Fused heterocyclic amido compounds as anti-hepatitis C virus agents

Article abstract of DOI:10.1016/j.bmc.2011.03.002

We identified a fused heteroaromatic amido structure based on the phenanthridine skeleton as a superior scaffold for candidate drugs with potent anti-HCV activity. Among the compounds synthesized, a phenanthridine analogue with a 1,3-dioxolyl group (24) possessed the most potent anti-HCV activity (EC₅₀ value: 50 nM), with acceptable cytotoxicity. The structural development and structure-activity relationships of these compounds are described.

Full text of DOI:10.1016/j.bmc.2011.03.002

Bioorganic & Medicinal Chemistry 19 (2011) 2675–2687
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fused heterocyclic amido compounds as anti-hepatitis C virus agents
Hiroshi Aoyama a, , Kazuyuki Sugita a, Masahiko Nakamura a, Atsushi Aoyama a, Mohammed T. A. Salim b,
⇑
a
Mika Okamoto b, Masanori Baba b, , Yuichi Hashimoto
⇑
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka,
Kagoshima 890-8544, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We identified a fused heteroaromatic amido structure based on the phenanthridine skeleton as a superior
scaffold for candidate drugs with potent anti-HCV activity. Among the compounds synthesized, a
phenanthridine analogue with a 1,3-dioxolyl group (24) possessed the most potent anti-HCV activity
(EC50 value: 50 nM), with acceptable cytotoxicity. The structural development and structure–activity
relationships of these compounds are described.
Received 17 February 2011
Accepted 2 March 2011
Available online 11 March 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Hepatitis C virus
Phenanthridine
Anti-HCV agent
Methoxy
1,3-Dioxolyl
1. Introduction
activity (Fig. 1). The c-carboline analogue 1 showed powerful
inhibitory activity for bovine viral diarrhea virus (BVDV) replica-
tion, with an EC50 value of 3.5 nM. Although BVDV belongs to the
Flaviviridae family, as HCV does,17 and is thought to be a surrogate
model for HCV,18–20 compound 1 showed only very weak activity
against HCV. On the other hand, phenanthridine analogue 2, which
has a fused heterocyclic amido structure, exhibited moderate,
dose-dependent HCV replication-inhibitory activity,12 supporting
the idea that phenanthridine analogues could be lead compounds
in the development of anti-HCV drugs.
Hepatitis C virus (HCV: a member of the Flaviviridae family) is
thought to be a major cause of human hepatitis,1,2 and it is esti-
mated that at least 170 million people worldwide are chronically
infected with this virus.3 Most infections become persistent, and
about 60% of cases progress to chronic liver disease, which in turn
can lead to cirrhosis, hepatocellular carcinoma, and liver failure.4,5
Currently, no vaccine is available to prevent HCV infection, and the
standard treatment for chronic hepatitis C consists of pegylated
interferon (IFN)-
a
in combination with the nucleoside analogue
In this paper, we describe the synthesis of a series of fused het-
erocyclic amido compounds based on the structure of phenanthri-
dine 2, as well as the evaluation of their anti-HCV activity, and the
creation of potent anti-HCV agents possessing one or more meth-
oxy group(s) and/or a 1,3-dioxolyl group.
ribavirin (1-b- -ribofuranosyl-1,2,4-triazole-3-carboxamide). How-
D
ever, the virus cannot be eliminated from approximately half of in-
fected patients treated with these agents.6 In addition, the side ef-
fects of these agents are sometimes serious and unacceptable to
patients. A number of molecules are being studied in clinical tri-
als,7 but none of them has yet been approved. Therefore, alterna-
tive agents for the treatment and prevention of HCV infection are
urgently needed.
Recently, we have succeeded in the development of antiviral
agents with polyphyletic skeletons.8–16 Among them, several
fused-heterocyclic compounds, including 3,4,5-trimethyl-c-carbo-
line (1)9 and 5-butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)phenanthridin-6(5H)-one (2),12 exhibited anti-flavivirus
⇑
Corresponding authors. Tel.: +81 3 5841 7848; fax: +81 3 5841 8495 (H.A.);
tel.: +81 99 275 5930; fax: +81 99 275 5932 (M.B.).
E-mail addresses: aoyama@iam.u-tokyo.ac.jp (H. Aoyama), m-baba@vanilla.ocn.
ne.jp (M. Baba).
Figure 1. Antiviral activity of fused heterocyclic compounds.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.002

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H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
Table 1
2. Results and discussion
2.1. Electronic effects of substituents
Effects of substituents of phenanthridine on anti-HCV activities and cytotoxicities
Our previous study12 on the anti-HCV activity of phenanthri-
dine analogues indicated that a 1,1,1,3,3,3-hexafluoro-2-hydroxy-
propyl group at the 2-position is essential for inhibition of HCV
replication. As for the substituent on the amido nitrogen, a butyl
group is appropriate in terms of size and electric charge. Indeed,
introduction of a polar methoxyethyl (3) or bulky 2-naphthyl (4)
group at this position did not result in improvement of anti-HCV
activity in further investigations (Fig. 2). On the basis of these find-
ings, we designed new phenanthridine analogues (derived from 2)
modified at the 3- or 4-position with three kinds of substituents (F,
Me and OMe) possessing different properties, and examined their
antiviral activity. These compounds were synthesized as shown
in Scheme 1. Briefly, N-butylaniline derivatives 28a–f, prepared
from the corresponding anilines, were treated with hexafluoroace-
tone trihydrate to give 1,1,1,3,3,3-hexafluoro-2-hydroxypropyl
derivatives 29a–f, which were condensed with 2-iodobenzoyl
chloride. The resulting anilides 30a–f were cyclized in the presence
of Pd catalyst to give compounds 5–10.
As shown in Table 1, introduction of substituents at the 4-posi-
tion enhanced the anti-HCV activity in the order of fluoro
(6) < methyl (8) < methoxy (10), whereas introduction of substitu-
ents at the 3-position did not improve the anti-HCV activity, com-
pared with unsubstituted 2. The analogues with a non-polar
methyl group, that is, compounds 7 and 8, showed almost the same
anti-HCV activity as the analogues substituted at the correspond-
ing site (3- or 4-position) with an electron-withdrawing fluoro
group, that is, compounds 5 and 6, respectively. On the other hand,
analogues with an electron-donating methoxy group (compounds
9 and 10) exhibited higher anti-HCV activity than the correspond-
ing methyl- and/or fluoro-substituted analogues. As regards cyto-
toxicity, electron-withdrawing character of the substituent seems
R1
EC50
(l
M)
CC50 (lM)
2
5
6
7
8
H
3-F
4-F
3-CH3
4-CH3
3-OCH3
4-OCH3
3.2
5.3
2.4
5.6
2.1
3.4
1.1
15.8
25.5
12.1
28.9
31.9
>40
9
10
22.3
to be unfavorable, that is, introduction of a fluoro group at the 4-
position (6) resulted in an increase of the cytotoxicity compared
to other 4-substituted compounds, 8 and 10. These results suggest
that the substituent effect is determined more by electronic char-
acter than by the steric factor, and an electron-donating group is
favorable for anti-HCV activity. Therefore, we selected the methoxy
group as a fixed substituent for further structural development
studies.
2.2. Regioisomers of methoxyl-substituted analogues
Based on our previous structure–activity relationship studies of
anti-BVDV c-carboline analogues, which indicated that regioselec-
tive methyl substitution dramatically influenced the antiviral
activity,8,9 we next investigated the effect of methoxy substitution.
For this purpose, we synthesized all regioisomers of methoxy-
substituted phenanthridine analogues 9–15 by using the same
method as in Scheme 1 (see Scheme 2).
The anti-HCV activity and cytotoxicity of prepared compounds
9–15 are summarized in Table 2. The effect of methoxylation on
the anti-HCV activity seems to be dependent on the position at
which the methoxy group is introduced. Introduction of a methoxy
group at positions 1, 7, 9 and 10 decreased the anti-HCV activity
compared to methoxy-unsubstituted analogue 2, whereas intro-
duction at positions 3, 4 and 8 increased the activity. The 8-meth-
oxylated analogue (13) showed the most potent anti-HCV activity
among this series of compounds, with the EC50 value of 0.98
lM. As
for cytotoxicity, the introduction of the methoxy group at positions
Figure 2. Antiviral efficacy of 3 and 4 against HCV proliferation.
Scheme 1. Synthesis of 5–10.

H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
2677
Scheme 2. Synthesis of 11–15.
28h was prepared by means of metal-catalyzed reaction, as shown
in Scheme 3. Briefly, 1-iodo-2,3-dimethoxybenzene (34a),
prepared from 1,2-dimethoxybenzene by the literature method,21
was coupled with butylamine by amino acid-promoted CuI-cata-
lyzed C–N bond formation reaction.22
Table 2
Anti-HCV activities and cytotoxicities of regioisomers of methoxyphenanthridine
9–15
As shown in Table 3, among the dimethoxylated compounds, 3,8-
disubstituted (18) and 4,8-disubstituted analogues (19) exhibited
anti-HCV activity several-fold higher than that of the monomethoxy
compounds: the EC50 values of 18 and 19 were 0.18 and 0.51 lM,
respectively. Two other dimethoxylated compounds, 1,4-disubsti-
tuted (16) and 3,4-disubstituted (17) analogues, showed anti-HCV
potency similar to that of the monomethoxy compounds. On the
other hand, trimethoxylated compounds 20 and 21 showed only a
slight improvement in the anti-HCV activity compared to the mono-
methoxy compounds. As for cytotoxicity, analogues possessing a
methoxy group at the 1-position, 16 and 20, exhibited almost no
cytotoxicity, while other polymethoxylated compounds were more
cytotoxic than the monomethoxylated compounds. These results
suggest that introduction of two methoxy groups on the same phe-
nyl ring is unfavorable for anti-HCV activity: n,4-dimethoxy ana-
logues (n = 1 or 3: 16 and 17, respectively) show weaker anti-HCV
activity than 4-methoxy analogue 10, and n,4,8-trimethoxy ana-
logues (n = 1 or 3: 20 and 21, respectively) show weaker anti-HCV
activity than 4-methoxy (10) or 8-methoxy (13) analogues. In addi-
tion, it was suggested that introduction of a methoxy group at the 1-
position reduced the cytotoxicity.
R1
R2
EC50
(lM)
CC50 (lM)
2
11
9
10
12
13
14
15
H
H
H
H
H
3.2
7.2
3.4
1.1
9.96
0.98
16.6
5.0
15.8
25.3
>40
22.3
12.6
9.6
1-OCH3
3-OCH3
4-OCH3
H
H
H
H
7-OCH3
8-OCH3
9-OCH3
10-OCH3
28.9
6.9
7, 8 and 10 increased the cytotoxicity, while introduction at other
positions decreased the cytotoxicity or had no effect.
2.3. Dimethoxyl- and trimethoxyl-substituted analogues
The results for the regioisomers of the monomethoxy-substi-
tuted analogues indicate that the introduction of a methoxy group
at the 3, 4, or 8 position of the phenanthridine skeleton enhanced
the anti-HCV activity, compared to unsubstituted analogue 2. In
addition, introduction of a methoxy group at the 1-position seems
to have potential for the reduction of cytotoxicity, without affect-
ing the anti-HCV activity. On the basis of these results, we expected
that dimethoxylation and/or trimethoxylation at a combination of
the 1, 3, 4 and 8-positions might lead to more potent anti-HCV
agents. Various compounds, except for analogues possessing meth-
oxy groups at both the 1 and 3 positions, were prepared by means
of a method similar to that used for the synthesis of 5–10, as illus-
trated in Schemes 4 and 5. The commercially unavailable aniline
2.4. [1,3]Dioxolophenanthridine analogues
Although 3,8-(18) and 4,8-dimethoxyphenanthridine (19) ana-
logues showed potent anti-HCV activity, which seems to arise from
a synergistic effect of the methoxy groups, 3,4,8-trimethoxy-
substituted phenanthridine analogue (21) showed enhancement
of only the cytotoxicity, but not the anti-HCV activity. This result
suggests that the unfavorable effect of steric hindrance between
adjacent methoxy groups at the 3 and 4 positions is larger than
the cumulative antiviral activity-enhancing effect of these meth-
oxy groups. This hypothesis prompted us to examine the introduc-
tion of a 1,3-dioxolyl group (so-called methylenedioxy group)

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H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
Scheme 3. Synthesis of 29h–k.
3. Conclusion
Table 3
Anti-HCV activities and cytotoxicities of dimethoxy and trimethoxyphenanthridine
16–21
We have developed a series of potent anti-HCV agents consist-
ing of fused heterocyclic amido structure with good selectivity
for virus replication-inhibitory activity over cytotoxicity. Among
them, compound 24 showed the most potent anti-HCV activity,
having an EC50 value of 50 nM, with
(SI: CC50/EC50) value of 128.
a selectivity index
4. Experimental
R1
R2
EC50
(l
M)
CC50 (lM)
4.1. General comments
16
17
18
19
20
21
1,4-(OCH3)2
3,4-(OCH3)2
3-OCH3
4-OCH3
1,4-(OCH3)2
3,4-(OCH3)2
H
H
5.7
2.6
0.18
0.51
6.1
>100
10.4
7.6
11.1
>100
6.8
Melting points were determined by using a Yanagimoto hot-
stage melting point apparatus and are uncorrected. 1H NMR spectra
were recorded on a JEOL JNM-GX500 (500 MHz) spectrometer.
Chemical shifts are expressed in d (ppm) values with tetramethylsil-
ane (TMS) as an internal reference. Mass spectra (MS) and high-res-
olution mass spectra (HRMS) were recorded on a JEOL JMS-DX303
spectrometer. Elemental analyses were carried out in the Microana-
lytical Laboratory, Faculty of Pharmaceutical Sciences, The Univer-
sity of Tokyo, and were within 0.4% of the theoretical values.
8-OCH3
8-OCH3
8-OCH3
8-OCH3
1.8
instead of the adjacent methoxy structure of analogue (21), and we
prepared various related analogues with a 1,3-dioxolyl group as
illustrated in Schemes 3, 6 and 7.
As expected, the compounds with a 1,3-dioxolyl group at the 3/
4-positions, that is, compounds 23 and 24, exhibited potent anti-
4.2. Chemistry
HCV activity, with EC50 values of 0.29 and 0.050 lM, respectively
(Table 4). As with the dimethoxylated compounds, introduction
of a methoxy group at the 8-position enhanced the anti-HCV activ-
ity. Although introduction of a methoxy group at the 1-position in
this series of compounds, 26 and 27, greatly decreased the cytotox-
icity, it did not affect the anti-HCV activity. The compound possess-
ing a 1,3-dioxolyl group at the 8/9-positions (22) showed moderate
anti-HCV activity with almost no cytotoxicity, whereas introduc-
tion of an additional 1,3-dioxolyl group at the 3/4-positions (25)
resulted in enhancement of the cytotoxicity.
4.2.1. General procedure A: synthesis of phenylhexafluoropro-
panol analogues
4.2.1.1.
oropropan-2-ol (29a).
2-(4-Butylamino-2-fluorophenyl)-1,1,1,3,3,3-hexaflu-
To a mixture of hexafluoroacetone
trihydrate (707 mg, 3.21 mmol) and toluene (5.0 ml) were added
N-butyl-3-fluoroaniline (28a) (358 mg, 2.14 mmol) and p-
TsOHÁH2O (81.5 mg, 428
lmol), then the mixture was stirred at
120 °C for 10 h. After cooling to room temperature, the mixture
was diluted with ethyl acetate and then washed with satd NaHCO3

H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
2679
Scheme 4. Synthesis of 16, 17, 20, 21.
Scheme 5. Synthesis of 18, 19.
aqueous solution and brine, and dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by column
chromatography (n-hexane/AcOEt = 1:0–2:1) to give 29a (263 mg,
0.790 mmol, 37%) as a colorless oil. 1H NMR (500 MHz, CDCl3) d
7.40–7.24 (m, 1H), 6.41–6.38 (m, 1H), 6.31–6.27 (m, 1H), 4.30 (br
s, 1H), 4.02 (br s, 1H), 3.11 (t, 2H, J = 6.1 Hz), 1.61 (quin, 2H,
J = 7.3 Hz), 1.43 (sext, 2H, J = 7.3 Hz), 0.97 (t, 3H, J = 7.3 Hz); MS
(FAB) m/z 334 (M+H)+.
4.2.1.3. 2-(4-Butylamino-2-methylphenyl)-1,1,1,3,3,3-hexaflu-
oropropan-2-ol (29c). Prepared from N-butyl-3-methylani-
line (28c) in accordance with the general procedure A. Colorless oil
(39%); 1H NMR (500 MHz, CDCl3) d 7.50 (d, 1H, J = 7.9 Hz), 7.09 (d,
1H, J = 7.9 Hz), 7.07 (s, 1H), 3.52 (br s, 1H), 3.03 (t, 2H, J = 7.3 Hz),
2.37 (s, 3H), 1.65 (quin, 2H, J = 7.3 Hz), 1.52 (s, 1H), 1.44 (sext, 2H,
J = 7.3 Hz), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 330 (M+H)+.
4.2.1.4. 2-(4-Butylamino-3-methylphenyl)-1,1,1,3,3,3-hexaflu-
4.2.1.2.
oropropan-2-ol (29b).
2-(4-Butylamino-3-fluorophenyl)-1,1,1,3,3,3-hexaflu-
Prepared from N-butyl-2-fluoroani-
oropropan-2-ol (29d).
Prepared from N-butyl-2-methylani-
line (28d) in accordance with the general procedure A. Colorless
oil (93%); 1H NMR (500 MHz, CDCl3) d 7.41 (d, 1H, J = 8.5 Hz),
7.33 (s, 1H), 6.61 (d, 1H, J = 8.5 Hz), 3.65 (br s, 1H), 3.32 (br s,
1H), 3.17 (t, 2H, J = 7.3 Hz), 2.15 (s, 3H), 1.66 (quin, 2H,
J = 7.3 Hz), 1.45 (sext, 2H, J = 7.3 Hz), 0.98 (t, 3H, J = 7.3 Hz); MS
(FAB) m/z 330 (M+H)+.
line (28b) in accordance with the general procedure A. Colorless
oil (74%); 1H NMR (500 MHz, CDCl3) d 7.33–7.29 (m, 2H), 6.71–
6.67 (m, 1H), 4.06 (br s, 1H), 3.70 (br s, 1H), 3.19–3.12 (m, 2H),
1.65 (quin, 2H, J = 7.3 Hz), 1.44 (sext, 2H, J = 7.3 Hz), 0.97 (t, 3H,
J = 7.3 Hz); MS (FAB) m/z 334 (M+H)+.

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H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
Scheme 6. Synthesis of 22–25.
4.2.1.5. 2-(4-Butylamino-2-methoxyphenyl)-1,1,1,3,3,3-hexaflu-
oropropan-2-ol (29e). Prepared from N-butyl-3-methoxyani-
a solution of 28h (167 mg, 0.800 mmol) in toluene (1.6 mL), p-
TsOHÁH2O (15.2 mg, 79.9 mol), and MS4A (600 mg), and the mix-
l
line (28e) in accordance with the general procedure A. Colorless oil
(35%); 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, 1H), 3.12 (m, 2H), 1.61 (quin, 2H,
J = 7.3 Hz), 1.43 (sext, 2H, J = 7.3 Hz), 0.97 (t, 3H, J = 7.3 Hz); MS
(FAB) m/z 346 (M+H)+.
ture was stirred at 120 °C for 10 h. After cooling to room tempera-
ture, the mixture was diluted with ethyl acetate, and then filtered
off under suction and washed with ethyl acetate. The filtrate was
washed with water and brine, and dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by column
chromatography (n-hexane/CH2Cl2 = 7:1) to give 29i (115 mg,
0.306 mmol, 38%) as a colorless oil. 1H NMR (DMSO-d6, 500 MHz) d
8.00 (s, 1H), 7.18 (d, J = 9.2 Hz, 1H), 6.41 (d, J = 9.2 Hz, 1H), 5.52 (t,
J = 6.1 Hz, 1H), 3.79 (s, 3H), 3.63 (s, 3H), 3.08 (td, J = 6.1, 6.7 Hz,
2H), 1.52 (tt, J = 6.7, 7.3 Hz, 2H), 1.34 (qt, J = 7.3, 7.3 Hz, 2H), 0.90
(t, J = 7.3 Hz, 3H); MS (FAB) m/z 375 (M)+.
4.2.1.6. 2-(4-Butylamino-3-methoxyphenyl)-1,1,1,3,3,3-hexaflu-
oropropan-2-ol (29f).
Prepared from N-butyl-2-methoxyani-
line (28f) in accordance with the 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), 3.95 (br s, 1H), 3.85 (s,
3H), 3.14 (t, 2H, J = 7.3 Hz), 1.64 (quin, 2H, J = 7.3 Hz), 1.44 (sext,
2H, J = 7.3 Hz), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 346 (M+H)+.
4.2.1.9. 4-(Butylamino)benzo[d][1,3]dioxole-2-(1,1,1,3,3,3-hexa-
fluoro)propan-2-ol (29j).
Prepared from 28i in accordance
with the synthesis of 29i. White solid (81%); mp 60.5–62.0 °C; 1H
NMR (DMSO-d6, 500 MHz) d 8.19 (s, 1H), 6.93 (d, J = 9.2 Hz, 1H),
6.33 (d, J = 9.2 Hz, 1H), 5.91 (s, 2H), 5.47 (t, J = 6.7 Hz, 1H), 3.08
(td, J = 6.7, 7.3 Hz, 2H), 1.52 (tt, J = 7.3, 7.9 Hz, 2H), 1.34 (qt,
J = 7.3, 7.9 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 359 (M)+.
4.2.1.7.
2-(4-Butylamino-2,5-dimethoxyphenyl)-1,1,1,3,3,3-
Prepared from 2,5-dime-
hexafluoropropan-2-ol (29h).
thoxy-N-butylaniline (28g) in accordance with the general proce-
dure A. Colorless oil (47%); 1H NMR (500 MHz, CDCl3) d 7.74 (s,
1H), 6.80 (s, 1H), 6.21 (s, 1H), 4.46 (br s, 1H), 3.92 (s, 3H), 3.80 (s,
3H), 3.14 (m, 2H), 1.66 (quin, 2H, J = 7.3 Hz), 1.45 (sext, 2H,
J = 7.3 Hz), 0.98 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 376 (M+H)+.
4.2.1.10.
4-(Butylamino)-6-methoxybenzo[d][1,3]dioxole-2-
Prepared from
(1,1,1,3,3,3-hexafluoro)propan-2-ol (29k).
28j in accordance with the synthesis of 29i. Yellow amorphous so-
lid (65%); 1H NMR (DMSO-d6, 500 MHz) d 7.55–7.85 (br, 1H), 5.98
(br s, 1H), 5.84 (s, 2H), 5.40–5.80 (br, 1H), 3.72 (br s, 2H), 3.13 (td,
J = 6.7, 6.7 Hz [with NH–CH2 coupling], 2H), 1.50 (tt, J = 6.7, 7.3 Hz,
2H), 1.33 (qt, J = 6.7, 7.3 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H); MS (FAB)
m/z 389 (M)+.
4.2.1.8.
2-(4-Butylamino-2,3-dimethoxyphenyl)-1,1,1,3,3,3-
Prepared from 28b in accor-
hexafluoropropan-2-ol (29i).
dance with a slight modification of the general procedure A. To a
mixture of hexafluoroacetone sesquihydrate (309 mg, 1.60 mmol)
and hexafluoroacetone trihydrate (352 mg, 1.60 mmol) were added

H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
2681
Scheme 7. Synthesis of 26 and 27.
6.87 (dd, J = 7.9, 1.2 Hz, 1H), 6.78 (dd, J = 7.9, 7.9 Hz, 1H), 3.84 (s,
3H), 3.81 (s, 3H); MS (FAB) m/z 264 (M)+.
Table 4
Anti-HCV activities and cytotoxicities of phenanthridine analogues with 1,3-dioxolyl
group (22–27)
4.2.3. N-Butyl-2,3-dimethoyxaniline (28h)
To a mixture of 34a (396 mg, 1.50 mmol) and K2CO3 (415 mg,
3.00 mmol) were added a solution of CuI (85.7 mg, 0.450 mmol)
and
L-proline (104 mg, 0.900 mmol) in DMSO (2.0 mL) and n-butyl-
amine (600
ll, 6.07 mmol), and the mixture was stirred at 90 °C for
15 h. After cooling to room temperature, the mixture was diluted
with ethyl acetate, and then washed with water and brine. The or-
ganic layer was dried over Na2SO4, and concentrated under re-
duced pressure. The residue was purified by column
chromatography (n-hexane/ethyl acetate/CH2Cl2 = 50:1:1 to
30:1:1) to give 28h (174 mg, 0.833 mmol, 56%) as a colorless oil.
1H NMR (CDCl3, 500 MHz) d 6.85–6.96 (m, 3H), 3.87 (s, 3H), 3.83
(s, 3H), 3.15 (t, J = 7.3 Hz, 2H), 1.65 (tt, J = 7.3, 7.3 Hz, 2H), 1.38
(qt, J = 7.3, 7.3 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 209
(M)+, 210 (M+H)+.
R1
R2
EC50
(
l
M) CC50
(l
M) SIa
23 3,4-(OCH2O)
H
0.29
2.3
9.8
0.050
12.0
1.73
24.2
>100
93.9
6.4
>100
15.9
83.3
22
H
8,9-(OCH2O)
H
8-OCH3
>43.1
9.56
128
>8.31
9.18
26 1-OCH3 3,4-(OCH2O)
24 3,4-(OCH2O)
27 1-OCH3 3,4-(OCH2O) 8-OCH3
25 3,4-(OCH2O)
8,9-(OCH2O)
a
Selectivity index (CC50/EC50).
4.2.4. 4-Iodobenzo[d][1,3]dioxole (34b)
4.2.2. 3-Iodo-1,2-dimethoxybenzene (34a)
To a solution of 34a (792 mg, 3.00 mmol) in CH2Cl2 (10.0 mL)
was added BBr3 (1.0 M CH2Cl2 solution, 14.0 mL, 14.0 mmol) at
À78 °C under Ar atmosphere, and the mixture was stirred at the
room temperature. After 42 h, the reaction mixture was poured
into ice, and volatile materials were removed under reduced pres-
sure. The residue was extracted with ethyl acetate. The combined
organic layer was washed with brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was dissolved in
DMF (30 mL), and to this were added Cs2CO3 (1.00 g, 3.07 mmol)
and CH2I2 (250 mL, 3.10 mmol) and the mixture was stirred at
120 °C for 1 h under Ar atmosphere. After the reaction mixture
was cooled to room temperature, DMF was removed under re-
duced pressure. To the residue were added ethyl acetate and water,
and the resulting insoluble material was removed by filtration and
washed with ethyl acetate. The filtrate was extracted with ethyl
To a solution of veratrol (1.80 g, 13.0 mmol) in THF (10.0 mL)
was added n-butyllithium (1.65 M hexane solution, 8.70 mL,
14.4 mmol) at À10 °C, and the mixture was stirred at the room
temperature for 2 h. After the reaction mixture was cooled to
À45 °C, to this was added a solution of I2 (3.63 g, 14.3 mmol) in
THF (10.0 mL), and the mixture was stirred at the room tempera-
ture for 1.5 h. The reaction was quenched by the addition of satu-
rated aqueous NH4Cl solution and then extracted with ethyl
acetate. The combined organic extracts were washed with 10%
aqueous Na2S2O3 solution, brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by silica
gel chromatography (n-hexane to n-hexane/ethyl acetate = 29:1)
to give 34a (2.44 g, 9.25 mmol, 71%) as a white solid. Mp 35.0–
35.5 °C; 1H NMR (CDCl3, 500 MHz) d 7.33 (dd, J = 7.9, 1.2 Hz, 1H),

2682
H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
acetate, and the combined organic layer was washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by silica gel chromatography (n-hexane/ethyl
acetate = 50:1) to give 34b [563 mg, 2.27 mmol, 76% (2 steps)] as a
white solid. Mp 35.5–37.0 °C; 1H NMR (CDCl3, 500 MHz) d 7.11 (dd,
J = 1.2, 8.6 Hz, 1H), 6.75 (dd, J = 1.2, 7.9 Hz, 1H), 6.78 (dd, J = 7.9,
8.6 Hz, 1H), 5.99 (s, 2H); MS (FAB) m/z 248 (M)+.
4.2.7.5. N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)-3-methoxyphenyl]-2-iodobenzamide (30e). Prepared
from 29e in accordance with the general procedure B. White solid
(quant.); 1H NMR (500 MHz, CDCl3) d 7.72 (d, 2H, J = 7.9 Hz), 7.41
(d, 1H, J = 9.1 Hz), 7.21–6.97 (m, 2H), 6.95–6.83 (m, 3H), 6.80 (s,
1H), 4.85 (br s, 1H), 4.12–3.85 (m, 2H), 3.77 (s, 3H), 1.72–1.60
(m, 2H), 1.52–1.37 (m, 2H), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB) m/z
448 (M+H)+.
4.2.5. 4-(Butylamino)benzo[d][1,3]dioxole (28i)
Prepared from 34b in accordance with the synthesis of 28h. Col-
orless oil (81%); 1H NMR (CDCl3, 500 MHz) d 6.73 (t, J = 7.9 Hz, 1H),
6.36–6.53 (m, 2H), 5.92 (s, 2H), 3.19 (t, J = 6.7 Hz, 2H), 1.64 (tt,
J = 6.7, 8.0 Hz, 2H), 1.40 (qt, J = 7.3, 8.0 Hz, 2H), 0.92 (t, J = 7.3 Hz,
3H); MS (FAB) m/z 193 (M+H)+.
4.2.7.6. N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)-2-methoxyphenyl]-2-iodobenzamide (30f).
Prepared
from 29f in accordance with the general procedure B. White solid
(82%); 1H NMR (500 MHz, CDCl3) d 7.63 (d, 2H, 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), 4.71 (br s, 1H), 4.22–4.07 (m, 1H),
3.85 (s, 1H), 3.48–3.34 (m, 1H), 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)+.
4.2.6. 4-(Butylamino)-6-methoxybenzo[d][1,3]dioxole (28j)
Prepared
from
3-bromo-5-methoxybenzo[d][1,3]dioxole
(34c)23 in accordance with the synthesis of 28h. Pale yellow oil
(81%); 1H NMR (DMSO-d6, 500 MHz) d 5.89 (d, J = 2.5 Hz, 1H),
5.81 (s, 2H), 5.77(d, J = 2.5 Hz, 1H), 5.14 (t, J = 6.1 Hz, 1H), 3.61 (s,
3H), 3.05 (dt, J = 6.1, 6.7 Hz, 2H), 1.47 (tt, J = 6.7, 7.3 Hz, 2H), 1.32
(qt, J = 7.3, 7.3 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 223
(M)+.
4.2.7.7. N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)-2,5-dimethoxyphenyl]-2-iodobenzamide (30k).
Pre-
pared from 29h in accordance with the general procedure B. White
solid (78%); 1H NMR (500 MHz, CDCl3) d 7.67 (d, 1H, J = 7.9 Hz),
7.34 (br s, 1H), 7.04 (dd, 1H, J = 7.3, 6.7 Hz), 7.01 (s, 1H), 6.93 (d,
1H, J = 7.3 Hz), 6.91 (s, 1H), 6.87 (dd, 1H, J = 7.9, 6.7 Hz), 4.30–
4.20 (m, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 3.44–3.33 (m, 1H), 1.75–
1.50 (m, 2H), 1.50–1.40 (m, 2H), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB)
m/z 606 (M+H)+.
4.2.7. General procedure B: synthesis of benzamide analogues
(I)
4.2.7.1.
hydroxypropan-2-yl)phenyl]-2-iodobenzamide (30a).
solution of 29a (100 mg, 300 mol) in CH2Cl2 (5.0 mL) were added
dropwise 2-iodobenzoyl chloride (88.0 mg, 330 mol) and Et3N
(82.5 l, 750 mol) at 0 °C, then the mixture was stirred for 3 h,
N-Butyl-N-[3-fluoro-4-(1,1,1,3,3,3-hexafluoro-2-
To a
l
4.2.7.8. N-Butyl-N-[7-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
l
2-yl)benzo[d][1,3]dioxol-4-yl]-2-iodobenzamide (30r).
Pre-
l
l
pared from 29j in accordance with the general procedure B. White
solid (81%); mp 113.0–115.0 °C; 1H NMR (DMSO-d6, 500 MHz,
50 °C) d 8.52 (s, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.20 (dd, J = 7.3,
7.9 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 6.96 (dd, J = 7.3, 8.6 Hz, 1H),
6.86 (d, J = 8.6 Hz, 1H), 5.98 (s, 2H), 3.79 (t, J = 6.7 Hz, 2H), 1.54
(tt, J = 6.7, 8.0 Hz, 2H), 1.39 (dd, J = 7.3, 8.0 Hz, 2H), 6.86 (t,
J = 7.3 Hz, 3H); MS (FAB) m/z 590 (M+H)+.
and was allowed to warm to room temperature. After cooling to
0 °C, the mixture was diluted with ethyl acetate and then water
was added. The organic layer was separated and washed with
brine, and dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
(n-hexane/AcOEt = 1:0–1:1) to give 30a (144 mg, 256 lmol, 85%)
as a colorless oil. 1H NMR (500 MHz, CDCl3) d 7.90–6.80 (m, 7H),
4.05–3.80 (m, 2H), 3.50 (br s, 1H), 1.76–1.57 (m, 2H), 1.57–1.35
(m, 2H), 1.02–0.83 (m, 3H); MS (FAB) m/z 436 (M+H)+.
4.2.8. General procedure C: synthesis of benzamide analogues
(II)
4.2.8.1. N-Butyl-2-bromo-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro-
4.2.7.2.
N-Butyl-N-[2-fluoro-4-(1,1,1,3,3,3-hexafluoro-2-
Pre-
xypropan-2-yl)phenyl]-6-methoxybenzamide (30g).
solution of 2-bromo-6-methoxybenzoic acid (104 mg, 397
To
lmol)
a
hydroxypropan-2-yl)phenyl]-2-iodobenzamide (30b).
pared from 29b in accordance with the general procedure B. White
solid (87%); 1H NMR (500 MHz, CDCl3) d 7.54 (d, 2H, J = 7.3 Hz),
7.52–7.25 (m, 3H), 7.02–6.93 (m, 2H), 6.89–6.81 (m, 1H), 4.20–
4.00 (m, 1H), 3.73–3.50 (m, 1H), 3.50–3.37 (m, 1H), 1.72–1.53
(m, 2H), 1.53–1.37 (m, 2H), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB) m/z
436 (M+H)+.
in CH2Cl2 (3.0 mL) were added chloromethlenedimethylimini-
um chloride (50.8 mg, 397 mol) at 0 °C under Ar atmosphere,
then the mixture was stirred at room temperature for 1 h.
l
At 0 °C, 2-(4-butylaminophenyl)-1,1,1,3,3,3-hexafluoropropan-2-
ol (29g)24 (50.0 mg, 159
lmol) and Et3N (166 ll, 1.19 mmol) then
the mixture was stirred for 3 h, and was allowed to warm to room
temperature. At 0 °C, the mixture was diluted with ethyl acetate
and then water was added. The organic layer was separated and
washed with brine, and dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chromatog-
4.2.7.3. N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)-3-methylphenyl]-2-iodobenzamide
(30c).
Prepared
from 29c in accordance with the general procedure B. White solid
(81%); 1H NMR (500 MHz, CDCl3) d 7.65 (d, 2H, J = 7.9 Hz), 7.10–
6.95 (m, 5H), 6.80–6.77 (m, 1H), 4.85 (br s, 1H), 3.93–3.82 (m,
2H), 2.23 (s, 3H), 1.70–1.55 (m, 2H), 1.49–1.30 (m, 2H), 0.93 (t,
3H, J = 7.3 Hz); MS (FAB) m/z 432 (M+H)+.
raphy (n-hexane/AcOEt = 1:0–1:1) to give 30g (73.5 mg, 139 lmol,
88%) as a colorless oil. 1H NMR (500 MHz, CDCl3) d 7.50 (d, 1H,
J = 8.5 Hz), 7.30 (d, 1H, 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), 4.75–4.67 (m, 1H),
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)+.
4.2.7.4. N-Butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)-2-methylphenyl]-2-iodobenzamide
(30d).
Prepared
from 29d in accordance with the general procedure B. White solid
(85%); 1H NMR (500 MHz, CDCl3) d 7.69 (d, 1H, J = 7.9 Hz), 7.48 (s,
1H), 7.28 (s, 2H), 6.96 (dd, 1H, J = 7.9, 7.3 Hz), 6.82–6.76 (m, 2H),
4.47–4.38 (m, 2H), 3.18–3.10 (m, 1H), 2.38 (s, 3H), 1.83–1.76 (m,
1H),1.67–1.52 (m, 1H), 1.52–1.30 (m, 2H), 0.95 (t, 3H, J = 7.3 Hz);
MS (FAB) m/z 432 (M+H)+.
4.2.8.2. N-Butyl-2-bromo-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro-
xypropan-2-yl)phenyl]-5-methoxybenzamide (30h).
Pre-
pared from 29g and 2-bromo-5-methoxybenzoic acid in accor-
dance with the general procedure C. White solid (60%); 1H NMR
(500 MHz, CDCl3) d 7.27 (d, 1H, J = 9.1 Hz), 7.21 (d, 1H, J = 8.5 Hz),
7.13 (s, 1H), 7.07 (d, 1H, J = 8.5 Hz), 6.56 (d, 1H, J = 3.1 Hz), 6.53

H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
2683
(dd, 1H, J = 9.1, 3.1 Hz), 4.25 (br s, 1H), 4.25–4.12 (m, 1H), 3.85 (s,
3H), 3.56 (s, 3H), 3.50–3.35 (m, 1H), 1.72–1.50 (m, 2H), 1.50–1.35
(m, 2H), 0.94 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 558 (M+H)+.
123.0–125.5 °C; 1H NMR (DMSO-d6, 500 MHz, 1:0.3 mixture of
amide conformers) d 8.74 (s, 0.3H), 8.66 (s, 1H), 7.78 (d, J = 9.2 Hz,
0.3H), 7.55 (d, J = 9.2 Hz, 1H), 7.22 (d, J = 9.2 Hz, 0.3H), 7.07 (d,
J = 9.2 Hz, 0.3H), 6.97 (d, J = 9.2 Hz, 1H), 6.95 (d, J = 3.1 Hz, 0.3H),
6.85 (d, J = 9.2 Hz, 1H), 6.84 (dd, J = 3.1, 9.2 Hz, 0.3H), 6.63 (d,
J = 3.1 Hz, 1H), 6.59 (dd, J = 3.1, 9.2 Hz, 1H), 6.13 (br s, 0.6H), 5.99
(br s, 2H), 3.72–3.82 (m, [2+0.6]H), 3.57 (s, [3+0.9]H), 1.52 (tt,
J = 7.3, 8.0 Hz, [2+0.6]H), 1.38 (qd, J = 7.3, 8.0 Hz, [2+0.6]H), 0.90 (t,
J = 7.3 Hz, [3+0.9]H); MS (FAB) m/z 620 (M+H)+.
4.2.8.3.
hydroxypropan-2-yl)phenyl]-4-methoxybenzamide
(30i). Prepared from 29g and 2-bromo-4-methoxybenzoic
N-Butyl-2-bromo-N-[4-(1,1,1,3,3,3-hexafluoro-2-
acid in accordance with the 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), 1.70–1.50 (m, 2H), 1.50–1.33 (m, 2H), 0.99–0.78 (m,
3H); MS (FAB) m/z 528 (M+H)+.
4.2.8.10.
pan-2-yl)benzo[d][1,3]dioxol-4-yl]-6-iodobenzo[d][1,3]dioxole-
5-carboxamide (30t). Prepared from 29j and 6-
N-Butyl-N-[7-(1,1,1,3,3,3-hexafluoro-2-hydroxypro-
4.2.8.4.
hydroxypropan-2-yl)phenyl]-3-methoxybenzamide
(30j).
N-Butyl-2-bromo-N-[4-(1,1,1,3,3,3-hexafluoro-2-
iodobenzo[d][1,3]dioxole-5-carboxylic acid27 in accordance with
the general procedure C. Pale yellow foam (47%); 1H NMR
(DMSO-d6, 500 MHz, 1:0.3 mixture of amide conformers) d 8.75
(s, 0.3H), 8.68 (s, 1H), 7.45 (s, 0.3H), 7.24 (s, 1H), 7.21 (d,
J = 9.2 Hz, 0.3H), 7.05 (d, J = 9.2 Hz, 0.3H), 7.01 (s, 0.3H), 6.99 (d,
J = 9.2 Hz, 1H), 6.85 (d, J = 9.2 Hz, 1H), 6.69 (s, 1H), 6.12 (br s,
1.2H), 6.01 (s, 2H), 5.93 (s, 2H), 3.65–3.85 (br, 2H), 3.35–3.55 (br,
0.6H), 1.50 (tt, J = 7.3, 7.3 Hz, [2+0.6]H), 1.35 (qd, J = 7.3, 7.3 Hz,
[2+0.6]H), 0.87 (t, J = 7.3 Hz, [3+0.9]H); MS (FAB) m/z 634 (M+H)+.
Prepared from 29g and 2-bromo-3-methoxybenzoic acid
in accordance with the general procedure C. White solid (84%); 1H
NMR (500 MHz, CDCl3) d 7.51 (d, 1H, J = 8.5 Hz), 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)+.
4.2.8.5.
hydroxypropan-2-yl)-2,5-dimethoxyphenyl]-5-methoxybenz-
amide (30m). Prepared from 29h and 2-bromo-5-methoxy-
2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-
4.2.9. General procedure D: synthesis of
benzyloxyhexafluoropropane analogues
benzoic acid in accordance with the general procedure C. White
solid (71%); 1H NMR (500 MHz, CDCl3) d 7.38 (br s, 1H), 7.26 (d, 1H,
J = 9.2 Hz), 6.97 (s, 1H), 6.92 (s, 1H), 6.58 (dd, 1H, J = 9.2, 3.1 Hz),
6.55 (d, 1H, J = 3.1 Hz), 4.28–4.18 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H),
3.59 (s, 3H), 3.48–3.35 (m, 1H), 1.72–1.50 (m, 2H), 1.50–1.37 (m,
2H), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 588 (M+H)+.
4.2.9.1. N-[4-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-
N-butyl-3-methoxyphenyl]-2-iodobenzamide (31a).
solution of 30e (41.0 mg, 71.3 mol) in DMF (3.0 mL) was added
55% NaH (3.73 mg, 85.5 mol) at 0 °C under Ar atmosphere, then
the mixture was stirred for 0.5 h at room temperature. At 0 °C, ben-
zyl bromide (12.7 l, 107 mol) was added, then the mixture was
To a
l
l
l
l
stirred for 3 h, and allow to warm to room temperature. At 0 °C, the
mixture was diluted with ethyl acetate and then water was added.
The organic layer was separated and washed with brine, and dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by column chromatography (n-hexane/ethyl ace-
4.2.8.6.
hydroxypropan-2-yl)-3-methoxyphenyl]-5-methoxybenzamide
(30o). Prepared from 29e and 2-bromo-5-methoxybenzoic
acid in accordance with the general procedure C. White solid
(quant.); 1H NMR (500 MHz, CDCl3) d 7.42 (d, 1H, J = 8.5 Hz), 7.30
(d, 2H, J = 9.2 Hz), 7.13 (br s, 1H), 6.92 (d, 1H, J = 8.5 Hz), 6.80 (s,
1H), 6.63 (d, 1H, J = 9.2 Hz), 6.50 (s, 1H), 4.10–3.85 (m, 1H), 3.77
(s, 3H), 3.61 (s, 3H), 1.70–1.59 (m, 2H), 1.50–1.38 (m, 2H), 0.96
(t, 3H, J = 7.3 Hz); MS (FAB) m/z 478 (M+H)+.
2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-
tate = 1:0–2:1) to give 31a (48.5 mg, 71.3 lmol, quant.) as a white
solid. 1H NMR (500 MHz, CDCl3) d 7.75–7.70 (m, 1H), 7.40–7.28 (m,
6H), 7.15–7.05 (m, 1H), 6.95–6.80 (m, 3H), 6.76 (s, 1H), 4.70 (s, 2H),
4.10–3.75 (m, 2H), 3.61 (s, 3H), 1.77–1.70 (m, 2H), 1.55–1.38 (m,
2H), 1.03–0.90 (m, 3H); MS (FAB) m/z 666 (M+H)+.
4.2.8.7. 2-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-hydro-
xypropan-2-yl)-2-methoxyphenyl]-5-methoxybenzamide
4.2.9.2. N-[4-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-
2,5-dimethoxyphenyl]-N-butyl-2-iodobenzamide
(30p).
Prepared from 29f and 2-bromo-5-methoxybenzoic
(31b).
Prepared from 30k in accordance with the general pro-
acid in accordance with the general procedure C. White solid
(68%); 1H NMR (500 MHz, CDCl3) d 7.27 (d, 1H, J = 9.1 Hz), 7.21
(d, 1H, J = 8.5 Hz), 7.13 (s, 1H), 7.07 (d, 1H, J = 8.5 Hz), 6.56 (d,
1H, J = 3.1 Hz), 6.53 (dd, 1H, J = 9.1, 3.1 Hz), 4.25 (br s, 1H), 4.25–
4.12 (m, 1H), 3.85 (s, 3H), 3.56 (s, 3H), 3.50–3.35 (m, 1H), 1.72–
1.50 (m, 2H), 1.50–1.35 (m, 2H), 0.94 (t, 3H, J = 7.3 Hz); MS (FAB)
m/z 558 (M+H)+.
cedure D. White solid (92%); 1H NMR (500 MHz, CDCl3) d 7.67 (d,
1H, J = 7.9 Hz), 7.45–7.25 (m, 5H), 7.01 (dd, 1H, J = 7.3, 6.7 Hz),
6.96 (s, 1H), 6.92 (d, 1H, J = 7.3 Hz), 6.90–6.80 (m, 2H), 4.56 (d,
1H, J = 12.1 Hz), 4.39 (d, 1H, J = 12.1 Hz), 4.25–4.15 (m, 1H), 3.65
(s, 3H), 3.45–3.32 (m, 1H), 3.41 (s, 3H), 1.75–1.50 (m, 2H), 1.50–
1.37 (m, 2H), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB) m/z 696 (M+H)+.
4.2.9.3. N-[4-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-
2,5-dimethoxyphenyl]-2-bromo-N-butyl-5-methoxybenzamide
4.2.8.8.
6-Bromo-N-butyl-N-[4-(1,1,1,3,3,3-hexafluoro-2-
hydroxypropan-2-yl)phenyl]benzo[d][1,3]dioxole-5-carboxam-
(31d).
Prepared from 30m in accordance with the general
ide (30q).
Prepared from 29g and 6-bromobenzo[d][1,3]diox-
procedure D. White solid (94%); 1H NMR (500 MHz, CDCl3) d
7.45–7.28 (m, 5H), 7.24 (d, 1H, J = 8.5 Hz), 6.93 (s, 1H), 6.84 (s,
1H), 6.57 (dd, 1H, J = 8.5, 3.0 Hz), 6.54 (d, 1H, J = 3.0 Hz), 4.58 (d,
1H, J = 11.6 Hz), 4.36 (d, 1H, J = 11.6 Hz), 4.25–4.18 (m, 1H), 3.35
(s, 3H), 3.55 (s, 3H), 3.48–3.35 (m, 1H), 3.41 (s, 3H), 1.75–1.50
(m, 2H), 1.50–1.40 (m, 2H), 0.96 (t, 3H, J = 7.3 Hz); MS (FAB) m/z
598 (M+H)+.
ole-5-carboxylic acid25 in accordance with the general procedure
C. White solid (80%); 1H NMR (500 MHz, CDCl3) d 7.70–7.50 (m,
2H), 7.25–7.13 (m, 2H), 6.83–6.77 (m, 1H), 6.50–6.45 (m, 1H), 5.85
(s, 1H), 4.50–4.30 (m, 1H), 4.00–3.80 (m, 2H), 1.70–1.50 (m, 2H),
1.50–1.25 (m, 2H), 1.05–0.80 (m, 3H); MS (FAB) m/z 542 (M+H)+.
4.2.8.9. N-Butyl-N-[7-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)benzo[d][1,3]dioxol-4-yl]-2-iodo-5-methoxbenzamide
(30s).
in accordance with the general procedure C. White solid (99%); mp
4.2.9.4. N-[7-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-6-
methoxybenzo[d][1,3]dioxol-4-yl]-N-butyl-2-iodobenzamide
Prepared from 29j and 2-iodo-5-methoxybenzoic acid26
(31f).
Prepared from 29k in accordance with the general pro-

2684
H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
cedure B and subsequent the general procedure D. Yellow oil [90%
(2 steps)]; 1H NMR (DMSO-d6, 500 MHz, 1:0.15 mixture of amide
conformers) d 7.74 (d, J = 8.0 Hz, 1H), 7.26–7.46 (m, [5+1.2]H),
7.18–7.26 (m, [1+0.15]H), 7.15 (dd, J = 1.2, 7.3 Hz, 1H), 7.00 (ddd,
J = 1.8, 7.3, 8.0 Hz, 1H), 6.73 (s, 0.15H), 6.44 (s, 1H), 6.07 (s, 0.3H),
5.90 (s, 2H), 4.65 (s, 0.3H), 4.37 (s, 2H), 3.75–3.95 (br, 2H), 3.76
(s, 0.3H), 3.55 (s, [3+0.45]H), 1.59 (tt, J = 7.3, 8.0 Hz, [2+0.3]H),
1.40 (qd, J = 7.3, 8.0 Hz, [2+0.3]H), 0.92 (t, J = 7.3 Hz, [3+0.45]H);
MS (FAB) m/z 709 (M)+, 710 (M+H)+.
MS (FAB) m/z 598 (M+H)+; HRMS (FAB) calcd for C30H30F6NO5
598.2028; found: 598.2045 (M+H)+.
4.2.10.4. 2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5-
butyl-3,4,8-trimethoxyphenanthridin-6(5H)-one
(33e).
Prepared from 29i and 2-iodo-5-methoxybenzoic acid
in accordance with the synthesis of 30s and subsequent the general
procedures D and E. Pale yellow oil (64%, 3 steps). 1H NMR (DMSO-
d6, 500 MHz) d 7.97 (s, 1H), 7.71 (d, J = 2.5 Hz, 1H), 7.41–7.51 (m,
5H), 7.32 (d, J = 9.2 Hz, 1H), 7.19 (dd, J = 2.5, 9.2 Hz, 1H), 4.75 (s,
2H), 4.48 (t, J = 8.0 Hz, 2H), 3.92 (s, 3H), 3.88 (s, 3H), 3.77 (s, 3H),
1.68 (tt, J = 7.3, 8.0 Hz, 2H), 1.32 (qt, J = 7.3, 7.3 Hz, 2H), 0.91 (t,
J = 7.3 Hz, 3H); MS (FAB) m/z 598 (M+H)+.
4.2.9.5. N-[7-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-6-
methoxybenzo[d][1,3]dioxol-4-yl]-N-butyl-2-iodo-5-methoxy-
benzamide (31g).
Prepared from 29k and 2-iodo-5-methoxy-
benzoic acid in accordance with the synthesis of 30s and
subsequent the general procedure D. Yellow solid [92% (2 steps)];
mp 123.0–125.5 °C; 1H NMR (DMSO-d6, 500 MHz, 1:0.135 mixture
of amide conformers) d 7.90 (d, J = 8.6 Hz, 0.135H), 7.59 (d,
J = 8.6 Hz, 1H), 7.25–7.43 (m, [5+0.675]H), 7.01 (d, J = 3.1 Hz,
0.135H), 6.97 (dd, J = 3.1, 8.6 Hz, 0.135H), 6.75 (s, 0.135H), 6.68
(d, J = 3.1 Hz, 1H), 6.61 (dd, J = 3.1, 8.6 Hz, 1H), 6.47 (s, 1H), 6.07
(br s, 0.27H), 5.89 (br s, 2H), 4.64 (s, 0.27H), 4.37 (s, 1H), 3.75–
3.90 (br, 2H), 3.80 (s, 0.405H), 3.79 (s, 0.405H), 3.76 (s, 0.27H),
3.56 (s, 3H), 3.55 (s, 3H), 1.60 (tt, J = 7.3, 8.0 Hz, [2+0.27]H), 1.41
(qd, J = 7.3, 8.0 Hz, [2+0.27]H), 0.92 (t, J = 7.3 Hz, [3+0.405]H); MS
(FAB) m/z 739 (M)+.
4.2.10.5. 11-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-4-
butyl-10-methoxy-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-one
(33f).
Prepared from 31f in accordance with the general pro-
cedure E. White solid (32%); mp 165.0–167.0 °C; 1H NMR (DMSO-
d6, 500 MHz) d 8.68 (d, J = 8.6 Hz, 1H), 8.33 (dd, J = 1.2, 7.9 Hz, 1H),
7.84 (ddd, J = 1.2, 7.4, 8.6 Hz, 1H), 7.61 (dd, J = 7.4, 7.9 Hz, 1H),
7.31–7.44 (m, 5H), 6.15 (s, 1H), 6.05 (s, 1H), 4.64 (d, J = 9.8 Hz,
1H), 4.59 (d, J = 9.8 Hz, 1H), 4.42–4.52 (m, 1H), 4.29–4.40 (m,
1H), 3.55 (s, 3H), 1.62–1.79 (m, 2H), 1.36 (qt, J = 7.3, 7.3 Hz, 2H),
0.93 (t, J = 7.3 Hz, 3H); HRMS (FAB) calcd for
C29H26F6NO5
582.1715; found: 582.1704 (M+H)+.
4.2.10. General procedure E: synthesis of phenanthridine
analogues by Pd-catalyzed cyclization
4.2.10.6. 11-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-4-
butyl-7,10-dimethoxy-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-
4.2.10.1. 2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5-
one (33g).
Prepared from 31g in accordance with the general
butyl-1,4-dimethoxyphenanthridin-6(5H)-one (33b).
solution of 31b (50.0 mg, 71.9 mol) in DMA (3.0 mL) were
added PCy3ÁHBF4 (13.2 mg, 35.9 mol), Cs2CO3 (141 mg,
431 mol) and Pd(OAc)2 (4.04 mg, 18.0 mol) under Ar atmo-
To
a
procedure E. White solid (46%); mp 134.5–136.0 °C; 1H NMR
(DMSO-d6, 500 MHz) d 1H NMR (DMSO-d6, 500 MHz) d 8.63 (d,
J = 9.2 Hz, 1H), 7.77 (d, J = 3.1 Hz, 1H), 7.45 (dd, J = 3.1, 9.2 Hz,
1H), 7.31–7.43 (m, 5H), 6.14 (s, 1H), 6.03 (s, 1H), 4.63 (d,
J = 9.8 Hz, 1H), 4.57 (d, J = 9.8 Hz, 1H), 4.43–4.52 (m, 1H), 4.31–
4.41 (m, 1H), 3.90 (s, 3H), 3.54 (s, 3H), 1.70 (ttd, J = 6.7, 7.3,
7.3 Hz, 2H), 1.36 (qt, J = 7.3, 7.3 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H);
MS (FAB) m/z 611 (M)+, 612 (M+H)+.
l
l
l
l
sphere, then the mixture was stirred for 3 h at 120 °C. At 0 °C,
the mixture was diluted with ethyl acetate and then water
was added. The organic layer was separated and washed with
brine, and dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
(n-hexane/AcOEt = 1:0–2:1) to give 33b (36.7 mg, 41.0
lmol,
4.2.10.7. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
90%) as a white solid. Mp 98.0–100.0 °C; 1H NMR (500 MHz,
CDCl3) d 8.90 (d, J = 8.5 Hz, 1H), 8.50 (d, J = 7.9 Hz, 1H), 7.72
(dd, J = 8.5, 7.3 Hz, 1H), 7.59 (dd, J = 7.9, 7.3 Hz, 1H), 7.47–7.25
(m, 5H), 7.12 (s, 1H), 4.85–4.80 (m, 1H), 4.60–4.52 (m, 1H),
4.52–4.40 (m, 1H), 4.40–4.25 (m, 1H), 3.61 (s, 3H), 3.51 (s,
3H), 1.95–1.75 (m, 2H), 1.42 (sext, J = 7.3 Hz, 2H), 0.97 (t,
J = 7.3 Hz, 3H); MS (FAB) m/z 568 (M+H)+; HRMS (FAB) calcd
for C29H28F6NO4 568.1923; found: 568.1891 (M+H)+.
yl)-3-fluorophenanthridin-6(5H)-one (5).
Prepared from
30a in accordance with the general procedure E. White solid
(36%); mp 182.0–184.5 °C; 1H NMR (500 MHz, CDCl3) d 8.65 (d,
J = 9.7 Hz, 1H), 8.58 (d, J = 7.9 Hz, 1H), 7.94 (dd, J = 9.1, 8.5 Hz,
1H), 7.77 (dd, J = 9.7, 7.9 Hz, 1H), 7.63 (dd, J = 7.9, 7.9 Hz, 1H),
7.29 (d, J = 9.1 Hz, 1H), 4.66 (s, 1H), 4.37 (t, J = 7.3 Hz, 2H), 1.77
(quin, J = 7.3 Hz, 2H), 1.51 (sext, J = 7.3 Hz, 2H), 1.02 (t, J = 7.3 Hz,
3H); MS (FAB) m/z 436 (M+H)+; Anal. Calcd for C20H16F7NO2: C,
55.18; H, 3.70; N, 3.22. Found: C, 55.18; H, 3.78; N, 3.37.
4.2.10.2. 2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5-
butyl-3,4-dimethoxyphenanthridin-6(5H)-one (33c).
Pre-
4.2.10.8. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
pared from 29i in accordance with the general procedure B and
subsequent the general procedures D and E. Pale yellow oil (70%,
3 steps). 1H NMR (DMSO-d6, 500 MHz) d 8.26–8.31 (m, 1H), 8.06
(s, 1H), 7.56–7.61 (m, 2H), 7.43–7.50 (m, 5H), 7.34–7.40 (m, 1H),
4.77 (s, 2H), 4.47 (t, J = 7.3 Hz, 2H), 3.94 (s, 3H), 3.77 (s, 3H), 1.68
(tt, J = 7.3, 7.9 Hz, 2H), 1.31 (qt, J = 7.3, 7.9 Hz, 2H), 0.91 (t,
J = 7.3 Hz, 3H); MS (FAB) m/z 568 (M+H)+.
yl)-4-fluorophenanthridin-6(5H)-one (6).
Prepared from
30b in accordance with the general procedure E. White solid
(73%); mp 188.5–190.0 °C; 1H NMR (500 MHz, CDCl3) d 8.47 (d,
J = 9.8 Hz, 1H), 8.46 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 7.75 (dd,
J = 8.5, 7.9 Hz, 1H), 7.60 (dd, J = 9.8, 8.5 Hz, 1H), 7.60 (s, 1H), 4.98
(s, 1H), 4.46 (s, 2H), 1.82 (quin, J = 7.3 Hz, 2H), 1.47 (sext,
J = 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 436 (M+H)+;
Anal. Calcd for C20H16F7NO2: C, 55.18; H, 3.70; N, 3.22. Found: C,
54.80; H, 3.75; N, 3.40.
4.2.10.3. 2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5-
butyl-1,4,8-trimethoxyphenanthridin-6(5H)-one
(33d).
Prepared from 31d in accordance with the general pro-
4.2.10.9. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
cedure E. White solid (90%); 1H NMR (500 MHz, CDCl3) d 8.84 (d,
J = 9.2 Hz, 1H), 7.93 (d, J = 3.1 Hz, 1H), 7.46–7.26 (m, 6H), 7.07 (s,
1H), 4.85–4.80 (m, 1H), 4.60–4.52 (m, 1H), 4.52–4.40 (m, 1H),
4.40–4.30 (m, 1H), 3.96 (s, 3H), 3.60 (s, 3H), 3.52 (s, 3H), 1.95–
1.75 (m, 2H), 1.43 (sext, J = 7.3 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H);
yl)-3-methylphenanthridin-6(5H)-one (7).
Prepared from
30c in accordance with the general procedure E. White solid
(37%); mp 64.0–67.0 °C; 1H NMR (500 MHz, CDCl3) d 8,56 (d,
J = 7.9 Hz, 1H), 8.29 (d, J = 7.9 Hz, 1H), 7.77 (dd, J = 7.9, 7.9 Hz,
1H), 7.64 (dd, J = 7.9, 7.9 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.19 (d,

H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
2685
J = 7.9 Hz, 1H), 4.03 (t, J = 7.3 Hz, 2H), 2.98 (s, 3H), 1.56 (s, 1H), 1.56
(quin, J = 7.3 Hz, 2H), 1.13 (sext, J = 7.3 Hz, 2H), 0.82 (t, J = 7.3 Hz,
J = 7.3 Hz, 2H), 1.48 (sext, J = 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H);
MS (FAB) m/z 448 (M+H)+; Anal. Calcd for C21H19F6NO3ÁH2O: C,
54.20; H, 4.55; N, 3.01. Found: C, 54.32; H, 4.54; N, 3.13.
3H); HRMS (FAB) calcd for
C21H20F6NO2 432.1398; found:
432.1350 (M+H)+.
4.2.10.16. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
4.2.10.10. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-4-methylphenanthridin-6(5H)-one (8). Prepared from
yl)-10-methoxyphenanthridin-6(5H)-one
(15).
Prepared
from 30j in accordance with the 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, J = 7.9 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H), 7.50 (dd,
J = 7.9, 7.9 Hz, 1H), 7.38 (d, J = 9.2 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H),
4.68 (br s, 1H), 4.30 (t, J = 7.3 Hz, 2H), 4.01 (s, 3H), 1.74 (quin,
J = 7.3 Hz, 2H), 1.49 (sext, J = 7.3 Hz, 2H), 1.00 (t, J = 7.3 Hz, 3H);
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.
30d in accordance with the general procedure E. White solid
(81%); mp 133.0–135.0 °C; 1H NMR (500 MHz, CDCl3) d 8.48 (s,
1H), 8.47 (d, J = 7.9 Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 7.73 (dd,
J = 7.9, 7.9 Hz, 1H), 7.61 (s, 1H), 7.57 (dd, J = 7.9, 7.9 Hz, 1H), 4.45
(t, J = 7.3 Hz, 2H), 4.43 (s, 1H), 2.69 (s, 3H), 1.65 (quin, J = 7.3 Hz,
2H), 1.26 (sext, J = 7.3 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H); MS (FAB)
m/z 432 (M+H)+; Anal. Calcd for C21H19F6NO2: C, 58.47; H, 4.44;
N, 3.25. Found: C, 58.37; H, 4.50; N, 3.24.
4.2.10.17. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
4.2.10.11. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-3,8-dimethoxyphenanthridin-6(5H)-one (18).
Prepared
yl)-3-methoxyphenanthridin-6(5H)-one (9).
Prepared from
from 30o in accordance with the general procedure E. White solid
(55%); mp 184.5–186.5 °C; 1H NMR (500 MHz, CDCl3) d 8.38 (s, 1H),
8.04 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 3.1 Hz, 1H), 7.33 (dd, J = 8.5,
3.1 Hz, 1H), 7.32 (s, 1H), 4.56 (t, J = 7.3 Hz, 2H), 4.34 (s, 1H), 3.97
(s, 3H), 3.95 (s, 3H), 1.85 (quin, J = 7.3 Hz, 2H), 1.45 (sext,
J = 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 478 (M+H)+;
Anal. Calcd for C22H21F6NO4: C, 55.35; H, 4.43; N, 2.93. Found: C,
55.17; H, 4.39; N, 2.93.
30e in accordance with the general procedure E. White solid
(27%); mp 101.0–104.0 °C; 1H NMR (500 MHz, CDCl3) d 8.52 (d,
J = 7.9 Hz, 1H), 8.47 (s, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.79 (dd,
J = 8.6, 6.7 Hz, 1H), 7.59 (dd, J = 7.9, 6.7 Hz, 1H), 6.98 (s, 1H), 4.38
(t, J = 7.3 Hz, 2H), 4.12 (s, 3H), 1.82 (quin, J = 7.3 Hz, 2H), 1.54 (sext,
J = 7.3 Hz, 2H), 1.05 (t, J = 7.3 Hz, 3H); HRMS (FAB) calcd for
C
21H20F6NO3 448.1347; found: 448.1350 (M+H)+.
4.2.10.12. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-4-methoxyphenanthridin-6(5H)-one (10). Prepared
4.2.10.18. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-4,8-dimethoxyphenanthridin-6(5H)-one (19).
Prepared
from 30f in accordance with the general procedure E. White solid
(73%); mp 91.0–95.0 °C; 1H NMR (500 MHz, CDCl3) d 8.50 (d,
J = 7.9 Hz, 1H), 8.26 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 7.75 (dd,
J = 7.9, 7.3 Hz, 1H), 7.59 (dd, J = 7.9, 7.3 Hz, 1H), 7.36 (s, 1H), 4.54
(t, J = 7.3 Hz, 1H), 4.03 (s, 1H), 3.97 (s, 3H), 1.84 (quin, J = 7.3 Hz,
2H), 1.44 (sext, J = 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H); MS (FAB)
m/z 448 (M+H)+; Anal. Calcd for C21H19F6NO3Á1/2H2O: C, 55.30;
H, 4.42; N, 3.07. Found: C, 55.27; H, 4.46; N, 3.09.
from 30p in accordance with the general procedure E. White solid
(82%); mp 177.0–181.0 °C; 1H NMR (500 MHz, CDCl3) d 8.18 (s, 1H),
8.14 (d, J = 9.1 Hz, 1H), 7.92 (d, J = 3.1 Hz, 1H), 7.38 (dd, J = 8.5,
3.1 Hz, 1H), 7.23 (br s, 1H), 6.98 (s, 1H), 4.39 (t, J = 7.3 Hz, 2H),
4.11 (s, 3H), 3.96 (s, 3H), 1.82 (quin, J = 7.3 Hz, 2H), 1.55 (sext,
J = 7.3 Hz, 2H), 1.05 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 478 (M+H)+;
HRMS (FAB) calcd for C22H22F6NO4 478.1453; found: 478.1412
(M+H)+.
4.2.10.13. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
4.2.10.19. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-7-methoxyphenanthridin-6(5H)-one
(12).
Prepared
yl)-[1,3]dioxolo[4,5-j]phenanthridin-6(5H)-one (22).
Pre-
from 30g in accordance with the 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, J = 9.1 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 2.4 Hz,
1H), 7.37 (d, J = 8.5 Hz, 1H), 7.10 (dd, J = 9.1, 2.4 Hz, 1H), 5.09 (br
s, 1H), 4.26 (t, J = 7.3 Hz, 2H), 3.94 (s, 3H), 1.72 (quin, J = 7.3 Hz,
2H), 1.44 (sext, J = 7.3 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H); MS (FAB)
m/z 448 (M+H)+; Anal. Calcd for C21H19F6NO3Á3/4H2O: C, 54.73;
H, 4.48; N, 3.04. Found: C, 54.71; H, 4.51; N, 2.91.
pared from 30q in accordance with the general procedure E. White
solid (78%); mp 214.5–215.5 °C; 1H NMR (500 MHz, CDCl3) d 8.43
(s, 1H), 7.84 (s, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.55 (s, 1H), 7.41 (d,
J = 9.1 Hz, 1H), 6.13 (s, 2H), 4.45 (s, 1H), 4.31 (t, J = 7.3 Hz, 2H),
1.75 (quin, J = 7.3 Hz, 2H), 1.50 (sext, J = 7.3 Hz, 2H), 1.01 (t,
J = 7.3 Hz, 3H); MS (FAB) m/z 462 (M+H)+; Anal. Calcd for
C21H17F6NO4: C, 54.67; H, 3.71; N, 3.04. Found: C, 54.45; H, 3.72;
N, 3.12.
4.2.10.14. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
4.2.10.20. 4-Butyl-11-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
yl)-8-methoxyphenanthridin-6(5H)-one
(13).
Prepared
2-yl)-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-one
from 30h in accordance with the general procedure E. White solid
(75%); mp 195.0–197.5 °C; 1H NMR (500 MHz, CDCl3) d 8.59 (s, 1H),
8.18 (d, J = 9.2 Hz, 1H), 7.94 (d, J = 3.0 Hz, 1H), 7.79 (d, J = 9.2 Hz,
1H), 7.44 (d, J = 9.2 Hz, 1H), 7.34 (dd, J = 9.2, 3.0 Hz, 1H), 4.59 (s,
1H), 4.38 (t, J = 7.3 Hz, 2H), 1.77 (quin, J = 7.3 Hz, 2H), 1.52 (sext,
J = 7.3 Hz, 2H), 1.02 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 448 (M+H)+;
Anal. Calcd for C21H19F6NO3ÁH2O: C, 54.20; H, 4.55; N, 3.01. Found:
C, 53.89; H, 4.12; N, 2.96.
(23).
Prepared from 30r in accordance with the general proce-
dure E. White solid. Mp 186.5–188.5 °C; 1H NMR (DMSO-d6,
500 MHz) d 8.98 (s, 1H), 8.32 (dd, J = 7.9, 1.2 Hz, 1H), 8.27 (s, 1H),
8.26 (d, J = 7.9 Hz, 1H), 7.85 (td, J = 7.3, 1.2 Hz, 1H), 7.62 (t,
J = 7.3 Hz, 1H), 6.19 (s, 2H), 4.43 (t, J = 7.9 Hz, 2H), 1.68 (tt, J = 7.3,
7.9 Hz, 2H), 1.37 (qt, J = 7.3, 7.3 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H);
HRMS (FAB) calcd for C21H18F6NO4 462.1140; found: 462.1169
(M+H)+.
4.2.10.15. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
4.2.10.21. 4-Butyl-11-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
yl)-9-methoxyphenanthridin-6(5H)-one
(14).
Prepared
2-yl)-7-methoxy-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-one
from 30i in accordance with the general procedure E. White solid
(84%); mp 85.5–87.0 °C; 1H NMR (500 MHz, CDCl3) d 8.51 (s, 1H),
7.83 (d, J = 9.1 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.60 (dd, J = 7.9,
7.9 Hz, 1H), 7.33 (d, J = 9.1 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 5.22
(br s, 1H), 4.20 (t, J = 7.3 Hz, 2H), 4.04 (s, 3H), 1.68 (quin,
(24).
Prepared from 30s in accordance with the general proce-
dure E. White solid (51%); mp 196.0–197.5 °C; 1H NMR (DMSO-d6,
500 MHz) d 8.94 (s, 1H), 8.20 (d, J = 9.2 Hz, 1H), 8.16 (s, 1H), 7.74 (d,
J = 3.1 Hz, 1H), 7.46 (dd, J = 9.2, 3.1 Hz, 1H), 6.16 (s, 2H), 4.44 (t,
J = 7.9 Hz, 2H), 3.89 (s, 3H), 1.68 (tt, J = 7.3, 7.9 Hz, 2H), 1.36 (qt,

2686
H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
J = 7.3, 7.9 Hz, 2H), 0.92 (t, J = 7.9 Hz, 3H); HRMS (FAB) calcd for
22H20F6NO5 492.1246; found: 492.1257 (M+H)+.
(dd, J = 9.2, 3.0 Hz, 1H), 7.13 (s, 1H), 4.55–4.45 (m, 1H), 4.40–4.30
(m, 1H), 3.97 (s, 3H), 3.92 (s, 3H), 3.76 (s, 3H), 1.92–1.80 (m, 1H),
1.45 (sext, J = 7.3 Hz, 2H), 1.00 (t, J = 7.3 Hz, 3H); MS (FAB) m/z
508 (M+H)+; HRMS (FAB) calcd for C23H24F6NO5 508.1559; found:
508.1605 (M+H)+.
C
4.2.10.22. 4-Butyl-12-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)-bis[1,3]dioxolo[4,5-c:4’,5’-j]phenanthridin-5(4H)-one
(25).
Prepared from 30t in accordance with the general proce-
dure E. White solid (95%); mp 236.0–237.0 °C; 1H NMR (DMSO-d6,
500 MHz) d 8.89 (s, 1H), 8.10 (s, 1H), 7.70 (s, 1H), 7.63 (s, 1H), 6.22
(s, 2H), 6.16 (s, 2H), 4.40 (t, J = 7.9 Hz, 2H), 1.66 (tt, J = 7.3, 7.9 Hz,
2H), 1.35 (qt, J = 7.3, 7.3 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H); HRMS
(FAB) calcd for C22H18F6NO6 506.1038; found: 506.1010 (M+H)+.
4.2.11.6. 4-Butyl-11-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-10-methoxy-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-one
(26).
Prepared from 33f in accordance with the general proce-
dure F. White solid (93%); mp 160.0–161.5 °C; 1H NMR (DMSO-d6,
500 MHz) d 8.67 (d, J = 8.6 Hz, 1H), 8.35 (s, 1H), 8.31 (d, J = 8.0 Hz,
1H), 7.81 (dd, J = 7.3, 8.6 Hz, 1H), 7.59 (dd, J = 7.3, 8.0 Hz, 1H), 6.10
(s, 2H), 4.22–4.55 (br, 2H), 3.54 (s, 3H), 1.66 (tt, J = 7.3, 7.9 Hz, 2H),
1.35 (qt, J = 7.3, 7.9 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H); HRMS (FAB)
calcd for C22H20F6NO5 492.1246; found: 492.1261 (M+H)+.
4.2.11. General procedure F: synthesis of phenanthridine
analogues via debenzylation by hydrogenation
4.2.11.1. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-3,4,8-trimethoxyphenanthridin-6(5H)-one (21).
To
a
solution of 33e (19.2 mg, 32.1 mol) in ethanol (0.5 mL) was added
l
4.2.11.7. 4-Butyl-11-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
10% Pd/C (2.2 mg), and the mixture was stirred for 3 h at room
temperature under an H2 atmosphere. The reaction mixture was
filtered through a pad of CeliteÒ, and the filtrate was concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (n-hexane/ethyl acetate = 9:1) to give 21 (95%)
yl)-7,10-dimethoxy-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-
one (27).
Prepared from 33c in accordance with the general
procedure F. White solid (82%); mp 138.5–140.0 °C; 1H NMR
(DMSO-d6, 500 MHz) d 8.62 (d, J = 9.2 Hz, 1H), 8.34 (s, 1H), 7.76
(d, J = 3.1 Hz, 1H), 7.43 (dd, J = 3.1, 9.2 Hz, 1H), 6.08 (s, 2H), 4.28–
4.53 (br, 2H), 3.89 (s, 3H), 3.53 (s, 3H), 1.66 (tt, J = 7.3, 7.3 Hz,
2H), 1.35 (qt, J = 7.3, 7.3 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H); HRMS
(FAB) calcd for C23H22F6NO6 522.1351; found: 522.1361 (M+H)+.
as
a
white solid. Mp 108.0–110.0 °C; 1H NMR (DMSO-d6,
500 MHz) d 8.72 (s, 1H), 8.40 (s, 1H), 8.21 (d, J = 9.2 Hz, 1H), 7.75
(d, J = 3.1 Hz, 1H), 7.48 (dd, J = 9.2, 3.1 Hz, 1H), 4.50 (t, J = 7.3 Hz,
2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.73 (s, 3H), 1.67 (tt, J = 7.3, 7.3 Hz,
2H), 1.28 (qt, J = 7.3, 7.3 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H); HRMS
(FAB) calcd for C23H24F6NO5 508.1559; found: 508.1549 (M+H)+.
4.2.12. N-[4-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-N-
butyl-3-hydroxyphenyl]-2-iodobenzamide (32)
To a solution of 31a (40.0 mg, 60.1
added dropwise PCy3ÁHBF4 (5.53 mg, 15.0
210 mol) and Pd(OAc)2 (1.75 mg, 7.81
lmol) in DMA (3.0 mL) were
4.2.11.2. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
l
mol), Cs2CO3 (68.6 mg,
yl)-1-methoxyphenanthridin-6(5H)-one
(11).
Prepared
l
l
mol) under Ar atmo-
from 33a in accordance with the general procedure F. White solid
(90%); mp 123.0–123.5 °C; 1H NMR (500 MHz, CDCl3) d 8.88 (d,
J = 8.5 Hz, 1H), 8.59 (d, J = 6.7 Hz, 1H), 8.27 (br s, 1H), 7.79 (dd,
J = 8.5, 6.7 Hz, 1H), 7.73 (d, J = 9.8 Hz, 1H), 7.65 (dd, J = 8.5,
6.7 Hz, 1H), 7.29 (d, J = 9.8 Hz, 1H), 4.48–4.40 (m, 1H), 4.35–
4.4.22 (m, 1H), 3.86 (s, 3H), 1.90–1.73 (m, 2H), 1.60–0.97 (m,
2H), 1.03 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 448 (M+H)+; HRMS
(FAB) calcd for C21H20F6NO3 448.1347; found: 448.1394 (M+H)+.
sphere, then the mixture was stirred for 2 h at 165 °C. At 0 °C,
the mixture was diluted with ethyl acetate and then water was
added. The organic layer was separated and washed with brine,
and dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by column chromatography (n-hexane/
AcOEt = 1:0–2:1) to give 32 (11.0 mg, 21.0 lmol, 35%) as a white
solid. 1H NMR (500 MHz, CDCl3) d 9.26 (d, J = 8.5 Hz, 1H), 8.92 (s,
1H), 8.63 (d, J = 7.9 Hz, 1H), 7.74 (dd, J = 8.5, 7.3 Hz, 1H), 7.60 (dd,
J = 7.9, 7.3 Hz, 1H), 7.53 (d, 1H, J = 9.2 Hz), 7.46 (s, 5H), 7.11 (d,
1H, J = 9.2 Hz), 4.83 (s, 2H), 4.45–4.35 (m, 2H), 1.82 (quin, 2H,
J = 7.3 Hz), 1.54 (sext, J = 7.3 Hz, 2H), 1.04 (t, J = 7.3 Hz, 3H); MS
(FAB) m/z 524 (M+H)+.
4.2.11.3. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-1,4-dimethoxyphenanthridin-6(5H)-one (16).
Prepared
from 33b in accordance with the general procedure F. White solid
(77%); mp 134.0–134.5 °C; 1H NMR (500 MHz, CDCl3) d 8.80 (d,
J = 7.9 Hz, 1H), 8.53 (d, J = 7.9 Hz, 1H), 8.39 (br s, 1H), 7.74 (dd,
J = 7.9, 6.7 Hz, 1H), 7.63 (dd, J = 7.9, 6.7 Hz, 1H), 7.18 (s, 1H),
4.55–4.45 (m, 1H), 4.40–4.30 (m, 1H), 3.92 (s, 3H), 3.78 (s, 3H),
1.92–1.80 (m, 1H), 1.44 (sext, J = 7.3 Hz, 2H), 1.00 (t, J = 7.3 Hz,
3H); MS (FAB) m/z 478 (M+H)+; HRMS (FAB) calcd for C22H22F6NO4
478.1453; Found: 478.1420 (M+H)+.
4.2.13. 2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5-
butyl-1-methoxyphenanthridin-6(5H)-one (33a)
To a solution of 32 (10.0 mg, 19.1
added dropwise methyl iodide (1.78
(3.96 mg, 28.7 mol) at 0 °C, then the mixture was stirred for 3 h
lmol) in CH2Cl2 (0.5 mL) were
l
l, 28.7 mol) and K2CO3
l
l
and was allowed to warm to room temperature. At 0 °C, the mix-
ture was diluted with ethyl acetate and then water was added.
The organic layer was separated and washed with brine, and dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by column chromatography (n-hexane/
4.2.11.4. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-3,4-dimethoxyphenanthridin-6(5H)-one (17).
Prepared
from 33c in accordance with the general procedure F. White solid
(99%); mp 106.0–107.0 °C; 1H NMR (DMSO-d6, 500 MHz) d 8.76 (s,
1H), 8.50 (s, 1H), 8.33 (d, J = 8.6 Hz, 1H), 8.28 (d, J = 7.3 Hz, 1H),
7.87 (t, J = 8.6 Hz, 1H), 7.64 (t, J = 7.3 Hz, 2H), 4.49 (t, J = 7.3 Hz,
2H), 3.91 (s, 3H), 3.73 (s, 3H), 1.67 (tt, J = 7.3, 7.3 Hz, 2H), 1.28
(qt, J = 7.3, 7.3 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H); HRMS (FAB) calcd
for C22H22F6NO4 478.1453; found: 478.1445 (M+H)+.
AcOEt = 1:0–2:1) to give 33a (13.5 mg, 19.1 lmol, quant.) as a
white solid. 1H NMR (500 MHz, CDCl3) d 8.91 (d, J = 7.9 Hz, 1H),
8.56 (d, J = 6.7 Hz, 1H), 7.78 (dd, J = 7.9, 7.3 Hz, 1H), 7.71 (d,
J = 9.2 Hz, 1H), 7.62 (dd, 1H, J = 7.3, 6.7 Hz), 7.41 (s, 5H), 7.21 (d,
1H, J = 9.2 Hz), 4.76–4.25 (m, 2H), 3.69 (s, 3H), 1.82–1.74 (m, 2H),
1.52 (sext, J = 7.3 Hz, 2H), 1.02 (t, J = 7.3 Hz, 3H); MS (FAB) m/z
538 (M+H)+.
4.2.11.5. 5-Butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-
yl)-1,4,8-trimethoxyphenanthridin-6(5H)-one
(20).
Pre-
4.2.14. 2-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)-5-(2-
methoxyethyl)phenanthridin-6(5H)-one (3)
Prepared from N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)phenyl]-2-iodobenzamide and 2-chloroethyl methyl ether in
pared from 33d in accordance with the general procedure F. White
solid (80%); mp 156.0–157.0 °C; 1H NMR (500 MHz, CDCl3) d 8.73
(d, J = 9.2 Hz, 1H), 8.42 (br s, 1H), 7.96 (d, J = 3.0 Hz, 1H), 7.31

H. Aoyama et al. / Bioorg. Med. Chem. 19 (2011) 2675–2687
2687
accordance with the literature method.28 White solid (84%); mp
174.0–178.0 °C; 1H NMR (500 MHz, CDCl3) d 8.65 (s, 1H), 8.52 (d,
J = 7.9 Hz, 1H), 8.29 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 9.2 Hz, 1H),
7.76 (dd, J = 8.5, 8.5 Hz, 1H), 7.67 (d, J = 9.2 Hz, 1H), 7.61 (dd,
J = 8.5, 7.9 Hz, 1H), 4.60 (t, J = 5.5 Hz, 2H), 3.38 (s, 3H); MS (FAB)
m/z 420 (M+H)+; HRMS (FAB) calcd for C19H16F6NO3 420.1034;
found: 420.0997 (M+H)+.
Technology Agency (JST), Japan and The Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan, and a Grant from the
Japan Society for the Promotion of Science.
References and notes
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Heterocycles 2008, 76, 137.
4.2.15. 2-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)-5-(2-
naphthylmethyl)phenanthridin-6(5H)-one (4)
Prepared from N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)phenyl]-2-iodobenzamide and 2-(bromomethyl)naphthalene
in accordance with the literature method.28 White solid (48%);
mp 128.0–129.5 °C; 1H NMR (500 MHz, CDCl3) d 8.68 (s, 1H),
8.64 (d, J = 7.9 Hz, 1H), 8.34 (d, J = 8.5 Hz, 1H), 7.85–7.77 (m, 3H),
7.73–57 (m, 4H), 7.49–7.33 (m, 4H), 5.76 (br s, 2H), 4.39 (s, 1H);
MS (FAB) m/z 502 (M+H)+; Anal. Calcd for C27H17F6NO2Á2/3H2O:
C, 63.16; H, 3.60; N, 2.73. Found: C, 63.16; H, 3.66; N, 2.79.
4.3. Bioassay
Subgenome HCV RNA replicon cells containing the luciferase
gene (LucNeo#2)29 were cultured and maintained in Dulbecco’s
Modified Eagle Medium supplemented with 10% fetal bovine ser-
um, antibiotics, and 1 mg/ml geneticin (Wako, Osaka, Japan). For
anti-HCV assays, the cells were suspended (50,000 cells/ml) in
the culture medium without geneticin and inoculated into a
mictotiter plate. After incubation for 24 h, the cells were further
incubated with fresh culture medium containing various concen-
trations of test compounds. After 3 days, the cells were washed
with phosphate-buffered saline and treated with a cell-lysis solu-
tion for 10 min by intermittent shaking. The cell lysate (25 ll)
was transferred to a white microtiter plate. One hundred microlitre
of a luciferase assay reagent (Promega, Madison, WI) was added
into each well, and its luciferase activity was measured by a lumi-
nometer. For cell viability assay, 10 ll of a tetrazolium dye solution
(TetraColor OneÒ, Seikagaku Biobusiness Corp., Tokyo, Japan) was
added into each well. After incubation for 1 h, specific absorbance
(450 nm) was measured by a microplate reader.
Acknowledgments
29. Goto, M.; Watashi, K.; Murata, T.; Hishiki, T.; Hijikata, M.; Shimotohno, K.
Biochem. Bipphys. Res. Commun. 2006, 343, 879.
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from the Science and Technol-
ogy Incubation Program in Advanced Regions, Japan Science and