Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 8. Pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics

Article abstract of DOI:10.1021/jm030166l

The optimization of the pharmacokinetic performance of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors following oral administration to either beagle dogs or CM-monkeys is described. The molecules described in this work are composed of a 2-pyridone-containing peptidomimetic binding determinant and an α,β-unsaturated ester Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. Modification of the ester contained within these compounds is detailed along with alteration of the P ₂ substituent present in the peptidomimetic portion of the inhibitors. The pharmacokineties of several inhibitors in both dogs and monkeys are described (7 h plasma concentrations after oral administration) along with their human plasma stabilities, stabilities in incubations with human, dog, and monkey microsomes and hepatocytes, Caco-2 permeabilities, and aqueous solubilities. Compounds containing an α,β-unsaturated ethyl ester fragment and either an ethyl or propargyl P₂ moiety displayed the most promising combination of 3C enzyme inhibition (k_obs/[Il 170 000-223 000 M^-1 s^-1), antiviral activity (EC₅₀ = 0.047-0.058 μM, mean vs seven HRV serotypes), and pharmacokinetics following oral administration (7 h dog plasma levels = 0.248-0.682 μM; 7 h CM-monkey plasma levels = 0.057-0.896 μM).

Full text of DOI:10.1021/jm030166l

4572
J . Med. Chem. 2003, 46, 4572-4585
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. 8. P h a r m a cologica l Op tim iza tion of
Or a lly Bioa va ila ble 2-P yr id on e-Con ta in in g P ep tid om im etics
Peter S. Dragovich,* Thomas J . Prins,† Ru Zhou, Theodore O. J ohnson, Ye Hua, Hiep T. Luu, Sylvie K. Sakata,
Edward L. Brown, Fausto C. Maldonado, Tove Tuntland, Caroline A. Lee, Shella A. Fuhrman, Leora S. Zalman,‡
Amy K. Patick, David A. Matthews, Ellen Y. Wu, Ming Guo, Bennett C. Borer, Naresh K. Nayyar,
Terence Moran, Lijian Chen, Paul A. Rejto, Peter W. Rose, Mark C. Guzman, Elena Z. Dovalsantos, Steven Lee,
Kevin McGee,§ Michael Mohajeri, Andreas Liese, J unhua Tao, Maha B. Kosa, Bo Liu, Minerva R. Batugo,
J ean-Paul R. Gleeson, Zhen Ping Wu, J ia Liu, J ames W. Meador, III, and Rose Ann Ferre
Pfizer Global Research and Development-La J olla, 10777 Science Center Drive, San Diego, California 92121-1111
Received April 10, 2003
The optimization of the pharmacokinetic performance of various 2-pyridone-containing human
rhinovirus (HRV) 3C protease (3CP) inhibitors following oral administration to either beagle
dogs or CM-monkeys is described. The molecules described in this work are composed of a
2-pyridone-containing peptidomimetic binding determinant and an R,â-unsaturated ester
Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine
residue of the 3C enzyme. Modification of the ester contained within these compounds is detailed
along with alteration of the P2 substituent present in the peptidomimetic portion of the
inhibitors. The pharmacokinetics of several inhibitors in both dogs and monkeys are described
(7 h plasma concentrations after oral administration) along with their human plasma stabilities,
stabilities in incubations with human, dog, and monkey microsomes and hepatocytes, Caco-2
permeabilities, and aqueous solubilities. Compounds containing an R,â-unsaturated ethyl ester
fragment and either an ethyl or propargyl P2 moiety displayed the most promising combination
of 3C enzyme inhibition (kobs/[I] 170 000-223 000 M-1 s-1), antiviral activity (EC50 ) 0.047-
0.058 µM, mean vs seven HRV serotypes), and pharmacokinetics following oral administration
(7 h dog plasma levels ) 0.248-0.682 µM; 7 h CM-monkey plasma levels ) 0.057-0.896 µM).
In tr od u ction
efforts led to the identification of a novel class of
substrate-inspired 2-pyridone-containing peptidomimet-
ics which incorporate a Michael acceptor moiety capable
of irreversibly forming a covalent adduct with the active
site cysteine residue of the 3C enzyme (Figure 1).9,10
These efforts culminated with the identification of
several optimized pyridones which display potent in
vitro antiviral activity against multiple rhinovirus se-
rotypes (e.g., 1 and 2, Figure 1 and Table 1,) and one
such molecule (2) was shown to be orally bioavailable
in the dog.9
The human rhinoviruses (HRVs) are members of the
picornavirus family and are the single most significant
cause of the common cold.1-3 Presently, no marketed
antiviral agents exist for treating rhinovirus-related
illnesses, and the large number of HRV serotypes (>100)
makes the development of a vaccine seem unlikely.4 In
contrast to earlier attempts at antirhinoviral identifica-
tion,5 our research efforts have focused on inhibiting a
critical virus-encoded enzyme whose activity is essential
for completion of the HRV replication cycle. This protein,
the human rhinovirus 3C protease (3CP), is a cysteine
protease which catalyzes the proteolytic processing of
the large polypeptide produced by cellular translation
of the rhinovirus RNA genome.6 In addition, the 3CP
enzyme exhibits structural similarity to the trypsin
protein family, but displays minimal homology to preva-
lent mammalian enzymes.7 Due to its importance in the
viral replication cycle, 3CP is an ideal target for the
development of novel antirhinoviral agents, and several
examples of 3CP inhibitors have recently appeared in
the literature.8 Our previous 3CP inhibitor discovery
As previously detailed, compound 2 exhibited good
pharmacokinetics in beagle dogs following administra-
tion as an oral solution with 7 h plasma levels of the
molecule in excess of its average in vitro antiviral
potency (Table 1).9,11,12 Similar compound exposures
were also noted when 2 was administered to dogs using
a suspension formulation (Table 1). In stark contrast,
the inhibitor was poorly bioavailable when orally de-
livered in solution to CM-monkeys with 7 h postadmin-
istration plasma levels well below the compound’s
average in vitro antiviral activity (Table 1). This dra-
matic species difference may result from the greater in
vitro metabolism of 2 observed upon exposure to simian
hepatocytes relative to that noted during analogous
canine experiments (Table 2). Since no animal model
of HRV infection exists which strongly correlates human
symptomatology with in vitro antirhinoviral activity, the
significance of the observed differences in compound 2
* To whom correspondence should be addressed. Phone: (858) 622-
7918; Fax: (858) 622-7998; e-mail: peter.dragovich@pfizer.com.
† Present address: Anadys Pharmaceuticals, 9050 Camino Santa
Fe, San Diego, CA 92121.
‡ Present address: Alta Analytical Laboratories, Inc., 5627 Oberlin
Drive, San Diego, CA 92121.
§ Present address: Neurocrine Biosciences, Inc., 10555 Science
Center Drive, San Diego, CA 92121.
10.1021/jm030166l CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

Irreversible Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4573
with similar reductions noted during earlier studies
conducted with related peptidyl molecules.17 Despite the
anti-3CP potency alteration, compound 3 retained good
absolute antiviral properties when tested against seven
distinct rhinovirus serotypes in cell culture (Table 1).
In addition, the molecule exhibited good stability toward
human plasma (Table 1) and displayed improved stabil-
ity in human and canine microsome and/or hepatocyte-
based metabolism experiments relative to the benzyl-
containing molecule 1 (Table 2). The former result
suggested that large, lipophilic 3CP inhibitors such as
1 tended to exhibit greater affinity for human plasma
esterases relative to the more hydrophilic compound 3.
The latter observations were consistent with improve-
ments in oxidative metabolic lability effected through
reduced cytochrome P450 association and/or removal of
the metabolically labile P2 benzyl substituent from the
inhibitor series.19 The permeability of compound 3
through Caco-2 cell monolayers was also examined, and
its aqueous solubility was determined (Table 2). The
permeability of 3 was observed to be somewhat reduced
relative to that noted for compounds 1 and 2, but its
solubility was surprisingly enhanced. Importantly, the
results of these physical property assessments col-
lectively suggested that 3 might exhibit good systemic
exposures in animals following oral administration.20
F igu r e 1. Design of 2-pyridone-containing HRV 3CP inhibi-
tors.
Encouragingly, compound 3 was orally bioavailable
in the dog when administered in a solution formulation
and afforded 7 h plasma concentrations in excess of its
average in vitro antiviral activity (Table 1). Nearly
identical 7 h plasma concentrations were observed when
the molecule was administered orally to dogs using a
suspension vehicle as well. As predicted by in vitro
simian metabolic stability experiments (Table 2), solu-
tion-based oral administration of compound 3 to CM-
monkeys afforded poorer 7 h plasma concentration
levels relative to those observed in dog (Table 1).
However, absolute plasma levels of the molecule 7 h post
administration in the monkey substantially exceeded
those previously noted for compound 2 and were nearly
identical to the average antiviral activity of 3 as
determined by cell culture testing. These promising
results prompted a more detailed exploration of 3CP
inhibitors which incorporated a propargyl substituent
at the P2 position in lieu of the benzyl fragment present
in the parent molecules.
exposure between dog and monkey is not known with
certainty. However, data obtained from in vitro human
hepatocyte metabolism studies conducted with 2 more
closely paralleled results from the corresponding simian
experiments (Table 2). We therefore sought to identify
3CP inhibitors which exhibited good oral bioavailability
and pharmacokinetics in both dogs and monkeys in an
effort to maximize their potential to display promising
human exposures.13
Resu lts a n d Discu ssion
To accomplish our goal, we envisioned that further
modification of the 3CP inhibitors identified by the
project to date would be required. As mentioned above,
rapid metabolism of compound 2 was suspected to be
the cause of the poor oral bioavailability exhibited by
the molecule in CM-monkeys. Accordingly, we sought
to improve upon such parameters by reducing the
lipophilicity of 2 in an effort to minimize its suspected
association with metabolizing cytochrome P450 enzymes
and/or esterases.14,15 Our previous structure-activity
studies also indicated that the Michael acceptor moiety,
the P1 γ-lactam, the P3 pyridone, and the P4 isoxazole
fragment were all critical for imparting potent antiviral
activity to this series of 3CP inhibitors.9,16 We therefore
focused our compound modification efforts on altering
the lipophilic P2 benzyl substituent present in both
compounds 1 and 2. Prior structure-activity studies
conducted with related peptidyl molecules had also
demonstrated that this substituent could be truncated
to some degree without drastic loss of either 3CP
inhibitory properties or antiviral activity.17
Accordingly, the ester moiety present in this class of
3CP inhibitors was extensively modified in the hopes
of further improving the esterase-related metabolic
stability of the molecules (Table 1, compounds 4-11).
Such improvements were noted during our previous
studies involving 2-pyridone anti-3CP agents containing
P2 benzyl substituents although they were achieved
with a simultaneous worsening of 3CP inhibition prop-
erties (cf., compare compounds 1 and 2, Table 1).9 As
expected, incorporation of sterically demanding ester
groups into the P2-propargyl-pyridone inhibitor design
typically afforded molecules which displayed reduced
anti-3CP potencies relative to the ethyl ester-containing
compound 3. Unfortunately, and in contrast to results
obtained with inhibitor 2, the majority of the non-ethyl
ester-containing analogues of 3 exhibited weak (mean
EC50 > 0.20 µM) absolute antirhinoviral potency when
tested against seven HRV serotypes in cell culture
Replacement of the P2 benzyl substituent present in
1 with a propargyl moiety afforded a molecule (3) which
displayed somewhat reduced anti-3CP properties (Table
1).18 This loss in 3CP inhibitor potency was consistent

4574 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Ta ble 1. 2-Pyridone-Containing HRV 3CP Inhibitors
Dragovich et al.
compd
no.
kobs/[I]
plasma t1/2 C7h (µM) C7h (µM)
c
R1
R2
prepa
formulab
(M-1 s-1
)
EC50 (µM)d
(h)e
(dog)f
(monkey)g
1
2
CH2CH3
CH(CH3)2
CH2(3,4-F)Ph ref 9 C30H31F2N5O7
CH2(3,4-F)Ph ref 9 C31H33F2N5O7‚0.75H2O
1 800 000
548 000
0.011
0.078
1.4
9.7
NA
0.815
0.959h
0.248
0.190h
NA
NA
NA
0.073
0.160
NA
NA
0.012
3
CH2CH3
CH2C≡CH
B
C26H29N5O7‚0.75H2O
223 400
0.058
6.6
0.057
4
5
6
7
8
9
10
11
13
CH(CH3)2
C(CH3)3
CH2C≡CH
CH2C≡CH
B
A
B
B
B
B
B
B
B
C27H31N5O7‚0.50H2O
C28H33N5O7
100 000
60 500
127 500
207 100
186 000
34 000
47 000
300 000
170 000
0.174i
0.361
0.276
0.090
0.174
0.406j
0.812j
0.036
0.047
ND
ND
ND
3.3
12.5
ND
ND
4.3
NA
NA
NA
ND
ND
NA
NA
0.003
0.896
CH2C(CH3)3 CH2C≡CH
C29H35N5O7‚0.25H2O
C28H31N5O7‚0.25H2O
C29H33N5O7‚0.75H2O
C30H35N5O7‚0.75H2O
C31H37N5O7‚0.30H2O
C31H31N5O7‚0.50H2O
C25H31N5O7‚0.50H2O
cyclobutyl
cyclopentyl
cyclohexyl
cycloheptyl
CH2Ph
CH2C≡CH
CH2C≡CH
CH2C≡CH
CH2C≡CH
CH2C≡CH
CH2CH3
NA
0.563
0.682
<0.010h
NA
NA
NA
0.222
NA
NA
CH2CH3
5.6
14
15
16
17
18
19
20
CH(CH3)2
C(CH3)3
CH2C(CH3)3 CH2CH3
cyclobutyl
cyclohexyl
cycloheptyl
CH2Ph
CH2CH3
CH2CH3
B
A
B
B
B
B
B
C26H33N5O7‚0.50H2O
C27H35N5O7‚0.50H2O
C28H37N5O7‚0.50H2O
C27H33N5O7‚0.75H2O
C29H37N5O7‚0.50H2O
C30H39N5O7‚0.80H2O
C30H33N5O7‚0.50H2O
ref 31
36 000
45 100
38 200
170 000
23 000
15 000
210 000
NA
0.322j
0.405
0.310
0.076
0.313j
ND
0.064
0.031k
0.136
ND
ND
ND
9.2
ND
ND
2.5
NA
NA
NA
ND
NA
NA
ND
CH2CH3
CH2CH3
CH2CH3
CH2CH3
0.156
Pirodavir
Pleconaril
ref 32
NA
a
b
Method of preparation: see Schemes 1 and 2. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical
values. c Inhibition activity against HRV-14 3C protease; see ref 30 for assay method and error. Antirhinoviral activity (mean of EC50
d
values determined against HRV serotypes 2, 3, 9, 14, 16, 25, and 39 unless otherwise noted); cytotoxicity values for all compounds were
>10 µM; see ref 30 for assay method and error. e Half-life of compound upon exposure to human plasma; see Experimental Section for
additional details. f Compound plasma concentration in dogs 7 h after oral administration (30 mg/kg, vehicle ) 80:20 propylene glycol:
g
H2O); see Experimental Section for additional details. Compound plasma concentration in CM-monkeys 7 h after oral administration
h
(50 mg/kg, vehicle ) 80:20 propylene glycol:H2O); see Experimental Section for additional details. Vehicle ) 0.5% CMC suspension.
i
j
Compound was inactive when tested to 10 µM against HRV 39 (value is mean of activity against other six serotypes). Mean of EC50
k
values determined against HRV serotypes 9, 14, and 25. Compound was inactive when tested to 10 µM against HRV 25 (value is mean
of activity against other six serotypes). ND ) not determined. NA ) not applicable.
(Table 1). A similar potency reduction was also exhibited
by a molecule containing a methyl substituent adjacent
to the Michael acceptor ester moiety (compound 12),
exhibited relatively poor pharmacokinetics in the dog
following oral administration. In each case, 7 h plasma
concentrations were noted below the respective mean
in vitro antiviral EC50s of the molecules (Table 1). The
poorer canine pharmacokinetic performance of 7 and 8
relative to 3 may be related to the increased in vitro
canine metabolism and/or the lower water solubility
exhibited by the former molecules (Table 2). In addition,
the stability profiles of 7 and 8 toward canine and
human liver microsomes were not significantly different
from each other and suggested that dramatic pharma-
cokinetic improvement would not occur in the latter
species (Table 2). These two molecules were therefore
not examined further and were not studied in simian
oral bioavailability experiments.
In contrast, the benzyl ester-containing inhibitor 11
displayed acceptable human plasma stability properties
(half-life >3 h) and exhibited excellent exposure and
pharmacokinetics after oral administration in the dog
with 7 h plasma concentrations of the molecule far
exceeding its in vitro antiviral potency (Table 1). These
excellent pharmacokinetics were not obviously sug-
gested by in vitro experiments which indicated a
reduced stability of compound 11 toward canine mi-
crosomes and hepatocytes along with lesser aqueous
thus precluding this alternate approach to improving
esterase-related metabolic stability. The three notable
exceptions which emerged from the above activities were
derivatives of 3 which incorporated cyclobutyl, cyclo-
pentyl, and benzyl esters in lieu of the ethyl fragment
(compounds 7, 8, and 11, respectively). Although both
inhibitors 7 and 8 displayed acceptable stability profiles
toward human plasma (half-life >3 h, Table 1), they also

Irreversible Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4575
Ta ble 2. In Vitro Properties of 2-Pyridone-Containing HRV 3CP Inhibitors
dog (% metab)a
monkey (% metab)a
human (% metab)a
protein binding (% free)e
compd
no.
H2O soln
Caco-2 Papp
M
H
M
H
M
H
(µg/mL)b (×10-6 cm/s)c ClogPd
dog
human
1
2
3
7
57
57
45
53
76
67
37
46
69
ND
16f
12
ND
ND
61
14
ND
55
ND
ND
55
ND
92f
79
81
56
40
52
59
52
61
49
56
94
87f
85
ND
ND
51
82
ND
27
25
21
377
25
15
22
38
192
41
35
36
6
14
36
18
5
ND
2.07
0.18
0.57
1.12
1.38
0.66
1.04
1.86
ND
8-19
54
ND
4-9
34
ND
ND
ND
61
ND
ND
ND
ND
69
ND
20
ND
7
8
11
13
17
20
25
13
76g
ND
ND
ND
42
ND
33
10
11
20
8
a
M ) loss of compound (% metabolized) after 30 min exposure to liver microsomes from indicated species (25 µM compound concentration);
see Experimental Section for additional details. H ) loss of compound (% metabolized) after 2 h exposure to hepatocytes from indicated
b
species (25 µM compound concentration); see Experimental Section for additional details. Thermodynamic aqueous solubility; see
Experimental Section for additional details. c Apical to basolateral permeability through Caco-2 cell monolayers; see Experimental Section
d
for additional details. LogP values calculated using Biobyte ClogP 4.0. e Plasma protein binding (% free); see Experimental Section for
g
additional details. f 4 h exposure. 5 µM compound concentration. ND ) not determined.
solubility relative to inhibitor 3 (Table 2). Although
compound 11 performed well in the dog, the molecule
was poorly bioavailable in CM-monkeys and provided
7 h simian plasma levels that were near the limits of
detection. The strikingly poor performance of compound
11 in the monkey relative to 3 was also not easily
predicted by in vitro experiments which suggested
roughly equivalent hepatocyte stability and improved
gut permeability of the former molecule (Table 2). Thus,
a precise explanation of the oral pharmacokinetics
observed for compound 11 in dogs and monkeys remains
elusive but may involve additional metabolism of the
molecule not easily detected using microsome and/or
hepatocyte-based stability assessments. These results
underscore the difficulty in accurately predicting the
oral exposures of these peptidomimetic 3CP inhibitors
using in vitro data and reaffirmed our desire to identify
compounds which demonstrated good pharmacokinetic
properties in both dogs and monkeys.
assessed with relatively simple in vitro metabolism
assays. A potential concern with compound 13 was
noted when the molecule was orally administered to
dogs using a suspension formulation. In stark contrast
to the solution-based experiment, suspension adminis-
tration afforded very poor 7 h plasma levels of the
compound (Table 1). This result suggested that the
aqueous solubility of 13 may limit its oral exposure
potential relative to 3 in the absence of a solution-based
formulation and a significant solubility difference was
indeed noted between the two compounds (Table 2).
However, inhibitor 2 displayed aqueous solubility simi-
lar to that exhibited by 13 yet was readily bioavailable
in the dog following oral suspension dosing (Table 1).
Thus, the precise reasons for the poor exposures of 13
following suspension administration are not known with
certainty.
As noted above, oral administration of compound 17
to the dog afforded 7 h plasma levels in excess of the
molecule’s in vitro antiviral potency (Table 1). The
compound also demonstrated excellent in vitro stability
toward human plasma (Table 1). However, the ratio of
dog 7 h levels to antiviral potency was relatively low
(<4-fold) compared to some of the other compounds
described in this work (cf., compounds 2, 3, 11, and 13)
and similar metabolic stability was observed for the
compound in both human and canine in vitro microsome
experiments (Table 2). Because of these limitations,
inhibitor 17 was not selected for subsequent profiling
in simian oral exposure assessments. Compound 20 also
exhibited a relatively poor ratio between dog 7 h plasma
concentration and antiviral potency and displayed unac-
ceptable stability when exposed to human plasma as
well (half-life <3 h; Table 1). The compound was
therefore not selected for further experimentation. The
latter result was particularly unfortunate given the
molecule’s good in vitro stability profile when exposed
to human liver microsomes or hepatocytes (Table 2). The
origins of the poor human plasma stability of compound
20 are presently not known.
Accordingly, we also examined a series of 3CP inhibi-
tors in which the P2 benzylic substituent present in the
lead compound 2 was replaced with an ethyl fragment
(compounds 13-20, Table 1). As was observed for the
propargyl-containing molecules above, inhibitors which
incorporated ethyl, cyclobutyl, or benzyl esters displayed
the most potent 3CP inhibitory properties and in vitro
antirhinoviral activities (compounds 13, 17, and 20,
respectively). Compound 13 also exhibited excellent
exposure when orally administered to dogs using a
solution formulation with 7 h plasma concentrations far
exceeding its mean in vitro antirhinoviral activity (Table
1). Even greater exposures of the compound were noted
in corresponding simian experiments which employed
a solution-based delivery vehicle. Once again, the excel-
lent pharmacokinetics of compound 13 in the monkey
relative to the dog were not easily predicted from in vitro
metabolic stability assessments which indicated that the
molecule was more stable toward canine microsomes
and hepatocytes (Table 2). Since additional assessments
of 3 and 13 suggested comparable in vitro simian
metabolism, equivalent Caco-2 permeabilities, and im-
proved aqueous solubility of 3, they did not readily
clarify the >10-fold exposure difference between the two
that was observed in the monkey (Tables 1 and 2). As
mentioned above, we suspect that the in vivo metabo-
lism of the compounds described in this work is complex
and most likely involves processes that are not easily
Because of the difficulties encountered in predicting
the in vivo pharmacokinetics of the 2-pyridone-contain-
ing 3CP inhibitors from the available in vitro data, we
retrospectively examined whether extensive tissue com-
partmentalization and/or metabolism of the more lipo-
philic compounds might explain the molecules’ observed
plasma levels. No correlation was noted between the

4576 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Dragovich et al.
Sch em e 1a
a
Synthetic Method A. Reagents and conditions (DMB ) 2,4-dimethoxybenzyl, Tf ) trifluoromethanesulfonyl: (a) NaNO2, 1.0 M H2SO4,
0 f 23 °C, 16 h, 66%; (b) HCl, CH3OH, 23 °C, 16 h, 79%; (c) 1.9 equiv of 2,6-lutidine, 1.7 equiv of Tf2O, CH2Cl2, 0 °C, 25 min; (d) 1.1 equiv
of 25, 0.95 equiv of NaH, THF, 23 °C, 30 min, then 1.0 equiv of 24, 23 °C, 30 min, 90%; (e) 2.5 equiv of LiI, pyridine, reflux, 30 min, 97%;
i
(f) 1.1 equiv of 28, HCl, 1,4-dioxane, 23 °C, 2 h, then 1.0 equiv of 27, 1.4 equiv of HOBt, 7.0 equiv of Pr2NEt, 1.3 equiv of EDC, CH2Cl2,
0 f 23 °C, 16 h, 33%; (g) 1.15 equiv of Dess-Martin periodinane, CH2Cl2, 0 °C, 75 min; (h) 1.1 equiv of Ph3PdCHCO2tBu, THF, reflux,
45 min, 64%; (i) 4.2 equiv of DDQ, 10:1 CHCl3:H2O, 65 °C, 4 h, 71%.
compounds’ calculated logP values (Table 2) and their
measured 7 h canine plasma concentrations (R ) -0.16;
not graphically depicted). Comparison of calculated
polar surface area and canine plasma levels resulted in
a similar lack of correlation (R ) 0.44; data not shown).
The calculated logP values did qualitatively parallel the
propensity of the compounds to bind to plasma proteins
in both dog and human (more lipophilic ) smaller free
fraction; Table 2), but the relevance of this observation
to in vivo predictions is not known at this time. Thus,
somewhat contrary to our original hypothesis and
strategy, good in vivo pharmacokinetics were not always
imparted to the 2-pyridone-containing 3CP inhibitors
simply by reducing their lipophilicties.
the P2 benzyl-containing compound 2 (very low monkey
exposures) and suggested that truncation of the P2
benzyl substituent may enhance oral exposure of related
molecules in these species. Thus, compounds 3 and 13
represent additional attractive options for the develop-
ment of orally bioavailable human rhinovirus 3C pro-
tease inhibitors.
Syn th esis
The 2-pyridone-containing 3CP inhibitors described
in this study were prepared by two related synthetic
methods (A and B). The particular method employed to
synthesize a given compound is indicated in Table 1,
and representative examples of each are given below.
These syntheses differ primarily in the nature of the
P1 fragment utilized to complete each sequence and
employ several synthetic transformations and interme-
diates from previously reported preparations of related
pyridone-containing 3CP inhibitors.9 The first method
(Method A) is illustrated in Scheme 1 with the prepara-
tion of compound 5 and involves coupling of a P1 amino-
alcohol moiety to a P4-P3-P2 fragment followed by
oxidation and olefin formation.16 Thus, commercially
available D-propargylglycine (21) was converted to
intermediate 22 using a known process for the trans-
formation of R-amino acids to the corresponding R-hy-
droxy compounds.21 This entity was subsequently es-
terified under acidic conditions to afford methyl ester
23 in good yield. The alcohol present in 23 was then
Despite the above-mentioned uncertainties surround-
ing the use of in vitro data to predict in vivo pharma-
cokinetics, the optimization studies described above did
succeed in achieving our primary objective of identifying
potent 3CP inhibitors and antirhinoviral agents which
display good exposures in both dogs and monkeys
following oral administration. In particular, compounds
3 and 13 exhibited plasma concentrations in the dog 7
h after an oral administration more than 4-fold greater
than their corresponding in vitro antirhinoviral poten-
cies. Similarly, oral administration of these molecules
to CM-monkeys afforded 7 h plasma concentrations that
either far exceeded (compound 13) or approximated
(compound 3) their respective in vitro antiviral EC50
values. The collective performance of both compounds
in dogs and monkeys was superior to that exhibited by

Irreversible Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4577
Sch em e 2a
a
Synthetic Method B. Reagents and conditions (DMB ) 2,4-dimethoxybenzyl): (a) 1.1 equiv of Dess-Martin periodinane, CH2Cl2,
0 f 23 °C, 1.5 h; (b) 1.2 equiv of Ph3PdCHCO2CH2tBu, THF, reflux, 1.5 h; (c) 4.2 equiv of DDQ, 10:1 CHCl3:H2O, 60 °C, 4 h, 67%; (d)
TFA, CH2Cl2, 23 °C, 35 min; (e) 1.0 equiv of 27, 1.4 equiv of HOBt, 1.25 equiv of EDC, 7.0 equiv of iPr2NEt, CH2Cl2, 0 f 23 °C, 24 h, 44%;
(f) 0.97 equiv of Ph3PdCHCO2cyclobutyl, THF, reflux, 1.5 h, 44%; (g) 1.0 equiv of 27, 1.4 equiv of HOBt, 1.25 equiv of EDC, 7.0 equiv of
iPr2NEt, CH2Cl2, 0 f 23 °C, 16 h, 46%.
converted to the corresponding triflate (24) for use in
the following coupling reaction. Although the described
sequence to prepare 24 from D-propargylglycine was
executed without purification, it routinely provided the
desired triflate in quantities and purities sufficient for
subsequent transformations.
pound 15 employing P4-P3-P2 intermediate 38 (see
Scheme 3 below) in lieu of carboxylic acid 27.
The acid-labile esters present in inhibitors 5 and 15
necessitated the introduction of such functionalities
after assembly of the P4-P3-P2-P1 inhibitor skeleton.
In contrast, the remainder of the other 2-pyridone-
containing 3CP inhibitors described in this work were
prepared by coupling an olefin-containing P1 fragment
with an appropriate P4-P3-P2 moiety (synthetic Method
B).16 This method was operationally simpler than
Method A above since no compound modifications were
required after effecting the described coupling. A rep-
resentative example of Method B is illustrated in
Scheme 2 with the preparation of compound 6. Thus,
γ-lactam 2824 was subjected to an oxidation/olefination
sequence employing (triphenyl-λ5-phosphanylidene)ace-
tic acid 2,2-dimethylpropyl ester (see below and Experi-
mental Section) to provide the trans-olefination product
31 in moderate yield. The dimethoxybenzyl moiety
present in 31 was then removed under oxidative27
conditions to afford γ-lactam 32. This intermediate was
subjected to an acidic Boc deprotection protocol, and the
resulting amine salt (not shown) was coupled with
carboxylic acid 27 to provide inhibitor 6 as a single
diastereomer following silica gel purification.25
Accordingly, condensation of 24 with the sodium salt
of hydroxypyridine 259 afforded coupling product 26 in
good yield (95% ee). In direct analogy to our previous
work, the described preparation of intermediate 26
predominantly provided the N-alkylated pyridone prod-
uct as evidenced by TLC analysis of the reaction mixture
1
and H NMR analysis of the coupling product.22,23 The
methyl ester present in 26 was subsequently converted
to the corresponding carboxylic acid (27) by treatment
with LiI/pyridine. This reagent combination was chosen
in order to minimize racemization of the chiral center
present in 26 that was noted when employing more
basic saponification conditions (e.g., NaOH). Coupling
of 27 with the amine derived from deprotection of
γ-lactam 2824 then afforded alcohol 29 as a single
diastereomer in good yield following flash column
purification.25 This intermediate was then oxidized
using the Dess-Martin reagent,26 and the resulting
crude aldehyde (not shown) was converted to olefin 30
in good overall yield. As was encountered during previ-
ous syntheses of peptidyl and peptidomimetic 3CP
inhibitors,17,24 the above olefination process afforded the
desired trans-isomer with <5% of the corresponding cis-
isomer as determined by 1H NMR analysis of the crude
reaction mixture. Oxidative removal27 of the dimethoxy-
benzyl protecting group present in 30 afforded the
desired 3CP inhibitor 5 in good yield after purification
by silica gel chromatography. The described inhibitor
preparation method was also used to synthesize com-
A variation of synthetic Method B is also illustrated
in Scheme 2 with the preparation of inhibitor 7. This
variation utilizes γ-lactam 3328 in lieu of 28 and
therefore did not require a debenzylation step analogous
to the conversion of 31 to 32 described above. Thus,
oxidation/olefination of 33 employing (triphenyl-λ5-
phosphanylidene)acetic acid cyclobutyl ester (see below
and Experimental Section) afforded the trans-olefination
product 34 in moderate yield. Acidic deprotection of this
material followed by carbodiimide-mediated coupling of

4578 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Dragovich et al.
Sch em e 3a
a
Synthetic Method B. Reagents and conditions: (a) 2.0 equiv of 2,6-lutidine, 1.9 equiv of Tf2O, CH2Cl2, 0 °C, 25 min; (b) 1.1 equiv of
25, 1.0 equiv of NaH, THF, 23 °C, 30 min, then 1.0 equiv of 36, 23 °C, 30 min, 59%; (c) TFA, CH2Cl2, 23 °C, 30 min; (d) (from 39) TFA,
i
CH2Cl2, 23 °C, 2 h, then 1.0 equiv of 38, 1.2 equiv of HOBt, 1.1 equiv of EDC, 7.0 equiv of Pr2NEt, CH2Cl2, 0 f 23 °C, 18 h, 58%.
the resulting amine salt (not shown) with carboxylic acid
27 provided inhibitor 7 in good yield after purification
by flash column chromatography.25
hibitors of the human rhinovirus 3C protease can
function as potent, broad-spectrum, orally bioavailable
antirhinoviral agents. Because of an apparent complex
interplay between the metabolism profiles, gut perme-
abilities, and aqueous solubilities of these compounds,
the physiochemical and biological properties which
determine their pharmacokinetic performance following
oral administration in both dogs and monkeys were not
rigorously defined by our efforts. Two potent 3CP
inhibitors were nevertheless identified which displayed
promising pharmacokinetics in both dogs and monkeys
following oral administration. Importantly, the plasma
concentrations of these molecules in both species equaled
or exceeded their average antirhinoviral activity as
determined by cell culture assays (EC50, 7 HRV sero-
types) for at least 7 h postadministration. Collectively,
these results suggest that additional examination of
2-pyridone-containing HRV 3CP inhibitors is warranted
and that such study may lead to the identification of
related molecules suitable for clinical development as
orally delivered agents.
Another application of Method B is provided in
Scheme 3 with the preparation of inhibitor 13 and
involves the synthesis of a P4-P3-P2 intermediate
containing a P2-ethyl moiety. Thus, commercially avail-
able (R)-2-hydroxybutyric acid tert-butyl ester was
converted to the corresponding triflate (36). In direct
analogy with synthetic Method A above, condensation
of crude 36 with the sodium salt of hydroxypyridine 259
afforded coupling product 37 as a single enantiomer in
good yield.22,23 Subsequent acidic deprotection of 37 then
provided carboxylic acid 38 which was used without
purification in the next step. Coupling of 38 with the
amine salt (not shown) derived from γ-lactam 3928 gave
inhibitor 13 in good yield after purification by flash
column chromatography.25
The remainder of the 3CP inhibitors described in this
work were prepared by utilization of synthetic Method
B to couple either carboxylic acid 27 (compounds 3 and
4 as well as 8-12) or 38 (compounds 14-20) to ap-
propriately derivatized P1 fragments. The phosphorus
ylides required to effect these syntheses, along with
those illustrated in Schemes 1, 2, and 3, were either
commercially available (compounds 3, 5, 11-13, 15, and
Exp er im en ta l Section
General descriptions of experimental procedures, reagent
purifications, and instrumentation along with conditions and
uncertainties for enzyme and antiviral assays are provided
elsewhere.30 1H NMR chemical shifts are reported in ppm (δ)
downfield relative to internal tetramethylsilane, and coupling
constants are given in hertz. The following abbreviations also
apply: HATU [O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetrameth-
yluronium hexafluorophosphate], HOBt (1-hydroxybenzotria-
zole hydrate), EDC [1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride], CDI (1,1′-carbonyldiimidazole), MTBE
(tert-butyl methyl ether), DDQ (2,3-dichloro-5,6-dicyano-1,4-
benzoquinone), TFA (trifluoroacetic acid), DMAP [4-(dimeth-
20), known in the literature (compounds 4,29 and 1429
)
or were prepared by slight modifications of known
syntheses29 (compounds 6-10, and 16-19; see Experi-
mental Section).
Con clu sion s
The studies presented above further demonstrate that
peptidomimetic, 2-pyridone-containing irreversible in-

Irreversible Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4579
ylamino)pyridine]. Pirodavir31 was kindly provided by J anssen
Pharmaceuticals. Pleconaril was prepared as described in the
literature.32
Rep r esen ta tive Exa m p le of Syn th esis Meth od A. Syn -
th esis of tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Meth ylisoxazole-
3′′′-ca r b on yl)a m in o]-2′′-oxo-2′′H -p yr id in -1′′-yl}p en t -4′-
yn oylam in o)-5-(2′′′′-oxopyr r olidin -3′′′′-yl)pen t-2-en oic Acid
ter t-Bu tyl Ester (5).
(R)-2-Hyd r oxyp en t-4-yn oic Acid (22). D-Propargylglycine
(21) (1.50 g, 13.3 mmol, 1 equiv) was dissolved in 1 M H2SO4
(52 mL) and cooled to -2 °C. A solution of sodium nitrite (40%
aq, 6.6 mL) was added dropwise, keeping the temperature
below 10 °C. After addition was complete, the reaction mixture
was held at 0 °C for 3 h and then allowed to warm to 23 °C
and stirred 16 h more. The reaction mixture was extracted
with MTBE (3 × 75 mL), and the organic phases were dried
over Na2SO4 and evaporated to give 22 as a yellow oil (1.0 g,
66%) which was used without further purification.
(R)-2-Hyd r oxyp en t-4-yn oic Acid Meth yl Ester (23).
Compound 22 (0.714 g, 6.26 mmol, 1 equiv) was dissolved in
CH3OH (20 mL). A solution of HCl in 1,4-dioxane (4.0 M, 0.3
mL) was added. The reaction mixture was stirred 16 h, diluted
with EtOAc (250 mL), washed with aq NaHCO3 and brine (70
mL each), dried over Na2SO4, and evaporated to give 23 as a
yellow oil (0.630 g, 79%) which was used without further
purification.
Compound 27 (1.82 g, 5.77 mmol, 1 equiv), HOBt (1.09 g, 8.07
mmol, 1.4 equiv), iPr2NEt (7.03 mL, 40.3 mmol, 7.0 equiv), and
EDC (1.44 g, 7.51 mmol, 1.3 equiv) were added successively.
The reaction mixture was allowed to warm to 23 °C and stirred
16 h then diluted with CH2Cl2 (600 mL) and washed with 2.5%
KHSO4, brine, aq NaHCO3 and brine (100 mL each). After
being dried over Na2SO4, the organic phase was concentrated,
and the residue was purified by flash column chromatography
(3% CH3OH in CH2Cl2) to give 29 as a foam (1.16 g, 33%):
Rf ) 0.20 (5% CH3OH in CH2Cl2); IR (cm-1) 3389, 1649, 1596,
1531; 1H NMR (CDCl3) δ 1.48-1.69 (m, 2H), 1.90-2.02 (m,
2H), 2.11-2.22 (m, 1H), 2.39-2.53 (m, 1H), 2.50 (d, 3H, J )
0.7), 3.05-3.16 (m, 4H), 3.55-3.71 (m, 2H), 3.77 (s, 3H), 3.78
(s, 3H), 3.85-3.91 (m, 1H), 3.98-4.08 (m, 1H), 4.16 (d, 1H,
J ) 14.5), 4.40 (d, 1H, J ) 14.5), 5.32-5.39 (m, 1H), 6.31
(t, 1H, J ) 7.3), 6.40-6.44 (m, 2H), 6.47 (s, 1H), 6.99-7.03
(m, 1H), 7.28 (dd, 1H, J ) 7.3, 1.7), 8.35 (s, 1H, J ) 7.0), 8.44
(dd, 1H, J ) 7.3, 1.7), 9.57 (s, 1H); Anal. (C31H35N5O8‚0.5H2O)
C, H, N.
tr a n s-(2′′′S,3′S,4S)-5-[1′-(2′′,4′′-Dim et h oxyb en zyl)-2′-
oxopyrrolidin-3′-yl]-4-(2′′′-{3′′′′-[(5′′′′′-methylisoxazole-3′′′′′-carbonyl)-
am in o]-2′′′′-oxo-2′′′′H-pyr idin -1′′′′-yl}pen t-4′′′-yn oylam in o)-
p en t-2-en oic Acid ter t-Bu tyl Ester (30). Dess-Martin
periodinane (Lancaster, 0.917 g, 2.15 mmol, 1.15 equiv) was
added to a 0 °C solution of 29 (1.13 g, 1.87 mmol, 1 equiv) in
CH2Cl2 (20 mL). The reaction vessel was then warmed to
23 °C and stirred 75 min. The volatiles were evaporated, and
the residue was evaporated from toluene (3 × 5 mL). The
residue was then dissolved in THF (40 mL). (tert-Butoxycar-
bonylmethylene)triphenylphosphorane (0.773 g, 2.05 mmol, 1.1
equiv) was added, and the reaction mixture was refluxed 45
min and then cooled and concentrated. The residue was
purified by flash column chromatography (2% CH3OH in
CHCl3) to give 30 as a foam (0.844 g, 64%): Rf ) 0.38 (5%
CH3OH in CH2Cl2); IR (cm-1) 3331, 1678, 1649, 1596, 1531;
1H NMR (CDCl3) δ 1.46 (s, 9H), 1.50-1.70 (m, 1H), 1.83-1.96
(m, 1H), 2.00 (t, 1H, J ) 2.6), 2.11-2.22 (m, 1H), 2.40-2.53
(m, 1H), 2.50 (s, 3H), 3.07-3.14 (m, 4H), 3.75 (s, 3H), 3.78 (s,
3H), 4.10 (d, 1H, J ) 14.5), 4.38 (d, 1H, J ) 14.5), 4.44-4.55
(m, 1H), 5.29-5.36 (m, 1H), 5.92 (dd, 1H, J ) 15.6, 1.4), 6.32
(t, 1H, J ) 7.2), 6.37-6.44 (m, 2H), 6.47 (s, 1H), 6.70 (dd, 1H,
J ) 15.6, 5.6), 7.00 (d, 1H, J ) 8.6), 7.27 (dd, 1H, J ) 7.2, 1.6),
8.45 (dd, 1H, J ) 7.2, 1.6), 8.68 (d, 1H, J ) 6.0), 9.59 (s, 1H);
Anal. (C37H42N5O9‚0.75H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
am in o)-5-(2′′′′-oxopyr r olidin -3′′′′-yl)pen t-2-en oic Acid ter t-
Bu tyl Ester (5). DDQ (0.372 g, 1.64 mmol, 1.4 equiv) was
added to a solution of 30 (0.821 g, 1.17 mmol, 1 equiv) in a
mixture of CHCl3 and H2O (10:1, 29 mL). The reaction vessel
was placed in an oil bath maintained at 65 °C. After stirring
1.5 h, additional DDQ (0.372 g, 1.64 mmol, 1.4 equiv) was
added. After an additional 1.5 h, still more DDQ (0.372 g, 1.64
mmol, 1.4 equiv) was added. After 4 h total, the reaction
mixture was allowed to cool, diluted with EtOAc (500 mL),
washed with a mixture of 10% KHSO4 and brine (1:1, 100 mL)
and a mixture of saturated NaHCO3 and brine (1:1, 100 mL),
and then dried over MgSO4 and evaporated. The residue was
purified by flash column chromatography (2% CH3OH in CH2-
Cl2) to provide 5 (0.458 g, 71%) as a solid: mp ) 195 °C, dec;
Rf ) 0.22 (5% CH3OH in CH2Cl2); IR (cm-1) 3295, 1690, 1649;
1H NMR (CDCl3) δ 1.47 (s, 9H), 1.50-1.79 (m, 2H), 2.04 (t,
1H, J ) 2.6), 2.05-2.37 (m, 3H), 2.49 (s, 3H), 2.92 (ddd, 1H,
J ) 17.0, 8.6, 2.6), 3.02 (ddd, 1H, J ) 17.0, 6.7, 2.6), 3.20-
3.37 (m, 2H), 4.41-4.52 (m, 1H), 5.65-5.73 (m, 1H), 5.94 (dd,
1H, J ) 15.6, 1.4), 6.33 (t, 1H, J ) 7.3), 6.46 (s, 1 H), 6.70 (s,
1 H), 6.73 (dd, 1 H, J ) 15.6, 5.3), 7.48 (dd, 1 H, J ) 7.3, 1.7),
8.41 (dd, 1 H, J ) 7.3, 1.7), 8.62 (d, 1 H, J ) 6.6), 9.53 (s, 1H);
Anal. (C28H33N5O7) C, H, N.
(R)-2-Tr iflu or om eth a n esu lfon yloxyp en t-4-yn oic Acid
Meth yl Ester (24). Compound 23 (0.460 g, 3.59 mmol, 1
equiv) was dissolved in CH2Cl2 (27 mL) and cooled to 0 °C.
2,6-Lutidine (0.795 mL, 6.83 mmol, 1.9 equiv) and trifluo-
romethanesulfonic anhydride (1.03 mL, 6.12 mmol, 1.7 equiv)
were added successively. After being stirred 25 min, the
reaction mixture was diluted with MTBE (250 mL), washed
with a mixture of brine and 1 N HCl (3:1, 70 mL then 40 mL
× 2), dried over MgSO4, and evaporated to give 24 as a residue
which was used without further purification.
(2S)-2-{3′-[(5′′-Meth ylisoxa zole-3′′-ca r bon yl)a m in o]-2′-
oxo-2′H -p yr id in -1′-yl}p en t -4-yn oic Acid Met h yl E st er
(26). 5-Methylisoxazole-3-carboxylic acid (2′-hydroxypyridin-
3′-yl)amide (259) (0.866 g, 3.95 mmol, 1.1 equiv) was dissolved
in THF (15 mL). Sodium hydride (60% dispersion in mineral
oil, 0.136 g, 3.4 mmol, 0.95 equiv) was added in portions, and
the reaction mixture was stirred 30 min. To this mixture was
added a solution of 24 prepared above (3.59 mmol theoretical)
in THF (15 mL). After being stirred 30 min more, the reaction
mixture was diluted with EtOAc (300 mL), washed with brine
(2 × 75 mL), dried over Na2SO4, and evaporated. The residue
was purified by flash column chromatography (40% EtOAc in
hexanes) to give the product as an off-white solid (0.776 g,
90%): mp ) 162-164 °C; Rf ) 0.42 (50% EtOAc in hexanes);
1
IR (cm-1) 3342, 3284, 1748, 1697, 1650, 1597, 1533; H NMR
(CDCl3) δ 2.01 (t, 1H, J ) 2.7), 2.50 (d, 3H, J ) 0.7), 3.06 (ddd,
1H, J ) 17.6, 4.5, 2.7), 3.21 (ddd, 1H, J ) 17.6, 9.6, 2.7), 3.78
(s, 3H), 5.11 (dd, 1H, J ) 9.6, 4.5), 6.33 (t, 1H, J ) 7.3), 6.48
(s, 1H), 7.16 (dd, 1H, J ) 7.3, 1.7), 8.49 (dd, 1H, J ) 7.3, 1.7),
9.56 (s, 1H); Anal. (C16H15N3O5‚0.25H2O) C, H, N.
(2S)-2-{3′-[(5′′-Meth ylisoxa zole-3′′-ca r bon yl)a m in o]-2′-
oxo-2′H-p yr id in -1′-yl}p en t-4-yn oic Acid (27). Lithium io-
dide (3.13 g, 23.4 mmol, 2.5 equiv) and compound 26 (3.08 g,
9.35 mmol, 1 equiv) were combined in pyridine (12 mL) and
refluxed 30 min. After being cooled, the reaction mixture was
poured into 1 M HCl (300 mL) and extracted with CH2Cl2
(3 × 300 mL). The combined organic phases were washed with
a mixture of brine and 1 M HCl (10:1, 3 × 50 mL), dried over
MgSO4, and evaporated to provide crude 27 (2.86 g, 97%)
which was used without further purification.
(1′′S,1′′′S,3′′′′S)-5-Meth ylisoxa zole-3-ca r boxylic a cid [1′-
(1′′-{1′′′-[1′′′′-(2′′′′′,4′′′′′-d im eth oxyben zyl)-2′′′′-oxop yr r oli-
d in -3′′′′-ylm eth yl]-2′′′-h yd r oxyeth ylca r ba m oyl}bu t-3′′-yn -
yl)-2′-oxo-1′,2′-d ih yd r op yr id in -3′-yl]a m id e (29). Compound
2824 (2.59 g, 6.34 mmol, 1.1 equiv) was dissolved in a solution
of HCl in 1,4-dioxane (2.0 M, 54 mL) and stirred 2 h. The
volatiles were evaporated to give the crude amine salt which
was then dissolved in CH2Cl2 (140 mL) and cooled to 0 °C.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Eth yl Ester
(15). Rf ) 0.27 (5% CH3OH in CH2Cl2); IR (cm-1) 3331, 3295,
1
1690, 1649, 1590, 1531, 1455, 1155; H NMR (CDCl3) δ 0.93

4580 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Dragovich et al.
(t, 3H, J ) 7.3), 1.40-1.60 (m, 1H), 1.49 (s, 9H), 1.62-1.78
(m, 1H), 1.87-2.02 (m, 1H), 2.09-2.36 (m, 4H), 2.50 (s, 3H),
3.26-3.43 (m, 2H), 4.39-4.50 (m, 1H), 5.78 (dd, 1H, J ) 8.9,
6.7), 5.89 (dd, 1H, J ) 15.7, 1.3), 6.35 (t, 1H, J ) 7.3), 6.47 (s,
1H), 6.76 (dd, 1H, J ) 15.7, 5.9), 7.33 (s, 1H), 7.64 (dd, 1H,
J ) 7.3, 1.8), 8.43 (dd, 1H, J ) 7.3, 1.8), 8.59 (d, 1H, J ) 6.8),
9.57 (s, 1H); Anal. (C27H35N5O7‚0.5H2O) C, H, N.
Rep r esen ta tive Exa m p le of Ylid e P r ep a r a tion . Syn -
th esis of (Tr ip h en yl-λ5-p h osp h a n ylid en e)a cetic Acid Cy-
clobu tyl Ester . EDC (14.4 g, 75.1 mmol, 1.1 equiv) and DMAP
(0.833 g, 6.82 mmol, 0.1 equiv) were added sequentially to a
solution of bromoacetic acid (9.48 g, 68.2 mmol, 1.0 equiv) and
cyclobutanol (4.92 g, 68.2 mmol, 1 equiv) in CH2Cl2 at 0 °C.
The reaction mixture was warmed to 23 °C, was stirred at that
temperature for 16 h, and then was partitioned between water
(150 mL) and CH2Cl2 (100 mL). The aqueous layers were
subsequently extracted with a 1:1 mixture of EtOAc and
hexanes (150 mL). The combined organic layers were dried
over Na2SO4 and concentrated under reduced pressure to a
volume of approximately 35 mL (no heat was applied during
this concentration in order to minimize loss of the desired
intermediate).
The crude cyclobutyl ester thus obtained was dissolved in
benzene (100 mL) at 23 °C, and triphenylphosphine (16.1 g,
61.4 mmol, 0.9 equiv) was added. The reaction mixture was
then heated to 45 °C at which point a white precipitate began
to form. After being stirred 22 h at 45 °C, the mixture was
cooled to 23 °C. The precipitate was collected by filtration
through medium weight paper, washed with benzene (2 × 50
mL), and air-dried.
The material obtained in the previous step was suspended
in water (150 mL) at 23 °C. Sodium hydroxide (7.5 mL of a
2.0 M aqueous solution) was added via pipet over a 2 min
period, resulting in additional precipitate formation. The
reaction mixture was then transferred to a separatory funnel
and extracted with EtOAc (2 × 150 mL). The combined organic
layers were dried over Na2SO4 and concentrated to provide
slightly impure (triphenyl-λ5-phosphanylidene)acetic acid cy-
clobutyl ester (4.33 g, 19% from bromoacteic acid) as a colorless
oil. This crude material was subsequently used in the prepara-
tion of inhibitors 7 and 17. The ylides required for the
synthesis of compounds 6 and 16, 8, 9 and 18, and 10 and 19
were prepared in analogous manner utilizing neopentyl alco-
hol, cyclopentanol, cyclohexanol, and cycloheptanol, respec-
tively, in lieu of the cyclobutanol employed above.
equiv) was added. After an additional 1.5 h, still more DDQ
(0.925 g, 4.07 mmol, 1.4 equiv) was added. After 4 h total, the
reaction mixture was allowed to cool, diluted with EtOAc (500
mL), washed with a mixture of 10% KHSO4 and brine (1:1,
100 mL) and a mixture of saturated NaHCO3 and brine (1:1,
100 mL), then dried over MgSO4 and evaporated. The residue
was purified by flash column chromatography (2% CH3OH in
CH2Cl2) to provide 32 as a brown foam (0.717 g, 67%): Rf )
0.42 (5% CH3OH in CH2Cl2); IR (cm-1) 3295, 1713, 1690, 1666,
1272; 1H NMR (CDCl3) δ 0.95 (s, 9H), 1.44 (s, 9H), 1.55-1.66
(m, 1H), 1.78-1.86 (m, 1H), 1.95-2.06 (m, 1H), 2.39-2.56 (m,
2H), 3.30-3.40 (m, 2H), 3.83 (s, 2H), 4.31-4.42 (m, 1H), 5.32
(d, 1H, J ) 7.9), 5.98 (dd, 1H, J ) 15.7, 1.6), 6.38 (s, 1H), 6.86
(dd, 1H, J ) 15.7, 5.2); Anal. (C19H32N2O5‚0.25H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
a m in o)-5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid 2,2-
Dim eth ylp r op yl Ester (6). TFA (10 mL) was added to a
solution of 32 (0.702 g, 1.91 mmol, 1 equiv) in CH2Cl2 (14 mL)
and stirred 35 min at 23 °C. The volatiles were evaporated,
and the residue was concentrated from CCl4 (20 mL) to give
the crude amine salt. This material was combined with
intermediate 27 (0.601 g, 1.91 mmol, 1 equiv) and HOBt (0.360
g, 2.66 mmol, 1.4 equiv) in CH2Cl2 (34 mL) and cooled to 0 °C.
i
EDC (0.457 g, 2.38 g, 1.25 equiv) and Pr2NEt (2.32 mL, 13.3
mmol, 7.0 equiv) were then added sequentially. The reaction
mixture was allowed to warm to 23 °C, stirred 24 h, diluted
with EtOAc (550 mL), and washed with 2.5% KHSO4, brine,
aq NaHCO3, and brine (75 mL each). The organic phase was
dried over Na2SO4 and evaporated. The residue was purified
by flash column chromatography (2% CH3OH in CHCl3) to give
6 as a white amorphous powder (0.470 g, 44%): Rf ) 0.48 (5%
CH3OH in CH2Cl2); IR (cm-1) 3295, 1690, 1649, 1596, 1531;
1H NMR (CDCl3) δ 0.95 (s, 9H), 1.54-1.80 (m, 2H), 2.05 (t,
1H, J ) 2.6), 2.10-2.39 (m, 3H), 2.49 (s, 3H), 2.91 (ddd, 1H,
J ) 17.0, 8.2, 2.6), 3.01 (ddd, 1H, J ) 17.0, 6.8, 2.6), 3.22-
3.38 (m, 2H), 3.82 (s, 2H), 4.45-4.56 (m, 1H), 5.72-5.79 (m,
1H), 6.06 (dd, 1H, J ) 15.7, 1.5), 6.33 (t, 1H, J ) 7.2), 6.46 (s,
1H), 6.86 (dd, 1H, J ) 15.7, 5.4), 6.93 (s, 1H), 7.51 (dd, 1H,
J ) 7.2, 1.7), 8.41 (dd, 1H, J ) 7.2, 1.7), 8.69 (d, 1H, J ) 6.6),
9.52 (s, 1H); Anal. (C29H35N5O7‚0.25H2O) C, H, N.
Rep r esen ta tive Exa m p le of Syn th etic Meth od B. Syn -
th esis of tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Meth ylisoxazole-
3′′′-ca r b on yl)a m in o]-2′′-oxo-2′′H -p yr id in -1′′-yl}p en t -4′-
yn oylam in o)-5-(2′′′′-oxopyr r olidin -3′′′′-yl-pen t-2-en oic Acid
Cyclobu tyl Ester (7).
Rep r esen ta tive Exa m p le of Syn th esis Meth od B. Syn -
th esis of tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Meth ylisoxazole-
3′′′-ca r b on yl)a m in o]-2′′-oxo-2′′H -p yr id in -1′′-yl}p en t -4′-
yn oylam in o)-5-(2′′′′-oxopyr r olidin -3′′′′-yl)pen t-2-en oic Acid
2,2-Dim eth ylp r op yl Ester (6).
tr a n s-(3′S,4S)-4-ter t-Bu t oxyca r b on yla m in o-5-(2′-oxo-
p yr r olid in -3′-yl)p en t-2-en oic Acid Cyclobu tyl Ester (34).
Dess-Martin periodinane (1.16 g, 2.72 mmol, 1 equiv) was
added to a 0 °C solution of 3328 (0.700 g, 2.71 mmol, 1 equiv)
in CH2Cl2 (25 mL). The reaction vessel was then warmed to
23 °C and stirred 1.5 h. The volatiles were evaporated, and
the residue was evaporated from toluene (2 × 10 mL). The
residue was then dissolved in THF (50 mL). (Triphenyl-λ5-
phosphanylidene)acetic acid cyclobutyl ester (0.982 g, 2.62
mmol, 0.97 equiv) was added, and the reaction mixture was
refluxed 1.5 h and then cooled and concentrated. The residue
was filtered through a plug of silica gel (5% CH3OH in CH2-
Cl2 as eluent) to provide 34 (0.389 g, 41%) which was used
without further purification.
tr a n s-(3′S,4S)-4-ter t-Bu toxyca r bon yla m in o-5-[1′-(2′′,4′′-
dim eth oxyben zyl)-2′-oxopyr r olidin -3′-yl]pen t-2-en oic Acid
2,2-Dim eth ylp r op yl Ester (31). Dess-Martin periodinane
(2.30 g, 5.38 mmol, 1.1 equiv) was added to a 0 °C solution of
2824 (2.00 g, 4.90 mmol, 1 equiv) in CH2Cl2 (50 mL). The
reaction vessel was then warmed to 23 °C and stirred 1.5 h.
The volatiles were evaporated, and the residue was evaporated
from toluene (2 × 20 mL). The residue was then dissolved in
THF (100 mL). (Triphenyl-λ5-phosphanylidene)acetic acid 2,2-
dimethylpropyl ester (prepared using the general ylide syn-
thesis described above, 2.29 g, 5.86 mmol, 1.2 equiv) was
added, and the reaction mixture was refluxed 1.5 h, cooled,
and evaporated. The residue was purified by flash column
chromatography (3% CH3OH in CHCl3 then 40% EtOAc in
hexanes) to give slightly impure 31 (1.50 g). This material was
not characterized and was instead used directly in the next
step.
tr a n s-(3′S,4S)-4-ter t-Bu t oxyca r b on yla m in o-5-(2′-oxo-
p yr r olid in -3′-yl)p en t-2-en oic Acid 2,2-Dim eth ylp r op yl
Ester (32). DDQ (0.925 g, 4.07 mmol, 1.4 equiv) was added to
a solution of 31 (1.50 g, 2.89 mmol, 1 equiv) in a mixture of
CHCl3 and H2O (10:1, 55 mL). The reaction vessel was placed
in an oil bath maintained at 60 °C. After the mixture was
stirred for 1.5 h, additional DDQ (0.925 g, 4.07 mmol, 1.4
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
a m in o)-5-(2′′′′-oxop yr r olid in -3′′′′-yl-p en t-2-en oic Acid Cy-
clobu tyl Ester (7). TFA (3 mL) was added to a solution of 34
(0.389 g, 1.10 mmol, 1 equiv) in CH2Cl2 (4 mL) and stirred 35
min. The volatiles were evaporated, and the residue was
concentrated from CCl4 (5 mL) to provide the crude amine salt.
This material was combined with 27 (0.348 g, 1.10 mmol, 1
equiv) and HOBt (0.209 g, 1.55 mmol, 1.4 equiv) in CH2Cl2
(20 mL) and cooled to 0 °C. EDC (0.265 g, 1.38 g, 1.25 equiv)
i
and Pr2NEt (1.35 mL, 7.75 mmol, 7.0 equiv) were added. The
reaction mixture was allowed to warm to 23 °C, was stirred
16 h, and then was partitioned between CH2Cl2 (3 × 125 mL)
and a mixture of NaHCO3 and brine (1:1, 100 mL). The

Irreversible Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4581
combined organic phases were washed with a mixture of
NaHCO3 and brine (1:1, 100 mL), dried over MgSO4, and
evaporated. The residue was purified by flash column chro-
matography (2% CH3OH in CHCl3) to give 7 as a glass (0.281
g, 46%): Rf ) 0.33 (5% CH3OH in CH2Cl2); IR (cm-1) 3507,
3331, 3295, 1690, 1649, 1596, 1531; 1H NMR (CDCl3) δ 1.52-
1.87 (m, 4H), 2.01-2.41 (m, 8H), 2.50 (s, 3H), 2.92 (ddd, 1H,
J ) 17.0, 8.5, 2.6), 3.02 (ddd, 1H, J ) 17.0, 6.8, 2.6), 3.21-
3.37 (m, 2H), 4.43-4.54 (m, 1H), 4.96-5.08 (m, 1H), 5.68-
5.76 (m 1H), 6.00 (dd, 1H, J ) 15.7, 1.5), 6.33 (t, 1H, J ) 7.3),
6.45-6.48 (m, 1H), 6.83 (s, 1H), 6.84 (dd, 1H, J ) 15.7, 5.4),
7.49 (dd, 1H, J ) 7.3, 1.7), 8.41 (dd, 1H, J ) 7.3, 1.7), 8.68
(d, 1H, J ) 6.6), 9.52 (s, 1H); Anal. (C28H31N5O7‚0.25H2O)
C, H, N.
(CDCl3) δ 0.92 (t, 3H, J ) 7.3), 1.29 (t, 3H, J ) 7.1), 1.47-
1.58 (m, 1H), 1.62-1.77 (m, 1H), 1.85-2.00 (m, 1H), 2.08-
2.33 (m, 4H), 2.49 (s, 3H), 3.25-3.42 (m, 2H), 4.19 (q, 2H, J )
7.1), 4.39-4.50 (m, 1H), 5.73 (dd, 1H, J ) 8.8, 6.8), 5.97 (dd,
1H, J ) 15.7, 1.4), 6.31-6.37 (m, 1H), 6.46 (s, 1H), 6.86 (dd,
1H, J ) 15.7, 5.9), 7.18 (s, 1H), 7.57-7.62 (m, 1H), 8.40-8.44
(m, 1H), 8.58-8.62 (m, 1H), 9.56 (s, 1); Anal. (C25H31N5O7‚
0.5H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
am in o)-5-(2′′′′-oxopyr r olidin -3′′′′-yl)pen t-2-en oic Acid Eth -
yl Ester (3). Rf ) 0.31 (5% CH3OH in CH2Cl2); IR (cm-1) 3295,
1
1684, 1649, 1596, 1531; H NMR (CDCl3) δ 1.27 (t, 3H, J )
7.1), 1.52-1.62 (m, 1H), 1.64-1.80 (m, 1H), 2.04 (t, 1H, J )
2.6), 2.08-2.38 (m, 3H), 2.49 (s, 3H), 2.91 (ddd, 1H, J ) 17.0,
8.4, 2.6), 3.01 (ddd, 1H, J ) 17.0, 6.8, 2.6), 3.22-3.39 (m, 2H),
4.18 (q, 2H, J ) 7.1), 4.44-4.55 (m, 1H), 5.71-5.78 (m, 1H),
6.03 (dd, 1H, J ) 15.6, 1.5), 6.32 (t, 1H, J ) 7.2), 6.46 (s, 1H),
6.85 (dd, 1H, J ) 15.6, 5.4), 6.89 (s, 1H), 7.49 (dd, 1H, J ) 7.2,
1.7), 8.41 (dd, 1H, J ) 7.2, 1.7), 8.68 (d, 1H, J ) 6.8), 9.52
(s, 1H); Anal. (C26H29N5O7‚0.75H2O) C, H, N.
Rep r esen ta tive Exa m p le of Syn th etic Meth od B. Syn -
th esis of tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Meth ylisoxazole-
3′′′-ca r b on yl)a m in o]-2′′-oxo-2′′H -p yr id in -1′′-yl}b u t yr yl-
am in o)-5-(2′′′′-oxopyr r olidin -3′′′′-yl)pen t-2-en oic Acid Eth yl
Ester (13).
(R)-2-Tr iflu or om eth a n esu lfon yloxybu tyr ic Acid ter t-
Bu tyl Ester (36). (R)-2-Hydroxybutyric acid tert-butyl ester
(35) (0.133 g, 0.830 mmol, 1 equiv) was dissolved in CH2Cl2
(7 mL) and cooled to 0 °C. 2,6-Lutidine (0.193 mL, 1.66 mmol,
2.0 equiv) and trifluoromethanesulfonic anhydride (0.265 mL,
1.58 mmol, 1.9 equiv) were added successively. After being
stirred 25 min, the reaction mixture was diluted with MTBE
(100 mL) and washed with a mixture of brine and 1 N HCl
(3:1, 3 × 30 mL) and then dried over MgSO4 and concentrated
to give 36 as a residue. This material was used in the next
step without further purification.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
am in o)-5-(2′′′′′-oxopyr r olidin -3′′′′-yl)pen t-2-en oic Acid Iso-
p r op yl Ester (4). Rf ) 0.45 (10% CH3OH in CH2Cl2); IR (cm-1
)
3304, 1692, 1650; 1H NMR (CDCl3) δ 1.25 (d, 6H, J ) 6.2),
1.54-1.78 (m, 2H), 2.03 (t, 1H, J ) 2.5), 2.06-2.33 (m, 3H),
2.49 (s, 3H), 2.88-3.07 (m, 2H), 4.47-4.49 (m, 1H), 5.00-5.08
(m, 1H), 5.66-5.71 (m, 1H), 6.00 (dd, 1H, J ) 15.6, 1.4), 6.33
(t, 1H, J ) 7.2), 6.46 (s, 1H), 7.73 (br, s, 1H), 6.83 (dd, 1H,
J ) 15.7, 5.4), 7.48 (dd, 1H, J ) 7.2, 1.7), 8.41 (dd, 1H, J )
7.2, 1.7), 8.65 (d, 1H, J ) 6.59), 9.53 (s, 1H); Anal. (C27H31N5O7‚
0.50H2O) C, H, N.
(2S)-2-{3′-[(5′′-Meth ylisoxa zole-3′′-ca r bon yl)a m in o]-2′-
oxo-2′H-p yr id in -1′-yl}bu tyr ic Acid ter t-Bu tyl Ester (37).
5-Methylisoxazole-3-carboxylic acid (2′-hydroxypyridin-3′-yl)-
amide (259) (0.200 g, 0.912 mmol, 1.1 equiv) was dissolved in
THF (6 mL). Sodium hydride (60% dispersion in mineral oil,
0.0332 g, 0.83 mmol, 1.0 equiv) was added, and the reaction
mixture was stirred 30 min. To this mixture was added a
solution of crude 36 prepared above (0.830 mmol theoretical)
in THF (7 mL). After being stirred 30 min more, the reaction
mixture was diluted with EtOAc (100 mL), washed with brine
(2 × 50 mL), dried over Na2SO4, and evaporated. The residue
was purified by flash column chromatography (25% EtOAc in
hexanes) to give 37 as an oil (0.178 g, 59%): Rf ) 0.30 (25%
EtOAc in hexanes); IR (cm-1) 3331, 3131, 1731, 1690, 1649,
1602, 1531, 1455; 1H NMR (CDCl3) δ 0.93 (t, 3H, J ) 7.3),
1.45 (s, 9), 1.83-2.01 (m, 1H), 2.17-2.31 (m, 1H), 2.50 (s, 3H),
5.44-5.51 (m, 1H), 6.32 (t, 1H, J ) 7.2), 6.48 (s, 1H), 7.10 (dd,
1H, J ) 7.2, 1.8), 8.45 (dd, 1H, J ) 7.2, 1.8), 9.64 (s, 1H); Anal.
(C18H23N3O5) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
a m in o)-5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Cy-
clop en tyl Ester (8). Rf ) 0.54 (10% CH3OH in CH2Cl2); IR
1
(cm-1) 3302, 1690, 1649, 1632; H NMR (CDCl3) δ 1.62-1.80
(m, 7H), 1.87-1.92(m, 2H), 2.01-2.12 (m, 2H), 2.26-2.40 (m,
2H), 2.53 (s, 3H), 2.95-3.12 (m, 2H), 3.26-3.36 (m, 2H), 4.50-
4.53 (m, 1H), 5.20-5.25 (m, 1H), 5.54-5.60 (m, 1H), 6.01 (dd,
1H, J ) 15.6, 1.5), 6.31 (t, 2H, J ) 7.2), 6.40 (s, br, 1H), 6.49
(s, 1H), 6.83 (dd, 1H, J ) 15.6, 5.4), 7.43 (dd, 1H, J ) 6.9, 1.8),
8.44 (dd, 1H, J ) 6.9, 1.5), 8.58 (d, 1H, J ) 6.6), 9.56 (s, 1H);
Anal. (C29H33N5O7‚0.75H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
a m in o)-5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Cy-
cloh exyl Ester (9). Rf ) 0.48 (10% CH3OH in CH2Cl2); IR
(cm-1) 3305, 1685, 1650, 1597, 1534; 1H NMR (CDCl3) δ 1.24-
1.98 (m, 12H), 2.03 (t, 1H, J ) 2.5), 2.05-2.39 (m, 3H), 2.50
(s, 3H), 2.90-3.08 (m, 2H), 3.20-3.34 (m, 2H), 4.45-4.52 (m,
1H), 4.75-4.83 (m, 1H), 5.59-5.64 (m, 1H), 6.01 (dd, 1H, J )
15.6, 1.2), 6.33 (t, 1H, J ) 7.2), 6.46 (s, 1H), 6.54 (br, s, 1H),
6.82 (dd, 1H, J ) 15.6, 5.3), 7.44 (dd, 1H, J ) 7.2, 1.7), 8.41
(dd, 1H, J ) 7.2, 1.7), 8.59 (d, 1H, J ) 6.6), 9.53 (s, 1H); Anal.
(C30H35N5O7‚0.75H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Eth yl Ester
(13). TFA (2 mL) was added to a solution of 3928 (0.130 g, 0.398
mmol, 1 equiv) in CH2Cl2 (3 mL) and stirred 30 min. The
volatiles were evaporated, and the residue was concentrated
from CCl4 (5 mL) to provide the crude amine salt which was
set aside.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
a m in o)-5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Cy-
cloh ep tyl Ester (10). 1H NMR (CDCl3) δ 1.30-1.90 (m, 15H),
2.01 (t, 1H, J ) 2.4), 2.20-2.40 (m, 2H), 2.49 (s, 3H), 2.90-
3.10 (m, 2H), 3.20-3.30 (m, 2H), 4.45-4.56 (m, 1H), 4.92-
5.03 (m, 1H), 5.48 (t, 1H, J ) 7.3), 5.98 (d, 1H, J ) 15.6), 6.21
(s, 1H), 6.32 (t, 1H, J ) 7.4), 6.46 (s, 1H), 6.80 (dd, 1H, J )
15.6, 5.3), 7.35-7.38 (dd, 1H, J ) 7.0, 1.5), 8.39-8.43 (m, 2H),
9.52 (s, 1H); Anal. (C31H37N5O7‚0.3H2O) C, H, N.
TFA (2 mL) was added to a solution of 37 (0.143 g, 0.397
mmol, 1.0 equiv) in CH2Cl2 (3 mL) and stirred 2 h. The
volatiles were evaporated to provide the crude carboxylic acid
(38) which was combined with the crude amine salt prepared
above in CH2Cl2 (4 mL) and cooled to 0 °C. HOBt (0.064 g,
i
0.47 mmol, 1.2 equiv), Pr2NEt (0.484 mL, 2.78 mmol, 7.0
equiv), and EDC (0.084 g, 0.44 mmol, 1.1 equiv) were added
successively. The reaction mixture was allowed to warm to 23
°C, stirred 18 h, and then partitioned between CH2Cl2 (3 × 30
mL) and a mixture of NaHCO3 and brine (1:1, 30 mL). The
combined organic phases were washed with a mixture of
NaHCO3 and brine (1:1, 10 mL), dried over MgSO4, and
evaporated. The residue was purified by flash column chro-
matography (gradient elution, 2 to 3% CH3OH in CH2Cl2) to
give 13 as a white foam (0.119 g, 58%): Rf ) 0.46 (10% CH3-
OH in CHCl3); IR (cm-1) 3331, 1684, 1649, 1590, 1531; 1H NMR
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-4′-yn oyl-
am in o)-5-(2′′′′-oxopyr r olidin -3′′′′-yl)pen t-2-en oic Acid Ben -
zyl Ester (11). Rf ) 0.36 (10% CH3OH in CH2Cl2); IR (cm-1
3298, 1685, 1650, 1596, 1534; H NMR (CDCl3) δ 1.54-1.74
(m, 2H), 1.97 (t, 3H, J ) 2.7), 2.00-2.19 (m, 1H), 2.22-2.39
)
1

4582 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Dragovich et al.
(m, 2H), 2.49 (s, 3H), 2.88-3.07 (m, 2H), 3.19-3.32 (m, 2H),
4.44-4.52 (m, 1H), 5.16 (s, 2H), 5.55-5.60 (m, 1H), 6.07 (dd,
1H, J ) 15.7, 1.5), 6.32 (t, 1H, J ) 7.2), 6.45 (s, 1H), 6.48 (br,
s, 1H), 6.89 (dd, 1H, J ) 15.7, 5.3), 7.29-7.41 (m, 5H), 7.44
(dd, 1H, J ) 6.2, 1.7), 8.40 (dd, 1H, J ) 7.5, 1.7), 8.61 (d, 1H,
J ) 6.6), 9.52 (s, 1H); Anal. (C31H31N5O7‚0.50H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-2-Meth yl-4-(2′-{3′′-[(5′′′-m eth ylisox-
a zole-3′′′-ca r bon yl)a m in o]-2′′-oxo-2′′H-p yr id in -1′′-yl}p en t-
4′-yn oyla m in o)-5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t -2-en o-
ic Acid Eth yl Ester (12). Rf ) 0.50 (10% CH3OH in CHCl3);
IR (cm-1) 3307, 1690, 1649; 1H NMR (CDCl3) δ 1.29 (t, 3H,
J ) 7.1), 1.36-1.47 (m, 1H), 1.65-1.80 (m, 1H), 1.95-2.00
(m, 4H), 2.14-2.40 (m, 3H), 2.50 (s, 3H), 2.87-3.03 (m, 2H),
3.23-3.38 (m, 2H), 4.18 (q, 2H, J ) 7.1), 4.56-4.68 (m, 1H),
5.63-5.72 (m, 1H), 6.34 (t, 1H, J ) 7.3), 6.47 (s, 1H), 6.52-
6.58 (m, 1H), 6.81 (s, 1H), 7.46 (dd, 1H, J ) 7.3, 1.6), 8.42 (dd,
1H, J ) 7.3, 1.6), 8.65 (d, 1H, J ) 6.4), 9.54 (s, 1H); Anal.
(C27H31N5O7‚0.5H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Isop r op yl
Ester (14). IR (cm-1) 3333, 1681, 1531, 1276; 1H NMR (CDCl3)
δ 0.98 (t, 3H, J ) 7.2), 1.29 (d, 6H, J ) 6.6), 1.46-1.73 (m,
1H), 1.89-2.07 (m, 1H), 2.13-2.37 (m, 4H), 2.52 (s, 3H), 3.30-
3.43 (m, 3H), 4.48 (m, 1H), 5.02-5.14 (m, 1H), 5.69 (t, 1H,
J ) 6.9), 5.96 (d, 1H, J ) 15.6), 6.39 (t, 1H, J ) 7.5), 6.49 (s,
1H), 6.86 (dd, 1H, J ) 15.6, 6.0), 6.91 (s, br. 1H), 7.56 (d, 1H,
J ) 7.2), 8.44 (d, 1H, J ) 7.5), 8.53 (d, 1H, J ) 6.3), 9.59 (s,
1H); Anal. (C26H33N5O7‚0.5H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Ben zyl Es-
ter (20). Rf
) 0.35 (10% CH3OH in CH2Cl2); IR (cm-1) 3335,
1
1685, 1649, 1595, 1533; H NMR (CDCl3) δ 0.91 (t, 3H, J )
7.4), 1.50-1.97 (m, 3H), 2.11-2.31 (m, 4H), 2.48 (s, 3H), 3.27-
3.38 (m, 2H), 4.45-4.47 (m, 1H), 5.17 (s, 2H), 5.65-5.70 (m,
1H), 6.01 (dd, 1H, J ) 15.6, 1.5), 6.33 (t, 1H, J ) 7.1), 6.46 (s,
1H), 6.90 (dd, 1H, J ) 15.6, 5.8), 7.00 (br, s, 1H), 7.30-7.39
(m, 5H), 7.54 (dd, 1H, J ) 7.1, 1.8), 8.42 (dd, 1H, J ) 7.4, 1.8),
8.56 (d, 1H, J ) 6.8), 9.56 (s, 1H); Anal. (C30H33N5O7‚0.50H2O)
C, H, N.
P h a r m a cok in etic Exp er im en ts. The in vivo pharmaco-
kinetic results described in this work were determined after
either intravenous (15 mg/kg, n ) 3) and oral (30 mg/kg, n )
3) single agent dosing of the appropriate molecules to male
beagle dogs (BW ∼9.3 kg) or intravenous (25 mg/kg, n ) 3)
and oral (50 mg/kg, n ) 3) single agent dosing to male CM-
monkeys (BW ∼3.9 kg). The compounds were typically admin-
istered in a vehicle consisting of 80% propylene glycol and 20%
sterile water at a concentration of 15.0 mg/mL, although
animals from several oral studies were also dosed using a 0.5%
carboxymethyl cellulose suspension formulation. In each case,
blood samples were collected periodically for up to 24 h
postdosing, and the plasma concentrations of each test com-
pound were determined by LCMS. Pharmacokinetic param-
eters were estimated by noncompartmental analysis of the
individual plasma concentration-time data.
P la sm a Sta bility Stu d ies. Pooled heparinized human
plasma from 10 individuals was obtained from Golden West
Biologicals, Temecula, CA, and frozen at -70 °C until analysis.
The plasma stability experiment was initiated by transferring
1990 µL of plasma (n ) 3) or 100 mM potassium phosphate
pH 7.4 buffer (n ) 3) into separate test tubes. A 10 µL aliquot
of a freshly made 5 mM acetonitrile solution of the individual
test compounds was transferred into the plasma or buffer to
achieve a final compound concentration of 25 µM. The test
tubes were incubated at 37 °C for 1-3 h and 200 µL samples
were collected at regular intervals. The samples were mixed
with 2 mL of acetonitrile and vortexed to ensure protein
precipitation and immediate termination of metabolic trans-
formations. After centrifugation for 10 min at 4000 rpm at
10 °C in a Sorvall RT 7 centrifuge, the clear supernatant was
decanted into a new set of tubes, and the volatiles were
removed under a stream of nitrogen using a Dri-block sample
concentrator (Techne, Princeton, NJ ). The samples were
reconstituted in 250 µL of mobile phase (60% 25 mM pH )
5.1 NH4H2PO4 buffer and 40% CH3CN). Chromatographic
analysis was performed using a 1100 Hewlett-Packard HPLC
with a Primesphere reversed phase column (5 µm, 4.6 × 15
mm, Phenomenex, Torrance, CA) at a flow rate of 1 mL/min
using a gradient elution. A volume of 100 µL was injected onto
the column, and the test molecules were detected by UV
absorption at 212 nm. The standard curve of the compounds
ranged from 0.05 µg/mL to 40 µg/mL. The half-life of a given
compound’s metabolic conversion rate was determined by
linear regression (WinNonlin Professional, Pharsight, Moun-
tainview, CA) of the mean plasma concentration-time data
obtained from the incubation studies.
Micr osom e Sta bility Stu d ies. Canine, simian, or human
pooled liver microsomes (1 mg/mL, consisting of 6-8 livers)
and 2 mM NADPH were incubated in 100 mM potassium
phosphate buffer, pH 7.4, at 37 °C in a shaking water bath
for 1 min. The reaction was initiated by the addition of 25 µM
test compound and proceeded for 30 min followed by the
addition of 500 µL acetonitrile to terminate the reaction. The
incubation volume was 500 µL. Separate control samples were
also prepared in a manner similar to the time zero samples
with 500 µL of acetonitrile added to the test tube prior to the
test compound. All samples were vortexed (2 min) on a SP
Multi-tube Vortexer and then centrifuged at 2500 × g for 20
min. The supernatant was concentrated and then analyzed by
HPLC. Chromatographic separation was achieved for the test
compounds using either a Hewlett-Packard 1050 HPLC with
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid 2,2-Dim eth -
ylp r op yl Ester (16). Rf ) 0.27 (5% CH3OH in CH2Cl2); IR
1
(cm-1) 3331, 3295, 1690, 1649; H NMR (CDCl3) δ 0.87-0.99
(m, 12H), 1.51-1.61 (m, 1H), 1.64-1.79 (m, 1H), 1.83-2.00
(m, 1H), 2.10-2.37 (m, 4H), 2.49 (s, 3H), 3.26-3.43 (m, 2H),
3.83 (s, 2H), 4.43-4.54 (m, 1H), 5.71 (dd, 1H, J ) 8.7, 6.9),
6.00 (dd, 1H, J ) 15.7, 1.3), 6.35 (t, 1H, J ) 7.2), 6.46 (s, 1H),
6.86 (dd, 1H, J ) 15.7, 5.9), 7.39 (s, 1H), 7.58 (dd, 1H, J ) 7.2,
1.7), 8.42 (dd, 1H, J ) 7.2, 1.7), 8.53 (d, 1H, J ) 7.0), 9.55 (s,
1H); Anal. (C28H37N5O7‚0.5H2O) C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Cyclobu tyl
Ester (17). IR (cm-1) 3334, 1690, 1632; 1H NMR (CDCl3) δ
0.95 (t, 3H, J ) 7.5), 1.55-1.85 (m, 5H), 1.88-2.01 (m, 1H),
2.04-2.44 (m, 7H), 2.52 (s, 3H), 3.31-3.41 (m, 2H), 4.48 (m,
1H), 5.01-5.11 (m, 1H), 5.63-5.71 (m, 1H), 5.96 (dd, 1H, J )
15.0, 1.5), 6.36 (t, 1H, J ) 7.5), 6.49 (s, 1H), 6.83-6.90 (m,
2H), 7.54 (d, 1H, J ) 7.2), 8.45 (dd, 1H, J ) 7.5, 1.8), 8.53 (dd,
1H, J ) 6.6, 1.8), 9.59 (s, 1H); Anal. (C27H33N5O7‚0.75H2O) C,
H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Cycloh exyl
Ester (18). Rf ) 0.37 (10% CH3OH in CH2Cl2); IR (cm-1) 3334,
1
1687, 1649, 1595, 1532; H NMR (CDCl3) δ 0.92 (t, 3H, J )
7.4), 1.24-1.99 (m, 14H), 2.06-2.30 (m, 3H), 2.49 (s, 3H),
3.28-3.39 (m, 2H), 4.45-4.47 (m, 1H), 4.77-4.84 (m, 1H),
5.65-5.70 (m, 1H), 5.95 (dd, 1H, J ) 15.7, 1.1), 6.34 (t, 1H,
J ) 7.3), 6.46 (s, 1H), 6.83 (dd, 1H, J ) 15.7, 5.8), 6.93 (br, s,
1H), 7.54 (dd, 1H, J ) 7.3, 1.7), 8.42 (dd, 1H, J ) 7.3, 1.7),
8.49 (d, 1H, J ) 6.8), 9.57 (s, 1H); Anal. (C29H37N5O7‚0.50H2O)
C, H, N.
tr a n s-(2′S,3′′′′S,4S)-4-(2′-{3′′-[(5′′′-Met h ylisoxa zole-3′′′-
car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -1′′-yl}bu tyr ylam in o)-
5-(2′′′′-oxop yr r olid in -3′′′′-yl)p en t-2-en oic Acid Cycloh ep -
1
tyl Ester (19). H NMR (CDCl3) δ 0.85-2.35 (m, 22H), 2.49
(s, 3H), 3.23-3.38 (m, 2H), 4.46-4.57 (m, 1H), 4.93-5.03 (m,
1H), 5.44-5.52 (m, 1H), 5.93 (dd, 1H, J ) 15.6, 1.2), 6.12 (s,
br, 1H), 6.32 (1H, t, J ) 7.2), 6.46 (s, 1H), 6.80 (dd, 1H, J )
15.7, 5.7), 7.35 (dd, 1H, J ) 7.2, 1.7), 8.15 (d, 1H, J ) 6.8),
8.41 (dd, 1H, J ) 7.4, 1.6), 9.58 (s, 1H); Anal. (C30H39N5O7‚
0.80H2O) C, H, N.

Irreversible Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4583
a Primesphere C18 HC reversed phase column (5µ, 4.6 × 150
mm) or a Zorbax Eclipse XDB C18 column (5 µm, 4.6 × 150
mm). Analyte elution was conducted with the former using
25 mM NH4H2PO4 buffer, pH 4.5, with constant 5% methanol
and a time gradient for acetonitrile of 0-2 min: 5%; 2-10
min: 5-50%; 10-20 min: 50%; 20-21 min: 50-5%; 21-24
min 5%, with total run time of 24 min. Alternatively, com-
pounds examined using the Zorbax column were eluted using
25 mM NH4H2PO4 buffer, pH 4.5 with constant 10% methanol
and a time gradient for acetonitrile of time 0-10 min: 10-
55%; 10-16 min: 55-75%; 16-18 min: 75-10%; 18-20
min: 10%, with a total run time of 25 min. All molecules were
monitored at 215 nm.
Ca co-2 P er m ea bility Stu d ies. Caco-2 cells were cultivated
under aseptic conditions at 37 °C in an atmosphere of 90%
relative humidity (95% air and 5% CO2). The culture medium
consisted of 10% fetal bovine serum, 100 IU/mL penicillin,
100 µg/mL streptomycin, and 1% MEM nonessential amino
acid solution. The culture medium was replaced every 3-4
days, and cells were harvested with trypsin-EDTA (0.25%)
upon reaching 70-80% confluency. The Caco-2 cells from
passages 30-50 were seeded onto six-well transwell plates
(63 000 cells/cm2) and grown for 21-25 days until formation
of a confluent monolayer was apparent (TEER values between
400 and 600 Ω). Sanpwell filters were soaked in Hanks
balanced salt solution (HBSS) for 1 h and then were mounted
onto the diffusion chamber.
RC 5C with a swinging bucket rotor SH-3000. Following
centrifugation, 100 µL was removed from the ultrafiltrate side
and transferred to a test tube containing methanol (1 mL).
An internal standard, pentoxifylline (50 µL of 20 µg/mL
solution in methanol), was added to all tubes. All samples were
vortexed (5 min) on a SP Multi-tube Vortexer (Baxter, McGaw
Park, IL) and centrifuged at 2500 × g for 30 min. The organic
layer was removed and evaporated under a gentle stream of
nitrogen using a Dri-Block sample concentrator (Techne,
Princeton, NJ ) at 45 °C. Samples were reconstituted with 100
mL of a 1:1 mixture of methanol and 0.1% formic acid. All
samples were vortexed a second time for 5 min and centrifuged
at 2500 × g for 20 min. The supernatants were transferred
into a 96-well plate for analysis by LCMS. The protein binding
value (percent free) was calculated by dividing the measured
amount of compound present in the filtrate by the amount
detected in whole plasma and multiplying the product by 100.
Three replicates were evaluated per assay for each species at
each concentration.
Wa ter Solu bility Deter m in a tion s. To a buffer solution
(pH ) 7.0, 50 mM phosphate, 1 mL) in a vial was added 1-2
mg of test compound. The mixture was stirred at room
temperature for 4 h and then was filtered using a 0.45 µm
syringe filter. The concentration of the drug in the filtrate was
determined by HPLC analysis employing a standard solution
of a known concentration.
The apical to basolateral permeability of test compounds
was studied using the above apparatus as follows. A donor
buffer consisting of HBSS with 25 mM mannitol (pH 6.5), 0.5%
DMSO, and 2% ethanol was prepared as a vehicle for the
molecules under study. Independently, 5 mL of a buffer
solution containing the test compound (100 µM) or perme-
ability reference markers (metoprolol and atenolol; 100 µM
each) was added to the apical side of the cell monolayer (n )
3). Blank HBSS receiver buffer containing 25 mM glucose
(5 mL, pH ) 7.4) was quickly added to the basolateral side of
the monolayer, and 1 mL aliquots were subsequently removed
from this compartment at 5, 45, 90, and 120 min. After removal
of each aliquot, 1 mL of blank HBSS was added to the
basolateral side of the monolayer. Buffer solutions were
maintained at 37 °C during the course of the experiment and
were bubbled with a mixture of air-CO2 (95/5) to facilitate
oxygenation and stirring. The concentrations of the test
compounds and permeability marker (atenolol) present in the
samples were determined by HPLC and Papp values were
calculated as described in the literature.33 Using the above
apparatus and procedure, the Papp of internal atenolol was
determined to be 1.0 ( 0.1 × 10-6 cm/s while that of metoprolol
(measured independently of test compounds) was found to be
11.1 ( 0.5 × 10-6 cm/s.
Ack n ow led gm en t. We are grateful for many helpful
discussions throughout the course of this work with
Prof. Larry Overman and Drs. Stephen Worland and
Steven Bender.
Refer en ces
(1) For part 7 in this series, see: Dragovich, P. S.; Prins, T. J .; Zhou,
R.; J ohnson, T. O.; Brown, E. L.; Maldonado, F. C.; Fuhrman,
S. A.; Zalman, L. S.; Patick, A. K.; Matthews, D. A.; Hou, X.;
Meador, J . W., III; Ferre, R. A.; Worland, S. T. Structure-Based
Design, Synthesis, and Biological Evaluation of Irreversible
Human Rhinovirus 3C Protease Inhibitors. 7. Structure-Activ-
ity Studies of Bicyclic 2-Pyridone-Containing Peptidomimetics.
Bioorg. Med. Chem. Lett. 2002, 12, 733-738.
(2) (a) Couch, R. B. Rhinoviruses. In Fields Virology, 4th ed.; Knipe,
D. M., Howley, P. M., et al., Eds.; Lippincott, Williams, & Wilkins
Publishers: Philadelphia, 2001; Vol. 1, Chapter 25, pp 777-797.
(b) McKinlay, M. A.; Pevear, D. C.; Rossmann, M. G. Treatment
of the Picornavirus Common Cold by Inhibitors of Viral Uncoat-
ing and Attachment. Annu. Rev. Microbiol. 1992, 46, 635-654.
(c) Phillpotts, R. J .; Tyrrell, D. A. J . Rhinovirus Colds. Br. Med.
Bull. 1985, 41, 386-390. (d) Gwaltney, J . M. The Common Cold.
In Principles and Practices of Infectious Diseases; Mandell, G.
L., Douglas, R. G., Bennett, J . E., Eds.; J ohn Wiley & Sons: New
York, 1985, Chapter 38, pp 351-355. (e) Gwaltney, J . M.
Rhinoviruses. In Viral Infections of Humans; Evans, A. S., Ed.;
Plenum Publishing Corp.: New York, 1982; Chapter 20, pp 491-
517.
(3) (a) Rueckert, R. R. Picornaviridae: The Viruses and Their
Replication. In Fields Virology, 4th ed.; Knipe, D. M., Howley,
P. M., et al., Eds.; Lippincott, Williams, & Wilkins Publishers:
Philadelphia, 2001; Vol. 1, Chapter 23, pp 685-722. (b) Kra¨us-
slich, H.-G.; Wimmer, E. Viral Proteinases. Annu. Rev. Biochem.
1988, 57, 701-754.
(4) 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.
At the end of the transport experiment, 14C-radiolabeled
mannitol (1.7 µCi) was added to each donor chamber to
evaluate the integrity of the cell monolayers. Samples were
collected from the donor (100 µL) and receiver (1 mL) side at
0 and 60 min after adding 14C-mannitol. These samples were
diluted by adding 5 mL of liquid scintillation cocktail, and the
amount of 14C-mannitol present in each was measures using
a liquid scintillation counter. The Papp of internal mannitol was
determined to be 0.07 × 10-6 cm/s.
P la sm a P r otein Bin d in g Deter m in a tion s. For each
tested compound, 1 mM and 0.1 mM stock solutions (contain-
ing 50% acetonitrile, 20% DMSO, and 30% 100 mM potassium
phosphate buffer, pH 7.4) were prepared, and 10 µL of each
was added to 1 mL of heparinized dog or human plasma or
1 mL buffer (100 mM potassium phosphate pH 7.4; final
compound concentrations of 1 and 10 µM, respectively). The
samples were incubated at 37 °C in a shaking water bath for
1 h. An aliquot (100 µL) of plasma or buffer was removed and
placed in separate test tubes containing methanol (1 mL) for
estimation of total compound concentration. The remainder
(900 µL) of each plasma or buffer sample was then transferred
to a Millipore Centrifree device. The Millipore tubes were
centrifuged at 2200 rpm for 20 min using a Sorvall Centrifuge
(5) (a) Turner, R. B.; Wecker, M. T.; Pohl, G.; Witek, T. J .; McNally,
E.; St. George, R.; Winther, B.; Hayden, F. G. Efficacy of
Tremacamra, a Soluble Intercellular Adhesion Molecule 1, for
Experimental Rhinovirus Infection. J AMA 1999, 281, 1797-
1804. (b) Rogers, J . M.; Diana, G. D.; McKinlay, M. A. Pleconaril,
a Broad Spectrum Antipicornaviral Agent. Antiviral Chemother.
1999, 5, 69-76. (c) Hamdouchi, C.; Ezquerra, J .; Vega, J . A.;
Vaquero, J . J .; Alvarez-Builla, J .; Heinz, B. A. Short Synthesis
and Anti-Rhinoviral Activity of Imidazo[1,2-a]Pyridines: The
Effect of Acyl Groups at 3-Position. Bioorg. Med. Chem. Lett.
1999, 9, 1391-1394. (d) Carrasco, L. Picornavirus Inhibitors.
Pharmacol. Ther. 1994, 64, 215-290.

4584 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Dragovich et al.
(6) (a) Bergmann, E. M.; J ames, M. N. G. Proteolytic Enzymes of
the Viruses of the Family Picornaviridae. Proteases Infect. Agents
1999, 139-163. (b) Cordingley, M. G.; Callahan, P. L.; Sardana,
V. V.; Garsky, V. M.; Colonno, R. J . Substrate Requirements of
Human Rhinovirus 3C Protease for Peptide Cleavage in Vitro.
J . Biol. Chem. 1990, 265, 9062-9065. (c) Orr, D. C.; Long, A.
C.; Kay, J .; Dunn, B. M.; Cameron, J . M. Hydrolysis of a Series
of Synthetic Peptide Substrates by the Human Rhinovirus 14
3C Proteinase, Cloned and Expressed in Escherichia coli. J . Gen.
Virol. 1989, 70, 2931-2942. (d) Cordingley, M. G.; Register, R.
B.; Callahan, P. L.; Garsky, V. M.; Colonno, R. J . Cleavage of
Small Peptides In Vitro by Human Rhinovirus 14 3C Protease
Expressed in Escherichia coli. J . Virol. 1989, 63, 5037-5045.
(7) (a) Matthews, D. A.; Smith, W. W.; Ferre, R. A.; Condon, B.;
Budahazi, G.; Sisson, W.; Villafranca, J . E.; J anson, C. A.;
McElroy, H. E.; Gribskov, C. L.; Worland, S. Structure of Human
Rhinovirus 3C Protease Reveals a Trypsin-like Polypeptide Fold,
RNA-Binding Site, and Means for Cleaving Precursor Polypro-
tein. Cell 1994, 77, 761-771. (b) Allaire, M.; Chernaia, M. M.;
Malcolm, B. A.; J ames, M. N. G. Picornaviral 3C Cysteine
Proteinases Have a Fold Similar to Chymotrypsin-like Serine
Proteinases. Nature 1994, 369, 72-76. (c) Bazan, J . F.; Flett-
erick, R. J . Viral Cysteine Proteases are Homologous to the
Trypsin-like Family of Serine Proteases: Structural and Func-
tional Implications. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7872-
7876. (d) Gorbalenya, A. E.; Blinov, V. M.; Donchenko, A. P.
Poliovirus-encoded Proteinase 3C: A Possible Evolutionary Link
Between Cellular Serine and Cysteine Proteinase Families.
FEBS Lett. 1986, 194, 253-257.
(8) (a) Dragovich, P. S. Recent Advances in the Development of
Human Rhinovirus 3C Protease Inhibitors. Exp. Opin. Ther. Pat.
2001, 11, 177-184. (b) Wang, Q. M. Protease Inhibitors as
Potential Antiviral Agents for the Treatment of Picornaviral
Infections. Prog. Drug Res. 1999, 52, 197-219. (c) Wang, Q. M.
Human Rhinovirus 3C Protease Inhibitors: Recent Develop-
ments. Exp. Opin. Ther. Pat. 1998, 8, 1151-1156.
(9) Dragovich, P. S.; Prins, T. J .; Zhou, R.; Brown, E. L.; Maldonado,
F. C.; Fuhrman, S. A.; Zalman, L. S.; Tuntland, T.; Lee, C. A.;
Patick, A. K.; Matthews, D. A.; Hendrickson, T. F.; Kosa, M. B.;
Liu, B.; Batugo, M. R.; Gleeson, J .-P. R.; Sakata, S. K.; Chen,
L.; Guzman, M. C.; Meador, J . W., III; Ferre, R. A.; Worland, S.
T. Structure-Based Design, Synthesis, and Biological Evaluation
of Irreversible Human Rhinovirus 3C Protease Inhibitors. 6.
Structure-Activity Studies of Orally Bioavailable, 2-Pyridone-
Containing Peptidomimetics. J . Med. Chem. 2002, 45, 1607-
1623.
(10) For other examples of Michael acceptor-containing cysteine
protease inhibitors, see: (a) Roush, W. R.; Gwaltney, S. L., II;
Cheng, J .; Scheidt, K. A.; McKerrow, J . H.; Hansell, E. Vinyl
Sulfonate Esters and Vinyl Sulfonamides: Potent, Irreversible
Inhibitors of Cysteine Proteases. J . Am. Chem. Soc. 1998, 120,
10994-10995. (b) McGrath, M. E.; Klaus, J . L.; Barnes, M. G.;
Bro¨mme, D. Crystal Structure of Human Cathepsin K Com-
plexed with a Potent Inhibitor. Nature Struct. Biol. 1997, 4,
105-109. (c) Bro¨mme, D.; Klaus, J . L.; Okamoto, K.; Rasnick,
D.; Palmer, J . T. Peptidyl Vinyl Sulphones: A New Class of
Potent and Selective Cysteine Protease Inhibitors. Biochem. J .
1996, 315, 85-89. (d) Palmer, J . T.; Rasnick, D.; Klaus, J . L.;
Bro¨mme, D. Vinyl Sulfones as Mechanism-Based Cysteine
Protease Inhibitors. J . Med. Chem. 1995, 38, 3193-3196. (e) Liu,
S.; Hanzlik, R. P. Structure-Activity Relationships for Inhibition
of Papain by Peptide Michael Acceptors. J . Med. Chem. 1992,
35, 1067-1075. (f) Hanzlik, R. P.; Thompson, S. A. Vinylogous
Amino Acid Esters: A New Class of Inactivators for Thiol
Proteases. J . Med. Chem. 1984, 27, 711-712.
(14) (a) de Groot, M. J .; Ekins, S. Pharmacophore Modeling of
Cytochromes P450. Adv. Drug Deliv. Rev. 2002, 54, 367-383.
(b) Lewis, D. F. V.; Dickins, M. Factors Influencing Rates and
Clearance in P450-Mediated Reactions: QSARs for Substrates
of the Xenobiotic-metabolizing Hepatic Microsomal P450s. Toxi-
cology 2002, 170, 45-53. (c) Lewis, D. F. V. Structural Charac-
teristics of Human P450s Involved in Drug Metabolism: QSARs
and Lipophilicity Profiles. Toxicology 2000, 144, 197-203. (d)
Rendic, S.; Di Carlo, F. J . Human Cytochrome P450 Enzymes:
A Status Report Summarizing Their Reactions, Substrates,
Inducers, and Inhibitors. Drug Met. Rev. 1997, 29, 413-580.
(15) In vitro microsome experiments conducted in both the presence
and absence of the NADPH cytochrome cofactor suggested that
the metabolism of the majority of the compounds described in
this study was effected by a combination of P450-mediated
oxidative transformations and esterase-mediated ester hydroly-
sis (Lee, C. A., unpublished results).
(16) The nomenclature used for describing the individual amino acid
residues of a peptide substrate (P2, P1, P1′, P2′, etc.) and the
corresponding enzyme subsites (S2, S1, S1′, S2′, etc.) is described
in: Schechter, I.; Berger, A. On the Size of the Active Site in
Proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967, 27,
157-162.
(17) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Reich, S. H.; Marakovits, J . T.;
Prins, T. J .; Zhou, R.; Tikhe, J .; Littlefield, E. S.; Bleckman, T.
M.; Wallace, M. B.; Little, T. L.; Ford, C. E.; Meador, J . W., III;
Ferre, R. A.; Brown, E. L.; Binford, S. L.; DeLisle, D. M.;
Worland, S. T. Structure-Based Design, Synthesis, and Biological
Evaluation of Irreversible Human Rhinovirus 3C Protease
Inhibitors. 2. Peptide Structure-Activity Studies. J . Med. Chem.
1998, 41, 2819-2834.
(18) In the subsequent discussion of structure-activity relationships,
the notation “3CP” indicates 3C protease derived from HRV-14
unless otherwise specified.
(19) Our previous research indicated that the P2 benzyl substituent
present in 2-pyridone-containing 3CP inhibitors such as 1 and
2
is subject to facile P450-mediated oxidative metabolism.
Fluorination of this fragment was observed to improve the in
vitro metabolic stability profiles of compounds which contain it.9
Full details of these metabolism studies will be published
elsewhere (Lee, C. A., manuscript in preparation).
(20) For some general discussions concerning the physiochemical
attributes of organic molecules which lead to favorable oral
bioavailability properties, see: (a) Veber, D. F.; J ohnson, S. R.;
Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D. Molecular
Properties That Influence the Oral Bioavailability of Drug
Candidates. J . Med. Chem. 2002, 45, 2615-2623. (b) Egan, W.
J .; Lauri, G. Prediction of Intestinal Permeability. Adv. Drug.
Deliv. Rev. 2002, 54, 273-289. (c) Stenberg, P.; Norinder, U.;
Luthman, K.; Artursson, P. Experimental and Computational
Screening Models for the Prediction of Intestinal Drug Absorp-
tion. J . Med. Chem. 2001, 44, 1927-1937. (d) Lipinsky, C. A.;
Lombardo, F.; Dominy, B. W.; Feeney, P. J . Experimental and
Computational Approaches to Estimate Solubility and Perme-
ability in Drug Discovery and Development Settings. Adv. Drug.
Deliv. Rev. 1997, 23, 3-25. (e) Chan, O. H.; Stewart, B. H.
Physiochemical and Drug-Delivery Considerations for Oral Drug
Bioavailability. Drug Discuss. Today 1996, 1, 461-473.
(21) Cahiez, G.; Metais, E. Enantioselective Preparation of R-Acyloxy
Ketones from R-Hydroxy and R-Amino Acids. Tetrahedron Lett.
1995, 36, 6449-6452 and references therein.
(22) No rigorous effort was made to detect and/or quantitate the
possible O-alkylation reaction product using more stringent
analytical methods.
(11) The 7 h plasma concentration of a given compound observed in
the dog and monkey following oral administration was chosen
as a convenient measure of both oral bioavailability and phar-
macokinetic performance in these species. In general, this
parameter correlated well with Cmax and AUC values across the
series of molecules under study. Since no correlation between
3CP inhibitor in vitro antirhinoviral activity, human plasma
level, and clinical efficacy has been established, the relevance
of the 7 h concentration values toward assessing the therapeutic
efficacy potential of the described 3CP inhibitors is not known.
(12) The absolute oral bioavailabilities (defined as the ratios of AUCs
observed following oral and IV administration) of the compounds
described in this work varied from 27 to 55% in the dog and
from 1 to 25% in the monkey. Such absolute bioavailability
values did not correlate well with observed pharmacokinetics
(i.e., 7 h plasma levels) and were therefore determined not to
be a reliable predictor of potential compound exposures in other
species.
(23) For general references on the alkylation of 2-hydroxypyridines,
see: (a) Scriven, E. F. V. In Comprehensive Heterocyclic Chem-
istry; Katritzky, A. R., Rees, C. W., Eds.; Pergamon Press:
Oxford, 1984; Vol. 2, pp 165-314. (b) Tieckelmann, H. In
Pyridine and its Derivatives; Abramovitch, R. A., Ed.; Wiley-
Interscience: New York, 1974; Vol. 14, Supplement 3, Chapter
12, pp 597-1180.
(24) Dragovich, P. S.; Prins, T. J .; Zhou, R.; Webber, S. E.; Marako-
vits, J . T.; Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.; Lee,
C. A.; Ford, C. E.; Burke, B. J .; Rejto, P. A.; Hendrickson, T. F.;
Tuntland, T.; Brown, E. L.; Meador, J . W., III; Ferre, R. A.; Harr,
J . E. V.; Kosa, M. B.; Worland, S. T. Structure-Based Design,
Synthesis, and Biological Evaluation of Irreversible Human
Rhinovirus 3C Protease Inhibitors. 4. Incorporation of P1 Lactam
Moieties as L-Glutamine Replacements. J . Med. Chem. 1999, 42,
1213-1224.
(25) The described coupling of the propargyl- and ethyl-containing
P4-P3-P2 moieties with the P1 fragments typically resulted in
greater racemization (20-30%) of the P2 stereocenter than was
noted in our previous work employing analogues which incor-
porated P2 benzyl substituents.9 Thus, careful flash column
chromatography was often required to obtain the 3CP inhibitors
described in the present work in diastereomerically pure form.
(13) Representative examples of the 2-pyridone-containing 3CP
inhibitors described in this work were shown to be highly
unstable toward rodent plasma due to facile ester hydrolysis.9
Pharmacological evaluation of these molecules was therefore not
conducted in rodents.

Irreversible Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4585
In all cases, the purified compounds were shown to be configu-
rationally stable when subjected to routine handling and biologi-
cal testing procedures. The cause of the differing racemization
rates observed during the synthesis of the various 2-pyridone-
containing 3CP inhibitors is not known.
Inhibitors of Human Rhinovirus 3C Protease: Design, Synthesis,
Biological Evaluation, and Cocrystal Structure Solution of P1
Glutamine Isosteric Replacements. J . Med. Chem. 1998, 41,
2786-2805.
(31) Andries, K.; Dewindt, B.; Snoeks, J .; Willebrords, R.; Van
Eemeren, K.; Stokbroekx, R.; J anssen, P. A. J . In Vitro Activity
of Pirodavir (R 77975), a Substituted Phenoxy-Pyridazinamine
with Broad-Spectrum Antipicornaviral Activity. Antimicrob.
Agents Chemother. 1992, 36, 100-107.
(26) Dess, D. B.; Martin, J . C. A Useful 12-I-5 Triacetoxyperiodinane
(the Dess-Martin Periodinane) for the Selective Oxidation of
Primary or Secondary Alcohols and a Variety of Related 12-I-5
Species. J . Am. Chem. Soc. 1991, 113, 7277-7287.
(27) (a) Mori, S.; Iwakura, H.; Takechi, S. A New Amidoalkynylation
Using Alkynlyzinc Reagent. Tetrahedron Lett. 1988, 29, 5391-
5394 and references therein. (b) Overman, L. E.; Osawa, T. A
Convenient Synthesis of 4-Unsubstituted â-Lactams. J . Am.
Chem. Soc. 1985, 107, 1698-1701 and references therein.
(28) (a) Tian, Q.; Nayyar, N. K.; Babu, S.; Chen, L.; Tao, J .; Lee, S.;
Tibbetts, A.; Moran, T.; Liou, J .; Guo, M.; Kennedy, T. An
Efficient Synthesis of a Key Intermediate for the Preparation
of the Rhinovirus Protease Inhibitor AG7088 via Asymmetric
Dianionic Cyanomethylation of N-Boc-L-(+)-Glutamic Acid Di-
methyl Ester. Tetrahedron Lett. 2001, 42, 6807-6809. (b) Tian,
Q.; Nayyar, N. K.; Babu, S.; Tao, J .; Moran, T. J .; Dagnino, R.,
J r.; Remarchuk, T. P.; Melnick, M. J .; Mitchell, L. J ., J r.; Bender,
S. L. WO 01/14329, 2001.
(29) Okuma, K.; Tachibana, Y.; Sakata, J .-i.; Komiya, T.; Kaneko,
I.; Komiya, Y.; Yamasaki, Y.; Yamamoto, S.-i.; Ohta, H. Reac-
tions of Wittig Reagents With Episulfides or Elemental Sulfur.
Bull. Chem. Soc. J pn. 1988, 61, 4323-4327 and references
therein.
(30) Webber, S. E.; Okano, K.; Little, T. L.; Reich, S. H.; Xin, Y.;
Fuhrman, S. A.; Matthews, D. A.; Love, R. A.; Hendrickson, T.
F.; Patick, A. K.; Meador, J . W., III; Ferre, R. A.; Brown, E. L.;
Ford, C. E.; Binford, S. L.; Worland, S. T. Tripeptide Aldehyde
(32) 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: Trifluoromethyl Substitution Pro-
vides a Global Protective Effect Against Hepatic Metabolism.
J . Med. Chem. 1995, 38, 1355-1371 and references therein.
(33) (a) Artursson, P.; Palm, K.; Luthman, K. Caco-2 Monolayers in
Experimental and Theoretical Predictions of Drug Transport.
Adv. Drug Deliv. Rev. 2001, 46, 27-43. (b) Yee, S. In Vitro
Permeability Across Caco-2 Cells (Colonic) can Predict In Vivo
(Small Intestinal) Absorption in Man-Fact or Myth. Pharm. Res.
1997, 14, 763-766. (c) Delie, F.; Rubas, W. A Human Colonic
Cell Line Sharing Similarities With Enterocytes as a Model to
Examine Oral Absorption: Advantages and Limitations of the
Caco-2 Model. Crit. Rev. Ther. Drug Car. Sys. 1997, 14, 221-
286. (d) Audus, K. L.; Bartel, R. L.; Hidalgo, I. J .; Borchardt, R.
T. The Use of Cultured Epithelial and Endothelial Cells for Drug
Transport and Metabolism Studies. Pharm. Res. 1990, 7,
435-451.
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