Pseudoxazolones, a new class of inhibitors for cysteine proteinases: Inhibition of hepatitis A virus and human rhinovirus 3C proteinases

Article abstract of DOI:10.1039/b109095j

Monophenyl and diphenyl pseudoxazolone derivatives of glycine and alanine were prepared and found to be time-dependent inhibitors of hepatitis A virus (HAV) 3C and human rhinovirus (HRV) 3C proteinases with IC₅₀ values in the micromolar range.

Full text of DOI:10.1039/b109095j

Pseudoxazolones, a new class of inhibitors for cysteine proteinases:
inhibition of hepatitis A virus and human rhinovirus 3C proteinases
Yeeman K. Ramtohul,a Nathaniel I. Martin,a Lara Silkin,a Michael N. G. James‡b and John C.
Vederas*†a
a Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
E-mail: john.vederas@ualberta.ca; Fax: +1 (780) 492 8231; Tel: +1 (780) 492 5475
b Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
E-mail: michael.james@ualberta.ca; Fax: +1 (780) 492 0886; Tel: +1 (780) 492 4550
Received (in Cambridge, UK) 9th October 2001, Accepted 14th November 2001
First published as an Advance Article on the web 6th December 2001
Monophenyl and diphenyl pseudoxazolone derivatives of
glycine and alanine were prepared and found to be time-
dependent inhibitors of hepatitis A virus (HAV) 3C and
human rhinovirus (HRV) 3C proteinases with IC50 values in
the micromolar range.
Cysteine proteinases have attracted much attention as ther-
apeutic targets because of their implication in many diseases
such as osteoporosis, Alzheimer’s, and arthritis.1 As part of our
ongoing effort on the inhibition of hepatitis A virus (HAV) and
human rhinovirus (HRV) 3C cysteine proteinases, we have
explored pseudoxazolones as a new motif for inhibition of these
enzymes.
Scheme 1 Reagents and conditions: (i) 2-chloro-2,2-diphenylacetyl chlo-
ride, propylene oxide, ethyl acetate, D (ii) DCC, propylene oxide, CH3CN,
80% over 2 steps. (iii) Ac2O, pyridine, 68% over 2 steps.
HAV causes an acute infection of the liver whereas HRV is
the major causative agent for the common cold in humans.2 Like
other picornaviruses, the HAV and HRV genome consists of a
small positive strand RNA, which upon translation produces a
single polyprotein precursor. This polyprotein undergoes multi-
ple proteolytic cleavages by the 2A and/or 3C proteinases to
produce the structural and nonstructural viral components.3 In
HAV, these cleavages are mediated solely by the 3C enzyme, a
cysteine proteinase present in all picornaviruses.4 Crystal
structures of HAV 3C5 and HRV 3C6 have revealed that these
enzymes are distinct from the papain cysteine proteinases and
have folds that closely resemble the chymotrypsin serine
proteinases.
Several potent inhibitors of HAV 3C and HRV 3C protei-
nases have been reported by our group7 and by others.8 The
ability of pseudoxazolones to react with thiols at the imine9
position suggests that these compounds can be used to inhibit
thiol containing enzymes (Fig. 1). We first explored the
diphenyl pseudoxazolone analogue of glycine 1 and alanine 2.
These compounds are readily prepared using a modified
literature procedure.10 Condensation of glycine 3 or alanine 4
with 2-chloro-2,2-diphenylacetyl chloride (Scheme 1) gives the
corresponding halo adducts, which upon reaction with acetic
anhydride–pyridine or DCC undergo cyclization followed by
elimination of HCl to afford the desired compounds 1 and 2.
Testing of these compounds against HAV 3C C24S mutant§
indicates that 1 is a time dependent inhibitor with an IC50 of 33
mM (Table 1). However, the alanine derivative 2 was found to
Table 1 Inhibition data for HAV 3C and HRV 3C proteinases
Compound
HAV 3C IC50 (mM)a
HRV 3C IC50 (mM)b
1
2
5a
5b
6a
6b
33
> 100
6
4
26
38
> 100
16
17
43
> 100
> 100
a Fluorometric assay conditions: 0.1 mM HAV 3C, 10 mM Dabcyl-
GLRTQSFS-Edans, 2 mM EDTA, 0.1 mg/mL BSA, 100 mM KH2PO4–
K2HPO4, 1% DMF, pH 7.5, 5 min pre-incubation of the enzyme with
inhibitor. b Continuous UV assay conditions: 0.4 mM HRV 3C, 250 mM
EALFQ-pNA, 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% DMF, pH
7.5, 5 min pre-incubation of the enzyme with inhibitor.
be less active with an IC50 > 100 mM, possibly because the
methyl group in 2 offers some steric interaction to the
nucleophilic thiol of the enzyme. Our next approach was to
prepare the monophenyl pseudoxazolone analogues of glycine
5a,b and alanine 6a,b. These compounds can be synthesised as
depicted in Scheme 2.10b,c Although these pseudoxazolones
have previously been reported as a mixture of E and Z isomers,
we were able to separate each isomer by flash column
chromatography for full characterisation. The E and Z config-
urations were assigned based on the proton (1H) chemical shift
of the olefinic hydrogen. The E isomer has its olefinic proton
Fig. 1 Possible mode of inactivation by cysteine proteinase.
Scheme 2 Reagents and conditions: (i) DL-2-chloro-2-phenylacetyl chlo-
ride, NaOH, H2O, 0–5 °C; (ii) Ac2O, pyridine, the yields reported are over
two steps.
† Canada Research Chair in Bioorganic and Medicinal Chemistry.
‡ Canada Research Chair in Protein Structure and Function.
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Chem. Commun., 2001, 2740–2741
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b109095j

aligned with the oxazolone ring oxygen and is more deshielded
(Fig. 2) compared to the Z isomer which is aligned with the
imine nitrogen. These observations were further supported by
two X-ray crystal structures of 5b and 6a¶ (Fig. 3). Although
interconversion of the E and Z double bond is possible, this was
not observed for the monophenyl pseudoxazolones. Assay of 5a
and 5b against HAV 3C proteinase gives time dependent
inhibition with IC50 of 6 mM and 4 mM, respectively. However,
compounds 6a and 6b display different levels of inhibition. The
E isomer 6a is a time dependent inhibitor with an IC50 of 26 mM.
However, the Z isomer 6b displays weaker inhibition, with an
IC50 > 100 mM, indicating that the enzyme has a selectivity for
the E over the Z isomer when there is substitution at the imine
carbon. Other derivatives with different substitution at the imine
position (data not shown) display the same type of behaviour.
This is possibly due to some unfavourable electronic interaction
of the phenyl group of 6b in the active site of the enzyme.
Assays of these pseudoxazolones against HRV 3C proteinase
(Table 1) also show the same pattern of inhibition as that
observed for the HAV 3C enzyme. Although the inhibition
levels are weaker for the HRV 3C proteinase, it should be noted
that the amount of enzyme used in the HRV assay (0.4 mM) is
more than that for HAV (0.1 mM) because of the limitations of
the UV assay used for the former.
versity of Alberta) for enzyme preparation and helpful sugges-
tions. We are grateful to Dr Robert McDonald for crystallo-
graphic analyses. These investigations were supported by the
Natural Sciences and Engineering Research Council of Canada
(scholarship to N. I. M.; summer studentship to L. S.) and the
Alberta Heritage Foundation for Medical Research (scholarship
to Y. K. R.).
Notes and references
§ Enzyme inhibition studies for HAV 3C employed a recombinant C24S
mutant in which a non-catalytic external cysteine (Cys24) was replaced by
serine and which displays catalytic properties indistinguishable from the
wild type enzyme. For an ideal peptide substrate mimicking the 2B/2C
junction of the large precursor polyprotein with glutamine preferred at the
P1 residue: the kcat of this enzyme is about 1.8 s21 with a Km of 2.1 mM at
pH 7.5.4 HRV 3C assays used the 3C proteinase from serotype 14.7a
¶ Crystal data for 5b: C10H7NO2: M = 173.17, triclinic, a = 5.5390(6), b
= 7.2645(8), c = 10.4291(11) Å, a = 83.673(2), b = 83.789(2), g =
¯
80.624(8)°, U = 409.77(8) Å3, T = 193 K, space group P1 (No. 2), Z = 2,
m(Mo-Ka) = 0.100 mm21, 2179 reflections measured, 1641 unique (Rint
=
0.0164) which were used in all least square calculations, R1(F) = 0.0374
2
(for 1365 reflections with Fo ! 2s(Fo2)), wR2(F2) = 0.1037 (for all unique
relections).
Crystal data for 6a: C11H9NO2: M = 187.19, triclinic, a = 6.0008(8), b
In summary, we have shown that pseudoxazolones are potent
inhibitors of HAV and HRV 3C enzymes with micromolar IC50
values in the range. Although the monophenyl pseudoxazolones
of glycine 5a and 5b show comparable inhibition, this is not the
case with the alanine derivatives, indicating that the enzymes
have selectivity for the E 6a over the Z isomer 6b. X-Ray crystal
structures of 5b and 6a have helped to assign the geometrical
configuration of the monophenyl pseudoxazolone analogues.
The inhibition data and mass spectrometry suggest that the
enzyme forms a covalent bond with the inhibitors. Further
mechanistic studies involving 13C labelled analogues and
pseudoxazolones with other substitution patterns are in pro-
gress. The use of pseudoxazolones for inhibition of other
cysteine proteinases is also being explored.
= 7.2370(11), c = 10.9282(16) Å, a = 95.403(2), b = 101.029(3), g =
¯
102.046(3)°, U = 451.10(11) Å3, T = 193 K, space group P1 (No. 2), Z =
2, m(Mo-Ka) = 0.096 mm21, 2409 reflections measured, 1827 unique (Rint
= 0.0422) which were used in all least square calculations, R1(F) = 0.0449
2
(for 1207 reflections with Fo ! 2s(Fo2)), wR(F2) = 0.1113 for all unique
relections).
CCDC 172532 and 172533. See http://www.rsc.org/suppdata/cc/b1/
b109095j/ for crystallographic data in CIF or other electronic format.
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Bergmann and Craig Garen (Biochemistry Department, Uni-
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Fig. 2 1H Chemical shift of the glycine pseudoxazolone olefinic proton in
acetone-d6 (300 MHz).
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Fig. 3 Crystal structure of 5b (top) and 6a (bottom).
Chem. Commun., 2001, 2740–2741
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