Synthesis and anti-HCV activity evaluation of anilinoquinoline derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.11.121

Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. Here, we have synthesized a

Full text of DOI:10.1016/j.bmcl.2011.11.121

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1107–1110
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-HCV activity evaluation of anilinoquinoline derivatives
Huang-Kai Peng a, Chun-Kuang Lin b, Shiang-Yu Yang c, Chin-Kai Tseng b, Cherng-Chyi Tzeng d,
a,
Jin-Ching Lee b, , Shyh-Chyun Yang
⇑
⇑
a School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
d Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and
hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against
viral replication. Here, we have synthesized a series of anilinoquinoline derivatives. Based on a cell-based
HCV replicon system, we observed that 2-(30-nitroanilino)quinoline (18) exhibited anti-HCV activity with
Received 20 September 2011
Revised 25 November 2011
Accepted 28 November 2011
Available online 4 December 2011
a 50% effective concentration (EC50) value of 7 lM and a selective index (SI) value of 10. In addition, com-
pound 18 possessed the inhibitory effect on HCV NS3/4A protease activity. Therefore, we concluded that
the compound 18 possessed a potent activity against HCV replication and could provide as a new lead
compound as anti-HCV inhibitor.
Keywords:
Anilinoquinolines
Hepatitis C virus
Anti-HCV activity
NS3/4A protease
Ó 2011 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is an enveloped virus that is classified
in the hepacivirus genus of the Flaviviridae family.1 It has a 9.6-
kb positive-single strand RNA genome that comprises an open
reading frame (ORF) flanked by highly structured 50 and 30
untranslated regions (UTRs).2 The virus RNA genome encodes a
polyprotein, which is posttranslationally processed by host and
virus proteases into mature proteins, including structural pro-
teins (C, E1, E2, and p7) and nonstructural proteins (NS2, NS3,
NS4A, NS4B, NS5A, and NS5B).3 Among the nonstructural pro-
teins, NS3/4A protease has been considered as a prime target
to eradicate HCV infection due to possessing multiple functions,
including RNA helicase activity for catalyzing the unwinding of
RNA–RNA and proteolytic activity for post-translational process-
ing of polyprotein in viral replication.4 HCV is a worldwide infec-
tious pathogen that causes chronic liver diseases, including
hepatic fibrosis, hepatic cirrhosis and hepatocellular carcinoma
(HCC).5 At present, medical treatment of HCV-infected patients
novel agents targeting HCV replication are under investigation
through the replicon system, which is based on Huh7 hepatoma
cell lines containing stably replicating HCV subgenomic RNA.8
A
number of potent anti-HCV agents have been developed for tar-
geting HCV RNA replication, protease, and RNA polymerase activ-
ity.4 Nevertheless, in addition to the dependence of therapeutic
effectiveness on virus genotype, drug resistance is an inevitable
problem due to the high mutation rate of HCV and its rapid rep-
lication.9,10 Therefore, it is extremely desirable to further develop
new, safer and even more effective agents against HCV infection
and resistance emergence.
Since the natural products and their derivatives play important
roles in drug discovery, most of the synthetic and semi-synthetic
compounds and theirs pharmacophores were designed from natu-
ral products.11 Studies have shown that quinoline derivatives had
diverse of biological activities, including antifungal, antibacterial,
and antiplatelet activities.12–15 Besides, the 2-phenylquinoline
derivative (1) were consists of a large number of antitumor com-
pounds.16–18 In recent years, increased attention has been devoted
to the antiviral activity of quinoline derivatives. The naturally
occurring quinolone alkaloid, buchapine (2), and its synthetic
derivative (3) exhibited potent inhibitory activities against HIV.19
Similarly, a 4-hydroxyquinolon-3-yl-benzothiadiazine derivative
(4) was evaluated as an inhibitor of genotype 1 HCV polymerase
and also demonstrated good antiviral activity20 (Fig. 1). Although
there are many studies that reported good antiviral activity for
the non-nucleotide inhibitors of the heterocyclic backbone, to date,
is
a combination of pegylated interferon-a (IFN-a) and the
nucleoside analog ribavirin. However, the therapy usually in-
volves adverse side effects and yields a limited sustained viro-
logical response (SVR) of approximately 50% for predominant
genotype 1 infected patients.6,7 Furthermore, there is no avail-
able vaccine or specific agents against HCV infection. Currently,
⇑
Corresponding authors. Tel.: +886 7 3121101x2662; fax: +886 7 3210683.
(S.-C.Y.); tel.: +886 7 3121101x2369; fax: +886 7 3125339 (J.-C.L.).
E-mail addresses: jclee@kmu.edu.tw (J.-C. Lee), scyang@kmu.edu.tw (S.-C. Yang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.121

1108
H.-K. Peng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1107–1110
O
O
OH
N
N
OH
N
H
O
O
N
H
OH
1
2
3
O
N
S
OH
N
H
N
O
NH
4
Figure 1. Chemical structure of quinoline derivatives.
there has been no research on the anti-HCV activity of quinoline
derivatives.21–36 Therefore, we were interested in designing and
synthesizing an aniline moiety incorporated into a natural quino-
line pharmacophore to generate a new anilinoquinoline lead
compound. In this study, we examined the anti-HCV activity of
synthesized anilinoquinoline derivatives. Quantitative RT-PCR
and western blot analyses were performed to quantify the intracel-
lular HCV RNAs and protein expression, respectively.
The synthesized series of anilinoquinoline derivatives was pre-
pared by simple amination. Reaction of the starting material,
2-chloroquinoline (5), with various anilines in the presence of eth-
anol produced 6–23 with overall yields of 34–95%. The preparation
of anilinoquinoline derivatives is described in Scheme 1. Designed
series of compounds 6–23 were characterized by 1H NMR, 13C
NMR, IR spectra, and HRMS techniques, and their purity by element
analysis.
To examine the effects of various anilinoquinoline derivatives
on HCV replication, Huh7 cells harboring the HCV subgenomic
replicon,8 designated Ava5 cells, were treated with anilinoquino-
line derivatives at concentrations of 0.5–150 lM. After three days,
total cellular RNAs of compound-treated cells were subjected to
quantitative RT-PCR (RT-qPCR) with specific primers correspond-
ing to HCV RNA. In parallel, cytotoxicity was determined by the
MTS assay. The results for the anilinoquinoline derivatives are
summarized in Table 1. Among them, compounds 6, 11, 18, 19,
20, 21, and 23 exhibited anti-HCV activity at concentrations
<30
lM. Compounds 17, 18, 20, and 22 were found to be less cyto-
toxic (>70
l
M). Based on the above CC50 (concentration of the
compound at which cell viability is reduced by 50% when com-
pared to the mock control, 0.1% DMSO-treated cells) and EC50 (con-
centration of the compound at which HCV RNA level is reduced by
50% when compared to the mock control) results, the selective
R
R
i
+
H2N
N
N
H
N
Cl
5
6 ~ 23
6, R = H
15, R = 4'-OMe
7, R = 4'-Me
16, R = 3', 5'-diOMe
17, R = 3', 4', 5'-triOMe
18, R = 3'-NO2
8, R = 2', 4'-diMe
9, R = 3', 5'-diMe
10, R = 2', 6'-diMe
19, R = 4'-NO2
11 , R = 4'-Cl, 6'-Me 20, R = 3'-NO2, 4'-OH
12, R = 4'-Cl
21, R = 4'-CN
13, R = 2'-Cl, 4'-Br
14, R = 2'-Br
22, R = 4'-CO2Et
23, R = 3'-OCH2Ph
Scheme 1. Reagent and condition: (i) EtOH, reflux (34–95%).

H.-K. Peng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1107–1110
1109
Table 1
Inhibition of HCV by anilinoquinoline derivatives
Compds
HCV
a
b
CC50
(
lM)
EC50
(lM)
SIc
1.96
1
1
1
1
3.4
1.1
1
1
1
1
3
6
7
8
45
45
35
31
40
43
60
48
61
46
33
77
71
48
84
50
147
47
23
43
35
31
40
12.5
53
45
60
43
33
25
7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
10
20
2.4
3.8
3.5
2.9
3.1
21.7
14.3
50
15.1
a
Concentration of the compound at which cell viability was reduced by 50%
when compared to the 0.1% DMSO-treated cells, which served as a mock control.
b
Inhibitory concentration that reduced viral replication by 50%.
The selective index was calculated as the ratio of CC50 versus EC50
c
.
Figure 2. Inhibition of HCV replication and protein expression in Ava5 cells by
compound 18. Ava5 cells were incubated with compound 18 at the indicated
concentration (from 2.5 to 15 lM). The non-linear regression graphs and an
effective concentration of 50% inhibition (EC50) was calculated by the GraphPad
Prism5.0 program (Version 5.0, GraphPad Software, Inc. La Jolla, CA). Treatment
with 0.1% DMSO served as a mock control. (A). After three days of incubation, total
RNA was extracted from the Ava5 cells to quantify HCV RNA levels by RT-qPCR. HCV
RNA expression was normalized by cellular GAPDH mRNA (left axis). Cell viability
was performed simultaneously by the MTS assay (right axis). The results are
expressed as the means standard deviations (error bars) of triplicate experiments.
(B). After four days of incubation, cell lysates were extracted and analyzed by
western blotting with anti-NS5B and anti-GAPDH antibody (a loading control).
index (SI; CC50/EC50) values for 11, 17, 18, 20, 21, and 23 were
greater than those for the other compounds. The results indicated
that 2-(30-nitroanilino)quinoline (18) displayed the best SI value
compared to the other compounds in terms of anti-HCV activity,
with a SI value of 10. As shown in Figure 2A, a dose-dependent
reduction of HCV RNA levels by compound 18 was also observed,
which exhibited an EC50 value of 7 0.3 lM, as normalized by
cellular GAPDH mRNA. To further clarify the inhibitory effect of
compound 18 on the synthesis of viral protein, cell lysates were
Treatment with 100 U/ml of interferon-
inhibition of HCV protein synthesis.
a (IFN-a) served as positive control of
Figure 3. Cell-based HCV NS3/4A protease activity assay. (A) Schematic diagram of the HCV NS3/4A protease reporter construct, pEG(
contains the HCV NS3/4A protease gene and a dual-functional EG( 4A/B)SEAP reporter gene harboring a cleavage site of NS3/4A protease (designed
the egfp and the seap genes. Both genes are controlled by the human cytomegalovirus immediate-early (CMV IE) promoter. (B) Transient reporter assay of NS3/4A protease.
D
4A/B)SEAP-NS3/4A. The construct
D
D
4A/B) inserted between
Huh-7 cells were transfected with 0.5 lg of pEG(D4AB)SEAP-NS3/4A plasmid for 6 h, and then were treated with compound 18 at concentration of 5, 10, 12.5 and 15 lM, in
which treatment of 10 nM BILN 2061, a HCV protease inhibitor,40 and 0.1% DMSO (mock) served as positive and negative control, respectively. After three days, the culture
media were harvested to measure the SEAP activity. The efficacy of compound 18 against NS3/4A protease was reflected by the reductive percentage of SEAP activity
compared with the mock control. The results are expressed as the means standard deviations (error bars) of triplicate experiments. ⁄P < 0.05.

1110
H.-K. Peng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1107–1110
4. Kwong, A. D.; McNair, L.; Jacobson, I.; George, S. Curr. Opin. Pharmacol. 2008, 8,
522.
5. Pawlotsky, J. M. Hepatology 2006, 43, 207.
6. Manns, M. P.; Foster, G. R.; Rockstroh, J. K.; Zeuzem, S.; Zoulim, F.; Houghton, M.
Nat. Rev. Drug Disc. 2007, 6, 991.
subjected to western blot analysis with anti-HCV NS5B antibody, in
which the level of GAPDH served as a loading control. The synthe-
sis of HCV NS5B proteins was inhibited by compound 18 in a dose-
dependent manner, confirming that compound 18 was a potential
lead with anti-HCV activity (Fig. 2B). As the results showed, the
inhibition of HCV replication of compound 18 is similar to the lit-
erature compound 4.20 This could be explained by the presence of
the same core structure of quinoline ring. Comparable both EC50
and CC50 activities were observed by the peripheral substitution
on the phenyl ring that plays an important role in the SI value of
anti-HCV activity. Replacement of the electron-donating group
and weak electron-withdrawing group on the phenyl ring showed
the lower SI value (<1.1). On the contrary, introduction of strong
electron-withdrawing group on the phenyl ring showed the higher
SI value (2.4–10). Based on the structure of anilinoquinolines, we
found that the features for optimum anti-HCV activities are: (1)
substituent on the phenyl ring should be a strong electron-with-
drawing group; (2) substituent at C-3 on phenyl ring is crucial in
which the potency decreased in an order of 20 > 19 > 18. To
further analyze the effect of compound 18 on viral targets, such
as internal ribosome entry site (IRES), NS3/4A protease activity,
or NS5B polymerase activity, several reporter-based assays were
performed.37–39 The results indicated that compound 18 possessed
anti-NS3/4A protease activity (Fig. 3B). In contrast, there no signif-
icant inhibition on IRES-mediated translation and NS5B polymer-
ase activity (data not shown). However, we observed that the
inhibitory effect of compound 18 on NS3/4A protease (ꢀ40%) is
not consistent with that on HCV RNA level (ꢀ80%) at concentration
7. Ferenci, P. Minerva Gastroenterol. Dietol. 2006, 52, 157.
8. Blight, K. J.; Kolykhalov, A. A.; Rice, C. M. Science 2000, 290, 1972.
9. De Francesco, R.; Migliaccio, G. Nature 2005, 436, 953.
10. Sarrazin, C.; Zeuzem, S. Gastroenterology 2010, 138, 447.
11. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461.
12. Ramesh, E.; Manian, R. D. R. S.; Raghunathan, R.; Sainath, S.; Raghunathan, M.
Bioorg. Med. Chem. 2009, 17, 660.
13. Musiol, R.; Jampilek, J.; Kralova, K.; Richardson, D. R.; Kalinowski, D.; Podeszwa,
B.; Finster, J.; Niedbala, H.; Palka, A.; Polanski, J. Bioorg. Med. Chem. 2007, 15,
1280.
14. Musiol, R.; Jampilek, J.; Buchta, V.; Silva, L.; Niedbala, H.; Podeszwa, B.; Palka,
A.; Majerz-Maniecka, K.; Oleksyn, B.; Polanski, J. Bioorg. Med. Chem. 2006, 14,
3592.
15. Priya, N.; Gupta, A.; Chand, K.; Singh, P.; Kathuria, A.; Raj, H. G.; Parmar, V. S.;
Sharma, S. K. Bioorg. Med. Chem. 2010, 18, 4085.
16. Kuo, S. C.; Lee, H. Z.; Juang, J. P.; Lin, Y. T.; Wu, T. S.; Chang, J. J.; Lednicer, D.;
Paull, K. D.; Lin, C. M. J. Med. Chem. 1993, 36, 1146.
17. Vázquez, M. T.; Romero, M.; Pujol, M. D. Bioorg. Med. Chem. 2004, 12, 949.
18. Chang, F. S.; Chen, W.; Wang, C.; Tzeng, C. C.; Chen, Y. L. Bioorg. Med. Chem.
2010, 18, 124.
19. Ahmed, N.; Brahmbhatt, K. G.; Sabde, S.; Mitra, D.; Singh, I. P.; Bhutani, K. K.
Bioorg. Med. Chem. 2010, 18, 2872.
20. Melagraki, G.; Afantitis, A.; Sarimveis, H.; Koutentis, P. A.; Markopoulos, J.;
Igglessi-Markopoulou, O. Bioorg. Med. Chem. 2007, 15, 7237.
21. Rostom, S. A. F.; Shalaby, M. A.; El-Demellawy, M. A. Eur. J. Med. Chem. 2003, 38,
959.
22. Corona, P.; Vitale, G.; Loriga, M.; Paglietti, G.; La Colla, P.; Collu, G.; Sanna, G.;
Loddo, R. Eur. J. Med. Chem. 2006, 41, 1102.
23. Zhang, P.; Zhang, N.; Buckwold, V. E.; Hosmane, R. S. Bioorg. Med. Chem. 2007,
15, 4933.
24. Ismail, N. S. M.; El Dine, R. S.; Hattori, M.; Takahashi, K.; Ihara, M. Bioorg. Med.
Chem. 2008, 16, 7877.
25. Tonelli, M.; Vazzana, I.; Tasso, B.; Boido, V.; Sparatore, F.; Fermeglia, M.; Paneni,
M. S.; Posocco, P.; Pricl, S.; Colla, P. L.; Ibba, C.; Secci, B.; Collu, G.; Loddo, R.
Bioorg. Med. Chem. 2009, 17, 4425.
26. Farghaly, T. A.; Abdalla, M. M. Bioorg. Med. Chem. 2009, 17, 8012.
27. Ontoria, J. M.; Rydberg, E. H.; Di Marco, S.; Tomei, L.; Attenni, B.; Malancona, S.;
Martin Hernando, J. I.; Gennari, N.; Koch, U.; Narjes, F.; Rowley, M.; Summa, V.;
Carroll, S. S.; Olsen, D. B.; De Francesco, R.; Altamura, S.; Migliaccio, G.; Carfi, A.
J. Med. Chem. 2009, 52, 5217.
28. Mhaske, S. B.; Ksebati, B.; Prichard, M. N.; Drach, J. C.; Zemlicka, J. Bioorg. Med.
Chem. 2009, 17, 3892.
29. Chen, C. S.; Chiou, C. T.; Chen, G. S.; Chen, S. C.; Hu, C. Y.; Chi, W. K.; Chu, Y. D.;
Hwang, L. H.; Chen, P. J.; Chen, D. S.; Liaw, S. H.; Chern, J. W. J. Med. Chem. 2009,
52, 2716.
of 15 lM, suggesting that there were multiple targets influenced
by compound 18 for enhancement of anti-HCV activity.
We have synthesized and evaluated the anti-HCV activities of
anilinoquinoline derivatives. Results indicated that introduction
of a strong electron-withdrawing substituent such as NO2 on the
2-anilinoquinolie pharmacophore is crucial for the suppression of
HCV replication. Among them, 2-(30-nitroanilino)quinoline (18)
exhibited anti-HCV activity with an EC50 value of 7 lM and an SI
value of 10. Inhibition of HCV NS3/4A protease may be partly
responsible for the anti-HCV properties of compound 18. This
study discovered a new lead molecule to design more potent
anti-HCV agents. Further structural modification of compound 18
as an anti-HCV candidate is currently in progress.
30. Tonelli, M.; Simone, M.; Tasso, B.; Novelli, F.; Boido, V.; Sparatore, F.; Paglietti,
G.; Pricl, S.; Giliberti, G.; Blois, S.; Ibba, C.; Sanna, G.; Loddo, R.; La Colla, P.
Bioorg. Med. Chem. 2010, 18, 2937.
31. Farghaly, T. A.; Abdel Hafez, N. A.; Ragab, E. A.; Awad, H. M.; Abdalla, M. M. Eur.
J. Med. Chem. 2010, 45, 492.
32. Riyadh, S. M.; Farghaly, T. A.; Abdallah, M. A.; Abdalla, M. M.; Abd El-Aziz, M. R.
Eur. J. Med. Chem. 2010, 45, 1042.
Acknowledgments
33. El-Sabbagh, O. I.; Baraka, M. M.; Ibrahim, S. M.; Pannecouque, C.; Andrei, G.;
Snoeck, R.; Balzarini, J.; Rashad, A. A. Eur. J. Med. Chem. 2009, 44, 3746.
Financial support of this work by the National Science Council
of the Republic of China is gratefully acknowledged (Grant
Number: NSC96-2745-M-037-003-URD, NSC98-2119-M-037-001-
MY3, and NSC 97-2311-B-037-001-MY3). We also thank the
National Center for High-Performance Computing for providing
computer resources and chemical database services.
´
34. Stankiewicz-Drogon, A.; Dörner, B.; Erker, T.; Boguszewska-Chachulska, A. M. J.
Med. Chem. 2010, 53, 3117.
35. Malancona, S.; Donghi, M.; Ferrara, M.; Martin Hernando, J. I.; Pompei, M.;
Pesci, S.; Ontoria, J. M.; Koch, U.; Rowley, M.; Summa, V. Bioorg. Med. Chem.
2010, 18, 2836.
36. Nakamura, M.; Aoyama, A.; Salim, M. T. A.; Okamoto, M.; Baba, M.; Miyachi, H.;
Hashimoto, Y.; Aoyama, H. Bioorg. Med. Chem. 2010, 18, 2402.
37. Lee, J. C.; Wu, T. Y.; Huang, C. F.; Yang, F. M.; Shih, S. R.; Hsu, J. A. Biotechnol.
Bioeng. 2005, 90, 656.
Supplementary data
38. Lee, J. C.; Shih, Y. F.; Hsu, S. P.; Chang, T. Y.; Chen, L. H.; Hsu, J. T. Anal. Biochem.
2003, 316, 162.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.121.
39. Lee, J. C.; Tseng, C. K.; Chen, K. J.; Huang, K. J.; Lin, C. K.; Lin, Y. T. Anal. Biochem.
2010, 403, 52.
40. Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.;
Bos, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A. M.;
Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagace, L.; LaPlante, S. R.; Narjes, H.;
Poupart, M. A.; Rancourt, J.; Sentjens, R. E.; St George, R.; Simoneau, B.;
Steinmann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C. L.; Llinas-
Brunet, M. Nature 2003, 426, 186.
References
1. Lindenbach, B. D.; Rice, C. M. Nature 2005, 436, 933.
2. Yi, M.; Lemon, S. M. J. Virol. 2003, 77, 3557.
3. Penin, F.; Dubuisson, J.; Rey, F. A.; Moradpour, D.; Pawlotsky, J. M. Hepatology
2004, 39, 5.