Discovery of potent nucleotide-mimicking competitive inhibitors of hepatitis C virus NS3 helicase

Article abstract of DOI:10.1016/j.bmcl.2010.09.002

Among the enzymes involved in the life cycle of HCV, the non-structural protein NS3, with its double function of protease and NTPase/helicase, is essential for the virus replication. Exploiting our previous knowledge in the development of nucleotide-mimic

Full text of DOI:10.1016/j.bmcl.2010.09.002

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2776–2779
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent nucleotide-mimicking competitive inhibitors of hepatitis C
virus NS3 helicase
Sandra Gemma a,b, Stefania Butini , Giuseppe Campiani
a,b
a,b,
⇑
, Margherita Brindisi , Samantha Zanoli a,c,
a,b
a,b
a,b
a,b
a,b
a,b
Maria Pia Romano , Pierangela Tripaldi , Luisa Savini , Isabella Fiorini , Giuseppe Borrelli
,
a,d
a,c
Ettore Novellino , Giovanni Maga
a
European Research Centre for Drug Discovery and Development, Università di Siena, via Aldo Moro, 53100 Siena, Italy
Department of Pharmaceutical and Applied Chemistry, Università di Siena, via Aldo Moro, 53100 Siena, Italy
Istituto di Genetica Molecolare—CNR, via Abbiategrasso 207, 27100 Pavia, Italy
b
c
d
Dipartimento di Chimica Farmaceutica e Tossicologica Università di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2010
Revised 31 August 2010
Accepted 1 September 2010
Available online 6 September 2010
Among the enzymes involved in the life cycle of HCV, the non-structural protein NS3, with its double
function of protease and NTPase/helicase, is essential for the virus replication. Exploiting our previous
knowledge in the development of nucleotide-mimicking NS3 helicase (NS3h) inhibitors endowed with
key structural and electronic features necessary for an optimal ligand–enzyme interaction, we developed
the tetrahydroacridinyl derivative 3a as the most potent NS3h competitive inhibitor reported to date
i
(HCV NS3h K = 20 nM).
Keywords:
HCV
Ó 2010 Elsevier Ltd. All rights reserved.
Helicase
Hydrazides
Hepatitis C virus (HCV) is a positive-stranded RNA virus of the
We previously reported the discovery of the first NS3h nucleot-
ido-mimetic inhibitor (1, QU663, Fig. 1), which inhibits the helicase
activity by competitively binding the DNA recognition site of the
1
Flaviviridae family. More than 170 million people are chronically
2
infected with HCV worldwide. At the moment, the only drugs ap-
1
9
proved for treatment of chronic HCV infection are interferon-alpha
i
enzyme (K = 0.75 lM). In our studies the NS3h inhibitory activ-
(
pegylated and conventional) and ribavirin, administered alone or
ity of 1 was explained by considering its peculiar electronic and
3
in combination therapy. However, the current treatments for HCV
infection are far from being ideal, mainly due to efficacy and safety
issues. Consequently, the development of specifically targeted
antiviral therapies for HCV is urgently needed. Among the en-
zymes involved in the HCV life cycle, the non-structural protein
NS3 is a multifunctional enzyme essential for virus replication.
The N-terminal domain of NS3 is a serine protease whereas the
C-terminal domain has NTPase and helicase activities and unwinds
both DNA and RNA.
conformational features resembling those calculated for RNA–
1
9
DNA nucleotides. Indeed, the heterocyclic system of 1 presents
a partially negative charged area around the hydrazide moiety
positioned at 5.92–6.23 Å away from the basic quinoline nitrogen,
thus reproducing the electronic distribution of purine and pyrimi-
dine nucleotides, which have a positively charged area ꢀ5.8 and
ꢀ7.3 Å from the negatively charged phosphate moiety, respec-
3
4,5
6
,7
8
,9
Inhibition of NS3 helicase (NS3h) is a challenging approach for
the development of innovative anti-HCV drugs.10 Drugs that target
unwinding activity could act in different ways: by inhibiting ATP-
ase activity by interfering with ATP binding or hydrolysis, by pre-
venting nucleic acid (NA) binding and thus blocking the
N
H
N
H
N
Ar
HN
N
HN
N
A
O
1
1–14
R
unwinding process, or by intercalating into the NA chains.
Few NS3h inhibitors competing for the NA binding site have been
EtO
N
Me
Me
2a-i
1
2,15–18
QU663, 1
so far reported,
but most of them suffer from modest po-
H
N
Ar
tency, inappropriate pharmacokinetic properties or toxicity.
HN
A
⇑
Corresponding author. Tel.: +39 0577 234172; fax: +39 0577 234333.
E-mail address: campiani@unisi.it (G. Campiani).
R
N
3
a-d, A = CO, SO
2
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.002
Figure 1. Reference and title compounds.

S. Gemma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2776–2779
2777
Table 1
Inhibition of HCV NS3h by inhibitors 1, 2a–i and 3a–d
Compds
Structure
NS3h
Compds
Structure
NS3h
K (lM)
i
a
a
K
i
(l
M)
2
a
125
2h
>300
>300
2
b
>300
>300
2i
2
c
3a
0.020
2
d
250
3b
>300
2
2
e
5.0
75
3c
0.16
0.10
f
3d
2
g
0.30
1b
0.75
a
Values are means of three experiments, standard deviation is within 10% of the mean.
See Ref. 19.
b
tively.19 Moreover, docking studies revealed that compound 1 was
Sulfonic hydrazides 2e–g and 3c,d were obtained in reasonable
yield by reacting hydrazines 4c or 4e with the suitable arylsulfonyl
chloride in pyridine at 25 °C. Hydrazine 4e, necessary for the syn-
thesis of compounds 3a,c,d, was prepared as described in Scheme 2,
following classic literature protocols, starting from 3-ethoxyaniline
able to engage several interactions with key amino acids (e.g.,
1
9
Trp501) important for NA binding and for helicase activity. Based
on this rational, and with the aim to further investigate this class of
compounds for their potential role as anti-HCV drugs, we synthe-
sized and biochemically characterized quinolylhydrazides 2a–i,
and 1,2,3,4-tetrahydroacridinylhydrazides 3a–d (Fig. 1 and Table 1)
as potent NS3h inhibitors.
2
0
5 and 2-ethoxycarboxylcyclohexanone 6.
Inhibition of HCV NS3h by the synthesized inhibitors was mea-
sured by using the helicase assay previously described19 and re-
sults are reported in Table 1.
We first investigated the role of the aromatic hydrazide moiety
for enzyme affinity. The pyrazine system of 1 proved to have a key
role for the interaction with the enzyme. Indeed, substitution of
the pyrazine system by a phenyl ring (2a) led to a 170-fold drop
in inhibitory activity with respect to 1. Also introduction of
The synthesis of target inhibitors 2a–i and 3a–d is reported in
Scheme 1. Hydrazines 4a–e, prepared following reported proce-
1
9,20
dures,
were coupled with the appropriate carboxylic acid using
aldrithiol and triphenylphosphine as activating reagents in dichlo-
1
9
rometane, to afford the desired compounds in 35–80% overall
yields after chromatographic purification and re-crystallization.

2
778
S. Gemma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2776–2779
Ar
OH
EtO C
NHNH2
HN
HN
2
a)
65%
3
R
O
a)
5-80%
3
R1
3
O
R
EtO
NH2
6
EtO
N
3
N
R2
R1
5
7
2
R2
4
4
4
4
4
a, R1 = H, R = 2-Me, R =H
b)
N
85%
1
2
3
b, R = 6-OEt, R = 2-Me, R =H
2a-d,h,i
3a,b
1
2
3
NHNH2
c, R = 7-OEt, R = 2-Me, R =H
Cl
N
1
2
3
d, R = H, R ,R =-(CH ) -
Ar
SO2
2
4
H
N
1
2
3
c)
70%
EtO
e, R = 7-OEt, R ,R =-(CH ) -
2
4
HN
EtO
N
4
R3
e
b)
4
c,4e
R1
8
1
5-35%
N
e-g
c,d
R2
Scheme 2. Synthesis of hydrazine intermediate 4e. Reagents and conditions: (a)
Ph O, reflux, 3 h; (b) POCl , 25 °C, 1 h; (c) NH NH ꢁH O, 5 min, 150 W, 150 °C.
2
2
3
2
2
2
3
Scheme 1. Synthesis of inhibitors 2a–i and 3a–d. Reagents and conditions: (a) (i)
ArCO H, PPh , aldrithiol, DCM, 25 °C, 2–3 h, (ii) 4a–e, DCM, 25 °C, 18 h; (b) ArSO Cl,
pyridine, 25 °C, 12 h. Structures of compounds 2a–i and 3a–d as displayed in
C6 (2i). Contrastingly, modification at C2–C3 of the quinoline moi-
ety, and in particular replacing the quinoline system of 1 by a
substituted 1,2,3,4-tetrahydroacridine ring resulted in the synthe-
2
3
2
Table 1.
i
sis of 3a, which displayed a K of 0.020 lM, thus being 37.5 times
more potent than our prototype compound 1, and representing
the most potent NS3h inhibitor known to date. Compounds that
lack a 6-OEt substituent displayed a marked decrease in inhibitory
activity (3b vs 3a). With compound 3a in our hand, the synthesis of
sulfonyl derivatives 3c and 3d was a logical choice in an attempt to
further improve the inhibitory potency of 3a. As expected, sulfonyl
derivatives 3c and 3d resulted more potent than their quinoline
counterparts (2e and 2g, respectively). However, 3d proved to be
less potent than 3a, probably due to the lack of synergism among
the key pharmacophoric elements for enzyme interaction, namely
the sulfonyl and the 1,2,3,4-tetrahydroacridine moieties.
Due to its exceedingly high inhibitory potency, compound 3a
was selected for further studies aimed at confirming its mecha-
nism of inhibitory activity. As shown in Figure 2A, titrating increas-
ing amounts of compound 3a resulted in a dose-dependent
reduction of the reaction products, visualized as displaced single
five-membered heterocycles such as a 2-furyl (2b) or a (R)-4-thia-
zolidinyl (2c) was detrimental for affinity, as well as the bulkier 2-
isoquinoline system of 2d. More encouraging results were obtained
when a sulfonyl group replaced the carbonyl group of 2a. Accord-
ingly, 2e, bearing a benzenesulfonyl moiety, was 25 times more po-
tent than 2a, although 7 times less potent than 1. While the p-
toluensulfonyl derivative 2f was less potent than 2e, probably
due to steric constraints, a significant increase of inhibitory activity
was achieved with the 2-pyridylsulfonyl derivative 2g, this latter
showing a 2.5-fold potency increase with respect to our prototype
compound 1.
Subsequently, we investigated substitutions at the quinoline
system. The 7-OEt substituent of 1 revealed to be an important
determinant for inhibitory activity. Accordingly, a marked decrease
of potency was observed when it was removed (2h), or shifted at
Figure 2. Compound 3a is a competitive inhibitor of the NS3h activity. Experiments were performed as described in Ref. 19. (A) Native PAGE analysis of the reaction products
0
of the NS3h activity in the presence of 5 nM NA template 5 -FAM-18/66-mer and in the absence (lane 6) or in the presence of increasing amounts of 3a (lanes 1–4). Lane 5,
control reaction in the absence of the enzyme. Lane 7, boiled substrate. (B) As in panel A, but in the presence of 10 nM NA template. (C) As in panel A, but in the presence of
2
5 nM of 3a. (D) Dose–response curves for the inhibition of the NS3h activity by 3a in the presence of increasing fixed NA concentrations. Values are the means of three
independent experiments. Error bars are ±SD.

S. Gemma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2776–2779
2779
inhibitors of NS3h. This work could lead to a novel class of prom-
ising anti-HCV agents, typified by 3a, potentially useful in the fight
against this viral infection, although further biological assays to
characterize these selective inhibitors are necessary.
Acknowledgment
S.Z. is the recipient of a Fondazione Buzzati–Traverso Fellow-
ship. Authors thank NatSynDrugs for financial support.
References and notes
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i
) for 3a to
the NS3h was 0.02 ± 0.002 M. As shown in Figure 3, the apparent
l
1
1
inhibitory constant (Kiapp) values for both 3a and 1 increased as the
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1
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slopes ðꢂ K =Km Þ of the curves. Similarly to 1, compound 3a did
i
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not show inhibition towards the ATPase activity of NS3 up to
00 M, nor revealed any significant NA intercalating ability (data
not shown).
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1
l
understanding of the key structural and electron distribution fea-
tures necessary to reproduce in a small organic molecule the elec-
2
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1
9
tron distribution of purine and pyrimidine nucleotides,
we
discovered 3a as the most potent NS3h competitive inhibitor re-
ported to date. As its simplified analog 1, 3a represents the proto-
typic compound of the class of nucleotide-mimicking competitive