Potent inhibition of norovirus by dipeptidyl α-hydroxyphosphonate transition state mimics

Article abstract of DOI:10.1016/j.bmcl.2013.08.073

The design, synthesis, and evaluation of a series of dipeptidyl α-hydroxyphosphonates is reported. The synthesized compounds displayed high anti-norovirus activity in a cell-based replicon system, as well as high enzyme selectivity.

Full text of DOI:10.1016/j.bmcl.2013.08.073

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5941–5944
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent inhibition of norovirus by dipeptidyl
-hydroxyphosphonate transition state mimics
a
Sivakoteswara Rao Mandadapu a, Mallikarjuna Reddy Gunnam a, Anushka C. Galasiti Kankanamalage a,
Roxanne Adeline Z. Uy a, Kevin R. Alliston a, Gerald H. Lushington b, Yunjeong Kim c, Kyeong-Ok Chang c,
William C. Groutas a,
⇑
a Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
b LiS Consulting, Lawrence, KS 66046, USA
c Department of Diagnostic Medicine and 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:
The design, synthesis, and evaluation of a series of dipeptidyl
synthesized compounds displayed high anti-norovirus activity in a cell-based replicon system, as well
as high enzyme selectivity.
a-hydroxyphosphonates is reported. The
Received 29 June 2013
Revised 12 August 2013
Accepted 15 August 2013
Available online 22 August 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
a-Hydroxyphosphonates
Transition state mimics
Norovirus 3CL protease
Inhibitors
Noroviruses are the most common cause of acute viral gastro-
enteritis in the US, and worldwide,1–3 accounting for ꢀ21 million
cases of gastroenteritis annually in the US alone.4 Noroviruses
are very stable in the environment and refractory to many com-
mon disinfectants, with only a few virions required to initiate virus
infection. Therefore, norovirus outbreaks are hard to contain using
routine sanitation, and even implementation of aggressive sanitary
measures often fails to prevent subsequent norovirus outbreaks.
The problem is further compounded by the lack of norovirus-
specific antiviral agents or effective vaccines.5 The challenges
associated with vaccine development, such as high viral diversity
and short-term immunity,6 and the need for aggressive sanitary
measures for combating continuous outbreaks of norovirus-associ-
ated gastroenteritis, render norovirus infection a serious public
health problem and underscores the importance of developing
small molecule anti-norovirus therapeutics and prophylactics.
Noroviruses have a single-stranded, positive sense 7–8 kb RNA
genome that encodes a polyprotein precursor that is processed
by a virus-encoded 3C-like cysteine protease (3CLpro) to generate
mature non-structural proteins. Processing of the polyprotein by
3CLpro is essential for virus replication.7 We have recently de-
scribed the structure-based design, synthesis, and evaluation of
transition state inhibitors of norovirus 3CLpro, including peptidyl
aldehydes,8 peptidyl
a-ketoamides and a-ketoheterocycles and
their corresponding bisulfite salts,9,10 and macrocyclic inhibitors.11
These studies have demonstrated that representative members of
these classes of compounds potently inhibit norovirus 3CL prote-
ases from various genogroups and exhibit significant anti-
norovirus activity in
a
cell-based replicon system.12 Taken
together, the results of these studies are strongly supportive of
the notion that norovirus 3CLpro is a druggable target that is
well-suited to the discovery and development of anti-norovirus
small molecule therapeutics and prophylactics.
In continuing our endeavors in this area,8–13 we describe herein
the results of preliminary studies related to the inhibition of
3CLpro by peptidyl
To our knowledge, this is the first time that
a
-hydroxyphosphonates (Fig. 1, structure (I)).
-hydroxyphospho-
a
nate transition state mimics have been used in the inhibition of a
viral cysteine protease.
The design of inhibitor (I) rested on the following consider-
ations: (a) previous studies have shown that the
a
-hydroxyester14
and
a
-hydroxyphosphonate15 moieties function as effective transi-
tion state mimics which yield highly potent inhibitors when linked
to a peptidyl recognition element that is tailored to the substrate
specificity of a target protease. This approach has been successfully
used in the design of highly effective inhibitors of human renin;15
(b) NV 3CLpro is a cysteine endoprotease with a chymotrypsin-like
fold, a His-Cys-Glu triad, and an extended binding site.12,16–18 Map-
ping of the active site of 3CLpro using chromogenic and fluorogenic
⇑
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 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.073

5942
S. R. Mandadapu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5941–5944
lithium borohydride, followed by oxidation with Dess–Martin peri-
O
R1
OH
R2
R2
H
N
O
odinane reagent,25 yielded the corresponding dipeptidyl aldehyde
which was reacted with an array of dialkyl phosphites and diiso-
propylethylamine in dichloromethane to yield the desired com-
pounds as mixtures of epimers26,27 (listed in Table 1). The
reaction sequence chosen is highly tractable and permits facile
modification of the cap by reaction with an array of structurally-
diverse alcohols. This is of paramount importance in terms of
optimizing pharmacological activity, ADMET, and PK via cap
modifications.28 The inhibitory activity of the generated
O
N
H
P
O
O
O
NH
O
(I)
Figure 1. General structure of inhibitor (I).
a
-hydroxyphosphonate derivatives was evaluated against norovi-
substrates has shown that the protease has a strong preference for
a -D/E-F/Y-X-L-Q-G- sequence, where X is H, E or Q, and cleavage is
at the Q-G (P1-P1’)19 bond;16,20–22 (c) we have previously demon-
strated that the presence of a P2 cyclohexyl alanine residue results
in a significant enhancement in potency and cellular permeabil-
ity.10,23 Based on the aforementioned considerations, it was envis-
aged that an inhibitor represented by general structure (I) (Fig. 1)
may display high anti-norovirus activity in a cell-based replicon
system mediated via the inhibition of NV 3CLpro.
Inhibitor (I) was readily constructed by transforming commer-
cially available (L) cyclohexyl alanine to the corresponding ester
and subsequent conversion of the ester to the isocyanate by reflux-
ing with trichloromethyl chloroformate in dioxane (Scheme 1).
Subsequent reaction with an appropriate alcohol yielded the
N-protected amino acid ester which was hydrolyzed with LiOH
in aqueous THF to yield the corresponding acid, which was then
coupled to the methyl ester of a previously-reported P1 glutamine
surrogate.24 Reduction of the N-protected amino acid ester with
rus in a cell-based replicon system.29–32 The selectivity of a repre-
sentative member of this series of compounds was also assessed
using a panel of proteases (Table 2).
It is clearly evident from the results summarized in Table 1 that
-hydroxyphosphonates exhibit potent anti-norovirus activity in a
a
cell-based replicon system. Consistent with previous findings,10
the replacement of the P2 leucine with cyclohexyl alanine en-
hanced the inhibitory activity of the compounds (compare 7a/7d
and 7b/7e in Table 1). The effect of varying R2 (assumed to be pro-
jecting toward the S’ subsites) on potency was also explored. With
the exception of compound 7e, variations in R2 yielded compounds
with submicromolar EC50 values, with compounds 7d and 7g hav-
ing the highest potency.
Toxicity arising from off-target effects is a potential caveat in
viral protease drug development since the inhibitors may also
inhibit host proteases. Thus, the selectivity profile of
a-hydroxy-
phosphonate (7d) was investigated using a representative panel
of proteases (Table 2). Compound 7d was devoid of any inhibitory
O
R1
R1
O
R1
a, b
OH
OCH3
c
OCH3
O
O
N
H
O
N
H
H2N
(HCl)
O
O
O
2
1
R1
R1
H
N
O
H
N
d
e
O
N
H
OH
NH
COOCH3
NH
O
N
H
O
O
H2N
(HCl)
COOCH3
O
O
O
5
4
NH
O
3
R1
O
OH
R1
O
O
R2
R2
H
H
N
N
O
N
P O
g
f
O
N
H
H
H
O
O
O
O
P
O
R2
R2
H
NH
NH
O
O
O
7a-h
6
R2 = ethyl (7a, 7d)
methyl (7b, 7e)
H (7c)
R1 = isobutyl (7a-c)
cyclohexylmethyl (
7d-h)
n-butyl (7f)
trifluoroethyl (7g)
benzyl (7h)
1
Reagents
: (a) CCl3O(C=O)Cl/dioxane; (b) Triethylamine/R OH; (c) LiOH/THF/H2O; (d) EDCI/HOBt/DIEA/DMF;
(e) LiBH4/THF; (f) Dess-Martin periodinane/DCM; (g) DIEA/DCM
Scheme 1. Synthesis of compounds 7a–h.

S. R. Mandadapu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5941–5944
5943
Table 1
Anti-norovirus activity of inhibitor (I)
R1
OH
O
R2
H
N
O
R2
O
N
P O
H
O
O
(I)
NH
O
Compound
R1
R2
IC50
l
Ma
EC50 l
Ma
7a
7b
7c
7d
7e
7f
Isobutyl
Isobutyl
Isobutyl
Cyclohexylmethyl
Cyclohexylmethyl
Cyclohexylmethyl
Cyclohexylmethyl
Cyclohexylmethyl
Ethyl
Methyl
H
Ethyl
Methyl
n-Butyl
Trifluoroethyl
Benzyl
11.5
20.2
NDb
3.5
0.8
3.5
7.5
0.25
2.8
0.5
8.3
NDb
6.5
7g
7h
0.35
0.6
NDb
a
Determined as described in Ref. 12.
ND: not determined.
b
Table 2
Enzyme selectivity of compound 7d
Enzyme
HNEb,36
Inhibitiona (%)
15 ([If] = 17.5
0 ([If] = 2.5 M)
0 ([If] = 125 M)
M)
M)
l
M)c
a
-Chymotrypsin37
l
l
l
Trypsin38
Carboxypeptidase A39
Thrombin40
0 ([If] = 43
Figure 2. Computationally predicted conformers for noncovalent tetrahedral
mimics A (a-OH) (CPK colored sticks, with green carbons) and B (b-OH) (CPK
0 ([If] = 2.75
l
sticks; purple carbons) bound to the catalytic site of NV 3C protease (Connolly
surface colored as follows: yellow = nonpolar groups; white = weakly polar alkyl
and aryl groups; cyan = polar H’s, blue = polar N’s and red = polar O’s). In both cases,
the predicted conformer of the corresponding tetrahedral adduct (thin sticks; CPK
colors with black carbons is shown for reference.
a
Determined by incubating enzyme and inhibitor in
buffer solution for 30 min at an [I]/[E] ratio of 250.
b
HNE, human neutrophil elastase.
Final inhibitor concentration.
c
Acknowledgment
activity against basic serine (trypsin, thrombin) and metallo- (car-
boxypeptidase A) proteases, as well as no or very low activity
The generous financial support of this work by the National
Institutes of Health (AI081891) is gratefully acknowledged.
against neutral serine proteases (a-chymotrypsin, human neutro-
phil elastase), attesting to the high enzyme selectivity of this class
of compounds.
References and Notes
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anti-norovirus activity and enzyme selectivity of a series of dipep-
tidyl a-hydroxyphosphonates.

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