Design, synthesis, and anti-HCV activity of thiourea compounds

Article abstract of DOI:10.1016/j.bmcl.2009.02.048

A series of thiourea derivatives were synthesized and their antiviral activity was evaluated in a cell-based HCV subgenomic replicon assay. SAR studies revealed that the chain length and the position of the alkyl linker largely influenced the in vitro anti-HCV activity of this class of potent antiviral agents. Among this series of compounds synthesized, the thiourea derivative with a six-carbon alkyl linker at the meta-position of the central phenyl ring (10) was identified as the most potent anti-HCV inhibitor (EC₅₀ = 0.047 μM) with a selectivity index of 596.

Full text of DOI:10.1016/j.bmcl.2009.02.048

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1950–1955
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and anti-HCV activity of thiourea compounds
*
*
Iou-Jiun Kang, Li-Wen Wang, Chung-Chi Lee, Yen-Chun Lee, Yu-Sheng Chao, Tsu-An Hsu , 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 thiourea derivatives were synthesized and their antiviral activity was evaluated in a cell-based
HCV subgenomic replicon assay. SAR studies revealed that the chain length and the position of the alkyl
linker largely influenced the in vitro anti-HCV activity of this class of potent antiviral agents. Among this
series of compounds synthesized, the thiourea derivative with a six-carbon alkyl linker at the meta-posi-
tion of the central phenyl ring (10) was identified as the most potent anti-HCV inhibitor
Received 5 November 2008
Revised 9 February 2009
Accepted 12 February 2009
Available online 20 February 2009
(EC50 = 0.047 lM) with a selectivity index of 596.
Keyword:
Ó 2009 Elsevier Ltd. All rights reserved.
HCV inhibitor
Hepatitis C virus (HCV) is a blood-borne virus that was identi-
fied in 1989.1 Six genotypes with several subtypes within each
genotype have been described for this member of the Flaviviridae
family.2 It is estimated that more than 170 million people world-
wide are infected by HCV.3 Most of those with acute hepatitis C de-
Ava5 cell lines. Preliminary structure-activity relationships against
HCV are reported (Tables 1 and 2).
The thiourea compounds 1 and 5–20 were prepared by the
method summarized in Scheme 1. Nucleophilic substitution of
phenol (or aniline) 2 with phenylalkyl bromide using potassium
carbonate as a base in 1-methyl-2-pyrrolidine (NMP) at 90 °C for
3 h afforded compound 3. Subsequent nitro reduction of com-
pound 3 in the presence of tin(II) chloride in ethanol at 70 °C over-
night gave the aniline 4 in excellent yields. The aniline 4 was
treated with 1,10-(thiocarbonyl)diimidazole (TCDI) in dichloro-
methane at room temperature followed by reaction with 25%
ammonia solution or the appropriate primary amines to give the
desired compounds 1 and 5–20.
Compounds 25–53 were synthesized by the method summa-
rized in Scheme 2. In the presence of potassium carbonate, alcohol
21 can undergo nucleophilic substitution with dibromo compound
to give compound 22. Subsequent O-alkylation of 3-nitrophenol
with compound 22 in the presence of potassium carbonate in
NMP at 90 °C gave compound 23. A solution of compound 23 in
ethyl acetate is hydrogenated in the presence of 10% Pd/C with
hydrogen filled in a balloon at room temperature overnight to give
the aniline 24 in excellent yields. The aniline 24 was treated with
1,10-(thiocarbonyl)diimidazole (TCDI) in dichloromethane at room
velop
a
chronic infection. Chronic hepatitis
C
is often
asymptomatic but may lead to liver cirrhosis and hepatocellular
carcinoma (HCC) within the next decade.4 Liver failure related to
HCV infection is, in many countries, the leading cause of liver
transplantation.5 There is currently no vaccine or a direct antiviral
agent for HCV.6 The standard of care for treating HCV is a combina-
tion of pegylated interferon and ribavirin. However, this therapy in
patients with genotype 1 is not very successful and associated with
serious side effects.7 Therefore, there is an urgent need to discover
and develop new anti-HCV agents that are more effective and bet-
ter tolerated by patients.
Recently, an antiviral screening program was initiated to search
for new HCV inhibitors via HCV replicon assay8 in our laboratory.
Interestingly, a thiourea compound 1 was identified as a potential
anti-HCV agent, the structure of which is illustrated in Figure 1.9
This compound was found to possess antiviral activity against
HCV with an EC50 of 0.494 lM. Therefore many structurally related
thiourea compounds were synthesized and evaluated for their
anti-HCV activity. Details of this investigation will be described
herein.
In this Letter, we described the synthesis of antiviral thiourea
compounds via the key nucleophilic substitution of phenol or ani-
line with phenylalkyl bromide, compound 22 and ditosylate 56
(Schemes 1–3). All the resulting compounds were then submitted
for anti-HCV testing as well as cytotoxicity evaluation in the
S
H2N
N
H
O
1
EC50 = 0.494 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. Structure of thiourea compound 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.048

I.-J. Kang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1950–1955
1951
K CO , KI
2
3
XH
X
(CH )
2 n
+
NMP, 90 ºC, 3 h
O N
2
(CH ) Br
O N
2
2 n
2
3
X = O, NH
25% NH (aq)
3
1
or R NH
2
SnCl .2H O
TCDI
2
2
X
(CH )
2 n
EtOH, 70 ºC
overnight
CH Cl , r.t., 1h
r.t., overnight
H N
2
2
2
4
1
10. R = H, X = O, n = 6, meta
1
S
11. R = H, X = O, n = 7, meta
X
(CH )
2 n
1
12. R = H, X = O, n = 5, ortho
1
R
N
H
N
H
1
13. R = H, X = O, n = 5, para
1, 5-20
1
14. R = H, X = NH, n = 5, meta
1
1
1. R = H, X = O, n = 0, meta
15. R = Me, X = O, n = 5, meta
1
1
5. R = H, X = O, n = 1, meta
16. R = Et, X = O, n = 5, meta
1
1
6. R = H, X = O, n = 2, meta
17. R = n-Pr, X = O, n = 5, meta
1
1
7. R = H, X = O, n = 3, meta
18. R = Ph, X = O, n = 5, meta
1
1
8. R = H, X = O, n = 4, meta
19. R = CH Ph, X = O, n = 5, meta
2
1
1
9. R = H, X = O, n = 5, meta
20. R = CH CH Ph, X = O, n = 5, meta
2
2
Scheme 1. General synthetic route to thiourea compounds 1 and 5–20.
2
R
2
R
K CO , KI
2
3
Ar
+
Br
Br
Ar OH
O
Br
NMP, 90 ºC, 3 h
21
22
2
R
H , 10% Pd/C
2
HO
NO
2
O N
O
O Ar
EtOAc
r.t., overnight
2
K CO , KI
NMP, 90 ºC, 3 h
2
3
23
2
R
25% NH (aq)
3
TCDI
CH Cl , r.t., 1h
H N
O
O Ar
2
r.t., overnight
2
2
24
2
2
S
R
41. R = H, Ar = 4-pyrrolidinylPh
2
42. R = H, Ar = 4-piperidinylPh
H N
N
H
O
O Ar
2
2
43. R = H, Ar = 4-morpholinylPh
2
25-53
44. R = H, Ar = 4-pyrrolinylPh
2
45. R = H, Ar = 4-(2-furyl)Ph
2
2
2
33. R = H, Ar = 3-PhPh
34. R = H, Ar = 2-PhPh
35. R = H, Ar = 1-Naphthyl
36. R = H, Ar = 3-CH PhPh 49. R = H, Ar = 4-pyridyl
46. R = H, Ar = 4-(2-thienyl)Ph
25. R = H, Ar = Ph
2
2
2
47. R = H, Ar = 2-pyridyl
26. R = Me, Ar = Ph
2
2
2
48. R = H, Ar = 3-pyridyl
27. R = H, Ar = 4-FPh
2
2
2
28. R = H, Ar = 4-ClPh
2
2
2
2
37. R = H, Ar = 3-OPhPh
50. R = H, Ar = 5-isoquinolyl
29. R = H, Ar = 4-BrPh
2
2
2
30. R = H, Ar = 4-OMePh 38. R = H, Ar = 3-NHPhPh
31. R = H, Ar = 4-CO HPh 39. R = H, Ar = 3-COPhPh
51. R = H, Ar = 5-quinolyl
2
2
2
52. R = H, Ar = 4-quinolyl
2
2
2
2
32. R = H, Ar = 4-PhPh
40. R = H, Ar = 4-NEt Ph
53. R = H, Ar = 4-indolyl
2
Scheme 2. General synthetic route to thiourea compounds 25–53.
temperature followed by reaction with 25% ammonia solution gave
the target compounds 25–53.
nol with 2 equiv of the ditosylate 56 using potassium carbonate as
a base at refluxing acetonitrile gave compound 57, which was cou-
pled with phenol in the presence of potassium carbonate in aceto-
nitrile to give the compound 58. A solution of compound 58 in
ethyl acetate is hydrogenated in the presence of 10% Pd/C with
hydrogen filled in a balloon at room temperature overnight to give
the aniline 59 in excellent yields. The aniline 59 was treated with
Compound 60 was synthesized by the method summarized in
Scheme 3. Reduction of the glutaric acid 54 with lithium aluminum
hydride in dry ether gave the diol 55, which was then reacted with
p-toluenesulfonyl chloride in pyridine to give the corresponding
ditosylate 56. Subsequent nucleophilic substitution of 3-nitrophe-

1952
I.-J. Kang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1950–1955
O
O
CH3
H C
3
H3C CH3
LiAlH4
TsCl
HO
OH
HO
OH
dry ether
r.t., 4h
pyridine
r.t., 1h
54
55
H3C CH3
O2N
OH
H3C CH3
TsO
OTs
K2CO3, CH3CN
reflux, overnight
O2N
O
OTs
56
57
OH
H2, 10% Pd/C
H3C CH3
EtOAc
r.t., overnight
K2CO3, CH3CN
reflux, 3h
O2N
O
O
58
25% NH3 (aq)
r.t., overnight
TCDI
H3C CH3
CH2Cl2, r.t., 1h
H2N
H2N
O
O
59
S
H3C CH3
N
H
O
O
60
Scheme 3. Synthesis of thiourea compound 60.
Table 1
Anti-HCV activity and cytotoxicity for compounds 1 and 5–20
S
X
(CH2)n
R1
N
H
N
H
Compound
R1
X
Isomer
n
1b EC50
(l
M)a
CC50
(l
M)a
SIb
1
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
H
H
H
H
H
H
H
H
H
H
H
Me
Et
n-Pr
Ph
O
O
O
O
O
O
O
O
O
O
NH
O
O
O
O
O
O
Meta
Meta
Meta
Meta
Meta
Meta
Meta
Meta
Ortho
Para
Meta
Meta
Meta
Meta
Meta
Meta
Meta
0
1
2
3
4
5
6
7
5
5
5
5
5
5
5
5
5
0.494
0.561
0.335
0.231
0.159
0.048
0.047
0.097
1.012
0.197
0.143
0.822
0.685
0.708
31.497
7.238
13.388
>50
>50
>50
>50
>50
22
28
23
31
10
>50
>50
>50
32
35
32
>101
>89
>149
>216
>314
458
596
237
31
51
>350
>61
>73
45
1
4
2
CH2Ph
CH2CH2Ph
33
a
Mean of triplicate well values. All experiments were performed at least twice.
In vitro selectivity index (CC50/EC50).
b
1,10-(thiocarbonyl)diimidazole (TCDI) in dichloromethane at room
temperature followed by reaction with 25% ammonia solution gave
the target compound 60.
for the activity of compounds. Compound 5 with a chain length of
one carbon was found to be less active compared to lead com-
pound 1. On the other hand compounds 6–10 with a chain length
of two, three, four, five and six carbons increased the potency
about 1.5- to 10-fold with respect to compound 1. However, going
to longer seven-carbon homologue (11) was not favorable for
activity. These significant results demonstrated that the five and
six-carbon homologues (9, 10) in this series were more active
The thiourea compounds described herein were tested in an
HCV subgenomic replicon assay with a SEcreted Alkaline Phospha-
tase (SEAP) reporter. Compounds 1, 5–53 and 60 were submitted
for anti-HCV testing as well as cytotoxicity evaluation in the
Ava5 cell lines. As shown in Table 1, compared to the lead com-
pound 1, several thiourea compounds with various tether lengths
(5–11) were synthesized in order to determine the optimal spacing
between the oxygen atom and the distal phenyl group. Interest-
ingly, the chain length was found to be of considerable importance
against HCV (EC50 = 0.048 and 0.047 lM, respectively) than their
corresponding longer or shorter compounds. These observations
provide remarkable evidence that the hydrophobic interaction
and conformational flexibility of the alkyl linker influence anti-

I.-J. Kang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1950–1955
1953
Table 2
Anti-HCV activity and cytotoxicity for compounds 25–53 and 60
2
3
R
R
S
H N
2
N
H
O
O Ar
Compound
R2
H
R3
H
Ar
1b EC50
0.059
(
l
M)a
CC50
>50
(l
M)a
SIb
25
>847
26
60
27
28
29
30
31
32
Me
Me
H
H
0.104
0.105
0.072
0.072
0.072
0.218
>50
>50
13
>481
124
69
Me
H
5
F
H
H
7
97
Cl
H
H
19
264
>229
>1
Br
H
H
>50
>50
>50
OMe
CO2H
H
H
H
H
0.191
>262
33
34
H
H
H
H
0.214
0.097
13
13
61
134
35
36
37
38
H
H
H
H
H
H
H
H
0.058
0.058
0.107
0.177
14
13
18
11
241
224
168
62
O
N
H
(continued on next page)

1954
I.-J. Kang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1950–1955
Table 2 (continued)
Compound
R2
H
R3
H
Ar
1b EC50
0.218
(l
M)a
CC50
13
(
l
M)a
SIb
60
39
40
41
42
43
44
45
46
47
48
49
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.594
0.913
0.407
0.485
0.255
0.136
0.121
0.279
1.134
2.992
>50
16
>84
18
N
N
N
N
N
12
29
>50
>50
11
>103
>196
81
O
O
S
>50
>50
>50
>50
>413
>179
>44
>17
N
N
N
50
51
52
H
H
H
H
H
H
H
H
0.481
0.250
0.411
0.355
26
54
N
15
60
N
N
24
58
53
>50
>141
NH
a
Mean of triplicate well values. All experiments were performed at least twice.
HCV activity of these thiourea compounds. The position of the alkyl
linker at the central phenyl ring of five-carbon homologues (9, 12,
13) impacted anti-HCV activity. The activity increased from ortho-
substitution (12) to para-substitution (13), and to meta-substitu-
tion (9), the latter being the most preferred pattern for sterically
demanding substituents. Replacing the oxygen linker (9) with a

I.-J. Kang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1950–1955
1955
NH unit (14) resulted in a threefold decrease in activity, perhaps
caused by a conformational change of the side chain. Substitution
at the NH2 of the thiourea moiety with methyl (15), ethyl (16), n-
propyl (17), phenyl (18), benzyl (19) and phenethyl (20) groups
showed a 14- to 656-fold loss in activity compared to the corre-
sponding compound 9. The results of antiviral testing indicate that
a free NH2 is required for maintaining activity.
results demonstrated that the endocyclic nitrogen of the pyridyl,
isoquinolyl and quinolyl moieties was not tolerated (compare 25
to 47–49 and 35 to 50–52). This unexpected biological result is
not fully understood and is worthy of further study.
In conclusion, from the compound screening using a cell-based
HCV subgenomic replicon assay, we identified a lead compound 1
with an EC50 of 0.494 lM. We explored the structure-activity rela-
In this study, we observed that the thiourea compound 10, in
terms of potency and selectivity index (SI = 596), appear to be the
most promising candidate for further development as an anti-HCV
agent. On the basis of the skeleton of compound 10, several thiourea
compounds 25–53 and 60 were thus synthesized (Schemes 2 and 3).
The results are shown in Table 2 and are compared to the compound
10. The simple replacement of a carbon atom by oxygen (25) in the
alkyl linker of compound 10 showed a much better selectivity index
tionship for this class of inhibitors. A free NH2 group of the thiourea
moiety is critical for activity. The activities of this series of com-
pounds were very sensitive to the variation in the chain length of
the alkyl linker. Attempts to replace the oxygen linker with nitro-
gen caused a loss of activity. On the basis of these biological results,
compound 10 with a chain length of six carbons was found to ex-
hibit the most potent antiviral activity against HCV
(EC50 = 0.047 lM). Further SAR studies and mechanistic studies
(SI > 847) in the replicon assay with anEC50 of 0.059
l
Mand no cyto-
on this class of antiviral compounds are currently under active
toxicity up to 50 M. Introduction of one (26) or two (60) methyl
l
investigation and will be reported in due course.
groups at the 3-position of the alkyl linker resulted in a 1.7-fold de-
crease in activity compared to compound 25. This effect might be
due to their drastically conformational change and steric require-
ment of the alkyl linker of these thiourea compounds. Introduction
of a substituent (F, Cl, Br, OMe) at the para-position of the distal phe-
nyl ring of compound 25, such as compounds 27–30, resulted in a
1.2- to 3.7-fold decrease in activity. It is also interesting to note that
the methoxy analogue 30 was considerably less active than the halo
analogues 27–29. On the other hand, the carboxylic acid analogue
31 showed a total loss of activity. Among the biphenyl analogues (
32–34), the ortho-biphenyl analogue 34 showed better activity
Acknowledgements
We gratefully acknowledge the financial support of the National
Health Research Institutes in Taiwan (ROC).
References and notes
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(EC50 = 0.097 lM) than the para- and the meta-analogues (32, 33).
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36, where the phenyl ring (25) was replaced with naphthalene
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moiety by an oxygen atom (37), amino group (38) and carbonyl
group (39) resulted in a slight loss of activity, indicating that meth-
ylene linker is optimal for activity. Additionally, replacement of the
phenyl group (32) in the para-position of biphenyl moiety with
diethyl amino (40), pyrrolidinyl (41), piperidinyl (42), morpholinyl
(43) and pyrrolyl (44) groups caused a loss of activity. This effect
might be due to their distinct differences of hydrophobic proper-
ties. A comparison of the furan-containing compound (45) with
thiophene analogue (46) reveals some interesting differences.
The thienyl compound 46 exhibited 1.1 times more potent anti-
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HCV activity (EC50 = 0.121 lM), 5 times higher selective index
(SI > 413) than the furyl compound 45. Among the pyridyl ana-
logues (47–49), the 2-pyridyl analogue 47 was more active than
3- and 4-pyridyl analogues (48, 49) without significant cytotoxicity
(CC50 > 50 lM). Replacement of the naphthyl group of compound
35 with isoquinolyl (50), quinolyl (51, 52) and indolyl (53) groups
8. Lee, J. C.; Chang, C. F.; Chi, Y. H.; Hwang, D. R.; Hsu, J. T. A. J. Virol. Methods 2004,
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Expert Opin. Ther. Patents 2003, 13, 1707; (b) Wyles, D. L.; Kaihara, K. A.;
Schooley, R. T. Antimicrob. Agents Chemother. 2008, 52, 1862; (c) Yang, W.; Zhao,
Y.; Fabrycki, J.; Hou, X.; Nie, X.; Sanchez, A.; Phadke, A.; Deshpande, M.; Agarwal,
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resulted in a 4.3- to 8.3-fold decrease in activity. The significant