Peptide inhibitors of dengue virus NS3 protease. Part 1: Warhead

Article abstract of DOI:10.1016/j.bmcl.2005.09.062

Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluorom

Full text of DOI:10.1016/j.bmcl.2005.09.062

Bioorganic & Medicinal Chemistry Letters 16 (2006) 36–39
Peptide inhibitors of dengue virus NS3 protease. Part 1: Warhead
Zheng Yin,* Sejal J. Patel, Wei-Ling Wang, Gang Wang, Wai-Ling Chan,
K. R. Ranga Rao, Jenefer Alam, Duraiswamy A. Jeyaraj, Xinyi Ngew, Viral Patel,
David Beer, Siew Pheng Lim, Subhash G. Vasudevan and Thomas H. Keller
Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore 138670, Singapore
Received 15 July 2005; revised 11 September 2005; accepted 21 September 2005
Available online 21 October 2005
Abstract—Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the
Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde,
trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a Ki of 43 nM.
Ó 2005 Elsevier Ltd. All rights reserved.
The mosquito-borne dengue virus is endemic to most
tropical and sub-tropical regions throughout the world,
making dengue fever the most important mosquito-
borne viral disease affecting humans. Its global distribu-
tion is comparable to that of malaria, and an estimated
2.5 billion people live in areas at risk for epidemic trans-
mission.1 The four serotypes of dengue cause a self-lim-
iting viral fever which can lead to life-threatening
conditions such as dengue hemorrhagic fever (DHF)
and dengue shock syndrome (DSS). Due to the public
health threat posed by dengue virus infection and the
lack of vaccines or antiviral therapies, there is an urgent
need to develop new chemotherapeutic approaches to
counter this emerging infectious disease.
Classical inhibitors of serine proteases are ineffective or
only effective at high concentration against the dengue
NS3 protease, except for aprotinin, which is a large pro-
tein likely to prevent to the substrate from accessing the
protease active site by enveloping the enzyme.4a Addi-
tionally, only a few peptidic and non-peptidic inhibitors
of the dengue serine protease with moderate activities
have been reported (Fig. 1).4,5 In view of the general lack
of information about dengue protease inhibitors, it was
desirable to identify peptides with good inhibitory activ-
ities that can be used as prototype enzyme inhibitors for
future drug discovery efforts.
Unlike trypsin, dengue protease has a marked cleavage
site preference for dibasic residues. Two high affinity
non-prime side substrates: Bz-Nle-Lys-Arg-Arg (S1)
(Km 12.42 lM) and Bz-Nle-Lys-Thr-Arg (S2) (Km
33.9 lM), have been recently identified through sub-
strate profiling of dengue protease using positional scan-
ning tetrapeptides.6 We sought to capitalize on the
substrate information of NS3 protease to develop potent
small molecule inhibitors of the dengue serine protease.
In order to rapidly map the active site, we decided, as a
first step, to examine several well-established serine
The dengue virus genome contains a trypsin-like prote-
ase with a classical serine protease catalytic triad
(His51, Asp75, and Ser135) which constitutes part of
the nonstructural protein 3 (NS3). The enzymatic activ-
ity of NS3 protease is enhanced by interactions with the
NS2B protein, which acts as an essential cofactor.2 NS3
protease is vital for the post-translational proteolytic
processing of the polyprotein precursor and is essential
for viral replication and maturation of infectious dengue
virons.3 Therefore, dengue NS3 protease is an attractive
therapeutic target for dengue virus infections.
Keywords: Dengue virus NS3 protease; Peptide inhibitors.
*
Corresponding author. Tel.: +65 6722 2981; fax: +65 6722 2918;
e-mail: zheng.yin@novartis.com
Figure 1. Inhibitors of dengue virus NS3 protease.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.062

Z. Yin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 36–39
37
protease warheads. In this paper, we describe the design,
synthesis, and evaluation of dengue protease inhibitors
based on the tetrapeptide substrates. The importance
of the warheads and their contributions in the binding
to the NS3 protease will also be discussed.
Peptides including compounds 1, 2, Bz-Nle-Lys(Boc)-
Arg(Pbf)-OH and Bz-Nle-Lys(Boc)-Thr(t-Bu)-OH, were
either synthesized according to standard solution-phase
peptide chemistry7 or acquired from commercial sourc-
es. The synthesis of compounds 3 and 4 was accom-
plished by standard solid-phase peptide chemistry
using rink amide resin as solid support.8 Trifluoroacyl-
sulfonamide 5 was obtained from tetrapeptide 1 and tri-
fluoromethylsulfonamide using EDC. Tetrapeptide
aldehydes 6 and 7 were prepared according to previously
reported methods.9
Scheme 2. Reagents: (a) AcCl, EtOH; (b) 2-aminophenol, CH2Cl2; (c)
piperidine, DMF; (d) Bz-Nle-Lys(Boc)-Arg(Pbf)-OH, EDC, HOBt,
DIEA, DMF; (e) Dess–Martin periodinane, CH2Cl2; (f) 95% TFA,
CH2Cl2; (g) thiazole, BuLi, THF, À78°C; (h) 20% TFA, CH2Cl2.
[AA1 = Bz-Nle-Lys-Arg.]
Various substrate-based tetrapeptide inhibitors possess-
ing a-ketoamides were synthesized as shown in Scheme
1. Protected Arg-OH was converted to corresponding
ꢀreducedꢁ warheads using previously reported methodol-
ogies from Weinreb amides9b,10 and resulting intermedi-
ates were used for subsequent coupling reactions.
Tetrapeptide a-ketoamides 10-a and 10-b were prepared
by Dess–Martin periodinane oxidation11 of the corre-
sponding alcohols followed by removal of protecting
groups. Tetrapeptide a-hydroxyamide 11 was also
synthesized by coupling 9-b with tripeptide followed
by simple removal of protecting group.
benzoxazole 13-a was achieved by oxidation of the
corresponding alcohol. Meanwhile, a-ketothiazole 13-b
was prepared through Arg a-ketothiazole, which was
constructed through reaction of arginine Weinreb amide
14 with excess 2-lithiothiazole at low temperature.13
Scheme 3 outlines the synthesis of substrate-based tetra-
peptide trifluoromethyl ketone 18. The fluorinated
building block 16 was prepared in a few steps starting
with a modified Dakin–West reaction.14 The fluorinated
alcohol 17 was assembled through guanylation using
bis-Boc-pyrazole-1-carboxamidine.15 Subsequently, the
tetrapeptide trifluoromethyl ketone 18 was achieved
through Dess–Martin periodinane oxidation of tetra-
peptide fluorinated alcohol followed by removal of pro-
tecting groups.
Peptide a-ketobenzoxazole 13-a and a-ketothiazole 13-b
were prepared as shown in Scheme 2. Treatment of cya-
nohydrin 8-a with anhydrous HCl and ethanol in a Pin-
ner reaction afforded the intermediate imino ether
hydrochloride, which was then cyclized with o-amino-
phenol to afford a-hydroxybenzoxazole 12.12 a-Keto-
Finally, a peptide boronic acid inhibitor was synthesized
through known intermediate 19 as shown in Scheme 4.16
All tetrapeptide inhibitors were evaluated in an enzyme
inhibition assay against the viral NS3 protease Den 2
CF40ÆNS3pro, a truncated dengue 2 NS3 enzyme fused
via a flexible linker to a 47 amino acid region of NS2B.17
The activity of the inhibitors is summarized in Table 1.
A non-covalent, charged warhead, such as a carboxylic
acid, theoretically could provide binding efficiency via
electrostatic interactions. A product based peptide acid
was reported as a competitive inhibitor of the hepatitis
Scheme 1. Reagents: (a) HCl, CH2Cl2; (b) piperidine, DMF; (c)
Bz-Nle-Lys(Boc)-Arg(Pbf)-OH, EDC, HOBt, DIEA, DMF; (d) Dess–
Martin periodinane, CH2Cl2; (e) 95% TFA, CH2Cl2; (f) H2, Pd/C,
AcOH/MeOH; (g) HCl, MeOH; (h) (Boc)2O, NaHCO3, THF; (i)
LiOH, THF/H2O; (j) Benzylamine, EDC, HOBt, DIEA, DMF.
[AA1 = Bz-Nle-Lys-Arg.]
Scheme 3. Reagents: (a) Fmoc-Arg(Pbf)-OH, EDC, HOBt, DIEA,
DMF; (b) 20% TFA, CH2Cl2; (c) bis-Boc-pyrazole-1-carboxamidine,
DMAP, THF; (d) piperidine, DMF; (e) Bz-Nle-Lys(Boc)-OH, EDC,
HOBt, DIEA, DMF; (f) Dess–Martin periodinane, CH2Cl2; (g) 95%
TFA, CH2Cl2. [AA1 = Bz-Nle-Lys-Arg.]

38
Z. Yin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 36–39
Scheme 4. Reagents: (a) Bz-Nle-Lys(Boc)-Arg(Pbf)-OH, IBCF, THF;
(b) NaN3, DMF; (c) H2, Pd(OH)2/C, MeOH; (d) bis-Boc-pyrazole-1-
carboxamidine, DMAP, MeOH; (e) TFA, CH2Cl2; (f) PhB(OH)2,
diethyl ether/water. [AA1 = Bz-Nle-Lys-Arg.]
Table 1. Inhibition of dengue virus NS3 protease by tetrapeptide
analogs containing different warheads
Figure 2. Lineweaver–Burk plot of aldehyde 7.
Compound
Ki (lM)a
micromolar concentration. The reason for this unusual
result was found to be the quick degradation of a-keto-
amides in the enzyme assay buffer medium (pH 8.5) dur-
ing incubation to a seven-membered ring lactam (22),
which presumably, is inactive because of the loss of
the key interaction between P1(Arg) side-chain and the
enzyme. To provide additional structural evidence for
the formation of lactam, a model study was performed
as shown in Scheme 5 and the structure of lactam 24
was confirmed by 2D NMR.
1
Bz-Nle-Lys-Thr-Arg-OH
Bz-Nle-Lys-Arg-Arg-OH
>500
>500
>500
127.5
>500
>500
5.8
2
3
Bz-Nle-Lys-Thr-Arg-NH2
Bz-Nle-Lys-Arg-Arg-NH2
Bz-Nle-Lys-Arg-Arg-NHSO2CF3
Bz-Nle-Lys-Thr-Arg-H
4
5
6
7
Bz-Nle-Lys-Arg-Arg-H
11
13-a
13-b
18
21
Bz-Nle-Lys-Arg-Arg(OH)-CONH-Bn
Bz-Nle-Lys-Arg-Arg-Benzoxazole
Bz-Nle-Lys-Arg-Arg-Thiazole
Bz-Nle-Lys-Arg-Arg-CF3
178
82.9
42.8
0.85
0.043
Bz-Nle-Lys-Arg-Arg-B(OH)2
Inhibitors incorporating the a-keto heterocycle moiety
like 13-a and 13-b proved to be less active than aldehyde
7. These heterocyclic groups gave potent inhibitors with
the related serine proteases elastase, chymase, and
thrombin.22 We surmise that their relatively poor activ-
ity compared to aldehyde 7 may be due to either the dif-
ference of carbonyl reactivities or some steric restriction
in the active site of the enzyme, which prevented optimal
binding.
a Each Ki is the average of at least two independent determinations.
C virus NS3-4A serine protease with a Ki of 0.6 lM.18 In
our hands, carboxylic acids 1 and 2 failed to show activ-
ity against dengue serine protease. Furthermore, peptide
analog 5 containing trifluoroacetylsulfonamide, which
is a known isostere for carboxylic acids, was not
active as well.19 Simple amides are known to be relative-
ly inert toward certain serine proteases and can serve
as substrate-like enzyme inhibitors.20 As a consequence,
substrate-based tetrapeptide amides 3 and 4 were
explored. Amide 4 corresponding to the best substrate
S1 showed activity at high micromolar concentration.
In general, peptides without well-established serine
protease electrophilic warheads did not show useful
inhibition of dengue serine protease.
Potent inhibitors were identified by incorporating triflu-
oromethyl ketone and boronic acid onto the substrate
peptide. Tetrapeptide boronic acid 21 proved to be the
most potent inhibitor of dengue virus NS3 protease with
a Ki of 43 nM. The affinity of trifluromethyl ketone 18
was intermediate between those of peptide aldehyde 7
and peptide boronic acid 21.
In the present investigation, we have examined the
major requirements for nanomolar inhibition of the
dengue NS3 protease. Starting with the best known
Peptide analogs containing electrophilic warheads are,
in general, good inhibitors of serine proteases.21 There-
fore, the effect of different electrophilic warheads on
the inhibition of dengue protease was studied. We ini-
tially chose to study the substrate-based peptide alde-
hydes due to the ease of synthesis, which resulted in
the identification of low micromolar inhibitors (e.g.,
aldehyde 7, Ki 5.8 lM). Not surprisingly, this aldehyde
(7) was found to be a competitive inhibitor of dengue
NS3 protease as shown by Lineweaver–Burk plot
(Fig. 2). However, the aldehdye analog (6), correspond-
ing to the second best substrate S2,6 did not show any
activity against dengue protease.
Subsequently, a-ketoamides were synthesized to deter-
mine their efficacy against dengue protease and they
showed no effect up to 500 lM concentration, whereas
a-hydroxyamides showed inhibitory activity at high
Scheme 5. Degradation of a-ketoamides. Reagents and conditions:
(a) Enzyme assay buffer medium, pH 8.5.

Z. Yin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 36–39
39
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contrast to HCV NS3 protease, cleavage products and
their analogs do not appreciably inhibit the Dengue
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