Inhibition of noroviruses by piperazine derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.10.122

There is currently an unmet need for the development of small-molecule therapeutics for norovirus infection. The piperazine scaffold, a privileged structure embodied in many pharmacological agents, was used to synthesize an array of structurally-diverse derivatives which were screened for anti-norovius activity in a cell-based replicon system. The studies described herein demonstrate for the first time that functionalized piperazine derivatives possess anti-norovirus activity. Furthermore, these studies have led to the identification of two promising compounds (6a and 9l) that can be used as a launching pad for the optimization of potency, cytotoxicity, and drug-like characteristics.

Full text of DOI:10.1016/j.bmcl.2011.10.122

Bioorganic & Medicinal Chemistry Letters 22 (2012) 377–379
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of noroviruses by piperazine derivatives
Dengfeng Dou a, Guijia He a, Sivakoteswara Rao Mandadapu a, Sridhar Aravapalli a,
Yunjeong Kim b, Kyeong-Ok Chang b, William C. Groutas a,
⇑
a Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
b Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
There is currently an unmet need for the development of small-molecule therapeutics for norovirus infec-
tion. The piperazine scaffold, a privileged structure embodied in many pharmacological agents, was used to
synthesize an array of structurally-diverse derivatives which were screened for anti-norovius activity in a
cell-based replicon system. The studies described herein demonstrate for the first time that functionalized
piperazine derivatives possess anti-norovirus activity. Furthermore, these studies have led to the identifi-
cation of two promising compounds (6a and 9l) that can be used as a launching pad for the optimization of
potency, cytotoxicity, and drug-like characteristics.
Received 27 September 2011
Revised 27 October 2011
Accepted 31 October 2011
Available online 15 November 2011
Keywords:
Norovirus
Inhibition
Ó 2011 Elsevier Ltd. All rights reserved.
Piperazine derivatives
Noroviruses belong to the Norovirus genus of the Caliciviridae
family and are the most common cause of acute viral gastroenteritis
in the US and worldwide.1–3 Noroviruses are highly contagious and
outbreaks in cruise ships, schools, hospitals and nursing homes, are
associated with significant morbidity and mortality. Although
infection by noroviruses is generally self-limiting, the disease con-
stitutes an important health problem and a potential bioterrorism
threat because of its highly contagious nature and morbidity. The
problem is further compounded by a dearth of small-molecule ther-
apeutics or vaccines. Indeed, only a limited number of studies
aimed at the discovery of therapeutics for norovirus infection have
been reported in the literature.4–6
R
N
N
N
X
(I)
O
S O
O
O
O
X =
R5
N
N
...
N
R3
R2
R4
R1
O
We have recently described the inhibition of noroviruses by
cyclosulfamide derivatives and have used a scaffold hopping strat-
egy to identify additional series of compounds with anti-norovirus
activity.7–9 During the course of those studies, a cyclosulfamide-
based piperazine hit was identified that exhibited noteworthy
anti-norovirus activity. The piperazine scaffold is a privileged struc-
ture10–12 capable of binding to multiple receptors with high affinity.
It is a recurring structural motif in a large number of biologically
active molecules.13 Based on the forgoing, we hypothesized that
functionalized piperazine derivatives may exhibit anti-norovirus
activity. To explore this hypothesis, small, focused libraries of piper-
azine derivatives were synthesized and screened for anti-norovirus
activity using a replicon-based system. We describe herein the
results of synthetic and biochemical studies related to the discovery
of piperazine derivatives (structure (I), Fig. 1) as anti-norovirus
agents.
Figure 1. General structure of piperazine derivatives.
A series of structurally-diverse piperazine derivatives was syn-
thesized in order to develop preliminary structure–activity relation-
ship studies and to identify a hit suitable for use in a hit-to-lead
optimization campaign.14,15 The anti-norovirus effects of the syn-
thesized compounds16 were examined in NV replicon-harboring
cells (HG23 cells)17–20 and the results are summarized in Table 1.
Benzyl piperazine was initially coupled to a series of carboxylic
acids to generate compounds 1a–i (Scheme 1) which were subse-
quently screened in a cell-based replicon system. A few of the com-
pounds had low
lM anti-norovirus activity (compounds 1a–1b
and 1f, Table 1), with compound 1f having a better therapeutic in-
dex than the other two compounds. Furthermore, anti-norovirus
activity was found to be very sensitive to the nature of the ring
substituent. These observations provided preliminary support of
the hypothesis that suitably-functionalized piperazine derivatives
possess anti-norovirus activity.
⇑
Corresponding author. Tel.: +1 316 978 7374; fax: +1 316 978 3431.
E-mail address: bill.groutas@wichita.edu (W.C. Groutas).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.122

378
D. Dou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 377–379
Table 1
Anti-norovirus and cytotoxic effects of piperazine derivatives
a
a
Compound
ED50
(
lM)
TD50
(lM)
Compound
ED50
(lM)
TD50 (lM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
3a
3b
3c
3d
3e
6a
8.2
2.8
18.5
11.4
N/Db
N/D
N/D
95.3
N/D
N/D
N/D
62.5
N/D
N/D
N/D
N/D
6b
6c
6d
9a
9b
9c
9d
9f
9g
9h
9i
9j
9k
9l
>20
>20
>20
3.8
N/D
N/D
N/D
23.5
15.6
N/D
N/D
N/D
25.4
N/D
75.4
N/D
31.5
>20
>20
>20
7.8
>20
>20
>20
8.5
>20
>20
>20
>20
7.2
5.5
>20
>20
>20
4.5
>20
5.6
>20
8.5
6.7
121.2
118.7
155.4
9m
12.5
a
Average of two independent experiments.
N/D: not done due to high ED50 values.
b
A series of substituted 1H-1,2,3-triazole-4-carboxylic acids 2a–e
were coupled to 1-benzyl piperazine to give compounds 6a–d
(Scheme 2) of which the (p-methoxyphenyl) substituted derivative
6a had a therapeutic index of ꢀ22. Thus, the replacement of the tri-
were then prepared using click chemistry methodology21–23 from
propargylic acid and the corresponding azides. Subsequent coupling
to 1-benzyl piperazine dihydrobromide gave compounds 3a–e
(Scheme 1) which were found to be inactive.
azole ring by a c-lactam ring was highly beneficial.
To further probe how the nature of the substituents attached
to the piperazine scaffold affects anti-norovirus activity, a series
of (m-phenoxy)phenyl substituted piperazine derivatives was
synthesized (compounds 9a–m, Scheme 3). Reductive amination
of m-phenoxybenzaldehyde and 1-Boc-piperazine, followed by
The triazole ring was then replaced by
c-lactam ring. Thus,
compounds 4a–d were constructed using dimethyl itaconate and
the corresponding primary amines.24 Subsequent hydrolysis of
4a–d with 10% potassium hydroxide gave compounds 5a–d which
N
N
N
i
i
N
N
N
N
R1
N
N
N
R2
HN
OH
N
(2HBr)
O
O
R2
R1= o-phenoxyphenyl
m-phenoxyphenyl
p-phenoxyphenyl
m-Fluorophenyl
O
R2= p-methoxyphenyl
m-Fluorophenyl
p-Fluorophenyl
phenylthio
3a
3b
3c
3d
3e
1a
1b
1c
1d
1e
2a-e
ii
m-Chlorophenyl
p-C6H4COOCH3
p-n-propoxyphenyl 1f
1g
1h
1i
2-furanyl
OH
p-methoxybenzyl
N(CH2CH2)2O
O
Scheme 1. Reagents and reaction conditions: (i) R1COOH or 2/EDCI/DMF, then 1-benzyl piperazine dihydrobromide/TEA; (ii) R2CH2N3/sodium ascorbate/CuSO4/t-BuOH/H2O.
OCH3
O
OH
OCH3
O
O
N
O
O
i
ii
O
O
iii
N
N
N
N
R3
R3
R3
O
H3CO
R3= p-methoxyphenyl 4a
6a-d
5a-d
4b
4c
4d
phenyl
benzyl
2-furanyl
Scheme 2. Reagents and reaction conditions: (i) R3CH2NH2/CH3OH/rt to reflux; (ii) 10% KOH/THF; (iii) EDCl/DMF/1-benzyl-piperazine dihydrobromide/TEA.

D. Dou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 377–379
379
X
NBoc
ii
NH
iii
O
H
N
R4
N
N
N
i
O
O
O
O
X = CO
CH2
SO2
8
7
9a-m
R4
X
R4
X
CO
CO
CO
CO
CO
CO
CO
p-phenoxyphenyl
m-phenoxyphenyl
p-hydroxyphenyl
p-isopropoxyphenyl
p-chlorophenyl
p-cyanophenyl
p-methoxyphenyl
9a
CO
p-chlorobenzyl
m-phenoxyphenyl
m-hydroxyphenyl
p-isopropoxyphenyl
n-butyl
9h
9i
9j
9k
9l
9m
9b
9c
9d
9e
9f
CH2
CH2
CH2
SO2
SO2
p-cyanophenyl
9g
Scheme 3. Reagents and reaction conditions: (i) 1-Boc-piperazine/NaHB(OAc)3/CH2Cl2; (ii) TFA/CH2Cl2; (iii) EDCI/R4COOH/DMF or R4CHO/NaHB(OAc)3/CH2Cl2 or R4SO2Cl/
TEA/CH2Cl2.
removal of the Boc with trifluoroacetic acid, gave compound 8.
Compound 8 was either acylated with EDCI activated carboxylic
acid, or alkylated using reductive amination with substituted
benzaldehyde and sodium triacetoxyborohydride, or sulfonylated
with sulfonyl chloride in the presence of triethylamine to give
compounds 9a–m (Scheme 3). Several derivatives were found
to possess anti-norovirus activity, however, potency and toxicity
were highly sensitive to structural variations. The best com-
pound in this group, tertiary sulfonamide 9l, had a therapeutic
index of 18.
In summary, this preliminary report demonstrates for the first
time that functionalized piperazine compounds exhibit notewor-
thy anti-norovirus activity in a cell-based system. Two first-gener-
ation piperazine derivatives (compounds 6a and 9l) have been
identified that could potentially serve as a starting point for further
optimization studies in conjunction with mechanism of action
studies aimed at identifying the molecular target(s) with which
these compounds interact. Taken together, these results hold sig-
nificant promise for the development of inhibitors directed against
norovirus infection.
References and notes
1. Bergmann, R. J.; Gebhardt, J.; Gould, E.; Tucker, P.; Unge, T.; Hilgenfeld, R.;
Neyts, J. Antiviral Res. 2010, 87, 162.
2. Koo, H. L.; Ajami, N.; Atmar, R. L.; DuPont, H. L. Discov. Med. 2010, 10, 61.
3. Atmar, R. L. Food Environ. Virol. 2011, 2, 117.
4. Guiard, J.; Fiege, B.; Kitov, P. I.; Peters, T.; Bundle, D. R. Chemistry 2011, 17, 7438.
5. Rademacher, C.; Guiard, J.; Kitov, P. I.; Fiege, B.; Dalton, K. P.; Parra, F.; Bubdle,
D. R.; Peters, T. Chemistry 2011, 17, 7442.
6. Rohayem, J.; Bergmann, M.; Gebhardt, J.; Gould, E.; Tucker, P.; Mattevi, A.;
Unge, T.; Higenfeld, R.; Neyts, J. Antiviral Res. 2010, 87, 162.
7. Dou, D.; Tiew, K.-C.; He, G.; Mandadapu, S. R.; Aravapalli, S.; Alliston, K. R.; Kim,
Y.; Chang, K.-O.; Groutas, W. C. Bioorg. Med. Chem. 2011, 19, 5975.
8. Dou, D.; Mandadapu, S. R.; Alliston, K. R.; Kim, Y.; Chang, K.-O.; Groutas, W. C.
Bioorg. Med. Chem. 2011, 19, 5749.
9. Dou, D.; Mandadapu, S. R.; Alliston, K. R.; Kim, Y.; Chang, K.-O.; Groutas, W. C.
Eur. J. Med. Chem. 2011. doi: 10.1016/j.ejmech.2011.10.019.
10. DeSimone, R. W.; Currie, K. S.; Mitchell, S. A.; Darrow, J. W.; Pippin, D. A. Comb.
Chem. High Throughput Screening 2004, 7, 473.
11. Welsch, M. E.; Snyder, S. A.; Stockwell, B. R. Curr. Opin. Chem. Biol. 2010, 14, 347.
12. Barelier, S.; Krimm, I. Curr. Opin. Chem. Biol. 2011, 15, 469.
13. Berkheij, M.; van der Sluis, L.; Sewing, C.; den Boer, D. J.; Terpstra, J. W.;
Hiemstra, H.; Iwema Bakker, W. I.; van den Hoogenband, A.; van Maarseveen, J.
H. Tetrahedron Lett. 2005, 46, 2369.
14. Gillespie, P.; Goodnow, R. A. Ann. Rep. Med. Chem. 2004, 39, 293.
15. Wunberg, T.; Hendrix, M.; Hillisch, A.; Lobell, M.; Meier, H.; Schmeck, C.; Wild,
H.; Hinzen, B. Drug Discovery Today 2006, 11, 175.
16. All compounds were characterized by 1H NMR and HRMS and had a >95%
purity.
Acknowledgments
17. Chang, K. O.; Sosnovtsev, S. V.; Belliot, G.; King, A. D.; Green, K. Y. Virology 2006,
2, 463.
18. Chang, K. O.; George, D. W. J. Virol. 2007, 22, 12111.
The generous financial support of this work by the National
Institutes of Health (AI081891) is gratefully acknowledged.
19. Chang, K. O. J. Virol. 2009, 83, 8587.
20. Kim, Y.; Thapa, M.; Hua, D. H.; Chang, K.-O. Antiviral Res. 2011, 89, 165.
21. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
22. Moses, J. E.; Moorhouse, A. D. Chem. Soc. Rev. 2007, 36, 1249.
23. Shen, J.; Woodward, R.; Kedenburg, J. P.; Liu, X.; Chen, M.; Fang, L.; Sun, D.;
Wang, P. G. J. Med. Chem. 2008, 51, 7417.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.122.
24. Mansour, T. S.; Jin, H. Bioorg. Med. Chem. Lett. 1991, 1, 757.