T0901317
Carba-T0901317 analog
Figure 2. Structural development of candidate ROR ligands.
Table 1
Inverse agonistic activities of phenanthridin-6(5H)-one analogues 1 and 2a–j
Compound
R
IC50 (lM)
ROR
a
RORb
RORc
T0901317
1
—
>20
9.9
NA
6.5
9.0
9.3
5.8
>20
>20
>20
6.0
4.2
3.9
4.7
>20
CH2CF3
CH2-c-Pr
CH2-c-Hex
H
Me
Et
i-Pr
n-Pr
n-Bu
n-C6H13
n-C9H19
10.1
11.8
8.8
>20
>20
>20
9.7
8.2
6.5
5.4
>20
2a
2b
2c
2d
2e
2f
2g
2h
2i
13.6
10.3
>20
>20
>20
12.3
10.8
9.8
7.6
>20
2j
9.0 lM, RORb; 7.9 lM, RORc; 9.2 lM), the activity toward RORc
was reduced by half as compared to that of the lead compound
2h. These results indicated that the bulky hexafluoroisopropanol
moiety is important for ROR ligand recognition.
2.3. Synthesis and evaluation of monomethoxy-substituted
phenanthridin-6(5H)-one analogues
Figure 3. Superposition of X-ray crystal structures of the LBDs of ROR
PDB ID: 3KYT) and LXRb (cyan, PDB ID: 1PQC). Orange represents H12 of ROR
blue represents H12 of LXRb.
c
(yellow,
and
c
Based on the evaluation of the lead compound, we next investi-
gated the effect of methoxy substitution and synthesized regioisom-
ers of methoxy-substituted phenanthridin-6(5H)-one analogues
2.2. Synthesis and evaluation of 2-substituted phenanthridin-
6(5H)-one analogues
Next, we examined whether
a
1,1,1,3,3,3-hexafluoro-2-
hydroxypropyl group at the 2-position is essential for inverse ago-
nistic activity towards ROR. We prepared various compounds in
which the hexafluoroisopropanol moiety was replaced with an al-
kyl group, as shown in Scheme 1. Briefly, p-alkyl anilines 6 were
condensed with o-Br/I benzoic chloride. After introduction of an
n-butyl group on the nitrogen atom, intramolecular cyclization of
8a–h proceeded smoothly with Pd(OAc)2–PCy3ÁHBF4–Cs2CO3/
K2CO3 as the coupling reagent24 to afford 3a,c–i. Compound 3b
was obtained by reduction of 3a.
Table 2
Inverse agonistic activities of phenathridin-6(5H)-one analogues 2h and 3b–i
The synthesized compound 3b showed weak inhibitory activity,
suggesting that the ditrifluoromethyl group might be associated
with more potent repressive activity and the hydroxyl group might
Compound
R
IC50 (lM)
ROR
a
RORb
RORc
2h
3b
3c
3d
3e
3f
3g
3h
3i
C(CF3)2(OH)
CH2OH
H
Me
Et
i-Pr
t-Bu
n-C6H13
n-C12H25
9.8
>20
10.2
17.6
9.0
16.7
11.8
10.3
NA
6.5
3.9
>20
not form a strong hydrogen bond with histidine in ROR
trast to the case of LXR and LXRb (Table 2). This idea is consistent
with the reported co-crystal structure of T0901317 complexed
with RORc 26
The analogues 3c–g showed moderate ROR inverse
c, in con-
>20
13.4
17.1
7.9
17.0
12.8
11.3
NA
a
14.5
16.8
9.2
11.9
10.4
13.0
NA
.
agonistic activities, whereas an analog with a large group at the
2-position, that is, 2-n-dodecyl analog 3i, was inactive.
Although compound 3e exhibited strong inverse agonistic activ-
ity towards ROR in this series of compounds (IC50 values: RORa;

2802
Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
Scheme 1.
Table 3
4a–f as shown in Scheme 2. Briefly, N-butylamine derivatives 9a–c,
which are commercially available anilines, were treated with hexa-
fluoroacetone sesquihydrate to afford hexafluoropropanol deriva-
tives 10a–c. They were condensed with 2-iodobenzoyl chloride or
regioisomers of methoxyl-substituted 2-bromobenzoic acid and the
resulting anilides 11a,b and 12a–d were cyclized in the presence of
Pd catalyst to give compounds 4a–f.
Inverse agonistic activities of phenanthridin-6(5H)-one analogues 2h and 4a–f
The effect of methoxylation on the ROR inverse agonistic activ-
ity seemed to depend on the position at which the methoxy group
is introduced. Introduction of a methoxy group at the 3-, 9- or 10-
position repressed ROR transcriptional activities (4a, 4e, and 4f,
respectively) (Table 3). Compounds 4c and 4d, with a monometh-
oxy group at the 7- and 8-position, respectively, were cytotoxic at
Compound
R
IC50 (lM)
ROR
a
RORb
RORc
2h
4a
4b
4c
4d
4e
4f
—
9.8
7.8
>10
Toxic
Toxic
5.3
6.5
3.9
9.2
>10
Toxic
Toxic
1.1
3-OMe
4-OMe
7-OMe
8-OMe
9-OMe
10-OMe
10.9
>10
Toxic
Toxic
7.7
20
lM. Interestingly, 4e showed 5-fold higher activity towards
ROR
c
than ROR (IC50 values: ROR ; 5.3 M, ROR ; 1.1 M).
a
a
l
c
l
2.4. Synthesis and evaluation of 9-substituted phenanthridin-
6(5H)-one analogues
9.1
7.5
5.7
Since a methoxy group at the 9-position provided selectivity for
RORc, we next synthesized 9-substituted phenathridin-6(5H)-one
analogues 5a–d to investigate the effects of substituent bulkiness
and electronic factors (Scheme 3). Briefly, 10c was condensed with
2-bromo-4-substituted benzoic acids and the products were cy-
clized by using the same methods as in Schemes 1 and 2. As 2-bro-
mo-4-(trifluoromethyl)benzoic acid is not commercially available,
we prepared it from 2-amino-4-(trifluoromethyl)benzoic acid by
means of the Sandmeyer reaction, as reported.27
As shown in Table 4, introduction of a hydroxyl group at the
9-position (5d) reduced the inverse agonistic effect on ROR
c,
whereas introduction of a chloro group (5b) enhanced it. In
Scheme 2.

Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
2803
Scheme 3.
Table 4
Inverse agonistic activities of phenanthridin-6(5H)-one analogues 4e and 5a–d
These results suggest that ROR
for the 9-chloro group on the phenanthridin-6(5H)-one scaffold,
c
might contain a binding pocket
resulting in high selectivity for RORc (see Fig. 4).
2.5. Selectivity for LXRs
Since our compounds were originally developed from LXR li-
gands, compound 5b might possess LXR agonistic activity. There-
fore, we investigated the LXR-agonistic activity using a reporter
gene assay as previously reported.28 As shown in Figure 5, we con-
firmed that T0901317 showed agonistic activity toward both LXR
and LXRb. However, 5b exhibited no agonistic effect towards either
of these receptors, like ROR inverse agonist SR1001. On the
other hand, compound 5b also exhibited potent antagonistic activ-
ity, with IC50 value for LXRb of 0.68 M (Table 5). For these reasons,
although compound 5b has LXR antagonistic activity, we obtained
an ROR -selective inverse agonist that lacks LXR agonistic activity.
a
Compound
R
IC50 (lM)
ROR
a
RORb
RORc
a/c
4e
5a
5b
5c
5d
OMe
Me
Cl
CF3
OH
5.3
7.6
5.6
5.5
6.4
7.7
7.1
4.9
5.6
5.5
1.1
1.0
0.69
2.6
4.1
l
c
3. Conclusion
Based on the similarity of the fold structures of LXR and ROR,
we speculated that our LXR ligand library might contain ROR li-
gands. Screening assay identified compound 1 as an ROR pan in-
verse agonist. Then, SAR study enabled us to develop a series of
nuclear receptor ROR inverse agonists based on the phenanthri-
din-6(5H)-one skeleton. Among them, compound 5b was the most
potent ROR inverse agonist. Further, it showed ROR
c selectivity
and lacked LXR-agonistic activity, as determined by luciferase re-
porter gene assay. Further optimization of the structure of this
compound should provide useful tools for elucidation of ROR
c
function and candidate agents for treatment of insulin resistance
and type 2 diabetes.
4. Experimental section
4.1. Biology
Figure 4. Compound 5b inhibited ROR transcriptional activities in a dose-depen-
dent manner.
4.1.1. Cell culture condition
addition, 5b was highly selective for ROR
5b: 8-fold). Introduction of a methyl group (5a) or a trifluoro-
methyl group (5c) did not enhance the inverse agonistic effect.
c
over ROR
a
(4e: 5-fold,
Human embryonic kidney (HEK) 293 cells were cultured in
DMEM supplemented with 5% FBS at 37 °C in a humidified
atmosphere containing 5% CO2.

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Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
Figure 5. Compound 5b showed no LXR-agonistic activity. (a) LXR
a
agonist assay. (b) LXRb agonist assay.
Table 5
Agonistic activity (EC50) and antagonistic activity (IC50) of T0901317, SR1001 and
compound 5b on LXRs
recorded on a JEOL JNM-GX500 or JNM-ECA500 (500 MHz) spec-
trometer. Chemical shifts are expressed in d (ppm) values, with tet-
ramethylsilane (TMS) as an internal reference. Fast atom
bombardment mass spectra (FAB-MS) and high-resolution mass
spectra (HRMS) were recorded on a JEOL JMS-HX110 spectrometer
with m-nitrobenzyl alcohol as the matrix.
Compound
LXR
a
LXRb
EC50
(l
M)
IC50
(lM)
EC50
(lM)
IC50 (lM)
T0901317
SR1001
5b
0.29
NA
NA
NA
NA
>3.0
0.12
NA
NA
NA
NA
0.68
4.2.2. General procedure A: synthesis of phenylhexafluoropro-
panol analogues
4.2.2.1. 2-(4-Butylamino-2-methoxyphenyl)-1,1,1,3,3,3-hexaflu-
4.1.2. ROR inverse agonistic activity assay
oropropan-2-ol (10a).
trihydrate (707 mg, 3.21 mmol) in toluene (5.00 mL) were added
N-butyl-3-methoxyaniline (9a) (384 mg, 2.14 mmol) and p-TsOH-
To a solution of hexafluoroacetone
HEK293 cells were seeded in 96-well culture plates (1.0 Â 104
cells per well) the day before transfection. Each cell was co-trans-
fected with 30 ng of
(pcDNA3.1(À)-hROR 1; accession no. NM_134261, pcDNA3.1(À)-
hRORb; accession no. NM_006914, or pcDNA3.1(À)-hROR 1;
a
nuclear receptor expression plasmid
H2O (81.5 mg, 428 lmol), and the mixture was stirred at 120 °C
a
for 10 h. After cooling to room temperature, the mixture was di-
luted with ethyl acetate, washed with satd NaHCO3 aqueous solu-
tion and brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
(n-hexane/AcOEt = 1:0–2:1) to give 10a (258 mg, 0.749 mmol,
35%) as a colorless oil. 1H NMR (500 MHz, CDCl3) d 7.38 (s, 1H),
7.27 (d, 1H, J = 8.5 Hz), 6.26 (dd, 1H, J = 8.5, 2.4 Hz), 6.18 (d, 1H,
J = 2.4 Hz), 4.15 (br s, 1H), 3.91 (s, 3H), 3.12 (m, 2H), 1.61 (tt, 2H,
J = 7.3, 7.3 Hz), 1.43 (tq, 2H, J = 7.3, 7.3 Hz), 0.97 (t, 3H,
J = 7.3 Hz); MS (FAB) m/z 346 (M+H)+.
c
accession no. NM_005060), 50 ng of a luciferase reporter (RORE-
TK-Luc) and 10 ng of CMX-b-galactosidase expression vector.
Transfections were performed according to the calcium phosphate
precipitation method. At 8 h post-transfection, the cells were trea-
ted with dimethyl sulfoxide (DMSO) or compounds for 16 h. Trea-
ted cells were assayed for luciferase activity in a luminometer. The
luciferase activity of each well was normalized by the level of b-
galactosidase activity. Each sample was carried out in triplicate
and the experiments were repeated at least three times.
4.2.2.2. 2-(4-Butylamino-3-methoxyphenyl)-1,1,1,3,3,3-hexaflu-
4.1.3. LXR agonistic/antagonistic activity assay
oropropan-2-ol (10b).
Prepared from N-butyl-2-methoxyan-
HEK293 cells were seeded in 96-well culture plates (1.0 Â 104
cells per well) the day before transfection. Each cell was co-trans-
fected with 30 ng of a nuclear receptor expression plasmid (CMX-
iline (9b) in accordance with general procedure A. Colorless oil
(76%); 1H NMR (500 MHz, CDCl3) d 7.16 (d,1H, J = 8.5 Hz), 7.05 (s,
1H), 6.58 (d, 1H, J = 8.5 Hz), 4.36 (br s, 1H), 3.85 (s, 3H), 3.14 (t,
2H, J = 7.3 Hz), 1.64 (tt, 2H, J = 7.3, 7.3 Hz), 1.44 (tq, 2H, J = 7.3,
7.3 Hz), 0.98 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 376 (M+H)+.
GAL4N hLXRa, or CMX-Gal4N hLXRb), 50 ng of a luciferase reporter
(TK-MH100x4-Luc) and 10 ng of CMX-b-galactosidase expression
vector. Transfections were performed according to the calcium
phosphate precipitation method. At 24 h post-transfection, the
cells were treated with dimethyl sulfoxide (DMSO) or compounds
for 16 h. For antagonistic activity assay, cells were treated test
4.2.2.3.
pan-2-ol (10c).
2-(4-(Butylamino)phenyl)-1,1,1,3,3,3-hexafluoropro-
Prepared from N-butylaniline (9c) using a
slight modification of general procedure A. To a solution of hexa-
fluoroacetone sesquihydrate (1.16 g, 3.00 mmol) and N-butylani-
line (8c) (448 mg, 3.00 mmol) in toluene (20.0 mL) was added
p-TsOH-H2O (62.1 mg, 0.360 mmol), and the mixture was stirred
at 100 °C for 3 h. After cooling to room temperature, the mixture
was diluted with ethyl acetate, washed with satd NaHCO3 aqueous
solution and brine, dried over Na2SO4, and concentrated under re-
duced pressure. The residue was purified by column chromatogra-
phy (n-hexane/AcOEt = 10:1) to give 10c (414 mg, 1.31 mmol, 44%)
as a white solid. 1H NMR (500 MHz, CDCl3) d 7.39 (d, 2H, J = 8.0 Hz),
6.54 (d, 2H, J = 9.2 Hz), 3.05 (t, 2H, J = J = 7.2 Hz), 1.57–1.51 (m, 2H),
1.37–1.34 (m, 2H), 0.89 (t, 3H, J = 7.4 Hz).
compound in the presence of T0901317 (for LXR
a; 0.30 lM, for
LXRb; 0.10 M). Treated cells were assayed for luciferase activity
l
in a luminometer. The luciferase activity of each well was normal-
ized by the level of b-galactosidase activity. Each sample was car-
ried out in triplicate.
4.2. Chemistry
4.2.1. General comments
Melting points were determined on a Yanagimoto hot-stage
melting point apparatus, without correction. 1H NMR spectra were

Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
2805
4.2.3. General procedure B: synthesis of benzamide analogues 7
and 11
J = 7.5, 1.5 Hz), 7.19 (d, 2H, J = 8.5 Hz), 2.59 (t, 2H, J = 8.0 Hz),
1.64–1.52 (m, 2H), 1.35–1.22 (m, 18H), 0.88 (t, 3H, J = 7.0 Hz);
MS (FAB) m/z 444,446 (M+H)+.
4.2.3.1. N-(4-Acetylphenyl)-2-bromobenzamide (7a).
To a
solution of 4-aminoacetophenone (41.0 mg, 0.300 mmol) in CH2Cl2
(5.00 mL) were added dropwise 2-bromobenzoyl chloride
4.2.3.9. N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
(72.4 mg, 0.333 mmol) and Et3N (82.5
l
l, 750
l
mol) at 0 °C, and
2-yl)-3-methoxyphenyl]-2-iodo-benzamide (11a).
solution of 10a (104 mg, 0.300 mmol) in CH2Cl2 (5.00 mL) were
added dropwise Et3N (82.5 l, 750 mol) and 2-iodobenzoyl chlo-
To
a
the mixture was stirred for 3 h, then allowed to warm to room
temperature. After cooling to 0 °C, the mixture was diluted with
ethyl acetate and water was added. The organic layer was sepa-
rated, washed with brine, dried over Na2SO4, and concentrated un-
der reduced pressure. The residue was purified by column
chromatography (n-hexane/AcOEt = 1:0–1:1) to give 7a (85.0 mg,
0.270 mmol, 89%) as a white solid; 1H NMR (500 MHz, CDCl3) d
8.07 (d, 1H, J = 8.5 Hz), 7.83 (br s, 1H), 7.73 (d, 2H, J = 8.5 Hz),
7.68 (d, 1H, J = 7.5 Hz), 7.66 (d, 1H, J = 7.5 Hz), 7.44 (t, 1H,
J = 7.5 Hz), 7.36 (td, 1H, J = 7.5, 1.5 Hz), 3.92 (s, 3H).
l
l
ride (88.0 mg, 0.330 mmol) at 0 °C, and the mixture was stirred for
3 h, then allowed to warm to room temperature. After cooling
again to 0 °C, the mixture was diluted with ethyl acetate, and water
was added. The organic layer was separated, washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography (n-hexane/
AcOEt = 1:0–1:1) to give 11a (quant.) as a white solid. 1H NMR
(500 MHz, CDCl3) d 7.72 (d, 1H, J = 7.3 Hz), 7.41 (d, 1H, J = 7.9 Hz),
7.11 (t, 1H, J = 7.3 Hz), 6.93–6.90 (m, 3H), 6.80 (s, 1H), 4.01–3.98
(br m, 2H), 3.78 (s, 3H), 1.69–1.67 (m, 2H), 1.46–1.43 (m, 2H),
0.96 (t, 3H, J = 7.0 Hz).
4.2.3.2. 2-Iodo-N-phenylbenzamide (7b).
Prepared from ani-
line and 2-iodobenzoyl chloride in accordance with general proce-
dure B. White solid (99%); 1H NMR (500 MHz, CDCl3) d 7.90 (d, 1H,
J = 7.9 Hz), 7.62 (d, 2H, J = 7.3 Hz), 7.52 (dd, 1H, J = 8.0, 1.9 Hz), 7.40
(m, 4H), 7.15 (m, 2H).
4.2.3.10. N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan
-2-yl)-2-methoxyphenyl]-2-iodobenzamide (11b).
Prepared
from 10b in accordance with general procedure B. White solid
(82%); 1H NMR (500 MHz, CDCl3) d 7.63 (d, 1H, J = 7.9 Hz), 7.30
(d, 1H, J = 8.5 Hz), 7.14 (s, 1H), 7.06 (d, 1H, J = 8.5 Hz), 6.99–6.92
(m, 2H), 6.80–6.77 (m, 1H), 3.85 (s, 3H), 3.60–3.28 (m, 2H), 1.72–
1.51 (m, 2H), 1.51–1.32 (m, 2H), 0.94 (t, 3H, J = 7.3 Hz); MS (FAB)
m/z 576 (M+H)+.
4.2.3.3. 2-Bromo-N-p-tolylbenzamide (7c).
Prepared from p-
toluidinein accordance with general procedure B. White solid
(74%); 1H NMR (500 MHz, CDCl3) d 7.66 (dd, 1H, J = 7.6, 1.8 Hz),
7.64 (d, 1H, J = 7.6 Hz), 7.58 (br s, 1H), 7.53 (d, 1H, J = 8.0 Hz),
7.42 (t, 1H, J = 7.6 Hz), 7.33 (td, 1H, J = 7.6, 1.8 Hz), 7.19 (d, 2H,
J = 8.0 Hz), 2.35 (s, 3H); MS (FAB) m/z 290, 292 (M+H)+.
4.2.4. General procedure C: synthesis of benzamide analogues
4.2.3.4. 2-Bromo-N-(4-ethylphenyl)benzamide (7d).
Pre-
12
pared from p-ethylaniline in accordance with general procedure
B. White solid (99%); 1H NMR (500 MHz, CDCl3) d 7.65 (ddd, 2H,
J = 10.0, 8.0, 1.5 Hz), 7.59 (br s, 1H), 7.55 (d, 2H, J = 8.0 Hz), 7.42
(td, 1H, J = 8.0, 1.5 Hz), 7.32 (td, 1H, J = 8.0, 1.5 Hz), 7.22 (d, 2H,
J = 8.0 Hz), 2.65 (q, 2H, J = 7.5 Hz), 1.24 (t, 3H, J = 7.5 Hz); MS
(FAB) m/z 304, 306 (M+H)+.
4.2.4.1. 2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro
xypropan-2-yl)phenyl]-6-methoxybenzamide (12a).
To
a
solution of 2-bromo-6-methoxybenzoic acid (104 mg, 397
lmol)
in CH2Cl2 (3.00 mL) was added chloromethylenedimethyliminium
chloride (50.8 mg, 397 mol) at 0 °C under an Ar atmosphere,
l
and the mixture was stirred at room temperature for 1 h. Then,
2-(4-butylaminophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (10c)
4.2.3.5. 2-Iodo-N-(4-isopropylphenyl)benzamide (7e).
Pre-
(50.0 mg, 159 lmol) and Et3N (166 ll, 1.19 mmol) were added at
pared from 4-isopropylaniline and 2-iodobenzoyl chloride in
accordance with general procedure B. White solid (99%); 1H NMR
(500 MHz, CDCl3) d 7.89 (d, 1H, J = 8.0 Hz), 7.54 (d, 2H, J = 8.6 Hz),
7.51 (dd, 1H, J = 5.0, 1.9 Hz), 7.41 (td, 1H, J = 7.3, 1.3 Hz), 7.23 (d,
2H, J = 8.5 Hz), 7.13 (td, 1H, J = 7.9, 1.9 Hz), 2.89 (sep, 1H,
J = 7.3 Hz), 1.23 (d, 6H, J = 6.7 Hz).
0 °C. Stirring was continued for 3 h, then the mixture was allowed
to warm to room temperature, and diluted with ethyl acetate.
Water was added, and the organic layer was separated, washed
with brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
(n-hexane/AcOEt = 1:0–1:1) to give 12a (73.5 mg, 139 lmol, 88%)
as a colorless oil. 1H NMR (500 MHz, CDCl3) d 7.50 (d, 2H,
J = 8.5 Hz), 7.30 (d, 2H, J = 8.5 Hz), 6.96–6.93 (m, 2H), 6.51 (d, 1H,
J = 7.3 Hz), 4.20–4.08 (m, 1H), 4.06 (br s, 1H), 3.75–3.67 (m, 2H),
3.65 (s, 3H), 1.81–1.54 (m, 2H), 1.52–1.34 (m, 2H), 0.94 (t, 3H,
J = 7.3 Hz); MS (FAB) m/z 528 (M+H)+.
4.2.3.6.
2-Bromo-N-(4-tert-butylphenyl)benzamide
(7f)
Prepared from 4-tert-butylaniline in accordance with general pro-
cedure B. Colorless oil (90%); 1H NMR (500 MHz, CDCl3) d 7.65 (t,
2H, J = 8.0 Hz), 7.59 (br s, 1H), 7.57 (d, 2H, J = 8.0 Hz), 7.42 (t 1H,
J = 8.0 Hz), 7.41 (d, 2H, J = 8.0 Hz), 7.33 (td, 1H, J = 8.0, 1.8 Hz),
1.33 (s, 9H); MS (FAB) m/z 332, 334 (M+H)+.
4.2.4.2. 2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro
xypropan-2-yl)phenyl]-5-methoxybezamide
(12b).
Pre-
4.2.3.7. 2-Bromo-N-(4-hexylphenyl)benzamide (7g).
Pre-
pared from 10c and 2-bromo-5-methoxybenzoic acid in accor-
dance with general procedure C. White solid (60%); 1H NMR
(500 MHz, CDCl3) d 7.54 (d, 2H, J = 7.9 Hz), 7.25 (s, 1H), 7.22 (d,
2H), 6.57 (dd, 1H, J = 8.9, 2.7 Hz), 6.51 (d, 1H, J = 2.4 Hz), 3.95 (br
s, 2H), 3.59 (s, 3H), 1.67–1.59 (m, 2H), 1.42 (tq, 2H, J = 7.3,
7.3 Hz), 0.94 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 528 (M+H)+.
pared from p-hexylaniline in accordance with general procedure
B. White solid (93%); 1H NMR (500 MHz, CDCl3) d 7.65 (td, 2H,
J = 8.0, 1.5 Hz), 7.60 (br s, 1H), 7.54 (d, 2H, J = 8.5 Hz), 7.41 (td,
1H, J = 8.0, 1.5 Hz), 7.32 (td, 1H, J = 8.0, 1.5 Hz), 7.19 (d, 2H,
J = 8.5 Hz), 2.60 (t, 2H, J = 7.6 Hz), 1.62–1.53 (m, 2H), 1.37–1.26
(m, 6H), 0.89 (t, 3H, J = 7.0 Hz); MS (FAB) m/z 360, 362 (M+H)+.
4.2.4.3. 2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro
4.2.3.8. 2-Bromo-(4-dodecylphenyl)benzamide (7h).
Pre-
xypropan-2-yl)phenyl]-4-methoxybenzamide
(12c).
Pre-
pared from p-dodecylaniline and 2-bromobenzoyl chloride in
accordance with general procedure B. White solid (65%); 1H NMR
(500 MHz, CDCl3) d 7.65 (td, 2H, J = 8.5, 1.5 Hz), 7.59 (br s, 1H),
7.54 (d, 2H, J = 7.5 Hz), 7.41 (td, 1H, J = 7.5, 1.5 Hz), 7.32 (td, 1H,
pared from 10c and 2-bromo-4-methoxybenzoic acid in accor-
dance with general procedure C. White solid (70%); 1H NMR
(500 MHz, CDCl3) d 7.70–7.47 (m, 2H), 7.32–7.10 (m, 3H), 7.10–
6.85 (m, 1H), 6.70–6.48 (m, 1H), 4.05–3.78 (m, 3H), 3.70 (s, 3H),

2806
Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
1.70–1.50 (m, 2H), 1.50–1.33 (m, 2H), 0.99–0.78 (m, 3H); MS (FAB)
4.2.5.5. N-Butyl-2-iodo-N-(4-isopropylphenyl)benzamide (8e)
Prepared from 7e in accordance with general procedure D. Color-
less oil (98%); 1H NMR (500 MHz, CDCl3) d 7.63 (d, 1H, J = 8.0 Hz),
7.29 (s, 1H), 7.04 (t, 1H, J = 8.5 Hz), 7.00 (d, 1H, J = 8.5 Hz), 6.92
(dd, 1H, J = 7.3, 1.8 Hz), 6.78 (td, 1H, J = 7.9, 1.8 Hz), 3.88 (br s,
2H), 2.75 (sep, 1H, J = 6.7 Hz), 1.62 (m, 2H), 1.40 (m, 2H), 1.12 (d,
6H, J = 6.7 Hz), 0.91 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 422 (M+H)+.
m/z 528 (M+H)+.
4.2.4.4. 2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro
xypropan-2-yl)phenyl]-3-methoxybenzamide (12d).
Pre-
pared from 10c and 2-bromo-3-methoxybenzoic acid in accor-
dance with general procedure C. White solid (84%); 1H NMR
(500 MHz, CDCl3) d 7.51 (d, 2H, J = 8.5 Hz), 7.22 (m, 2H), 7.00 (dd,
1H, J = 7.9, 7.9 Hz), 6.66 (d, 1H, J = 7.9 Hz), 6.59 (d, 1H, J = 7.9 Hz),
4.05–3.90 (m, 2H), 3.80 (s, 3H), 1.73–1.63 (m, 2H), 1.50–1.35 (m,
2H), 0.94 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 528 (M+H)+.
4.2.5.6. 2-Bromo-N-butyl-N-(4-tert-butylphenyl)benzamide (8f)
Prepared from 7f in accordance with general procedure D. Color-
less oil (quant.); MS (FAB) m/z 388, 390 (M+H)+.
4.2.4.5. 2-Bromo-N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro
4.2.5.7. 2-Bromo-N-butyl-N-(4-hexylphenyl)benzamide (8g)
Prepared from 7g in accordance with general procedure D. Color-
less oil (quant.); MS (FAB) m/z 416, 418 (M+H)+.
xypropan-2-yl)phenyl]-4-methylbenzamide
(12e).
Pre-
pared from 10c and 2-bromo-4-methylbenzoic acid in accordance
with general procedure C. Colorless oil (58%); MS (FAB) m/z 512,
514 (M+H)+.
4.2.5.8. 2-Bromo-N-butyl-N-(4-dodecylphenyl)benzamide (8h)
Prepared from 8h in accordance with general procedure D. Color-
less oil (quant.); MS (FAB) m/z 500, 502 (M+H)+.
4.2.4.6. 2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro
xypropan-2-yl)phenyl]-4-chlorobenzamide (12f).
Prepared
from 10c and 2-bromo-4-chlorobenzoic acid in accordance
with general procedure C. Colorless oil (56%); 1H NMR (500 MHz,
CDCl3) d 7.49 (d, 2H, J = 8.6 Hz), 7.35 (s, 1H), 7.14 (d, 2H,
J = 8.6 Hz), 6.97 (d, 1H, J = 5.2 Hz), 6.86 (d, 1H, J = 8.0 Hz),
3.93–3.81 (m, 2H), 1.60–1.52 (m, 2H), 1.39–1.30 (m, 2H), 0.88 (t,
3H, J = 7.5 Hz).
4.2.6. General procedure E: synthesis of phenanthridine analogues
by Pd-catalyzed cyclization
4.2.6.1. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-3-methoxyphenanthridin-6(5H)-one (4a).
To a solution
mol) in DMA (3.00 mL) were added PCy3-
mol), Cs2CO3 (141 mg, 431 mol) and
mol) under an Ar atmosphere, and the
of 11a (41.4 mg, 71.9
HBF4 (13.2 mg, 35.9
Pd(OAc)2 (4.04 mg, 18.0
l
l
l
l
4.2.4.7. 2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro
mixture was stirred for 3 h at 120 °C. Then, the mixture was cooled
to 0 °C, and diluted with ethyl acetate. Water was added, and the
organic layer was separated, washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography (n-hexane/AcOEt = 1:0–2:1) to
xypropan-2-yl)phenyl]-4-(trifluoromethyl)benzamide
(12g)
Prepared from 10c and 2-bromo-4-(trifluoromethyl)benzoic acid
in accordance with general procedure C. 2-Bromo-4-(trifluoro-
methyl)benzoic acid was prepared by the reported method. Color-
less oil (64%); 1H NMR (500 MHz, CDCl3) d 7.63 (s, 1H), 7.53 (d, 2H,
J = 8.6 Hz), 7.29 (d, 1H, J = 7.4 Hz), 7.21 (d, 2H, J = 8.6 Hz), 7.08 (d,
1H, J = 8.0 Hz), 3.94 (br s, 2H), 1.67–1.60 (m, 2H), 1.41 (tq, 2H,
J = 7.4, 7.4 Hz), 0.94 (t, 3H, J = 7.4 Hz).
give 4a (8.7 mg, 19.4 lmol, 27%) as a white solid; mp 101.0–
104.0 °C; 1H NMR (500 MHz, CDCl3) d 8.52 (d, 1H, J = 7.9 Hz), 8.47
(s, 1H), 8.12 (d, 1H, J = 8.6 Hz), 7.79 (dd, 1H, J = 8.6, 6.7 Hz), 7.59
(dd, 1H, J = 7.9, 6.7 Hz), 6.98 (s, 1H), 4.38 (t, 2H, J = 7.3 Hz), 4.12
(s, 3H), 1.82 (tt, 2H, J = 7.3, 7.3 Hz), 1.54 (tq, 2H, J = 7.3, 7.3 Hz),
4.2.5. General procedure D: introduction of butyl group
4.2.5.1. N-(4-Acetylphenyl)-2-bromo-N-butylbenzamide (8a)
To a solution of 7a (104 mg, 1.00 mmol) in DMF (2.00 mL) was
added sodium hydride (40.0 mg, 1.67 mmol) at 0 °C under an Ar
atmosphere, and the mixture was stirred at room temperature
for 30 min. Then, 1-iodobutane (276 mg, 1.50 mmol) was added
at 0 °C. The mixture was stirred overnight, and allowed to warm
to room temperature. Then, it was diluted with ethyl acetate, and
water was added. The organic layer was separated, washed with
brine, dried over Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by column chromatography (n-hex-
ane/AcOEt = 4:1) to give 8a (348 mg, 0.930 mmol, 93%) as a
colorless oil.
1.05 (t, 3H, J = 7.3 Hz); HRMS (FAB) calcd for C21H20F6NO3
448.1347; found: 448.1350 (M+H)+.
4.2.6.2. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-4-methoxyphenanthridin-6(5H)-one (4b). Prepared from
11b in accordance with general procedure E. White solid (73%); mp
91.0–95.0 °C; 1H NMR (500 MHz, CDCl3) d 8.50 (d, 1H, J = 7.9 Hz),
8.26 (s, 1H), 8.22 (d, 1H, J = 7.9 Hz), 7.75 (dd, 1H, J = 7.9, 7.3 Hz),
7.59 (dd, 1H, J = 7.9, 7.3 Hz), 7.36 (s, 1H), 4.54 (t, 2H, J = 7.3 Hz),
4.03 (s, 1H), 3.97 (s, 3H), 1.84 (tt, 2H, J = 7.3, 7.3 Hz), 1.44 (tq, 2H,
J = 7.3, 7.3 Hz), 0.99 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 448 (M+H)+;
Anal. calcd for C21H19F6NO3-1/2H2O: C, 55.30; H, 4.42; N, 3.07.
Found: C, 55.27; H, 4.46; N, 3.09.
4.2.5.2. N-Butyl-2-iodo-N-phenylbenzamide (8b).
Prepared
4.2.6.3. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-
from 7b in accordance with general procedure D. Colorless oil
(98%); 1H NMR (500 MHz, CDCl3) d 7.63 (d, 1H, J = 7.3 Hz), 7.16
(d, 4H, J = 4.3 Hz), 7.09 (m, 1H), 7.04 (dd, 1H, J = 7.5, 1.2 Hz), 6.95
(dd, 1H, J = 7.4, 1.3 Hz), 6.79 (td, 1H, J = 7.4, 1.3 Hz), 3.91 (br s,
2H), 1.60 (m, 2H), 1.40 (m, 2H), 0.91 (t, 3H, J = 7.4 Hz).
7-methoxyphenanthridin-6(5H)-one (4c).
Prepared from
12a in accordance with general procedure E. White solid (85%);
mp 226.0–230.0 °C; 1H NMR (500 MHz, CDCl3) d 8.64 (s, 1H), 8.37
(d, 1H, J = 9.1 Hz), 7.82 (d, 1H, J = 8.5 Hz), 7.48 (d, 1H, J = 2.4 Hz),
7.37 (d, 1H, J = 8.5 Hz), 7.10 (dd, 1H, J = 9.1, 2.4 Hz), 5.09 (br s,
1H), 4.26 (t, 2H, J = 7.3 Hz), 3.94 (s, 3H), 1.72 (tt, 2H, J = 7.3,
7.3 Hz), 1.44 (tq, 2H, J = 7.3, 7.3 Hz), 0.95 (t, 3H, J = 7.3 Hz); MS
(FAB) m/z 448 (M+H)+; Anal. calcd for C21H19F6NO3-3/4H2O: C,
54.73; H, 4.48; N, 3.04. Found: C, 54.71; H, 4.51; N, 2.91.
4.2.5.3. 2-Bromo-N-butyl-N-p-tolylbenzamide (8c).
Pre-
pared from 7c in accordance with general procedure D. Oily solid
(quant.); MS (FAB) m/z 346, 348 (M+H)+.
4.2.5.4. 2-Bromo-N-butyl-N-(4-ethylphenyl)benzamide (8d)
Prepared from 7d in accordance with general procedure D. Color-
less oil (quant.); MS (FAB) m/z 360, 362 (M+H)+.
4.2.6.4. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-8-methoxyphenanthridin-6(5H)-one
(4d).
Prepared
from 12b in accordance with general procedure E. White solid

Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
2807
(75%); mp 195.0–197.5 °C; 1H NMR (500 MHz, CDCl3) d 8.59 (s, 1H),
8.18 (d, 1H, J = 9.2 Hz), 7.94 (d, 1H, J = 3.0 Hz), 7.79 (d, 1H,
J = 9.2 Hz), 7.44 (d, 1H, J = 9.2 Hz), 7.34 (dd, 1H, J = 9.2, 3.0 Hz),
4.59 (s, 1H), 4.38 (t, 2H, J = 7.3 Hz), 3.95 (s, 3H), 1.77 (tt, 2H,
J = 7.3, 7.3 Hz), 1.52 (tq, 2H, J = 7.3, 7.3 Hz), 1.02 (t, 3H,
J = 7.3 Hz); MS (FAB) m/z 448 (M+H)+; Anal. calcd for C21H19NO3–
H2O: C, 54.20; H, 4.55; N, 3.01. Found: C, 53.89; H, 4.12; N, 2.96.
7.3 Hz), 7.44 (d, 1H, J = 9.2 Hz), 4.39 (t, 2H, J = 7.9 Hz), 2.69 (s,
3H), 1.77 (tt, 2H, J = 7.9, 7.9 Hz), 1.52 (tq, 2H, J = 7.3, 7.3 Hz), 1.01
(t, 3H, J = 7.3 Hz); HRMS (FAB) calcd for C19H19NO2 294.1494.
Found: 294.1501 (M+H)+.
4.2.6.11. 5-Butylphenanthridin-6(5H)-one (3c).
Prepared
from 8b in accordance with general procedure E. Colorless oil
(98%, two steps); 1H NMR (500 MHz, CDCl3) d 8.54 (dd, 1H,
J = 7.9, 1.3 Hz), 8.29 (dd, 1H, J = 8.6, 1.3 Hz), 8.27 (d, 1H,
J = 8.6 Hz), 7.74 (ddd, 1H, J = 8.0, 7.4, 1.2 Hz), 7.57 (t,
1H, J = 7.4 Hz), 7.53 (ddd, 1H, J = 8.5, 7.4, 1.2 Hz), 7.40 (d, 1H,
J = 8.5 Hz), 7.30 (dd, 1H, J = 8.0, 7.4 Hz), 4.38 (t, 2H, J = 7.9 Hz),
1.78 (tt, 2H, J = 7.9, 7.3 Hz), 1.52 (tq, 2H, J = 7.3, 7.3 Hz), 1.00 (t,
3H, J = 7.3 Hz); HRMS (FAB) calcd for C17H17NO 252.1388. Found:
252.1349 (M+H)+.
4.2.6.5. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-9-methoxyphenanthridin-6(5H)-one
(4e).
Prepared
from 12c in accordance with general procedure E. White solid
(84%); mp 152.0–154.0 °C; 1H NMR (500 MHz, CDCl3) d 8.51 (s,
1H), 7.83 (d, 1H, J = 9.1 Hz), 7.81 (d, 1H, J = 7.9 Hz), 7.60 (dd, 1H,
J = 7.9, 7.9 Hz), 7.33 (d, 1H, J = 9.1 Hz), 7.06 (d, 1H, J = 7.9 Hz),
5.22 (br s, 1H), 4.20 (t, 2H, J = 7.3 Hz), 4.04 (s, 3H), 1.68 (tt, 2H,
J = 7.3, 7.3 Hz), 1.48 (tq, 2H, J = 7.3, 7.3 Hz), 0.99 (t, 3H,
J = 7.3 Hz); MS (FAB) m/z 448 (M+H)+; Anal. calcd for C21H19F6NO3–
H2O: C, 54.20; H, 4.55; N, 3.01. Found: C, 54.32; H, 4.54; N, 3.13.
4.2.6.12.
5-Butyl-2-methylphenanthridin-6(5H)-one
(3d)
Prepared from 8c in accordance with general procedure E. Colorless
oil (89%, two steps); 1H NMR (500 MHz, CDCl3) d 8.55 (dd, 1H,
J = 8.0, 1.2 Hz), 8.28 (d, 1H, J = 8.0 Hz), 8.10 (s, 1H), 7.74 (td, 1H,
J = 8.0, 1.2 Hz), 7.57 (m, 1H), 7.37–7.30 (d, 1H, J = 7.6 Hz), 4.38 (t,
2H, J = 7.6 Hz), 2.49 (s, 3H), 1.82–1.75 (m, 3H), 1.56–1.48 (m, 2H),
1.01 (t, 3H, J = 7.3 Hz); HRMS (FAB) calcd for C18H19NO 266.1545.
Found: 266.1584 (M+H)+.
4.2.6.6. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-10-methoxyphenanthridin-6(5H)-one
(4f).
Prepared
from 12d in accordance with general procedure E. White solid
(79%); mp 175.5–177.0 °C; 1H NMR (500 MHz, CDCl3) d 9.75 (s,
1H), 8.16 (d, 1H, J = 7.9 Hz), 7.84 (d, 1H, J = 9.2 Hz), 7.50 (dd, 1H,
J = 7.9, 7.9 Hz), 7.38 (d, 1H, J = 9.2 Hz), 7.22 (d, 1H, J = 7.9 Hz),
4.68 (br s, 1H), 4.30 (t, 2H, J = 7.3 Hz), 4.01 (s, 3H), 1.74 (tt, 2H,
J = 7.3, 7.3 Hz), 1.49 (tq, 2H, J = 7.3, 7.3 Hz), 1.00 (t, 3H,
J = 7.3 Hz); MS (FAB) m/z 448 (M+H)+; Anal. calcd for C21H19F6NO3:
C, 56.38; H, 4.28; N, 3.13. Found: C, 56.15; H, 4.41; N, 3.14.
4.2.6.13. 5-Butyl-2-ethylphenathridin-6(5H)-one (3e).
Pre-
pared from 8d in accordance with general procedure E. Pale brown
oil (87%, two steps); 1H NMR (500 MHz, CDCl3) d 8.55 (dd, 1H,
J = 8.0, 1.5 Hz), 8.30 (d, 1H, J = 8.0 Hz), 8.12 (d, 1H, J = 1.5 Hz),
7.75 (td, 1H, J = 8.0, 1.5 Hz), 7.58 (t, 1H, J = 8.0 Hz), 7.39 (dd, 1H,
J = 8.0, 1.5 Hz), 7.34 (d, 1H, J = 8.0 Hz), 4.39 (t, 2H, J = 8.0 Hz), 2.79
(q, 2H, J = 8.0 Hz), 1.83–1.76 (m, 2H), 1.57–1.49 (m, 2H), 1.34 (t,
3H, J = 7.6 Hz), 1.02 (t, 3H, J = 7.3 Hz); HRMS (FAB) calcd for
4.2.6.7. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-9-methylphenanthridin-6(5H)-one (5a).
Prepared from
12e in accordance with general procedure E. White solid (13%);
mp 194.5–196.0 °C; 1H NMR (500 MHz, CDCl3) d 8.58 (d, 1H,
J = 2.3 Hz), 8.35 (d, 1H, J = 8.0 Hz), 7.98 (s, 1H), 7.75 (d, 1H,
J = 9.2 Hz), 7.39–7.36 (m, 2H), 4.30 (dd, 2H, J = 7.7, 3.9 Hz), 2.51
(s, 3H), 1.75–1.69 (m, 2H), 1.48–1.42 (m, 2H), 0.95 (t, 3H,
J = 7.4 Hz); HRMS (FAB) calcd for C21H19F6NO2 431.1320. Found:
431.1320 (M)+.
C
19H21NO 280.1701. Found: 280.1696 (M+H)+.
4.2.6.14. 5-Butyl-2-isopropylphenanthridin-6(5H)-one
(3f)
Prepared from 8e in accordance with general procedure E. Color-
less oil (98%, two steps); 1H NMR (500 MHz, CDCl3) d 8.53 (dd,
1H, J = 7.9, 1.3 Hz), 8.30 (d, 1H, J = 8.0 Hz), 8.12 (d, 1H, J = 1.9 Hz),
7.73 (t, 1H, J = 7.8 Hz), 7.55 (t, 1H, J = 7.8 Hz), 7.41 (dd, 1H, J = 8.7,
1.9 Hz), 7.34 (d, 1H, J = 8.6 Hz), 4.36 (t, 2H, J = 7.9 Hz), 3.04 (sep,
1H, J = 7.0 Hz), 1.77 (m, 2H), 1.51 (m, 2H), 1.33 (d, 3H, J = 7.3 Hz),
1.00 (t, 3H, J = 7.3 Hz); HRMS (FAB) calcd for C20H23NO 294.1858.
Found: 294.1851 (M+H)+.
4.2.6.8. 5-Butyl-9-chloro-2-(1,1,1,3,3,3-hexafluoro-2-hydroxy-
propan-2-yl)phenanthridin-6(5H)-one (5b).
Prepared from
12f in accordance with general procedure E. White solid (29%);
mp 194.0–198.5 °C; 1H NMR (500 MHz, CDCl3) d 8.56 (d, 1H,
J = 2.3 Hz), 8.46 (d, 1H, J = 8.6 Hz), 8.22 (d, 1H, J = 1.7 Hz), 7.84 (d,
1H, J = 9.2 Hz), 7.55 (dd, 1H, J = 8.6, 2.3 Hz), 7.45 (d, 1H,
J = 8.6 Hz), 4.35 (t, 2H, J = 8.0 Hz), 1.80–1.74 (m, 2H), 1.53–1.47
(m, 2H), 1.01 (t, 3H, J = 7.4 Hz); HRMS (FAB) calcd for C20H17ClF6-
NO2 452.0852. Found: 452.0868 (M+H)+.
4.2.6.15. 2-tert-Butyl-5-butylphenanthridin-6(5H)-one (3g)
Prepared from 8f in accordance with general procedure E. Pale
brown oil (67%, two steps); 1H NMR (500 MHz, CDCl3) d 8.56 (dd,
1H, J = 8.0, 1.5 Hz), 8.32 (d, 1H, J = 8.0 Hz), 8.31 (d, 1H, J = 1.5 Hz),
7.78–7.74 (m, 1H), 7.60 (dd, 1H, J = 8.0, 1.5 Hz), 7.57 (d, 1H,
J = 8.0 Hz), 7.37 (d, 1H, J = 8.0 Hz), 4.39 (t, 2H, J = 7.6 Hz), 1.83–
1.76 (m, 2H), 1.561.50 (m, 2H), 1.44 (s, 9H), 1.02 (t, 3H,
J = 7.3 Hz); HRMS (FAB) calcd for C21H25NO 308.2014. Found:
308.2008 (M+H)+.
4.2.6.9.
5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
Pre-
2-yl)-9-(trifluoromethyl)phenathridin-6(5H)-one (5c).
pared from 12g in accordance with general procedure E. White
solid (80%); mp 182.5–185.0 °C; 1H NMR (500 MHz, CDCl3) d
8.61 (dd, 2H, J = 10.0, 5.4 Hz), 8.46 (s, 1H), 7.83 (d, 1H, J = 9.2 Hz),
7.76 (d, 1H, J = 8.6 Hz), 7.44 (d, 1H, J = 9.2 Hz), 4.33 (t, 2H,
J = 8.0 Hz), 1.77–1.71 (m, 2H), 1.47 (td, 2H, J = 14.9, 7.4 Hz), 0.96
(t, 3H, J = 7.2 Hz); HRMS (FAB) calcd for C21H17F9NO2 486.1116.
Found: 486.1113 (M+H)+.
4.2.6.16.
5-Butyl-2-hexylphenanthridin-6(5H)-one
(3h)
Prepared from 8g in accordance with general procedure E. Color-
less oil (90%, two steps); 1H NMR (500 MHz, CDCl3) d 8.55 (d, 1H,
J = 8.0 Hz), 8.30 (d, 1H, J = 8.0 Hz), 8.09 (s, 1H), 7.75 (t, 1H,
J = 8.0 Hz), 7.57 (t, 1H, J = 8.0 Hz), 7.37 (dd, 1H, J = 8.0, 1.5 Hz),
7.33 (dd, 1H, J = 8.0, 1.5 Hz), 4.39 (t, 2H, J = 7.6 Hz), 2.74 (t, 2H,
J = 7.9 Hz), 1.83–1.76 (m, 2H), 1.73–1.66 (m, 2H), 1.58–1.49 (m,
2H), 1.41–1.32 (m, 6H), 1.02 (t, 3H, J = 7.3 Hz), 0.90 (t, 3H,
J = 7.0 Hz); HRMS (FAB) calcd for C23H29NO 336.2327. Found:
336.2310 (M+H)+.
4.2.6.10. Methyl 5-butyl-6-oxo-5,6-dihydrophenanthridine-2-
carboxylate (3a).
Prepared from 8a in accordance with gen-
eral procedure E. White solid (77%, two steps); mp 114.0–
118.0 °C; 1H NMR (500 MHz, CDCl3) d 8.92 (d, 1H, J = 1.8 Hz),
8.53 (d, 1H, J = 7.3 Hz), 8.37 (d, 1H, J = 7.9 Hz), 8.10 (dd, 1H,
J = 9.2, 1.8 Hz), 7.79 (t, 1H, J = 7.3 Hz), 7.61 (dd, 1H, J = 7.9,

2808
Y. Nishiyama et al. / Bioorg. Med. Chem. 22 (2014) 2799–2808
4.2.6.17.
5-Butyl-2-dodecylphenanthridin-6(5H)-one
(3i)
References and notes
Prepared from 8h in accordance with general procedure E. Color-
less oil (66%, two steps); 1H NMR (500 MHz, CDCl3) d 8.55 (dd,
1H, J = 8.0, 1.5 Hz), 8.30 (d, 1H, J = 8.0 Hz), 8.09 (d, 1H, J = 1.5 Hz),
7.75 (ddd, 1H, J = 8.0, 6.5, 1.5 Hz), 7.57 (td, 1H, J = 8.0, 1.5 Hz),
7.36 (dd, 1H, J = 8.0, 1.5 Hz), 7.33 (d, 1H, J = 8.0 Hz), 4.38 (t, 2H,
J = 7.6 Hz), 2.74 (t, 2H, J = 7.6 Hz), 1.83–1.76 (m, 2H), 1.73–1.66
(m, 2H), 1.57–1.49 (m, 2H), 1.45–1.20 (m, 18H), 1.02 (t, 3H,
J = 7.3 Hz), 0.88 (t, 3H, J = 7.0 Hz); HRMS (FAB) calcd for
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4.2.7. 5-Butyl-2-hydroxymethylphenanthridin-6(5H)-one (3b)
To a solution of 3a (288 mg, 0.770 mmol) in THF (5.00 mL) was
added lithium borohydride (86.8 mg, 4.00 mmol) at 0 °C. The mix-
ture was stirred overnight, and then allowed to warm to room tem-
perature and diluted with ethyl acetate. Water was added, and the
organic layer was separated, washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified
by column chromatography (n-hexane/AcOEt = 2:1) to give 3b
(193 mg, 0.680 mmol, 89%) as a white solid; mp 150.0–153.0 °C;
1H NMR (500 MHz, CDCl3) d 8.54 (d, 1H, J = 8.0 Hz), 8.28–8.26 (m,
2H), 7.75 (t, 1H, J = 8.0 Hz), 7.59 (t, 1H, J = 8.0, 1.8 Hz), 7.52 (dd,
1H, J = 8.0, 1.8 Hz), 7.36 (d, 1H, J = 8.0 Hz), 4.83 (d, 2H, J = 6.0 Hz),
4.37 (t, 2H, J = 7.5 Hz), 1.99–1.94 (m, 1H), 1.81–1.74 (m, 2H), 1.55–
1.48 (m, 2H), 1.01 (t, 3H, J = 7.5 Hz); Anal. calcd for C18H19NO2: C,
76.84; H, 6.81; N, 4.98. Found: C, 76.94; H, 6.71; N, 5.01.
4.2.8. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-
9-hydroxyphenathridin-6(5H)-one (5d)
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ature, then NaHCO3 was added to it. Water was added, and the or-
ganic layer was separated, washed with brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography (n-hexane/AcOEt = 2:1) to give
5d (45.9 mg, 0.106 mmol, 85%) as a white solid; mp 250.0–
252.5 °C; 1H NMR (500 MHz, DMSO) d 8.50 (d, 1H, J = 1.7 Hz),
8.22 (d, 1H, J = 9.2 Hz), 7.81 (d, 1H, J = 8.6 Hz), 7.69 (d, 1H,
9.2 Hz), 7.64 (d, 1H, J = 2.3 Hz), 7.10 (dd, 1H, J = 8.6, 2.3 Hz), 4.30
(t, 2H, J = 7.7 Hz), 1.66–1.60 (m, 2H), 1.41 (tq, 2H, J = 7.4, 7.4 Hz),
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0.94 (t, 3H, J = 7.4 Hz); HRMS (FAB) calcd for
C20H17F6NO3
433.1113. Found: 433.1118 (M)+.
Acknowledgements
The work described in this paper was supported in part by a
Grant-in-aid for Scientific Research from the Ministry of Education,
Culture, Sports, Sciences and Technology, Japan, and a Grant from
the Japan Society for the Promotion of Science, Japan.
28. Aoyama, A.; Endo-Umeda, K.; Kishida, K.; Ohgane, K.; Noguchi-Yachide, T.;
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Chem. 2012, 55, 7360.