Structure-based design of a parallel synthetic array directed toward the discovery of irreversible inhibitors of human rhinovirus 3C protease

Article abstract of DOI:10.1021/jm010435c

Utilizing the tools of parallel synthesis and structure-based design, a new class of Michael acceptor-containing, irreversible inhibitors of human rhinovirus 3C protease (HRV 3CP) was discovered. These inhibitors are shown to inhibit HRV-14 3CP with rates

Full text of DOI:10.1021/jm010435c

2016
J . Med. Chem. 2002, 45, 2016-2023
Str u ctu r e-Ba sed Design of a P a r a llel Syn th etic Ar r a y Dir ected Tow a r d th e
Discover y of Ir r ever sible In h ibitor s of Hu m a n Rh in ovir u s 3C P r otea se
Theodore O. J ohnson,* Ye Hua, Hiep T. Luu, Edward L. Brown, Fora Chan, Shao Song Chu,†
Peter S. Dragovich, Brian W. Eastman,‡ Rose Ann Ferre, Shella A. Fuhrman, Thomas F. Hendrickson,§
Fausto C. Maldonado, David A. Matthews, J ames W. Meador, III, Amy K. Patick, Siegfried H. Reich,
Donald J . Skalitzky, Stephen T. Worland,| Michelle Yang, and Leora S. Zalman
Pfizer Global R&D-La J olla/ Agouron Pharmaceuticals, Inc., 10770 Science Center Road, San Diego, California 92121
Received September 17, 2001
Utilizing the tools of parallel synthesis and structure-based design, a new class of Michael
acceptor-containing, irreversible inhibitors of human rhinovirus 3C protease (HRV 3CP) was
discovered. These inhibitors are shown to inhibit HRV-14 3CP with rates of inactivation ranging
from 886 to 31 400 M-1 sec-1. These inhibitors exhibit antiviral activity when tested against
HRV-14 infected H1-HeLa cells, with EC50 values ranging from 1.94 to 0.15 µM. No cytotoxicity
was observed at the limits of the assay concentration. A crystal structure of one of the more
potent inhibitors covalently bound to HRV-2 3CP is detailed. These compounds were also tested
against HRV serotypes other than type 14 and were found to have highly variable activities.
In tr od u ction
The human rhinoviruses (HRVs) are members of the
picornavirus family and are the single most causative
agent of the common cold.1 Because over 100 serotypes
of the virus exist, immunization would be an impractical
approach to prevention of infection. Rhinoviruses con-
tain a positive-sense strand of RNA that is translated
to a large polyprotein in infected cells. This polyprotein
is cleaved by viral proteases to yield mature viral
enzymes and structural proteins. The 3C protease (3CP)
does the majority of the proteolytic processing. Inhibi-
F igu r e 1. Selection of a core of parallel synthesis.
tion of this viral protease by a small molecule agent
retains its negligible cytotoxicity. Tripeptidyl aldehydes5
should stop viral replication and thus control the extent
and ketones6 have been reported, but these achieve only
of infection.
modest levels of antiviral activity. Many analogues of
Much work has been previously described regarding
the unsaturated ester, such as unsaturated ketones,
the design and development of substrate-derived trip-
amides, and cyclic analogues have been tested, but
eptidyl, Michael acceptor-containing HRV 3CP inhibi-
unacceptable losses in antiviral activity were encoun-
tors,2 such as compound 1.2b This molecule is comprised
tered.7 Stability tests such as incubation in the presence
of a substrate-derived tripeptide binding determinant
of dog and human blood plasma esterases have shown
that provides affinity and selectivity for the target
that this unsaturated ester has a degree of stability that
protease and a Michael acceptor moiety that forms a
may lead to resistance toward metabolic degradation.2e
The Michael acceptor-ethyl ester compound 1 also did
not react when incubated with high concentrations of
covalent adduct with the active site cysteine (Cys-147)
of the enzyme. It has been our goal to develop low
molecular weight, nonpeptidic HRV 3CP inhibitors.
dithiothreitol (5 mM), suggesting that the inhibitor does
Other nonpeptidic inhibitors such as isatins3 and ho-
not react readily with nonenzymatic thiols.2e
The benefits of converting the P1 glutamine side chain
mophthalimides4 have been described in the literature.
Typically, these molecules have suffered from problems
into a lactam have been discussed in a previous paper.2e
such as cellular toxicity and modest antiviral activity.
Cyclization of the P1 glutamine side chain into a lactam
generally increases enzyme activity and antiviral activ-
ity, presumably by favorably altering the change in
entropy of the formation of the inhibitor-protein com-
plex. This modification also improves the physical
Efforts have been made toward finding a replacement
for the Michael acceptor-ethyl ester of 1 that maintains
antiviral potency, increases metabolic stability, and
* To whom correspondence should be addressed. Tel.: 858-622-7629.
properties (less peptidelike) of the resulting inhibitors.
With these factors in mind, we sought to develop a small
molecular version of 1, using the ethyl ester Michael
acceptor P1 lactam 2 as a core for high-throughput
synthesis of a large number of potential inhibitor
molecules. Specifically, we sought to replace the P2-
P4 portion of 1 with a smaller, nonpeptidic substituent.
Fax: 858-678-8156. E-mail: tjohnson@agouron.com.
† Current address: Quorex Pharmaceuticals, Inc., 2075 Corte del
Nogal, Suite J , Carlsbad, CA 92009.
‡ Current address: Merck Research Laboratories, 505 Coast Blvd.
So., La J olla, CA 92037.
§ Current address: Schro¨dinger, Inc., 3655 Nobel Dr., Suite 550,
San Diego, CA 92122-1003.
| Current address: Anadys Pharmaceuticals, Inc., 9050 Camino
Santa Fe, San Diego, CA 92121.
10.1021/jm010435c CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/12/2002

Inhibitors of Human Rhinovirus 3C Protease
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2017
Sch em e 1. Synthesis of 2a
a
Reagents: (a) DIBAH; MeOH; 6, 60%. (b) TBAF. (c) K2CO3, MeOH, 73% from 5. (d) S-BINAP-RuCl, H2 (1200 psi) 70%. (e) IBX;
Ph3PdCH-CO2Et. (f) HCl. (g)K2CO3, 60% from 3.
Str u ctu r e-Ba sed Design of a P a r a llel Syn th etic
Ar r a y
Ch em istr y
The synthesis of compounds such as 2 was described
in a previous paper;2e however, we sought to make this
preparation more amenable to scale-up by setting the
side chain chirality through the use of an asymmetric
hydrogenation. This change results in a shorter overall
route and eliminates the need for a stoichiometric
amount of chiral auxiliary that was used in the previous
route.
It was envisioned that a large number of compounds
could be produced in 96 well plate format by a solution
phase coupling reaction between the selected carboxylic
acids and the terminal nitrogen of the P1-Michael
acceptor compound 2, mediated by a resin-bound cou-
pling reagent. Because a large number of carboxylic
acids are commercially available, we sought to condense
this list to a more manageable number by selecting
reagents based on information from the cocrystal struc-
ture of the covalently bound complex between 1 and
HRV-2 3CP.8 Our goal was not to attempt the difficult
task of predicting the most active candidates but rather
to eliminate carboxylic acids that had little chance of
leading to a potent inhibitor, for reasons such as large
steric clashes with the target protein.
A virtual library of two-dimensional structures was
derived by coupling a list of carboxylic acids obtained
from the Available Chemicals Directory (ACD)9 to the
P1-Michael acceptor core 2 using proprietary software.
The resulting set of two-dimensional structures was
converted to three-dimensional structures using
CORINA version 1.7.10 These structures were then
energy-minimized in the Batchmin module of Macro-
model,11 using the AMBER* force field. The minimized
structures were docked into the target protein using an
in-house automated docking program.12 The P1-Michael
acceptor core of each docked structure was fixed in the
position observed in the cocrystal structure of 1 (active
site Cys-147 covalently bound to the â-carbon of the
former Michael acceptor at this point), and the remain-
ing portion of the molecule was docked into the S2-
S3-S4 sites of the protein. The docking procedure was
reiterated eight times, a process that resulted in some
molecules having several docked conformations. The
resulting set of docked structures was evaluated by
criteria such as number of hydrogen-bonding inter-
actions, hydrophobic interactions, and steric inter-
actions. For compounds with several docked conforma-
tions, only the lowest energy docked structure was
retained. Structures that led to high energy interactions
in the docking process were removed from consideration
for inclusion in the parallel synthetic array.
The synthetic route is shown in Scheme 1. Compound
8, derived from D-serine, is reduced with diisobutyl-
aluminum hydride (DIBAH) to the corresponding alde-
hyde 7 and then treated with Wittig reagent 6 to give
compound 5. This sequence was analogous to the one
described by Hanessian for the synthesis of Kainic acid
derivatives.13 The lactam Wittig reagent 6 was prepared
using a modified procedure of Ikuta.14 Compound 5 was
deprotected with potassium carbonate and methanol to
remove the acetate, followed by desilylation with TBAF
to produce 4. Several attempts were made at utilizing
a directed hydrogenation of 4 to set the side chain
chirality.15 These reactions gave low yields and low
selectivities. The use of S-2,2′-bis(diphenylphophino)-
1,1′-binaphthyl (BINAP)‚RuCl16 as a chiral catalyst in
the hydrogenation reaction produced 3 in good yields
with excellent diastereomeric purity. This reaction could
be easily carried out in 10 g scale using a Paar bomb
hydrogenator.17 The alcohol 3 was converted to the
desired product 2 by a one pot IBX18 oxidation-Wittig
sequence,19 followed by HCl-mediated deprotection of
the Boc-amine. The stereochemistry of 2 was confirmed
by correlation to material prepared by the literature
route2e and ultimately by examination of the crystal
structure of an inhibitor-protein complex (within this
paper).
A mixture of 2 and a selected carboxylic acid was
coupled using polystyrene-bound carbodiimide.25 No
attempts were made to purify the products. The product
purity was examined by liquid chromatography-mass
spectrometry (LC-MS) for a selection of representative
wells (the diagonals) and was typically found to be
greater than 80%. Synthesis of selected hits in larger
scale was carried out utilizing O-(7-azabenzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium (HATU) as a cou-

2018 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Ta ble 1. Active Compounds
J ohnson et al.
Ta ble 2. Multiple HRV Serotypes
compd
no.
HRV
serotype
kobs/[I]
EC50
(µM)
(M-1 s-1
)
17
14
16
2
1A
10
14
2
19 700
2200
385
ND
ND
0.16
ND
0.65
5.10
0.46
0.18
ND
18
25 000
372
1A
10
14
2
14
16
89
2
ND
ND
1090
110
25 000
6529
3439
2000
ND
5.10
0.55
0.15
1.40
0.54
2.3
6.3
1.6
0.6
19
1
10
out of a high-throughput screen based on their rates of
inactivation of HRV-14 3CP.2f,2g,3,5 All of the compounds
9-21, with additional information from an X-ray co-
crystal structure of 17, are predicted to occupy S2 of
HRV 3CP, analogous to the Phe side chain of 1. All of
the compounds 9-21 contain planar, hydrophobic,
aromatic moieties, reflective of the hydrophobic nature
of S2. No highly active compound was found that
appeared to have a substituent that extended to S3 or
S4. Compounds 16, 18, and 21 were not found in the
parallel array but were synthesized as follow-ups to
compounds 17 and 19, to test the effects of halogenation.
The halogenation pattern of the P2 substituent is shown
to have a large influence on activity. Comparison
between 16, 17, and 18 and between 19 and 21 shows
that a properly placed halogen can increase the rate of
enzyme inactivation by as much as 30-fold against HRV-
14 3CP.
The antiviral activities of compounds 9-21 loosely
follow the order of their rates of inactivation. Compound
19 appears to be an anomaly, having much better
antiviral activity than would be predicted by its rate of
enzyme inactivation.20 All of the compounds in Table 1
were nontoxic (CC50) to the cell line used in the antiviral
assay, at the limits of the testing concentrations.
Compounds 17, 18, and 21 show rates of inactivation
comparable to the peptide lead 1 but have an increased
degree of antiviral activity. This may reflect the im-
proved physical properties, leading to improved cellular
permeability, of the smaller molecules as compared to
the larger peptidic compound 1.
Compounds 17-19 were tested against several other
HRV serotypes with highly variable results (Table 2).
The activity against HRV-14, the serotype used in the
high-throughput assay, was consistently the highest.
The activity against other serotypes fell off dramatically.
The enzyme inhibitory activity of the tripeptide lead 1
varied approximately 10-fold over the serotypes tested.
Examination of the cocrystal structure of the HRV-2
3CP-17 covalent complex leads to a plausible explana-
tion of the differences in activities across different
serotypes, as discussed below. Despite their reduced
enzyme activity across other serotypes, 17-19 still
maintained better antiviral activity in comparable se-
rotypes than the larger, more peptidic 1.
* ND ) not determined.
pling reagent to react 2 with the desired carboxylic acid.
These resynthesized compounds were all purified by
preparative reverse phase high-performance liquid chro-
matography (HPLC).
Resu lts
Table 1 shows a selection of the most active 3CP
inhibitors in this series. The library hits were picked

Inhibitors of Human Rhinovirus 3C Protease
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2019
F igu r e 2. (a) Cocrystal structure of complex between 17 and HRV-2 3CP. (b) Map of complex between 17 and HRV-2 3CP.
X-r a y An a lysis of a Cocr ysta l Str u ctu r e
with the backbone carbonyl oxygen of Ser-128 and is
probably important for stabilizing the â-hairpin turn
that forms one wall of the S2 pocket.
A crystal structure was obtained for the covalent
complex between 17 and HRV-2 3CP (Figure 2). Inhibi-
tor-protein complexes of serotypes other than type 2
have not produced crystals of high enough quality to
be used for X-ray crystallography. As expected, the
Michael acceptor and the lactam groups of 17 are
oriented in a fashion similar to the corresponding
cocrystal structure of 1.8 Cys-147 is covalently linked
to the inhibitor’s unsaturated ester â-carbon. The lactam
lies in the S1 specificity pocket where it makes hydrogen
bonds with His-161 and Thr-142 residues. The bulky
bicyclic ring substituent is coplanar with the inhibitor’s
carboxamide group and binds deep in the S2 pocket.
Although the extended substrate binding site for HRV
3CP is highly conserved among various rhinoviral
serotypes, the differences that do exist involve residues
that form the back portion of the S2 specificity pocket.8
It is likely that these differences account for much of
the variability in the enzyme inhibitory activity ob-
served for 17 against the proteases from different HRV
serotypes. The two nonconserved residues in the S2
pocket of HRV-2 3CP are Lys-69 and Asn-130.
The Cl of bound 17 is buried underneath the side
chain carboxylate of Glu-71, where it forces the side
chain of Lys-69 to adopt a conformation not seen in
other HRV-2 3CP cocrystal structures with inhibitors
having less bulky S2 substituents. There is also a close
approach (3.55 Å) of the 7-position of the chromene ring
to the side chain amide nitrogen of Asn-130, which is
also expected to be unfavorable for the binding of 17 to
HRV-2 3CP. This Asn-130 side chain hydrogen bonds
Unfavorable steric interactions between the P2 sub-
stituent of 17 and the residues at the back of the S2
pocket also force the inhibitor’s amide NH away from
the backbone carbonyl oxygen of Val-162, disrupting a
potential hydrogen bond. In the current structure, this
distance has increased to 3.7 Å as compared to 3.0 Å
for the corresponding distance in the complex between
1 and HRV-2 3CP.8
The two nonconserved residues in the S2 pocket of
HRV-2 3CP are Lys-69 and Asn-130. These residues are
replaced by less bulky amino acids (Asn and Thr,
respectively) in HRV-14 3CP. The increased inhibitory
activity for 17 against HRV-14 3CP is most likely a
result of the fact that the larger S2 pocket can accom-
modate the bulky P2 substituent without unfavorable
steric interactions, while allowing the formation of the
hydrogen bond between the inhibitor’s amide and Val-
162. This hydrogen bond is probably important for
proper alignment of the Michael acceptor to facilitate
the Michael reaction with the active site cysteine.
HRV-16 3CP has an S2 pocket containing intermedi-
ate size amino acids. Residues 69 and 130 are Lys and
Thr, respectively. Consistent with these arguments, the
potency of 17 against HRV 3CP serotypes increases in
the order type 2 > type 16 > type 14.
Con clu sion s
The use of parallel synthesis guided with information
from target structural data led to the discovery of a new

2020 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
J ohnson et al.
(1 L) was cooled to 0 °C and treated with a solution of lithium
bis(trimethylsilylamide) (40.9 g, 244.6 mmol) in THF (200 mL).
The solution was held at 0 °C for 2.5 h and then poured into
brine (800 mL), extracted with ethyl acetate (2 L), and then
dried (MgSO4). Evaporation yielded 25.5 g of 3-bromo-pyrro-
lidin-2-one as a brown oil. This material was treated with Ac2O
(76 mL) and refluxed for 5 h. Evaporation followed by purifica-
tion (silica gel filtration, EtOAc elutant) yielded 28 g of
1-acetyl-3-bromo-pyrrolidin-2-one as a dark oil. THF (272 mL)
and triphenylphosphine (42.8 g, 163.3 mmol) were added, and
the resulting solution was refluxed for 8 h. After it was cooled
to room temperature, a precipitate of 1-acetyl-3-(triphenylphos-
phanyl)pyrrolidin-2-one bromide formed and was collected by
filtration (27.1 g). Concentration of the mother liquor, followed
by cooling to 0 °C, yielded an additional 6.6 g. The combined
material in CH2Cl2 (1 L) was washed with 1 N NaOH (100
mL) and then brine (2 × 100 mL). Evaporation of the organic
layer yielded 26.9 g (37% overall) of a tan oil. 1H NMR
(CDCl3): δ 7.76-7.32 (15H, m), 3.90-3.85 (2H, m), 2.50 (3H,
s), 2.56-2.30 (2H, m).
2R-ter t-Bu toxyca r bon yl-3-(ter t-bu tyld im eth ylsila n ox-
y)p r op ion ic Acid Meth yl Ester (8). Boc-D-serine methyl
ester (20.0 g, 91.2 mmol) in DMF (300 mL) was treated with
imidazole (18.6 g, 273.7 mmol) and then TBSCl (13.0 g, 86.7
mmol). The solution was held at room temperature for 8 h and
then was washed with saturated aqueous ammonium chloride
(300 mL) and then extracted with ethyl acetate (800 mL). The
organic layer was washed with brine (300 mL) and then dried
(MgSO4) to yield 30.2 g (100%) of a colorless oil. 1H NMR
(CDCl3): δ 5.32 (1H, d, J ) 8.3), 4.33 (1H, dt, J ) 8.8, 2.7),
4.02 (1H, dd, J ) 10.1, 2.6), 3.80 (1H, dd, J ) 9.8, 3.1), 3.72
(3H, s), 1.43 (9H, s), 0.85 (9H, s), 0.00 (6H, s).
[1S-(1-Acetyl-2-oxop yr r olid in -3-ylid en em eth yl)-2-(ter t-
bu tyld im eth ylsila n yloxy)eth yl]ca r ba m ic Acid ter t-Bu tyl
Ester (5). Compound 8 (12.7 g, 38.0 mmol) in toluene (190
mL) was cooled to -78 °C and treated with a solution of
diisobutylaluminum hydride (15.6 mL, 87.4 mmol) in toluene
(175 mL). Internal temperature was kept below -70 °C. The
solution was held at -78 °C for an additional 90 min and then
methanol (7.7 mL, 190 mmol) was added. 1-Acetyl-3-(triph-
enylphosphanylidine)pyrrolidin-2-one (6, 11.1 g, 28.6 mmol)
in CH2Cl2 (50 mL) was added at -78 °C, and the resulting
solution was allowed to warm to room temperature and held
for 30 min. A solution of sodium potassium tartrate (150 g) in
water (600 mL) was added and stirred vigorously for 30 min.
The mixture was extracted with ethyl acetate (4 × 250 mL),
dried (MgSO4), and evaporated. Purification by silica gel
chromatography yielded 7.04 g (60%) of a colorless oil. 1H NMR
(CDCl3): δ 6.59 (1H, dt, J ) 8.7, 2.9), 4.98 (1H, d, J ) 6.8),
4.37-4.25 (1H, m), 3.77 (2H, t, J ) 7.3), 3.70-3.58 (2H, m),
2.90-2.80 (1H, m), 2.75-2.60 (1H, m), 5.53 (3H, s), 1.41 (9H,
s), 0.87 (9H, s), 0.04 (6H, s).
class of HRV-3CP inhibitors. We have used this strategy
to focus high diversity on a small portion of the protein
target (S2), while conserving a known binding element
of the inhibitor (the P1-Michael acceptor moiety). Using
available structural information also served to reduce
the number of potential synthons to a manageable
number. We have shown that this technique is a
powerful tool to optimize the biological activity of a lead
compound.
We have shown that for HRV-14 serotype, it is
possible to obtain inhibitors with potent antiviral activ-
ity without having to build the molecules into S3 and
S4. For type 14 serotype, protein-inhibitor interactions
at S2 can have a large beneficial impact on inhibitor
activity; however, because of the problem of high sero-
type variability at S2, broad spectrum activity may be
a problem. Despite reduced enzyme inhibition against
serotypes other than type 14, these new small molecule
inhibitors still maintain slightly better antiviral activity
than the larger, peptidic 1.
Exp er im en ta l Section
General descriptions of experimental procedures, reagent
purifications, and instrumentation, along with conditions and
uncertainties for enzyme and antiviral assays, are provided
elsewhere.2f,2g,3,5 1H nuclear magnetic resonance (NMR) cou-
pling constants are given in Hz. Analytical HPLC analysis was
performed on a Hewlett-Packard Series 1100, running a 20-
100% acetonitrile-water gradient elution over 10 min. A
Zorbax SB-C18 4.6 mm × 15 cm column was used. Preparative
HPLC was performed using a Gilson 215 liquid handling robot,
along with the Gilson 306 pump, and a Gilson 119 detector
using 215 and 254 nm wavelengths. A Phenomenex LUNA C8-
(2) 250 mm × 21 mm column was used, with an acetonitrile-
water gradient elution. The method used for analysis of
proteolytic processing has been described previously.20 Ab-
breviations: DMSO, dimethyl sulfoxide; ADA, N-(2-acetami-
do)iminodiacetic acid; DTT, dithiothreitol; THF, tetrahydro-
furan; TFA, trifluoroacetic acid.
P r otein Cr ysta llogr a p h y. HRV-2 3CP was incubated with
a 4-fold molar excess of 17 in the presence of 2% (vol/vol)
DMSO for 24 h at 4 °C. The complex was concentrated to 10
mg/mL and then passed through a 0.22 µm cellulose-acetate
filter. Crystals were grown at 13 °C, using a hanging drop
vapor diffusion method in which equal volumes (3 uL) of the
protein-ligand complex and reservoir solution were mixed on
plastic coverslips and sealed over individual wells filled with
1 mL of reservoir solution containing 2 M sodium acetate, 0.1
M ADA, pH 6.5, and 10 mM DTT.
A single crystal measuring 0.3 mm × 0.2 mm × 0.1 mm
(space group P21212; a ) 62.6, b ) 77.9, c ) 34.1 Å) was
prepared for low-temperature X-ray data collection by transfer
to an artificial mother liquor solution consisting of 300 µL of
the reservoir solution mixed with 100 µL of glycerol and then
flash-freezing in liquid nitrogen. X-ray diffraction data were
collected at -180 °C on a MAR Research 345 mm imaging
plate and processed with DENZO and SCALEPACK.21 Dif-
fraction data were 95.4% complete to a resolution of 1.73 Å
with R(sym) ) 3.1%. Protein atomic coordinates from an
isomorphous cocrystal structure of HRV-2 3CP7 were used to
initiate rigid body refinement in X-PLOR22 followed by simu-
lated annealing and conjugate gradient minimization protocols.
Placement of the inhibitor, addition of ordered solvent, and
further refinement proceeded as described previously.5a The
final R factor was 20.9% (16 017 reflections with F > 2σ(F)).
The root mean square deviations from ideal bond lengths and
angles were 0.014 Å and 2.6 degrees, respectively. The final
model consisted of all atoms for residues 1-180 (excluding side
chains of residues 12, 45, and 167), the inhibitor, and 175
ordered waters.
[2-Hyd r oxy-1S-(2-oxop yr r olid in -3-ylid en em eth yl)eth -
yl]ca r ba m ic Acid ter t-Bu tyl Ester (4). Compound 5 (9.18
g, 22.2 mmol) in THF (150 mL) was treated with tetrabuty-
lammonium fluoride (22.2 mL of a 1 M solution in THF, 22.2
mmol) at 0 °C and held for 1 h. A solution of saturated aqueous
ammonium chloride was added and stirred for 10 min and
then, the solution was extracted with ethyl acetate (2 × 200
mL). The organic layer was dried (MgSO4) and then evapo-
rated. Purification by silica gel chromatography yielded 4.82
g (73%) of a colorless oil. This material was taken up in
methanol (160 mL), treated with potassium carbonate (223 mg,
1.62 mmol), and held for 1 h at room temperature. The mixture
was then treated with solid citric acid (311 mg, 1.62 mmol),
and ethyl acetate (800 mL) was added. The solution was
filtered through silica gel. Evaporation yielded 4.30 g (73%
1
overall) of a colorless oil. H NMR (CDCl3): δ 7.03 (1H, br s),
6.35 (1H, dt, J ) 8.6, 2.6), 5.37 (1H, d, J ) 6.5), 4.40-4.20
(1H, m), 3.66 (br s, 3H), 3.40 (2H, t, J ) 6.7), 3.10-2.80 (1H,
m), 2.80-2.70 (1H, m), 1.41 (9H, s).
[2-Hydr oxy-1S-(2-oxopyr r olidin -3S-ylm eth yl)eth yl]car -
ba m ic Acid ter t-Bu tyl Ester (3). Compound 4 (9.56 g, 37.3
mmol) in methanol (230 mL) was treated with (S)-BINAP-
1-Acet yl-3-(t r ip h en ylp h osp h a n ylid in e)p yr r olid in -2-
on e (6). 2,4-Dibromobutyride14 (46.1 g, 188.2 mmol) in THF

Inhibitors of Human Rhinovirus 3C Protease
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2021
Compound 11 was prepared according to the representative
RuCl23 (69 mg, 74 µmol) and then put under a hydrogen
atmosphere (1200 psi) at room temperature for 72 h. The
solution was evaporated and then taken up in EtOAc (200 mL)
and allowed to precipitate. The precipitate was filtered and
dried to give 6.6 g (69%) of a white powder. This material was
determined by 1H NMR to consist of a single diastereomer.
1H NMR (CDCl3): δ 6.66 (1H, br s), 5.51 (1H, d, J ) 8.2), 3.72-
3.57 (3H, m), 3.34-3.31 (2H, m), 2.52-2.33 (2H, m), 2.20-
1.86 (1H, m), 1.86-1.70 (1H, m), 1.62-1.50 (1H, m), 1.40 (9H,
s).
1
experimental, using 3-bromo-4-fluorocinnamic acid. H NMR
(CDCl3): δ 8.02 (1H, d, J ) 6.6), 7.71 (1H, d, J ) 8.6), 7.52
(1H, d, J ) 15.6), 7.42-7.39 (1H, m), 7.11 (1H, t, J ) 8.3),
6.88 (1H, dd, J ) 15.6, 5.4), 6.41 (1H, d, J ) 15.6), 5.97 (1H,
d, J ) 15.7), 5.94 (1H, s), 4.75-4.61 (1H, m), 4.17 (2H, q, J )
7.1), 3.41-3.37 (2H, m), 2.59-2.45 (2H, m), 2.17-1.82 (3H,
m), 1.27 (3H, t, J ) 7.2). MS (FAB): 453.0812 (MH+, calcd.
453.0825), 475 (MNa+).
4S-[3-(2,5-Dibr om op h en yl)a cr yloyla m in o]-5-(2-oxop yr -
r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (12). Com-
pound 12 was prepared according to the representative
experimental, using 2,5-dibromocinnamic acid. 1H NMR
(CDCl3): δ 8.15 (1H, d, J ) 6.4), 7.92 (1H, d, J ) 15.6), 7.71
(1H, s), 7.47 (1H, d, J ) 8.6), 7.32 (1H, d, J ) 8.6), 6.93 (1H,
dd, J ) 15.6, 5.4), 6.43 (1H, d, J ) 15.6), 6.03 (1H, d, J ) 15.6),
5.62 (1H, s), 4.68-4.63 (1H, m), 4.22 (2H, q, J ) 7.1), 3.42-
3.37 (2H, m), 2.54-2.44 (1H, m), 2.05-1.58 (4H, m), 1.30 (3H,
t, J ) 7.1). MS (FAB): 515.0021 (MH+, calcd. 515.0005).
4S-(3-Ben zo[1,3]dioxol-5-yl-acr yloylam in o)-5-(2-oxopyr -
r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (13). Com-
pound 13 was prepared according to the representative
experimental, using 3,4-methylenedioxycinnamic acid. 1H
NMR (CDCl3): δ 7.54 (1H, d, J ) 6.3), 7.51 (1H, d, J ) 15.1),
7.01 (1H, s), 6.97 (1H, d, J ) 8.1), 6.89 (1H, dd, J ) 15.7, 5.3),
6.78 (1H, d, J ) 7.9) 6.35 (1H, d, J ) 15.6), 6.01-5.93 (4H,
m), 4.74-4.72 (1H, m), 4.20 (2H, q, J ) 7.1), 3.37-3.34 (2H,
m), 2.53-2.42 (2H, m), 2.04-1.70 (3H, m), 1.26 (3H, t, J )
7.1). MS (FAB): 423.1545 (MNa+, calcd. 423.1532), 423
(MNa+).
4S-Am in o-5-(2-oxop yr r olid in -3S-yl)p en t-2-en oic Acid
Eth yl Ester (2). Compound 3 (2.02 g, 7.83 mmol) in DMSO
(78 mL) was treated with IBX18 (3.30 g, 11.7 mmol) and held
at room temperature for 1.5 h. Carbethoxymethylene triph-
enylphosphorane (5.45 g, 15.7 mmol) was added, and the
solution was held at room temperature for 1 h. Brine (500 mL)
was added, and the solution was extracted with ethyl acetate
(4 × 150 mL) and then dried (MgSO4). Evaporation yielded
8.50 g of a brown oil. Purification by silica gel chromatography
yielded 1.54 g (60%) of a colorless oil. 1H NMR (CDCl3): δ 6.85
(1H, dd, J ) 15.6, 5.2), 5.97 (1 H, dd, J ) 15.6, 1.6), 4.5-4.3
(1H, m), 4.19 (2H, q, J ) 7.1), 3.40-3.25 (2H, m), 2.50-2.35
(1H, m), 2.35-2.24 (1H, m), 2.11-1.55 (3H, m), 1.43 (9H, s),
1.28 (3H, t, J ) 7.1). MS (FAB) 349 (MNa+) 327 (MH+). 4S-
tert-Butoxycarbonylamino-5-(2-oxopyrrolidin-3S-yl)pent-2-eno-
ic acid ethyl ester (2.13 g, 6.53 mmol) was treated with EtOH
saturated with HCl gas (100 mL), held at room temperature
for 1 h, and then evaporated. The residue was taken up in
CHCl3 (50 mL), then washed with 10% K2CO3-H2O (100 mL),
followed by brine (50 mL). The organic layer was dried over
K2CO3 and then evaporated to give 1.50 g (100%) of the
deprotected amine. 1H NMR (CDCl3): δ 6.89 (1H, dd, J ) 15.7,
6.7), 5.96 (1H, d, J ) 15.7), 4.18 (2H, q, J ) 7.1), 3.74-3.63
(1H, m), 3.38-3.29 (2H, m), 2.65-2.53 (1H, m), 2.40-2.28 (1H,
m), 2.23-1.92 (1H, m), 1.85-1.70 (1H, m), 1.60-1.48 (1H, m),
1.26 (3H, t, J ) 7.1). Anal. (C11H18N2O3) C, H, N.
4S-[(Na p h th a len e-2-ca r bon yl)a m in o]-5-(2-oxop yr r oli-
d in -3S-yl)p en t-2-en oic Acid Eth yl Ester (19). Rep r esen -
ta tive Exp er im en ta l. Compound 2 (30 mg, 0.13 mmol) in
DMF (1 mL) was treated with diisopropylethylamine (0.07 mL,
0.40 mmol), 2-naphthoic acid (22 mg, 0.13 mmol), and HATU
(49 mg, 0.13 mmol) and held at room temperature for 1 h. The
solution was washed with brine (5 mL) and extracted with
EtOAc (10 mL). Evaporation yielded 34 mg of crude product.
Purification by preparative reverse phase chromatography
(CH3CN-H2O gradient) yielded 20 mg (41%) of product. 1H
NMR (CDCl3): δ 8.48 (1H, s), 8.01-7.85 (4H, m), 7.58-7.50
(2H, m), 6.98 (1H, dd, J ) 15.6, 5.3), 6.04 (1H, d, J ) 15.8),
4.85-4.78 (1H, m), 4.17 (2H, q, J ) 7.0), 3.39-3.34 (2H, m),
2.64-2.47 (2H, m), 2.17-2.06 (1H, m), 1.97-1.82 (3H, m), 1.34
(3H, t, J ) 7.0). MS (FAB): 381 (MH+), 403 (MNa+). Anal.
(C22H24N2O4‚0.5H2O) C, H, N.
4S-[3′-(6′-Br om o-ben zo[1,3]d ioxol-5-yl)a cr yloyla m in o]-
5-(2-oxop yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester
(14). Compound 14 was prepared according to the representa-
tive experimental, using 2-bromo-3,4-methylenedioxycinnamic
1
acid. H NMR (CDCl3): δ 7.91 (1H, d, J ) 15.5), 7.78 (1H, d,
J ) 6.8), 7.05 (1H, s), 7.03 (1H, s), 6.89 (1H, dd, J ) 15.6, 5.4),
6.29 (1H, d, J ) 15.5,), 6.03 (2H, s), 6.01, (1H, s), 5.98 (1H, d,
J ) 14.5), 4.72-4.67 (1H, m), 4.16 (2H, q, J ) 7.1), 3.49-3.36
(2H, m), 2.56-2.43 (2H, m), 2.17-1.82 (3H, m), 1.28 (3H, t, J
) 7.1). MS (FAB): 479.0807 (MH+, calcd. 479.0818), 501
(MNa+). Anal. (C21H23BrN2O6‚0.6H2O) C, H, N.
4S-[(2-Met h yl-5-p h en ylfu r a n -3-ca r bon yl)a m in o]-5-(2-
oxop yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (15).
Compound 15 was prepared according to the representative
experimental, using 2-methyl-5-phenyl-furan-3-carboxylic acid.
1H NMR (CDCl3): δ 8.16 (1H, d, J ) 5.8), 7.69-7.62 (2H, m),
7.42-7.34 (2H, m), 7.29-7.22 (1H, m), 6.93 (1H, s), 6.92 (1H,
dd, J ) 15.6, 5.3), 6.01 (1H, d, J ) 5.6), 5.68 (1H, s), 4.74-
4.63 (1H, m), 4.18 (2H, q, J ) 7.1), 3.42-3.34 (2H, m), 2.68
(3H, s), 2.62-2.42 (2H, m), 2.10-1.77 (3H, m), 1.27 (3H, t, J
) 7.1). MS (FAB): 411.1929 (MH+, calcd 411.1920), 433
(MNa+).
4S-{3-(3-Br om op h en yl)a cr yloyla m in o]-5-(2-oxop yr r o-
lid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (9). Compound
9 was prepared according to the representative experimental,
using 3-bromocinnamic acid. 1H NMR (CDCl3): δ 7.67 (1H,
s), 7.53 (1H, d, J ) 15.6), 7.50-7.38 (2H, m), 7.22 (1H, t, J )
7.8), 6.89 (1H, dd, J ) 15.6, 5.3), 6.47 (1H, d, J ) 15.7), 5.98
(1H, d, J ) 15.6), 4.74-4.63 (1H, m), 4.17 (2H, q, J ) 7.1),
3.42-3.35 (2H, m), 2.60-2.40 (2H, m), 2.10-1.70 (3H, m), 1.26
(3H, t, J ) 7.1). MS (FAB): 435 (MH+), 457 (MNa+). Anal.
(C20H23BrN2O4‚0.3H2O) C, H, N.
4S-[3-(3-Br om o-4-m eth yl-p h en yl)-a cr yloyla m in o]-5-(2-
oxop yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (10).
Compound 10 was prepared according to the representative
experimental, using 3-bromo-4-methylcinnamic acid. 1H NMR
(CDCl3): δ 7.81 (1H, d, J ) 7.0), 7.65 (1H, s), 7.52 (1H, d, J )
15.6), 7.35-7.20 (2H, m), 6.89 (1H, dd, J ) 15.6, 5.3), 6.46
(1H, d, J ) 15.7), 6.15 (1H, s), 5.95 (1H, d, J ) 15.6), 4.78-
4.65 (1H, m), 4.17 (2H, q, J ) 7.1), 3.40-3.30 (2H, m), 2.60-
2.35 (5H, m), 2.10-1.70 (3H, m), 1.27 (3H, t, J ) 7.1). MS
(FAB): 449.1090 (MH+, calcd 449.1076), 471 (MNa+).
4S-[(2H-Ch r om en e-3-ca r b on yl)a m in o]-5-(2-oxop yr r o-
lid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (16). Compound
16 was prepared according to the representative experimental,
using 2H-chromene-3-carboxylic acid. 1H NMR (CDCl3): δ 8.46
(1H, d, J ) 5.4), 7.23-7.12 (3H, m), 6.94-6.81 (3H, m), 5.97
(1H, dd, J ) 15.6, 1.4), 5.62 (1H, s), 5.03 (1H, d, J ) 1.2), 4.64-
4.53 (1H, m), 4.17 (2H, q, J ) 7.2), 3.42-3.38 (2H, m), 2.58-
2.40 (2H, m), 2.03-1.75 (3H, m), 1.27 (3H, t, J ) 7.2). MS
(FAB): 385.1774 (MH+, calcd. 385.1763).
4S-[(6-Ch lor o-2H -ch r om en e-3-ca r b on yl)a m in o]-5-(2-
oxop yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (17).
Compound 17 was prepared according to the representative
experimental, using 6-chloro-2H-chromene-3-carboxylic acid.
1H NMR (CDCl3): δ 8.61 (1H, d, J ) 5.1), 7.18-7.09 (3H, m),
6.86 (1H, dd, J ) 15.6, 5.6), 6.76 (1H, d, J ) 8.2), 5.96 (1H, d,
J ) 15.6), 5.88 (1H, s), 5.03 (2H, s), 4.60-4.50 (1H, m), 4.18
(2H, q, J ) 7.2), 3.43-3.36 (2H, m), 2.59-2.40 (2H, m), 2.04-
1.76 (3H, m), 1.27 (3H, t, J ) 7.1). MS (FAB): 419.1361 (MH+,
calcd 419.1374), 441 (MNa+). Anal. (C21H23ClN2O5‚0.7H2O) C,
H, N.
4S-[3-(3-Br om o-4-flu or op h en yl)a cr yloyla m in o]-5-(2-
oxop yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (11).
4S-[(6-Br om o-2H -ch r om en e-3-ca r b on yl)a m in o]-5-(2-
oxop yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (18).

2022 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
J ohnson et al.
Human Rhinovirus 3C Protease Inhibitors. J . Med. Chem. 1998,
41, 2819-2834. (c) Dragovich, P. S.; Webber, S. E.; Prins, T. J .;
Zhou, R.; Marakovits, J . T.; Tikhe, J . G.; Fuhrman, S. A.; Patick,
A. K.; Matthews, D. A.; Ford, C. E.; Brown, E. L.; Binford, S. L.;
Meador, J . W., III; Ferre, R.; Worland, S. T. Structure-Based
Design of Irreversible, Tripeptidyl Human Rhinovirus 3C Pro-
tease Inhibitors Containing N-Methyl Amino Acids. Bioorg. Med.
Chem. Lett. 1999, 9, 2189-2194. (d) Dragovich, P. S.; Prins, T.
J .; Zhou, R.; Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.;
Ford, C. E.; Meador, J . W., III; Ferre, R.; Worland, S. T.
Structure-Based Design, Synthesis, and Biological Evaluation
of Irreversible Human Rhinovirus 3C Protease Inhibitors. 3.
Structure-Activity Studies of Ketomethylene-Containing Pep-
tidomimetics. J . Med. Chem. 1999, 42, 1203-1212. (e) Dragovich,
P. S.; Prins, T. J .; Zhou, R.; Webber, S. E.; Marakovits, J . T.;
Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.; Lee, C. A.; Ford,
C. E.; Burke, B. J .; Rejto, P. A.; Hendrickson, T. F.; Tuntland,
T.; Brown, E. L.; Meador, J . W., III; Ferre, R.; Harr, J . E. V.;
Kosa, M. B.; Worland, S. T. Structure-Based Design, Synthesis,
and Biological Evaluation of Irreversible Human Rhinovirus 3C
Protease Inhibitors. 4. Incorporation of P1 Lactam Moieties as
L-Glutamine Replacements. J . Med. Chem. 1999, 42, 1213-1224.
(f) Dragovich, P. S.; Zhou, R.; Skalitzky, D. J .; Fuhrman, S. A.;
Patick, A. K.; Ford, C. E.; Meador, J . W., III; Worland, S. T. Solid-
phase Synthesis of Irreversible Human Rhinovirus 3C Protease
Inhibitors. Part 1: Optimization of Tripeptides Incorporating
N-terminal Amides. Bioorg. Med. Chem. 1999, 7, 589-598. (g)
Reich, S. H.; J ohnson, T. O.; Wallace, M. B.; Kephart, S. E.;
Fuhrman, S. A.; Worland, S. T.; Matthews, D. A.; Hendrickson,
T. F.; Chan, F.; Meador, J . W., III; Ferre, R.; Brown, E. L.;
Delisle, D. M.; Patick, A. K.; Binford, S. L.; Ford, C. E.
Substituted Benzamide Inhibitors of Human Rhinovirus 3C
Protease: Structure-Based Design, Synthesis, and Biological
Evaluation. J . Med. Chem. 2000, 43, 1670-1683.
Compound 18 was prepared according to the representative
experimental, using 6-bromo-2H-chromene-3-carboxylic acid.
1H NMR (CDCl3): δ 8.72 (1H, d, J ) 5.2), 7.28-7.23 (2H, m),
7.16 (1H, s), 6.86 (1H, dd, J ) 15.6, 5.6), 6.72 (1H, d, J ) 9.1),
5.96 (1H, dd, J ) 5.6, 1.4), 5.02 (d, 2H, J ) 1.2), 4.60-4.49
(1H, m), 4.17 (2H, q, J ) 7.1), 3.43-3.36 (2H, m), 2.59-2.40
(2H, m), 2.04-1.76 (3H, m), 1.27 (3H, t, J ) 7.1). MS (FAB):
463.0883 (MH+, calcd 463.0869), 485 (MNa+). Anal. (C21H23
-
BrN2O5‚0.6H2O) C, H, N.
4S-[(6-Meth yl-n aph th alen e-2-car bon yl)am in o]-5-(2-oxo-
p yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (20). Com-
pound 20 was prepared according to the representative
experimental, using 6-methyl-2-naphthoic acid. 1H NMR
(CDCl3): δ 8.42 (1H, s), 7.95 (1H, dd, J ) 8.6, 1.7), 7.84 (1H,
d, J ) 8.4), 7.79 (1H, d, J ) 8.6), 7.63 (1H, s), 7.36 (1H, dd, J
) 8.6, 1.4), 6.97 (1H, dd, J ) 15.6, 5.3), 6.01 (1H, d, J ) 15.6),
5.99 (1H, s), 4.90-4.78 (1H, m), 4.17 (2H, q, J ) 7.1), 3.43-
3.30 (2H, m), 2.70-2.60 (2H, m), 2.52 (3H, s), 2.20-2.05 (1H,
m), 2.00-1.80 (2H, m), 1.26 (3H, t, J ) 1.7). MS (FAB):
395.1964 (MH+, calcd. 395.1971), 417 (MNa+).
4S-[(7-Br om on a p h th a len e-2-ca r bon yl)a m in o]-5-(2-oxo-
p yr r olid in -3S-yl)p en t-2-en oic Acid Eth yl Ester (21). Com-
pound 21 was prepared according to the representative
experimental, using 7-bromo-2-naphthoic acid.24 1H NMR
(CDCl3): δ 8.82 (1H, d, J ) 5.7), 8.39 (1H, s), 8.09 (1H, d, J )
1.6), 8.02 (1H, dd, J ) 8.6, 1.6), 7.85 (1H, d, J ) 8.6), 7.73
(1H, d, J ) 8.7), 7.61 (1H, dd, J ) 8.7, 1.9), 6.96 (1H, dd, J )
15.6, 5.4), 6.06 (1H, s), 6.03 (1H, d, J ) 15.6), 4.85-4.70 (1H,
m), 4.17 (2H, q, J ) 7.1), 3.45-3.30 (2H, m), 2.70-2.40 (2H,
m), 2.20-1.80 (3H, m), 1.27 (3H, t, J ) 7.1). MS (FAB):
(3) Webber, S. E.; Tikhe, J .; Worland, S. T.; Fuhrman, S. A.;
Hendrickson, T. F.; Matthews, D. A.; Love, R. A.; Patick, A. K.;
Meador, J . W.; Ferre, R.; Brown, E. L.; DeLisle, D. M.; Ford, C.
E.; Binford, S. L. Design, Synthesis, and Evaluation of Nonpep-
tidic Inhibitors of Human Rhinovirus 3C Protease. J . Med.
Chem. 1996, 39, 5072-5082.
459.0906 (MH+, calcd. 459.0919), 481 (MNa+). Anal. (C22H23
BrN2O4) C, H, N.
-
P a r a llel Syn th esis
PS-carbodiimide25 (15.9 umol, 1.59 mmol/g, approxi-
mately 10 mg) was loaded into each well of a filter
bottom 96 well plate (Charybdis Calypso NXT-75 reac-
tion block system26) using a resin-loading device (Mil-
lipore).27 Compound 2 (5 µmol, 0.5 mL of a 10 mM
solution in DMF) was subsequently loaded into each
well using a multichannel pipet. A solution of the
selected carboxylic acid (7.5 µmol, 0.1 mL of a 75 mM
solution in DMF) was added to each well using an
automated liquid handler (Charybdis Iliad PS2).26 The
plate was agitated overnight at room temperature, then
filtered into a second solid-bottom 96 well plate, and
then evaporated using a Genevac Atlas HT-1228 evapo-
rator.
(4) (a) J ungheim, L. A.; Cohen, J . D.; J ohnson, R. B.; Villarreal, E.
C.; Wakulchik, M.; Loncharich, R. J .; Wang, Q. M. Inhibition of
Human Rhinovirus 3C Protease by Homophthalimides. Bioorg.
Med. Chem. Lett. 1997, 7, 1589-1594. (b) Wang, Q. M.; J ohnson,
R. B.; J ungheim, L. A.; Cohen, J . D.; Villarreal, E. C. Dual
Inhibition of Human Rhinovirus 2A and 3C Proteases by
Homophthalimides. Antimicrob. Agents Chemother. 1998, 42,
916-920.
(5) (a) Webber, S. E.; Okano, K.; Little, T. L.; Reich, S. H.; Xin, Y.;
Fuhrman, S. A.; Matthews, D. A.; Love, R. A.; Hendrickson, T.
F.; Patick, A. K.; Meador, J . W., III; Ferre, R. A.; Brown, E. L.;
Ford, C. E.; Binford, S. L.; Worland, S. T. Tripeptide Aldehyde
Inhibitors of Human Rhinovirus 3C Protease: Design, Synthesis,
Biological Evaluation, and Cocrystal Structure Solution of P1
Glutamine Isosteric Replacements. J . Med. Chem. 1998, 41,
2786-2805. (b) Kaldor, S. W.; Hammond, M.; Dressman, B. A.;
Labus, J . M.; Chadwell, F. W.; Kline, A. D.; Heinz, B. A.
Glutamine-Derived Aldehydes for the Inhibition of Human
Rhinovirus 3C Protease. Bioorg. Med. Chem. Lett. 1995, 5, 2021-
2026.
(6) Dragovich, P. S.; Zhou, R.; Webber, S. E.; Prins, T. J .; Kwok, A.
K.; Okano, K.; Fuhrman, S. A.; Zalman, L. S.; Maldonado, F.
C.; Brown, E. L.; Meador, J . W., III; Patick, A. K.; Ford, C. E.;
Brothers, M. A.; Binford, S. L.; Matthews, D. A.; Ferre, R.;
Worland, S. T. Structure-Based Design of Ketone-Containing,
Tripeptidyl Human Rhinovirus 3C Protease Inhibitors. Bioorg.
Med. Chem. Lett. 2000, 10, 45-48.
(7) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Lee, C. A.; Reich, S. H.; Prins,
T. J .; Marakovits, J . T.; Littlefield, E. S.; Zhou, R.; Tikhe, J .;
Ford, C. E.; Wallace, M. B.; Meador, J . W., III; Ferre, R.; Brown,
E. L.; Binford, S. L.; Harr, J . E. V.; DeLisle, D. M.; Worland, S.
T. Structure-Based Design, Synthesis, and Biological Evaluation
of Irreversible Human Rhinovirus 3C Protease Inhibitors. 1.
Michael Acceptor Structure-Activity Studies. J . Med. Chem.
1998, 41, 2806-2818.
Refer en ces
(1) (a) Couch, R. B. Rhinoviruses. In Virology, 3rd ed.; Fields, B.
N., Knipe, D. M., Howley, P. M., et al., Eds.; Lippencott-Raven
Publishers: Philadelphia, 1996; Vol. 1, Chapter 22, pp 607-629.
(b) McKinlay, M. A.; Pevear, D. C.; Rossman, M. G. Treatment
of the Picornavirus Common Cold by Inhibitors of Viral Uncoat-
ing and Attachment. Annu. Rev. Microbiol. 1992, 46, 635-654.
(c) Phillpotts, R. J .; Tyrrell, D. A. Rhinovirus Colds. Br. Med.
Bull. 1985, 41, 386-390. (d) Gwaltney, J . M. Rhinoviruses. In
Viral Infections of Humans; Evans, S. A., Ed.; Plunem Publishing
Corp.: New York, 1982; Chapter 20, pp 491-517. (e) Rueckert,
R. R. Picornaviridae and Their Replication. In Virology, 3rd ed.;
Fields, B. N., Knipe, D. M., Howley, P. M., et al., Eds.; Lippincott-
Raven Publishers: Philadelphia, 1996; Vol. 1, Chapter 21, pp
609-654. (f) Kra¨usslich, H.-G.; Wimmer, E. Viral Proteinases.
Annu. Rev. Biochem. 1988, 57, 701-754.
(8) Matthews, D. A.; Dragovich, P. S.; Webber, S. E.; Fuhrman, S.
A.; Patick, A. K.; Zalman, L. S.; Hendrickson, T. F.; Love, R. A.;
Prins, T. J .; Marakovits, J . T.; Zhou, R.; Tikhe, J .; Ford, C. E.;
Meador, J . W.; Ferre, R. A.; Brown, E. L.; Binford, S. L.;
Brothers, M. A.; DeLisle, D. M.; Worland, S. T. Structure-
Assisted Design of Mechanism-Based Irreversible Inhibitors of
Human Rhinovirus 3C Protease With Potent Antiviral Activity
Against Multiple Rhinovirus Serotypes. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 11000-11007.
(2) Kong, J .-S.; Venkatraman, S.; Furness, K.; Nimkar, S.; Shepard,
T. A.; Wang, Q. M.; Aub’e, J .; Hanzlick, R. P. Synthesis and
Evaluation of Peptidyl Michael Acceptors That Inactivate Hu-
man Rhinovirus 3C Protease and Inhibit Virus Replication. J .
Med. Chem. 1998, 41, 2579-2587. (b) Dragovich, P. S.; Webber,
S. E.; Babine, R. E.; Fuhrman, S. A.; Patick, A. K.; Matthews,
D. A.; Reich, S. H.; Marakovits, J . T.; Prins, T. J .; Zhou, R.;
Tikhe, J .; Littlefield, E. S.; Bleckman, T. M.; Wallace, M. B.;
Little, T. L.; Ford, C. E.; Meador, J . W., III; Ferre, R.; Brown, E.
L.; Binford, S. L.; DeLisle, D. M.; Worland, S. T. Structure-Based
Design, Synthesis, and Biological Evaluation of Irreversible
(9) Available Chemicals Directory supplied through ISIS Database
2.0 by MDL Information System, San Leandro, CA.

Inhibitors of Human Rhinovirus 3C Protease
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2023
(10) Sadowski, J .; Gesteiger, J . CORINA 3D-Structure Generator,
version 1.7; April, 1996.
(11) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MacroModel-An Integrated Software System for Modeling
Organic and Bioorganic Molecules Using Molecular Mechanics.
J . Comput. Chem. 1990, 11, 440.
(12) Gehlhaar, D. K.; Verkhivker, G.; Rejto, P. A.; Sherman, C. A.;
Fodel, D. A.; Fogel, L. J .; Freer, S. T. Molecular Recognition of
the Inhibitor AG-1343 by HIV-1 Protease: Conformationally
Flexible Docking by Evolutionary Programming. Chem. Biol.
1995, 5, 317.
(13) Hanessian, S.; Ninkovic, S.; Reinhold, U. The Synthesis of 4,5-
Methano Congeners of R-Kainic and R-allo-Kainic Acids as
Probes for Glutamate Receptors. Tetrahedron Lett. 1996, 37,
8971-8974.
γ-Butyrolactones and Cyclopentanones. Tetrahedron Lett. 1992,
33, 635-638. (c) Chung, J . Y. L.; Zhao, D.; Hughes, D. L.;
McNamara, J . M.; Grabowski, E. J . J .; Reider, P. J . Asymmetric
Hydrogenation of 3-Alkylidine-2-Piperidones Using Noyori’s
Catalyst. Effect of N-Substituents on the Enantioselectivity.
Tetrahedron Lett. 1995, 36, 7379-7382.
(17) Use of R-BINAP gave a 9:1 mixture favoring the opposite
diastereomer (2-hydroxy-1S-(2-oxopyrrolidin-3R-ylmethyl)ethyl]-
carbamic acid tert-butyl ester). Incorporation of this diastereomer
into compounds related to 9-21 afforded inhibitors with sig-
nificantly reduced 3CP inhibition activity.
(18) Frigeria, M.; Santagostino, M.; Sputore, S.; Palmisano, G.
Oxidation os Alcohols with o-Iodoxybenzoic Acid (IBX) in
DMSO: A New Insight into an Old Hypervalent Iodine Reagent.
J . Org. Chem. 1995, 60, 7272-7276.
(19) Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. Dess-Martin
Periodinane Oxidation of Alcohols in the Presence of Stabilized
Phosphorus Ylides: A Convenient Method for the Homologation
of Alcohols via Unstable Aldehydes. J . Org. Chem. 1997, 62,
9376-9378.
(20) It is possible that other mechanisms are contributing to the
antiviral activity in addition to protease inhibition.
(21) Otwinoski, Z.; Minor, W. Processing of X-ray Diffraction Data
Collected in Oscillation Mode. Methods Enzymol. 1997, 26, 307-
326.
(14) Ikuta, H.; Shirota, H.; Kobayashi, S.; Yamagishi, Y.; Yamada,
K.; Yamatsu, I.; Katayama, K. Synthesis and Antiinflammatory
Activities of 3-(3,5-Di-tert-butyl-4-hydroxybenzylidine)pyrrolidin-
2-ones. J . Med. Chem. 1987, 30, 1995-1998.
(15) (a) Brown, M. J .; Evans, P. L.; J ames, A. P. Directed Homoge-
neous Hydrogenation: Methyl anti-3-Hydroxy-2-Methylpen-
tanoate. Org. Synth. 1989, 68, 64-75. (b) Evans, D. A.; Morris-
sey, M. M. Rhodium(I)-Catalyzed Hydrogenation of Olefins. The
Documentation of Hydroxyl-Directed Stereochemical Control in
Cyclic and Acyclic Systems. J . Am. Chem. Soc. 1984, 106, 3866-
3868. (c) Evans, D. A.; Morrissey, M. M. Hydroxyl-Directed
Olefin Hydrogenation with Iridium Catalysts. The Documenta-
tion of Catalyst: Substrate Stoichiometry as a Variable in
Reaction Diastereoselection. Tetrahedron Lett. 1984, 25, 4637-
4640. (d) Evans, D. A.; Morrissey, M. M.; Dow, R. L. Hydroxyl-
Directed Hydrogenation of Homoallylic Alcohols. Effects of
Achiral and Chiral Rhodium Catalysts on 1,3-Stereocontrol.
Tetrahedron Lett. 1985, 26, 6005-6008.
(22) Bru¨nger, A. T. X-PLOR, version 3.1 Manual; Yale University
Press: New Haven, CT, 1992.
(23) This material was obtained as a p-cymene complex from Aldrich
Chemical Co. and used with no further purification.
(24) (a) Adcock, W.; Wells, P. R. Substituent Effects in Naphthalene.
Aust. J . Chem. 1965, 18, 1351-1364. (b) Gray, G. W.; J ones, B.
The Preparation of 4- and 5-n-Alkoxy-1-naphthoic and 6- and
7-n-Alkoxy-2-naphthoic Acids. J . Chem. Soc. 1954, 678-682.
(25) Argonaut Technologies, 887 Industry Road, San Carlos, CA
94070.
(16) (a) Takaya, H.; Ohta, T.; Inoue, S.; Tokunaga, M.; Kitamura,
M.; Noyori, R. Asymmetric Hydrogenation of Allylic Alcohols
Using BINAP-Ruthenium Complexes: (S)-(-)-Citronellol. Org.
Synth. 1993, 72, 74-85. (b) Ohta, T.; Miyake, T.; Seido, N.;
Kumobayashi, H.; Akutagawa, S.; Takaya, H. Asymmetric
Hydrogenation of Unsaturated Carbonyl Compounds Catalyzed
by BINAP-Ru(II) Complexes. Enantioselective Synthesis of
(26) Charybdis Technologies, Inc., 5925 Priestly Drive, Carlsbad, CA
92008.
(27) Millipore, 80 Ashby Road, Bedford, MA 01730.
(28) Genevac Inc., 711 Executive Blvd., Suite H, Valley Cottage, NY
10989.
J M010435C