Synthesis, activity, and pharmacokinetic properties of a series of conformationally-restricted thiourea analogs as novel hepatitis C virus inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.07.002

A series of novel conformationally-restricted thiourea analogs were designed, synthesized, and evaluated for their anti-HCV activity. Herein we report the synthesis, structure-activity relationships (SARs), and pharmacokinetic properties of this new class of thiourea compounds that showed potent inhibitory activities against HCV in the cell-based subgenomic HCV replicon assay. Among compounds tested, the fluorene compound 4b was found to possess the most potent activity (EC₅₀ = 0.3 μM), lower cytotoxicity (CC₅₀ > 50 μM), and significantly better pharmacokinetic properties compared to its corresponding fluorenone compound 4c.

Full text of DOI:10.1016/j.bmc.2010.07.002

Bioorganic & Medicinal Chemistry 18 (2010) 6414–6421
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, activity, and pharmacokinetic properties of a series
of conformationally-restricted thiourea analogs as novel hepatitis C virus
inhibitors
Iou-Jiun Kang, Li-Wen Wang, Teng-Kuang Yeh, Chung-Chi Lee, Yen-Chun Lee, Sheng-Ju Hsu, Yen-Shian Wu,
*
*
Jing-Chyi Wang, Yu-Sheng Chao, Andrew Yueh , Jyh-Haur Chern
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel conformationally-restricted thiourea analogs were designed, synthesized, and evaluated
for their anti-HCV activity. Herein we report the synthesis, structure–activity relationships (SARs), and
pharmacokinetic properties of this new class of thiourea compounds that showed potent inhibitory activ-
ities against HCV in the cell-based subgenomic HCV replicon assay. Among compounds tested, the fluo-
Received 21 May 2010
Revised 30 June 2010
Accepted 1 July 2010
Available online 8 July 2010
rene compound 4b was found to possess the most potent activity (EC50 = 0.3
(CC50 > 50 M), and significantly better pharmacokinetic properties compared to its corresponding flu-
orenone compound 4c.
lM), lower cytotoxicity
l
Keywords:
Hepatitis C virus
HCV inhibitors
Anti-HCV agents
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
development of the HCV replicon provides a cell-based assay sys-
tem for the evaluation of antiviral agents targeted to viral and host
proteins involved in HCV replication.9 Moreover, the use of combi-
nations of anti-HCV agents with different mechanisms of action
seems to be an important strategy to prevent viral resistance.8
Recently, the arylthiourea compound 110 (Fig. 1) has been re-
Hepatitis C is a liver disease caused by the hepatitis C virus
(HCV) that was first discovered in 1989.1 HCV, a single-stranded
positive RNA virus in the family Flaviviridae, has six genotypes
and several subtypes with varying geographic distribution.2 An
estimated more than 170 million people worldwide are chronically
infected with this virus.3 HCV infection is associated with severe li-
ver disease, including cirrhosis, liver cancer, and liver failure, a
leading indication for liver transplantation.4 Currently, no vaccine
is available to prevent HCV infection and treatments for chronic
ported to exert strong anti-HCV activity (EC50 = 0.49 lM) in a
cell-based subgenomic HCV replicon assay.11 By maintaining the
arylthiourea moiety of 1, the initial structure–activity relationships
of this class of compounds was explored. Tough the mechanism of
action of this class of compounds is not yet fully understood,12
some arylthiourea derivatives were found to possess potent activ-
ity with nanomolar range in a cell-based HCV replicon assay.
Unfortunately, these compounds showed significant cytotoxicity
and poor pharmacokinetic properties which have high clearance,
low drug exposure and poor oral bioavailabilities in rats from our
previous studies. In continuing efforts to discover new HCV
hepatitis C are limited.5 Interferon-alpha (IFN-
a), alone or in com-
bination with ribavirin, is the currently approved treatment for
chronic hepatitis C.6 Unfortunately, response rates to this treat-
ment are significantly lower in patients infected with HCV geno-
type 1, the most common type in the United States and Europe.7
In addition, this treatment is frequently associated with significant
side effects that can lead to discontinuation of therapy in approx-
imately 20% of patients.7 Thus there is an urgent need for the
development of more efficacious anti-HCV agents with fewer lim-
itations and less side effects.
X
2
R
1
R
S
N
H
N
H
According to the literature review, many emerging antiviral
agents are targeted against specific HCV enzymes, such as NS3 ser-
ine protease and NS5B RNA-dependent RNA polymerase.8 The
Y
H N
2
N
H
O
2
1
X = O, S
Y = CH2,O, NR, CO
EC50 = 0.49 µM
* Corresponding authors. Tel.: +886 37 246 166x35716; fax: +886 37 586 456
(J.-H.C.).
E-mail address: jhchen@nhri.org.tw (J.-H. Chern).
Figure 1. Arylthiourea compound 1 and its tricyclic analog 2.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.002

I.-J. Kang et al. / Bioorg. Med. Chem. 18 (2010) 6414–6421
6415
inhibitors with high potency, low cytotoxicity and suitable phar-
macokinetic properties, we elected to extend our optimization
strategy to conformationally-restricted scaffold 2, such as fluorene,
fluorenone, dibenzofuran, and carbazole (Fig. 1). In this letter, we
would like to report the synthesis, activity, and pharmacokinetic
properties of these new scaffolds as potential HCV inhibitors. De-
tails of this investigation will be described herein.
coupling reaction with commercially available isothiocyanate in
dichloromethane.
In addition, a variety of 7-aminofluorene, 7-alkylaminofluorene,
and 7-dialkylaminofluorene analogs 15–23 were prepared accord-
ing to a general synthetic method shown in Scheme 3 starting from
commercially available 2,7-diaminofluorene. N-protection with di-
tert-butyl dicarbonate in the presence of sodium carbonate pro-
duced the N-Boc derivative 13, which was reacted with TCDI and
then treated with 25% ammonia solution, followed by deprotection
with trifluoroacetic acid (TFA) to give (7-amino-9H-fluoren-2-yl)-
thiourea 15. On the other hand, N-alkylation of 13 with a variety
of alkyl iodides (n-Pr, n-Bu, and CH2Ph) in the presence of potas-
sium carbonate in acetonitrile gave a mixture of the N-alkylated
and N,N-dialkylated derivatives, which were reacted with TFA to
give the corresponding amines 14. It is worth to mention that
the N-alkylation of 13 with highly reactive alkylating agents such
as methyl iodide and ethyl iodide provided only N,N-dialkylated
derivatives under the same reaction condition in good yields. Sub-
sequent treatment of 14 with TCDI followed by reaction with 25%
ammonia solution gave the desired thiourea compounds 16–23.
2. Results and discussion
2.1. Chemistry
A series of conformationally-restricted thiourea analogs 4a–k
and urea 5a, including tricyclic and bicyclic scaffolds, have been
synthesized according to the procedure outlined in Scheme 1. In
general, the starting material of amine 3 was obtained from
commercial sources or prepared by reduction of the corresponding
nitro compounds with tin (II) chloride.10 The amine 3 was subse-
quently reacted with 1,10-thiocarbonyldiimidazole (TCDI) or 1,10-
carbonyldiimidazole (CDI) in dichloromethane at room tempera-
ture followed by reaction with 25% ammonia solution to give the
desired compounds 4a–k and 5a.
2.2. Bioactivity
As shown in Scheme 2, several 7-bromofluorene analogs 6–12
were prepared from 2-amino-7-bromofluorene in one step by
All compounds prepared above were tested for anti-HCV activ-
ity using an in vitro assay system that is suitable for monitoring
O
CDI
25% NH3 (aq)
R
N
H
NH2
5a
rt, overnight
CH2Cl2, rt, 3 h
S
25% NH3 (aq)
rt, overnight
TCDI
R
R
NH2
N
H
NH2
CH2Cl2, rt, 3 h
3
4a-k
O
N
N
a. R =
b. R =
O
N
e. R =
f. R =
i. R =
j. R =
H
N
Br
Br
N
N
H
H
N
O
O
N
N
c. R =
d. R =
k. R =
g. R =
O
O
S
N
h. R =
Scheme 1. Synthesis of compounds 4a–k and 5a.
Br
R
N C S
Br
S
R
CH2Cl2, rt, overnight
N
H
N
H
H2N
6. R = Ph
7. R = CH2Ph
10. R = 3-Pyridyl
11. R = Me
8. R = 1-Naphthyl
12. R = Et
9. R = 4-MorpholinylPh
Scheme 2. Synthesis of compounds 6–12.

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I.-J. Kang et al. / Bioorg. Med. Chem. 18 (2010) 6414–6421
S
TFA, CH Cl , rt, 1 h
NH
2
2
2
H N
2
N
S
H
1. TCDI, CH Cl , rt, 3 h
NH
2
2
2
BocHN
N
15
H
2. 25% NH (aq)
3
rt, overnight
R
(Boc) O, Na CO
3
2
2
BocHN
N
H
BocHN
NH
2
H N
NH
2
2
Dioxane/H O
2
R = n-Pr, n-Bu, CH Ph
2
rt, 6 h
13
RI, K CO
2
3
o
CH CN, 60 C, 3 h
3
R
R
BocHN
N
R = Me, Et, n-Pr, n-Bu, CH Ph
2
S
3
R
H N
2
N
N
H
4
R
3
R
25% NH (aq)
3
TFA
TCDI
CH Cl , rt, 3 h
H N
2
N
4
16. R3 = R4 = Me
17. R3 = R4 = Et
20. R3 = R4 = CH2Ph
21. R3 = H, R4 = n-Pr
rt, overnight
CH Cl , rt, 1 h
2
2
2
2
R
18. R3 = R4 = n-Pr 22. R3 = H, R4 = n-Bu
14
19. R3 = R4 = n-Bu 23. R3 = H, R4 = CH2Ph
Scheme 3. Synthesis of compounds 15–23.
anti-HCV activities of compounds. This system is composed of a
human hepatocarcinoma cell line (Huh-7) supporting multiplica-
tion of a HCV replicon.11 In our previous paper,10 it was demon-
strated that the arylthiourea compound 1 was identified as a
tuted thiourea analogs (6–12) is probably due to their increases
in lipophilic, steric, and electrostatic properties caused by the sub-
stituents. However, the underlying cause of this biological result is
not fully understood and worthy of further study.
potent inhibitor of HCV replication (EC50 = 0.49
l
M). On the basis
By keeping the free thiourea group fixed at the 2-position on the
fluorene ring, we explored a series of fluorene derivatives with
structural variations at the 7-position (Table 3). When the bromo
group at position 7 of fluorene ring of 4b was replaced with an
amino group, the compound 15 was completely inactive. This ef-
fect might be due to its drastically lipophilic change at the 7-posi-
tion of the fluorene ring. Changing the C7-amino group of 15 to a
dimethylamino group resulted in compound 16 with an EC50 of
of these encouraging results, the diphenyl ether moiety of 1 was
reasonably converted to a conformationally-restricted tricyclic
moiety 2 as shown in Figure 1. Preliminary SAR studies of this class
of compounds are summarized in Table 1. Replacement of the di-
phenyl ether moiety of 1 with a fluorene ring (4a) resulted in a dra-
matic decrease in inhibitory activity (EC50 = 0.49 lM vs 2.09 lM
for 1 and 4a, respectively). Importantly, introduction of a bromo
group at the 7-position of the fluorene ring of 4a improved activity
1.11 lM. Replacement of the dimethylamino group of 16 with a
by seven-fold (4a vs 4b, 2.09
l
M vs 0.3
l
M). This effect might be
variety of dialkylamino groups such as a diethylamino (17), dipro-
pylamino (18), dibutylamino (19), or dibenzyl amino (20) group
led to further improvement in inhibitory activity. Additionally,
we have found that the corresponding monoalkylamino analogs
(21–23, respectively) displayed comparable activity, and they were
due to the hydrophobic interaction of the bromo group at the 7-po-
sition. Replacement of the methylene group (4b, fluorene analog)
with a carbonyl (4c, fluorenone analog) slightly reduced activity
(4b vs 4c, 0.3 lM vs 0.57 lM). Unfortunately, the other fluorenone
analog 4d showed only weak activity against HCV. It is also inter-
esting to note that the dibenzofuran 4e and carbazole 4f analogs
were considerably less active than the fluorene compound 4b. On
the other hand, the heterocyclic analogs 4g–k showed a total loss
of activity. This unexpected result is not fully understood and is
worthy of further study. In addition, these compounds showed
less cytotoxic (CC50 > 50 lM) than dialkylamino analogs. These
observations provide remarkable evidence that the hydrophobic
interaction and conformational flexibility of the alkylamino groups
largely influence anti-HCV activity of these compounds.
2.3. Pharmacokinetic study
no cytotoxicity up to 50 lM. With these results, the fluorene com-
pound 4b exhibited the best potency in this series, and it was se-
lected as a potential lead for further SAR study.
On the basis of these biological results, the fluorene compound
4b and its corresponding fluorenone compound 4c were selected
for further pharmacokinetic evaluation since they appeared to
demonstrate potent inhibitory activity against HCV. To determine
the pharmacokinetic behavior in vivo, compounds 4b and 4c were
administered intravenously and orally to rats (n = 3) at 5 and
25 mg/kg, respectively. The plasma was analyzed by LC/MS/MS
for the concentration of 4b and 4c, and the calculated pharmacoki-
netic parameters are summarized in Tables 4 and 5. Compound 4c
is cleared rapidly following intravenous administration and has a
relatively shorter half-life (t1/2) when compared with 4b (Table
4). The apparent volume of distribution is large relative to the
Using compound 4b as the reference compound, we then re-
placed the thiourea moiety of 4b with urea (5a), which resulted
in a complete loss of activity (Table 2). This finding indicates that
the sulfur atom of the thiourea is critical for anti-HCV activity.
Introduction of a phenyl group (6) at the NH2 of the thiourea moi-
ety of 4b led to a drastic decrease in activity. Other large (7, 8), po-
lar (9, 10), or small (11, 12) groups were also not tolerated,
emphasizing the importance of a free NH2 group there. However,
significant cytotoxicity was observed for morpholinyl (9) and pyr-
idyl (10) analogs. The lack of anti-HCV activity of the N,N0-substi-

I.-J. Kang et al. / Bioorg. Med. Chem. 18 (2010) 6414–6421
6417
Table 1
Table 3
Anti-HCV activity and cytotoxicity for compounds 4a–k
Anti-HCV activity and cytotoxicity for compounds 15–23
R3
a
a
Compound
Structure
1b EC50
(lM)
CC50
>50
(lM)
N
S
R4
S
7
1
0.49
H2N
N
H
O
H2N
N
H
2
S
S
S
4a
4b
4c
2.09
0.3
>50
>50
>50
a
a
Compound
R3
R4
1b EC50
(lM)
CC50
(lM)
H2N
H2N
N
H
1
—
—
H
Me
Et
n-Pr
n-Bu
CH2Ph
H
—
—
H
Me
0.49
0.3
>50
>50
>50
>50
>50
>50
Br
4b
15
16
17
18
19
20
21
22
23
N
H
1.11
0.83
0.44
0.72
0.39
0.42
0.38
0.49
Et
Br
n-Pr
n-Bu
CH2Ph
n-Pr
n-Bu
CH2Ph
19.49
40.69
35.57
0.57
H2N
H2N
N
H
O
O
>50
>50
>50
S
S
H
H
4d
4e
20.97
5.51
>50
>50
N
H
1b, there are several genotypes in HCV, our assay employed genotype 1b subge-
nomic replicon.
O
a
Mean of triplicate well values. All experiments were performed at least twice.
O
H N
2
N
H
N
S
S
Table 4
4f
7.55
>50
>50
>50
H2N
H2N
N
H
Pharmacokinetic parameters of 4b and 4c following iv administrationa to ratsb
Parameter
Compound
O
O
N
4g
4b
17.0 0.9
2.8 0.2
2.6 0.2
4c
N
H
CL (mL/min/kg)
Vss (L/kg)
t1/2 (h)
127.4 10.7
2.0 0.6
S
N
S
S
4h
4i
>50
>50
>50
>50
>50
>50
H N
2
N
H
1.4 0.2
AUC (ng/mL h)
4937 255
655 51
N
a
Compound was formulated as a solution in DMA/propyl glycol (20:80, v/v) and
N
H N
2
N
H
H
N
administered at 5 mg/kg.
b
n = 3.
S
S
N
4j
N
H
H2N
N
H
H
N
Table 5
Pharmacokinetic parameters of 4b and 4c following oral administrationa to ratsb
N
4k
>50
>50
H N
2
N
H
Parameter
Compound
4b
4c
1b, there are several genotypes in HCV, our assay employed genotype 1b subge-
nomic replicon.
Cmax (ng/mL)
Tmax (h)
t1/2 (h)
AUC (ng/mL h)
Bioavailability (%)
106.5 22.5
0.7 0.3
3.8 3.1
294 73
1.2
65.3 39.5
0.25 0.0
1.7 0.4
43 16
1.3
a
Mean of triplicate well values. All experiments were performed at least twice.
Table 2
Anti-HCV activity and cytotoxicity for compounds 5a and 6–12
a
Compound was formulated as a solution in DMA/propyl glycol (20:80, v/v) and
Br
X
7
administered at 25 mg/kg.
R
b
n = 3.
HN
N
H
2
a
a
Compound
X
R
1b EC50
(l
M)
CC50 (lM)
pounds 4b and 4c. Additionally, compound 4b exhibited a much
higher AUC than 4c in rats (AUC = 4937 vs 655 ng/mL h for 4b
and 4c, respectively). These results indicated that the fluorine com-
pound 4b is metabolically more stable than the corresponding flu-
orenone compound 4c.
Following oral administration (25 mg/kg) of 4b and 4c to rats,
the maximum concentration (Cmax) was 106.5 and 65.3 ng/mL,
and the time to reach the maximum concentration (Tmax) was 0.7
1
—
S
O
S
S
S
S
S
S
S
—
H
H
Ph
CH2Ph
1-Naphthyl
4-MorpholinylPh
3-Pyridyl
Me
0.49
0.3
>50
42.84
>50
15.38
34.14
>50
>50
>50
>50
4b
5a
6
7
8
46.22
>50
>50
9
18.10
19.71
>50
10
11
12
>50
>50
and 0.25 h, respectively (Table 5). The oral plasma half-lives (t1/2
)
Et
>50
of 4b and 4c were 3.8 and 1.7 h, respectively. Although compound
4b showed approximately seven-fold higher oral exposure than
that achieved by 4c (AUC = 294 vs 43 ng/mL h for 4b and 4c,
respectively), both compounds 4b and 4c showed poor oral bio-
availability (F < 2%) and therefore are not suitable for oral use.
The underlying cause of this effect is not fully understood and wor-
thy of further study.
1b, there are several genotypes in HCV, our assay employed genotype 1b subge-
nomic replicon.
a
Mean of triplicate well values. All experiments were performed at least twice.
plasma volume of the rat, indicating extensive distribution into tis-
sues which is consistent with the lipophilic properties of both com-

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I.-J. Kang et al. / Bioorg. Med. Chem. 18 (2010) 6414–6421
7.34 (dd, J = 1.8, 8.4 Hz, 1H), 7.53 (dd, J = 2.0, 8.0 Hz, 1H), 7.65 (s,
1H), 7.75–7.85 (m, 3H), 9.78 (s, 1H). 13C NMR (300 MHz, DMSO-
3. Conclusion
d6)
d 36.44, 119.32, 119.88, 120.44, 121.48, 122.11, 128.09,
In summary, we have discovered a series of conformationally-
restricted thiourea analogs as novel HCV inhibitors. According to
our SAR investigation, introduction of a bromo group at the 7-posi-
tion of the fluorene ring can significantly enhance the anti-HCV
activity. The presence of a free thiourea group at the 2-position
of the fluorene ring was essential for their anti-HCV activity.
Replacement of the bromo group at the 7-position of the fluorene
ring with a variety of alkylamino substituents resulted in an inter-
esting pattern of activity. The pharmacokinetic properties of the
fluorene compound 4b and its corresponding fluorenone com-
pound 4c after iv and po dosing to rats were dramatically different.
Among compounds synthesized, compound 4b was found to exhi-
bit promising in vitro activity in a cell-based subgenomic HCV rep-
licon assay. In addition, this compound showed lower cytotoxicity
and better pharmacokinetic properties compared to the arylthiou-
rea derivatives from our previous studies following iv dosing,
although its poor oral bioavailability required further optimization.
Further SAR studies as well as mechanistic and pharmacokinetic
studies on this class of compounds are currently under active
investigation and will be reported in due course.
129.67, 136.42, 138.31, 140.12, 143.55, 145.55, 180.90. MS (ESI+)
m/z calcd for C14H11BrN2S: 319.98; found: 320.0 (M+H), 342.0
(M+Na).
4.1.1.3. (7-Bromo-9-oxo-9H-fluoren-2-yl)-thiourea (4c). Com-
pound 4c was prepared from corresponding amine 3 in 65% yield
as a orange solid, mp 220–221 °C. 1H NMR (300 MHz, DMSO-d6)
d 7.57 (dd, J = 1.2, 8.0 Hz, 1H), 7.68–7.80 (m, 4H), 7.86 (s, 1H),
9.98 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d 118.30, 121.60,
121.76, 122.79, 126.65, 128.63, 133.29, 135.46, 137.64, 138.31,
141.01, 142.93, 181.20, 191.42. MS (ESI+) m/z calcd for
C14H9BrN2OS: 333.96; found: 334.9 (M+H), 356.9 (M+Na).
4.1.1.4. (9-Oxo-9H-fluoren-3-yl)-thiourea (4d). Compound 4d
was prepared from corresponding amine 3 in 41% yield as a yellow
solid, mp 211–212 °C. 1H NMR (300 MHz, DMSO-d6) d 7.35–7.43
(m, 2H), 7.54–7.61 (m, 3H), 7.71 (d, J = 7.2 Hz, 1H), 7.96 (d,
J = 1.8 Hz, 1H), 10.11 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d
114.13, 120.99, 121.56, 123.56, 124.84, 128.28, 129.59, 134.07,
134.96, 143.06, 144.88, 145.91, 181.12, 191.81. MS (ESI+) m/z calcd
for C14H10N2OS: 254.05; found: 255.0 (M+H), 277.0 (M+Na).
4. Experimental
4.1. Chemistry
4.1.1.5. (2-Methoxy-dibenzofuran-3-yl)-thiourea (4e). Compound
4e was prepared from corresponding amine 3 in 35% yield as a
white solid, mp 191–192 °C. 1H NMR (300 MHz, DMSO-d6) d 3.97
(s, 3H), 7.37 (dd, J = 7.2, 8.1 Hz, 1H), 7.46 (dd, J = 7.2, 8.0 Hz, 1H),
7.64 (d, J = 8.0 Hz, 1H), 7.80 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H), 8.54 (s,
1H), 9.27 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d 56.41, 102.66,
107.14, 111.50, 119.28, 120.68, 122.84, 124.08, 126.72, 128.09,
147.89, 148.93, 155.98, 181.23. MS (ESI+) m/z calcd for C14H12N2O2S:
272.06; found: 273.1 (M+H), 295.0 (M+Na).
All commercial chemicals and solvents are reagent grade and
were used without further treatment unless otherwise noted. 1H
NMR spectra were obtained with a Varian Mercury-300 or a Varian
Mercury-400 spectrometer. Chemical shifts were recorded in parts
per million (ppm, d) and were reported relative to the solvent peak
or TMS. LC/MS data were measured on an Agilent MSD-1100 ESI-
MS/MS System. Flash column chromatography was done using sil-
ica gel (Merck Kieselgel 60, No. 9385, 230–400 mesh ASTM). Reac-
tions were monitored by TLC using Merck 60 F254 silica gel glass
backed plates; zones were detected visually under ultraviolet irra-
diation (254 nm) or by spraying with phosphomolybdic acid re-
agent (Aldrich) followed by heating at 80 °C. Melting points were
determined on an Electrothermal IA9000 Series Digital Melting
Point Apparatus.
4.1.1.6. (9-Ethyl-9H-carbazol-3-yl)-thiourea (4f). Compound 4f
was prepared from corresponding amine 3 in 83% yield as a white
solid, mp 199–200 °C. 1H NMR (300 MHz, DMSO-d6) d 1.30 (t,
J = 7.2 Hz, 3H), 4.44 (q, J = 7.2 Hz, 2H), 7.19 (dd, J = 7.2, 7.5 Hz,
1H), 7.32 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 7.5, 7.8 Hz, 1H), 7.57–
7.61 (m, 2H), 8.04 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 9.64 (s, 1H). 13C
NMR (300 MHz, DMSO-d6) d 13.74, 37.01, 109.17, 117.00, 118.69,
120.60, 122.02, 122.22, 123.64, 125.90, 130.14, 137.46, 139.97,
181.31. MS (ESI+) m/z calcd for C15H15N3S: 269.10; found: 270.2
(M+H), 292.1 (M+Na).
4.1.1. General procedure for the preparation of compounds 4a–
k
1,10-Thiocarbonyldiimidazole (TCDI, 1.2 mmol) was added to a
stirred solution of amine 3 (1.0 mmol) in CH2Cl2 (10 mL) at room
temperature. The reaction mixture was treated with excess 25%
ammonia solution about 3 h later and continually stirred overnight
at room temperature. The precipitate was filtered in vacuo and
washed with methanol and water to give the corresponding prod-
ucts 4a–k.
4.1.1.7. (6-Oxo-6H-benzo[c]chromen-2-yl)-thiourea (4g). Com-
pound 4g was prepared from corresponding amine 3 in 54% yield
as a white solid, mp 209–210 °C. 1H NMR (300 MHz, DMSO-d6) d
7.40 (d, J = 8.7 Hz, 1H), 7.50 (dd, J = 2.4, 8.7 Hz, 1H), 7.69 (dd,
J = 7.6, 8.0 Hz, 1H), 7.97 (ddd, J = 1.4, 7.6, 8.0 Hz, 1H), 8.26 (dd,
J = 1.4, 8.0 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H),
9.80 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d 117.53, 117.59,
118.32, 120.49, 122.64, 126.79, 129.48, 129.82, 134.06, 135.47,
135.84, 147.66, 160.22, 181.47. MS (ESI+) m/z calcd for
4.1.1.1. (9H-Fluoren-2-yl)-thiourea (4a). Compound 4a was pre-
pared from corresponding amine 3 in 68% yield as a white solid,
mp 213–214 °C. 1H NMR (300 MHz, DMSO-d6) d 3.89 (s, 2H),
7.24–7.38 (m, 3H), 7.55 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H), 7.82 (d,
J = 8.1 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 9.75 (s, 1H). 13C NMR
(300 MHz, DMSO-d6) d 36.49, 119.73, 120.05, 120.12, 122.07,
125.09, 126.39, 126.80, 137.62, 137.82, 140.78, 142.98, 143.65,
180.88. MS (ESI+) m/z calcd for C14H12N2S: 240.07; found: 241.0
(M+H), 263.0 (M+Na).
C14H10N2O2S: 270.05; found: 271.0 (M+H).
4.1.1.8. (2,3-Dihydro-benzo[d]imidazo[2,1-b]thiazol-6-yl)-thio-
urea (4h). Compound 4h was prepared from corresponding amine
3 in 86% yield as a yellow solid, mp 250–251 °C. MS (ESI+) m/z calcd
for C10H10N4S2: 250.03; found: 251.1 (M+H).
4.1.1.9. (1H-Indazol-6-yl)-thiourea (4i). Compound 4i was pre-
pared from corresponding amine 3 in 84% yield as a white solid,
mp 194–195 °C. 1H NMR (300 MHz, DMSO-d6) d 6.96 (dd, J = 1.5,
8.4 Hz, 1H), 7.52 (br s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H),
4.1.1.2. (7-Bromo-9H-fluoren-2-yl)-thiourea (4b). Compound 4b
was prepared from corresponding amine 3 in 93% yield as a white
solid, mp 218–219 °C. 1H NMR (300 MHz, DMSO-d6) d 3.91 (s, 2H),

I.-J. Kang et al. / Bioorg. Med. Chem. 18 (2010) 6414–6421
6419
7.99 (s, 1H), 9.80 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d 103.14,
117.30, 119.97, 120.64, 133.34, 137.13, 140.05, 180.99. MS (ESI+)
m/z calcd for C8H8N4S: 192.05; found: 193.1 (M+H), 215.1 (M+Na).
4.1.3.3. 1-(7-Bromo-9H-fluoren-2-yl)-3-naphthalen-1-yl-thio-
urea (8). Compound 8 was prepared from 1-naphthalenyl isothio-
cyanate in 45% yield as a white solid, mp 206–207 °C. 1H NMR
(300 MHz, DMSO-d6) d 3.92 (s, 2H), 7.47–7.60 (m, 6H), 7.75–7.86
(m, 5H), 7.94–8.00 (m, 2H), 9.87 (s, 1H), 9.89 (s, 1H). 13C NMR
(300 MHz, DMSO-d6) d 36.38, 119.29, 120.08, 120.86, 121.48,
123.10, 123.14, 125.37, 125.64, 126.08, 126.16, 126.72, 128.08,
129.65, 129.97, 133.91, 135.16, 136.53, 138.86, 140.15, 143.13,
145.57, 181.20. MS (ESI+) m/z calcd for C24H17BrN2S: 444.03;
found: 467.0 (M+Na).
4.1.1.10. (3H-Benzotriazol-5-yl)-thiourea (4j). Compound 4j was
prepared from corresponding amine 3 in 81% yield as a brown so-
lid, mp 203–204 °C. 1H NMR (300 MHz, DMSO-d6) d 7.29 (dd,
J = 1.5, 9.0 Hz, 1H), 7.58 (br s, 1H), 7.87 (d, J = 9.0 Hz, 1H), 8.08 (s,
1H), 9.90 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d 106.44, 116.35,
121.37, 122.33, 137.21, 181.38. MS (ESI+) m/z calcd for C7H7N5S:
193.04; found: 194.3 (M+H), 216.2 (M+Na).
4.1.3.4. 1-(7-Bromo-9H-fluoren-2-yl)-3-(4-morpholin-4-yl-phe-
nyl)-thiourea (9). Compound 9 was prepared from 4-morpholinyl-
phenyl isothiocyanate in 25% yield as a yellow solid, mp 200–
201 °C. 1H NMR (300 MHz, DMSO-d6) d 3.07 (br t, J = 4.7 Hz, 4H),
3.72 (br t, J = 4.7 Hz, 4H), 3.92 (s, 2H), 6.91 (d, J = 8.8 Hz, 2H),
7.28 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.1 Hz,
1H), 7.73 (s, 1H), 7.75 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.83 (d,
J = 8.1 Hz, 1H), 9.60 (s, 1H), 9.68 (s, 1H). 13C NMR (300 MHz,
DMSO-d6) d 36.40, 48.65, 66.10, 115.01, 119.22, 120.10, 120.34,
121.42, 122.64, 125.34, 128.06, 129.65, 131.02, 136.24, 138.95,
140.20, 143.16, 145.53, 148.43, 179.50. MS (ESI+) m/z calcd for
C24H22BrN3OS: 479.07; found: 480.0 (M+H).
4.1.1.11. (1H-Indazol-5-yl)-thiourea (4k). Compound 4k was pre-
pared from corresponding amine 3 in 85% yield as a violet solid, mp
192–193 °C. 1H NMR (300 MHz, DMSO-d6) d 7.22 (dd, J = 1.5,
8.7 Hz, 1H), 7.35 (br s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.67 (s, 1H),
8.04 (s, 1H), 9.62 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d 110.31,
115.59, 122.83, 124.51, 131.57, 133.65, 137.88, 181.35. MS (ESI+)
m/z calcd for C8H8N4S: 192.05; found: 193.3 (M+H), 215.3 (M+Na).
4.1.2. Preparation of (7-bromo-9H-fluoren-2-yl)-urea (5a)
1,10-Carbonyldiimidazole (CDI, 1.2 mmol) was added to a stir-
red solution of 2-amino-7-bromofluorene (1.0 mmol) in CH2Cl2
(10 mL) at room temperature. The reaction mixture was treated
with excess 25% ammonia solution about 3 h later and continually
stirred overnight at room temperature. The precipitate was filtered
in vacuo and washed with methanol and water to give product 5a
in 90% yield as a white solid, mp 274–275 °C. 1H NMR (300 MHz,
DMSO-d6) d 3.87 (s, 2H), 5.88 (s, 2H), 7.30 (d, J = 8.7 Hz, 1H), 7.49
(d, J = 8.4 Hz, 1H), 7.69–7.75 (m, 4H), 8.65 (s, 1H). 13C NMR
(300 MHz, DMSO-d6) d 36.41, 114.33, 116.64, 118.38, 120.41,
120.77, 127.87, 129.47, 133.23, 140.22, 140.66, 143.78, 145.07,
155.96. MS (ESI+) m/z calcd for C14H11BrN2O: 302.01; found:
303.0 (M+H).
4.1.3.5.
(10). Compound 10 was prepared from 3-pyridinyl isothiocyanate
in 90% yield as
yellow solid, mp 170–171 °C. 1H NMR
1-(7-Bromo-9H-fluoren-2-yl)-3-pyridin-3-yl-thiourea
a
(300 MHz, DMSO-d6) d 3.94 (s, 2H), 7.36 (dd, J = 5.1, 8.1 Hz, 1H),
7.45 (dd, J = 1.2, 8.3 Hz, 1H), 7.55 (dd, J = 1.4, 8.4 Hz, 1H), 7.73 (s,
1H), 7.76 (s, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H),
7.94 (br d, J = 8.4 Hz, 1H), 8.31 (d, J = 5.1 Hz, 1H), 8.61 (d,
J = 2.7 Hz, 1H), 9.87 (s, 1H), 10.13 (s, 1H). 13C NMR (300 MHz,
DMSO-d6)
d 36.43, 119.41, 120.34, 120.58, 121.54, 122.87,
123.11, 128.11, 129.69, 131.48, 136.39, 136.79, 138.33, 140.07,
143.39, 145.26, 145.51, 145.59, 180.15. MS (ESI+) m/z calcd for
4.1.3. General procedure for the preparation of compounds 6–
10 and 12
C19H14BrN3S: 395.01; found: 395.9 (M+H), 417.9 (M+Na).
Commercially available isothiocyanate (1.2 mmol) was added to
stirred solution of 2-amino-7-bromofluorene (1.0 mmol) in
CH2Cl2 (10 mL). The reaction mixture was stirred overnight at
room temperature. The precipitate was filtered in vacuo and
washed with ether/CH2Cl2 (10:1) to give the corresponding prod-
ucts 6–10 and 12.
4.1.3.6. 1-(7-Bromo-9H-fluoren-2-yl)-3-ethyl-thiourea (12). Com-
pound 12 was prepared from ethyl isothiocyanate in 90% yield as
a white solid, mp 209–210 °C. 1H NMR (300 MHz, DMSO-d6) d 1.1
(t, J = 7.2 Hz, 3H), 3.36 (br s, 1H), 3.44–3.53 (m, 2H), 3.91 (s, 2H),
7.34 (dd, J = 1.8, 8.4 Hz, 1H), 7.54 (dd, J = 2.0, 8.1 Hz, 1H), 7.64 (s,
1H), 7.75 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H),
9.53 (br s, 1H). 13C NMR (300 MHz, DMSO-d6) d 14.22, 36.41,
38.74, 119.21, 119.99, 120.35, 121.40, 122.25, 128.06, 129.65,
136.15, 138.52, 140.17, 143.44, 145.49, 179.98. MS (ESI+) m/z calcd
for C16H15BrN2S: 346.01; found: 346.9 (M+H), 368.9 (M+Na).
a
4.1.3.1. 1-(7-Bromo-9H-fluoren-2-yl)-3-phenyl-thiourea (6). Com-
pound 6 was prepared from phenyl isothiocyanate in 77% yield as a
white solid, mp 189–190 °C. 1H NMR (300 MHz, DMSO-d6) d 3.93 (s,
2H), 7.09–7.14 (m, 1H), 7.30–7.35 (m, 2H), 7.43–7.50 (m, 3H), 7.54
(dd, J = 1.2, 8.1 Hz, 1H), 7.74 (s, 1H), 7.75 (s, 1H), 7.80 (d, J = 8.4 Hz,
1H), 7.85 (d, J = 8.1 Hz, 1H), 9.81 (s, 1H), 9.90 (s, 1H). 13C NMR
4.1.4. Preparation of 1-(7-bromo-9H-fluoren-2-yl)-3-methyl-
thiourea (11)
(300 MHz, DMSO-d6)
d 36.40, 119.29, 120.18, 120.38, 121.46,
1,10-Thiocarbonyldiimidazole (TCDI, 1.2 mmol) was added to a
stirred solution of 2-amino-7-bromofluorene (1.0 mmol) in CH2Cl2
(10 mL). The reaction mixture was treated with excess 40% methyl
amine aqueous solution about 3 h later and continually stirred
overnight at room temperature. After concentrating in vacuo, the
crude product was recrystallized with methanol and CH2Cl2 (1:5)
to give pure product 11 in 28% yield as a white solid, mp 186–
187 °C. 1H NMR (300 MHz, DMSO-d6) d 2.36 (q, J = 5.6 Hz, 1H),
2.92 (d, J = 5.6 Hz, 3H), 3.91 (s, 2H), 7.34 (d, J = 8.3 Hz, 1H), 7.54
(dd, J = 1.5, 8.1 Hz, 1H), 7.63 (s, 1H), 7.75 (d, J = 1.5 Hz, 1H), 7.80
(d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 9.69 (br s, 1H). 13C
NMR (300 MHz, DMSO-d6) d 31.23, 36.41, 119.22, 119.92, 120.39,
121.42, 122.11, 128.08, 129.65, 136.20, 138.51, 140.17, 143.47,
145.50, 181.01. MS (ESI+) m/z calcd for C15H13BrN2S: 332.00;
found: 333.0 (M+H), 354.9 (M+Na).
122.68, 123.70, 124.42, 128.08, 128.43, 129.66, 136.44, 138.74,
139.49, 140.15, 143.24, 145.56, 179.51. MS (ESI+) m/z calcd for
C20H15BrN2S: 394.01; found: 394.9 (M+H), 416.9 (M+Na).
4.1.3.2. 1-Benzyl-3-(7-bromo-9H-fluoren-2-yl)-thiourea (7). Com-
pound 7 was prepared from benzyl isothiocyanate in 43% yield as
1
a white solid, mp 183–184 °C. H NMR (300 MHz, DMSO-d6) d 3.92
(s, 2H), 4.74 (d, J = 5.7 Hz, 2H), 7.22–7.29 (m, 1H), 7.33 (s, 2H),
7.35 (s, 2H), 7.38 (dd, J = 1.2, 8.4 Hz, 1H), 7.54 (dd, J = 0.9, 8.1 Hz,
1H), 7.68 (s, 1H), 7.75 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.85 (d,
J = 8.1 Hz, 1H), 8.21 (br t, 1H), 9.71 (br s, 1H). 13C NMR (300 MHz,
DMSO-d6) d 36.42, 47.24, 119.28, 120.22, 120.39, 121.45, 122.47,
126.87, 127.43, 128.08, 128.27, 129.67, 136.38, 138.41, 139.04,
140.14, 143.47, 145.52, 180.74. MS (ESI+) m/z calcd for C21H17BrN2S:
408.03; found: 409.0 (M+H), 430.9 (M+Na).

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I.-J. Kang et al. / Bioorg. Med. Chem. 18 (2010) 6414–6421
4.1.5. Preparation of (7-amino-9H-fluoren-2-yl)-carbamic acid
tert-butyl ester (13)
4.1.7.2. (7-Diethylamino-9H-fluoren-2-yl)-thiourea (17). Com-
pound 17 was prepared from compound 13 for three steps in
21% overall yield as a white solid, mp 172–173 °C. 1H NMR
(400 MHz, DMSO-d6) d 1.10 (t, J = 7.0 Hz, 6H), 3.34 (q, J = 7.0 Hz,
4H), 3.77 (s, 2H), 6.66 (dd, J = 2.2, 8.4 Hz, 1H), 6.85 (s, 1H), 7.18
(d, J = 8.0 Hz, 1H), 7.44 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.57 (d,
J = 8.4 Hz, 1H), 9.66 (s, 1H). 13C NMR (400 MHz, DMSO-d6) d
12.56, 36.63, 43.97, 108.21, 110.66, 118.06, 120.30, 120.50,
122.26, 128.63, 135.51, 139.00, 142.35, 144.98, 147.00, 180.81.
MS (ESI+) m/z calcd for C18H21N3S: 311.15; found: 312.1 (M+H),
334.1 (M+Na).
2,7-Diaminofluorene (5.0 mmol) and sodium carbonate
(10.0 mmol) were suspended in a solution of 1,4-dioxane (20 mL)
and H2O (10 mL). Di-tert-butyl dicarbonate (6.0 mmol) was added
dropwise to the suspension solution at ice-bath. The reaction mix-
ture was warmed to room temperature and stirred for 6 h. The
mixture was diluted with CH2Cl2 and washed sequentially with
saturated ammonium chloride. The organic layer was dried over
MgSO4, concentrated in vacuo and purified by column chromatog-
raphy using EtOAc/hexane (1:3) as an eluant to give desired prod-
uct 13 in 41% yield as a off-white solid, mp 172–173 °C. 1H NMR
(300 MHz, DMSO-d6) d 1.47 (s, 9H), 3.67 (s, 2H), 5.09 (s, 2H), 6.53
(dd, J = 1.8, 8.1 Hz, 1H), 6.72 (d, J = 1.8 Hz, 1H), 7.29 (dd, J = 1.8,
8.4 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.60
(s, 1H), 9.25 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d 28.21,
36.31, 78.79, 110.55, 112.72, 115.03, 116.87, 117.85, 119.79,
129.77, 136.44, 136.65, 142.23, 144.18, 147.65, 152.91. MS (ESI+)
m/z calcd for C18H20N2O2: 296.15; found: 297.1 (M+H).
4.1.7.3. (7-Dipropylamino-9H-fluoren-2-yl)-thiourea (18). Com-
pound 18 was prepared from compound 13 for three steps in
51% overall yield as a yellow solid, mp 166–167 °C. 1H NMR
(400 MHz, DMSO-d6) d 0.89 (t, J = 7.4 Hz, 6H), 1.52–1.57 (m, 4H),
3.24–3.31 (m, 4H), 3.77 (s, 2H), 6.63 (dd, J = 2.2, 8.8 Hz, 1H), 6.82
(s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.43 (s, 1H), 7.55 (d, J = 8.0 Hz,
1H), 7.56 (d, J = 8.8 Hz, 1H), 9.66 (s, 1H). 13C NMR (400 MHz,
DMSO-d6) d 11.31, 20.12, 36.67, 52.32, 108.07, 110.56, 118.08,
120.32, 120.47, 122.28, 128.51, 135.48, 139.03, 142.39, 144.96,
147.42, 180.78. MS (ESI+) m/z calcd for C20H25N3S: 339.18; found:
340.2 (M+H), 362.2 (M+Na).
4.1.6. Preparation of (7-amino-9H-fluoren-2-yl)-thiourea (15)
1,10-Thiocarbonyldiimidazole (TCDI, 1.2 mmol) was added to a
stirred solution of compound 13 (1.0 mmol) in CH2Cl2 (10 mL).
The reaction mixture was treated with excess 25% ammonia solu-
tion about 3 h later and continually stirred overnight at room tem-
perature. After filtering and washing with methanol, the
precipitate was dissolved in CH2Cl2 (2 mL) and introduced by TFA
(2 mL) at ice-bath. The reaction mixture was warmed to room tem-
perature and stirred for 1 h. The mixture was diluted with CH2Cl2
and neutralized by saturated sodium bicarbonate. The organic
layer was washed by brine, dried over MgSO4, and concentrated
in vacuo. The precipitate was washed by methanol to give the pure
product 15 in 54% yield as a off-white solid, mp 250–251 °C. 1H
NMR (400 MHz, DMSO-d6) d 3.71 (s, 2H), 5.17 (s, 2H), 6.56 (dd,
J = 2.0, 8.2 Hz, 1H), 6.73 (s, 1H), 7.18 (br d, J = 8.0 Hz, 1H), 7.43
(br s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.2 Hz, 1H), 9.74 (br
s, 1H). 13C NMR (400 MHz, DMSO-d6) d 36.27, 110.43, 112.83,
117.87, 120.29, 120.34, 122.24, 129.30, 135.48, 139.18, 142.17,
144.65, 148.14, 180.83. MS (ESI+) m/z calcd for C14H13N3S:
255.08; found: 256.1 (M+H).
4.1.7.4. (7-Dibutylamino-9H-fluoren-2-yl)-thiourea (19). Com-
pound 19 was prepared from compound 13 for three steps in
15% overall yield as a white solid. 1H NMR (300 MHz, DMSO-d6)
d 0.91 (t, J = 7.4 Hz, 6H), 1.28–1.36 (m, 4H), 1.46–1.54 (m, 4H),
3.29 (t, J = 7.5 Hz, 4H), 3.77 (s, 2H), 6.63 (dd, J = 2.4, 8.7 Hz, 1H),
6.82 (s, 1H), 7.17 (dd, J = 1.5, 8.1 Hz, 1H), 7.44 (s, 1H), 7.55 (d,
J = 8.1 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 9.63 (s, 1H). 13C NMR
(300 MHz, DMSO-d6) d 13.91, 19.75, 29.11, 36.64, 50.25, 108.13,
110.63, 118.04, 120.30, 120.44, 122.26, 128.51, 135.44, 139.01,
142.36, 144.92, 147.41, 180.78. MS (ESI+) m/z calcd for
C22H29N3S: 367.21; found: 368.1 (M+H), 390.1 (M+Na).
4.1.7.5. (7-Dibenzylamino-9H-fluoren-2-yl)-thiourea (20). Com-
pound 20 was prepared from compound 13 for three steps in 8%
overall yield as a white solid, mp 181–182 °C. 1H NMR (300 MHz,
DMSO-d6) d 3.70 (s, 2H), 4.73 (s, 4H), 6.68 (dd, J = 2.0, 8.4 Hz,
1H), 6.91 (d, J = 1.5 Hz, 1H), 7.16–7.35 (m, 11H), 7.43 (s, 1H), 7.52
(d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 9.68 (s, 1H). 13C NMR
4.1.7. General Procedure for the preparation of compounds 16–
23
(300 MHz, DMSO-d6)
d 36.58, 54.34, 109.01, 111.53, 118.28,
120.14, 120.29, 122.15, 126.66, 126.73, 128.50, 129.77, 135.76,
138.58, 138.98, 142.44, 144.64, 147.80, 180.72. MS (ESI+) m/z calcd
for C28H25N3S: 435.18; found: 436.3 (M+H).
Alkyl iodide (3.0 mmol) was added to the mixture of compound
13 (1.0 mmol) and potassium carbonate (1.2 mmol) in acetonitrile
(5 mL). The reaction mixture was stirred at 60 °C for 3 h and then
extracted with EtOAc. The organic layer was washed with brine,
dried over MgSO4 and concentrated in vacuo to give the crude
product which was used without further purification. The crude
product in CH2Cl2 (2 mL) was deprotected with TFA (2 mL) at ice-
bath and followed by treating with TCDI (1.2 mmol) and excess
25% ammonia solution. The final product was purified by column
chromatography using EtOAc/hexane (1:3) as an eluant to give
the desired products 16–23.
4.1.7.6. (7-Propylamino-9H-fluoren-2-yl)-thiourea (21). Com-
pound 21 was prepared from compound 13 for three steps in
11% overall yield as a brown solid, mp 193–194 °C. 1H NMR
(300 MHz, DMSO-d6) d 0.94 (t, J = 7.4 Hz, 3H), 1.54–1.61 (m, 2H),
3.00 (dt, J = 5.7, 6.9 Hz, 2H), 3.73 (s, 2H), 5.70 (t, J = 5.7 Hz, 1H),
6.57 (dd, J = 1.8, 8.3 Hz, 1H), 6.74 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H),
7.42 (s, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 9.65
(br s, 1H). 13C NMR (300 MHz, DMSO-d6) d 11.70, 21.96, 36.39,
44.91, 108.05, 111.18, 117.87, 120.30, 120.35, 122.28, 128.93,
135.28, 139.26, 142.18, 144.72, 148.62, 180.77. MS (ESI+) m/z calcd
for C17H19N3S: 297.13; found: 298.1 (M+H), 320.1 (M+Na).
4.1.7.1. (7-Dimethylamino-9H-fluoren-2-yl)-thiourea (16). Com-
pound 16 was prepared from compound 13 for three steps in 32%
overall yield as a pink solid, mp 196–197 °C. 1H NMR (300 MHz,
DMSO-d6) d 2.94 (s, 6H), 3.79 (s, 2H), 6.73 (dd, J = 1.8, 8.4 Hz,
1H), 6.93 (s, 1H), 7.19 (d, J = 8.1 Hz, 1H), 7.47 (s, 1H), 7.60 (d,
J = 8.1 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 9.65 (s, 1H). 13C NMR
4.1.7.7.
(7-Butylamino-9H-fluoren-2-yl)-thiourea (22). Com-
pound 22 was prepared from compound 13 for three steps in
14% overall yield as a off-white solid, mp 195–196 °C. 1H NMR
(300 MHz, DMSO-d6) d 0.91 (t, J = 7.4 Hz, 3H), 1.35–1.43 (m, 2H),
1.50–1.57 (m, 2H), 3.03 (dt, J = 5.4, 6.9 Hz, 2H), 3.73 (s, 2H), 5.67
(t, J = 5.4 Hz, 1H), 6.56 (dd, J = 2.0, 8.4 Hz, 1H), 6.73 (d, J = 1.2 Hz,
1H), 7.16 (d, J = 8.1 Hz, 1H), 7.42 (s, 1H), 7.50 (d, J = 8.1 Hz, 1H),
(300 MHz, DMSO-d6)
d 36.60, 40.49, 108.99, 111.40, 118.38,
120.23, 120.34, 122.29, 129.60, 135.70, 138.85, 142.53, 144.64,
149.90, 180.74. MS (ESI+) m/z calcd for C16H17N3S: 283.11; found:
284.1 (M+H), 306.1 (M+Na).

I.-J. Kang et al. / Bioorg. Med. Chem. 18 (2010) 6414–6421
6421
7.53 (d, J = 8.4 Hz, 1H), 9.63 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d
13.85, 19.88, 30.92, 36.42, 42.74, 108.06, 111.19, 117.89, 120.32,
120.35, 122.29, 128.95, 135.30, 139.27, 142.20, 144.73, 148.66,
180.78. MS (ESI+) m/z calcd for C18H21N3S: 311.15; found: 312.1
(M+H), 334.1 (M+Na).
30 min and 1, 2, 4, 6, 8, and 24 h after dosing, a blood sample
(ꢁ150 mL) was taken from each animal via the jugular-vein can-
nula and stored in ice (0–4 °C). The processing of the plasma and
analysis by high performance liquid chromatography–tandem
mass spectrometry (HPLC–MS/MS) was carried out as described.13
The plasma concentration data were analyzed with a standard
non-compartmental method.
4.1.7.8. (7-Benzylamino-9H-fluoren-2-yl)-thiourea (23). Com-
pound 23 was prepared from compound 13 for three steps in
15% overall yield as a yellow solid, mp 197–198 °C. 1H NMR
(400 MHz, DMSO-d6) d 3.70 (s, 2H), 4.30 (d, J = 6.0 Hz, 2H), 6.37
(t, J = 6.0 Hz, 1H), 6.59 (dd, J = 2.0, 8.1 Hz, 1H), 6.77 (s, 1H), 7.15–
7.41 (m, 7H), 7.48 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 9.65
(s, 1H). 13C NMR (400 MHz, DMSO-d6) d 37.07, 47.33, 109.35,
112.15, 118.69, 120.98, 121.01, 122.97, 127.33, 127.89, 128.98,
130.11, 136.11, 139.79, 140.96, 142.93, 145.31, 148.91, 181.46.
MS (ESI+) m/z calcd for C21H19N3S: 345.13; found: 346.3 (M+H).
Acknowledgment
The authors gratefully acknowledge the financial support of the
National Health Research Institutes in Taiwan (ROC).
References and notes
1. Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Science 1989, 244, 359.
2. (a) Simmonds, P.; Holmes, E. C.; Cha, T. A.; Chan, S. W.; McOmish, F.; Irvine, B.;
Beall, E.; Yap, P. L.; Kolberg, J.; Urdea, M. S. J. Gen. Virol. 1993, 74, 2391; (b)
Simmonds, P. J. Hepatol. 1999, 31, 54; (c) Simmonds, P. J. Gen. Virol. 2004, 85,
3173.
4.2. Biology
Huh-7 cells containing HCV subgenomic replicons (Ava5) were
provided by Apath, LLC (St. Louis, MO). The reporter-based HCV
subgenomic replicon, Ava5-EG(D4AB)SEAP, has previously been
described.11 Cell culture reagents were obtained from Life Technol-
ogies (Gaithersburg, MD). Cell viability was determined by the MTS
assay that was essentially as described.
3. (a) Weekly Epidemiological Record, 1997; Vol. 72, p. 341; (b) Lavanchy, D. J. Viral
Hepat. 1999, 6, 35.; (c) Wasley, A.; Alter, M. J. Semin. Liver Dis. 2000, 20, 1; (d)
Armstrong, G. L.; Wasley, A.; Simard, E. P.; McQuillan, G. M.; Kuhnert, W. L.;
Alter, M. J. Ann. Intern. Med. 2006, 144, 705; (e) Sy, T.; Jamal, M. M. Int. J. Med.
Sci. 2006, 3, 41; (f) Esteban, J. I.; Sauleda, S.; Quer, J. J. Hepatol. 2008, 48, 148.
4. (a) Cuthbert, J. A. J. Clin. Microbiol. Rev. 1994, 7, 505; (b) El-Serag, H.; Mason, A.
Arch. Int. Med. 2000, 160, 3227; (c) Bartenschlagar, R. Antiviral Chem. Chemother.
1997, 8, 281; (d) Dymock, B. W. Emerg. Drugs 2001, 6, 13; (e) Dymock, B. W.;
Jones, P. S.; Wilson, F. X. Antiviral Chem. Chemother. 2000, 11, 79; (f) Hoofnagle,
J. H.; Di Bisceglie, A. M. N. Eng. J. Med. 1997, 336, 347; (g) Hoofnagle, J. H.
Hepatology 2002, 36, S21; (h) Seeff, L. B. Hepatology 2002, 36, S35; (i) Pawlotsky,
J. M. Currr. Opin. Infect. Dis. 2003, 16, 587; (j) Boyer, N.; Marcellin, P. J. Hepatol.
2000, 32, 98.
5. (a) Abrignani, S.; Houghton, M.; Hsu, H. H. J. Hepatol. 1999, 31, 259; (b) Prince,
A. M.; Shata, M. T. Clin. Liver Dis. 2001, 5, 1091; (c) Forns, X.; Payette, P. J.; Ma,
X.; Satterfield, W.; Eder, G.; Mushahwar, I. K.; Govindarajan, S.; Davis, H. L.;
Emerson, S. U.; Purcell, R. H.; Bukh, J. Hepatology 2000, 32, 618; (d) Houghton,
M. Curr. Top. Microbiol. Immunol. 2000, 242, 327.
6. (a) Manns, M. P.; McHutchison, J. G.; Gordon, S. C.; Rustgi, V. K.; Shiffman, M.;
Reindollar, R.; Goodman, Z. D.; Koury, K.; Ling, M.; Albrecht, J. K. Lancet 2001,
358, 958; (b) Kjaergard, L. L.; Krogsgaard, K.; Gluud, C. BMJ 2001, 323, 1151.
7. (a) Hoofnagle, J. H.; Seeff, L. B. N. Eng. J. Med. 2006, 355, 2444; (b) Zoulim, F.;
Chevallier, M.; Maynard, M.; Trepo, C. Rev. Med. Virol. 2003, 13, 57; (c)
McHutchinson, J. G.; Gordon, S. C.; Schiff, E. R.; Shiffman, M. L.; Lee, W. M.;
Rustgi, V. K.; Goodman, Z. D.; Ling, M. H.; Cort, S.; Albrecht, J. K. N. Eng. J. Med.
1998, 339, 1485.
4.2.1. Subgenomic HCV inhibitory assay
In 96-well plates, Ava5-EG(D4AB)SEAP cells were seeded at a
density of 7 ꢀ 103 cells per well. After incubation at 37 °C for
1 day, cells were treated with various drugs at final 10 lM. Two
days later, culture medium was replaced with fresh phenol red-
free DMEM/10% FBS containing the same concentration of drugs
and cells were incubated for one more day. Culture supernatants
were collected from each well and SEAP activities were measured
using Phospha-Light assay kit (Tropix, Foster City, CA), according
to the manufacturer’s instruction.
4.3. Pharmacokinetic study
4.3.1. Pharmacokinetic analysis of 4b and 4c in Sprague–Dawley
rats
8. (a) Gordon, C. P.; Keller, P. A. J. Med. Chem. 2005, 48, 1; (b) Beaulieu, P. L.;
Tsantrizos, Y. S. Curr. Opin. Invest. Drugs 2004, 5, 838.
9. Bartenschlager, R.; Lohmann, V. Antiviral Res. 2001, 52, 1.
10. Kang, I. J.; Wang, L. W.; Lee, C. C.; Lee, Y. C.; Chao, Y. S.; Hsu, T. A.; Chern, J. H.
Bioorg. Med. Chem. Lett. 2009, 19, 1950.
11. Lee, J. C.; Chang, C. F.; Chi, Y. H.; Hwang, D. R.; Hsu, J. T. A. J. Virol. Methods 2004,
116, 27.
12. Recently, structurally related acylthiourea compound ACH-806 was reported
to show potent anti-HCV activity with a unique mechanism of action. It
selectively binds to the HCV NS4A protein, resulting in altered protein
composition and inactivation of the replicase complex, see: (a) Wyles, D. L.;
Kaihara, K. A.; Schooley, R. T. Antimicrob. Agents Chemother. 2008, 52, 1862; (b)
Yang, W.; Zhao, Y.; Fabrycki, J.; Hou, X.; Nie, X.; Sanchez, A.; Phadke, A.;
Deshpande, M.; Agarwal, A.; Huang, M. Antimicrob. Agents Chemother. 2008, 52,
2043.
The SD rats for the pharmacokinetic study were obtained from
BioLASCO Taiwan Co., Ltd (Ilan, Taiwan, ROC), and housed in the
animal facility at the National Health Research Institutes, Taiwan,
ROC. The animal studies were performed according to committee
approved procedures. Male rats, each weighing 330–380 g (9–
10 weeks old), were quarantined for 1 week before use. The ani-
mals were surgically implanted with
a jugular-vein cannula
1 day before treatment, and were fasted before treatment. The
compound was given to the rats (n = 4) as an intravenous (5 mg/
kg) or oral (20 mg/kg) dose prepared in a mixture of dosing vehi-
cles. The volume of the dosing solution given was adjusted accord-
ing to the body weight recorded before the drug was administered.
At 0 (immediately before dosing), 2, 5 (intravenous only), 15 and
13. Yao, H. T.; Wu, Y. S.; Chang, Y. W.; Hsieh, H. P.; Chen, W. C.; Lan, S. J.; Chen, C. T.;
Chao, Y. S.; Chang, L.; Sun, H. Y.; Yeh, T. K. Drug Metab. Dispos. 2007, 35, 1042.