Solid-phase synthesis of irreversible human rhinovirus 3C protease inhibitors. Part 1: Optimization of tripeptides incorporating N-terminal amides

Article abstract of DOI:10.1016/S0968-0896(99)00005-X

The optimization of a series of irreversible human rhinovirus (HRV) 3C protease (3CP) inhibitors is described. These inhibitors are comprised of an l-Leu-l-Phe-l-Gln tripeptide containing an N-terminal amide moiety and a C-terminal ethyl propenoate Michae

Full text of DOI:10.1016/S0968-0896(99)00005-X

Bioorganic & Medicinal Chemistry 7 (1999) 589±598
Solid-phase Synthesis of Irreversible Human Rhinovirus 3C
Protease Inhibitors. Part 1: Optimization of Tripeptides
Incorporating N-terminal Amides
Peter S. Dragovich,* Ru Zhou, Donald J. Skalitzky, Shella A. Fuhrman, Amy K. Patick,
Cli€ord E. Ford, James W. Meador, III and Stephen T. Worland
Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, CA 92121, USA
Received 14 September 1998
AbstractÐThe optimization of a series of irreversible human rhinovirus (HRV) 3C protease (3CP) inhibitors is described. These
inhibitors are comprised of an l-Leu-l-Phe-l-Gln tripeptide containing an N-terminal amide moiety and a C-terminal ethyl pro-
penoate Michael acceptor. Examination of approximately 500 compounds with varying N-terminal amides utilizing solid-phase
synthesis and high-throughput assay techniques is described along with the solution phase preparation of several highly active
molecules. A tripeptide Michael acceptor containing an N-terminal amide derived from 5-methylisoxazole-3-carboxylic acid is
shown to exhibit potent, irreversible anti-3CP activity (kobs/[I]=260,000 M 1 s 1; type-14 3CP) and broad-spectrum antirhinoviral
properties (average EC50=0.47 mM against four di€erent HRV serotypes). # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
Results and Discussion
The human rhinoviruses (HRVs) are members of the
picornavirus family and are the single most signi®cant
cause of the common cold.1,2 We recently described a
series of compounds that inhibit the HRV 3C protease
(3CP), a virally encoded cysteine protease essential for
replication, and which exhibit antirhinoviral activity in
cell culture.3 These inhibitors contain a substrate-
derived tripeptide binding determinant4,5 that provides
anity for the target protein and a Michael acceptor
which irreversibly forms a covalent adduct with the
active site cysteine residue of the 3C enzyme (Fig. 1).3 In
our earlier work, we found that inclusion of an N-
terminal P4 thiocarbamate moiety in the inhibitor
design signi®cantly improved anti-3CP and antirhino-
viral activity relative to similar carbamate-containing
molecules (compare compounds 1 and 2, Fig. 1).3b
However, we were concerned that the thiocarbamate
moiety might undergo facile in vivo metabolism, and we
therefore sought to replace this functionality with
amides which also imparted good levels of activity to
the resulting inhibitors (Chart 1). Below we describe the
identi®cation of such amide moieties through the utili-
zation of solid-phase synthesis and high-throughput
assay techniques.6
The solid-phase synthesis of tripeptide-derived, irrever-
sible HRV 3CP inhibitors is illustrated in Scheme 1.
Although several Michael acceptor±tripeptide combina-
tions are known to exhibit potent anti-3CP activity,3 the
ethyl propenoate Michael acceptor and l-Leu-l-Phe-l-
Gln binding element were selected for this study due to
their ease of synthesis. In addition, all potential 3CP
inhibitors prepared on solid phase were synthesized as
single entities utilizing Chiron Multipins27 in order to
facilitate the identi®cation of active molecules. Thus,
PyBOP-mediated8 coupling of Fmoc-protected glutamic
acid derivative 33b with pin-supported Rink Amide9
linker a€orded intermediate 4. Removal of the Fmoc
protecting group from 4 by exposure to 2% DBU10 in
CH2Cl2 and HATU-e€ected11 condensation of the
resulting amine with Fmoc-l-Phe-OH gave dipeptide
intermediate 5 (not shown). Treatment of 5 with 2%
DBU in CH2Cl2 and coupling of the liberated amine
with Fmoc-l-Leu-OH then provided tripeptide inter-
mediate 6. The purity of each coupling adduct (4, 5 and
6) was evaluated by cleavage from the solid support
(TFA/H2O) and HPLC analysis of the resulting mono-,
di- or tripeptide. Although such analysis indicated that
intermediates 4 and 5 were produced in >90% purity,
the incomplete formation of the tripeptide 6 after a
single Fmoc-l-Leu-OH coupling reaction was also
detected. This latter transformation was therefore
repeated in order to improve the purity of 6 (88% after
second coupling).
Key words: Rhinovirus; irreversible; cysteine; protease; inhibitor.
*Corresponding author.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00005-X

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P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
activity against HRV-14 3CP in an assay system custom-
ized for microtiter plate use (8Â12 well). As no
attempt was made to quantitate the amount of a given
compound produced by the above solid-phase synthesis,
assay results were compared from well to well assuming
a nominal product yield of 50%. Compounds 1 and 2
(Fig. 1) were included as positive controls, and assay
conditions were chosen such that the inhibitory activ-
ity exhibited by 2 corresponded to the maximum
measurable (100% 3CP inhibition, see Experimental).
Since it was anticipated that most, if not all, tripeptides
8 would display some level of anti-3CP activity,13 com-
pounds were considered worthy of further examination
only if their 3CP inhibition exceeded that displayed
by compound 1 (75%, see Experimental). Using the
above method for inhibitor evaluation, several highly-
active molecules (8a±g, Table 2) were identi®ed for
solution-phase resynthesis and additional biological
evaluation.14,15
Figure 1. Irreversible HRV 3CP inhibitors (aSerotype-14).
The solution-phase preparation of tripeptide-derived
3CP inhibitors 8a±g is illustrated in Scheme 2, and
directly follows that previously described for the syn-
thesis of closely related molecules.3 Thus, Boc-protected
glutamine derivative 93a was deprotected under acidic
conditions (HCl) and the resulting amine salt was cou-
pled with Boc-l-Phe-OH to a€ord dipeptide 10 (not
shown). Acid-mediated deprotection of 10 and sub-
sequent coupling with Boc-l-Leu-OH uneventfully pro-
vided tripeptide 11. Removal of the N-terminal Boc
moiety from 11 and derivatization of the amine salt thus
obtained with an appropriate acid or acid chloride
a€orded the tripeptides 12a±g. The trityl protecting
groups present in 12a±g were removed by short expo-
sure to TFA in the presence of triisopropylsilane to give
the desired compounds 8a±g in good yields. As was
described previously,3 the tripeptide inhibitors prepared
by the above solution-phase method were isolated as
white solids by removal of the volatiles from the detri-
tylation reaction mixtures, trituration of the resulting
oils with Et2O, and subsequent ®ltration.
Chart 1. Design of thiocarbamate replacements.
Having assembled the tripeptide 6 on solid support, we
then focussed our e€orts on N-terminal amide synthesis
and diversi®cation (Scheme 1). Accordingly, the Fmoc
group was removed from 6 as described above, and the
newly-formed N-terminal amine was independently
coupled with a variety of carboxylic acids and acid
chlorides to a€ord approximately 500 unique inter-
mediates 7. Subsequent exposure of these intermediates
to aqueous TFA e€ected cleavage from the solid sup-
port and provided the desired tripeptidyl compounds 8.
The acids and acid chlorides utilized in this study were
selected from the ACD12 based on their commercial
availability, low molecular weight (<300), and lack of
highly reactive functional groups other than the desired
acyl moiety (e.g. a-bromo-carboxylic acids were not
included). Prior to coupling the complete set of acids
and acid chlorides, several representative derivatization/
cleavage reactions were performed (data not shown).
HPLC analysis indicated that the tripeptide products 8
were typically obtained in the highest purities from
coupling reactions utilizing acid chlorides, but some
reactions employing carboxylic acids and certain acid
chlorides were incomplete. Therefore, when synthesizing
the entire complement of intermediates 7, all couplings
were performed twice in order to increase the purity of
the desired products. A random sample (approximately
5%) of all tripeptides 8 thus prepared on solid phase
was subjected to HPLC and mass spectral analysis, and
several examples are presented in Table 1. In most cases,
the target compounds were produced in >50% purity
and all exhibited a major peak corresponding to the
correct molecular ion in the EI mass spectrum.
The biological activities of the resynthesized compounds
8a±g are depicted in Table 2. As expected, these mol-
ecules irreversibly inhibited HRV-14 3C protease with
many exhibiting signi®cantly greater activity than the
control compound 1 (Fig. 1). Compounds 8a±g also
displayed various levels of antiviral activity without
observed cytotoxicity when tested against HRV-14 in
cell culture. The most-active inhibitor (8e) was also
examined against several HRV serotypes other than 14
and, as illustrated in Table 3, exhibited broad-spectrum
antiviral activity. Combination of the N-terminal isox-
azole moiety present in 8e with similar tripeptide or
peptidomimetic 3CP inhibitors is thus anticipated to
a€ord additional potent anti-3CP and antirhinoviral
agents.16
Conclusions
The N-terminal optimization of tripeptide-derived
HRV-3CP inhibitors was accomplished utilizing solid-
phase synthesis and high-throughput assay techniques.
The completed series of approximately 500 derivatized
tripeptide Michael acceptors (8) was then tested for

P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
591
Scheme 1. Reagents and conditions: (a) 0.2 equiv solid-supported Rink-Amide linker, 2.0 equiv DIEA, 0.10 equiv HOBt, 1.0 equiv PyBOP, DMF,
23 ꢀC, 1 h; (b) 2% DBU in CH2Cl2, 23 ꢀC, 30 min; (c) 5.0 equiv Fmoc-l-Phe-OH, 10.0 equiv collidine, 5.0 equiv HATU, DMF, 23 ꢀC, 1 h; (d) 5.0
equiv Fmoc-l-Leu-OH, 10.0 equiv collidine, 5.0 equiv HATU, DMF, 23 ꢀC, 1 h, 2 times; (e) excess RC(O)Cl and collidine, CHCl3, 23 ꢀC, 1 h or
excess RCO2H, DIEA, HATU, DMF, 23 ꢀC, 1 h, 2 times; (f ) 95:5 TFA:H2O, 23 ꢀC, 1 h.
Such optimization identi®ed a molecule (8e) containing
an N-terminal 5-methylisoxazole-3-carboxamide as a
potent 3CP inhibitor and broad-spectrum antirhinoviral
agent. Incorporation of such carboxamides into other
tripeptide or peptidomimetic HRV-3CP inhibitors will
be described in future publications.
(CH2Cl2) was distilled from calcium hydride prior to
use. Flash column chromatography17 was performed
using silica gel 60 (Merck Art 9385). H NMR spectra
1
were recorded at 300 MHz utilizing a Varian UNI-
TYplus 300 spectrometer. Chemical shifts are reported
in ppm (d) down®eld relative to internal tetra-
methylsilane and coupling constants are given in Hertz.
Infrared absorption spectra were recorded using a
Perkin±Elmer 1600 series FTIR. HPLC purity analyses
were conducted utilizing a Hewlett Packard Series 1100
HPLC with UV detection (222 nm) equipped with a
ZORBAX RX-C18 analytical column (4.6Â150 mm).
Elemental analyses were performed by Atlantic Micro-
lab, Inc., Norcross, GA. Melting points were determined
using a Mel-Temp II apparatus and are uncorrected.
SynPhase2 crowns, deepwell microtiter plates (8Â12),
and Multipin2 stems were purchased from Chiron
Technologies, San Diego, CA.
Experimental
General
All reactions were performed in septum-sealed ¯asks
under a slight positive pressure of argon unless otherwise
noted. All commercial reagents were used as received
from their respective suppliers with the following excep-
tions. Tetrahydrofuran (THF) was distilled from sodium-
benzophenone ketyl prior to use. Dichloromethane

592
P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
Table 1. Characterization of representative tripeptides 8 prepared on solid phase
Entry
1
RC(O)X
HPLC area (%)
78
Mass spectrum Entry
RC(O)X
HPLC area (%)
61
Mass spectrum
621(MH)
587(MNa)
6
2
80
605(MNa)
7
93
579(MH)
3
4
5
61
51
93
711/713(MH)
531(MH)
8
9
90
80
32
607(MH)
599(MNa)
625(MH)
559(MH)
10
A simpli®ed naming system employing amino acid
abbreviations is used to identify intermediates and ®nal
products. When utilizing this naming system, italicized
amino acid abbreviations represent modi®cations at the
C-terminus of that residue where acrylic acid esters are
reported as `e' (trans) propenoates. The following
abbreviations are also utilized: TFA (tri¯uoroacetic
acid), DIEA (N,N-diisopropylethylamine), DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene), HOBt (1-hydroxy-
benzotriazole hydrate), PyBOP (benzotriazole-1-yl-oxy-
Multipin2 stems to support the pins. The following
standard puri®cation procedures were utilized. For
reactions run in round bottom ¯asks, the pins were
washed sequentially with DMF (100 mL) and CH2Cl2
(100 mL) and the solvent was decanted away. The
washing sequence was repeated two additional times,
and the pins were then dried under vacuum to give the
solid-phase intermediates. For reactions run in deepwell
microtiter plates, the stems were suspended in a plastic
wash bath, and the attached pins were washed sequen-
tially with DMF (100 mL) and CH2Cl2 (100 mL). The
washing sequence was repeated two additional times,
and the pins were then air-dried to give the solid-phase
intermediates.
tris-pyrrolidino-phosphonium
hexa¯uorophosphate),
HATU [O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetrameth-
yluronium hexa¯uorophosphate], EDC [1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride].
Intermediate and product purities were determined as
follows. A single pin was stirred in a mixture of TFA
and H2O (95:5, 1 mL) for 15 min at 23 ꢀC. After removal
of the pin from the reaction ¯ask, the volatiles were
evaporated under reduced pressure and the residue was
dissolved in CH3CN (1 mL). The resulting solution was
then analysed by HPLC utilizing a mixture of CH3CN,
H2O, and TFA (50%, 49.9%, and 0.1%, respectively)
¯owing isocratically at 1.0 mL/min as the mobile phase.
Speci®c retention times and purities are indicated below.
Preparation of tripeptides 8 on solid support. General
solid-phase synthesis procedures.
Solid-phase synthesis was performed using SynPhase2-
PS (polystyrene) O-series crowns (pins) derivatized with
Fmoc-protected Rink Amide Linkers (RAM). The pre-
parations of intermediates 4, 5, and 6 as well as the
subsequent deprotection of 6 were performed in round
bottom ¯asks. Intermediates 7 and ®nal products 8 were
synthesized in deepwell microtiter plates (8Â12) utilizing

P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
Table 2. Biological activity of tripeptidyl HRV 3CP inhibitors
593
under vacuum. The pins were suspended in 2% DBU in
CH2Cl2 (100 mL) and stirred for 30 min at 23 ꢀC, then
were puri®ed by the standard procedure. The depro-
tected pins were resuspended in DMF (100 mL), and
Fmoc-l-Phe-OH (3.13 g, 8.08 mmol, ꢁ5.0 equiv), colli-
dine (2.02 mL, 16.2 mmol, ꢁ10 equiv), and HATU
(3.07 g, 8.08 mmol, ꢁ5.0 equiv) were added sequentially.
The reaction mixture was stirred for 1 h at 23 ꢀC after
which the solvent was decanted away. Standard puri-
®cation then provided dipeptide intermediate 5 (HPLC
Anal. 6.82 min, 98% purity). Prior to the next synthetic
step, the derivatized pins were treated with 5% Ac2O in
CH2Cl2 (100 mL) for 30 min at 23 ꢀC in order to acylate
any unreacted amine.
Compound
P4
kobs/[I]
(M 1 s
EC50 (mM)a CC50 (mM)
1 a
)
8ab
95,000
1.4
>100
Ethyl-3-[Fmoc-L-Leu-L-Phe-L-(N-pin)Gln]-E-propenoate
(6). Pins containing dipeptide intermediate 5 (700 pins
at ꢁ2.3 mmol/pin, ꢁ1.61 mmol) were washed with
CH2Cl2 and dried under vacuum. The pins were then
suspended in 2% DBU in CH2Cl2 (100 mL) and stirred
for 30 min at 23 ꢀC. After puri®cation by the standard
procedure, the deprotected pins were resuspended in
DMF (100 mL) and Fmoc-l-Leu-OH (2.84 g, 8.04 mmol,
ꢁ5.0 equiv), DIEA (2.80 mL, 16.1 mmol, ꢁ10 equiv),
and HATU (3.06 g, 8.04 mmol, ꢁ5.0 equiv) were added
sequentially. The reaction mixture was stirred for 1 h at
23 ꢀC, after which the solvent was decanted away.
Standard puri®cation was performed and the pins were
then subjected to a second Fmoc-l-Leu-OH coupling
reaction using the above conditions. A second standard
puri®cation a€orded tripeptide intermediate 6 (HPLC
Anal. 12.81 min, 88% purity). Prior to the next synthetic
step, the derivatized pins were treated with 5% Ac2O in
CH2Cl2 (100 mL) for 30 min at 23 ꢀC in order to acylate
any unreacted amine.
8b
8c
8d
8e
50,000
66,000
27,000
260,000
2.0
>100
>100
>100
>100
0.55
25
0.25
2.8
8f
28,000
47,000
>100
>100
Ethyl-3-[Amide-L-Leu-L-Phe-L-(N-pin)Gln]-E-propenoate
(7). Acid chloride couplings. Pins containing tripeptide
intermediate 6 (697 pins at ꢁ2.3 mmol/pin, ꢁ1.61 mmol)
were washed with CH2Cl2 and dried under vacuum. The
pins were suspended in 2% DBU in CH2Cl2 (100 mL)
and stirred for 30 min at 23 ꢀC, then were puri®ed by the
standard procedure and axed to Multipin2 stems
(8Â12). Solutions of an appropriate acid chloride (10%
w/v acid chloride in ethanol-free CHCl3, 200 mL) and
collidine (10% v/v in ethanol-free CHCl3, 200 mL) were
added sequentially to deepwell microtiter plate wells
(8Â12), and the stems/pins containing the deprotected
tripeptide were then placed in the wells. The assembled
plates were gently shaken on a platform rotator at 23 ꢀC
for 1 h, after which the pins were puri®ed by the stan-
dard procedure. The coupling and puri®cation proce-
dures were then repeated to give intermediates 7
(representative HPLC purities given in Table 1).
8g
1.6
aSerotype-14.
b1:1 mixture of diastereomers.
Ethyl-3-[Fmoc-L-(N-pin)Gln]-E-propenoate (4). Fmoc-
Rink Amide-derivatized pins (710 pins at ꢁ2.3 mmol/
pin, ꢁ1.63 mmol) were washed with CH2Cl2 and dried
under vacuum, then suspended in 2% DBU in CH2Cl2
(100 mL) and stirred for 30 min at 23 ꢀC. After puri®ca-
tion by the standard procedure, the pins were suspended
in DMF (100 mL) and 33b (3.45 g, 8.15 mmol, 5.0 equiv),
DIEA (2.84 mL, 16.3 mmol, 10 equiv), HOBt (0.11 g,
0.815 mmol, 0.50 equiv) and PyBOP (4.24 g, 8.15 mmol,
5.0 equiv) were added sequentially. The reaction
mixture was stirred for 1 h at 23 ꢀC after which the
solvent was decanted away. Standard puri®cation then
provided intermediate 4 (HPLC Anal. 4.04 min, 94%
purity).
Ethyl-3-[Amide-L-Leu-L-Phe-L-(N-pin)Gln]-E-propenoate
(7). Carboxylic acid couplings. Solutions of appro-
priate carboxylic acids (0.115 mM in DMF, 100 mL),
DIEA (0.23 mM in DMF, 100 mL), and HATU
(0.058 mM in DMF, 200 mL) were added sequentially to
deepwell microtiter plate wells (8Â12) and the stems/
pins containing the deprotected tripeptide intermediate
(prepared as described in the preceding experimental)
Ethyl-3-[Fmoc-L-Phe-L-(N-pin)Gln]-E-propenoate (5).
Pins containing intermediate 4 (704 pins @ ꢁ2.3 mmol/
pin, ꢁ1.62 mmol) were washed with CH2Cl2 and dried

594
P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
Scheme 2. Reagents and conditions (Tr=CPh3): (a) HCl in 1,4-dioxane, 23 ꢀC, 2 h; (b) 1.0 equiv Boc-l-Phe-OH, 1.5 equiv HOBt, 3.0 equiv NMM,
1.5 equiv EDC, CH2Cl2, 23 ꢀC, overnight, 85%; (c) 1.2 equiv Boc-l-Leu-OH, 1.5 equiv HOBt, 3.0 equiv NMM, 1.5 equiv EDC, CH2Cl2, 23 ꢀC,
overnight, 87%; (d) excess RC(O)Cl and Et3N, CH2Cl2, 0 ꢀC, 30 min or excess RCO2H, HOBt, NMM and EDC, CH2Cl2, 23 ꢀC, overnight; (e)
excess (i-Pr)3SiH, 1:1 TFA:CH2Cl2, 23 ꢀC, 30 min.
added to a solution of 93a (3.26 g, 6.01 mmol, 1 equiv) in
the same solvent (15 mL) at 23 ꢀC. After 2 h, the vola-
Table 3. Antirhinoviral activity of compound 8e
Serotype
EC50 (mM)
CC50 (mM)
tiles were removed under reduced pressure. The residue
was dissolved in CH2Cl2 (60 mL) and Boc-l-Phe-OH
(1.59 g, 6.01 mmol, 1.0 equiv), HOBt (1.22 g, 9.02 mmol,
1.5 equiv), 4-methylmorpholine (1.98 mL, 18.03 mmol,
3.0 equiv), and EDC (1.73 g, 9.02 mmol, 1.5 equiv) were
added sequentially. The reaction mixture was stirred at
23 ꢀC overnight then was partitioned between water
(100 mL) and CH2Cl2 (2Â100 mL). The combined
organic layers were dried over Na2SO4, concentrated,
and the residue was puri®ed by ¯ash column chroma-
tography (40% EtOAc in hexane) to a€ord 10 (3.55 g,
85%) as white foam: Rf=0.52 (50% EtOAc in hexanes);
IR (cm 1) 3306, 1706, 1661; 1H NMR (CDCl3) d 1.29 (t,
3H, J=7.2), 1.38 (s, 9H), 1.65±1.76 (m, 1H), 1.87±1.99
(m, 1H), 2.25±2.27 (m, 2H), 2.94±3.01 (m, 2H), 4.14±
4.26 (m, 3H), 4.48±4.53 (m, 1H), 4.95 (s, br, 1H), 5.64
(d, 1H, J=15.8), 6.29 (d, 1H, J=8.1), 6.64 (dd, 1H,
J=15.8, 5.4), 6.80 (s, br, 1H), 7.14±7.32 (m, 20H); Anal.
calcd for C42H47N3O6: C, 73.13; H, 6.87; N, 6.09.
Found: C, 72.94; H, 6.97; N, 6.00.
14
1A
2
0.25
1.0
0.41
0.22
>100
>100
>100
>100
89
were then placed in the wells. The assembled plates were
gently shaken on a platform rotator at 23 ꢀC for 1 h,
after which the pins were puri®ed by the standard pro-
cedure. The coupling and puri®cation procedures were
then repeated to give intermediates 7 (representative
HPLC purities given in Table 1).
Ethyl-3-(Amide-L-Leu-L-Phe-L-Gln)-E-propenoate (8). A
mixture of TFA and H2O (95:5, 400 mL) was added to
deepwell microtiter plate wells (8Â12) and the stems/
pins containing the intermediates 7 were then placed in
the wells. The assembled plates were gently shaken on a
platform rotator at 23 ꢀC for 1 h. The volatiles were
removed from the plates by evaporation utilizing a
Speed Vac (2 h) to provide tripeptide products 8 (repre-
sentative HPLC purities given in Table 1).
Ethyl-3-[Boc-L-Leu-L-Phe-L-(Tr-Gln)]-E-propenoate (11).
A solution of HCl in 1,4-dioxane (4.0 M, 15 mL) was
added to a solution of 10 (6.40 g, 9.28 mmol, 1 equiv) in
the same solvent (15 mL) at 23 ꢀC. After 2 h, the vola-
tiles were removed under reduced pressure. The residue
was dissolved in CH2Cl2 (100 mL) and Boc-l-Leu-OH
(2.58 g, 11.1 mmol, 1.2 equiv), HOBt (1.88 g, 13.9 mmol,
1.5 equiv), 4-methylmorpholine (3.06 mL, 27.8 mmol,
Representative examples of solution phase synthesis
Preparation of (20R/S)-ethyl-3-[(10,20,30,40-tetrahydro-
naphthalene-20-carbonyl)-L-Leu-L-Phe-L-Gln]-E-propenoate
(8a). Ethyl-3-[Boc-L-Phe-L-(Tr-Gln)]-E-propenoate (10).
A solution of HCl in 1,4-dioxane (4.0 M, 15 mL) was

P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
595
3.0 equiv), and EDC (2.67 g, 13.92 mmol, 1.5 equiv)
were added sequentially. The reaction mixture was
stirred at 23 ꢀC overnight then was partitioned between
water (100 mL) and CH2Cl2 (2Â100 mL). The com-
bined organic layers were dried over Na2SO4, con-
centrated, and the residue was puri®ed by ¯ash column
chromatography (2% CH3OH in CH2Cl2) to a€ord 11
(6.46 g, 87%) as white foam: Rf=0.40 (50% EtOAc in
hexanes); IR (cm 1) 3284, 1651, 1515; 1H NMR
(CDCl3) d 0.86 (d, 3H, J=6.0), 0.89 (d, 3H, J=6.0),
1.29 (t, 3H, J=7.2), 1.34 (s, 9H), 1.38±1.60 (m, 3H),
1.62±1.89 (m, 1H), 1.95±1.97 (m, 1H), 2.28±2.30 (m,
2H), 3.06±3.08 (m, 2H), 3.92±3.94 (m, 1H), 4.17 (q, 2H,
J=7.2), 4.48±4.51 (m, 2H), 4.67 (m, 1H), 5.66 (d, 1H,
J=15.9), 6.51±6.57 (m, 2H), 6.69 (dd, 1H, J=15.6, 5.1),
4H), 2.84±2.91 (m, 1H), 2.95±3.03 (m, 1H), 4.11 (q, 2H,
J=7.2), 4.20±4.27 (m, 1H), 4.39 (m, 1H), 4.43±4.50 (m,
1H), 5.66 (dd, 1H, J=15.6, 1.5), 6.66±6.69 (m, 1H),
6.71±6.76 (m, 1H), 7.05±7.07 (m, 4H), 7.18±7.23 (m,
6H), 7.88±7.94 (m, 1H), 7.98±8.05 (m, 2H); Anal. calcd
for C35H46N4O6: C, 67.94; H, 7.49; N, 9.05. Found: C,
67.69; H, 7.43; N, 9.00.
Preparation of ethyl-3-[(50-methylisoxazole-30-carbonyl)-
L-Leu-L-Phe-L-Gln]-E-propenoate (8e). Ethyl-3-[(50-meth-
ylisoxazole-30-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln)]-E-pro-
penoate (12e). A solution of HCl in 1,4-dioxane (4.0 M,
3 mL), was added to a solution of 11 (0.216 g,
0.27 mmol, 1 equiv) in the same solvent (3 mL) at 23 ꢀC.
After 2 h, the volatiles were removed under reduced
pressure. The residue was dissolved in CH2Cl2 (15 mL),
cooled to 0 ꢀC, and triethylamine (0.112 mL, 0.81 mmol,
3.0 equiv) and 5-methylisoxazole-3-carbonyl chloride
(0.058 g, 0.40 mmol, 1.5 equiv) were added sequentially.
The reaction mixture was stirred at 0 ꢀC for 30 min, then
was partitioned between water (50 mL) and CH2Cl2
(2Â50 mL). The combined organic layers were dried
over Na2SO4, concentrated, and the residue was puri®ed
by ¯ash column chromatography (2% CH3OH in
CH2Cl2) to a€ord 12e (0.199 g, 91%) as a white foam:
Rf=0.87 (10% CH3OH in CH2Cl2); IR (cm 1) 3286,
.
7.10±7.33 (m, 21H); Anal. calcd for C48H58N4O7 0.33H2O:
C, 71.27; H, 7.31; N, 6.93. Found: C, 70.99; H, 7.29; N,
7.26.
(20R/S)-Ethyl-3-[(10,20,30,40-tetrahydronaphthalene-20-carb-
onyl)-L-Leu-L-Phe-L-(Tr-Gln)]-E-propenoate (12a).
A
solution of HCl in 1,4-dioxane (4.0 M, 3 mL) was added
to a solution of 11 (0.218 g, 0.27 mmol, 1 equiv) in the
same solvent (3 mL) at 23 ꢀC. After 2 h, the volatiles
were removed under reduced pressure. The residue was
dissolved in CH2Cl2 (15 mL) and 1,2,3,4-tetrahydro-2-
naphthoic acid (0.071 g, 0.41 mmol, 1.5 equiv), HOBt
(0.055 g, 0.41 mmol, 1.5 equiv), 4-methylmorpholine
(0.089 mmol, 0.81 mmol, 3.0 equiv), and EDC (0.078 g,
0.41 mmol, 1.5 equiv) were added sequentially. The
reaction mixture was stirred at 23 ꢀC overnight then was
partitioned between water (50 mL) and CH2Cl2
(2Â50 mL). The combined organic layers were dried
over Na2SO4, concentrated, and the residue was puri®ed
by ¯ash column chromatography (2% CH3OH in
CH2Cl2) to a€ord 12a (0.207 g, 89%) as a white foam:
Rf=0.88 (10% CH3OH in CH2Cl2); IR (cm 1) 3277,
1635, 1547; 1H NMR (CDCl3) d 0.84±0.91 (m, 6H), 1.28
(t, 3H, J=7.2), 1.39±1.47 (m, 2H), 1.54±1.76 (m, 3H),
1.83±1.97 (m, 2H), 2.20±2.39 (m, 2H), 2.71±2.82 (m,
4H), 2.99±3.08 (m, 2H), 4.17 (q, 2H, J=7.2), 4.24±4.25
(m, 1H), 4.48±4.55 (m, 2H), 5.68 (d, 1H, J=15.9), 5.88±
5.92 (m, 2H), 6.52±6.55 (m, 1H), 6.62±6.65 (m, 1H), 6.69
(dd, 1H, J=15.6, 5.1), 6.70±7.32 (m, 25H); Anal. calcd
1
1650, 1541; H NMR (CDCl3) d 0.86 (d, 3H, J=5.4),
0.89 (d, 3H, J=5.7), 1.28 (t, 3H, J=7.2), 1.43±1.59 (m,
2H), 1.67±1.75 (m, 1H), 1.95±1.99 (m, 2H), 2.28 (t, 2H,
J=7.2), 2.41 (s, 3H), 2.97±3.04 (m, 1H), 3.06±3.13 (m,
1H), 4.17 (q, 2H, J=7.2), 4.31±4.33 (m, 1H), 4.48±4.52
(m, 2H), 5.72 (d, 1H, J=15.9), 6.19 (s, 1H), 6.41 (d, 1H,
J=7.5), 6.59 (d, 1H, J=8.1), 6.71 (dd, 1H, J=15.3,
6.0), 6.95 (d, 1H, J=6.6), 7.09±7.21 (m, 21H); Anal.
.
calcd for C48H53N5O7 H2O: C, 69.84; H, 6.58; N, 8.63.
Found: C, 69.80; H, 6.54; N, 8.57.
Ethyl-3-[(50-methylisoxazole-30-carbonyl)-L-Leu-L-Phe-L-
Gln]-E-propenoate (8e). Triisopropylsilane (0.077 mL,
0.376 mmol, 1.8 equiv) and tri¯uoroacetic acid (3 mL)
were added sequentially to a solution of 12e (0.185 g,
0.21 mmol, 1 equiv) in CH2Cl2 (3 mL) at 23 ꢀC produ-
cing a bright yellow solution. The reaction mixture was
stirred for 30 min at 23 ꢀC during which time it became
colorless. The volatiles were removed under reduced
pressure, and the resulting white solid was triturated
with Et2O (10 mL), ®ltered, and air-dried to give 8e
(0.087 g, 81%) as a white solid: mp=223±225 ꢀC;
Rf=0.56 (10% CH3OH in CH2Cl2); IR (cm 1) 3298,
.
for C54H60N4O6 0.5H2O: C, 74.54; H, 7.07; N, 6.44.
Found: C, 74.47; H, 7.06; N, 6.46.
(20R/S)-Ethyl-3-[(10,20,30,40-tetrahydronaphthalene-20-carb-
onyl)-L-Leu-L-Phe-L-Gln]-E-propenoate (8a). Triisopro-
pylsilane (0.077 mL, 0.376 mmol, 1.8 equiv) and tri-
¯uoroacetic acid (3 mL) were added sequentially to
a solution of 12a (0.185 g, 0.21 mmol, 1 equiv) in
CH2Cl2 (3 mL) at 23 ꢀC producing a bright yellow solu-
tion. The reaction mixture was stirred for 30 min at
23 ꢀC during which time it became colorless. The vola-
tiles were removed under reduced pressure, and the
resulting white solid was triturated with Et2O (10 mL),
®ltered, and air-dried to give 8a (0.111 g, 85%):
mp=238±239 ꢀC; Rf=0.67 (10% CH3OH in CH2Cl2);
1
1662, 1544, 1457, 1278; H NMR (DMSO-d6) d 0.81 (d,
3H, J=6.0), 0.85 (d, 3H, J=6.3), 1.23 (t, 3H, J=6.9),
1.38±1.42 (m, 1H), 1.48±1.77 (m, 4H), 2.04 (t, 2H,
J=7.2), 2.46 (s, 3H), 2.78±2.86 (m, 1H), 2.93±3.00 (m,
1H), 4.11 (q, 2H, J=7.2), 4.36±4.54 (m, 3H), 5.63 (d,
1H, J=15.6), 6.56 (s, 1H), 6.68 (dd, 1H, J=15.9, 5.4),
6.76 (s, br, 1H), 7.19 (m, 6H), 8.09 (d, 1H, J=8.1), 8.14
(d, 1H, J=7.8), 8.58 (d, 1H, J=7.5); Anal. calcd for
C29H39N5O7: C, 61.15; H, 6.90; N, 12.29. Found: C,
60.98; H, 6.94; N, 12.17.
1
IR (cm 1) 3289, 1654, 1544; H NMR (DMSO-d6) d
0.81 (d, 3H, J=6.0), 0.86 (d, 3H, J=6.6), 1.21 (t, 3H,
J=7.2), 1.38±1.41 (m, 2H), 1.54±1.75 (m, 4H), 1.90 (m,
1H), 2.02±2.09 (m, 2H), 2.57 (m, 1H), 2.75±2.78 (m,
The following compounds were prepared using one of
the general solution-phase synthesis procedures descri-
bed above.

596
P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
(20S)-Ethyl-3-[(20-chloro-40-methylpentane-10-carbonyl)-
L-Leu-L-Phe-L-(Tr-Gln)]-E-propenoate (12b). Rf=0.90
(10% CH3OH in CH2Cl2); IR (cm 1) 3296, 1654, 1524;
1H NMR (CDCl3) d 0.84±0.94 (m, 12H), 1.29 (t, 3H,
J=7.2), 1.42±2.02 (m, 7H), 2.28 (t, 2H, J=7.2), 2.96±
3.11 (m, 2H), 3.61±3.71 (m, 1H), 3.75±3.79 (m, 1H),
4.09±4.23 (m, 3H), 4.45±4.52 (m, 2H), 5.70 (d, 1H,
J=15.6), 6.47 (d, 1H, J=7.5), 6.59 (d, 1H, J=8.1), 6.69
(dd, 1H, J=15.9, 5.7), 6.75 (d, 1H, J=7.2), 7.05±7.33
2H, J=7.2), 4.42±4.48 (m, 3H), 5.69 (d, 1H, J=15.3),
6.65 (s, br, 1H), 6.66 (dd, 1H, J=15.9, 5.4), 6.76±6.79
(m, 2H), 7.00±7.31 (m, 22H), 8.24 (s, 1H); Anal. calcd
for C47H51N5O7 0.75H2O: C, 69.57; H, 6.52; N, 8.63.
Found: C, 69.58; H, 6.51; N, 8.63.
.
(20S)-Ethyl-3-[(20-chloro-40-methylpentane-10-carbonyl)-
L-Leu-L-Phe-L-Gln]-E-propenoate (8b). mp=230±
232 ꢀC; Rf=0.60 (10% CH3OH in CH2Cl2); IR (cm
)
1
1
.
(m, 21H); Anal. calcd for C49H59N4O6 0.75H2O: C,
69.32; H, 7.18; N, 6.60. Found: C, 69.36; H, 7.15; N,
6.62.
3288, 1640, 1544; H NMR (DMSO-d6) d 0.81±0.89 (m,
12H), 1.21 (t, 3H, J=7.2), 1.38±1.43 (m, 2H), 1.51±1.76
(m, 5H), 2.04 (t, 2H, J=7.5), 2.80±2.88 (m, 1H), 2.91±
2.98 (m, 1H), 4.11 (q, 2H, J=7.2), 4.27±4.35 (m, 3H),
4.43±4.50 (m, 2H), 5.61 (d, 1H, J=15.6), 6.69 (dd, 1H,
J=15.9, 5.4), 6.74 (s, br, 1H), 7.17±7.25 (m, 6H), 8.05
(d, 1H, J=8.1), 8.14 (d, 1H, J=7.8), 8.36 (d, 1H,
Ethyl-3-[(30-thiophen-20-yl-acrylic-10-carbonyl)-L-Leu-L-
Phe-L-(Tr-Gln)]-E-propenoate (12c). Rf=0.87 (10%
1
CH3OH in CH2Cl2); IR (cm 1) 3290, 1654, 1526; H
.
NMR (CDCl3) d 0.85 (d, 3H, J=5.7), 0.89 (d, 3H,
J=6.0), 1.28 (t, 3H, J=7.2), 1.38±1.47 (m, 1H), 1.53±
1.57 (m, 1H), 1.97±2.08 (m, 1H), 2.26±2.35 (m, 2H),
3.00±3.07 (m, 1H), 3.12±3.19 (m, 1H), 3.62±3.78 (m,
2H), 4.16 (q, 2H, J=7.2), 4.25 (m, 1H), 4.46±4.53 (m,
2H), 5.75 (d, 1H, J=15.9), 5.82 (d, 1H, J=6.3), 5.98 (d,
1H, J=15.6), 6.66 (d, 1H, J=7.8), 6.73 (dd, 1H,
J=15.6, 5.1), 6.81 (d, 1H, J=8.4), 7.02 (t, 1H, J=3.6),
7.09±7.14 (m, 3H), 7.20±7.34 (m, 20H), 7.54 (d, 1H,
J=8.1); Anal. calcd for C30H45ClN4O6 0.75H2O: C,
59.39; H, 7.73; N, 9.24. Found: C, 59.37; H, 7.57; N,
9.18.
Ethyl-3-[(30-thiophen-20-yl-acrylic-10-carbonyl)-L-Leu-L-
Phe-L-Gln]-E-propenoate (8c). mp=250±251 ꢀC; Rf=0.59
(10% CH3OH in CH2Cl2); IR (cm 1) 3288, 1654, 1543;
1H NMR (DMSO-d6) d 0.80 (d, 3H, J=6.6), 0.84 (d,
3H, J=6.6), 1.21 (t, 3H, J=6.9), 1.34±1.39 (m, 2H),
1.48±1.54 (m, 1H), 1.63±1.81 (m, 2H), 2.05 (t, 2H,
J=7.2), 2.81±2.89 (m, 1H), 2.98±3.04 (m, 1H), 4.07 (t,
2H, J=6.9), 4.24±4.49 (m, 3H), 5.67 (dd, 1H, J=15.6,
1.2), 6.47 (d, 1H, J=15.6), 6.71 (dd, 1H, J=15.6, 5.4),
6.77 (s, br, 1H), 7.09±7.24 (m, 7H), 7.38 (d, 1H, J=3.0),
7.57 (d, 1H, J=15.3), 7.60 (d, 1H, J=4.2), 7.96 (d, 1H,
J=8.1), 8.07 (d, 1H, J=8.1), 8.26 (d, 1H, J=7.5); Anal.
calcd for C31H40N4O6S: C, 62.40, H, 6.76; N, 9.39.
Found: C, 62.24; H, 6.85; N, 9.35.
.
J=15.3); Anal. calcd for C50H54N4O6S 0.5H2O: C,
70.81; H, 6.54; N, 6.61. Found: C, 70.50; H, 6.50; N,
6.96.
(20S)-Ethyl-3-[(20,50-dihydro-10H-pyrrole-20-carbonyl)-L-
Leu-L-Phe-L-(Tr-Gln)]-E-propenoate (12d). Rf=0.88
(10% CH3OH in CH2Cl2); IR (cm 1) 3280, 1645, 1541;
1H NMR (CDCl3) d 0.80 (d, 3H, J=5.7), 0.86 (d, 3H,
J=6.0), 1.27 (t, 3H, J=7.2), 1.47 (s, 9H), 1.75±1.86 (m,
2H), 2.00±2.07 (m, 1H), 2.26±2.41 (m, 2H), 2.88±2.96
(m, 2H), 3.44±3.50 (m, 2H), 3.62±3.71 (m, 2H), 3.76±
3.80 (m, 1H), 4.16 (q, 2H, J=7.2), 4.58 (m, 1H), 4.75
(m, 2H), 5.37 (m, 1H), 5.75±5.77 (m, 1H), 5.82 (d, 1H,
J=15.6), 6.49 (d, 1H, J=4.8), 6.78±6.85 (m, 2H), 7.12±
7.32 (m, 20H), 7.45 (s, 1H); Anal. calcd for C53H62
(20S)-Ethyl-3-[(20,50-dihydro-10H-pyrrole-20-carbonyl)-L-
Leu-L-Phe-L-Gln]-E-propenoate (8d). mp=184±188 ꢀC;
Rf=0.35 (10% CH3OH in CH2Cl2); IR (cm 1) 3279,
1
1657, 1560, 1202; H NMR (DMSO-d6) d 0.87 (t, 6H,
J=7.2), 1.21 (t, 3H, J=7.2), 1.41±1.45 (m, 2H), 1.50±
1.76 (m, 3H), 2.03 (t, 2H, J=7.8), 2.78±2.85 (m, 1H),
2.91±2.97 (m, 1H), 4.04 (m, 2H), 4.11 (q, 2H, J=7.2
Hz), 4.34±4.41 (m, 2H), 4.43±4.50 (m, 1H), 4.97 (s, br,
1H), 5.60 (dd, 1H, J=15.6, 1.2), 5.77±5.79 (m, 1H),
5.99±6.01 (m, 1H), 6.67 (dd, 1H, J=15.9, 5.4), 6.78 (s,
br, 1H), 7.17±7.24 (m, 7H), 8.12 (d, 1H, J=8.1), 8.27 (d,
1H, J=7.8), 8.74 (d, 1H, J=8.1); Anal. calcd for
.
N5O8 1.0H2O: C, 69.56; H, 7.05; N, 7.65. Found: C,
69.46; H, 6.98; N, 7.80.
Ethyl-3-[(30 -phenylpropyl-10 -carbonyl)-L-Leu-L-Phe-L-
(Tr-Gln)]-E-propenoate (12f). Rf=0.83 (10% CH3OH in
CH2Cl2); IR (cm 1) 3280, 1645, 1541; 1H NMR
(CDCl3) d 0.77 (d, 3H, J=6.0), 0.81 (d, 3H, J=6.3),
1.28 (t, 3H, J=7.2), 1.34±1.44 (m, 1H), 1.62 (m, 2H),
1.70±1.76 (m, 1H), 1.98±2.00 (m, 1H), 2.23±2.37 (m,
4H), 2.77 (t, 2H, J=7.2), 2.92±2.97 (m, 1H), 3.06±3.13
(m, 1H), 4.09±4.14 (m, 1H), 4.17 (q, 2H, J=7.4), 4.42±
4.50 (m, 2H), 5.56 (d, 1H, J=6.3), 5.72 (dd, 1H,
J=15.6, 1.5), 6.32 (d, 1H, J=7.8), 6.62 (d, 1H, J=7.8),
6.71 (dd, 1H, J=15.9, 5.1), 7.08±7.12 (m, 5H), 7.18±7.31
(m, 21H); Anal. calcd for C52H58N4O6: C, 74.79; H,
7.00; N, 6.71. Found: C, 74.52; H, 6.97; N, 6.82.
.
C29H41N5O6 1.0H2O: C, 60.72; H, 7.55; N, 12.21.
Found: C, 60.96; H, 7.34; N, 12.34.
Ethyl-3-[(30 -phenylpropyl-10 -carbonyl)-L-Leu-L-Phe-L-
Gln]-E-propenoate (8f). mp=245±246 ꢀC; Rf=0.54
(10% CH3OH in CH2Cl2); IR (cm 1) 3389, 1640, 1545,
1280, 1188; 1H NMR (DMSO-d6) d 0.74 (d, 3H,
J=6.3), 0.80 (d, 3H, J=6.3), 1.21 (t, 3H, J=6.9), 1.29±
1.32 (m, 2H), 1.36±1.40 (m, 1H), 1.62±1.77 (m, 2H), 2.06
(t, 2H, J=7.5), 2.32±2.44 (m, 2H), 2.75±2.80 (m, 2H),
2.84±2.89 (m, 1H), 2.96±3.02 (m, 1H), 4.11 (q, 2H,
J=7.2), 4.18±4.22 (m, 1H), 4.38±4.45 (m, 2H), 5.62 (d,
1H, J=15.6), 6.71 (dd, 1H, J=15.9, 5.4), 6.76 (s, br,
1H), 7.17±7.27 (m, 11H), 7.97±7.80 (m, 3H); Anal. calcd
for C33H44N4O6: C, 66.87; H, 7.48; N, 9.45. Found: C,
66.61; H, 7.44; N, 9.37.
Ethyl-3-[(isoxazole-50-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln)]-
E-propenoate (12g). Rf=0.76 (10% CH3OH in CH2Cl2);
IR (cm 1) 3282, 1643, 1530; 1H NMR (CDCl3) d 0.87 (t,
6H, J=6.6), 1.29 (t, 3H, J=7.2), 1.49±1.64 (m, 3H),
1.69±1.80 (m, 1H), 1.90±1.96 (m, 1H), 2.30 (t, 2H,
J=7.2), 2.92±2.96 (m, 1H), 3.02±3.09 (m, 1H), 4.17 (q,

P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
597
Ethyl-3-[(isoxazole-50-carbonyl)-L-Leu-L-Phe-L-Gln]-E-
propenoate (8g). mp=217±220 ꢀC; Rf=0.51 (10%
days later, XTT/PMS was added to the test plates and
the amount of formazan produced was quanti®ed spec-
trophotometrically at 450/650 nm. The EC50 was calcu-
lated as the concentration of drug that increased the
percentage of formazan 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 minimize false
positives due to cytotoxicity, only compounds display-
ing CC50s greater than 10Â the observed EC50s were
considered to be active antiviral agents. Using the above
method, the EC50 of a representative compound (1) was
calculated to be 0.54‹0.27 mM (range 0.18 to 1 mM).
The EC50 of the known antirhinoviral agent Pirodavir
was similarly determined to be 0.02‹0.01 mM (range
0.01 to 0.05 mM) and was comparable to the 0.03 mM
minimal inhibitory concentration value previously
reported.21
1
CH3OH in CH2Cl2); IR (cm 1) 3302, 1655, 1541; H
NMR (DMSO-d6) d 0.81 (d, 3H, J=6.0), 0.86 (d, 3H,
J=6.0), 1.21 (t, 3H J=7.2), 1.42±1.75 (m, 5H), 2.04 (t,
2H, J=7.2), 2.78±2.87 (m, 1H), 2.94±3.01 (m, 1H), 4.11
(q, 2H, J=7.2), 4.37 (m, 1H), 4.41±4.52 (m, 2H), 5.64
(d, 1H, J=15.6), 6.68 (dd, 1H, J=15.9, 5.4), 6.76 (s, br,
1H), 7.12±7.19 (m, 7H), 8.02 (d, 1H, J=8.1), 8.20 (d,
1H, J=8.1), 8.74 (d, 1H, J=1.8), 8.94 (d, 1H, J=7.8);
Anal. calcd for C28H37N5O7: C, 60.53; H, 6.71; N,
12.60. Found: C, 60.44; H, 6.66; N, 12.48.
Enzyme assays
The general conditions of the ¯uorescence resonance
energy transfer assay utilized to assess 3CP activity are
described in the literature.18 Continuous ¯uorometric
enzyme assays were conducted using a Perkin±Elmer
LS50B spectro¯uorimeter 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 mM substrate and
varying inhibitor concentrations. Typically, 3±5 con-
centrations were examined and data from the con-
tinuous assays were directly analysed with the nonlinear
regression analysis program ENZFITTER19 to obtain
®rst order rate constants for enzyme inactivation at each
inhibitor concentration. The slope of a graph of kobs(I)
versus [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%.
Acknowledgements
We thank Dorothy DeLisle and Dr Siegfried Reich for
helpful discussions throughout the course of this work.
References and Notes
1. (a) Couch, R. B. Rhinoviruses. In Virology, 2nd Ed.;
Fields, B. N.; Knipe, D. M., Eds.; Raven Press: New York,
1990; Vol. 1, Chapter 22, pp 607±629. (b) McKinlay, M. A.;
Pevear, D. C.; Rossmann, M. G. Annu. Rev. Microbiol. 1992,
46, 635. (c) Phillpotts, R. J.; Tyrrell, D. A. J. Br. Med. Bull.
1985, 41, 386. (d) Gwaltney, J. M. In Principles and Practices
of Infectious Diseases; Mandell, G. L., Douglas, R. G., Ben-
nett, J. E., Eds.; John Wiley & Sons: New York, 1985, Chapter
38, pp 351±355. (e) Gwaltney, J. M. In Viral Infections of
Humans; Evans, A. S., Ed.; Plenum Publishing Corp.: New
York, 1982; Chapter 20, pp 491±517.
Enzyme assays (high-throughput)
Compounds were assayed in a 96 (8Â12) well format,
using the previously described18 peptide substrate.
Assays contained 2 mM (nominal) test compound, 1 mM
substrate, 20 mM potassium phosphate bu€er (pH 7.5),
2% DMSO and 0.2 mM HRV-14 3CP. Assays were
initiated by addition of enzyme, and ¯uorescence was
measured on a DYNEX Fluorolite 1000 plate reader
prior to and 40 and 390 s after addition of enzyme.
Control wells contained either 2% DMSO or 2.5 mM of
the known 3CP inhibitors 1 and 2 (Fig. 1) and displayed
approximately 0%, 75%, and 100% inhibition in this
assay, respectively.
2. (a) Rueckert, R. R. In Virology, 2nd Ed.; Fields, B. N.,
Knipe, D. M., Eds.; Raven Press: New York, 1990; Vol. 1,
Chapter 20, pp 507±548. (b) Krausslich, H.-G.; Wimmer, E.
Annu. Rev. Biochem. 1988, 57, 701.
3. (a) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhr-
man, S. A.; Patick, A. K.; Matthews, D. A.; Lee, C. A.; Reich,
S. R.; Prins, T. J.; Marakovits, J. T.; Little®eld, E. S.; Zhou,
R.; Tikhe, J.; Ford, C. E.; Wallace, M. B.; Meador, III J. W.;
Ferre, R. A.; Brown, E. L.; Binford, S. L.; Harr, J. E. V.;
DeLisle, D. M.; Worland, S. T. J. Med. Chem. 1998, 41, 2806.
(b) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhrman,
S. A.; Patick, A. K.; Matthews, D. A.; Reich, S. H.; Mara-
kovits, J. T.; Prins, T. J.; Zhou, R.; Tikhe, J.; Little®eld, E. S.;
Bleckman, T. M.; Wallace, M. B.; Little, T. L.; Ford, C. E.;
Meador, III, J. W.; Ferre, R. A.; Brown, E. L.; Binford, S. L.;
DeLisle, D. M.; Worland, S. T. J. Med. Chem. 1998, 41, 2819.
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Garsky, V. M.; Colonno, R. J. J. Biol. Chem. 1990, 265, 9062.
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Antiviral assays
All strains of human rhinovirus (HRV) were purchased
from American Type Culture Collection (ATCC). HRV
stocks were propagated and antiviral assays were per-
formed 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.20 Brie¯y, H1-HeLa cells were infected with
HRV-14 at a multiplicity of infection (m.o.i.) of 0.08 or
mock-infected with medium only. Infected or uninfected
cells were resuspended at 8Â105 cells per mL and incu-
bated with appropriate concentrations of drug. Two
5. The nomenclature used for describing the individual amino
0
acid residues of a peptide substrate (P2, P1, P1 , P2 , etc.) and
0

598
P. S. Dragovich et al. / Bioorg. Med. Chem. 7 (1999) 589±598
0
0
the corresponding enzyme subsites (S2, S1, S1 , S2 , etc.) is
described in: Schechter, I.; Berger, A. Biochem. Biophys. Res.
Commun. 1967, 27, 157.
variety of N-terminal moieties were tolerated in the tripeptide
3CP inhibitor design. See ref. 3b above.
14. Wells containing compounds 8a±g displayed inhibition
values ranging from 85% to 97% when tested in the high
throughput 3CP enzyme assay system.
15. A high-throughput antiviral assay was also utilized to exam-
ine compounds 8 prepared on solid phase. However, such exam-
ination did not reveal any molecules worthy of solution-phase
resynthesis other than those identi®ed by the anti-3CP assay.
16. The incorporation of additional N-terminal carboxamides
derived from aromatic heterocyles into tripeptidyl 3CP inhibi-
tors is ongoing.
6. The optimization of a series of peptidyl thrombin inhibitors
utilizing a similar approach was recently described. See:
Brady, S. F.; Stau€er, K. J.; Lumma, W. C.; Smith, G. M.;
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13. Previous structure±activity studies demonstrated that a