Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 1. Michael acceptor structre-activity studies

Article abstract of DOI:10.1021/jm980068d

The structure-based design, chemical synthesis, and biological evaluation of peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds incorporate various Michael acceptor moieties and are shown to irreversibly bin

Full text of DOI:10.1021/jm980068d

2806
J . Med. Chem. 1998, 41, 2806-2818
Str u ctu r e-Ba sed Design , Syn th esis, a n d Biologica l Eva lu a tion of Ir r ever sible
Hu m a n Rh in ovir u s 3C P r otea se In h ibitor s. 1. Mich a el Accep tor
Str u ctu r e-Activity Stu d ies
Peter S. Dragovich,* Stephen E. Webber, Robert E. Babine, Shella A. Fuhrman, Amy K. Patick,
David A. Matthews, Caroline A. Lee, Siegfried H. Reich, Thomas J . Prins, J oseph T. Marakovits,
Ethel S. Littlefield, Ru Zhou, J ayashree Tikhe, Clifford E. Ford, Michael B. Wallace, J ames W. Meador, III,
Rose Ann Ferre, Edward L. Brown, Susan L. Binford, J ames E. V. Harr, Dorothy M. DeLisle, and
Stephen T. Worland
Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, California 92121
Received J anuary 29, 1998
The structure-based design, chemical synthesis, and biological evaluation of peptide-derived
human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds
incorporate various Michael acceptor moieties and are shown to irreversibly bind to HRV
serotype 14 3CP with inhibition activities (kobs/[I]) ranging from 100 to 600 000 M-1 s-1. These
inhibitors are also shown to exhibit antiviral activity when tested against HRV-14-infected
H1-HeLa cells with EC50’s approaching 0.50 µM. Extensive structure-activity relationships
developed by Michael acceptor alteration are reported along with the evaluation of several
compounds against HRV serotypes other than 14. A 2.0 Å crystal structure of a peptide-derived
inhibitor complexed with HRV-2 3CP is also detailed.
In tr od u ction
strate composition was previously determined to be
H2N-Thr-Leu-Phe-Gln-Gly-Pro-CO2H with cleavage oc-
curring between the Gln and Gly residues (Figure 1).11,12
It was reasoned that a truncated 3CP peptide substrate
in which the scissile amide carbonyl was replaced with
a Michael acceptor might function as an irreversible
3CP inhibitor (Figure 1). Similar analyses have pro-
duced potent, irreversible inhibitors of several other
cysteine proteases.13 Accordingly, incorporation of a
trans-R,â-unsaturated methyl ester moiety into a sub-
strate-derived, Cbz-protected tripeptide afforded a com-
pound which displayed relatively potent irreversible
inhibition of HRV-14 3CP (1, Table 1).14 Equally
important, compound 1 exhibited moderate antiviral
activity (EC50) when tested against H1-HeLa cells
infected with HRV-14 and was nontoxic (CC50) to the
limits of its solubility.15,16 In addition, the inhibitory
properties of the molecule were not affected by short
exposure to high concentrations of dithiothreitol (see the
Experimental Section), suggesting that Michael accep-
tors of this type would not react readily with ubiquitous
biological thiols (e.g., glutathione). These encouraging
results prompted an extensive SAR study of peptide-
derived Michael acceptors to better define this class of
3CP inhibitors.
The human rhinoviruses (HRVs) belong to the picor-
navirus family and are the single most significant cause
of the common cold.1,2 These viruses translate a posi-
tive-strand RNA genome into a large polyprotein which
undergoes co- and posttranslational processing by vi-
rally encoded proteases to produce the structural and
enzymatic proteins required for viral replication.3 Specif-
ically, cleavage between several glutamine-glycine
residues is effected by the human rhinovirus 3C pro-
tease (3CP),3 a cysteine protease with structural simi-
larity to the trypsin protein family but possessing
minimal homology to prevalent mammalian enzymes.4,5
Importantly, the activity of 3CP is essential for viral
replication, and its active site residues are believed to
be highly conserved throughout the more than 100
known rhinovirus serotypes.4-7
Currently, no effective therapy exists to directly treat
rhinoviral infections, and the numerous viral serotypes
make the development of a vaccine unlikely.1a,1e Previ-
ous approaches toward the identification of antirhinovi-
ral therapeutics include the use of interferon,1a,c,e dis-
ruption of virus-receptor (ICAM-1) interactions,1b the
examination of capsid-binding antipicornaviral com-
pounds,8 and the use of other miscellaneous agents.7,9
In addition, several examples of 3CP inhibitors have
recently been described in the literature, and some of
these compounds exhibit antiviral properties.10 Due to
the importance of the 3C protease in viral replication,
we also sought to develop inhibitors of this enzyme
which might function as broad-spectrum antirhinoviral
agents.
The corresponding cis-R,â-unsaturated methyl ester
(2) exhibited 10-fold lower anti-3CP activity and reduced
antiviral properties relative to 1 (Table 1). Further
efforts were therefore focused on molecules containing
Michael acceptors with trans geometry. Inclusion of an
ethyl ester in the inhibitor design (3) produced both a
better enzyme inhibitor and a more potent antiviral
agent, while incorporation of an R-fluoro substituent
drastically reduced 3CP inhibitory activity (4). Impor-
tantly, compound 3 irreversibly inhibited 3CPs obtained
from several rhinovirus serotypes other than HRV-14
In h ibitor Design a n d Str u ctu r e-Activity
Stu d ies
Inhibition studies commenced by examination of the
peptide sequence of 3CP substrates. A minimal sub-
S0022-2623(98)00068-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/30/1998

Human Rhinovirus 3C Protease Inhibitors. 1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2807
3CP inhibitors did not substantially improve from that
exhibited by the unsubstituted parent compound 20. In
addition to the indolines described above, amides de-
rived from N,O-dialkylhydroxylamines were also potent
3CP inhibitors (21, 23, and 24). As expected, an
indoline-containing compound displayed good stability
toward rat plasma, but a related N,O-dimethylhydroxy-
lamine amide was surprisingly short-lived (20 and 21,
respectively, Table 2).
Several molecules incorporating aliphatic and aryl
R,â-unsaturated ketones were also evaluated as 3CP
inhibitors (39-47, Table 1). Most of these compounds
displayed increased anti-3CP activity relative to the
ester-containing inhibitors described above. However,
the inhibitory activity of all ketone-derived Michael
acceptors examined was substantially reduced or elimi-
nated by short exposure to dithiothreitol, suggesting
that such compounds might react rapidly with biological
thiols (e.g., glutathione). In addition, the observed
EC50’s of the ketones were usually worse than those
exhibited by related ester-containing compounds (see
above) and were often not significantly distinct from
toxic concentrations to be considered true antiviral
activity (CC50 > 10EC50). The ketone-containing mol-
ecules were also significantly more toxic than similar
esters, possibly due to the greater reactivity of the
ketones toward nonenzymatic thiols.
F igu r e 1. Design of irreversible HRV 3CP inhibitors. EWG
) electron-withdrawing group.
and also exhibited antiviral properties when tested
against these serotypes in cell culture. The related R,â-
unsaturated carboxylic acid (10) was a poor 3CP inhibi-
tor and did not exhibit antiviral activity at the highest
concentration examined (100 µM). Compounds contain-
ing the simple esters described above were rapidly
converted to the corresponding carboxylic acids upon
incubation in rat plasma (Table 2), and efforts were
therefore directed toward ester modification or replace-
ment in order to increase the biological stability of the
inhibitors under study.
Analysis of the X-ray crystal structure of compound
3 complexed with HRV-2 3CP (see below) suggested that
additional substitution could be tolerated at the R-posi-
tion of the Michael acceptor. Accordingly, inclusion of
an R-methyl group in the inhibitor design (5) increased
stability toward rat plasma (Table 2) but also reduced
anti-3CP activity relative to compound 3. The crystal-
lographic analysis also indicated that additional func-
tionality could be incorporated in the vicinity of the ethyl
ester. Therefore, several other ester-derived Michael
acceptors were evaluated in which the sterics opposite
the R,â-unsaturated carbonyl moiety were altered (6-
9). Most of these molecules displayed relatively good
anti-3CP activity, particularly the benzyl analogue 8,
but some exhibited reduced stability toward rat plasma
(Table 2, compound 7). Importantly, the half-lives in
rat plasma of all ester-containing compounds examined
were sufficiently short to warrant the development of
3CP inhibitors which did not incorporate this functional
group.
A variety of miscellaneous Michael acceptors were
prepared in addition to the compounds described above
(Table 1). Vinyl sulfones (48 and 49) displayed low
levels of anti-3CP inhibition and were poor antiviral
agents. Similar properties were exhibited by molecules
which incorporated a vinyl nitrile (50), vinyl phospho-
nate (53), vinyl oxime (52), and several vinyl hetero-
cycles (54-56). In contrast, a compound containing an
R,â-unsaturated nitro moiety (51) demonstrated potent,
irreversible 3CP inhibitory activity. However, this
molecule was inactivated by exposure to dithiothreitol
and did not display measurable antiviral properties.
Likewise, compounds incorporating acyl lactam, acyl
oxazolidinone, and acyl urea functionalities were also
potent anti-3CP agents but were inactivated by expo-
sure to nonenzymatic thiols (57, 58, and 59, respec-
tively).
Since the preparation of compound 5 demonstrated
that hydrocarbon substitution could be tolerated at the
Michael acceptor R-position, the possibility of utilizing
exocyclic R,â-unsaturated lactones as 3CP inhibitors was
also examined (Table 4). The γ-lactone 60 exhibited
irreversible 3CP inhibitory activity similar to that
observed for ester 5 above and was stable toward rat
plasma for much greater periods of time (Table 2).
Surprisingly, the corresponding δ-lactone (61) displayed
potent, reversible 3CP inhibition. The factors which
determine whether a given lactone functions as a
reversible or irreversible 3CP inhibitor are currently not
understood. A molecule containing an N-acyl lactam-
derived Michael acceptor (62) was a potent, irreversible
3CP inhibitor and exhibited good antiviral properties.
However, the related carbamate and N-methoxy deriva-
tives displayed reduced anti-3CP activity and were poor
antiviral agents (compounds 63 and 64, respectively).
A series of amide-containing Michael acceptors was
therefore also investigated (11-26, Table 1). These
inhibitors typically displayed reduced anti-3CP activity,
poorer antiviral activity, and/or increased toxicity com-
pared to the esters described above. One exception was
a molecule incorporating an indoline-derived Michael
acceptor (20), and this discovery prompted an examina-
tion of inhibitors containing substituted indolines (27-
38, Table 3). Although proper indoline modification
increased anti-3CP activity to that observed for the
corresponding ethyl ester (compare 3 and 29), the
antiviral activity displayed by all indoline-containing
Finally, compound 65 was prepared in an effort to
assess the affinity of the peptidyl portion of the above

2808 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
Ta ble 1.

Human Rhinovirus 3C Protease Inhibitors. 1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2809
Ta ble 1. (Continued)
a
b
Method of preparation: A ) Scheme 1, B ) Scheme 2, C ) Scheme 3, D ) see the Experimental Section. Elemental analyses (C, H,
N) of all compounds agreed to within (0.4% of theoretical values unless otherwise noted. c Serotype 14 unless otherwise noted. See the
d
Experimental Section for assay method and error. e Indicates partial or complete loss of 3CP inhibitory activity after exposure of compound
to 5 mM dithiothreitol for 2-3 min at 23 °C. f C, calcd 62.50, found 63.31; N, calcd 11.76, found 11.20. 1:1 mixture of diastereomers. ND
g
) not determined.
Ta ble 2. Stability of Several 3CP Inhibitors in Rat Plasma
series of shallow grooves on the protein surface N-
terminal to the scissile amide bond of the substrate (S1-
S4).12 As expected, a covalent bond was observed
between the 3CP active site cysteine residue (Cys-147)
compd
t1/2 rat plasma (min)
compd
t1/2 rat plasma (min)
3
5
7
10
20
4
20
21
60
>80
12
>240
and the â-carbon of the Michael acceptor of 3. A
hydrogen bond between the carbonyl moiety of the
Michael acceptor and the 3CP Cys-147 amide NH was
also evident in the above complex. It is uncertain
whether this interaction facilitates Michael addition of
the cysteine to 3 or merely arises after the covalent
adduct has been formed. Interestingly, two equally
populated conformations were observed for two 3CP
residues flanking the enzyme active site. These resi-
dues (Ser-144 and Gly-145) are believed to help stabilize
the tetrahedral transition state which arises during
peptide hydrolysis. One conformation, in which the Gly-
145 amide NH pointed toward 3, resembled that ob-
served crystallographically for the corresponding resi-
dues of many trypsin-like serine proteases complexed
with peptidyl inhibitors (Figure 2, shown in yellow).19
The other involved rotation of Ser-144 so that its
carbonyl moiety pointed toward the inhibitor and the
Gly-145 amide NH faced the solvent. Similar alternate
conformations of related residues have been observed
crystallographically in other protease-inhibitor com-
plexes,10a,19 but the relevance of this conformational
variance toward 3CP inhibitor design has not been fully
evaluated.
inhibitors for the target protease. This compound was
a relatively weak, reversible 3CP inhibitor (Ki ) 20 µM)
and did not display antiviral properties when tested to
its solubility limit (100 µM). These results demonstrate
the importance of the Michael acceptor moiety in the
design of potent 3CP inhibitors and suggest that cova-
lent 3CP modification is required for obtaining such
inhibitors which function as highly active antirhinoviral
agents.
X-r a y Str u ctu r e An a lysis
Several other important protein-ligand interactions
were observed in addition to those noted in the vicinity
of the active site cysteine. The glutamine amide of 3
formed hydrogen bonds with the His-161 side chain and
both the Thr-142 side chain and amide carbonyl in the
bottom and on the edge of the 3CP S1 binding pocket,
respectively.12 The inhibitor phenylalanine residue
bound to 3CP in a canyon formed by the side chains of
Leu-127, Ser-128, and Asn-130 on one side and His-40
on the other. In contrast, the leucine side chain of 3
was quite solvent exposed and did not make appreciable
contacts with the enzyme. This observation suggested
that a variety of amino acids could be tolerated at this
location within the inhibitor design.20 The N-terminal
Cbz moiety of the inhibitor was situated in a hydropho-
bic pocket on the surface of the protein formed by the
side chains of Tyr-122, Ile-125, and Phe-170. However,
the interaction of this portion of 3 with 3CP appeared
to be suboptimal as evidenced by significant gaps
between the protein and the ligand.21 An antiparallel
â-sheet hydrogen bonding interaction was also noted
The iterative analysis of protein-ligand interactions
by X-ray crystallography is an essential element of a
structure-based inhibitor design program.17 Accord-
ingly, several crystal structures of 3CP-inhibitor-
complexes were obtained during the development of the
Michael acceptor-containing compounds described above.
These structures confirmed the binding geometries that
the inhibitors adopted when complexed with 3CP and
suggested locations for Michael acceptor modification.
However, the X-ray data were utilized somewhat cau-
tiously for inhibitor design since the structures depicted
the covalent protein-inhibitor product and not the
presumably more-relevant transition state for adduct
formation. The specific details of one protein-inhibitor
complex are discussed below.
The 2.0 Å X-ray crystal structure of the covalent
adduct formed between compound 3 and HRV-2 3CP18
is shown in Figure 2, and key protein-inhibitor interac-
tions are illustrated in Figure 3. In general, the
inhibitor bound to the enzyme in a manner similar to
that predicted by substrate modeling4a,10c and filled a

2810 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
Ta ble 3.
c
compd no.
X
Y
Z
prepa
formulab
kobs/[I] (M-1 s-1
)
DTT inhib?d EC50 (µM)c CC50 (µM)c
20
27
28
29
30
31
32
33
34
35
36
37
38
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
NO2
H
H
H
H
F
H
Cl
Br
OCH3
H
C
C
C
C
C
C
C
C
C
C
C
C
C
C38H45N5O6‚1.0H2O
C38H44N6O8‚0.50H2O
C38H44N6O8‚0.50H2O
C40H50N6O8S‚0.50H2O
C42H52N6O8S‚0.25H2O
C39H47N5O8S‚0.75H2O
6 500
12 000
29 000
28 300
22 700
12 900
5 400
5 400
450
5 800
3 400
,1 000
300
no
no
no
no
ND
ND
ND
ND
ND
ND
ND
ND
ND
16
17
>320
>320
>320
ND
>320
>320
>320
>320
56
>320
>320
>320
ND
NO2
SO2N(CH3)2
SO2Pyrrolidine
SO2CH3
H
H
H
H
H
H
H
>320
>100
>320
>320
11
56
5.6
47
>320
>320
ND
C
C
C
C
38H44FN5O6‚0.50H2O
38H44ClN5O6‚0.50H2O
38H44ClN5O6‚0.50H2O
38H44BrN5O6
C39H47N5O7‚0.50H2O
C39H47N5O8S‚1.0H2O
C39H47N5O6S‚1.8H2O
SO2CH3
SCH3
H
a
b
Method of preparation: C ) Scheme 3. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values.
c Serotype 14; see the Experimental Section for assay method and error. Indicates partial or complete loss of 3CP inhibitory activity
d
after exposure of compound to 5 mM dithiothreitol for 2-3 min at 23 °C. ND ) not determined.
Ta ble 4.
d
compd no.
X
R
n
prepa
formulab
C32H40N4O7
serotypec kobs/[I] (M-1 s-1
)
DTT inhib?e EC50 (µM)d CC50 (µM)d
60
61
62
O
O
N
1
2
1
A
A
A
10 900
Ki ) 30 nM
155 500
no
ND
no
5.2
16
0.71
ND
>320
>100
>100
C
33H42N4O7‚0.50H2O
C(O)CH3
C34H43N5O7‚0.50H2O
16
2
39 000
13 000
8 400
800
ND
28
18
63
64
N
N
CO2CH3
OCH3
1
1
A
A
C34H43N5O8‚0.75H2O
C33H43N5O7‚0.50H2O
no
ND
>100
>100
a
b
Method of preparation: A ) Scheme 1. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values.
c Serotype 14 unless otherwise noted. See the Experimental Section for assay method and error. e Indicates partial or complete loss of
d
3CP inhibitory activity after exposure of compound to 5 mM dithiothreitol for 2-3 min at 23 °C. ND ) not determined.
between the leucine residue and Gly-164 (Figure 3).
Similar interactions were previously observed in crystal
structures of several trypsin-like serine proteases com-
plexed with peptidyl inhibitors.19 The two other back-
bone amide NHs present in 3 formed hydrogen bonds
with 3CP Val-162 and Ser-128, while an additional
hydrogen bond between the inhibitor Cbz carbonyl
moiety and the 3CP Ser-128 amide NH was also evident
(Figure 3). Thus, a total of nine hydrogen bonds
involving every inhibitor amino acid residue were
observed in the complex between compound 3 and
HRV-2 3CP.
Analysis of the above complex suggested several
possibilities for Michael acceptor modification. The
Michael acceptor R-position was solvent exposed and did
not appreciably contact 3CP. Inclusion of additional
functionalities at this location in the inhibitor design
was therefore predicted not to significantly diminish
anti-3CP activity. This prediction was verified with the
preparation of compounds 5, 60, and 62 described above.
Likewise, replacement of the ethyl ester present in 3
with larger moieties was anticipated to have a minimal
effect on inhibitor potency. Again, this expectation was
confirmed by the synthesis of many potent 3CP inhibi-
tors containing Michael acceptors other than an R,â-
unsaturated ethyl ester (Tables 1 and 3).
Syn th esis
Most of the peptide-derived Michael acceptors utilized
in this study were prepared by three related methods
(A, B, and C). The particular method used to synthesize
a given compound is indicated in Tables 1, 3, and 4.
Representative synthetic examples are described below
and are detailed in the Experimental Section. Prepara-
tions of compounds which either did not involve one of
the general methods described above or included ad-
ditional transformations are also given in the Experi-
mental Section (Table 1, method D).
The first synthetic route (A) is illustrated by the
preparation of compound 3 (Scheme 1). N-R-Boc-γ-
trityl-L-glutamine was transformed into aldehyde 67 by
reduction of the corresponding Weinreb amide (66).22,23

Human Rhinovirus 3C Protease Inhibitors. 1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2811
F igu r e 2. Stereoview of the crystal structure of 3 complexed with HRV-2 3CP. A portion of the water-accessible surface of the
protein is shown in gray. An alternate conformation observed for several 3CP residues is superimposed and is shown in yellow.
This method allowed Michael acceptor diversification
to be effected at a later synthetic step than in the
preparation described above for compound 3. Thus,
N-R-Cbz-γ-trityl-L-glutamine was converted in several
steps to aldehyde 70 by the reported literature method.10a
Crude 70 was then subjected to a Wittig reaction
employing (carbethoxyethylidene)triphenylphosphorane
to provide olefin 71 after purification on silica gel. As
before, the trityl protecting group was removed by short
treatment of 71 with TFA to give compound 5 in
moderate yield. As noted for method A, a variety of
Michael acceptors could be prepared in this manner by
utilization of the appropriate triphenylphosphoranes for
reaction with aldehyde 70 (Table 1).27
The final general synthesis method (C) is depicted by
the preparations of compounds 6 and 13 (Scheme 3).
This method was employed to prepare esters and amides
for which the appropriate phosphonoacetate or triph-
F igu r e 3. Schematic diagram of 3 bound in the HRV-2 3CP
active site. Hydrogen bonds are represented as dashed lines,
and the residues which make up the enzyme binding subsites
are depicted.
enylphosphorane was not commercially available. Ethyl
ester 68 was carefully hydrolyzed under basic conditions
to afford carboxylic acid 72 with a minimal amount of
racemization.28 This material was not purified, but was
esterified with cyclopentanol to provide ester 73 in
moderate yield. Again, removal of the Boc protecting
group under acidic conditions and coupling of the
resulting amine salt with Cbz-L-Leu-L-Phe-OH afforded
the corresponding tripeptide (74) in moderate yield after
purification on silica gel. Alternatively, carboxylic acid
72 was condensed with aniline to give amide 75 which
was subsequently transformed into tripeptide 76 in a
manner analogous to that used for the preparation of
74. Compounds 6 and 13 were prepared from interme-
diates 74 and 76, respectively, by brief exposure to TFA
in the presence of triisopropylsilane. A variety of R,â-
unsaturated esters and amides could be prepared via
method C by derivitization of carboxylic acid 72 with
an appropriate alcohol or amine (Tables 1 and 3).
The crude aldehyde thus obtained was converted to the
desired Michael acceptor by reaction with the sodium
enolate of triethyl phosphonoacetate to afford ethyl ester
68 in good yield. For the preparation of compounds in
Tables 1 and 4 other than 3, the desired olefination was
accomplished by reaction of 67 with an appropriate
phosphonate anion or phosphonium ylide.24 Removal
of the Boc protecting group from 68 under acidic
conditions (HCl) and carbodiimide-mediated coupling of
the resulting crude amine salt with commercially avail-
able Cbz-L-Leu-L-Phe-OH provided tripeptide 69 in
moderate yield.25 The trityl protecting group was
removed by short exposure of 69 to trifluoroacetic acid
(TFA) in the presence of triisopropylsilane to give
compound 3 in good yield.26 The tripeptide inhibitors
prepared in this study were typically isolated as white
solids by removal of the volatiles from the detritylation
reaction mixture, trituration of the resulting oil with
Et2O, and subsequent filtration.
Con clu sion s
The ability of peptide-derived Michael acceptors to
function as potent inhibitors of human rhinovirus 3C
protease was demonstrated by the synthesis and bio-
logical evaluation of the compounds described above.
An illustration of the second preparative method (B)
is provided by the synthesis of compound 5 (Scheme 2).

2812 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
Sch em e 1a
a
Reagents and conditions (Tr ) CPh3): (a) 1.0 of equiv isobutyl chloroformate, 1.0 equiv of HCl‚HN(OCH3)CH3, 2.0 equiv of NMM,
CH2Cl2, 0 °C, 20 min f 23 °C, 2 h, 82%; (b) 2.5 equiv DIBAL, THF, -78 °C, 4 h, 97%; (c) 1.0 equiv of (EtO)2POCH2CO2Et, 1.0 equiv of
NaN(TMS)2, THF, -78 °C, 20 min, then 67, -78 °C, 2 h f 0 °C, 10 min, 88%; (d) HCl in 1,4-dioxane, 23 °C, 3 h; (e) 1.0 equiv of Cbz-L-
Leu-L-Phe-OH, 1.5 equiv of HOBT, 4.0 equiv of NMM, 1.5 equiv of EDC, CH2Cl2, 23 °C, 18 h, 83%; (f) 5.0 equiv of (i-Pr)3SiH, 1:1 TFA:
CH2Cl2, 23 °C, 20 min, 81%.
Sch em e 2a
a
Reagents and conditions (Tr ) CPh3): (a) ref 10a; (b) 1.1 equiv of Ph3PdC(CH3)CO2Et, THF, 23 °C, overnight; (c) 1:10 TFA:CH2Cl2,
23 °C, 4 h, 35%.
Sch em e 3a
a
Reagents and conditions (Tr ) CPh3, Cy ) cyclopentyl): (a) 2.3 equiv of NaOH, EtOH, 23 °C, 3 h, 98%; (b) 1.1 equiv of cyclopentanol,
0.10 equiv of DMAP, 1.05 equiv of EDC, CH2Cl2, 23 °C, overnight, 20%; (c) HCl in 1,4-dioxane, 23 °C, 2 h; (d) 1.0 equiv of Cbz-L-Leu-L-
Phe-OH, 1.5 equiv of HOBT, 4.0 equiv of NMM, 1.5 equiv of EDC, CH2Cl2, 23 °C, overnight, 59%; (e) 1.0 equiv of isobutyl chloroformate,
2.0 equiv of NMM, 1.0 equiv of PhNH2, CH2Cl2, 0 °C, 20 min f 23 °C, 24 h, 20%; (f) 5.0 equiv of (i-Pr)3SiH, 1:1 TFA:CH2Cl2, 23 °C, 1 h,
56%.
These molecules irreversibly inhibited 3CP’s from sev-
eral HRV serotypes and exhibited antiviral activity
when tested against these serotypes in cell culture.
Crystallographic analysis of an enzyme-inhibitor com-
plex confirmed the binding orientation of these com-
pounds and suggested modifications for inhibitor design.

Human Rhinovirus 3C Protease Inhibitors. 1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2813
(CDCl3) δ 1.44 (s, 9H), 1.65-1.75 (m, 1H), 2.17-2.23 (m, 1H),
2.31-2.54 (m, 2H), 4.11 (s, br, 1H), 5.38-5.40 (m, 1H), 7.11
(s, 1H), 7.16-7.36 (m, 15H), 9.45 (s, 1H).
The use of such structural information to further
improve this class of 3CP inhibitors is described in the
following paper.
Eth yl 3-[Boc-L-(Tr -Gln )]-(E)-p r op en oa te (68). Sodium
bis(trimethylsilyl)amide (22.9 mL of a 1.0 M solution in THF,
22.9 mmol, 1.0 equiv) was added to a solution of triethyl
phosphonoacetate (5.59 g, 22.9 mmol, 1.0 equiv) in THF (200
mL) at -78 °C, and the resulting solution was stirred for 20
min at that temperature. Crude 67 (10.8 g, 22.9 mmol, 1
equiv) in THF (50 mL) was added via cannula, and the reaction
mixture was stirred for 2 h at -78 °C, warmed to 0 °C for 10
min, and partitioned between 0.5 M HCl (150 mL) and a 1:1
mixture of EtOAc and hexanes (2 × 150 mL). The combined
organic layers were dried over Na2SO4 and were concentrated.
Purification of the residue by flash column chromatography
(40% EtOAc in hexanes) provided 68 (10.9 g, 88%) as a white
foam: Rf ) 0.60 (50% EtOAc in hexanes); IR (cm-1) 3321, 1710;
1H NMR (CDCl3) δ 1.27 (t, 3H, J ) 7.2), 1.42 (s, 9H), 1.70-
1.78 (m, 1H), 1.80-1.96 (m, 1H), 2.35 (t, 2H, J ) 7.0), 4.18 (q,
2H, J ) 7.2), 4.29 (s, br, 1H), 4.82-4.84 (m, 1H), 5.88 (dd, 1H,
J ) 15.7, 1.6), 6.79 (dd, 1H, J ) 15.7, 5.3), 6.92 (s, 1H), 7.19-
7.34 (m, 15H). Anal. (C33H38N2O5) C, H, N.
E t h yl 3-[Cb z-L-Leu -L-P h e-L-(Tr -Gln )]-(E)-p r op en oa t e
(69). A solution of HCl in 1,4-dioxane (4.0 M, 20 mL) was
added to a solution of 68 (1.00 g, 1.84 mmol, 1 equiv) in the
same solvent (20 mL) at 23 °C. After 3 h, the volatiles were
removed under reduced pressure. The residue was dissolved
in CH2Cl2 (50 mL), and Cbz-L-Leu-L-Phe-OH (0.759 g, 1.84
mmol, 1.0 equiv), 1-hydroxybenzotriazole hydrate (HOBt, 0.373
g, 2.76 mmol, 1.5 equiv), 4-methylmorpholine (0.809 mL, 7.36
mmol, 4.0 equiv), and 1-(3-(dimethylamino)propyl)-3-ethylcar-
bodiimide hydrochloride (EDC, 0.529 g, 2.76 mmol, 1.5 equiv)
were added sequentially. The reaction mixture was stirred
at 23 °C for 18 h and then was partitioned between water (150
mL) and EtOAc (2 × 150 mL). The combined organic layers
were dried over Na2SO4 and were concentrated. Flash chro-
matographic purification of the residue (5% CH3OH in CH2-
Cl2) afforded 69 (1.25 g, 83%) as a white solid: mp 192-194
°C; Rf ) 0.33 (5% CH3OH in CH2Cl2); IR (cm-1) 3295, 1696,
1678, 1655, 1519; 1H NMR (CDCl3) δ 0.84 (d, 3H, J ) 6.5),
0.86 (d, 3H, J ) 6.5), 1.24-1.32 (m, 1H), 1.28 (t, 3H, J ) 7.2),
1.43-1.75 (m, 3H), 1.91-2.06 (m, 1H), 2.20-2.38 (m, 2H),
2.93-3.02 (m, 1H), 3.07-3.18 (m, 1H), 3.95-4.02 (m, 1H), 4.17
(q, 2H, J ) 7.2), 4.43-4.55 (m, 2H), 4.82-4.95 (m, 2H), 5.69
(d, 1H, J ) 15.7), 6.46 (d, 1H, J ) 7.5), 6.60 (d, 1H, J ) 8.1),
6.69 (dd, 1H, J ) 15.7, 5.1), 7.09-7.38 (m, 27H). Anal.
(C51H56N4O7) C, H, N.
Eth yl 3-(Cbz-L-Leu -L-P h e-L-Gln )-(E)-p r op en oa te (3).
Trifluoroacetic acid (20 mL) was added to a solution of 69 (1.25
g, 1.49 mmol, 1 equiv) and triisopropylsilane (1.53 mL, 7.47
mmol, 5.0 equiv) in CH2Cl2 (20 mL) at 23 °C, producing a
bright yellow solution. The reaction mixture was stirred for
20 min at 23 °C during which time it became colorless. Carbon
tetrachloride (20 mL) was added, and the volatiles were
removed under reduced pressure. The residue was triturated
with Et2O (20 mL), and the resulting white solid was collected
by vacuum filtration, washed with Et2O (3 × 50 mL), and air-
dried to afford 3 (0.717 g, 81%): mp 219-221 °C; Rf ) 0.17
(5% CH3OH in CH2Cl2); IR (cm-1) 3300, 1672, 1535; 1H NMR
(DMSO-d6) δ 0.78 (d, 3H, J ) 6.8), 0.82 (d, 3H, J ) 6.5), 1.21
(t, 3H, J ) 7.0), 1.25-1.37 (m, 2H), 1.42-1.54 (m, 1H), 1.58-
1.80 (m, 2H), 2.02-2.09 (m, 2H), 2.84 (dd, 1H, J ) 13.2, 8.9),
2.97 (dd, 1H, J ) 13.2, 5.8), 3.93-4.01 (m, 1H), 4.11 (q, 2H, J
) 7.0), 4.33-4.52 (m, 2H), 4.97 (d, 1H, J ) 12.3), 5.04 (d, 1H,
J ) 12.3), 5.64 (d, 1H, J ) 15.9), 6.69 (dd, 1H, J ) 15.9, 5.4),
6.76 (s, 1H), 7.13-7.37 (m, 11H), 7.43 (d, 1H, J ) 7.8), 7.99
(d, 1H, J ) 8.1), 8.04 (d, 1H, J ) 8.1). Anal. (C32H42N4O7) C,
H, N.
Exp er im en ta l Section
Gen er a l. All reactions were performed in septum-sealed
flasks under a slight positive pressure of argon unless other-
wise noted. All commercial reagents were used as received
from their respective suppliers with the following exceptions.
Tetrahydrofuran (THF) was distilled from sodium-benzophe-
none ketyl prior to use. Dichloromethane (CH2Cl2) was
distilled from calcium hydride prior to use. Flash column
chromatography29 was performed using silica gel 60 (Merck
Art 9385). 1H NMR spectra were recorded at 300 MHz
utilizing either a Varian UNITYplus 300 or a General Electric
QE-300 spectrometer equipped with Techmag operating soft-
ware. Chemical shifts are reported in ppm (δ) downfield
relative to internal tetramethylsilane, and coupling constants
are given in hertz. Infrared absorption spectra were recorded
using either a MIDAC Corp. or a Perkin-Elmer 1600 series
FTIR. Elemental analyses were performed by Atlantic Mi-
crolab, Inc., Norcross, GA. Melting points were determined
using a Mel-Temp II apparatus and are uncorrected.
A simplified naming system employing amino acid ab-
breviations is used to identify some intermediates and final
products. In this naming system, italicized amino acid ab-
breviations represent modifications at the C-terminus of that
residue where the following apply: (1) acrylic acid esters are
reported as “E” (trans) propenoates; (2) acrylamides are
reported as “E” propenamides. The N,O-dialkylhydroxy-
lamines required for the preparation of compounds 23 and 24
were prepared by literature methods.30 The noncommercially
available indolines utilized in the synthesis of compounds 27-
38 were prepared by reduction of the corresponding indoles
(34-36),31 lithiation of 1-Boc-indolines (33, 37, and 38),32 or
substitution of 1-acyl-5-bromoindoline (29-31).33 The tri-
phenylphosphoranes required for the preparation of com-
pounds 42-47 and 54-56 were synthesized from the corre-
sponding R-bromo ketones,34 while those required for inhibitors
60-64 were prepared by related literature methods.35,36 The
phosphonates utilized in the synthesis of compounds 48 and
49 were also prepared by literature methods.37
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od A.
Syn th esis of Eth yl 3-(Cbz-L-Leu -L-P h e-L-Gln )-(E)-p r op en -
oa te (3). [Boc-L-(Tr -Gln )]-N(OMe)Me (66). Isobutyl chlo-
roformate (4.77 mL, 36.8 mmol, 1.0 equiv) was added to a
solution of N-R-Boc-γ-trityl-L-glutamine (18.7 g, 36.7 mmol, 1
equiv) and 4-methylmorpholine (8.08 mL, 73.5 mmol, 2.0
equiv) in CH2Cl2 (250 mL) at 0 °C. The reaction mixture was
stirred at 0 °C for 20 min, and then N,O-dimethylhydroxy-
lamine hydrochloride (3.60 g, 36.7 mmol, 1.0 equiv) was added.
The resulting solution was stirred at 0 °C for 20 min and at
23 °C for 2 h and then was partitioned between water (150
mL) and CH2Cl2 (2 × 150 mL). The combined organic layers
were dried over Na2SO4 and were concentrated. Purification
of the residue by flash column chromatography (gradient
elution, 40 f 20% hexanes in EtOAc) provided 66 (16.1 g, 82%)
as a white foam: Rf ) 0.22 (50% EtOAc in hexanes); IR (cm-1
)
3411, 3329, 3062, 1701, 1659; 1H NMR (CDCl3) δ 1.42 (s, 9H),
1.63-1.77 (m, 1H), 2.06-2.17 (m, 1H), 2.29-2.43 (m, 2H), 3.17
(s, 3H), 3.64 (s, 3H), 4.73 (s, br, 1H), 5.38-5.41 (m, 1H), 7.20-
7.31 (m, 15H). Anal. (C31H37N3O5) C, H, N.
[Boc-L-(Tr -Gln )]-H (67). Diisobutylaluminum hydride
(50.5 mL of a 1.5 M solution in toluene, 75.8 mmol, 2.5 equiv)
was added to a solution of 66 (16.1 g, 30.3 mmol, 1 equiv) in
THF at -78 °C, and the reaction mixture was stirred at -78
°C for 4 h. Methanol (4 mL) and 1.0 M HCl (10 mL) were
added sequentially, and the mixture was warmed to 23 °C.
The resulting suspension was diluted with Et2O (150 mL) and
was washed with 1.0 M HCl (3 × 100 mL), half-saturated
NaHCO3 (100 mL), and water (100 mL). The organic layer
was dried over MgSO4, filtered, and concentrated to give crude
67 (13.8 g, 97%) as a white solid: mp 114-116 °C; Rf ) 0.42
(50% EtOAc in hexanes); IR (cm-1) 3313, 1697, 1494; 1H NMR
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od B.
Eth yl 2-Meth yl-3-(Cbz-L-Leu -L-P h e-L-Gln )-(E)-pr open oate
(5). (Carbethoxyethylidene)triphenylphosphorane (0.29 g, 0.78
mmol, 1.1 equiv) was added to a solution of crude 7010a (0.54
g, 0.71 mmol, 1 equiv) in THF (14 mL) at 23 °C. After the
mixture was stirred overnight, the volatiles were removed

2814 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
under reduced pressure and the residue was chromatographed
on silica gel (gradient elution, 0 f 2% CH3OH in CHCl3) to
give 71 (0.52 g) contaminated with triphenylphosphine oxide.
This material was dissolved in CH2Cl2 (12 mL) at 23 °C, and
trifluoroacetic acid (1.2 mL) was added. After 4 h of stirring
at 23 °C, the reaction mixture was concentrated and the
residue was triturated with a 1:1 mixture of EtOAc and Et2O
(10 mL). The resulting white precipitate was filtered, washed
with Et2O (2 × 40 mL), and dried under vacuum to give 5 (0.15
g, 35%) as an off-white solid: mp 187-188 °C; Rf ) 0.24 (5%
CH3OH in CHCl3); IR (cm-1) 1707, 1647, 1541, 1261; 1H NMR
(DMSO-d6) δ 0.79 (d, 3H, J ) 6.3), 0.82 (d, 3H, J ) 6.6), 1.22
(t, 3H, J ) 7.0), 1.29 (m, 2H), 1.43 (m, 1H), 1.61 (m, 1H), 1.75
(m, 1H), 1.79 (s, 3H), 2.02 (m, 2H), 2.83 (m, 1H), 2.92 (m, 1H),
3.99 (m, 1H), 4.13 (q, 2H, J ) 7.0), 4.41 (m, 1H), 4.49 (m, 1H),
4.99 (d, 1H, J ) 12.5), 5.04 (d, 1H, J ) 12.1), 6.31 (d, 1H, J )
8.8), 6.73 (s, br, 1H), 7.15 (m, 6H), 7.34 (m, 5H), 7.45 (d, 1H,
J ) 7.7), 7.91 (d, 1H, J ) 7.7), 8.04 (d, 1H, J ) 8.1). Anal.
(C33H44N4O7‚0.75H2O) C, H, N.
6.65 (dd, 1H, J ) 15.9, 5.4), 7.04-7.35 (m, 25H). Anal.
(C54
H60N4O7‚H2O) C, H, N.
Cyclop en tyl 3-(Cbz-L-Leu -L-P h e-L-Gln )-(E)-p r op en oa te
(6). Trifluoroacetic acid (6 mL) was added to a solution of 74
(0.150 g, 0.171 mmol, 1 equiv) and triisopropylsilane (0.175
mL, 0.854 mmol, 5.0 equiv) in CH2Cl2 (7 mL) at 23 °C,
producing a bright yellow solution. The reaction mixture was
stirred at 23 °C until it became colorless (1 h), CCl4 (4 mL)
was added, and the volatiles were removed under reduced
pressure. The residue was triturated with Et2O (10 mL), and
the resulting white solid was collected by vacuum filtration,
washed with Et2O (3 × 5 mL), and air-dried to afford 6 (0.061
g, 56%) as a white solid: mp 188-190 °C; Rf ) 0.5 (10% CH3-
OH in CHCl3); IR (cm-1) 3389, 3295, 1707; 1H NMR (acetone-
d6) δ 0.83-0.87 (m, 6H), 1.06-1.09 (m, 1H), 1.46-1.51 (m, 1H),
1.60-1.70 (m, 11H), 1.83-1.94 (m, 1H), 2.20-2.25 (m, 2H),
2.90-3.01 (m, 1H), 3.18 (dd, 1H, J ) 13.9, 5.8), 4.00 (d, 1H, J
) 6.8), 4.05-4.10 (m, 1H), 4.55-4.62 (m 2H), 4.97-5.16 (m,
4H), 5.76 (d, 1H, J ) 15.3), 6.71 (m, 2H), 7.15-7.41 (m, 10H),
7.51 (d, 1H, J ) 7.8). Anal. (C35H40N4O7) C, H, N.
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od C.
Syn th esis of Cyclop en tyl-3-(Cbz-L-Leu -L-P h e-L-Gln )-(E)-
p r op en oa t e (6). 3-[Boc-L-(Tr -Gln )]-(E)-p r op en oic Acid
(72). Sodium hydroxide (4.6 mL of a 1.0 M aqueous solution,
4.6 mmol, 2.3 equiv) was added to a 23 °C solution of 68 (1.09
g, 2.10 mmol, 1 equiv) in EtOH (10 mL) dropwise via addition
funnel over 20 min. The resulting white suspension was
stirred at 23 °C for 3 h and then was partitioned between H2O
(100 mL) and Et2O (100 mL). The aqueous layer was acidified
to pH ) 2 with 1.0 M HCl and was extracted with EtOAc (2 ×
100 mL). The organic layers were dried over Na2SO4 and were
concentrated to provide crude 72 (1.01 g, 98%) as a white
solid: 1H NMR (CDCl3) δ 1.42 (s, 9H), 1.72-1.80 (m, 1H),
1.83-1.98 (m, 1H), 2.37 (t, 2H, J ) 7.0), 4.22-4.37 (m, 1H),
4.86-4.89 (m, 1H), 5.85 (d, 1H, J ) 15.3), 6.86 (dd, 1H, J )
15.5, 5.1), 6.92 (s, 1H), 7.18-7.32 (m, 15H).
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od C.
Syn t h esis of N-P h en yl-3-(Cb z-L-Leu -L-P h e-L-Gln )-(E)-
p r op en a m id e (13). N-P h en yl-3-[Boc-L-(Tr -Gln )]-(E)-p r o-
p en a m id e (75). Isobutyl chloroformate (0.075 mL, 0.578
mmol, 1.0 equiv) was added to a solution of 72 (0.296 g, 0.575
mmol, 1 equiv) and 4-methylmorpholine (0.126 mL, 1.15 mmol,
2.0 equiv) in CH2Cl2 (15 mL) at 0 °C. The reaction mixture
was stirred at 0 °C for 30 min, and then aniline (0.052 mL,
0.571 mmol, 1.0 equiv) was added. The resulting suspension
was stirred at 0 °C for 20 min and at 23 °C for 24 h and then
was vacuum filtered through medium paper. The solid thus
obtained was washed with CH2Cl2 (10 mL) and Et2O (10 mL)
and was air-dried to provided 75 (0.065 g, 20%) as a white
solid: mp 241-243 °C; Rf ) 0.31 (5% CH3OH in CH2Cl2); IR
1
(cm-1) 3264, 1715, 1660; H NMR (DMSO-d6) δ 1.40 (s, 9H),
1.62-1.65 (m, 1H), 2.27-2.35 (m, 1H), 4.11 (s, br, 1H), 6.13
(d, 1H, J ) 15.3), 6.64 (dd, 1H, J ) 15.3, 5.9), 7.01-7.08 (m,
2H), 7.12-7.32 (m, 20H), 7.64 (d, 1H, J ) 7.8), 8.62 (s, 1H).
Anal. (C37H39N3O4) C, H, N.
Cyclop en t yl-3-[Boc-L-(Tr -Gln )]-(E)-p r op en oa t e (73).
1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochlo-
ride (0.372 g, 1.94 mmol, 1.05 equiv) was added to a solution
of 72 (0.951 g, 1.85 mmol, 1 equiv), cyclopentanol (0.185 mL,
2.04 mmol, 1.1 equiv), and 4-(dimethylamino)pyridine (0.038
g, 0.185 mmol, 0.10 equiv) in CH2Cl2 (30 mL) at 23 °C. The
reaction mixture was stirred at 23 °C overnight and then was
concentrated under reduced pressure. The residue was puri-
fied by flash column chromatography (50% EtOAc in hexanes)
to provide 73 (0.215 g, 20%) as a white foam: Rf ) 0.70 (50%
N-P h en yl-3-[Cbz-L-Leu -L-P h e-L-(Tr -Gln )]-(E)-p r op en a -
m id e (76). A solution of HCl in 1,4-dioxane (4.0 M, 6 mL)
was added to a solution of 75 (0.060 g, 0.102 mmol, 1 equiv)
in the same solvent (6 mL) at 23 °C. After 1 h, the volatiles
were removed under reduced pressure. The residue was
dissolved in CH2Cl2 (10 mL), and Cbz-L-Leu-L-Phe-OH (0.042
g, 0.102 mmol, 1.0 equiv), 1-hydroxybenzotriazole hydrate
(0.021 g, 0.155 mmol, 1.5 equiv), 4-methylmorpholine (0.034
mL, 0.309 mmol, 3 equiv), and 1-(3-(dimethylamino)propyl)-
3-ethylcarbodiimide hydrochloride (0.029 g, 0.151 mmol, 1.5
equiv) were added sequentially. The reaction mixture was
stirred at 23 °C for 26 h and then was partitioned between
water (100 mL) and EtOAc (2 × 100 mL). The combined
organic layers were dried over Na2SO4 and were concentrated.
Flash chromatographic purification of the residue (5% CH3-
OH in CH2Cl2) afforded 76 (0.049 g, 55%) as a white foam: Rf
) 0.47 (10% CH3OH in CH2Cl2); IR (cm-1) 3278, 1642, 1536;
1H NMR (CDCl3) δ 0.82 (d, 3H, J ) 11.4), 0.84 (d, 3H, J )
11.4), 1.23-1.30 (m, 1H), 1.34-1.49 (m, 2H), 1.76-1.83 (m,
1H), 1.97 (s, br, 1H), 2.28 (t, 2H, J ) 7.0), 2.87-2.94 (m, 1H),
3.17-3.21 (m, 1H), 3.96-4.03 (m, 1H), 4.58 (s, br, 2H), 4.84-
4.94 (m, 2H), 5.77 (d, 1H, J ) 15.9), 6.53 (d, 1H, J ) 7.8), 6.70-
6.75 (m, 1H), 6.96-7.32 (m, 31H), 7.57 (d, 2H, J ) 7.8), 7.70
(s, 1H). Anal. (C55H57N5O6‚0.50H2O) C, H, N.
N-P h en yl-3-(Cbz-L-Leu -L-P h e-L-Gln )-(E)-p r op en a m id e
(13). Trifluoroacetic acid (4 mL) was added to a solution of
76 (0.042 g, 0.048 mmol, 1 equiv) and triisopropylsilane (0.100
mL, 0.488 mmol, 10 equiv) in CH2Cl2 (4 mL) at 23 °C,
producing a bright yellow solution. The reaction mixture was
stirred for 20 min at 23 °C, during which time it became
colorless. Carbon tetrachloride (6 mL) was added, and the
volatiles were removed under reduced pressure. The residue
was triturated with Et2O (6 mL), and the resulting white solid
was collected by vacuum filtration, washed with Et2O (3 × 6
mL), and air-dried to afford 13 (0.023 g, 74%): mp 240 °C dec;
1
EtOAc in hexanes); IR (cm-1) 3319, 1708; H NMR (CDCl3) δ
1.25-1.29 (m, 2H), 1.44 (s, 9H), 1.59-1.89 (m, 8H), 2.38 (t,
2H, J ) 7.2), 4.32 (s, br, 1H), 4.54-4.56 (m, 1H), 5.20-5.23
(m, 1H), 5.87 (d, 1H, J ) 15.6), 6.77 (dd, 1H, J ) 15.1, 4.1),
6.90 (s, br, 1H), 7.20-7.33 (m, 15H). Anal. (C36H42N2O5‚
0.50H2O) C, H, N.
Cyclop en t yl-3-[Cb z-L-Leu -L-P h e-L-(Tr -Gln )]-(E)-p r o-
p en oa te (74). A 4.0 M solution of HCl in 1,4-dioxane (3.5
mL) was added to a solution of 73 (0.215 g, 0.369 mmol, 1
equiv) in the same solvent (3.5 mL) at 23 °C. After 2 h of
stirring, the reaction mixture was concentrated under reduced
pressure. The residue was dissolved in CH2Cl2 (30 mL), and
Cbz-L-Leu-L-Phe-OH (0.152 g, 0.369 mmol, 1.0 equiv), 4-meth-
ylmorpholine (0.163 mL, 1.48 mmol, 4.0 equiv), 1-hydroxy-
benzotriazole hydrate (0.075 g, 0.554 mmol, 1.5 equiv), and
1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochlo-
ride (0.106 g, 0.554 mmol, 1.5 equiv) were added sequentially.
The resulting solution was stirred overnight at 23 °C and then
was partitioned between 1 M HCl (100 mL) and CH2Cl2 (2 ×
100 mL). The combined organic layers were dried over MgSO4,
filtered, and concentrated. Purification of the residue by flash
chromatography (50% EtOAc in hexanes) afforded 74 (0.191
g, 59%) as a white foam: Rf ) 0.4 (50% EtOAc in hexanes);
IR (cm-1) 3401, 3319, 1708; 1H NMR (CDCl3) δ 0.78-0.90 (m,
6H), 1.01-1.10 (m, 1H), 1.23-1.33 (m, 1H), 1.46-1.71 (m, 9H),
1.82-1.88 (m, 1H), 2.24-2.32 (m, 2H), 2.98-3.12 (m, 4H),
3.95-4.02 (m 1H), 4.46-4.48 (m, 2H), 4.83-5.21 (m, 4H), 5.65
(d 1H, J ) 15.9), 6.50 (d, 1H, J ) 7.2) 6.59 (d, 1H, J ) 8.1),

Human Rhinovirus 3C Protease Inhibitors. 1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2815
Rf ) 0.29 (10% CH3OH in CH2Cl2); IR (cm-1) 3267, 1647; H
NMR (DMSO-d6) δ 0.78 (d, 3H, J ) 6.2), 0.82 (d, 3H, J ) 6.5),
1.29-1.38 (m, 2H), 1.46-1.48 (m, 1H), 1.73-1.75 (m, 2H),
2.07-2.12 (m, 2H), 2.84 (dd, 1H, J ) 13.6, 9.2), 3.04 (dd, 1H,
J ) 13.6, 4.7), 3.92-4.00 (m, 1H), 4.37-4.51 (m, 2H), 4.98 (d,
1H, J ) 12.5), 5.05 (d, 1H, J ) 12.5), 6.13 (d, 1H, J ) 15.3),
6.60 (dd, 1H, J ) 15.3, 6.4), 6.77 (s, 1H), 7.04 (t, 2H, J ) 7.3),
7.12-7.38 (m, 12H), 7.43 (d, 1H, J ) 8.1), 7.64 (d, 2H, J )
7.8), 7.93 (d, 1H, J ) 8.1), 8.14 (d, 1H, J ) 8.1), 10.05 (s, 1H).
Anal. (C36H43N5O6‚0.25H2O) C, H, N.
Exa m p les of P r ep a r a tion Meth od D. Syn th esis of
P yr r ole 25, Nitr o Com p ou n d 51, a n d Oxim e 52. 1-(P yr -
r ol-1-yl)-3-(Cbz-Leu -L-P h e-L-Gln )-(E)-p r op en a m id e (25).
A solution of 1-(3-pyrrolin-1-yl)-3-[Cbz-Leu-L-Phe-L-(Tr-Gln)]-
(E)-propenamide (prepared utilizing general method C, 0.424
g, 0.493 mmol, 1 equiv) and 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (0.17 g, 0.75 mmol, 1.5 equiv) in benzene (30
mL) were stirred rapidly for 72 h at 23 °C. The volatiles were
then removed under vacuum, and the residue was purified by
flash column chromatography (3% CH3OH in CHCl3) to yield
1-(3-pyrrol-1-yl)-3-[Cbz-Leu-L-Phe-L-(Tr-Gln)]-(E)-propen-
amide (0.31 g, 73%) as a white solid: 1H NMR (DMSO-d6) δ
0.76 (d, 3H, J ) 6.6), 0.80 (d, 3H, J ) 6.3), 1.30 (m, 2H), 1.44
(m, 1H), 1.63 (m, 2H), 2.28 (m, 2H), 2.90 (m, 1H), 2.99 (m,
1H), 3.95 (m, 1H), 4.49 (m, 2H), 4.98 (q, 2H, J ) 13.4), 6.35 (s,
br, 2H), 6.66 (d, 1H, J ) 15.8), 6.96 (dd, 1H, J ) 14.9, 5.0),
7.06-7.40 (m, 25H), 7.47 (m, 3H), 8.04 (m, 2H), 8.58 (s, 1H).
This material was dissolved in CH2Cl2 (11 mL) and cooled
to 0 °C, and trifluoroacetic acid (0.7 mL) was added. The
reaction mixture was warmed to 23 °C and was stirred for 30
min. The mixture was then concentrated under reduced
pressure, and the residue was dissolved in EtOAc (50 mL) and
washed sequentially with 10% aqueous NaHCO3 (50 mL) and
brine (50 mL). The organic layer was dried over Na2SO4 and
was concentrated. Purification of the residue by flash column
chromatography (3% CH3OH in CH2Cl2) provided 25 (0.16 g,
74%) as a white solid: 1H NMR (DMSO-d6) δ 0.77 (d, 3H, J )
6.6), 0.82 (d, 3H, J ) 6.3), 1.32 (m, 2H), 1.45 (m, 1H), 1.77 (m,
2H), 2.10 (t, 2H, J ) 7.5), 2.89 (dd, 1H, J ) 14.3, 9.7), 3.01
(dd, 1H, J ) 9.7, 5.5), 3.94 (m, 1H), 4.47 (m, 2H), 5.01 (q, 2H,
J ) 12.9), 6.35 (s, br, 2H), 6.68 (d, 1H, J ) 15.1), 6.77 (s, br,
1H), 6.96 (dd, 1H, J ) 14.7, 5.2), 7.07 (s, br, 1H), 7.19 (m, 6H),
7.34 (m, 5H), 7.51 (s, br 2H), 8.05 (m, 2H). Anal. (C34H41N5O6)
C, H, N.
3.95-4.03 (m, 1H), 4.43-4.48 (m, 2H), 4.96 (d, 1H, J ) 12.9),
5.02 (d, 1H, J ) 12.9), 6.86 (d, 1H, J ) 13.6), 7.06 (dd, 1H, J
) 13.6, 5.5), 7.13-7.32 (m, 25H), 7.67 (d, 1H, J ) 7.7), 8.03
(d, 1H, J ) 7.7), 8.15 (d, 1H, J ) 8.5), 8.56 (s, 1H).
1
Nitr o Com p ou n d 51. Trifluoroacetic acid (0.25 mL) was
added to a solution of 78 (0.095 g, 0.12 mmol) in CH2Cl2 (2.5
mL), and the reaction mixture was stirred at room tempera-
ture for 6 h. The volatiles were removed under reduced
pressure, and the residue was triturated with a 1:1 mixture
of EtOAc/Et2O (5 mL). The resulting white solid was filtered
and washed with Et2O (20 mL) to afford 51 (0.022 g, 33%) as
a white solid: IR (cm-1) 1647, 1530; 1H NMR (DMSO-d6) δ
0.79 (d, 3H, J ) 6.6), 0.82 (d, 3H, J ) 7.0), 1.28-1.30 (m, 2H),
1.47-1.53 (m, 1H), 1.67-1.82 (m, 2H), 2.04-2.09 (m, 2H),
2.89-2.92 (m, 2H), 3.97-4.02 (m, 1H), 4.41-4.48 (m, 2H), 4.97
(d, 1H, J ) 12.5), 5.04 (d, 1H, J ) 12.5), 6.78 (s, br, 1H), 6.88
(d, 1H, J ) 13.6), 7.07 (dd, 1H, J ) 13.6, 5.5), 7.14-7.34 (m,
11H), 7.44 (d, 1H, J ) 7.0), 8.07 (d, 1H, J ) 7.7), 8.15 (d, 1H,
J ) 8.1). Anal. (C29H37N5O7‚0.85CH2Cl2) C, H, N.
Oxim e 52. (Triphenylphosphoranylidene)acetaldehyde (0.26
g, 0.82 mmol, 1.1 equiv) was added to a solution of 7010a (0.576
g, 0.75 mmol, 1 equiv) in THF (15 mL), and the reaction was
stirred at room temperature overnight. The solvent was
removed under reduced pressure, and the residue was dis-
solved in pyridine (8 mL). Methoxylamine hydrochloride
(0.064 g, 0.75 mmol, 1.0 equiv) was added, and the reaction
was again stirred at room temperature overnight. The solvent
was removed in vacuo, and the residue was dissolved in EtOAc
(50 mL) and washed with saturated NaHCO3 (50 mL), water
(50 mL), and brine (50 mL). The organic layer was dried over
MgSO4 and concentrated. Purification of the residue by flash
column chromatography (gradient elution, 0 f 1% CH3OH in
CHCl3) gave intermediate 79 (0.334 g, contaminated with
triphenylphosphine oxide) which was used without further
purification.
Trifluoroacetic acid (1.0 mL) was added to a solution of 79
in CH2Cl2 (10 mL), and the reaction mixture was stirred at
room temperature for 6 h. The volatiles were removed under
reduced pressure, and the residue was triturated with a 1:1
mixture of EtOAc/Et2O (20 mL). The resulting white solid was
filtered and washed with Et2O (30 mL) to afford 52 (0.104 g,
24%, mixture of oxime isomers) as a white solid: Rf ) 0.10
1
(3% CH3OH/CHCl3); IR (cm-1) 1645, 1539; H NMR (DMSO-
d6, major isomer) δ 0.77-0.83 (m, 6H), 1.26-1.30 (m, 2H),
1.43-1.46 (m, 1H), 1.65-1.68 (m, 2H), 2.03-2.08 (m, 2H),
2.78-2.86 (m, 1H), 2.94-3.00 (m, 1H), 3.76 (s, 3H), 3.92-3.94
(m, 1H), 4.26-4.31 (m, 1H), 4.45-4.47 (m, 1H), 4.98 (d, 1H, J
) 12.9), 5.04 (d, 1H, J ) 12.5), 5.96-5.98 (m, 1H), 6.47-6.55
(m, 1H), 6.75 (s, 1H), 7.11-7.43 (m, 13H), 8.01-8.06 (m, 2H).
Anal. (C31H41N5O6) C, H, N.
Nitr o Alcoh ol 77. Potassium fluoride (0.0014 g, 0.02 mmol,
0.04 equiv) was added to a solution of 7010a (0.361 g, 0.47 mmol,
1 equiv) in a 1:1 mixture of CH3NO2 and i-PrOH (4 mL). The
reaction mixture was stirred overnight at room temperature
and then was concentrated under reduced pressure. The
residue was purified by flash column chromatography (gradi-
ent elution, 0 f 2% CH3OH in CHCl3) to give 77 (0.32 g, 83%,
2:1 mixture of diastereomers) as a glassy solid: Rf ) 0.13 (2%
P r ep a r a tion of Com p ou n d 65. A suspension of ethyl
3-[Boc-L-(Tr-Gln)]-(E)-propenoate (68) (0.383 g, 0.706 mmol,
1 equiv) and Pd/C (10%, 0.060 g) in EtOH (20 mL) was stirred
under a hydrogen atmosphere (balloon) at 23 °C for 16 h. The
reaction mixture was filtered through Celite, and the filtrate
was concentrated to afford a colorless oil. This material was
dissolved in 1,4-dioxane (10 mL), and HCl (4.0 M solution in
1,4-dioxane, 10 mL) was added. The resulting solution was
stirred at 23 °C for 1.5 h, and then the volatiles were removed
under reduced pressure. The residue was dissolved in CH2-
Cl2 (20 mL), and Cbz-L-Leu-L-Phe-OH (0.291 g, 0.705 mmol,
1.0 equiv), 1-hydroxybenzotriazole hydrate (0.114 g, 0.844
mmol, 1.2 equiv), 4-methylmorpholine (0.310 mL, 2.82 mmol,
4.0 equiv), and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiim-
ide hydrochloride (0.162 g, 0.845 mmol, 1.2 equiv) were added
sequentially. The reaction mixture was stirred at 23 °C for
21 h and then was partitioned between water (100 mL) and
EtOAc (2 × 100 mL). The combined organic layers were dried
over Na2SO4 and were concentrated. Flash chromatographic
purification of the residue (3% CH3OH in CH2Cl2) afforded the
product tripeptide (80) (0.281 g, 47%) as a white foam: Rf )
0.53 (10% CH3OH in CH2Cl2); IR (cm-1) 3293, 1708, 1647; 1H
NMR (CDCl3) δ 0.85 (t, 6H, J ) 6.2), 1.24 (t, 3H, J ) 7.2),
1.29-1.39 (m, 1H), 1.42-1.58 (m, 4H), 1.70-1.84 (m, 2H),
1
CH3OH in CHCl3); IR (cm-1) 3500-3200 (br), 1659, 1518; H
NMR (DMSO-d6, major diastereomer) δ 0.71-0.80 (m, 6H),
1.28-1.35 (m, 2H), 1.1.48-1.60 (m, 2H), 1.64-1.95 (m, 1H),
2.19-2.30 (m, 2H), 2.78-2.87 (m, 1H), 2.96-3.03 (m, 1H),
3.72-3.82 (m, 1H), 3.84-4.03 (m, 2H), 4.10-4.20 (m, 1H),
4.33-4.38 (m, 1H), 4.46-4.54 (m, 1H), 4.95 (d, 1H, J ) 12.5),
5.01 (d, 1H, J ) 12.5), 5.64-5.68 (m, 1H), 7.15-7.41 (m, 26H),
7.72 (d, 1H, J ) 8.1), 7.97 (d, 1H, J ) 6.6), 8.51 (s, 1H). Anal.
(C48H53N5O8) C, H, N.
Vin yln itr o Com pou n d 78. Methanesulfonyl chloride (0.027
mL, 0.35 mmol, 1.0 equiv) and Et3N (0.20 mL, 1.40 mmol, 4.0
equiv) were added sequentially to a solution of 77 (0.292 g,
0.35 mmol, 1 equiv) in CH2Cl2 (4 mL) at 0 °C. The reaction
was allowed to warm to room temperature and was stirred
overnight. Additional methanesulfonyl chloride (0.10 mL) was
then added, and the reaction mixture was stirred for 2 h at
23 °C. The volatiles were then removed in vacuo, and the
product was purified by flash chromatography (0.5% CH3OH
in CHCl3) to afford 78 (0.11 g, 39%) as an off-white, glassy
1
solid: Rf ) 0.18 (3% CH3OH in CHCl3); H NMR (DMSO-d6)
δ 0.65-0.81 (m, 6H), 1.28-1.36 (m, 2H), 1.47-1.62 (m, 1H),
1.70-1.80 (m, 2H), 2.24-2.40 (m, 2H), 2.84-2.94 (m, 2H),

2816 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
2.04-2.33 (m, 4H), 2.98 (dd, 1H, J ) 13.9, 7.0), 3.12 (dd, 1H,
J ) 13.9, 6.7), 3.75-3.82 (m, 1H), 3.95-4.02 (m, 1H), 4.09 (q,
2H, J ) 7.2), 4.45-4.53 (m, 1H), 4.85-4.94 (m, 2H), 6.17 (d,
1H, J ) 9.0), 6.45 (d, 1H, J ) 7.8), 7.14-7.39 (m, 27H). Anal.
(C51H58N4O7) C, H, N.
Two days later, XTT/PMS was added to the test plates, and
the amount of formazan produced was quantified spectropho-
tometrically at 450/650 nm. The EC50 was calculated as the
concentration of drug that increased the percentage of forma-
zan production in drug-treated, virus-infected cells to 50% of
that produced by drug-free, uninfected cells. The 50% cytotoxic
concentration (CC50) was calculated as the concentration of
drug that decreased the percentage of formazan produced in
drug-treated, uninfected cells to 50% of that produced in drug-
free, uninfected cells. The reported values were obtained from
either a single antiviral determination or the mean of two or
more experiments. To avoid false positives due to toxicity, only
compounds displaying CC50’s greater than 10 times the
observed EC50’s were considered to be truly active antiviral
agents. Using the above method, the EC50 of a representative
compound (3) was calculated to be 0.54 ( 0.27 µM (range
0.18-1 µM). The EC50 of the known antirhinoviral agent
Pirodavir was similarly determined to be 0.02 ( 0.01 µM
(range 0.01-0.05 µM) and was comparable to the 0.03 µM
minimal inhibitory concentration value previously reported.40
Trifluoroacetic acid (8 mL) was added to a solution of 80
(0.270 g, 0.322 mmol, 1 equiv) and triisopropylsilane (0.330
mL, 1.61 mmol, 5.0 equiv) in CH2Cl2 (10 mL) at 23 °C,
producing a bright yellow solution. The reaction mixture was
stirred for 20 min at 23 °C during which time it became
colorless. Carbon tetrachloride (6 mL) was added, and the
volatiles were removed under reduced pressure. The residue
was triturated with Et2O (10 mL), and the resulting white solid
was collected by vacuum filtration, washed with Et2O (3 × 30
mL), and air-dried to afford 65 (0.171 g, 89%): mp 210-215
°C; Rf ) 0.45 (10% CH3OH in CH2Cl2); IR (cm-1) 3267, 1689,
1643; 1H NMR (DMSO-d6) δ 0.78 (d, 3H, J ) 6.5), 0.82 (d, 3H,
J ) 6.5), 1.17 (t, 3H, J ) 7.2), 1.22-1.63 (m, 7H), 1.97-2.05
(m, 4H), 2.82 (dd, 1H, J ) 13.5, 8.4), 2.96 (dd, 1H, J ) 13.5,
6.2), 3.62 (s, br, 1H), 3.94-4.05 (m, 3H), 4.39-4.47 (m, 1H),
4.98 (d, 1H, J ) 12.6), 5.05 (d, 1H, J ) 12.6), 6.70 (s, br, 1H),
7.14-7.34 (m, 11H), 7.42 (d, 1H, J ) 7.8), 7.64 (d, 1H, J )
8.7), 7.97 (d, 1H, J ) 8.1). Anal. (C32H44N4O7) C, H, N.
En zym e Assa ys. The general conditions of the fluorescence
resonance energy transfer assay utilized to assess 3CP activity
and procedures for reversible inhibitor Ki determinations are
described in ref 10c. Continuous fluorometric enzyme assays
were conducted using a Perkin-Elmer LS50B spectrofluorim-
eter with a four place motorized cuvette holder. The kobs/[I]
values for the irreversible inhibitors were obtained from
reactions initiated by addition of 50 nM 3CP, containing 1 µM
substrate and varying inhibitor concentrations. Typically,
three to five concentrations were examined, and three related
methods were utilized for subsequent data analysis. For
reactions in which less than 20% of the substrate was
processed when 100% inactivation was observed, data from
the continuous assays were directly analyzed with the non-
linear regression analysis program ENZFITTER38 to obtain
first-order rate constants for enzyme inactivation at each
inhibitor concentration. The slope of a graph of kobs(I) vs [I]
was calculated using ENZFITTER and reported as kobs/[I]. The
error associated with this determination is less than 10% of a
given value and is often less than 5%. For very fast irrevers-
ible inhibitors (kobs/[I] > 400 000 M-1 s-1), sufficient data points
could not be obtained to determine kobs/[I] by the above slope
method. These “fast” values were therefore calculated directly
from a single kobs number by dividing by [I]. The error
associated with this determination is less than 20% of a given
value. For very slow irreversible inhibitors (kobs/[I] < 1000 M-1
s-1) and/or when >20% consumption of substrate was observed
at 100% 3CP inactivation, the primary data was adjusted to
account for expected changes in velocity due to changes in
substrate concentration. The transformed data was used to
calculate kobs(I) values and the kobs/[I] figure was determined
utilizing the slope method described above.
Ra t P la sm a Sta bility Stu d ies. Rat plasma was isolated
from blood collected in vacutainers containing sodium heparin
as the anticoagulant via a cardiac stick from Spraque-Dawley
rats (Hilltop, Scottsdale, PA). This plasma (1:1 dilution with
100 mM potassium phosphate buffer, pH 7.4) was incubated
in a final volume of 4 mL at 37 °C in a shaking water bath for
5 min. A DMSO solution of a given 3CP inhibitor (25 µM)
was then added such that the amount of organic solvent in
the final incubations was 0.5% (v/v). After 0, 20, 40, 60, 120,
180, and 240 min, a 200 µL aliquot of the incubate was
removed and transferred to a test tube containing 2.0 mL of
CH3CN. Samples were vortexed for 30 s and subsequently
centrifuged for 20 min at 2000 rpm. The supernatant (2 mL)
was transferred to a clean test tube and dried under N2 at 40
°C. The dried residue was reconstituted in 200 µL of 1:1 CH3-
CN:potassium phosphate buffer (pH 3.0), and the amount of
a given 3CP inhibitor present was determined by HPLC
analysis at 23 °C utilizing a Hewlett-Packard 1100 HPLC
equipped with a Rainin Microsorb MV C8 reverse phase
column (5 µm, 4.6 mm × 150 mm) and UV detection at 210
nm. The inhibitors were eluted using a solvent gradient
beginning with 25%:25%:50% CH3OH:CH3CN:25 mM potas-
sium phosphate (pH 3.0) for 3 min and then increasing over
10 min to a 70%:5%:25% ratio and subsequently returning to
the initial conditions over 7 min all at a flow rate of 1.0 mL/
min.
P r otein -Liga n d Cr ysta l Str u ctu r e Deter m in a tion .
Serotype 2 human rhinovirus 3C protease was incubated with
a 3-fold molar excess of compound 3 and the complex concen-
trated to a final protein concentration of 12.5 mg/mL. Equal
volumes of the protein-ligand stock solution and a 2.0 M
solution of ammonium sulfate buffered with 100 mM ADA pH
6.5 were mixed and allowed to incubate at 21 °C for 1 h. This
solution was then passed through a 0.45 µm Centrex centrifu-
gal filter and used immediately for crystallization experiments.
Crystals were grown at 21 °C using a hanging drop vapor
diffusion method in which 10 µL drops were placed on plastic
coverslips and sealed over individual wells filled with 1 mL of
a reservoir solution containing 2.0 M ammonium sulfate, 100
mM ADA pH 6.5, plus 2.5% (v/v) 1,4-dioxane. Crystals
typically grew as large rectangular blocks but were invariably
twinned. Careful use of a microscope with crossed polarizers
and a dissecting needle facilitated extraction of a single crystal
measuring 0.7 × 0.25 × 0.15 mm from such a binary twin.
The crystallographic space group was determined to be P21212
with a ) 63.49, b ) 78.56, c ) 34.50 Å with one protease
molecule in the crystallographic asymmetric unit.
The sensitivity of inhibitors to nonenzymatic thiols was
evaluated using reactions similar to those described in the
previous paragraph, but with the addition of 5 mM dithio-
threitol (DTT) to the inhibitor-containing mixture 2-3 min
before the addition of enzyme. Inhibitor DTT sensitivity was
indicated by a significantly reduced kobs for enzyme inactiva-
tion compared to that obtained in a control reaction lacking
DTT.
An tivir a l Assa ys. All strains of human rhinovirus (HRV)
were purchased from American Type Culture Collection
(ATCC). HRV stocks were propagated, and antiviral assays
were performed in H1-HeLa cells (ATCC). Cells were grown
in Minimal Essential Medium with 10% fetal bovine serum.
The ability of compounds to protect cells against HRV infection
was measured by the XTT dye reduction method.39 Briefly,
H1-Hela cells were infected with HRV-14 at a multiplicity of
infection (moi) of 0.08 or mock-infected with medium only.
Infected or uninfected cells were resuspended at 8 × 105 cells
per mL and incubated with appropriate concentrations of drug.
Cu KR X-ray diffraction data were recorded from one crystal
of the serotype 2 3CP-3 complex at 4 °C using a 9 kW rotating
anode source and two Xuong-Hamlin multiwire area detector
systems. These data were 90% complete to 1.97 Å resolution
with R(sym)(I) ) 4.65% and an average of 4.5 measurements
per symmetry-unique reflection. Rotation and translation
search procedures implemented in X-PLOR41 were used to

Human Rhinovirus 3C Protease Inhibitors. 1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2817
Natl. Acad. Sci. U.S.A. 1987, 84, 2605-2609. (e) Werner, G.;
Rosenwirth, B.; Bauer, E.; Siefert, J .-M.; Werner, F.-J .; Besemer,
J . Molecular Cloning and Sequence Determination of the Ge-
nomic Regions Encoding Protease and Genome-Linked Protein
of Three Picornaviruses. J . Virol. 1986, 57, 1084-1093. (f) Skern,
T.; Sommergruber, W.; Blaas, D.; Gruendler, P.; Fraundorfer,
F.; Pieler, C.; Fogy, I.; Kuechler, E. Human Rhinovirus 2:
Complete Nucleotide Sequence and Proteolytic Processing Sig-
nals in the Capsid Protein Region. Nucleic Acids Res. 1985, 13,
2111-2126. (g) Callahan, P. L.; Mizutani, S.; Colonno, R. J .
Molecular Cloning and Complete Sequence Determination of
RNA Genome of Human Rhinovirus Type 14. Proc. Natl. Acad.
Sci. U.S.A. 1985, 82, 732-736. (h) Stanway, G.; Hughes, P. J .;
Mountford, R. C.; Minor, P. D.; Almond, J . W. The Complete
Nucleotide Sequence of a Common Cold Virus: Human Rhi-
novirus 14. Nucleic Acids Res. 1984, 12, 7859-7875.
orient and position a search model of serotype 2 3CP whose
structure had been previously determined in another crystal
form.42 Refinement was initiated using the rigid body option
in X-PLOR followed by simulated annealing and conjugate
gradient minimization protocols using all data in the 10 to 2
Å resolution range. Atomic coordinates for compound 3 were
then fit to difference electron density maps and the protein-
ligand model refined. This was followed by sequential addition
of ordered solvent in several cycles involving stereochemically
reasonable placement of candidate waters into difference maps
and subsequent refinement of the combined model. The final
R factor was 19.1% for data between 8 and 2 Å resolution
(10236 reflections with F > 2σ(F)). The root-mean-square
deviations from ideal bond lengths and angles were 0.016 Å
and 3.0°, respectively. The final model contained all atoms
for residues 1-180 (excluding side chains for residues 12, 45,
and 65) plus 104 water molecules.
(6) Hamparian, V. V.; Colonno, R. J .; Cooney, M. K.; Dick, E. C.;
Gwaltney, J . M., J r.; Hughes, J . H.; J ordan, W. S., J r.; Kapikian,
A. Z.; Mogabgab, W. J .; Monto, A.; Phillips, C. A.; Rueckert, R.
R.; Schieble, J . H.; Stott, E. J .; Tyrrell, D. A. J . A Collaborative
Report: Rhinoviruses-Extension of the Numbering System From
89 to 100. Virology 1987, 159, 191-192.
Ack n ow led gm en t. We are grateful for many helpful
discussions throughout the course of this work with
Prof. Larry Overman. We also thank Dr. Kim Albizati,
Dr. Srinivasan Babu, and Terence Moran for the large-
scale syntheses of several intermediates and Dr. Xinjun
Hou for assistance with the preparation of Figure 2.
(7) Carrasco, L. Picornavirus Inhibitors. Pharmacol. Ther. 1994, 64,
215-290.
(8) (a) Oren, D. A.; Zhang, A.; Nesvadba, H.; Rosenwirth, B.; Arnold,
E. Synthesis and Activity of Piperazine-containing Antirhinovi-
ral Agents and Crystal Structure of SDZ 880-061 Bound to
Human Rhinovirus 14. J . Mol. Biol. 1996, 259, 120-134 and
references therein. (b) Diana, G. D.; Rudewicz, P.; Pevear, D.
C.; Nitz, T. J .; Aldous, S. C.; Aldous, D. J .; Robinson, D. T.;
Draper, T.; Dutko, F. J .; Aldi, C.; Gendron, G.; Oglesby, R. C.;
Volkots, D. L.; Reuman, M.; Bailey, T. R.; Czerniak, R.; Block,
T.; Roland, R.; Oppermann, J . Picornavirus Inhibitors: Trifluo-
romethyl Substitution Provides a Global Protective Effect Against
Hepatic Metabolism. J . Med. Chem. 1995, 38, 1355-1371 and
references therein.
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(3) (a) Orr, D. C.; Long, A. C.; Kay, J .; Dunn, B. M.; Cameron, J . M.
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