Structural variations in keto-glutamines for improved inhibition against hepatitis A virus 3C proteinase

Article abstract of DOI:10.1016/j.bmcl.2004.05.021

A series of keto-glutamine tetrapeptide analogs containing a 2-oxo-pyrrolidine ring as a glutamine side chain mimic were synthesized with both R and S configuration at the β-carbon. Compounds bearing a phthalhydrazide moiety show improved reversible inhib

Full text of DOI:10.1016/j.bmcl.2004.05.021

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3655–3658
Structural variations in keto-glutamines for improved inhibition
against hepatitis A virus 3C proteinase
Rajendra P. Jain and John C. Vederas*
Department of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2
Received 12 April 2004; revised 11 May 2004; accepted 12 May 2004
Available online
Abstract—A series of keto-glutamine tetrapeptide analogs containing a 2-oxo-pyrrolidine ring as a glutamine side chain mimic were
synthesized with both R and S configuration at the b-carbon. Compounds bearing a phthalhydrazide moiety show improved
reversible inhibition of HAV 3C proteinase in the low micromolar range.
Ó 2004 Elsevier Ltd. All rights reserved.
Hepatitis A virus (HAV) is a causative agent of an acute
form of infectious hepatitis,1 and belongs to the picor-
navirus family that contains more than 200 known
members, including other pathogens such as human
rhinovirus (HRV), foot and mouth disease virus,
poliovirus (PV), and encephalomyocarditis virus
(EMCV). Picornaviruses possess a small positive single-
stranded RNA genome whose translation in the host
cells produces a single ꢀ250 kDa polyprotein, proteo-
lytic processing of which is a core feature of the viral
replication strategy. In hepatitis A virus, the 3C cysteine
proteinase is the key enzyme necessary for cleavage of
the primary polyprotein.2 Hence, potent and selective
inhibitors of 3C proteinase could serve as attractive
targets for the development of new antiviral therapeutic
agents.
Figure 1. HAV 3C and HRV 3C proteinase inhibitors.
Our previous studies on picornaviral 3C proteinases
have focused on synthesis and evaluation of several
glutamine analogs as effective inhibitors of these en-
as intramolecular hydrogen bonding to the carbonyl,
which makes it more electrophilic and potentially sus-
ceptible to hemithioacetal formation with the active site
cysteine. We now report synthesis and evaluation of
structurally modified keto-glutamines that exhibit im-
proved inhibition of the HAV 3C proteinase.
zymes from human rhinovirus-14 (HRV-14) and hepa-
titis A virus (HAV).3 In particular, we have recently
reported3b that the keto-glutamine analog 1 (Fig. 1) is a
good reversible inhibitor of HAV 3C proteinase, with an
IC50 value in the low micromolar range (13 lM). This is
probably due to the combined effect of the b and b0
amino functionalities adjacent to the keto group as well
Our attention focused on the 2-oxo-pyrrolidine ring in
the glutamine side chain of AG7088 (2, Fig. 1), a potent,
nontoxic antirhinoviral agent that irreversibly inhibits
HRV 3C proteinase from different virus serotypes.4
Based on the close structural and mechanistic similari-
ties of various 3C viral proteinases, it appeared that
Keywords: Cysteine protease; Hepatitis A virus; Inhibitors; Glutamine
analogs; Phthalhydrazides.
* Corresponding author. Tel.: +1-780-492-5475; fax: +1-780-492-8231;
e-mail: john.vederas@ualberta.ca
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.021

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R. P. Jain, J. C. Vederas / Bioorg. Med. Chem. Lett. 14 (2004) 3655–3658
incorporation of the 2-oxo-pyrrolidine ring moiety of 2
into the P1 position of 1 could generate potent inhibitors
of the HAV 3C enzyme.
spectrofluorometer. Samples were pre-incubated with
inhibitor for 15 min. Initial rates were calculated using
the first 3 min of the progress curves. The results (Table
1) indicate that compounds 8a–c show considerable
improvement of the reversible inhibition (IC50 ¼ 1.6–
Thus, N-Boc-L-glutamic acid dimethyl ester (3) was
converted into cyclic glutamine derivative 4 by following
the literature route.5 This protocol involved stereose-
lective b-alkylation6 of 3 with bromoacetonitrile fol-
lowed by reduction of nitrile and base promoted ring
closure to form 2-oxo-pyrrolidine ring with ‘S’ stereo-
chemistry (final cyclization step simplified: satd aq
NaHCO3/CH2Cl2, rt, 70% yield). Hydrolysis of methyl
ester in 4 (LiOH/THF, 0 °C) produced the correspond-
ing acid in quantitative yield. This was then converted
into diazo ketone 5 in 85% yield. Treatment of 5 with aq
HBr generated the bromomethyl ketone 6 (89% yield)
that was reacted with sodium phthalhydrazide in
DMF3b to produce 7a–c in 15–33% isolated yield. Re-
moval of Boc group in 7a–c (TFA/CH2Cl2) and cou-
pling with the tripeptide Ac-Leu-Ala-Ala-OH (HBTU
or HATU, DIPEA, DMF, rt) afforded the tetrapeptides
8a–c in 39–55% yield (Scheme 1).
2.5 lM) compared to the parent compound
(IC50 ¼ 13 lM).
1
In order to probe the effect of S stereocenter in the
pyrrolidone ring of 8a–c on the inhibition of the HAV
3C proteinase, compound 15 was synthesized with ‘R’
configuration at the ring stereocenter, as outlined in
Scheme 2. Thus, L-pyroglutamic acid (9) was converted
into pyrrolidone 10 as reported.8 This involved reduc-
tion of carboxylic acid moiety of 9 followed by silyl
protection of the resulting alcohol and Boc protection of
amide nitrogen. Alkylation of 10 with LiHMDS/
bromoacetonitrile gave compound 11 as 12:1 diaste-
reomeric mixture (by 1H NMR analysis) from which the
desired major anti-isomer was readily separated by silica
gel chromatography (85% yield). Hydrogenation of the
nitrile functionality in 11 (PtO2, 50 psi H2, EtOH/
CHCl3) generated the amine 12 that upon treatment
with LiOH (THF/H2O) undergoes intramolecular acyl
transfer to afford 13. Removal of TBDPS group (TBAF,
THF/AcOH) and oxidation of resulting alcohol (RuCl3/
NaIO4) produced the carboxylic acid 14 that was con-
verted into 15 by following steps similar to those used
for synthesis of 8a–c.
Assay of 8a–c for inhibition of HAV 3C proteinase
employed an overexpressed C24S mutant in which the
nonessential surface cysteine is replaced with serine and
which displays catalytic parameters indistinguishable
from the wild-type proteinase.7 The enzyme activity is
monitored using a continuous fluorometric assay.
Cleavage reactions (700 lL) were performed at 22 °C in
a solution containing 100 mM KH2PO4/K2HPO4 at
pH 7.5, 2 mM EDTA, 0.1 mg/mL bovine serum albumin
Other heterocyclic keto-glutamines, such as triazole
derivatives 18, 20, and 21, which lack the activation of
ketone carbonyl group by hydrogen bonding, were also
synthesized as outlined in Scheme 3. Thus, bromide 6
was converted into the corresponding azide 16 with
NaN3/KF (58% yield). Treatment of 16 with dimethyl
acetylenedicarboxylate generated the triazole 17 in
quantitative yield, which upon removal of Boc-protec-
tion (TFA/CH2Cl2) and coupling with Ac-Leu-Ala-Ala-
OH (HATU, DIPEA, DMF, rt) gave 18 in 65% yield.
Similarly, bromide 19 was converted into 20 in 58%
overall yield. Hydrolysis of methyl esters in 20 (LiOH,
MeOH/H2O) produced the diacid 21 in 80% yield. Assay
of 15, 18, 20, and 21 for inhibition of HAV 3C pro-
teinase were done as described for 8a–c (Table 1).
(BSA),
10 lM
fluorogenic
substrate
Dabcyl-
GLRTQSFS-Edans (Bachem), 0.1 lM of proteinase and
1% DMF. Increase in fluorescence (kex 336 nm, kem
472 nm) was monitored using a Shimadzu RF5301
As outlined in Table 1, the keto-glutamine 15
(IC50 ¼ 62.6) with R configuration at pyrrolidone ring
shows 25-fold weaker reversible inhibition of the HAV
3C enzyme as compared to the corresponding S diaste-
reomer 8a (IC50 ¼ 2.5 lM). The triazoles 18, 20, and 21
that lack ketone activation by intramolecular hydrogen
bonding exhibit very poor inhibition (1–14% inhibition
at 100 lM inhibitor concentration), despite being dia-
mino ketones. These results indicate that the cyclic
glutamines with S configuration at pyrrolidone stereo-
center are good reversible inhibitors of HAV 3C pro-
teinase and support the proposal3b that the activation of
‘keto-glutamine’ war-head by intramolecular H-bonding
may be essential for strong inhibition. Although struc-
tural studies on these enzyme–inhibitor complexes are
not yet available, it seems likely that the enzyme’s active
site thiol (Cys172) adds reversibly to the keto function-
Scheme 1. Reagents and conditions: (a) LiOH, THF/H2O, 0 °C,
quant.; (b) EtOCOCl, Et3N, THF, ꢁ30 °C, then CH2N2/Et2O, ꢁ30 °C
to rt, 85%; (c) 48% aq HBr, THF, 0 °C, 89%; (d) phthalhydrazide,
NaH, DMF, rt, 15–33%; (e) TFA–CH2Cl2 1:1, 0 °C, 1.5 h, quant.; (f)
Ac-Leu-Ala-Ala-OH, HBTU, or HATU, DIPEA, DMF, rt, 39–55%.

R. P. Jain, J. C. Vederas / Bioorg. Med. Chem. Lett. 14 (2004) 3655–3658
Table 1. Evaluation of compounds 8a–c, 15, 18, and 20–21 as
reversible inhibitors of HAV 3C proteinase
3657
Compound
IC50
% Inhi-
valuesa bitiona;b
(lM)
H
O
N
O
O
O
O
H
AcHN
N
N
H
N
N
2.5
H
O
O
HN
8a
H
N
O
O
O
O
O
H
1.6
2.0
AcHN
N
N
H
N
N
H
O
O
HN
NO
2
8b
H
Scheme 2. Reagents and conditions: (a) BrCH2CN, LiHMDS, THF,
ꢁ78 °C, 85%; (b) PtO2, 50 psi H2, EtOH/CHCl3, rt, 48 h; (c) LiOH,
THF/H2O, rt, 12 h, 63% over two steps; (d) TBAF, THF/AcOH, 0 °C–
rt, 18 h, 85%; (e) RuCl3/NaIO4, MeCN/CCl4/H2O, 0 °C–rt, 64%;
(f) EtOCOCl, Et3N, THF, ꢁ30 °C, then CH2N2/Et2O, ꢁ30 °C to rt,
82%; (g) 48% aq HBr, THF, 0 °C, 80%; (h) phthalhydrazide, NaH,
DMF, rt, 32%; (i) TFA–CH2Cl2 1:1, 0 °C, 1.5 h, quant.; (j) Ac-Leu-
Ala-Ala-OH, HATU, DIPEA, DMF, rt, 45%.
O
N
O
O
O
F
F
H
AcHN
N
F
F
N
H
N
H
N
O
O
HN
O
8c
H
O
N
O
O
O
O
H
N
62.6
AcHN
N
H
N
H
N
O
O
O
O
O
HN
15
H
O
N
O
O
H
N
14
AcHN
N
N
H
N
N
N
H
O
MeO C
2
CO Me
2
18
CONMe
2
O
O
H
N
AcHN
N
9
N
H
N
N
N
H
O
MeO C
2
CO Me
2
20
CONMe
2
O
O
H
N
AcHN
N
1
N
H
N
H
N
N
O
HO C
2
Scheme 3. Reagents and conditions: (a) NaN3, KF, DMF, rt, 1 h,
58%; (b) dimethyl acetylenedicarboxylate, toluene, rt, 3 d, quant.; (c)
TFA–CH2Cl2 1:1, 0 °C, 1.5 h, quant.; (d) Ac-Leu-Ala-Ala-OH,
HATU, DIPEA, DMF, rt, 65%; (e) LiOH, MeOH/H2O, 80%.
CO
H
2
21
a HAV 3C (0.1 lM), Dabcyl-GLRTQSFS-Edans (10 lM), EDTA
(2 mM), BSA (0.1 mg/mL), KH2PO4/K2HPO4 (100 mM) at pH 7.5,
22 °C, 15 min preincubation of the enzyme with inhibitor. The IC50
values reported are within ꢂ10% error.
modifications in the glutamine side chain as well as by
substitutions on the phthalhydrazide ring. Investigations
on the detailed structure of the inhibitor enzyme com-
plexes and on improved inhibitors are in progress.
b Inhibition at [I] 100 lM.
ality of the keto-glutamine P1 mimic in these substrate
analogs to transiently form a hemithioacetal.
Acknowledgements
In conclusion, inhibition studies of HAV 3C proteinase
were performed with various keto-glutamine inhibitors
and the structural factors affecting the inhibition were
examined. These studies indicate that there is further
scope of improvement in inhibition of HAV 3C by
We are grateful to Prof. Lindsay Eltis (Departments of
Microbiology and Biochemistry, University of British
Columbia), Michael James (Department of Biochemis-
try, University of Alberta) and David Wishart

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(Department of Biological Sciences) for helpful discus-
sions. We thank Prof. Lindsay Eltis for providing HAV
3C (C24S) proteinase. Financial support from the Nat-
ural Sciences and Engineering Research Council of
Canada (NSERC), the Protein Engineering Network of
Centres of Excellence (PENCE), and the Canada Re-
search Chair in Bioorganic and Medicinal Chemistry is
gratefully acknowledged.
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